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List features of the Kaufman Oculocerebrofacial Syndrome. | Clinical features of the Kaufman Oculocerebrofacial Syndrome include hypotonia, developmental delay, intellectual disability, low cholesterol levels, microcephaly, long narrow face, ocular anomalies, and long thin hands and feet. | Two unrelated Mexican girls, aged 14 months and 6 years respectively, with
Kaufman oculocerebrofacial syndrome, are reported. Both showed psychomotor
retardation, microcephaly, blepharophimosis and delayed growth as the main
features; the infant also presented preauricular tags and large clitoris.
Comparative analysis with previous cases reveals a heterogeneous syndrome in
which the micro-brachycephaly, the mongoloid slanted eyes with different
anomalies, the micrognathia and the neonatal respiratory distress are the most
typical characteristics of this mental retardation syndrome. A pair of sisters was ascertained for multiple congenital defects, including
marked craniofacial dysmorphisms with blepharophimosis, and severe psychomotor
delay. Two novel compound heterozygous mutations in UBE3B were identified in
both the sisters by exome sequencing. These mutations include c.1A>G, which
predicts p.Met1?, and a c.1773delC variant, predicted to cause a frameshift at
p.Phe591fs. UBE3B encodes a widely expressed protein ubiquitin ligase E3B,
which, when mutated in both alleles, causes Kaufman oculocerebrofacial syndrome.
We report on the thorough clinical examination of the patients and review the
state of art knowledge of this disorder. |
What is the role of DNA Repair Cofactors ATMIN and NBS1? | The DNA double-strand break signaling kinase ATM and its cofactor NBS1 are required during T cell development and for the maintenance of genomic stability. The role of a second ATM cofactor, ATMIN (also known as ASCIZ) in T cells is much less clear, and whether ATMIN and NBS1 function in synergy in T cells is unknown. | Proper development of the immune system is an intricate process dependent on
many factors, including an intact DNA damage response. The DNA double-strand
break signaling kinase ATM and its cofactor NBS1 are required during T cell
development and for the maintece of genomic stability. The role of a second
ATM cofactor, ATMIN (also known as ASCIZ) in T cells is much less clear, and
whether ATMIN and NBS1 function in synergy in T cells is unknown. Here, we
investigate the roles of ATMIN and NBS1, either alone or in combination, using
murine models. We show loss of NBS1 led to a developmental block at the
double-positive stage of T cell development, as well as reduced TCRα
recombination, that was unexpectedly neither exacerbated nor alleviated by
concomitant loss of ATMIN. In contrast, loss of both ATMIN and NBS1 enhanced DNA
damage that drove spontaneous peripheral T cell hyperactivation, proliferation
as well as excessive production of proinflammatory cytokines and chemokines,
leading to a highly inflammatory environment. Intriguingly, the disease causing
T cells were largely proficient for both ATMIN and NBS1. In vivo this resulted
in severe intestinal inflammation, colitis and premature death. Our findings
reveal a novel model for an intestinal bowel disease phenotype that occurs upon
combined loss of the DNA repair cofactors ATMIN and NBS1. |
The MMR vaccine protects against what 3 viruses? | The MMR vaccine provides immunity to measles, mumps and rubella. | Measles, mumps, rubella (MMR) vaccine is a live vaccine preparation containing
attenuated strains of all 3 viruses. MMR vaccine is widely used throughout the
world, with the US having the widest experience with the vaccine. In countries
where the vaccine has been introduced successfully, significant reductions in
all 3 diseases for which it is protective have occurred. The vaccine has been
shown to be highly immunogenic, with seroconversion rates of 95 to 100% being
achieved for each of the 3 component vaccines. This immunity appears to be
long-lasting and may even be lifelong. Minor adverse effects may occur
approximately 1 week after immunisation. Rarely, mumps vaccine-induced
meningitis (milder than that associated with wild mumps virus) may occur, its
frequency varying with the strain of attenuated mumps virus contained in any
particular vaccine. Clinically, the vaccine is indicated for infants aged
between 12 and 15 months, and should be administered by intramuscular or deep
subcutaneous injection. A few specific contraindications exist, including a
genuine hypersensitivity to eggs, and to the aminoglycoside antibiotics
kanamycin and neomycin. An increasing number of countries are now adopting a
2-stage MMR policy in an attempt to prevent epidemics among those who remain
unprotected, and to move towards eventual disease eradication. Uptake rates for the combined measles, mumps and rubella (MMR) vaccine have been
below the required 95% in the UK since a retracted and discredited article
linking the MMR vaccine with autism and inflammatory bowel disease was released
in 1998. This study undertook semi-structured telephone interviews among parents
or carers of 47 unvaccinated measles cases who were aged between 13 months and 9
years, during a large measles outbreak in Merseyside. Results showed that
concerns over the specific links with autism remain an important cause of
refusal to vaccinate, with over half of respondents stating this as a reason. A
quarter stated child illness during scheduled vaccination time, while other
reasons included general safety concerns and access issues. Over half of
respondents felt that more information or a discussion with a health
professional would help the decision-making process, while a third stated
improved access. There was clear support for vaccination among respondents when
asked about current opinions regarding MMR vaccine. The findings support the
hypothesis that safety concerns remain a major barrier to MMR vaccination, and
also support previous evidence that experience of measles is an important
determit in the decision to vaccinate. |
List cardinal features of the Triple A syndrome. | Triple A syndrome, also known as Allgrove syndrome, is a rare disease, and presents mainly in children. Its cardinal symptoms are achalasia, alacrima, and adrenal insufficiency. | The triple A syndrome or Allgrove syndrome (MIM*231550) is characterized by
adrenocorticotropic hormone (ACTH) resistant Adrenal insufficiency, Achalasia of
the cardia and Alacrima. In addition to the main features, patients frequently
suffer from neurological disturbances. Dermatological abnormalities such as
palmoplantar hyperkeratosis as well as other signs like short stature,
microcephaly and osteoporosis point to the multisystemic character of the
disorder. The molecular defect of the autosomal recessively inherited triple A
syndrome is not known. We initially performed a systematic genome linkage scan
in eight triple A families and were able to map the syndrome to a 6 cM interval
on human chromosome 12q13 near the type II keratin gene cluster. A refinement of
the triple A critical region was achieved by detailed haplotype analysis in a
further 37 families from different ethnic backgrounds. There was no indication
of genetic heterogeneity. The achalasia-alacrima (AA) syndrome which has been
defined as a distinct clinical entity (MIM 200440) is most likely a variant of
the triple A syndrome as shown by haplotype analysis in three AA families. We
constructed a high-resolution BAC/PAC-based transcript map of the region which
will greatly facilitate the identification of the triple A syndrome gene. The
considerable intra- and interfamilial variability of the severity of the
disorder implies a variable expression of an impaired pleiotropically acting
gene. The triple A or Allgrove syndrome is an autosomal-recessive disease (MIM*231550)
characterized by the triad of achalasia, alacrima and adrenocorticotropic
hormone (ACTH)-resistant adrenal insufficiency. Associated features of the
syndrome are neurological and dermatological abnormalities. Until the discovery
of the AAAS gene as the responsible gene in triple A syndrome, the diagnosis was
based on characteristic clinical features. Here we present the clinical and
molecular genetic data which demonstrated the marked phenotypic variability in
three unrelated patients with triple A syndrome. The final diagnosis of triple A
syndrome was confirmed by molecular analysis. In one patient with isolated
achalasia, the diagnosis of triple A syndrome could only be made on the basis of
the molecular genetic analysis of the AAAS gene. We therefore suggest that the
diagnosis of triple A syndrome should be considered in patients who exhibit only
one or two of the main symptoms (i.e. alacrima, achalasia or adrenal
insufficiency). These patients require careful neurological investigation, and
mutation analysis of the AAAS gene should be performed. BACKGROUND: Triple-A syndrome (Allgrove syndrome) is an autosomal recessive
disorder characterized by adrenal insufficiency, alacrima, achalasia, and -
occasionally - autonomic instability. Mutations have been found in the AAAS gene
on 12q13.
CASE PRESENTATION: We present the case of a 12 year-old boy with classic
systemic features of triple-A syndrome and several prominent ophthalmic
features, including: accommodative spasm, dry eye, superficial punctate
keratopathy, and pupillary hypersensitivity to dilute pilocarpine. MRI showed
small lacrimal glands bilaterally. DNA sequencing of PCR-amplified fragments
from the 16 exons of the AAAS gene revealed compound heterozygosity for a new,
out-of-frame 5-bp deletion in exon 15, c1368-1372delGCTCA, and a
previously-described nonsense mutation in exon 9, c938C>T, R286X.
CONCLUSIONS: In addition to known ophthalmic manifestations, triple-A syndrome
can present with accommodative dysregulation and ocular signs of autonomic
dysfunction. Allgrove syndrome (or triple-A syndrome) is a rare autosomal recessive disorder
characterized by alacrima, achalasia, adrenal insufficiency (glucocorticoid in
the majority of cases) and autonomic/neurological abnormalities. This disease is
now known to be caused by mutation in the AAAS gene located on chromosome 12q13.
Diagnosis should be readily available when the full-blown features are there,
but it becomes less apparent when presentation is atypical or in the evolving
process. We present a brother and sister (12 and 19 y old, respectively) born to
consanguineous parents of Palestinian origin with Allgrove syndrome. The index
patient was erroneously diagnosed to be a case of familial dysautonomia before
the diagnosis of adrenal insufficiency was made at the age of 7.5 y, while his
elder sister had only alacrima from birth and developed achalasia at the age of
15 y. She started to develop early evidence of adrenal disease at the age of 19
y. Both of them had neuroautonomic dysfunction. The diagnosis of Allgrove
syndrome was confirmed in these two patients by studying the gene mutation in
the family. The sequencing of the AAAS gene in the two patients identified a
novel homozygous mutation within intron 5 (IVS5+1G-->A). Both parents as well as
all three other children were heterozygous for the same mutation.
CONCLUSION: These two cases illustrate the heterogenous nature and the
intrafamilial phenotypic variability of Allgrove syndrome. OBJECTIVE: Allgrove syndrome is a rare autosomal recessive disorder
characterized by the triad of adrenal insufficiency, achalasia and alacrima and
many cases have multi-systems disorder: endocrine, gastrointestinal tract, eyes
and nervous system. This syndrome is also known as
achalasia-addisonianism-alacrima syndrome or triple A syndrome. Allgrove
syndrome is now known to be caused by mutations of AAAS gene encoding the aladin
protein. In the present paper, we report a Chinese mainland girl with Allgrove
syndrome with mutations in the AAAS gene.
METHOD: The patient was a 7-year-old girl complained of coma and dark skin; she
was treated as Addison disease for 2 years and had vomiting for 9 months before
the second admission. Gene analysis was performed after extracting genomic DNA
by amplification and sequencing of the specific fragments of AAA gene.
RESULTS: The patient was confirmed to have adrenal insufficiency at the age of 5
years and 6 months. During the second hospitalization, she was found to have a
remarkable brisk reflexion, bilateral optic nerve atrophy, alacrima and
achalasia besides ACTH resistance. The girl was born to consanguineous parents.
Based on these findings, she was diagnosed as having Allgrove syndrome. Mutation
analysis revealed a novel homozygous deletion of a single G, c.771delG, in exon
8 of the AAAS gene. This frame shift mutation was predicted to create a
premature stop codon at locus 290, p.R258GfsX33, leading to a truncated and
non-functioning aladin protein. Both the parents were heterozygous for the
mutation.
CONCLUSION: The clinical manifestations and AAAS gene mutations analysis
confirmed the diagnosis of Allgrove syndrome. Gene analysis indicated that this
syndrome is an autosomal recessive inherent disorder. ALADIN is significant for
the normal cell function. When compared with reported cases, it seems that there
are no remarkable relation between gene mutation loci and clinical
manifestations in Allgrove syndrome. BACKGROUND: Triple A syndrome, also known as Allgrove syndrome, is a rare
autosomal recessive disorder characterized by three cardinal symptoms: adrenal
insufficiency due to ACTH insensitivity, achalasia and alacrima. Various
progressive neurological abnormalities and skin changes have been described in
association with the syndrome. The disease is caused by mutation in the AAAS
gene on chromosome 12q13. AAAS encodes a protein named ALADIN which is part of
the nuclear pore complex (NPC). The mislocalization of mutated ALADIN proteins
in the cytoplasm and/or the nucleus results in an impaired protein function.
Phenotypes of previously reported patients with triple A syndrome varied within
and between affected families so that no genotype-phenotype could be
established.
METHODS: Genetic analysis was performed in two unrelated patients, their parents
and one sister. AAAS coding sequences including exon-intron boundaries were
amplified and sequenced using an ABI 3100 sequencing machine.
PATIENTS: We present two unrelated Swiss patients with triple A syndrome
demonstrating similar phenotypic characteristics. Both showed a progression of
the disease presenting with adrenal insufficiency and alacrima in early
childhood. At the age between 30-40 years they developed symptomatic achalasia.
The pattern and severity of progressive neurological and autonomic dysfunction
was comparable. In both patients molecular genetic analysis revealed an
identical novel homozygous mutation (c.618delC, p.Ser207fs) in the AAAS gene.
CONCLUSION: Recent genotype/phenotype studies showed a marked inter- and
intrafamiliar variability in triple A syndrome. Here we present a rather tight
genotype/phenotype correlation in two unrelated patients carrying the identical
novel p.Ser207fs mutation in the AAAS gene. Triple-A or Allgrove syndrome is a rare multisystem disease classically
associated with esophageal achalasia, adrenal insufficiency and alacrima. Here,
we describe the poorly understood neurological characteristics often associated
with this condition, through the clinical and electrophysiological analysis of
eight patients. All patients were genetically confirmed and had a mutation in
the ALADIN gene. They all displayed a classical picture of Triple-A syndrome:
all suffered from achalasia and alacrima and half of them from adrenal
insufficiency. However, all harbored a neurological picture characterized by a
recognizable pattern of peripheral neuropathy. Other neurological features
included cognitive deficits, pyramidal syndrome, cerebellar dysfunction,
dysautonomia, neuro-ophthalmological signs and bulbar and facial symptoms. This
neurological picture was prominent in all patients and misled the initial
diagnosis in six of them, which had a late onset. We then review the previous
neurological reports of this disease, to improve the understanding of this rare
condition. Diagnosis of late-onset Triple-A syndrome is difficult when the
clinical picture is mainly neurological and when endocrine or gastrointestinal
signs are minor. The characteristics of the peripheral neuropathy, among other
neurological signs, can be of help. Triple A syndrome (alacrima, achalasia, adrenal failure, progressive
neurodegenerative disease) is caused by mutations in the AAAS gene which encodes
the protein alacrima achalasia adrenal insufficiency neurologic disorder
(ALADIN). Our investigation suggests that low bone mineral density (BMD) for
age/osteoporosis could be a common but overlooked symptom of unexplained
etiology in this rare multisystemic disease.
INTRODUCTION: The purpose of this study is to evaluate incidence and etiology of
BMD for age/osteoporosis, a possibly overlooked symptom in triple A syndrome.
METHODS: Dual-energy X-ray absorptiometry (DXA) of the femoral neck, total hip,
lumbar spine, and radius, bone turnover markers, minerals, total alkaline
phosphatase (ALP), 25-hydroxy vitamin D (25-OHD), 1,25-dihydroxy vitamin D
(1,25-OH2D), intact parathyroid hormone (PTH), and adrenal androgens
(dehydroepiandrosterone sulfate (DHEAS) and androstenedione) were measured in
five male and four female patients.
RESULTS: At time of diagnosis, low BMD for age was suspected on X-ray in seven
of nine patients aged 2-11 years (not performed in two patients); normal levels
of minerals and ALP were found in nine patients and low levels of adrenal
androgens in eight patients (not measured in one patient). Reevaluation
5-35 years after introduction of 12 mg/m(2)/day hydrocortisone showed low BMD
for age in two children, osteopenia in one, and osteoporosis in six adults.
Normal levels of minerals, ALP, PTH, 1,25-OH2D, procollagen type 1, crosslaps,
and osteocalcin were found in all patients. Low levels of adrenal androgens were
found in all and 25OHD deficiency in six patients. Body mass index was <25 % for
age and sex in eight of nine patients.
CONCLUSION: Low BMD for age/osteoporosis in our patients probably is not a
result of glucocorticoid therapy but could be the consequence of low level of
adrenal androgens, neurological impairment causing physical inactivity,
inadequate sun exposure, and protein malnutrition secondary to achalasia.
Considering ubiquitous ALADIN expression, low BMD/osteoporosis may be a primary
phenotypic feature of the disease. Besides optimizing glucocorticoid dose,
physical activity, adequate sun exposure, appropriate nutrition, and vitamin D
supplementation, therapy with DHEA should be considered. Allgrove (Triple A) syndrome is a rare autosomal recessive disorder
characterized by cardinal features of adrenal insufficiency due to
adrenocorticotropic hormone (ACTH) resistance, achalasia, and alacrimia. It is
frequently associated with neurological manifestations like polyneuropathy.
Since its first description by Allgrove in 1978, approximately 100 cases have
been reported in the literature. Here we report an 18-year-old boy diagnosed as
having Allgrove syndrome, with ACTH resistant adrenal insufficiency, achalasia,
alacrimia, and severe motor polyneuropathy. Alacrimia was the earliest feature
evident at the age of 8 years. He presented with achalasia and adrenal
insufficiency at 12 and 18 years respectively and developed neurological
symptoms in the form of severe muscle wasting at the age of 15 years. Patients
with Allgrove syndrome usually manifest adrenal insufficiency and achalasia
during first decade of life. Our patient manifested adrenal insufficiency and
achalasia in the second decade and manifested neurological dysfunction before
adrenal dysfunction. BACKGROUND: Oesophageal achalasia is well-recognized but relatively rare in
children, occasionally appearing as the "triple A" syndrome (with adrenal
insufficiency and alacrima). Treatment modalities, as in adult practice, are not
curative, often needing further interventions and spurring the search for better
management. The outcome for syndromic variants is unknown. We sought to define
the efficacy of treatments for children with achalasia with and without triple A
syndrome.
METHODS: We conducted a retrospective analysis of presentation and outcomes for
42 children with achalasia presenting over three decades to a major pediatric
referral center. Long term impact of the diagnosis was assessed by
questionnaire.
RESULTS: We identified 42 children including six with triple A syndrome. The
median overall age at diagnosis was 10.8 years and median follow-up 1593 days.
Initial Heller myotomy in 17 required further interventions in 11 (65%), while
initial treatment with botulinum toxin (n = 20) was ultimately followed by
myotomy in 17 (85%). Ten out of 35 patients who underwent myotomy required a
repeat myotomy (29%). Patients with triple A syndrome developed symptoms
earlier, but had delayed diagnosis, were more underweight at diagnosis and at
last follow up. Questionnaire results suggested a significant long term
deleterious impact on the quality of life of children and their families.
CONCLUSION: Many children with achalasia relapse after initial treatment,
undergoing multiple, different procedures, despite which symptoms persist and
impact on quality of life. Symptoms develop earlier in patients with triple A
syndrome, but the diagnosis is delayed and this has substantial nutritional
impact. INTRODUCTION: Allgrove syndrome (AS) is a rare autosomal recessive disorder
characterized by achalasia cardia, alacrimia, and adrenocorticotropic
hormone-resistant adrenal insufficiency which is sometimes associated with
autonomic dysfunction. It has also been referred to as the triple A syndrome in
view of the cardinal symptoms described above. First described by Allgrove et al
in 1978, the disorder usually presents mostly during the first decade of life.
These patients have the threat of adrenal crisis, shock, and hypoglycemia and
are usually on steroid supplementation.
CASE REPORT: The anesthesiologist's encounter with such patients, although rare,
is mostly for repair of the achalasia cardia. We thus report a similar case of
AS in a 2-year-old girl who was scheduled to undergo Heller myotomy along with
the preoperative evaluation and intraoperative management of the same.
CONCLUSION: Being aware of the pathophysiology of AS gives useful insight about
the disease and successful perioperative management in the form of the triple S
strategy, that is, stress dose of steroids, slow induction and positioning, and
finally maintece of stable hemodynamics and euglycemia. BACKGROUND Allgrove syndrome, or triple "A" syndrome (3A syndrome), is a rare
autosomal recessive syndrome with variable phenotype, and an estimated
prevalence of 1 per 1,000,000 individuals. Patients usually display the triad of
achalasia, alacrima, and adrenocorticotropin (ACTH) insensitive adrenal
insufficiency, though the presentation is inconsistent. CASE REPORT Here, the
authors report a case of Allgrove syndrome in a pediatric patient with delayed
diagnosis in order to raise awareness of this potentially fatal disease as a
differential diagnosis of alacrima. CONCLUSIONS The prevalence of Allgrove
syndrome may be much higher as a result of underdiagnosis and missed diagnosis
due to the variable presentation and sudden unexplained childhood death from
adrenal crisis. The authors review the characteristic symptoms of Allgrove
syndrome in relation to the case study in order to avoid missed or delayed
diagnosis, potentially decreasing morbidity, and mortality in those affected by
this disease. |
Describe the applicability of Basset in the context of deep learning | Basset is an open source package which applies CNNs to learn the functional activity of DNA sequences from genomics data. Basset was trained on a compendium of accessible genomic sites mapped in 164 cell types by DNase-seq, and demonstrated greater predictive accuracy than previous methods. Basset predictions for the change in accessibility between variant alleles were far greater for Genome-wide association study (GWAS) SNPs that are likely to be causal relative to nearby SNPs in linkage disequilibrium with them. With Basset, a researcher can perform a single sequencing assay in their cell type of interest and simultaneously learn that cell's chromatin accessibility code and annotate every mutation in the genome with its influence on present accessibility and latent potential for accessibility. Thus, Basset offers a powerful computational approach to annotate and interpret the noncoding genome. | The complex language of eukaryotic gene expression remains incompletely
understood. Despite the importance suggested by many noncoding variants
statistically associated with human disease, nearly all such variants have
unknown mechanisms. Here, we address this challenge using an approach based on a
recent machine learning advance-deep convolutional neural networks (CNNs). We
introduce the open source package Basset to apply CNNs to learn the functional
activity of DNA sequences from genomics data. We trained Basset on a compendium
of accessible genomic sites mapped in 164 cell types by DNase-seq, and
demonstrate greater predictive accuracy than previous methods. Basset
predictions for the change in accessibility between variant alleles were far
greater for Genome-wide association study (GWAS) SNPs that are likely to be
causal relative to nearby SNPs in linkage disequilibrium with them. With Basset,
a researcher can perform a single sequencing assay in their cell type of
interest and simultaneously learn that cell's chromatin accessibility code and
annotate every mutation in the genome with its influence on present
accessibility and latent potential for accessibility. Thus, Basset offers a
powerful computational approach to annotate and interpret the noncoding genome. |
Does the Abelson-related gene (ARG) gene encode for a serine kinase? | No, the ARG gene encodes for a nonreceptor tyrosine kinase. | Arg encodes a protein highly related to the c-abl gene product with regard to
overall structural architecture as well as the amino acid sequences of their
tyrosine kinase, and src-homologous 2 and 3 domains. The two genes form a
distinct subfamily of non-receptor tyrosine kinases and share a common homolog
in Drosophila. In this study we characterized the arg protein product by
expression of its coding sequence in bacteria. The recombit arg protein was
detected in bacterial lysates by immunoblotting and exhibited a molecular mass
of 145 kDa. Phosphoamino acid analysis of the arg gene product following an
immune complex autokinase reaction revealed tyrosine phosphorylation and
established that it possesses tyrosine kinase activity. High-titer antibody
capable of detecting the cellular arg gene product was generated by expressing a
carboxy-terminal segment of arg in bacteria and using the recombit protein as
an immunogen. The arg gene product was identified in cultured human cells as a
145 kDa protein that exhibited autokinase activity. Analysis of arg expression
in murine tissues revealed that arg, like c-abl, is widely expressed, further
extending the similarities between the two genes, and suggesting that arg
probably functions in signaling pathways fundamental to many cell types. We studied the relationship of direct karyotypes, determined at diagnosis and
remission, to Abelson-related tyrosine kinase activity and the cytogenetic
features of erythroid and myeloid colonies derived from remission marrow of six
children with acute lymphoblastic leukemia (ALL). These patients had either the
characteristic Philadelphia chromosome (Ph1) [t(9;22)(q34;q11)] or
cytogenetically similar variants with a 22q11 breakpoint but no detectable
cytogenetic involvement of 9q34. The findings suggested two distinct subtypes of
ALL: one defined by t(9;22)(q34;q11) and expression of P185BCR-ABL tyrosine
kinase and one with variant karyotypes and no P185BCR-ABL expression. The former
comprises cases with Ph1 + marrow cells and Ph1 + erythroid and (or) myeloid
colonies in remission marrow and others in which the t(9;22) is undetectable in
remission marrow cells. In the latter subgroup, the disease may reflect more
extreme mosaicism with a similar stem cell that is cytogenetically undetectable.
Variant karyotypes included a del(22)(q11) in one patient and a t(6;22;15;9)
(q21;q11;q?22;q21) in another; in both instances, the maligt blast cells
lacked P185BCR-ABL expression. Thus ALL with t(9;22)(q34;q11) should be
distinguished from ALL with other involvement of the 22q11 breakpoint by
molecular studies including protein expression. The diversity of karyotypic
findings in cases with involvement of 22q11 suggests at least two mechanisms of
leukemogenesis in patients with ALL defined by this breakpoint. The products of the human ARG gene and the human ABL gene characterize the
Abelson family of non-receptor tyrosine protein kinases. Both genes are
ubiquitously expressed. The interactions of these two similar protein kinases
are still not well known, although it has been suggested that they could
cooperate, with redundant actions, to provide intracellular signals in the
cells. Lymphopenia occurs in mice with homozygous disruption of c-abl,
indicating that in certain tissues Arg is unable to substitute c-abl functions.
In B and T lymphoid cell lines at different stages of differentiation, we
studied, by a reverse transcriptase-competitive polymerase chain reaction and
Western blotting, Arg and c-abl in order to evaluate whether the expression
pattern of the two genes could give insight as to why they do not exhibit
overlapping roles in lymphocytes and whether the product levels of the two genes
are related to lymphoid differentiation. The data showed that their expression
is differently modified in lymphoid B cell lines. The highest Arg transcript and
protein levels are in the mature B cells. The human ABL2 (or ARG) gene codes for a nonreceptor tyrosine kinase is involved
in translocation with the ETV6 gene in human leukemia and has an altered
expression in several human carcinomas. Two isoforms of Arg with different
N-termini (1A and 1B) have been described. The C-terminal domain of Arg contains
two F-actin-binding sequences that perform a number of actions related to cell
morphology and motility by interacting with actin filaments. We have identified
different-sized specific cDNAs in hematopoietic, epithelial, nervous, and
fibroblastic cells by means of the reverse transcription (RT)-polymerase chain
reaction (PCR) analysis of human Arg mRNA. Some of these cDNAs showed an
adjunctive alternative splice event involving the 63 bp sequence of exon II,
thus leading to four cDNA types with different N-termini: 1A long and short, and
1B long and short. Other cDNAs lacked a 309 bp sequence in the last exon
involving one of the C-terminal F-actin binding domains, thus giving rise to two
cDNA types: C-termini long and short. Quantified by real-time PCR-quantitative
RT-PCR-these Arg transcript isoforms have specific expression patterns not only
in different normal and tumor cell types, but also during cell differentiation
and growth arrest. These isoforms maintained the open reading frames, and eight
putative proteins were predicted. The different C-termini isoforms seem to
retain the same quantitative reciprocal ratio of their respective transcripts.
The Arg protein isoforms with different C-terminal actin-binding domains and
different N-termini might have specific cellular localizations/concentrations,
and differently regulated catalytic activity with different implications in
normal and neoplastic cells. To investigate the expression profile of protein tyrosine kinases (PTKs) in
normal human epidermal keratinocytes (NHEK) in response to UVA and UVB we
employed a reversed transcriptase polymerase chain reaction (PCR) approach using
degenerate primers derived from the conserved catalytic domain of PTKs.
Quantitative real-time PCR with specific primers was used to confirm the
influence of UV on the expression of the identified PTKs. Arg (Abelson-related
gene, Abl2) was the PTK with the highest prevalence (30% of all PTKs) and UVA
led to a further induction of Arg expression reaching nine-fold mRNA baseline
expression at 17 h after irradiation. UVB was followed by an initial
downregulation and a subsequent increase in Arg mRNA reaching five-fold baseline
levels after 24 h. We conclude that UVA and UVB differentially modify the
expression of PTKs in NHEK, and that Arg appears to have a major role in the
response of keratinocytes to UV. These results provide a basis for further
studies of PTK in UV-induced signaling that regulates protective responses, cell
growth and carcinogenesis in the skin. The human Arg (Abl2) nonreceptor tyrosine kinase has a role in cytoskeletal
rearrangements by its C-terminal F-actin- and microtubule-binding sequences. We
have previously identified Arg transcripts with different 5'- and 3'-ends, named
respectively long and short 1A and 1B (1AL, 1AS, 1BL, 1BS) and long and short
C-termini (CTL and CTS), that have different expression patterns in various cell
types. The combination of the different ends permits to predict eight putative
full-length Arg transcripts and corresponding proteins. By Reverse
Transcription-Long PCR we show here that all eight full-length transcripts are
endogenously expressed in Caki-1 cells and the two bands, approximately 10 kDa
different, shown by 1-D Western blots of Hek293T and Caki-1 lysates correspond
to the full-length Arg protein isoforms with different C-termini. 2-D Western
blot analysis evidenced different high molecular weight and slight acidic
specific spots in Hek293T and Caki-1 lysates. The cellular localization of two
Arg isoforms (1BLCTL and 1BLCTS) transfected in Caki-1 and Hek293T cells was
cytoplasmic, and some differences in cytoskeleton interactions have been
evidenced. Moreover, in Hek293T cells only the transfected 1BLCTS isoform gives
rise to a large intracytoplasmic cylindrical structure containing
phalloidin-positive amorphous actin aggregates. The presence of eight
full-length Arg isoforms with different cellular expression may imply a diverse
functional role in normal and neoplastic cells. The CCAAT/enhancer-binding protein beta (C/EBPbeta) is a critical transcription
factor that regulates gene expression during numerous biological processes,
including differentiation, metabolism, homeostasis, proliferation,
tumorigenesis, inflammation, and apoptosis. In this study, interactions between
C/EBPbeta and either the Abelson murine leukemia viral oncogene homolog 1
(c-Abl) or the Abl-related gene (Arg) were demonstrated in vitro and in vivo
with a direct binding assay and by co-immunoprecipitation, respectively. The Y79
amino acid residue of C/EBPbeta was phosphorylated by c-Abl or Arg. The
phosphorylation of C/EBPbeta resulted in an increased C/EBPbeta stability and a
potentiation of C/EBPbeta transcription activation activity in cells. Expression
of the C/EBPbeta(Y79F) mutant in HEK293, and K562, and in other cell lines,
resulted in less of a delay in the cell cycle compared to the wild type
C/EBPbeta; furthermore, the C/EBPbeta (Y79F) mutant induced an increased
apoptosis compared to the wild type C/EBPbeta. These findings suggest that the
c-Abl family non-receptor tyrosine kinases have a role in the regulation of the
C/EBPbeta transcription factor. ABL2/ARG (ABL-related gene) belongs to the ABL (Abelson tyrosine-protein kinase)
family of tyrosine kinases. ARG plays important roles in cell morphogenesis,
motility, growth and survival, and many of these biological roles overlap with
the cellular functions of the ABL kinase. Chronic myeloid leukemia (CML) is
associated with constitutive ABL kinase activation resulting from fusion between
parts of the breakpoint cluster region (BCR) and ABL1 genes. Similarly, fusion
of the ETV6 (Tel) and ARG genes drives some forms of T-cell acute lymphoblastic
leukemia (T-ALL) and acute myeloid leukemia (AML). Dasatinib is a tyrosine
kinase inhibitor used for the treatment of CML by inhibiting ABL, and while it
also inhibits ARG, there is currently no structure of ARG in complex with
dasatinib. Here, the co-crystal structure of the mouse ARG catalytic domain with
dasatinib at 2.5 Å resolution is reported. Dasatinib-bound ARG is found in the
DFG-in conformation although it is nonphosphorylated on the activation-loop
tyrosine. In this structure the glycine-rich P-loop is found in a relatively
open conformation compared with other known ABL family-inhibitor complex
structures. |
What organism causes tularemia? | Francisella tularensis, the agent of tularemia, is a Gram-negative coccobacillus primarily pathogen for animals and occasionally for humans. F. tularensis is the causative agent of zoonotic tularemia. | Francisella tularensis is a highly virulent bacterium that causes tularemia, a
disease that is often fatal if untreated. A live vaccine strain (LVS) of this
bacterium is attenuated for virulence in humans but produces lethal disease in
mice. F. tularensis has been classified as a Category A agent of bioterrorism.
Despite this categorization, little is known about the components of the
organism that are responsible for causing disease in its hosts. Here, we report
the deletion of a well-characterized lipoprotein of F. tularensis, designated
LpnA (also known as Tul4), in the LVS. An LpnA deletion mutant was comparable to
the wild-type strain in its ability to grow intracellularly and cause lethal
disease in mice. Additionally, mice inoculated with a sublethal dose of the
mutant strain were afforded the same protection against a subsequent lethal
challenge with the LVS as were mice initially administered a sublethal dose of
the wild-type bacterium. The LpnA-deficient strain showed an equivalent ability
to promote secretion of chemokines by human monocyte-derived macrophages as its
wild-type counterpart. However, recombit LpnA potently stimulated primary
cultures of human macrophages in a Toll-like receptor 2-dependent manner.
Although human endothelial cells were also activated by recombit LpnA, their
response was relatively modest. LpnA is clearly unnecessary for multiple
functions of the LVS, but its inflammatory capacity implicates it and other
Francisella lipoproteins as potentially important to the pathogenesis of
tularemia. Francisella tularensis is a highly infectious intracellular bacterium that
causes the fulminating disease tularemia, which can be transmitted between
mammals by arthropod vectors. Genomic studies have shown that the F. tularensis
has been undergoing genomic decay with the most virulent strains having the
lowest number of functional genes. Entry of F. tularensis into macrophages is
mediated by looping phagocytosis and is associated with signalling through Syk
tyrosine kinase. Within macrophages and arthropod-derived cells, the
Francisella-containing phagosome matures transiently into an acidified late
endosome-like phagosome with limited fusion to lysosomes followed by rapid
bacterial escape into the cytosol within 30-60 min, and bacterial proliferation
within the cytosol. The Francisella pathogenicity island, which potentially
encodes a putative type VI secretion system, is essential for phagosome
biogenesis and bacterial escape into the cytosol within macrophages and
arthropod-derived cells. Initial sensing of F. tularensis in the cytosol
triggers IRF-3-dependent IFN-beta secretion, type I IFNR-dependent signalling,
activation of the inflammasome mediated by caspase-1, and a pro-inflammatory
response, which is suppressed by triggering of SHIP. The past few years have
witnessed a quantum leap in our understanding of various aspects of this
organism and this review will discuss these remarkable advances. Tularemia, caused by the bacterium Francisella tularensis, where F. tularensis
subspecies holarctica has long been the cause of endemic disease in parts of
northern Sweden. Despite this, our understanding of the natural life-cycle of
the organism is still limited. During three years, we collected surface water
samples (n = 341) and sediment samples (n = 245) in two areas in Sweden with
endemic tularemia. Real-time PCR screening demonstrated the presence of F.
tularenis lpnA sequences in 108 (32%) and 48 (20%) of the samples, respectively.
The 16S rRNA sequences from those samples all grouped to the species F.
tularensis. Analysis of the FtM19InDel region of lpnA-positive samples from
selected sampling points confirmed the presence of F. tularensis subspecies
holarctica-specific sequences. These sequences were detected in water sampled
during both outbreak and nonoutbreak years. Our results indicate that diverse F.
tularensis-like organisms, including F. tularensis subsp. holarctica, persist in
natural waters and sediments in the investigated areas with endemic tularemia. The tularemia-causing bacterium Francisella tularensis is a facultative
intracellular organism with a complex intracellular lifecycle that ensures its
survival and proliferation in a variety of mammalian cell types, including
professional phagocytes. Because this cycle is essential to Francisella
pathogenesis and virulence, much research has focused on deciphering the
mechanisms of its intracellular survival and replication and characterizing both
bacterial and host determits of the bacterium's intracellular cycle. Studies
of various strains and host cell models have led to the consensual paradigm of
Francisella as a cytosolic pathogen, but also to some controversy about its
intracellular cycle. In this review, we will detail major findings that have
advanced our knowledge of Francisella intracellular survival strategies and also
attempt to reconcile discrepancies that exist in our molecular understanding of
the Francisella-phagocyte interactions. Tularemia is a bacterial zoonotic disease that is caused by Francisella
tularensis and among the infectious reasons that cause fever of unknown origin
(FUO) in children. Typhoidal or pneumonic tularemia can manifest predomitly
as FUO. However, presentation of oropharyngeal tularemia as FUO is uncommon.
Here, we report a case of an 11-month-old infant with oropharyngeal tularemia
presenting as FUO. To the best of our knowledge, this clinical presentation of
oropharyngeal tularemia has not been previously reported in literature. Francisella tularensis causes disease (tularemia) in a large number of mammals,
including man. We previously demonstrated enhanced efficacy of conventional
antibiotic therapy for tularemia by postexposure passive transfer of immune sera
developed against a F. tularensis LVS membrane protein fraction (MPF). However,
the protein composition of this immunogenic fraction was not defined. Proteomic
approaches were applied to define the protein composition and identify the
immunogens of MPF. MPF consisted of at least 299 proteins and 2-D Western blot
analyses using sera from MPF-immunized and F. tularensis LVS-vaccinated mice
coupled to liquid chromatography-tandem mass spectrometry identified 24
immunoreactive protein spots containing 45 proteins. A reverse vaccinology
approach that applied labeling of F. tularensis LVS surface proteins and
bioinformatics was used to reduce the complexity of potential target immunogens.
Bioinformatics analyses of the immunoreactive proteins reduced the number of
immunogen targets to 32. Direct surface labeling of F. tularensis LVS resulted
in the identification of 31 surface proteins. However, only 13 of these were
reactive with MPF and/or F. tularensis LVS immune sera. Collectively, this use
of orthogonal proteomic approaches reduced the complexity of potential
immunogens in MPF by 96% and allowed for prioritization of target immunogens for
antibody-based immunotherapies against tularemia. Tularemia is a zoonosis caused by Francisella tularensis that can be transmitted
by several ways to human being and cause different clinical manifestations. We
report three clinical cases of tularemia with ulceroglandular presentation in
young males acquired during outdoor activities in Southern Belgium. Confirmation
of the diagnosis was established by serology. Only three cases of tularemia have
been reported in Belgium between 1950 and 2012 by the National Reference
Laboratory CODA-CERVA (Ref Lab CODA-CERVA) but re-emergence of tularemia is
established in several European countries and F. tularensis is also well known
to be present in animal reservoirs and vectors in Belgium. The diagnosis of
tularemia has to be considered in case of suggestive clinical presentation
associated with epidemiological risk factors. |
Is Prochlorococcus the most abundant photosynthetic organism? | Yes, the marine cyanobacterium Prochlorococcus is the smallest and most abundant photosynthetic organism on Earth. | The oceanic picoplankton Prochlorococcus - probably the most abundant
photosynthetic organism on our planet - can grow at great depths where light
intensity is very low. We have found that the chlorophyll-binding proteins in a
deep-living strain of this oxyphotobacterium form a ring around a trimer of the
photosystem I (PS I) photosynthetic reaction centre, a clever arrangement that
maximizes the capture of light energy in such dim conditions. The marine cyanobacterium Prochlorococcus is the most abundant photosynthetic
organism in oligotrophic regions of the oceans. The inability to assimilate
nitrate is considered an important factor underlying the distribution of
Prochlorococcus, and thought to explain, in part, low abundance of
Prochlorococcus in coastal, temperate, and upwelling zones. Here, we describe
the widespread occurrence of a genomic island containing nitrite and nitrate
assimilation genes in uncultured Prochlorococcus cells from marine surface
waters. These genes are characterized by low GC content, form a separate
phylogenetic clade most closely related to marine Synechococcus, and are located
in a different genomic region compared with an orthologous cluster found in
marine Synechococcus strains. This sequence distinction suggests that these
genes were not transferred recently from Synechococcus. We demonstrate that the
nitrogen assimilation genes encode functional proteins and are expressed in the
ocean. Also, we find that their relative occurrence is higher in the Caribbean
Sea and Indian Ocean compared with the Sargasso Sea and Eastern Pacific Ocean,
which may be related to the nitrogen availability in each region. Our data
suggest that the ability to assimilate nitrite and nitrate is associated with
microdiverse lineages within high- and low-light (LL) adapted Prochlorococcus
ecotypes. It challenges 2 long-held assumptions that (i) Prochlorococcus cannot
assimilate nitrate, and (ii) only LL adapted ecotypes can use nitrite. The
potential for previously unrecognized productivity by Prochlorococcus in the
presence of oxidized nitrogen species has implications for understanding the
biogeography of Prochlorococcus and its role in the oceanic carbon and nitrogen
cycles. The marine cyanobacterium Prochlorococcus is the smallest and most abundant
photosynthetic organism on Earth. In this Review, we summarize our understanding
of the diversity of this remarkable phototroph and describe its role in ocean
ecosystems. We discuss the importance of interactions of Prochlorococcus with
the physical environment, with phages and with heterotrophs in shaping the
ecology and evolution of this group. In light of recent studies, we have come to
view Prochlorococcus as a 'federation' of diverse cells that sustains its broad
distribution, stability and abundance in the oceans via extensive genomic and
phenotypic diversity. Thus, it is proving to be a useful model system for
elucidating the forces that shape microbial populations and ecosystems. The marine cyanobacterium Prochlorococcus is the numerically domit
photosynthetic organism in the oligotrophic oceans, and a model system in marine
microbial ecology. Here we report 27 new whole genome sequences (2 complete and
closed; 25 of draft quality) of cultured isolates, representing five major
phylogenetic clades of Prochlorococcus. The sequenced strains were isolated from
diverse regions of the oceans, facilitating studies of the drivers of microbial
diversity-both in the lab and in the field. To improve the utility of these
genomes for comparative genomics, we also define pre-computed clusters of
orthologous groups of proteins (COGs), indicating how genes are distributed
among these and other publicly available Prochlorococcus genomes. These data
represent a significant expansion of Prochlorococcus reference genomes that are
useful for numerous applications in microbial ecology, evolution and
oceanography. |
List 3 features of IRVAN syndrome. | Idiopathic retinal vasculitis, aneurysms, and neuroretinitis is coined as IRVAN syndrome. | PURPOSE: The authors describe the clinical feature of ten patients with a new
syndrome characterized by the presence of retinal vasculitis, multiple
macroaneurysms, neuro-retinitis, and peripheral capillary nonperfusion.
METHODS: The authors evaluated ten patients identified to have clinical features
compatible with the syndrome of idiopathic retinal vasculitis, aneurysms and
neuroretinits (IRVAN). Clinical examination findings, sequential funds
photographs (when available), fluorescein angiograms, systemic investigations,
response to therapy, and visual outcomes were reviewed.
RESULTS: Seven eyes of four patients sustained a marked decrease in visual
acuity of 20/200 or worse. Visual loss was due to a combination of an exudative
maculopathy and sequelae of retinal ischemia. Capillary nonperfusion was seen in
all ten patients and was severe enough to warrant panretinal laser
photocoagulation in six patients. Systemic investigations were uniformly
noncontributory. Oral prednisone appears to have little beneficial effects on
patients with this disorder.
CONCLUSIONS: Patients with IRVAN have characteristic retinal features that
readily identify this syndrome. An increased awareness of this rare syndrome may
help to identify sight-threatening complications at an earlier stage. The
authors caution against extensive medical investigations. PURPOSE: To describe an unusual case of idiopathic retinal vasculitis,
aneurysms, and neuroretinitis (IRVAN) syndrome with rapid dynamics in the number
and appearance of the aneurysms.
DESIGN: Observational case report.
METHODS: Clinical and angiographic data of the patient were reviewed.
RESULTS: In the course of only 6 months, preexisting retinal aneurysms resolved
while new ones appeared. Changes were observed in the shape and size of
preexisting lesions. The resolution of lesions in eyes previously untreated by
laser is reported for the first time.
CONCLUSIONS: Vascular lesions in IRVAN syndrome may show an unusually rapid
turnover. The resolution of aneurysms is a part of the natural course of the
disease and may occur without previous retinal laser photocoagulation. The idiopathic retinal vasculitis, aneurysms, and neuroretinitis (IRVAN)
syndrome typically occurs in young patients and may produce multiple retinal
macroaneurysms, neuroretinitis, and peripheral capillary nonperfusion. Optic
disc edema has been described, but elevated intracranial pressure has not been
previously documented. We report a case of a 12-year-old girl who presented with
bilateral disc swelling and peripapillary hemorrhage. Brain magnetic resoce
imaging (MRI) was normal, but lumbar puncture yielded an opening pressure of 360
mm H2O with normal constituents. Fluorescein angiography delineated saccular
aneurysms of the retinal arteriolar vasculature, and IRVAN syndrome was
diagnosed. MR venography disclosed poor filling of both transverse venous
sinuses. Acetazolamide treatment of 14 months did not alter the fundus findings.
IRVAN syndrome may present initially with optic nerve swelling and elevated
intracranial pressure with subsequent development of the characteristic retinal
vascular abnormalities. PURPOSE: We report our experience in treating 2 patients of idiopathic retinal
vasculitis, aneurysm, and neuroretinitis (IRVAN) syndrome with antitumor
necrosis factor agent, infliximab, who showed a very favorable response to
treatment.
METHODS: Two patients with clinical diagnosis of IRVAN syndrome were included in
the study. The visual acuity was affected due to ocular inflammation and
presence of macular edema due to exudation around the optic nerve.
RESULTS: The patients did not respond to initial treatment with oral steroids,
and visual acuity continued to deteriorate due to macular exudation. Infliximab
therapy resulted in prompt resolution of the inflammatory reaction and retinal
exudation, with improvement in visual acuity, that was subsequently maintained
with maintece therapy. The intravenous infliximab infusions were scheduled at
0, 4, 8, and 12 weeks initially, and every 2 months thereafter. Retinal
neovasculariztion in each patient was managed by pan retinal photocoagulation.
CONCLUSION: Infliximab therapy may be useful in reducing inflammation and
leakage from the optic nerve in patients with IRVAN syndrome. This may help
preserve or improve visual acuity. However, further studies are required to
evaluate the long-term benefits of this therapy. PURPOSE: To report a case of idiopathic retinal vasculitis, aneurysms and
neuroretinitis (IRVAN) syndrome associated with positive perinuclear
antineutrophil cytoplasmic antibody (P-ANCA).
CASE REPORT: A 51-year-old man presented with loss of vision in his right eye
since many years ago and blurred vision in his left eye over the past year.
Ophthalmologic examination revealed optic atrophy and old vascular sheathing in
the right eye and blurred disc margin, macular exudation, flame shaped
hemorrhages, retinal vascular sheathing and multiple aneurysms at arterial
bifurcation sites in the left eye, findings compatible with IRVAN syndrome. On
systemic workup, the only notable finding was P-ANCA positivity.
CONCLUSION: IRVAN syndrome may be a retinal component of P-ANCA associated
vasculitis. CASE REPORT: A 55 year old woman presented with retinal vasculitis, multiple
aneurysms, macular exudation and widespread retinal nonperfusion and was
diagnosed with IRVAN. She was treated with panretinal laser photocoagulation.
After 3 years of follow up visual acuity remains stable and there are no
complications due to ischaemic sequelae.
DISCUSSION: IRVAN syndrome with neovascularisation can progress rapidly despite
laser treatment. Panretinal laser photocoagulation has to be considered in the
early stages as it is effective in stopping the progression of ischaemia. 1. BACKGROUND: Idiopathic retinal vasculitis, aneurysms, and neuroretinitis (IRVAN)
syndrome presents with characteristic clinical manifestations such as aneurysms
at arteriolar bifurcations and optic nerve and retinal vascular inflammation.
Regression of such features on treatment with anti-tubercular therapy (ATT)
combined with corticosteroids has not been reported in literature.
FINDINGS: A 30-year-old female with sudden painless decreased vision in the left
eye was referred with a diagnosis of presumed tuberculous retinal vasculitis and
a positive tuberculin skin test. Based on the clinical and angiographic features
of the right eye, a diagnosis of IRVAN syndrome was made. In the left eye, the
patient had vitreous hemorrhage for which pars plana vitrectomy was performed.
The vitreous sample was positive for Mycobacterium tuberculosis using multiplex
polymerase chain reaction, and the patient was started on standard four-drug ATT
and oral corticosteroids. At 6-month follow-up, vanishing of retinal arterial
aneurysms was observed.
CONCLUSIONS: The pathogenesis of IRVAN syndrome is uncertain. One of the
postulates is that the features of arterial aneurysms and other retinal vascular
alterations occur secondary to acquired inflammatory reaction. We hypothesize
that IRVAN syndrome may be a morphological diagnosis possibly associated with
various entities, one of which could be ocular tuberculosis. It may be prudent
to rule out intraocular tuberculosis in cases labeled as IRVAN syndrome in an
endemic population. |
Which are the Proprotein Convertase Subtilisin Kexin 9 (PCSK9) inhibitors that are FDA approved? | The PCSK9 inhibitors that are FDA approved are:
1) Alirocumab and
2) Evolocumab. | INTRODUCTION: Reduction of plasma low-density lipoprotein (LDL) cholesterol
concentration with statins reduces adverse cardiovascular outcomes. However,
lack of efficacy and intolerance of statins in many patients requires
alternative treatments. Currently available non-statin alternatives include bile
acid sequestrants, the cholesterol absorption inhibitor ezetimibe, niacin-based
preparations and fibrates; however, each of these has limitations. Newer agents
for LDL cholesterol reduction include the cholesterol ester transfer protein
inhibitors, the microsomal triglyceride transfer protein inhibitor lomitapide,
the apolipoprotein B antisense oligonucleotide mipomersen and several molecules
that inhibit or interfere with proprotein convertase subtilisin/kexin 9 (PCSK9).
AREAS COVERED: Among the various PCSK9 inhibitors, human data are available for
monoclonal antibodies against PCSK9 of which the two most advanced are
alirocumab (SAR236553/REGN727) and AMG 145. Phase II studies of these agents as
monotherapy or in combination with statins have shown reductions of LDL
cholesterol by > 70%, with acceptable safety and tolerability so far.
EXPERT OPINION: Despite their biochemical efficacy, clinical efficacy, reflected
by reduction of cardiovascular end points, remains to be shown for two leading
monoclonal antibodies against PSCK9. Other issues to be evaluated with these
agents over the longer term include development of rare adverse effects and
potential attenuation of efficacy. Proprotein convertase subtilisin kexin type 9 (PCSK9) belongs to the proprotein
convertase family. Several studies have demonstrated its involvement in the
regulation of low-density lipoprotein (LDL) cholesterol levels by inducing the
degradation of the LDL receptor (LDLR). However, experimental, epidemiologic,
and pharmacologic data provide important evidence on the role of PCSK9 also on
high-density lipoproteins (HDLs). In mice, PCSK9 regulates the HDL cholesterol
(HDL-C) levels by the degradation of hepatic LDLR, thus inhibiting the uptake of
apolipoprotein (Apo)E-containing HDLs. Several epidemiologic and genetic studies
reported positive relationship between PCSK9 and HDL-C levels, likely by
reducing the uptake of the ApoE-containing HDL particles. PCSK9 enhances also
the degradation of LDLR's closest family members, ApoE receptor 2, very
low-density lipoprotein receptor, and LDLR-related protein 1. This feature
provides a molecular mechanism by which PCSK9 may affect HDL metabolism.
Experimental studies demonstrated that PCSK9 directly interacts with HDL by
modulating PCSK9 self-assembly and its binding to the LDLR. Finally, the
inhibition of PCSK9 by means of monoclonal antibodies directed to PCSK9 (ie,
evolocumab and alirocumab) determines an increase of HDL-C fraction by 7% and
4.2%, respectively. Thus, the understanding of the role of PCSK9 on HDL
metabolism needs to be elucidated with a particular focus on the effect of PCSK9
on HDL-mediated reverse cholesterol transport. At the present time there are novel hypolipidemics registered globally
(alirocumab was the first drug of this group in the world registered by an
American drug agency FDA) and in Europe, which in many ways differ from the
medicines administered until now. They are bringing another advancement in the
treatment of disorders of lipid metabolism and in preventive cardiology.
Alirocumab is a fully human monoclonal antibody to PCSK-9 enzyme (proprotein
convertase subtilisin kexin-9). PCSK-9 enzyme plays an important role in the
metabolism of LDL-cholesterol through affecting the breakdown and eventually the
amount and activity of LDL-receptors. From the clinical point of view it is
essential that drugs from this group are administered parenterally, as a
subcutaneous injection. In the case of Praluent® the interval between
administration is two weeks. The dose is then 75 or 150 mg in a 1 ml injection.
From the clinical point of view it is particularly important that alirocumab
decreases LDL-C concentrations by 50-60%, it decreases Lp/a/ levels by 25-30%,
and it also positively influences other components of lipid metabolism and,
above all, is very likely to have a potential to decrease a cardiovascular risk.
Although the resuIts of morbidity and mortality studies are expected in the
coming years, initial analyses strongly indicate a clinically significant
reduction of CV events. Alirocumab, Praluent can be administered as monotherapy
(mainly to statin-intolerant patients), however it will be primarily
administered in combination with the other hypolipidemic drugs (in particular
statins) where the effort to reach target values has not succeeded. A new class of lipid-lowering drugs, inhibitors of PCSK9 has been generating
impressive clinical trial data over the last several years, and alirocumab
(Praluent) has become the first to be approved by the US FDA. Alirocumab has
been shown to lower low density lipoprotein cholesterol by 45-62% with a safety
profile generally comparable to placebo. Alirocumab is a monoclonal antibody to
PCSK9 administered subcutaneously and has been evaluated in 16 Phase III
clinical trials, the majority of which have been enrolled or completed. This
article will be a review of the available Phase III safety and efficacy data of
the ODYSSEY studies including a brief description of each of the 16 studies. Lowering of low-density lipoprotein (LDL) cholesterol reduces coronary heart
disease morbidity and mortality, not only in secondary but also in primary
prevention. Statins are generally accepted as a treatment of choice for this.
However, still many high-risk and very-high-risk patients fail to achieve target
LDL cholesterol values. Therefore, a new class of lipid-lowering drugs was
recently developed-inhibitors of proprotein convertase subtilisin/kexin type 9
(PCSK9). Alirocumab was the first drug in this class to be approved by the U.S.
Food and Drug Administration (FDA) and recently also by the European Medicines
Agency (EMA). Alirocumab has been shown to lower LDL cholesterol by up to 60%
with a safety profile comparable to that of placebo. Large outcome studies are
still on the way and their first results will be available in 2017. This review
focuses on alirocumab, discussing currently available hard evidence on the
beneficial effects of this drug in the treatment of hypercholesterolemia. INTRODUCTION: Statins are currently the most commonly used agents for treatment
of hypercholesterolemia in patients with atherosclerotic cardiovascular disease.
However, some patients on statins do not achieve their treatment goals or are
intolerant to statins. Therefore, new therapies for treatment of
hypercholesterolemia are under investigation.
AREAS COVERED: This article reviews the new emerging medications for the
treatment of hypercholesterolemia and discusses their efficacy and safety
profile based on literature searches that included human studies published on
PubMed and reported clinical trials.
EXPERT OPINION: Inhibition of the PCSK9 protein by monoclonal antibodies results
in a dramatic 40%-60% lowering of serum low-density lipoprotein cholesterol
(LDL-C). This is in addition to LDL-C lowering achieved by statins. Multiple
clinical studies have demonstrated the high selectivity of these antibodies for
the PCSK9 pathway and their long-term safety and efficacy. Alirocumab and
evolocumab have been approved by the FDA for the treatment of patients with
heterozygous familial hypercholesterolemia and patients with clinical
atherosclerotic cardiovascular disease) who do not achieve their LDL-C target on
maximal tolerated statin treatment and dietary modification. In addition,
evolocumab has been approved by the FDA for homozygous familial
hypercholesterolemia. However, the long-term efficacy and safety of PCSK9
inhibitors are unknown. Proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitors are novel
agents indicated for the treatment of hyperlipidemia. Inhibition of PCSK9
produces an increase in surface low-density lipoprotein (LDL) receptors and
increases removal of LDL from the circulation. Alirocumab (Praluent;
Sanofi/Regeneron, Bridgewater, NJ) and evolocumab (Repatha; Amgen, Thousand
Oaks, CA) are currently available and approved for use in patients with
heterozygous familial hypercholesterolemia, homozygous familial
hypercholesterolemia, and clinical atherosclerotic cardiovascular disease.
Bococizumab (RN316; Pfizer, New York, NY) is currently being studied in similar
indications, with an estimated approval date in late 2016. The pharmacodynamic
effects of PCSK9 inhibitors have been extensively studied in various patient
populations. They have been shown to produce significant reductions in LDL and
are well tolerated in clinical studies, but they are very costly when compared
with statins, the current mainstay of hyperlipidemia treatment. Clinical outcome
studies are underway, but not yet available; however, meta-analyses have pointed
to a reduction in cardiovascular death and cardiovascular events with the use of
PCSK9 inhibitors. This review will discuss the novel mechanism of action of
PCSK9 inhibitors, the results of clinical studies, and the clinical
considerations of these agents in current therapy. Author information:
(1)Professor of Internal Medicine, Medical School, National and Kapodistrian
University of Athens, Athens, Greece.
(2)Professor of Internal Medicine-Endocrinology, Medical School, University of
Patras, Patras, Greece.
(3)Professor of Cardiology, Medical School, University of Patras, Patras,
Greece.
(4)Associate Professor of Internal Medicine, Past President of the Hellenic
Atherosclerosis Society Aristotle University of Thessaloniki, Thessaloniki,
Greece.
(5)Assistant Professor of Internal Medicine-Endocrinology, Medical School,
University of Thessaly, Larissa, Greece.
(6)Consultant-Cardiology, Head of Lipid Outpatient Clinic, "Tzaneio" General
Hospital of Piraeus, Piraeus, Greece.
(7)Consultant Cardiology, 1st University Cardiology Clinic, "Hippokratio"
General Hospital of Athens, Athens, Greece.
(8)Assistant Professor of Metabolic Pediatrics, Medical School, National and
Kapodistrian University of Athens, Athens, Greece.
(9)Professor of Internal Medicine, Medical School, Past President of the
Hellenic Atherosclerosis Society, University of Ioannina, Ioannina, Greece.
(10)Professor of Internal Medicine, Medical School, Past President of the
Hellenic Atherosclerosis Society, University of Crete, Heraklion, Greece.
(11)Professor of Cardiology, Medical School, University of Ioannina, Ioannina,
Greece.
(12)Consultant-Internal Medicine, Head of the Diabetes and Obesity Outpatient
Clinics, General Hospital of Nea Ionia "Konstantopouleio-Patission", Athens,
Greece.
(13)Consultant-Cardiology, Head of LDL Apheresis Unit and Lipid Outpatient
Clinics, Past President of the Hellenic Atherosclerosis Society, President of
the Hellenic College of Treatment of Atherosclerosis, Onassis Cardiac Surgery
Center, Athens, Greece.
(14)Assistant Professor of Internal Medicine, Aristotle University of
Thessaloniki, Thessaloniki, Greece.
(15)Professor of Cardiology, Medical School, National and Kapodistrian
University of Athens, Athens, Greece.
(16)Assistant Professor of Internal Medicine, Medical School, University of
Ioannina, Ioannina, Greece.
(17)Consultant-Internal Medicine, Coordinator-Director of 1st Internal Medicine
Clinic, "Tzaneio" General Hospital of Piraeus, Pireaus, Greece.
(18)Consultant-Cardiology, "Korgialenio Benakio" "HRC" Hospital, Athens, Athens,
Greece.
(19)Assistant Professor of Internal Medicine, Medical School, University of
Thessaly, Larissa, Greece.
(20)Associate Professor of Internal Medicine, Medical School, Democritus
University of Thrace, Alexandroupolis, Greece.
(21)Internist-Diabetologist, President of the Institute for the Study, Research
and Training on Diabetes Mellitus and Metabolic Diseases, Athens, Athens,
Greece.
(22)Emeritus Professor of Cardiology, Medical School, National and Kapodistrian
University of Athens, Athens, Greece.
(23)Associate Professor of Cardiology, Medical School, University of Athens,
Athens, Greece.
(24)Director of Cardiology Department, Athens Euroclinic, President of the
Hellenic Society of Lipidology and Atherosclerosis, Athens, Athens, Greece.
(25)Cosultant-Cardiology, Head of the Lipid Unit of the 1st University Clinic,
"Hippokratio" General Hospital of Athens, Athens, Greece.
(26)Associate Professor of Internal Medicine, Medical School, National and
Kapodistrian University of Athens, Athens, Greece.
(27)Professor of Biochemistry-Clinical Chemistry, President of the Hellenic
Atherosclerosis Society, University of Ioannina, Ioannina, Greece.
(28)Associate Professor of Cardiology, National and Kapodistrian University of
Athens, Athens, Greece.
(29)Professor of Cardiology, Medical School, Democritus University of Thrace,
Alexandroupolis, Greece.
(30)Assistant Professor of Medicine, Medical School, Aristotle University of
Thessaloniki, Thessaloniki, Greece.
(31)Professor of Cardiology, Medical School, Past President of the European
Cardiology Society, University of Crete, Heraklion, Greece.
(32)Associate Professor of Cardiology, President of the European Society of
Cardiology working Group for Peripheral Circulation, National and Kapodistrian
University of Athens, Athens, Greece.
(33)Associate Professor of Nephrology, National and Kapodistrian University of
Athens, Athens, Greece. In 2015 the U.S. Food and Drug Administration approved the first two proprotein
convertase subtilisin/kexin type 9 (PCSK9) inhibitors, alirocumab (Praluent®;
Sanofi/ Regeneron) and evolocumab (Repatha®; Amgen), for use in patients with
heterozygous and homozygous familial hypercholesterolemia and for patients
intolerant of statins or those with a major risk of cardiovascular disease (CVD)
but unable to lower their LDL cholesterol (LDL-C) to optimal levels with statins
and ezetimibe. Numerous randomized clinical trials have demonstrated that these
inhibitors cause a fall in LDL-C levels of 50-60% as well as causing a decline
in lipoprotein(a) and an increase in HDL cholesterol. They are effective in
reducing levels of LDL-C to 1.8 mmol/L or less in almost all patients in the
groups listed above except for those with homozygous familial
hypercholesterolemia. In the latter case, many patients will still have LDL-C
levels well above optimal levels despite the use of statins and a PCSK9
inhibitor. To date these inhibitors have not caused major adverse effects.
However, the results of ongoing long-term randomized clinical trials are needed
to determine whether they cause a significant reduction in CVD events including
deaths from CVD. These studies will also demonstrate whether the PCSK9
inhibitors have any unexpected adverse effects and/or effects resulting from the
loss of PCSK9 functions at other sites in the body, in particular regarding
neurocognition. A further major concern is the high cost of PCSK9 inhibitors and
their effect on healthcare costs and health insurance premiums. The 2 or 4‑week subcutaneous therapy with the recently approved antibodies
alirocumab and evolocumab for inhibition of proprotein convertase
subtilisin-kexin type 9 (PCSK9) reduces low-density lipoprotein cholesterol
(LDL-C) in addition to statins and ezetimibe by 50-60 %. The therapy is
well-tolerated. The safety profile in the published studies is comparable to
placebo. Outcome data and information on long-term safety and the influence on
cardiovascular events are not yet available but the results of several large
trials are expected in 2016-2018. At present (spring 2016) PCSK9 inhibitors
represent an option for selected patients with a high cardiovascular risk and
high LDL-C despite treatment with the maximum tolerated oral lipid-lowering
therapy. This group includes selected patients with familial
hypercholesterolemia and high-risk individuals with statin-associated muscle
symptoms (SAMS). IMPORTANCE: Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors
were recently approved for lowering low-density lipoprotein cholesterol in
heterozygous familial hypercholesterolemia (FH) or atherosclerotic
cardiovascular disease (ASCVD) and have potential for broad ASCVD prevention.
Their long-term cost-effectiveness and effect on total health care spending are
uncertain.
OBJECTIVE: To estimate the cost-effectiveness of PCSK9 inhibitors and their
potential effect on US health care spending.
DESIGN, SETTING, AND PARTICIPANTS: The Cardiovascular Disease Policy Model, a
simulation model of US adults aged 35 to 94 years, was used to evaluate
cost-effectiveness of PCSK9 inhibitors or ezetimibe in heterozygous FH or ASCVD.
The model incorporated 2015 annual PCSK9 inhibitor costs of $14,350 (based on
mean wholesale acquisition costs of evolocumab and alirocumab); adopted a
health-system perspective, lifetime horizon; and included probabilistic
sensitivity analyses to explore uncertainty.
EXPOSURES: Statin therapy compared with addition of ezetimibe or PCSK9
inhibitors.
MAIN OUTCOMES AND MEASURES: Lifetime major adverse cardiovascular events (MACE:
cardiovascular death, nonfatal myocardial infarction, or stroke), incremental
cost per quality-adjusted life-year (QALY), and total effect on US health care
spending over 5 years.
RESULTS: Adding PCSK9 inhibitors to statins in heterozygous FH was estimated to
prevent 316,300 MACE at a cost of $503,000 per QALY gained compared with adding
ezetimibe to statins (80% uncertainty interval [UI], $493,000-$1,737,000). In
ASCVD, adding PCSK9 inhibitors to statins was estimated to prevent 4.3 million
MACE compared with adding ezetimibe at $414,000 per QALY (80% UI,
$277,000-$1,539,000). Reducing annual drug costs to $4536 per patient or less
would be needed for PCSK9 inhibitors to be cost-effective at less than $100,000
per QALY. At 2015 prices, PCSK9 inhibitor use in all eligible patients was
estimated to reduce cardiovascular care costs by $29 billion over 5 years, but
drug costs increased by an estimated $592 billion (a 38% increase over 2015
prescription drug expenditures). In contrast, initiating statins in these
high-risk populations in all statin-tolerant individuals who are not currently
using statins was estimated to save $12 billion.
CONCLUSIONS AND RELEVANCE: Assuming 2015 prices, PCSK9 inhibitor use in patients
with heterozygous FH or ASCVD did not meet generally acceptable incremental
cost-effectiveness thresholds and was estimated to increase US health care costs
substantially. Reducing annual drug prices from more than $14,000 to $4536 would
be necessary to meet a $100,000 per QALY threshold. Control of lipid levels is one of the most effective strategies for
cardiovascular (CV) event prevention. In fact, many clinical trials have clearly
demonstrated that low-density lipoprotein cholesterol (LDL-C) lowering,
primarily with statins, reduces major CV events and mortality. The evidence from
these trials has been useful in designing the cholesterol treatment guidelines,
which are mainly aimed at preventing and managing cardiovascular disease (CVD).
However, available data indicate that a large proportion of patients fail to
achieve lipid goals, and this is particularly frequent in patients at high or
very high CV risk. Furthermore, owing to side effects, a significant percentage
of patients cannot tolerate statin treatment. Hence, researchers have focused
their attention on novel LDL-C-lowering agents that act via mechanisms distinct
from that of statins. Among the new compounds under investigation, the
monoclonal antibodies to proprotein convertase subtilisin/kexin type 9 (PCSK9)
seem particularly promising, having recently been shown to be well tolerated and
highly effective at lowering LDL-C, with a possible effect on the occurrence of
CV events. Currently, alirocumab is approved by the US Food and Drug
Administration (FDA) as an adjunct to diet and maximally tolerated statin
therapy for use in adults with heterozygous familial hypercholesterolemia (FH)
or those with atherosclerotic CV disease who require additional LDL-C lowering;
it has also been recently approved by the European Medicines Agency (EMA) for
use in patients with heterozygous FH, non-familial hypercholesterolemia or mixed
dyslipidemia in whom statins are ineffective or not tolerated. Evolocumab is
approved by the FDA as an adjunct to diet and maximally tolerated statins for
adults with hetero- and homozygous FH and those with atherosclerotic CV disease
who require additional lowering of LDL-C, and by the EMA in adults with primary
hypercholesterolemia or mixed dyslipidemia, as an adjunct to diet, in
combination with a statin or a statin with other lipid lowering therapies in
patients unable to reach LDL-C goals with the maximum tolerated dose of a
statin; alone or in combination with other lipid lowering therapies in patients
who are statin-intolerant, or those for whom a statin is contraindicated.
Evolocumab is also indicated in adults and adolescents aged 12 years and over
with homozygous familial hypercholesterolemia in combination with other
lipid-lowering therapies. PURPOSE OF REVIEW: Proprotein convertase subtilisin kexin type 9 (PCSK9)
inhibitors are promising therapies that inhibit the degradation of low-density
lipoprotein (LDL) receptors in the hepatocyte and thus increase LDL cholesterol
(LDL-C) uptake from the blood. This review summarizes main findings in the field
of PCSK9 inhibitors, from basic mechanism to clinical studies, and aims to
provide a contemporary and practical overview of the clinical implication and
future directions with PCSK9 inhibitors.
RECENT FINDINGS: Monoclonal antibodies that inhibit PCSK9 reduce LDL-C levels by
40-70% across a wide range of patients with various LDL-C levels, and with
different lipid-lowering regimens. These agents significantly reduce
apolipoprotein B and lipoprotein (a), may have a potential role in plaque
stabilization in acute coronary syndromes, and are safe and tolerable, even
among statin-intolerant patients. Preliminary data with evolocumab and
alirocumab demonstrate the potential reduction of cardiovascular (CV) events.
These PCSK9 inhibitors were recently approved for clinical use, and recommended
in the 2016 American College of Cardiology expert consensus document for
nonstatin therapy for LDL-C lowering.
SUMMARY: PCSK9 inhibitors are novel promising therapies to reduce LDL-C. Ongoing
phase 3 clinical trials with more than 70 000 high-risk patients will examine
their safety and efficacy in reducing cardiovascular disease. |
Is dupilumab an antibody targeting the IL-1 receptor? | No, Dupilumab is a fully human monoclonal antibody directed against the IL-4 receptor α subunit that blocks the signaling of IL-4 and IL-13, both key cytokines in Th2-mediated pathways. | Collaborators: Nikolova-Pavlova E, Stoyanova B, Vlaeva T, Alavi A, Gauvreau G,
Henein S, Poulos E, Yang W, Lepage F, Wiseman M, Bissonnette R, Agner T,
Deleuran M, Jemec G, Skov L, Kingo K, Konno P, Pender K, Põder A, Vahlberg A,
Oksman R, Pasternack R, Remitz A, Bieber T, Dominicus R, Gerlach B, Kardorff B,
Toader AL, Kleinheinz A, Gellrich S, Kreutzer K, Leitz N, Offers M, Pauser S,
Radtke M, Roloff E, Rosenbach T, Schwarz B, Sell S, Simon JC, Staubach P, Weigel
US, Werfel T, Wohlrab J, Wollenberg A, Rothenberger C, Walter A, Yazdi A, Aihara
M, Hide M, Kataoka Y, Katoh N, Kawashima M, Kobayashi S, Mitsui H, Nakahara T,
Saeki H, Sueki H, Arai S, Ikeda M, Kabashima K, Kawachi Y, Kume A, Moriwaki S,
Natsuaki Y, Ogata F, Omi T, Seishima M, Sugaya M, Tsukamoto K, Tsuruta D, Urano
S, Watanabe D, Yoshioka A, Furukawa F, Katoh A, Ang CC, Aw DC, Tang M, Lee HY,
Orpinell FB, Hernández GC, De La Cueva P, Foraster CF, Iranzo P, Serra AJ, Luna
PL, Moya SM, Ramírez DM, Muñoz JP, Carazo JS, Soong W, Hull C, Johnson S, Bhatia
N, Limova M, Raikhel M, Sher L, Sofen H, Spector S, Tan R, Yamauchi P, Weber R,
Kimura S, Nelson C, Randhawa S, Rendon M, Trevino M, Ling M, Rice Z, Silverberg
J, Siri D, Fretzin S, Fowler JF, Boh E, Merola J, Murakawa G, Korenblat P,
Campbell J, Bagel J, Beck L, Hazan C, Kalb R, Smith C, Bardelas J, Gawchik S,
Schenkel E, Krause R, Allison D, Browning J, Davis S, Lee M, Duffin K, Fisher
CT, Pariser D, Gower RG, Adams S, Sapijaszko MJ, Wasel N, Albrecht L, Hong CH,
Gulliver W, Landells I, Adam D, Gooderham M, Lomaga M, Lynde C, Rosoph L, Raman
M, Robern M, Sapra S, Toth D, Poulin Y, Bagot M, Barbarot S, Grob JJ, Guillet G,
Lacour JP, Khemis A, Misery L, Staumont-Sallé D, Brüning H, Darsow U,
Ekanayake-Bohlig S, Herbst R, Hoffmann M, Homey B, Niesmann J, Pinter A, Radny
P, Reich K, Sattler G, Sebastian M, Thaçi D, Weidinger S, Wildfeuer T, Worm M,
Chan H, Chan J, Amerio P, Carlesimo M, Di Lernia V, Emilia R, Didona B, Fargnoli
M, Ferrucci SM, Naldi L, Papini M, Parodi A, Pellacani G, Peris K, Pimpinelli N,
Romanelli M, Talamonti M, Bylaite-Bucinskiene M, Cesiene J, Narbutas R,
Sidlauskiene RB, Kucinskiene V, Adamski Z, Bystrzanowska D, Dyczek A, Hofman T,
Leszniewska L, Nowicki R, Owczarek W, Slowinska M, Sobieszek-Kundro A, Weglowska
J, Zakrzewski M, Ahn HH, Ahn KJ, Chang SE, Choi GS, Kim MB, Kim KH, Lee KH, Park
YM, Park CW, Park GH, Nahm DH, Park YL, Roh J, Seo SJ, Ameen M, Ardern-Jones M,
Bewley A, Cooper H, Cork MJ, Guha-Niyogi B, Khan M, Marshall M, Foerster J,
Smith C, Appell M, Elewski B, Haynes S, Jazayeri SS, Crowley J, Dhawan S, Ellis
M, Kim S, Meltzer S, Mitchell J, Pearlman D, Moss J, Ehrlich A, Forman S,
Kuttner B, Penate F, Vaca C, Hamilton T, Paull W, Weisman J, Glazer S, Mehlis S,
Guenthner S, Lockshin B, Kimball A, Rosmarin D, Pickett-Baisden T, Halverson P,
Kaiser H, Martin A, Stone M, Davis K, Mirkil V, Nossa R, Bretton E, Alexis A,
Guttman-Yassky E, Peredo M, Weinberg J, Fleischer A, George R, Lugo-Somolinos A,
Nasir A, Hussain I, Blauvelt A, Simpson E, Kalafer M, Hampton M, Humeniuk JM,
Rupp N, Carrasco D, MacGillivray B, Moore A, Teller C, Tyring S, Harris D,
Jenkin P. |
List active ingredients of the Stribild polypill. | Active ingredients of Stribild are elvitegravir, cobicistat, emtricitabine and tenofovir. It is used for treatment of HIV infection. | OBJECTIVE: To review the clinical trials, pharmacologic characteristics, safety,
and efficacy of the elvitegravir/cobicistat/emtricitabine/tenofovir single
tablet formulation (Stribild).
DATA SOURCES: Literature searches were performed in MEDLINE (1948-September
2012) and PubMed (1966-September 2012) using the search terms GS-9137,
elvitegravir, GS 9350, cobicistat, quad pill, Stribild, and integrase
inhibitors. Abstracts from HIV/AIDS conferences were reviewed.
STUDY SELECTION AND DATA EXTRACTION: Phase 3 studies evaluating the safety and
efficacy of Stribild were preferentially evaluated, as well as relevant
references from the published studies.
DATA SYNTHESIS: Stribild contains complete antiretroviral therapy for HIV-1
infection in a single tablet. It is the first once-daily therapy option
available with an integrase inhibitor and a novel pharmacokinetic boosting
agent. Stribild has shown noninferiority in viral load suppression at 48 weeks
when compared with dual nucleoside/nucleotide reverse transcriptase inhibitor
and either a ritonavir-boosted protease inhibitor or nonnucleoside reverse
transcriptase inhibitor regimen. Stribild was well tolerated, but some patients
experienced increases in serum creatinine early in treatment that stabilized
over time.
CONCLUSIONS: Stribild is the first single-tablet regimen for HIV-1 infection
treatment containing an integrase inhibitor. It is expected to have a prominent
place in the formularies of health plans providing care for individuals with
HIV-1 infection. A new single-tablet, fixed-dose formulation consisting of elvitegravir, an HIV-1
integrase strand transfer inhibitor (INSTI); cobicistat, a pharmacokinetic
enhancer; emtricitabine, a nucleoside reverse transcriptase inhibitor; and
tenofovir disoproxil fumarate (tenofovir DF), a nucleotide reverse transcriptase
inhibitor (elvitegravir/cobicistat/emtricitabine/tenofovir DF 150 mg/150 mg/200
mg/300 mg; Stribild®) is available in some countries for the once-daily
treatment of HIV-1 infection in antiretroviral therapy-naïve adults.
Elvitegravir/cobicistat/emtricitabine/tenofovir DF is the first INSTI-based
single-tablet regimen available for the complete initial treatment of adults
with HIV-1 infection. In two large, randomized, double-blind, phase III trials,
once-daily treatment with elvitegravir/cobicistat/emtricitabine/tenofovir DF was
effective in reducing plasma HIV-1 RNA levels to <50 copies/mL at the week 48
assessment and showed virological efficacy noninferior to that of the
efavirenz/emtricitabine/tenofovir DF single-tablet regimen or a once-daily
regimen of atazanavir plus ritonavir (ritonavir-boosted atazanavir) plus the
fixed-dose combination of emtricitabine/tenofovir DF.
Elvitegravir/cobicistat/emtricitabine/tenofovir DF also showed durable efficacy
in terms of achieving sustained suppression of HIV-1 RNA levels to <50 copies/mL
for up to 144 weeks in both of the phase III trials.
Elvitegravir/cobicistat/emtricitabine/tenofovir DF is an important addition to
the group of simplified once-daily single-tablet regimens currently available
for the effective treatment of HIV-1 infection in antiretroviral therapy-naïve
patients and is among the preferred regimens recommended for use as initial
treatment. It offers advantages over more complex multiple-tablet regimens that
may impair treatment adherence, which is fundamental to the successful
management of HIV-1 infection. BACKGROUND: Coformulated elvitegravir, cobicistat, emtricitabine, and tenofovir
disoproxil fumarate (EVG/COBI/FTC/TDF; Stribild(®)) is a recommended integrase
inhibitor-based regimen in treatment guidelines from the US Department of Health
and Human Services and the British HIV Association. The purpose of this analysis
was to determine the change in patient-reported symptoms over time among
HIV-infected adults who switch to Stribild(®) versus those continuing on a
protease inhibitor (PI) with FTC/TDF.
METHODS: A secondary analysis was conducted on the STRATEGY-PI study
(GS-US-236-0115, ClinicalTrials.gov NCT01475838), a randomized, open-label,
phase 3b trial of HIV-infected adults taking a PI with FTC/TDF who were randomly
assigned (2:1) either to Stribild(®) (switch) or continuation of their existing
regimen (no-switch). Logistic regressions and longitudinal modeling were
conducted to evaluate the relationship of treatment with bothersome symptoms.
RESULTS: At week 4 as compared with baseline, the switch group experienced a
statistically significantly lower prevalence in five symptoms (diarrhea/loose
bowels, bloating/pain/gas in stomach, pain/numbness/tingling in hands/feet,
nervous/anxious, and trouble remembering). The lower prevalence of
diarrhea/loose bowels, bloating/pain/gas in stomach, and pain/numbness/tingling
in hands/feet observed at week 4 was maintained over time. While there were no
significant differences between groups in the prevalence of sad/down/depressed
and problems with sex at week 4 or week 48, longitudinal models indicated the
switch group had a statistically significantly decreased prevalence in both
symptoms from week 4 to week 48. As compared with the no-switch group, higher
levels of satisfaction with treatment were experienced by patients in the switch
group at the first follow-up visit and at week 24.
CONCLUSIONS: In this study sample, a switch from a ritonavir-boosted PI, FTC,
and TDF regimen to coformulated EVG/COBI/FTC/TDF was associated with more
treatment satisfaction and a reduction in the prevalence of patient-reported
diarrhea/loose bowel symptoms, which was maintained over the 48-week study
period. OBJECTIVES: To evaluate dolutegravir and elvitegravir/cobicistat
pharmacokinetics in HIV-negative volunteers up to 10 days after drug cessation.
METHODS: Healthy volunteers received 50 mg of dolutegravir once-daily for 10
days, then underwent a 9 day wash-out period, and then received
elvitegravir/cobicistat as part of Stribild(®) (245 mg of tenofovir, 200 mg of
emtricitabine, 150 mg of elvitegravir and 150 mg of cobicistat) for 10 days.
Serial pharmacokinetic (PK) sampling occurred prior to the final dose of each
course and at regular intervals for up to 216 h (10 days) after drug cessation.
Concentrations were determined by LC-MS/MS, and PK parameters were illustrated
as geometric mean and 90% CI.
RESULTS: Seventeen volunteers completed the study. For dolutegravir, plasma
terminal elimination t1/2 to the last measurable concentration (within 216 h)
was longer than its t1/2 within the dosing interval (0-24 h): 14.3 h (12.9-15.7
h) versus 23.1 h (19.7-26.6 h); conversely, the terminal elimination t1/2 for
elvitegravir was lower than its t1/2 within the dosing interval (0-24 h): 10.8 h
(9.7-13.0 h) versus 5.2 h (4.7-6.1 h). Dolutegravir concentrations were above
the protein-adjusted (PA) IC90 (64 ng/mL) in 100% of subjects after 36 and 48 h
and in 94% after 60 and 72 h. All subjects had detectable dolutegravir
concentrations at 96 h, a mean of 23.5% above the IC90. Elvitegravir
concentrations were above the PA IC95 (45 ng/mL) in 100% of subjects at 24 h,
65% at 36 h but 0% after 48 h.
CONCLUSIONS: Our data show marked differences in the elimination rates of
dolutegravir and elvitegravir following treatment interruption, which is likely
to impact the extent to which drug doses can be delayed or missed. They suggest
that clinical differences may emerge in patients who have suboptimal adherence. |
List clinical features of the IMAGe syndrome. | Clinical features of IMAGe syndrome include intra-uterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital abnormalities. It is s caused by gain-of-function mutations of maternally expressed gene CDKN1C on chromosome 11p15.5. | IMAGe syndrome (intrauterine growth restriction, metaphyseal dysplasia, adrenal
hypoplasia congenita, genital abnormalities; MIM 300290) is a multisystem
disorder with a broad phenotype, which, if unrecognized, may result in major and
possibly life-threatening complications. Initial clinical features overlap with
those of Russell-Silver syndrome (RSS) and isolated growth hormone (GH)
deficiency, conditions from which it must be distinguished. We report an
Australian male with adrenal hypoplasia congenita (AHC) in association with
IMAGe syndrome. The patient had intrauterine growth restriction (IUGR) and
dysmorphic features comprising small, low-set ears, micrognathia, bilateral
cryptorchidism, micropenis, and skeletal abnormalities. Signs of adrenal
insufficiency occurred at aged 4.6 years. Our patient differs from those
previously described by the late onset of adrenal insufficiency and the presence
of GH deficiency. IMAGe is a complex syndrome involving dysmorphic features;
disorders of growth, gonadal, and adrenal function; and skeletal abnormalities. Adrenal hypoplasia congenita (AHC) is a rare condition and causes primary
adrenal insufficiency. X-linked (OMIM 300200) and autosomal recessive (OMIM
240200) forms are recognized. Recently, an association between Intrauterine
growth restriction, Metaphyseal dysplasia, Adrenal hypoplasia congenita, and
Genital abnormalities (IMAGe syndrome; OMIM 300290) has been described. We
present the clinical features of two sisters with intrauterine growth
restriction, AHC, and dysmorphic features. Interesting histopathologic findings
of one sister are also presented. We suggest that IMAGe syndrome is the most
plausible diagnosis and that autosomal recessive inheritance is likely. We
analyzed genes that were postulated candidates for IMAGe syndrome (SF1, DAX-1,
and STAR), and no mutations were found. Other cases of IMAGe syndrome are
reviewed. IMAGe syndrome is a rare condition, first reported by Vilain et al., in 1999,
characterized by intrauterine growth restriction, metaphyseal dysplasia,
congenital adrenal hypoplasia, and genital anomalies. Patients with this
condition may present shortly after birth with severe adrenal insufficiency,
which can be life-threatening if not recognized early and commenced on steroid
replacement therapy. Other reported features in this condition include,
hypercalciuria and/or hypercalcemia, craniosynostosis, cleft palate, and
scoliosis. We report on a 7-year-old boy with IMAGe syndrome, who in addition to
the features in the acronym also has bilateral sensorineural hearing loss which
has not been reported in previously published cases of IMAGe syndrome. We
discuss the clinical presentation in our patient and review the literature in
this rare multisystem disorder. IMAGe syndrome (intrauterine growth restriction, metaphyseal dysplasia, adrenal
hypoplasia congenita and genital anomalies) is an undergrowth developmental
disorder with life-threatening consequences. An identity-by-descent analysis in
a family with IMAGe syndrome identified a 17.2-Mb locus on chromosome 11p15 that
segregated in the affected family members. Targeted exon array capture of the
disease locus, followed by high-throughput genomic sequencing and validation by
dideoxy sequencing, identified missense mutations in the imprinted gene CDKN1C
(also known as P57KIP2) in two familial and four unrelated patients. A familial
analysis showed an imprinted mode of inheritance in which only maternal
transmission of the mutation resulted in IMAGe syndrome. CDKN1C inhibits
cell-cycle progression, and we found that targeted expression of
IMAGe-associated CDKN1C mutations in Drosophila caused severe eye growth defects
compared to wild-type CDKN1C, suggesting a gain-of-function mechanism. All
IMAGe-associated mutations clustered in the PCNA-binding domain of CDKN1C and
resulted in loss of PCNA binding, distinguishing them from the mutations of
CDKN1C that cause Beckwith-Wiedemann syndrome, an overgrowth syndrome. OBJECTIVE: Arboleda et al. have recently shown that IMAGe (intra-uterine growth
restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital
abnormalities) syndrome is caused by gain-of-function mutations of maternally
expressed gene CDKN1C on chromosome 11p15.5. However, there is no other report
describing clinical findings in patients with molecularly studied IMAGe
syndrome. Here, we report clinical and molecular findings in Japanese patients.
PATIENTS: We studied a 46,XX patient aged 8·5 years (case 1) and two 46,XY
patients aged 16·5 and 15·0 years (cases 2 and 3).
RESULTS: Clinical studies revealed not only IMAGe syndrome-compatible phenotypes
in cases 1-3, but also hitherto undescribed findings including relative
macrocephaly and apparently normal pituitary-gonadal endocrine function in cases
1-3, familial glucocorticoid deficiency (FGD)-like adrenal phenotype and the
history of oligohydramnios in case 2, and arachnodactyly in case 3. Sequence
analysis of CDKN1C, pyrosequencing-based methylation analysis of KvDMR1 and
high-density oligonucleotide array comparative genome hybridization analysis for
chromosome 11p15.5 were performed, showing an identical de novo and maternally
inherited CDKN1C gain-of-function mutation (p.Asp274Asn) in cases 1 and 2,
respectively, and no demonstrable abnormality in case 3.
CONCLUSIONS: The results of cases 1 and 2 with CDKN1C mutation would argue the
following: [1] relative macrocephaly is consistent with maternal expression of
CDKN1C in most tissues and biparental expression of CDKN1C in the foetal brain;
[2] FGD-like phenotype can result from CDKN1C mutation; and [3] genital
abnormalities may primarily be ascribed to placental dysfunction. Furthermore,
lack of CDKN1C mutation in case 3 implies genetic heterogeneity in IMAGe
syndrome. IMAGe syndrome (OMIM 300290) is a rare multisystem disorder that has a broad
phenotypic presentation. Though variable, this disorder mainly consists of
Intrauterine growth retardation, Metaphyseal dysplasia, Adrenal hypoplasia
congenita, and Genital abnormalities. Patients with IMAGe syndrome present as an
uncommon yet important challenge for dentists and anesthesiologists due to their
wide range of dysmorphic facial features, adrenal insufficiency, electrolyte
imbalances, and need for steroid replacement. The purpose of this case report is
to describe the successful anesthetic management of a pediatric patient
diagnosed with IMAGe syndrome who presented for full mouth dental
rehabilitation. CDKN1C (also known as P57 (kip2) ) is a cyclin-dependent kinase inhibitor that
functions as a negative regulator of cell proliferation through G1 phase cell
cycle arrest. Recently, our group described gain-of-function mutations in the
PCNA-binding site of CDKN1C that result in an undergrowth syndrome called IMAGe
Syndrome (Intrauterine Growth Restriction, Metaphyseal dysplasia, Adrenal
hypoplasia, and Genital anomalies), with life-threatening consequences.
Loss-of-function mutations in CDKN1C have been identified in 5-10% of
individuals with Beckwith-Wiedemann syndrome (BWS), an overgrowth disorder with
features that are the opposite of IMAGe syndrome. Here, we investigate the
effects of IMAGe-associated mutations on protein stability, cell cycle
progression and cell proliferation. Mutations in the PCNA-binding site of CDKN1C
significantly increase CDKN1C protein stability and prevent cell cycle
progression into the S phase. Overexpression of either wild-type or BWS-mutant
CDKN1C inhibited cell proliferation. However, the IMAGe-mutant CDKN1C protein
decreased cell growth significantly more than both the wild-type or BWS protein.
These findings bring new insights into the molecular events underlying IMAGe
syndrome. |
Which gene mutations are predictive of response to anti-TNF therapy in Rheumatoid Arthritis patients? | Μutations in TLR5 and TLR1 genes contribute to differential response to anti-TNF treatment in RA. Variation at FCGR2A and functionally related genes such as DHX32 and RGS12 is also associated with the response to anti-TNF therapy in rheumatoid arthritis. | The introduction of anti-TNF therapy has dramatically improved the outlook for
patients suffering from a number of inflammatory conditions including rheumatoid
arthritis and inflammatory bowel disease. Despite this, a substantial proportion
of patients (approximately 30-40%) fail to respond to these potentially toxic
and expensive therapies. Treatment response is likely to be multifactorial;
however, variation in genes or their expression may identify those most likely
to respond. By targeted testing of variants within candidate genes, potential
predictors of anti-TNF response have been reported; however, very few markers
have replicated consistently between studies. Emerging genome-wide association
studies suggest that there may be a number of genes with modest effects on
treatment response rather than a few genes of large effect. Other potential
serum biomarkers of response have also been explored including cytokines and
autoantibodies, with antibodies developing to the anti-TNF drugs themselves
being correlated with treatment failure. OBJECTIVE: Anti-TNF therapies have been highly efficacious in the management of
rheumatoid arthritis (RA), but 25-30% of patients do not show a significant
clinical response. There is increasing evidence that genetic variation at the Fc
receptor FCGR2A is associated with the response to anti-TNF therapy. We aimed to
validate this genetic association in a patient cohort from the Spanish
population, and also to identify new genes functionally related to FCGR2A that
are also associated with anti-TNF response.
METHODS: A total of 348 RA patients treated with an anti-TNF therapy were
included and genotyped for FCGR2A polymorphism rs1081274. Response to therapy
was determined at 12 weeks, and was tested for association globally and
independently for each anti-TNF drug (infliximab, etanercept and adalimumab).
Using gene expression profiles from macrophages obtained from synovial fluid of
RA patients, we searched for genes highly correlated with FCGR2A expression. Tag
SNPs were selected from each candidate gene and tested for association with the
response to therapy.
RESULTS: We found a significant association between FCGR2A and the response to
adalimumab (P=0.022). Analyzing the subset of anti-CCP positive RA patients
(78%), we also found a significant association between FCGR2A and the response
to infliximab (P=0.035). DHX32 and RGS12 were the most consistently correlated
genes with FCGR2A expression in RA synovial fluid macrophages (P<0.001). We
found a significant association between the genetic variation at DHX32
(rs12356233, corrected P=0.019) and a nominally significant association between
RGS12 and the response to adalimumab (rs4690093, uncorrected P=0.040). In the
anti-CCP positive group of patients, we also found a nominally significant
association between RGS12 and the response to infliximab (rs2857859, uncorrected
P=0.042).
CONCLUSIONS: In the present study we have validated the FCGR2A association in an
independent population, and we have identified new genes associated with the
response to anti-TNF therapy in RA. |
Which method is used for prediction of novel microRNA genes in cancer-associated genomic regions? | SSCprofiler is a computational tool utilizing a probabilistic method based on Profile Hidden Markov Models to predict novel miRNA precursors. Via the simultaneous integration of biological features such as sequence, structure and conservation, SSCprofiler achieves a performance accuracy of 88.95% sensitivity and 84.16% specificity on a large set of human miRNA genes. The trained classifier is used to identify novel miRNA gene candidates located within cancer-associated genomic regions and rank the resulting predictions using expression information from a full genome tiling array. SSCprofiler is freely available as a web service at http://www.imbb.forth.gr/SSCprofiler.html. | The majority of existing computational tools rely on sequence homology and/or
structural similarity to identify novel microRNA (miRNA) genes. Recently
supervised algorithms are utilized to address this problem, taking into account
sequence, structure and comparative genomics information. In most of these
studies miRNA gene predictions are rarely supported by experimental evidence and
prediction accuracy remains uncertain. In this work we present a new
computational tool (SSCprofiler) utilizing a probabilistic method based on
Profile Hidden Markov Models to predict novel miRNA precursors. Via the
simultaneous integration of biological features such as sequence, structure and
conservation, SSCprofiler achieves a performance accuracy of 88.95% sensitivity
and 84.16% specificity on a large set of human miRNA genes. The trained
classifier is used to identify novel miRNA gene candidates located within
cancer-associated genomic regions and rank the resulting predictions using
expression information from a full genome tiling array. Finally, four of the top
scoring predictions are verified experimentally using northern blot analysis.
Our work combines both analytical and experimental techniques to show that
SSCprofiler is a highly accurate tool which can be used to identify novel miRNA
gene candidates in the human genome. SSCprofiler is freely available as a web
service at http://www.imbb.forth.gr/SSCprofiler.html. |
Does the histone chaperone ASF1 interact with histones H1/H2? | No, the histone chaperone ASF1 interacts with histones H3/H4. | In this issue of Cell, English et al. present the first crystal structure of a
histone chaperone (Asf1) bound to histones (the H3/H4 heterodimer). The
structure provides insights into how histone chaperones participate in
nucleosome disassembly. It reveals that Asf1 physically blocks (H3/H4)(2)
tetramer formation and that the C terminus of H4 undergoes a dramatic
conformational change upon binding to Asf1. Anti-silencing function 1 (Asf1) is a highly conserved chaperone of histones
H3/H4 that assembles or disassembles chromatin during transcription,
replication, and repair. We have found that budding yeast lacking Asf1 has
greatly reduced levels of histone H3 acetylated at lysine 9. Lysine 9 is
acetylated on newly synthesized budding yeast histone H3 prior to its assembly
onto newly replicated DNA. Accordingly, we found that the vast majority of H3
Lys-9 acetylation peaked in S-phase, and this S-phase peak of H3 lysine 9
acetylation was absent in yeast lacking Asf1. By contrast, deletion of ASF1 has
no effect on the S-phase specific peak of H4 lysine 12 acetylation; another
modification carried by newly synthesized histones prior to chromatin assembly.
We show that Gcn5 is the histone acetyltransferase responsible for the
S-phase-specific peak of H3 lysine 9 acetylation. Strikingly, overexpression of
Asf1 leads to greatly increased levels of H3 on acetylation on lysine 56 and
Gcn5-dependent acetylation on lysine 9. Analysis of a panel of Asf1 mutations
that modulate the ability of Asf1 to bind to histones H3/H4 demonstrates that
the histone binding activity of Asf1 is required for the acetylation of Lys-9
and Lys-56 on newly synthesized H3. These results demonstrate that Asf1 does not
affect the stability of the newly synthesized histones per se, but instead
histone binding by Asf1 promotes the efficient acetylation of specific residues
of newly synthesized histone H3. Histone chaperones that escort histones during their overall lifetime from
synthesis to sites of usage can participate in various tasks. Their requirement
culminates in the dynamic processes of nucleosome assembly and disassembly. In
this context, it is important to define the exact role of the histone chaperone
Asf1. In mammals, Asf1 interacts with two other chaperones, CAF-1 and HIRA,
which are critical in DNA synthesis-coupled and synthesis-uncoupled nucleosome
assembly pathways, respectively. A key issue is whether Asf1 is able or not to
deposit histones onto DNA by itself in both pathways. Here, to delineate the
precise role of Asf1 in chromatin assembly, we used Xenopus egg extracts as a
powerful system to assay de novo chromatin assembly pathways in vitro. Following
characterization of both Xenopus Asf1 and p60 (CAF-1), we used immunodepletion
strategies targeting Asf1, HIRA, or CAF-1. Strikingly, the depletion of Asf1 led
to the simultaneous depletion of HIRA and consequently impaired the DNA
synthesis-independent nucleosome assembly pathway. The rescue of nucleosome
assembly capacity in such extracts was effective when adding HIRA along with
H3/H4 histones, yet addition of Asf1 along with H3/H4 histones did not work.
Moreover, nucleosome assembly coupled to DNA repair was not affected in these
Asf1/HIRA-depleted extracts, a pathway impaired by CAF-1 depletion. Thus, these
data show that Asf1 is not directly involved in de novo histone deposition
during DNA synthesis-independent and synthesis-dependent pathways in egg
extracts. Based on our results, it becomes important to consider the
implications for Asf1 function during early development in Xenopus. DNA replication in eukaryotes requires nucleosome disruption ahead of the
replication fork and reassembly behind. An unresolved issue concerns how histone
dynamics are coordinated with fork progression to maintain chromosomal
stability. Here, we characterize a complex in which the human histone chaperone
Asf1 and MCM2-7, the putative replicative helicase, are connected through a
histone H3-H4 bridge. Depletion of Asf1 by RNA interference impedes DNA
unwinding at replication sites, and similar defects arise from overproduction of
new histone H3-H4 that compromises Asf1 function. These data link Asf1 chaperone
function, histone supply, and replicative unwinding of DNA in chromatin. We
propose that Asf1, as a histone acceptor and donor, handles parental and new
histones at the replication fork via an Asf1-(H3-H4)-MCM2-7 intermediate and
thus provides a means to fine-tune replication fork progression and histone
supply and demand. Histone acetylation and nucleosome remodeling regulate DNA damage repair,
replication and transcription. Rtt109, a recently discovered histone
acetyltransferase (HAT) from Saccharomyces cerevisiae, functions with the
histone chaperone Asf1 to acetylate lysine K56 on histone H3 (H3K56), a
modification associated with newly synthesized histones. In vitro analysis of
Rtt109 revealed that Vps75, a Nap1 family histone chaperone, could also
stimulate Rtt109-dependent acetylation of H3K56. However, the molecular function
of the Rtt109-Vps75 complex remains elusive. Here we have probed the molecular
functions of Vps75 and the Rtt109-Vps75 complex through biochemical, structural
and genetic means. We find that Vps75 stimulates the kcat of histone acetylation
by approximately 100-fold relative to Rtt109 alone and enhances acetylation of
K9 in the H3 histone tail. Consistent with the in vitro evidence, cells lacking
Vps75 showed a substantial reduction (60%) in H3K9 acetylation during S phase.
X-ray structural, biochemical and genetic analyses of Vps75 indicate a unique,
structurally dynamic Nap1-like fold that suggests a potential mechanism of
Vps75-dependent activation of Rttl09. Together, these data provide evidence for
a multifunctional HAT-chaperone complex that acetylates histone H3 and deposits
H3-H4 onto DNA, linking histone modification and nucleosome assembly. The eukaryotic genome forms a chromatin structure that contains repeating
nucleosome structures. Nucleosome packaging is regulated by chromatin remodeling
factors such as histone chaperones. The Saccharomyces cerevisiae H3/H4 histone
chaperones, CAF-1 and Asf1, regulate DNA replication and chromatin assembly.
CAF-1 function is largely restricted to non-transcriptional processes in
heterochromatin, whereas Asf1 regulates transcription together with another
H3/H4 chaperone, HIR. This study examined the role of the yeast H3/H4 histone
chaperones, Asf1, HIR, and CAF-1 in chromatin dynamics during transcription.
Unexpectedly, CAF-1 was recruited to the actively transcribed region in a
similar way to HIR and Asf1. In addition, the three histone chaperones
genetically interacted with Set2-dependent H3 K36 methylation. Similar to
histone chaperones, Set2 was required for tolerance to excess histone H3 but not
to excess H2A, suggesting that CAF-1, Asf1, HIR, and Set2 function in a related
pathway and target chromatin during transcription. To restore chromatin on new DNA during replication, recycling of histones
evicted ahead of the fork is combined with new histone deposition. The Asf1
histone chaperone, which buffers excess histones under stress, is a key player
in this process. Yet how histones handled by human Asf1 are modified remains
unclear. Here we identify marks on histones H3-H4 bound to Asf1 and changes
induced upon replication stress. In S phase, distinct cytosolic and nuclear
Asf1b complexes show ubiquitous H4K5K12diAc and heterogeneous H3 marks,
including K9me1, K14ac, K18ac, and K56ac. Upon acute replication arrest, the
predeposition mark H3K9me1 and modifications typical of chromatin accumulate in
Asf1 complexes. In parallel, ssDNA is generated at replication sites, consistent
with evicted histones being trapped with Asf1. During recovery, histones stored
with Asf1 are rapidly used as replication resumes. This shows that replication
stress interferes with predeposition marking and histone recycling with
potential impact on epigenetic stability. Co-expression offers an important strategy for producing multiprotein complexes
for biochemical and biophysical studies. We have found that co-expression of
histones H2A and H2B (from yeast, chicken or Drosophila) leads to production of
soluble heterodimeric H2AH2B complexes. Drosophila histones H3 and H4 can also
be produced as a soluble (H3H4)(2) heterotetrameric complex if they are
co-expressed with the histone chaperone Asf1. The soluble H2AH2B and (H3H4)(2)
can be purified by simple chromatographic techniques and have similar properties
to endogenous histones. Our methods should facilitate histone production for
studies of chromatin structure and regulatory proteins that interact with
histones. We describe a simple strategy for constructing co-expression plasmids,
based on the T7 RNA polymerase system, which is applicable to other systems. It
offers several advantages for quickly creating plasmids to express two or more
proteins and for testing different combinations of proteins for optimal complex
production, solubility or activity. The nucleosome, which is composed of DNA wrapped around a histone octamer, is a
fundamental unit of chromatin and is duplicated during the eukaryotic DNA
replication process. The evolutionarily conserved histone chaperone cell cycle
gene 1 (CCG1) interacting factor A/anti-silencing function 1 (CIA/Asf1) is
involved in histone transfer and nucleosome reassembly during DNA replication.
CIA/Asf1 has been reported to split the histone (H3-H4)(2) tetramer into histone
H3-H4 dimer(s) in vitro, raising a possibility that, in DNA replication,
CIA/Asf1 is involved in nucleosome disassembly and the promotion of
semi-conservative histone H3-H4 dimer deposition onto each daughter strand in
vivo. Despite numerous studies on the functional roles of CIA/Asf1, its
mechanistic role(s) remains elusive because of lack of biochemical analyses. The
biochemical studies described here show that a V94R CIA/Asf1 mutant, which lacks
histone (H3-H4)(2) tetramer splitting activity, does not form efficiently a
quaternary complex with histones H3-H4 and the minichromosome maintece 2
(Mcm2) subunit of the Mcm2-7 replicative DNA helicase. Interestingly, the mutant
enhances nascent DNA strand synthesis in a cell-free chromosomal DNA replication
system using Xenopus egg extracts. These results suggest that CIA/Asf1 in the
CIA/Asf1-H3-H4-Mcm2 complex, which is considered to be an intermediate in
histone transfer during DNA replication, negatively regulates the progression of
the replication fork. The promoter activity of yeast genes can depend on lysine 56 (K56) acetylation
of histone H3. This modification of H3 is performed by lysine acetylase Rtt109
acting in concert with histone chaperone Asf1. We have examined the
contributions of Rtt109, Asf1, and H3 K56 acetylation to nutrient regulation of
a well-studied metabolic gene, ARG1. As expected, Rtt109, Asf1, and H3 K56
acetylation are required for maximal transcription of ARG1 under inducing
conditions. However, Rtt109 and Asf1 also inhibit ARG1 under repressing
conditions. This inhibition requires Asf1 binding to H3-H4 and Rtt109 KAT
activity, but not tail acetylation of H3-H4 or K56 acetylation of H3. These
observations suggest the existence of a unique mechanism of transcriptional
regulation by Rtt109. Indeed, chromatin immunoprecipitation and genetic
interaction studies support a model in which promoter-targeted Rtt109 represses
ARG1 by silencing a pathway of transcriptional activation that depends on ASF1.
Collectively, our results show that ARG1 transcription intensity at its induced
and repressed set points is controlled by different mechanisms of functional
interplay between Rtt109 and Asf1. The histone H3-H4 chaperone Asf1 is involved in chromatin assembly (or
disassembly), histone exchange, regulation of transcription, and chromatin
silencing in several organisms. To investigate the essential functions of Asf1
in Schizosaccharomyces pombe, asf1-ts mutants were constructed by random
mutagenesis using PCR. One mutant (asf1-33(ts)) was mated with mutants in 77
different kinase genes to identify synthetic lethal combinations. The asf1-33
mutant required the DNA damage checkpoint factors Chk1 and Rad3 for its survival
at the restrictive temperature. Chk1, but not Cds1, was phosphorylated in the
asf1-33 mutant at the restrictive temperature, indicating that the DNA damage
checkpoint was activated in the asf1-33 mutant. DNA damage occured in the
asf1-33 mutant, with degradation of the chromosomal DNA observed through
pulse-field gel electrophoresis and the formation of Rad22 foci. Sensitivity to
micrococcal nuclease in the asf1-33 mutant was increased compared to the asf1(+)
strain at the restrictive temperature, suggesting that asf1 mutations also
caused a defect in overall chromatin structure. The Asf1-33 mutant protein was
mislocalized and incapable of binding histones. Furthermore, histone H3 levels
at the centromeric outer repeat region were decreased in the asf1-33 mutant and
heterochromatin structure was impaired. Finally, sim3, which encodes a CenH3
histone chaperone, was identified as a strong suppressor of the asf1-33 mutant.
Taken together, these results clearly indicate that Asf1 plays an essential role
in maintaining genomic stability in S. pombe. The histone chaperone Asf1 and the checkpoint kinase Rad53 are found in a
complex in budding yeast cells in the absence of genotoxic stress. Our data
suggest that this complex involves at least three interaction sites. One site
involves the H3-binding surface of Asf11 with an as yet undefined surface of
Rad53. A second site is formed by the Rad53-FHA1 domain binding to Asf1-T(270)
phosphorylated by casein kinase II. The third site involves the C-terminal 21
amino acids of Rad53 bound to the conserved Asf1 N-terminal domain. The
structure of this site showed that the Rad53 C-terminus binds Asf1 in a
remarkably similar manner to peptides derived from the histone cochaperones HirA
and CAF-I. We call this binding motif, (R/K)R(I/A/V) (L/P), the AIP box for
Asf1-Interacting Protein box. Furthermore, C-terminal Rad53-F(820) binds the
same pocket of Asf1 as does histone H4-F(100). Thus Rad53 competes with histones
H3-H4 and cochaperones HirA/CAF-I for binding to Asf1. Rad53 is phosphorylated
and activated upon genotoxic stress. The Asf1-Rad53 complex dissociated when
cells were treated with hydroxyurea but not methyl-methane-sulfonate, suggesting
a regulation of the complex as a function of the stress. We identified a rad53
mutation that destabilized the Asf1-Rad53 complex and increased the viability of
rad9 and rad24 mutants in conditions of genotoxic stress, suggesting that
complex stability impacts the DNA damage response. Ascomycetes develop four major types of fruiting bodies that share a common
ancestor, and a set of common core genes most likely controls this process. One
way to identify such genes is to search for conserved expression patterns. We
analysed microarray data of Fusarium graminearum and Sordaria macrospora,
identifying 78 genes with similar expression patterns during fruiting body
development. One of these genes was asf1 (anti-silencing function 1), encoding a
predicted histone chaperone. asf1 expression is also upregulated during
development in the distantly related ascomycete Pyronema confluens. To test
whether asf1 plays a role in fungal development, we generated an S. macrospora
asf1 deletion mutant. The mutant is sterile and can be complemented to fertility
by transformation with the wild-type asf1 and its P. confluens homologue. An
ASF1-EGFP fusion protein localizes to the nucleus. By tandem-affinity
purification/mass spectrometry as well as yeast two-hybrid analysis, we
identified histones H3 and H4 as ASF1 interaction partners. Several
developmental genes are dependent on asf1 for correct transcriptional
expression. Deletion of the histone chaperone genes rtt106 and cac2 did not
cause any developmental phenotypes. These data indicate that asf1 of S.
macrospora encodes a conserved histone chaperone that is required for fruiting
body development. Anti-silencing function 1 (Asf1) is a conserved key eukaryotic histone H3/H4
chaperone that participates in a variety of DNA and chromatin-related processes.
These include the assembly and disassembly of histones H3 and H4 from chromatin
during replication, transcription, and DNA repair. In addition, Asf1 is required
for H3K56 acetylation activity dependent on histone acetyltransferase Rtt109.
Thus, Asf1 impacts on many aspects of DNA metabolism. To gain insights into the
functional links of Asf1 with other cellular machineries, we employed mass
spectrometry coupled to tandem affinity purification (TAP) to investigate novel
physical interactions of Asf1. Under different TAP-MS analysis conditions, we
describe a new repertoire of Asf1 physical interactions and novel Asf1
post-translational modifications as ubiquitination, methylation and acetylation
that open up new ways to regulate Asf1 functions. Asf1 co-purifies with several
subunits of the TREX-2, SAGA complexes, and with nucleoporins Nup2, Nup60, and
Nup57, which are all involved in transcription coupled to mRNA export in
eukaryotes. Reciprocally, Thp1 and Sus1 interact with Asf1. Albeit mRNA export
and GAL1 transcription are not affected in asf1Δ a strong genetic interaction
exists between ASF1 and SUS1. Notably, supporting a functional link between Asf1
and TREX-2, both Sus1 and Thp1 affect the levels of Asf1-dependent histone H3K56
acetylation and histone H3 and H4 incorporation onto chromatin. Additionally, we
provide evidence for a role of Asf1 in histone H2B ubiquitination. This work
proposes a functional link between Asf1 and TREX-2 components in histone
metabolism at the vicinity of the nuclear pore complex. MCM2 is a subunit of the replicative helicase machinery shown to interact with
histones H3 and H4 during the replication process through its N-terminal domain.
During replication, this interaction has been proposed to assist disassembly and
assembly of nucleosomes on DNA. However, how this interaction participates in
crosstalk with histone chaperones at the replication fork remains to be
elucidated. Here, we solved the crystal structure of the ternary complex between
the histone-binding domain of Mcm2 and the histones H3-H4 at 2.9 Å resolution.
Histones H3 and H4 assemble as a tetramer in the crystal structure, but MCM2
interacts only with a single molecule of H3-H4. The latter interaction exploits
binding surfaces that contact either DNA or H2B when H3-H4 dimers are
incorporated in the nucleosome core particle. Upon binding of the ternary
complex with the histone chaperone ASF1, the histone tetramer dissociates and
both MCM2 and ASF1 interact simultaneously with the histones forming a 1:1:1:1
heteromeric complex. Thermodynamic analysis of the quaternary complex together
with structural modeling support that ASF1 and MCM2 could form a chaperoning
module for histones H3 and H4 protecting them from promiscuous interactions.
This suggests an additional function for MCM2 outside its helicase function as a
proper histone chaperone connected to the replication pathway. The HAT-B enzyme complex is responsible for acetylating newly synthesized
histone H4 on lysines K5 and K12. HAT-B is a multisubunit complex composed of
the histone acetyltransferase 1 (Hat1) catalytic subunit and the Hat2 (rbap46)
histone chaperone. Hat1 is predomitly localized in the nucleus as a member of
a trimeric NuB4 complex containing Hat1, Hat2, and a histone H3-H4 specific
histone chaperone called Hif1 (NASP). In addition to Hif1 and Hat2, Hat1
interacts with Asf1 (anti-silencing function 1), a histone chaperone that has
been reported to be involved in both replication-dependent and -independent
chromatin assembly. To elucidate the molecular roles of the Hif1 and Asf1
histone chaperones in HAT-B histone binding and acetyltransferase activity, we
have characterized the stoichiometry and binding mode of Hif1 and Asf1 to HAT-B
and the effect of this binding on the enzymatic activity of HAT-B. We find that
Hif1 and Asf1 bind through different modes and independently to HAT-B, whereby
Hif1 binds directly to Hat2, and Asf1 is only capable of interactions with HAT-B
through contacts with histones H3-H4. We also demonstrate that HAT-B is
significantly more active against an intact H3-H4 heterodimer over a histone H4
peptide, independent of either Hif1 or Asf1 binding. Mutational studies further
demonstrate that HAT-B binding to the histone tail regions is not sufficient for
this enhanced activity. Based on these data, we propose a model for
HAT-B/histone chaperone assembly and acetylation of H3-H4 complexes. Vps75 is a histone chaperone that has been historically characterized as
homodimer by X-ray crystallography. In this study, we present a crystal
structure containing two related tetrameric forms of Vps75 within the crystal
lattice. We show Vps75 associates with histones in multiple oligomers. In the
presence of equimolar H3-H4 and Vps75, the major species is a reconfigured Vps75
tetramer bound to a histone H3-H4 tetramer. However, in the presence of excess
histones, a Vps75 dimer bound to a histone H3-H4 tetramer predominates. We show
the Vps75-H3-H4 interaction is compatible with the histone chaperone Asf1 and
deduce a structural model of the Vps75-Asf1-H3-H4 (VAH) co-chaperone complex
using the Pulsed Electron-electron Double Resoce (PELDOR) technique and
cross-linking MS/MS distance restraints. The model provides a molecular basis
for the involvement of both Vps75 and Asf1 in Rtt109 catalysed histone H3 K9
acetylation. In the absence of Asf1 this model can be used to generate a complex
consisting of a reconfigured Vps75 tetramer bound to a H3-H4 tetramer. This
provides a structural explanation for many of the complexes detected
biochemically and illustrates the ability of Vps75 to interact with dimeric or
tetrameric H3-H4 using the same interaction surface. |
Is Hepatic mesenchymal hamartoma usually a malignant tumor? | Mesenchymal hamartoma of the liver (MHL) is an uncommon benign hepatic tumor typically affecting children under 2 years of age. | A case of a prenatally recognized hepatic mesenchymal hamartoma is presented and
the literature reviewed. These tumors are benign and usually present in early
infancy with symptoms that are related to the mass effect on adjacent organs.
Radiologic methods used in the past to image this tumor include angiography and
ultrasound. However, there is no specific radiologic finding, and, therefore,
the diagnosis is usually made during surgery. Once the tumor is removed, the
prognosis is generally good. With the increasing use of high resolution
ultrasound in prenatal diagnosis, this rare tumor should be considered in the
differential diagnosis of any multicystic mass found in the fetal abdomen. The
recognition of a mass should then alert the physician to the need for early
neonatal intervention. Primary tumours of the liver are uncommon in childhood. Of these, more than
two-thirds are maligt. As such, benign hepatic tumours are often not
considered in the differential diagnosis of a hepatic mass in childhood. We
report a case of hepatic mesenchymal hamartoma, a rare benign tumour, in a
10-month-old infant. This tumour is characterised by an admixture of ductal
structures within a copious loose connective tissue stroma. Only approximately
160 cases had been reported in the literature. Awareness of the ultrasound (U/S)
and computed tomography (CT) features, although not diagnostic, is helpful in
distinguishing it from the more common maligt tumours. A correct preoperative
diagnosis is important as surgical excision is often curative. This review on the pathology of hepatic tumors in childhood, from a personal
series of 245 tumors, focuses on incidence, management, description of frequent
tumors such as hepatoblastoma, fibrolamellar carcinoma, and undifferentiated
sarcoma for maligt tumors, focal nodular hyperplasia, hepatocellular adenoma,
and mesenchymal hamartoma for benign tumors. Maligt and benign entities of
recent description, including the following: crowded, small cell
undifferentiated and cholangioblastic variants of hepatoblastomas, mesenchymal
hamartoma miming hepatoblastoma, liver adenoma and adenomatosis in diabete MODY3
families, gastrointestinal stromal tumor with liver metastasis associated to
Carney triad, macronodules in non-cirrhotic portal fibrosis are reviewed. For
each entity, the clinical presentation, the diagnostic criteria and the
differential diagnosis are described. The role of immunohistochemistry and
molecular biology in the diagnosis and identification of new molecular
mechanisms triggered by oncogenic activation with new prognostic markers, and
therapeutic targets is emphasized. Hepatic mesenchymal hamartoma is a rare benign tumour in children. It is often
large and centrally located in the liver at diagnosis, making surgical resection
difficult; thus non-radical resection has been proposed in the past as
acceptable management. However, a literature survey and a case with recurrence
associated with cytogenetic anomalies suggest that radical liver surgery
(resection with a margin of normal liver parenchyma, as for maligt tumour)
should be recommended for mesenchymal hamartoma. Mesenchymal hamartoma is a rare and benign tumor.. Representing 5 to 8 % of
children's hepatic tumors, it is rarely described in adults. Authors report a
new case of hepatic mesenchymal hamartoma in a 21-year-old woman, diagnosed
after a sudden onser of clinical and biological cholestasis. Abdominal US and
CTscan exminations showed a medial liver tumor with cystic formations suggestive
of a hydatid cyst. The diagnosis of hepatic mesenchymal hamartoma was based onn
hitology of the resected liver specimen. Radiological findings can suggest the
diagnosis but only histology can confirm it. Treatment is surgical involving in
most of the cases; a wide hepatic resection because of the size of the tumor.
Progosis is excellent when complete exeresis is possible. Hepatic mesenchymal hamartoma is a rare benign tumor in children, and infantile
hepatic hemangioendothelioma is also a rare liver neoplasm. We report a female
newborn with an abdominal mass noted by the regular maternal ultrasound at 32
weeks of gestation. After birth, a liver mass was detected by computed
tomography and magnetic resoce cholangiopancreatography. Frequent
postprandial vomiting and progressive abdominal distension occurred 4 months
later. Three tumor masses were detected this time, and the serum
alpha-fetoprotein (AFP) was 6700 ng/mL. Segmental resection was performed
initially and complete resection of these tumors and left lobectomy were
performed 21 days later. Pathologic examination of these liver masses revealed
mesenchymal hamartoma combined with infantile hepatic hemangioendothelioma.
After half a year of regular follow-up, the AFP level decreased gradually to
79.5 ng/mL, without evidence of tumor recurrence. Mesenchymal hamartoma of the liver (MHL) is an uncommon benign tumor found
primarily in children younger than 2 years of age. We report a rare case of MHL
with a daughter nodule and atypical histological findings in a 14-month-old
girl. On admission, computed tomography, magnetic resoce imaging, and
angiography showed a solid hypovascular mass with a central cystic area in the
liver. Laparotomy revealed a tumor, 8 cm in size, occupying segment 5 and parts
of segments 4 and 6 of the liver, and a small nodule, 10 mm in size, in segment
7. Thus, we performed a partial hepatic resection (S4-6) and tumor extirpation
(S7). The histological findings of both tumors were the same, but atypical of
MHL. Recent studies on the pathogenesis of this tumor have found neoplastic
features such as genetic anomalies and maligt transformation. These findings
suggest that the conventional approach of completely resecting the tumor
whenever possible is the best treatment. BACKGROUND/PURPOSE: Although hepatic tumors are uncommon in the perinatal period
they are associated with significant morbidity and mortality in affected
patients. The purpose of this review is to focus on the fetus and neonate in an
attempt to determine the various ways liver tumors differ clinically and
pathologically from those found in the older child and adult and to show that
certain types of tumors have a better prognosis than others.
METHODS: The author conducted a retrospective review of perinatal hepatic tumors
reported in the literature and of patients treated and followed up at Children's
Hospital San Diego and Children's Hospital Los Angeles. Only fetuses and infants
younger than 2 months with adequate clinical and pathologic data ere accepted
for review. The period of patient accrual was from 1970 to 2005. Length of
follow-up varied from 1 week to more than 5 years. Elevated alpha-fetoprotein
level was defined as one significantly higher than that of the reporting
institution's normal level for age group; laboratory values for this protein
vary from one institution to the next and therefore it was not possible to
assign one figure as a standard reference number. Discussion of the differential
diagnosis and pathologic findings of hepatic tumors in the fetus and neonate are
described elsewhere and will not be discussed here in detail (Perspect Pediatr
Pathol 1978;4:217; Weinberg AG, Finegold MJ. Primary hepatic tumors in
childhood. In: Finegold M, editor. Pathology of neoplasia in children and
adolescents. Philadelphia, PA: WB Saunders, 1986; Am J Surg Pathol 1982;6:693;
Pediatr Pathol 1983;1:245; Arch Surg 1990;125:598; Semin Neonatol 2003;8:403;
Pediatr Pathol 1985;3:165; Isaacs H Jr. Liver tumors. In: Isaacs H Jr, editor.
Tumors of the fetus and newborn. Philadelphia, PA: WB Saunders, 1997; Isaacs H
Jr. Liver tumors. In: Isaacs H Jr, editor. Tumors of the fetus and infant: an
atlas. Philadelphia, PA: WB Saunders, 2002).
RESULTS: One hundred ninety-four fetuses and neonates presented with hepatic
tumors diagnosed prenatally (n = 56) and in the neonatal period (n = 138). The
study consisted of 3 main tumors: hemangioma (117 cases, 60.3%), mesenchymal
hamartoma (45 cases, 23.2%), and hepatoblastoma (32 cases, 16.5%). The most
common initial finding was a mass found either by antenatal sonography or by
physical examination during the neonatal period. Overall, hydramnios was next
followed by fetal hydrops, respiratory distress, and congestive heart failure,
which were often related to the cause of death. Half of the fetuses and neonates
with hepatoblastoma had abnormally elevated serum alpha-fetoprotein levels
compared with 16 (14%) of 117 of those with hemangioma and 1 neonate with
mesenchymal hamartoma. There were 76 (65%) examples of solitary (unifocal)
hemangiomas and 41 (35%) of multifocal (which included the entity diffuse
hemangiomatosis) with 86% and 71% survival rates, respectively. Of 45 patients
with mesenchymal hamartoma, of the 29 (64%) who had surgical resections, 23
(79%) survived. Patients with hepatoblastoma had the worst outcome of the group,
for only 8 (25%) of 32 were alive. Half of patients with either stage 1 or 3
hepatoblastoma died; no patient with stage 4 survived. There was some
relationship between histologic type and prognosis. For example, half of the
patients with the pure fetal hepatoblastoma histology survived compared with
those with fetal and embryonal histology where 30% survived. Fifteen of 32
hepatoblastoma patients received surgical resection with or without
chemotherapy, resulting in 7 (47%) of 15 cures. The 56 fetuses and 138 neonates
with hepatic tumors (hemangioma, mesenchymal hamartoma, and hepatoblastoma) had
survival rates of 75%, 64%, and 25%, respectively. The overall survival of the
entire group consisting of 194 tumors was 125 or 64%.
CONCLUSIONS: The study shows that clinical findings in fetuses and neonates with
hepatic tumors are less well defined than in older children. Survival rates are
much lower as well. When the clinical course is complicated by associated
conditions such as stillbirth, fetal hydrops, congestive heart failure, severe
anemia, or thrombocytopenia, the mortality rate is much greater. If the patient
is mature enough and in a clinical condition where he or she can be operated on,
survival figures approach those of the older child. Some hepatic tumors have a
better prognosis than others. Neonates with focal (solitary) hepatic hemangiomas
have the best outcome and fetuses with hepatoblastoma the worst. Although
infantile hemangioma undergoes spontaneous regression, it may be life
threatening when congestive heart failure and/or consumptive coagulopathy occur.
Mesenchymal hamartoma is a benign lesion best treated by surgical resection,
which usually results in cure. However, there are fatal complications associated
with this tumor, ie, fetal hydrops, respiratory distress, and circulatory
problems owing to a large space occupying abdominal lesion and sometimes
stillbirth, all contributing to the death rate. Hepatoblastoma, the major
maligcy of the fetus and neonate, is treated primarily by surgical resection.
Pre- or postoperative chemotherapy is reserved for those patients with
unresectable tumors or metastatic disease. The survival rate is much lower than
that reported by multigroup prospective trials. Patients die from the mass
effect caused by the tumor, which lead to abdominal distension, vascular
compromise, anemia, hydrops, respiratory distress, and stillbirth. Metastases to
the abdominal cavity, lungs, and placenta are other causes of death. Because of
the danger of labor-induced rupture of the tumor and potentially fatal
intraabdominal hemorrhage, cesarean delivery is recommended when a hepatic tumor
is found on prenatal ultrasound. BACKGROUND: Mesenchymal hamartoma of the liver is a rare benign liver tumor in
children, usually arising from the right liver lobe and represents about 5 to 6%
of all primary hepatic tumors. Complete surgical resection of the tumor is
curative.
CLINICAL CASE: A 30 months old male presented with epigastrium abdominal pain
and a palpable mass over a period of two days with no other symptom. The mass
was excised completely. Postoperatively the patient recovered with an uneventful
course and was discharge 13 days following surgery. All microscopic findings
were consistent with the diagnosis of mesenchymal hamartoma of the liver.
CONCLUSIONS: Approximately 75% of mesenchymal hamartoma of the liver occur in
the right lobe of the liver. Several diagnostic considerations should be
elucidated to differentiate these type of tumors in the left lobe from other
benign liver tumors. Sometimes a multidisciplinary approach is necessary to
complete a successful complete surgical excision. Our case exemplifies a rare
entity in a rare location, an adequate treatment in a third level reference
hospital setting. Mesenchymal hamartoma of the liver (MHL) is a benign and rare hepatic lesion,
with an uncertain etiology and a potential for developing into an
undifferentiated distant embryonal sarcoma after an incomplete resection. It
mainly presents as progressive abdominal distension with normal blood works.
Most cases are diagnosed in the first two years of life, with a higher frequency
in boys and on the right liver. We report the case of a mesenchymal hamartoma of
the left liver in an 18-month-old girl, with a rough evolution and a literature
review. There were performed an abdominal computed tomography (CT) scan and
resection of the lesion. The macroscopic and histological examination described
a 16.5×17.9×10.5 cm multicystic mass as a MHL lesion. MHLs may have a maligt
potential and in the clinical presence of a "neoplastic" syndrome there requires
a good diagnosis and drastic surgical treatment. |
List clinical features of EEM syndrome. | EEM syndrome is characterized by ectodermal dysplasia, ectrodactyly and macular dystrophy. | The authors reported a 41-year-old female patient with EEM (ectodermal
dysplasia, ectrodactyly and macular dystrophy) syndrome with hypotrichosis,
teeth anomaly, split hand complex and retinal changes with prominent
pigmentations located in the posterior pole of the retina. Retinal degeneration
had shown minimal progression during 11 years. A longer follow-up period was
necessary to make a definite diagnosis of these fundus changes. This is an
isolated case born from a consanguineous marriage. We report five patients with ectodermal dysplasia, ectrodactyly associated with
syndactyly or cleft hand or both, and, in addition, macular dystrophy which was
presumed to be progressive, in an isolated population on a remote island in
Japan. The heredity of this syndrome was thought to be autosomal recessive.
Three cases have been reported so far with a combination of the same
abnormalities. The parents in these cases were consanguineous. We report on a Brazilian kindred in which two sibs presented with the complete
form of EEM (ectodermal dysplasia, ectrodactyly, and macular dystrophy) syndrome
with hypotrichosis, dental anomalies, syndactyly, and retinal changes with
prominent pigmentation in the posterior pole of the retina. In this family, we
also observed another sib with syndactyly, as well as a first cousin with
ectrodactyly. A 10-year follow-up demonstrated gradually decreasing visual
acuity and progression of retinal degenerative anomalies. We report a brother and sister with ectodermal dysplasia, ectrodactyly, and
macular dystrophy (the EEM syndrome). Both children had abnormalities of the
hands and the hair, and bilateral macular degeneration. The clinical picture in
both is similar to, but less severe than, that described in the previously
reported cases of this rare syndrome. Even though the parents are not related,
they are both of Jewish Yemenite origin, and the possibility of a common
ancestor cannot be ruled out. This would suggest autosomal recessive
inheritance. The clinical picture in these patients suggests either variable
expression or genetic heterogeneity in the EEM syndrome and further delineates
the clinical and genetic spectrum of this condition. BACKGROUND: EEM syndrome is the rare association of ectodermal dysplasia,
ectrodactyly, and macular dystrophy.
METHODS: We here demonstrate through molecular analysis that EEM is caused by
distinct homozygous CDH3 mutations in two previously published families.
RESULTS: In family 1, a missense mutation (c.965A-->T) causes a change of amino
acid 322 from asparagine to isoleucine; this amino acid is located in a highly
conserved motif likely to affect Ca2+ binding affecting specificity of the
cell-cell binding function. In family 2, a homozygous frameshift deletion
(c.829delG) introduces a truncated fusion protein with a premature stop codon at
amino acid residue 295, expected to cause a non-functional protein lacking both
its intracellular and membrane spanning domains and its extracellular cadherin
repeats 3-5. Our mouse in situ expression data demonstrate that Cdh3 is
expressed in the apical ectodermal ridge from E10.5 to E12.5, and later in the
interdigital mesenchyme, a pattern compatible with the EEM phenotype.
Furthermore, we discuss possible explanations for the phenotypic differences
between EEM and congenital hypotrichosis with juvenile macular dystrophy (HJMD),
which is also caused by CDH3 mutations.
CONCLUSIONS: In summary, we have ascertained a third gene associated with
ectrodactyly and have demonstrated a hitherto unrecognised role of CDH3 in
shaping the human hand. EEM syndrome is a rare condition characterised by ectodermal dysplasia,
ectrodactyly and macular dystrophy. Additional abnormalities such as alopecia,
cataract, absent eyebrows, and oligodontia may occur. We report two brothers and
a sister born to consanguineous parents with EEM syndrome. EEM syndrome differs
from other ectrodactly syndromes by the characteristic findings in the ocular
fundus showing extensive retinochoroidal atrophy with diffuse retinal
pigmentation and mild arteriolar attenuation at the posterior pole. In contrast
to other ectrodactyly syndromes autosomal recessive inheritance is most likely. Adherens junctions (AJs) are one of the major intercellular junctions in various
epithelia including the epidermis and the follicular epithelium. AJs connect the
cell surface to the actin cytoskeleton and comprise classic transmembrane
cadherins, such as P-cadherin, armadillo family proteins, and actin
microfilaments. Loss-of-function mutations in CDH3, which encodes P-cadherin,
result in two allelic autosomal recessive disorders: hypotrichosis with juvenile
macular dystrophy (HJMD) and ectodermal dysplasia, ectrodactyly, and macular
dystrophy (EEM) syndromes. Both syndromes feature sparse hair heralding
progressive macular dystrophy. EEM syndrome is characterized in addition by
ectodermal and limb defects. Recent studies have demonstrated that, together
with its involvement in cell-cell adhesion, P-cadherin plays a crucial role in
regulating cell signaling, maligt transformation, and other major
intercellular processes. Here, we review the roles of P-cadherin in skin and
hair biology, with emphasize on human hair growth, cycling and pigmentation. |
How many times is CLAST faster than BLAST? | was capable of identifying sequence similarities ~80.8 times faster than blast and 9.6 times faster than blat . | BACKGROUND: Metagenomics is a powerful methodology to study microbial
communities, but it is highly dependent on nucleotide sequence similarity
searching against sequence databases. Metagenomic analyses with next-generation
sequencing technologies produce enormous numbers of reads from microbial
communities, and many reads are derived from microbes whose genomes have not yet
been sequenced, limiting the usefulness of existing sequence similarity search
tools. Therefore, there is a clear need for a sequence similarity search tool
that can rapidly detect weak similarity in large datasets.
RESULTS: We developed a tool, which we named CLAST (CUDA implemented large-scale
alignment search tool), that enables analyses of millions of reads and thousands
of reference genome sequences, and runs on NVIDIA Fermi architecture graphics
processing units. CLAST has four main advantages over existing alignment tools.
First, CLAST was capable of identifying sequence similarities ~80.8 times faster
than BLAST and 9.6 times faster than BLAT. Second, CLAST executes global
alignment as the default (local alignment is also an option), enabling CLAST to
assign reads to taxonomic and functional groups based on evolutionarily distant
nucleotide sequences with high accuracy. Third, CLAST does not need a
preprocessed sequence database like Burrows-Wheeler Transform-based tools, and
this enables CLAST to incorporate large, frequently updated sequence databases.
Fourth, CLAST requires <2 GB of main memory, making it possible to run CLAST on
a standard desktop computer or server node.
CONCLUSIONS: CLAST achieved very high speed (similar to the Burrows-Wheeler
Transform-based Bowtie 2 for long reads) and sensitivity (equal to BLAST, BLAT,
and FR-HIT) without the need for extensive database preprocessing or a
specialized computing platform. Our results demonstrate that CLAST has the
potential to be one of the most powerful and realistic approaches to analyze the
massive amount of sequence data from next-generation sequencing technologies. |
Which are the most common methods for circular RNA detection from RNASeq? | The main algorithms are circRNA_finder, find_circ, CIRCexplorer, CIRI, and MapSplice. | CircRNAs are novel members of the non-coding RNA family. For several decades
circRNAs have been known to exist, however only recently the widespread
abundance has become appreciated. Annotation of circRNAs depends on sequencing
reads spanning the backsplice junction and therefore map as non-linear reads in
the genome. Several pipelines have been developed to specifically identify these
non-linear reads and consequently predict the landscape of circRNAs based on
deep sequencing datasets. Here, we use common RNAseq datasets to scrutinize and
compare the output from five different algorithms; circRNA_finder, find_circ,
CIRCexplorer, CIRI, and MapSplice and evaluate the levels of bona fide and false
positive circRNAs based on RNase R resistance. By this approach, we observe
surprisingly dramatic differences between the algorithms specifically regarding
the highly expressed circRNAs and the circRNAs derived from proximal splice
sites. Collectively, this study emphasizes that circRNA annotation should be
handled with care and that several algorithms should ideally be combined to
achieve reliable predictions. Large-scale RNAseq has substantially changed the transcriptomics field, as it
enables an unprecedented amount of high resolution data to be acquired. However,
the analysis of these data still poses a challenge to the research community.
Many tools have been developed to overcome this problem, and to facilitate the
study of miRNA expression profiles and those of their target genes. While a few
of these enable both kinds of analysis to be performed, they also present
certain limitations in terms of their requirements and/or the restrictions on
data uploading. To avoid these restraints, we have developed a suite that offers
the identification of miRNA, mRNA and circRNAs that can be applied to any
sequenced organism. Additionally, it enables differential expression, miRNA-mRNA
target prediction and/or functional analysis. The miARma-Seq pipeline is
presented as a stand-alone tool that is both easy to install and flexible in
terms of its use, and that brings together well-established software in a single
bundle. Our suite can analyze a large number of samples due to its multithread
design. By testing miARma-Seq in validated datasets, we demonstrate here the
benefits that can be gained from this tool by making it readily accessible to
the research community. |
From which cell type is leptin secreted? | leptin is mainly produced and secreted by adipocytes, but other tissues and gastric glands have also recently been shown to produce it in a dual (endocrine and exocrine) mode. | Leptin is a circulating hormone secreted by adipose and a few other tissues. The
leptin receptor consists of a single transmembrane-spanning polypeptide that is
present as a long physiologically important form as well as in several short
isoforms. Recent studies have suggested that the anterior pituitary may have a
role in the regulatory effects of leptin in animal models. To test this
possibility in human pituitaries, we examined the expression of leptin and OB-R
in normal and neoplastic pituitaries, and the possible functions of leptin in
the pituitary were also analyzed. Leptin was present in 20-25% of anterior
pituitary cells and was expressed in most normal anterior pituitary cells,
including ACTH (70% of ACTH cells), GH (21%), FSH (33%), LH (29%), TSH (32%),
and folliculo-stellate cells (64%), but was colocalized with very few PRL cells
(3%), as detected by double labeling immunohistochemistry with two different
antileptin antibodies. In addition, leptin expression was detected by RT-PCR in
some pituitary tumors, including ACTH (three of four), GH (one of four), null
cells (two of four), and gonadotroph (one of four) tumors as well as in normal
pituitary. Immunohistochemical staining showed greater immunoreactivity for
leptin in normal pituitaries compared to adenomas. Treatment of an immortalized
cultured anterior pituitary cell line, HP75, with leptin stimulated
pancreastatin secretion in vitro. Leptin also inhibited cell growth in the human
HP75 and in the rat pituitary GH3 cell lines. Both long (OB-Rb) and common
(OB-Ra) forms of the leptin receptor messenger ribonucleic acid and leptin
receptor protein were expressed in normal and neoplastic anterior pituitary
cells. These findings show for the first time that leptin is expressed by most
human anterior pituitary cell types and that there is decreased leptin protein
immunoreactivity in pituitary adenomas compared to that in normal pituitary
tissues. We also show that OB-Rb is widely expressed by normal and neoplastic
anterior pituitary cells, implicating an autocrine/paracrine loop in the
production and regulation of leptin in the pituitary. White adipose tissue plays an integral role in energy metabolism and is governed
by endocrine, autocrine, and neural signals. Neural control of adipose
metabolism is mediated by sympathetic neurons that innervate the tissue. To
investigate the effects of this innervation, an ex vivo system was developed in
which 3T3-L1 adipocytes are cocultured with sympathetic neurons isolated from
the superior cervical ganglia of newborn rats. In coculture, both adipocytes and
neurons exhibit appropriate morphology, express cell-type-specific markers, and
modulate key metabolic processes in one another. Lipolysis (stimulated by
beta-adrenergic agents) and leptin secretion by adipocytes are down-regulated by
neurons in coculture, effects apparently mediated by neuropeptide Y (NPY).
Secretion of NPY by neurons is up-regulated dramatically by the presence of
adipocytes in coculture and appears to be mediated by an adipocyte-derived
soluble factor. Insulin, an antilipolytic agent, down-regulates NPY secretion.
Our findings suggest that an adipocyte-derived factor(s) up-regulates the
secretion of NPY by sympathetic neurons, which, in turn, attenuates lipolytic
energy mobilization by adipocytes. Leptin is a 16 kDa protein that exerts important effects on the regulation of
food intake and energy expenditure by interacting with the leptin receptor in
the brain and in many other tissues. Although leptin is produced mainly by white
adipose tissue, several laboratories have shown low levels of leptin production
by a growing number of tissues including the anterior pituitary gland. Many
studies have implicated leptin in anterior pituitary function including the
observation that homozygous mutations of the leptin receptor gene led to morbid
obesity, lack of pubertal development and decreased GH and TSH secretion. In
addition, leptin functions as a neuroendocrine hormone and regulates many
metabolic activities. Leptin also interacts with and regulates the
hypothalamic-pituitary-adrenal, the hypothalamic-pituitary-thyroid and the
hypothalamic-pituitary-gonadal axes. All of the anterior pituitary cell types
express the leptin receptor. However, leptin has been localized in specific
subtypes of anterior pituitary cells indicating cell type-specific production of
leptin in the anterior pituitary. Subcellular localization of leptin indicates
co-storage with secretory granules and implicates hypothalamic releasing
hormones in leptin secretion from anterior pituitary hormone cells. Leptin
signal transduction in the anterior pituitary has been shown to involve the
janus protein-tyrosine kinase (JAK)/signal transducer and activation of
transcription (STAT) as well as suppressor of cytokine signalling (SOCS). These
proteins are activated by tyrosine-phosphorylation in anterior pituitary cells.
The various steps in pituitary leptin signal transduction remain to be
elucidated. Leptin, primarily secreted by adipocytes, is a peripheral hormonal signal
involved in the hypothalamic integration of energy homeostasis. We report that
plasma leptin levels fluctuated in a pulsatile fashion in gonad-intact adult
female and male rats. Whereas in male rats leptin was secreted in the form of
low-amplitude, high-frequency pulses, in female rats high-amplitude pulses were
secreted at only a slightly lower frequency. Consequently, plasma leptin
concentrations were higher in female than in male rats. Gonadectomy decreased
leptin secretion but the sexually dimorphic leptin pulsatility pattern
persisted. These results show that there is a distinct female-type and male-type
leptin pulsatility pattern and each is amenable to augmentation by gonadal
steroids either involving mechanisms that impart leptin pulsatility patterns
directly at the level of adipocytes and/or at hypothalamic target sites. Leptin, the adipocyte-secreted hormone, exerts its main function as regulator of
food intake and energy expenditure through central effects at the hypothalamic
level. However, it appeared that this cytokine-like peptide has also direct
effects on other peripheral tissues and cell types. Remarkable effects have been
demonstrated on the immune function in vivo and in vitro. Monocytes are one of
the target cells of leptin, and we have demonstrated that secretion of L-1Ra, an
IL-1 receptor antagonist, is induced by leptin. In human obesity leptin and
IL-1Ra levels are elevated, and these levels are decreased after weight loss. It
is discussed that IL-1Ra may contribute to central leptin resistance. Adipokines (leptin, adiponectin, and hepatocyte growth factor (HGF)) secreted
from adipose tissue have come to be recognized for their contribution to the
mechanisms by which obesity and related metabolic disorders influence breast
cancer risk. In this review, we discuss the direct and indirect effects of these
protein factors on the biological and clinical aspects of breast cancer biology,
and emphasize their distinctive modes of action through endocrine-, paracrine-,
and autocrine-mediated pathways. The stimulatory effects of leptin on breast
cancer growth were considered to occur primarily via activation of the estrogen
receptor; however, new evidence suggests that leptin may be acting on downstream
cell signaling pathways in both estrogen-dependent and -independent cell types.
Another secretory adipokine, HGF, may act largely not only to promote tumor cell
invasion, but also to enhance tumor growth indirectly by stimulating
angiogenesis. In contrast, adiponectin, an endogenous insulin sensitizer, exerts
a direct growth-inhibitory effect on tumor cells by downregulating cell
proliferation and upregulating apoptosis, and also inhibits tumor-related
angiogenesis. Leptin, a circulating hormone secreted mainly from adipose tissues, possesses
protective effects on many cell types. Serum leptin concentration increases in
patients with chronic renal failure and those undergoing maintece dialysis.
Gentamicin, a widely used antibiotic for the treatment of bacterial infection,
can cause nephrotoxicity. In the present study, we intended to investigate the
influence of leptin on apoptotic pathways and its mechanism in rat renal tubular
cells treated with gentamicin. By using Annexin V-FITC/propidium iodide double
staining, we found that leptin expressed a dose-dependent protective effect
against gentamicin-induced apoptosis in rat renal tubular cells (NRK-52E) within
24h. Pretreatment of the cells with 50 or 100 ng/ml of leptin induced Bcl-2 and
Bcl-x(L), increased the phosphorylation of Bad, and decreased the cleaved
caspase-3 and caspase-9 in gentamicin-treated NRK-52E cells. Leptin also
suppressed the activation of the transcription factor NF-κB and upregulated Akt
activation in gentamicin-treated NRK-52E cells. We found that leptin activated
the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling
pathway as demonstrated by the suppression of the anti-apoptotic effect of
leptin by wortmannin. The treatment of wortmannin suppressed the leptin-induced
phospho-Akt, Bcl-2, phospho-Bad as well as Bcl-x(L), and recovered the
leptin-reduced cleaved caspase-3 and caspase-9. Based on our results, we
suggested that leptin can attenuate gentamicin-induced apoptotic injury in rat
renal tubular cells through PI3K/Akt signaling pathway. PURPOSE: Leptin, an adipose secreted cytokine, is implicated in mammary cancer
stem cell self-renewal and tumor growth in murine mammary tumor virus
(MMTV)-Wnt-1 transgenic mice. In vitro studies indicate that leptin induces
expression of cyclin D1, a cell-cycle control protein necessary for mammary
tumor development. The aim of the present study was to assess cyclin D1
expression in spontaneous tumors that develop in the MMTV-Wnt-1 transgenic mice
and interrogate the in vivo effect of leptin.
MATERIALS AND METHODS: Cells derived from spontaneous MMTV-Wnt-1 tumors were
orthotopically transplanted into wild-type, leptin-deficient, and
hyperleptinemic mice. After 6 weeks, tumors were collected and formalin fixed.
Immunoflurescence staining was used to assess cyclin D1, keratin 8, α-SMA,
phospho-AKT expression.
RESULTS: Cyclin D1 is expressed exclusively in luminal keratin 8 immunoreactive
tumor cells and is dependent on the adipose secreted hormone leptin. Tumor cell
transplant into leptin-deficient mice resulted in approximately an 80 %
reduction of cyclin D1 immunoreactivity in keratin 8 luminal epithelial cells,
and this was independent of Akt activation.
CONCLUSIONS: These data and our previous findings indicate that inhibition of
leptin signaling provides an excellent therapeutic target for breast cancer. The
current data indicate that in luminal mammary tumors, leptin antagonists would
potentially inhibit growth in a cyclin D1-dependent mechanism. In contrast, in
basal mammary tumors, leptin antagonists would inhibit growth in an
Akt-dependent manner leading to reduction in cancer stem cell self-renewal.
Thus, leptin therapeutics may inhibit breast cancer via distinct mechanisms
related to tumor type. Leptin, an adipose-secreted hormone, links metabolism and immunity. Our aim was
to determine whether leptin affects the alloimmune response. We used an
allogeneic skin transplant model as a means to analyze the allograft immune
response in Lep(ob/ob) and wild-type mice. Leptin deficiency results in an
increased frequency of Treg and Th2 cells and a prolonged graft survival. These
effects of leptin deficiency indicate the importance of leptin and obesity in
modulating the allograft immune responses. Our data suggest a possible
explanation for the increased susceptibility of hyperleptinemic obese patients
to acute and chronic graft rejection. Leptin, a 16-kDa protein that is mainly secreted by adipocytes, plays a
protective role in many cell types. It has been shown that leptin acts in the
central and peripheral immune system to protect thymocytes. Cytosolic
phospholipase A(2) (cPLA(2)) is an enzyme that can specifically initiate the
release of arachidonic acid (AA) to produce eicosanoids, which regulate
inflammation and immune responses. Our previous work has shown that leptin is
important to prevent apoptosis of thymocytes. However, the role of cPLA(2) is
still unclear, and the precise mechanism also remains to be elucidated. In this
work, we demonstrated that leptin inhibited the LPS-induced toxicity and
apoptosis of thymocytes. Western blot and RT-PCR showed that leptin led to a
reduction of cPLA(2) activity and mRNA level, as well as caspase-3 cleavage.
Moreover, we found that leptin could decrease the activation of p38 MAPK.
Accordingly, we pre-treated apoptotic thymocytes with the p38 MAPK inhibitor,
SB203580 and observed an effect similar to the leptin alone treated group.
SB203580 also suppressed expression of cPLA(2) and cleavage of caspase-3. Based
on these results, we suggest that leptin could attenuate LPS-induced apoptotic
injury in mouse thymocyte cells, mainly through the p38/cPLA(2) signalling
pathway. The study of the regulatory role of leptin in LPS-induced thymocyte
apoptosis can help to explain the role of leptin in the immune system and may
provide a novel treatment option in cases of severe trauma, infection, shock,
organ failure and autoimmune disease caused by thymic atrophy. STUDY QUESTION: Do the adipocytokines, leptin and adiponectin affect the
granulosa cell expression of anti-Mullerian hormone (AMH) and its receptor
(AMHR-II)?
SUMMARY ANSWER: Leptin suppresses AMH mRNA levels in human luteinized granulosa
cells through the JAK2/STAT3 pathway, while adiponectin has no such effect.
WHAT IS KNOWN ALREADY: AMH is one of the most reliable markers of ovarian
reserve. Serum AMH levels decline with obesity. Obesity is associated with
elevated leptin and reduced adiponectin levels.
STUDY DESIGN, SIZE AND DURATION: This prospective study included 60 infertile
women undergoing fresh IVF and ICSI cycles utilizing autologous oocytes at
Montefiore's Institute for Reproductive Medicine and Health between July 2010
and April 2012.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Follicular fluid was collected from
small (SFs; <14 mm) and large follicles (LFs; ≥14 mm) from 38 participants.
Total RNA was extracted separately from mural and cumulus granulosa cells and
mRNA levels were measured by RT-PCR. In an additional group of participants (N =
22), primary cumulus and mural granulosa cells (pooled SFs and LFs) were
cultured in media alone or with addition of either leptin (N = 7), adiponectin
(N = 8) or JAK2/STAT3 inhibitor + leptin (N = 7), and AMH and AMHR-II mRNA
levels measured. Levels of AMH, leptin and adiponectin protein were measured in
follicular fluid.
MAIN RESULTS AND THE ROLE OF CHANCE: AMH and AMHR-II mRNA and follicular fluid
AMH protein levels were inversely correlated with age. AMH mRNA expression was
six times higher in cumulus compared with mural granulosa cells in SFs (P< 0.05)
and eight times higher in cumulus compared with mural granulosa cells in LFs (P
< 0.001). In follicular fluid, leptin protein level positively correlated (r =
0.7, P = 0.03), while adiponectin protein level inversely correlated (r = -0.46,
P = 0.02) with BMI. Leptin treatment suppressed AMH and AMHR-II mRNA in both
cumulus and mural granulosa cells (all P < 0.05). In the presence of JAK2/STAT3
inhibitor, leptin treatment did not alter AMH but continued to suppress AMHR-II
mRNA in cumulus cells (P = 0.02). Adiponectin treatment did not alter AMH or
AMHR-II mRNA levels.
LIMITATIONS, REASONS FOR CAUTION: This study included a luteinized granulosa
cell model as these cells were collected from women who were hyperstimulated
with gonadotrophins. The results obtained may not fully extrapolate to
non-luteinized granulosa cells.
WIDER IMPLICATIONS OF THE FINDINGS: Leptin may program abnormal AMH signaling,
thereby resulting in ovarian dysfunction. This study opens a new perspective for
understanding the low ovarian reserve seen in obese women and provides new
insights into potential mechanisms that explain the lower AMH seen in obese
women. Whether our findings explain the worse response to ovulation induction
observed in obese women needs to be further elucidated. BACKGROUND: Leptin, the adipocyte-secreted hormone that regulates weight, is
known to link lipid metabolism with inflammation in various cell types. However,
its role in human sebocytes has not yet been investigated.
OBJECTIVES: The purpose of this study was to investigate the effects of leptin
in human sebaceous gland biology.
METHODS: Expression of the long form of the leptin receptor (Ob-Rb) was detected
by real-time quantitative reverse transcriptase polymerase chain reaction
(qRT-PCR) and immunochemistry. Lipid analysis was by high-performance thin-layer
chromatography, gas chromatography-mass spectrometry and time-of-flight mass
spectrometer mass detection. Lipid bodies were visualized by BODIPY staining
using fluorescent microscopy and measured by flow cytometry. Interleukin (IL)-6
and IL-8 mRNA levels were assessed by real-time qRT-PCR and their release was
evaluated by enzyme-linked immunosorbent assay. Cyclooxygenase (COX)-2 and
5-lipooxygenase (LOX) protein expression and phosphorylation of p65 and signal
transducer and activator of transcription (STAT)-3 were determined by Western
blot analysis.
RESULTS: Expression of Ob-Rb was detected in human sebaceous glands and in
cultured human SZ95 sebocytes. The treatment of SZ95 sebocytes with leptin led
to enlarged intracellular lipid bodies, increased ratios of
unsaturated/saturated fatty acids and decreased vitamin E levels. Further
supporting a proinflammatory role, leptin induced COX-2 and 5-LOX expression in
SZ95 sebocytes and augmented the production of IL-6 and IL-8 cytokines. On
leptin treatment, the STAT-3 and nuclear factor-κB pathways were activated,
indicating that these known leptin signalling pathways are active in human
sebocytes.
CONCLUSIONS: Our findings suggest that leptin signalling may be involved in the
proinflammatory regulation of sebaceous lipid metabolism and the induction of
inflammatory enzymes and cytokines. |
Which tool is used for the identification of recurrent variants in noncoding regions? | LARVA is an integrative framework for large-scale analysis of recurrent variants in noncoding annotations. It integrates variants with a comprehensive set of noncoding functional elements, modeling the mutation counts of the elements with a β-binomial distribution to handle overdispersion. LARVA, moreover, uses regional genomic features such as replication timing to better estimate local mutation rates and mutational hotspots. Furthermore, LARVA highlights several novel highly mutated regulatory sites that could potentially be noncoding drivers. | In cancer research, background models for mutation rates have been extensively
calibrated in coding regions, leading to the identification of many driver
genes, recurrently mutated more than expected. Noncoding regions are also
associated with disease; however, background models for them have not been
investigated in as much detail. This is partially due to limited noncoding
functional annotation. Also, great mutation heterogeneity and potential
correlations between neighboring sites give rise to substantial overdispersion
in mutation count, resulting in problematic background rate estimation. Here, we
address these issues with a new computational framework called LARVA. It
integrates variants with a comprehensive set of noncoding functional elements,
modeling the mutation counts of the elements with a β-binomial distribution to
handle overdispersion. LARVA, moreover, uses regional genomic features such as
replication timing to better estimate local mutation rates and mutational
hotspots. We demonstrate LARVA's effectiveness on 760 whole-genome tumor
sequences, showing that it identifies well-known noncoding drivers, such as
mutations in the TERT promoter. Furthermore, LARVA highlights several novel
highly mutated regulatory sites that could potentially be noncoding drivers. We
make LARVA available as a software tool and release our highly mutated
annotations as an online resource (larva.gersteinlab.org). |
What body parts are also known as phalanges? | The anatomical structure of each finger is comprised of four phalanges (distal, middle, proximal, and metacarpal phalange). Toes are also known as phalages | The legal systems of the Germanic tribes in the early Middle Ages elaborated
detailed catalogs of forfeits in compensation for certain physical injuries. The
perpetrator had to pay the forfeit to the injured person, or in case of
manslaughter, to the tribe of the dead. By doing so he could avert the feud
which otherwise faced him. These catalogs of forfeits exactly reflect the
relative value that was appointed to certain parts of the body and to sensory
functions. The catalog of the Lex Saxonum (c. 802), in which physical injuries
are listed, ranging from loss of single phalanges, differentiated between thumb,
forefinger, small finger, and the other fingers, to death, is compared with
modern grades of disability. There are surprising parallels and interesting
contrasts. Bilateral deafness is put on a level with bilateral blindness, the
loss of both hands, both feet, both testicles, and death. Cartilage-Hair-Hypoplasia is a rare form of metaphyseal chondrodystrophia. Its
clinical picture is characterized by dysproportionate deficient growth shift to
length of upper part of the body. The hair diameter is reduced, and the eyebrows
are defectly marked. After stimulation by insulin, the levels of somatotropic
hormone are found in the acromegalic range. The bone structure is rarefied at
the distal metaphyses of the metacarpals and the proximal metaphyses of the
finger basal phalanges. The most important roentgenologic symptoms to be found
are clowdy, cystic rarefactions at the distal femoral metaphyses. As to the
pathophysiology, deficient proliferation of cartilaginous cells is mentioned in
literature. Careful examination of foot impressions can provide important evidences and
clues in a crime scene investigation. The present study is conducted on a
cross-sectional sample of 1040 adult male Gujjars inhabiting the sub-Himalayan
region of North India. The study describes the utility of individualizing
characteristics of footprints in forensic examinations. Various features of the
toes, humps in the toe line, phalange marks, flatfoot condition, pits, cracks,
corns, etc., were studied. Frequency of some of these characters has also been
recorded. The frequency of the tibialis-type foot is the highest, followed by
fibularis-type, then intermediate-type and midularis-type is found to be least
frequent among the sample. Three humps have been found most often in footprints,
followed by two humps, four humps, and then five humps and one hump are found to
be least frequent. Flatfoot condition is found to be present in 1.54% of the
sample population and the trait also shows bilateral variation. Phalange marks,
crease marks, pits, deformity, etc., are also demonstrated with suitable
examples in the present population. These characteristic features can provide
useful clues to establish personal identity whenever complete or partial
footprints are recovered at the crime scene and can help in including or
excluding the possible presence of individual at the scene of crime. The interaction between the handle and operator's hand affects the comfort and
safety of tool and machine operations. In most of the previous studies, the
investigators considered only the normal contact forces. The effect of friction
on the joint moments in fingers has not been analyzed. Furthermore, the observed
contact forces have not been linked to the internal musculoskeletal loading in
the previous experimental studies. In the current study, we proposed a universal
model of a hand to evaluate the joint moments in the fingers during grasping
tasks. The hand model was developed on the platform of the commercial software
package AnyBody. Only four fingers (index, long, ring, and little finger) were
included in the model. The anatomical structure of each finger is comprised of
four phalanges (distal, middle, proximal, and metacarpal phalange). The
simulations were performed using an inverse dynamics technique. The joint angles
and the normal contact forces on each finger section reported by previous
researchers were used as inputs, while the joint moments of each finger were
predicted. The predicted trends of the dependence of the distal interphalangeal
(DIP) and proximal interphalangeal (PIP) joint moments on the cylinder diameter
agree with those of the contact forces on the fingers observed in the previous
experimental study. Our results show that the DIP and PIP joint moments reach
their maximums at a cylinder diameter of about 31mm, which is consistent with
the trend of the finger contact forces measured in the experiments. The proposed
approach will be useful for simulating musculoskeletal loading in the hand for
occupational activities, thereby optimizing tool-handle design. |
Are selenium supplements recommended for prostate cancer prevention? | No. The SELECT study failed to show any significant risk reduction for prostate cancers ascribable to selenium and vitamin E supplementations. | BACKGROUND: Few studies have evaluated the relation between selenium
supplementation after diagnosis and prostate cancer outcomes.
METHODS: We prospectively followed 4459 men initially diagnosed with
nonmetastatic prostate cancer in the Health Professionals Follow-Up Study from
1988 through 2010 and examined whether selenium supplement use (from
selenium-specific supplements and multivitamins) after diagnosis was associated
with risk of biochemical recurrence, prostate cancer mortality, and,
secondarily, cardiovascular disease mortality and overall mortality, using Cox
proportional hazards models. All P values were from two-sided tests.
RESULTS: We documented 965 deaths, 226 (23.4%) because of prostate cancer and
267 (27.7%) because of cardiovascular disease, during a median follow-up of 8.9
years. In the biochemical recurrence analysis, we documented 762 recurrences
during a median follow-up of 7.8 years. Crude rates per 1000 person-years for
prostate cancer death were 5.6 among selenium nonusers and 10.5 among men who
consumed 140 or more μg/day. Crude rates per 1000 person-years were 28.2 vs 23.5
for all-cause mortality and 28.4 vs 29.3 for biochemical recurrence, for nonuse
vs highest-dose categories, respectively. In multivariable analyses, men who
consumed 1 to 24 μg/day, 25 to 139 μg/day, and 140 or more μg/day of
supplemental selenium had a 1.18 (95% confidence interval [CI] = 0.73 to 1.91),
1.33 (95% CI = 0.77 to 2.30), and 2.60-fold (95% CI = 1.44 to 4.70) greater risk
of prostate cancer mortality compared with nonusers, respectively, P trend =
.001. There was no statistically significant association between selenium
supplement use and biochemical recurrence, cardiovascular disease mortality, or
overall mortality.
CONCLUSION: Selenium supplementation of 140 or more μg/day after diagnosis of
nonmetastatic prostate cancer may increase risk of prostate cancer mortality.
Caution is warranted regarding usage of such supplements among men with prostate
cancer. There are several studies that relate oxidative damage as possible mechanism for
many cancers. Many studies have also shown that anti-oxidants like selenium and
vitamin E decrease the risk for prostate cancer. The main objective of the
Selenium and Vitamin E Cancer Prevention Trial (SELECT) study was to look for
the benefits of selenium and vitamin E supplementation on prostate cancer. The
study had a large sample size, stringent experimental conditions, very long
duration, standardized laboratories for biochemical analyses and other factors
that contribute to high external validity. The SELECT study failed to show any
significant risk reduction for prostate cancers ascribable to selenium and
vitamin E supplementations. Because of these conflicting results, many
researchers argue about the methods used, supplementations administered
(selenium and vitamin E) and indicators used for assessing levels of
supplementations. We reviewed many epidemiological studies, clinical trials, and
pre-clinical studies. With corroborative evidences we justify that SELECT study
has a sound methodology and rationale. In lieu of the contrary results of the
select study, researchers should focus on the probable mechanisms for these
contrary findings and continue their search for newer and effective agents for
prevention of prostate cancer. BACKGROUND/OBJECTIVES: Selenium was thought to have a role in cardiovascular
disease (CVD) owing to its antioxidant properties; however, evidence from
observational studies and randomized controlled trials (RCTs) has been
inconsistent and controversial. We thus conducted a meta-analysis to assess the
discrepancies between observational and randomized trial evidence.
SUBJECTS/METHODS: We searched MEDLINE and EMBASE for eligible prospective
studies regarding the relationship between selenium and CVD up to 15 December
2013 and finally included 16 prospective observational studies and 16 RCTs.
Random effects model was used to estimate the pooled relative risk (RR).
Generalized least-squares trend test and restricted cubic spline model were
performed to assess a linear and a nonlinear dose-response relationship.
RESULTS: Our meta-analysis of prospective studies showed a nonlinear
relationship of CVD risk with blood selenium concentrations across a range of
30-165 μg/l and a significant benefit of CVD within a narrow selenium range of
55-145 μg/l. Our meta-analyses of RCTs showed that oral selenium supplements
(median dose: 200 μg/day) for 2 weeks to 144 months significantly raised the
blood selenium concentrations by 56.4 μg/l (95% confidence interval (CI): 40.9,
72.0 μg/l), whereas oral selenium supplements (median: 100 μg/day) for 6 to 114
months caused no effect on CVD (RR=0.91; 95% CI: 0.74, 1.10).
CONCLUSIONS: Our meta-analysis in prospective studies demonstrated a significant
inverse association between selenium status and CVD risk within a narrow
selenium range and a null effect of selenium supplementation on CVD was observed
in RCTs. These findings indicate the importance of considering selenium status,
dose and safety in health assessment and future study design. Prevention is an important treatment strategy for diminishing prostate cancer
morbidity and mortality and is applicable to both early- and late-stage disease.
There are three basic classifications of cancer prevention: primary (prevention
of incident disease), secondary (identification and treatment of preclinical
disease), and tertiary (prevention of progression or recurrence). Based on level
I evidence, 5-alpha reductase inhibitors (5-ARIs) should be considered in
selected men to prevent incident prostate cancer. Level I evidence also supports
the consideration of dutasteride, a 5-ARI, for tertiary prevention in active
surveillance and biochemical recurrence patients. Vitamins and supplements,
including selenium or vitamin E, have not been proven in clinical trials to
prevent prostate cancer and in the case of Vitamin E has been found to increase
the risk of incident prostate cancer. Ongoing and future trials may further
elucidate the role of diet and immunotherapy for prevention of prostate cancer. |
Which disease is treated with lucinactant? | Lucinactant us used for the prevention of respiratory distress syndrome in premature infants. | BACKGROUND AND OBJECTIVE: Evidence suggests that synthetic surfactants
consisting solely of phospholipids can be improved through the addition of
peptides, such as sinapultide, that mimic the action of human surfactant
protein-B (SP-B). A synthetic surfactant containing a mimic of SP-B may also
reduce the potential risks associated with the use of animal-derived products.
Our objective was to compare the efficacy and safety of a novel synthetic
surfactant containing a functional SP-B mimic (lucinactant; Discovery
Laboratories, Doylestown, PA) with those of a non-protein-containing synthetic
surfactant (colfosceril palmitate; GlaxoSmithKline, Brentford, United Kingdom)
and a bovine-derived surfactant (beractant; Abbott Laboratories, Abbott Park,
IL) in the prevention of neonatal respiratory distress syndrome (RDS) and
RDS-related death.
METHODS: We assigned randomly (double-masked) 1294 very preterm infants,
weighing 600 to 1250 g and of < or =32 weeks gestational age, to receive
colfosceril palmitate (n = 509), lucinactant (n = 527), or beractant (n = 258)
within 20 to 30 minutes after birth. Primary outcome measures were the rates of
RDS at 24 hours and the rates of death related to RDS during the first 14 days
after birth. All-cause mortality rates, bronchopulmonary dysplasia (BPD) rates,
and rates of other complications of prematurity were prespecified secondary
outcomes. Primary outcomes, air leaks, and causes of death were assigned by an
independent, masked, adjudication committee with prespecified definitions. The
study was monitored by an independent data safety monitoring board.
RESULTS: Lucinactant reduced significantly the incidence of RDS at 24 hours,
compared with colfosceril (39.1% vs 47.2%; odds ratio [OR]: 0.68; 95% confidence
interval [CI]: 0.52-0.89). There was no significant difference in comparison
with beractant (33.3%). However, lucinactant reduced significantly RDS-related
mortality rates by 14 days of life, compared with both colfosceril (4.7% vs
9.4%; OR: 0.43; 95% CI: 0.25-0.73) and beractant (10.5%; OR: 0.35; 95% CI:
0.18-0.66). In addition, BPD at 36 weeks postmenstrual age was significantly
less common with lucinactant than with colfosceril (40.2% vs 45.0%; OR: 0.75;
95% CI: 0.56-0.99), and the all-cause mortality rate at 36 weeks postmenstrual
age was lower with lucinactant than with beractant (21% vs 26%; OR: 0.67; 95%
CI: 0.45-1.00).
CONCLUSIONS: Lucinactant is a more effective surfactant preparation than
colfosceril palmitate for the prevention of RDS. In addition, lucinactant
reduces the incidence of BPD, compared with colfosceril palmitate, and decreases
RDS-related mortality rates, compared with beractant. Therefore, we conclude
that lucinactant, the first of a new class of surfactants containing a
functional protein analog of SP-B, is an effective therapeutic option for
preterm infants at risk for RDS. BACKGROUND: Available therapeutic surfactants are either animal-derived or
non-protein-containing synthetic products. Animal-derived surfactants contain
variable amounts of surfactant apoproteins, whereas the older-generation
synthetic products contain only phospholipids and lack surfactant proteins
(SPs). Both decrease morbidity and mortality rates associated with respiratory
distress syndrome (RDS) among preterm infants, compared with placebo. However,
excess mortality rates have been observed with non-protein-containing synthetic
surfactants, compared with the animal-derived products. Evidence suggests that
synthetic surfactants consisting solely of phospholipids can be improved with
the addition of peptides that are functional analogs of SPs. Lucinactant is a
new synthetic peptide-containing surfactant that contains sinapultide, a novel,
21-amino acid peptide (leucine and lysine repeating units, KL4 peptide) designed
to mimic human SP-B. It is completely devoid of animal-derived components.
OBJECTIVE: We hypothesized that the outcomes for premature infants treated with
lucinactant and poractant alfa would be similar. Therefore, we compared
lucinactant (Surfaxin; Discovery Laboratories, Doylestown, PA) with
porcine-derived, poractant alfa (Curosurf; Chiesi Farmaceutici, Parma, Italy) in
a trial to test for noninferiority.
METHODS: A total of 252 infants born between 24 and 28 weeks of completed
gestation, with birth weights between 600 and 1250 g, were assigned randomly in
a multicenter, multinational, noninferiority, randomized, controlled study to
receive either lucinactant (n = 124) or poractant alfa (n = 128) within 30
minutes of life. The primary outcome was the incidence of being alive without
bronchopulmonary dysplasia (BPD) through 28 days of age. Key secondary outcomes
included death at day 28 and 36 weeks postmenstrual age (PMA), air leaks,
neuroimaging abnormalities, and other complications related to either
prematurity or RDS. An independent, international, data and safety monitoring
committee monitored the trial.
RESULTS: The treatment difference between lucinactant and poractant alfa for
survival without BPD through 28 days was 4.75% (95% confidence interval [CI]:
-7.3% to 16.8%) in favor of lucinactant, with the lower boundary of the 95% CI
for the difference, ie, -7.3%, being greater than the prespecified
noninferiority margin of -14.5%. At 28 days, 45 of 119 infants given lucinactant
were alive without BPD (37.8%; 95% CI: 29.1-46.5%), compared with 41 of 124
given poractant alfa (33.1%; 95% CI: 24.8-41.3%); at 36 weeks PMA, the rates
were 64.7% and 66.9%, respectively. The corresponding mortality rate through day
28 for the lucinactant group was lower than that for the poractant alfa group
(11.8% [95% CI: 6.0-17.6%] vs 16.1% [95% CI: 9.7-22.6%]), as was the rate at 36
weeks PMA (16% and 18.5%, respectively). There were no differences in major
dosing complications. In addition, no significant differences were observed in
the incidences of common complications of prematurity, including
intraventricular hemorrhage (grades 3 and 4) and cystic periventricular
leukomalacia (lucinactant: 14.3%; poractant alfa: 16.9%).
CONCLUSIONS: Lucinactant and poractant alfa were similar in terms of efficacy
and safety when used for the prevention and treatment of RDS among preterm
infants. The ability to enhance the performance of a synthetic surfactant with
the addition of a peptide that mimics the action of SP-B, such as sinapultide,
brings potential advantages to exogenous surfactant therapy. Lucinactant, formerly known as KL(4) surfactant, is a novel synthetic lung
surfactant containing phospholipids and an engineered peptide, sinapultide,
which is designed to mimic the actions of human surfactant protein B. It has
been developed for use in the prevention or treatment of respiratory distress
syndrome (RDS), a common problem in premature infants, which results from a
deficiency or degradation of pulmonary surfactant. Lucinactant is administered
intratracheally soon after birth as a replacement surfactant. In the pivotal
randomized, double-blind, prophylaxis trial in premature infants, the incidence
of RDS at 24 hours after birth was significantly lower in lucinactant recipients
than in recipients of colfosceril palmitate, a synthetic non-protein-containing
surfactant. RDS-related mortality at 14 days was significantly lower in
lucinactant recipients than in recipients of colfosceril palmitate or beractant,
a bovine-derived surfactant. In another randomized, double-blind, prophylaxis
trial in premature infants, the rate of survival without bronchopulmonary
dysplasia at 28 days of age in lucinactant recipients was not inferior to that
in recipients of poractant alfa, a porcine-derived surfactant. Lucinactant was
generally well tolerated. Adverse events were transient and related to the
administration procedure. There were no differences in the incidences of
complications of prematurity between lucinactant and the other surfactants. BACKGROUND: Animal-derived, protein-containing surfactants seem to be superior
to protein-free surfactants. Lucinactant, a synthetic surfactant containing a
surfactant protein-B peptide analog, has been shown to be effective in animal
models and phase II clinical trials. To date, lucinactant has not been compared
with an animal-derived surfactant in a premature animal model.
OBJECTIVE: The objective was to compare the acute and sustained effects of
lucinactant among premature lambs with respiratory distress syndrome (RDS) with
the effects of a natural porcine surfactant (poractant-alpha).
METHODS: After 5 minutes of mechanical ventilation twin premature lambs were
assigned randomly to the lucinactant group (30 mg/mL, 5.8 mL/kg) or the
poractant-alpha group (80 mg/mL, 2.2 mL/kg). Heart rate, systemic arterial
pressure, arterial pH, blood gas values, and lung mechanics were recorded for 12
hours.
RESULTS: Baseline fetal pH values were similar for the 2 groups (pH 7.27). After
5 minutes of mechanical ventilation, severe RDS developed (pH: <7.08; Paco2: >80
mm Hg; Pao2: <40 mm Hg; dynamic compliance: <0.08 mL/cm H2O per kg). After
surfactant instillation, similar improvements in gas exchange and lung mechanics
were observed for the lucinactant and poractant-alpha groups at 1 hour (pH: 7.3
+/- 0.1 vs 7.4 +/- 0.1; Paco2: 8 +/- 18 mm Hg vs 40 +/- 8 mm Hg; Pao2: 167 +/-
52 mm Hg vs 259 +/- 51 mm Hg; dynamic compliance: 0.3 +/- 0.1 mL/cm H2O per kg
vs 0.3 +/- 0.1 mL/cm H2O per kg). The improvements in lung function were
sustained, with no differences between groups. Cardiovascular profiles remained
stable in both groups.
CONCLUSIONS: Among preterm lambs with severe RDS, lucinactant produced
improvements in gas exchange and lung mechanics similar to those observed with a
porcine-derived surfactant. There are numerous pulmonary conditions in which qualitative or quantitative
anomalies of the surfactant system have been demonstrated. In premature newborns
with immature lungs, a functional deficit in surfactant is the main
physiopathologic mechanism of the neonatal respiratory distress syndrome (RDS).
Since the landmark pilot study of Fujiwara, published more than 20 years ago,
the efficacy of exogenous surfactant for the treatment of neonatal RDS has been
established by numerous controlled studies and meta-analyses. Enlightened by a
growing insight into both the structure and function of the different surfactant
components, a new generation of synthetic surfactants has been developed.
Various complementary approaches have confirmed the fundamental role of the two
hydrophobic proteins, SP-B and SP-C, in the surfactant system, thus opening the
way to the design of analogues, either by chemical synthesis or expression in a
prokaryotic system. An example of these peptide-containing synthetic surfactant
preparations, lucinactant (Surfaxin), has been recently tested in comparison to
a synthetic surfactant that does not contain protein as well as to animal
derived surfactant preparations. Major clinical outcomes between lucinactant and
animal-derived surfactant preparations were fund similar in two randomized
controlled trials, opening the way to a new generation of synthetic surfactants
in the near future. BACKGROUND: Acute inflammatory responses to supplemental oxygen and mechanical
ventilation have been implicated in the pathophysiological sequelae of
respiratory distress syndrome (RDS). Although surfactant replacement therapy
(SRT) has contributed to lung stability, the effect on lung inflammation is
inconclusive. Lucinactant contains sinapultide (KL4), a novel synthetic peptide
that functionally mimics surfactant protein B, a protein with anti-inflammatory
properties. We tested the hypothesis that lucinactant may modulate lung
inflammatory response to mechanical ventilation in the management of RDS and may
confer greater protection than animal-derived surfactants.
METHODS: Preterm lambs (126.8 ± 0.2 SD d gestation) were randomized to receive
lucinactant, poractant alfa, beractant, or no surfactant and studied for 4 h.
Gas exchange and pulmonary function were assessed serially. Lung inflammation
biomarkers and lung histology were assessed at termination.
RESULTS: SRT improved lung compliance relative to no SRT without significant
difference between SRT groups. Lucinactant attenuated lung and systemic
inflammatory response, supported oxygenation at lower ventilatory requirements,
and preserved lung structural integrity to a greater degree than either no SRT
or SRT with poractant alfa or beractant.
CONCLUSION: These data suggest that early intervention with lucinactant may more
effectively mitigate pulmonary pathophysiological sequelae of RDS than the
animal-derived surfactants poractant alfa or beractant. The key feature of respiratory distress syndrome (RDS) is the insufficient
production of surfactant in the lungs of preterm infants. As a result,
researchers have looked into the possibility of surfactant replacement therapy
as a means of preventing and treating RDS. We sought to identify the role of
surfactant in the prevention and management of RDS, comparing the various types,
doses, and modes of administration, and the recent development. A PubMed search
was carried out up to March 2012 using phrases: surfactant, respiratory distress
syndrome, protein-containing surfactant, protein-free surfactant, natural
surfactant, animal-derived surfactant, synthetic surfactant, lucinactant,
surfaxin, surfactant protein-B, surfactant protein-C.Natural, or animal-derived,
surfactant is currently the surfactant of choice in comparison to protein-free
synthetic surfactant. However, it is hoped that the development of
protein-containing synthetic surfactant, such as lucinactant, will rival the
efficacy of natural surfactants, but without the risks of their possible side
effects. Administration techniques have also been developed with nasal
continuous positive airway pressure (nCPAP) and selective surfactant
administration now recommended; multiple surfactant doses have also reported
better outcomes. An aerosolised form of surfactant is being trialled in the hope
that surfactant can be administered in a non-invasive way. Overall, the
advancement, concerning the structure of surfactant and its mode of
administration, offers an encouraging future in the management of RDS. OBJECTIVE: The use of exogenous surfactants among preterm infants for the
prevention and treatment of respiratory distress syndrome (RDS) has led to
economic and cost-effectiveness evaluations of these products. Lucinactant
(Surfaxin), a novel, peptide-based, synthetic surfactant, has been shown to
significantly reduce RDS-related mortality, compared with the most commonly
prescribed animal-derived surfactant, beractant (Survanta). Infants who survive
expend significant healthcare resources; therefore, the impact of improved
survival through 1-year corrected age was evaluated in a prospectively defined
pharmacoeconomic analysis. The objectives of this study were to estimate the
healthcare resource utilization, economic impact, and cost-effectiveness of
lucinactant versus beractant for the prevention of RDS among surviving very low
birth weight (VLBW) preterm infants weighing 600 to 1250 grams.
METHODS: A decision-analytic model was developed to compare the healthcare
resource utilization, economic impact, and cost-effectiveness of lucinactant
versus beractant.
RESULTS: Infants who received lucinactant had fewer neonatal intensive care unit
(NICU) days and fewer NICU days on mechanical ventilation compared with infants
who received beractant. Total healthcare costs for the initial stay in the NICU
were lower by $8,803 among infants who received lucinactant compared with
infants who received beractant. The incremental cost per life saved was $40,309
for lucinactant compared with beractant.
CONCLUSIONS: Administration of lucinactant to surviving VLBW preterm infants
resulted in fewer NICU days and fewer NICU days on mechanical ventilation
compared with beractant. Fewer NICU days translates into lower total costs among
infants who received lucinactant. This comprehensive pharmacoeconomic analysis
indicates that lucinactant is a cost-effective therapy for the prevention of RDS
among preterm infants. Respiratory distress syndrome (RDS) is the leading cause of neonatal morbidity
and mortality in premature infants. It is caused by surfactant deficiency and
lung immaturity. Lucinactant is a synthetic surfactant containing sinapultide, a
bioengineered peptide mimic of surfactant-associated protein B. A meta-analysis
of clinical trials demonstrates that lucinactant is as effective as
animal-derived surfactants in preventing RDS in premature neonates, and in vitro
studies suggest it is more resistant to oxidative and protein-induced
inactivation. Its synthetic origin confers lower infection and inflammation
risks as well other potential benefits, which may make lucinactant an
advantageous alternative to its animal-derived counterparts, which are presently
the standard treatment for RDS. |
Which proteins control the degradation of cryptic unstable transcripts (CUTs) in yeast? | Termination of cryptic unstable transcripts is directed by yeast RNA-binding proteins Nrd1 and Nab3. These cryptic unstable transcripts (CUTs) are rapidly degraded by the nuclear exosome. Key substrates for exosomal degradation include aberrant functional RNAs and cryptic unstable transcripts (CUTs). Yeast RNA binding proteins Nrd1 and Nab3 direct termination of sn/snoRNAs and recently have also been implicated in premature transcription termination of the NRD1 gene. These results suggest that transcription termination of CUTs directed by Nrd1 and Nab3 is a prerequisite for rapid degradation by the nuclear exosome. | Studies of yeast transcription have revealed the widespread distribution of
intergenic RNA polymerase II transcripts. These cryptic unstable transcripts
(CUTs) are rapidly degraded by the nuclear exosome. Yeast RNA binding proteins
Nrd1 and Nab3 direct termination of sn/snoRNAs and recently have also been
implicated in premature transcription termination of the NRD1 gene. In this
paper, we show that Nrd1 and Nab3 are required for transcription termination of
CUTs. In nrd1 and nab3 mutants, we observe 3'-extended transcripts originating
from CUT promoters but failing to terminate through the Nrd1- and Nab3-directed
pathway. Nrd1 and Nab3 colocalize to regions of the genome expressing antisense
CUTs, and these transcripts require yeast nuclear exosome and TRAMP components
for degradation. Dissection of a CUT terminator reveals a minimal element
sufficient for Nrd1- and Nab3-directed termination. These results suggest that
transcription termination of CUTs directed by Nrd1 and Nab3 is a prerequisite
for rapid degradation by the nuclear exosome. Pervasive and hidden transcription is widespread in eukaryotes, but its global
level, the mechanisms from which it originates and its functional significance
are unclear. Cryptic unstable transcripts (CUTs) were recently described as a
principal class of RNA polymerase II transcripts in Saccharomyces cerevisiae.
These transcripts are targeted for degradation immediately after synthesis by
the action of the Nrd1-exosome-TRAMP complexes. Although CUT degradation
mechanisms have been analysed in detail, the genome-wide distribution at the
nucleotide resolution and the prevalence of CUTs are unknown. Here we report the
first high-resolution genomic map of CUTs in yeast, revealing a class of
potentially functional CUTs and the intrinsic bidirectional nature of eukaryotic
promoters. An RNA fraction highly enriched in CUTs was analysed by a 3'
Long-SAGE (serial analysis of gene expression) approach adapted to deep
sequencing. The resulting detailed genomic map of CUTs revealed that they derive
from extremely widespread and very well defined transcription units and do not
result from unspecific transcriptional noise. Moreover, the transcription of
CUTs predomitly arises within nucleosome-free regions, most of which
correspond to promoter regions of bona fide genes. Some of the CUTs start
upstream from messenger RNAs and overlap their 5' end. Our study of glycolysis
genes, as well as recent results from the literature, indicate that such
concurrent transcription is potentially associated with regulatory mechanisms.
Our data reveal numerous new CUTs with such a potential regulatory role.
However, most of the identified CUTs corresponded to transcripts divergent from
the promoter regions of genes, indicating that they represent by-products of
divergent transcription occurring at many and possibly most promoters.
Eukaryotic promoter regions are thus intrinsically bidirectional, a fundamental
property that escaped previous analyses because in most cases divergent
transcription generates short-lived unstable transcripts present at very low
steady-state levels. Genome-wide studies have identified abundant small, noncoding RNAs, including
small nuclear RNAs, small nucleolar RNAs (snoRNAs), cryptic unstable transcripts
(CUTs), and upstream regulatory RNAs (uRNAs), that are transcribed by RNA
polymerase II (pol II) and terminated by an Nrd1-dependent pathway. Here, we
show that the prolyl isomerase Ess1 is required for Nrd1-dependent termination
of noncoding RNAs. Ess1 binds the carboxy-terminal domain (CTD) of pol II and is
thought to regulate transcription by conformational isomerization of Ser-Pro
bonds within the CTD. In ess1 mutants, expression of approximately 10% of the
genome was altered, due primarily to defects in termination of snoRNAs, CUTs,
stable unotated transcripts, and uRNAs. Ess1 promoted dephosphorylation of
Ser5 (but not Ser2) within the CTD, most likely by the Ssu72 phosphatase. We
also provide evidence for a competition between Nrd1 and Pcf11 for CTD binding
that is regulated by Ess1. These data indicate that a prolyl isomerase is
required for specifying the "CTD code." Non-coding transcripts originating from bidirectional promoters have been
reported in a wide range of organisms. In yeast, these divergent transcripts can
be subdivided into two classes. Some are designated Cryptic Unstable Transcripts
(CUTs) because they are terminated by the Nrd1-Nab3-Sen1 pathway and then
rapidly degraded by the nuclear exosome. This is the same processing pathway
used by yeast snoRNAs. Whereas CUTs are only easily observed in cells lacking
the Rrp6 or Rrp47 subunits of the nuclear exosome, Stable Uncharacterized
Transcripts (SUTs) are present even in wild-type cells. Here we show that SUTs
are partially susceptible to the nuclear exosome, but are primarily degraded by
cytoplasmic 5' to 3' degradation and nonsense-mediated decay (NMD). Therefore,
SUTs may be processed similarly to mRNAs. Surprisingly, both CUTs and SUTs were
found to produce 3' extended species that were also subject to cytoplasmic
degradation. The functions, if any, of these extended CUTs and SUTs are unknown,
but their discovery suggests that yeasts generate transcripts reminiscent of
long non-coding RNAs found in higher eukaryotes. Nuclear RNA degradation pathways are highly conserved across eukaryotes and play
important roles in RNA quality control. Key substrates for exosomal degradation
include aberrant functional RNAs and cryptic unstable transcripts (CUTs). It has
recently been reported that the nuclear exosome is inactivated during meiosis in
budding yeast through degradation of the subunit Rrp6, leading to the
stabilisation of a subset of meiotic unotated transcripts (MUTs) of unknown
function. We have analysed the activity of the nuclear exosome during meiosis by
deletion of TRF4, which encodes a key component of the exosome targeting complex
TRAMP. We find that TRAMP mutants produce high levels of CUTs during meiosis
that are undetectable in wild-type cells, showing that the nuclear exosome
remains functional for CUT degradation, and we further report that the meiotic
exosome complex contains Rrp6. Indeed Rrp6 over-expression is insufficient to
suppress MUT transcripts, showing that the reduced amount of Rrp6 in meiotic
cells does not directly cause MUT accumulation. Lack of TRAMP activity
stabilises ∼ 1600 CUTs in meiotic cells, which occupy 40% of the binding
capacity of the nuclear cap binding complex (CBC). CBC mutants display defects
in the formation of meiotic double strand breaks (DSBs), and we see similar
defects in TRAMP mutants, suggesting that a key function of the nuclear exosome
is to prevent saturation of the CBC complex by CUTs. Together, our results show
that the nuclear exosome remains active in meiosis and has an important role in
facilitating meiotic recombination. Cryptic unstable transcripts (CUTs) are rapidly degraded by the nuclear exosome.
However, the mechanism by which they are recognized and targeted to the exosome
is not fully understood. Here we report that the MTREC complex, which has
recently been shown to promote degradation of meiotic mRNAs and regulatory
ncRNAs, is also the major nuclear exosome targeting complex for CUTs and
unspliced pre-mRNAs in Schizosaccharomyces pombe. The MTREC complex specifically
binds to CUTs, meiotic mRNAs and unspliced pre-mRNA transcripts and targets
these RNAs for degradation by the nuclear exosome, while the TRAMP complex has
only a minor role in this process. The MTREC complex physically interacts with
the nuclear exosome and with various RNA-binding and RNA-processing complexes,
coupling RNA processing to the RNA degradation machinery. Our study reveals the
central role of the evolutionarily conserved MTREC complex in RNA quality
control, and in the recognition and elimination of CUTs. |
Is there any involvement of L1 retrotransposition in the Rett syndrome? | Yes. Recent studies indicate that long interspersed nuclear element-1 (L1) are mobilized in the genome of human neural progenitor cells and enhanced in Rett syndrome and ataxia telangiectasia. | Long interspersed element-1 (L1) retrotransposons compose ∼20% of the mammalian
genome, and ongoing L1 retrotransposition events can impact genetic diversity by
various mechanisms. Previous studies have demonstrated that endogenous L1
retrotransposition can occur in the germ line and during early embryonic
development. In addition, recent data indicate that engineered human L1s can
undergo somatic retrotransposition in human neural progenitor cells and that an
increase in human-specific L1 DNA content can be detected in the brains of
normal controls, as well as in Rett syndrome patients. Here, we demonstrate an
increase in the retrotransposition efficiency of engineered human L1s in cells
that lack or contain severely reduced levels of ataxia telangiectasia mutated, a
serine/threonine kinase involved in DNA damage signaling and neurodegenerative
disease. We demonstrate that the increase in L1 retrotransposition in ataxia
telangiectasia mutated-deficient cells most likely occurs by conventional
target-site primed reverse transcription and generate either longer, or perhaps
more, L1 retrotransposition events per cell. Finally, we provide evidence
suggesting an increase in human-specific L1 DNA copy number in postmortem brain
tissue derived from ataxia telangiectasia patients compared with healthy
controls. Together, these data suggest that cellular proteins involved in the
DNA damage response may modulate L1 retrotransposition. Author information:
(1)Department of Molecular Psychiatry, Graduate School of Medicine, The
University of Tokyo, Tokyo 113-8655, Japan; Laboratory for Molecular Dynamics of
Mental Disorders, RIKEN Brain Science Institute, Saitama 351-0198, Japan.
(2)Laboratory for Molecular Psychiatry, RIKEN Brain Science Institute, Saitama
351-0198, Japan.
(3)Department of Physiology, Keio University School of Medicine, Tokyo 160-8582,
Japan.
(4)Laboratory for Molecular Dynamics of Mental Disorders, RIKEN Brain Science
Institute, Saitama 351-0198, Japan.
(5)Department of Molecular Psychiatry, Graduate School of Medicine, The
University of Tokyo, Tokyo 113-8655, Japan.
(6)Department of Psychiatry, Nara Medical University, Nara 634-8521, Japan.
(7)Department of Pathology, Brain Research Institute, Niigata University,
Niigata 951-8585, Japan.
(8)Department of Neuropsychiatry, Keio University School of Medicine, Tokyo
160-8582, Japan.
(9)Department of Neuropsychiatry, Graduate School of Medicine, The University of
Tokyo, Tokyo 113-8655, Japan.
(10)Department of Molecular Neurobiology, Brain Research Institute, Niigata
University, Niigata 951-8585, Japan.
(11)Laboratory for Molecular Dynamics of Mental Disorders, RIKEN Brain Science
Institute, Saitama 351-0198, Japan. Electronic address: [email protected].
(12)Department of Molecular Psychiatry, Graduate School of Medicine, The
University of Tokyo, Tokyo 113-8655, Japan; PRESTO, Japan Science and Technology
Agency, Saitama 332-0012, Japan. Electronic address: [email protected]. |
Is Downs syndrome associated with decreased risk of leukemia? | No, multiple studies have established the incidence of leukemia in Down's syndrome patients to be 10- to 20-fold higher than that in the general population. | The association of Down's syndrome and leukemia has been documented for over 50
years. Multiple studies have established the incidence of leukemia in Down's
syndrome patients to be 10- to 20-fold higher than that in the general
population. The age of onset for leukemia in these children is bimodal, peaking
first in the newborn period and again at 3-6 years. This increased risk extends
into adulthood. All cytogenetic types of Down's syndrome apparently predispose
to leukemia. The proportion of acute lymphoblastic leukemia and acute
nonlymphoblastic leukemia in patients with Down's syndrome is similar to
non-Down's syndrome leukemia patients matched for age. There are case reports in
which leukemia, Down's syndrome, and other chromosomal aberrations cluster
within a family. In these kindreds, there may be a familial tendency toward
nondisjunction. Congenital leukemia also occurs with increased frequency in
Down's syndrome patients, and is characterized by a preponderance of acute
nonlymphoblastic leukemia (similar to non-Down's syndrome patients). Transient
leukemoid reactions have been observed in Down's syndrome patients, as well as
in phenotypically normal children with constitutional trisomy 21 mosaicism. The
transient leukemoid reactions are characterized by a high spontaneous remission
rate. However, in some Downs syndrome patients with apparent transient leukemoid
reaction, leukemia relapse following periods of spontaneous remission have been
reported. Cytogenetic studies of leukemic cells in Down's syndrome patients show
a tendency toward hyperdiploidy. Besides trisomy 21, there is no other specific
cytogenetic abnormality that is characteristic of the leukemia cells in Down's
syndrome patients. The possible mechanisms for leukemogenesis in Down's syndrome
patients may involve factors at the levels of the organism, the organ/system,
the cell, the chromosomes or the DNA. We describe the clinical, hematological and histomorphological features in
children of primary myelodysplastic syndrome (MDS) seen at the All India
Institute of Medical Sciences over three years (Jan 2001-Jan 2004). Twenty-one
patients of primary MDS aged 17 year or less were classified using the latest
proposed WHO classification for Pediatric MDS. The median age was 9 years with
male predomice (80%). Pallor was present in all the cases while fever and
bleeding diathesis was present in more than 50% of the cases. Morphological
assessment of the peripheral blood showed macrocytosis in 50%, pancytopenia in
15% and blast cells in 45% of cases. A complete analysis of clinical features in
conjunction with the bone marrow profile revealed 8 cases of refractory
cytopenia (RC), 3 cases of refractory anemia with excess blasts (RAEB), 5 cases
of refractory anemia with excess blasts in transformation (RAEB-T), 4 cases of
Juvenile myelomonocytic leukemia (JMML) and a solitary cases of acute myeloid
leukemia (AML) in Downs syndrome. These children were followed up from 1-36
months (mean 15 months). Three patients of RAEB-T progressed to AML within 3-4
months. RC had the best prognosis and all are alive and under regular follow up.
The solitary case of AML of Downs syndrome died 1.5 months after initial
diagnosis. All 3 cases of RAEB are under regular follow-up and doing well. Three
cases of RAEB-T died (all had progressed to AML); the remaining 2 cases were
lost to follow up. Of the 4 cases of JMML 1 died within 6 months of diagnosis;
the other 3 cases are under regular follow up of whom 1 has a progressively
increasing blast count. We conclude that the latest proposed WHO classification
for Pediatric MDS can be successfully applied to all cases of primary MDS. A new born with a mongoloid slant, brachycephaly and low-set ears presented at
birth with a total leucocyte count of 57 x 10(3)/microL and the differential
leucocyte count revealed 70% of these to be blasts. The morphology of the blasts
was not characteristic of myeloid and lymphoid lineage. Cytochemistry showed
myeloperoxidase (MPO), Sudan Black B (SBB), periodic acid-Schiff (PAS) and
non-specific esterase (NSE) negativity. Flowcytometry showed blasts that were
positive for CD-33, CD-34 and CD-7. On second follow-up on the 10th day, the
same picture persisted on morphology. On subsequent follow-up, the blasts
disappeared. This was thus confirmed to be a case with transient leukemia with
Downs syndrome. Congenital leukemia is a rare but a well-documented disease in which leukemic
process is detected at birth or very shortly thereafter (Philip McCoy and Roy
Overton, Commun Clin Cytom 22:85-88, 1995). These leukemias represent
approximately 0.8 % of all childhood leukemias. We present a case of congenital
acute myeloid leukemia manifesting from the very first day of birth. Diagnosis
of acute myeloid leukemia was suspected by the presence of blasts in the
peripheral blood smear and was confirmed on bone marrow by flowcytometry.
Karyotyping revealed Trisomy 21. |
Which enzyme is inhibited by ixazomib? | Ixazomib is proteasome inhibitor. It is used for treatment of multiple myeloma. | Medicinal chemists try to avoid certain organic functional groups, summarized in
an ever-growing list, in order to avoid the potential bioactivation to reactive
metabolites. To add to that alert list, we report herein that boronic
acid-containing compound structures, such as those found in proteasome
inhibitors bortezomib and ixazomib, can become bioactivated to chemically
reactive imine amide metabolites. Test compounds, ixazomib and bortezomib, were
incubated in vitro using human liver fractions containing cytosol and microsomes
(S9) under conventional conditions in the presence of GSH. Metabolites were then
analyzed using LC-MS(n) with or without online hydrogen-deuterium exchange (HDX)
liquid chromatography coupled with an LTQ-Orbitrap. The exact mass measurements
of both the precursor and product ions were acquired through data dependent
acquisition and compared with theoretical values of proposed fragment ions. Upon
deboronation catalyzed by cytochrome P450 enzymes, both test compounds formed
imine amide metabolites that were identified by high resolution exact mass
measurements in both normal aqueous and HDX HPLC-MS analysis. GSH conjugates
were also identified and were postulated as nucleophilic addition of GSH to the
imine amide metabolites. All mass spectrometric and HDX measurements of these
GSH conjugates proved that the GSH unit was added to the carbon atom of the
imine amide partial structure, hence demonstrating the electrophilic property of
these imine amide metabolites. The awareness of the formation of electrophilic
imine amide metabolites from boronic acid-containing compounds, where the boron
atom is bonded to a carbon atom adjacent to an amide nitrogen, should help in
drug candidate design and optimization with regard to avoiding potential
bioactivation. Evading apoptosis is a cancer hallmark that remains a serious obstacle in
current treatment approaches. Although proteasome inhibitors (PIs) have
transformed management of multiple myeloma (MM), drug resistance emerges through
induction of the aggresome+autophagy pathway as a compensatory protein clearance
mechanism. Genome-wide profiling identified microRNAs (miRs) differentially
expressed in bortezomib-resistant myeloma cells compared with drug-naive cells.
The effect of individual miRs on proteasomal degradation of short-lived
fluorescent reporter proteins was then determined in live cells. MiR-29b was
significantly reduced in bortezomib-resistant cells as well as in cells
resistant to second-generation PIs carfilzomib and ixazomib. Luciferase reporter
assays demonstrated that miR-29b targeted PSME4 that encodes the proteasome
activator PA200. Synthetically engineered miR-29b replacements impaired the
growth of myeloma cells, patient tumor cells and xenotransplants. MiR-29b
replacements also decreased PA200 association with proteasomes, reduced the
proteasome's peptidase activity and inhibited ornithine decarboxylase turnover,
a proteasome substrate degraded through ubiquitin-independent mechanisms.
Immunofluorescence studies revealed that miR-29b replacements enhanced the
bortezomib-induced accumulation of ubiquitinated proteins but did not reveal
aggresome or autophagosome formation. Taken together, our study identifies
miR-29b replacements as the first-in-class miR-based PIs that also disrupt the
autophagy pathway and highlight their potential to synergistically enhance the
antimyeloma effect of bortezomib. Proteasome inhibition represents one of the more important therapeutic targets
in the treatment of multiple myeloma (MM), since by suppressing nuclear
factor-κB activity, which promotes myelomagenesis, it makes plasma cells
susceptible to proapoptotic signals. Bortezomib, the first proteasome inhibitor
approved for MM therapy, has been shown to increase response rate and improve
outcome in patients with relapsed/refractory disease and in the frontline
setting, particularly when combined with immunomodulatory drugs and alkylating
agents. Among second-generation proteasome inhibitors, ixazomib (MLN9708) is the
first oral compound to be evaluated for the treatment of MM. Ixazomib has shown
improved pharmacokinetic and pharmacodynamic parameters compared with
bortezomib, in addition to similar efficacy in the control of myeloma growth and
prevention of bone loss. Ixazomib was found to overcome bortezomib resistance
and to trigger synergistic antimyeloma activity with dexamethasone,
lenalidomide, and histone deacetylase inhibitors. Phase I/II studies using
ixazomib weekly or twice weekly in relapsed/refractory MM patients suggested
antitumor activity of the single agent, but more promising results have been
obtained with the combination of ixazomib, lenalidomide, and dexamethasone in
newly diagnosed MM. Ixazomib has also been used in systemic amyloidosis as a
single agent, showing important activity in this difficult-to-treat plasma-cell
dyscrasia. More frequent side effects observed during administration of ixazomib
were thrombocytopenia, nausea, vomiting, diarrhea, fatigue, and rash, whereas
severe peripheral neuropathy was rare. Here, we review the chemical
characteristics of ixazomib, as well as its mechanism of action and results from
preclinical and clinical trials. AIMS: This population pharmacokinetic analysis of the investigational oral
proteasome inhibitor ixazomib assessed the feasibility of switching from body
surface area (BSA)-based to fixed dosing, and the impact of baseline covariates
on ixazomib pharmacokinetics.
METHODS: Data were pooled from 226 adult patients with multiple myeloma,
lymphoma or solid tumours in four phase 1 studies, in which ixazomib dosing
(oral/intravenous, once/twice weekly) was based on BSA. Population
pharmacokinetic modelling was undertaken using nonmem version 7.2.
RESULTS: Ixazomib pharmacokinetics were well described by a three compartment
model with first order absorption and linear elimination. Ixazomib was absorbed
rapidly (Ka 0.5 h(-1)), with dose- and time-independent pharmacokinetics.
Estimated absolute bioavailability and clearance were 60% and 2l h(-1),
respectively. Although a small effect of BSA (range 1.3-2.6 m(2)) was observed
on the peripheral volume of distribution (V4), reducing the corresponding
inter-individual variability by 12.9%, there was no relationship between BSA and
ixazomib clearance (the parameter that dictates total systemic exposure
following fixed dosing). Consistently, based on simulations (n = 1000), median
AUCs (including interquartile range) were similar after BSA-based (2.23 mg
m(-2)) and fixed (4 mg) oral dosing with no trend in simulated AUC vs. BSA for
fixed dosing (P = 0.42). No other covariates, including creatinine clearance
(22-213.7 ml min(-1)) and age (23-86 years), influenced ixazomib
pharmacokinetics.
CONCLUSIONS: This analysis supports a switch from BSA-based to fixed dosing,
without dose modification for mild/moderate renal impairment or age, in future
adult studies of ixazomib, simplifying dosing guidance and clinical development. BACKGROUND: The combination of bortezomib, lenalidomide, and dexamethasone is a
highly effective therapy for newly diagnosed multiple myeloma. Ixazomib is an
investigational, oral, proteasome inhibitor with promising anti-myeloma effects
and low rates of peripheral neuropathy. In a phase 1/2 trial we aimed to assess
the safety, tolerability, and activity of ixazomib in combination with
lenalidomide and dexamethasone in newly diagnosed multiple myeloma.
METHODS: We enrolled patients newly diagnosed with multiple myeloma aged 18
years or older with measurable disease, Eastern Cooperative Oncology Group
performance status 0-2, and no grade 2 or higher peripheral neuropathy, and
treated them with oral ixazomib (days 1, 8, 15) plus lenalidomide 25 mg (days
1-21) and dexamethasone 40 mg (days 1, 8, 15, 22) for up to 12 28-day cycles,
followed by maintece therapy with ixazomib alone. In phase 1, we gave
patients escalating doses of ixazomib (1·68-3·95 mg/m(2)) to establish the
recommended dose for phase 2. The primary endpoints were maximum tolerated dose
for phase 1, and the rate of very good partial response or better for phase 2.
Safety analyses were done in all patients who received at least one dose of
study drug; efficacy analyses were done in all patients who received at least
one dose of study drug at the phase 2 dose, had measurable disease at baseline,
and had at least one post-baseline response assessment. This study is registered
at ClinicalTrials.gov, number NCT01217957.
FINDINGS: Between Nov 22, 2010, and Feb 28, 2012, we enrolled 65 patients (15 to
phase 1 and 50 to phase 2). Four dose-limiting toxic events were noted in phase
1: one at a dose of ixazomib of 2·97 mg/m(2) and three at 3·95 mg/m(2). The
maximum tolerated dose of ixazomib was established as 2·97 mg/m(2) and the
recommended phase 2 dose was 2·23 mg/m(2), which was converted to a 4·0 mg fixed
dose based on population pharmacokinetic results. Grade 3 or higher adverse
events related to any drug were reported in 41 (63%) patients, including skin
and subcutaneous tissue disorders (11 patients, 17%), neutropenia (eight
patients, 12%), and thrombocytopenia (five patients, 8%); drug-related
peripheral neuropathy of grade 3 or higher occurred in four (6%) patients. Five
patients discontinued because of adverse events. In 64 response-evaluable
patients, 37 (58%, 95% CI 45-70) had a very good partial response or better.
INTERPRETATION: The all-oral combination of weekly ixazomib plus lenalidomide
and dexamethasone was generally well tolerated and appeared active in newly
diagnosed multiple myeloma. These results support the phase 3 trial development
of this combination for multiple myeloma.
FUNDING: Millennium Pharmaceuticals, a wholly owned subsidiary of Takeda
Pharmaceutical International Company. INTRODUCTION: Proteasome inhibition is a mainstay in the treatment of multiple
myeloma (MM). Bortezomib, the first proteasome inhibitor (PI) approved for MM
therapy, has shown efficacy in relapsed/refractory patients and in the
front-line setting. Among second-generation PIs, MLN9708 ( ixazomib ) is the
first oral compound to be evaluated in MM treatment and has shown improvement in
pharmacokinetic and pharmacodynamic parameters compared with bortezomib with a
similar efficacy in the control of myeloma growth and in the prevention of bone
loss.
AREAS COVERED: In this review, the authors discuss the rationale for use of PIs.
They then summarize the clinical development of ixazomib in MM, from initial
Phase I to Phase II studies as a monotherapy and in combination with other
chemotherapeutics.
EXPERT OPINION: Preliminary data of Phase I/II trials showed that ixazomib had a
good safety profile and exerted anti-myeloma activity as a single agent in
relapsed/refractory patients. Furthermore, ixazomib also had efficacy in
patients who were refractory to bortezomib. Its use in combination with
lenalidomide and dexamethasone was shown to be an effective and well-tolerated
regimen in up-front treatment leading to minimal residual disease negativity in
a significant number of patients. Results of Phase III trials, evaluating
ixazomib in induction or maintece therapy, are awaited. BACKGROUND: Ixazomib is the first oral, proteasome inhibitor to reach phase III
trials. Here, we present an integrated nonclinical and clinical assessment of
ixazomib's effect on QTc intervals.
METHODS: Nonclinical studies assessed (1) the in vitro binding of ixazomib to
the hERG channel and (2) its effect on QT/QTc in dogs (N = 4) via telemetry.
Pharmacokinetic-matched triplicate electrocardiograms were collected in four
clinical phase I studies of intravenous (0.125-3.11 mg/m(2), N = 125, solid
tumors/lymphoma) or oral (0.24-3.95 mg/m(2), N = 120, multiple myeloma)
ixazomib. The relationship between ixazomib plasma concentration and heart rate
(HR)-corrected QT using Fridericia (QTcF) or population (QTcP) methods was
analyzed using linear mixed-effects models with fixed effects for day and time.
RESULTS: In vitro binding potency for ixazomib to the hERG channel was weak (K i
24.9 μM; IC50 59.6 μM), and nonclinical telemetry studies showed no QT/QTc
prolongation at doses up to 4.2 mg/m(2). In cancer patients, ixazomib, when
evaluated at doses yielding various plasma concentrations (with 26 % of data
greater than mean C max for the 4 mg phase 3 dose), had no meaningful effect on
QTc based on model-predicted mean change in QTcF/QTcP from baseline. There was
no relationship between ixazomib concentration and RR, suggesting no effect on
HR.
CONCLUSIONS: Ixazomib has no clinically meaningful effects on QTc or HR.
Integrating preclinical data and concentration-QTc modeling of phase 1 data may
obviate the need for a dedicated QTc study in oncology. A framework for QT
assessment in oncology drug development is proposed. The development of proteasome inhibitors (PIs) and immunomodulatory drugs has
significantly improved outcomes for patients with relapsed/refractory multiple
myeloma (RRMM); however, not all patients benefit from treatment with these
agents and some patients can become drug refractory over time. Due to the
largely incurable nature of multiple myeloma, the development of newer agents is
ongoing and includes new oral PIs (ixazomib), immunotherapies (e.g., CD38- or
SLAMF7-targeted antibodies), and small molecules. This review provides an
overview of the advances in targeted therapy for patients with RRMM, including
recently approved agents, with a focus on monotherapy and combined targeted
therapies. PURPOSE: This study was performed to determine whether the investigational
proteasome inhibitor ixazomib demonstrated selective antineoplastic activity
against acute myelogenous leukemia cells expressing a mutated nucleophosmin-1
gene and to gain a better understanding of its mechanisms of action.
EXPERIMENTAL DESIGN: The cytotoxic effects of ixazomib treatment were analyzed
in human acute myelogenous leukemia (AML) cell lines and primary AML samples
expressing wild-type or mutated NPM1 (NPMc(+)). The potential roles of oxidative
stress in mediating cytotoxic activity were determined using flow cytometry,
enzyme-based assays, and Western blots.
RESULTS: Apoptosis induced by ixazomib was abrogated by knockdown of
NPM1/NPMc(+)expression using an inducible shRNA construct and enhanced by
NPMc(+)overexpression. Cytotoxicity was associated with superoxide generation
and was reduced by the addition of the antioxidant N-acetylcysteine. AML cells
expressing NPMc(+)had significantly reduced levels of intracellular glutathione
and NADPH associated with reduced antioxidant responses to drug treatment.
Treatment of 3 patients with relapsed NPMc(+)AML resulted in an antileukemic
effect in 1 patient as demonstrated by a marked reduction of leukemic blasts in
the peripheral blood. Efficacy was associated with superoxide generation,
reduced glutathione levels, and reduced mRNA and protein expression of
antioxidant effectors in responding cells.
CONCLUSIONS: In this study, a direct association was observed between
NPMc(+)expression in AML, reduced antioxidant responses, and enhanced
sensitivity to an oral proteasome inhibitor that induces oxidative stress. These
data suggest that intracellular determits of antioxidant responses may be
good predictors of therapeutic response to ixazomib. In the last few weeks, the FDA approved three new therapies for multiple
myeloma: ixazomib, the first oral proteasome inhibitor; and daratumumab and
elotuzumab, two monoclonal antibodies that target CD38 and SLAMF7, respectively. Inhibition of the proteasome has emerged as a clinically effective anticancer
therapeutic approach in recent years. Bortezomib (Velcade®) showed extremely
high potency against a wide range of cancer cell lines. Ixazomib
(MLN9708-MLN2238), the second-generation proteasome inhibitor, selectivity and
potency were similar to that of bortezomib, is currently being investigated in
phase I studies. It shows superior antitumor activity in hematologic maligcy,
especially multiple myelomas. In this study, for the first time, we evaluated
and compared the antiproliferative and apoptotic effects of the novel proteasome
inhibitor MLN2238 (the active form of MLN9708) with bortezomib using in vitro
chronic myeloid leukemia. Cytotoxic and apoptotic effects of MLN2238 and
bortezomib were determined by trypan blue dye exclusion assays, WST-1 cell
proliferation assay, increased AnnexinV-PI binding capacity, changes in
caspase-3 activity and loss of mitochondrial membrane potential (JC-1).
Associated with proteasome pathway NFκB1 and c-myc mRNA expression levels were
examined by the qRT-PCR method. We observed that cytotoxic and apoptotic effects
on K562 cells were started at 5 μm of MLN2238 and 1 μm of bortezomib after 24
and 48 h. Also, MLN2238 and bortezomib downregulated NFκB1 and c-myc mRNA
expression at 24 h. Our result revealed that MLN22238 and bortezomib had
significant cytotoxic and apoptotic effects on K562 cells. Here, we first
demonstrate in vitro data that support the development of MLN2238, by direct
comparison with bortezomib on K562 cells. Despite the significant therapeutic advances achieved with proteasome inhibitors
(PIs) such as bortezomib and carfilzomib in prolonging the survival of patients
with multiple myeloma, the development of drug resistance, peripheral
neuropathy, and pharmacokinetic limitations continue to pose major challenges
when using these compounds. Ixazomib is a second-generation PI with improved
activity over other PIs. Unlike bortezomib and carfilzomib, which are
administered by injection, ixazomib is the first oral PI approved by US Food and
Drug Administration. This review discusses the biochemical properties,
mechanisms of action, preclinical efficacy, and clinical trial results leading
to the US Food and Drug Administration approval of ixazomib. Ixazomib is the first oral proteasome inhibitor to be investigated in the
clinic. This clinical study assessed whether the pharmacokinetics of ixazomib
would be altered if administered after a high-calorie, high-fat meal. In a
2-period, 2-sequence, crossover study design, adult patients with advanced solid
tumors or lymphoma received a 4-mg oral dose of ixazomib as immediate-release
capsules on day 1 without food (fasted, administered following an overnight
fast) or with food (fed, following consumption of a high-calorie, high-fat
meal), followed by another dose on day 15 in the alternate food intake condition
(fasted to fed or fed to fasted). Twenty-four patients were enrolled; of these,
15 were included in the pharmacokinetic-evaluable population. Administration of
ixazomib after a high-fat meal reduced both the rate and extent of absorption of
ixazomib. Under fed conditions, the median time to peak plasma concentration
(Tmax ) of ixazomib was delayed by approximately 3 hours compared with
administration in the fasted state (1.02 hours vs 4.0 hours), and there was a
28% reduction in total systemic exposure (area under the curve, AUC) and a 69%
reduction in peak plasma concentration (Cmax ). Together, the results support
the administration of ixazomib on an empty stomach, at least 1 hour before or at
least 2 hours after food. These recommendations are reflected in the United
States Prescribing Information for ixazomib (clinicaltrials.gov identifier
NCT01454076). Bone disease is a characteristic feature of multiple myeloma, a maligt plasma
cell dyscrasia. In patients with multiple myeloma, the normal process of bone
remodeling is dysregulated by aberrant bone marrow plasma cells, resulting in
increased bone resorption, prevention of new bone formation, and consequent bone
destruction. The ubiquitin-proteasome system, which is hyperactive in patients
with multiple myeloma, controls the catabolism of several proteins that regulate
bone remodeling. Clinical studies have reported that treatment with the
first-in-class proteasome inhibitor bortezomib reduces bone resorption and
increases bone formation and bone mineral density in patients with multiple
myeloma. Since the introduction of bortezomib in 2003, several next-generation
proteasome inhibitors have also been used clinically, including carfilzomib,
oprozomib, ixazomib, and delanzomib. This review summarizes the available
preclinical and clinical evidence regarding the effect of proteasome inhibitors
on bone remodeling in multiple myeloma. Firefly luciferase-based reporter gene assays are the most commonly used assays
to investigate the transcriptional regulation of gene expression. However,
direct interaction of tested compounds with the firefly luciferase leading to
altered enzymatic activity may lead to misinterpretation of experimental data.
When investigating the proteasome inhibitors bortezomib, carfilzomib, and
ixazomib, we observed increased luminescence for bortezomib and ixazomib, but
not for carfilzomib, in a prege-X-receptor (PXR) reporter gene assay, which
was inconsistent with the mRNA expression levels of the main PXR target gene
CYP3A4. To further scrutinize this phenomenon, we performed experiments with
constitutively expressed firefly luciferase and demonstrated that the increase
in cellular firefly luciferase activity is independent from PXR activation or
CYP3A4 promoter. Using cell-free assays with recombit firefly luciferase
enzyme, we made the counterintuitive observation that firefly luciferase
activity is inhibited by bortezomib and ixazomib in a reversible and competitive
manner. This inhibition stabilizes the firefly luciferase enzyme against
proteolytic degradation (e.g., toward trypsin), thereby increasing its half-life
with subsequent enhancement of total cellular luminescence that eventually
mimicked PXR-driven luciferase induction. These data show that particular
compounds can strikingly interfere with firefly luciferase and once more
illustrate the importance of careful interpretation of data obtained from
luciferase-based assays. Proteasome inhibitors have become an integral part of myeloma therapy.
Considerable efforts have gone into optimizing this therapeutic approach to
obtain maximal proteasome inhibition with least toxicity. Ixazomib is the first
oral proteasome inhibitor to enter the clinic and has been studied as a single
agent as well as in various combinations. The current trial was designed to
examine the efficacy and toxicity of combining 2 different doses of ixazomib (4
mg and 5.5 mg given weekly for 3 of 4 weeks) with 40 mg weekly of dexamethasone,
in relapsed myeloma. Seventy patients were enrolled, 35 patients randomly
assigned to each ixazomib dose. Overall, 30 (43%; 95% confidence interval,
31-55) of the patients achieved a confirmed partial response or better, with 31%
achieving a response with 4 mg and 54% with 5.5 mg of ixazomib. The median
event-free survival (EFS) for the entire study population was 8.4 months; 1-year
overall survival was 96%. The EFS was 5.7 months for patients with prior
bortezomib exposure and 11.0 months for bortezomib-naïve patients. A grade 3 or
4 adverse event considered at least possibly related to treatment was seen in 11
(32%) patients at 4 mg and in 21 (60%) at 5.5 mg. Dose reductions were more
frequent with 5.5 mg dose. Overall, the ixazomib with dexamethasone has good
efficacy in relapsed myeloma, is well-tolerated and with higher response rate at
5.5 mg, albeit with more toxicity. This study was registered at
www.clinicaltrials.gov as #NCT01415882. |
Does PCSK9 (Proprotein convertase subtilisin/kexin type 9) binds with HDL-receptor (HDL-R)? | No, Proprotein Convertase Subtilisin Kexin 9 (PCSK9) binds with LDL-receptor (LDL-R) causing its degradation in the lysosome with the result of LDL-C accumulating in the blood. | Mutations within PCSK9 (proprotein convertase subtilisin/kexin type 9) are
associated with domit forms of familial hyper- and hypocholesterolemia.
Although PCSK9 controls low density lipoprotein (LDL) receptor (LDLR) levels
post-transcriptionally, several questions concerning its mode of action remain
uswered. We show that purified PCSK9 protein added to the medium of human
endothelial kidney 293, HepG2, and Chinese hamster ovary cell lines decreases
cellular LDL uptake in a dose-dependent manner. Using this cell-based assay of
PCSK9 activity, we found that the relative potencies of several PCSK9 missense
mutants (S127R and D374Y, associated with hypercholesterolemia, and R46L,
associated with hypocholesterolemia) correlate with LDL cholesterol levels in
humans carrying such mutations. Notably, we found that in vitro wild-type PCSK9
binds LDLR with an approximately 150-fold higher affinity at an acidic endosomal
pH (K(D) = 4.19 nm) compared with a neutral pH (K(D) = 628 nm). We also
demonstrate that wild-type PCSK9 and mutants S127R and R46L are internalized by
cells to similar levels, whereas D374Y is more efficiently internalized,
consistent with their affinities for LDLR at neutral pH. Finally, we show that
LDL diminishes PCSK9 binding to LDLR in vitro and partially inhibits the effects
of secreted PCSK9 on LDLR degradation in cell culture. Together, the results of
our biochemical and cell-based experiments suggest a model in which secreted
PCSK9 binds to LDLR and directs the trafficking of LDLR to the lysosomes for
degradation. BACKGROUND: Proprotein convertase subtilisin kexin type 9 (PCSK9) is gaining
attention as a key regulator of serum LDL-cholesterol (LDLC). This novel serine
protease causes the degradation of hepatic LDL receptors by an unknown
mechanism. In humans, gain-of-function mutations in the PCSK9 gene cause a form
of familial hypercholesterolemia, whereas loss-of-function mutations result in
significantly decreased LDLC and decreased cardiovascular risk. Relatively
little is known about PCSK9 in human serum.
METHODS: We used recombit human PCSK9 protein and 2 different anti-PCSK9
monoclonal antibodies to build a sandwich ELISA. We measured PCSK9 and lipids in
55 human serum samples and correlated the results. We used the anti-PCSK9
antibodies to assay lipoprotein particle fractions separated by sequential
flotation ultracentrifugation.
RESULTS: Serum concentrations of PCSK9 ranged from 11 to 115 microg/L and were
directly correlated with serum concentrations of LDLC (r = 0.45, P = 0.001) and
total cholesterol (r = 0.50, P = 0.0003), but not with triglycerides (r = 0.15,
P = 0.28) or HDL cholesterol concentrations (r = 0.13, P = 0.36). PCSK9 was not
detectable in any lipoprotein particle fraction, including LDL.
CONCLUSIONS: PCSK9 is present in human serum, likely not associated with
specific lipoprotein particles. The circulating concentrations of human PCSK9
are directly correlated with LDL and total cholesterol concentrations. BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the
degradation of the LDL receptor (LDLr) in hepatocytes, and its expression in
mouse liver has been shown to decrease with fenofibrate treatment.
METHODS: We developed a sandwich ELISA using recombit human PCSK9 protein and
2 affinity-purified polyclonal antibodies directed against human PCSK9. We
measured circulating PCSK9 concentrations in 115 diabetic patients from the
FIELD (Fenofibrate Intervention and Event Lowering in Diabetes) study before and
after fenofibrate treatment.
RESULTS: We found that plasma PCSK9 concentrations correlate with total (r =
0.45, P = 0.006) and LDL (r = 0.54, P = 0.001) cholesterol but not with
triglycerides or HDL cholesterol concentrations in that cohort. After 6 weeks of
treatment with comicronized fenofibrate (200 mg/day), plasma PCSK9
concentrations decreased by 8.5% (P = 0.041 vs pretreatment). This decrease
correlated with the efficacy of fenofibrate, as judged by a parallel reduction
in plasma triglycerides (r = 0.31, P = 0.015) and LDL cholesterol concentrations
(r = 0.27, P = 0.048).
CONCLUSIONS: We conclude that this decrease in PCSK9 explains at least in part
the LDL cholesterol-lowering effects of fenofibrate. Fenofibrate might be of
interest to further reduce cardiovascular risk in patients already treated with
a statin. Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the extracellular
domain of the low density lipoprotein receptor (LDLR) at the cell surface, and
disrupts the normal recycling of the LDLR. However, the exact mechanism by which
the LDLR is re-routed for lysosomal degradation remains to be determined. To
clarify the role of the cytoplasmic domain of the LDLR for re-routing to the
lysosomes, we have studied the ability of PCSK9 to degrade a chimeric receptor
which contains the extracellular and transmembrane domains of the LDLR and the
cytoplasmic domain of the transferrin receptor. These studies were performed in
CHO T-REx cells stably transfected with a plasmid encoding the chimeric receptor
and a novel assay was developed to study the effect of PCSK9 on the LDLR in
these cells. Localization, function and stability of the chimeric receptor were
similar to that of the wild-type LDLR. The addition of purified gain-of-function
mutant D374Y-PCSK9 to the culture medium of stably transfected CHO T-REx cells
showed that the chimeric receptor was degraded, albeit to a lower extent than
the wild-type LDLR. In addition, a mutant LDLR, which has the three lysines in
the intracellular domain substituted with arginines, was also degraded by
D374Y-PCSK9. Thus, the mechanism for the PCSK9-mediated degradation of the LDLR
does not appear to involve an interaction between the endosomal sorting
machinery and LDLR-specific motifs in the cytoplasmic domain. Moreover,
ubiquitination of lysines in the cytoplasmic domain does not appear to play a
critical role in the PCSK9-mediated degradation of the LDLR. BACKGROUND: Familial hypercholesterolemia (FH) is an autosomal disorder
associated with elevated plasma low density lipoprotein (LDL) levels leading to
premature coronary heart disease (CHD). As a result of long-term hyperlipemia,
FH patients will present endarterium thickening and artherosclerosis. In the
present study we scanned the related gene of a clinically diagnosed autosomal
genetic hypercholesterolemia family for the possible mutations and established
eukaryotic expression vector of mutation of proprotein convertase
subtilisin/kexin type 9 (PCSK9) gene with gene recombination technique to
investigate the contributions of the variation on low density lipoprotein
receptor (LDL-R) metabolism and function alternation.
METHODS: Mutation detection was conducted for LDL-R, apolipoprotein B(100)
(apoB(100)) and PCSK9 gene with nucleotide sequencing in a Chinese FH family.
The full-length cDNA of wild type PCSK9 gene (WT-PCSK9) was obtained from
Bel-7402. Site mutagenesis was used to establish the recombit eukaryotic
expression vector carrying pathogenic type of PCSK9 gene and the inserted
fragment was sequenced. With the blank vector as control, liposome transfection
method was used to transfect the Bel-7402 cells with recombit plasmid. The
expression of LDL-R mRNA was examined by RT-PCR. PCSK9 and the expression of
LDL-R protein were determined by Western blotting.
RESULTS: The G-->T mutation at the 918 nucleotide of PCSK9 gene resulted in the
substitution of the arginine by a serine at the codon 306 of exon 6. After
sequencing, it was confirmed that the inserted fragment of established
expression vector had correct size and sequence and the mutant was highly
expressed in Bel-7402 cells. There was no significant variation in the levels of
LDL-R mRNA. LDL-R mature protein was decreased by 57% after the cells were
transfected by WT-PCSK9 plasmid. Mature LDL-R was significantly decreased by 12%
after the cells were transfected by R306S mutant as evidenced by gray scale
scanning, suggesting that the new mutant R306S can significantly decrease the
expression of mature LDL-R protein.
CONCLUSIONS: A novel missense mutation of PCSK9 gene, R306S, was found and the
eukaryotic expression vectors of mutant and wild-type of PCSK9 gene were
established. There was no significant variation in the levels of LDL-R mRNA. The
R306S mutation could significantly lead to the decrease of LDL-R mature protein
expression, which might be the pathogenic gene of the FH family. Proprotein convertase, subtilisin/kexin type 9 (PCSK9), a key regulator of
plasma LDL-cholesterol (LDL-c) and cardiovascular risk, is produced in liver and
secreted into plasma where it binds hepatic LDL receptors (LDLR), leading to
their degradation. PCSK9 is transcriptionally activated by sterol response
element-binding protein (SREBP)-2, a transcription factor that also activates
all genes for cholesterol synthesis as well as the LDLR. Here we investigated
the relationship between plasma PCSK9 levels and the lathosterol-to-cholesterol
ratio, a marker of cholesterol biosynthesis, in 18 healthy subjects during a 48
h fast. In all individuals, plasma PCSK9 levels declined steadily during the
fasting period, reaching a nadir at 36 h that was ∼58% lower than levels
measured in the fed state (P < 0.001). Similarly, the lathosterol-to-cholesterol
ratio declined in parallel with plasma PCSK9 concentrations during the fast,
reaching a nadir at 36 h that was ∼28% lower than that measured in the fed state
(P = 0.024). In summary, fasting has a marked effect on plasma PCSK9
concentrations, which is mirrored by measures of cholesterol synthesis in
humans. Inasmuch as cholesterol synthesis and PCSK9 are both regulated by
SREBP-2, these results suggest that plasma PCSK9 levels may serve as a surrogate
marker of hepatic SREBP-2 activity in humans. PURPOSE: Apolipoprotein M (apoM) retards atherosclerosis development in murine
models, and may be regulated by pathways involved in LDL metabolism. Proprotein
convertase subtilisin-kexin type 9 (PCSK9) plays a key role in LDL receptor
processing. We determined the extent to which plasma apoM is related to PCSK9
levels in subjects with varying degrees of obesity.
METHODS: We sought correlations between plasma apoM and PCSK9, measured using
recently developed ELISAs, in 79 non-diabetic subjects.
RESULTS: ApoM and PCSK9 levels were both correlated positively with total
cholesterol, non-HDL cholesterol, LDL cholesterol and apoB (P < 0.05 to P <
0.001). ApoM correlated positively with PCSK9 in lean individuals (n = 37, r =
0.337, P = 0.041), but not in overweight subjects (n = 32, r = 0.125, P = 0.50)
and in obese subjects (n = 10, r = -0.055, P = 0.88).
CONCLUSIONS: The PCSK9 pathway may contribute to plasma apoM regulation in
humans. The influence of PCSK9 on circulating apoM appears to be modified by
adiposity. OBJECTIVE: Type 2 diabetes mellitus (T2DM) is associated with elevated plasma
apolipoprotein B and triglycerides levels, reduced HDL cholesterol and the
presence of small-dense LDL particles. The present study was conducted to
investigate the role of plasma proprotein convertase subtilisin kexin type 9
(PCSK9) levels, a regulator of LDL-receptor expression, in the occurrence of
diabetic dyslipidemia.
METHODS: Plasma PCSK9 was measured in a cohort of subjects with normal glucose
metabolism (NGM; n=288), impaired glucose metabolism (IGM; n=121) and type 2
diabetes mellitus (T2DM; n=139) to study whether its relation with plasma
apolipoprotein B, triglycerides, total cholesterol, non-HDL cholesterol, LDL
cholesterol and HDL cholesterol differed by levels of glucose metabolism status.
RESULTS: Plasma PCSK9 levels were not different between the three groups (82, 82
and 80 ng/mL in NGM, IGM and T2DM, respectively). PCSK9 was positively
associated with total cholesterol, non-HDL cholesterol, LDL cholesterol,
apolipoprotein B and triglycerides levels in all subgroups. The regression
slopes for the associations with non-HDL cholesterol were steeper among
individuals with T2DM than with NGM (β = 0.016 versus β=0.009,
p-interaction=0.05). Similar results were obtained for the relation with
apolipoprotein B (β = 0.004 versus β = 0.002, p-interaction=0.09).
CONCLUSIONS: Although glucose metabolism status per se is not associated with
plasma PCSK9 levels, the presence of T2DM may modify the relation between plasma
PCSK9 and non-HDL cholesterol and apolipoprotein B. These observations should be
regarded as hypothesis generating for further studies aimed at elucidating the
role of PCSK9 in the pathogenesis and treatment of diabetic dyslipidemia. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that
regulates the number of cell surface low-density lipoprotein receptors (LDLRs)
and the levels of low-density lipoprotein cholesterol in plasma. Intact cells
have not previously been used to determine the characteristics of binding of
PCSK9 to LDLR. Using PCSK9 iodinated by the tyramine cellobiose (TC) method
([(125)I]TC-PCSK9), we measured the affinity and kinetics of binding of PCSK9 to
LDLR on HepG2 cells at 4 °C. The extent of [(125)I]TC-PCSK9 binding increased as
cell surface LDLR density increased. Unlabeled wild-type and two
gain-of-function mutants of PCSK9 reduced binding of [(125)I]TC-PCSK9. The
Scatchard plot of the binding-inhibition curve was curvilinear, indicative of
high-affinity and low-affinity sites for PCSK9 binding on HepG2 cells. Nonlinear
regression analysis of the binding data also indicated that a two-site model
better fitted the data. The time course of [(125)I]TC-PCSK9 binding showed two
phases in the association kinetics. Dissociation of [(125)I]TC-PCSK9 also
occurred in two phases. Unlabeled PCSK9 accelerated the dissociation of
[(125)I]TC-PCSK9. At low pH, only one phase of dissociation was apparent.
Furthermore, the dissociation of [(125)I]TC-PCSK9 under pre-equilibrium
conditions was faster than under equilibrium conditions. Overall, the data
suggest that PCSK9 binding to cell surface LDLR cannot be described by a simple
bimolecular reaction. Possible interpretations that can account for these
observations are discussed. OBJECTIVE: Proprotein convertase subtilisin kexin type 9 (PCSK9) is an important
regulator of hepatic low-density lipoprotein (LDL)-cholesterol levels. Although
PCSK9 is mainly of hepatic origin, extra-hepatic tissues significantly
contribute to PCSK9 production and, potentially, local regulation of LDL
receptor expression.
METHODS AND RESULTS: In the present study we show that, among vascular cells,
PCSK9 is expressed in smooth muscle cells (SMCs) but not in endothelial cells,
macrophages and monocytes. PCSK9 was also detectable in human atherosclerotic
plaques. Conditioned media from SMCs significantly reduced LDLR expression in
human macrophage and in the macrophage cell line J774. Co-culture experiments
also demonstrated the influence of SMCs on LDLR expression in J774. PCSK9
released from SMCs directly regulated LDLR expression in macrophages as
demonstrated by retroviral overexpression or knockdown of PCSK9 with small
interfering RNA and by using recombit PCSK9. Moreover, the proteolytic
activity of PCSK9 was not required for LDLR downregulation since cultured media
containing either the catalytic inactive PCSK9 or PCSK9 WT had a similar effect
on LDLR in J774. Finally, conditioned media from SMCs affected β-VLDL
cholesterol uptake and PCSK9 expression reduced both LDLR and LDL uptake in
J774.
CONCLUSIONS: Taken together our data indicate that PCSK9 secreted by human SMCs
is functionally active and capable of reducing LDLR expression in macrophages. A
possible direct role for this protein in foam cell formation and atherogenesis
is suggested. BACKGROUND AND AIMS: Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is a
pro-atherogenic phospholipase A(2), which is predomitly complexed to
low-density lipoprotein (LDL) particles. Proprotein convertase subtilisin-kexin
type 9 (PCSK9) provides a key step in LDL metabolism by stimulating LDL receptor
degradation. We determined relationships between plasma PCSK9 and Lp-PLA(2)
mass.
METHODS: Lp-PLA(2) mass (turbidimetric immunoassay), PCSK9 (enzyme-linked
immunosorbent assay) and (apo) lipoproteins were measured in 53 nondiabetic
subjects (27 women) with body mass index <30 kg/m(2).
RESULTS: Lp-PLA(2) and PCSK9 levels were both correlated positively with LDL
cholesterol and non-high-density lipoprotein (HDL) cholesterol (r = 0.330 to r =
0.382, p ≤0.02). Remarkably, Lp-PLA(2) was inversely related to PCSK9 (r =
-0.388, p = 0.004). The Lp-PLA(2)/apolipoprotein B ratio, as a measure of the
Lp-PLA(2) content in apolipoprotein B-containing lipoproteins, was also
inversely correlated with PCSK9 (r = -0.575, p <0.001). The inverse
relationships of Lp-PLA(2) (p = 0.023) and the Lp-PLA(2)/apolipoprotein B ratio
(p = 0.001) with PCSK9 levels remained significant after controlling for age,
gender, triglycerides and HDL cholesterol.
CONCLUSIONS: Despite increasing effects on LDL cholesterol, higher PCSK9 levels
are unlikely to confer impaired Lp-PLA(2) metabolism. We propose to evaluate the
possible influence of PCSK9 inhibiting strategies on Lp-PLA(2) regulation and
vice versa to determine effects of Lp-PLA(2) inhibitors on the PCSK9 pathway. OBJECTIVE: proprotein convertase subtilisin/kexin type 9 (PCSK9) negatively
regulates the low-density lipoprotein (LDL) receptor (LDLR) in hepatocytes and
therefore plays an important role in controlling circulating levels of
LDL-cholesterol. To date, the relationship between PCSK9 and metabolism of
apolipoprotein B (apoB), the structural protein of LDL, has been controversial
and remains to be clarified.
METHODS AND RESULTS: We assessed the impact of PCSK9 overexpression (≈400-fold
above baseline) on apoB synthesis and secretion in 3 mouse models: wild-type
C57BL/6 mice and LDLR-null mice (Ldlr(-/-) and Ldlr(-/-)Apobec1(-/-)).
Irrespective of LDLR expression, mice transduced with the PCSK9 gene invariably
exhibited increased levels of plasma cholesterol, triacylglycerol, and apoB.
Consistent with these findings, the levels of very-low-density lipoprotein and
LDL were also increased whereas high-density lipoprotein levels were unchanged.
Importantly, we demonstrated that endogenous PCSK9 interacted with apoB in
hepatocytes. The PCSK9/apoB interaction resulted in increased production of
apoB, possibly through the inhibition of intracellular apoB degradation via the
autophagosome/lysosome pathway.
CONCLUSIONS: We propose a new role for PCSK9 that involves shuttling between
apoB and LDLR. The present study thus provides new insights into the action of
PCSK9 in regulating apoB metabolism. Furthermore, our results indicate that
targeting PCSK9 expression represents a new paradigm in therapeutic intervention
against hyperlipidemia. Secreted proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the
low-density lipoprotein receptor (LDLR) at the cell surface and disrupts the
normal recycling of the LDLR. When human PCSK9 is injected into LDLR-deficient
mice, PCSK9 is still rapidly cleared by the liver. This finding may suggest that
PCSK9 is physiologically also cleared by receptors other than the LDLR. An
alternative explanation could be that PCSK9 has undergone modifications during
purification and is cleared by scavenger receptors on liver endothelial
sinusoidal cells when injected into mice. If the only mechanism for clearing
PCSK9 in humans is through the LDLR, one would expect that differences in the
number of LDLRs would affect the plasma levels of low-density lipoprotein
cholesterol (LDLC) and PCSK9 in a similar fashion. In this study, levels of LDLC
and PCSK9 were measured in familial hypercholesterolemia (FH) homozygotes, FH
heterozygotes, and normocholesterolemic subjects. The ratio between the levels
of LDLC and PCSK9 was 1.7-fold higher in FH heterozygotes and 3-fold higher in
FH homozygotes than in the normocholesterolemic subjects. Thus, defective LDLRs
have a greater impact on the levels of LDLC than on the levels of PCSK9. By
assuming that the rate of PCSK9 synthesis is similar in the 3 groups, this
finding suggests that in humans, plasma PCSK9 is also cleared by
LDLR-independent mechanisms. BACKGROUND: Effects of thyroid function status on lipoprotein metabolism may
extend into the euthyroid range. Low-density lipoprotein (LDL) metabolism is
governed by proprotein convertase subtilisin-kexin type 9 (PCSK9), which
down-regulates LDL receptor expression, resulting in higher LDL cholesterol
(LDL-C). Here, we tested whether plasma PCSK9 correlates with thyroid function
in nonobese and obese euthyroid subjects.
METHODS: We assessed the extent to which plasma PCSK9 is determined by
thyrotropin (TSH) in 74 euthyroid subjects (31 women; TSH between 0.5 and
4.0 mU/L and free thyroxine [FT4] between 11.0 and 19.5 pM) with varying degrees
of obesity (body mass index [BMI] ranging from 20.2 to 40.4 kg/m(2)).
RESULTS: TSH, FT4, PCSK9, non-high-density lipoprotein cholesterol (non-HDL-C),
LDL-C, and apolipoprotein B (apoB) levels were not different between 64 nonobese
subjects (BMI<30 kg/m(2)) and 10 obese subjects (BMI≥30 kg/m(2); p>0.20 for
each). PCSK9 correlated positively with TSH in nonobese subjects (r=0.285,
p=0.023). In contrast, PCSK9 was not associated positively with TSH in obese
subjects (r=-0.249, p=0.49). The relationship of PCSK9 with TSH was different
between nonobese and obese subjects when taking age, sex, FT4, and the presence
of anti-thyroid antibodies into account (multiple linear regression analysis:
β=-0.320, p=0.012 for the interaction term between the presence of obesity and
TSH on PCSK9), and was also modified by BMI as a continuous trait (β=-0.241,
p=0.062 for the interaction term between BMI and TSH on PCSK9). Non-HDL-C,
LDL-C, and apoB levels were dependent on PCSK9 in nonobese subjects (p≤0.01 for
each), but not in obese subjects (p>0.50), Accordingly, BMI interacted
negatively with PCSK9 on non-HDL-C (p=0.028) and apoB (p=0.071).
CONCLUSIONS: This study suggests that circulating PCSK9 levels correlate with
thyroid function even in the normal range. This relationship appears to be
blunted by obesity. Thyroid functional status may influence cholesterol
metabolism through the PCSK9 pathway. BACKGROUND: LDL cholesterol (LDL-C) is a well established risk factor for
cardiovascular disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9)
binds LDL receptors, targeting them for degradation. We therefore assessed the
efficacy, safety, and tolerability of AMG 145, a human monoclonal IgG2 antibody
against PCSK9, in stable patients with hypercholesterolemia on a statin.
METHODS: In a phase 2, dose-ranging study done in 78 centres in the USA, Canada,
Denmark, Hungary, and Czech Republic, patients (aged 18-80 years) with LDL-C
greater than 2·2 mmol/L on a stable dose of statin (with or without ezetimibe),
were randomly assigned equally, through an interactive voice response system, to
subcutaneous injections of AMG 145 70 mg, 105 mg, or 140 mg, or matching placebo
every 2 weeks; or subcutaneous injections of AMG 145 280 mg, 350 mg, or 420 mg,
or matching placebo every 4 weeks. Everyone was masked to treatment assignment
within the every 2 weeks and every 4 weeks schedules. The primary endpoint was
the percentage change in LDL-C concentration from baseline after 12 weeks.
Analysis was by modified intention to treat. This study is registered with
ClinicalTrials.gov, number NCT01380730.
FINDINGS: 631 patients with hypercholesterolaemia were randomly assigned to AMG
145 70 mg (n=79), 105 mg (n=79), or 140 mg (n=78), or matching placebo (n=78)
every 2 weeks; or AMG 145 280 mg (n=79), 350 mg (n=79), and 420 mg (n=80), and
matching placebo (n=79) every 4 weeks. At the end of the dosing interval at week
12, the mean LDL-C concentrations were reduced generally dose dependently by AMG
145 every 2 weeks (ranging from 41·8% to 66·1%; p<0·0001 for each dose vs
placebo) and AMG 145 every 4 weeks (ranging from 41·8% to 50·3%; p<0·0001). No
treatment-related serious adverse events occurred. The frequencies of
treatment-related adverse events were similar in the AMG 145 and placebo groups
(39 [8%] of 474 vs 11 [7%] of 155); none of these events were severe or
life-threatening.
INTERPRETATION: The results suggest that PCSK9 inhibition could be a new model
in lipid management. Inhibition of PCSK9 warrants assessment in phase 3 clinical
trials.
FUNDING: Amgen. OBJECTIVE: LDL-receptor deficiency may provide a mechanism which contributes to
atherogenic lipoprotein abnormalities in experimental nephrosis and in humans
with glomerular proteinuria. The proprotein convertase subtilisin-kexin type 9
(PCSK9) pathway plays a key role in lipoprotein metabolism by promoting
LDL-receptor degradation. We tested whether plasma PCSK9 is elevated in
proteinuric states, and determined relationships of PCSK9 with lipoprotein
responses to proteinuria reduction.
METHODS: Thirty-nine kidney patients (e-GFR 61 ± 29 mL/min/1.73 m(2),
proteinuria 1.9 [0.9-3.3] g/day; 19 on statin treatment) were studied during 2
randomized double-blind 6-week periods on either lisinopril (40 mg/day) and a
regular sodium diet (194 ± 49 mmol Na+/day; baseline treatment) or lisinopril
plus valsartan (320 mg/day) and a low sodium diet (102 ± 52 mmol Na(+)/day;
maximal treatment), and compared to age- and sex-matched controls. Maximal
treatment decreased proteinuria to 0.5 [0.3-1.1] g/day (P < 0.001).
RESULTS: Plasma PCSK9 was increased at baseline in proteinuric subjects (213
[161-314] vs. 143 [113-190] ug/L in controls, P ≤ 0.001), irrespective of statin
use, e-GFR and BMI. PCSK9 correlated with proteinuria at baseline (R = 0.399,
P = 0.018) and at maximal antiproteinuric treatment (R = 0.525, P = 0.001), but
did not decrease during proteinuria reduction (P = 0.84). Individual changes in
total cholesterol (R = 0.365, P = 0.024), non-HDL cholesterol (R = 0.333,
P = 0.041), and LDL cholesterol (R = 0.346, P = 0.033) were correlated
positively with individual PCSK9 responses. PCSK9 at baseline independently
predicted the total/HDL cholesterol ratio response to treatment (P = 0.04).
CONCLUSION: Plasma PCSK9 was elevated in proteinuria, predicted lipoprotein
responses to proteinuria reduction but remained unchanged after proteinuria
reduction. Inhibition of the PCSK9 pathway may provide a novel treatment
strategy in proteinuric subjects. Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a secreted protein that
binds to the epidermal growth factor-like-A domain of the low density
lipoprotein receptor (LDLR) and mediates LDLR degradation in liver.
Gain-of-function mutations in PCSK9 are associated with autosomal domit
hypercholesterolemia in humans. Size-exclusion chromatography of human plasma
has shown PCSK9 to be partly associated with undefined high molecular weight
complexes within the LDL size range. We used density gradient centrifugation to
isolate LDL in plasma pooled from 5 normolipidemic subjects and report that >40%
of total PCSK9 was associated with LDL. Binding of fluorophore-labeled
recombit PCSK9 to isolated LDL in vitro was saturable with a K(D) ∼ 325 nM.
This interaction was competed >95% by excess unlabeled PCSK9, and competition
binding curves were consistent with a one-site binding model. An N-terminal
region of the PCSK9 prodomain (amino acids 31-52) was required for binding to
LDL in vitro. LDL dose-dependently inhibited binding and degradation of cell
surface LDLRs by exogenous PCSK9 in HuH7 cells. LDL also inhibited PCSK9 binding
to mutant LDLRs defective at binding LDL. These data suggest that association of
PCSK9 with LDL particles in plasma lowers the ability of PCSK9 to bind to cell
surface LDLRs, thereby blunting PCSK9-mediated LDLR degradation. Elevated LDL-cholesterol (LDLc) levels are a major risk factor for
cardiovascular disease and atherosclerosis. LDLc is cleared from circulation by
the LDL receptor (LDLR). Proprotein convertase subtilisin/kexin 9 (PCSK9)
enhances the degradation of the LDLR in endosomes/lysosomes, resulting in
increased circulating LDLc. PCSK9 can also mediate the degradation of LDLR
lacking its cytosolic tail, suggesting the presence of as yet undefined
lysosomal-targeting factor(s). Herein, we confirm this, and also eliminate a
role for the transmembrane-domain of the LDLR in mediating its PCSK9-induced
internalization and degradation. Recent findings from our laboratory also
suggest a role for PCSK9 in enhancing tumor metastasis. We show herein that
while the LDLR is insensitive to PCSK9 in murine B16F1 melanoma cells, PCSK9 is
able to induce degradation of the low density lipoprotein receptor-related
protein 1 (LRP-1), suggesting distinct targeting mechanisms for these receptors.
Furthermore, PCSK9 is still capable of acting upon the LDLR in CHO 13-5-1 cells
lacking LRP-1. Conversely, PCSK9 also acts on LRP-1 in the absence of the LDLR
in CHO-A7 cells, where re-introduction of the LDLR leads to reduced
PCSK9-mediated degradation of LRP-1. Thus, while PCSK9 is capable of inducing
degradation of LRP-1, the latter is not an essential factor for LDLR regulation,
but the LDLR effectively competes with LRP-1 for PCSK9 activity. Identification
of PCSK9 targets should allow a better understanding of the consequences of
PCSK9 inhibition for lowering LDLc and tumor metastasis. PCSK9 (proprotein convertase subtilisin/kexin type 9) binds to the LDLR
(low-density lipoprotein receptor) at the cell surface and disrupts recycling of
the LDLR. However, PCSK9 also interacts with the LDLR in the ER (endoplasmic
reticulum). In the present study we have investigated the role of PCSK9 for the
transport of the LDLR from the ER to the cell membrane. A truncated LDLR
consisting of the ectodomain (ED-LDLR) was used for these studies to avoid
PCSK9-mediated degradation of the LDLR. The amount of secreted ED-LDLR was used
as a measure of the amount of ED-LDLR transported from the ER. From
co-transfection experiments of various PCSK9 and ED-LDLR plasmids, PCSK9
increased the amount of WT (wild-type) ED-LDLR in the medium, but not of an
ED-LDLR lacking the EGF (epidermal growth factor)-A repeat or of a Class 2a
mutant ED-LDLR which fails to exit the ER. Mutant PCSK9s which failed to undergo
autocatalytic cleavage or failed to exit the ER, failed to increase the amount
of WT-ED-LDLR in the medium. These mutants also reduced the amount of WT-ED-LDLR
intracellularly, which could partly be prevented by the proteasome inhibitor
lactacystine. WT-ED-LDLR promoted autocatalytic cleavage of pro-PCSK9. The
findings of the present study indicate that the binding of WT-ED-LDLR to
pro-PCSK9 in the ER promotes autocatalytic cleavage of PCSK9, and
autocatalytically cleaved PCSK9 acts as a chaperone to promote the exit of
WT-ED-LDLR from the ER. PCSK9 proprotein convertase subtilisin/kexin type (PCSK9) protein plays an
important role in LDL cholesterol (LDL-C) metabolism, due to its role in the
degradation of the LDL receptor. Preliminary clinical data of PCSK9 inhibition
are quite promising and indicate that PCSK9 inhibition may be a novel strategy
for the treatment of dyslipidemia particularly for those with refractory
hypercholesterolemia, statin intolerance, or an elevated lipoprotein (a) level
and associated cardiovascular diseases. Furthermore, development of PCSK9
inhibitor is an excellent example of "bench to bedside" concept where discovery
of a genetic mutation was translated into a novel therapy to address unmet
clinical needs. Although several approaches have been attempted to inhibit PCSK9
activity including small molecules, gene silencing and inhibitory antibodies,
the most promising approach appears to be the use of monoclonal antibodies with
a 50 -70% LDL cholesterol reduction on top of maximal doses of statins. In this
article, we review the pharmacology of PCSK9 and summarize findings from key
clinical studies using PCSK9 inhibitors. BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a regulator
of LDL-cholesterol receptor homeostasis and emerges as a therapeutic target in
the prevention of cardiovascular (CV) disease. This prospective cohort study
analyzes risk prediction with PCSK9 serum concentrations in patients with stable
coronary artery disease (CAD) on statin treatment.
METHODS AND RESULTS: Fasting PCSK9 concentrations were measured in 504
consecutive patients with stable CAD confirmed by angiography. Oral glucose
tolerance tests were performed in all patients without known diabetes. Patients
were followed up for 48months. The primary outcome was the composite of
cardiovascular death and unplanned cardiovascular hospitalization. Serum
concentrations of PCSK9 predicted CV outcomes (PCSK9>622 ng/ml vs. <471 ng/ml:
HR 1.55, 95%-CI 1.11-2.16, p=0.009). Higher PCSK9 concentrations were associated
with female gender, hypertension, statin treatment, C-reactive protein, HbA1c,
insulin, total cholesterol and fasting triglycerides, but not with LDL- or
HDL-cholesterol. The association of PCSK9 levels with CV events was reduced
after adjustment for fasting TG.
CONCLUSION: PCSK9 concentrations predict cardiovascular events in patients with
coronary artery disease on statin treatment. Serum triglycerides are correlated
with PCSK9 and modify risk prediction by PCSK9. BACKGROUND: Proprotein convertase subtilisin kexin type 9 (PCSK9) promotes the
degradation of the low-density lipoprotein (LDL) receptor (LDLR), and its
deficiency in humans results in low plasma LDL cholesterol and protection
against coronary heart disease. Recent evidence indicates that PCSK9 also
modulates the metabolism of triglyceride-rich apolipoprotein B (apoB)
lipoproteins, another important coronary heart disease risk factor. Here, we
studied the effects of physiological levels of PCSK9 on intestinal
triglyceride-rich apoB lipoprotein production and elucidated for the first time
the cellular and molecular mechanisms involved.
METHODS AND RESULTS: Treatment of human enterocytes (CaCo-2 cells) with
recombit human PCSK9 (10 μg/mL for 24 hours) increased cellular and secreted
apoB48 and apoB100 by 40% to 55% each (P<0.01 versus untreated cells), whereas
short-term deletion of PCSK9 expression reversed this effect. PCSK9 stimulation
of apoB was due to a 1.5-fold increase in apoB mRNA (P<0.01) and to enhanced
apoB protein stability through both LDLR-dependent and LDLR-independent
mechanisms. PCSK9 decreased LDLR protein (P<0.01) and increased cellular apoB
stability via activation of microsomal triglyceride transfer protein. PCSK9 also
increased levels of the lipid-generating enzymes FAS, SCD, and DGAT2 (P<0.05).
In mice, human PCSK9 at physiological levels increased intestinal microsomal
triglyceride transfer protein levels and activity regardless of LDLR expression.
CONCLUSIONS: PCSK9 markedly increases intestinal triglyceride-rich apoB
production through mechanisms mediated in part by transcriptional effects on
apoB, microsomal triglyceride transfer protein, and lipogenic genes and in part
by posttranscriptional effects on the LDLR and microsomal triglyceride transfer
protein. These findings indicate that targeted PCSK9-based therapies may also be
effective in the management of postprandial hypertriglyceridemia. Author information:
(1)Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne,
United Kingdom; Faculty of Health and Life Sciences, Northumbria University,
Newcastle upon Tyne, United Kingdom. Electronic address:
[email protected].
(2)Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne,
United Kingdom; Institute of Translational & Stratified Medicine, Plymouth
University Peninsula School of Medicine & Dentistry, United Kingdom.
(3)Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne,
United Kingdom; Inserm U1110, University of Strasbourg and Center for Liver and
Digestive Diseases, Strasbourg University Hospitals, 3 Rue Koeberlé, F-67000
Strasbourg, France.
(4)Liver Unit, Department of Medicine, Imperial College London, St Mary's
Hospital Campus, Praed Street, London, United Kingdom.
(5)Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne,
United Kingdom.
(6)Faculty of Health and Life Sciences, Northumbria University, Newcastle upon
Tyne, United Kingdom.
(7)Hyperlipidemia and Atherosclerosis Research Group, Clinical Research
Institute of Montréal (IRCM), Montréal, Canada; University of Montréal,
Montréal, Canada.
(8)Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of
Montréal, Montréal, Canada; University of Montréal, Montréal, Canada.
(9)Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne,
United Kingdom; Department of Clinical Biochemistry, Newcastle upon Tyne
Hospitals NHS Foundation Trust, Royal Victoria Infirmary, United Kingdom.
(10)Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne,
United Kingdom. Electronic address: [email protected]. The proprotein convertase subtilisin/kexin type 9 (PCSK9) gene regulates
cholesterol homoeostasis by accelerating low-density lipoprotein receptor (LDLR)
degradation resulting in the decreased catabolism of low-density lipoprotein
(LDL) leading to hypercholesterolaemia. PCSK9 has also been related to other
metabolic risk factors such as triglycerides (TGs) and glucose levels and body
mass index (BMI). Therefore, our aim was to study the relationship between the
PCSK9 and the lipid and lipoprotein profile. We studied 267 diabetic and
metabolic syndrome patients who were not receiving any lipid-lowering therapy.
We measured circulating lipids, cholesterol in remt lipoproteins (RLPc) and
PCSK9 levels. A detailed lipoprotein profile was determined based on NMR. Plasma
PCSK9 levels were significantly and positively correlated with TG (r=0.136,
P=0.033), total cholesterol (r=0.219, P<0.001) and apoB (apolipoprotein B;
r=0.226, P=0.006) circulating levels and with an atherogenic profile of
lipoprotein subclasses. In further detail, circulating PCSK9 levels were
positively correlated with large very-low density lipoprotein (VLDL) particles,
(r=0.210, P=0.001) and with their remts, the intermediate-density lipoprotein
(IDL) particles (r=0.206, P=0.001); positively correlated with smaller LDL
particles (for small LDL: r=0.224, P<0.001; for medium small LDL: r=0.235,
P<0.001; and for very small LDL: r=0.220, P<0.001); and with high-density
lipoprotein (HDL) particles (r=0.146, P<0.001), which is mainly explained by the
PCSK9 correlation with the smallest HDL particles (r=0.130, P=0.037). In
addition, circulating PCSK9 levels were positively correlated with the
pro-atherogenic circulating RLPc levels (r=0.171, P=0.006). All of the
correlations were adjusted by age, gender and BMI. PCSK9 levels are
significantly and positively correlated with atherogenic lipoproteins such as
large VLDL, IDL, the smallest LDL, the smallest HDL particles and RLPc levels. Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation
of the hepatic low-density lipoprotein receptor (LDL-R) and is therefore a
prominent therapeutic target for reducing LDL-cholesterol. The C-terminal domain
of PCSK9 is unlikely to be involved in a direct extracellular interaction with
the LDL-R. We probed the importance of the C-terminus for the degradation of the
LDL-R by designing seven de novo mutants of PCSK9 that fill potential druggable
cavities. The mutants were tested for their ability to diminish LDL uptake in
human HepG2 cells and for affinity towards a calcium independent mutant of the
EGF(A) domain of the human LDL-R. The later was done by a newly developed
surface plasmon resoce-based assay format. We identified three mutant
proteins (G517R, V610R and V644R) with decreased ability to block LDL uptake
into HepG2 cells. These mutations define areas outside the direct interaction
area between PCSK9 and the LDL-R that could be targeted to inhibit the PCSK9
triggered degradation of the LDL-R. We also describe the mechanistic
rationalisation of the affinity changes seen with the natural occurring human
D374Y (gain of function) mutation causing severe hypercholesterolaemia. The
action of this mutant is due to a significantly decreased dissociation rate
constant, whereas the mutation does not affect the association rate constant. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a soluble protein that
directs membrane-bound receptors to lysosomes for degradation. In the most
studied example of this, PCSK9 binding leads to the degradation of low density
lipoprotein receptor (LDLR), significantly affecting circulating LDL-C levels.
The mechanism mediating this degradation, however, is not completely understood.
We show here that LDLR facilitates PCSK9 interactions with amyloid precursor
like protein 2 (APLP2) at neutral pH leading to PCSK9 internalization, although
direct binding between PCSK9 and LDLR is not required. Moreover, binding to
APLP2 or LDLR is independently sufficient for PCSK9 endocytosis in hepatocytes,
while LDL can compete with APLP2 for PCSK9 binding to indirectly mediate PCSK9
endocytosis. Finally, we show that APLP2 and LDLR are also required for the
degradation of another PCSK9 target, APOER2, necessitating a general role for
LDLR and APLP2 in PCSK9 function. Together, these findings provide evidence that
PCSK9 has at least two endocytic epitopes that are utilized by a variety of
internalization mechanisms and clarifies how PCSK9 may direct proteins to
lysosomes. Reduction in low-density lipoprotein cholesterol (LDL-C), mainly with statins,
has decreased the risk of cardiovascular events over the last few decades.
However, there are several patient populations that warrant further decrease in
LDL-C by additional cholesterol-lowering therapy other than statins. Proprotein
convertase subtilisin/kexin type 9 (PCSK9) inhibitors are a new class of drugs
that have been shown to further decrease LDL-C by 50-70% when administered as a
monotherapy or on a background therapy with statins. Proprotein convertase
subtilisin/kexin type 9 inhibitors are also an excellent example of drug
development in which discovery of gene mutations and its clinical effects have
rapidly progressed into successful preclinical and clinical studies with
multiple Phases 1-3 clinical trials completed or ongoing to date. This review
summarizes the rapid evolution of the drug from genetic discovery to
identification of targets for the drugs, to animal and human testing, and to
large clinical outcomes trials, followed by discussion on foreseeable challenges
of PCSK9 inhibitors. OBJECTIVE: Proprotein convertase subtilisin/kexin type 9 (PCSK9), which binds
the low-density lipoprotein receptor and targets it for degradation, has emerged
as an important regulator of serum cholesterol levels and cardiovascular disease
risk. Although much work is currently focused on developing therapies for
inhibiting PCSK9, the endogenous regulation of PCSK9, particularly by insulin,
remains unclear. The objective of these studies was to determine the effects of
insulin on PCSK9 in vitro and in vivo.
APPROACH AND RESULTS: Using rat hepatoma cells and primary rat hepatocytes, we
found that insulin increased PCSK9 expression and increased low-density
lipoprotein receptor degradation in a PCSK9-dependent manner. In parallel,
hepatic Pcsk9 mRNA and plasma PCSK9 protein levels were reduced by 55% to 75% in
mice with liver-specific knockout of the insulin receptor; 75% to 88% in mice
made insulin-deficient with streptozotocin; and 65% in ob/ob mice treated with
antisense oligonucleotides against the insulin receptor. However, antisense
oligonucleotide-mediated knockdown of insulin receptor in lean, wild-type mice
had little effect. In addition, we found that fasting was able to reduce PCSK9
expression by 80% even in mice that lack hepatic insulin signaling.
CONCLUSIONS: Taken together, these data indicate that although insulin induces
PCSK9 expression, it is not the sole or even domit regulator of PCSK9 under
all conditions. Proprotein convertase subtilisin/kexin 9 (PCSK9) is the ninth member of the
proprotein convertase family. It is an important regulator of cholesterol
metabolism. PCSK9 can bind to low-density lipoprotein receptors (LDLRs) and
induce the degradation of these receptors through the endosome/lysosome pathway,
thus decreasing the LDLR levels on the cell surface of hepatocytes, resulting in
increased serum low-density lipoprotein cholesterol (LDL-C) concentrations.
Recent studies have found that gene polymorphisms of PCSK9 are associated with
hypercholesterolemia, risk of atherosclerosis, and ischemic stroke. Furthermore,
monoclonal antibodies, peptide mimetics, small molecule inhibitors and gene
silencing agents that are associated with PCSK9 are some of the newer
pharmaceutical therapeutic strategies and approaches for lowering serum LDL-C
levels. In this review, we will discuss recent advances in PCSK9 research, which
show that PCSK9 is correlated with lipid metabolism, atherosclerosis, and, in
particular, ischemic stroke. We will also discuss the current state of PCSK9
therapeutics and their potential in modulating these diseases. Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low-density
lipoprotein receptor, escorting it to its destruction in the lysosome and
thereby preventing the recirculation of the low-density lipoprotein receptor to
the hepatocyte cell surface. Both gain-of-function mutations in PCSK9 (causing
marked increases in low-density lipoprotein cholesterol [LDL-C] concentration
and premature atherosclerosis) and loss-of-function mutations (causing modest
LDL-C reduction with low rates of coronary heart disease) have been described.
Several monoclonal antibodies to PCSK9 have achieved LDL-C reductions of 50% to
70% across various patient populations and background lipid therapies. Phase 2/3
trials have demonstrated good tolerability without clear drug-related toxicity,
although the number and duration of patients treated to date is modest.
Currently, 4 phase 3 trials involving >70,000 patients are testing whether these
drugs reduce cardiovascular events. The U.S. Food and Drug Administration is
currently reviewing the existing data to determine whether these agents could be
made available prior to the completion of these cardiovascular endpoint trials
expected in 2018. PURPOSE: Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low
density lipoprotein receptor (LDLR) and promotes degradation of the LDLR.
Inhibition of PCSK9 either by reducing its expression or by blocking its
activity results in the upregulation of the LDLR and subsequently lowers the
plasma concentration of LDL-cholesterol. As a modality to inhibit PCSK9 action,
we searched the chemical library for small molecules that block the binding of
PCSK9 to the LDLR.
MATERIALS AND METHODS: We selected 100 chemicals that bind to PCSK9 where the
EGF-AB fragment of the LDLR binds via in silico screening of the ChemBridge
chemical library, using the computational GOLD algorithm analysis. Effects of
chemicals were evaluated using the PCSK9-LDLR binding assay, immunoblot
analysis, and the LDL-cholesterol uptake assay in vitro, as well as the fast
performance liquid chromatography assay for plasma lipoproteins in vivo.
RESULTS: A set of chemicals were found that decreased the binding of PCSK9 to
the EGF-AB fragment of the LDLR in a dose-dependent manner. They also increased
the amount of the LDLR significantly and subsequently increased the uptake of
fluorescence-labeled LDL in HepG2 cells. Additionally, one particular molecule
lowered the plasma concentration of total cholesterol and LDL-cholesterol
significantly in wild-type mice, while such an effect was not observed in Pcsk9
knockout mice.
CONCLUSION: Our findings strongly suggest that in silico screening of small
molecules that inhibit the protein-protein interaction between PCSK9 and the
LDLR is a potential modality for developing hypercholesterolemia therapeutics. To date HMG-CoA-reductase inhibitors are the most effective drugs for reduction
of LDL-cholesterol levels and for prevention of cardiovascular events.
Inhibition of the enzyme PCSK9 (proprotein convertase subtilisin/kexin type 9),
which is involved in depletion of the LDL-receptor, is a new pharmacologic
approach. Inhibition of PCSK9 by monoclonal antibodies provokes an additional
reduction of LDL-cholesterol levels by 50-60 % in addition to statins. Previous
phase III studies indicate good compatibility. Ongoing long-term studies will
answer questions of safety and influence on cardiovascular events. Although
those results are not available yet, alirocumab and evolocumab have already been
recommendd for approval. BACKGROUND: Site-1 protease (S1P) is the key enzyme required for activation of
the sterol regulatory element binding proteins (SREBPs) that govern lipid
synthesis. While S1P has been speculated to influence plasma apoB-containing
lipoprotein (Blp) metabolism, there has been little investigative work. LDL
receptor (LDLR) is the major receptor for clearing plasma LDL cholesterol
(LDL-c). Proprotein convertase subtilisin kexin type 9 (PCSK9) modulates LDL-c
through post-translational degradation of the LDLR.
METHODS: A hepatic-specific knockdown (KD) of S1P was achieved using floxed S1P
mouse models (S1P(f/f) and LDLR(-/-)S1P(f/f)) and hepatic expression of Cre
recombinase. Lipids were measured in total plasma and size fractionated plasma
using colorimetric assays. Realtime polymerase chain reaction, western blotting
and ELISA were used to determine hepatic expression of key genes/protein.
Plasmid mediated overexpression and siRNA mediated knockdown of genes were
performed in mouse primary hepatocytes to determine the mechanistic basis of
PCSK9 gene regulation.
RESULTS: A hepatic-specific KD of S1P resulted in a 45 % and 38 % reduction in
plasma total cholesterol and triglyceride levels, respectively. Hepatic S1P KD
had a minimal effect on plasma Blp cholesterol (Blp-c) in S1P(f/f) mice, despite
significantly reducing VLDL secretion. Notably, hepatic S1P KD decreased the LDL
receptor (LDLR) mRNA expression by 50 %. However, the reduction in LDLR protein
levels was less than that of mRNA expression, especially under fed conditions.
Further assessment of hepatic S1P deficiency revealed that it increased LDLR
protein stability in vivo. Mechanistically, hepatic S1P KD was shown to decrease
the liver and plasma levels of the protein proprotein convertase
subtilisin/kexin type 9 (PCSK9), which degrades LDLR protein. This effect was
more prominent in the fed condition and sufficient to account for the
discordance in LDLR mRNA and protein levels. Furthermore, hepatic S1P was shown
to regulate PCSK9 expression through activation of the SREBPs. In the LDLR(-/-)
background, hepatic S1P KD significantly reduced Blp-c levels.
CONCLUSION: Hepatic S1P is a physiological modulator of plasma Blp metabolism
through its regulation of LDLR and PCSK9. Hepatic S1P is a valid target for
lowering plasma Blp-c levels in the situation where LDLR function is
compromised. Proprotein convertase subtilisin kexin type 9 (PCSK9) belongs to the proprotein
convertase family. Several studies have demonstrated its involvement in the
regulation of low-density lipoprotein (LDL) cholesterol levels by inducing the
degradation of the LDL receptor (LDLR). However, experimental, epidemiologic,
and pharmacologic data provide important evidence on the role of PCSK9 also on
high-density lipoproteins (HDLs). In mice, PCSK9 regulates the HDL cholesterol
(HDL-C) levels by the degradation of hepatic LDLR, thus inhibiting the uptake of
apolipoprotein (Apo)E-containing HDLs. Several epidemiologic and genetic studies
reported positive relationship between PCSK9 and HDL-C levels, likely by
reducing the uptake of the ApoE-containing HDL particles. PCSK9 enhances also
the degradation of LDLR's closest family members, ApoE receptor 2, very
low-density lipoprotein receptor, and LDLR-related protein 1. This feature
provides a molecular mechanism by which PCSK9 may affect HDL metabolism.
Experimental studies demonstrated that PCSK9 directly interacts with HDL by
modulating PCSK9 self-assembly and its binding to the LDLR. Finally, the
inhibition of PCSK9 by means of monoclonal antibodies directed to PCSK9 (ie,
evolocumab and alirocumab) determines an increase of HDL-C fraction by 7% and
4.2%, respectively. Thus, the understanding of the role of PCSK9 on HDL
metabolism needs to be elucidated with a particular focus on the effect of PCSK9
on HDL-mediated reverse cholesterol transport. Single domain antibodies (sdAbs) correspond to the antigen-binding domains of
camelid antibodies. They have the same antigen-binding properties and
specificity as monoclonal antibodies (mAbs) but are easier and cheaper to
produce. We report here the development of sdAbs targeting human PCSK9
(proprotein convertase subtilisin/kexin type 9) as an alternative to anti-PCSK9
mAbs. After immunizing a llama with human PCSK9, we selected four sdAbs that
bind PCSK9 with a high affinity and produced them as fusion proteins with a
mouse Fc. All four sdAb-Fcs recognize the C-terminal Cys-His-rich domain of
PCSK9. We performed multiple cellular assays and demonstrated that the selected
sdAbs efficiently blocked PCSK9-mediated low density lipoprotein receptor (LDLR)
degradation in cell lines, in human hepatocytes, and in mouse primary
hepatocytes. We further showed that the sdAb-Fcs do not affect binding of PCSK9
to the LDLR but rather block its induced cellular LDLR degradation. Pcsk9
knock-out mice expressing a human bacterial artificial chromosome (BAC)
transgene were generated, resulting in plasma levels of ∼300 ng/ml human PCSK9.
Mice were singly or doubly injected with the best sdAb-Fc and analyzed at day 4
or 11, respectively. After 4 days, mice exhibited a 32 and 44% decrease in the
levels of total cholesterol and apolipoprotein B and ∼1.8-fold higher liver LDLR
protein levels. At 11 days, the equivalent values were 24 and 46% and ∼2.3-fold
higher LDLR proteins. These data constitute a proof-of-principle for the future
usage of sdAbs as PCSK9-targeting drugs that can efficiently reduce
LDL-cholesterol, and as tools to study the Cys-His-rich domain-dependent sorting
the PCSK9-LDLR complex to lysosomes. |
Hy's law measures failure for what organ? | Hy's law correlates enzyme elevations with liver injury ad subsequent failure. | BACKGROUND AND AIM: The genotype-phenotype interaction in drug-induced liver
injury (DILI) is a subject of growing interest. Previous studies have linked
amoxicillin-clavulanate (AC) hepatotoxicity susceptibility to specific HLA
alleles. In this study we aimed to examine potential associations between HLA
class I and II alleles and AC DILI with regards to phenotypic characteristics,
severity and time to onset in Spanish AC hepatotoxicity cases.
METHODS: High resolution genotyping of HLA loci A, B, C, DRB1 and DQB1 was
performed in 75 AC DILI cases and 885 controls.
RESULTS: The distributions of class I alleles A*3002 (P/Pc = 2.6E-6/5E-5, OR
6.7) and B*1801 (P/Pc = 0.008/0.22, OR 2.9) were more frequently found in
hepatocellular injury cases compared to controls. In addition, the presence of
the class II allele combination DRB1*1501-DQB1*0602 (P/Pc = 5.1E-4/0.014, OR
3.0) was significantly increased in cholestatic/mixed cases. The A*3002 and/or
B*1801 carriers were found to be younger (54 vs 65 years, P = 0.019) and were
more frequently hospitalized than the DRB1*1501-DQB1*0602 carriers. No
additional alleles outside those associated with liver injury patterns were
found to affect potential severity as measured by Hy's Law criteria. The
phenotype frequencies of B*1801 (P/Pc = 0.015/0.42, OR 5.2) and
DRB1*0301-DQB1*0201 (P/Pc = 0.0026/0.07, OR 15) were increased in AC DILI cases
with delayed onset compared to those corresponding to patients without delayed
onset, while the opposite applied to DRB1*1302-DQB1*0604 (P/Pc = 0.005/0.13, OR
0.07).
CONCLUSIONS: HLA class I and II alleles influence the AC DILI signature with
regards to phenotypic expression, latency presentation and severity in Spanish
patients. INTRODUCTION: The most reliable liver safety signal in a clinical trial is
considered to be 'Hy's Law cases' defined as subjects experiencing
hepatocellular injury and serum bilirubin elevations with no more likely cause
than study drug. However, there is little published data to support the current
biochemical criteria for Hy's Law cases or their use to estimate postmarketing
risk of severe liver injury.
OBJECTIVES: The primary objective of this study was to identify and characterize
Hy's Law cases in patients treated for tuberculosis (TB). A secondary objective
was to identify patient risk factors for drug-induced liver injuries.
METHODS: We utilized eDISH (evaluation of Drug-Induced Serious Hepatoxicity) to
retrospectively analyze data from 517 patients treated for activeTB, a regimen
well known to be capable of causing severe hepatotoxicity.
RESULTS: We identified two Hy's Law cases, which is consistent with the
treatment's known risk of liver failure. Despite monthly monitoring, neither
Hy's Law case experienced a documented elevation in serum alanine
aminotransferase exceeding 10 × upper limits of normal. Hepatoprotectant use and
infection with chronic hepatitis B were associated with increased risk of liver
injury.
CONCLUSIONS: Our observations support the current biochemical criteria for Hy's
Law cases and their use to estimate postmarketing risk. Author information:
(1)Unidad de Gestión Clínica de Enfermedades Digestivas, Servicio de
Farmacología Clínica, Instituto de Investigación Biomédica de Málaga (IBIMA),
Hospital Universitario Virgen de la Victoria, Universidad de Málaga, Málaga,
Spain; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y
Digestivas, Barcelona, Spain.
(2)Unidad de Gestión Clínica de Enfermedades Digestivas, Servicio de
Farmacología Clínica, Instituto de Investigación Biomédica de Málaga (IBIMA),
Hospital Universitario Virgen de la Victoria, Universidad de Málaga, Málaga,
Spain; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y
Digestivas, Barcelona, Spain. Electronic address: [email protected].
(3)University of Southern California Research Center for Liver Diseases, Keck
School of Medicine, Los Angeles, California.
(4)Unidad de Gestión Clínica de Enfermedades Digestivas, Servicio de
Farmacología Clínica, Instituto de Investigación Biomédica de Málaga (IBIMA),
Hospital Universitario Virgen de la Victoria, Universidad de Málaga, Málaga,
Spain.
(5)Servicio de Farmacia, Hospital de Torrecardenas, Almeria, Spain.
(6)Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y
Digestivas, Barcelona, Spain; Unidad de Gestión Clínica de Enfermedades
Digestivas, Hospital Universitario de Valme, Sevilla, Spain.
(7)Unidad de Gestión Clínica de Enfermedades Digestivas, Instituto de
Investigación Biomédica de Málaga (IBIMA), Hospital Regional Universitario
Carlos Haya, Málaga, Spain.
(8)Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y
Digestivas, Barcelona, Spain; Instituto de Enfermedades Digestivas y
Metabolismo, Hospital Clinic, Barcelona, Spain.
(9)Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y
Digestivas, Barcelona, Spain; Unidad de Gestión Clínica de Enfermedades
Digestivas, Hospital La Fe, Valencia, Spain.
(10)Facultad de Ciencias Médicas, Servicio de Gastroenterología y Hepatología,
Hospital Provincial del Centenario, Universidad Nacional de Rosario, Rosario,
Argentina.
(11)Hospital de Clínicas, Clínica de Gastroenterología, Facultad de Medicina,
Universidad de la Republica, Montevideo, Uruguay.
(12)Departamento de Gastroenterología, Facultad de Medicina Pontificia,
Universidad Católica de Chile, Santiago, Chile. Drug-induced liver injury (DILI) remains a leading reason why new compounds are
dropped from further study or are the subject of product warnings and regulatory
actions. Hy's Law of drug-induced hepatocellular jaundice causing a
case-fatality rate or need for transplant of 10% or higher has been validated in
several large national registries, including the ongoing, prospective U.S.
Drug-Induced Liver Injury Network. It serves as the basis for stopping rules in
clinical trials and in clinical practice. Because DILI can mimic all known
causes of acute and chronic liver disease, establishing causality can be
difficult. Histopathologic findings are often nonspecific and rarely, if ever,
considered pathognomonic. A daily drug dose >50-100 mg is more likely to be
hepatotoxic than does <10 mg, especially if the compound is highly lipophilic or
undergoes extensive hepatic metabolism. The quest for a predictive biomarker to
replace alanine aminotransferase is ongoing. Markers of necrosis and apoptosis
such as microRNA-122 and keratin 18 may prove useful in identifying patients at
risk for severe injury when they initially present with a suspected
acetaminophen overdose. Although a number of drugs causing idiosyncratic DILI
have HLA associations that may allow for pre-prescription testing to prevent
hepatotoxicity, the cost and relatively low frequency of injury among affected
patients limit the current usefulness of such genome-wide association studies.
Alanine aminotransferase monitoring is often recommended but has rarely been
shown to be an effective method to prevent serious DILI. Guidelines on the
diagnosis and management of DILI have recently been published, although specific
therapies remain limited. The LiverTox Web site has been introduced as an
interactive online virtual textbook that makes the latest information on more
than 650 agents available to clinicians, regulators, and drug developers alike. Author information:
(1)Division of Infectious Diseases, Department of Medicine, Perelman School of
Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; Center for
Clinical Epidemiology and Biostatistics, Department of Biostatistics and
Epidemiology, Perelman School of Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania; Center for Pharmacoepidemiology Research and
Training, Perelman School of Medicine, University of Pennsylvania, Philadelphia,
Pennsylvania. Electronic address: [email protected].
(2)Center for Clinical Epidemiology and Biostatistics, Department of
Biostatistics and Epidemiology, Perelman School of Medicine, University of
Pennsylvania, Philadelphia, Pennsylvania; Center for Pharmacoepidemiology
Research and Training, Perelman School of Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania.
(3)Center for Clinical Epidemiology and Biostatistics, Department of
Biostatistics and Epidemiology, Perelman School of Medicine, University of
Pennsylvania, Philadelphia, Pennsylvania; Center for Pharmacoepidemiology
Research and Training, Perelman School of Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania; Division of Gastroenterology, Department of
Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia,
Pennsylvania.
(4)Center for Clinical Epidemiology and Biostatistics, Department of
Biostatistics and Epidemiology, Perelman School of Medicine, University of
Pennsylvania, Philadelphia, Pennsylvania; Division of Gastroenterology,
Department of Medicine, Perelman School of Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania.
(5)Center for Clinical Epidemiology and Biostatistics, Department of
Biostatistics and Epidemiology, Perelman School of Medicine, University of
Pennsylvania, Philadelphia, Pennsylvania.
(6)Center for Pharmacoepidemiology Research and Training, Perelman School of
Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; Division of
Gastroenterology, Department of Medicine, Perelman School of Medicine,
University of Pennsylvania, Philadelphia, Pennsylvania.
(7)Division of Research, Kaiser Permanente Northern California, Oakland,
California.
(8)Center for Clinical Epidemiology and Biostatistics, Department of
Biostatistics and Epidemiology, Perelman School of Medicine, University of
Pennsylvania, Philadelphia, Pennsylvania; Center for Pharmacoepidemiology
Research and Training, Perelman School of Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania; Rutgers Biomedical & Health Sciences, Rutgers, the
State University of New Jersey, Newark, New Jersey. Drug-induced liver injury (DILI), a relatively rare condition, is nevertheless a
major reason for not approving a drug in development or for removing one already
marketed. With a specific diagnostic biomarker lacking, finding elevated serum
enzyme [alanine aminotransferase (ALT), aspartate aminotransferase and alkaline
phosphatase] activities remains an initial signal for incipient liver injury.
Enzyme elevations alone may not be harmful, but if caused by a drug and followed
by jaundice (called 'Hy's law') there is a high possibility of serious DILI. In
1997 several drugs were approved by the Food and Drug Administration (FDA) of
the USA that were later withdrawn from the market for serious liver toxicity.
New drugs in development are now required to be monitored for liver injury, and
the data is to be considered in the approval decision. A program called e-DISH
(evaluation of drug-induced serious hepatotoxicity) was introduced in 2004 to
aid medical reviewers to select from all subjects studied those few who show
nontrivial liver injury and estimate the most likely cause. The threshold of
enzyme elevation comprising a warning for possibly serious DILI is uncertain,
although generally accepted as 3-5 times the 'upper limit of normal'. The new
direct-acting antiviral agents for treating chronic hepatitis C virus, which
often lead to a reduction of elevated ALTs, mandate that a later increase
without viral breakthrough be compared to the new on-treatment level of values.
The drug may be discontinued or interrupted for evaluation to exclude other
possible causes of liver injury. The FDA has approved no drug since 1997 that
has been withdrawn later because of serious hepatotoxicity. |
Is apremilast effective for psoriatic arthritis? | Yes, apremilast, an oral phosphodiesterase 4 inhibitor, is effective for psoriatic arthritis. | BACKGROUND: Discoid lupus erythematosus (DLE) is a chronic inflammatory disorder
mediated by Th1 cells. Apremilast is a novel oral PDE4 enzyme inhibitor capable
of blocking leukocyte production of IL-12, IL-23, TNF-a, INF- with subsequent
suppression of Th1 and Th17-mediated immune responses, and proven clinical
efficacy for psoriasis as well as rheumatoid and psoriatic arthritis.
OBSERVATIONS: Cutaneous Lupus Erythematosus Disease Area and Severity Index
(CLASI) showed a significant (P<0.05) decrease after 85 days of treatment with
apremilast 20 mg twice daily in 8 patients with active discoid lupus. The
adverse events related to the drug were mild and transient.
CONCLUSIONS: This is the first open label study to use apremilast as a treatment
modality for discoid lupus. Our observations indicate that apremilast may
constitute a safe and effective therapeutic option for DLE. OBJECTIVES: Apremilast, an oral phosphodiesterase 4 inhibitor, regulates
inflammatory mediators. Psoriatic Arthritis Long-term Assessment of Clinical
Efficacy 1 (PALACE 1) compared apremilast with placebo in patients with active
psoriatic arthritis despite prior traditional disease-modifying antirheumatic
drug (DMARD) and/or biologic therapy.
METHODS: In the 24-week, placebo-controlled phase of PALACE 1, patients (N=504)
were randomised (1:1:1) to placebo, apremilast 20 mg twice a day (BID) or
apremilast 30 mg BID. At week 16, patients without ≥20% reduction in swollen and
tender joint counts were required to be re-randomised equally to either
apremilast dose if initially randomised to placebo or remained on their initial
apremilast dose. Patients on background concurrent DMARDs continued stable doses
(methotrexate, leflunomide and/or sulfasalazine). Primary outcome was the
proportion of patients achieving 20% improvement in modified American College of
Rheumatology response criteria (ACR20) at week 16.
RESULTS: At week 16, significantly more apremilast 20 mg BID (31%) and 30 mg BID
(40%) patients achieved ACR20 versus placebo (19%) (p<0.001). Significant
improvements in key secondary measures (physical function, psoriasis) were
evident with both apremilast doses versus placebo. Across outcome measures, the
30-mg group generally had higher and more consistent response rates, although
statistical comparison was not conducted. The most common adverse events were
gastrointestinal and generally occurred early, were self-limiting and
infrequently led to discontinuation. No imbalance in major adverse cardiac
events, serious or opportunistic infections, maligcies or laboratory
abnormalities was observed.
CONCLUSIONS: Apremilast was effective in the treatment of psoriatic arthritis,
improving signs and symptoms and physical function. Apremilast demonstrated an
acceptable safety profile and was generally well tolerated.
CLINICAL TRIAL REGISTRATION NUMBER: NCT01172938. Apremilast (Otezla(®)), an oral small molecule inhibitor of type-4 cyclic
nucleotide phosphodiesterase (PDE-4), is under development with Celgene
Corporation for the treatment of psoriatic arthritis, psoriasis, ankylosing
spondylitis, Behçet's syndrome, atopic dermatitis, and rheumatoid arthritis.
Apremilast is indicated for the treatment of active psoriatic arthritis in
adults. Apremilast has received its first global approval for this indication in
the USA. Regulatory submissions for approval in this indication are under review
in Canada and Europe. Regulatory filings have also been submitted for apremilast
in the treatment of plaque psoriasis in the USA and Europe. This article
summarizes the milestones in the development of apremilast leading to its first
approval for the treatment of psoriatic arthritis. PURPOSE OF REVIEW: The purpose of this study is to give an overview of the new
treatments approved by the U.S. Food and Drug Administration (FDA) for use in
psoriatic arthritis (PsA).
RECENT FINDINGS: FDA has approved three new drugs for PsA: Certolizumab-pegol: a
PEGylated Fc-free tumour necrosis factor inhibitor (TNFi); ustekinumab: an anti
interleukin (IL)-12 and IL-23 mAb; and apremilast and oral phosphodiesterase 4
inhibitor. On well designed and extensive developing programmes, all three drugs
proved to be effective for the treatment of most PsA manifestations, including
peripheral arthritis, skin involvement, enthesitis, dactylitis, quality of life
and radiographic progression in patients failing traditional disease modifying
drugs (DMARDs) and TNFi. Safety profile of all three drugs seems to be
reassuring until now, although long-term data are still not available. Although
Certolizumab-pegol is likely to be placed among the other TNFi, ustekinumab and
apremilast, due to lower efficacy on arthritis, are being more frequently used
as second-line therapy after TNFi failure, especially among rheumatologists.
SUMMARY: There are new therapeutic options approved for the treatment of PsA.
For the first time, well proved effective therapies with a different mechanism
of action than the inhibition of TNF alpha are available for the treatment of
this progressive disease. INTRODUCTION: Apremilast is an orally available small molecule that targets
PDE4. PDE4 modulates intracellular signaling and thereby can impact various
proinflammatory and anti-inflammatory mediators. Apremilast has been approved by
the USA FDA for the treatment of active psoriatic arthritis (PsA) and
moderate-to-severe psoriasis (PsO). Although there are several therapies
approved and used for the treatment of PsA, there is still an unmet need for
additional effective and well-tolerated therapeutic options. In PsA clinical
trials, apremilast has been shown to be efficacious and to have an acceptable
safety profile.
AREAS COVERED: This review article covers the mechanism of action of apremilast,
its efficacy in clinical trials and a detailed focus on its safety profile,
mainly from Phase III clinical trials.
EXPERT OPINION: Based on the available literature, apremilast has proven to be
an efficacious therapy for PsA and PsO. It may offer some advantage as compared
to other therapeutic options given its favorable safety profile, including a
lack of need for routine laboratory monitoring. Apremilast, an oral phosphodiesterase 4 inhibitor, demonstrated effectiveness
(versus placebo) for treatment of active psoriatic arthritis in the psoriatic
arthritis long-term assessment of clinical efficacy (PALACE) phase III clinical
trial program. Pharmacodynamic effects of apremilast on plasma biomarkers
associated with inflammation were evaluated in a PALACE 1 substudy. Of 504
patients randomized in PALACE 1, 150 (placebo: n = 51; apremilast 20 mg BID: n =
51; apremilast 30 mg BID: n = 48) provided peripheral blood plasma samples for
analysis in a multiplexed cytometric bead array assay measuring 47 proteins
associated with systemic inflammatory immune responses. Association between
biomarker levels and achievement of 20% improvement from baseline in modified
American College of Rheumatology (ACR20) response criteria was assessed by
logistic regression. At Week 24, IL-8, TNF-α, IL-6, MIP-1β, MCP-1, and ferritin
were significantly reduced from baseline with apremilast 20 mg BID or 30 mg BID
versus placebo. ACR20 response correlated with change in TNF-α level with both
apremilast doses. At Week 40, IL-17, IL-23, IL-6, and ferritin were
significantly decreased and IL-10 and IL-1 receptor antagonists significantly
increased with apremilast 30 mg BID versus placebo. In patients with active
psoriatic arthritis, apremilast reduced circulating levels of Th1 and Th17
proinflammatory mediators and increased anti-inflammatory mediators. Apremilast (Otezla(®)) is an oral phosphodiesterase 4 inhibitor indicated for
the twice-daily treatment of adults with psoriasis and psoriatic arthritis
(PsA). Its use in these patient populations has been assessed in two phase III
clinical trial programmes (ESTEEM and PALACE). At 16 weeks in the two ESTEEM
trials, apremilast reduced the severity and extent of moderate to severe plaque
psoriasis, including nail, scalp and palmoplantar manifestations, versus placebo
in adults, with these benefits generally being sustained over 52 weeks of
treatment. Similarly, in three PALACE trials (PALACE 1-3), apremilast improved
the signs and symptoms of PsA relative to placebo at 16 weeks in adults with
active disease despite treatment with conventional synthetic and/or biologic
disease-modifying anti-rheumatic drugs. These PsA benefits were generally
sustained for up to 104 weeks of treatment; skin involvement, enthesitis and
dactylitis also improved with the drug. Apremilast was generally well tolerated,
with the most common adverse events being diarrhoea and nausea in the first year
of treatment (usually occurring in the first 2 weeks after the first dose and
resolving within 4 weeks) and nasopharyngitis and upper respiratory tract
infection with continued treatment. Although further longer-term and comparative
efficacy and tolerability data would be beneficial, the current clinical data
indicate that apremilast is an effective and well tolerated option for the
management of psoriasis and PsA in adults. Psoriatic arthritis (PsA) is a chronic inflammatory disease of the joints that
occurs in patients with psoriasis. The spectrum of PsA includes arthritis,
dactylitis, enthesitis, axial involvement, and skin lesions. Non-biologic
disease-modifying anti-rheumatic drugs (DMARDs) such as methotrexate and
leflunomide, and biologic DMARDs such as tumor necrosis factor (TNF) antagonists
and ustekinumab, have been used to treat PsA. Apremilast is a novel therapy that
inhibits phosphodiesterase 4, increases intracellular cAMP levels, and modulates
expression of inflammatory mediators in favor of anti-inflammatory activity. It
decreases the pro-inflammatory cytokines TNF-α, IFN-γ, IL-17, and IL-23 and
increases the anti-inflammatory cytokine IL-10 under certain conditions. One
phase II and four phase III clinical trials as well as long-term extension
studies showed significant and sustained clinical efficacy and an adequate
safety profile for apremilast in patients with active psoriatic arthritis. Author information:
(1)Sorbonne Universités, UPMC Univ Paris 06, Institut Pierre Louis
d'Epidémiologie et de Santé Publique, GRC-UPMC 08 (EEMOIS), Paris, France
Department of rheumatology, AP-HP, Pitié Salpêtrière Hospital, Paris, France.
(2)Division of Rheumatology, Department of Medicine 3, Medical University of
Vienna, Vienna, Austria Second Department of Medicine, Hietzing Hospital,
Vienna, Austria.
(3)Department of Rheumatology, Leiden University Medical Centre, Leiden, The
Netherlands.
(4)EULAR, representing People with Arthritis/Rheumatism in Europe (PARE),
London, UK.
(5)Research Laboratory and Clinical Division of Rheumatology, Department of
Internal Medicine, University of Genova, Viale Benedetto, Italy.
(6)Medicine Faculty, Paris Descartes University, Paris, France Rheumatology B
Department, APHP, Cochin Hospital, Paris, France.
(7)Leeds NIHR Musculoskeletal Biomedical Research Unit, LTHT, Leeds, UK Leeds
Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds,
UK.
(8)Department of Clinical Immunology & Rheumatology, Amsterdam Rheumatology
Center, Amsterdam, The Netherlands Atrium Medical Center, Heerlen, The
Netherlands.
(9)North Devon, UK.
(10)Division of Rheumatology, Department of Medicine 3, Medical University of
Vienna, Vienna, Austria.
(11)Rheumazentrum Ruhrgebiet, Herne and Ruhr-Universität Bochum, Herne, Germany.
(12)Department of Rheumatology and Clinical Immunology, Charité-University
Medicine Berlin, Germany.
(13)Arthritis Unit, Department of Rheumatology, Hospital Clínic and IDIBAPS,
Barcelona, Spain.
(14)Belgrade University School of Medicine, Belgrade, Serbia.
(15)Department of Rheumatology, St. Vincent's University Hospital and Conway
Institute, University College Dublin, Dublin, Ireland.
(16)Section of Rheumatology, Department of Clinical Sciences, Lund University,
Lund, Sweden Sweden and School of Business, Engineering and Science, Halmstad
University, Halmstad, Sweden.
(17)Section of Musculoskeletal Disease, Leeds Institute of Molecular Medicine,
University of Leeds, Leeds, UK.
(18)Department of Rheumatology, Diakonhjemmet Hospital, Oslo, Norway.
(19)Laboratory of Tissue Homeostasis and Disease, Skeletal Biology and
Engineering Research Center, KU Leuven, Belgium Division of Rheumatology,
University Hospitals Leuven, Leuven, Belgium.
(20)Department of Dermatology, University Hospital Münster, Münster, Germany.
(21)A.DI.PSO. (Associazione per la Difesa degli Psoriasici)-PE.Pso.POF (Pan
European Psoriasis Patients' Organization Forum), Rome, Italy.
(22)Institute of Infection, Immunity and Inflammation, University of Glasgow,
Glasgow, UK.
(23)Rheumatology Department of Lucania, San Carlo Hospital of Potenza and
Madonna delle Grazie Hospital of Matera, Potenza, Italy.
(24)Institute and Clinic of Rheumatology Charles University Prague, Czech
Republic.
(25)Department of Internal Medicine 3, University of Erlangen-Nuremberg,
Erlangen, Germany.
(26)Department of Rheumatology, Campus Benjamin Franklin, Charité, Berlin,
Germany.
(27)Ghent University Hospital, Ghent, Belgium.
(28)Centre for Arthritis and Rheumatic Disease, Dublin Academic Medical Centre,
St. Vincent's University Hospital, Dublin, Ireland.
(29)Schoen Klinik Hamburg, Rheumatology and Clinical Immunology, Hamburg,
Germany.
(30)Department of Rheumatology and Clinical Immunology, German Rheumatism
Research Centre Berlin, Charité-University Medicine Berlin, Germany. OBJECTIVE: To update the evidence on the efficacy and safety of pharmacological
agents in psoriatic arthritis (PsA).
METHODS: Systematic literature review of randomised controlled trials comparing
pharmacological interventions in PsA: non-steroidal anti-inflammatory drugs,
glucocorticoid, synthetic disease modifying antirheumatic drugs (sDMARDs) either
conventional or targeted, biologicals (bDMARDs), placebo or any combination.
Main outcomes were American College of Rheumatology (ACR)20-50, Psoriasis Area
Severity Index 75, radiographic progression, and withdrawals due to adverse
events (AEs). Multiple studies of the same intervention were meta-analysed using
random effects.
RESULTS: In total, 25 papers and 12 abstracts were included. The efficacy of
tumour necrosis factor inhibitors (including the recently added golimumab and
certolizumab pegol) was confirmed and 16 articles/abstracts focused on 3 drugs
with new modes of action: ustekinumab (UST), secukinumab (SEC) and apremilast
(APR). All were placebo-compared trials and met their primary end point, ACR20.
In 2 studies with UST ACR20 was met by 50% and 44% of patients with UST 90 mg,
42% and 44% with UST 45 mg vs 23% and 20% with placebo, respectively. In two
studies with SEC ACR20 ranged 54% (SEC 300 mg), 50-51% (SEC 150 mg), 29-51% (SEC
75 mg) and 15-17% (placebo). In four studies with APR, ACR20 ranged 32-43% (APR
30 mg), 29-38% (APR 20 mg) and 17-20% (placebo). For all three drugs, no more
withdrawals due to AEs than placebo were seen and, in general, safety appeared
satisfactory. A strategy trial, TIght COntrol of Psoriatic Arthritis (TICOPA),
showed better ACR responses with treatment adaptations upon tight control
compared with standard care.
CONCLUSIONS: UST, SEC and APR are new drugs with efficacy demonstrated for the
treatment of PsA. No major safety signals arise, but long-term studies are
needed. This review informed about the European League Against Rheumatism
recommendations for management of PsA. OBJECTIVE: To review the pharmacology, efficacy, and safety of apremilast and
determine its role relative to other agents in the treatment of psoriasis and
psoriatic arthritis.
DATA SOURCES: A PubMed search (1946 to December 2015) using the terms apremilast
and CC-10004 was conducted to identify relevant articles.
STUDY SELECTION AND DATA EXTRACTION: In vitro or in vivo evaluations of
apremilast published in the English language were eligible for inclusion.
Controlled clinical trials that involved psoriasis or psoriatic arthritis were
selected for review.
DATA SYNTHESIS: Four trials were identified on the treatment of psoriasis. In
those that involved doses of 30 mg twice daily, a significantly greater
percentage of patients receiving apremilast (28.8% to 40.9%) compared with
placebo (5.3% to 5.8%) achieved at least 75% improvement from baseline in
Psoriasis Area and Severity Index score at 16 weeks. Two trials were identified
on the treatment of psoriatic arthritis. In the one that involved a dose of 30
mg twice daily, a significantly greater percentage of patients receiving
apremilast (38.1%) compared with placebo (19.0%) achieved the American College
of Rheumatology criteria for 20% improvement at 16 weeks. In all trials, the
drug had an acceptable safety profile, with the most common adverse effects of
diarrhea, nausea, and headache.
CONCLUSIONS: Apremilast has a novel mechanism of action and is safe and
effective for the management of psoriasis and psoriatic arthritis. At this time,
apremilast should be reserved for patients unable to take disease-modifying
antirheumatic drugs. OBJECTIVE: To evaluate apremilast treatment in patients with active psoriatic
arthritis, including current skin involvement, despite prior therapy with
conventional disease-modifying antirheumatic drugs and/or biologic agents.
METHODS: Patients (N=505) were randomised (1:1:1) to placebo, apremilast 20 mg
twice daily, or apremilast 30 mg twice daily. Rescue therapy with apremilast was
designated at week 16 for placebo patients not achieving 20% improvement in
swollen and tender joint counts. At week 24, the remaining placebo patients were
then randomised to apremilast 20 mg twice daily or 30 mg twice daily. The
efficacy and safety of apremilast were assessed over 52 weeks.
RESULTS: At week 16, significantly more patients receiving apremilast 20 mg
twice daily (28%) and 30 mg twice daily (41%) achieved 20% improvement in
American College of Rheumatology response criteria versus placebo (18%; p=0.0295
and p<0.0001, respectively), and mean decrease in the Health Assessment
Questionnaire-Disability Index score was significantly greater with apremilast
30 mg twice daily (-0.20) versus placebo (-0.07; p=0.0073). In patients with
baseline psoriasis body surface area involvement ≥3%, significantly more
apremilast 30 mg twice daily patients achieved 50% reduction from baseline
Psoriasis Area and Severity Index score (41%) versus placebo (24%; p=0.0098) at
week 16. At week 52, observed improvements in these measures demonstrated
sustained response with continued apremilast treatment. Most adverse events were
mild to moderate in severity; the most common were diarrhoea, nausea, headache
and upper respiratory tract infection.
CONCLUSIONS: Apremilast demonstrated clinically meaningful improvements in
psoriatic arthritis and psoriasis at week 16; sustained improvements were seen
with continued treatment through 52 weeks. Apremilast was generally well
tolerated and demonstrated an acceptable safety profile.
TRIAL REGISTRATION NUMBER: NCT01212770. Phosphodiesterases 4 (PDE4) act as proinflammatory enzymes via degradation of
cAMP, whereas PDE4 inhibitors play an anti-inflammatory role in vitro and in
vivo. In particular, apremilast has been recently approved for the treatment of
psoriasis and psoriatic arthritis. However, little is known on the expression
pattern of PDE4 in psoriasis. We report that PDE4B and PDE4D mRNA are
overexpressed in peripheral blood mononuclear cells (PBMC) from psoriasis, as
compared with normal controls, while apremilast reduces PBMC production of a
number of pro-inflammatory cytokines and increases the levels of
anti-inflammatory mediators. PDE4 expression is up-regulated in psoriatic dermis
as compared with normal skin, with particular regard to fibroblasts. This is
confirmed in vitro, where both dermal fibroblasts (DF) and, to a greater extent,
myofibroblasts (DM) express all PDE4 isoforms at the mRNA and protein level.
Because PDE4 interacts with the nerve growth factor (NGF) receptor CD271 in lung
fibroblasts, we evaluated the relationship and function of PDE4 and CD271 in
normal human skin fibroblasts. All PDE4 isoforms co-immunoprecipitate with CD271
in DM, while apremilast inhibits apoptosis induced by β-amyloid, a CD271 ligand,
in DM. Furthermore, apremilast significantly reduces NGF- and transforming
growth factor-β1 (TGF-β1)-induced fibroblast migration, and inhibits DF
differentiation into DM mediated by NGF or TGF-β1. Finally, in DM, apremilast
significantly reduces cAMP degradation induced by treatment with β-amyloid.
Taken together, these results indicate that PDE4 play an important role in
psoriasis. In addition, the study reveals that the PDE4/CD271 complex could be
important in modulating fibroblast functions. INTRODUCTION: The majority of Psoriatic Arthritis patients experience a good
clinical response to anti-Tumor Necrosis Factor (TNF)-α therapies. However,
treatment failure with anti-TNF-α can represent a relevant clinical problem.
AREAS COVERED: We review the efficacy and safety profile of biological therapies
that have been reported from randomized, controlled trials in phase II and phase
III available in Pubmed Database for agents targeting IL-12/23p40 antibody
(ustekinumab) and IL-17 (secukinumab), inhibitor of phosphodiesterase 4,
(apremilast), and of JAK/STAT pathways (tofacitinib) and CTLA4 co-stimulation
(abatacept) in Psoriatic Arthritis.
EXPERT OPINION: In Psoriatic Arthritis, main emerging drugs are represented by
the fully human monoclonal IL-12/23p40 antibody, ustekinumab, the agent
targeting IL-17, secukinumab, and the inhibitor of phosphodiesterase 4,
apremilast. Results on T cell co-stimulation inhibition by abatacept are
insufficient both in psoriasis and in PsA. In vitro investigations on JAK/STAT
pathways in PsA suggest that tofacitinib could represent a further valuable
therapeutic option. Emerging biological treatments other than anti-TNF agents,
ustekinumab, secukinumab and apremilast appear promising for Psoriatic Arthritis
and recent studies have showed a good efficacy and an acceptable safety profile;
however, further and long-term studies are advocated. Several classes of new oral therapy are in use or in development for the
treatment of psoriasis. Despite the high efficacy of biologics, new oral
therapies remain important as patients generally prefer this mode of
administration and they offer an alternative risk-benefit profile. In this
review, we discuss the novel modes of action of these drugs, including
modulation of cellular pathways involving diverse targets such as Janus kinase,
phosphodiesterase 4, sphingosine 1-phosphate, A3 adenosine receptor and
rho-associated kinase 2. We review the available evidence around licensed drugs
(apremilast) and drugs that are advanced (tofacitinib) or early (ponesimod,
baricitinib, peficitinib, INCB039110, CF101, KD025) in the development pipeline.
The key limitations of these oral therapies are their modest efficacy profile
(apremilast, ponesimod) and the limitations of their safety profile
(tofacitinib, ponesimod), while the evidence for the early pipeline drugs are at
phase II level only. Potential niches of current unmet needs include apremilast
for patients with concomitant psoriatic arthritis, as combination treatments
with biologic therapies, and/or for patients in whom multiple biologic therapies
have failed due to immunogenicity and secondary inefficacy. The present
knowledge gap regarding these novel drugs includes the need for longer clinical
trials or observational studies to evaluate safety, and randomised phase III
trials for the early pipeline drugs. We conclude that further research and data
are necessary to conclusively establish the role of these agents in the current
psoriasis treatment paradigm. PURPOSE OF REVIEW: Over the last several years, novel immunologic pathways
pivotal in the development of the pathobiology of psoriasis and psoriatic
arthritis (PsA) have been revealed. These discoveries catalyzed a search for new
treatment targets resulting in many new therapies that are now available for
patients with psoriatic disease.
RECENT FINDINGS: Helper T cells that secrete interleukin-17 (TH17) along with
CD8+ cells, innate lymphocyte cells, and gamma delta T cells are important in
the pathogenesis of psoriasis and PsA. Recently, agents that target
interleukin-17, the interleukin-17 receptor, and interleukin-23 (antip19) have
been approved or are in clinical trials. Apremilast, a new oral agent, was
approved for the treatment of psoriasis and PsA.
SUMMARY: Secukinumab, an interleukin-17A antibody, has been approved for
treatment of psoriasis and PsA in the United States. It is effective with a good
safety profile. Ixekizumab, another anti-interleukin-17A antibody, is currently
in clinical trials and brodalumab, an interleukin-17 receptor antagonist, was
removed from clinical trials because of safety concerns despite demonstrated
efficacy in psoriasis and PsA. Targeting interleukin-23 with antibodies to p19
is another approach with encouraging results in psoriasis. Apremilast, an oral
agent, approved to treat psoriasis and PsA demonstrates moderate efficacy with
an excellent safety record. The role of tofacitinib in psoriatic disease remains
to be determined pending a safety review in psoriasis and completion of PsA
trials. We report a 67-year-old Caucasian man with a long-term history of recalcitrant
plaque psoriasis and psoriatic arthritis who was initiated on a treatment
regimen of apremilast and secukinumab after failing multiple topical, photo, and
systemic therapies. This combination provided significant skin improvement with
minimal drug side effects. <br /><br /> <em>J Drugs Dermatol.
</em>2016;15(5):648-649. As part of the National Institute for Health and Clinical Excellence (NICE)
single technology appraisal (STA) process, the manufacturer of apremilast was
invited to submit evidence for its clinical and cost effectiveness for the
treatment of active psoriatic arthritis (PsA) for whom disease-modifying
anti-rheumatic drugs (DMARDs) have been inadequately effective, not tolerated or
contraindicated. The Centre for Reviews and Dissemination and Centre for Health
Economics at the University of York were commissioned to act as the independent
Evidence Review Group (ERG). This paper provides a description of the ERG review
of the company's submission, the ERG report and submission and summarises the
NICE Appraisal Committee's subsequent guidance (December 2015). In the company's
initial submission, the base-case analysis resulted in an incremental
cost-effectiveness ratio (ICER) of £14,683 per quality-adjusted life-year (QALY)
gained for the sequence including apremilast (positioned before tumour necrosis
factor [TNF]-α inhibitors) versus a comparator sequence without apremilast.
However, the ERG considered that the base-case sequence proposed by the company
represented a limited set of potentially relevant treatment sequences and
positions for apremilast. The company's base-case results were therefore not a
sufficient basis to inform the most efficient use and position of apremilast.
The exploratory ERG analyses indicated that apremilast is more effective (i.e.
produces higher health gains) when positioned after TNF-α inhibitor therapies.
Furthermore, assumptions made regarding a potential beneficial effect of
apremilast on long-term Health Assessment Questionnaire (HAQ) progression, which
cannot be substantiated, have a very significant impact on results. The NICE
Appraisal Committee (AC), when taking into account their preferred assumptions
for HAQ progression for patients on treatment with apremilast, placebo response
and monitoring costs for apremilast, concluded that the addition of apremilast
resulted in cost savings but also a QALY loss. These cost savings were not high
enough to compensate for the clinical effectiveness that would be lost. The AC
thus decided that apremilast alone or in combination with DMARD therapy is not
recommended for treating adults with active PsA that has not responded to prior
DMARD therapy, or where such therapy is not tolerated. Chronic plaque psoriasis presents clinically as an inflammatory disease of the
skin, which is often associated with comorbidities and responsible for a poor
quality of life. It can widely vary among patients because of different age of
onset, type of symptoms, areas of involvement, and disease severity. The choice
of the treatment of psoriasis should be personalized according to the specific
needs of the patients. Apremilast is a well-tolerated and effective
phosphodiesterase type 4 inhibitor that is indicated for the treatment of
moderate-to-severe plaque psoriasis and psoriatic arthritis. In this article,
the pharmacological, clinical, and safety aspects of apremilast are reviewed.
Based on these data, apremilast could be indicated for patients with a Psoriasis
Area and Severity Index score <10 but with a significant impact on quality of
life and seems to be an appropriate treatment for elderly patients also. BACKGROUND: Apremilast, an oral phosphodiesterase 4 inhibitor, has an acceptable
safety profile and is effective for treatment of plaque psoriasis and psoriatic
arthritis.
OBJECTIVES: To evaluate the impact of apremilast on health-related quality of
life (HRQOL), general functioning and mental health using patient-reported
outcome (PRO) assessments among patients with moderate to severe plaque
psoriasis in the ESTEEM 1 and 2 trials.
METHODS: A total of 1255 patients were randomized (2 : 1) to apremilast 30 mg
BID or placebo for 16 weeks; all received apremilast through Week 32. PRO
assessments included the Dermatology Life Quality Index (DLQI), 36-Item
Short-Form Health Survey version 2 mental/physical component summary scores
(SF-36v2 MCS/PCS), Patient Health Questionnaire-8 (PHQ-8), EuroQol-5D (EQ-5D)
and Work Limitations Questionnaire-25 (WLQ-25). Post hoc analyses examined
relationships between Psoriasis Area and Severity Index (PASI) scores and PHQ-8
in the apremilast-treated population at Week 16.
RESULTS: Treatment with apremilast improved all HRQOL PROs at Week 16 (vs.
placebo), except the SF-36v2 PCS, and improvements were sustained through Week
32. Mean DLQI and SF-36v2 MCS improvements exceeded minimal clinically important
differences. Changes at Week 16 in PHQ-8 and PASI were weakly correlated, and
only 35.8% of patients who achieved a ≥75% reduction from baseline in PASI score
(PASI-75) with apremilast treatment also achieved PHQ-8 scores of 0-4.
CONCLUSIONS: Apremilast led to improvements in HRQOL PROs vs. placebo in
patients with moderate to severe plaque psoriasis. INTRODUCTION: Psoriatic arthritis (PsA) is a spondyloarthritis that occurs in up
to 30% of psoriasis patients. Patients with PsA are at risk for decreased
quality of life due to both joint and skin symptoms, impaired physical function
and disease progression. Treatments include non-steroidal anti-inflammatory
drugs, conventional systemic disease-modifying anti-rheumatic drugs (DMARDs)
such as methotrexate, and biologic agents, including tumor necrosis factor-α
inhibitors. The most recently introduced treatment option is apremilast, an oral
phosphodiesterase 4 inhibitor.
METHODS: This review provides an in-depth discussion of apremilast's mechanism
of action, and evidence of its clinical efficacy and safety from the Psoriatic
Arthritis Long-term Assessment of Clinical Efficacy (PALACE) phase III pivotal
clinical trials (PALACE 1, 2, and 3).
RESULTS: These trials demonstrate that apremilast is effective for the treatment
of active PsA, despite prior conventional DMARDs or biologic treatment. The
primary efficacy end point, a 20% improvement from baseline in modified American
College of Rheumatology response criteria at Week 16, was achieved by
significantly greater proportions of patients treated with apremilast 20 mg
twice daily (BID) and apremilast 30 mg BID versus placebo in PALACE 1, 2, and 3.
Improvements in this and other clinical and patient-reported end points,
including swollen and tender joint counts, Psoriasis Area and Severity Index
score, physical function, and quality of life, were maintained, extending over
52 weeks of treatment among patients initially randomized to apremilast.
Apremilast's safety profile has been acceptable, with diarrhea and nausea being
the most common adverse events, with no evidence for an increased risk of
infection or need for laboratory monitoring. The PALACE pivotal data indicate
that apremilast presents a new option for the treatment of PsA that may be
appropriate for use early in the treatment ladder. Ongoing PALACE open-label
extension trials of up to 4 years will characterize the long-term clinical
effects and safety of apremilast therapy.
FUNDING: Celgene Corporation, Summit, NJ, USA. Psoriatic arthritis (PsA) is a heterogeneous disease that can involve a variety
of distinct anatomical sites including a patient's peripheral and axial joints,
entheses, skin and nails. Appropriate management of PsA requires early
diagnosis, monitoring of disease activity, and utilization of cutting edge
therapies. To accomplish the former there are a variety of PsA-specific tools
available to screen, diagnose, and assess patients. This review will outline the
recently developed PsA screening tools, including the Toronto Psoriatic
Arthritis Screening Questionnaire (TOPAS), the Psoriasis Epidemiology Screening
Tool (PEST), the Psoriatic Arthritis Screening and Evaluation (PASE), and the
Psoriasis and Arthritis Screening Questionnaire (PASQ). We will also review the
Classification Criteria for Psoriatic Arthritis (CASPAR) and current PsA disease
severity measures, such as the Disease Activity index for Psoriatic Arthritis
(DAPSA), the Psoriatic Arthritis Joint Activity Index (PsAJAI) and the Composite
Psoriatic Disease Activity Index (CPDAI). As is the case for PsA screening and
assessment tools, there are also a variety of new therapies available for PsA.
Historically, patients with PsA were treated with NSAIDS and traditional
disease-modifying anti-rheumatic drugs (DMARDs). However, the ability of these
medications to slow down the radiographic progression of joint disease has not
been demonstrated. In contrast, anti-TNF agents, such as etanercept, infliximab,
adalimumab, golimumab and certolizumab, are effective in this regard. Emerging
PsA treatments include an oral phosphodiesterase 4 inhibitor, apremilast; a
Janus kinase (JAK) inhibitor, tofacitinib; and several new biologics that target
the IL-23/IL-17 pathway including secukinumab, brodalumab, ixekizumab, and
ustekinumab. Herein we will review the mechanisms of action of these drugs,
their results in clinical trials, and guidelines for administration. Lastly,
treatment recommendations from the European League Against Rheumatism (EULAR)
and The Group for Research and Assessment of Psoriasis and Psoriatic Arthritis
(GRAPPA) will be discussed. |
Is there any role of TBR1 in autism? | Yes. Exome sequencing studies have identified multiple genes harboring de novo loss-of-function (LoF) variants in individuals with autism spectrum disorders (ASD), including T-Brain-1 (TBR1), a master regulator of cortical development. T-brain-1 (TBR1) is a brain-specific T-box transcription factor. In 1995, Tbr1 was first identified from a subtractive hybridization that compared mouse embryonic and adult telencephalons. Previous studies of Tbr1 (-∕-) mice have indicated critical roles for TBR1 in the development of the cerebral cortex, amygdala, and olfactory bulb. Neuronal migration and axonal projection are two important developmental features controlled by TBR1. | The neuron-specific transcription factor T-box brain 1 (TBR1) regulates brain
development. Disruptive mutations in the TBR1 gene have been repeatedly
identified in patients with autism spectrum disorders (ASDs). Here, we show that
Tbr1 haploinsufficiency results in defective axonal projections of amygdalar
neurons and the impairment of social interaction, ultrasonic vocalization,
associative memory and cognitive flexibility in mice. Loss of a copy of the Tbr1
gene altered the expression of Ntng1, Cntn2 and Cdh8 and reduced both inter- and
intra-amygdalar connections. These developmental defects likely impair neuronal
activation upon behavioral stimulation, which is indicated by fewer
c-FOS-positive neurons and lack of GRIN2B induction in Tbr1(+/-) amygdalae. We
also show that upregulation of amygdalar neuronal activity by local infusion of
a partial NMDA receptor agonist, d-cycloserine, ameliorates the behavioral
defects of Tbr1(+/-) mice. Our study suggests that TBR1 is important in the
regulation of amygdalar axonal connections and cognition. Next-generation sequencing recently revealed that recurrent disruptive mutations
in a few genes may account for 1% of sporadic autism cases. Coupling these novel
genetic data to empirical assays of protein function can illuminate crucial
molecular networks. Here we demonstrate the power of the approach, performing
the first functional analyses of TBR1 variants identified in sporadic autism. De
novo truncating and missense mutations disrupt multiple aspects of TBR1
function, including subcellular localization, interactions with co-regulators
and transcriptional repression. Missense mutations inherited from unaffected
parents did not disturb function in our assays. We show that TBR1 homodimerizes,
that it interacts with FOXP2, a transcription factor implicated in
speech/language disorders, and that this interaction is disrupted by pathogenic
mutations affecting either protein. These findings support the hypothesis that
de novo mutations in sporadic autism have severe functional consequences.
Moreover, they uncover neurogenetic mechanisms that bridge different
neurodevelopmental disorders involving language deficits. The activity-regulated gene expression of transcription factors is required for
neural plasticity and function in response to neuronal stimulation. T-brain-1
(TBR1), a critical neuron-specific transcription factor for forebrain
development, has been recognized as a high-confidence risk gene for autism
spectrum disorders. Here, we show that in addition to its role in brain
development, Tbr1 responds to neuronal activation and further modulates the
Grin2b expression in adult brains and mature neurons. The expression levels of
Tbr1 were investigated using both immunostaining and quantitative reverse
transcription polymerase chain reaction (RT-PCR) analyses. We found that the
mRNA and protein expression levels of Tbr1 are induced by excitatory synaptic
transmission driven by bicuculline or glutamate treatment in cultured mature
neurons. The upregulation of Tbr1 expression requires the activation of both
α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and
N-methyl-D-aspartate (NMDA) receptors. Furthermore, behavioral training triggers
Tbr1 induction in the adult mouse brain. The elevation of Tbr1 expression is
associated with Grin2b upregulation in both mature neurons and adult brains.
Using Tbr1-deficient neurons, we further demonstrated that TBR1 is required for
the induction of Grin2b upon neuronal activation. Taken together with the
previous studies showing that TBR1 binds the Grin2b promoter and controls
expression of luciferase reporter driven by Grin2b promoter, the evidence
suggests that TBR1 directly controls Grin2b expression in mature neurons. We
also found that the addition of the calcium/calmodulin-dependent protein kinase
II (CaMKII) antagonist KN-93, but not the calcium-dependent phosphatase
calcineurin antagonist cyclosporin A, to cultured mature neurons noticeably
inhibited Tbr1 induction, indicating that neuronal activation upregulates Tbr1
expression in a CaMKII-dependent manner. In conclusion, our study suggests that
Tbr1 plays an important role in adult mouse brains in response to neuronal
activation to modulate the activity-regulated gene transcription required for
neural plasticity. T-Brain-1 (TBR1), a causative gene in autism spectrum disorders (ASDs), encodes
a brain-specific T-box transcription factor. It is therefore possible that TBR1
controls the expression of other autism risk factors. The downstream genes of
TBR1 have been identified using microarray and promoter analyses. In this study,
we annotated individual genes downstream of TBR1 and investigated any
associations with ASDs through extensive literature searches. Of 124 TBR1 target
genes, 23 were reported to be associated with ASDs. In addition, one gene,
Kiaa0319, is a known causative gene for dyslexia, a disorder frequently
associated with autism. A change in expression level in 10 of these 24 genes has
been previously confirmed. We further validated the alteration of RNA expression
levels of Kiaa0319, Baiap2, and Gad1 in Tbr1 deficient mice. Among these 24
genes, four transcription factors Auts2, Nfia, Nr4a2, and Sox5 were found,
suggesting that TBR1 controls a transcriptional cascade relevant to autism
pathogenesis. A further five of the 24 genes (Cd44, Cdh8, Cntn6, Gpc6, and
Ntng1) encode membrane proteins that regulate cell adhesion and axonal
outgrowth. These genes likely contribute to the role of TBR1 in regulation of
neuronal migration and axonal extension. Besides, decreases in Grin2b expression
and increases in Gad1 expression imply that neuronal activity may be aberrant in
Tbr1 deficient mice. These analyses provide direction for future experiments to
reveal the pathogenic mechanism of autism. Exome sequencing studies have identified multiple genes harboring de novo
loss-of-function (LoF) variants in individuals with autism spectrum disorders
(ASD), including TBR1, a master regulator of cortical development. We performed
ChIP-seq for TBR1 during mouse cortical neurogenesis and show that TBR1-bound
regions are enriched adjacent to ASD genes. ASD genes were also enriched among
genes that are differentially expressed in Tbr1 knockouts, which together with
the ChIP-seq data, suggests direct transcriptional regulation. Of the nine ASD
genes examined, seven were misexpressed in the cortices of Tbr1 knockout mice,
including six with increased expression in the deep cortical layers. ASD genes
with adjacent cortical TBR1 ChIP-seq peaks also showed unusually low levels of
LoF mutations in a reference human population and among Icelanders. We then
leveraged TBR1 binding to identify an appealing subset of candidate ASD genes.
Our findings highlight a TBR1-regulated network of ASD genes in the developing
neocortex that are relatively intolerant to LoF mutations, indicating that these
genes may play critical roles in normal cortical development. |
What is the role of the MCM2-7 complex? | The MCM2-7 complex is a ring-shaped heterohexamer helicase, that unwinds the DNA double helix ahead of the other replication machinery. During pre-replication complex (pre-RC) formation, origin recognition complex (ORC), Cdc6, and Cdt1 cooperatively load a double-hexameric MCM2-7 complex onto DNA. Loading of MCM2-7 is a prerequisite for licensing of eukaryotic DNA replication. During S phase MCM2-7 functions as part of the replicative helicase but within the pre-RC MCM2-7 is inactive. | To maintain genome integrity in eukaryotes, DNA must be duplicated precisely
once before cell division occurs. A process called replication licensing ensures
that chromosomes are replicated only once per cell cycle. Its control has been
uncovered by the discovery of the CDKs (cyclin dependent kinases) as master
regulators of the cell cycle and the initiator proteins of DNA replication, such
as the Origin Recognition Complex (ORC), Cdc6/18, Cdt1 and the MCM complex. At
the end of mitosis, the MCM complex is loaded on to chromatin with the aid of
ORC, Cdc6/18 and Cdt1, and chromatin becomes licensed for replication. CDKs,
together with the Cdc7 kinase, trigger the initiation of replication, recruiting
the DNA replicating enzymes on sites of replication. The activated MCM complex
appears to play a key role in the DNA unwinding step, acting as a replicating
helicase and moves along with the replication fork, at the same time bringing
the origins to the unlicensed state. The cycling of CDK activity in the cell
cycle separates the two states of replication origins, the licensed state in
G1-phase and the unlicensed state for the rest of the cell cycle. Only when CDK
drops at the completion of mitosis, is the restriction on licensing relieved and
a new round of replication is allowed. Such a CDK-regulated licensing control is
conserved from yeast to higher eukaryotes, and ensures that DNA replication
takes place only once in a cycle. Xenopus laevis and mammalian cells have an
additional system to control licensing. Geminin, whose degradation at the end of
mitosis is essential for a new round of licensing, has been shown to bind Cdt1
and negatively regulate it, providing a new insight into the regulation of DNA
replication in higher eukaryotes. The MCM2-7 complex is believed to function as the eukaryotic replicative DNA
helicase. It is recruited to chromatin by the origin recognition complex (ORC),
Cdc6, and Cdt1, and it is activated at the G(1)/S transition by Cdc45 and the
protein kinases Cdc7 and Cdk2. Paradoxically, the number of chromatin-bound MCM
complexes greatly exceeds the number of bound ORC complexes. To understand how
the high MCM2-7:ORC ratio comes about, we examined the binding of these proteins
to immobilized linear DNA fragments in Xenopus egg extracts. The minimum length
of DNA required to recruit ORC and MCM2-7 was approximately 80 bp, and the
MCM2-7:ORC ratio on this fragment was approximately 1:1. With longer DNA
fragments, the MCM2-7:ORC ratio increased dramatically, indicating that MCM
complexes normally become distributed over a large region of DNA surrounding
ORC. Only a small subset of the chromatin-bound MCM2-7 complexes recruited Cdc45
at the onset of DNA replication, and unlike Cdc45, MCM2-7 was not limiting for
DNA replication. However, all the chromatin-bound MCM complexes may be
functional, because they were phosphorylated in a Cdc7-dependent fashion, and
because they could be induced to support Cdk2-dependent Cdc45 loading. The data
suggest that in Xenopus egg extracts, origins of replication contain multiple,
distributed, initiation-competent MCM2-7 complexes. The six minichromosome maintece proteins (Mcm2-7) are required for both the
initiation and elongation of chromosomal DNA, ensuring that DNA replication
takes place once, and only once, during the S phase. Here we report on the
cloning of a new human Mcm gene (hMcm8) and on characterisation of its protein
product. The hMcm8 gene contains the central Mcm domain conserved in the Mcm2-7
gene family, and is expressed in a range of cell lines and human tissues. hMcm8
mRNA accumulates during G(1)/S phase, while hMcm8 protein is detectable
throughout the cell cycle. Immunoprecipitation-based studies did not reveal any
participation of hMcm8 in the Mcm3/5 and Mcm2/4/6/7 subcomplexes. hMcm8
localises to the nucleus, although it is devoid of a nuclear localisation
signal, suggesting that it binds to a nuclear protein. In the nucleus, the hMcm8
structure-bound fraction is detectable in S, but not in G(2)/M, phase, as for
hMcm3. However, unlike hMcm3, the hMcm8 structure-bound fraction is not
detectable in G(1) phase. Overall, our data identify a new Mcm protein, which
does not form part of the Mcm2-7 complex and which is only structure-bound
during S phase, thus suggesting its specific role in DNA replication. The MCM2-7 complex, which may act as a replicative helicase during DNA
synthesis, plays a central role in S-phase genome stability. MCM proteins are
required for processive DNA replication and are a target of S-phase checkpoints.
Loss of MCM function causes DNA damage and genome instability. MCM expression is
upregulated in proliferating cells, providing a diagnostic marker for both
cancerous cells and cells with the potential to become maligt. The role of
the MCM complex in genome integrity reflects its activity both at active
replication forks and away from forks. To maintain chromosome stability in eukaryotic cells, replication origins must
be licensed by loading mini-chromosome maintece (MCM2-7) complexes once and
only once per cell cycle. This licensing control is achieved through the
activities of geminin and cyclin-dependent kinases. Geminin binds tightly to
Cdt1, an essential component of the replication licensing system, and prevents
the inappropriate reinitiation of replication on an already fired origin. The
inhibitory effect of geminin is thought to prevent the interaction between Cdt1
and the MCM helicase. Here we describe the crystal structure of the mouse
geminin-Cdt1 complex using tGeminin (residues 79-157, truncated geminin) and
tCdt1 (residues 172-368, truncated Cdt1). The amino-terminal region of a
coiled-coil dimer of tGeminin interacts with both N-terminal and
carboxy-terminal parts of tCdt1. The primary interface relies on the steric
complementarity between the tGeminin dimer and the hydrophobic face of the two
short N-terminal helices of tCdt1 and, in particular, Pro 181, Ala 182, Tyr 183,
Phe 186 and Leu 189. The crystal structure, in conjunction with our biochemical
data, indicates that the N-terminal region of tGeminin might be required to
anchor tCdt1, and the C-terminal region of tGeminin prevents access of the MCM
complex to tCdt1 through steric hindrance. The DNA replication (or origin) licensing system ensures precise duplication of
the genome in each cell cycle and is a powerful regulator of cell proliferation
in metazoa. Studies in yeast, Drosophila melanogaster and Xenopus laevis have
characterised the molecular machinery that constitutes the licensing system, but
it remains to be determined how this important evolutionary conserved pathway is
regulated in Homo sapiens. We have investigated regulation of the origin
licensing factors Cdc6, Cdt1, Mcm2 and Geminin in human somatic and germ cells.
Cdc6 and Cdt1 play an essential role in DNA replication initiation by loading
the Mcm2-7 complex, which is required for unwinding the DNA helix, onto
chromosomal origins. Geminin is a repressor of origin licensing that blocks
Mcm2-7 loading onto origins. Our studies demonstrate that Cdc6, Cdt1 and Mcm2
play a central role in coordinating growth during the
proliferation-differentiation switch in somatic self-renewing systems and that
Cdc6 expression is rate-limiting for acquisition of replication competence in
primary oocytes. In striking contrast, we show that proliferation control during
male gametogenesis is not linked to Cdc6 or Mcm2, but appears to be coordinated
by the negative regulator Geminin with Cdt1 becoming rate-limiting in late
prophase. Our data demonstrate a striking sexual dimorphism in the mechanisms
repressing origin licensing and preventing untimely DNA synthesis during meiosis
I, implicating a pivotal role for Geminin in maintaining integrity of the male
germline genome. During late mitosis and early G1, replication origins are licensed for
subsequent replication by loading heterohexamers of the mini-chromosome
maintece proteins (Mcm2-7). To prevent re-replication of DNA, the licensing
system is down-regulated at other cell cycle stages. A small protein called
geminin plays an important role in this down-regulation by binding and
inhibiting the Cdt1 component of the licensing system. We examine here the
organization of Xenopus Cdt1, delimiting regions of Cdt1 required for licensing
and regions required for geminin interaction. The C-terminal 377 residues of
Cdt1 are required for licensing and the extreme C-terminus contains a domain
that interacts with an Mcm(2,4,6,7) complex. Two regions of Cdt1 interact with
geminin: one at the N-terminus, and one in the centre of the protein. Only the
central region binds geminin tightly enough to successfully compete with
full-length Cdt1 for geminin binding. This interaction requires a predicted
coiled-coil domain that is conserved amongst metazoan Cdt1 homologues. Geminin
forms a homodimer, with each dimer binding one molecule of Cdt1. Separation of
the domains necessary for licensing activity from domains required for a strong
interaction with geminin generated a construct, whose licensing activity was
partially insensitive to geminin inhibition. DNA replication, as with all macromolecular synthesis steps, is controlled in
part at the level of initiation. Although the origin recognition complex (ORC)
binds to origins of DNA replication, it does not solely determine their
location. To initiate DNA replication ORC requires Cdc6 to target initiation to
specific DNA sequences in chromosomes and with Cdt1 loads the ring-shaped
mini-chromosome maintece (MCM) 2-7 DNA helicase component onto DNA. ORC and
Cdc6 combine to form a ring-shaped complex that contains six AAA+ subunits. ORC
and Cdc6 ATPase mutants are defective in MCM loading, and ORC ATPase mutants
have reduced activity in ORC x Cdc6 x DNA complex formation. Here we analyzed
the role of the Cdc6 ATPase on ORC x Cdc6 complex stability in the presence or
absence of specific DNA sequences. Cdc6 ATPase is activated by ORC, regulates
ORC x Cdc6 complex stability, and is suppressed by origin DNA. Mutations in the
conserved origin A element, and to a lesser extent mutations in the B1 and B2
elements, induce Cdc6 ATPase activity and prevent stable ORC x Cdc6 formation.
By analyzing ORC x Cdc6 complex stability on various DNAs, we demonstrated that
specific DNA sequences control the rate of Cdc6 ATPase, which in turn controls
the rate of Cdc6 dissociation from the ORC x Cdc6 x DNA complex. We propose a
mechanism explaining how Cdc6 ATPase activity promotes origin DNA sequence
specificity; on DNA that lacks origin activity, Cdc6 ATPase promotes
dissociation of Cdc6, whereas origin DNA down-regulates Cdc6 ATPase resulting in
a stable ORC x Cdc6 x DNA complex, which can then promote MCM loading. This
model has relevance for origin specificity in higher eukaryotes. Sequence-dependent DNA flexibility is an important structural property
originating from the DNA 3D structure. In this paper, we investigate the DNA
flexibility of the budding yeast (S. Cerevisiae) replication origins on a
genome-wide scale using flexibility parameters from two different models, the
trinucleotide and the tetranucleotide models. Based on analyzing average
flexibility profiles of 270 replication origins, we find that yeast replication
origins are significantly rigid compared with their surrounding genomic regions.
To further understand the highly distinctive property of replication origins, we
compare the flexibility patterns between yeast replication origins and
promoters, and find that they both contain significantly rigid DNAs. Our results
suggest that DNA flexibility is an important factor that helps proteins
recognize and bind the target sites in order to initiate DNA replication.
Inspired by the role of the rigid region in promoters, we speculate that the
rigid replication origins may facilitate binding of proteins, including the
origin recognition complex (ORC), Cdc6, Cdt1 and the MCM2-7 complex. The essential S-phase kinase Cdc7-Dbf4 acts at eukaryotic origins of replication
to trigger a cascade of protein associations that activate the Mcm2-7
replicative helicase. Also known as Dbf4-dependent kinase (DDK), this kinase
preferentially targets chromatin-associated Mcm2-7 complexes that are assembled
on the DNA during prereplicative complex (pre-RC) formation. Here we address the
mechanisms that control the specificity of DDK action. We show that
incorporation of Mcm2-7 into the pre-RC increased the level and changes the
specificity of DDK phosphorylation of this complex. In the context of the
pre-RC, DDK preferentially targets a conformationally distinct subpopulation of
Mcm2-7 complexes that is tightly linked to the origin DNA. This targeting
requires DDK to tightly associate with Mcm2-7 complexes in a Dbf4-dependent
manner. Importantly, we find that DDK association with and phosphorylation of
origin-linked Mcm2-7 complexes require prior phosphorylation of the pre-RC. Our
findings provide insights into the mechanisms that ensure that DDK action is
spatially and temporally restricted to the origin-bound Mcm2-7 complexes that
will drive replication fork movement during S phase and suggest new mechanisms
to regulate origin activity. Genome integrity in eukaryotes depends on licensing mechanisms that prevent
loading of the minichromosome maintece complex (MCM2-7) onto replicated DNA
during S phase. Although the principle of licensing appears to be conserved
across all eukaryotes, the mechanisms that control it vary, and it is not clear
how licensing is regulated in plants. In this work, we demonstrate that subunits
of the MCM2-7 complex are coordinately expressed during Arabidopsis (Arabidopsis
thaliana) development and are abundant in proliferating and endocycling tissues,
indicative of a role in DNA replication. We show that endogenous MCM5 and MCM7
proteins are localized in the nucleus during G1, S, and G2 phases of the cell
cycle and are released into the cytoplasmic compartment during mitosis. We also
show that MCM5 and MCM7 are topologically constrained on DNA and that the MCM
complex is stable under high-salt conditions. Our results are consistent with a
conserved replicative helicase function for the MCM complex in plants but not
with the idea that plants resemble budding yeast by actively exporting the MCM
complex from the nucleus to prevent unauthorized origin licensing and
rereplication during S phase. Instead, our data show that, like other higher
eukaryotes, the MCM complex in plants remains in the nucleus throughout most of
the cell cycle and is only dispersed in mitotic cells. Origin licensing builds a fundamental basis for genome stability in DNA
replication. Recent studies reported that deregulation of origin licensing is
associated with replication stress in precancerous lesions. The heterohexameric
complex of minichromosome maintece proteins (MCM2-7 complex) plays an
essential role in origin licensing. Previously, we reported the recovery of the
first viable Mcm mutant allele (named Mcm4(Chaos3)) in mice. The Mcm4(Chaos3)
allele destabilizes the MCM2-7 complex, leading to chromosome instability and
the formation of spontaneous tumors in Mcm4(Chaos3) homozygous mice. Supporting
our finding, a recent study reported that mice with reduced expression of MCM2
die with lymphomas within the first few months after birth. These data strongly
suggest that mutant Mcm2-7 genes are cancer-causing genes with nearly complete
penetrance in mice. This could be the case for humans as well. Nevertheless,
related investigations have not been undertaken due to the essential nature of
the MCM2-7 genes. To circumvent this problem, we focused on the variant alleles
of human MCM2-7 genes derived from single nucleotide polymorphisms. We created a
total of 14 variant alleles in the corresponding genes in Saccharomyces
cerevisiae. The phenotypic consequence was assayed for minichromosome loss, a
surrogate phenotype for genome instability and cancer susceptibility. This
screen identified a MCM5 variant allele with pathogenic potential. This allele
deserves further investigations on its effect on cancer development in human
populations. The essential minichromosome maintece (Mcm) proteins Mcm2 through Mcm7 likely
comprise the replicative helicase in eukaryotes. In addition to Mcm2-7, other
subcomplexes, including one comprising Mcm4, Mcm6, and Mcm7, unwind DNA. Using
Mcm4/6/7 as a tool, we reveal a role for nucleotide binding by Saccharomyces
cerevisiae Mcm2 in modulating DNA binding by Mcm complexes. Previous studies
have shown that Mcm2 inhibits DNA unwinding by Mcm4/6/7. Here, we show that
interaction of Mcm2 and Mcm4/6/7 is not sufficient for inhibition; rather, Mcm2
requires nucleotides for its regulatory role. An Mcm2 mutant that is defective
for ATP hydrolysis (K549A), as well as ATP analogues, was used to show that ADP
binding by Mcm2 is required to inhibit DNA binding and unwinding by Mcm4/6/7.
This Mcm2-mediated regulation of Mcm4/6/7 is independent of Mcm3/5. Furthermore,
the importance of ATP hydrolysis by Mcm2 to the regulation of the native complex
was apparent from the altered DNA binding properties of Mcm2(KA)-7. Moreover,
together with the finding that Mcm2(K549A) does not support yeast viability,
these results indicate that the nucleotide-bound state of Mcm2 is critical in
regulating the activities of Mcm4/6/7 and Mcm2-7 complexes. In eukaryotes, the activation of the prereplicative complex and assembly of an
active DNA unwinding complex are critical but poorly understood steps required
for the initiation of DNA replication. In this report, we have used bimolecular
fluorescence complementation assays in HeLa cells to examine the interactions
between Cdc45, Mcm2-7, and the GINS complex (collectively called the CMG
complex), which seem to play a key role in the formation and progression of
replication forks. Interactions between the CMG components were observed only
after the G(1)/S transition of the cell cycle and were abolished by treatment of
cells with either a CDK inhibitor or siRNA against the Cdc7 kinase. Stable
association of CMG required all three components of the CMG complex as well as
RecQL4, Ctf4/And-1, and Mcm10. Surprisingly, depletion of TopBP1, a homologue of
Dpb11 that plays an essential role in the chromatin loading of Cdc45 and GINS in
yeast cells, did not significantly affect CMG complex formation. These results
suggest that the proteins involved in the assembly of initiation complexes in
human cells may differ somewhat from those in yeast systems. Accurate DNA replication requires a complex interplay of many regulatory
proteins at replication origins. The CMG (Cdc45·Mcm2-7·GINS) complex, which is
composed of Cdc45, Mcm2-7, and the GINS (Go-Ichi-Ni-San) complex consisting of
Sld5 and Psf1 to Psf3, is recruited by Cdc6 and Cdt1 onto origins bound by the
heterohexameric origin recognition complex (ORC) and functions as a replicative
helicase. Trypanosoma brucei, an early branched microbial eukaryote, appears to
express an archaea-like ORC consisting of a single Orc1/Cdc6-like protein.
However, unlike archaea, trypanosomes possess components of the eukaryote-like
CMG complex, but whether they form an active helicase complex, associate with
the ORC, and regulate DNA replication remains unknown. Here, we demonstrated
that the CMG complex is formed in vivo in trypanosomes and that Mcm2-7 helicase
activity is activated by the association with Cdc45 and the GINS complex in
vitro. Mcm2-7 and GINS proteins are confined to the nucleus throughout the cell
cycle, whereas Cdc45 is exported out of the nucleus after DNA replication,
indicating that nuclear exclusion of Cdc45 constitutes one mechanism for
preventing DNA re-replication in trypanosomes. With the exception of Mcm4, Mcm6,
and Psf1, knockdown of individual CMG genes inhibits DNA replication and cell
proliferation. Finally, we identified a novel Orc1-like protein, Orc1b, as an
additional component of the ORC and showed that both Orc1b and Orc1/Cdc6
associate with Mcm2-7 via interactions with Mcm3. All together, we identified
the Cdc45·Mcm2-7·GINS complex as the replicative helicase that interacts with
two Orc1-like proteins in the unusual origin recognition complex in
trypanosomes. Eukaryotic origins of replication are selected by loading a head-to-head double
hexamer of the Mcm2-7 replicative helicase around origin DNA. Cdt1 plays an
essential but transient role during this event; however, its mechanism of action
is unknown. Through analysis of Cdt1 mutations, we demonstrate that Cdt1
performs multiple functions during helicase loading. The C-terminus of Cdt1
binds Mcm2-7, and this interaction is required for efficient origin recruitment
of both proteins. We show that origin recognition complex (ORC) and Cdc6 recruit
multiple Cdt1 molecules to the origin during helicase loading, and disruption of
this multi-Cdt1 intermediate prevents helicase loading. Although dispensable for
loading Mcm2-7 double hexamers that are topologically linked to DNA, the
essential N-terminal domain of Cdt1 is required to load Mcm2-7 complexes that
are competent for association with the Cdc45 and GINS helicase-activating
proteins and replication initiation. Our data support a model in which
origin-bound ORC and Cdc6 recruit two Cdt1 molecules to initiate double-hexamer
formation prior to helicase loading and demonstrate that Cdt1 influences the
replication competence of loaded Mcm2-7 helicases. In most organisms, DNA replication is initiated by DNA primases, which
synthesize primers that are elongated by DNA polymerases. In this study, we
describe the isolation and biochemical characterization of the DNA primase
complex and its subunits from the archaeon Thermococcus kodakaraensis. The T.
kodakaraensis DNA primase complex is a heterodimer containing stoichiometric
levels of the p41 and p46 subunits. The catalytic activity of the complex
resides within the p41 subunit. We show that the complex supports both DNA and
RNA synthesis, whereas the p41 subunit alone marginally produces RNA and
synthesizes DNA chains that are longer than those formed by the complex. We
report that the T. kodakaraensis primase complex preferentially interacts with
dNTP rather than ribonucleoside triphosphates and initiates RNA as well as DNA
chains de novo. The latter findings indicate that the archaeal primase complex,
in contrast to the eukaryote homolog, can initiate DNA chain synthesis in the
absence of ribonucleoside triphosphates. DNA primers formed by the archaeal
complex can be elongated extensively by the T. kodakaraensis DNA polymerase
(Pol) B, whereas DNA primers formed by the p41 catalytic subunit alone were not.
Supplementation of reactions containing the p41 subunit with the p46 subunit
leads to PolB-catalyzed DNA synthesis. We also established a rolling circle
reaction using a primed 200-nucleotide circle as the substrate. In the presence
of the T. kodakaraensis minichromosome maintece (MCM) 3' → 5' DNA helicase,
PolB, replication factor C, and proliferating cell nuclear antigen, long leading
strands (>10 kb) are produced. Supplementation of such reactions with the DNA
primase complex supported lagging strand formation as well. The origin recognition complex (ORC) of Saccharomyces cerevisiae binds origin
DNA and cooperates with Cdc6 and Cdt1 to load the replicative helicase MCM2-7
onto DNA. Helicase loading involves two MCM2-7 hexamers that assemble into a
double hexamer around double-stranded DNA. This reaction requires ORC and Cdc6
ATPase activity, but it is unknown how these proteins control MCM2-7 double
hexamer formation. We demonstrate that mutations in Cdc6 sensor-2 and Walker A
motifs, which are predicted to affect ATP binding, influence the ORC-Cdc6
interaction and MCM2-7 recruitment. In contrast, a Cdc6 sensor-1 mutant affects
MCM2-7 loading and Cdt1 release, similar as a Cdc6 Walker B ATPase mutant.
Moreover, we show that Orc1 ATP hydrolysis is not involved in helicase loading
or in releasing ORC from loaded MCM2-7. To determine whether Cdc6 regulates
MCM2-7 double hexamer formation, we analysed complex assembly. We discovered
that inhibition of Cdc6 ATPase restricts MCM2-7 association with origin DNA to a
single hexamer, while active Cdc6 ATPase promotes recruitment of two MCM2-7
hexamer to origin DNA. Our findings illustrate how conserved Cdc6 AAA+ motifs
modulate MCM2-7 recruitment, show that ATPase activity is required for MCM2-7
hexamer dimerization and demonstrate that MCM2-7 hexamers are recruited to
origins in a consecutive process. Hexameric complexes of the six related Mcm2-7 proteins form the core of the
replicative helicase. Two other proteins, Mcm8 and Mcm9, with significant
homology to Mcm2-7 were first shown to play distinct roles during DNA
replication in Xenopus laevis egg extract. Recent work has revealed that Mcm8
and 9 form a complex that plays a role during homologous recombination in human,
chicken and mouse cells. We have therefore re-examined the behavior of the
Xenopus homologs of these proteins. We show that Mcm8 and Mcm9 form a dimeric
complex in Xenopus egg extract. They both associate with chromatin at later
stages of DNA replication, and this association is stimulated by DNA damage,
suggesting that their function is analogous to the one described in higher
eukaryotes. In contrast to previous reports, we do not find Mcm9 essential for
loading of Mcm2-7 complex onto chromatin during origin licensing nor detect its
interaction with Cdt1 origin licensing factor. Altogether, we conclude that the
role Mcm8 and Mcm9 play in Xenopus egg extract is not different from recent
findings in higher eukaryotes, consistent with an evolutionary conservation of
their function. Cyclin-dependent kinase (CDK) that plays a central role in preventing
re-replication of DNA phosphorylates several replication proteins to inactivate
them. MCM4 in MCM2-7 and RPA2 in RPA are phosphorylated with CDK in vivo. There
are inversed correlations between the phosphorylation of these proteins and
their chromatin binding. Here, we examined in vitro phosphorylation of human
replication proteins of MCM2-7, RPA, TRESLIN, CDC45 and RECQL4 with
CDK2/cyclinE, CDK2/cyclinA, CDK1/cyclinB, CHK1, CHK2 and CDC7/DBF4 kinases.
MCM4, RPA2, TRESLIN and RECQL4 were phosphorylated with CDKs. Effect of the
phosphorylation by CDK2/cyclinA on DNA-binding abilities of MCM2-7 and RPA was
examined by gel-shift analysis. The phosphorylation of RPA did not affect its
DNA-binding ability but that of MCM4 inhibited the ability of MCM2-7. Change of
six amino acids of serine and threonine to alanines in the amino-terminal region
of MCM4 rendered the mutant MCM2-7 insensitive to the inhibition with CDK. These
biochemical data suggest that phosphorylation of MCM4 at these sites by CDK
plays a direct role in dislodging MCM2-7 from chromatin and/or preventing
re-loading of the complex to chromatin. The MINICHROMOSOME MAINTENANCE 2-7 (MCM2-7) complex, a ring-shaped
heterohexamer, unwinds the DNA double helix ahead of the other replication
machinery. Although there is evidence that individual components might have
other roles, the essential nature of the MCM2-7 complex in DNA replication has
made it difficult to uncover these. Here, we present a detailed analysis of
Arabidopsis thaliana mcm2-7 mutants and reveal phenotypic differences. The
MCM2-7 genes are coordinately expressed during development, although MCM7 is
expressed at a higher level in the egg cell. Consistent with a role in the egg
cell, heterozygous mcm7 mutants resulted in frequent ovule abortion, a phenotype
that does not occur in other mcm mutants. All mutants showed a maternal effect,
whereby seeds inheriting a maternal mutant allele occasionally aborted later in
seed development with defects in embryo patterning, endosperm nuclear size, and
cellularization, a phenotype that is variable between subunit mutants. We
provide evidence that this maternal effect is due to the necessity of a maternal
store of MCM protein in the central cell that is sufficient for maintaining seed
viability and size in the absence of de novo MCM transcription. Reducing MCM
levels using endosperm-specific RNAi constructs resulted in the up-regulation of
DNA repair transcripts, consistent with the current hypothesis that excess
MCM2-7 complexes are loaded during G1 phase, and are required during S phase to
overcome replicative stress or DNA damage. Overall, this study demonstrates the
importance of the MCM2-7 subunits during seed development and suggests that
there are functional differences between the subunits. Eukaryotic cells license each DNA replication origin during G1 phase by
assembling a prereplication complex that contains a Mcm2-7 (minichromosome
maintece proteins 2-7) double hexamer. During S phase, each Mcm2-7 hexamer
forms the core of a replicative DNA helicase. However, the mechanisms of origin
licensing and helicase activation are poorly understood. The helicase loaders
ORC-Cdc6 function to recruit a single Cdt1-Mcm2-7 heptamer to replication
origins prior to Cdt1 release and ORC-Cdc6-Mcm2-7 complex formation, but how the
second Mcm2-7 hexamer is recruited to promote double-hexamer formation is not
well understood. Here, structural evidence for intermediates consisting of an
ORC-Cdc6-Mcm2-7 complex and an ORC-Cdc6-Mcm2-7-Mcm2-7 complex are reported,
which together provide new insights into DNA licensing. Detailed structural
analysis of the loaded Mcm2-7 double-hexamer complex demonstrates that the two
hexamers are interlocked and misaligned along the DNA axis and lack ATP
hydrolysis activity that is essential for DNA helicase activity. Moreover, we
show that the head-to-head juxtaposition of the Mcm2-7 double hexamer generates
a new protein interaction surface that creates a multisubunit-binding site for
an S-phase protein kinase that is known to activate DNA replication. The data
suggest how the double hexamer is assembled and how helicase activity is
regulated during DNA licensing, with implications for cell cycle control of DNA
replication and genome stability. All organisms ensure once and only once replication during S phase through a
process called replication licensing. Cdt1 is a key component and crucial
loading factor of Mcm complex, which is a central component for the eukaryotic
replicative helicase. In higher eukaryotes, timely inhibition of Cdt1 by Geminin
is essential to prevent rereplication. Here, we address the mechanism of DNA
licensing using purified Cdt1, Mcm and Geminin proteins in combination with
replication in Xenopus egg extracts. We mutagenized the 223th arginine of mouse
Cdt1 (mCdt1) to cysteine or serine (R-S or R-C, respectively) and 342nd and
346th arginines constituting an arginine finger-like structure to alanine
(RR-AA). The RR-AA mutant of Cdt1 could not only rescue the DNA replication
activity in Cdt1-depleted extracts but also its specific activity for DNA
replication and licensing was significantly increased compared to the wild-type
protein. In contrast, the R223 mutants were partially defective in rescue of DNA
replication and licensing. Biochemical analyses of these mutant Cdt1 proteins
indicated that the RR-AA mutation disabled its functional interaction with
Geminin, while R223 mutations resulted in ablation in interaction with the
Mcm2∼7 complex. Intriguingly, the R223 mutants are more susceptible to the
phosphorylation-induced inactivation or chromatin dissociation. Our results show
that conserved arginine residues play critical roles in interaction with Geminin
and Mcm that are crucial for proper conformation of the complexes and its
licensing activity. |
In which fields of DNA sequencing are Bloom filters applied? | A novel algorithm, fast and accurate classification of sequences (FACSs), is introduced that can accurately and rapidly classify sequences as belonging or not belonging to a reference sequence. Classification of DNA sequences using Bloom filters Lighter is a fast, memory-efficient tool for correcting sequencing errors. | MOTIVATION: New generation sequencing technologies producing increasingly
complex datasets demand new efficient and specialized sequence analysis
algorithms. Often, it is only the 'novel' sequences in a complex dataset that
are of interest and the superfluous sequences need to be removed.
RESULTS: A novel algorithm, fast and accurate classification of sequences
(FACSs), is introduced that can accurately and rapidly classify sequences as
belonging or not belonging to a reference sequence. FACS was first optimized and
validated using a synthetic metagenome dataset. An experimental metagenome
dataset was then used to show that FACS achieves comparable accuracy as BLAT and
SSAHA2 but is at least 21 times faster in classifying sequences.
AVAILABILITY: Source code for FACS, Bloom filters and MetaSim dataset used is
available at http://facs.biotech.kth.se. The Bloom::Faster 1.6 Perl module can
be downloaded from CPAN at http://search.cpan.org/ approximately
palvaro/Bloom-Faster-1.6/
CONTACTS: [email protected]; [email protected]
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
online. BACKGROUND: De Brujin graphs are widely used in bioinformatics for processing
next-generation sequencing data. Due to a very large size of NGS datasets, it is
essential to represent de Bruijn graphs compactly, and several approaches to
this problem have been proposed recently.
RESULTS: In this work, we show how to reduce the memory required by the data
structure of Chikhi and Rizk (WABI'12) that represents de Brujin graphs using
Bloom filters. Our method requires 30% to 40% less memory with respect to their
method, with insignificant impact on construction time. At the same time, our
experiments showed a better query time compared to the method of Chikhi and
Rizk.
CONCLUSION: The proposed data structure constitutes, to our knowledge, currently
the most efficient practical representation of de Bruijn graphs. MOTIVATION: New sequencing technologies generate larger amount of short reads
data at decreasing cost. De novo sequence assembly is the problem of combining
these reads back to the original genome sequence, without relying on a reference
genome. This presents algorithmic and computational challenges, especially for
long and repetitive genome sequences. Most existing approaches to the assembly
problem operate in the framework of de Bruijn graphs. Yet, a number of recent
works use the paradigm of string graph, using a variety of methods for storing
and processing suffixes and prefixes, like suffix arrays, the Burrows-Wheeler
transform or the FM index. Our work is motivated by a search for new approaches
to constructing the string graph, using alternative yet simple data structures
and algorithmic concepts.
RESULTS: We introduce a novel hash-based method for constructing the string
graph. We use incremental hashing, and specifically a modification of the
Karp-Rabin fingerprint, and Bloom filters. Using these probabilistic methods
might create false-positive and false-negative edges during the algorithm's
execution, but these are all detected and corrected. The advantages of the
proposed approach over existing methods are its simplicity and the incorporation
of established probabilistic techniques in the context of de novo genome
sequencing. Our preliminary implementation is favorably comparable with the
first string graph construction of Simpson and Durbin (2010) (but not with
subsequent improvements). Further research and optimizations will hopefully
enable the algorithm to be incorporated, with noticeable performance
improvement, in state-of-the-art string graph-based assemblers. BACKGROUND: Data from large Next Generation Sequencing (NGS) experiments present
challenges both in terms of costs associated with storage and in time required
for file transfer. It is sometimes possible to store only a summary relevant to
particular applications, but generally it is desirable to keep all information
needed to revisit experimental results in the future. Thus, the need for
efficient lossless compression methods for NGS reads arises. It has been shown
that NGS-specific compression schemes can improve results over generic
compression methods, such as the Lempel-Ziv algorithm, Burrows-Wheeler
transform, or Arithmetic Coding. When a reference genome is available, effective
compression can be achieved by first aligning the reads to the reference genome,
and then encoding each read using the alignment position combined with the
differences in the read relative to the reference. These reference-based methods
have been shown to compress better than reference-free schemes, but the
alignment step they require demands several hours of CPU time on a typical
dataset, whereas reference-free methods can usually compress in minutes.
RESULTS: We present a new approach that achieves highly efficient compression by
using a reference genome, but completely circumvents the need for alignment,
affording a great reduction in the time needed to compress. In contrast to
reference-based methods that first align reads to the genome, we hash all reads
into Bloom filters to encode, and decode by querying the same Bloom filters
using read-length subsequences of the reference genome. Further compression is
achieved by using a cascade of such filters.
CONCLUSIONS: Our method, called BARCODE, runs an order of magnitude faster than
reference-based methods, while compressing an order of magnitude better than
reference-free methods, over a broad range of sequencing coverage. In high
coverage (50-100 fold), compared to the best tested compressors, BARCODE saves
80-90% of the running time while only increasing space slightly. De novo assembly of the genome of a species is essential in the absence of a
reference genome sequence. Many scalable assembly algorithms use the de Bruijn
graph (DBG) paradigm to reconstruct genomes, where a table of subsequences of a
certain length is derived from the reads, and their overlaps are analyzed to
assemble sequences. Despite longer subsequences unlocking longer genomic
features for assembly, associated increase in compute resources limits the
practicability of DBG over other assembly archetypes already designed for longer
reads. Here, we revisit the DBG paradigm to adapt it to the changing sequencing
technology landscape and introduce three data structure designs for spaced seeds
in the form of paired subsequences. These data structures address memory and run
time constraints imposed by longer reads. We observe that when a fixed distance
separates seed pairs, it provides increased sequence specificity with increased
gap length. Further, we note that Bloom filters would be suitable to implicitly
store spaced seeds and be tolerant to sequencing errors. Building on this
concept, we describe a data structure for tracking the frequencies of observed
spaced seeds. These data structure designs will have applications in genome,
transcriptome and metagenome assemblies, and read error correction. MOTIVATION: The deluge of current sequenced data has exceeded Moore's Law, more
than doubling every 2 years since the next-generation sequencing (NGS)
technologies were invented. Accordingly, we will able to generate more and more
data with high speed at fixed cost, but lack the computational resources to
store, process and analyze it. With error prone high throughput NGS reads and
genomic repeats, the assembly graph contains massive amount of redundant nodes
and branching edges. Most assembly pipelines require this large graph to reside
in memory to start their workflows, which is intractable for mammalian genomes.
Resource-efficient genome assemblers combine both the power of advanced
computing techniques and innovative data structures to encode the assembly graph
efficiently in a computer memory.
RESULTS: LightAssembler is a lightweight assembly algorithm designed to be
executed on a desktop machine. It uses a pair of cache oblivious Bloom filters,
one holding a uniform sample of [Formula: see text]-spaced sequenced [Formula:
see text]-mers and the other holding [Formula: see text]-mers classified as
likely correct, using a simple statistical test. LightAssembler contains a light
implementation of the graph traversal and simplification modules that achieves
comparable assembly accuracy and contiguity to other competing tools. Our method
reduces the memory usage by [Formula: see text] compared to the
resource-efficient assemblers using benchmark datasets from GAGE and
Assemblathon projects. While LightAssembler can be considered as a gap-based
sequence assembler, different gap sizes result in an almost constant assembly
size and genome coverage.
AVAILABILITY AND IMPLEMENTATION:
https://github.com/SaraEl-Metwally/LightAssembler CONTACT:
[email protected] information: Supplementary data are
available at Bioinformatics online. Using a sequence's k-mer content rather than the full sequence directly has
enabled significant performance improvements in several sequencing applications,
such as metagenomic species identification, estimation of transcript abundances,
and alignment-free comparison of sequencing data. As k-mer sets often reach
hundreds of millions of elements, traditional data structures are often
impractical for k-mer set storage, and Bloom filters (BFs) and their variants
are used instead. BFs reduce the memory footprint required to store millions of
k-mers while allowing for fast set containment queries, at the cost of a low
false positive rate (FPR). We show that, because k-mers are derived from
sequencing reads, the information about k-mer overlap in the original sequence
can be used to reduce the FPR up to 30 × with little or no additional memory and
with set containment queries that are only 1.3 - 1.6 times slower.
Alternatively, we can leverage k-mer overlap information to store k-mer sets in
about half the space while maintaining the original FPR. We consider several
variants of such k-mer Bloom filters (kBFs), derive theoretical upper bounds for
their FPR, and discuss their range of applications and limitations. |
Which 2 medications are included in the Qsymia pill? | Qsymia pill includes phentermine and topiramate. It is used for treatment of obesity. | Obesity is a world-wide epidemic associated with significant morbidity and
mortality which costs billions of dollars per year. The associated related
conditions are many and include heart disease, stroke, type II diabetes
mellitus, sleep apnea and certain types of cancer. Given that it is a
multifactorial problem, the treatments must also address the numerous causes
associated with the development of obesity. The neurohormonal regulation of
feeding and energy is a complex system often necessitating modification through
more than 1 pathway to achieve weight loss. Therefore, in addition to lifestyle
changes, attenuation of caloric intake and increase in caloric expenditure,
pharmacotherapies, including combination medications, may prove beneficial in
its treatment. Adding to the current available pharmacotherapies for obesity,
the Food and Drug Administration has recently approved 2 new combination
medications known as lorcaserin (Belviq) and phentermine-topiramate (Qsymia). As
with these and other medications used for weight loss, clinical cautions, side
effects, precise review of patients' medical history and selecting the
appropriate medication are imperative. Additionally, close follow-up is
necessary in patients undergoing treatment for weight loss. As weight loss
progresses, patients who are currently undergoing concomitant treatment for
comorbid diabetes and hypertension need to be monitored for appropriate changes
in medications used to treat those conditions. Weight loss is often accompanied
by improvement in blood pressure and glucose levels and therefore resting blood
pressure and fasting and/or postprandial plasma glucose levels should be
monitored at follow-up. Although unique to each individual, the benefits of
weight loss are substantial and can improve well-being and physical health. After a long period of failure in development, two new medications
(phentermine/topiramate ER - Qsymia™ and lorcaserin - Belviq®) have been
approved by the US Food and Drug Administration for long-term weight management
in persons with obesity (BMI ≥ 30 kg/m(2)) or in overweight persons (BMI ≥ 27
kg/m(2)) with comorbidities. Another medication, bupropion/naltrexone, is
undertaking a cardiovascular outcomes trial and an analysis in 2014 will
determine its approval and release. The most widely prescribed drug for obesity,
phentermine, used since 1959 for short-term weight management, has been released
in a new formulation. This paper reviews these new medications, and other
important events in the landscape for management of obesity, with an eye to the
interests of physicians who manage hypertension. All the new drugs under
discussion are re-fittings of old agents or fresh approaches to old targets;
thus, what is old is new again in the pharmacotherapy of obesity. BACKGROUND: Phase 3 clinical trial results reveal that Qsymia is a clinically
effective long-term treatment for obesity, but whether this treatment is
cost-effective compared to a diet and lifestyle intervention has yet to be
explored.
OBJECTIVE: To quantify the incremental cost-effectiveness of Qsymia (phentermine
and topiramate extended-release) for health-related quality of life
improvements.
STUDY DESIGN AND METHODS: Estimates are based on cost and quality of life
outcomes from a 56-week, multicenter, placebo-controlled, phase 3 clinical trial
undertaken in 93 health centers in the US. Participants were overweight and
obese adults (aged 18-70 years) with a body-mass index of 27-45 kg/m(2) and two
or more comorbidities (hypertension, dyslipidemia, diabetes or pre-diabetes or
abdominal obesity). The intervention was diet and lifestyle advice plus the
recommended dose of Qsymia (phentermine 7.5 mg plus topiramate 46.0 mg) vs.
control, which included diet and lifestyle advice plus placebo. The study was
from the payer perspective. Costs included the prescription cost, medication
cost offsets and physician appointment costs. Effectiveness was measured in
terms of quality-adjusted life years gained (QALYs). The main outcome measure
was incremental cost per QALY gained of the intervention relative to control.
RESULTS: Our base-case model, in which participants take Qsymia for 1 year with
benefits linearly decaying over the subsequent 2 years, generates an incremental
cost-effectiveness ratio (ICER) of $48,340 per QALY gained. Using the base-case
assumptions, probabilistic sensitivity analyses reveal that the ICER is below
$50,000 per QALY in 54 % of simulations. However, results are highly dependent
on the extent to which benefits are maintained post medication cessation. If
benefits persist for only 1 year post cessation, the ICER increases to $74,480.
CONCLUSION: Although base-case results suggest that Qsymia is cost-effective,
this result hinges on the time on Qsymia and the extent to which benefits are
maintained post medication cessation. This should be an area of future research. BACKGROUND: Obesity is a serious and costly disease that is growing in epidemic
proportions. Obesity-related hospitalizations have nearly tripled from 1996 to
2009. If the current trend in the growth of obesity continues, the total
healthcare costs attributable to obesity could reach $861 billion to $957
billion by 2030. The American Medical Association has officially recognized
obesity as a disease. Obesity is a public health crisis affecting approximately
more than 33% of Americans and costing the healthcare system more than $190
billion annually.
OBJECTIVES: To review the 2 new drugs that were recently approved by the US Food
and Drug Administration (FDA) for the treatment of obesity, lorcaserin HCl
(Belviq) and phentermine/topiramate (Qsymia) and their potential impact on the
treatment of obese patients.
DISCUSSION: Lifestyle modification is the first and mainstay treatment for
obesity. Antiobesity drugs are indicated as adjuncts to a healthy, low-fat,
low-calorie diet and an exercise plan. Currently, 4 drugs are approved by the
FDA for the treatment of obesity, 2 of which were approved after June 2012.
These 2 drugs, Belviq and Qsymia, have added new tools for the treatment of
obesity. In addition to reducing body mass index, these drugs have been shown to
reduce hemoglobin A1c levels in patients with diabetes and blood pressure levels
in patients with hypertension, as well as to decrease lipid levels in patients
with hyperlipidemia. This article reviews the drugs' mechanisms of action,
evaluates landmark clinical studies leading to the FDA approval of the 2 drugs,
their common side effects, and the benefits these new drugs can provide toward
the management of the obesity epidemic that are different from other medications
currently available.
CONCLUSION: The weight loss seen in patients who are using the 2 new medications
has been shown to further improve other cardiometabolic health parameters,
including blood pressure, blood glucose levels, and serum lipid levels. Based on
clinical trials evidence, it is likely that many obese patients could benefit
from these therapies, if used appropriately. The pharmacotherapy of obesity has historically recorded an overall poor safety
and efficacy profile largely because of the complex mechanisms involved in the
pathophysiology of obesity. It is hoped that a better understanding of the
regulation of body weight will lead us to the development of effective and safer
drugs. Recent advances in our understanding of the regulation of energy
homeostasis has allowed the design of novel anti-obesity drugs targeting
specific molecules crucial for the modulation of energy balance, including drugs
that induce satiety, modulate nutrient absorption or influence metabolism or
lipogenesis. Almost a decade after the Food and Drug Administration approved the
first weight loss medication, it recently approved two novel anti-obesity drugs
Belviq (lorcaserin) and Qsymia (topiramate and phentermine), thus signalling the
beginning of a new era in the pharmacotherapy of obesity. It is believed that
the next generation of weight-loss drugs will be based on combination treatments
with gut hormones in a manner that mimics the changes underlying surgically
induced weight loss thus introducing the so called 'bariatric pharmacotherapy'.
An in-depth understanding of the interrelated physiological and behavioural
effects of these new molecules together with the development of new treatment
paradigms is needed so that future disappointments in the field of obesity
pharmacotherapy may be avoided. |
Is sonidegib effective for basal cell carcinoma? | Yes. Sonidegib, an oral smoothened antagonist, is indicated for the treatment of adults with locally advanced basal cell carcinoma (laBCC) who are not candidates for surgery or radiation therapy, or adults with recurrent laBCC following surgery or radiation therapy. | PURPOSE: This phase I trial was undertaken to determine the maximum tolerated
dose (MTD), dose-limiting toxicities (DLT), safety, tolerability,
pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of the
novel smoothened inhibitor sonidegib (LDE225), a potent inhibitor of hedgehog
signaling, in patients with advanced solid tumors.
EXPERIMENTAL DESIGN: Oral sonidegib was administered to 103 patients with
advanced solid tumors, including medulloblastoma and basal cell carcinoma (BCC),
at doses ranging from 100 to 3,000 mg daily and 250 to 750 mg twice daily,
continuously, with a single-dose pharmacokinetics run-in period. Dose
escalations were guided by a Bayesian logistic regression model. Safety,
tolerability, efficacy, pharmacokinetics, and biomarkers in skin and tumor
biopsies were assessed.
RESULTS: The MTDs of sonidegib were 800 mg daily and 250 mg twice daily. The
main DLT of reversible grade 3/4 elevated serum creatine kinase (18% of
patients) was observed at doses ≥ the MTD in an exposure-dependent manner.
Common grade 1/2 adverse events included muscle spasm, myalgia, gastrointestinal
toxicities, increased liver enzymes, fatigue, dysgeusia, and alopecia. Sonidegib
exposure increased dose proportionally up to 400 mg daily, and displayed
nonlinear pharmacokinetics at higher doses. Sonidegib exhibited
exposure-dependent reduction in GLI1 mRNA expression. Tumor responses observed
in patients with medulloblastoma and BCC were associated with evidence of
hedgehog pathway activation.
CONCLUSIONS: Sonidegib has an acceptable safety profile in patients with
advanced solid tumors and exhibits antitumor activity in advanced BCC and
relapsed medulloblastoma, both of which are strongly associated with activated
hedgehog pathway, as determined by gene expression. INTRODUCTION: Basal cell carcinoma (BCC) is a maligcy that is driven by an
activated Hedgehog (Hh) pathway. Smoothened inhibitors are a new promising
treatment option for patients with locally advanced or metastatic BCC or basal
cell nevus syndrome. But long-term data are still limited, the optimal treatment
duration is not yet defined and there are already documented cases with acquired
resistance.
AREAS COVERED: Treatment modalities with Hh inhibitors, side effects and
potential pharmacological combination options are discussed. The current
literature, including PubMed, Cochrane database and registered trials on
ClinicalTrials.gov, was searched.
EXPERT OPINION: BCCs typically regress during therapy with Hh inhibitors. Muscle
toxicity, dysgeusia and hair loss can be considered as on target adverse
reactions. Muscle toxicity is the dose-limiting toxicity of sonidegib. It was
not seen with vismodegib because of its high binding to plasma protein α-1-acid
glycoprotein. Sonidegib is different and shows a clear dose-toxicity
relationship, which allows to address the question of whether there is a dose
dependency of regression rate, cure rate and progression-free survival. In
addition, basic research has offered strategies to enhance efficacy by the
combination with other molecules, such as EGFR inhibitors, MEK inhibitors or
immunotherapy. The Hedgehog (Hh) signaling pathway is critical for embryonic development. In
adult tissues, Hh signaling is relatively quiescent with the exception of roles
in tissue maintece and repair. Aberrant activation of Hh signaling is
implicated in multiple aspects of transformation, including the maintece of
the cancer stem cell (CSC) phenotype. Preclinical studies indicate that CSCs
from many tumor types are sensitive to Hh pathway inhibition and that
Hh-targeted therapeutics block many aspects of transformation attributed to
CSCs, including drug resistance, relapse, and metastasis. However, to date, Hh
inhibitors, specifically those targeting Smoothened [such as vismodegib,
BMS-833923, saridegib (IPI-926), sonidegib/erismodegib (LDE225), PF-04449913,
LY2940680, LEQ 506, and TAK-441], have demonstrated good efficacy as monotherapy
in patients with basal cell carcinoma and medulloblastoma, but have shown
limited activity in other tumor types. This lack of success is likely due to
many factors, including a lack of patient stratification in early trials,
cross-talk between Hh and other oncogenic signaling pathways that can modulate
therapeutic response, and a limited knowledge of Hh pathway activation
mechanisms in CSCs from most tumor types. Here, we discuss Hh signaling
mechanisms in the context of human cancer, particularly in the maintece of
the CSC phenotype, and consider new therapeutic strategies that hold the
potential to expand considerably the scope and therapeutic efficacy of
Hh-directed anticancer therapy. BACKGROUND: Patients with advanced basal cell carcinoma have limited treatment
options. Hedgehog pathway signalling is aberrantly activated in around 95% of
tumours. We assessed the antitumour activity of sonidegib, a Hedgehog signalling
inhibitor, in patients with advanced basal cell carcinoma.
METHODS: BOLT is an ongoing multicentre, randomised, double-blind, phase 2
trial. Eligible patients had locally advanced basal cell carcinoma not amenable
to curative surgery or radiation or metastatic basal cell carcinoma. Patients
were randomised via an automated system in a 1:2 ratio to receive 200 mg or 800
mg oral sonidegib daily, stratified by disease, histological subtype, and
geographical region. The primary endpoint was the proportion of patients who
achieved an objective response, assessed in the primary efficacy analysis
population (patients with fully assessable locally advanced disease and all
those with metastatic disease) with data collected up to 6 months after
randomisation of the last patient. This trial is registered with
ClinicalTrials.gov, number NCT01327053.
FINDINGS: Between July 20, 2011, and Jan 10, 2013, we enrolled 230 patients, 79
in the 200 mg sonidegib group, and 151 in the 800 mg sonidegib group. Median
follow-up was 13·9 months (IQR 10·1-17·3). In the primary efficacy analysis
population, 20 (36%, 95% CI 24-50) of 55 patients receiving 200 mg sonidegib and
39 (34%, 25-43) of 116 receiving 800 mg sonidegib achieved an objective
response. In the 200 mg sonidegib group, 18 (43%, 95% CI 28-59) patients who
achieved an objective response, as assessed by central review, were noted among
the 42 with locally advanced basal cell carcinoma and two (15%, 2-45) among the
13 with metastatic disease. In the 800 mg group, 35 (38%, 95% CI 28-48) of 93
patients with locally advanced disease had an objective response, as assessed by
central review, as did four (17%, 5-39) of 23 with metastatic disease. Fewer
adverse events leading to dose interruptions or reductions (25 [32%] of 79
patients vs 90 [60%] of 150) or treatment discontinuation (17 [22%] vs 54 [36%])
occurred in patients in the 200 mg group than in the 800 mg group. The most
common grade 3-4 adverse events were raised creatine kinase (five [6%] in the
200 mg group vs 19 [13%] in the 800 mg group) and lipase concentration (four
[5%] vs eight [5%]). Serious adverse events occurred in 11 (14%) of 79 patients
in the 200 mg group and 45 (30%) of 150 patients in the 800 mg group.
INTERPRETATION: The benefit-to-risk profile of 200 mg sonidegib might offer a
new treatment option for patients with advanced basal cell carcinoma, a
population that is difficult to treat.
FUNDING: Novartis Pharmaceuticals Corporation. PURPOSE: To assess the tumor response to the smoothened (SMO) inhibitor,
sonidegib (LDE225), in patients with an advanced basal cell carcinoma (BCC)
resistant to treatment with vismodegib (GDC0449).
EXPERIMENTAL DESIGN: Nine patients with an advanced BCC that was previously
resistant to treatment with vismodegib were given sonidegib in this
investigational, open-label study. Tumor response was determined using the
response evaluation criteria in solid tumors. SMO mutations were identified
using biopsy samples from the target BCC location.
RESULTS: The median duration of treatment with sonidegib was 6 weeks (range,
3-58 weeks). Five patients experienced progressive disease with sonidegib. Three
patients experienced stable disease and discontinued sonidegib either due to
adverse events (n = 1) or due to election for surgery (n = 2). The response of
one patient was not evaluable. SMO mutations with in vitro data suggesting
resistance to Hh pathway inhibition were identified in 5 patients, and none of
these patients experienced responses while on sonidegib.
CONCLUSIONS: Patients with advanced BCCs that were previously resistant to
treatment with vismodegib similarly demonstrated treatment resistance with
sonidegib. Patients who have developed treatment resistance to an SMO inhibitor
may continue to experience tumor progression in response to other SMO
inhibitors. Nonmelanoma skin cancer (NMSC) is the most common cancer in patients and
includes basal cell carcinoma (BCC) and squamous cell carcinoma (SCC).
Treatments useful for SCC and BCC include surgical, topical, and in advanced
cases systemic chemo-radiation. This review of the literature aims to describe
previous and current treatment options for oral therapy in locally advanced and
metastatic NMSC otherwise unamenable to standard treatment. Oral Smoothened
(Smo) inhibitors Vismodegib, Sonidegib, and Taladegib have shown to be effective
in several trials. Oral tyrosine kinase inhibitors Erlotinib and Gefitinib,
which target epidermal growth factor receptor (EGFR), have early supporting data
and are currently undergoing large multicenter trials. Other less studied oral
therapies which have shown at least partial efficacy include 5-Fluorouracil,
capecitabine, and picropodophyllin. In vitro studies have elucidated new targets
for dual combination oral therapy targeting both EGFR and insulin-like growth
factor 1 receptor (IGF-1R). It is important to stratify treatment options based
on patient risk of advanced disease, failure of conservative treatment, and
ill-tolerated intravenous chemotherapy adverse events. Oral therapy in NMSC is
useful in high risk patients with recurrent and aggressive disease who may not
tolerate other systemic therapies. The advent of more sophisticated studies published has clarified the
understating of the root cause of various skin cancers or basal cell carcinomas
(BCCs). The remarkable role is played by the comprehensive work done on
unraveling the mechanism controlling the function of hedgehog (Hh) pathway. The
defective Hh pathway has been found as the major cause for BCCs as activated Hh
signaling within primary cilia plays a key role in the pathogenesis of BCCs. The
BCC accounts for up to 40% of all cancers in the US, with growing incidences in
other countries as well. Thus, it is considered to be utmost important by the
researchers all over the world developing drugs for the treatment of skin
cancers targeting Hh pathway. Fewer drugs like vismodegib, itraconazole and
sonidegib have shown promising results inhibiting the awry function of Hh
pathway resulting in treatment of different forms of skin cancers. These drugs
have shown positive results but failed to prove their potential as expected.
Vismodegib and sonidegib are better but fail in case of resistant tumors. This
review article describes the mechanism of actions of these Hh pathway inhibitors
and provides the rationale for their effectiveness/non-effectiveness for the
treatment of metastatic or locally advanced BCC. PURPOSE OF REVIEW: We summarize the concept of a locally advanced basal cell
carcinoma (laBCC) and present the current consensus definition. We also review
the key pieces of primary research undertaken in the past year and how these
affect the use of smoothened inhibitors in a clinical setting.
RECENT FINDINGS: Medium term follow-up (30 months) of patients treated with
vismodegib shows an improvement in response rates for patients with laBCC. The
safety profile of vismodegib demonstrated in the original ERIVANCE study has
been replicated in a larger patient cohort in a repeat study. Sonidegib is a new
smoothened inhibitor currently under investigation for treatment of laBCC, which
demonstrates a comparable safety profile to vismodegib. The side-effects of
smoothened inhibitors appear related to both dose and duration of treatment. The
durability of response to vismodegib is uncertain, but has been observed to last
for over a year following discontinuation of treatment.
SUMMARY: The understanding of the efficacy and safety of vismodegib has improved
since its introduction in 2012. A broadening evidence base supports its use as a
valid treatment for laBCC. However, questions remain as to how to integrate its
use into existing pathways for treating laBCC and its long-term efficacy. Author information:
(1)UniversitätsSpital Zürich-Skin Cancer Center, University Hospital, Zürich,
Switzerland. Electronic address: [email protected].
(2)Royal North Shore Hospital, Sydney, Australia.
(3)Medizinische Hochschule Hannover, Hannover, Germany.
(4)Sint-Augustinus Ziekenhuis, Antwerp, Belgium.
(5)Division of Medical Oncology, School of Medicine, University of Colorado,
Aurora, Colorado.
(6)Anti Cancer Institute, Léon Bérard, Lyon, France.
(7)Glasgow Royal Infirmary, Glasgow, United Kingdom.
(8)University Hospital Jena, Jena, Germany; SRH Wald-Klinikum Gera GmbH, Gera,
Germany.
(9)University Medical Center Mainz, Mainz, Germany.
(10)Department of Dermatology, Andreas Sygros Hospital, University of Athens,
Athens, Greece.
(11)Fachklinik Hornheide, Münster, Germany.
(12)Northern Centre for Cancer Care, Freeman Hospital, Newcastle upon Tyne,
United Kingdom.
(13)Novartis Pharma AG, Oncology Global Development, Basel, Switzerland.
(14)Novartis Pharmaceuticals Corporation, East Hanover, New Jersey.
(15)Stanford University School of Medicine, Redwood City, California.
(16)Cliniques Universitaires Saint-Luc, Université Catholique de Louvain,
Brussels, Belgium.
(17)Winship Cancer Institute at Emory University, Atlanta, Georgia.
(18)Dermatologikum Berlin, Berlin, Germany.
(19)Manchester Academic Health Science Centre, University of Manchester,
Manchester, United Kingdom.
(20)Departments of Dermatology and Head and Neck Surgery, University of Texas MD
Anderson Cancer Center, Houston, Texas. The Hedgehog inhibitors are promising alternative for patients with advanced
basal cell carcinoma that are not amenable to radiotherapy or surgery.
Sonidegib, also known as LDE225, is an orally available SMO antagonist that was
recently approved by the US FDA for the treatment of patients with locally
advanced basal cell carcinoma. This article will provide an overview of the
pharmacology and pharmacokinetics of sonidegib and in-depth analysis of the BOLT
trial with additional data from the 12-month update. The present challenges
associated with Hedgehog inhibitors will also be discussed. INTRODUCTION: Advanced and metastatic basal cell carcinomas (BCCs) are rare but
still present a severe medical problem. These tumors are often disfiguring and
impact the quality of life by pain or bleeding. Based on discovery of the
hedgehog (Hh) signaling pathway and its role in the pathogenesis of BCCs,
smoothened (SMO) inhibitors have been developed with Sonidegib being the 2nd in
class. It is the only Hh pathway inhibitor investigated in a randomized trial
accompanied by pharmacodynamic investigations. Also, the disease assessment
criteria applied were more stringent than those used in trials of 1st developed
SMO inhibitor - vismodegib, and required annotated photographs, MRI as well as
multiple biopsies in case of regression.
AREAS COVERED: All available papers from Medline and the abstracts of the most
important dermato-oncology meetings were included.
EXPERT OPINION: Sonidegib is a promising medication for advanced BCC and other
maligcies, driven by Hh signaling. It presents favorable pharmacokinetic
properties and causes class specific toxicity with dose dependent adverse events
in muscular and taste bud homeostasis, gastrointestinal symptoms and hair
growth. Early after treatment initiation, it impacts the immunesusceptibility of
the tumor lesions. Sonidegib deserves further development in combination with
other drugs or antibodies, or alternative dosing schedules. Basal cell carcinoma (BCC) is the most common nonmelanoma skin cancer. If left
untreated, BCCs can become locally aggressive or even metastasize. Currently
available treatments include local destruction, surgery, and radiation. Systemic
options for advanced disease are limited. The Hedgehog (Hh) pathway is
aberrantly activated in a majority of BCCs and in other cancers. Hh pathway
inhibitors are targeted agents that inhibit the aberrant activation of the Hh
pathway, with smoothened being a targeted component. Sonidegib is a novel
smoothened inhibitor that was recently approved by the US Food and Drug
Administration. This review focuses on BCC pathogenesis and the clinical
efficacy of sonidegib for the treatment of advanced BCC. |
Which R package could be used for the identification of pediatric brain tumors? | MethPed | |
Describe the usefulness of CAMUR in The Cancer Genome Atlas (TCGA) | CAMUR is a new method that extracts multiple and equivalent classification models. CAMUR iteratively computes a rule-based classification model, calculates the power set of the genes present in the rules, iteratively eliminates those combinations from the data set, and performs again the classification procedure until a stopping criterion is verified. CAMUR includes an ad-hoc knowledge repository (database) and a querying tool. Three different types of RNA-seq data sets (Breast, Head and Neck, and Stomach Cancer) were analyzed from The Cancer Genome Atlas (TCGA) and CAMUR and its models were validated also on non-TCGA data. Experimental results show the efficacy of CAMUR by obtaining several reliable equivalent classification models, from which the most frequent genes, their relationships, and the relation with a particular cancer are deduced. | MOTIVATION: Nowadays, knowledge extraction methods from Next Generation
Sequencing data are highly requested. In this work, we focus on RNA-seq gene
expression analysis and specifically on case-control studies with rule-based
supervised classification algorithms that build a model able to discriminate
cases from controls. State of the art algorithms compute a single classification
model that contains few features (genes). On the contrary, our goal is to elicit
a higher amount of knowledge by computing many classification models, and
therefore to identify most of the genes related to the predicted class.
RESULTS: We propose CAMUR, a new method that extracts multiple and equivalent
classification models. CAMUR iteratively computes a rule-based classification
model, calculates the power set of the genes present in the rules, iteratively
eliminates those combinations from the data set, and performs again the
classification procedure until a stopping criterion is verified. CAMUR includes
an ad-hoc knowledge repository (database) and a querying tool.We analyze three
different types of RNA-seq data sets (Breast, Head and Neck, and Stomach Cancer)
from The Cancer Genome Atlas (TCGA) and we validate CAMUR and its models also on
non-TCGA data. Our experimental results show the efficacy of CAMUR: we obtain
several reliable equivalent classification models, from which the most frequent
genes, their relationships, and the relation with a particular cancer are
deduced.
AVAILABILITY AND IMPLEMENTATION: dmb.iasi.cnr.it/camur.php
CONTACT: [email protected]
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
online. |
Which markers are screened with the triple test for the detection of syndromes in fetus? | The markers that are screened with the triple test for the detection of syndromes in fetus are:
1) alpha-fetoprotein (AFP),
2) beta-chorionic gonadotrophin (beta-CG) and
3) unconjugated oestriol (uE3). | OBJECTIVE: Our purpose was to compare the efficacy of triple-marker screening
(alpha-fetoprotein, unconjugated estriol, human chorionic gonadotropin) with
alpha-fetoprotein plus free beta-human chorionic gonadotropin.
STUDY DESIGN: Free beta-human chorionic gonadotropin was concurrently assayed in
2349 maternal serum samples. Trivariate and bivariate algorithms were used to
calculate the risk for fetal Down syndrome by the two protocols. Free beta-human
chorionic gonadotropin from 12 cases of fetal Down syndrome previously screened
with the triple marker was retrospectively assayed.
RESULTS: Mean maternal age of our study was 29.8 years (range 14 to 51 years).
The initial screen-positive rate with the triple marker was 8.0% compared with
12.8% for alpha-fetoprotein plus free beta-human chorionic gonadotropin. All
three cases of fetal Down syndrome ascertained in our prospective study were
detected by the triple marker; in contrast, one of three was detected by
alpha-fetoprotein plus free beta-human chorionic gonadotropin. By adding 12
additional cases of fetal Down syndrome, 12 of 15 (80%) were screen positive
with triple marker and nine of 15 (60%) were screen positive with
alpha-fetoprotein plus free beta-human chorionic gonadotropin.
CONCLUSION: The detection rate of fetal Down syndrome was greater by use of a
triple marker screen than when using alpha-fetoprotein plus free beta-human
chorionic gonadotropin. Our data do not support the claims of other studies that
suggest that alpha-fetoprotein plus free beta-human chorionic gonadotropin is
superior to triple markers. OBJECTIVE: To develop an artificial intelligent diagnostic system with neural
networks to determine genetical disorders and fetal health problems by using
maternal serum markers ('Triple Test') and maternal age.
STUDY DESIGN: A total of 112 pregt women were referred to Fetal Medicine Unit
of Hacettepe University Hospital for fetal ultrasonography and chromosome
analysis with different indications. All patients underwent genetic
amniocentesis or fetal blood sampling under ultrasound guidance. Gross
malformations and hydrops fetalis were detected in 15 and 5 fetuses,
respectively. We have found chromosomal abnormality in 7 cases. 'Triple Test' is
offered to all patients and serum levels of alpha-fetoprotein, human chorionic
gonadotropin and unconjugated estriol were analyzed by radioimmunoassay. In this
study, we have used supervised artificial neural network structure to develop a
diagnostic system. Our system's input parameters are maternal age, gestational
age and 'Triple Test' results. Our system consists of two different artificial
neural network modules whose decision-making logics are different. One of them
is designed to search genetical disorders while the other one is for the
assessment of fetal well-being. Confusion matrix is used for statistical
evaluation.
RESULTS: The discriminatory power of the artificial neural network to search
genetical disorders and fetal well-being is found to be highly significant (z =
10.583 and z = 10.424, respectively).
CONCLUSION: This system brings objectively to the evaluation of 'Triple Test'
results and can be used both for the detection of genetical disorders and fetal
well-being. Nevertheless, the analysis program's performance is limited to input
information and knowledge and medical expert expert can not get more than he or
she has donated the system. OBJECTIVE: This study was undertaken to compare the Down syndrome screening
efficiency of elevated maternal urine level of the beta-core fragment of human
chorionic gonadotropin with that of the traditional serum triple test.
STUDY DESIGN: Urinary beta-core fragment and serum analyte levels were measured
prospectively in women with singleton pregcies who were undergoing
second-trimester genetic amniocentesis. Urinary analyte levels were measured
within a week of specimen collection. In some cases only alpha-fetoprotein was
measured initially and human chorionic gonadotropin and unconjugated estriol
levels were subsequently determined from the stored serum specimens. The Down
syndrome screening efficiency of urinary concentration of beta-core fragment
plus maternal age was compared with that of the traditional triple test.
Receiver operating characteristic curves were generated for each algorithm and
the areas under the curves were compared to determine which algorithm was
superior.
RESULTS: There were a total of 926 study patients, of whom 21 (2.3%) carried
fetuses with Down syndrome. The mean (+/-SD) gestations at amniocentesis were
16.6 +/- 1.5 weeks for the fetuses without Down syndrome and 17.7 +/- 2.3 for
the fetuses with Down syndrome. A total of 539 women (4 of whom carried fetuses
with Down syndrome) had serum alpha-fetoprotein alone measured initially.
Urinary concentration of beta-core fragment had a 61.9% detection rate with a
4.9% false-positive rate for Down syndrome, whereas the values for the triple
screen were 57. 1% and 11.2%, respectively. The areas under the
receiver-operating characteristic curves were 0.8744 for elevated urinary
beta-core fragment level and 0.7504 for the triple screen (P =.1116). When the
false-positive rate was fixed at an ideal threshold value (</=5%) the urine test
was superior (area under the curve, 0.0212 vs 0.0133, P <.05). Similarly, when
we considered only cases in which the complete triple screen was performed
prospectively (17 fetuses with Down syndrome and 431 fetuses without Down
syndrome), the urine test was significantly better (area under the curve, 0.873
vs 0.624, P =.012).
CONCLUSION: In this first reported direct comparison we consistently observed
higher sensitivity values for screening with urinary levels of beta-core
fragment than for serum triple screen, suggesting an equivalent or superior Down
syndrome screening performance for the urinary analyte. It is important that
freezing and prolonged urine storage before testing be avoided. The reduced cost
(single- versus triple-analyte testing) and excellent screening performance
support large-scale testing and evaluation of maternal urinary beta-core
fragment measurement as an alternative to the traditional serum triple test. OBJECTIVE: Both modest screening performance and declining patient and physician
acceptance have stimulated interest in alternative markers to the triple screen
for the detection of Down syndrome. Our purpose was to compare the concentration
of a single urinary analyte, hyperglycosylated human chorionic gonadotropin,
with the serum triple screen (alpha-fetoprotein, human chorionic gonadotropin,
and unconjugated estriol concentrations combined with age) for second-trimester
Down syndrome detection.
STUDY DESIGN: Urine and blood were obtained from pregt women in the second
trimester undergoing genetic amniocentesis. Urinary hyperglycosylated human
chorionic gonadotropin concentration and serum triple-screen values were
measured. Individuals undergoing amniocentesis because of abnormal triple-screen
results were excluded. Individual Down syndrome risks on the basis of urinary
hyperglycosylated human chorionic gonadotropin concentration plus maternal age
and on the basis of the triple-screen results were calculated. For each
algorithm the sensitivity and false-positive rate for Down syndrome detection at
different risk thresholds were determined. From these values receiver operating
characteristic curves were constructed, and the area under the curve was
determined for each algorithm. Finally, the performance of a new combination in
which urinary hyperglycosylated human chorionic gonadotropin concentration
replaced serum human chorionic gonadotropin concentration in the triple screen
was ascertained.
RESULTS: We studied 24 pregcies complicated by Down syndrome and 500
unaffected pregcies between 14 and 22 weeks' gestation in a mostly white
(93.5%) population undergoing amniocentesis primarily because of advanced
maternal age. The sensitivity and false-positive rate for urinary
hyperglycosylated human chorionic gonadotropin concentration were 75. 0% and
5.6%, respectively, whereas those for the triple screen were 75.0% and 33.2%,
respectively. Urinary hyperglycosylated human chorionic gonadotropin
concentration was superior to the triple screen (area under the curve, 0.9337 vs
0.7887; P =.02). The substitution of urinary hyperglycosylated human chorionic
gonadotropin concentration for serum human chorionic gonadotropin concentration
in the triple screen resulted in a 91.7% sensitivity at a 10.0% false-positive
rate, versus a 54.2% sensitivity for the traditional triple screen at the same
false-positive rate.
CONCLUSION: The performance of urinary hyperglycosylated human chorionic
gonadotropin concentration was statistically superior to that of the serum
triple screen in a high-risk population. The use of urinary hyperglycosylated
human chorionic gonadotropin concentration as an alternative test or
substitution of this measurement for serum human chorionic gonadotropin
concentration in the triple screen would improve diagnostic accuracy and address
many current concerns related to the triple screen. The aim of this prospective study was to compare triple test screening
(alpha-fetoprotein, beta-chorionic gonadotrophin and unconjugated oestriol) with
amniocentesis in the detection of fetuses with Down's syndrome in women of 35
years of age or more. Between 1992 and 1996, maternal serum markers were
evaluated in 1406 women who had amniocentesis for prenatal diagnosis of
chromosomal abnormalities related to a maternal age of 35 or more years. Sixteen
fetuses with Down's syndrome were identified in the whole group by amniocentesis
and karyotyping. The group with negative triple test screening consisted of 919
pregcies and included two fetuses with trisomy-21 (false negatives). With
triple test screening in the age group over 35, there was a detection rate of
87.5% for cut-off points ranging from 1:200 up to 1:350, with corresponding
false positive rates ranging between 23% and 34%. In our population, if we had
practiced the policy of offering amniocentesis only to women screening positive
for the ages of 35 and 36 and to all pregt women of 37 or more, we would have
carried out 30% less amniocenteses. In this group of 1406 women, 33 abnormal
karyotypes were detected with amniocentesis (16 Down's syndrome included) and
equal number of elective abortions were carried out. Nevertheless, 19 healthy
fetuses and neonates were lost after amniocentesis. Considering the high
detection rates that can be achieved with triple test screening, the existing
procedure related risk of amniocentesis (0.5-1.0%), and the facts that
conception in women over 35 years of age is usually more difficult and the
background loss usually higher than in younger women, we believe that in the
future women over 35 should be offered a choice between non-invasive and
invasive procedures after being thoroughly informed. Second-trimester maternal serum markers (triple test) is common used to estimate
of the fetal risk of genetic abnormalities and open neural tube defects.
Positive results of the triple test concomitant with the normal fetus karyotype
pattern can also predict the adverse pregcy outcome. Many authors have been
indicated such false positive results of the triple test in the cases of the
uterine myomas, PIH, IUGR, and IUD.
OBJECTIVE: The purpose of this study was to determine the association between
abnormal second trimester Down syndrome screening markers and adverse pregcy
outcome.
MATERIAL AND METHODS: A total of 775 pregt women underwent maternal serum
screening. Pregcy complications were studied in the groups of pregcies
with structurally and chromosomally normal fetuses--with: elevated AFP >
1,89MoM, elevated beta-hCG > 1,69MoM or low beta-hCG < 0,48MoM.
RESULTS: Increased maternal serum AFP > 1,89MoM were found to be significantly
associated with IUGR, PIH and placental pathology. Increased beta-hCG > 1,69MoM
were significantly associated with PIH and IUGR. Finally decreased beta-hCG <
0,48MoM were found to be significantly associated with IUGR, PIH and IUD.
CONCLUSION: Triple test can be used not only for the detection of fetal
chromosomal and NTD abnormalities but also for the detection of high-risk
pregcies. AIM: The purpose of this article was to evaluate the reliability of maternal
serum triple marker screening of alpha-fetoprotein, human chorionic
gonadotropin, and unconjugated estriol for the prenatal diagnosis of fetal
chromosomal abnormalities in Turkish pregt women.
METHOD: Medical records were used to analyze indications of amniocentesis and
quantitative fluorescent-polymerase chain reaction. Anomaly screening was
performed for all patients between 13 and 22 weeks of pregcy. A total of 1725
pregcies with chromosomal abnormality risk according to triple test screening
were accepted for fetal chromosome analysis and quantitative
fluorescent-polymerase chain reaction.
RESULTS: Chromosomal aberrations were observed in 56 (3.2%) cases. About 44.6%
of the abnormalities detected were numerical aberrations; however, 55.3% of the
abnormalities were structural aberrations. Abnormalities detected were inversion
of chromosome 9 in 20 cases, trisomy 21 in 14 cases, 46,XX/47,XX, +21 in 1 case,
trisomy 18 in 2 cases, trisomy 13 in 1 case, 47,XXY, in 1 case, 45,X, in 1 case,
structural abnormalities in 12 cases, and mosaic or tetraploidy in 6 cases.
CONCLUSION: Second trimester triple test is an effective screening tool for
detecting fetal Down syndrome in Turkish women. BACKGROUND: The incidence of Down syndrome (DS) in Egypt varies between 1:555
and 1:770 and its screening by triple test is becoming increasingly popular
nowadays. Results, however, seem inaccurate due to the lack of Egyptian-specific
information needed for risk calculation and a clear policy for programme
implementation. Our study aimed at calculation and validation of the triple
marker medians used in screening Egyptian females as well as to recommend
programme conventions to unify screening in this country.
METHODS: The study was conducted on 668 Egyptian women, in weeks 15-20 of
pregcy as proven by sonar. Chorionic gonadotropin (CG), α-fetoprotein (AFP)
and unconjugated oestriol (uE3) were measured on Siemens Immulite analyzer.
Medians of the three parameters were calculated, regressed against gestational
age (GA) and weighted by the number of participants/week. Equations were derived
to adjust each parameter to the maternal weight and were centered on the median
Egyptian weight. Prisca software was fed with the above data,
multiples-of-median (MoM) and DS risks were calculated and the screening
performance was evaluated at a mid-trimester risk cutoff of 1:270.
RESULTS: Log-linear [AFP/uE3 = 10(A+B*GA)] and exponential equations [CG = A*e
(B*GA)] were derived and the regressed medians were found to follow similar
patterns to other Asian and Western medians. Oestriol was always lowest (even
halved) while CG and AFP were intermediate. A linear reciprocal model best
fitted weight distribution among Egyptians and successfully adjusted each
parameter to a weight of 78.2 kg. Epidemiological monitoring of these
recommendations revealed satisfactory performance in terms of 6.7% initial
positive rate and 1.00 grand MoM.
CONCLUSIONS: Adoption of the above recommendations is hoped to pave the way to a
successful DS screening programme tailored to Egyptian peculiarities. |
What is the role of TAD protein domain? | TAD domain is a transcription activation domain found in transcription factors. | The amino terminus of the avian myelocytomatosis virus MC29 v-Myc oncoprotein
contains sequences that are essential for cellular transformation (S. Farina, et
al. J. Virol., 66: 2698-2708, 1992; S. Min and E. J. Taparowsky. Oncogene,
7:1531-1540, 1992) and for the ability to activate gene transcription (S. Min
and E. J. Taparowsky. Oncogene, 7:1531-1540, 1992). To investigate the molecular
interactions that mediate these v-Myc-associated activities, we performed
competition assays in which various regions of the v-Myc amino terminal
transcription activation domain (TAD) were examined for their ability to inhibit
transcription activation by v-Myc, VP16, and the myogenic regulatory factor
MyoD. Overexpression of these transcriptional activators also was used to
investigate whether Myc-interacting proteins were required for cellular
transformation and cell proliferation events. Our results demonstrate that at
least two distinct cellular activities interact with the v-Myc TAD and that it
is the synergism between these activities that is required for v-Myc to function
fully as a transcriptional activator. In addition, v-Myc activators squelch
VP16- and MyoD-dependent transcription activation, suggesting that the v-Myc TAD
interacts with a component of the general transcription machinery. In support of
this observation, we found that overexpression of the v-Myc TAD inhibits
ras-mediated cellular transformation as well as cell proliferation, underscoring
the critical role these amino terminal Myc-interacting factors play in
regulating the physiology of both normal and transformed cells. Basic helix-loop-helix (bHLH) transcription factors play diverse roles in
controlling many developmental events. Although a great deal is understood about
how bHLH factors activate gene transcription via E-box DNA consensus sequences,
studies of bHLH factor function in higher eukaryotes often have been hindered by
the presence of multiple family members. As a first step in developing a
simplified in vivo system to examine bHLH factor activities, we examined whether
the bHLH muscle regulatory factors MRF4 and MyoD function appropriately in
yeast. We show that Gal4-MRF4 fusion proteins, or native MRF4 proteins, activate
expression of an E-box HIS3 reporter gene whereas MyoD proteins remain inactive.
Deletion of the MRF4 transcription activation domain (TAD) or point mutations
that abolish MRF4 DNA interactions inhibit HIS3 expression. Substitution of the
MRF4 TAD with the Gal4 TAD also produces a functional protein, demonstrating
that these transcription activation domains are functionally equivalent in
yeast. Replacement of the MRF4 TAD with the related MyoD TAD, however, generates
an inactive protein, suggesting that some specificity exists between bHLH family
members. Using this experimental system, we also demonstrate that mammalian cDNA
libraries can be screened successfully for cDNAs encoding novel bHLH proteins
that interact with E-box targets. Thus, this in vivo yeast system provides a
novel approach to facilitate functional studies of bHLH factor regulation. In the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related
chemicals, the Ah receptor nuclear translocator (Arnt) forms a heterodimeric
complex with the ligand-bound Ah receptor, leading to recognition of
dioxin-responsive elements within the enhancer of the CYP1A1 gene and
transcription activation by an unknown mechanism. To understand the role of Arnt
in transcription activation by the Ah receptor-Arnt heterodimer, we performed a
deletion analysis of Arnt to locate domains that are directly involved in
transcription activation. We showed that the C-terminal 34 amino acids of Arnt
encode a transcription activation domain (TAD) that functions independently of
other sequences in the Ah receptor complex when attached to the heterologous
Gal4 DNA binding domain. Deletion of the C-terminal acidic-rich 14 amino acids
completely abolishes activity. Sequences important in Arnt TAD function were
independent of the glutamine-rich region which is an important structural
feature in the TAD of other transcription factors. The strength of the Arnt TAD
when compared with the strong TAD from the herpes simplex virus VP16 protein was
cell-type specific. Both the Arnt and VP16 TAD were equally strong in COS-1
cells, but the Arnt TAD had weak activity in an Arnt-deficient mouse hepatoma
cell line and was not needed for restoration of CYP1A1 activation. These results
imply that for CYP1A1 activation the Ah receptor provides the domit
activation function for the heterodimer in hepatoma cells. The potential of the
Arnt TAD to contribute to activation by the Ah receptor complex is likely
determined by availability or activity of cell-specific factors with which the
TAD interacts. Potent induction of the gene coding for human prointerleukin 1beta (il1b)
normally requires a far-upstream inducible enhancer in addition to a minimal
promoter located between positions -131 and +12. The transcription factor Spi-1
(also called PU.1) is necessary for expression and binds to the minimal
promoter, thus providing an essential transcription activation domain (TAD). In
contrast, infection by human cytomegalovirus (HCMV) can strongly activate il1b
via the expression of immediate early (IE) viral proteins and eliminates the
requirement for the upstream enhancer. Spi-1 has been circumstantially
implicated as a host factor in this process. We report here the molecular basis
for the direct involvement of Spi-1 in HCMV activation of il1b. Transfection of
Spi-1-deficient HeLa cells demonstrated both the requirement of Spi-1 for IE
activity and the need for a shorter promoter (-59 to +12) than that required in
the absence of IE proteins. Furthermore, in contrast to normal,
enhancer-dependent il1b expression, which absolutely requires both the Spi-1
winged helix-turn-helix (wHTH) DNA-binding domain and the majority of the Spi-1
TAD, il1b expression in the presence of IE proteins does not require the Spi-1
TAD, which plays a synergistic role. In addition, we demonstrate that a single
IE protein, IE2, is critical for the induction of il1b. Protein-protein
interaction experiments revealed that the wing motif within the Spi-1 wHTH
domain directly recruits IE2. In turn, IE2 physically associates with the Spi-1
wing and requires the integrity of at least one region of IE2. Functional
analysis demonstrates that both this region and a carboxy-terminal acidic TAD
are required for IE2 function. Therefore, we propose a protein-tethered
transactivation mechanism in which the il1b promoter-bound Spi-1 wHTH tethers
IE2, which provides a TAD, resulting in the transactivation of il1b. DBP, HLF and TEF comprise a distinct subfamily of mammalian bZIP proteins that
plays an important role in regulation of tissue-specific gene expression,
particularly in the liver. In this report we demonstrate that DBP contains a 38
amino acid TAD which is highly homologous to the HLF and TEF TADs that we have
delineated previously. Deletion of this domain completely abrogates
transcriptional activity of native DBP and GAL4-DBP fusion proteins. This domain
functions as a modular TAD that is a potent transcriptional activator when fused
to the GAL4 DBD. While DBP itself is a liver-specific transactivator, the DBP
TAD is active in a variety of cell types, indicating that liver-specific
activity is not an intrinsic property of the TAD and must be conferred by other
regions of the protein. Using GAL4-HLF fusion proteins, we further refine the
core TAD of PAR proteins to a region of 13 amino acids. Recently described
PAR-bZIP proteins from Drosophila and zebrafish also contain domains that share
strong homology with the TAD of mammalian PAR proteins, making this one of the
most highly evolutionarily conserved TADs identified to date. The c-Myc oncoprotein (Myc) is a DNA sequence-specific transcription factor that
regulates transcription of a wide variety of genes involved in the control of
cell growth, proliferation, differentiation, and apoptosis and its deregulated
expression is implicated in many types of human cancer. Myc has an N-terminal
transcription activation domain (TAD) that interacts with various coactivators
and a C-terminal basic-helix-loop-helix-leucine zipper (bHLHZip) domain required
for E box-specific DNA-binding and heterodimerization with its obligatory
bHLHZip protein partner Max. The analysis of the mechanisms by which the Myc:Max
complex regulates transcription at the molecular level in vitro has been
hampered by the difficulty in obtaining highly pure recombit Myc:Max
heterodimers that contain full-length Myc with its complete TAD domain and that
have sequence-specific DNA-binding activity. Here, we describe a simple method
to reconstitute recombit Myc:Max complexes from highly purified full-length
proteins expressed in Escherichia coli that are soluble and highly active in E
box-specific DNA-binding in vitro. The reconstituted Myc:Max complexes are
stable and lack Max:Max homodimers. This procedure should facilitate the
characterization of the DNA-binding and transcription activation functions of
full-length Myc:Max complexes in vitro and in particular the role of Myc
TAD-interacting cofactors and Myc:Max post-translational modifications. The aryl hydrocarbon receptor (AhR) is an intracellular receptor protein that
regulates gene transcription in response to both man-made and natural ligands. A
modular transactivaton domain (TAD) has been mapped to the 304 C-terminal amino
acids and consists of acidic, Q-rich, and P/S/T-rich subdomains. We have used
steady-state intrinsic tryptophan fluorescence and circular dichroism
spectroscopy to investigate the conformation of the acidic Q-rich region. The
results reveal that this region of the protein is structurally flexible but
adopts a more folded conformation in the presence of the natural osmolyte
trimethylamine N-oxide (TMAO) and the solvent trifluoroethanol (TFE). In
protein-protein interaction studies, the acidic Q-rich region bound to
components of the general transcription machinery [TATA-binding protein (TBP),
TAF4, and TAF6] as well as the coactivator proteins SRC-1a and TIF2. The binding
site for TBP mapped to the acidic subdomain, while SRC-1a bound preferentially
to the Q-rich sequence. Significantly, the binding of TBP was modulated by
induced folding of the TAD with TMAO. The results indicate that the AhR TAD
makes multiple interactions with the transcriptional machinery and protein
conformation plays a critical role in receptor function. Taken together, these
findings support a role for protein folding in AhR action and suggest possible
mechanisms of receptor-dependent gene activation. The proto-oncogene c-myc governs the expression of a number of genes targeting
cell growth and apoptosis, and its expression levels are distorted in many
cancer forms. The current investigation presents an analysis by proteolysis,
circular dichroism, fluorescence and Biacore of the folding and ligand-binding
properties of the N-terminal transactivation domain (TAD) in the c-Myc protein.
A c-Myc sub-region comprising residues 1-167 (Myc1-167) has been investigated
that includes the unstructured c-Myc transactivation domain (TAD, residues
1-143) together with a C-terminal segment, which appears to promote increased
folding. Myc1-167 is partly helical, binds both to the target proteins Myc
modulator-1 (MM-1) and TATA box-binding protein (TBP), and displays the
characteristics of a molten globule. Limited proteolysis divides Myc1-167 in two
halves, by cleaving in a predicted linker region between two hotspot mutation
regions: Myc box I (MBI) and Myc box II (MBII). The N-terminal half (Myc1-88) is
unfolded and does not alone bind to target proteins, whereas the C-terminal half
(Myc92-167) has a partly helical fold and specifically binds both MM-1 and TBP.
Although this might suggest a bipartite organization in the c-Myc TAD, none of
the N and C-terminal fragments bind target protein with as high affinity as the
entire Myc1-167, or display molten globule properties. Furthermore, merely
linking the MBI with the C-terminal region, in Myc38-167, is not sufficient to
achieve binding and folding properties as in Myc1-167. Thus, the entire N and
C-terminal regions of c-Myc TAD act in concert to achieve high specificity and
affinity to two structurally and functionally orthogonal target proteins, TBP
and MM-1, possibly through a mechanism involving molten globule formation. This
hints towards understanding how binding of a range of targets can be
accomplished to a single transactivation domain. Signal transducer and activator of transcription (Stat5) is a transcription
factor, which transduces extracellular cytokine and growth-factor signals to the
nuclei of mammalian cells. As a major mediator of prolactin action, it is
involved in the regulation of the development, function, and survival of mammary
epithelial cells. The carboxyl terminal of Stat5 encodes a transactivation
domain (TAD), which interacts with coactivators and is crucial for the
transcriptional induction of Stat5 target genes. To study the role of the Stat5
TAD in mediating Stat5 functions, a carboxy terminally truncated Stat5 variant
(Stat5Delta750) was directed for expression in the mammary glands of transgenic
mice by regulatory sequences of the beta-lactoglobulin (BLG) gene. Expression of
Stat5Delta750 in mammary tissue reduced the rates of cell proliferation at mid
and late pregcy. Subsequently, morphological signs of milk secretion upon
parturition were delayed. In double-transgenic mice, expression of Stat5Delta750
drastically decreased BLG/luciferase activity during lactation, but did not
affect the expression and secretion of the endogenous beta-casein or
alpha-lactalbumin into the milk. Expression of Stat5Delta750 also caused an
increase in the number of apoptotic cells during mammary involution by a factor
of 3 relative to control glands. Our data established a role for the Stat5 TAD
in mediating the effects of Stat5 on mammary development, regulation of milk
protein gene activity, and cell survival. The full effects of Stat5Delta750 may
be partially buffered by the expression of endogenous wild-type Stat5 and the
formation of truncated and wild-type heterodimers. The p53 protein exerts its tumor suppressive function mainly by acting as a
transcription activator. Two transactivation domains (TADs) located at the
amino-terminus of p53 are required for transcription activation, and the
activity of TADs is tightly regulated by post-translational modifications, such
as phosphorylation. We attempted to dissect the functions of the two TADs and
phosphorylation within the TADs by analyzing p53 target genes induced by
full-length p53 (FL-p53), N-terminally deleted p53 isoform lacking the first TAD
(Delta1stTAD) and p53 carrying point mutations at all serine residues within the
two TADs (TAD-S/A). By performing a comprehensive survey by employing microarray
expression analysis, the induction of target genes by FL-p53, Delta1stTAD and
TAD-S/A was analyzed. All p53s showed different target gene induction patterns,
suggesting the importance of the two TADs and phosphorylation within the TADs in
target gene induction. Although Delta1stTAD showed a marked decrease in the
ability to induce genes induced by FL-p53, Delta1stTAD induced many
apoptosis-related genes that were not induced by FL-p53, suggesting the roles of
these Delta1stTAD-induced genes in Delta1stTAD-dependent apoptosis.
Approximately 80% of genes induced by FL-p53 were not induced by TAD-S/A,
including 29 previously reported p53 target genes such as Hdm2 and Bax,
emphasizing the importance of phosphorylation within the TADs. These results
demonstrate the significance of the regulation and differential roles of the
N-terminal TADs in p53 transcriptional activity. p300/cyclic AMP-responsive element binding protein-binding protein (CBP) are
general coactivators for multiple transcription factors involved in various
cellular processes. Several highly conserved domains of p300/CBP serve as
interacting sites for transcription factors and regulatory proteins.
Particularly, the intrinsic histone acetyltransferase (HAT) activity and
transactivation domains (TAD) play essential roles for their coactivating
function. Autoacetylation of p300/CBP is commonly observed in cell-free HAT
assays and has been implicated in the regulation of their HAT activity. Here, we
show that six lysine-rich regions in several highly conserved functional domains
of p300 are targeted by p300HAT for acetylation in cell-free systems. We show
that p300 is susceptible to acetylation in cultured tumor cells and that its
acetylation status is affected by histone deacetylase inhibitor trichostatin A.
We further show that either treatment with deacetylase inhibitors or
coexpression of Gal4-p300HAT, which alone has no transactivation activity,
stimulates the activity of the COOH-terminal TAD of p300 (p300C-TAD). We have
defined the minimal p300C-TAD and show that it is sufficient to respond to
deacetylase inhibitors and is a substrate for p300HAT. Finally, we show that
acetylated p300 possesses enhanced ability to interact with p53. Taken together,
our data suggest that acetylation regulates p300C-TAD and that acetylation of
p300/CBP may contribute to the dynamic regulation of their complex formation
with various interacting partners. ERM, PEA3 and ETV1 belong to the PEA3 group of ETS transcription factors. They
are involved in many developmental processes and are transcriptional regulators
in metastasis. The PEA3 group members share an N-terminal transactivation domain
(TAD) whose activity is inhibited by a flanking domain named the negative
regulatory domain (NRD). The mechanism of this inhibition is still unknown. Here
we show that the NRD maps to residues 73 to 298 in ERM and contains three of the
five SUMO sites previously identified in the protein. We demonstrate that these
three SUMO sites are responsible for NRD's inhibitory function in the Gal4
system. Although the presence of the three sites is required to obtain maximal
inhibition, only one SUMO site is sufficient to repress transcription whatever
its localization within the NRD. We also show that NRD is a SUMO-dependent
repression domain that can act in cis and in trans to downregulate the powerful
TAD of the VP16 viral protein. In addition, we find that the SUMO sites outside
the NRD also play a role in the negative regulation of full-length ERM activity.
We thus postulate that each SUMO site in ERM may function as an inhibitory
motif. Molecular interactions between the tumor suppressor p53 and the transcriptional
coactivators CBP/p300 are critical for the regulation of p53 transactivation and
stability. The transactivation domain (TAD) of p53 binds directly to several
CBP/p300 domains (TAZ1, TAZ2, NCBD, and KIX). Here we map the interaction
between the p53 TAD and the CBP KIX domain using isothermal titration
calorimetry and NMR spectroscopy. KIX is a structural domain in CBP/p300 that
can simultaneously bind two polypeptide ligands, such as the activation domain
of MLL and the kinase-inducible activation domain (pKID) of CREB, using distinct
interaction surfaces. The p53 TAD consists of two subdomains (AD1 and AD2);
peptides corresponding to the isolated AD1 and AD2 subdomains interact with KIX
with relatively low affinity, but a longer peptide containing both subdomains
binds KIX tightly. In the context of the full-length p53 TAD, AD1 and AD2 bind
synergistically to KIX. Mapping of the chemical shift perturbations onto the
structure of KIX shows that isolated AD1 and AD2 peptides bind to both the MLL
and pKID sites. Spin-labeling experiments show that the complex of the
full-length p53 TAD with KIX is disordered, with the AD1 and AD2 subdomains each
interacting with both the MLL and pKID binding surfaces. Phosphorylation of the
p53 TAD at Thr18 or Ser20 increases the KIX binding affinity. The affinity is
further enhanced by simultaneous phosphorylation of Thr18 and Ser20, and the
specificity of the interaction is increased. The p53 TAD simultaneously occupies
the two distinct sites that have been identified on the CBP KIX domain and
efficiently competes for these sites with other known KIX-binding transcription
factors. Escherichia coli BL21 (DE3) is commonly used for the overproduction of fusion
proteins. Using this system, we recently reported the overproduction of
histidine-tagged mouse estrogen receptor (ER) alpha-ligand binding domain as an
intact 30 kD protein and its inhibitory effect on the growth of bacteria.
However, when GST-tagged mouse ERalpha transactivation domain (TAD) was
overproduced using this system, it showed no effect on the growth of bacteria
but was specifically degraded during its expression and purification. Here we
report the expression of 47 kD GST-tagged mouse ERalpha-TAD protein, which was
degraded partially and specifically into 46 and 43 kD fragments. This fusion
protein was further degraded into 37, 31, 29 and 26 kD fragments during its
purification by affinity chromatography. Such specific degradation of GST-tagged
mouse ERalpha-TAD during its overproduction in E. coli and purification
indicates the induction of specific protease and suggests the modification of
expression system. E proteins are a special class of basic helix-loop-helix (bHLH) proteins that
heterodimerize with many bHLH activators to regulate developmental decisions,
such as myogenesis and neurogenesis. Daughterless (Da) is the sole E protein in
Drosophila and is ubiquitously expressed. We have characterized two
transcription activation domains (TADs) in Da, called activation domain 1 (AD1)
and loop-helix (LH), and have evaluated their roles in promoting peripheral
neurogenesis. In this context, Da heterodimerizes with proneural proteins, such
as Scute (Sc), which is dynamically expressed and also contributes a TAD. We
found that either one of the Da TADs in the Da/Sc complex is sufficient to
promote neurogenesis, whereas the Sc TAD is incapable of doing so. Besides its
transcriptional activation role, the Da AD1 domain serves as an interaction
platform for E(spl) proteins, bHLH-Orange family repressors which antagonize
Da/Sc function. We show that the E(spl) Orange domain is needed for this
interaction and strongly contributes to the antiproneural activity of E(spl)
proteins. We present a mechanistic model on the interplay of these bHLH factors
in the context of neural fate assignment. The proteins belonging to the nuclear factor of activated T cells (NFAT) family
of transcription factors are expressed in several cell types and regulate genes
involved in differentiation, cell cycle and apoptosis. NFAT proteins share two
conserved domains, the NFAT-homology region (NHR) and a DNA-binding domain
(DBD). The N- and C-termini display two transactivation domains (TAD-N and
TAD-C) that have low sequence similarity. Due to the high sequence conservation
in the NHR and DBD, NFAT members have some overlapping roles in gene regulation.
However, several studies have shown distinct roles for NFAT proteins in the
regulation of cell death. The TAD-C shows low sequence similarity among NFAT
family members, but its contribution to specific NFAT1-induced phenotypes is
poorly understood. Here, we described at least two regions of NFAT1 TAD-C that
confer pro-apoptotic activity to NFAT1. These regions extend from amino acids
699 to 734 and 819 to 850 of NFAT1. We also showed that the NFAT1 TAD-C is
unable to induce apoptosis by itself and requires a functional DBD. Furthermore,
we showed that when fused to NFAT1 TAD-C, NFAT2, which is associated with cell
transformation, induces apoptosis in fibroblasts. Together, these results
suggest that the NFAT1 TAD-C includes NFAT death domains that confer to
different NFAT members the ability to induce apoptosis. NF-κB plays a vital role in cellular immune and inflammatory response, survival,
and proliferation by regulating the transcription of various genes involved in
these processes. To activate transcription, RelA (a prominent NF-κB family
member) interacts with transcriptional co-activators like CREB-binding protein
(CBP) and its paralog p300 in addition to its cognate κB sites on the
promoter/enhancer regions of DNA. The RelA:CBP/p300 complex is comprised of two
components--first, DNA binding domain of RelA interacts with the KIX domain of
CBP/p300, and second, the transcriptional activation domain (TAD) of RelA binds
to the TAZ1 domain of CBP/p300. A phosphorylation event of a well-conserved
RelA(Ser276) is prerequisite for the former interaction to occur and is
considered a decisive factor for the overall RelA:CBP/p300 interaction. The role
of the latter interaction in the transcription of RelA-activated genes remains
unclear. Here we provide the solution structure of the latter component of the
RelA:CBP complex by NMR spectroscopy. The structure reveals the folding of
RelA-TA2 (a section of TAD) upon binding to TAZ1 through its well-conserved
hydrophobic sites in a series of grooves on the TAZ1 surface. The structural
analysis coupled with the mechanistic studies by mutational and isothermal
calorimetric analyses allowed the design of RelA-mutants that selectively
abrogated the two distinct components of the RelA:CBP/p300 interaction. Detailed
studies of these RelA mutants using cell-based techniques, mathematical
modeling, and genome-wide gene expression analysis showed that a major set of
the RelA-activated genes, larger than previously believed, is affected by this
interaction. We further show how the RelA:CBP/p300 interaction controls the
nuclear response of NF-κB through the negative feedback loop of NF-κB pathway.
Additionally, chromatin analyses of RelA target gene promoters showed
constitutive recruitment of CBP/p300, thus indicating a possible role of
CBP/p300 in recruitment of RelA to its target promoter sites. Infection with the Epstein-Barr virus (EBV) can lead to a number of human
diseases including Hodgkin's and Burkitt's lymphomas. The development of these
EBV-linked diseases is associated with the presence of nine viral latent
proteins, including the nuclear antigen 2 (EBNA2). The EBNA2 protein plays a
crucial role in EBV infection through its ability to activate transcription of
both host and viral genes. As part of this function, EBNA2 associates with
several host transcriptional regulatory proteins, including the Tfb1/p62
(yeast/human) subunit of the general transcription factor IIH (TFIIH) and the
histone acetyltransferase CBP(CREB-binding protein)/p300, through interactions
with its C-terminal transactivation domain (TAD). In this manuscript, we examine
the interaction of the acidic TAD of EBNA2 (residues 431-487) with the Tfb1/p62
subunit of TFIIH and CBP/p300 using nuclear magnetic resoce (NMR)
spectroscopy, isothermal titration calorimeter (ITC) and transactivation studies
in yeast. NMR studies show that the TAD of EBNA2 binds to the pleckstrin
homology (PH) domain of Tfb1 (Tfb1PH) and that residues 448-471 (EBNA2₄₄₈₋₄₇₁)
are necessary and sufficient for this interaction. NMR structural
characterization of a Tfb1PH-EBNA2₄₄₈₋₄₇₁ complex demonstrates that the
intrinsically disordered TAD of EBNA2 forms a 9-residue α-helix in complex with
Tfb1PH. Within this helix, three hydrophobic amino acids (Trp458, Ile461 and
Phe462) make a series of important interactions with Tfb1PH and their importance
is validated in ITC and transactivation studies using mutants of EBNA2. In
addition, NMR studies indicate that the same region of EBNA2 is also required
for binding to the KIX domain of CBP/p300. This study provides an atomic level
description of interactions involving the TAD of EBNA2 with target host
proteins. In addition, comparison of the Tfb1PH-EBNA2₄₄₈₋₄₇₁ complex with
structures of the TAD of p53 and VP16 bound to Tfb1PH highlights the versatility
of intrinsically disordered acidic TADs in recognizing common target host
proteins. The molecular circadian clock in mammals is generated from transcriptional
activation by the bHLH-PAS transcription factor CLOCK-BMAL1 and subsequent
repression by PERIOD and CRYPTOCHROME (CRY). The mechanism by which CRYs repress
CLOCK-BMAL1 to close the negative feedback loop and generate 24-h timing is not
known. Here we show that, in mouse fibroblasts, CRY1 competes for binding with
coactivators to the intrinsically unstructured C-terminal transactivation domain
(TAD) of BMAL1 to establish a functional switch between activation and
repression of CLOCK-BMAL1. TAD mutations that alter affinities for co-regulators
affect the balance of repression and activation to consequently change the
intrinsic circadian period or eliminate cycling altogether. Our results suggest
that CRY1 fulfills its role as an essential circadian repressor by sequestering
the TAD from coactivators, and they highlight regulation of the BMAL1 TAD as a
critical mechanism for establishing circadian timing. Posttranslational modifications have critical roles in diverse biological
processes through interactions. Tumor-suppressor protein p53 and nucleotide
excision repair factor XPC each contain an acidic region, termed the acidic
transactivation domain (TAD) and acidic fragment (AF), respectively, that binds
to the pleckstrin homology (PH) domain of the p62 subunit of the transcription
factor TFIIH. Human p53-TAD contains seven serine and two threonine residues,
all of which can be phosphorylated. Similarly, XPC-AF contains six serine and
two threonine residues, of which Thr117, Ser122 and Ser129 have been reported as
phosphorylation sites in vivo, although their phosphorylation roles are unknown.
Phosphorylation of Ser46 and Thr55 of p53-TAD increases its binding ability;
however, the role of XPC-AF phosphorylation remains elusive. Here we describe a
system for real-time and simultaneous monitoring of the phosphorylation and
p62-PH affinity of p53-TAD and XPC-AF using nuclear magnetic resoce (NMR)
spectroscopy. Unexpectedly, among seven reported kinases that presumably
phosphorylate Ser46 and/or Thr55 of p53-TAD, only two specific and
high-efficiency enzymes were identified: JNK2α2 for Ser46 and GRK5 for Thr55.
During interaction with p62-PH, four different affinity complexes resulting from
various phosphorylation states of p53-TAD by the kinases were identified. The
kinetics of the site-specific phosphorylation reaction of p53-TAD and its
affinity for p62-PH were monitored in real-time using the NMR system. Isothermic
calorimetry showed that phosphorylation of Ser129 of XPC-AF increases binding to
p62-PH. Although CK2 was predicted to phosphorylate Ser122, Ser129 and Ser140
from its sequence context, it specifically and efficiently phosphorylated only
Ser129. Simultaneous monitoring of the phosphorylation and augmentation in
p62-PH binding identified a key residue of p62-PH for contacting phosphorylated
Ser129. In summary, we have established an NMR system for real-time and
simultaneous monitoring of site-specific phosphorylation and enhancement of
affinity between phosphorylation domains and their target. The system is also
applicable to other posttranslational modifications. The NF-κB transcription factor family plays a central role in innate immunity
and inflammation processes and is frequently dysregulated in cancer. We
developed an NF-κB functional assay in yeast to investigate the following
issues: transactivation specificity of NF-κB proteins acting as homodimers or
heterodimers; correlation between transactivation capacity and in vitro DNA
binding measurements; impact of co-expressed interacting proteins or of small
molecule inhibitors on NF-κB-dependent transactivation. Full-length p65 and p50
cDNAs were cloned into centromeric expression vectors under inducible GAL1
promoter in order to vary their expression levels. Since p50 lacks a
transactivation domain (TAD), a chimeric construct containing the TAD derived
from p65 was also generated (p50TAD) to address its binding and transactivation
potential. The p50TAD and p65 had distinct transactivation specificities towards
seventeen different κB response elements (κB-REs) where single nucleotide
changes could greatly impact transactivation. For four κB-REs, results in yeast
were predictive of transactivation potential measured in the human MCF7 cell
lines treated with the NF-κB activator TNFα. Transactivation results in yeast
correlated only partially with in vitro measured DNA binding affinities,
suggesting that features other than strength of interaction with naked DNA
affect transactivation, although factors such as chromatin context are kept
constant in our isogenic yeast assay. The small molecules BAY11-7082 and
ethyl-pyruvate as well as expressed IkBα protein acted as NF-κB inhibitors in
yeast, more strongly towards p65. Thus, the yeast-based system can recapitulate
NF-κB features found in human cells, thereby providing opportunities to address
various NF-κB functions, interactions and chemical modulators. The immediate early 62 protein (IE62) of varicella-zoster virus (VZV), a major
viral trans-activator, initiates the virus life cycle and is a key component of
pathogenesis. The IE62 possesses several domains essential for trans-activation,
including an acidic trans-activation domain (TAD), a serine-rich tract (SRT),
and binding domains for USF, TFIIB, and TATA box binding protein (TBP).
Transient-transfection assays showed that the VZV IE62 lacking the SRT
trans-activated the early VZV ORF61 promoter at only 16% of the level of the
full-length IE62. When the SRT of IE62 was replaced with the SRT of equine
herpesvirus 1 (EHV-1) IEP, its trans-activation activity was completely
restored. Herpes simplex virus 1 (HSV-1) ICP4 that lacks a TAD very weakly
(1.5-fold) trans-activated the ORF61 promoter. An IE62 TAD-ICP4 chimeric protein
exhibited trans-activation ability (10.2-fold), indicating that the IE62 TAD
functions with the SRT of HSV-1 ICP4 to trans-activate viral promoters. When the
serine and acidic residues of the SRT were replaced with Ala, Leu, and Gly,
trans-activation activities of the modified IE62 proteins IE62-SRTΔSe and
IE62-SRTΔAc were reduced to 46% and 29% of wild-type activity, respectively.
Bimolecular complementation assays showed that the TAD of IE62, EHV-1 IEP, and
HSV-1 VP16 interacted with Mediator 25 in human melanoma MeWo cells. The SRT of
IE62 interacted with the nucleolar-ribosomal protein EAP, which resulted in the
formation of globular structures within the nucleus. These results suggest that
the SRT plays an important role in VZV viral gene expression and replication.
IMPORTANCE: The immediate early 62 protein (IE62) of varicella-zoster virus
(VZV) is a major viral trans-activator and is essential for viral growth. Our
data show that the serine-rich tract (SRT) of VZV IE62, which is well conserved
within the alphaherpesviruses, is needed for trans-activation mediated by the
acidic trans-activation domain (TAD). The TADs of IE62, EHV-1 IEP, and HSV-1
VP16 interacted with cellular Mediator 25 in bimolecular complementation assays.
The interaction of the IE62 SRT with nucleolar-ribosomal protein EAP resulted in
the formation of globular structures within the nucleus. Understanding the
mechanisms by which the TAD and SRT of IE62 contribute to the function of this
essential regulatory protein is important in understanding the gene program of
this human pathogen. Sox9 plays an important role in a large variety of developmental pathways in
vertebrates. It is composed of three domains: high-mobility group box (HMG box),
dimerization (DIM) and transactivation (TAD). One of the main processes for
regulation and variability of the pathways involving Sox9 is the self-gene
expression regulation of Sox9. However, the subsequent roles of the Sox9 domains
can also generate regulatory modulations. Studies have shown that TADs can bind
to different types of proteins and its function seems to be influenced by DIM.
Therefore, we hypothesized that both domains are directly associated and can be
responsible for the functional variability of Sox9. We applied a method based on
a broad phylogenetic context, using sequences of the HMG box domain, to ensure
the homology of all the Sox9 copies used herein. The data obtained included
4,921 sequences relative to 657 metazoan species. Based on coevolutionary and
selective pressure analyses of the Sox9 sequences, we observed coevolutions
involving DIM and TADs. These data, along with the experimental data from
literature, indicate a functional relationship between these domains. Moreover,
DIM and TADs may be responsible for the functional plasticity of Sox9 because
they are more tolerant for molecular changes (higher Ka/Ks ratio than the HMG
box domain). This tolerance could allow a differential regulation of target
genes or promote novel targets during transcriptional activation. In conclusion,
we suggest that DIM and TADs functional association may regulate differentially
the target genes or even promote novel targets during transcription activation
mediated by Sox9 paralogs, contributing to the subfunctionalization of Sox9a and
Sox9b in teleosts. ERK5, the last MAP kinase family member discovered, is activated by the upstream
kinase MEK5 in response to growth factors and stress stimulation. MEK5-ERK5
pathway has been associated to different cellular processes, playing a crucial
role in cell proliferation in normal and cancer cells by mechanisms that are
both dependent and independent of its kinase activity. Thus, nuclear ERK5
activates transcription factors by either direct phosphorylation or acting as
co-activator thanks to a unique transcriptional activation TAD domain located at
its C-terminal tail. Consequently, ERK5 has been proposed as an interesting
target to tackle different cancers, and either inhibitors of ERK5 activity or
silencing the protein have shown antiproliferative activity in cancer cells and
to block tumor growth in animal models. Here, we review the different mechanisms
involved in ERK5 nuclear translocation and their consequences. Inactive ERK5
resides in the cytosol, forming a complex with Hsp90-Cdc37 superchaperone. In a
canonical mechanism, MEK5-dependent activation results in ERK5 C-terminal
autophosphorylation, Hsp90 dissociation, and nuclear translocation. This
mechanism integrates signals such as growth factors and stresses that activate
the MEK5-ERK5 pathway. Importantly, two other mechanisms, MEK5-independent, have
been recently described. These mechanisms allow nuclear shuttling of
kinase-inactive forms of ERK5. Although lacking kinase activity, these forms
activate transcription by interacting with transcription factors through the TAD
domain. Both mechanisms also require Hsp90 dissociation previous to nuclear
translocation. One mechanism involves phosphorylation of the C-terminal tail of
ERK5 by kinases that are activated during mitosis, such as Cyclin-dependent
kinase-1. The second mechanism involves overexpression of chaperone Cdc37, an
oncogene that is overexpressed in cancers such as prostate adenocarcinoma, where
it collaborates with ERK5 to promote cell proliferation. Although some ERK5
kinase inhibitors have shown antiproliferative activity it is likely that those
tumors expressing kinase-inactive nuclear ERK5 will not respond to these
inhibitors. |
What is the function of lncRNA? | Long noncoding RNAs (lncRNAs) are involved in a variety of biological processes, including the epigenetic control of gene expression, post-transcriptional regulation of mRNA, and cellular proliferation and differentiation | BACKGROUND: Though most of the transcripts are long non-coding RNAs (lncRNAs),
little is known about their functions. lncRNAs usually function through
interactions with proteins, which implies the importance of identifying the
binding proteins of lncRNAs in understanding the molecular mechanisms underlying
the functions of lncRNAs. Only a few approaches are available for predicting
interactions between lncRNAs and proteins. In this study, we introduce a new
method lncPro.
RESULTS: By encoding RNA and protein sequences into numeric vectors, we used
matrix multiplication to score each RNA-protein pair. This score can be used to
measure the interactions between an RNA-protein pair. This method effectively
discriminates interacting and non-interacting RNA-protein pairs and predicts
RNA-protein interactions within a given complex. Applying this method on all
human proteins, we found that the long non-coding RNAs we collected tend to
interact with nuclear proteins and RNA-binding proteins.
CONCLUSIONS: Compared with the existing approaches, our method shortens the time
for training matrix and obtains optimal results based on the model being used.
The ability of predicting the associations between lncRNAs and proteins has also
been enhanced. Our method provides an idea on how to integrate different
information into the prediction process. Long non-coding RNAs (lncRNAs) are increasingly being recognized as epigenetic
regulators of gene transcription. The diversity and complexity of lncRNA genes
means that they exert their regulatory effects by a variety of mechanisms.
Although there is still much to be learned about the mechanism of lncRNA
function, general principles are starting to emerge. In particular, the
application of high throughput (deep) sequencing methodologies has greatly
advanced our understanding of lncRNA gene function. lncRNAs function as adaptors
that link specific chromatin loci with chromatin-remodeling complexes and
transcription factors. lncRNAs can act in cis or trans to guide
epigenetic-modifier complexes to distinct genomic sites, or act as scaffolds
which recruit multiple proteins simultaneously, thereby coordinating their
activities. In this review we discuss the genomic organization of lncRNAs, the
importance of RNA secondary structure to lncRNA functionality, the multitude of
ways in which they interact with the genome, and what evolutionary conservation
tells us about their function. Long non-coding RNAs (lncRNAs) were recently shown to regulate chromatin
remodelling activities. Their function in regulating gene expression switching
during specific developmental stages is poorly understood. Here we describe a
nuclear, non-coding transcript responsive for the stage-specific activation of
the chicken adult α(D) globin gene. This non-coding transcript, named α-globin
transcript long non-coding RNA (lncRNA-αGT) is transcriptionally upregulated in
late stages of chicken development, when active chromatin marks the adult α(D)
gene promoter. Accordingly, the lncRNA-αGT promoter drives erythroid-specific
transcription. Furthermore, loss of function experiments showed that lncRNA-αGT
is required for full activation of the α(D) adult gene and maintece of
transcriptionally active chromatin. These findings uncovered lncRNA-αGT as an
important part of the switching from embryonic to adult α-globin gene
expression, and suggest a function of lncRNA-αGT in contributing to the
maintece of adult α-globin gene expression by promoting an active chromatin
structure. Long noncoding RNAs (lncRNAs) are emerging as a novel class of noncoding RNAs
and potent gene regulators, which play an important and varied role in cellular
functions. lncRNAs are closely related with the occurrence and development of
some diseases. High-throughput RNA-sequencing techniques combined with de novo
assembly have identified a large number of novel transcripts. The discovery of
large and 'hidden' transcriptomes urgently requires the development of effective
computational methods that can rapidly distinguish between coding and long
noncoding RNAs. In this study, we developed a powerful predictor (named as
lncRNA-MFDL) to identify lncRNAs by fusing multiple features of the open reading
frame, k-mer, the secondary structure and the most-like coding domain sequence
and using deep learning classification algorithms. Using the same human training
dataset and a 10-fold cross validation test, lncRNA-MFDL can achieve 97.1%
prediction accuracy which is 5.7, 3.7, and 3.4% higher than that of CPC, CNCI
and lncRNA-FMFSVM predictors, respectively. Compared with CPC and CNCI
predictors in other species (e.g., anole lizard, zebrafish, chicken, gorilla,
macaque, mouse, lamprey, orangutan, xenopus and C. elegans) testing datasets,
the new lncRNA-MFDL predictor is also much more effective and robust. These
results show that lncRNA-MFDL is a powerful tool for identifying lncRNAs. The
lncRNA-MFDL software package is freely available at for academic users. A large part of the mammalian genome is transcribed into noncoding RNAs. Long
noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators of gene
expression. Distinct molecular mechanisms allow lncRNAs either to activate or to
repress gene expression, thereby participating in the regulation of cellular and
tissue function. LncRNAs, therefore, have important roles in healthy and
diseased hearts, and might be targets for therapeutic intervention. In this
Review, we summarize the current knowledge of the roles of lncRNAs in cardiac
development and ageing. After describing the definition and classification of
lncRNAs, we present an overview of the mechanisms by which lncRNAs regulate gene
expression. We discuss the multiple roles of lncRNAs in the heart, and focus on
the regulation of embryonic stem cell differentiation, cardiac cell fate and
development, and cardiac ageing. We emphasize the importance of chromatin
remodelling in this regulation. Finally, we discuss the therapeutic and
biomarker potential of lncRNAs. OBJECTIVE: Long non-coding RNAs (lncRNAs) are emerging as key molecules in
cancers, yet their potential molecular mechanisms are not well understood. The
objective of this study is to examine the expression and functions of lncRNAs in
the development of colorectal cancer (CRC).
METHODS: LncRNA expression profiling of CRC, adenoma and normal colorectal
tissues was performed to identify tumour-related lncRNAs involved in colorectal
maligt transformation. Then, we used quantitative reverse transcription PCR
assays to measure the tumour-related lncRNA and to assess its association with
survival and response to adjuvant chemotherapy in 252 patients with CRC. The
mechanisms of CCAL function and regulation in CRC were examined using molecular
biological methods.
RESULTS: We identified colorectal cancer-associated lncRNA (CCAL) as a key
regulator of CRC progression. Patients whose tumours had high CCAL expression
had a shorter overall survival and a worse response to adjuvant chemotherapy
than patients whose tumours had low CCAL expression. CCAL promoted CRC
progression by targeting activator protein 2α (AP-2α), which in turn activated
Wnt/β-catenin pathway. CCAL induced multidrug resistance (MDR) through
activating Wnt/β-catenin signalling by suppressing AP-2α and further
upregulating MDR1/P-gp expression. In addition, we found that histone H3
methylation and deacetylases contributed to the upregulation of CCAL in CRC.
CONCLUSIONS: Our results suggest that CCAL is a crucial oncogenic regulator
involved in CRC tumorigenesis and progression. The heritability of schizophrenia has been reported to be as high as ~80%, but
the contribution of genetic variants identified to this heritability remains to
be estimated. Long non-coding RNAs (LncRNAs) are involved in multiple processes
critical to normal cellular function and dysfunction of lncRNA MIAT may
contribute to the pathophysiology of schizophrenia. However, the genetic
evidence of lncRNAs involved in schizophrenia has not been documented. Here, we
conducted a two-stage association analysis on 8 tag SNPs that cover the whole
MIAT locus in two independent Han Chinese schizophrenia case-control cohorts
(discovery sample from Shanxi Province: 1093 patients with paranoid
schizophrenia and 1180 control subjects; replication cohort from Jilin Province:
1255 cases and 1209 healthy controls). In discovery stage, significant genetic
association with paranoid schizophrenia was observed for rs1894720 (χ(2)=74.20,
P=7.1E-18), of which minor allele (T) had an OR of 1.70 (95% CI=1.50-1.91). This
association was confirmed in the replication cohort (χ(2)=22.66, P=1.9E-06,
OR=1.32, 95%CI 1.18-1.49). Besides, a weak genotypic association was detected
for rs4274 (χ(2)=4.96, df=2, P=0.03); the AA carriers showed increased disease
risk (OR=1.30, 95%CI=1.03-1.64). No significant association was found between
any haplotype and paranoid schizophrenia. The present studies showed that lncRNA
MIAT was a novel susceptibility gene for paranoid schizophrenia in the Chinese
Han population. Considering that most lncRNAs locate in non-coding regions, our
result may explain why most susceptibility loci for schizophrenia identified by
genome wide association studies were out of coding regions. The functions of long noncoding RNAs (lncRNAs) have mainly been studied using
cultured cell lines, and this approach has revealed the involvement of lncRNAs
in a variety of biological processes, including the epigenetic control of gene
expression, post-transcriptional regulation of mRNA, and cellular proliferation
and differentiation. Recently, increasing numbers of studies have investigated
the functions of lncRNAs using gene-targeted model mice, largely confirming the
physiological importance of lncRNA-mediated regulation in individual animals. In
some cases, however, the results obtained by studies using knockout mice have
been somewhat inconsistent with those of the preceding cell-based analyses. In
this review, I will summarize the lessons that we are learning from the
reverse-genetic studies of lncRNAs, namely the importance of noncoding DNA
elements, the weak correlation between expression level and phenotypic
prominence, the existence of tissue- and condition-specific phenotypes and
incomplete penetrance, and the function of lncRNAs as precursor molecules. This
article is part of a Special Issue entitled: Clues to long noncoding RNA
taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa. Over the last decade, long noncoding RNAs (lncRNAs) have emerged as a
fundamental molecular class whose members play pivotal roles in the regulation
of the genome. The observation of pervasive transcription of mammalian genomes
in the early 2000s sparked a revolution in the understanding of information flow
in eukaryotic cells and the incredible flexibility and dynamic nature of the
transcriptome. As a molecular class, distinct loci yielding lncRNAs are set to
outnumber those yielding mRNAs. However, like many important discoveries, the
road leading to uncovering this diverse class of molecules that act through a
remarkable repertoire of mechanisms, was not a straight one. The same
characteristic that most distinguishes lncRNAs from mRNAs, i.e. their
developmental-stage, tissue-, and cell-specific expression, was one of the major
impediments to their discovery and recognition as potentially functional
regulatory molecules. With growing numbers of lncRNAs being assigned to
biological functions, the specificity of lncRNA expression is now increasingly
recognized as a characteristic that imbues lncRNAs with great potential as
biomarkers and for the development of highly targeted therapeutics. Here we
review the history of lncRNA research and how technological advances and insight
into biological complexity have gone hand-in-hand in shaping this revolution. We
anticipate that as increasing numbers of these molecules, often described as the
dark matter of the genome, are characterized and the structure-function
relationship of lncRNAs becomes better understood, it may ultimately be feasible
to decipher what these non-(protein)-coding genes encode. This article is part
of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by
Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa. Benefiting from the fast development of sequencing technique and bioinformatics
methods, more and more new long non-coding RNAs (lncRNAs) are discovered and
identified. lncRNAs were firstly thought to be transcription noise that from
genome desert without biological function; however, as the discovery of lncRNA
XIST and HOTAIR uncovers the emerging roles of lncRNAs in development and
tumorigenesis. In the past decades, accumulating evidence have indicated that
lncRNAs involve in a wide range of biological functions, such as X-chromosome
inactivation, reprogramming stem cell pluripotency, regulation of the immune
response and carcinogenesis. Although lots of studies have demonstrated that
dysregulation of lncRNAs involve in diverse diseases including cancers, the
underlying molecular mechanisms of lncRNAs are not well documented.
Interestingly, our previous studies and others' have shown that numerous of
lncRNAs expression was misregulated in gastric cancer. In this review, we will
focus on the dysregulated lncRNAs and their biological function and underlying
pathways or mechanisms in GC. Finally, we will discuss the potential roles of
lncRNAs acting as biomarkers or therapeutic targets in GC patients. Protection against infection and maintece of homeostasis are the hallmarks of
the innate immune system. The complex signaling cascades that occur following
microbial infection have been studied intensely for a number of years and long
noncoding RNA (lncRNA) represent novel regulatory components of these pathways.
The catalogue of lncRNA present in our genomes continues to increase as deep
sequencing data becomes available. It is clear that they represent critical
regulatory steps in a large number of biological systems yet we currently
understand the functions for approximately 1% of all annotated lncRNA. This
review will cover the recent findings on the emerging roles for lncRNA in
controlling the inflammatory response and their mechanisms of action. Gaining a
better understanding of these processes could facilitate the development of
novel therapeutics to prevent damaging inflammation. Despite the breadth of knowledge that exists regarding the function of long
noncoding RNAs (lncRNAs) in biological phenomena, the role of lncRNAs in host
antiviral responses is poorly understood. Here, we report that lncRNA#32 is
associated with type I IFN signaling. The silencing of lncRNA#32 dramatically
reduced the level of IFN-stimulated gene (ISG) expression, resulting in
sensitivity to encephalomyocarditis virus (EMCV) infection. In contrast, the
ectopic expression of lncRNA#32 significantly suppressed EMCV replication,
suggesting that lncRNA#32 positively regulates the host antiviral response. We
further demonstrated the suppressive function of lncRNA#32 in hepatitis B virus
and hepatitis C virus infection. lncRNA#32 bound to activating transcription
factor 2 (ATF2) and regulated ISG expression. Our results reveal a role for
lncRNA#32 in host antiviral responses. Head and neck cancers (HNCs) include a series of maligt tumors arising in
epithelial tissues, typically oral cancer, laryngeal cancer, nasopharynx cancer
and thyroid cancer. HNCs are important contributors to cancer incidence and
mortality, leading to approximately 225,100 new patients and 77,500 deaths in
China every year. Determination of the mechanisms of HNC carcinogenesis and
progression is an urgent priority in HNC treatment. Long noncoding RNAs
(lncRNAs) are noncoding RNAs longer than 200 bps. lncRNAs have been reported to
participate in a broad scope of biological processes, and lncRNA dysregulation
leads to diverse human diseases, including cancer. In this review, we focus on
lncRNAs that are dysregulated in HNCs, summarize the latest findings regarding
the function and molecular mechanisms of lncRNAs in HNC carcinogenesis and
progression, and discuss the clinical application of lncRNAs in HNC diagnosis,
prognosis and therapy. |
What is the function of the exosome? | Exosomes are 40-100-nm vesicles released by most cell types after fusion of multivesicular endosomes with the plasma membrane. Exosomes contain proteins and RNA species and can mediate communication and immune responses. | Exosomes are small membrane vesicles originating from late endosomes and
secreted by hematopoietic and epithelial cells in culture. Exosome proteic and
lipid composition is unique and might shed some light into exosome biogenesis
and function. Exosomes secreted from professional antigen-presenting cells
(i.e., B lymphocytes and dendritic cells) are enriched in MHC class I and II
complexes, costimulatory molecules, and hsp70-90 chaperones, and have therefore
been more extensively studied for their immunomodulatory capacities in vitro and
in vivo. This review will present the main biological features pertaining to
tumor or DC-derived exosomes, will emphasize their immunostimulatory function,
and will discuss their implementation in cancer immunotherapy. The identification and characterization of the exosome complex has shown that
the exosome is a complex of 3' --> 5' exoribonucleases that plays a key role in
the processing and degradation of a wide variety of RNA substrates. Advances in
the understanding of exosome function have led to the identification of numerous
cofactors that are required for a selective recruitment of the exosome to
substrate RNAs, for their structural alterations to facilitate degradation, and
to aid in their complete degradation/processing. Structural data obtained by
two-hybrid interaction analyses and X-ray crystallography show that the core of
the exosome adopts a doughnut-like structure and demonstrates that probably not
all exosome subunits are active exoribonucleases. Despite all data obtained on
the structure and function of the exosome during the last decade, there are
still a lot of uswered questions. What is the molecular mechanism by which
cofactors select and target substrate RNAs to the exosome and modulate its
function for correct processing or degradation? How can the exosome discriminate
between processing or degradation of a specific substrate RNA? What is the
precise structure of exosome subunits and how do they contribute to its
function? Here we discuss studies that provide some insight to these questions
and speculate on the mechanisms that control the exosome. Exosomes are ometer-sized vesicles, secreted from most cell types, with
documented immune-modulatory functions. Exosomes can be purified from cultured
cells but to do so effectively, requires maintece of cells at high density in
order to obtain sufficient accumulation of exosomes in the culture medium, prior
to purification. Whilst high density cultures can be achieved with cells in
suspension, this remains difficult with adherent cells, resulting in low
quantity of exosomes for subsequent study. We have used the Integra CELLine
culture system, originally designed for hybridoma cultures, to achieve a
significant increase in obtainable exosomes from adherent and non-adherent
tumour cells. Traditional cultures of mesothelioma cells (cultured in 75 cm(2)
flasks) gave an average yield of 0.78 microg+/-0.14 microg exosome/ml of
conditioned medium. The CELLine Adhere 1000 (CLAD1000) flask, housing the same
cell line, increased exosome yield approximately 12 fold to 10.06 microg+/-0.97
microg/ml. The morphology, phenotype and immune function of these exosomes were
compared, and found to be identical in all respects. Similarly an 8 fold
increase in exosome production was obtained from NKL cells (a suspension cell
line) using a CELLine 1000 (CL1000) flask. The CELLine system also incurred ~5.5
fold less cost and reduced labour for cell maintece. This simple culture
system is a cost effective, useful method for significantly increasing the
quantity of exosomes available from cultured cells, without detrimental effects.
This tool should prove advantageous in future studies of exosome-immune
modulation in cancer and other settings. NKG2D is an activating receptor for NK, NKT, CD8(+), and gammadelta(+) T cells,
whose aberrant loss in cancer is a key mechanism of immune evasion. Soluble
NKG2D ligands and growth factors, such as TGFbeta1 emanating from tumors, are
mechanisms for down-regulating NKG2D expression. Cancers thereby impair the
capacity of lymphocytes to recognize and destroy them. In this study, we show
that exosomes derived from cancer cells express ligands for NKG2D and express
TGFbeta1, and we investigate the impact of such exosomes on CD8(+) T and NK cell
NKG2D expression and on NKG2D-dependent functions. Exosomes produced by various
cancer cell lines in vitro, or isolated from pleural effusions of mesothelioma
patients triggered down-regulation of surface NKG2D expression by NK cells and
CD8(+) T cells. This decrease was rapid, sustained, and resulted from direct
interactions between exosomes and NK cells or CD8(+) T cells. Other markers
(CD4, CD8, CD56, CD16, CD94, or CD69) remained unchanged, indicating the
selectivity and nonactivatory nature of the response. Exosomal NKG2D ligands
were partially responsible for this effect, as down-modulation of NKG2D was
slightly attenuated in the presence of MICA-specific Ab. In contrast,
TGFbeta1-neutralizing Ab strongly abrogated NKG2D down-modulation, suggesting
exosomally expressed TGFbeta as the principal mechanism. Lymphocyte effector
function was impaired by pretreatment with tumor exosomes, as these cells
exhibited poor NKG2D-dependent production of IFN-gamma and poor NKG2D-dependent
killing function. This hyporesponsiveness was evident even in the presence of
IL-15, a strong inducer of NKG2D. Our data show that NKG2D is a likely
physiological target for exosome-mediated immune evasion in cancer. The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is expressed in
multiple human maligcies and has potent effects on cell growth. It has been
detected in exosomes and shown to inhibit immune function. Exosomes are small
secreted cellular vesicles that contain proteins, mRNAs, and microRNAs (miRNAs).
When produced by maligt cells, they can promote angiogenesis, cell
proliferation, tumor-cell invasion, and immune evasion. In this study, exosomes
released from nasopharyngeal carcinoma (NPC) cells harboring latent EBV were
shown to contain LMP1, signal transduction molecules, and virus-encoded miRNAs.
Exposure to these NPC exosomes activated the ERK and AKT signaling pathways in
the recipient cells. Interestingly, NPC exosomes also contained viral miRNAs,
several of which were enriched in comparison with their intracellular levels.
LMP1 induces expression of the EGF receptor in an EBV-negative epithelial cell
line, and exosomes produced by these cells also contain high levels of EGF
receptor in exosomes. These findings suggest that the effects of EBV and LMP1 on
cellular expression also modulate exosome content and properties. The exosomes
may manipulate the tumor microenvironment to influence the growth of neighboring
cells through the intercellular transfer of LMP1, signaling molecules, and viral
miRNAs. The RNA exosome is responsible for a wide variety of RNA processing and
degradation reactions. The activity and specificity of the RNA exosome is
thought to be controlled by a number of cofactors. Mtr4 is an essential
RNA-dependent adenosine triphosphatase that is required for all of the nuclear
functions of the RNA exosome. The crystal structure of Mtr4 uncovered a domain
that is conserved in the RNA exosome cofactors Mtr4 and Ski2 but not in other
helicases, suggesting it has an important role related to exosome activation.
Rrp6 provides the nuclear exosome with one of its three nuclease activities, and
previous findings suggested that the arch domain is specifically required for
Rrp6 functions. Here, we report that the genetic interactions between the arch
domain of Mtr4 and Rrp6 cannot be explained by the arch domain solely acting in
Rrp6-dependent processing reactions. Specifically, we show that the arch domain
is not required for all Rrp6 functions, and that the arch domain also functions
independently of Rrp6. Finally, we show that the arch domain of Ski2, the
cytoplasmic counterpart of Mtr4, is required for Ski2's function, thereby
confirming that the arch domains of these cofactors function independently of
Rrp6. Integrin trafficking, including internalization, recycling, and lysosomal
degradation, is crucial for the regulation of cellular functions. Exosomes,
o-sized extracellular vesicles, are believed to play important roles in
intercellular communications. This study demonstrates that exosomes released
from human macrophages negatively regulate endothelial cell migration through
control of integrin trafficking. Macrophage-derived exosomes promote
internalization of integrin β1 in primary HUVECs. The internalized integrin β1
persistently accumulates in the perinuclear region and is not recycled back to
the plasma membrane. Experimental results indicate that macrophage-derived
exosomes stimulate trafficking of internalized integrin β1 to lysosomal
compartments with a corresponding decrease in the integrin destined for
recycling endosomes, resulting in proteolytic degradation of the integrin.
Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and
exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal
degradation inhibitor. Macrophage-derived exosomes were also shown to
effectively suppress collagen-induced activation of the mitogen-activated
protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC
migration, which are both dependent on integrin β1. These observations provide
new insight into the functional significance of exosomes in the regulation of
integrin trafficking. Aqueous humor (AH) is a dynamic intraocular fluid that supports the vitality of
tissues that regulate intraocular pressure. We recently discovered that
extracellular ovesicles called exosomes are a major constituent of AH.
Exosomes function in extracellular communication and contain proteins and small
RNA. Our goal was to characterize the physical properties of AH exosomes and
their exosomal RNA (esRNA) content. We isolated exosomes from human AH collected
during cataract surgery from five patients using serial ultracentrifugation. We
measured the size and concentration of AH exosomes in solution using
oparticle tracking analysis. We found a single population of vesicles having
a mean size of 121 ± 11 nm in the unprocessed AH. Data show that centrifugation
does not significantly affect the mean particle size (121 ± 11 nm versus
124 ± 21 nm), but does impact the final number of exosomes in solution (87% loss
from the unprocessed AH; n = 5). We extracted esRNA from the pooled human AH
samples using miRCURY RNA isolation kit from Exiqon. The quality of extracted
esRNA was evaluated using Agilent Bioanalyzer 2100 and was used to generate a
sequencing library for small RNA sequencing with Illumina MiSeq sequencer. More
than 10 different miRNAs were identified; abundant species included miR-486-5p,
miR-204, and miR-184. We found that the majority of extracellular vesicles in
the AH were in the exosome size range, suggesting that miRNAs housed within
exosomes may function in communication between AH inflow and outflow tissues. Directional cell movement through tissues is critical for multiple biological
processes and requires maintece of polarity in the face of complex
environmental cues. Here we use intravital imaging to demonstrate that secretion
of exosomes from late endosomes is required for directionally persistent and
efficient in vivo movement of cancer cells. Inhibiting exosome secretion or
biogenesis leads to defective tumour cell migration associated with increased
formation of unstable protrusions and excessive directional switching. In vitro
rescue experiments with purified exosomes and matrix coating identify adhesion
assembly as a critical exosome function that promotes efficient cell motility.
Live-cell imaging reveals that exosome secretion directly precedes and promotes
adhesion assembly. Fibronectin is found to be a critical motility-promoting
cargo whose sorting into exosomes depends on binding to integrins. We propose
that autocrine secretion of exosomes powerfully promotes directionally
persistent and effective cell motility by reinforcing otherwise transient
polarization states and promoting adhesion assembly. BACKGROUND AIMS: Exosomes, a key component of cell paracrine secretion, can
exert protective effects in various disease models. However, application of
exosomes in vascular repair and regeneration has rarely been reported. In this
study, we tested whether endothelial progenitor cell (EPC)-derived exosomes
possessed therapeutic effects in rat models of balloon-induced vascular injury
by accelerating reendothelialization.
METHODS: Exosomes were obtained from the conditioned media of EPCs isolated from
human umbilical cord blood. Induction of the endothelial injury was performed in
the rats' carotid artery, and the pro-re-endothelialization capacity of
EPC-derived exosomes was measured. The in vitro effects of exosomes on the
proliferation and migration of endothelial cells were investigated.
RESULTS: We found that the EPC-derived exosomes accelerated the
re-endothelialization in the early phase after endothelial damage in the rat
carotid artery. We also demonstrated that these exosomes enhanced the
proliferation and migration of endothelial cells in vitro. Moreover, endothelial
cells stimulated with these exosomes showed increased expression of
angiogenesis-related molecules.
CONCLUSIONS: Taken together, our results indicate that exosomes are an active
component of the paracrine secretion of human EPCs and can promote vascular
repair in rat models of balloon injury by up-regulating endothelial cells
function. Cells are able to produce and release different types of vesicles, such as
microvesicles and exosomes, in the extracellular microenvironment. According to
the scientific community, both microvesicles and exosomes are able to take on
and transfer different macromolecules from and to other cells, and in this way,
they can influence the recipient cell function. Among the different
macromolecule cargos, the most studied are microRNAs. MicroRNAs are a large
family of non-coding RNAs involved in the regulation of gene expression. They
control every cellular process and their altered regulation is involved in human
diseases. Their presence in mammalian follicular fluid has been recently
demonstrated, and here, they are enclosed within microvesicles and exosomes or
they can also be associated to protein complexes. The presence of microvesicles
and exosomes carrying microRNAs in follicular fluid could represent an
alternative mechanism of autocrine and paracrine communication inside the
ovarian follicle. The outcomes from these studies could be important in basic
reproductive research but could also be useful for clinical application. In
fact, the characterization of extracellular vesicles in follicular fluid could
improve reproductive disease diagnosis and provide biomarkers of oocyte quality
in ART (Assisted Reproductive Treatment). Intercellular communication of immune cells is critical to elicit efficient
inflammatory responses. In intestinal mucosa, imbalance in pro-inflammatory and
anti-inflammatory mediators, especially cytokines and chemokines, characterizes
the underlying immune mechanisms of inflammatory bowel disease. Exosomes, small
membrane vesicles secreted into the extracellular environment, are emerging as
another important intercellular messenger in immune responses. A major recent
breakthrough in this field unveils the capacity of exosomes to mediate the
functional transfer of genetic materials (mRNAs and miRNAs) between immune
cells. RAB27A and RAB27B are two small GTPases involved in exosome secretion.
With respect to intestinal mucosal immunity, increased number of RAB27A-positive
immune cells and RAB27B-positive immune cells are demonstrated in the colonic
mucosa of patients with active ulcerative colitis as compared with that of
healthy controls. This indicates the important role of exosome-mediated immune
responses in the pathogenesis of inflammatory bowel disease. Here, we will
discuss the immune properties of exosomes and recent advances in their function
with a special focus on intestinal mucosal immunity. Exosomes are extracellular vesicles of endosomal origin which have emerged as
key mediators of intercellular communication. All major cardiac cell
types-including cardiomyocytes, endothelial cells, and fibroblasts-release
exosomes that modulate cellular functions. Exosomes released from human cardiac
progenitor cells (CPCs) are cardioprotective and improve cardiac function after
myocardial infarction to an extent comparable with that achieved by their parent
cells. Cardiac progenitor cell-derived exosomes are enriched in cardioprotective
microRNAs, particularly miR-146a-3p. Circulating exosomes mediate remote
ischaemic preconditioning. Moreover, they currently are being investigated as
diagnostic markers. The discovery that cell-derived extracellular signalling
organelles mediate the paracrine effects of stem cells suggests that cell-free
strategies could supplant cell transplantation. This review discusses emerging
roles of exosomes in cardiovascular physiology, with a focus on cardioprotective
activities of CPC-derived exosomes. Exosomes are a particular type of extracellular vesicle, characterized by their
endosomal origin as intraluminal vesicles present in large endosomes with a
multivesicular structure. After these endosomes fuse with the plasma membrane,
exosomes are secreted into the extracellular space. The ability of exosomes to
carry and selectively deliver bioactive molecules (e.g., lipids, proteins, and
nucleic acids) confers on them the capacity to modulate the activity of receptor
cells, even if these cells are located in distant tissues or organs. Since
exosomal cargo depends on cell type, a detailed understanding of the mechanisms
that regulate the biochemical composition of exosomes is fundamental to a
comprehensive view of exosome function. Here, we review the latest advances
concerning exosome function and biogenesis in T cells, with particular focus on
the mechanism of protein sorting at multivesicular endosomes. Exosomes secreted
by specific T-cell subsets can modulate the activity of immune cells, including
other T-cell subsets. Ceramide, tetraspanins and MAL have been revealed to be
important in exosome biogenesis by T cells. These molecules, therefore,
constitute potential molecular targets for artificially modulating exosome
production and, hence, the immune response for therapeutic purposes. Exosomes-secreted microRNAs play an important role in metastatic spread. During
this process breast cancer cells acquire the ability to transmigrate through
blood vessels by inducing changes in the endothelial barrier. We focused on
miR-939 that is predicted to target VE-cadherin, a component of adherens
junction involved in vessel permeability. By in silico analysis miR-939 was
found highly expressed in the basal-like tumor subtypes and in our cohort of 63
triple-negative breast cancers (TNBCs) its expression significantly interacted
with lymph node status in predicting disease-free survival probability. We
demonstrated, in vitro, that miR-939 directly targets VE-cadherin leading to an
increase in HUVECs monolayer permeability. MDA-MB-231 cells transfected with a
miR-939 mimic, released miR-939 in exosomes that, once internalized in
endothelial cells, favored trans-endothelial migration of MDA-MB-231-GFP cells
by the disruption of the endothelial barrier. Notably, when up taken in
endothelial cells exosomes caused VE-cadherin down-regulation specifically
through miR-939 as we demonstrated by inhibiting miR-939 expression in
exosomes-releasing TNBC cells. Together, our data indentify an extracellular
pro-tumorigenic role for tumor-derived, exosome-associated miR-939 that can
explain its association with worse prognosis in TNBCs. |
Which factors drive replisome disassembly during DNA replication termination and mitosis? | CUL-2LRR-1 and UBXN-3. | Replisome disassembly is the final step of DNA replication in eukaryotes,
involving the ubiquitylation and CDC48-dependent dissolution of the CMG helicase
(CDC45-MCM-GINS). Using Caenorhabditis elegans early embryos and Xenopus laevis
egg extracts, we show that the E3 ligase CUL-2LRR-1 associates with the
replisome and drives ubiquitylation and disassembly of CMG, together with the
CDC-48 cofactors UFD-1 and NPL-4. Removal of CMG from chromatin in frog egg
extracts requires CUL2 neddylation, and our data identify chromatin recruitment
of CUL2LRR1 as a key regulated step during DNA replication termination.
Interestingly, however, CMG persists on chromatin until prophase in worms that
lack CUL-2LRR-1, but is then removed by a mitotic pathway that requires the
CDC-48 cofactor UBXN-3, orthologous to the human tumour suppressor FAF1. Partial
inactivation of lrr-1 and ubxn-3 leads to synthetic lethality, suggesting future
approaches by which a deeper understanding of CMG disassembly in metazoa could
be exploited therapeutically. |
Does RNA polymerase II have RNA cleavage activity? | In addition to RNA synthesis, multisubunit RNA polymerases (msRNAPs) support enzymatic reactions such as intrinsic transcript cleavage. The eukaryotic transcription factor TFIIS enhances elongation and nascent transcript cleavage activities of RNA polymerase II in a stalled elongation complex. | In addition to polynucleotide polymerization, DNA polymerases and bacterial RNA
polymerase can also remove nucleotides from the growing end of nucleic acid
chains. For DNA polymerases this activity is an important factor in establishing
fidelity in DNA synthesis. This report describes a novel in vitro activity of
RNA polymerase II whereby it cleaves an RNA chain contained within an active
elongation complex. These elongation complexes are arrested at a previously
identified, naturally occurring transcriptional pause site in a human gene. The
new 3'-end revealed by this cleavage remains associated with an active
elongation complex and is capable of being extended by RNA polymerase II.
Nascent RNA cleavage is evident after removal of free nucleotides and is
dependent upon a divalent metal cation and transcription elongation factor SII.
This function of SII could be important in its function as an activator of
transcription elongation. It is also possible that the transcript cleavage
activity of RNA polymerase II represents a proofreading function of the enzyme. Regulation of transcription elongation is an important mechanism in controlling
eukaryotic gene expression. SII is an RNA polymerase II-binding protein that
stimulates transcription elongation and also activates nascent transcript
cleavage by RNA polymerase II in elongation complexes in vitro (Reines, D.
(1992) J. Biol. Chem. 267, 3795-3800). Here we show that SII-dependent in vitro
transcription through an arrest site in a human gene is preceded by nascent
transcript cleavage. RNA cleavage appeared to be an obligatory step in the SII
activation process. Recombit SII activated cleavage while a truncated
derivative lacking polymerase binding activity did not. Cleavage was not
restricted to an elongation complex arrested at this particular site, showing
that nascent RNA hydrolysis is a general property of RNA polymerase II
elongation complexes. These data support a model whereby SII stimulates
elongation via a ribonuclease activity of the elongation complex. Obstacles incurred by RNA polymerase II during primary transcript synthesis have
been identified in vivo and in vitro. Transcription past these impediments
requires SII, an RNA polymerase II-binding protein. SII also activates a
nuclease in arrested elongation complexes and this nascent RNA shortening
precedes transcriptional readthrough. Here we show that in the presence of SII
and nucleotides, transcript cleavage is detected during SII-dependent elongation
but not during SII-independent transcription. Thus, under typical transcription
conditions, SII is necessary but insufficient to activate RNA cleavage. RNA
cleavage could serve to move RNA polymerase II away from the transcriptional
impediment and/or permit RNA polymerase II multiple attempts at RNA elongation.
By mapping the positions of the 3'-ends of RNAs and the elongation complex on
DNA, we demonstrate that upstream movement of RNA polymerase II is not required
for limited RNA shortening (seven to nine nucleotides) and reactivation of an
arrested complex. Arrested complexes become elongation competent after removal
of no more than nine nucleotides from the nascent RNA's 3'-end. Further cleavage
of nascent RNA, however, does result in "backward" translocation of the enzyme.
We also show that one round of RNA cleavage is insufficient for full readthrough
at an arrest site, consistent with a previously suggested mechanism of SII
action. In the absence of DNA, purified yeast RNA polymerase II can bind RNA to form a
binary complex. RNA in such RNA-RNA polymerase complexes undergoes reactions
previously thought to be unique to nascent RNA in ternary complexes with DNA,
including TFIIS-dependent cleavage and elongation by 3'-terminal addition of NMP
from NTP. Both of these reactions are inhibited by alpha-amanitin. Hence, by
several criteria the RNA in binary complexes is bound to the polymerase in a
manner quite similar to that in ternary complexes in which the catalytic site
for nucleotide addition is positioned at or near the 3'-OH terminus of the RNA.
These findings are consistent with a model for the RNA polymerase ternary
complex in which the RNA is bound at the 3' terminus through two protein-binding
sites located up to 10 nt apart. RNA chain elongation by RNA polymerase is a dynamic process. Techniques that
allow the isolation of active elongation complexes have enabled investigators to
describe individual steps in the polymerization of RNA chains. This article will
describe recent studies of elongation by RNA polymerase II (pol II). At least
four types of blockage to chain elongation can be overcome by elongation factor
SII: (a) naturally occurring "arrest" sequences, (b) DNA-bound protein, (c)
drugs bound in the DNA minor groove, and (d) chain-terminating substrates
incorporated into the RNA chain. SII binds to RNA polymerase II and stimulates a
ribonuclease activity that shortens nascent transcripts from their 3' ends. This
RNA cleavage is required for chain elongation from some template positions. As a
result, the pol II elongation complex can repeatedly shorten and reextend the
nascent RNA chain in a process we refer to as cleavage-resynthesis. Hence,
assembly of large RNAs does not necessarily proceed in a direct manner. The
ability to shorten and reextend nascent RNAs means that a transcription
impediment through which only half the enzyme molecules can proceed per
encounter, can be overcome by 99% of the molecules after six iterations of
cleavage-resynthesis. Surprisingly, the boundaries of the elongation complex do
not move upstream after RNA cleavage. The physico-chemical alterations in the
elongation complex that accompany RNA cleavage and permit renewed chain
elongation are not yet understood. Elongation factor SII (also known as TFIIS) is an RNA polymerase II binding
protein that allows bypass of template arrest sites by activating a nascent RNA
cleavage reaction. Here we show that SII contacts the 3'-end of nascent RNA
within an RNA polymerase II elongation complex as detected by photoaffinity
labeling. Photocross-linking was dependent upon the presence of SII,
incorporation of 4-thio-UMP into RNA, and irradiation and was sensitive to
treatment by RNase and proteinase. A transcriptionally active mutant of SII
lacking the first 130 amino acids was also cross-linked to the nascent RNA, but
SII from Saccharomyces cerevisiae, which is inactive in concert with mammalian
RNA polymerase II, failed to become photoaffinity labeled. SII-RNA contact was
not detected after a labeled oligoribonucleotide was released from the complex
by nascent RNA cleavage, demonstrating that this interaction takes place between
elongation complex-associated but not free RNA. This shows that the 3'-end of
RNA is near the SII binding site on RNA polymerase II and suggests that SII may
activate the intrinsic RNA hydrolysis activity by positioning the transcript in
the enzyme's active site. Budding yeast RNA polymerase III (Pol III) contains a small, essential subunit,
named C11, that is conserved in humans and shows a strong homology to TFIIS. A
mutant Pol III, heterocomplemented with Schizosaccharomyces pombe C11, was
affected in transcription termination in vivo. A purified form of the enzyme
(Pol III Delta), deprived of C11 subunit, initiated properly but ignored pause
sites and was defective in termination. Remarkably, Pol III Delta lacked the
intrinsic RNA cleavage activity of complete Pol III. In vitro reconstitution
experiments demonstrated that Pol III RNA cleavage activity is mediated by C11.
Mutagenesis in C11 of two conserved residues, which are critical for the
TFIIS-dependent cleavage activity of Pol II, is lethal. Immunoelectron
microscopy data suggested that C11 is localized on the mobile thumb-like stalk
of the polymerase. We propose that C11 allows the enzyme to switch between an
RNA elongation and RNA cleavage mode and that the essential role of the Pol III
RNA cleavage activity is to remove the kinetic barriers to the termination
process. The integration of TFIIS function into a specific Pol III subunit may
stem from the opposite requirements of Pol III and Pol II in terms of transcript
length and termination efficiency. During gene transcription, the RNA polymerase (Pol) active center can catalyze
RNA cleavage. This intrinsic cleavage activity is strong for Pol I and Pol III
but very weak for Pol II. The reason for this difference is unclear because the
active centers of the polymerases are virtually identical. Here we show that Pol
II gains strong cleavage activity when the C-terminal zinc ribbon domain
(C-ribbon) of subunit Rpb9 is replaced by its counterpart from the Pol III
subunit C11. X-ray analysis shows that the C-ribbon has detached from its site
on the Pol II surface and is mobile. Mutagenesis indicates that the C-ribbon
transiently inserts into the Pol II pore to complement the active center. This
mechanism is also used by transcription factor IIS, a factor that can bind Pol
II and induce strong RNA cleavage. Together with published data, our results
indicate that Pol I and Pol III contain catalytic C-ribbons that complement the
active center, whereas Pol II contains a non-catalytic C-ribbon that is
immobilized on the enzyme surface. Evolution of the Pol II system may have
rendered mRNA transcript cleavage controllable by the dissociable factor
transcription factor IIS to enable promoter-proximal gene regulation and
elaborate 3'-processing and transcription termination. In addition to RNA synthesis, multisubunit RNA polymerases (msRNAPs) support
enzymatic reactions such as intrinsic transcript cleavage. msRNAP active sites
from different species appear to exhibit differential intrinsic transcript
cleavage efficiency and have likely evolved to allow fine-tuning of the
transcription process. Here we show that a single amino-acid substitution in the
trigger loop (TL) of Saccharomyces RNAP II, Rpb1 H1085Y, engenders a gain of
intrinsic cleavage activity where the substituted tyrosine appears to
participate in acid-base chemistry at alkaline pH for both intrinsic cleavage
and nucleotidyl transfer. We extensively characterize this TL substitution for
each of these reactions by examining the responses RNAP II enzymes to catalytic
metals, altered pH, and factor inputs. We demonstrate that TFIIF stimulation of
the first phosphodiester bond formation by RNAP II requires wild type TL
function and that H1085Y substitution within the TL compromises or alters RNAP
II responsiveness to both TFIIB and TFIIF. Finally, Mn(2+) stimulation of H1085Y
RNAP II reveals possible allosteric effects of TFIIB on the active center and
cooperation between TFIIB and TFIIF. |
Is there a sequence bias in MNase digestion patterns? | The cutting preference of MNase in combination with size selection generates a sequence-dependent bias in the resulting fragments. | We have mapped sequence-directed nucleosome positioning on genomic DNA molecules
using high-throughput sequencing. Chromatins, prepared by reconstitution with
either chicken or frog histones, were separately digested to mononucleosomes
using either micrococcal nuclease (MNase) or caspase-activated DNase (CAD). Both
enzymes preferentially cleave internucleosomal (linker) DNA, although they do so
by markedly different mechanisms. MNase has hitherto been very widely used to
map nucleosomes, although concerns have been raised over its potential to
introduce bias. Having identified the locations and quantified the strength of
both the chicken or frog histone octamer binding sites on each DNA, the results
obtained with the two enzymes were compared using a variety of criteria. Both
enzymes displayed sequence specificity in their preferred cleavage sites,
although the nature of this selectivity was distinct for the two enzymes. In
addition, nucleosomes produced by CAD nuclease are 8-10 bp longer than those
produced with MNase, with the CAD cleavage sites tending to be 4-5 bp further
out from the nucleosomal dyad than the corresponding MNase cleavage sites.
Despite these notable differences in cleavage behaviour, the two nucleases
identified essentially equivalent patterns of nucleosome positioning sites on
each of the DNAs tested, an observation that was independent of the histone
type. These results indicate that biases in nucleosome positioning data
collected using MNase are, under our conditions, not significant. BACKGROUND: The organization of eukaryotic DNA into chromatin has a strong
influence on the accessibility and regulation of genetic information. The
locations and occupancies of a principle component of chromatin, nucleosomes,
are typically assayed through use of enzymatic digestion with micrococcal
nuclease (MNase). MNase is an endo-exo nuclease that preferentially digests
naked DNA and the DNA in linkers between nucleosomes, thus enriching for
nucleosome-associated DNA. To determine nucleosome organization genome-wide, DNA
remaining from MNase digestion is sequenced using high-throughput sequencing
technologies (MNase-seq). Unfortunately, the results of MNase-seq can vary
dramatically due to technical differences and this confounds comparisons between
MNase-seq experiments, such as examining condition-dependent chromatin
organizations.
RESULTS: In this study we use MNase digestion simulations to demonstrate how
MNase-seq signals can vary for different nucleosome configuration when
experiments are performed with different extents of MNase digestion. Signal
variation in these simulations reveals an important DNA sampling bias that
results from a neighborhood effect of MNase digestion techniques. The presence
of this neighborhood effect ultimately confounds comparisons between different
MNase-seq experiments. To address this issue we present a standardized chromatin
preparation which controls for technical variance between MNase-based chromatin
preparations and enables the collection of similarly sampled (matched) chromatin
populations. Standardized preparation of chromatin includes a normalization step
for DNA input into MNase digestions and close matching of the extent of
digestion between each chromatin preparation using gel densitometry analysis.
The protocol also includes directions for successful pairing with multiplex
sequencing reactions.
CONCLUSIONS: We validated our method by comparing the experiment-to-experiment
variation between biological replicates of chromatin preparations from S.
cerevisiae. Results from our matched preparation consistently produced MNase-seq
datasets that were more closely correlated than other unstandardized approaches.
Additionally, we validated the ability of our approach at enabling accurate
downstream comparisons of chromatin structures, by comparing the specificity of
detecting Tup1-dependent chromatin remodeling events in comparisons between
matched and un-matched wild-type and tup1Δ MNase-seq datasets. Our matched
MNase-seq datasets demonstrated a significant reduction in non-specific
(technical) differences between experiments and were able to maximize the
detection of biologically-relevant (Tup1-dependent) changes in chromatin
structure. Cellular processes mediated through nuclear DNA must contend with chromatin.
Chromatin structural assays can efficiently integrate information across diverse
regulatory elements, revealing the functional noncoding genome. In this study,
we use a differential nuclease sensitivity assay based on micrococcal nuclease
(MNase) digestion to discover open chromatin regions in the maize genome. We
find that maize MNase-hypersensitive (MNase HS) regions localize around active
genes and within recombination hotspots, focusing biased gene conversion at
their flanks. Although MNase HS regions map to less than 1% of the genome, they
consistently explain a remarkably large amount (∼40%) of heritable phenotypic
variance in diverse complex traits. MNase HS regions are therefore on par with
coding sequences as annotations that demarcate the functional parts of the maize
genome. These results imply that less than 3% of the maize genome (coding and
MNase HS regions) may give rise to the overwhelming majority of phenotypic
variation, greatly narrowing the scope of the functional genome. |
Is NEMO a zinc finger protein? | NEMO function is mediated by two distal ubiquitin binding domains located in the regulatory C-terminal domain of the protein: the coiled-coil 2-leucine zipper (CC2-LZ) domain and the zinc finger (ZF) domain. | Molecular dynamics (MD) simulation methods have seen significant improvement
since their inception in the late 1950s. Constraints of simulation size and
duration that once impeded the field have lessened with the advent of better
algorithms, faster processors, and parallel computing. With newer techniques and
hardware available, MD simulations of more biologically relevant timescales can
now sample a broader range of conformational and dynamical changes including
rare events. One concern in the literature has been under which circumstances it
is sufficient to perform many shorter timescale simulations and under which
circumstances fewer longer simulations are necessary. Herein, our simulations of
the zinc finger NEMO (2JVX) using multiple simulations of length 15, 30, 1000,
and 3000 ns are analyzed to provide clarity on this point. NF-κB essential modulator (NEMO) and cylindromatosis protein (CYLD) are
intracellular proteins that regulate the NF-κB signaling pathway. Although mice
with either CYLD deficiency or an alteration in the zinc finger domain of NEMO
(K392R) are born healthy, we found that the combination of these two gene
defects in double mutant (DM) mice is early embryonic lethal but can be rescued
by the absence of TNF receptor 1 (TNFR1). Notably, NEMO was not recruited into
the TNFR1 complex of DM cells, and consequently NF-κB induction by TNF was
severely impaired and DM cells were sensitized to TNF-induced cell death.
Interestingly, the TNF signaling defects can be fully rescued by reconstitution
of DM cells with CYLD lacking ubiquitin hydrolase activity but not with CYLD
mutated in TNF receptor-associated factor 2 (TRAF2) or NEMO binding sites.
Therefore, our data demonstrate an unexpected non-catalytic function for CYLD as
an adapter protein between TRAF2 and the NEMO zinc finger that is important for
TNF-induced NF-κB signaling during embryogenesis. |
Does TFIIS affect nucleosome positioning? | Transcript cleavage factor TFIIS reactivates the backtracked complexes and promotes pol II transcription through the nucleosome. The same nucleosomes transcribed in the opposite orientation form a weaker, more diffuse barrier that is largely relieved by higher salt, TFIIS, or FACT | Transcriptional elongation involves dynamic interactions among RNA polymerase
and single-stranded and double-stranded nucleic acids in the ternary complex. In
prokaryotes its regulation provides an important mechanism of genetic control.
Analogous eukaryotic mechanisms are not well understood, but may control
expression of proto-oncogenes and viruses, including the human immunodeficiency
virus HIV-1 (ref. 8). The highly conserved eukaryotic transcriptional elongation
factor TFIIS enables RNA polymerase II (RNAPII) to read though pause or
termination sites, nucleosomes and sequence-specific DNA-binding proteins. Two
distinct domains of human TFIIS, which bind RNAPII and nucleic acids, regulate
read-through and possibly nascent transcript cleavage. Here we describe the
three-dimensional NMR structure of a Cys4 nucleic-acid-binding domain from human
TFIIS. Unlike previously characterized zinc modules, which contain an
alpha-helix, this structure consists of a three-stranded beta-sheet. Analogous
Cys4 structural motifs may occur in other proteins involved in DNA or RNA
transactions, including RNAPII itself. This new structure, designated the Zn
ribbon, extends the repertoire of Zn-mediated peptide architectures and
highlights the growing recognition of the beta-sheet as a motif of nucleic-acid
recognition. In the cell, RNA polymerase II (pol II) efficiently transcribes DNA packaged
into nucleosomes, but in vitro encounters with the nucleosomes induce catalytic
inactivation (arrest) of the pol II core enzyme. To determine potential
mechanisms making nucleosomes transparent to transcription in vivo, we analyzed
the nature of the nucleosome-induced arrest. We found that the arrests have been
detected mostly at positions of strong intrinsic pause sites of DNA. The
transient pausing makes pol II vulnerable to arrest, which involves backtracking
of the elongation complex for a considerable distance on DNA. The histone-DNA
contacts reestablished in front of pol II stabilize backtracked conformation of
the polymerase. In agreement with this mechanism, blocking of backtracking
prevents nucleosome-induced arrest. Transcript cleavage factor TFIIS reactivates
the backtracked complexes and promotes pol II transcription through the
nucleosome. Our findings establish the crucial role of elongation factors that
suppress pol II pausing and backtracking for transcription in the context of
chromatin. Nucleosomes uniquely positioned on high-affinity DNA sequences present a polar
barrier to transcription by human and yeast RNA polymerase II (Pol II). In one
transcriptional orientation, these nucleosomes provide a strong, factor- and
salt-insensitive barrier at the entry into the H3/H4 tetramer that can be
recapitulated without H2A/H2B dimers. The same nucleosomes transcribed in the
opposite orientation form a weaker, more diffuse barrier that is largely
relieved by higher salt, TFIIS, or FACT. Barrier properties are therefore
dictated by both the local nucleosome structure (influenced by the strength of
the histone-DNA interactions) and the location of the high-affinity DNA region
within the nucleosome. Pol II transcribes DNA sequences at the entry into the
tetramer much less efficiently than the same sequences located distal to the
nucleosome dyad. Thus, entry into the tetramer by Pol II facilitates further
transcription, perhaps due to partial unfolding of the tetramer from DNA. In mammalian cells RNA polymerase II efficiently transcribes nucleosome-packaged
DNA. In this regard, a fundamental question concerns the nature and mechanism of
action of the accessory factors that are necessary and sufficient for, or
enhance, transcription through nucleosomal arrays by RNA polymerase II. Here we
describe a highly purified system that allows for efficient activator-dependent
transcription by RNA polymerase II from the promoter through several contiguous
nucleosomes on defined chromatin templates. The system contains natural or
recombit histones, chromatin assembly factors, the histone-acetyltransferase
p300, all components of the general transcription machinery, general
coactivators and the elongation factor SII (TFIIS). As examples of the
applicability of this system for mechanistic analyses of these and other
factors, representative experiments show (i) that activated transcription from
chromatin templates is concomitantly dependent on the activator, p300-mediated
histone acetylation and elongation factor SII/TFIIS. (ii) that SII/TFIIS acts in
a highly synergistic manner with p300 (and histone acetylation) at a step
subsequent to preinitiation complex (PIC) formation and (iii) that SII/TFIIS
works directly at the elongation step of chromatin transcription. Here we
describe purification methods for the different factors employed and the
specific transcriptional assays that led to the above-mentioned conclusions.
This purified system will be very useful as an assay system for the discovery of
new factors or the mechanistic analysis of known or candidate factors involved
in transcription initiation or elongation on chromatin templates, including
factors that effect specific histone modifications or nucleosomal remodeling. The nucleosome is generally found to be a strong barrier to transcript
elongation by RNA polymerase II (pol II) in vitro. The elongation factors TFIIF
and TFIIS have been shown to cooperate in maintaining pol II in the
catalytically competent state on pure DNA templates. We now show that although
TFIIF or TFIIS alone is modestly stimulatory for nucleosome traversal, both
factors together increase transcription through nucleosomes in a synergistic
manner. We also studied the effect of TFIIF and TFIIS on transcription of
nucleosomes containing a Sin mutant histone. The Sin point mutations reduce
critical histone-DNA contacts near the center of the nucleosome. Significantly,
we found that nucleosomes with a Sin mutant histone are traversed to the same
extent and at nearly the same rate as equivalent pure DNA templates if both
TFIIS and TFIIF are present. Thus, the nucleosome is not necessarily an
insurmountable barrier to transcript elongation by pol II. If unfolding of
template DNA from the nucleosome surface is facilitated and the tendency of pol
II to retreat from barriers is countered, transcription of nucleosomal templates
can be rapid and efficient. |
Which two cotransporters are inhibited by sotagliflozin? | Sotagliflozin works by inhibiting sodium-glucose cotransporter 1 (SGLT1) and sodium-glucose cotransporter 2 (SGLT2). It is used for treatment of diabetes. | The sodium-dependent glucose transporter 2 (SGLT2) inhibitors are an important
emerging class for the treatment of diabetes. Development of SGLT2 inhibitors
has been oriented around a desire for high selectivity for the SGLT2 protein
relative to the SGLT1 protein. More recently, genetic and pharmacology research
in mice has indicated that gastrointestinal SGLT1 inhibition may also be an
appropriate therapeutic target to treat diabetes. Combining SGLT1 and SGLT2
inhibition in a single molecule would provide complementary insulin-independent
mechanisms to treat diabetes. Therefore, sotagliflozin (LX4211) has been
developed as a dual inhibitor of SGLT1 and SGLT2. The differentiating clinical
features of dual inhibitor of SGLT1 and SGLT2 include a large postprandial
glucose reduction, elevation of glucagon-like peptide 1 and modest urinary
glucose excretion. These features may have clinical implications for the use of
sotagliflozin in the treatment of both type 1 and type 2 diabetes. PURPOSE: Oral agents are needed that improve glycemic control without increasing
hypoglycemic events in patients with type 1 diabetes (T1D). Sotagliflozin may
meet this need, because this compound lowers blood glucose through the
insulin-independent mechanisms of inhibiting kidney SGLT2 and intestinal SGLT1.
We examined the effect of sotagliflozin on glycemic control and rate of
hypoglycemia measurements in T1D mice maintained on a low daily insulin dose,
and compared these results to those from mice maintained in better glycemic
control with a higher daily insulin dose alone.
MATERIALS AND METHODS: Nonobese diabetes-prone mice with
cyclophosphamide-induced T1D were randomized to receive one of four daily
treatments: 0.2 U insulin/vehicle, 0.05 U insulin/vehicle, 0.05 U insulin/2
mg/kg sotagliflozin or 0.05 U insulin/30 mg/kg sotagliflozin. Insulin was
delivered subcutaneously by micro-osmotic pump; the day after pump implantation,
mice received their first of 22 once-daily oral doses of sotagliflozin or
vehicle. Glycemic control was monitored by measuring fed blood glucose and
hemoglobin A1c levels.
RESULTS: Blood glucose levels decreased rapidly and comparably in the 0.05 U
insulin/sotagliflozin-treated groups and the 0.2 U insulin/vehicle group
compared to the 0.05 U insulin/vehicle group, which had significantly higher
levels than the other three groups from day 2 through day 23. A1c levels were
also significantly higher in the 0.05 U insulin/vehicle group compared to the
other three groups on day 23. Importantly, the 0.2 U insulin/vehicle group had,
out of 100 blood glucose measurements, 13 that were <70 mg/dL compared to one of
290 for the other three groups combined.
CONCLUSION: Sotagliflozin significantly improved glycemic control, without
increasing the rate of hypoglycemia measurements, in diabetic mice maintained on
a low insulin dose. This sotagliflozin-mediated improvement in glycemic control
was comparable to that achieved by raising the insulin dose alone, but was not
accompanied by the increased rate of hypoglycemia measurements observed with the
higher insulin dose. OBJECTIVE: Review available data on adjunctive therapies for type 1 diabetes
(T1D), with a special focus on newer antihyperglycemic agents.
METHODS: Published data on hypoglycemia, obesity, mortality, and goal attainment
in T1D were reviewed to determine unmet therapeutic needs. PubMed databases and
abstracts from recent diabetes meetings were searched using the term "type 1
diabetes" and the available and investigational sodium-glucose cotransporter
(SGLT) inhibitors, glucagon-like peptide 1 (GLP-1) receptor agonists, dipeptidyl
peptidase 4 inhibitors, and metformin.
RESULTS: The majority of patients with T1D do not meet glycated hemoglobin (A1C)
goals established by major diabetes organizations. Hypoglycemia risks and a
rising incidence of obesity and metabolic syndrome featured in the T1D
population limit optimal use of intensive insulin therapy. Noninsulin
antihyperglycemic agents may enable T1D patients to achieve target A1C levels
using lower insulin doses, which may reduce the risk of hypoglycemia. In pilot
studies, the SGLT2 inhibitor dapagliflozin and the GLP-1 receptor agonist
liraglutide reduced blood glucose, weight, and insulin dose in patients with
T1D. Phase 2 studies with the SGLT2 inhibitor empagliflozin and the dual SGLT1
and SGLT2 inhibitor sotagliflozin, which acts in the gut and the kidney, have
demonstrated reductions in A1C, weight, and glucose variability without an
increased incidence of hypoglycemia.
CONCLUSION: Newer antihyperglycemic agents, particularly GLP-1 agonists, SGLT2
inhibitors, and dual SGLT1 and SGLT2 inhibitors, show promise as adjunctive
treatment for T1D that may help patients achieve better glucose control without
weight gain or increased hypoglycemia. INTRODUCTION: SGLT1 is the primary transporter responsible for the absorption of
glucose and galactose in the intestine, while SGLT2 and SGLT1 are both involved
in the renal reabsorption of glucose. SGLT2 inhibitors are a new class of oral
antidiabetic drugs, acting by increasing urinary glucose excretion (UGE). They
offer the advantages of a reduced risk of hypoglycaemia, a decrease in body
weight and blood pressure and an efficacy at all stages of type 2 diabetes
(T2DM).
AREAS COVERED: Herein, the authors focus specifically on sotagliflozin (LX4211),
the first-in-class dual SGLT1/SGLT2 inhibitor. Original publications in English
were selected as the basis of this review. Clinical trials were identified using
the Clinicaltrial.gov database.
EXPERT OPINION: By a potential additional mechanism of action on intestinal
glucose absorption linked to SGLT1 inhibition, sotagliflozin differentiates from
SGLT2 inhibitors by reducing postprandial glucose excursion and insulin
secretion, as well as by increasing GLP-1 secretion. Despite a weaker effect on
UGE than selective SGLT2 inhibitors, sotagliflozin is as effective as SGLT2
inhibitors on HbA1C reduction, with a similar safety profile in short-term
studies. While sotagliflozin was first assessed in T2DM, it is now in phase 3
development as an adjuvant treatment in patients with T1DM after positive
results from a pilot study. Type 2 Diabetes Mellitus (T2DM) and Alzheimer's disease (AD) are the two
disorders which are known to share pertinent pathological and therapeutic links.
Sodium glucose co-transporter-2 (SGLT2) and Acetylcholinesterase (AChE) are
established inhibition targets for T2DM and AD treatments, respectively. Reports
suggest that anti-diabetic drugs could be used for AD treatment also. The
present study used molecular docking by Autodock4.2 using our
"Click-By-Click"-protocol, Ligplot1.4.3 and "change in accessible surface area
(ΔASA)-calculations" to investigate the binding of two investigational
anti-diabetic drugs, Ertugliflozin and Sotagliflozin to an established target
(SGLT2) and a research target (human brain AChE). Sotagliflozin appeared more
promising for SGLT2 as well as AChE-inhibition with reference to ΔG and Ki
values in comparison to Ertugliflozin. The ΔG and Ki values for
"Sotagliflozin:AChE-binding" were -7.16 kcal/mol and 5.6 μM, respectively while
the same were found to be -8.47 kcal/mol and 0.62 μM, respectively for its
interaction with SGLT2. Furthermore, "Sotagliflozin:SGLT2-interaction" was
subjected to (un)binding simulation analyses by "Molecular-Motion-Algorithms."
This information is significant as the exact binding mode, interacting amino
acid residues and simulation results for the said interaction have not been
described yet. Also no X-ray crystal is available for the same. Finally, the
results described herein indicate that Sotagliflozin could have an edge over
Ertugliflozin for treatment of Type 2 diabetes. Future design of drugs based on
Sotagliflozin scaffolds for treatment of Type 2 and/or Type 3 diabetes are
highly recommended. As these drugs are still in late phases of clinical trials,
the results described herein appear timely. J. Cell. Biochem. 118: 3855-3865,
2017. © 2017 Wiley Periodicals, Inc. The sotagliflozin molecule exhibits two fundamentally different molecular
conformations in form 1 {systematic name:
(2S,3R,4R,5S,6R)-2-[4-chloro-3-(4-ethoxybenzyl)phenyl]-6-(methylsulfanyl)tetrahydro-2H-pyran-3,4,5-triol,
C21H25ClO5S, (I)} and the monohydrate [C21H25ClO5S·H2O, (II)]. Both crystals
display hydrogen-bonded layers formed by intermolecular interactions which
involve the three -OH groups of the xyloside fragment of the molecule. The layer
architectures of (I) and (II) contain a non-hydrogen-bonded molecule-molecule
interaction along the short crystallographic axis (a axis) whose total PIXEL
energy exceeds that of each hydrogen-bonded molecule-molecule pair. The
hydrogen-bonded layer of (I) has the topology of the 4-connected sql net and
that formed by the water and sotagliflozin molecules of (II) has the topology of
a 3,7-connected net. BACKGROUND: In most patients with type 1 diabetes, adequate glycemic control is
not achieved with insulin therapy alone. We evaluated the safety and efficacy of
sotagliflozin, an oral inhibitor of sodium-glucose cotransporters 1 and 2, in
combination with insulin treatment in patients with type 1 diabetes.
METHODS: In this phase 3, double-blind trial, which was conducted at 133 centers
worldwide, we randomly assigned 1402 patients with type 1 diabetes who were
receiving treatment with any insulin therapy (pump or injections) to receive
sotagliflozin (400 mg per day) or placebo for 24 weeks. The primary end point
was a glycated hemoglobin level lower than 7.0% at week 24, with no episodes of
severe hypoglycemia or diabetic ketoacidosis after randomization. Secondary end
points included the change from baseline in glycated hemoglobin level, weight,
systolic blood pressure, and mean daily bolus dose of insulin.
RESULTS: A significantly larger proportion of patients in the sotagliflozin
group than in the placebo group achieved the primary end point (200 of 699
patients [28.6%] vs. 107 of 703 [15.2%], P<0.001). The least-squares mean change
from baseline was significantly greater in the sotagliflozin group than in the
placebo group for glycated hemoglobin (difference, -0.46 percentage points),
weight (-2.98 kg), systolic blood pressure (-3.5 mm Hg), and mean daily bolus
dose of insulin (-2.8 units per day) (P≤0.002 for all comparisons). The rate of
severe hypoglycemia was similar in the sotagliflozin group and the placebo group
(3.0% [21 patients] and 2.4% [17], respectively). The rate of documented
hypoglycemia with a blood glucose level of 55 mg per deciliter (3.1 mmol per
liter) or below was significantly lower in the sotagliflozin group than in the
placebo group. The rate of diabetic ketoacidosis was higher in the sotagliflozin
group than in the placebo group (3.0% [21 patients] and 0.6% [4], respectively).
CONCLUSIONS: Among patients with type 1 diabetes who were receiving insulin, the
proportion of patients who achieved a glycated hemoglobin level lower than 7.0%
with no severe hypoglycemia or diabetic ketoacidosis was larger in the group
that received sotagliflozin than in the placebo group. However, the rate of
diabetic ketoacidosis was higher in the sotagliflozin group. (Funded by Lexicon
Pharmaceuticals; inTandem3 ClinicalTrials.gov number, NCT02531035 .). |
What is the administration route of IVIG in Alzheimer's disease patients? | IVIG is administered intravenously. | |
What is metaSPAdes? | MetaSPAdes is a new versatile metagenomic assembler. | MOTIVATION: We present Faucet, a two-pass streaming algorithm for assembly graph
construction. Faucet builds an assembly graph incrementally as each read is
processed. Thus, reads need not be stored locally, as they can be processed
while downloading data and then discarded. We demonstrate this functionality by
performing streaming graph assembly of publicly available data, and observe that
the ratio of disk use to raw data size decreases as coverage is increased.
RESULTS: Faucet pairs the de Bruijn graph obtained from the reads with
additional meta-data derived from them. We show these metadata-coverage counts
collected at junction k-mers and connections bridging between junction
pairs-contain most salient information needed for assembly, and demonstrate they
enable cleaning of metagenome assembly graphs, greatly improving contiguity
while maintaining accuracy. We compared Fauceted resource use and assembly
quality to state of the art metagenome assemblers, as well as leading
resource-efficient genome assemblers. Faucet used orders of magnitude less time
and disk space than the specialized metagenome assemblers MetaSPAdes and
Megahit, while also improving on their memory use; this broadly matched
performance of other assemblers optimizing resource efficiency-namely, Minia and
LightAssembler. However, on metagenomes tested, Faucet,o outputs had 14-110%
higher mean NGA50 lengths compared with Minia, and 2- to 11-fold higher mean
NGA50 lengths compared with LightAssembler, the only other streaming assembler
available.
AVAILABILITY AND IMPLEMENTATION: Faucet is available at
https://github.com/Shamir-Lab/Faucet.
CONTACT: [email protected] or [email protected].
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
online. |
What is measured through the NOMe-Seq methodology? | We have developed a method (NOMe-seq) that uses a GpC methyltransferase (M.CviPI) and next generation sequencing to generate a high resolution footprint of nucleosome positioning genome-wide using less than 1 million cells while retaining endogenous DNA methylation information from the same DNA strand. DNaseI-seq and NOMe-seq. | Despite the fact that 45% of all human gene promoters do not contain CpG
islands, the role of DNA methylation in control of non-CpG island promoters is
controversial and its relevance in normal and pathological processes is poorly
understood. Among the few studies which investigate the correlation between DNA
methylation and expression of genes with non-CpG island promoters, the majority
do not support the view that DNA methylation directly leads to transcription
silencing of these genes. Our reporter assays and gene reactivation by
5-aza-2'-deoxycytidine, a DNA demethylating agent, show that DNA methylation
occurring at CpG poor LAMB3 promoter and RUNX3 promoter 1(RUNX3 P1) can directly
lead to transcriptional silencing in cells competent to express these genes in
vitro. Using Nucleosome Occupancy Methylome- Sequencing, NOMe-Seq, a
single-molecule, high-resolution nucleosome positioning assay, we demonstrate
that active, but not inactive, non-CpG island promoters display a
nucleosome-depleted region (NDR) immediately upstream of the transcription start
site (TSS). Furthermore, using NOMe-Seq and clonal analysis, we show that in
RUNX3 expressing 623 melanoma cells, RUNX3 P1 has two distinct chromatin
configurations: one is unmethylated with an NDR upstream of the TSS; another is
methylated and nucleosome occupied, indicating that RUNX3 P1 is monoallelically
methylated. Together, these results demonstrate that the epigenetic signatures
comprising DNA methylation, histone marks and nucleosome occupancy of non-CpG
island promoters are almost identical to CpG island promoters, suggesting that
aberrant methylation patterns of non-CpG island promoters may also contribute to
tumorigenesis and should therefore be included in analyses of cancer
epigenetics. 5-Aza-2'-deoxycytidine, approved by the FDA for the treatment of myelodysplastic
syndrome (MDS), is incorporated into the DNA of dividing cells where it
specifically inhibits DNA methylation by forming covalent complexes with the DNA
methyltransferases (DNMTs). In an effort to study the correlations between DNA
methylation, nucleosome remodeling, and gene reactivation, we investigate the
integrated epigenetic events that worked coordinately to reprogram the
methylated and closed promoters back to permissive chromatin configurations
after 5-Aza-2'-deoxycytidine treatment. The ChIP results indicate that H2A.Z is
deposited at promoter regions by the Snf2-related CBP activator protein (SRCAP)
complex following DNA demethylation. According to our genome-wide expression and
DNA methylation profiles, we find that the complete re-activation of silenced
genes requires the insertion of the histone variant H2A.Z, which facilitates the
acquisition of regions fully depleted of nucleosome as demonstrated by NOMe-seq
(Nucleosome Occupancy Methylome-sequencing) assay. In contrast, SRCAP-mediated
H2A.Z deposition is not required for maintaining the active status of
constitutively expressed genes. By combining Hpa II digestion with NOMe-seq
assay, we show that hemimethylated DNA, which is generated following drug
incorporation, remains occupied by nucleosomes. Our data highlight H2A.Z as a
novel and essential factor involved in 5-Aza-2'-deoxycytidine-induced gene
reactivation. Furthermore, we elucidate that chromatin remodeling translates the
demethylation ability of DNMT inhibitors to their downstream efficacies,
suggesting future therapeutic implications for chromatin remodelers. DNA methylation and nucleosome positioning work together to generate chromatin
structures that regulate gene expression. Nucleosomes are typically mapped using
nuclease digestion requiring significant amounts of material and varying enzyme
concentrations. We have developed a method (NOMe-seq) that uses a GpC
methyltransferase (M.CviPI) and next generation sequencing to generate a high
resolution footprint of nucleosome positioning genome-wide using less than 1
million cells while retaining endogenous DNA methylation information from the
same DNA strand. Using a novel bioinformatics pipeline, we show a striking
anti-correlation between nucleosome occupancy and DNA methylation at CTCF
regions that is not present at promoters. We further show that the extent of
nucleosome depletion at promoters is directly correlated to expression level and
can accommodate multiple nucleosomes and provide genome-wide evidence that
expressed non-CpG island promoters are nucleosome-depleted. Importantly,
NOMe-seq obtains DNA methylation and nucleosome positioning information from the
same DNA molecule, giving the first genome-wide DNA methylation and nucleosome
positioning correlation at the single molecule, and thus, single cell level,
that can be used to monitor disease progression and response to therapy. It is well established that cancer-associated epigenetic repression occurs
concomitant with CpG island hypermethylation and loss of nucleosomes at
promoters, but the role of nucleosome occupancy and epigenetic reprogramming at
distal regulatory elements in cancer is still poorly understood. Here, we
evaluate the scope of global epigenetic alterations at enhancers and insulator
elements in prostate and breast cancer cells using simultaneous genome-wide
mapping of DNA methylation and nucleosome occupancy (NOMe-seq). We find that the
genomic location of nucleosome-depleted regions (NDRs) is mostly cell type
specific and preferentially found at enhancers in normal cells. In cancer cells,
however, we observe a global reconfiguration of NDRs at distal regulatory
elements coupled with a substantial reorganization of the cancer methylome.
Aberrant acquisition of nucleosomes at enhancer-associated NDRs is associated
with hypermethylation and epigenetic silencing marks, and conversely, loss of
nucleosomes with demethylation and epigenetic activation. Remarkably, we show
that nucleosomes remain strongly organized and phased at many facultative distal
regulatory elements, even in the absence of a NDR as an anchor. Finally, we find
that key transcription factor (TF) binding sites also show extensive peripheral
nucleosome phasing, suggesting the potential for TFs to organize NDRs
genome-wide and contribute to deregulation of cancer epigenomes. Together, our
findings suggest that "decommissioning" of NDRs and TFs at distal regulatory
elements in cancer cells is accompanied by DNA hypermethylation susceptibility
of enhancers and insulator elements, which in turn may contribute to an altered
genome-wide architecture and epigenetic deregulation in maligcy. Author information:
(1)Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer
Center, Keck School of Medicine, University of Southern California, Los Angeles,
California 90033, USA; Program in Genetic, Molecular and Cellular Biology, Keck
School of Medicine, University of Southern California, Los Angeles, California
90033, USA;
(2)Program in Genetic, Molecular and Cellular Biology, Keck School of Medicine,
University of Southern California, Los Angeles, California 90033, USA; USC
Epigenome Center, University of Southern California, Los Angeles, California
90033, USA;
(3)Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer
Center, Keck School of Medicine, University of Southern California, Los Angeles,
California 90033, USA;
(4)Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer
Center, Keck School of Medicine, University of Southern California, Los Angeles,
California 90033, USA; Van Andel Institute, Grand Rapids, Michigan 49503, USA;
[email protected] [email protected].
(5)USC Epigenome Center, University of Southern California, Los Angeles,
California 90033, USA; Department of Preventive Medicine, University of Southern
California, Los Angeles, California 90033, USA [email protected]
[email protected]. DNA methylation and nucleosome positioning are two key mechanisms that
contribute to the epigenetic control of gene expression. During carcinogenesis,
the expression of many genes is altered alongside extensive changes in the
epigenome, with repressed genes often being associated with local DNA
hypermethylation and gain of nucleosomes at their promoters. However the
spectrum of alterations that occur at distal regulatory regions has not been
extensively studied. To address this we used Nucleosome Occupancy and
Methylation sequencing (NOMe-seq) to compare the genome-wide DNA methylation and
nucleosome occupancy profiles between normal and cancer cell line models of the
breast and prostate. Here we describe the bioinformatic pipeline and methods
that we developed for the processing and analysis of the NOMe-seq data published
by (Taberlay et al., 2014 [1]) and deposited in the Gene Expression Omnibus with
accession GSE57498. The study of epigenetic heterogeneity at the level of individual cells and in
whole populations is the key to understanding cellular differentiation,
organismal development, and the evolution of cancer. We develop a statistical
method, epiG, to infer and differentiate between different epi-allelic
haplotypes, annotated with CpG methylation status and DNA polymorphisms, from
whole-genome bisulfite sequencing data, and nucleosome occupancy from NOMe-seq
data. We demonstrate the capabilities of the method by inferring allele-specific
methylation and nucleosome occupancy in cell lines, and colon and tumor samples,
and by benchmarking the method against independent experimental data. Gaining insights into the regulatory mechanisms that underlie the
transcriptional variation observed between individual cells necessitates the
development of methods that measure chromatin organization in single cells. Here
I adapted Nucleosome Occupancy and Methylome-sequencing (NOMe-seq) to measure
chromatin accessibility and endogenous DNA methylation in single cells
(scNOMe-seq). scNOMe-seq recovered characteristic accessibility and DNA
methylation patterns at DNase hypersensitive sites (DHSs). An advantage of
scNOMe-seq is that sequencing reads are sampled independently of the
accessibility measurement. scNOMe-seq therefore controlled for fragment loss,
which enabled direct estimation of the fraction of accessible DHSs within
individual cells. In addition, scNOMe-seq provided high resolution of chromatin
accessibility within individual loci which was exploited to detect footprints of
CTCF binding events and to estimate the average nucleosome phasing distances in
single cells. scNOMe-seq is therefore well-suited to characterize the chromatin
organization of single cells in heterogeneous cellular mixtures. |
What is the function of the dormancy survival regulator (DosR) in Mycobacterium tuberculosis? | During this phase, at least 48 genes, collectively named Dormancy survival regulator (DosR) regulon, are important for the long-term survival of bacilli under a non-respiring state. | Mycobacterium tuberculosis residing within pulmonary granulomas and cavities
represents an important reservoir of persistent organisms during human latent
tuberculosis infection. We present a novel in vivo model of tuberculosis
involving the encapsulation of bacilli in semidiffusible hollow fibers that are
implanted subcutaneously into mice. Granulomatous lesions develop around these
hollow fibers, and in this microenvironment, the organisms demonstrate an
altered physiologic state characterized by stationary-state colony-forming unit
counts and decreased metabolic activity. Moreover, these organisms show an
antimicrobial susceptibility pattern similar to persistent bacilli in current
models of tuberculosis chemotherapy in that they are more susceptible to the
sterilizing drug, rifampin, than to the bactericidal drug isoniazid. We used
this model of extracellular persistence within host granulomas to study both
gene expression patterns and mutant survival patterns. Our results demonstrate
induction of dosR (Rv3133c) and 20 other members of the DosR regulon believed to
mediate the transition into dormancy, and that rel(Mtb) is required for
Mycobacterium tuberculosis survival during extracellular persistence within host
granulomas. Interestingly, the dormancy phenotype of extracellular M.
tuberculosis within host granulomas appears to be immune mediated and
interferon-gamma dependent. In Mycobacterium tuberculosis, the sensor kinases DosT and DosS activate the
transcriptional regulator DosR, resulting in the induction of the DosR regulon,
which is important for anaerobic survival and perhaps latent infection. The
individual and collective roles of these sensors have been postulated
biochemically, but their roles in vivo have remained unclear. This work
demonstrates distinct and additive roles for each sensor during anaerobic
dormancy. Both sensors are necessary for wild-type levels of DosR regulon
induction, and concomitantly, full induction of the regulon is required for
wild-type anaerobic survival. In the anaerobic model, DosT plays an early role,
responding to hypoxia. DosT then induces the regulon and with it DosS, which
sustains and further induces the regulon. DosT then loses its functionality as
oxygen becomes limited, and DosS alone maintains induction of the genes from
that point forward. Thus, M. tuberculosis has evolved a system whereby it
responds to hypoxic conditions in a stepwise fashion as it enters an anaerobic
state. Mycobacterium tuberculosis (Mtb), the pathogen causing tuberculosis, continues
to elude a cure. Latent Mtb forms are present in human population for extended
periods and have the potential to be re-activated into an active form. The
prophylactic vaccine, live-attenuated Mycobacterium bovis
Bacillus-Calmette-Guerin (BCG) vaccine is not effective in preventing latent
infection. The failure of BCG in prevention/protection against latent forms of
Mtb calls for efforts to curb latent Mtb infection. The inclusion of
latency/dormancy antigens in the classical antigen preparation is surmised as a
strategy. DosR (Dormancy Survival Regulator, Rv3133c) regulon genes are
expressed under the conditions of latency/dormancy. Previous bioinformatics
analyses have pointed towards their role as probable vaccine candidates. Since
nearly 60% of DosR regulon genes are unotated, efforts towards elucidating
their functional role will prove valuable. The study presented here provides an
in-depth in silico 3D-structure prediction and functional analyses of the first
member of the DosR regulon group, the hypothetical protein, Rv0079. A
combination of approaches such as: homology modeling and threading using
SWISS-MODEL workspace, Phyre and BioInfo bank Metaserver; protein localization
predictions using PSORTb, LOCtree, TMHMM and TMpred; function prediction using
ProFunc, epitope prediction using NetCTL and others was implemented. Evidence
gathered from a combination of bioinformatics tools supports the hypothesis that
Mtb Rv0079 protein is a likely cytoplasmic translation factor. Experimental
validation will help provide more insight into its actual function. With 2 million deaths per year, TB remains the most significant bacterial
killer. The long duration of chemotherapy and the large pool of latently
infected people represent challenges in disease control. To develop drugs that
effectively eradicate latent infection and shorten treatment duration, the
pathophysiology of the causative agent Mycobacterium tuberculosis needs to be
understood. The discovery that the tubercle bacillus can develop a drug-tolerant
dormant form and the identification of the underlying genetic program 10 years
ago paved the way for a deeper understanding of the life of the parasite inside
human lesions and for new approaches to antimycobacterial drug discovery. Here,
we summarize what we have learnt since the discovery of the master regulator of
dormancy, DosR, and the key gaps in our knowledge that remain. Furthermore, we
discuss a possible wider clinical relevance of DosR for 'nontuberculous
mycobacteria'. It is thought that during latent infection, Mycobacterium tuberculosis bacilli
are retained within granulomas in a low-oxygen environment. The dormancy
survival (Dos) regulon, regulated by the response regulator DosR, appears to be
essential for hypoxic survival in M. tuberculosis, but it is not known how the
regulon promotes survival. Here we report that mycobacteria, in contrast to
enteric bacteria, do not form higher-order structures (e.g. ribosomal dimers)
upon entry into stasis. Instead, ribosomes are stabilized in the associated form
(70S). Using a strategy incorporating microfluidic, proteomic, and ribosomal
profiling techniques to elucidate the fate of mycobacterial ribosomes during
hypoxic stasis, we show that the dormancy regulator DosR is required for optimal
ribosome stabilization. We present evidence that the majority of this effect is
mediated by the DosR-regulated protein MSMEG_3935 (a S30AE domain protein),
which is associated with the ribosome under hypoxic conditions. A Δ3935 mutant
phenocopies the ΔdosR mutant during hypoxia, and complementation of ΔdosR with
the MSMEG_3935 gene leads to complete recovery of dosR mutant phenotypes during
hypoxia. We suggest that this protein is named ribosome-associated factor under
hypoxia (RafH) and that it is the major factor responsible for DosR-mediated
hypoxic survival in mycobacteria. Mycobacterium tuberculosis is a major human pathogen that has evolved survival
mechanisms to persist in an immune-competent host under a dormant condition. The
regulation of M. tuberculosis metabolism during latent infection is not clearly
known. The dormancy survival regulon (DosR regulon) is chiefly responsible for
encoding dormancy related functions of M. tuberculosis. We describe functional
characterization of an important gene of DosR regulon, Rv0079, which appears to
be involved in the regulation of translation through the interaction of its
product with bacterial ribosomal subunits. The protein encoded by Rv0079,
possibly, has an inhibitory role with respect to protein synthesis, as revealed
by our experiments. We performed computational modelling and docking simulation
studies involving the protein encoded by Rv0079 followed by in vitro translation
and growth curve analysis experiments, involving recombit E. coli and Bacille
Calmette Guérin (BCG) strains that overexpressed Rv0079. Our observations
concerning the interaction of the protein with the ribosomes are supportive of
its role in regulation/inhibition of translation. We propose that the protein
encoded by locus Rv0079 is a 'dormancy associated translation inhibitor' or
DATIN. One of the challenges faced by Mycobacterium tuberculosis (M. tuberculosis) in
dormancy is hypoxia. DosR/DevR of M. tuberculosis is a two component dormancy
survival response regulator which induces the expression of 48 genes. In this
study, we have used DosR regulon proteins of M. tuberculosis H37Rv as the query
set and performed a comprehensive homology search against the non-redundant
database. Homologs were found in environmental mycobacteria, environmental
bacteria and archaebacteria. Analysis of genomic context of DosR regulon
revealed that they are distributed as nine blocks in the genome of M.
tuberculosis with many transposases and integrases in their vicinity. Further,
we classified DosR regulon proteins into eight functional categories. One of the
hypothetical proteins Rv1998c could probably be a methylisocitrate lyase or a
phosphonomutase. Another hypothetical protein, Rv0572 was found only in
mycobacteria. Insights gained in this study can potentially aid in the
development of novel therapeutic interventions. Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), which claims
approximately two million people annually, remains a global health concern. The
non-replicating or dormancy like state of this pathogen which is impervious to
anti-tuberculosis drugs is widely recognized as the culprit for this scenario.
The dormancy survival regulator (DosR) regulon, composed of 48 co-regulated
genes, is held as essential for Mtb persistence. The DosR regulon is regulated
by a two-component regulatory system consisting of two sensor kinases-DosS
(Rv3132c) and DosT (Rv2027c), and a response regulator DosR (Rv3133c). The
underlying regulatory mechanism of DosR regulon expression is very complex. Many
factors are involved, particularly the oxygen tension. The DosR regulon enables
the pathogen to persist during lengthy hypoxia. Comparative genomic analysis
demonstrated that the DosR regulon is widely distributed among the mycobacterial
genomes, ranging from the pathogenic strains to the environmental strains.
In-depth studies on the DosR response should provide insights into its role in
TB latency in vivo and shape new measures to combat this exceeding recalcitrant
pathogen. Upon oxygen shift-down, Mycobacterium tuberculosis complex bacteria can induce a
genetic program characterized by halted duplication, which is called
Non-replicating persistence (NRP). During this phase, at least 48 genes,
collectively named Dormancy survival regulator (DosR) regulon, are important for
the long-term survival of bacilli under a non-respiring state, a condition that
bacilli encounter inside granulomatous lesions. It remains unclear whether
expression of NRP genes occurs within the tissue of Mycobacterium bovis
naturally infected cattle. In order to start dissecting this question, total RNA
from bovine lymph node tissues of sacrificed tuberculin reacting animals was
isolated and transcription of genes required for in vivo duplication (esxB and
fbpB) and in vitro NRP (hspX, pfkB, and mb2660c) were analyzed by RT-PCR
approaches. Detection of transcripts was positive in bovine tissue samples for
genes hspX, pfkB, and mb2660c in 84, 32, and 21%, respectively. NRP genes were
upregulated even in animals with a negative IFN-γ in vitro test, and the
expression of NRP genes occurred more often than expression of the esxB gene. The latency global regulator DosR regulon of Mycobacterium tuberculosis, which
is stimulated by hypoxia, comprises approximately fifty genes including ctpF
(Rv1997), which encodes a putative alkali/alkaline earth ion transporter of the
plasma membrane. In this work, the influence of hypoxia and M. tuberculosis DosR
on the ATPase activity of mycobacterial plasma membrane was assessed. We
performed bioinformatic analyses which indicated that the pma1 gene product is
the M. smegmatis ortholog of the M. tuberculosis cation transporter CtpF. In
addition, a possible Na(+), K(+) and/or Ca(2+) pumping mediated by Pma1 was also
predicted. Enzymatic analyses indicated that the basal ATPase activity of plasma
membrane vesicles from M. smegmatis cells cultured under hypoxia and
over-expressing DosR, decreased 30 and 40 % respectively in comparison to
oxygenated cells. In contrast, the specific Na(+)/K(+) and Ca(2+) ATPase
activities of the plasma membrane increased 2.8- and 3.5-fold, respectively,
under hypoxia, similar to that observed for cells over-expressing the DosR
regulator. In agreement, RT-qPCR experiments demonstrated that the transcription
level of the pma1 gene increased under hypoxia at levels similar to that of M.
smegmatis cells over-expressing the M. tuberculosis DosR regulator. The entire
findings suggest that hypoxia stimulates Na(+)/K(+) and Ca(2+) ATPase activities
in the mycobacterial plasma membrane, and this is possibly mediated by the
dormancy regulator DosR. Latent tuberculosis infection (LTBI) is evidence of immunological control of
tuberculosis. Dormancy survival regulator (DosR) regulon-encoded proteins may
have a role in the maintece of LTBI. T cell responses to Rv1733c, Rv0081,
Rv1735c, and Rv1737c DosR regulon-encoded proteins were found to be most
frequent among household contacts of TB cases from Uganda compared to other DosR
proteins, but antibody responses were not described. We characterized antibody
responses to these proteins in individuals from Uganda. Antibodies to Rv1733c,
Rv0081, Rv1735c, and Rv1737c DosR regulon-encoded proteins were measured in 68
uninfected individuals, 62 with LTBI, and 107 with active pulmonary tuberculosis
(APTB) cases. There were no differences in the concentrations of antibodies to
Rv0081, Rv1735c, and Rv1737c DosR regulon-encoded proteins between individuals
with LTBI and APTB and those who were uninfected. LTBI was associated with
higher concentrations of antibodies to Rv1733c in female participants [adjusted
geometric mean ratio: 1.812, 95% confidence interval (CI): 1.105 2.973, and p =
0.019] but not in males (p value for interaction = 0.060). Antibodies to the
four DosR regulon-encoded proteins investigated may not serve as good biomarkers
of LTBI in the general population. More of the M.tb proteome needs to be
screened to identify proteins that induce strong antibody responses in LTBI. |
Which are the effects of ALDH2 deficiency? | In alcohol drinkers, ALDH2-deficiency is a well-known risk factor for upper aerodigestive tract cancers, i.e., head and neck cancer and esophageal cancer. Diabetic patients with ALDH2 mutations are predisposed to worse diastolic dysfunction.
These data demonstrate that ALDH2 deficiency enhances EtOH-induced disruption of intestinal epithelial tight junctions, barrier dysfunction, and liver damage. | Acetaldehyde (ACH) associated with alcoholic beverages is Group 1 carcinogen to
humans (IARC/WHO). Aldehyde dehydrogenase (ALDH2), a major ACH eliminating
enzyme, is genetically deficient in 30-50% of Eastern Asians. In alcohol
drinkers, ALDH2-deficiency is a well-known risk factor for upper aerodigestive
tract cancers, i.e., head and neck cancer and esophageal cancer. However, there
is only a limited evidence for stomach cancer. In this study we demonstrated for
the first time that ALDH2 deficiency results in markedly increased exposure of
the gastric mucosa to acetaldehyde after intragastric administration of alcohol.
Our finding provides concrete evidence for a causal relationship between
acetaldehyde and gastric carcinogenesis. A plausible explanation is the gastric
first pass metabolism of ethanol. The gastric mucosa expresses alcohol
dehydrogenase (ADH) enzymes catalyzing the oxidation of ethanol to acetaldehyde,
especially at the high ethanol concentrations prevailing in the stomach after
the consumption of alcoholic beverages. The gastric mucosa also possesses the
acetaldehyde-eliminating ALDH2 enzyme. Due to decreased mucosal ALDH2 activity,
the elimination of ethanol-derived acetaldehyde is decreased, which results in
its accumulation in the gastric juice. We also demonstrate that ALDH2
deficiency, proton pump inhibitor (PPI) treatment, and L-cysteine cause
independent changes in gastric juice and salivary acetaldehyde levels,
indicating that intragastric acetaldehyde is locally regulated by gastric
mucosal ADH and ALDH2 enzymes, and by oral microbes colonizing an achlorhydric
stomach. Markedly elevated acetaldehyde levels were also found at low
intragastric ethanol concentrations corresponding to the ethanol levels of many
foodstuffs, beverages, and dairy products produced by fermentation. A capsule
that slowly releases L-cysteine effectively eliminated acetaldehyde from the
gastric juice of PPI-treated ALDH2-active and ALDH2-deficient subjects. These
results provide entirely novel perspectives for the prevention of gastric
cancer, especially in established risk groups. BACKGROUND: Acetaldehyde, the toxic ethanol (EtOH) metabolite, disrupts
intestinal epithelial barrier function. Aldehyde dehydrogenase (ALDH) detoxifies
acetaldehyde into acetate. Subpopulations of Asians and Native Americans show
polymorphism with loss-of-function mutations in ALDH2. We evaluated the effect
of ALDH2 deficiency on EtOH-induced disruption of intestinal epithelial tight
junctions and adherens junctions, gut barrier dysfunction, and liver injury.
METHODS: Wild-type and ALDH2-deficient mice were fed EtOH (1 to 6%) in
Lieber-DeCarli diet for 4 weeks. Gut permeability in vivo was measured by
plasma-to-luminal flux of FITC-inulin, tight junction and adherens junction
integrity was analyzed by confocal microscopy, and liver injury was assessed by
the analysis of plasma transaminase activity, histopathology, and liver
triglyceride.
RESULTS: EtOH feeding elevated colonic mucosal acetaldehyde, which was
significantly greater in ALDH2-deficient mice. ALDH2(-/-) mice showed a drastic
reduction in the EtOH diet intake. Therefore, this study was continued only in
wild-type and ALDH2(+/-) mice. EtOH feeding elevated mucosal inulin permeability
in distal colon, but not in proximal colon, ileum, or jejunum of wild-type mice.
In ALDH2(+/-) mice, EtOH-induced inulin permeability in distal colon was not
only higher than that in wild-type mice, but inulin permeability was also
elevated in the proximal colon, ileum, and jejunum. Greater inulin permeability
in distal colon of ALDH2(+/-) mice was associated with a more severe
redistribution of tight junction and adherens junction proteins from the
intercellular junctions. In ALDH2(+/-) mice, but not in wild-type mice, EtOH
feeding caused a loss of junctional distribution of tight junction and adherens
junction proteins in the ileum. Histopathology, plasma transaminases, and liver
triglyceride analyses showed that EtOH-induced liver damage was significantly
greater in ALDH2(+/-) mice compared to wild-type mice.
CONCLUSIONS: These data demonstrate that ALDH2 deficiency enhances EtOH-induced
disruption of intestinal epithelial tight junctions, barrier dysfunction, and
liver damage. Humans are cumulatively exposed to acetaldehyde from various sources including
alcoholic beverages, tobacco smoke, foods and beverages. The
genetic-epidemiologic and biochemical evidence in ALDH2-deficient humans
provides strong evidence for the causal relationship between
acetaldehyde-exposure due to alcohol consumption and cancer of the upper
digestive tract. The risk assessment has so far relied on thresholds based on
animal toxicology with lower one-sided confidence limit of the benchmark dose
values (BMDL) typically ranging between 11 and 63 mg/kg bodyweight (bw)/day
dependent on species and endpoint. The animal data is problematic for regulatory
toxicology for various reasons (lack in study quality, problems in animal models
and appropriateness of endpoints - especially cancer - for transfer to humans).
In this study, data from genetic epidemiologic and biochemical studies are
reviewed. The increase in the daily exposure dose to acetaldehyde in
alcohol-consuming ALDH2-deficients vs. ALDH2-actives was about twofold. The
acetaldehyde increase due to ALDH2 inactivity was calculated to be
6.7 μg/kg bw/day for heavy drinkers, which is associated with odds ratios of up
to 7 for head and neck as well as oesophageal cancer. Previous animal toxicology
based risk assessments may have underestimated the risk of acetaldehyde. Risk
assessments of acetaldehyde need to be revised using this updated evidence. |
What is the role of the positive effector of transcription (pet) in the hepatitis B virus? | This element, which we have named pet (positive effector of transcription), exerts its effect in cis in a position and orientation-dependent manner, suggesting that it may function as part of the nascent pregenome transcript. In the presence of this region, deletion of pet activates transcription from downstream promoters, suggesting that pregenome transcription complexes fail to reach the downstream promoters. In vitro transcription experiments support the model that pet is required for transcription elongation on the DHBV template. We speculate that pet is required to suppress transcription termination during the first passage of pregenome transcription complexes through a viral termination region on the circular viral DNA. | We report the presence of two elements, pet and net, that are required for
proper transcription of the duck hepatitis B virus (DHBV). These regions were
previously identified by using plasmid clones of the virus in transient
expression assays (M. Huang and J. Summers, J. Virol. 68:1564-1572, 1994). In
this study, we further analyzed these regions by using in vitro-synthesized
circular DHBV DNA monomers to mimic the authentic transcriptional template. We
observed that pet was required for pregenome transcription from circular viral
monomers, and in the absence of pet-dependent transcription, expression of the
viral envelope genes was increased. We found that deletion of net in
circularized DNA monomers led to the production of abnormally long transcripts
due to a failure to form 3' ends during transcription. In addition, we report
the presence of a net-like region in the mammalian hepadnavirus woodchuck
hepatitis virus. These results are consistent with a model that net is a region
involved in transcription termination and that in DHBV, pet is required for
transcription complexes to read through this region during the first pass
through net. |
What is the role of Kmt5a in liver? | H4K20 monomethylation maintains genome integrity by regulating proper mitotic condensation, DNA damage response, and replication licensing. In non-dividing hepatic cells, H4K20Me1 is specifically enriched in active gene bodies and dynamically regulated by the antagonistic action of Kmt5a methylase and Kdm7b demethylase. In liver-specific Kmt5a-deficient mice, reduced levels of H4K20Me1 correlated with reduced RNA Pol II release from promoter-proximal regions. Genes regulating glucose and fatty acid metabolism were most sensitive to impairment of RNA Pol II release. Downregulation of glycolytic genes resulted in an energy starvation condition partially compensated by AMP-activated protein kinase (AMPK) activation and increased mitochondrial activity. This metabolic reprogramming generated a highly sensitized state that, upon different metabolic stress conditions, quickly aggravated into a senescent phenotype due to ROS overproduction-mediated oxidative DNA damage. | H4K20 monomethylation maintains genome integrity by regulating proper mitotic
condensation, DNA damage response, and replication licensing. Here, we show
that, in non-dividing hepatic cells, H4K20Me1 is specifically enriched in active
gene bodies and dynamically regulated by the antagonistic action of Kmt5a
methylase and Kdm7b demethylase. In liver-specific Kmt5a-deficient mice, reduced
levels of H4K20Me1 correlated with reduced RNA Pol II release from
promoter-proximal regions. Genes regulating glucose and fatty acid metabolism
were most sensitive to impairment of RNA Pol II release. Downregulation of
glycolytic genes resulted in an energy starvation condition partially
compensated by AMP-activated protein kinase (AMPK) activation and increased
mitochondrial activity. This metabolic reprogramming generated a highly
sensitized state that, upon different metabolic stress conditions, quickly
aggravated into a senescent phenotype due to ROS overproduction-mediated
oxidative DNA damage. The results illustrate how defects in the general process
of RNA Pol II transition into a productive elongation phase can trigger specific
metabolic changes and genome instability. |
Is tretinoin effective for photoaging? | Yes, Tretinoin is commonly used topically in the treatment of photoaging. | Tretinoin was shown in the late 1960s to be useful for the treatment of
disorders associated with abnormal epithelial differentiation; however, because
of irritation, retinoids were only slowly accepted. In the 1970s, evidence
accumulated to show that topical tretinoin could modulate many of the
abnormalities in the epidermis and dermis associated with photoageing. It has
been shown in hairless mice that tretinoin can reverse dermal elastosis with the
formation of new collagen and this has led to clinical trials being carried out
in man. Randomized, controlled trials have shown that topical tretinoin is
effective in the treatment of photoaged skin. In a 16-week randomized, double-blind, vehicle-controlled study of topical
tretinoin in the treatment of photoaging, all patients applied topical tretinoin
to one forearm and vehicle cream to the other. Half of the patients received
tretinoin to the face, and half received vehicle cream. All 30 patients who
completed the study showed statistically significant improvement in photoaging
on the tretinoin-treated forearms, but not on the vehicle-treated forearms.
Fourteen of the 15 patients who received tretinoin to the face had improvement
in photoaging, whereas none of the vehicle-treated patients' faces improved, a
statistically significant difference in response between the two groups.
Statistically significant histologic changes were seen in forearm skin treated
with tretinoin, but not with vehicle cream. Side effects were limited to
irritation of tretinoin-exposed skin. BACKGROUND AND DESIGN: The efficacy of topical tretinoin (all-trans-retinoic
acid) in treating photoaging is well established. Questions that remain are (1)
whether irritation causes all or part of the improvement; (2) the concentration
of tretinoin that maximizes clinical response with minimal side effects; and (3)
the effects of long-term treatment on components of the cutaneous immune system.
To address these issues, 99 photoaged patients completed a 48-week study using
0.1% tretinoin cream (n = 32), 0.025% tretinoin (n = 35), or vehicle (n = 32)
once daily in a double-blind manner. Before and after treatment, we assessed
histologic features, keratinocyte expression of HLA-DR and intercellular
adhesion molecule-1, numbers of epidermal Langerhans' cells and epidermal and
dermal T lymphocytes, and vascularity as measured by dermal endothelial cell
area.
RESULTS: Both 0.1% and 0.025% tretinoin produced statistically significant
overall improvement in photoaging of the face compared with vehicle; there were
no clinically or statistically significant differences in efficacy between the
two concentrations of tretinoin. After 48 weeks, 0.1% and 0.025% tretinoin
produced similar statistically significant epidermal thickening (by 30% and 28%,
respectively) compared with vehicle (11% decrease) and increased vascularity (by
100% and 89%, respectively) compared with vehicle (9% decrease). By various
analyses, irritant side effects (erythema and scaling) were statistically
significantly greater with 0.1% tretinoin than with 0.025% tretinoin. No
significant changes occurred in any immunologic markers when tretinoin and
vehicle treatments were compared.
CONCLUSIONS: Tretinoin 0.1% and 0.025% produce similar clinical and histologic
changes in patients with photoaging, despite significantly greater incidence of
irritation with the higher concentration. The separation between clinical
improvement and irritation suggests that mechanisms other than irritation
dominate tretinoin-induced repair of photoaging in humans. The appearance of photoaged skin is cosmetically unacceptable to many in our
society. Ostensibly, avoidance of ultraviolet light and sunlight from early
childhood is most desirable but not likely to happen in our culture. Tretinoin
is the only pharmacologic compound shown to partially reverse some signs of
photoaging. Improvement with tretinoin therapy has been quantified clinically
and histologically. A major degree of improvement occurs in 6 to 12 months, and
maintece treatment one to three times per week may continue this response.
Tretinoin therapy should optimally be used with daily moisturizer and sunscreen
applications. Psychosocial benefits of tretinoin therapy, use of tretinoin for
intrinsically aged or non-Caucasian skin, and higher-strength tretinoin therapy
for severely photoaged skin need to be further explored. It is possible that
some subsets of patients with photoaged skin may respond better than others. BACKGROUND: Topical tretinoin is effective treatment for both acne and
photoaging. This creates a problem for insurers that cover medication costs,
because treatment of acne is often covered but treatment of photoaging is not.
The age distributions of patients with acne or photoaging are likely to be very
different. Therefore, one approach insurers can use is an age cutoff for
covering the cost of topical tretinoin therapy.
OBJECTIVE: Our purpose was to determine at what age patients are more likely to
receive tretinoin for treatment of acne vulgaris versus other conditions to
provide a rational basis for insurers to set coverage cutoffs.
METHODS: National Ambulatory Medical Care Survey data for the years 1990 to 1994
were analyzed to ascertain the age distribution of acne vulgaris office visits
and treatment with topical acne agents including tretinoin. These data were
compared to office visits and tretinoin treatment of wrinkles, solar elastosis,
and other conditions.
RESULTS: The mean age (+/- standard deviation) of patients seen for acne
vulgaris was 24.3 +/-11.5 years old. The age distribution of topical tretinoin
treatment paralleled the age distribution of acne. Tretinoin treatment of acne
and of nonacne conditions were equal at an age of 44.
CONCLUSION: The distribution of outpatient visits for acne treatment is skewed
toward older patients and persists beyond age 40. A rational age cut-off for
coverage of topical tretinoin treatment is 40 years. Premature skin aging caused by repeated exposure to solar radiation is called
photoaging. Although once considered an irreversible process, it is now
established that photoaging can be treated by topical tretinoin. Both from
carefully designed controlled clinical studies; and basic investigations into
the mechanism by which tretinoin improves photoaged skin, our understanding of
photoaging has been enhanced. This article highlights some of these studies
which have contributed to our knowledge. Topical tretinoin is established as an effective treatment for photoaging. Yet
some confusion still exists about the proper way to use this medication,
confusion that can misguide physicians in their clinical approaches and patients
in their treatment regimens. Most of the misinformation about tretinoin has been
perpetuated from the early days of the drug, when its efficacy for treating the
effects of photoaging was still in dispute. Significant advances in clinical and
basic research in this area have dispelled much of the confusion, clearing the
way for an evidence-based medical approach to tretinoin therapy for photoaging.
This review summarizes recent relevant advances in tretinoin therapy to guide
physicians in treating patients with this safe and effective hormone. To date,
tretinoin remains the only therapeutic agent proved to repair photodamage. * A cream containing 0.05% tretinoin (Retinova((R)) is approved for treatment of
sun-induced skin damage ("photoaging").* Three trials comparing tretinoin with
the excipient show that the effects of tretinoin cream are at best limited and
slow to occur. Furthermore, they disappear on treatment cessation, necessitating
long-term use.* The 0.05% tretinoin cream has poor local tolerability: most
subjects develop irritation and fragile skin and require longer intervals
between each application. Systemic adverse effects occur in some circumstances.*
There are persistent doubts about whether it is safe to use tretinoin during
pregcy. Premature skin aging, or photoaging, results largely from repeated exposure to
ultraviolet (UV) radiation from the sun. Photoaging is characterized clinically
by wrinkles, mottled pigmentation, rough skin, and loss of skin tone; the major
histologic alterations lie in dermal connective tissue. In recent years, a great
deal of research has been done to explain the mechanism by which UV induces
dermal damage. This research has enabled the identification of rational targets
for photoaging prevention strategies. Moreover, studies that have elucidated
photoaging pathophysiology have produced significant evidence that topical
tretinoin (all-trans retinoic acid), the only agent approved so far for the
treatment of photoaging, also works to prevent it. This article summarizes
evidence mainly from studies of human volunteers that provide the basis for the
current model of photoaging and the effects of tretinoin. The efficacy of tretinoin is well established in the treatment of acne and
photoaged skin, however as a typical side effect of tretinoin treatment most
patients develop a low-grade irritant dermatitis. Since isotretinoin topical
treatment usually shows much lower incidence and intensity of adverse effects
than tretinoin topical treatment, histological studies are needed to
scientifically evaluate the effects of isotretinoin application on epidermis and
also to assess if it can be used in anti-aging products as an alternative to
tretinoin. Thus, the aim of this study was to compare the effects of topical use
of tretinoin or isotretinoin on hairless mice epidermis, using appropriate
histopathological and histometric techniques, in order to evaluate the influence
of isomerism on skin effects. For this, gel cream formulations containing or not
0.05% tretinoin or 0.05% isotretinoin were applied in the dorsum of hairless
mice, once a day for seven days. Histopathological evaluation, viable epidermal
and horny layer thicknesses as well as the number of epidermal cell layers were
determined. Our results showed that tretinoin and isotretinoin were effective in
the enhancement of viable epidermis thickness and number of epidermal cell
layers, suggesting that they could be used for stimulation of cellular renewal.
However isomerism influenced skin effects since isotretinoin had more pronounced
effects than tretinoin in viable epidermis. In addition only isotretinoin
treatment enhanced horny layer thickness when compared to the gel cream
treatment. INTRODUCTION: Topical tretinoin is considered the gold standard to treat
photoaged skin, but it is associated with side effects and only available upon
prescription.
AIM OF THE STUDY: To compare the efficacy, tolerance, and perception of a fixed
proprietary combination (Retinol 0.2%/LR2412 2%) vs. tretinoin 0.025% cream in
women with photoaged skin.
MATERIAL/METHODS: In this randomized, parallel, double-blind, controlled
clinical study, women applied to the entire face for 3 months in the morning a
SPF 50 sunscreen and in the evening either the association of Retinol
0.2%/LR2412 2% or tretinoin 0.025%. Clinical and instrumental parameters were
assessed at days 0, 28, 56, and 84. Subject perception of the efficacy,
tolerance and cosmeticity of the tested products were assessed at days 28, 56,
and 84.
RESULTS: A total of 120 women (60 to Retinol 0.2%/LR2412 2% cream and 60 to
tretinoin 0.025% cream) were included in the study. Both products improved
considerably wrinkles, mottled pigmentation, pores, and global photodamage. No
statistically significant differences were noted between Retinol 0.2%/LR2412 2%
cream and tretinoin 0.025% cream. Adverse effects were mostly graded mild.
Overall, Retinol 0.2%/LR2412 2% cream was better tolerated than tretinoin 0.025%
cream. At all visits, subject perception of the association of Retinol
0.2%/LR2412 2% was either comparable to or better than tretinoin 0.025% cream.
CONCLUSION: The treatment outcome of Retinol 0.2%/LR2412 2% cream does not
differ from the one of tretinoin 0.025% cream. Clinical results were not
statistically different. Furthermore, Retinol 0.2%/LR2412 2% cream is better
tolerated and better perceived by women used to rejuvenation procedures. INTRODUCTION: Research has shown that a disrupted stratum corneum permeability
barrier coupled with chronic inflammation induce signs of extrinsic aging
(photoaging). An novel herbal-based three product cosmeceutical regimen used to
reverse these two anomalies that does not contain retinol, soy, niacinamide,
tea, L-ascorbic acid or esters, hydroxy acids, tocopherol, or growth factors was
tested in six human clinical trials to determine effectiveness and safety in
reversing photoaging.
MATERIALS AND METHODS: Six randomized split face, double blind, prospective,
controlled clinical trials involving a total of 110 subjects compared a
cosmeceutical blend of novel herbs in regimens consisting of one to three
products to several common antiaging topical treatments. These comparative
products include prescription tretinoin, physician strength idebenone, kinetin,
polyhydroxy, lactic and glycolic acids in reversing signs of photoaging.
RESULTS: The novel cosmeceutical blend regimen showed superior efficacy and
safety in all six trials.
DISCUSSION: These trials substantiate that herbs not used in common antiaging
products effectively and safely mitigate and reverse photoaging signs and
symptoms. The novel concept of treating photoaging and preventing its
progression by repairing and optimizing the stratum corneum barrier, while
reversing and inhibiting chronic cutaneous inflammation, has now been proven. BACKGROUND: Salicylic acid (SA) and retinoids, tretinoin (all-trans retinoic
acid [ATRA]), and retinol (all-trans retinol) are widely used as topical agents
for the improvement of photodamage and acne vulgaris. They can be used in daily
take-home products or as part of an in-office procedure, combining the benefits
of a keratolytic (SA) and a retinoid.
OBJECTIVE: The objective of this research was to compare the efficacy for
ameliorating photodamage of topical tretinoin (0.25%) and retinol (0.25%) to
baseline and with each other when applied after a 30% salicylic acid peel on
human facial skin.
METHODS: Twenty female subjects received a full face 30% SA peel followed by the
overnight application of tretinoin to a 1 randomized half-face and retinol to
the opposite side (split-face study). The identical procedure was repeated at
week 2. Double-blinded subject and investigator assessments of the results were
captured at weeks 2 and 4.
RESULTS: By investigator evaluation, both peeling regimens were effective in
improving photodamage parameters compared to baseline. (ATRA P-values at week 4
were: P=.00008 texture, P=.00013 roughness, P=.00221 pores, P=.00098 dryness,
P=.02770 erythema, and P=.00008 overall appearance. Retinol P-values at week 4
were: P=.00019 texture, P=.00053 roughness, P=.00221 pores, P=.00147 dryness,
P=.02770 erythema, and P=.0043 overall appearance.) By subject self-assessment
compared with baseline, both tretinoin and retinol were effective in improving
overall appearance (ATRA P=.0229 and retinol P=.0190). By investigator
evaluation comparing tretinoin with retinol, tretinoin was slightly better than
retinol at week 4 in improving texture P=.00506, roughness P=.01171, and overall
appearance P=.00506. By subject self-assessment comparing tretinoin with
retinol, there was no difference in overall appearance (ATRA P=.2367 and retinol
P=.3613).
CONCLUSION: Either topical tretinoin (0.25%) or retinol (0.25%) can be used
safely and effectively when applied in office immediately after SA peeling to
ameliorate signs of photoaging. BACKGROUND: Topical retinoids are used to treat the visible signs of photoaging.
While efficacious, they are irritating.
OBJECTIVE: Evaluate the effectiveness and tolerability of a double-conjugate
retinoid cream (AlphaRet Overnight Cream; AHA-Ret) in improving visible signs of
photoaging vs 1.0% retinol or 0.025% tretinoin.
METHODS: A 12-week, split-face, randomized trial was conducted in 48 female
subjects, aged 30-65 years with mild to severe photodamage. AHA-Ret was applied
to one side of the face and either retinol (n=24) or tretinoin (n=24) to the
other side (PM). Expert blinded evaluation of images and Nova measurements
occurred at 4, 8, and 12 weeks. Tolerability was assessed throughout the study.
RESULTS: Forty-seven subjects completed the study. AHA-Ret demonstrated
significant reductions in average severity from baseline: Fine Lines/Wrinkles
(P<.001; all time points); Erythema (P=.004, P<.0001; 8 and 12 weeks,
respectively); Dyschromia (P<.0001; all time points); Skin Tone (P<.0001; all
time points), and Pore Size (P=.035, P<.0001; 8 and 12 weeks, respectively).
AHA-Ret induced less Erythema vs retinol at 8 (P=.008) and 12 (P<.02) weeks.
AHA-Ret was noninferior to prescription tretinoin in all categories at 4 and
8 weeks, and for Fine Lines/Wrinkles, Erythema, Dyschromia, and Skin Tone at
12 weeks. Improvements in Hydration occurred at every time point with AHA-Ret
only (P<.04, P<.03, P<.01). Less irritation was reported with AHA-Ret vs retinol
or tretinoin.
CONCLUSIONS: Treatment with a double-conjugate retinoid cream demonstrated early
reductions in photodamage and improvements in Hydration. AHA-Ret induced less
Erythema vs retinol and was more tolerable vs retinol and tretinoin. |
What is the difference between ganglion mother cells (GMC) and intermediate neural precursor cells (INP) in Drosophila? | GMC divides only once to give rise to two post-mitotic cells (neurons or glia), whereas the INP can also self-renew, albeit for fewer rounds than a NSC, and generate GMCs | The approximately 300 distinct neurons comprising each hemineuromere of the
Drosophila embryonic central nervous system are derived from a segmentally
reiterated array of approximately 30 progenitor cells, neuroblasts (NBs). Each
NB has a unique identity and undergoes repeated cell divisions to produce
several smaller secondary precursor cells, ganglion mother cells (GMCs); each
GMC divides once to produce two neurons and/or glia, thereby generating a
specific lineage of neurons/glia. Understanding the generation of neuronal
diversity requires not only elucidation of the molecules and mechanisms that
specify NB identity but also those that act to differentiate between the cell
types produced within one NB lineage. Here we show that the Drosophila Zn finger
protein Klumpfuss (Klu), which shows sequence similarities to the mammalian
Wilm's tumor suppressor (WT-1), acts to differentiate between the identities of
the first two secondary precursor cells produced from one NB lineage. Klu is
expressed in the NB4-2 lineage only after two rounds of NB cell division, in the
second born GMC (GMC4-2b). In loss-of-function mutant embryos, the first born
GMC (GMC4-2a) as well as its progeny neurons are duplicated; we show that this
duplication of the GMC4-2a sublineage arises because GMC4-2b adopts the identity
of GMC4-2a and divides to produce the GMC4-2a progeny. Moreover, when Klu is
ectopically expressed in GMC4-2a, it fails to acquire its normal identity and
fails to produce correctly specified progeny. klu therefore acts to specify the
identity of GMC4-2b and to make it distinct from GMC4-2a. Our findings further
suggest that the determination of GMC cell fate occurs in two steps; the initial
GMC identity is the consequence of inheritance from the maternal NB, however,
the subsequent stabilization of this identity requires functions like klu in the
GMC. Asymmetric cell division is a widespread mechanism in developing tissues that
leads to the generation of cell diversity. In the embryonic central nervous
system of Drosophila melanogaster, secondary precursor cells-ganglion mother
cells (GMCs)-divide and produce postmitotic neurons that take on different cell
fates. In this study, we show that binary fate decision of two pairs of sibling
neurons is accomplished through the interplay of Notch (N) signaling and the
intrinsic fate determit Numb. We show that GMCs have apical-basal polarity
and Numb localization and the orientation of division are coordinated to
segregate Numb to only one sibling cell. The correct positioning of Numb and the
proper orientation of division require Inscuteable (Insc). Loss of insc results
in the generation of equivalent sibling cells. Our results provide evidence that
sibling neuron fate decision is nonstochastic and normally depends on the
presence of Numb in one of the two siblings. Moreover, our data suggest that the
fate of some sibling neurons may be regulated by signals that do not require
lateral interaction between the sibling cells. The bipotential Ganglion Mother Cells, or GMCs, in the Drosophila CNS
asymmetrically divide to generate two distinct post-mitotic neurons. Here, we
show that the midline repellent Slit (Sli), via its receptor Roundabout (Robo),
promotes the terminal asymmetric division of GMCs. In GMC-1 of the RP2/sib
lineage, Slit promotes asymmetric division by down regulating two POU proteins,
Nubbin and Mitimere. The down regulation of these proteins allows the asymmetric
localization of Inscuteable, leading to the asymmetric division of GMC-1.
Consistent with this, over-expression of these POU genes in a late GMC-1 causes
mis-localization of Insc and symmetric division of GMC-1 to generate two RP2s.
Similarly, increasing the dosage of the two POU genes in sli mutant background
enhances the penetrance of the RP2 lineage defects whereas reducing the dosage
of the two genes reduces the penetrance of the phenotype. These results tie a
cell-non-autonomous signaling pathway to the asymmetric division of precursor
cells during neurogenesis. In the Drosophila CNS, neuroblasts undergo self-renewing asymmetric divisions,
whereas their progeny, ganglion mother cells (GMCs), divide asymmetrically to
generate terminal postmitotic neurons. It is not known whether GMCs have the
potential to undergo self-renewing asymmetric divisions. It is also not known
how precursor cells undergo self-renewing asymmetric divisions. Here, we report
that maintaining high levels of Mitimere or Nubbin, two POU proteins, in a GMC
causes it to undergo self-renewing asymmetric divisions. These asymmetric
divisions are due to upregulation of Cyclin E in late GMC and its unequal
distribution between two daughter cells. GMCs in an embryo overexpressing Cyclin
E, or in an embryo mutant for archipelago, also undergo self-renewing asymmetric
divisions. Although the GMC self-renewal is independent of inscuteable and numb,
the fate of the differentiating daughter is inscuteable and numb-dependent. Our
results reveal that regulation of Cyclin E levels, and asymmetric distribution
of Cyclin E and other determits, confer self-renewing asymmetric division
potential to precursor cells, and thus define a pathway that regulates such
divisions. These results add to our understanding of maintece and loss of
pluripotential stem cell identity. Drosophila larval neurogenesis is an excellent system for studying the balance
between self-renewal and differentiation of a somatic stem cell (neuroblast).
Neuroblasts (NBs) give rise to differentiated neurons and glia via intermediate
precursors called GMCs or INPs. We show that E(spl)mγ, E(spl)mβ, E(spl)m8 and
Deadpan (Dpn), members of the basic helix-loop-helix-Orange protein family, are
expressed in NBs but not in differentiated cells. Double mutation for the E(spl)
complex and dpn severely affects the ability of NBs to self-renew, causing
premature termination of proliferation. Single mutations produce only minor
defects, which points to functional redundancy between E(spl) proteins and Dpn.
Expression of E(spl)mγ and m8, but not of dpn, depends on Notch signalling from
the GMC/INP daughter to the NB. When Notch is abnormally activated in NB progeny
cells, overproliferation defects are seen. We show that this depends on the
abnormal induction of E(spl) genes. In fact E(spl) overexpression can partly
mimic Notch-induced overproliferation. Therefore, E(spl) and Dpn act together to
maintain the NB in a self-renewing state, a process in which they are assisted
by Notch, which sustains expression of the E(spl) subset. Neural progenitors of the Drosophila larval brain, called neuroblasts, can be
divided into distinct populations based on patterns of proliferation and
differentiation. Type I neuroblasts produce ganglion mother cells (GMCs) that
divide once to produce differentiated progeny, while type II neuroblasts produce
self-renewing intermediate neural progenitors (INPs) and thus generate lineages
containing many more progeny. We identified Taranis (Tara) as an important
determit of type I lineage-specific neural progenitor proliferation patterns.
Tara is an ortholog of mammalian SERTAD proteins that are known to regulate cell
cycle progression. Tara is differentially-expressed in neural progenitors, with
high levels of expression in proliferating type I neuroblasts but no detectable
expression in type II lineage INPs. Tara is necessary for cell cycle
reactivation in quiescent neuroblasts and for cell cycle progression in type I
lineages. Cell cycle defects in tara mutant neuroblasts are due to decreased
activation of the E2F1/Dp transcription factor complex and delayed progression
through S-phase. Mis-expression of tara in type II lineages delays INP cell
cycle progression and induces premature differentiation of INPs into GMCs.
Premature INP differentiation can also be induced by loss of E2F1/Dp function
and elevated E2F1/Dp expression suppresses Tara-induced INP differentiation. Our
results show that lineage-specific Tara expression is necessary for proper brain
development and suggest that distinct cell cycle regulatory mechanisms exist in
type I versus type II neural progenitors. |
What is the FIRE (Functional Inference of Regulators of Expression) tool? | FIRE (Functional Inference of Regulators of Expression) is a tool to score both noncoding and coding SNVs based on their potential to regulate the expression levels of nearby genes. | |
Does TUC.338 inhibit colorectal cancer? | No. TUC.338 is significantly up-regulated in colorectal cancers (CRC) tissue and CRC cell lines, and the up-regulated TUC.338 is associated with lymph node metastasis. TUC.338 acts as a novel oncogene by targeting the TIMP-1 gene thus promoting colorectal cancer cell migration and invasion. | |
Describe mechanism of action of Napabucasin. | Napabucasin (BBI608) is an orally administered small molecule that blocks stem cell activity in cancer cells by targeting the signal transducer and activator of transcription 3 (STAT3) pathway. | A small population of cells with stem cell-like properties in prostate cancer
(PCa), called prostate cancer stem cells (PrCSCs) or prostate stemness-high
cancer cells, displays highly tumorigenic and metastatic features and may be
responsible for the therapy resistance. A small molecule, napabucasin (BBI608),
recently have been identified with suppression of stemness-high cancer cells in
a variety of cancers. However, the effects of napabucasin on PCa cells as well
as PrCSCs isolated from PCa cells have not yet been defined. The effect of
napabucasin on PCa cells in cell proliferation, colony formation, and cell
migration in vitro were measured by MTS, colony formation assay, and Transwell,
respectively. Flow cytometry was employed to evaluate cell cycle and cell
apoptosis, and the effect on tumorigenesis in vivo was examined by tumor growth
assays. Furthermore, the role of napabucasin on self-renewal and survival of
PrCSCs was evaluated by their ability to grow spheres and cell viability assay,
respectively. Western Blot and qRT-PCR were used to determine the effect of
napabucasin on the expressions of stemness markers. Decrease in cell viability,
colony formation, migration, and survival with cell cycle arrest, higher
sensitivity to docetaxel in vitro, and repressed tumorigenesis in vivo was
observed upon napabucasin treatment. More importantly, napabucasin can obviously
inhibit spherogenesis and even kill PrCSCs in vitro. Downregulation of stemness
markers was observed after PrCSCs were treated with napabucasin. This study
demonstrates that napabucasin may be a novel approach in the treatment of
advanced PCa, specifically for castration-resistant prostate cancer (CRPC). Many pathogenic microorganisms have been demonstrated in atherosclerotic plaques
and in cerebral plaques in dementia. Hyperhomocysteinemia, which is a risk
factor for atherosclerosis and dementia, is caused by dysregulation of
methionine metabolism secondary to deficiency of the allosteric regulator,
adenosyl methionine. Deficiency of adenosyl methionine results from increased
polyamine biosynthesis by infected host cells, causing increased activity of
ornithine decarboxylase, decreased nitric oxide and peroxynitrate formation and
impaired immune reactions. The down-regulation of oxidative phosphorylation that
is observed in aging and dementia is attributed to deficiency of thioretinaco
ozonide oxygen complexed with nicotinamide adenine dinucleotide and phosphate,
which catalyzes oxidative phosphorylation. Adenosyl methionine biosynthesis is
dependent upon thioretinaco ozonide and adenosine triphosphate (ATP), and the
deficiency of adenosyl methionine and impaired immune function in aging are
attributed to depletion of thioretinaco ozonide from mitochondrial membranes.
Allyl sulfides and furanonaphthoquinones protect against oxidative stress and
apoptosis by increasing the endogenous production of hydrogen sulfide and by
inhibiting electron transfer to the active site of oxidative phosphorylation.
Diallyl trisulfide and napabucasin inhibit the signaling by the signal
transducer and activator of transcription 3 (Stat3), potentially enhancing
immune function by effects on T helper lymphocytes and promotion of apoptosis.
Homocysteine promotes endothelial dysfunction and apoptosis by the unfolded
protein response and endoplasmic reticulum stress through activation of the
N-methyl D-aspartate (NMDA) receptor, causing oxidative stress, calcium influx,
apoptosis and endothelial dysfunction. The prevention of atherosclerosis and
dementia may be accomplished by a proposed nutritional metabolic
homocysteine-lowering protocol which enhances immunity and corrects the altered
oxidative metabolism in atherosclerosis and dementia. |
Has ruxolitinib received FDA approval? | Yes, ruxolitinib is FDA approved. In 2011 the oral JAK2 kinase inhibitor ruxolitinib became the first Food and Drug Administration (FDA)-approved drug for the treatment of myelofibrosis. | Chronic myeloproliferative neoplasms (MPN) comprise a spectrum of clonal
neoplastic disorders characterized by overproduction of terminally
differentiated cells of the myeloid lineage. A common genetic basis for the
BCR-ABL-negative MPN disorders was elucidated in 2005 with the identification of
the JAK2V617F mutation in the majority of MPN patients. The discovery of
JAK2V617F had a dramatic impact on the diagnosis and treatment of MPN. Testing
for JAK2 mutations is now included in the World Health Organization (WHO)
criteria for the diagnosis of MPN, and in 2011 the oral JAK2 kinase inhibitor
ruxolitinib became the first Food and Drug Administration (FDA)-approved drug
for the treatment of myelofibrosis. The drug is now also approved in Europe and
Canada. |
Do origins of replication close to yeast centromeres fire early or late? | Epigenetically-inherited centromere and neocentromere DNA replicates earliest in S-phase we discovered that each centromere is associated with a replication origin that is the first to fire on its respective chromosome. | In the yeast Saccharomyces cerevisiae, origins of replication (autonomously
replicating sequences; ARSs), centromeres, and telomeres have been isolated and
characterized. The identification of these structures allows the construction of
artificial chromosomes in which the architecture of eukaryotic chromosomes may
be studied. A common feature of most, and possibly all, natural yeast
chromosomes is that they have an ARS within 2 kilobases of their physical ends.
To study the effects of such telomeric ARSs on chromosome maintece, we
introduced artificial chromosomes of approximately 15 and 60 kilobases into
yeast cells and analyzed the requirements for telomeric ARSs and the effects of
ARS-free chromosomal arms on the stability of these molecules. We find that
terminal blocks of telomeric repeats are sufficient to be recognized as
telomeres. Moreover, artificial chromosomes containing telomere-associated Y'
sequences and telomeric ARSs were no more stable during both mitosis and meiosis
than artificial chromosomes lacking terminal ARSs, indicating that
yeast-specific blocks of telomeric sequences are the only cis-acting requirement
for a functional telomere during both mitotic growth and meiosis. The results
also show that there is no requirement for an origin of replication on each arm
of the artificial chromosomes, indicating that a replication fork may
efficiently move through a functional centromere region. We isolated mutants of Saccharomyces cerevisiae that lose a 100 kb linear yeast
artificial chromosome (YAC) at elevated rates. Mutations in two of these LCS
(linear chromosome stability) genes had little or no effect on the loss rate of
a circular YAC that had the same centromere and origin of replication as present
on the linear YAC. Moreover, mutations in these LCS genes also increased the
loss rate of an authentic linear yeast chromosome, chromosome III, but had only
small effects on the loss rate of a circular derivative of chromosome III. As
these mutants preferentially destabilize linear chromosomes, they may affect
chromosome stability through interactions at telomeres. Telomeres are thought to
be essential for the protection and complete replication of chromosome ends. The
cytological properties of telomeres suggest that these structures may play
additional roles in chromosome function. The lengths of the terminal C1-3A
repeats at the ends of yeast chromosomes were unaltered in the linear
preferential lcs mutants, suggesting that these mutants do not affect the
replication or protection of telomeric DNA. Thus, the linear-preferential lcs
mutants may identify a role for telomeres in chromosome stability that is
distinct from their function in the replication and protection of chromosomal
termini. The fission yeast Schizosaccharomyces pombe normally has haploid cells of two
mating types, which differ at the chromosomal locus mat1. After two consecutive
asymmetric cell divisions, only one in four 'grand-daughter' cells undergoes a
'mating-type switch', in which genetic information is transferred to mat1 from
the mat2-P or mat3-M donor loci. This switching pattern probably results from an
imprinting event at mat1 that marks one sister chromatid in a strand-specific
manner, and is related to a site-specific, double-stranded DNA break at mat1.
Here we show that the genetic imprint is a strand-specific, alkali-labile DNA
modification at mat1. The DNA break is an artefact, created from the imprint
during DNA purification. We also propose and test the model that mat1 is
preferentially replicated by a centromere-distal origin(s), so that the
strand-specific imprint occurs only during lagging-strand synthesis. Altering
the origin of replication, by inverting mat1 or introducing an origin of
replication, affects the imprinting and switching efficiencies in predicted
ways. Two-dimensional gel analysis confirmed that mat1 is preferentially
replicated by a centromere-distal origin(s). Thus, the DNA replication machinery
may confer different developmental potential to sister cells. The budding yeast S phase checkpoint responds to hydroxyurea-induced nucleotide
depletion by preventing replication fork collapse and the segregation of
unreplicated chromosomes. Although the block to chromosome segregation has been
thought to occur by inhibiting anaphase, we show checkpoint-defective rad53
mutants undergo cycles of spindle extension and collapse after hydroxyurea
treatment that are distinct from anaphase cells. Furthermore, chromatid
cohesion, whose dissolution triggers anaphase, is dispensable for S phase
checkpoint arrest. Kinetochore-spindle attachments are required to prevent
spindle extension during replication blocks, and chromosomes with two
centromeres or an origin of replication juxtaposed to a centromere rescue the
rad53 checkpoint defect. These observations suggest that checkpoint signaling is
required to generate an inward force involved in maintaining preanaphase spindle
integrity during DNA replication distress. We propose that by promoting
replication fork integrity under these conditions Rad53 ensures centromere
duplication. Replicating chromosomes can then bi-orient in a cohesin-independent
manner to restrain untimely spindle extension. The centromeric regions of all Saccharomyces cerevisiae chromosomes are found in
early replicating domains, a property conserved among centromeres in fungi and
some higher eukaryotes. Surprisingly, little is known about the biological
significance or the mechanism of early centromere replication; however, the
extensive conservation suggests that it is important for chromosome maintece.
Do centromeres ensure their early replication by promoting early activation of
nearby origins, or have they migrated over evolutionary time to reside in early
replicating regions? In Candida albicans, a neocentromere contains an early
firing origin, supporting the first hypothesis but not addressing whether the
new origin is intrinsically early firing or whether the centromere influences
replication time. Because the activation time of individual origins is not an
intrinsic property of S. cerevisiae origins, but is influenced by surrounding
sequences, we sought to test the hypothesis that centromeres influence
replication time by moving a centromere to a late replication domain. We used a
modified Meselson-Stahl density transfer assay to measure the kinetics of
replication for regions of chromosome XIV in which either the functional
centromere or a point-mutated version had been moved near origins that reside in
a late replication region. We show that a functional centromere acts in cis over
a distance as great as 19 kb to advance the initiation time of origins. Our
results constitute a direct link between establishment of the kinetochore and
the replication initiation machinery, and suggest that the proposed higher-order
structure of the pericentric chromatin influences replication initiation. We generated a genome-wide replication profile in the genome of Lachancea
kluyveri and assessed the relationship between replication and base composition.
This species diverged from Saccharomyces cerevisiae before the ancestral whole
genome duplication. The genome comprises eight chromosomes among which a
chromosomal arm of 1 Mb has a G + C-content much higher than the rest of the
genome. We identified 252 active replication origins in L. kluyveri and found
considerable divergence in origin location with S. cerevisiae and with Lachancea
waltii. Although some global features of S. cerevisiae replication are
conserved: Centromeres replicate early, whereas telomeres replicate late, we
found that replication origins both in L. kluyveri and L. waltii do not behave
as evolutionary fragile sites. In L. kluyveri, replication timing along
chromosomes alternates between regions of early and late activating origins,
except for the 1 Mb GC-rich chromosomal arm. This chromosomal arm contains an
origin consensus motif different from other chromosomes and is replicated early
during S-phase. We showed that precocious replication results from the specific
absence of late firing origins in this chromosomal arm. In addition, we found a
correlation between GC-content and distance from replication origins as well as
a lack of replication-associated compositional skew between leading and lagging
strands specifically in this GC-rich chromosomal arm. These findings suggest
that the unusual base composition in the genome of L. kluyveri could be linked
to replication. |
Is scuba diving safe during pregnancy? | No, scuba diving should be avoided throughout pregnancy because the fetus is at an increased risk for decompression sickness during this activity. | Scuba diving is a leisure activity increasingly popular amongst women. Many
women are concerned about the risks associated with diving and a known or
planned pregcy. In order to advise these young women, we have reviewed the
literature concerning women and diving as well as animal studies on the subject.
The different international federations and the Undersea and Hyperbaric Medical
Society advise against scuba diving for pregt women or those planning a
pregcy, but no randomized trials or trials provide a solid scientific basis.
The fetal circulation is characterized by the exclusion of the pulmonary
circulation by 2 right to left shunts. As the lung appears to act as a filter
against the progression of micro-bubbles to the main circulation, the fetus may
be therefore particularly exposed to gas emboli. However, the placenta could
play this role in certain animal species. Nitrox diving appears to be
particularly promising, but studies on the subject are still insufficient to
recommend it for pregt women. The second trimester is the safest time for travelling, because the pregt
woman feels generally most at ease and the risk of spontaneous abortion and
pre-term labour is very low. Possible risks must be discussed with the
obstetrician before travelling. If the pregcy is uncomplicated most airlines
allow flying up to the 36th (domestic flights) and 35th (international flights)
week of gestation. Unless the fetal oxygen supply is already impaired at ground
level due to an underlying disease, flying does not pose a risk of fetal
hypoxia. Radiation exposure during a long distant flight is low compared to the
average annual exposure dosage, but the risk of thrombosis is increased.
Altitudes up to 2,500 m pose no problem. Sufficient time to acclimatize must be
taken when travelling to high altitudes and exercise kept to a minimum. Scuba
diving is contraindicated. Since only a few drugs are completely safe during
pregcy a thorough risk/benefit evaluation is mandatory. Treatment of
infections can be considerably complicated, but any necessary treatment should
not be withheld because of the fear of potential fetal injury. Good knowledge of
local medical resources is essential before travelling. Several personal
protective measures minimize the risk of infection: food and water precautions,
protection from insect bites and avoidance of crowds, unsafe sex and, if need
be, freshwater. Many vaccinations are recommended for travellers. However, live
vaccines are contraindicated in pregt women because of theoretical
considerations. Exceptionally a yellow fever vaccination may be given after the
first trimester. Killed, inactivated or polysaccharide vaccines can be given
after the first trimester after a thorough risk/benefit evaluation. Because of
the potentially devastating effect of malaria to the mother and the child,
travelling to endemic malaria regions should be avoided. If the risk of
infection is high chemoprophylaxis with mefloquine is indicated. In low-risk
countries mefloquine, in South-East-Asia artemisinin derivatives should be given
as stand-by treatment. |
Does deflazacort have more side effects than prednisone? | Deflazacort produces fewer side effects than Prednisone in DMD patients. | Though Deflazacort and prednisone improve clinical endpoints in Duchenne
muscular dystrophy (DMD) patients, Deflazacort produces fewer side effects. As
mechanisms of improvement and side effect differences remain unknown, we
evaluated effects of corticosteroid administration on gene expression in blood
of DMD patients. Whole blood was obtained from 14 children and adolescents with
DMD treated with corticosteroids (DMD-STEROID) and 20 DMD children and
adolescents naïve to corticosteroids (DMD). The DMD-STEROID group was further
subdivided into Deflazacort and prednisone groups. Affymetrix U133 Plus 2.0
expression microarrays were used to evaluate mRNA expression. Expression of 524
probes changed with corticosteroids, including genes in iron trafficking and the
chondroitin sulfate biosynthesis pathway. Deflazacort compared with prednisone
yielded 508 regulated probes, including many involved in adipose metabolism.
These genes and pathways help explain mechanisms of efficacy and side effects of
corticosteroids, and could provide new treatment targets for DMD and other
neuromuscular disorders. |
Does echinacea increase anaphylaxis risk? | Yes, there is evidence that echinacea use is associated with anaphylaxis. | A woman with atopy experienced anaphylaxis after taking, among other dietary
supplements, a commercial extract of echinacea. Hypersensitivity was confirmed
by skinprick and RAST testing. Regular ingestion of echinacea by up to 5% of
surveyed patients with atopy, combined with detection of echinacea-binding IgE
in atopic subjects (19% by skin testing; 20% with moderate to strong reactivity
by RAST testing), raises the possibility of severe allergic reactions, even with
first-time use, due to cross-reactivity with other structurally similar
allergens. Patients with atopy should be cautioned about the risk of developing
life-threatening reactions to complementary medicines, including echinacea. BACKGROUND: Fifty percent of Australians use complementary and alternative
medicines (other than vitamins) in any 12-month period, of which
echinacea-containing products are increasingly popular. Recent reports have
highlighted the risk of allergic reactions to complementary medicines in atopic
patients.
OBJECTIVE: To determine the characteristics of adverse reactions linked to use
of the popular herbal remedy echinacea.
METHODS: Five privately referred patients were evaluated by the authors in their
office practice via skin prick testing (SPT) on the volar aspect of the forearm
and radioallergosorbent test after adverse reactions to echinacea. As there was
little published information on adverse reactions to echinacea, reports to the
Australian Adverse Drug Reactions Advisory Committee were reviewed. Those
suggestive of possible allergic reactions were evaluated in greater detail by
anonymously surveying the healthcare professionals who had reported the cases
and from one unreported case. Serum was collected for further analysis where
possible.
RESULTS: Five cases of adverse reactions to echinacea were personally evaluated
by the authors. Two patients suffered anaphylaxis and a third had an acute
asthma attack 10 minutes after their first ever dose of echinacea. The fourth
patient suffered recurrent episodes of mild asthma each time echinacea was
ingested, and the fifth developed a maculopapular rash within 2 days of
ingestion which recurred when rechallenged. Three of the patients had positive
SPT results. Three reported repeated spontaneous "challenges" and symptoms after
further ingestion of echinacea. Fifty-one Australian adverse drug reports
implicating echinacea were also reviewed. There were 26 cases suggestive of
possible immunoglobulin E-mediated hypersensitivity (4 anaphylaxis, 12 acute
asthma, 10 urticaria/angioedema). Of these 26 patients, age ranged from 2 to 58
years, 78% were female and >50% were known to be atopic. Four were hospitalized,
4 reacted after their first known exposure, and 1 patient suffered multiple
progressive systemic reactions. Twenty percent of 100 atopic subjects who had
never taken echinacea also had positive SPT results to this substance when
tested by one of the authors in his office practice.
CONCLUSION: Some atopic subjects have positive SPT results to echinacea in the
absence of known exposure. Atopic subjects are also overrepresented in those
experiencing reactions to echinacea. The possibility that cross-reactivity
between echinacea and other environmental allergens may trigger allergic
reactions in "echinacea-naïve" subjects is supported by the Australian data.
Given its widespread (and largely unsupervised) community use, even rare adverse
events become inevitable. Atopic patients should be cautioned appropriately. PURPOSE OF REVIEW: Complementary and alternative medicine (CAM) use is
widespread across the world. Patients with asthma and allergy regularly use CAM
therapies. Allergic and anaphylactic reactions to CAM have been reported.
RECENT FINDINGS: Recent attempts to regulate and monitor adverse reaction to
these therapies have given us further insight into potential causes of severe
allergic reactions. Several culprits identified including Andrographis
paniculata, Echinacea species, bee products, Ginkgo biloba and Ginseng are
discussed here.
SUMMARY: Knowing the factors that increase the risk of anaphylaxis allows
reactions to be recognized, reported and further investigated. Research to
identify key causative allergens is necessary in the future. Collaboration
between the allergy community and CAM practitioners can allow better
understanding of allergy to these therapies. |
Are neurexins localized at pre-synapses? | Yes, neurexins are localized at pre-synapses. | Neurexins and neuroligins are two distinct families of single-pass transmembrane
proteins localized at pre- and postsynapses, respectively. They
trans-synaptically interact with each other and induce synapse formation and
maturation. Common variants and rare mutations, including copy number
variations, short deletions, and single or small nucleotide changes in neurexin
and neuroligin genes have been linked to the neurodevelopmental disorders, such
as autism spectrum disorders (ASDs). In this review, we summarize the structure
and basic synaptic function of neurexins and neuroligins, followed by behaviors
and synaptic phenotypes of knock-in and knock-out mouse of these family genes.
From the studies of these mice, it turns out that the effects of neurexins and
neuroligins are amazingly neural circuit dependent, even within the same brain
region. In addition, neurexins and neuroligins are commonly involved in the
endocannabinoid signaling. These finding may provide not only insight into
understanding the pathophysiology, but also the concept for strategy of
therapeutic intervention for ASDs. Neuroligins are postsynaptic cell-adhesion molecules that bind to presynaptic
neurexins. Mutations in neuroligin-3 predispose to autism, but how such
mutations affect synaptic function remains incompletely understood. Here we
systematically examined the effect of three autism-associated mutations, the
neuroligin-3 knockout, the R451C knockin, and the R704C knockin, on synaptic
transmission in the calyx of Held, a central synapse ideally suited for
high-resolution analyses of synaptic transmission. Surprisingly, germline
knockout of neuroligin-3 did not alter synaptic transmission, whereas the
neuroligin-3 R451C and R704C knockins decreased and increased, respectively,
synaptic transmission. These puzzling results prompted us to ask whether
neuroligin-3 mutant phenotypes may be reshaped by developmental plasticity.
Indeed, conditional knockout of neuroligin-3 during late development produced a
marked synaptic phenotype, whereas conditional knockout of neuroligin-3 during
early development caused no detectable effect, mimicking the germline knockout.
In canvassing potentially redundant candidate genes, we identified
developmentally early expression of another synaptic neurexin ligand,
cerebellin-1. Strikingly, developmentally early conditional knockout of
cerebellin-1 only modestly impaired synaptic transmission, whereas in contrast
to the individual single knockouts, developmentally early conditional double
knockout of both cerebellin-1 and neuroligin-3 severely decreased synaptic
transmission. Our data suggest an uticipated mechanism of developmental
compensation whereby cerebellin-1 and neuroligin-3 functionally occlude each
other during development of calyx synapses. Thus, although acute manipulations
more likely reveal basic gene functions, developmental plasticity can be a major
factor in shaping the overall phenotypes of genetic neuropsychiatric disorders. Perisomatic GABAergic synapses onto hippocampal pyramidal cells arise from two
populations of basket cells with different neurochemical and functional
properties. The presence of the dystrophin-glycoprotein complex in their
postsynaptic density (PSD) distinguishes perisomatic synapses from GABAergic
synapses on dendrites and the axon-initial segment. Targeted deletion of
neuroligin 2 (NL2), a transmembrane protein interacting with presynaptic
neurexin, has been reported to disrupt postsynaptic clustering of GABAA
receptors (GABAAR) and their anchoring protein, gephyrin, at perisomatic
synapses. In contrast, targeted deletion of Gabra2 disrupts perisomatic
clustering of gephyrin, but not of α1-GABAAR, NL2, or dystrophin/dystroglycan.
Unexpectedly, conditional deletion of Dag1, encoding dystroglycan, selectively
prevents the formation of perisomatic GABAergic synapses from basket cells
expressing cholecystokinin. Collectively, these observations suggest that
multiple mechanisms regulate formation and molecular composition of the
GABAergic PSD at perisomatic synapses. Here, we further explored this issue by
investigating the effect of targeted deletion of Gabra1 and NL2 on the
dystrophin-glycoprotein complex and on perisomatic synapse formation, using
immunofluorescence analysis with a battery of GABAergic pre- and postsynaptic
markers. We show that the absence of α1-GABAAR increases GABAergic synapses
containing the α2 subunit, without affecting the clustering of dystrophin and
NL2; in contrast, the absence of NL2 produces highly variable effects
postsynaptically, not restricted to perisomatic synapses and being more severe
for the GABAAR subunits and gephyrin than dystrophin. Altogether, the results
confirm the importance of NL2 as organizer of the GABAergic PSD and unravel
distinct roles for α1- and α2-GABAARs in the formation of GABAergic circuits in
close interaction with the dystrophin-glycoprotein complex. |
Is there any role of 5hmC in T-cell development and differentiation? | Yes. 5hmC is enriched in the gene body of highly expressed genes at all different stages of T-cell development in the thymus and that its presence correlates positively with gene expression. Further emphasizing the connection with gene expression, 5hmC is enriched in active thymus-specific enhancers and genes encoding key transcriptional regulators display high intragenic 5hmC levels in precursor cells at those developmental stages where they exert a positive effect. | Author information:
(1)Department of Signaling and Gene Expression, La Jolla Institute for Allergy
and Immunology, La Jolla, CA 92034;
(2)Department of Signaling and Gene Expression, La Jolla Institute for Allergy
and Immunology, La Jolla, CA 92034;Department of Information and Computer
Science, Aalto University School of Science, FI-00076 Aalto, Finland;
(3)Department of Signaling and Gene Expression, La Jolla Institute for Allergy
and Immunology, La Jolla, CA 92034;Sanford Consortium for Regenerative Medicine,
La Jolla, CA 92037;
(4)Howard Hughes Medical Institute,Department of Molecular, Cell, and
Developmental Biology, andEli and Edythe Broad Center of Regenerative Medicine
and Stem Cell Research, University of California, Los Angeles, CA 90095; and.
(5)Department of Information and Computer Science, Aalto University School of
Science, FI-00076 Aalto, Finland;
(6)Department of Signaling and Gene Expression, La Jolla Institute for Allergy
and Immunology, La Jolla, CA 92034;Sanford Consortium for Regenerative Medicine,
La Jolla, CA 92037;Department of Pharmacology and Moores Cancer Center,
University of California, San Diego, La Jolla, CA 92093 [email protected]. Author information:
(1)Centre for Personalized Medicine, Department of Pediatrics, Faculty of
Medicine, Linköping University, 581 85 Linköping, Sweden. Electronic address:
[email protected].
(2)Centre for Personalized Medicine, Department of Pediatrics, Faculty of
Medicine, Linköping University, 581 85 Linköping, Sweden.
(3)Bioinformatics, Department of Physics, Chemistry and Biology, Linköping
University, 581 83 Linköping, Sweden.
(4)MD Anderson Cancer Center, Houston, TX 77030, USA.
(5)MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine,
University of Edinburgh, Crewe Road, Edinburgh EH4 2XU, UK.
(6)Genomatix Software GmbH, 80335 Munich, Germany.
(7)Research Unit Protein Science, Helmholtz Zentrum München, German Research
Center for Environmental Health GmbH, 85764 Neuherberg, Germany.
(8)Else Kröner-Fresenius-Center for Nutritional Medicine, Chair of Nutritional
Medicine, MRI and ZIEL, Technische Universität München, 85354
Freising-Weihenstephan, Germany; German Center for Diabetes Research (DZD),
Clinical Cooperation Group Nutrigenomics and Type 2 Diabetes at the Helmholtz
Zentrum München, 85764 Neuherberg, Germany; Technische Universität München,
85354 Freising-Weihenstephan, Germany.
(9)Centre for Personalized Medicine, Department of Pediatrics, Faculty of
Medicine, Linköping University, 581 85 Linköping, Sweden. Electronic address:
[email protected]. |
Which are the properties of mammalian GA-sequences? | In this article we identify for the first time explicitly the GA-sequences as a class of fractal genomic sequences that are easy to recognize and to extract, and are scattered densely throughout the chromosomes of a large number of genomes from different species and kingdoms including the human genome. most GA-sequences [1] shared chains of tetra-GA-motifs and contained upstream poly(A)-segments. Although not integral parts of them, Alu-elements were found immediately upstream of all human and chimpanzee GA-sequences with an upstream poly(A)-segment The article hypothesizes that genome navigation uses these properties of GA-sequences in the following way The article describes DNA sequences of mammalian genomes that are longer than 50 bases, but consist exclusively of G's and A's ('pure GA-sequences'). With the exception of a small number of poly-A-, poly-G-, poly-GA-, and poly-GAAA-sequences (combined<0.5%), all pure GA-sequences of the mammals tested were unique individuals, contained several repeated short GA-containing motifs, and shared a common hexa-nucleotide spectrum. | Pyrimidine-purine DNAs with repeating sequences can be made to undergo a
reversible transition to possibly a tetra-stranded complex. Physicochemical
characterization of the new structures and model building are consistent with,
in the case of d(TC)n-d(GA)n, a tetra-stranded complex forming by the addition
of d(GA)n to the remaining space in the major groove of the triple-stranded
complex d(TC)n-d(GA)n-d(CT)n. A possible role for tetra-stranded complexes in
chromosome condensation is suggested by the natural occurrence of repeating
sequence pyrimidine-purine DNAs and the properties of condensed chromosomes. Alternating d(GA.TC)n sequences are highly structurally polymorphic. Most of
their conformational flexibility is likely to reside in the structural
properties of the individual strands themselves. In this paper the
conformational behaviour of the d(GA)20 and d(TC)20 oligonucleotides was
analysed. Formation of d(GA)20 intramolecular duplexes is observed at any pH
value, from 8.3 to 4.6. On the other hand, intramolecular d(TC)20 duplexes are
formed only under acidic conditions. The acid d(TC)20 intramolecular duplex is
likely to be stabilized through the formation of C+C pairs, the thymine residues
remaining unpaired. The d(GA)20 oligonucleotide also forms intermolecular
duplexes which coexist with the intramolecular forms at any pH, from 8.3 to 4.6.
The structural conformation adopted by the d(TC)20 oligonucleotide at neutral pH
is uncertain. Under these conditions, this oligonucleotide shows an
electrophoretic apparent molecular weight consistent with the formation of a
bimolecular complex. However, no hydrogen bonding was observed to occur under
these conditions. Implications of these results for an understanding of the
molecular principles behind the conformational flexibility of alternating
d(GA.TC)n sequences are discussed. The possible biological significance of these
results is also discussed. DNA sequences containing homopurine d(G1-3A)n tracts are known to be capable of
adopting non-B-DNA conformations. The structural polymorphism of these sequences
is a direct consequence of the structural properties of the homopurine d(G1-3A)n
tracts. Depending on the conditions, d(GA)n DNA sequences can form
antiparallel-and parallel-stranded homoduplexes, multistranded complexes, and
ordered single-stranded conformations. On the other hand, much less is known
about the structural properties of d(GGA)n and d(GGGA)n sequences. In this
paper, we show that d(GGA)n and d(GGGA)n repeats form antiparallel-stranded,
intramolecular hairpins. Under physiological salt and pH conditions, the thermal
stability of these hairpin forms is high, showing, at 50 mM NaCl, melting
temperatures in the range of 40-50 degrees C. The base-pairing interactions
involved in the formation of the d(GGA)n and d(GGGA)n hairpins are different.
G.A pairs importantly contribute to the stability of the d(GGA)n hairpins. On
the other hand, the d(GGGA)n hairpins are stabilized by the formation of G.G and
A.A, but not G.A pairs. Homopurine d(G1-3A)n tracts are frequently found at
genomic locations performing specialized chromosomal functions (i.e. telomeres,
centromeres, and recombination "hot-spots"). The molecular interactions
described here are relevant for the understanding of the peculiar structural and
biological properties of DNA sequences containing homopurine tracts. CD spectroscopy and PAGE were used to cooperatively analyze melting conformers
of DNA strands containing GA and TA dinucleotide repeats. The 20mer (GA)10
formed a homoduplex in neutral solutions containing physiological concentrations
of salts and this homoduplex was not destabilized even in the terminal (GA)3
hexamers of (GA)3(TA)4(GA)3, although the central (TA)4 portion of this
oligonucleotide preserved the conformation adopted by (TA)10. This observation
demonstrates that homoduplexes of alternating GA and TA sequences can co-exist
in a single DNA molecule. Another 20mer, (GATA)5, adopted as a whole either the
AT duplex, like (TA)10, or the GA duplex, like (GA)10, and switched between them
reversibly. The concentration of salt controlled the conformational switching.
Hence, guanine and thymine share significant properties regarding
complementarity to adenine, while the TA and GA sequences can stack in at least
two mutually compatible ways within the DNA duplexes analyzed here. These
properties extend our knowledge of non-canonical structures of DNA. Alternating polypurine d(GA)n, sequences exhibit a considerable polymorphism.
Here we report that alpha d(GA) x d(GA) sequences form an antiparallel stranded
duplex DNA at neutral pH. The spectroscopic, electrophoretic and thermodynamic
properties of the alpha/beta chimeric oligodeoxynucleotide, 5'-d(GA)4(T)4 alpha
d(AG)4T-3', support the formation of a hairpin structure with antiparallel
strands in the stem. The optical properties of this novel antiparallel structure
are different from the parallel stranded homoduplex formed by d(GA)G7. This
alpha/beta hairpin has a remarkably high Tm of 44.5 degrees C in 0.4 M NaCl with
a van't Hoff enthalpy comparable to that of a parallel d(GA)n duplex. Base
pairing was confirmed by T4 polynucleotide ligase catalyzed joining of the
alpha/beta hairpin to an antiparallel bimolecular duplex and by non-denaturing
gel electrophoresis using duplexes containing sequence constraints. Both support
the presence of alphaG-G and alphaA-A base pairing in the antiparallel
5'-d(GA)4(T)4 alpha d(AG)4T-3' intramolecular duplex. This study adds to the
polymorphic nature of alternating d(GA)n sequences as well as providing novel
homopurine base pairing approaches for probing polypurine polypyrimidine
sequences. To discover safe and effective topical skin-lightening agents, we have evaluated
alkyl esters of the natural product gentisic acid (GA), which is related to our
lead compound methyl gentisate (MG), and four putative tyrosinase inhibitors,
utilizing mammalian melanocyte cell cultures and cell-free extracts. Desirable
characteristics include the ability to inhibit melanogenesis in cells (IC50 <
100 microg/mL) without cytotoxicity, preferably due to tyrosinase inhibition. Of
the six esters synthesized, the smaller esters (e.g. methyl and ethyl) were more
effective enzyme inhibitors (IC50 approximately 11 and 20 microg/mL,
respectively). For comparison, hydroquinone (HQ), a commercial skin "bleaching"
agent, was a less effective enzyme inhibitor (IC50 approximately 72 microg/mL),
and was highly cytotoxic to melanocytes in vitro at concentrations substantially
lower than the IC50 for enzymatic inhibition. Kojic acid was a potent inhibitor
of the mammalian enzyme (IC50 approximately 6 microg/mL), but did not reduce
pigmentation in cells. Both arbutin and magnesium ascorbyl phosphate were
ineffective in the cell-free and cell-based assays. MG at 100 microg/mL
exhibited a minimal inhibitory effect on DHICA oxidase (TRP 1) and no effect on
DOPAchrome tautomerase (TRP-2), suggesting that MG inhibits melanogenesis
primarily via tyrosinase inhibition. MG and GA were non-mutagenic at the hprt
locus in V79 Chinese hamster cells, whereas HQ was highly mutagenic and
cytotoxic. The properties of MG in vitro, including (1) pigmentation inhibition
in melanocytes, (2) tyrosinase inhibition and selectivity, (3) reduced
cytotoxicity relative to HQ, and (4) lack of mutagenic potential in mammalian
cells, establish MG as a superior candidate skin-lightening agent. The genomic distribution of the abundant eukaryotic d(GA x TC)(n) DNA
microsatellite suggests that it could contribute to DNA recombination. Here, it
is shown that this type of microsatellite DNA sequence enhances DNA
recombination in SV40 minichromosomes, the rate of homologous DNA recombination
increasing by as much as two orders of magnitude in the presence of a d(GA x
TC)(22) sequence. This effect depends on the region of the SV40 genome at which
the d(GA x TC)(22) sequence is cloned. It is high when the sequence is located
proximal to the SV40 control region but no effect is observed when located 3.5
kb away from the SV40 ori. These results indicate that the recombination
potential of d(GA x TC)(n) sequences is likely linked to DNA replication and/or
transcription. The potential contribution of the structural properties of d(GA x
TC)(n) sequences to this effect is discussed. The article describes DNA sequences of mammalian genomes that are longer than 50
bases, but consist exclusively of G's and A's ('pure GA-sequences'). Although
their frequency of incidence should be 10(-16) or smaller, the chromosomes of
human, chimpanzee, dog, cat, rat, and mouse contained many tens of thousands of
them ubiquitously located along the chromosomes with a species-dependent
density, reaching sizes of up to 1300 [b]. With the exception of a small number
of poly-A-, poly-G-, poly-GA-, and poly-GAAA-sequences (combined <0.5%), all
pure GA-sequences of the mammals tested were unique individuals, contained
several repeated short GA-containing motifs, and shared a common hexa-nucleotide
spectrum. At most 2% of the human GA-sequences were transcribed into mRNAs; all
others were not coding for proteins. Although this could have made them less
subject to natural selection, they contained many [corrected] times fewer point
mutations than one should expect from the genome at large. As to the presence of
other sequences with similarly restricted base contents, there were
approximately as many pure TC-sequences as pure GA-sequences, but many fewer
pure AC-, TA, and TG-sequences. There were practically no pure GC-sequences. The
functions of pure GA-sequences are not known. Supported by a number of
observations related to heat shock phenomena, the article speculates that they
serve as genomic sign posts which may help guide polymerases and transcription
factors to their proper targets, and/or as spatial linkers that help generate
the 3-dimensional organization of chromatin. Introducing a new method to visualize large stretches of genomic DNA (see
Appendix S1) the article reports that most GA-sequences [1] shared chains of
tetra-GA-motifs and contained upstream poly(A)-segments. Although not integral
parts of them, Alu-elements were found immediately upstream of all human and
chimpanzee GA-sequences with an upstream poly(A)-segment. The article
hypothesizes that genome navigation uses these properties of GA-sequences in the
following way. (1) Poly(A) binding proteins interact with the upstream
poly(A)-segments and arrange adjacent GA-sequences side-by-side ('GA-ribbon'),
while folding the intervening DNA sequences between them into loops ('associated
DNA-loops'). (2) Genome navigation uses the GA-ribbon as a search path for
specific target genes that is up to 730-fold shorter than the full-length
chromosome. (3) As to the specificity of the search, each molecule of a target
protein is assumed to catalyze the formation of specific oligomers from a set of
transcription factors that recognize tetra-GA-motifs. Their specific
combinations of tetra-GA motifs are assumed to be present in the particular
GA-sequence whose associated loop contains the gene for the target protein. As
long as the target protein is abundant in the cell it produces sufficient
numbers of such oligomers which bind to their specific GA-sequences and,
thereby, inhibit locally the transcription of the target protein in the
associated loop. However, if the amount of target protein drops below a certain
threshold, the resultant reduction of specific oligomers leaves the
corresponding GA-sequence 'denuded'. In response, the associated DNA-loop
releases its nucleosomes and allows transcription of the target protein to
proceed. (4) The Alu-transcripts may help control the general background of
protein synthesis proportional to the number of transcriptionally active
associated loops, especially in stressed cells. (5) The model offers a new
mechanism of co-regulation of protein synthesis based on the shared segments of
different GA-sequences. The existence of fractal sets of DNA sequences have long been suspected on the
basis of statistical analyses of genome data. In this article we identify for
the first time explicitly the GA-sequences as a class of fractal genomic
sequences that are easy to recognize and to extract, and are scattered densely
throughout the chromosomes of a large number of genomes from different species
and kingdoms including the human genome. Their existence and their fractality
may have significant consequences for our understanding of the origin and
evolution of genomes. Furthermore, as universal and natural markers they may be
used to chart and explore the non-coding regions. |
What is the main focus of the CVE R/Bioconductor package? | The CVE package allows interactive variant prioritisation to expedite the analysis of cancer sequencing studies. | |
Are there ultraconserved genomic regions in the budding yeast? | Yes. In addition to some fundamental biological functions, ultraconserved genomic regions play an important role in the adaptation of S. cerevisiae to the acidic environment. | MOTIVATION: In the evolution of species, a kind of special sequences, termed
ultraconserved sequences (UCSs), have been inherited without any change, which
strongly suggests those sequences should be crucial for the species to survive
or adapt to the environment. However, the UCSs are still regarded as mysterious
genetic sequences so far. Here, we present a systematic study of ultraconserved
genomic regions in the budding yeast based on the publicly available genome
sequences, in order to reveal their relationship with the adaptability or
fitness advantages of the budding yeast.
RESULTS: Our results indicate that, in addition to some fundamental biological
functions, the UCSs play an important role in the adaptation of Saccharomyces
cerevisiae to the acidic environment, which is backed up by the previous
observation. Besides that, we also find the highly unchanged genes are enriched
in some other pathways, such as the nutrient-sensitive signaling pathway. To
facilitate the investigation of unique UCSs, the UCSC Genome Browser was
utilized to visualize the chromosomal position and related annotations of UCSs
in S.cerevisiae genome.
AVAILABILITY AND IMPLEMENTATION: For more details on UCSs, please refer to the
Supplementary information online, and the custom code is available on request.
CONTACT: [email protected].
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
online. |
Is davunetide being considered for the treatment of progressive supranuclear palsy? | Yes, Davunetide's efficacy and tolerability are being tested in a placebo-controlled study in PSP patients. | Progressive supranuclear palsy (PSP) is a rare neurodegenerative disease
characterized by the accumulation of tau protein aggregates in the basal
ganglia, brainstem and cerebral cortex leading to rapid disease progression and
death. The neurofibrillary tangles that define the neuropathology of PSP are
comprised of aggregated 4R tau and show a well-defined distribution.
Classically, PSP is diagnosed by symptoms that include progressive gait
disturbance, early falls, vertical ophthalmoparesis, akinetic-rigid features,
prominent bulbar dysfunction and fronto-subcortical dementia. There are
currently no effective therapies for the treatment of this rapidly degenerating
and debilitating disease. Davunetide is a novel neuroprotective peptide that is
thought to impact neuronal integrity and cell survival through the stabilization
of microtubules. Preclinical activity in models of tauopathy has been translated
to clinical studies, demonstrating pharmacologic activity that has supported
further development. Davunetide's efficacy and tolerability are being tested in
a placebo-controlled study in PSP patients, making it the most advanced drug
candidate in this indication. This review examines the disease characteristics
of PSP, the rationale for treating PSP with davunetide and assesses some of the
challenges of clinical trials in this patient population. |
What is the origin of XUT transcripts in yeast? | XUTs are a class of Xrn1-sensitive antisense regulatory non-coding RNA in yeast | Non-coding (nc)RNAs are key players in numerous biological processes such as
gene regulation, chromatin domain formation and genome stability. Large ncRNAs
interact with histone modifiers and are involved in cancer development,
X-chromosome inactivation and autosomal gene imprinting. However, despite recent
evidence showing that pervasive transcription is more widespread than previously
thought, only a few examples mediating gene regulation in eukaryotes have been
described. In Saccharomyces cerevisiae, the bona-fide regulatory ncRNAs are
destabilized by the Xrn1 5'-3' RNA exonuclease (also known as Kem1), but the
genome-wide characterization of the entire regulatory ncRNA family remains
elusive. Here, using strand-specific RNA sequencing (RNA-seq), we identify a
novel class of 1,658 Xrn1-sensitive unstable transcripts (XUTs) in which 66% are
antisense to open reading frames. These transcripts are polyadenylated and RNA
polymerase II (RNAPII)-dependent. The majority of XUTs strongly accumulate in
lithium-containing media, indicating that they might have a role in adaptive
responses to changes in growth conditions. Notably, RNAPII chromatin
immunoprecipitation followed by DNA sequencing (ChIP-seq) analysis of
Xrn1-deficient strains revealed a significant decrease of RNAPII occupancy over
273 genes with antisense XUTs. These genes show an unusual bias for H3K4me3
marks and require the Set1 histone H3 lysine 4 methyl-transferase for silencing.
Furthermore, abolishing H3K4me3 triggers the silencing of other genes with
antisense XUTs, supporting a model in which H3K4me3 antagonizes antisense ncRNA
repressive activity. Our results demonstrate that antisense ncRNA-mediated
regulation is a general regulatory pathway for gene expression in S. cerevisiae. Antisense long non-coding (aslnc)RNAs represent a substantial part of eukaryotic
transcriptomes that are, in yeast, controlled by the Xrn1 exonuclease.
Nonsense-Mediated Decay (NMD) destabilizes the Xrn1-sensitive aslncRNAs (XUT),
but what determines their sensitivity remains unclear. We report that 3'
single-stranded (3'-ss) extension mediates XUTs degradation by NMD, assisted by
the Mtr4 and Dbp2 helicases. Single-gene investigation, genome-wide RNA
analyses, and double-stranded (ds)RNA mapping revealed that 3'-ss extensions
discriminate the NMD-targeted XUTs from stable lncRNAs. Ribosome profiling
showed that XUT are translated, locking them for NMD activity. Interestingly,
mutants of the Mtr4 and Dbp2 helicases accumulated XUTs, suggesting that dsRNA
unwinding is a critical step for degradation. Indeed, expression of
anticomplementary transcripts protects cryptic intergenic lncRNAs from NMD. Our
results indicate that aslncRNAs form dsRNA that are only translated and targeted
to NMD if dissociated by Mtr4 and Dbp2. We propose that NMD buffers genome
expression by discarding pervasive regulatory transcripts. Antisense transcription can regulate sense gene expression. However, previous
annotations of antisense transcription units have been based on detection of
mature antisense long noncoding (aslnc)RNAs by RNA-seq and/or microarrays, only
giving a partial view of the antisense transcription landscape and incomplete
molecular bases for antisense-mediated regulation. Here, we used native
elongating transcript sequencing to map genome-wide nascent antisense
transcription in fission yeast. Strikingly, antisense transcription was detected
for most protein-coding genes, correlating with low sense transcription,
especially when overlapping the mRNA start site. RNA profiling revealed that the
resulting aslncRNAs mainly correspond to cryptic Xrn1/Exo2-sensitive transcripts
(XUTs). ChIP-seq analyses showed that antisense (as)XUT's expression is
associated with specific histone modification patterns. Finally, we showed that
asXUTs are controlled by the histone chaperone Spt6 and respond to meiosis
induction, in both cases anti-correlating with levels of the paired-sense mRNAs,
supporting physiological significance to antisense-mediated gene attenuation.
Our work highlights that antisense transcription is much more extended than
anticipated and might constitute an additional nonpromoter determit of gene
regulation complexity. |
How many genes constitute the DosR regulon, controlled by the dormancy survival regulator (DosR) in Mycobacterium tuberculosis? | The Mycobacterium dormancy survival regulator (DosR) regulon is composed of 48 co-regulated genes. | Mycobacterium bovis BCG is widely used as a vaccine against tuberculosis (TB),
despite its variable protective efficacy. Relatively little is known about the
immune response profiles following BCG vaccination in relation to protection
against TB. Here we tested whether BCG vaccination results in immune responses
to DosR (Rv3133c) regulon-encoded proteins. These so-called TB latency antigens
are targeted by the immune system during persistent Mycobacterium tuberculosis
infection and have been associated with immunity against latent M. tuberculosis
infection. In silico analysis of the DosR regulon in BCG and M. tuberculosis
showed at least 97% amino acid sequence homology, with 41 out of 48 genes being
identical. Transcriptional profiling of 14 different BCG strains, under hypoxia
and nitric oxide exposure in vitro, revealed a functional DosR regulon similar
to that observed in M. tuberculosis. Next, we assessed human immune responses to
a series of immunodomit TB latency antigens and found that BCG vaccination
fails to induce significant responses to latency antigens. Similar results were
obtained with BCG-vaccinated BALB/c mice. In contrast, responses to latency
antigens were observed in individuals with suspected exposure to TB (as
indicated by positive gamma interferon responses to TB-specific antigens ESAT-6
and CFP-10) and in mice vaccinated with plasmid DNA encoding selected latency
antigens. Since immune responses to TB latency antigens have been associated
with control of latent M. tuberculosis infection, our findings support the
development of vaccination strategies incorporating DosR regulon antigens to
complement and improve the current BCG vaccine. BACKGROUND: Low oxygen availability has been shown previously to stimulate M.
tuberculosis to establish non-replicative persistence in vitro. The two
component sensor/regulator dosRS is a major mediator in the transcriptional
response of M. tuberculosis to hypoxia and controls a regulon of approximately
50 genes that are induced under this condition. The aim of this study was to
determine whether the induction of the entire DosR regulon is triggered as a
synchronous event or if induction can unfold as a cascade of events as the
differential expression of subsets of genes is stimulated by different oxygen
availabilities.
RESULTS: A novel aspect of our work is the use of chemostat cultures of M.
tuberculosis which allowed us to control environmental conditions very tightly.
We exposed M. tuberculosis to a sudden drop in oxygen availability in chemostat
culture and studied the transcriptional response of the organism during the
transition from a high oxygen level (10% dissolved oxygen tension or DOT) to a
low oxygen level (0.2% DOT) using DNA microarrays. We developed a Bayesian
change point analysis method that enabled us to detect subtle shifts in the
timing of gene induction. It results in probabilities of a change in gene
expression at certain time points. A computational analysis of potential binding
sites upstream of the DosR-controlled genes shows how the transcriptional
responses of these genes are influenced by the affinity of these binding sites
to DosR. Our study also indicates that a subgroup of DosR-controlled genes is
regulated indirectly.
CONCLUSION: The majority of the dosR-dependent genes were up-regulated at 0.2%
DOT, which confirms previous findings that these genes are triggered by hypoxic
environments. However, our change point analysis also highlights genes which
were up-regulated earlier at levels of about 8% DOT indicating that they respond
to small fluctuations in oxygen availability. Our analysis shows that there are
pairs of divergent genes where one gene in the pair is up-regulated before the
other, presumably for a flexible response to a constantly changing environment
in the host. Increasing knowledge about DosR regulon-encoded proteins has led us to produce
novel Mycobacterium tuberculosis antigens for immunogenicity testing in human
populations in three countries in Africa to which tuberculosis (TB) is endemic.
A total of 131 tuberculin skin test-positive and/or ESAT-6/CFP10-positive, human
immunodeficiency virus-negative adult household contacts of active pulmonary TB
cases from South Africa (n = 56), The Gambia (n = 26), and Uganda (n = 49) were
tested for gamma interferon responses to 7 classical and 51 DosR regulon-encoded
M. tuberculosis recombit protein antigens. ESAT-6/CFP10 fusion protein evoked
responses in >75% of study participants in all three countries. Of the DosR
regulon-encoded antigens tested, Rv1733c was the most commonly recognized by
participants from both South Africa and Uganda and the third most commonly
recognized antigen in The Gambia. The four most frequently recognized DosR
regulon-encoded antigens in Uganda (Rv1733c, Rv0081, Rv1735c, and Rv1737c)
included the three most immunogenic antigens in South Africa. In contrast,
Rv3131 induced the highest percentage of responders in Gambian contacts (38%),
compared to only 3.4% of Ugandan contacts and no South African contacts.
Appreciable percentages of TB contacts with a high likelihood of latent M.
tuberculosis infection responded to several novel DosR regulon-encoded M.
tuberculosis proteins. In addition to significant similarities in antigen
recognition profiles between the three African population groups, there were
also disparities, which may stem from genetic differences between both pathogen
and host populations. Our findings have implications for the selection of
potential TB vaccine candidates and for determining biosignatures of latent M.
tuberculosis infection, active TB disease, and protective immunity. DevR regulon function is believed to be crucial for the survival of
Mycobacterium tuberculosis during dormancy. In this study, we undertook a
comprehensive analysis of the DevR regulon. All the regulon promoters were
assigned to four classes based on the number of DevR binding sites (Dev boxes).
A minimum of two boxes are essential for complete interaction and their tandem
arrangement is an architectural hallmark at all promoters. Initial interaction
of DevR with the conserved box is essential for its cooperative binding to
adjacent sites bearing low to very poor sequence conservation and is the
universal mechanism underlying DevR-mediated transcriptional induction. The
functional importance of tandem arrangement was established by analyzing
promoter variants harboring Dev boxes with altered spacing. Conserved sequence
logos were generated from 47 binding sequences which included 24 newly
discovered Dev boxes. In each half site of an 18-bp binding motif, G(5) and C(7)
are essential for DevR binding. Finally, we show that DevR regulon induction
occurs in a temporal manner and genes that are induced early are also usually
powerfully induced. The information theory-based approach along with binding and
temporal expression studies provide us with comprehensive insights into the
complex pattern of DevR regulon activation. One of the challenges faced by Mycobacterium tuberculosis (M. tuberculosis) in
dormancy is hypoxia. DosR/DevR of M. tuberculosis is a two component dormancy
survival response regulator which induces the expression of 48 genes. In this
study, we have used DosR regulon proteins of M. tuberculosis H37Rv as the query
set and performed a comprehensive homology search against the non-redundant
database. Homologs were found in environmental mycobacteria, environmental
bacteria and archaebacteria. Analysis of genomic context of DosR regulon
revealed that they are distributed as nine blocks in the genome of M.
tuberculosis with many transposases and integrases in their vicinity. Further,
we classified DosR regulon proteins into eight functional categories. One of the
hypothetical proteins Rv1998c could probably be a methylisocitrate lyase or a
phosphonomutase. Another hypothetical protein, Rv0572 was found only in
mycobacteria. Insights gained in this study can potentially aid in the
development of novel therapeutic interventions. Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), which claims
approximately two million people annually, remains a global health concern. The
non-replicating or dormancy like state of this pathogen which is impervious to
anti-tuberculosis drugs is widely recognized as the culprit for this scenario.
The dormancy survival regulator (DosR) regulon, composed of 48 co-regulated
genes, is held as essential for Mtb persistence. The DosR regulon is regulated
by a two-component regulatory system consisting of two sensor kinases-DosS
(Rv3132c) and DosT (Rv2027c), and a response regulator DosR (Rv3133c). The
underlying regulatory mechanism of DosR regulon expression is very complex. Many
factors are involved, particularly the oxygen tension. The DosR regulon enables
the pathogen to persist during lengthy hypoxia. Comparative genomic analysis
demonstrated that the DosR regulon is widely distributed among the mycobacterial
genomes, ranging from the pathogenic strains to the environmental strains.
In-depth studies on the DosR response should provide insights into its role in
TB latency in vivo and shape new measures to combat this exceeding recalcitrant
pathogen. Upon oxygen shift-down, Mycobacterium tuberculosis complex bacteria can induce a
genetic program characterized by halted duplication, which is called
Non-replicating persistence (NRP). During this phase, at least 48 genes,
collectively named Dormancy survival regulator (DosR) regulon, are important for
the long-term survival of bacilli under a non-respiring state, a condition that
bacilli encounter inside granulomatous lesions. It remains unclear whether
expression of NRP genes occurs within the tissue of Mycobacterium bovis
naturally infected cattle. In order to start dissecting this question, total RNA
from bovine lymph node tissues of sacrificed tuberculin reacting animals was
isolated and transcription of genes required for in vivo duplication (esxB and
fbpB) and in vitro NRP (hspX, pfkB, and mb2660c) were analyzed by RT-PCR
approaches. Detection of transcripts was positive in bovine tissue samples for
genes hspX, pfkB, and mb2660c in 84, 32, and 21%, respectively. NRP genes were
upregulated even in animals with a negative IFN-γ in vitro test, and the
expression of NRP genes occurred more often than expression of the esxB gene. |
Is autophagy modulated in a circadian fashion? | Yes, metabolic pathways, bile acid synthesis, and autophagic and immune/inflammatory processes are driven by the biological clock. | Autophagy is a highly conserved intracellular degradation system, and recently
was shown to display circadian rhythms in mice. The mechanisms underlying
circadian regulation of autophagy, however, are still unclear. Here, we observed
that numbers of autophagosomes and autolysosomes exhibit daily rhythms in the
zebrafish liver, and cebpb/(c/ebpβ) and various autophagy genes are rhythmically
expressed in zebrafish larvae but significantly upregulated in per1b and
TALEN-generated nr1d1/rev-erbα mutant fish, indicating that both Per1b and Nr1d1
play critical roles in autophagy rhythms. Luciferase reporter and ChIP assays
show that the circadian clock directly regulates autophagy genes through Nr1d1,
and also regulates transcription of cebpb through Per1b. We also found that
fasting leads to altered expression of both circadian clock genes and autophagy
genes in zebrafish adult peripheral organs. Further, transcriptome analysis
reveals multiple functions of Nr1d1 in zebrafish. Taken together, these findings
provide evidence for how the circadian clock regulates autophagy, imply that
nutritional signaling affects both circadian regulation and autophagy activities
in peripheral organs, and shed light on how circadian gene mutations act through
autophagy to contribute to common metabolic diseases such as obesity. Drosophila melanogaster is a common model used to study circadian rhythms in
behavior and circadian clocks. However, numerous circadian rhythms have also
been detected in non-clock neurons, especially in the first optic neuropil
(lamina) of the fly's visual system. Such rhythms have been observed in the
number of synapses and in the structure of interneurons, which exhibit changes
in size and shape in a circadian manner. Although the patterns of these changes
are known, the mechanism remains unclear. In the present study, we investigated
the role of the TOR signaling pathway and autophagy in regulating circadian
rhythms based on the behavior and structural plasticity of the lamina L2
monopolar cell dendritic trees. In addition, we examined the cyclic expression
of the TOR signaling pathway (Tor, Pi3K class 1, Akt1) and autophagy (Atg5 and
Atg7) genes in the fly's brain. We observed that Tor, Atg5 and Atg7 exhibit
rhythmic expressions in the brain of wild-type flies in day/night conditions (LD
12:12) that are abolished in per01 clock mutants. The silencing of Tor in per
expressing cells shortens a period of the locomotor activity rhythm of flies. In
addition, silencing of the Tor and Atg5 genes in L2 cells disrupts the circadian
plasticity of the L2 cell dendritic trees measured in the distal lamina. In
turn, silencing of the Atg7 gene in L2 cells changes the pattern of this rhythm.
Our results indicate that the TOR signaling pathway and autophagy are involved
in the regulation of circadian rhythms in the behavior and plasticity of neurons
in the brain of adult flies. Abnormal autophagy regulation affects the chemoresistance of ovarian cancer,
during which the circadian gene clock may play a major role. In this study, RNA
interference plasmid pSUPER-Clock and overexpression plasmid pcDNA3.1-Clock of
CLOCK were used to stably transfect the SKOV3/DDP cells by lipofection. Upon
screening, the in vitro transfected cell lines with pSUPER-Clock, the autophagy
level, and G0/G1 phase cells were significantly reduced, and the expression
levels of Clock, LC3, P-gp, and MRP2 were inhibited. In contrast, the autophagy
level and G0/G1 phase cells in cell lines transfected with pcDNA3.1-Clock were
significantly increased, and the expressions of Clock, LC3, P-gp, and MRP2 were
enhanced. In comparison with the untransfected control group showed the
percentage of apoptotic cells in SKOV3/DDP cell lines of Clock interfering
expression group after cisplatin treatment was significantly increased while the
survival was substantially reduced. These results indicated that inhibiting the
circadian gene Clock expression can reverse the cisplatin resistance of ovarian
cancer SKOV3/DDP cell lines by affecting the protein expression of drug
resistance genes during which autophagy plays an important role. The CLOCK gene
may be designated as a novel candidate for targeted gene therapy in
drug-resistant ovarian cancer. BACKGROUND: The mammalian circadian clock and its associated clock genes are
increasingly been recognized as critical components for a number of
physiological and disease processes that extend beyond hormone release, thermal
regulation, and sleep-wake cycles. New evidence suggests that clinical behavior
disruptions that involve prolonged shift work and even space travel may
negatively impact circadian rhythm and lead to multi-system disease.
METHODS: In light of the significant role circadian rhythm can hold over the
body's normal physiology as well as disease processes, we examined and discussed
the impact circadian rhythm and clock genes hold over lifespan,
neurodegenerative disorders, and tumorigenesis.
RESULTS: In experimental models, lifespan is significantly reduced with the
introduction of arrhythmic mutants and leads to an increase in oxidative stress
exposure. Interestingly, patients with Alzheimer's disease and Parkinson's
disease may suffer disease onset or progression as a result of alterations in
the DNA methylation of clock genes as well as prolonged pharmacological
treatment for these disorders that may lead to impairment of circadian rhythm
function. Tumorigenesis also can occur with the loss of a maintained circadian
rhythm and lead to an increased risk for nasopharyngeal carcinoma, breast
cancer, and metastatic colorectal cancer. Interestingly, the circadian clock
system relies upon the regulation of the critical pathways of autophagy, the
mechanistic target of rapamycin (mTOR), AMP activated protein kinase (AMPK), and
silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae)
(SIRT1) as well as proliferative mechanisms that involve the wingless pathway of
Wnt/β-catenin pathway to foster cell survival during injury and block tumor cell
growth.
CONCLUSION: Future targeting of the pathways of autophagy, mTOR, SIRT1, and Wnt
that control mammalian circadian rhythm may hold the key for the development of
novel and effective therapies against aging- related disorders,
neurodegenerative disease, and tumorigenesis. The molecular clockwork drives rhythmic oscillations of signaling pathways
managing intermediate metabolism; the circadian timing system synchronizes
behavioral cycles and anabolic/catabolic processes with environmental cues,
mainly represented by light/darkness alternation. Metabolic pathways, bile acid
synthesis, and autophagic and immune/inflammatory processes are driven by the
biological clock. Proper timing of hormone secretion, metabolism, bile acid
turnover, autophagy, and inflammation with behavioral cycles is necessary to
avoid dysmetabolism. Disruption of the biological clock and mistiming of body
rhythmicity with respect to environmental cues provoke loss of internal
synchronization and metabolic derangements, causing liver steatosis, obesity,
metabolic syndrome, and diabetes mellitus. High-glucose-induced cardiomyocyte injury is the major cause of diabetic
cardiomyopathy, but its regulatory mechanisms are not fully understood. Here, we
report that a circadian clock gene, brain and muscle Arnt-like 1 (Bmal1),
increases autophagy in high-glucose-induced cardiomyocyte injury. We constructed
a hyperglycemia model with cultured cardiomyocytes from neonatal rats.
High-glucose-induced inhibition of autophagy and cardiomyocyte injury were
attenuated by Bmal1 overexpression and aggravated by its knockdown. Furthermore,
autophagy stabilization by 3-methyladenine or rapamycin partially suppressed the
effects of altered Bmal1 expression on cardiomyocyte survival. Mechanistically,
Bmal1 mediated resistance to high-glucose-induced inhibition of autophagy at
least partly by inhibiting mTOR signaling activity. Collectively, our findings
suggest that the clock gene Bmal1 is a positive regulator of autophagy through
the mTOR signaling pathway and protects cardiomyocytes against high-glucose
toxicity. |
Is Drk essential for anesthesia-resistant memory (ARM) in Drosophila? | Yes. Drk, the Drosophila ortholog of the adaptor protein Grb2, is essential for ARM within adult mushroom body neurons in Drosophila. | Anesthesia-resistant memory (ARM) was described decades ago, but the mechanisms
that underlie this protein synthesis-independent form of consolidated memory in
Drosophila remain poorly understood. Whether the several signaling molecules,
receptors, and synaptic proteins currently implicated in ARM operate in one or
more pathways and how they function in the process remain unclear. We present
evidence that Drk, the Drosophila ortholog of the adaptor protein Grb2, is
essential for ARM within adult mushroom body neurons. Significantly, Drk signals
engage the Rho kinase Drok, implicating dynamic cytoskeletal changes in ARM, and
this is supported by reduced F-actin in the mutants and after pharmacological
inhibition of Drok. Interestingly, Drk-Drok signaling appears independent of the
function of Radish (Rsh), a protein long implicated in ARM, suggesting that the
process involves at least two distinct molecular pathways. Based on these
results, we propose that signaling pathways involved in structural plasticity
likely underlie this form of translation-independent memory. |
What is oclacitinib? | Oclacitinib (APOQUEL(®)) is a Janus kinase inhibitor with activity against cytokines involved in allergy. It is a potent inhibitor of JAK1. It effectively controls clinical signs associated with allergic skin disease in dogs. | |
Are TAD boundaries in Drosophila depleted in highly-expressed genes? | Drosophila inter-TADs harbor active chromatin and constitutively transcribed (housekeeping) genes. | The spatial arrangement of interphase chromosomes in the nucleus is important
for gene expression and genome function in animals and in plants. The recently
developed Hi-C technology is an efficacious method to investigate genome
packing. Here we present a detailed Hi-C map of the three-dimensional genome
organization of the plant Arabidopsis thaliana. We find that local chromatin
packing differs from the patterns seen in animals, with kilobasepair-sized
segments that have much higher intrachromosome interaction rates than
neighboring regions, representing a domit local structural feature of genome
conformation in A. thaliana. These regions, which appear as positive strips on
two-dimensional representations of chromatin interaction, are enriched in
epigenetic marks H3K27me3, H3.1, and H3.3. We also identify more than 400
insulator-like regions. Furthermore, although topologically associating domains
(TADs), which are prominent in animals, are not an obvious feature of A.
thaliana genome packing, we found more than 1000 regions that have properties of
TAD boundaries, and a similar number of regions analogous to the interior of
TADs. The insulator-like, TAD-boundary-like, and TAD-interior-like regions are
each enriched for distinct epigenetic marks and are each correlated with
different gene expression levels. We conclude that epigenetic modifications,
gene density, and transcriptional activity combine to shape the local packing of
the A. thaliana nuclear genome. Author information:
(1)Max Planck Institute for Molecular Genetics, RG Development & Disease, 14195
Berlin, Germany; Institute for Medical and Human Genetics, Charité
Universitätsmedizin Berlin, 13353 Berlin, Germany.
(2)Institute for Medical and Human Genetics, Charité Universitätsmedizin Berlin,
13353 Berlin, Germany.
(3)Medical Genetics Unit, Policlinico Tor Vergata University Hospital, 00133
Rome, Italy.
(4)Institute of Human Genetics Biozentrum, Julius Maximilian University of
Würzburg, 97070 Würzburg, Germany.
(5)Medical Genetics Department, Istanbul Medical Faculty, Istanbul University,
34093 Istanbul, Turkey.
(6)Department of Pediatrics, School of Medicine, University of Utah, Salt Lake
City, UT 84108, USA.
(7)Instituto de Genética Médica y Molecular (INGEMM), IdiPAZ, Hospital
Universitario La Paz, 28046 Madrid, Spain; U753 Centro de Investigación
Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III,
28046 Madrid, Spain.
(8)Service de Génétique, C.H.U. de Poitiers, 86021 Poitiers, France.
(9)Department Developmental Genetics, Max Planck Institute for Molecular
Genetics, 14195 Berlin, Germany.
(10)Max Planck Institute for Molecular Genetics, RG Development & Disease, 14195
Berlin, Germany.
(11)Department of Computational Molecular Biology, Max Planck Institute for
Molecular Genetics, 14195 Berlin, Germany.
(12)Genomics Division, MS 84-171, Lawrence Berkeley National Laboratory,
Berkeley, CA 94720, USA.
(13)Max Planck Institute for Molecular Genetics, Sequencing Core Facility, 14195
Berlin, Germany.
(14)Max Planck Institute for Molecular Genetics, RG Development & Disease, 14195
Berlin, Germany; Berlin-Brandenburg Center for Regenerative Therapies (BCRT),
Charité Universitätsmedizin Berlin, 13353 Berlin, Germany.
(15)Max Planck Institute for Molecular Genetics, RG Development & Disease, 14195
Berlin, Germany; Institute for Medical and Human Genetics, Charité
Universitätsmedizin Berlin, 13353 Berlin, Germany; Berlin-Brandenburg Center for
Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin,
Germany.
(16)Genomics Division, MS 84-171, Lawrence Berkeley National Laboratory,
Berkeley, CA 94720, USA; U.S. Department of Energy Joint Genome Institute,
Walnut Creek, CA 94598, USA; School of Natural Sciences, University of
California, Merced, CA 95343, USA.
(17)Max Planck Institute for Molecular Genetics, RG Development & Disease, 14195
Berlin, Germany; Institute for Medical and Human Genetics, Charité
Universitätsmedizin Berlin, 13353 Berlin, Germany; Berlin-Brandenburg Center for
Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin,
Germany. Electronic address: [email protected]. The three-dimensional organization of a genome plays a critical role in
regulating gene expression, yet little is known about the machinery and
mechanisms that determine higher-order chromosome structure. Here we perform
genome-wide chromosome conformation capture analysis, fluorescent in situ
hybridization (FISH), and RNA-seq to obtain comprehensive three-dimensional (3D)
maps of the Caenorhabditis elegans genome and to dissect X chromosome dosage
compensation, which balances gene expression between XX hermaphrodites and XO
males. The dosage compensation complex (DCC), a condensin complex, binds to both
hermaphrodite X chromosomes via sequence-specific recruitment elements on X (rex
sites) to reduce chromosome-wide gene expression by half. Most DCC condensin
subunits also act in other condensin complexes to control the compaction and
resolution of all mitotic and meiotic chromosomes. By comparing chromosome
structure in wild-type and DCC-defective embryos, we show that the DCC remodels
hermaphrodite X chromosomes into a sex-specific spatial conformation distinct
from autosomes. Dosage-compensated X chromosomes consist of self-interacting
domains (∼1 Mb) resembling mammalian topologically associating domains (TADs).
TADs on X chromosomes have stronger boundaries and more regular spacing than on
autosomes. Many TAD boundaries on X chromosomes coincide with the
highest-affinity rex sites and become diminished or lost in DCC-defective
mutants, thereby converting the topology of X to a conformation resembling
autosomes. rex sites engage in DCC-dependent long-range interactions, with the
most frequent interactions occurring between rex sites at DCC-dependent TAD
boundaries. These results imply that the DCC reshapes the topology of X
chromosomes by forming new TAD boundaries and reinforcing weak boundaries
through interactions between its highest-affinity binding sites. As this model
predicts, deletion of an endogenous rex site at a DCC-dependent TAD boundary
using CRISPR/Cas9 greatly diminished the boundary. Thus, the DCC imposes a
distinct higher-order structure onto X chromosomes while regulating gene
expression chromosome-wide. Dosage compensation mechanisms provide a paradigm to study the contribution of
chromosomal conformation toward targeting and spreading of epigenetic regulators
over a specific chromosome. By using Hi-C and 4C analyses, we show that
high-affinity sites (HAS), landing platforms of the male-specific lethal (MSL)
complex, are enriched around topologically associating domain (TAD) boundaries
on the X chromosome and harbor more long-range contacts in a sex-independent
manner. Ectopically expressed roX1 and roX2 RNAs target HAS on the X chromosome
in trans and, via spatial proximity, induce spreading of the MSL complex in cis,
leading to increased expression of neighboring autosomal genes. We show that the
MSL complex regulates nucleosome positioning at HAS, therefore acting locally
rather than influencing the overall chromosomal architecture. We propose that
the sex-independent, three-dimensional conformation of the X chromosome poises
it for exploitation by the MSL complex, thereby facilitating spreading in males. Recent advances enabled by the Hi-C technique have unraveled many principles of
chromosomal folding that were subsequently linked to disease and gene
regulation. In particular, Hi-C revealed that chromosomes of animals are
organized into topologically associating domains (TADs), evolutionary conserved
compact chromatin domains that influence gene expression. Mechanisms that
underlie partitioning of the genome into TADs remain poorly understood. To
explore principles of TAD folding in Drosophila melanogaster, we performed Hi-C
and poly(A)(+) RNA-seq in four cell lines of various origins (S2, Kc167,
DmBG3-c2, and OSC). Contrary to previous studies, we find that regions between
TADs (i.e., the inter-TADs and TAD boundaries) in Drosophila are only weakly
enriched with the insulator protein dCTCF, while another insulator protein
Su(Hw) is preferentially present within TADs. However, Drosophila inter-TADs
harbor active chromatin and constitutively transcribed (housekeeping) genes.
Accordingly, we find that binding of insulator proteins dCTCF and Su(Hw)
predicts TAD boundaries much worse than active chromatin marks do.
Interestingly, inter-TADs correspond to decompacted inter-bands of polytene
chromosomes, whereas TADs mostly correspond to densely packed bands.
Collectively, our results suggest that TADs are condensed chromatin domains
depleted in active chromatin marks, separated by regions of active chromatin. We
propose the mechanism of TAD self-assembly based on the ability of nucleosomes
from inactive chromatin to aggregate, and lack of this ability in acetylated
nucleosomal arrays. Finally, we test this hypothesis by polymer simulations and
find that TAD partitioning may be explained by different modes of
inter-nucleosomal interactions for active and inactive chromatin. Three-dimensional genome structure plays an important role in gene regulation.
Globally, chromosomes are organized into active and inactive compartments while,
at the gene level, looping interactions connect promoters to regulatory
elements. Topologically associating domains (TADs), typically several hundred
kilobases in size, form an intermediate level of organization. Major questions
include how TADs are formed and how they are related to looping interactions
between genes and regulatory elements. Here we performed a focused 5C analysis
of a 2.8 Mb chromosome 7 region surrounding CFTR in a panel of cell types. We
find that the same TAD boundaries are present in all cell types, indicating that
TADs represent a universal chromosome architecture. Furthermore, we find that
these TAD boundaries are present irrespective of the expression and looping of
genes located between them. In contrast, looping interactions between promoters
and regulatory elements are cell-type specific and occur mostly within TADs.
This is exemplified by the CFTR promoter that in different cell types interacts
with distinct sets of distal cell-type-specific regulatory elements that are all
located within the same TAD. Finally, we find that long-range associations
between loci located in different TADs are also detected, but these display much
lower interaction frequencies than looping interactions within TADs.
Interestingly, interactions between TADs are also highly cell-type-specific and
often involve loci clustered around TAD boundaries. These data point to key
roles of invariant TAD boundaries in constraining as well as mediating
cell-type-specific long-range interactions and gene regulation. |
List the two most important hematological features of the Evans syndrome | Evans syndrome is a rare autoimmune disorder, which is characterized by immune thrombocytopenia and autoimmune hemolytic anemia. | Evans' syndrome is an unusual illness of autoimmune etiology, characterized by
thrombocytopenia and hemolytic anemia. This is more frequent in females
throughout first half of the life and during pregcy. The present paper
describes two pregt women with Evans syndrome associated to preeclampsia.
This report emphasizes how the hematology and coagulation abnormalities of
preeclampsia could be added to those abnormalities observed in Evans' syndrome.
This association constitutes a severe disease of difficult treatment. Evans syndrome is an uncommon condition characterised by simultaneous or
sequential development of immune thrombocytopenia (ITP) and autoimmune
haemolytic anaemia (AIHA) with a positive direct antiglobulin test (DAT) in the
absence of a known underlying aetiology. The great majority of patients with
Evans syndrome have a chronic relapsing course despite treatment, which is
associated with significant morbidity and mortality. We reviewed the clinical
and laboratory features of six patients with Evans syndrome. All patients had
thrombocytopenia, bleeding symptoms and haemolytic anaemia with positive direct
Coombs test at presentation. We discuss the aetiopathogenic, clinical,
therapeutic and natural history of Evans syndrome. Objective To determine the overall prevalence of autoimmune hemolytic anemia
(AIHA), and to compare clinical and laboratory features in a large population of
children and adult lupus patients at diagnosis. Methods This retrospective study
evaluated the medical charts of 336 childhood-onset systemic lupus erythematosus
(cSLE) and 1830 adult SLE (aSLE) patients followed in the same tertiary
hospital. Demographic data, clinical features and disease activity were
recorded. AIHA was defined according to the presence of anemia (hemoglobin
<10 g/dL) and evidence of hemolysis (reticulocytosis and positive direct
antiglobulin test (DAT)/Coombs test) at SLE diagnosis. Evans syndrome (ES) was
defined by the combination of immune thrombocytopenia (platelet count
<100,000/mm3) and AIHA. Results The frequency of AIHA at diagnosis was
significantly higher in cSLE patients compared to aSLE (49/336 (14%) vs 49/1830
(3%), p = 0.0001), with similar frequency of ES (3/336 (0.9%) vs 10/1830 (0.5%),
p = 0.438). The median of hemoglobin levels was reduced in cSLE vs aSLE patients
(8.3 (2.2-10) vs 9.5 (6.6-10) g/dL, p = 0.002) with a higher frequency of
multiple hemorrhagic manifestations (41% vs 7%, p = 0.041) and erythrocyte
transfusion due to bleeding (24% vs 5%, p = 0.025). cSLE patients also had more
often constitutional involvement (84% vs 31%, p < 0.001), fever (65% vs 26%,
p < 0.001), weight loss > 2 kg (39% vs 6%, p < 0.001), reticuloendothelial
manifestations (48% vs 8%, p < 0.001), hepatomegaly (25% vs 2%, p < 0.001) and
splenomegaly (21% vs 2%, p = 0.004). Other major organ involvements were common
but with similar frequencies in cSLE and aSLE ( p > 0.05). Median systemic lupus
erythematosus disease activity index 2000 (SLEDAI-2 K) was comparable in cSLE
and aSLE (p = 0.161). Conclusions We identified that AIHA was not a common
condition in cSLE and aSLE, with distinct features characterized by a higher
prevalence/severity in children and concomitant constitutional symptoms in the
majority of them. Common variable immunodeficiency (CVID) is a heterogeneous group of diseases.
Our aim was to define sub-groups of CVID patients with similar phenotypes and
clinical characteristics. Using eight-color flow cytometry, we analyzed both B-
and T-cell phenotypes in a cohort of 88 CVID patients and 48 healthy donors. A
hierarchical clustering of probability binning "bins" yielded a separate cluster
of 22 CVID patients with an abnormal phenotype. We showed coordinated
proportional changes in naïve CD4+ T-cells (decreased), intermediate CD27- CD28+
CD4+ T-cells (increased) and CD21low B-cells (increased) that were stable for
over three years. Moreover, the lymphocytes' immunophenotype in this patient
cluster exhibited features of profound immunosenescence and chronic activation.
Thrombocytopenia was only found in this cluster (36% of cases, manifested as
Immune Thrombocytopenia (ITP) or Evans syndrome). Clinical complications more
frequently found in these patients include lung fibrosis (in 59% of cases) and
bronchiectasis (55%). The degree of severity of these symptoms corresponded to
more deviation from normal levels with respect to CD21low B-cells, naïve CD4+
and CD27− CD28+ CD4+ T-cells. Next-generation sequencing did not reveal any
common genetic background. We delineate a subgroup of CVID patients with
activated and immunosenescent immunophenotype of lymphocytes and distinct set of
clinical complications without common genetic background. BACKGROUND: Autoimmune cytopenias are characterized by immune-mediated
destruction of hematopoietic cell lines with immune thrombocytopenia (ITP)
affecting platelets and Evans syndrome (ES) affecting platelets and red blood
cells. For patients with persistent disease, limited options for effective and
well-tolerated therapies exist.
OBJECTIVES: Our aim is to describe our institution's experience with sirolimus
as therapy for pediatric patients with persistent ITP and ES.
DESIGN/METHOD: A retrospective analysis was performed in patients with
persistent ITP and ES treated with sirolimus. Responses were categorized as
complete response (CR), partial response, modest response, or no response.
RESULTS: Of the 17 patients treated, 12 had ITP and 5 had ES. Seventy-three
percent of ITP patients achieved a CR, 78% of them by 3 months. Only 2 patients
did not achieve a durable response. Eighty percent of ES patients had a
response, with 50% of them achieving CR and the other 50% an asymptomatic
partial response. One patient with ES achieved modest response, but discontinued
therapy due to an adverse effect. Of the patients that achieved CR, 90% remain
off all therapy for a median of 2 years.
CONCLUSIONS: Our data suggest that sirolimus is a safe and effective
steroid-sparing agent in the treatment of persistent ITP and ES. Primary Evans syndrome (ES) is defined by the concurrent or sequential
occurrence of immune thrombocytopenia and autoimmune hemolytic anemia in the
absence of an underlying etiology. The syndrome is characterized by a chronic,
relapsing, and potentially fatal course requiring long-term immunosuppressive
therapy. Treatment of ES is hardly evidence-based. Corticosteroids are the
mainstay of therapy. Rituximab has emerged as the most widely used second-line
treatment, as it can safely achieve high response rates and postpone
splenectomy. An increasing number of new genetic defects involving critical
pathways of immune regulation identify specific disorders, which explain cases
of ES previously reported as "idiopathic". Autoimmune hepatitis (AIH) is a progressive liver disease that is often
associated with extrahepatic autoimmune disorders. Evans syndrome (ES) is a rare
autoimmune disorder, which is characterized by immune thrombocytopenia and
autoimmune hemolytic anemia. Association of AIH with ES is rare, especially in
children. We report a 3-year-old female with a past medical history of ES who
presented with jaundice and significant transaminitis due to AIH type 1. She
required multiple treatments with steroids as well as azathioprine, intravenous
immunoglobulin and a course of rituximab. Evans syndrome (ES) is a rare autoimmune disorder whose exact pathophysiology is
unknown. It is characterized by the simultaneous or subsequent development of
autoimmune hemolytic anemia (AIHA) and immune thrombocytopenia (ITP).
Intravascular hemolysis, with hemoglobinemia, is known to produce acute kidney
injury; however, the development of intratubular hemoglobin casts (hemoglobin
cast nephropathy) in the setting of acute hemolysis is uncommon. Likewise, the
association of ES and acute renal failure is equally uncommon. We present a case
of a 7-year-old girl with ES who developed acute kidney injury in the setting of
intravascular hemolysis and had widespread intratubular hemoglobin casts. |
Is there increased recombination rate in human regulatory domains? | No. There is evidence of significantly reduced recombination rate compared to matched control regions at human regulatory domains. | |
Is enzastaurin effective treatment of glioblastoma? | No. Enzastaurin does not improve prognosis of glioblastoma patients. | The prognosis of patients with glioblastoma, anaplastic astrocytoma, and
anaplastic oligodendroglioma remains poor despite standard treatment with
radiotherapy and temozolomide. Molecular targeted therapy holds the promise of
providing new, more effective treatment options with minimal toxicity. However,
the development of targeted therapy for gliomas has been particularly
challenging. The oncogenetic process in such tumors is driven by several
signaling pathways that are differentially activated or silenced with both
parallel and converging complex interactions. Therefore, it has been difficult
to identify prevalent targets that act as key promoters of oncogenesis and that
can be successfully addressed by novel agents. Several drugs have been tested,
including epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors
(gefitinib and erlotinib), mammalian target of rapamycin (mTOR) inhibitors
(temsirolimus and everolimus), and vascular endothelial growth factor receptor
(VEGFR), protein kinase C-beta, and other angiogenesis pathways inhibitors
(vatalanib, bevacizumab, and enzastaurin). Although preliminary efficacy results
of most trials in recurrent disease have fallen short on expectations,
substantial advances have been achieved by associated translational research. In
this article, we seek to recapitulate the lessons learned in the development of
targeted therapy for gliomas, including challenges and pitfalls in the
interpretation of preclinical data, specific issues in glioma trial design,
insights provided by translational research, changes in paradigms, and future
perspectives. BACKGROUND AND PURPOSE: We review the indications and limitations of
chemotherapy in glioblastoma multiform (GBM), including the role played by
alkylating and other cytotoxic agents and the increased input of clinical
research on targeted agents in GBM management.
METHODS: In 2005, a phase III study clearly concluded in the benefit of adding
temozolomide during and after radiotherapy treatment in GBM and thus defined the
new standard of treatment in this devastating disease. This schedule increased
the median survival from 12.1 to 14 months and the two- and five-year survival
rates from 8 to 26%, and 3 to 10%, respectively, with a good tolerance profile.
Moreover, methylation of the promoter of the O6 methylguanine DNA transferase
(MGMT) gene exhibits a prognostic impact independently of therapeutic schedule
but may also predict the benefit of adding temozolomide to radiotherapy.
However, pitfalls in MGMT determination and lack of prospective validation have
to be solved before considering MGMT as a decisional marker. More recently,
antiangiogenic agents including enzastaurin, cediranib, bevacizumab, and others
that target mainly the VEGF pathway, have been evaluated in this highly
angiogenic disease. Among them, only bevacizumab has been associated with clear
anti-tumor activity, although the lack of control studies limits the impact of
the results to date.
CONCLUSIONS: Recent studies conducted in GBM, both in the adjuvant and recurrent
setting, have changed the natural course of the disease and opened a new area of
clinical research, including imaging and response evaluation, predictive
markers, and other targeted therapies. This open-label, single-arm, phase II study combined enzastaurin with
temozolomide plus radiation therapy (RT) to treat glioblastoma multiforme (GBM)
and gliosarcoma. Adults with newly diagnosed disease and Karnofsky performance
status (KPS) ≥ 60 were enrolled. Treatment was started within 5 weeks after
surgical diagnosis. RT consisted of 60 Gy over 6 weeks. Temozolomide was given
at 75 mg/m(2) daily during RT and then adjuvantly at 200 mg/m(2) daily for 5
days, followed by a 23-day break. Enzastaurin was given once daily during RT and
in the adjuvant period at 250 mg/day. Cycles were 28 days. The primary end point
was overall survival (OS). Progression-free survival (PFS), toxicity, and
correlations between efficacy and molecular markers analyzed from tumor tissue
samples were also evaluated. A prospectively planned analysis compared OS and
PFS of the current trial with outcomes from 3 historical phase II trials that
combined novel agents with temozolomide plus RT in patients with GBM or
gliosarcoma. Sixty-six patients were enrolled. The treatment regimen was well
tolerated. OS (median, 74 weeks) and PFS (median, 36 weeks) results from the
current trial were comparable to those from a prior phase II study using
erlotinib and were significantly better than those from 2 other previous studies
that used thalidomide or cis-retinoic acid, all in combination with temozolomide
plus RT. A positive correlation between O-6-methylguanine-DNA methyltransferase
promoter methylation and OS was observed. Adjusting for age and KPS, no other
biomarker was associated with survival outcome. Correlation of relevant
biomarkers with OS may be useful in future trials. BACKGROUND: This study's primary objective was evaluation of the
progression-free survival rate at 6 months (PFS-6) in patients with newly
diagnosed glioblastoma without O(6)-methylguanine-DNA-methyltransferase (MGMT)
promoter hypermethylation postsurgically treated with enzastaurin before and
concomitantly with radiation therapy, followed by enzastaurin maintece
therapy. PFS-6 of at least 55% was set to be relevant compared with the data of
the EORTC 26981/22981 NCIC CE.3 trial.
METHODS: Adult patients with a life expectancy of at least 12 weeks who were
newly diagnosed with a histologically proven supratentorial glioblastoma without
MGMT promoter hypermethylation were eligible. Patients were treated with
enzastaurin prior to, concomitantly with, and after standard partial brain
radiotherapy. Here we report on a multicenter, open-label, uncontrolled phase II
study of patients with newly diagnosed glioblastoma without MGMT promoter
hypermethylation treated with enzastaurin and radiation therapy within 4 study
periods.
RESULTS: PFS-6 was 53.6% (95% confidence interval [CI]: 39.8-65.6). The median
overall survival was 15.0 months (95% CI: 11.9-17.9) for all patients, 3.9
months (95% CI: 0.8-9.0) for patients with biopsy, 15.4 months (95% CI:
10.1-17.9) for patients with partial resection, and 18.9 months (95% CI:
13.9-28.5) for patients with complete resection. The safety profile in this
study was as expected from previous trials, and the therapy was well tolerated.
CONCLUSIONS: PFS-6 missed the primary planned outcome of 55%. The secondary
exploratory analysis according to resection status of the different subgroups of
patients with biopsies, partial resection, and complete resection demonstrates
the strong prognostic influence of resection on overall survival. We evaluated the efficacy of combination enzastaurin (LY317615) and bevacizumab
for recurrent maligt gliomas and explored serologic correlates. We enrolled
81 patients with glioblastomas (GBM, n = 40) and anaplastic gliomas (AG,
n = 41). Patients received enzastaurin as a loading dose of 1125 mg, followed by
500 or 875 mg daily for patients on non-enzyme-inducing or enzyme-inducing
antiepileptics, respectively. Patients received bevacizumab 10 mg/kg
intravenously biweekly. Clinical evaluations were repeated every 4 weeks.
Magnetic resoce imaging was obtained at baseline and every 8 weeks from
treatment onset. Phosphorylated glycogen synthase kinase (GSK)-3 levels from
peripheral blood mononuclear cells (PBMCs) were checked with each MRI. Median
overall survival was 7.5 and 12.4 months for glioblastomas and anaplastic glioma
cohorts, with median progression-free survivals of 2.0 and 4.4 months,
respectively. Of GBM patients, 3/40 (7.5 %) were not evaluable, while 8/37
(22 %) had partial or complete response and 20/37 (54 %) had stable disease for
2+ months. Of the 39 evaluable AG patients, 18 (46 %) had an objective response,
and 16 (41 %) had stable disease for 2+ months. The most common grade 3+
toxicities were lymphopenia (15 %), hypophosphatemia (8.8 %) and thrombotic
events (7.5 %). Two (2.5 %) GBM patients died suddenly; another death (1.3 %)
occurred from intractable seizures. Phosphorylated GSK-3 levels from PBMCs did
not correlate with treatment response. A minimally important improvement in
health-related quality of life was self-reported in 7-9/24 (29.2-37.5 %). Early
response based on Levin criteria was significantly associated with significantly
longer progression free survival for glioblastomas. Enzastaurin (LY317615) in
combination with bevacizumab for recurrent maligt gliomas is well-tolerated,
with response and progression-free survival similar to bevacizumab monotherapy. PURPOSE: To evaluate the time course and association with survival of anatomic
lesion volumes and diffusion imaging parameters for patients with newly
diagnosed glioblastoma who were treated with radiation and concurrently with
either temozolomide and enzastaurin (TMZ+enza cohort) or temozolomide,
erlotonib, and bevaciumab (TMZ+erl+bev cohort).
MATERIALS AND METHODS: Regions of interest corresponding to the
contrast-enhancing and hyperintense lesions on T2-weighted images were
generated. Diffusion-weighted images were processed to provide maps of apparent
diffusion coefficient, fractional anisotropy, and longitudinal and radial
eigenvalues. Histograms of diffusion values were generated and summary
statistics calculated. Cox proportional hazards models were employed to assess
the association of representative imaging parameters with survival with
adjustments for age, Karnofsky performance status, and extent of resection.
RESULTS: Although progression-free survival was significantly longer for the
TMZ+erl+bev cohort (12.8 vs 7.3 months), there was no significant difference in
overall survival between the two populations (17.0 vs 17.8 months). The median
contrast-enhancing lesion volumes decreased from 6.3 to 1.9 cm(3) from baseline
to the postradiotherapy scan for patients in the TMZ+enza cohort and from 2.8 to
0.9cm(3) for the TMZ+erl+bev cohort. Changes in the T2 lesion volumes were only
significant for the latter cohort (26.5 to 11.9 cm(3)). The median apparent
diffusion coefficient and related diffusion parameters were significantly
increased for the TMZ+enza cohort (1054 to 1225 μm(2)/s). More of the anatomic
parameters were associated with survival for the TMZ+enza cohort, whereas more
diffusion parameters were associated with survival for the TMZ+erl+bev cohort.
CONCLUSION: The early changes in anatomic and diffusion imaging parameters and
their association with survival reflected differences in the mechanisms of
action of the treatments that were being given. This suggests that integrating
diffusion metrics and anatomic lesion volumes into the Response Assessment in
Neuro-Oncology criteria would assist in interpreting treatment-induced changes
and predicting outcome in patients with newly diagnosed glioblastoma who are
receiving such combination treatments. INTRODUCTION: Glioblastoma, the most common maligt brain tumor, exhibits a
poor prognosis with little therapeutic progress in the last decade. Novel
treatment strategies beyond the established standard of care with
temozolomide-based radiotherapy are urgently needed.
AREAS COVERED: We reviewed the literature on glioblastoma with a focus on phase
III trials for pharmacotherapies and/or innovative concepts until December 2015.
EXPERT OPINION: In the last decade, phase III trials on novel compounds largely
failed to introduce efficacious pharmacotherapies beyond temozolomide in
glioblastoma. So far, inhibition of angiogenesis by compounds such as
bevacizumab, cediranib, enzastaurin or cilengitide as well as alternative dosing
schedules of temozolomide did not prolong survival, neither at primary diagnosis
nor at recurrent disease. Promising strategies of pharmacotherapy currently
under evaluation represent targeting epidermal growth factor receptor (EGFR)
with biomarker-stratified patient populations and immunotherapeutic concepts
including checkpoint inhibition and vaccination. The clinical role of the
medical device delivering 'tumor-treating fields' in newly diagnosed
glioblastoma which prolonged overall survival in a phase III study has remained
controversial. After failure of several phase III trials with previously
promising agents, improvement of concepts and novel compounds are urgently
needed to expand the still limited therapeutic options for the treatment of
glioblastoma. BACKGROUND: glioblastomas are highly vascularized tumors and various
antiangiogenic drugs have been investigated in clinical trials showing unclear
results. We performed a systematic review and a meta-analysis to clarify and
evaluate their effectiveness in glioblastoma patients.
PATIENTS AND METHODS: we searched relevant published and unpublished randomized
clinical trials analyzing antiangiogenic drugs versus chemotherapy in
glioblastoma patients from January 2006 to January 2016 in MEDLINE, WEB of
SCIENCE, ASCO, ESMO and SNO databases.
RESULTS: fourteen randomized clinical trials were identified (7 with
bevacizumab, 2 cilengitide, 1 enzastaurin, 1 dasatinib, 1 vandetanib, 1
temsirolimus, 1 cediranib) including 4330 patients. Antiangiogenic drugs showed
no improvement in overall survival with a pooled HR of 1.00, a trend for an
inferior outcome, in terms of overall survival, was observed in the group of
patients receiving antiangiogenic drug alone compared to cytotoxic drug alone
(HR=1.24, p=0.056). Bevacizumab did not improve overall survival. Twelve trials
(4113 patients) were analyzed for progression-free survival. Among
antiangiogenic drugs, only bevacizumab demonstrated an improvement of
progression-free survival (HR=0.63, p<0.001), both alone (HR=0.60, p=0.003) or
in combination to chemotherapy (HR=0.63; p<0.001), both as first-line treatment
(HR=0.70, p<0.001) or in recurrent disease (HR=0.52, p<0.001).
CONCLUSIONS: antiangiogenic drugs did not improve overall survival in
glioblastoma patients, either as first or second-line treatment, and either as
single agent or in combination with chemotherapy. Among antiangiogenic drugs,
only bevacizumab improved progression-free survival regardless of treatment
line, both as single agent or in combination with chemotherapy. |
Which yeast nucleosomes are preferentially marked by H2A.Z? | Yeast nucleosomes containing histone variant H2A.Z (Htz1p in yeast) are primarily composed of H4 K12ac and H3 K4me3. | Nucleosome positioning maps of several organisms have shown that Transcription
Start Sites (TSSs) are marked by nucleosome depleted regions flanked by strongly
positioned nucleosomes. Using genome-wide nucleosome maps and histone variant
occupancy in the mouse liver, we show that the majority of genes were associated
with a single prominent H2A.Z containing nucleosome in their promoter region. We
classified genes into clusters depending on the proximity of H2A.Z to the TSS.
The genes with no detectable H2A.Z showed lowest expression level, whereas H2A.Z
was positioned closer to the TSS of genes with higher expression levels. We
confirmed this relation between the proximity of H2A.Z and expression level in
the brain. The proximity of histone variant H2A.Z, but not H3.3 to the TSS, over
seven consecutive nucleosomes, was correlated with expression. Further, a
nucleosome was positioned over the TSS of silenced genes while it was displaced
to expose the TSS in highly expressed genes. Our results suggest that gene
expression levels in vivo are determined by accessibility of the TSS and
proximity of H2A.Z. Eukaryotes tune the transcriptional activity of their genome by altering the
nucleosome core particle through multiple chemical processes. In particular,
replacement of the canonical H2A histone with the variants macroH2A and H2A.Z
has been shown to affect DNA accessibility and nucleosome stability; however,
the processes by which this occurs remain poorly understood. In this study, we
elucidate the molecular mechanisms of these variants with an extensive molecular
dynamics study of the canonical nucleosome along with three variant-containing
structures: H2A.Z, macroH2A, and an H2A mutant with macroH2A-like L1 loops.
Simulation results show that variant L1 loops play a pivotal role in stabilizing
DNA binding to the octamer through direct interactions, core structural
rearrangements, and altered allosteric networks in the nucleosome. All variants
influence dynamics; however, macroH2A-like systems have the largest effect on
energetics. In addition, we provide a comprehensive analysis of allosteric
networks in the nucleosome and demonstrate that variants take advantage of
stronger interactions between L1 loops to propagate dynamics throughout the
complex. Furthermore, we show that posttranslational modifications are enriched
at key locations in these networks. Taken together, these results provide, to
our knowledge, new insights into the relationship between the structure,
dynamics, and function of the nucleosome core particle and chromatin fibers, and
how they are influenced by chromatin remodeling factors. Nucleosomes are implicated in transcriptional regulation as well as in packing
and stabilizing the DNA. Nucleosome positions affect the transcription by
impeding or facilitating the binding of transcription factors. The DNA sequence,
especially the periodic occurrences of dinucleotides, is a major factor that
affects the nucleosome positioning. We analyzed the Drosophila DNA sequences
bound by H2A and H2A.Z nucleosomes. Periodic patterns of dinucleotides
(weak-weak/strong-strong or purine-purine/pyrimidine-pyrimidine) were identified
as WW/SS and RR/YY nucleosome positioning sequence (NPS) patterns. The WW/SS NPS
pattern of the H2A nucleosome has a 10-bp period of weak-weak/strong-strong
(W = A or T; S = G or C) dinucleotides. The 10-bp periodicity, however, is
disrupted in the middle of the sequence. At the dyad, the SS dinucleotide is
preferred. On the other hand, the RR/YY NPS pattern has an 18-bp periodicity of
purine-purine/pyrimidine-pyrimidine (R = A or G; Y = T or C) dinucleotides. The
NPS patterns from H2A.Z nucleosomes differ from the NPS patterns from H2A
nucleosomes. The RR/YY pattern of H2A.Z nucleosomes has major peaks shifted by
10 bp deviated from the H2A nucleosome pattern. The H2A and H2A.Z nucleosomes
have different sequence preferences. The shifted peaks coincide with DNA regions
interacting with the histone loops. |
Is dasatinib effective for treatment of glioblastoma? | No, dasatinib is ineffective for treatment of glioblastoma and is associated with significant toxicity. | There is no effective treatment for recurrent glioblastoma (GBM) after
bevacizumab failure. Putative mechanisms of resistance to bevacizumab include
increased pericyte coverage, mediated partly by platelet-derived growth factor
receptor (PDGFR) signaling, and an infiltrative tumor growth pattern potentially
dependent on SRC. We explored the efficacy of dasatinib, a SRC, BCR-ABL, c-KIT,
EPHA2, and PDGFRβ inhibitor, in patients with recurrent GBM after bevacizumab
failure. Adult patients with histologically confirmed GBM who failed bevacizumab
therapy were treated with dasatinib 70-100 mg twice daily in combination with
bevacizumab (n = 14), until tumor progression or unacceptable toxicity. Fourteen
patients were treated. Median age was 55 years (range 32-66) and median KPS was
80 (range 50-90). All patients (100%) had glioblastomas. The median number of
prior regimens was 4 (range from 2 to 6). Of the thirteen evaluable patients,
none had a complete or partial response. Only one patient had stable disease
after an 8 week interval. Median progression-free survival (PFS) was 28 days
(95% confidence interval [CI] 26-35 days). Six month progression-free survival
(PFS6) was 0%. Median overall survival (OS) was 78 days (95% CI 41-137 days).
Treatment was moderately well-tolerated, although one patient sustained a grade
4 intracerebral hemorrhage. Dasatinib in conjunction with bevacizumab does not
appear to have activity in patients with recurrent, heavily pretreated GBM. The treatment of patients with recurrent glioblastoma remains a major oncologic
problem, with median survival after progression of 7-9 months. To determine the
maximum tolerated dose and dose-limiting toxicity (DLT), the combination of
dasatinib and cyclonexyl-chloroethyl-nitrosourea (CCNU) was investigated in this
setting. The study was designed as multicenter, randomized phase II trial,
preceded by a lead-in safety phase. The safety component reported here, which
also investigated pharmacokinetics and preliminary clinical activity, required
expansion and is therefore considered a phase I part to establish a recommended
dosing regimen of the combination of CCNU (90-110 mg/m(2)) and dasatinib
(100-200 mg daily). Overall, 28 patients were screened, and 26 patients were
enrolled. Five dose levels were explored. DLTs, mainly myelosuppression,
occurred in 10 patients. Grade 3 or 4 neutropenia was recorded in 7 patients
(26.9%) and thrombocytopenia in 11 patients (42.3%). No significant effect of
CCNU coadministration on dasatinib pharmacokinetics was found. Median
progression-free survival (PFS) was 1.35 months (95% confidence interval:
1.2-1.4) and 6-month PFS was 7.7%. In this phase I study of recurrent
glioblastoma patients, the combination of CCNU and dasatinib showed significant
hematological toxicities and led to suboptimal exposure to both agents. BACKGROUND: We conducted a phase II trial to evaluate the efficacy of dasatinib,
a multitargeted tyrosine kinase inhibitor, for adults with recurrent
glioblastoma (GBM).
METHODS: Eligibility requirements were Karnofsky performance status ≥ 60%; no
concurrent hepatic enzyme-inducing anticonvulsants; prior treatment with
surgery, radiotherapy, and temozolomide exclusively; and activation or
overexpression of ≥ 2 putative dasatinib targets in GBM (ie, SRC, c-KIT, EPHA2,
and PDGFR). Using a 2-stage design, 77 eligible participants (27 in stage 1, if
favorable, and then 50 in stage 2) were needed to detect an absolute improvement
in the proportion of patients either alive and progression-free patients at 6
months (6mPFS) or responding (any duration) from a historical 11% to 25%.
RESULTS: A high rate of ineligibility (27%) to stage 1 precluded a powered
assessment of efficacy, but there was also infrequent treatment-related toxicity
at 100 mg twice daily. Therefore, the study was redesigned to allow intrapatient
escalation by 50 mg daily every cycle as tolerated (stage 1B) before determining
whether to proceed to stage 2. Escalation was tolerable in 10 of 17 (59%)
participants evaluable for that endpoint; however, among all eligible patients
(stages 1 and 1B, n = 50), there were no radiographic responses, median overall
survival was 7.9 months, median PFS was 1.7 months, and the 6mPFS rate was 6%.
The clinical benefit was insufficient to correlate tested biomarkers with
efficacy. The trial was closed without proceeding to stage 2.
CONCLUSIONS: Intraparticipant dose escalation was feasible, but dasatinib was
ineffective in recurrent GBM. Clinical trials.gov identified. NCT00423735
(available at http://clinicaltrials.gov/ct2/show/NCT00423735). BACKGROUND: glioblastomas are highly vascularized tumors and various
antiangiogenic drugs have been investigated in clinical trials showing unclear
results. We performed a systematic review and a meta-analysis to clarify and
evaluate their effectiveness in glioblastoma patients.
PATIENTS AND METHODS: we searched relevant published and unpublished randomized
clinical trials analyzing antiangiogenic drugs versus chemotherapy in
glioblastoma patients from January 2006 to January 2016 in MEDLINE, WEB of
SCIENCE, ASCO, ESMO and SNO databases.
RESULTS: fourteen randomized clinical trials were identified (7 with
bevacizumab, 2 cilengitide, 1 enzastaurin, 1 dasatinib, 1 vandetanib, 1
temsirolimus, 1 cediranib) including 4330 patients. Antiangiogenic drugs showed
no improvement in overall survival with a pooled HR of 1.00, a trend for an
inferior outcome, in terms of overall survival, was observed in the group of
patients receiving antiangiogenic drug alone compared to cytotoxic drug alone
(HR=1.24, p=0.056). Bevacizumab did not improve overall survival. Twelve trials
(4113 patients) were analyzed for progression-free survival. Among
antiangiogenic drugs, only bevacizumab demonstrated an improvement of
progression-free survival (HR=0.63, p<0.001), both alone (HR=0.60, p=0.003) or
in combination to chemotherapy (HR=0.63; p<0.001), both as first-line treatment
(HR=0.70, p<0.001) or in recurrent disease (HR=0.52, p<0.001).
CONCLUSIONS: antiangiogenic drugs did not improve overall survival in
glioblastoma patients, either as first or second-line treatment, and either as
single agent or in combination with chemotherapy. Among antiangiogenic drugs,
only bevacizumab improved progression-free survival regardless of treatment
line, both as single agent or in combination with chemotherapy. |
Which algorithm is available for computing minimal absent words using external memory? | emMAW | |
Has IVIG been tested in clinical trials for the treatment of Alzheimer's disease? | Yes, IVIG has been tested in clinical trials for the treatment of Alzheimer's disease. | |
Which type of urinary incontinence is diagnosed with the Q tip test? | Stress urinary incontinence is diagnosed with the Q tip test. The test evaluates urethral mobility. | Thirty-two female patients with clinical and urodynamic findings of genuine
stress urinary incontinence were evaluated before and 6 months after surgery for
stress urinary incontinence. Twenty-nine control patients had identical
evaluations before and 6 months after surgery which did not involve the
urethrovesical junction. Twenty-four patients with primary bladder instability
had similar evaluations and served as a second control group. Anatomical
landmarks indicating support to the urethrovesical junction were evaluated by
the position of the urethra at the most dependent point in the bladder on
straining and the urethral descent on straining to beneath the posterior ramus
of the symphysis pubis on bead chain cystography. The urethrovesical junction
drop on straining was evaluated by transrectal ultrasonography. Cystographic and
ultrasonographic tests for the position of the urethrovesical junction at the
most dependent position in the bladder during straining were very sensitive in
women with stress urinary incontinence (94 and 87% respectively) but much less
specific (45 and 48% respectively). When evaluating anatomical support to the
urethrovesical junction and its descent on straining, these tests were both
highly sensitive (97 and 94% respectively) and specific (76 and 96%
respectively) in women with genuine stress urinary incontinence. Simple clinical
tests for support of the urethrovesical junction, such as the Q tip test, are
non-specific in patients with stress urinary incontinence. Transrectal
ultrasonography is a simple and quick out-patient procedure. The availability of
ultrasound equipment in most clinics and the high sensitivity and specificity of
the test make it an attractive and cost-effective alternative to X-ray
cystography in the pre-operative evaluation of anatomical support to the
urethrovesical junction. The Q-tip test was applied on 105 patients. Fifty-one had stress urinary
incontinency (SUI), 28 had bladder instability by clinical and urodynamic
criteria, and 36 had mild or moderate pelvic relaxation without urinary
pathology. More than 90% of the patients with SUI and no previous surgery had a
positive Q-tip test, with 90% test sensitivity in this group. More than
one-third of the patients with bladder instability and almost one-half of the
patients with pelvic relaxation and no urinary incontinence had a positive Q-tip
test, for low test specificity. The Q-tip test is a simple clinical tool for
diagnosing pelvic relaxation, which at times leads to SUI. Almost all patients
with primary SUI have pelvic relaxation. The Q-tip test alone does not stand as
a diagnostic test. When it is positive, the diagnosis of genuine stress
incontinence is possible although not absolute. A negative test should cause one
to question the diagnosis of genuine stress incontinence, and sophisticated and
more expensive tests should be ordered before establishing a final diagnosis. INTRODUCTION AND HYPOTHESIS: The aim of the study was to exclude neurovascular
damage due to prosthetic mini-invasive surgery using transobturator tape (TOT)
by pre- and postoperative electromyography (EMG) of the striated urethral
sphincter and a color Doppler ultrasonography evaluation of clitoral blood flow.
METHODS: A total of 25 women affected by clinical stress urinary incontinence
(SUI) were enrolled. After undergoing urodynamic assessment, pelvic organ
prolapse quantification, urine culture, Q-tip test, and stress test, each
subject underwent color Doppler ultrasonography to record clitoral blood flow
and EMG of the urethral sphincter with a needle electrode inserted through the
mucosa into the muscle tissue before surgery. A single urogynecologist performed
the TOT surgical technique for the treatment of all patients. Urogynecologic
examination, EMG, and color Doppler ultrasound follow-up were performed at 1 and
6 months after surgery.
RESULTS: At the urogynecologic examination performed 1 and 6 months after the
TOT approach the stress test was negative, urethral hypermobility was reduced,
and sling exposure was not observed for each patient. There was no statistically
significant difference in electromyographic values (p > 0.05) in both the
follow-ups with regard to baseline values. Pulsatility index (PI), resistance
index (RI), and peak systolic velocity (PSV) values increased during the first
follow-up (p < 0.01); PI and RI values increased during the second follow-up
with respect to baseline values (p < 0.01)
CONCLUSIONS: TOT prosthesis surgery, avoiding denervation and devascularization
of pelvic structures, does not damage the urethral sphincter. OBJECTIVE: To evaluate the impact of a more limited paraurethral dissection,
avoidance of perforating the obturator membrane with scissors or guide, and a
more medial trajectory of the trocar in positioning the TVT-O device on stress
urinary incontinence cure rates.
STUDY DESIGN: One hundred and ten patients were recruited for this randomized,
single blind, multicenter, non-inferiority study, with a 1:1 ratio to undergo
the traditional (n=55) or the modified (n=55) technique. Preoperatively,
patients underwent POP-Q staging, Q-tip test, challenge stress test and
urodynamics, and completed the I-QoL, PISQ-12, and PGI-S questionnaires. During
the post-operative period, patients attributed a pain VAS score 1, 3, 6, 12 and
24h after the procedure and were followed up at 12 months, undergoing the same
baseline evaluations. The primary outcome was the cure rate (absence of urine
leaks at the challenge stress test or urodynamic testing) one year after the
procedure. The primary outcome was evaluated using a non-inferiority test.
RESULTS: No differences were observed in cure rates (traditional technique 92.3%
vs. modified technique 88.8% and non-inferiority P<0.05) and in questionnaire
scores between the two groups. Post-operative pain was significantly lower in
the modified technique group at each time point assessed, with the exception of
12h post-operatively. No differences between the two groups were observed in the
number of analgesic vials administered.
CONCLUSIONS: The modified technique does not seem to reduce the efficacy of
TVT-O, but induces a reduction of post-operative pain. PURPOSE: To clarify the association between clinically defined simple stress
urinary incontinence (SUI) symptoms and urodynamic SUI, we examined the
relationship between Valsalva leak point pressure (VLPP) as measured by the
Q-tip test and Stamey grade in simple female SUI.
METHODS: Two hundred grade I or II female SUI patients with SUI symptom were
examined by reviewing medical history; physical examination; urethral mobility
as assessed by Q-tip test; stress test; and cystometry, including VLPP
measurement. On the basis of the VLPP, patients were classified into urethral
hypermobility [UH, subdivided into anatomical incontinence (AI) and equivocal
incontinence (EI)] or intrinsic sphincter deficiency groups for analysis of the
relationship between VLPP and Stamey grade and Q-tip angle.
RESULTS: Seventy-eight patients were included, and the mean patient age was 54 ±
7.5 years, mean SUI symptom duration 2.8 years (range 0.5-6 years), mean VLPP
103.6 ± 18.4 cm H2O, and mean Q-tip angle 28.6° ± 7.2°. Fifty-three patients
were categorized as Stamey grade I, 25 as Stamey grade II, 51 as AI, and 27 as
EI. VLPP was found to be negatively correlated with Q-tip angle (Rs = -0.798, Y
= -0.313X + 60.95, P < 0.001), and classifications of VLPP and Stamey grade have
positive correlation (χ (2) = 4.9130, P = 0.0267).
CONCLUSIONS: In simple female SUI, VLPP is associated with the Q-tip angle and
Stamey grade, which may help to reduce some of urodynamic items. OBJECTIVE: To compare the change of urethral mobility after midurethral sling
procedures in stress urinary incontinence with hypermobile urethra and assess
these findings with surgical outcomes.
STUDY DESIGN: 141 women who agreed to undergo midurethral sling operations due
to stress urinary incontinence with hypermobile urethra were enrolled in this
non-randomized prospective observational study. Preoperatively, urethral
mobility was measured by Q tip test. All women were asked to complete Urogenital
Distress Inventory Short Form (UDI-6) and Incontinence Impact Questionnaire
Short Form (IIQ-7) to assess the quality of life. Six months postoperatively, Q
tip test and quality of life assessment were repeated. The primary surgical
outcomes were classified as cure, improvement and failure. Transient urinary
obstruction, de novo urgency, voiding dysfunction were secondary surgical
outcomes.
RESULTS: Of 141 women, 50 (35. 5%) women underwent TOT, 91 (64.5%) underwent
TVT. In both TOT and TVT groups, postoperative Q tip test values, IIQ-7 and
UDI-6 scores were statistically reduced when compared with preoperative values.
Postoperative Q tip test value in TVT group was significantly smaller than in
TOT group [25°(15-45°) and 20° (15-45°), respectively]. When we compared the
Q-tip test value, IIQ-7 and UDI-6 scores changes, there were no statistically
significant changes between the groups. Postoperative urethral mobility was more
frequent in TOT group than in TVT group (40% vs 23.1%, respectively).
Postoperative primary and secondary outcomes were similar in both groups.
CONCLUSIONS: Although midurethral slings decrease the urethtal hypermobility,
postoperative mobility status of urethra does not effect surgical outcomes of
midurethral slings in women with preoperative urethral hypermobility. |
Does a tonsillectomy affect the patient's voice? | Some patients complaint for dry throat, foreign body sensation or voice change after tonsillectomy. Group B had a better awareness of tooth damage . There were no differences in secondary outcomes across treatment groups. The incidence rates of voice change, velopharyngeal insufficiency, bleeding, constipation, dehydration, and pain were measured. | This paper reports about a female mutational falsetto, that means an unusual
high (309 Hz base frequency) fundamental frequency of the speaking voice in a
19-year-old girl. The psychological background and the epicrisis as well as the
practical negative result of the hormon- and metabolism examination allow the
diagnosis of this rare disturbance of the voice, the formal classification of
which would on the other hand also allow the statement of a phononeurosis with a
too high fundamental frequency of the speaking voice because the behavior of the
voice from the menarche (12 years) up to the first examination does not seem
assured definately. The individual median fundamental frequency of the speaking
voice has inally been obtained at 270 Hz. Energy range of the speaking voice has
been measured, glottography and sonagraphy has been carried out, all of which
show the course of treatment clearly. It has been remarked critically that there
has to be found a method to better reproduce the hearing impression in a figure.
Investigations according to this have been made. The vocal tract from the glottis to the lips is considered to he a resonator and
the voice is changeable depending upon the shape of the vocal tract. In this
report, we examined the change in pharyngeal size and acoustic feature of voice
after tonsillectomy.
METHODS: Subjects were 20 patients. The distance between both anterior pillars
(glossopalatine arches), and between both posterior pillars (pharyngopalatine
arches) was measured weekly. For acoustic measurements, the five Japanese vowels
and Japanese conversational sentences were recorded and analyzed.
RESULTS: The distance between both anterior pillars became wider 2 weeks
postoperatively, and tended to become narrower thereafter. The distance between
both posterior pillars became wider even after 4 weeks postoperatively. No
consistent changes in F0, F1 and F2 were found after surgery. Although there was
a tendency for a decrease in F3, tonsillectomy did not appear to change the
acoustical features of the Japanese vowels remarkably. It was assumed that the
subject may adjust the shape of the vocal tract to produce consistent speech
sounds after the surgery using auditory feedback. Young adulthood is notable for rapid physical changes and psychosocial
instability. Care of the young adult professional voice requires knowledge of
the specific anatomic and physiologic changes associated with the mutational
voice, as well as the effects of general growth and maturation on the vocal
mechanism. The effects of psychological stresses common to young adulthood, such
as educational commitments and early career choices, must also be understood.
Upper respiratory infection and allergies are common in this age group.
Treatment of these conditions must be tailored in the professional voice user
because of the potential side effects of some medications and the performance
imperatives of the patient. Surgical indications for tonsillectomy in the young
voice patient are discussed. There are no special considerations in the
evaluation and treatment of laryngeal pathology in the young adult, with the
exception of limiting the use of sedative anesthesia. However, conservatism in
surgical decision-making is advised. The development of a stable, efficient
vocal technique and a mature professional background requires time, patience,
and hard work. BACKGROUND: Tonsillectomy is associated with postoperative nausea and vomiting
(PONV) if no prophylaxis is administered. Previous studies have shown that a
single dose of dexamethasone decreases the incidence of PONV. The most effective
dose of dexamethasone to affect clinical outcome is yet to be defined.
METHODS: One-hundred-twenty-five children were enrolled in a double-blind,
prospective, randomized, dose-escalating study of dexamethasone: 0.0625, 0.125,
0.25, 0.5, or 1 mg/kg, maximum dose 24 mg. Nonparametric ANOVA was used to
analyze the incidence of vomiting by treatment group for 0 to < or =5 h, >5 to
24 h. The Cox Proportional Likelihood Ratio Test was used to compare the time of
first vomit and time to first pain medication across treatment groups.
RESULTS: There was no difference in the incidence of vomiting for the five
escalating doses of dexamethasone in the time period. There were no differences
in secondary outcomes (analgesic requirements, time to first liquid, and change
in voice) across treatment groups.
CONCLUSION: We conclude that the lowest dose of dexamethasone (0.0625 mg/kg) was
as effective as the highest dose of dexamethasone (1.0 mg/kg) for preventing
PONV or reducing the incidence of other secondary outcomes following
tonsillectomy or adenotonsillectomy. There is no justification for the use of
high-dose dexamethasone for the prevention of PONV in this cohort of children. OBJECTIVE: To evaluate the effectiveness of follow-up telephone interviews and
questionnaires after tonsillectomy and adenoidectomy.
DESIGN: Cohort study and retrospective review of the outcomes of patients whose
follow-ups were conducted by telephone interview. Patients were contacted 2 to 4
weeks after surgery; responses were recorded on a standardized postoperative
questionnaire.
SETTING: Tertiary pediatric hospital.
PATIENTS: A total of 2554 consecutive patients who had undergone tonsillectomy,
adenoidectomy, or both procedures and completed a follow-up telephone interview
during the period of January 8, 2000, to September 23, 2004.
MAIN OUTCOME MEASURES: Time to return to normal diet and activities,
postoperative complications, pain management, postoperative visits, and
caregiver's evaluation of the follow-up telephone survey.
RESULTS: A total of 2554 patient outcomes were reviewed. The mean patient age
was 5.9 years. Follow-up contact occurred a mean of 24.1 days after surgery. Of
the surgical procedures performed, there were 1957 adenotonsillectomies, 235
adenoidectomies, and 362 tonsillectomies. At the time of follow-up, 2.7% of the
patients had undergone an additional surgical procedure to treat postoperative
bleeding, 96.9% had resumed eating a normal diet, and 96.2% had resumed normal
activities. Bleeding from the nose or mouth was reported to have occurred at
some point during the recovery period in 12.8%. On a pain scale of 1 to 10, a
mean pain peak of 6.7 was reported. For most patients, pain was highest on the
second day after surgery. The percentage of patients who had temporary voice
change was 62.7%, and 15.4% had a follow-up clinic visit. Regarding caregivers,
99.5% reported being given instructions for postoperative care, and 98.8%
reported that they felt well prepared to care for their child at home. There
were no adverse events reported from surgical intervention.
CONCLUSIONS: Compared with our previous experience with scheduled postsurgical
clinic follow-ups, telephone interviews and standardized postoperative
questionnaires pose no additional risk to patients. Considerable cost reduction
and patient convenience were realized with a reduction of patient visits. OBJECTIVE: To observe the long-term effect of tonsillectomy and provide clinical
evidence for tonsillectomy.
METHOD: One hundred and one patients undergoing tonsillectomy in our department
were included. Their satisfaction and symptom change were followed up by
telephone.
RESULT: 73.3% patients were satisfied with their surgery. Chief complaints such
as pharyngalgia, fever, snoring were significantly decreased after surgery,
while foreign body sensation still existed. Some patients complaint for dry
throat, foreign body sensation or voice change after tonsillectomy.
CONCLUSION: Most patients were satisfied with the tonsillectomy. While few of
them had new complaints after tonsillectomy. OBJECTIVE: To assess the effectiveness of preoperative phone counseling by
junior medical staff for improving the standard of informed consent for
tonsillectomy.
STUDY DESIGN: Prospective randomized controlled trial.
SETTING: District general hospital.
SUBJECTS AND METHODS: A total of 43 patients undergoing tonsillectomy were
randomly allocated to 2 groups. Group A (n = 25) underwent the conventional
consent process by the consultant ear, nose, and throat surgeon at the time of
assessment (which generally takes place 6 to 12 months prior to surgery due to
wait-list times). Group B (n = 18) underwent this same consent process but
received a structured preoperative phone call 2 to 3 weeks prior to the day of
surgery. A preoperative questionnaire assessing the knowledge of tonsillectomy,
perioperative course, and risks was completed on the day of surgery.
RESULTS: Group B had a better recall of the risks of tonsillectomy, recalling
7.1 of the 10 most significant risks, as compared with 4.6 for group A (P =
.017). Group B had a better awareness of tooth damage (78% vs 30% of patients, P
≤ .001), voice change (61 vs 19%, P = .005), and burns to lips and mouth (44% vs
8%, P = .005). Finally, 35% more patients from group B rated their understanding
of tonsillectomy as good or very good (P = .017).
CONCLUSION: Preoperative phone counseling by junior medical staff closer to the
time of surgery reinforces and clarifies the information previously provided by
senior consultants at the time of initial consent for tonsillectomy. |
List the four most important interferonopathies | Aicardi-Goutières syndrome
chilblain lupus
ubiquitin specific peptidase 18 (USP18)-deficiency
Singleton-Merten syndrome | Autoinflammatory disorders are sterile inflammatory conditions characterized by
episodes of early-onset fever and disease-specific patterns of organ
inflammation. Recently, the discoveries of monogenic disorders with strong type
I interferon (IFN) signatures caused by mutations in proteasome degradation and
cytoplasmic RNA and DNA sensing pathways suggest a pathogenic role of IFNs in
causing autoinflammatory phenotypes. The IFN response gene signature (IGS) has
been associated with systemic lupus erythematosus (SLE) and other autoimmune
diseases. In this review, we compare the clinical presentations and pathogenesis
of two IFN-mediated autoinflammatory diseases, CANDLE and SAVI, with Aicardi
Goutières syndrome (AGS) and monogenic forms of SLE (monoSLE) caused by
loss-of-function mutations in complement 1 (C1q) or the DNA nucleases, DNASE1
and DNASE1L3. We outline differences in intracellular signaling pathways that
fuel a pathologic type I IFN amplification cycle. While IFN amplification is
caused by predomitly innate immune cell dysfunction in SAVI, CANDLE, and AGS,
autoantibodies to modified RNA and DNA antigens interact with tissues and immune
cells including neutrophils and contribute to IFN upregulation in some SLE
patients including monoSLE, thus justifying a grouping of "autoinflammatory" and
"autoimmune" interferonopathies. Understanding of the differences in the
cellular sources and signaling pathways will guide new drug development and the
use of emerging targeted therapies. Type I interferon is a potent substance. As such, the induction, transmission,
and resolution of the type I interferon-mediated immune response are tightly
regulated. As defined, the type I interferonopathies represent discrete examples
of a disturbance of the homeostatic control of this system caused by Mendelian
mutations. Considering the complexity of the interferon response, the
identification of further monogenic diseases belonging to this disease grouping
seems likely, with the recognition of type I interferonopathies becoming of
increasing clinical importance as treatment options are developed based on an
understanding of disease pathology and innate immune signaling. Definition of
the type I interferonopathies indicates that autoinflammation can be both
interferon and noninterferon related, and that a primary disturbance of the
innate immune system can "spill over" into autoimmunity in some cases. Indeed,
that several non-Mendelian disorders, most particularly systemic lupus
erythematosus and dermatomyositis, are also characterized by an up-regulation of
type I interferon signaling suggests the possibility that insights derived from
this work will have relevance to a broader field of clinical medicine. OBJECTIVE: Type I interferon (IFN) is implicated in the pathogenesis of systemic
lupus erythematosus (SLE) and interferonopathies such as Aicardi-Goutières
syndrome. A recently discovered DNA-activated type I IFN pathway, cyclic GMP-AMP
synthase (cGAS), has been linked to Aicardi-Goutières syndrome and mouse models
of lupus. The aim of this study was to determine whether the cGAS pathway
contributes to type I IFN production in patients with SLE.
METHODS: SLE disease activity was measured by the Safety of Estrogens in Lupus
Erythematosus National Assessment version of the Systemic Lupus Erythematosus
Disease Activity Index. Expression of messenger RNA for cGAS and IFN-stimulated
genes (ISGs) was determined by quantitative polymerase chain reaction analysis.
Cyclic GMP-AMP (cGAMP) levels were examined by multiple reaction monitoring with
ultra-performance liquid chromatography tandem mass spectrometry.
RESULTS: Expression of cGAS in peripheral blood mononuclear cells (PBMCs) was
significantly higher in SLE patients than in normal controls (n = 51 and n = 20
respectively; P < 0.01). There was a positive correlation between cGAS
expression and the IFN score (P < 0.001). The expression of cGAS in PBMCs showed
a dose response to type I IFN stimulation in vitro, consistent with it being an
ISG. Targeted measurement of cGAMP by tandem mass spectrometry detected cGAMP in
15% of the SLE patients (7 of 48) but none of the normal (0 of 19) or rheumatoid
arthritis (0 of 22) controls. Disease activity was higher in SLE patients with
cGAMP versus those without cGAMP.
CONCLUSION: Increased cGAS expression and cGAMP in a proportion of SLE patients
indicates that the cGAS pathway should be considered as a contributor to type I
IFN production. Whereas higher cGAS expression may be a consequence of exposure
to type I IFN, detection of cGAMP in patients with increased disease activity
indicates potential involvement of this pathway in disease expression. Aicardi-Goutières syndrome (AGS) is an inflammatory disorder belonging to the
recently characterized group of type I interferonopathies. The most consistently
affected tissues in AGS are the central nervous system and skin, but various
organ systems and tissues have been reported to be affected, pointing to the
systemic nature of the disease. Here we describe a patient with AGS due to a
homozygous p.Arg114His mutation in the TREX1 gene. The histologically proven
inflammatory myopathy in our patient expands the range of clinical features of
AGS. Histological signs of muscle biopsies in the proband, and in two other AGS
patients described earlier, are similar to those seen in various autoimmune
myositises and could be ascribed to inapproapriate IFN I activation. In view of
signs of possible mitochondrial damage in AGS, we propose that mitochondrial DNA
could be a trigger of autoimmune responses in AGS. Type I interferons (IFNs), IFN-α and IFN-β, represent the major effector
cytokines of the host immune response against viruses and other intracellular
pathogens. These cytokines are produced via activation of numerous pattern
recognition receptors, including the Toll-like receptor signaling network,
retinoic acid-inducible gene-1 (RIG-1), melanoma differentiation-associated
protein-5 (MDA-5) and interferon gamma-inducible protein-16 (IFI-16). Whilst the
contribution of type I IFNs to peripheral immunity is well documented, they can
also be produced by almost every cell in the central nervous system (CNS).
Furthermore, IFNs can reach the CNS from the periphery to modulate the function
of not only microglia and astrocytes, but also neurons and oligodendrocytes,
with major consequences for cognition and behavior. Given the pleiotropic nature
of type I IFNs, it is critical to determine their exact cellular impact.
Inappropriate upregulation of type I IFN signaling and interferon-stimulated
gene expression have been linked to several CNS diseases termed
"interferonopathies" including Aicardi-Goutieres syndrome and ubiquitin specific
peptidase 18 (USP18)-deficiency. In contrast, in the CNS of mice with
virus-induced neuroinflammation, type I IFNs can limit production of other
cytokines to prevent potential damage associated with chronic cytokine
expression. This capacity of type I IFNs could also explain the therapeutic
benefits of exogenous type I IFN in chronic CNS autoimmune diseases such as
multiple sclerosis. In this review we will highlight the importance of a
well-balanced level of type I IFNs for healthy brain physiology, and to what
extent dysregulation of this cytokine system can result in brain
'interferonopathies'. The knowledge on systemic autoinflammatory disorders (SAID) is expanding rapidly
and new signalling pathways are being decrypted. The concept of autoinflammation
has been proposed since 1999, to define a group of diseases with abnormal innate
immunity activation. Since then, more than 30 monogenic SAID have been
described. In this review, we first describe inflammasomopathies and SAID
related to the interleukin-1 pathway. Recent insights into the pathogenesis of
familial Mediterranean fever and the function of Pyrin are detailed. In
addition, complex or polygenic SAID, such as Still's disease or PFAPA syndrome,
are also discussed. Then, major players driving autoinflammation, such as type-1
interferonopathies (including the recently described haploinsuffiency in A20 and
otulipenia), TNF-associated periodic syndromes, defects in ubiquitination, and
SAID with overlapping features of autoimmunity or immunodeficiency. Discoveries
of the pathogenic role of mosaicism, intronic defects coupled to the likelihood
to identify digenic or polygenic diseases are providing new challenges for
physicians and geneticists. This comprehensive review depicts the various SAID,
presenting them according to their predomit pathophysiological mechanism,
with a particular emphasis on recent findings. Epidemiologic data are also
presented. Finally, we propose a practical diagnostic approach to the most
common monogenic SAID, based on the most characteristic clinical presentation of
these disorders. PURPOSE OF REVIEW: We give an update on the etiology and potential treatment
options of rare inherited monogenic disorders associated with arterial
calcification and calcific cardiac valve disease.
RECENT FINDINGS: Genetic studies of rare inherited syndromes have identified key
regulators of ectopic calcification. Based on the pathogenic principles causing
the diseases, these can be classified into three groups: (1) disorders of an
increased extracellular inorganic phosphate/inorganic pyrophosphate ratio
(generalized arterial calcification of infancy, pseudoxanthoma elasticum,
arterial calcification and distal joint calcification, progeria, idiopathic
basal ganglia calcification, and hyperphosphatemic familial tumoral calcinosis;
(2) interferonopathies (Singleton-Merten syndrome); and (3) others, including
Keutel syndrome and Gaucher disease type IIIC. Although some of the identified
causative mechanisms are not easy to target for treatment, it has become clear
that a disturbed serum phosphate/pyrophosphate ratio is a major force triggering
arterial and cardiac valve calcification. Further studies will focus on
targeting the phosphate/pyrophosphate ratio to effectively prevent and treat
these calcific disease phenotypes. PURPOSE OF REVIEW: Familial chilblain lupus belongs to the group of type I
interferonopathies and is characterized by typical skin manifestations and acral
ischaemia. This review aims to give an overview of clinical signs and the
pathophysiological mechanisms.
RECENT FINDINGS: There are several mutations that can lead to this autosomal
domit disease. Most frequent is a mutation of the gene for TREX-1. However,
as well cases of families with mutations in the SAMHD1 gene and, recently, with
one for the gene that codes for the protein stimulator of interferon genes have
been described. These genes are involved in the process of the detection of
intracellular DNA, and their mutation results in an increased production of type
I interferons and their gene products, resulting in auto-inflammation and
auto-immunity. JAK inhibitors have been successfully used to treat this
disorder. Familial chilblain is a rare disorder with very distinct clinical
signs. Its pathophysiological mechanism gives insight into the process of
interferon-induced inflammation in auto-immune diseases. |
List the partners of budding yeast Cdc48 that are important for disassembly of ubiquitylated CMG helicase at the end of chromosome replication | The ubiquitin-binding Ufd1-Npl4 complex recruits Cdc48 to ubiquitylated CMG helicase at the end of chromosome replication. | Disassembly of the Cdc45-MCM-GINS (CMG) DNA helicase is the key regulated step
during DNA replication termination in eukaryotes, involving ubiquitylation of
the Mcm7 helicase subunit, leading to a disassembly process that requires the
Cdc48 "segregase". Here, we employ a screen to identify partners of budding
yeast Cdc48 that are important for disassembly of ubiquitylated CMG helicase at
the end of chromosome replication. We demonstrate that the ubiquitin-binding
Ufd1-Npl4 complex recruits Cdc48 to ubiquitylated CMG. Ubiquitylation of CMG in
yeast cell extracts is dependent upon lysine 29 of Mcm7, which is the only
detectable site of ubiquitylation both in vitro and in vivo (though in vivo
other sites can be modified when K29 is mutated). Mutation of K29 abrogates
in vitro recruitment of Ufd1-Npl4-Cdc48 to the CMG helicase, supporting a model
whereby Ufd1-Npl4 recruits Cdc48 to ubiquitylated CMG at the end of chromosome
replication, thereby driving the disassembly reaction. |
How are topologically associating domains (TAD) associated with replication timing? | Topologically associating domains and their long-range contacts are established during early G1 coincident with the establishment of the replication-timing program. Topologically associating domains are stable units of replication-timing regulation. | Mammalian genomes are partitioned into domains that replicate in a defined
temporal order. These domains can replicate at similar times in all cell types
(constitutive) or at cell type-specific times (developmental). Genome-wide
chromatin conformation capture (Hi-C) has revealed sub-megabase topologically
associating domains (TADs), which are the structural counterparts of replication
domains. Hi-C also segregates inter-TAD contacts into defined 3D spatial
compartments that align precisely to genome-wide replication timing profiles.
Determits of the replication-timing program are re-established during early
G1 phase of each cell cycle and lost in G2 phase, but it is not known when TAD
structure and inter-TAD contacts are re-established after their elimination
during mitosis. Here, we use multiplexed 4C-seq to study dynamic changes in
chromatin organization during early G1. We find that both establishment of TADs
and their compartmentalization occur during early G1, within the same time frame
as establishment of the replication-timing program. Once established, this 3D
organization is preserved either after withdrawal into quiescence or for the
remainder of interphase including G2 phase, implying 3D structure is not
sufficient to maintain replication timing. Finally, we find that developmental
domains are less well compartmentalized than constitutive domains and display
chromatin properties that distinguish them from early and late constitutive
domains. Overall, this study uncovers a strong connection between chromatin
re-organization during G1, establishment of replication timing, and its
developmental control. The combinatorial action of transcription factors drives cell-type-specific gene
expression patterns. However, transcription factor binding and gene regulation
occur in the context of chromatin, which modulates DNA accessibility.
High-resolution chromatin interaction maps have defined units of chromatin that
are in spatial proximity, called topologically associated domains (TADs). TADs
can be further classified based on expression activity, replication timing, or
the histone marks or non-histone proteins associated with them. Independently,
other chromatin domains have been defined by their likelihood to interact with
non-DNA structures, such as the nuclear lamina. Lamina-associated domains (LADs)
correlate with low gene expression and late replication timing. TADs and LADs
have recently been evaluated with respect to cell-type-specific gene expression.
The results shed light on the relevance of these forms of chromatin organization
for transcriptional regulation, and address specifically how chromatin
sequestration influences cell fate decisions during organismal development. The genome of metazoan cells is organized into topologically associating domains
(TADs) that have similar histone modifications, transcription level, and DNA
replication timing. Although similar structures appear to be conserved in
fission yeast, computational modeling and analysis of high-throughput chromosome
conformation capture (Hi-C) data have been used to argue that the small, highly
constrained budding yeast chromosomes could not have these structures. In
contrast, herein we analyze Hi-C data for budding yeast and identify 200-kb
scale TADs, whose boundaries are enriched for transcriptional activity.
Furthermore, these boundaries separate regions of similarly timed replication
origins connecting the long-known effect of genomic context on replication
timing to genome architecture. To investigate the molecular basis of TAD
formation, we performed Hi-C experiments on cells depleted for the Forkhead
transcription factors, Fkh1 and Fkh2, previously associated with replication
timing. Forkhead factors do not regulate TAD formation, but do promote
longer-range genomic interactions and control interactions between origins near
the centromere. Thus, our work defines spatial organization within the budding
yeast nucleus, demonstrates the conserved role of genome architecture in
regulating DNA replication, and identifies a molecular mechanism specifically
regulating interactions between pericentric origins. A current question in the high-order organization of chromatin is whether
topologically associating domains (TADs) are distinct from other hierarchical
chromatin domains. However, due to the unclear TAD definition in tradition, the
structural and functional uniqueness of TAD is not well studied. In this work,
we refined TAD definition by further constraining TADs to the optimal separation
on global intra-chromosomal interactions. Inspired by this constraint, we
developed a novel method, called HiTAD, to detect hierarchical TADs from Hi-C
chromatin interactions. HiTAD performs well in domain sensitivity, replicate
reproducibility and inter cell-type conservation. With a novel domain-based
alignment proposed by us, we defined several types of hierarchical TAD changes
which were not systematically studied previously, and subsequently used them to
reveal that TADs and sub-TADs differed statistically in correlating chromosomal
compartment, replication timing and gene transcription. Finally, our work also
has the implication that the refinement of TAD definition could be achieved by
only utilizing chromatin interactions, at least in part. HiTAD is freely
available online. |
How are cryptic unstable transcripts (CUTs) defined? | This resource includes deletions of small nuclear RNAs (snRNAs), transfer RNAs (tRNAs), small nucleolar RNAs (snoRNAs), and other annotated ncRNAs as well as the more recently identified stable unannotated transcripts (SUTs) and cryptic unstable transcripts (CUTs) whose functions are largely unknown There is extensive transcription throughout the eukaryotic genome resulting in both antisense transcripts from coding regions and cryptic unstable transcripts (CUTs) from intergenic regions These cryptic unstable transcripts (CUTs) are rapidly degraded by the nuclear exosome. These results suggest that transcription termination of CUTs directed by Nrd1 and Nab3 is a prerequisite for rapid degradation by the nuclear exosome. It is likely that many of these are cryptic unstable transcripts (CUTs), which are rapidly degraded and whose function(s) within the cell are still unclear, while others may be novel functional transcripts. These recently identified transcripts either exist stably in cells (stable unannotated transcripts, SUTs) or are rapidly degraded by the RNA surveillance pathway (cryptic unstable transcripts, CUTs) | Studies of yeast transcription have revealed the widespread distribution of
intergenic RNA polymerase II transcripts. These cryptic unstable transcripts
(CUTs) are rapidly degraded by the nuclear exosome. Yeast RNA binding proteins
Nrd1 and Nab3 direct termination of sn/snoRNAs and recently have also been
implicated in premature transcription termination of the NRD1 gene. In this
paper, we show that Nrd1 and Nab3 are required for transcription termination of
CUTs. In nrd1 and nab3 mutants, we observe 3'-extended transcripts originating
from CUT promoters but failing to terminate through the Nrd1- and Nab3-directed
pathway. Nrd1 and Nab3 colocalize to regions of the genome expressing antisense
CUTs, and these transcripts require yeast nuclear exosome and TRAMP components
for degradation. Dissection of a CUT terminator reveals a minimal element
sufficient for Nrd1- and Nab3-directed termination. These results suggest that
transcription termination of CUTs directed by Nrd1 and Nab3 is a prerequisite
for rapid degradation by the nuclear exosome. There is extensive transcription throughout the eukaryotic genome resulting in
both antisense transcripts from coding regions and cryptic unstable transcripts
(CUTs) from intergenic regions. In this issue, Camblong et al. (2007)
demonstrate in the budding yeast that antisense transcripts, if stabilized by
exosome impairment, are able to mediate gene silencing via the recruitment of
histone deacetylases. Cryptic unstable transcripts (CUTs) are short, 300-600-nucleotide (nt) RNA
polymerase II transcripts that are rapidly degraded by the nuclear RNA exosome
in yeast. CUTs are widespread and probably represent the largest share of hidden
transcription in the yeast genome. Similarly to small nucleolar and small
nuclear RNAs, transcription of CUT-encoding genes is terminated by the Nrd1
complex pathway. We show here that this termination mode and ensuing CUTs
degradation crucially depend on the position of RNA polymerase II relative to
the transcription start site. Notably, position sensing correlates with the
phosphorylation status of the polymerase C-terminal domain (CTD). The Nrd1
complex is recruited to chromatin via interactions with both the nascent RNA and
the CTD, but a permissive phosphorylation status of the latter is absolutely
required for efficient transcription termination. We discuss the mechanism
underlying the regulation of coexisting cryptic and mRNA-productive
transcription. A complete description of the transcriptome of an organism is crucial for a
comprehensive understanding of how it functions and how its transcriptional
networks are controlled, and may provide insights into the organism's evolution.
Despite the status of Saccharomyces cerevisiae as arguably the most well-studied
model eukaryote, we still do not have a full catalog or understanding of all its
genes. In order to interrogate the transcriptome of S. cerevisiae for low
abundance or rapidly turned over transcripts, we deleted elements of the RNA
degradation machinery with the goal of preferentially increasing the relative
abundance of such transcripts. We then used high-resolution tiling microarrays
and ultra high-throughput sequencing (UHTS) to identify, map, and validate
unotated transcripts that are more abundant in the RNA degradation mutants
relative to wild-type cells. We identified 365 currently unotated
transcripts, the majority presumably representing low abundance or short-lived
RNAs, of which 185 are previously unknown and unique to this study. It is likely
that many of these are cryptic unstable transcripts (CUTs), which are rapidly
degraded and whose function(s) within the cell are still unclear, while others
may be novel functional transcripts. Of the 185 transcripts we identified as
novel to our study, greater than 80 percent come from regions of the genome that
have lower conservation scores amongst closely related yeast species than 85
percent of the verified ORFs in S. cerevisiae. Such regions of the genome have
typically been less well-studied, and by definition transcripts from these
regions will distinguish S. cerevisiae from these closely related species. Genome-wide pervasive transcription has been reported in many eukaryotic
organisms, revealing a highly interleaved transcriptome organization that
involves hundreds of previously unknown non-coding RNAs. These recently
identified transcripts either exist stably in cells (stable unotated
transcripts, SUTs) or are rapidly degraded by the RNA surveillance pathway
(cryptic unstable transcripts, CUTs). One characteristic of pervasive
transcription is the extensive overlap of SUTs and CUTs with previously
annotated features, which prompts questions regarding how these transcripts are
generated, and whether they exert function. Single-gene studies have shown that
transcription of SUTs and CUTs can be functional, through mechanisms involving
the generated RNAs or their generation itself. So far, a complete transcriptome
architecture including SUTs and CUTs has not been described in any organism.
Knowledge about the position and genome-wide arrangement of these transcripts
will be instrumental in understanding their function. Here we provide a
comprehensive analysis of these transcripts in the context of multiple
conditions, a mutant of the exosome machinery and different strain backgrounds
of Saccharomyces cerevisiae. We show that both SUTs and CUTs display distinct
patterns of distribution at specific locations. Most of the newly identified
transcripts initiate from nucleosome-free regions (NFRs) associated with the
promoters of other transcripts (mostly protein-coding genes), or from NFRs at
the 3' ends of protein-coding genes. Likewise, about half of all coding
transcripts initiate from NFRs associated with promoters of other transcripts.
These data change our view of how a genome is transcribed, indicating that
bidirectionality is an inherent feature of promoters. Such an arrangement of
divergent and overlapping transcripts may provide a mechanism for local
spreading of regulatory signals-that is, coupling the transcriptional regulation
of neighbouring genes by means of transcriptional interference or histone
modification. Pervasive and hidden transcription is widespread in eukaryotes, but its global
level, the mechanisms from which it originates and its functional significance
are unclear. Cryptic unstable transcripts (CUTs) were recently described as a
principal class of RNA polymerase II transcripts in Saccharomyces cerevisiae.
These transcripts are targeted for degradation immediately after synthesis by
the action of the Nrd1-exosome-TRAMP complexes. Although CUT degradation
mechanisms have been analysed in detail, the genome-wide distribution at the
nucleotide resolution and the prevalence of CUTs are unknown. Here we report the
first high-resolution genomic map of CUTs in yeast, revealing a class of
potentially functional CUTs and the intrinsic bidirectional nature of eukaryotic
promoters. An RNA fraction highly enriched in CUTs was analysed by a 3'
Long-SAGE (serial analysis of gene expression) approach adapted to deep
sequencing. The resulting detailed genomic map of CUTs revealed that they derive
from extremely widespread and very well defined transcription units and do not
result from unspecific transcriptional noise. Moreover, the transcription of
CUTs predomitly arises within nucleosome-free regions, most of which
correspond to promoter regions of bona fide genes. Some of the CUTs start
upstream from messenger RNAs and overlap their 5' end. Our study of glycolysis
genes, as well as recent results from the literature, indicate that such
concurrent transcription is potentially associated with regulatory mechanisms.
Our data reveal numerous new CUTs with such a potential regulatory role.
However, most of the identified CUTs corresponded to transcripts divergent from
the promoter regions of genes, indicating that they represent by-products of
divergent transcription occurring at many and possibly most promoters.
Eukaryotic promoter regions are thus intrinsically bidirectional, a fundamental
property that escaped previous analyses because in most cases divergent
transcription generates short-lived unstable transcripts present at very low
steady-state levels. Non-coding transcripts originating from bidirectional promoters have been
reported in a wide range of organisms. In yeast, these divergent transcripts can
be subdivided into two classes. Some are designated Cryptic Unstable Transcripts
(CUTs) because they are terminated by the Nrd1-Nab3-Sen1 pathway and then
rapidly degraded by the nuclear exosome. This is the same processing pathway
used by yeast snoRNAs. Whereas CUTs are only easily observed in cells lacking
the Rrp6 or Rrp47 subunits of the nuclear exosome, Stable Uncharacterized
Transcripts (SUTs) are present even in wild-type cells. Here we show that SUTs
are partially susceptible to the nuclear exosome, but are primarily degraded by
cytoplasmic 5' to 3' degradation and nonsense-mediated decay (NMD). Therefore,
SUTs may be processed similarly to mRNAs. Surprisingly, both CUTs and SUTs were
found to produce 3' extended species that were also subject to cytoplasmic
degradation. The functions, if any, of these extended CUTs and SUTs are unknown,
but their discovery suggests that yeasts generate transcripts reminiscent of
long non-coding RNAs found in higher eukaryotes. Recent studies on yeast transcriptome have revealed the presence of a large set
of RNA polymerase II transcripts mapping to intergenic and antisense regions or
overlapping canonical genes. Most of these ncRNAs (ncRNAs) are subject to
termination by the Nrd1-dependent pathway and rapid degradation by the nuclear
exosome and have been dubbed cryptic unstable transcripts (CUTs). CUTs are often
considered as by-products of transcriptional noise, but in an increasing number
of cases they play a central role in the control of gene expression. Regulatory
mechanisms involving expression of a CUT are diverse and include attenuation,
transcriptional interference, and alternative transcription start site choice.
This review focuses on the impact of cryptic transcription on gene expression,
describes the role of the Nrd1-complex as the main actor in preventing
nonfunctional and potentially harmful transcription, and details a few systems
where expression of a CUT has an essential regulatory function. We also
summarize the most recent studies concerning other types of ncRNAs and their
possible role in regulation. It is well established that eukaryotic genomes are pervasively transcribed
producing cryptic unstable transcripts (CUTs). However, the mechanisms
regulating pervasive transcription are not well understood. Here, we report that
the fission yeast CENP-B homolog Abp1 plays an important role in preventing
pervasive transcription. We show that loss of abp1 results in the accumulation
of CUTs, which are targeted for degradation by the exosome pathway. These CUTs
originate from different types of genomic features, but the highest increase
corresponds to Tf2 retrotransposons and rDNA repeats, where they map along the
entire elements. In the absence of abp1, increased RNAPII-Ser5P occupancy is
observed throughout the Tf2 coding region and, unexpectedly, RNAPII-Ser5P is
enriched at rDNA repeats. Loss of abp1 also results in Tf2 derepression and
increased nucleolus size. Altogether these results suggest that Abp1 prevents
pervasive RNAPII transcription of repetitive DNA elements (i.e., Tf2 and rDNA
repeats) from internal cryptic sites. Eukaryotic genomes are extensively transcribed, generating many different RNAs
with no known function. We have constructed 1502 molecular barcoded ncRNA gene
deletion strains encompassing 443 ncRNAs in the yeast Saccharomyces cerevisiae
as tools for ncRNA functional analysis. This resource includes deletions of
small nuclear RNAs (snRNAs), transfer RNAs (tRNAs), small nucleolar RNAs
(snoRNAs), and other annotated ncRNAs as well as the more recently identified
stable unotated transcripts (SUTs) and cryptic unstable transcripts (CUTs)
whose functions are largely unknown. Specifically, deletions have been
constructed for ncRNAs found in the intergenic regions, not overlapping genes or
their promoters (i.e., at least 200 bp minimum distance from the closest gene
start codon). The deletion strains carry molecular barcodes designed to be
complementary with the protein gene deletion collection enabling parallel
analysis experiments. These strains will be useful for the numerous genomic and
molecular techniques that utilize deletion strains, including genome-wide
phenotypic screens under different growth conditions, pooled chemogenomic
screens with drugs or chemicals, synthetic genetic array analysis to uncover
novel genetic interactions, and synthetic dosage lethality screens to analyze
gene dosage. Overall, we created a valuable resource for the RNA community and
for future ncRNA research. |
How many Groucho-related genes (GRG) are contained in the mouse genome? | It spans approximately 7 kb on chromosome 10 and consists of seven exons. The groucho-related genes (Grg) of the mouse comprise at least four family members. | The Grg gene encodes a 197-amino-acid protein homologous to the amino-terminal
domain of the product of the groucho gene of the Drosophila Enhancer of split
complex. We describe here the genomic organization of the mouse Grg gene. It
spans approximately 7 kb on chromosome 10 and consists of seven exons. The 3'
region of the Grg gene contains two functional polyadenylation sites that give
rise to two transcripts that are differentially expressed among adult mouse
tissues. The promoter region is very GC rich and lacks TATA box and "initiator"
sequences. Primer extension analysis and ribonuclease protection assays show
that Grg has a major transcription start site situated down-stream of putative
binding motifs for the transcription factors Sp1, E2A, and PuF. The Grg gene encodes a 197 amino acid protein homologous to the amino-terminal
domain of the product of the groucho gene of the Drosophila Enhancer of split
complex. Analysis with a polyclonal antisera specific for the Grg protein
revealed that Grg is a 25 kd nuclear protein that can participate in specific
protein-protein interactions. A null mutation of the Grg gene was constructed by
gene targeting. Mice homozygous for this mutation completed embryogenesis and
were born, but exhibited varying degrees of post-natal growth deficiency. No
dosage-sensitive genetic interaction was detected between the Notch1 and Grg
genes in mice heterozygous for a Notch1 mutant allele and homozygous for the Grg
null mutation. The mouse Grg gene encodes a 197 amino acid nuclear protein homologous to the
amino-terminal domain of the product of the groucho (gro) gene of the Drosophila
Enhancer of split complex. Recent work has suggested that the gro protein
functions as a transcriptional corepressor during Drosophila development. We
therefore examined possible roles of the mouse Grg protein in DNA binding and in
vitro transcription. No sequence-specific DNA binding activity was detected by
polymerase chain reaction-DNA binding site selection nor was the glutamine-rich
Grg protein capable of acting as an activation domain in an in vivo
transactivation assay. However, depletion of Grg protein from HeLa nuclear
extracts inhibited the in vitro transcription activity of the extracts. We
suggest that Grg protein may interact with components of the basal transcription
machinery. The murine grg (Groucho-related gene) products are believed to interact with
transcription factors and repress transcription, thereby regulating cell
proliferation and differentiation. Most proteins in the grg family contain all
of the domains found in the Drosophila Groucho protein, including the S/P
(Ser-Pro-rich) domain required for interaction with transcription factors and
the WD40 domain, which is thought to interact with other proteins. However, at
least two Grg proteins contain only the amino-terminal Q (glutamine-rich)
domain. We examined whether the Q domain is used for dimerization between Grg
proteins, using the yeast two-hybrid system and binding assays with glutathione
S-transferase fusion proteins. We found that Grg proteins are able to dimerize
through the Q domain and that dimerization requires a core of 50 amino acids.
Surprisingly, the dimerization does not require the leucine zipper located
within the Q domain. We have isolated cDNAs representing multiple members of murine groucho
homologues, designated Grg for groucho-related genes. Among them, Grg3 appears
to produce two transcripts. One of the Grg3 transcripts contains coding sequence
for a complete Groucho protein homologue. The second transcript contains coding
sequence for only the two amino-terminal domains of the Groucho protein,
followed by a hydrophobic tail and a stop codon. We analyzed the expression of
both transcripts in mouse embryos using RNase protection and in situ
hybridization. Expression was detected during cell determination in the nervous
system and in somitic mesoderm, overlapping Notch1 expression and adjacent to
Mash1, MyoD and Myf5 expression. Thus, the expression pattern of Grg3 suggests a
conserved role in the Notch signalling pathway to regulate expression of basic
helix-loop-helix proteins and cell determination. Grg3 expression was also
consistently detected in epithelial structures undergoing mesenchyme induction. The human Transducin-like Enhancer of Split (TLE) and mouse homologue, Groucho
gene-related protein (GRG), represent a family of conserved non-DNA binding
transcriptional modulatory proteins divided into two subgroups based upon size.
The long TLE/GRGs consist of four pentadomain proteins that are dedicated
co-repressors for multiple transcription factors (TF). The second TLE/GRG
subgroup is composed of the Amino-terminal Enhancer of Split (AES) in humans and
its mouse homolog GRG5 (AES/GRG5). In contrast to the dedicated co-repressor
function of long TLE/GRGs, AES/GRG5 can both positively or negatively modulate
various TF as well as non-TF proteins in a long TLE/GRG-dependent or
-independent manner. Therefore, AES/GRG5 is a functionally dynamic protein that
is not exclusively defined by its role as a long TLE/GRG antagonist. AES/GRG5
may function in various developmental and pathological processes but the
functional characteristics of endogenous AES/GRG5 in a physiologically relevant
context remains to be determined. |
What is mechanism of action of galunisertib? | Galunisertib is a transforming growth factor-β receptor type I kinase inhibitor (TGF-βRI). It was tested for treatment of solid cancers, including glioblastoma and neuroblastoma, and liver fibrosis. | BACKGROUND: The combination of galunisertib, a transforming growth factor
(TGF)-β receptor (R)1 kinase inhibitor, and lomustine was found to have
antitumor activity in murine models of glioblastoma.
METHODS: Galunisertib (300 mg/day) was given orally 14 days on/14 days off
(intermittent dosing). Lomustine was given as approved. Patients were randomized
in a 2:1:1 ratio to galunisertib + lomustine, galunisertib monotherapy, or
placebo + lomustine. The primary objective was overall survival (OS); secondary
objectives were safety, pharmacokinetics (PKs), and antitumor activity.
RESULTS: One hundred fifty-eight patients were randomized: galunisertib +
lomustine (N = 79), galunisertib (N = 39), and placebo + lomustine (N = 40).
Baseline characteristics were: male (64.6%), white (75.3%), median age 58 years,
ECOG performance status (PS) 1 (63.3%), and primary glioblastoma (93.7%). The
PKs of galunisertib were not altered with lomustine, and galunisertib had a
median half-life of ∼8 hours. Median OS in months (95% credible interval [CrI])
for galunisertib + lomustine was 6.7 (range: 5.3-8.5), 8.0 (range: 5.7-11.7) for
galunisertib alone, and 7.5 (range: 5.6-10.3) for placebo + lomustine. There was
no difference in OS for patients treated with galunisertib + lomustine compared
with placebo + lomustine [P (HR < 1) = 26%]. Median progression-free survival of
∼2 months was observed in all 3 arms. Among 8 patients with IDH1 mutation, 7
patients were treated with galunisertib (monotherapy or with lomustine); OS
ranged from 4 to 17 months. Patients treated with galunisertib alone had fewer
drug-related grade 3/4 adverse events (n = 34) compared with lomustine-treated
patients (10% vs 26%). Baseline PS, post-discontinuation of bevacizumab, tumor
size, and baseline levels of MDC/CCL22 were correlated with OS.
CONCLUSIONS: Galunisertib + lomustine failed to demonstrate improved OS relative
to placebo + lomustine. Efficacy outcomes were similar in all 3 arms.
CLINICAL TRIAL REGISTRATION: NCT01582269, ClinicalTrials.gov. Evidence is accumulating highlighting the importance of extracellular miRNA as a
novel biomarker for diagnosing various kinds of maligcies. MiR-21 is one of
the most studied miRNAs and is over-expressed in cancer tissues. To explore the
clinical implications and secretory mechanisms of extracellular miR-21, we
firstly meta-analyzed the diagnostic efficiency of extracellular miR-21 in
different cancer types. Eighty-one studies based on 59 articles were finally
included. In our study, extracellular miR-21 was observed to exhibit an
outstanding diagnostic accuracy in detecting brain cancer (area under the
summary receiver operating characteristic curve or AUC = 0.94), and this
accuracy was more obvious in glioma diagnosis (AUC = 0.95). Our validation study
(n = 45) further confirmed the diagnostic and prognostic role of miR-21 in
cerebrospinal fluid (CSF) for glioma. These findings inspired us to explore the
biological function of miR-21. We next conducted mechanistic investigations to
explain the secretory mechanisms of extracellular miR-21 in glioma. TGF-β/Smad3
signaling was identified to participate in mediating the release of miR-21 from
glioma cells. Further targeting TGF-β/Smad3 signaling using galunisertib, an
inhibitor of the TGF-β type I receptor kinase, can attenuate the secretion of
miR-21 from glioma cells. Taken together, CSF-based miR-21 might serve as a
potential biomarker for diagnosing brain cancer, especially for patients with
glioma. Moreover, extracellular levels of miR-21 were affected by exogenous
TGF-β activity and galunisertib treatment. Author information:
(1)Immunopathology Group, BioCruces Health Research Institute, Barakaldo, Spain.
(2)Pediatric Oncology Group, BioCruces Health Research Institute, Barakaldo,
Spain.
(3)Pediatrics Service, Cruces University Hospital, Barakaldo, Spain.
(4)Department of Pediatrics, Faculty of Medicine and Dentistry, University of
the Basque Country, Leioa, Spain.
(5)Immunopathology Group, BioCruces Health Research Institute, Barakaldo, Spain.
[email protected].
(6)Immunotherapy Group, Center for Transfusion and Human Tissues, Galdakao,
Spain.
(7)Ikerbasque, Basque Foundation for Science, Bilbao, Spain. Transforming growth factor-beta (TGF-β) signaling has gained extensive interest
in hepatocellular carcinoma (HCC). The small molecule kinase inhibitor
galunisertib, targeting the TGF-β receptor I (TGF-βRI), blocks HCC progression
in preclinical models and shows promising effects in ongoing clinical trials. As
the drug is not similarly effective in all patients, this study was aimed at
identifying new companion diagnostics biomarkers for patient stratification.
Next-generation sequencing-based massive analysis of cDNA ends was used to
investigate the transcriptome of an invasive HCC cell line responses to TGF-β1
and galunisertib. These identified mRNA were validated in 78 frozen HCC samples
and in 26 ex-vivo HCC tissues treated in culture with galunisertib. Respective
protein levels in patients blood were measured by enzyme-linked immunosorbent
assay. SKIL, PMEPA1 ANGPTL4, SNAI1, Il11 and c4orf26 were strongly upregulated
by TGF-β1 and downregulated by galunisertib in different HCC cell lines. In the
78 HCC samples, only SKIL and PMEPA1 (P<0.001) were correlated with endogenous
TGF-β1. In ex-vivo samples, SKIL and PMEPA1 were strongly downregulated
(P<0.001), and correlated (P<0.001) with endogenous TGF-β1. SKIL and PMEPA1 mRNA
expression in tumor tissues was significantly increased compared with controls
and not correlated with protein levels in the blood of paired HCC patients. SKIL
and PMEPA1 mRNA levels were positively correlated with TGF-β1 mRNA
concentrations in HCC tissues and strongly downregulated by galunisertib. The
target genes identified here may serve as biomarkers for the stratification of
HCC patients undergoing treatment with galunisertib. Author information:
(1)Department of Neuropathology, Charité Universitätsmedizin Berlin,
Charitéplatz 1, 10117 Berlin, Germany. [email protected].
(2)Department of Neuropathology, University Hospital Heidelberg and Clinical
Cooperation Unit Neuropathology, German Cancer Consortium (DKTK), German Cancer
Research Center (DKFZ), 69120 Heidelberg, Germany.
[email protected].
(3)Medical Oncology Department, Bellaria-Maggiore Hospitals, Azienda USL-IRCCS
Institute of Neurological Sciences, 40139 Bologna, Italy. [email protected].
(4)Assistance Publique-Hôpitaux de Paris (AP-HP) & Paris 13 University, Hôpital
Avicenne, Service de Neurologie, 93009 Bobigny, France.
[email protected].
(5)UC San Diego Health System, La Jolla, CA 92103, USA. [email protected].
(6)Hospital Universitario 12 de Octubre, 28041 Madrid, Spain.
[email protected].
(7)Department of Oncology, Royal North Shore Hospital, St. Leonards, NSW 2065,
Australia. [email protected].
(8)CHU Hôpital De La Timone, Rue Saint Pierre, 13385 Marseille, France.
[email protected].
(9)Austin Hospital, Heidelberg, VIC 3084, Australia. [email protected].
(10)Dr. Senckenberg Institute of Neurooncology, University Hospital Frankfurt,
60590 Frankfurt, Germany. [email protected].
(11)Antwerp University Hospital, Wilrijkstraat 10, 2650 Edegem, Belgium.
[email protected].
(12)Medical Oncology, Vall d'Hebron University Hospital, Calle Natzaret,
115-117, 08035 Barcelona, Spain. [email protected].
(13)Eli Lilly and Company, Erl Wood Manor, Windlesham GU20 6PH, UK.
[email protected].
(14)Eli Lilly and Company, Erl Wood Manor, Windlesham GU20 6PH, UK.
[email protected].
(15)Eli Lilly and Company, Erl Wood Manor, Windlesham GU20 6PH, UK.
[email protected].
(16)Eli Lilly and Company, Erl Wood Manor, Windlesham GU20 6PH, UK.
[email protected].
(17)Eli Lilly and Company, Indianapolis, IN 46285, USA. [email protected].
(18)Eli Lilly and Company, Indianapolis, IN 46285, USA.
[email protected].
(19)Eli Lilly and Company, Indianapolis, IN 46285, USA.
[email protected].
(20)Eli Lilly and Company, Indianapolis, IN 46285, USA. [email protected].
(21)Department of Neurology, University Hospital Heidelberg, 69120 Heidelberg,
Germany. [email protected]. BACKGROUND: Glioblastoma multiforme (GBM) is characterized by lethal
aggressiveness and patients with GBM are in urgent need for new therapeutic
avenues to improve quality of life. Current studies on tumor invasion focused on
roles of cytokines in tumor microenvironment and numerous evidence suggests that
TGF-β2 is abundant in glioma microenvironment and vital for glioma invasion.
Autopagy is also emerging as a critical factor in aggressive behaviors of cancer
cells; however, the relationship between TGF-β2 and autophagy in glioma has been
poorly understood.
METHODS: U251, T98 and U87 GBM cell lines as well as GBM cells from a primary
human specimen were used in vitro and in vivo to evaluate the effect of TGF-β2
on autophagy. Western blot, qPCR, immunofluorescence and transmission-electron
microscope were used to detect target molecular expression. Lentivirus and siRNA
vehicle were introduced to establish cell lines, as well as mitotracker and
seahorse experiment to study the metabolic process in glioma. Preclinical
therapeutic efficacy was evaluated in orthotopic xenograft mouse models.
RESULTS: Here we demonstrated that TGF-β2 activated autophagy in human glioma
cell lines and knockdown of Smad2 or inhibition of c-Jun NH2-terminal kinase,
attenuated TGF-β2-induced autophagy. TGF-β2-induced autophagy is important for
glioma invasion due to the alteration of epithelial-mesenchymal transition and
metabolism conversion, particularly influencing mitochondria trafficking and
membrane potential (△Ψm). Autopaghy also initiated a feedback on TGF-β2 in
glioma by keeping its autocrine loop and affecting Smad2/3/7 expression. A
xenograft model provided additional confirmation on combination of TGF-β
inhibitor (Galunisertib) and autophagy inhibitor (CQ) to better "turn off" tumor
growth.
CONCLUSION: Our findings elucidated a potential mechanism of
autophagy-associated glioma invasion that TGF-β2 could initiate autophagy via
Smad and non-Smad pathway to promote glioma cells' invasion. |
What is a coligo? | Coligos are circularized oligodeoxynucleotides | Chemically synthesized DNA can carry small RNA sequence information but
converting that information into small RNA is generally thought to require large
double-stranded promoters in the context of plasmids, viruses and genes. We
previously found evidence that circularized oligodeoxynucleotides (coligos)
containing certain sequences and secondary structures can template the synthesis
of small RNA by RNA polymerase III in vitro and in human cells. By using
immunoprecipitated RNA polymerase III we now report corroborating evidence that
this enzyme is the sole polymerase responsible for coligo transcription. The
immobilized polymerase enabled experiments showing that coligo transcripts can
be formed through transcription termination without subsequent 3' end trimming.
To better define the determits of productive transcription, a
structure-activity relationship study was performed using over 20 new coligos.
The results show that unpaired nucleotides in the coligo stem facilitate
circumtranscription, but also that internal loops and bulges should be kept
small to avoid secondary transcription initiation sites. A polymerase
termination sequence embedded in the double-stranded region of a
hairpin-encoding coligo stem can antagonize transcription. Using lessons learned
from new and old coligos, we demonstrate how to convert poorly transcribed
coligos into productive templates. Our findings support the possibility that
coligos may prove useful as chemically synthesized vectors for the ectopic
expression of small RNA in human cells. Circularized oligonucleotides, or coligos, were previously found to serve as RNA
polymerase III (Pol III) templates in vitro and in human tissue culture cells.
Here we randomized the 12-nucleotide larger loop (L-loop) of a well
characterized coligo and found unexpectedly that in vitro transcription by
FLAG-Pol III was not significantly affected. This observation allowed us to test
the variable of coligo L-loop size separately from the variable of its sequence.
Transcription efficiency increased with L-loop size from 3 to 12 nucleotides of
randomized sequence, and the smallest loop forced initiation to move into the
stem region. To test further the need for any specific sequence we compared
seven nucleotide L-loops composed of random, abasic and abasic-acyclic
nucleotides, and all supported transcription by Pol III. Transcription of a
series of coligos containing twelve contiguous randomized nucleotides placed at
different locations within the coligo structure provided further evidence that
the stem-loop junction structure is important for precise initiation. Nearly the
same transcript pattern was formed in vitro by Pol III from yeast and human
cells. Overall, these experiments support structure, rather than L-loop
sequence, as the major determit of coligo transcription initiation by Pol
III. |
What is the relationship of fyn kinase and tau? | The Fyn kinase interacts with tau. The activated Fyn kinase hyperphosphorylates the tau protein. | The past decade has brought tremendous progress in unraveling the
pathophysiology of Alzheimer's disease (AD). While increasingly sophisticated
immunotherapy targeting soluble and aggregated brain amyloid-beta (Aβ) continues
to dominate clinical research in AD, a deeper understanding of Aβ physiology has
led to the recognition of distinct neuronal signaling pathways linking Aβ to
synaptotoxicity and neurodegeneration and to new targets for therapeutic
intervention. Identifying specific signaling pathways involving Aβ has allowed
for the development of more precise therapeutic interventions targeting the most
relevant molecular mechanisms leading to AD. In this review, I highlight the
discovery of cellular prion protein as a high-affinity receptor for Aβ
oligomers, and the downstream signaling pathway elucidated to date, converging
on nonreceptor tyrosine kinase Fyn. I discuss preclinical studies targeting Fyn
as a therapeutic intervention in AD and our recent experience with the safety,
tolerability, and cerebrospinal fluid penetration of the Src family kinase
inhibitor saracatinib in patients with AD. Fyn is an attractive target for AD
therapeutics, not only based on its activation by Aβ via cellular prion protein
but also due to its known interaction with tau, uniquely linking the two key
pathologies in AD. Fyn is also a challenging target, with broad expression
throughout the body and significant homology with other members of the Src
family kinases, which may lead to unintended off-target effects. A phase 2a
proof-of-concept clinical trial in patients with AD is currently under way,
providing critical first data on the potential effectiveness of targeting Fyn in
AD. |
Which are the main manifestations of Ohdo syndrome? | Severe ID, absent or deficient language, skeletal manifestations including bilateral patella dislocations. | We report on a girl with growth and mental retardation, peculiar face with
ptosis, epicanthus, broad nasal bridge, low-set and abnormal ears, cleft uvula,
congenital heart defect, and anal atresia. A similar condition was reported
previously by Wiedemann et al. [1982: An atlas of characteristic syndromes: a
visual aid to diagnosis, 2nd ed. p 114-115]. We confirm the existence of this
condition that, although similar to Ohdo syndrome, seems to be an independent
clinical entity. We propose that, based on the principal clinical
manifestations, this condition should be identified with the acronym ROCA
(retardation of growth and development, ocular ptosis, cardiac defect, and anal
atresia). We report a new case of Ohdo syndrome with bilateral patella dislocations where
surgical intervention has been indicated. A review of the skeletal
manifestations reported in the literature on Ohdo syndrome reveals that joint
laxity and skeletal deformities are important aspects of the phenotype. Mutations of the MED12 gene have been reported mainly in males with FG
(Opitz-Kaveggia), Lujan-Fryns, or X-linked Ohdo syndromes. Recently, a different
phenotype characterized by minor anomalies, severe intellectual disability (ID),
and absent language was reported in female and male patients belonging to the
same family and carrying a frameshift MED12 mutation (c.5898dupC). Here, we
report on two brothers and their niece affected by severe and mild ID,
respectively, where whole exome sequencing combined with variant analysis within
a panel of ID-related genes, disclosed a novel c.2312T>C (p.Ile771Thr) MED12
mutation. This variant, which has not been reported as a polymorphism, was not
present in a third unaffected brother, and was predicted to be deleterious by
five bioinformatic databases. This finding together with the phenotypic
analogies shared with the carriers of c.5898dupC mutation suggests the existence
of a fourth MED12-related disorder, characterized by severe ID, absent or
deficient language and, milder, clinical manifestation in heterozygotes. We have
reviewed the literature on MED12 heterozygotes, their clinical manifestations,
and discuss the possible biological causes of this condition. © 2016 Wiley
Periodicals, Inc. |
Which algorithm is used by the UCSC Genome Browser? | The UCSC Genome Browser organizes data and annotations (called tracks) around the reference sequences or draft assemblies of many eukaryotic genomes and presents them using a powerful web-based graphical interface. The database is optimized to support fast interactive performance with the web-based UCSC Genome Browser, a tool built on top of the database for rapid visualization and querying of the data at many levels. The annotations for a given genome are displayed in the browser as a series of tracks aligned with the genomic sequence. Sequence data and annotations may also be viewed in a text-based tabular format or downloaded as tab-delimited flat files. | The University of California Santa Cruz (UCSC) Genome Browser Database is an up
to date source for genome sequence data integrated with a large collection of
related annotations. The database is optimized to support fast interactive
performance with the web-based UCSC Genome Browser, a tool built on top of the
database for rapid visualization and querying of the data at many levels. The
annotations for a given genome are displayed in the browser as a series of
tracks aligned with the genomic sequence. Sequence data and annotations may also
be viewed in a text-based tabular format or downloaded as tab-delimited flat
files. The Genome Browser Database, browsing tools and downloadable data files
can all be found on the UCSC Genome Bioinformatics website
(http://genome.ucsc.edu), which also contains links to documentation and related
technical information. Comparative analysis of DNA sequence from multiple species can provide insights
into the function and evolutionary processes that shape genomes. The University
of California Santa Cruz (UCSC) Genome Bioinformatics group has developed
several tools and methodologies in its study of comparative genomics, many of
which have been incorporated into the UCSC Genome Browser
(http://genome.ucsc.edu), an easy-to-use online tool for browsing genomic data
and aligned annotation "tracks" in a single window. The comparative genomics
annotations in the browser include pairwise alignments, which aid in the
identification of orthologous regions between species, and conservation tracks
that show measures of evolutionary conservation among sets of multiply aligned
species, highlighting regions of the genome that may be functionally important.
A related tool, the UCSC Table Browser, provides a simple interface for
querying, analyzing, and downloading the data underlying the Genome Browser
annotation tracks. Here, we describe a procedure for examining a genomic region
of interest in the Genome Browser, analyzing characteristics of the region,
filtering the data, and downloading data sets for further study. The University of California Santa Cruz (UCSC) Genome Browser (genome.ucsc.edu)
is a popular Web-based tool for quickly displaying a requested portion of a
genome at any scale, accompanied by a series of aligned annotation "tracks". The
annotations-generated by the UCSC Genome Bioinformatics Group and external
collaborators-display gene predictions, mRNA and expressed sequence tag
alignments, simple nucleotide polymorphisms, expression and regulatory data, and
pairwise and multiple-species comparative genomics data. All information
relevant to a region is presented in one window, facilitating biological
analysis and interpretation. The database tables underlying the Genome Browser
tracks can be viewed, downloaded, and manipulated using another Web-based
application, the UCSC Table Browser. Users can upload personal data as custom
annotation tracks in both browsers for research or educational use. This unit
describes how to use the Genome Browser and Table Browser for genome analysis,
download the underlying database tables, and create and display custom
annotation tracks. The University of California Santa Cruz (UCSC) Genome Browser is a popular
Web-based tool for quickly displaying a requested portion of a genome at any
scale, accompanied by a series of aligned annotation "tracks." The
annotations-generated by the UCSC Genome Bioinformatics Group and external
collaborators-display gene predictions, mRNA and expressed sequence tag
alignments, simple nucleotide polymorphisms, expression and regulatory data,
phenotype and variation data, and pairwise and multiple-species comparative
genomics data. All information relevant to a region is presented in one window,
facilitating biological analysis and interpretation. The database tables
underlying the Genome Browser tracks can be viewed, downloaded, and manipulated
using another Web-based application, the UCSC Table Browser. Users can upload
data as custom annotation tracks in both browsers for research or educational
use. This unit describes how to use the Genome Browser and Table Browser for
genome analysis, download the underlying database tables, and create and display
custom annotation tracks. The University of California Santa Cruz (UCSC) Genome Browser is a popular
Web-based tool for quickly displaying a requested portion of a genome at any
scale, accompanied by a series of aligned annotation "tracks." The annotations
generated by the UCSC Genome Bioinformatics Group and external collaborators
include gene predictions, mRNA and expressed sequence tag alignments, simple
nucleotide polymorphisms, expression and regulatory data, phenotype and
variation data, and pairwise and multiple-species comparative genomics data. All
information relevant to a region is presented in one window, facilitating
biological analysis and interpretation. The database tables underlying the
Genome Browser tracks can be viewed, downloaded, and manipulated using another
Web-based application, the UCSC Table Browser. Users can upload personal
datasets in a wide variety of formats as custom annotation tracks in both
browsers for research or educational purposes. This unit describes how to use
the Genome Browser and Table Browser for genome analysis, download the
underlying database tables, and create and display custom annotation tracks. The UCSC Archaeal Genome Browser (http://archaea.ucsc.edu) offers a graphical
web-based resource for exploration and discovery within archaeal and other
selected microbial genomes. By bringing together existing gene annotations, gene
expression data, multiple-genome alignments, pre-computed sequence comparisons
and other specialized analysis tracks, the genome browser is a powerful
aggregator of varied genomic information. The genome browser environment
maintains the current look-and-feel of the vertebrate UCSC Genome Browser, but
also integrates archaeal and bacterial-specific tracks with a few graphic
display enhancements. The browser currently contains 115 archaeal genomes, plus
31 genomes of viruses known to infect archaea. Some of the recently developed or
enhanced tracks visualize data from published high-throughput RNA-sequencing
studies, the NCBI Conserved Domain Database, sequences from pre-genome
sequencing studies, predicted gene boundaries from three different protein gene
prediction algorithms, tRNAscan-SE gene predictions with RNA secondary
structures and CRISPR locus predictions. We have also developed a companion
resource, the Archaeal COG Browser, to provide better search and display of
arCOG gene function classifications, including their phylogenetic distribution
among available archaeal genomes. The University of California Santa Cruz (UCSC) Genome Browser is a popular
Web-based tool for quickly displaying a requested portion of a genome at any
scale, accompanied by a series of aligned annotation "tracks." The annotations
generated by the UCSC Genome Bioinformatics Group and external collaborators
include gene predictions, mRNA and expressed sequence tag alignments, simple
nucleotide polymorphisms, expression and regulatory data, phenotype and
variation data, and pairwise and multiple-species comparative genomics data. All
information relevant to a region is presented in one window, facilitating
biological analysis and interpretation. The database tables underlying the
Genome Browser tracks can be viewed, downloaded, and manipulated using another
Web-based application, the UCSC Table Browser. Users can upload personal
datasets in a wide variety of formats as custom annotation tracks in both
browsers for research or educational purposes. This unit describes how to use
the Genome Browser and Table Browser for genome analysis, download the
underlying database tables, and create and display custom annotation tracks. Electronic data resources can enable molecular biologists to quickly get
information from around the world that a decade ago would have been buried in
papers scattered throughout the library. The ability to access, query, and
display these data makes benchwork much more efficient and drives new
discoveries. Increasingly, mastery of software resources and corresponding data
repositories is required to fully explore the volume of data generated in
biomedical and agricultural research, because only small amounts of data are
actually found in traditional publications. The UCSC Genome Browser provides a
wealth of data and tools that advance understanding of genomic context for many
species, enable detailed analysis of data, and provide the ability to
interrogate regions of interest across disparate data sets from a wide variety
of sources. Researchers can also supplement the standard display with their own
data to query and share this with others. Effective use of these resources has
become crucial to biological research today, and this unit describes some
practical applications of the UCSC Genome Browser. Genomic data and annotations are rapidly accumulating in databases such as the
UCSC Genome Browser, NCBI, and Ensembl. Given the massive scale of these genomic
databases, it is important to be able to easily retrieve known data and
annotations of a specified genomic locus. For example, for a newly identified
cis-regulatory element bound by a transcription factor, questions that
immediately come to mind include whether the element is near a transcriptional
start site and, if so, the name of the corresponding gene, and whether the
histones or DNA at the locus are modified. The UCSC Genome Browser organizes
data and annotations (called tracks) around the reference sequences or draft
assemblies of many eukaryotic genomes and presents them using a powerful
web-based graphical interface. This protocol describes how to use the UCSC
Genome Browser to visualize selected tracks at specified genomic regions,
download the data and annotations for further analysis, and retrieve multiple
sequence alignments and their conservation scores. |
Which aminoacid position in the human CREB protein is phosphorylated? | pCREB is phosphorylated at its Serine 133. | Cyclic-AMP response element-binding protein (CREB) signaling has a critical role
in the formation of memories. CREB signaling is dysfunctional in the brains of
mouse models of Alzheimer's disease (AD), and evidence suggests that CREB
signaling may be disrupted in human AD brains as well. Here, we show that both
CREB and its activated form pCREB-Ser(133) (pCREB) are reduced in the prefrontal
cortex of AD patients. Similarly, the transcription cofactors CREB-binding
protein (CBP) and p300 are reduced in the prefrontal cortex of AD patients,
indicating additional dysfunction of CREB signaling in AD. Importantly, we show
that pCREB expression is reduced in peripheral blood mononuclear cells (PBMC) of
AD subjects. In addition, pCREB levels in PBMC positively correlated with pCREB
expression in the postmortem brain of persons with AD. These results suggest
that pCREB expression in PBMC may be indicative of its expression in the brain,
and thus offers the intriguing possibility of pCREB as a biomarker of cognitive
function and disease progression in AD. The ability to adequately measure the phosphorylation state of a protein has
major biological as well as clinical relevance. Due to its variable nature,
reversible protein phosphorylations are sensitive to changes in the tissue
environment. StabilizorTM T1 is a system for rapid inactivation of enzymatic
activity in biological samples. Enzyme inactivation is accomplished using
thermal denaturation in a rapid, homogeneous, and reproducible fashion without
the need of added chemical inhibitors. Using pCREB(Ser133) as a model system,
the applicability of the Stabilizor system to preserve a rapidly lost
phosphorylation is shown. |
Which gene is the paralog of yeast UPC2? | the related transcription factors Ecm22 and Upc2 play a crucial role in Saccharomyces cerevisiae filamentation. | We have expressed a cDNA to human apolipoprotein E (apoE) in Saccharomyces
cerevisiae. Secretion of apoE was achieved only by the use of a mutant (upc2)
strain of yeast with the phenotype of enhanced uptake and intracellular
esterification of exogenous cholesterol. Approximately 40 ng/ml apoE was
secreted by upc2 mutants in the absence of media cholesterol. ApoE secretion was
increased 2-3-fold upon the inclusion of cholesterol in the growth media. This
response to exogenous cholesterol was not mediated at the transcriptional level,
since apoE mRNA levels were constant under all culture conditions. Concomitant
with the increase in secretion following cholesterol uptake by upc2 strains,
approximately 5% of secreted apoE was associated with lipid; polar and non-polar
lipids were detected in this lipoprotein fraction. Intracellular degradation of
apoE in non-secreting strains of yeast was minimized by the presence of null
mutations in both vacuolar proteases with non-specific activity (pep4) and a
Golgi endopeptidase with specificity for paired basic residues (kex2). The
approach of expressing human apolipoproteins in yeast may identify factors that
mediate lipoprotein biosynthesis in higher cells. One such factor could be the
mammalian equivalent of the gene product of UPC2. Saccharomyces cerevisiae normally will not take up sterols from the environment
under aerobic conditions. A specific mutant, upc2-1, of the predicted
transcriptional activator UPC2 (YDR213w) has been recognized as a strain that
allows a high level of aerobic sterol uptake. Another predicted transcriptional
activator, the YLR228c gene product, is highly homologous to Upc2p. In fact, at
the carboxy terminus 130 of the last 139 amino acids are similar between the two
proteins. Since these proteins are very similar, the effect of mutations in the
YLR228c open reading frame (ORF) was compared with like alterations in UPC2.
First, the YLR228c ORF was insertionally inactivated and crossed with various
UPC2 constructs. Deletion of YLR228c and UPC2 in combination resulted in
nonviability, suggesting that the two proteins have some essential overlapping
function. The upc2-1 point mutation responsible for aerobic sterol uptake was
duplicated in the homologous carboxy region of the YLR228c ORF using
site-directed mutagenesis. This mutation on a high-copy vector resulted in an
increase in sterol uptake compared to an isogenic wild-type strain. The
combination of both point mutations resulted in the greatest level of aerobic
sterol uptake. When the YLR228c point mutation was expressed from a low-copy
vector there was little if any effect on sterol uptake. Gas chromatographic
analysis of the nonsaponifiable fractions of the various strains showed that the
major sterol for all YLR228c and UPC2 combinations was ergosterol, the consensus
yeast sterol. In the pathogenic yeast Candida albicans, the zinc cluster transcription factor
Upc2p has been shown to regulate the expression of ERG11 and other genes
involved in ergosterol biosynthesis upon exposure to azole antifungals. ERG11
encodes lanosterol demethylase, the target enzyme of this antifungal class.
Overexpression of UPC2 reduces azole susceptibility, whereas its disruption
results in hypersusceptibility to azoles and reduced accumulation of exogenous
sterols. Overexpression of ERG11 leads to the increased production of lanosterol
demethylase, which contributes to azole resistance in clinical isolates of C.
albicans, but the mechanism for this has yet to be determined. Using genome-wide
gene expression profiling, we found UPC2 and other genes involved in ergosterol
biosynthesis to be coordinately upregulated with ERG11 in a
fluconazole-resistant clinical isolate compared with a matched susceptible
isolate from the same patient. Sequence analysis of the UPC2 alleles of these
isolates revealed that the resistant isolate contained a single-nucleotide
substitution in one UPC2 allele that resulted in a G648D exchange in the encoded
protein. Introduction of the mutated allele into a drug-susceptible strain
resulted in constitutive upregulation of ERG11 and increased resistance to
fluconazole. By comparing the gene expression profiles of the
fluconazole-resistant isolate and of strains carrying wild-type and mutated UPC2
alleles, we identified target genes that are controlled by Upc2p. Here we show
for the first time that a gain-of-function mutation in UPC2 leads to the
increased expression of ERG11 and imparts resistance to fluconazole in clinical
isolates of C. albicans. The pathogenic yeast Candida albicans can develop resistance to the widely used
antifungal agent fluconazole, which inhibits ergosterol biosynthesis, by the
overexpression of genes encoding multidrug efflux pumps or ergosterol
biosynthesis enzymes. Zinc cluster transcription factors play a central role in
the transcriptional regulation of drug resistance. Mrr1 regulates the expression
of the major facilitator MDR1, Tac1 controls the expression of the ABC
transporters CDR1 and CDR2, and Upc2 regulates ergosterol biosynthesis (ERG)
genes. Gain-of-function mutations in these transcription factors result in
constitutive overexpression of their target genes and are responsible for
fluconazole resistance in many clinical C. albicans isolates. The transcription
factor Ndt80 contributes to the drug-induced upregulation of CDR1 and ERG genes
and also binds to the MDR1 and CDR2 promoters, suggesting that it is an
important component of all major transcriptional mechanisms of fluconazole
resistance. However, we found that Ndt80 is not required for the induction of
MDR1 and CDR2 expression by inducing chemicals. CDR2 was even partially
derepressed in ndt80Δ mutants, indicating that Ndt80 is a repressor of CDR2
expression. Hyperactive forms of Mrr1, Tac1, and Upc2 promoted overexpression of
MDR1, CDR1/CDR2, and ERG11, respectively, with the same efficiency in the
presence and absence of Ndt80. Mrr1- and Tac1-mediated fluconazole resistance
was even slightly enhanced in ndt80Δ mutants compared to wild-type cells. These
results demonstrate that Ndt80 is dispensable for the constitutive
overexpression of Mrr1, Tac1, and Upc2 target genes and the increased
fluconazole resistance of strains that have acquired activating mutations in
these transcription factors. Upc2, a zinc-cluster transcription factor, is a regulator of ergosterol
biosynthesis in yeast. In response to sterol levels, the transcriptional
activity of Upc2 is controlled by the C-terminal domain. In this study, the
C-terminal regulatory domain of Upc2 from Saccharomyces cerevisiae was purified
and crystallized by the vapour-diffusion method. To improve the diffraction
quality of Upc2 crystals, a Upc2 fusion protein in which 11 residues of the
variable loop (residues 715-725) were replaced by T4 lysozymes in Upc2
(Upc2-T4L) was engineered. The Upc2-T4L crystals diffracted to 2.9 Å resolution
using synchrotron radiation. The crystal was trigonal, belonging to space group
P3(2) with unit-cell parameters a = 67.2, b = 67.2, c = 257.5 Å. The Matthews
coefficient was determined to be 3.41 Å(3) Da(-1) with two molecules in the
asymmetric unit. Initial attempts to solve the structure by the single-anomalous
dispersion technique using selenomethionine were successful. Saccharomyces cerevisiae ergosterol biosynthesis, like cholesterol biosynthesis
in mammals, is regulated at the transcriptional level by a sterol feedback
mechanism. Yeast studies defined a 7-bp consensus sterol-response element (SRE)
common to genes involved in sterol biosynthesis and two transcription factors,
Upc2 and Ecm22, which direct transcription of sterol biosynthetic genes. The
7-bp consensus SRE is identical to the anaerobic response element, AR1c. Data
indicate that Upc2 and Ecm22 function through binding to this SRE site. We now
show that it is two novel anaerobic AR1b elements in the UPC2 promoter that
direct global ERG gene expression in response to a block in de novo ergosterol
biosynthesis, brought about by antifungal drug treatment. The AR1b elements are
absolutely required for auto-induction of UPC2 gene expression and protein and
require Upc2 and Ecm22 for function. We further demonstrate the direct binding
of recombit expressed S. cerevisiae ScUpc2 and pathogenic Candida albicans
CaUpc2 and Candida glabrata CgUpc2 to AR1b and SRE/AR1c elements. Recombit
endogenous promoter studies show that the UPC2 anaerobic AR1b elements act in
trans to regulate ergosterol gene expression. Our results indicate that Upc2
must occupy UPC2 AR1b elements in order for ERG gene expression induction to
take place. Thus, the two UPC2-AR1b elements drive expression of all ERG genes
necessary for maintaining normal antifungal susceptibility, as wild type cells
lacking these elements have increased susceptibility to azole antifungal drugs.
Therefore, targeting these specific sites for antifungal therapy represents a
novel approach to treat systemic fungal infections. In most eukaryotes, including the majority of fungi, expression of sterol
biosynthesis genes is regulated by Sterol-Regulatory Element Binding Proteins
(SREBPs), which are basic helix-loop-helix transcription activators. However, in
yeasts such as Saccharomyces cerevisiae and Candida albicans sterol synthesis is
instead regulated by Upc2, an unrelated transcription factor with a Gal4-type
zinc finger. The SREBPs in S. cerevisiae (Hms1) and C. albicans (Cph2) have lost
a domain, are not major regulators of sterol synthesis, and instead regulate
filamentous growth. We report here that rewiring of the sterol regulon, with
Upc2 taking over from SREBP, likely occurred in the common ancestor of all
Saccharomycotina. Yarrowia lipolytica, a deep-branching species, is the only
genome known to contain intact and full-length orthologs of both SREBP (Sre1)
and Upc2. Deleting YlUPC2, but not YlSRE1, confers susceptibility to azole
drugs. Sterol levels are significantly reduced in the YlUPC2 deletion. RNA-seq
analysis shows that hypoxic regulation of sterol synthesis genes in Y.
lipolytica is predomitly mediated by Upc2. However, YlSre1 still retains a
role in hypoxic regulation; growth of Y. lipolytica in hypoxic conditions is
reduced in a Ylupc2 deletion and is abolished in a Ylsre1/Ylupc2 double
deletion, and YlSre1 regulates sterol gene expression during hypoxia adaptation.
We show that YlSRE1, and to a lesser extent YlUPC2, are required for switching
from yeast to filamentous growth in hypoxia. Sre1 appears to have an ancestral
role in the regulation of filamentation, which became decoupled from its role in
sterol gene regulation by the arrival of Upc2 in the Saccharomycotina. Zinc cluster proteins are a large family of transcriptional regulators with a
wide range of biological functions. The zinc cluster proteins Ecm22, Upc2, Sut1
and Sut2 have initially been identified as regulators of sterol import in the
budding yeast Saccharomyces cerevisiae. These proteins also control adaptations
to anaerobic growth, sterol biosynthesis as well as filamentation and mating.
Orthologs of these zinc cluster proteins have been identified in several species
of Candida. Upc2 plays a critical role in antifungal resistance in these
important human fungal pathogens. Upc2 is therefore an interesting potential
target for novel antifungals. In this review we discuss the functions, mode of
actions and regulation of Ecm22, Upc2, Sut1 and Sut2 in budding yeast and
Candida. Budding yeast mating is an excellent model for receptor-activated cell
differentiation. Here we identify the related transcription factors Ecm22 and
Upc2 as novel regulators of mating. Cells lacking both ECM22 and UPC2 display
strong mating defects whereas deletion of either gene has no effect. Ecm22 and
Upc2 positively regulate basal expression of PRM1 and PRM4. These genes are
strongly induced in response to mating pheromone, which is also largely
dependent on ECM22 and UPC2. We further show that deletion of PRM4 like PRM1
results in markedly reduced mating efficiency. Expression of PRM1 but not of
PRM4 is also regulated by Ste12, a key transcription factor for mating. STE12
deletion lowers basal PRM1 expression, whereas STE12 overexpression strongly
increases PRM1 levels. This regulation of PRM1 transcription is mediated through
three Ste12-binding sites in the PRM1 promoter. Simultaneous deletion of ECM22
and UPC2 as well as mutation of the three Ste12-binding sites in the PRM1
promoter completely abolishes basal and pheromone-induced PRM1 expression,
indicating that Ste12 and Ecm22/Upc2 control PRM1 transcription through distinct
pathways. In summary, we propose a novel mechanism for budding yeast mating. We
suggest that Ecm22 and Upc2 regulate mating through the induction of the mating
genes PRM1 and PRM4. |
Where is the enzyme PM20D1 localized? | PM20D1 is enriched in UCP1+ adipocytes |
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