The purpose of this study was to determine the mid-term clinical, radiographic and health-related quality of life (HRQoL) outcomes and define the survival rate in patients who had undergone revision total knee arthroplasty (TKA) using the single rotating hinged knee (RHK) design.Between January 2004 and December 2013, 125 revision TKAs were performed at our institution using the single RHK implant. We conducted both a retrospective analysis of prospectively collected outcome data of these patients and a prospective follow-up study of all 39 living patients (41 knees). The follow-up phase included an optional extra follow-up visit, PROM questionnaires, and plain radiographs.The ten-year Kaplan-Meier survival rate of the revision RHK knees was 81.7% (95% CI 71.9–91.6%) with re-revision for any reason as the endpoint. Overall, 15 knees  underwent re-revision surgery during the follow-up. The median follow-up was 6.2 years  post-operatively for the baseline group. One mechanical hinge mechanism-related failure occurred without any history of trauma or infection. At the time of the final follow-up, the majority of patients evinced a fairly good clinical outcome measured with patient-reported outcome measures and none of the components were radiographically loose.We found that in patients undergoing complex revision TKA, fairly good functional outcome and quality of life can be achieved using an RHK implant. Further, it seems that in this type of patient cohort, revision TKA using an RHK implant relieves pain more than it improves ability to function. The NexGen® RHK design can be regarded as a suitable option in complex revision TKA.The online version contains supplementary material available at 10.1186/s12891-021-04205-9. In recent years, the incidence of total knee arthroplasty (TKA) has increased worldwide –4. Some Instability is one of the most frequent causes of knee revision and re-revision along with aseptic loosening and infections , 4, 8, 9Historically, aseptic loosening occurred more often with hinged knee implants that prohibited rotational motion. These models caused much unwanted stress at the prosthesis-bone interface or on the implant itself, leading to implant failure , 11. To In the literature, recently published rotating hinged knee implant studies are difficult to compare because of their heterogeneity , 13. IndThe purpose of this study was to determine the mid–term clinical, radiographic, and health-related quality of life (HRQoL) outcomes in patients who underwent revision TKA using the single rotating hinged knee (RHK) design at our institution between January 2004 and December 2013.From January 2004 to December 2013, 125 revision TKAs were performed at our institution using the NexGen® RHK implant. The study site is an academic high-volume tertiary referral center with an annual volume of approximately 150 revision TKAs.This study comprised three phases: first, a prospective follow-up study of all the surviving patients of this cohort was conducted. Second, a retrospective analysis of the prospectively collected outcome data recorded into the electronical joint replacement database at our institution was carried out. Third, information on possible revision surgeries that might have been performed elsewhere, and thus not recorded into our own database, was cross-checked from the Finnish Arthroplasty Register.All RHK revision knee arthroplasties were performed using the medial parapatellar approach and a tourniquet was also routinely used. TKAs were carried out under spinal anesthesia in combination with intravenous sedation. General anesthesia was only used if there was a contraindication to spinal anesthesia. Immediate, full weight-bearing was allowed, and all patients were mobilized on the first postoperative day. An antithrombotic prophylaxis with low-molecular-weight heparin, enoxaparin, was administered for 4 weeks postoperatively. All details of perioperative care and possible complications were recorded in the hospital’s electronical database in a routine manner. In 48 knees (38%), the patella was resurfaced in the revision TKA. In 25 knees (20%), the patella had already been resurfaced in the primary TKA operation. Cemented femoral and tibial components were used in all operations, and the choice between cemented and uncemented stems was based on surgeon preference. The trabecular metal cones were used in 13 cases (10%). At the baseline of the study, 119 patients (125 knees) were included in the retrospective analysis, where all the demographics, surgery reports, first post-operative visits (at 2–3 months), possible post-operative complications, and adverse events as well as reasons for revisions were obtained from the medical records and the hospital’s electronical clinical database. Information on possible revision surgeries performed on patients elsewhere who were lost to follow-up  was cross-checked from the Finnish Arthroplasty Register [In the prospective study phase, an extra follow-up visit was scheduled between 4 and 14 years post-operatively, depending on the year of the index operation. All of the 59 living and unrevised patients (61 knees) were recruited by telephone for an extra follow-up visit at our outpatient clinic  of the pre-operative radiographs was later reviewed by an experienced orthopaedic surgeon who was blinded to the original measurements.Radiographic findings, such as osteolytic defects and stability of the patella, were evaluated from the pre-operative radiographs. Pre-revision bone defects were classified according to Anderson Orthopaedic Research Institute (AORI) classification  and thesAll postoperative plain radiographs from the extra follow-up were evaluated by two senior orthopaedic surgeons (co-authors JN and AE). Radiographic evaluation was performed from standardized weight-bearing antero-posterior (AP), lateral, and skyline patellar views. Radiographs were assessed for the presence of radiolucent lines or osteolytic defects.Health-related quality of life (HRQoL) was measured using the comprehensive generic 15D instrument. The instrument combines the advantages of a profile and a preference-based single index measure. The 15D instrument includes the following 15 dimensions: mobility, vision, hearing, breathing, sleeping, eating, speech, excretion, usual activities, mental function, discomfort and symptoms, depression, distress, vitality, and sexual activity. For each dimension, the respondent chooses one of the five ordinal levels that best describe their state of health at the time . The single index score (15D score) represents the overall HRQoL on a 0–1 scale . The dimension level values reflect the goodness of the levels relative to no problems on the dimension (=1) and to being dead (=0). These values are then calculated from the health state descriptive system (questionnaire) by using a set of population-based preference or utility weights. Mean dimension level values are used to draw 15D profiles for groups .n = 4052) taken from the Health 2011 Survey carried out by the National Institute for Health and Welfare of Finland [We compared our study population’s 15D results to those of an age- and gender-standardized sample of the general Finnish population . Competing risk analysis was performed with R (version 4.0.2). The study was funded by an institutional grant from Zimmer Biomet Inc. . The study was approved by the local ethical committee (R17010).The Kaplan-Meier (K-M) analysis was performed to assess the survival rate of the RHK implant. Both survival rates and 95% confidence intervals (CI) were derived from K-M models. The independent samples t-test was used to test the statistical significance of the differences in the mean 15D results between the groups. Statistical significance was set at n = 28/39). The median follow-up was 7.3 years  at the time of the final follow-up. Demographics, revision indications, and pre-revision bone defects are summarized in Table n = 54, 43.2%). Pre-revision bone defects were mostly grade 1 both on the tibial  as well as on the femoral side  according to the AORI classification.The baseline study group consisted of 86 women 91 knees) and 33 men (34 knees) with a median age of 71.7 years  at the time of revision TKA. Five females and one male patient underwent bilateral revision TKA (RHK on both sides). In the final follow-up group, most of the patients were women . The median KOOS for Pain was 75 , Symptoms 73 , ADL 69 , Sport/Rec 18 , and QOL 44  at the time of final follow-up. The median FJS was 33 .OKS was good or excellent in a slight majority of patients , moderate in 6 knees, and poor in 14 knees at the time of the extra follow-up visit. The median OKS was 29 (p = 0.002). The mean level values of the dimensions of the patients compared to those of the age- and the gender-standardized general population are shown in Fig. The mean 15D score of the patients was 0.806  at the time of the final follow-up. The age- and gender-standardized control population average was 0.877 . This difference is both clinically important and statistically significant (p = 0.098). RHK patients were, however, statistically significantly worse off than the control population on the dimensions of mobility (p < 0.001), usual activities (p < 0.001), and discomfort and symptoms (p = 0.008).On average, there was a trend for RHK patients to be better off than the control population on the dimension of mental function, but the difference was not statistically significant . One patient (1 knee) had a radiolucent line in the cement-bone interface next to the medial condyle of the tibia; components were stable and no osteolysis was seen. Two patients (2 knees) had mild radiolucencies around the proximal part of the femoral stem, and one of them also had distal pedestal bone formation, which is typical for uncemented stems. None of the components were radiographically loose.In total, 15 knees  underwent re-revision surgery during the follow-up  with revision for any reason as the endpoint. The ten-year K-M survival rate was 82.4% (95% CI 72.6–92.2%) when those revisions, in which only patellar resurfacing was performed, were excluded. Competing risk analysis for overall survival probability of arthroplasty (40.0% at 10 years) and probability of re-revision (12.0% at 10 years) and death (36.8% at 10 years) as competing events is shown in Fig. During the follow-up, 31 postoperative complications were recorded  of the patients described their knee pain as either non-existent or very mild. However, HRQoL was worse in these patients than in the control population as measured with the 15D instrument. The ten-year K-M survival rate was 81.7% with revision for any reason as the endpoint. Overall, 15 knees  underwent re-revision surgery during the follow-up, and the most common reason for re-revision was prosthetic joint infection . One mechanical failure of the hinge mechanism led to re-revision. None of the RHK implants were radiographically loose at the time of the final follow-up.In our study, we demonstrated a fair mid-term clinical outcome and also acceptable implant survivorship in patients undergoing complex revision arthroplasty using the NexGen® RHK implant. A slight majority of the patients reported good or excellent OKS at the time of the final follow-up. Moreover, 39%  for revision arthroplasty in five-year mid-term follow-up for different single rotating hinge knee implants [n = 62) after two-year follow-up [The final follow-up group reported variable OKS results. It must be noted, however, that a slight majority reported good or excellent OKS, but the total median score was 29, which can be categorized as fair . Our OKSimplants . FurtherHelito et al. investigated single rotating hinge knee implants prospectively and reported the KOOS after one-year follow-up. The study included only nine patients, however, and six of them were primary rotating hinge knee implants, and therefore it is difficult to compare them with KOOS . We are There is a paucity of reported FJS in rotating hinge knee patients. Röhner et al. studied single hinge knee implants in primary arthroplasty and reported an FJS of 39 in the mean 20-months follow-up . This isn = 731) at the time of 12 month follow-up [The mean 15D score of our RHK cohort was statistically significantly and clinically importantly worse than in the control population. The patients were worse off, especially on the dimensions of mobility, usual activities, and discomfort and symptoms. It is reasonable therefore to assume that these kinds of complex revision arthroplasty patients have difficulties when compared with the age- and gender-standardized general population. Further, RHK patients were better only on the dimension of mental function. However, the difference was not statistically significant and this might be attributable to selection bias. Patients with severe neurodegenerative diseases were lost to follow-up and this could have biased our HRQoL results. Miettinen et al. reported a retrospective study, in which QoL was assessed the 15D instrument before and after primary TKA, but the type of implants remained unknown. After TKA, mean 15D score was 0.870  and of all the revisions 60% (n = 9/15). Moreover, 32% (n = 10/31) of all complications were related to extensor apparatus complications. It should be noted that only two of these had a preoperative well-functioning extensor mechanism  bears comparison with the current literature. In the current study, infections were a notable part of all the complications 29% n = 9/3 and of aThe 10-year implant survival rate of 81.7% is comparable with the rates reported in other rotating hinge knee studies. In their meta-analysis that included 12 different studies, Yoon et al. compared the survivorships and outcomes of RHK and condylar constrained knee prostheses. The rotating hinge knee implant models were variable. The meta-analysis revealed an overall survival rate of 81.3% for all RHK implants at the mid-term (5–10 years) follow-up .To conclude, we found the NexGen® RHK design to be a suitable option in complex revision TKA. This implant provided fairly good functional outcome and quality of life in our cohort of patients, and the mid-term implant survivorship was acceptable in this challenging patient group. Also mechanical problems in the hinge mechanism were rare. However, Nexgen RHK should only be used in complex cases, as complications are still frequent, and the results of this design are comparable, not superior, to those previously published of other rotating hinge knee designs.Additional file 1. Postoperative revision surgeries.


Inactivated coronaviruses, including severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and Middle East respiratory syndrome coronavirus (MERS-CoV), as potential vaccines have been reported to result in enhanced respiratory diseases (ERDs) in murine and nonhuman primate (NHP) pneumonia models after virus challenge, which poses great safety concerns of antibody-dependent enhancement (ADE) for the rapid wide application of inactivated SARS-CoV-2 vaccines in humans, especially when the neutralizing antibody levels induced by vaccination or initial infection quickly wane to nonneutralizing or subneutralizing levels over the time. With passive transfer of diluted postvaccination polyclonal antibodies to mimic the waning antibody responses after vaccination, we found that in the absence of cellular immunity, passive infusion of subneutralizing or nonneutralizing anti-SARS-CoV-2 antibodies could still provide some level of protection against infection upon challenge, and no low-level antibody-enhanced infection was observed. The anti-SARS-CoV-2 IgG-infused group and control group showed similar, mild to moderate pulmonary immunopathology during the acute phase of virus infection, and no evidence of vaccine-related pulmonary immunopathology enhancement was found. Typical immunopathology included elevated MCP-1, IL-8 and IL-33 in bronchoalveolar lavage fluid; alveolar epithelial hyperplasia; and exfoliated cells and mucus in bronchioles. Our results corresponded with the recent observations that no pulmonary immunology was detected in preclinical studies of inactivated SARS-CoV-2 vaccines in either murine or NHP pneumonia models or in large clinical trials and further supported the safety of inactivated SARS-CoV-2 vaccines. Antibody-dependent enhancement (ADE) effect, defined as the antibodies induced by initial infection or vaccination binding to viral surface proteins and promote viral invasion of host cells, is a major concern for all vaccine developers. Inactivated coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), as potential vaccines have been reported to result in enhanced respiratory diseases (ERDs) in murine and nonhuman primate (NHP) pneumonia models after virus challenge, which poses great safety concerns for the rapid wide application of inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines in humans ,2. No ER5 TCID50. Samples, including nasal swabs, pharyngeal swabs and blood samples, were collected daily for viral shedding and neutralizing titre analyses. Four days after SARS-CoV-2 infection, all animals were sacrificed after anesthesia. Bronchoalveolar lavage fluid (BALF) and the main tissues and organs were collected for proinflammatory mediator, viral load and histopathology analyses.Anti-serum with a neutralizing antibody geometric mean titre (GMT) > 1:128 was collected from rhesus macaque intramuscularly injected on days 0 and 28 with a formaldehyde and β-propiolactone double-inactivated SARS-CoV-2 vaccine developed by the Institute of Medical Biology, Chinese Academy of Medical Sciences (IMBCAMS) based on the KMS-1 strain (GenBank accession number MT226610.1) ,12. Six Although neutralization titres were detected only in 2 (No. 19256 and No. 19060) of the 4 passively immunized monkeys before virus challenge b, all ofAmong all of the inflammatory cytokines  and chemokines  tested in BALF, some monkeys in the anti-SARS-CoV-2 IgG-infused group showed relatively higher levels of MCP-1, IL-8 and IL-33 than the control group d, S1, alOne way in which SARS-CoV-2 is different from SARS and MERS is the prevalence of a large number of asymptomatic and mild infections. Studies have shown that asymptomatic and mild patients are also capable of producing neutralizing antibodies, the levels of which are positively correlated with disease severity . Recent Our study demonstrated that the antibodies purified in vitro from anti-serum of inactivated SARS-CoV-2 vaccination at subneutralizing or even nonneutralizing levels could offer protection from SARS-CoV-2 infection in vivo c. This r5 TCID50) administered in rhesus macaque pneumonia models may lead to excessive activation of the immune cascade in lung tissues, which results in pulmonary pathology. Studies of SARS immunization in animal models have yielded widely varying results in terms of protective efficacy and potential ADE, and different vaccine strategies may lead to differences in whether the vaccine enhances the disease. There is a general consensus that inducing higher titres of antibodies as well as antibodies that target the S protein more effectively can reduce the risk of ADE. It is encouraging, however, that the inactivated SARS-CoV-2 vaccine, which targets the full antigen of the virus, provides dose-dependent protection in a variety of animal models without producing evidence of enhanced immunopathological damage [On the other hand, the elimination of SARS-CoV-2 by macrophages is reported to be associated with the production of multiple antiviral and proinflammatory cytokines , which il damage . Our stuIn conclusion, our results showed that in the absence of cellular immunity, passive infusion of subneutralizing or nonneutralizing anti-SARS-CoV-2 antibodies could still provide some level of protection against infection upon challenge, and no low-level antibody-enhanced infection was observed. The anti-SARS-CoV-2 IgG-infused group and control group showed similar, mild to moderate pulmonary immunopathology during the acute phase of virus infection, and no evidence of vaccine-related pulmonary immunopathology enhancement was found. There results corresponded with the recent observations that no pulmonary immunology was detected in the preclinical studies of inactivated SARS-CoV-2 vaccines in both murine and NHP pneumonia models or in large clinical trials. Although more evidence is needed to confirm or deny the existence of ADE in SARS-CoV-2 infection and the immune damage caused by it, the available evidence has further supported the safety of the SARS-CoV-2 vaccine.Click here for additional data file.Click here for additional data file.


The absence of dystrophin has gave a massive impact on myotube development in Muscular Dystrophy pathogenesis. One of the conserved signaling pathways involved in skeletal muscle differentiation is the PI3K/Akt/mTOR pathway that plays a vital role in autophagy regulation. To further understand and establish targeted therapy in dystrophin-deficient myoblasts, protein expression profiling has been determined which provides information on perturbed autophagy modulation and activation.In this study, a dystrophin-deficient myoblast cell line established from the skeletal muscle of a dystrophic (mdx) mouse was used as a model. The dfd13 (dystrophin-deficient) and C2C12 (non-dystrophic) myoblasts were cultured in low mitogen conditions for 10 days to induce differentiation. The cells were subjected to total protein extraction prior to Western blotting assay technique. Protein sub-fractionation has been conducted to determine protein localization. The live-cell analysis of autophagy assay was done using a flow cytometer.In our culture system, the dfd13 myoblasts did not achieve terminal differentiation. PTEN expression was profoundly increased in dfd13 myoblasts throughout the differentiation day subsequently indicates perturbation of PI3K/Akt/mTOR regulation. In addition, rictor-mTORC2 was also found inactivated in this event. This occurrence has caused FoxO3 misregulation leads to higher activation of autophagy-related genes in dfd13 myoblasts. Autophagosome formation was increased as LC3B-I/II showed accumulation upon differentiation. However, the ratio of LC3B lipidation and autophagic flux were shown decreased which exhibited dystrophic features.Perturbation of the PTEN-PI3K/Akt pathway triggers excessive autophagosome formation and subsequently reduced autophagic flux within dystrophin-deficient myoblasts where these findings are of importance to understand Duchenne Muscular Dystrophy (DMD) patients. We believe that some manipulation within its regulatory signaling reported in this study could help restore muscle homeostasis and attenuate disease progression.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-021-00785-0. DMD is romosome . The absignaling , 4. One ignaling –7. PTEN  muscles , 9. The  muscles .mdx mouse [Similarly, PTEN has been reported to contribute to the DMD phenotype. A study by Feron et al. (2009) on the Golden Retriever muscular dystrophy (GRMD) dog demonstrated that PTEN was present at high levels, which led to a reduction of Akt1, GSK3β and p70S6K, also, ERK1/2 displayed decreased phosphorylation levels in GRMD dog muscle. The GRMD dog is characterized by rapidly progressive clinical dysfunction, severe muscle weakness, and displays a disease progression that is more similar to human DMD compared to the dx mouse .mdx mouse [mdx/utr−/− mouse model [mdx/utr−/− mice (α7βX2-mdx/utr-/-) showed increased phosphorylation of Akt at Ser473, indicating that α-integrin expression is connected to Akt. The mdx/utr−/− mouse lacks both dystrophin and utrophin and develops a severe pathology that closely resembles that seen in DMD [Akt plays a major role in cell signaling and its impairment leads to various defects in downstream protein signaling, including cell proliferation, differentiation, and apoptosis, autophagy, and protein synthesis perturbation. Activation of Akt is higher in the dx mouse , 11 as wdx mouse . Akt hasse model . Boppartn in DMD . That DMAutophagy is the process of engulfment of cargo into the double lipid formation known as an autophagosome, which eventually fuses with a lysosome and is degraded . Autophamdx mice [Col6a1−/− animals and long-term exposure to a low-protein diet [Col6a1−/− animals display impairment of basal autophagy, which determines the persistence of dysfunctional organelles in muscle fibers leading to muscle degeneration [Autophagy is defective in mdx mice  and DMD mdx mice . Restoraein diet , 15 can neration .Over a decade, many researchers have reported on impairment of Igf-1 pathway and its downstream protein regulation in muscular dystrophy. However, to further understand the impaired regulation of autophagy in dystrophin-deficient myoblast, the protein profiling of this pathway has been determined which sequentially provides information on autophagy modulation and activation. In the beginning, we reported the capacity of dystrophin-deficient myoblasts differentiation via analysis of myotube formation and the expression of specific myosin heavy chain as a terminal differentiation marker. Furthermore, we showed the elevation of PTEN perturbed PI3K/Akt/mTOR signaling thus excessively activate autophagy-related genes via FoxO3 but subsequently showed a reduction of autophagic flux in dystrophin-deficient myoblast. Overview of this event as illustrated at the end of this article.mdx mouse [The C2C12 myoblast cell line was used in this study and was established from an adult mouse myoblast C2 cell line derived from the thigh muscle of 2-month-old mice . The dfddx mouse , 22. MouProtein was extracted from cells cultured in a 10 cm dish. The medium was removed and the cells washed twice with PBS before adding approximately 300 µL of lysis buffer  containing a protease inhibitor . The cells were scraped and collected into a tube before being subjected to centrifugation at 14,000 rpm for 15 min at 4 °C. The pellet was discarded and the supernatant transferred to a new tube and stored at − 20 °C.The rapid efficient and practical (REAP) protocol was used for protein sub-fractionation . All reaEqual protein loading was prepared before resuspending with 2 × Laemmli sample buffer. The mixture was collected by short spin centrifugation and boiled for 5 min before loaded into SDS–polyacrylamide gel electrophoresis. Lysate was separated on SDS–polyacrylamide gel electrophoresis  with 200 V for 1 h. The separated protein was transferred onto the nitrocellulose membrane using the Transblot Turbo® Transfer System . The membranes were blocked in 5% reduced-fat milk for 1 h before incubate with primary antibodies. The antibodies are Fast-MyHC, α-tubulin, SSRP-1 (1:1000) and Desmin, (1:2000), from Sigma-Aldrich, UK. PTEN (1:1000) . PI3K, phospho-PI3K (Tyr458), Akt, phospho-Akt (Ser473), mTOR, phospho-mTOR (Ser2448), p70S6 Kinase, phospho-p70S6 Kinase, Rictor, phospho-rictor (Thr1135) and FoxO3a (1:1000) from Cell Signalling Technology, UK. Beclin1, Atg5, Atg7, and LC3B (1:1000) .The coverslips were acid-etched for 5 min with nitric acid. This will help myoblasts firmly attached to the coverslips before immunofluorescence. It has been sent for sterilization before myoblasts were seeded onto it. The myoblasts/myotubes were fixed using 4% PFA. After fixation cells were permeabilized with 0.25% Triton X-100 for 5 min to allow the antibody to bind specifically. The cells were then washed three times for 5 min with PBS before blocking with 5% BSA at room temperature for 30 min. The blocking solution was removed and the cells incubated with the primary antibody Fast-MyHC at 4 °C overnight. The primary antibody was removed the next day and the cells washed before probed using a secondary antibody (biotinylated) for 1 h at room temperature. After washing, the cells were incubated with streptavidin-Texas red (1:1000) at room temperature for 1 h in the dark. The cells were then counterstained with DAPI (1:100) and mounted onto slides using DakoCytomation. The slides were wrapped in aluminum foil and stored at 4 °C.5/mL myoblasts were added to every well in a 24-well plate and maintained via standard tissue culture practice. Cells were grown in a humidified incubator at 37 °C and 5% CO2; cells were not allowed to become over-crowded and the density should not exceed 1 × 106 /mL. In this experiment, 60 mM of chloroquine was used as a positive control, while unstained cells were utilized as a negative control. Myoblasts were treated with chloroquine for 4 h in a humidified incubator at 37 °C and 5% CO2. At the end of the treatment, cells were trypsinized and collected into a fresh 1.5 mL microtube. Cells were subjected to centrifugation for 5 min at1000 rpm at room temperature and washed by re-suspending in 1 × assay buffer and the centrifugation step was repeated. Each cell pellet was then re-suspended in 250 µL of 1 × assay buffer. Approximately 250 µL of the diluted CYTO-ID Green stain solution was added to all the cells except the negative control and incubated for 30 min at room temperature in the dark. The cells were collected by centrifugation, washed with 1 × assay buffer, and the pelleted cells resuspended in 500 µL of 1 × assay buffer. The samples were analyzed using the green (FL1) channel of a flow cytometer.An autophagy assay was performed using a CYTO-ID Autophagy Detection Kit  and 1 × 10Experiments were performed using a FACS Analyser CyAn B  at the Institute of Biomedical Research, University of Birmingham. Samples were run using Summit V4.3 software and all data were saved in the FCS format developed by the Society of Analytical Cytology, thereby allowing further analysis using other software packages. Data were analyzed using the software FCS Express 6 Plus  for three parameters; side scatter (SS), forward scatter (FS), and Fluorescein Isothiocyanate (FITC). Gating was performed based on the size and complexity of the myoblasts as illustrated in density plots. Gated data were further analyzed to count myoblasts labeled with FITC and the results presented in a histogram format.All Western blots and flow cytometry were repeated at least three times for each experiment. Statistical analyses were carried out using the student t-test (Microsoft Excel) and one-way ANOVA. The differences were considered significant when p < 0.05 and p < 0.01.In vitro differentiation of myoblasts can be induced in culture through the use of a low mitogen medium  for a few days. In this study, both C2C12 (non-dystrophic) and dfd13 (dystrophin-deficient) myoblasts were cultured in low mitogen medium  for 10 days before determining their terminal differentiation capacity. Immunofluorescence analyses  and then normalized against total nuclei. F-MyHC, also called ‘fast-twitch’ fibers, were chosen as the terminal differentiation marker based on the existence of a spectrum of fiber types, with type II being the most developed form of myosin: type 1 ↔ 1/2A ↔ 2A ↔ 2A/2X ↔ 2X ↔ 2X/2B ↔ 2B . The numImmunoblotting for F-MyHC, pan-myosin, and desmin was performed on days 4, 7, and 10 of differentiation  and day 10 (p < 0.01) when compared to the non-differentiated dfd13 myoblasts. There was no significant difference in total PI3K expression in C2C12 myoblasts throughout the differentiation period compared to non-differentiated myoblasts. Densitometry analysis showed that there were significant differences in total PI3K expression in dfd13 myoblasts on day 4 (p < 0.05), day 7 (p < 0.01), and day 10 when compared to differentiating C2C12 myoblasts , which is a target protein of Akt. FoxO3 is a transcription factor responsible for the activation of autophagy genes involved in the autophagy machinery. A previous study showed that FoxO3 controls autophagy in skeletal muscle in vivo and induced multiple autophagy genes, including LC3B transcription in skeletal muscle . In thisImmunoblot analyses Fig. a showed Akt plays a role in inhibiting FoxO3 through phosphorylation at Thr24, Ser256, and Ser319, which leads to nuclear exclusion and activation. As Akt is inactivated in dfd13 myoblasts, it is thought that FoxO3 is not phosphorylated. Therefore, the non-phosphorylated FoxO3 is translocated to the nucleus and activates the gene involved in autophagy. Akt represses FoxO3 via phosphorylation resulting in nuclear exclusion. As Akt is not activated in dfd13 myoblasts, it was speculated that unphosphorylated-FoxO3 translocates into the nucleus and binds to the promoter, thus up-regulating autophagy-related genes such as LC3B, Atg5, and Atg7. Therefore, FoxO3 expression and localization needed to be examined within subcellular fractions, i.e. the nucleus and cytoplasm.FoxO3 was found to be localized more to the nucleus of dfd13 myoblasts Fig. c, with aIt has been shown that FoxO3 is highly expressed and primarily localized to the nucleus of differentiating dfd13 myoblasts. Within the nucleus, FoxO3 acts as a transcriptional activator that can recognize and bind to DNA sequences resulting in the activation of genes involved in autophagy, such as those for Beclin1 and Atgs . It was Figure Atg5 was found to be not/less expressed in C2C12 myoblasts (C). It can be seen that Atg5 expression was increased upon differentiation in dfd13 myoblasts, and expression was significantly increased on days 4, 7, and 10 compared to the non-differentiated stage. Densitometry analysis also showed significant differences (p < 0.01) for dfd13 myoblasts at every time point compared to C2C12 myoblasts Fig. c.Densitometry analysis of Atg7 expression in dfd13 myoblasts showed its accumulation throughout the differentiation period; however, there was no significant difference in expression when compared to the non-differentiated stage in C2C12 myoblasts. Expression showed a significant increase in dfd13 myoblasts when compared to the non-differentiated stage and also significant on days 4, 7, and 10 compared to C2C12 myoblasts Fig. d.Autophagy related genes were highly activated, and the next step was to determine whether a double-membrane vesicle, known as autophagosome, had been formed. The conversion of Light Chain-3B (LC3B-I) to LC3B-II can be considered to represent total autophagosome formation in myoblasts during differentiation.As shown in Fig. LC3B-II levels correlate with the number of autophagosomes formed; however, this could indicate either the up-regulation of autophagosome formation or a blockage in autophagic degradation. Besides, it does not conclusively indicate autophagic degradation. Therefore, further analysis was performed to determine autophagic flux, with a lysosome inhibitor used as a positive control.To accurately determine autophagic activity, measuring the increase in the number of autophagosomes is required. Previous data showed that LC3B conversion was increased in C2C12 but reduced in dfd13 myoblasts upon differentiation; however, the results from measuring LC3B conversion alone could be inappropriately interpreted. LC3B-II itself has been reported to be degraded by autophagy and also tends to be more sensitive than LC3B-I during immunoblotting analysis . Autopha1–103) (X-axis) and then the percentage obtained.Figure Generally, both treated and non-treated myoblasts showed a reduction in autophagosome counts after 10 days of differentiation. Non-treated myoblasts had fewer autophagosomes when compared to treated myoblasts, which is due to chloroquine acting as a lysosome inhibitor, preventing autophagosome from fusing with lysosomes and causing the accumulation of autophagosomes . As illustrated in Fig. From these results, autophagic flux can be determined. Autophagic flux can be calculated by subtracting the chloroquine-treated from the untreated myoblasts, which enables the total number of non-fused autophagosomes to be measured and also the total number of autophagosomes formed. As depicted in Fig. Overall, autophagy activity was decreased upon differentiation in dystrophin-deficient myoblasts. Based on the flow cytometry analysis, autophagic flux was decreased, as the total number of autophagosomes detected was higher in dfd13 myoblasts (both non-differentiated and differentiated) when compared to C2C12 myoblasts. Besides, autophagy was twofold higher in non-differentiated myoblasts (proliferation state) compared to the differentiated state.In this experiment, it has been established that dystrophin-deficient myoblast differentiation is impaired when in a low mitogen medium for 10 days based on morphological analysis  inhibitor p21 and retinoblastoma protein (Rb) have been shown to play a critical role in establishing the post-mitotic state by permitting the transcription of the S-phase promoting gene during myogenesis . Most mi3 to produce PIP2. In contrast, PI3K reverses this event by phosphorylating PIP2 to become PIP3.The PI3K/Akt pathway is a highly conserved pathway for the regulation of skeletal muscle growth and is activated by the binding of Igf-1 to its receptor, Igf1-r. This binding leads to intrinsic tyrosine kinase activation, as well as auto-phosphorylation, thus activating PI3K/Akt signaling for myoblast differentiation via a series of phosphorylation events. This signaling is negatively regulated by PTEN. In this study PTEN was shown to be elevated in dfd13 myoblasts, which affected PI3K activation, as it was found to be decreased during differentiation Fig. d, indica2 phosphorylation. PIP3 helps to recruit Akt by binding the plekstrin homolog (PH) domain at the N-terminal of Akt to the cell membrane. PTEN in turn acts as a negative regulator by dephosphorylating PIP3 to form PIP2, and thereby reduces the available docking sites for Akt to bind to before activation. PTEN is mainly found in the cytosol and nucleus. The N-terminal possesses a PIP2-binding motif while the C-terminal contains a serine/threonine phosphorylation, Ser380, which regulates its stability and activity, as well as membrane recruitment. The main C2 domain contains basic residues that are essential for membrane binding. Phosphorylation of PTEN is considered to result in a closed conformation which is the inactive form. In this conformation, it has been proposed that the phosphorylated C-terminal interacts with the positively charged C2 domain and remains in the cytoplasm [2. From this data, it seems that high PTEN expression decreased PI3K activity, thus affecting its downstream protein activation.PI3K exists as a heterodimer consisting of two subunits; regulatory and catalytic. When cells are stimulated, p85 binds to tyrosine-phosphorylated IRS, and phosphorylated-p85 changes the conformation of p110 and thus mediates the p110 subunit to translocate to the membrane and increases its enzymatic activity such as PIPytoplasm . As the Feron and colleagues (2009) reported that increased PTEN expression caused the deregulation of the PI3K/Akt pathway in dystrophin-deficient muscle present in GRMD dogs. This was also observed in muscle sections from 3- to 36-month old animals and indicates that the PI3K/Akt pathway is a long-term alteration. A more recent study by Alexander et al. (2014) proposed that overexpression of micro-RNA-486 improved muscle physiology and performance in dystrophin-deficient mice . Micro-R3. Once bound, the conformation is changed and this event unmasks two residues for phosphorylation, Ser473, and Thr308. In this study, fewer Akt-phosphorylated at Ser473 was detected in dystrophin-deficient myoblasts, indicating that Akt is inactivated/less activated. According to Sarbassov et al. (2005), PDK1 has a better target on Akt phosphorylated at Ser473 than non-phosphorylated Akt [In this study, it was found that Akt is not/less activated in dfd13 myoblasts, as phosphorylation at Ser473 or Thr308 was not detected. However, in C2C12 myoblasts Akt was phosphorylated at Ser473, and the activated form accumulated upon differentiation. Immunofluorescence analysis showed that phosphorylated-Akt (Ser473) is localized to the membrane of C2C12 myotubes but found less often in dfd13 myoblasts. During biosynthesis, nascent Akt is phosphorylated at Thr450 within the turn motif site and localized to the cytosol when in its inactive conformation. In the presence of signals via PI3K activation, Akt is recruited to the membrane through the binding of plekstrin homolog (PH) to PIPated Akt . In lineAkt is a downstream protein of PI3K. It plays an important role and is recognized as one of the most critical pathways in the regulation of cell viability and maintenance in skeletal muscle mass . Therefo, and this facilitated Thr308 phosphorylation by PDK1 in drosophila [Since Akt was found to only be phosphorylated at Ser473 the protein responsible for this phosphorylation event was investigated. It is known that mTORC2, specifically, rictor, can directly phosphorylate Akt at Ser473 in vitroosophila . In thisosophila , HEK293 osophila . Recentlosophila . As PTENIn skeletal muscle, autophagy is transiently activated and continues for only a few days , 50; it In this study, it can be seen that autophagosome formation is modulated via FoxO3 in dfd13 myoblasts. Inactivation of Akt allowed the active-form of FoxO3 (unphosphorylated) to translocate into the nucleus and trigger the expression of autophagy-related genes. FoxO3 is required for the transcriptional regulation of LC3B and also for transcriptional regulation of MAFbx and MuRF1. This transcriptional regulation leads to protein degradation via the autophagy-lysosome pathway and the ubiquitin-proteosome pathway, respectively. However, autophagy in C2C12 myoblasts seems to be only partially FoxO3-mediated, as subsequent activation of Akt inhibited FoxO3 and suppressed its translocation to the nucleus and the targeting of autophagy-related gene activation. Activated-Akt, through phosphorylation at Ser473 by rictor-mTORC2, also contributes to autophagy activation during C2C12 myoblast differentiation , 51.Autophagy is responsible for removing unfolded proteins as well as dysfunctioning organelles and has been reported to be constantly active within skeletal muscle. Recently, increased autophagy has been reported to protect differentiating myoblasts from apoptotic cell death . SeveralLC3B is a subunit of microtubule-associated protein 1 (MAP1LC3B), and LC3 is cleaved by Atg4 to become cytosolic LC3B-I. LC3B-I is then converted to lipidated-LC3B-II through the conjugation of membrane lipid phosphatidylethanolamine (PE), which involves the E1-like enzyme ubiquitin, Atg7, and the E2-like enzyme, Atg10. LC3B-II then binds to the isolation membrane and mediates membrane elongation until the edges fuse to form an autophagosome. The isolation membrane appears when cells are placed under starvation conditions. Atg7 also conjugate Atg5 to Atg12 to form the Atg5-Atg12 complex and then bind to the isolation membrane with Atg16. This complex binding is necessary for autophagosome formation.In this study, the results showed that LC3B-I was increased in both types of myoblast due to the up-regulation of Beclin1 by nuclear-FoxO3, which is increased in dystrophin-deficient myoblasts. Beclin1 forms a complex with Vsp34 and becomes a core component during the pre-autophagosome stage. This complex then binds to the ULK1/Atg13/FIP200/Atg101 complex before entering the elongation stage, when Atg7 catalyzes the ligation of Atg5 to Atg12 to become the Atg5-Atg12 complex. With the aid of the Atg5-Atg12 complex, Atg7 catalyzes the transfer of PE to LC3B-I, converting it to LC3B-II, resulting in the accumulation of LC3B-II within dfd13 myoblasts  demonstrates excessive autophagosome formation in dfd13 myoblasts during differentiation; however, the conversion ratio of LC3B-I to LC3B-II was reduced in dystrophin-deficient myoblasts. Furthermore, although activation was higher, autophagic flux analysis showed a reduction upon differentiation and revealed that autophagy activity is decreased upon differentiation in dfd13 myoblasts Fig. . TherefoDMD is mostly characterized by ~ 2 years of age and it progresses until the early 20 s. Therefore, the applicability of this finding to the understating of DMD patients may fit with the actual scenario. During the early stage of life (newborn to below 2 years), deficient myoblasts survive as most cells are actively proliferating and high levels of autophagy activation can prevent apoptosis. As age increases (> 2 years old), most of the deficient myoblasts have been triggered for differentiation as the body develops and muscle size and functionality increase, but now autophagy starts to decrease and apoptosis begins. This is the most common period for when DMD is diagnosed. At the later stage (~ 10 years-old), there is a progressive disruption to the muscle due to loss/damage, and support (wheelchair) is needed for mobility and undertaking daily life tasks. This state will worsen and patients will eventually die, commonly as a complication of respiratory muscle damage and cardiomyopathy.PTEN-PI3K/Akt and its downstream proteins are perturbed in dystrophin-deficient myoblasts. From the data obtained, dystrophin-deficient myoblasts exhibit the high expression of autophagy-related proteins. However, a reduction in autophagy activity, as well as autophagy flux upon differentiation, indicates that autophagy is defective. This finding suggests a new mechanism for the reduction of autophagy in dystrophin-deficient myoblasts. All in all, we have come out with a schematic of autophagy modulation in dystrophin-deficient myoblasts Fig. . We show


Vandenboschia speciosa (Willd.) G. Kunkel). In addition to being a rare endangered Macaronesian-European endemism, this species has a huge genome (10.52 Gb) as well as particular biological features and extreme ecological requirements. These characteristics, together with the systematic position of ferns among vascular plants, make it of high interest for evolutionary, conservation and functional genomics studies. The transcriptome was constructed de novo and contained 36,430 transcripts, of which 17,706 had valid BLAST hits. A total of 19,539 transcripts showed at least one of the 7362 GO terms assigned to the transcriptome, whereas 6547 transcripts showed at least one of the 1359 KEGG assigned terms. A prospective analysis of functional annotation results provided relevant insights on genes involved in important functions such as growth and development as well as physiological adaptations. In this context, a catalogue of genes involved in the genetic control of plant development, during the vegetative to reproductive transition, in stress response as well as genes coding for transcription factors is given. Altogether, this study provides a first step towards understanding the gene expression of a significant fern species and the in silico functional and comparative analyses reported here provide important data and insights for further comparative evolutionary studies in ferns and land plants in general.We sequenced the sporophyte transcriptome of Killarney fern ( Vandenboschia speciosa (Willd.) G. Kunkel (=Trichomanes speciosum Willd.), is a tetraploid species  [V. speciosa is considered one of the most vulnerable fern species in Europe since it is threatened by habitat destruction and excessive collection and is listed in Annex I of the Berne Convention and Annex II of the Habitats Directive. At the national level, it is considered vulnerable in the Red List of Spanish Vascular Flora 2000. The two phases of its life cycle are perennial and capable of reproducing by vegetative propagation [Vittaria appalachiana, Hymenophyllum tayloriae and Vandenboschia intrincatum, the sporophytic phase of the life cycle has been completely eliminated, with the gametophyte surviving for a great length of time [The family Hymenophyllaceae has its origin in the Triassic and currently contains about 600 species, most of which emerged approximately 160 million years ago during the Jurassic period  and show0.52 Gb) ,8 that cpagation . The spopagation ,10,11. Tpagation ,10,11. Tpagation . The adapagation ,13,14,15 of time ,13,14,15V. speciosa would represent an important scientific resource for gene discovery and functional genomics studies. These studies might shed light on the knowledge of the genetic regulation of the adaptive response of both the sporophyte and the gametophyte and could be of use on the conservation genetics of this and similar species. In this context, the identification of the genes that regulate the growth and development of both phases of this species is also of special importance as it entails learning the genetic control of the transition between the vegetative and reproductive phases and the alternation between sporophyte and gametophyte. Here, we provide the de novo assembly and characterization of the sporophyte transcriptome of this vulnerable species, which represents an important step towards understanding the gene expression associated with phenotypes in a species with singular sporophytes and gametophytes, extreme ecological requirements and populations composed of independent gametophytes. In addition, the sporophyte transcriptome data and the functional analyses reported here provide an important platform for comparative evolutionary studies in ferns and land plants in general.A comprehensive transcriptome analysis in V. speciosa sporophytes were collected in April 2014 at one out of the seven populations located in the Alcornocales Natural Park : Valdeinfierno (VDI). We chose a population with a regular fern lifecycle of two free-living generations, gametophyte, and sporophyte. Sporophytes were frozen in liquid nitrogen in the field and stored at −80 °C. RNA was isolated from five sporophyte specimens using Spectrum™ Plant Total RNA Kit . RNAs were pooled and Next Generation Sequencing was carried out at Macrogen Inc.  based on the Illumina HiSeq 2000 Paired-end approach . Illumina sequencing data can be accessed at Short Read Archive (SRA) ENA database under the accession number ERX2079928.https://www.bioinformatics.babraham.ac.uk/projects/fastqc/; accessed on 10 April 2019). De novo transcriptome assembly was performed using Trinity v2.8 [Quality assessment of the resulting sequenced reads was performed using FastQC  [www.orthodb.org; accessed on 10 February 2021) to provide quantitative measures of the completeness of transcriptome assemblies in terms of expected gene content. We analyzed the percentage of conserved single-copy orthologues represented in three datasets , using the OrthoDB sets: Eukaryota_odb10, Viridiplantae_odb10 and Embryophyta_odb10. Plotting of the BUSCO results was performed using the script generate_plot.py—provided as companion script to BUSCO  that uses ggplot2 [We measured the completeness of the filtered transcriptome assembly using BUSCO v. 4.1.4  through ry 2021) . BUSCO s ggplot2  in R [28We estimated transcript abundance in a genome-free manner  with the “align_and_estimate_abundance” Perl script, using bowtie2  for the Candidate coding domain sequences (cds) within transcript sequences were identified with TransDecoder  using deV. speciosa generated after assembly and filtering was analyzed with BLASTx [https://www.ncbi.nlm.nih.gov/; accessed on 27 July 2020). Following the recommended default parameters [−3 e-value threshold. BLAST annotation was exported as a FASTA file to generate an annotated transcript database. The resulting database was deposited in FigShare . Gene Ontology (GO) terms  associated with the obtained BLAST hits were retrieved and GO annotation was carried out with BLAST2GO . In addition, InterPro annotations in BLAST2GO allowed retrieval of domain/motif information in a sequence-wise manner. Corresponding GO terms associated to InterPro results were then transferred to the sequences and merged with already existing GO terms. Augmented annotation by Annex was carried out. BLAST2GO integrates the second layer concept developed by the Norwegian University of Science and Technology [We used BLAST2GO software  for funch BLASTx  against rameters ,33, BLASchnology  for augmV. speciosa and a selected group of the following land plant species: the mosses Ceratodon purpureus (Hedw.) Brid.  and Physcomitrella patens (Hedw.) Bruch. and Schimp. , the ferns Azolla filiculoides Lam. and Salvinia cucullata Roxb. ex Bory  as well as Ceratopteris richardii Brongn. , the lycophyte Selaginella moellendorffii Hieron. , and the seed plant Arabidopsis thaliana (L.) Heynh., . Using reciprocal best-hits by the BLAST all-v-all algorithm, Orthofinder determined the number of shared putative orthologues between the eight species as well as species-specific transcripts. Orthofinder also infers gene trees for all orthogroups and a rooted species tree for the species being analyzed. Phylograms were visualized with Dendroscope [We used Orthofinder ,36 to iddroscope .Sequencing statistics are displayed in De novo transcriptome assembly statistics are shown in Polypodium amorphum Suksd. [Prunus salicina Lindl. [Ceratopteris richardii [V. speciosa transcripts gave identity to 7851 proteins of which 3196 (40.7% of transcripts) were covered in more than 90% of their lengths and 6444 (82% of transcripts) were covered in more than 50% of their lengths . Redundancy was further eliminated by clustering the filtered assembled transcripts using CD-HIT-EST at a nucleotide identity of 95%. Fewer than 22% of the transcripts were redundant and were therefore removed. The final filtered assembly contains 36,430 transcripts, with median and average transcript lengths of 1197 and 1437.37 nucleotides, respectively . Fifty pm Suksd.  and wereichardii . The N50 lengths .V. speciosa since there was a high recovery of conserved orthologues from eukaryotes in general and from plants in particular. However, the alignment against Embryophyta core genes was at the lower end of what it is considered a good quality transcriptome. This might be because we analyzed only the sporophyte phase, whereas complete general species transcriptomes should include sequencing from multiple tissues [V. speciosa sporophyte transcriptome is one of the most complete fern transcriptome hitherto published. Transcriptome assemblies from combined fern gametophyte and sporophyte tissues of Polypodium amorphum [Ceratopteris richardii [Lygodium japonicum (Thunb.) Sw. [Completeness of the filtered transcriptome assembly was measured using BUSCO . A total tissues ,46. Thusamorphum  and Ceraichardii  recoverenb.) Sw. ,47.TransDecoder determined that the total number of protein-coding transcripts among the final non-redundant transcripts was 29,220. Approximately 59.8% of the coding sequences were complete, 21.4% were 5′ partial, 10.4% were 3′ partial, and 8.4% were internal.e-values  genes involved in the vegetative growth in flowering plants were abundant within this list; (b) there is a remarkable presence of transcripts from genes involved in the synthesis of glucomannans, which constitute the type III primary cell wall in vascular plants and that are exclusively reported in some fern species ,51; (c) In addition to the annotated transcripts, 18,724 transcripts remained anonymous (not annotated using BLASTx). The latter had TPM values higher than 47.73 among the 1000 most expressed transcripts. Furthermore, the first four transcripts showed TPM values above the highest value among annotated transcripts and 449 transcripts had TPM higher than 88.11 . Seventy-nine of these transcripts had GO term annotation. In fact, 2816 of the total 18,724 anonymous transcripts showed an assigned GO term. Therefore, there is a number of potentially important hitherto unknown transcripts that merit future research—especially those with high TPM values.V. speciosa) were assigned to 26,875 orthogroups  from eight plant transcriptomes (including hogroups . Therefohogroups . Howeverectively . This isSellaginela moellendorffii) from the euphylophytes, which diverge in two major clades—the monilophytes or ferns (represented here by four species) and the spermatophytes or seed plants  [Azolla filiculoides and Salvinia cucullata), Cyatheales (tree ferns) and Polypodiales , form the large monophyletic clade of core leptosporangiates [V. speciosa), Gleicheniales and Schizaeales. The species tree based on single-copy orthologues of V. speciosa as a lineage that separates early from core leptosporangiate ferns [Azolla filiculoides and 0.25 Gb in the case of Salvinia cucullata, for example) [V. speciosa is an allotetraploid species with 2n = 144 chromosomes, which in part would explain its large genome (1C = 10.52 Gb) [A phylogenomic analysis based on the substitution rates of single-copy orthologues resulted in the species tree shown in haliana) . Among tangiates . The reste ferns ,57,58,59te ferns , whereasexample) . Polyploexample) ,62. In tV. speciosa transcriptome, a species that has biological and ecological relevance. The progressive accumulation of more information about the genomes of ferns and organisms from other basal branches of land plants will favor more accurate phylogenetic analyses [V. speciosa sporophyte transcriptome. Similarly, Sigel et al. [Monilophytes represent a phylogenetically important branch within the land plant lineage. However, there are few fern genomic studies, probably due to the huge sizes of fern genomes, especially in the case of leptosporangiate ferns (most ferns). In fact, there are only two complete fern genome assemblies, both belonging to two heterosporous ferns which have much smaller genomes  and someanalyses  as well analyses ,52,67,68analyses  analyzedanalyses , and frol et al.  also conA total of 5102 of the orthogroups (19%) were shared among the eight species. Of these, 101 were single-copy orthogroups, i.e., orthogroups with exactly one protein from each species . A totalV. speciosa (82%) were assigned to one of the 11,195 orthogroups  between analyzed . The resn mosses , but conn mosses . For exaichardii , 85% in aponicum  and 97.7amorphum . AdditioV. speciosa had an enriched number of transcripts. Among these orthogroups, transcripts representing genes encoding pentatricopeptide repeat-containing proteins were especially over-represented (1787), although they probably belong to a lower number of genes. Notwithstanding, Li et al. [Azolla (over 2000 PPR proteins) and Salvinia (over 1700 PPR proteins) genomes. They found that the large repertoire of PPRs correlates well with the extensive RNA editing observed in the organellar genomes of Salviniales [V. speciosa plastome [V. speciosa includes some other genes with interesting products, such as proteins involved in disease resistance, chromatin organization, several classes of transcription factors, different proteins controlling cell division, cell differentiation, cell wall formation, plant growth and plant development, proteins regulating dormancy or response to water deprivation, spliceosomal complex components or enzymes involved in lignin and mannan biosynthesis.We therefore focused our analysis specifically in those orthogroups for which i et al.  found thviniales , a phenoplastome . The lisA. thaliana transcriptome  against the V. speciosa transcriptome gave 456,516 significant hits at a 10−5 evalue cutoff  and 10,294 V. speciosa transcripts out of the 17,706 annotated transcripts of that species transcriptome (58.14%)—28.26%% if we consider all the 36,430 transcripts that form the whole V. speciosa transcriptome that we assembled as reference (annotated and with no known annotation). Such a large number of shared transcripts reflect the completeness of our transcriptome, whereas the transcripts of the A. thaliana transcriptome that do not appear in the V. speciosa transcriptome are very likely to be transcripts usually expressed in specific A. thaliana tissues, or developmental/physiological states not covered in our material. On the other hand, the transcripts present in our fern transcriptome but not in A. thaliana’s are probably fern specific genes. To that, we have to include the transcripts of proteins that have diverged too much as to be picked by our blast search. In fact, the mean percentage identity between the blast high-scoring parts of the sequences of both species is 47.68%, which reflects the evolutionary distance between both species.tBlastx of the e cutoff . When coV. speciosa. This is the first step towards understanding gene expression in a species that presents extreme ecological restrictions and biological particularities. Our study has unveiled outstanding clues about the transcriptomics of the adaptative response to hydric stress, the genetic control of sporophyte growth and development and the alternation between the vegetative and reproductive phases of this species. These results provide an important resource for further RNA sequencing studies of other fern transcriptomes, including that of the gametophyte of this species, to better understand the conservation genetics of ferns in general and, specifically, of V. speciosa.High-throughput RNA sequencing has emerged as a powerful tool for gene identification and gene expression analyses. In this work, we characterized and analyzed the transcriptome of the sporophyte (one of the two phases) of an endangered fern species,


Ultrasonic welding of thermoplastics is widely applied in automobile and aerospace industries. Increasing the weld area and avoiding thermal decomposition are contradictory factors in improving strength of ultrasonically welded polymers. In this study, relations among the loss modulus of carbon-fiber-reinforced polyamide 66 composite (CF/PA 66), time for obtaining stable weld area, and time for CF/PA 66 decomposition are investigated systematically. Then, a double-pulse ultrasonic welding process (DPUW) is proposed, and the temperature evolutions, morphologies and structures of fractured surfaces, and tensile and fatigue properties of the DPUWed joints are measured and assessed. Experimental results show the optimal welding parameters for DPUW include a weld time of 2.1 s for the first pulse, a cooling time of 12 s, and a weld time of 1.5 s for the second pulse. The DPUW process enlarged the weld area while avoided decomposition of CF/PA 66 under appropriate welding parameters. Compared to the single-pulse welded joint, the peak load, weld area, and endurance limit of the DPUWed joint increased by about 15%, 23% and 59%, respectively. DPUW also decreases the variance in strengths of the joints. Polymer composites play an increasingly significant role in automobile and aerospace industries due to low density, high specific strength, and corrosion resistance . JoiningIn ultrasonic welding of polymers without energy directors, the energy is not concentrated, and coulombic friction at the faying interface is less than that of the joint with energy directors. As a result, the workpieces need a longer time to melt, and the corresponding weld time is prolonged for UWed joints without energy directors ,8,9. HowIt is known that heat generation during ultrasonic welding depends on the loss modulus of polymer, while the loss modulus is closely related to temperature ,4,18. ThThe present study was undertaken to develop a method of double-pulse ultrasonic welding (DPUW) for improving the weld quality of carbon-fiber-reinforced polyamide 66. The mechanism of DPUW is evaluated thoroughly by analyzing the relations among the loss modulus of the material, time for obtaining stable weld area, and time for decomposition occurrence. The temperature evolution, peak load, and fatigue properties of the DPUWed joint are assessed systematically.3, and the injected CF/PA 66 exhibited a tensile strength of 99.2 MPa. During the ultrasonic welding process, a KZH 2026 welder with a nominal power of 2.6 kW, nominal frequency of 20 kHz, amplitude of 25 μm, and circular aluminum horn with a diameter of 18 mm were utilized. Time mode was chosen as welding mode.The material used in this research was carbon-fiber-reinforced polyamide 66 composite with 30 wt% fiber (CF/PA 66) , where the carbon fiber was about 2 mm in length with a diameter of 7 μm. All the coupons were injection-molded with dimensions of 132 × 38 × 4.0 mmTo analyze the weld formation during ultrasonic welding, temperature evolutions near the faying interface and in the middle of upper workpiece were measured. 3 were subjected to three-point bending with a span length of 20 mm. An oscillating force (≤4 N) was applied to give constant deflection amplitude of 30 μm. Measurements were conducted in the temperature range of 23–200 °C with a heating rate of 2 °C/min and fixed frequencies . The loss modulus at 20 kHz was extrapolated by the time temperature superposition. The cross-sectioned morphologies of the welds were characterized by scanning electron microscopy . All samples were sputter-coated with platinum for 50 s before SEM to induce conductivity. Chemical structures of the polymer were characterized using Fourier transform infrared spectroscopy (FTIR)  Perkin Elmer Spectrum One. Pellets made of CF/PA 66 powder were diluted in KBr, and FTIR spectra were recorded between 4000 and 400 cm−1. The powder was obtained by grinding in an agate mortar with a pestle under an infrared lamp. Shear tensile and fatigue tests were performed on an MTS 810 servohydraulic testing machine. To minimize the bending stresses inherent in the testing of single-lap weld specimens, filler plates were attached to both ends of the specimen using masking tape to accommodate the sample offset. The stroke rate was 2.0 mm/min for the tensile test, while the cyclic frequency was 30 Hz, and sinusoidal load cycles with a load ratio R = 0.1 were applied in fatigue test. Three replicates were tested, and the average values were reported for each welding condition.Loss modulus of CF/PA 66 was carried out by dynamic mechanical analysis . Specimens with dimensions of 38 × 8.5 × 4.0 mms and td, are also given in d is defined as the time when the workpiece starts to decompose and forms a porous area, while ts is the time when the joint obtains stable weld area. Numerous studies have shown that the weld area of the joints increases rapidly with weld time, and the weld area remains stable at a certain weld time [s, and the corresponding joint exhibits the highest strength. Further increasing the weld time, thermal decomposition emerges in the joint and the weld strength drops.There are two leading heating systems during ultrasonic welding of polymer composites: surface friction and viscoelastic dissipation. The loss modulus of CF/PA 66 composite is closely related to the condition of CF/PA 66, while the condition of CF/PA 66 rests on temperature. For ultrasonic welding process, the weld time only takes several seconds and the state transformation does not take place; therefore, the initial loss modulus at the very beginning of welding is identified as the loss modulus of CF/PA 66 during welding. The loss modulus of CF/PA 66 composite at 20 kHz (black curve) as a function of temperature is shown in eld time ,8,16. Ths and td are the results of extensive experiments (from preheating temperatures of 25 °C to 175 °C) [s and td decrease as the preheating temperature rises, and the peak values occur at approximately 75 °C, which is likely correlated with the preheating temperature and corresponding loss modulus of the workpiece. The decreases in ts and td are mainly because the preheating treatment provides energy for weld formation and workpiece decomposition. The peak and valley located at 75 °C are present because the maximum loss modulus of CF/PA 66 is at this temperature. The larger loss modulus leads to higher energy dissipation in the workpiece and relatively lower heat generation at the faying interface. Therefore, time for obtaining stable weld area prolongs, and the time for thermal decomposition shortens.t 175 °C) . Generals is smaller than td, implying the joint achieves its maximum weld area before thermal decomposition occurs in the joint. Accordingly, R1 and R3 are the appropriate regions, and ts is the proper weld time for joints with preheating temperatures ranging from 25–55 °C and from 95–145 °C. In regions R2 and R4, ts is larger than td, indicating the composite decomposes before the joint obtains stable weld area. Assuming the workpiece does not decompose, then the weld area of the joint is smaller, and the joint strength is lower [Based on the curves of time for stable weld area and time for CF/PA 66 decomposition in is lower . ConsequIn practical production, ultrasonic welding usually operates at room temperature, and the ambient temperature of 25 °C, which lies in R1, is selected for the first ultrasonic vibration. As shown in It takes about 8 s for the middle of the upper workpiece to cool to R3 region, as shown in Temperature histories for DPUWed joint at the faying interface and middle of the upper workpiece are measured, and the results are displayed in 2. After the applications of weld time for 2.9 s, preheating at 125 °C, and DPUW, the weld areas increase when compared to that of the joint welded with 2.1 s. Moreover, DPUWed joint exhibits increased peak load (higher by about 15%) and limited scatter, while the peak loads drop for the other two groups. These characteristics are probably related to the morphologies of the joints.−1 and N-H stretching at around 3300 and 3400 cm−1 decrease primarily, which indicates that these groups fracture, and the polymer decomposes severely [3, CO2, CO, which results in a fragile porous area and deteriorates the joint strength [Fractured surfaces of these joints are carefully examined and presented in severely ,24. The strength ,25.Similar to the joint with 2.9 s weld time, a porous area shows at the faying interface for the preheated joint, and peak load of the joint decreases. It is seen that extending the weld time and preheating treatment cannot enlarge the weld area significantly and improve weld quality. The weld area of the DPUWed joint increases by about 23% compared with that of the joint welded for 2.1 s. This phenomenon indicates the double-pulse ultrasonic welding process indeed increases the energy dissipation at the faying interface. In addition, overheating does not emerge, and no porous area occurs in the joint due to controlling the temperature distribution. These results indicate the selection of DPUW parameters based on the temperature in upper workpiece is reasonable. It is worth mentioning that the double-pulse ultrasonic welding process not only improves the weld quality but also decreases the variance in peak load, which is beneficial for producing continuous solid joints. This result is likely correlated with the contact condition at the faying interface. It is difficult to guarantee absolute flatness of the workpiece during manufacturing. Thus, the contacts between workpieces differ at the initial stage of welding. Surfaces with higher asperities usually generate more heat and obtain intimate contact between workpieces. By contrast, heat generation becomes limited for joints with loose contact and results in insufficient welds. However, the asperities suffer from melting and solidifying after application of the first ultrasonic pulse, which results in a relatively flat faying interface; thus, the second ultrasonic energy can be evenly distributed at the faying interface. Hence, the stable weld seam is obtained with the DPUW process and the spread in peak load narrows. The specific mechanism for the decreased scatter in double-pulse ultrasonic welding process will be further studied in future research.fN > 5 × 106. A decreasing trend in fatigue lives with increasing mean stresses is observed under all weld conditions. A power law relation between the S and fN is assumed in line with Basquin equation and can be expressed as [S is the stress, fN is the number of cycles to failure, and A and b are the fatigue strength coefficient and exponent, respectively. The approximated values of these coefficients, endurance limits, and correlation coefficients (R2) are summarized in 2 are close to 1, which shows good correspondence between tested and fitted results. The endurance limits of the joints mentioned above are 1.25 kN, 0.52 kN, 0.69 kN, and 1.99 kN. It is evident from The fatigue property of a joint is primarily important, because it inevitably suffers from complex and repeated mechanical loading during service. essed as ,27,28:S f), cooling time (Tc) and weld time of the second pulse (Ts). Three levels of 1.5 s, 2.1 s, 2.7 s for Tf; 6 s, 12 s, 28 s for Tc; and 0.9 s, 1.5 s, 2.1 s for Ts are adopted. Parametric values for each experimental run and the average peak load, weld area, and existence of an obvious porous area are given in f of 2.7 s irrespective of the cooling time and second weld time. This characteristic indicates that the weld time for the first ultrasonic pulse should be shorter than 2.7 s, and weld time of 1.5 s and 2.1 s are appropriate. It is seen from samples 12 to 18 that peak loads of joints with cooling intervals of 12 s are higher than those with cooling intervals of 6 s and 28 s. Combined with the temperature evolutions in To validate the correctness of the above analysis and results, experiments with three process parameters with three levels are conducted. The process parameters are weld time of the first pulse (T2, is 0.976, indicating the fitting is reliable. The relation can be described as:Normally, the peak load of a joint is proportional to the weld area for joint without porous area ,19,21,29Increasing the weld area and avoiding thermal decomposition are contradictory factors in improving strength of ultrasonically welded CF/PA 66. Ultrasonic welding of CF/PA 66 with different preheating temperatures was investigated thoroughly. The time for obtaining stable weld area and time for decomposition of CF/PA 66 decreased with increasing preheating temperature except for temperature around 75 °C. The occurrences of peak and valley values were mainly because the maximum loss modulus of CF/PA 66 was at this temperature. The proper temperature range for ultrasonic welding was R1 and R3, and the optimum temperature before application of the second ultrasonic pulse lay in the R3 region, i.e., 95–145 °C.A method of double-pulse ultrasonic welding of CF/PA 66 is proposed based on the relations among loss modulus of CF/PA 66, time for obtaining stable weld area and time for material decomposition. The appropriate welding parameters for DPUW include a weld time of 2.1 s for the first pulse, 12 s cooling, and a weld time of 1.5 s for the second pulse. Results show that the DPUW process enlarged the weld area and avoided decomposition of CF/PA 66 in the meantime. Compared with welds made with prolonged weld time and preheat treatment, DPUWed weld exhibits higher peak load, larger weld area, more compact microstructure, and better fatigue property. The DPUW schedule not only improves the weld quality of ultrasonically weld CF/PA 66 but also decreases the spread in tensile and fatigue properties.


Background: Positive airway pressure can be an effective and safe therapy for children with obstructive sleep apnea syndrome (OSAS). Few studies have assessed the safety and efficacy of autoCPAP in pediatric patients with obesity. Methods: This was a retrospective chart review of children with obesity (Body Mass Index (BMI) > 99th percentile), ages 2–18, diagnosed with OSAS (Obstructive Apnea-Hypopnea Index (OAHI) > 1/h) and used autoCPAP with 30-day adherence. Exclusion criteria included patients with complex comorbidities. Adherence was defined as autoCPAP use ≥4 h/night for at least 21/30 days. Baseline PSG OAHI was compared to the AHI from the 30-day autoCPAP compliance report. We also compared autoCPAP 30-day 95th percentile pressures with the pressures from PAP titration. Results: The study included 19 children, ranging 5–15 years old. The median BMI was 99.6th percentile and average adherence was 25/30 nights with mean of 7.3 h/night. The median OAHI was 12.3/h on baseline PSG and the 30-day autoCPAP download AHI decreased to 1.7/h. No adverse outcomes were identified. The average difference between 95th percentile autoCPAP pressure and PAP titration pressure was 0.89 cmH20. Conclusion: Our study suggests autoCPAP is effective and safe for the treatment of OSAS in pediatric patients with obesity. Using autoCPAP may reduce delays in treatment. Additional research is needed to verify the long-term effectiveness of autoCPAP in this population. Obstructive sleep apnea syndrome (OSAS) affects an estimated 2–4% of the general pediatric population . The cauIt is important to ensure that those patients who are already affected with obesity and OSAS are adequately screened, diagnosed and treated in order to maintain good health and avoid any of the associated disease. Untreated OSAS in children can lead to neurocognitive issues, behavior problems, difficulties in school, poor growth and increased cardiovascular risks ,8,9,10. The recommended first-line treatment for pediatric patients is tonsillectomy and adenoidectomy (T&A) in patients with adenoid and tonsillar hypertrophy ; howeverAutoCPAP is widely used in the adult population in place of in-laboratory PSG titration studies and has been shown to effectively decrease (Apnea-Hypopnea Index) AHI and improve symptoms related to OSA. It can provide variable pressure delivery to accommodate detected changes in flow limitation in the upper airway ,22. The A retrospective chart review was performed for children with obesity using autoCPAP at Valley Children’s Hospital between May 2016 to April 2021 (four patients before 2018 and the remaining from 2019 to present). The study was approved by the Valley Children’s Hospital institutional review board.Inclusion criteria were as follows: patients with OSAS and obesity, ages 2 to 18 years without significant comorbidities. OSAS was defined as an obstructive apnea-hypopnea index (OAHI) with more than one event per hour, obtained by in-lab diagnostic polysomnography using SomnoStar sleep system (PSG). PSG was scored, based on the most recent American Academy of Sleep Medicine (AASM) scoring rules. Obesity was defined as body mass index (BMI) at or above the 95th percentile. Patients were also required to have autoCPAP use greater than or equal to four hours per night for greater than or equal to 21 days out of a total 30-day period. The adherence criteria was based on adult health-insurance guidelines for compliance, which is defined as use of CPAP greater than or equal to four hours per night for greater than 70% of a given time frame (21 days out of a 30-day period). Pediatric adherence criteria are not established. Exclusion criteria were patients with complex comorbidities, including unrepaired congenital heart disease, chromosomal anomalies, and neuromuscular disorders.We then accessed the Cloud-based PAP compliance and therapy monitoring software from ResMed AirView to determine which patients were compliant to therapy for at least a 30-day period, using the criteria for adherence noted above. Among patients who had been adherent to therapy, the baseline PSG, PAP titration study and medical records were reviewed. All patients that met criteria were included in the study.We compared baseline polysomnography OAHI with the autoCPAP derived 30-day mean OAHI, reported via the cloud-based therapy data system. In order to compare the autoCPAP 95th percentile pressure to the in-lab PSG titration recommended pressure, we determined the mean difference between the autoCPAP-derived 95th percentile pressure and the PSG PAP titration pressure.There were 19 children who met the criteria aged from 5 to 15 years old with adherent autoCPAP use. The median BMI was in the 99.6th percentile. There were 15 patients that met criteria for severe obesity with BMI greater than or equal to the 99th percentile. Average autoCPAP use was 7.3 h per night, for an average of 25 days in a 30-day period. Each patient’s OAHI was obtained from their initial baseline polysomnography and compared to their autoCPAP 30-day compliance and therapy download. The comparison for these values can be seen in The cloud-based PAP compliance and therapy monitoring software-downloaded reports included the 95th percentile pressure from the patients’ autoCPAP use for 30 days. This value indicates that 95% of the time spent on autoCPAP is at this pressure or below. These pressures were compared to the PAP pressure settings recommended from the in-lab PSG PAP titration  for thosThe results from this study demonstrate that the use of autoCPAP among children with OSAS and obesity can reduce the number of obstructive apnea–hypopnea events. OSAS was defined as an OAHI of more than 1 event per hour, per the International Classification of Sleep Disorders by the American Academy of Sleep Medicine . Our stuGiven the growing prevalence of obesity among the pediatric population, this is a population that will need more and more attention. Effectively treating OSAS in pediatric patients with obesity should be considered as part of the overall medical treatment plan for these patients. In adult patients with OSAS and obesity, there is a correlation in the reduction of visceral fat  after CPAP treatment . While tTreatment with autoCPAP for OSAS can provide a wide range of pressures to facilitate adjustments for growth in children, weight changes, increase upper airway resistance against allergies and respiratory infections, and positional changes . The accAn abstract by DelRosso et al. found that autoCPAP use was well tolerated in 11 pediatric obese patients with adherent days of use ranging (use of 4+ h a night) from 53–100%, with an average of 5.5 h per night . In our Our study demonstrates that autoCPAP-derived pressures correlate with the titration PSG-derived pressures. The recommended PAP pressures obtained during in-lab PAP titration PSG were ≤2.1 (with six of the patients having ≤1 cmH20) pressure with a mean difference of 0.89 (0.1–2.1) when compared to the 95th percentile obtained on autoCPAP therapy download. Other studies in children have shown similar results, demonstrating a strong correlation between autoCPAP pressures and in-lab PSG PAP titration derived pressures ,24.Currently, the standard approach to starting children on CPAP therapy is to perform an in-lab PSG PAP titration study and then to prescribe a set CPAP pressure based on titration results . In-lab There were no adverse outcomes or side effects identified upon chart review during our study. This suggests that autoCPAP treatment can be both safe and effective when treating even severe OSAS in pediatric patients with obesity. Safety of autoCPAP use in the pediatric population has been established by previous studies ,24, in aLimitations of the study include that this is a retrospective study, and that the participant group is small. Some study subjects were excluded based on significant comorbidities, which makes this study useful for obese pediatric OSA patients without other complex health issues. We also studied compliance and therapy data for a 30-day period only, and did not study long term benefits and adherence. This study is also limited as it did not include patient’s satisfaction. A future study should not only include effectiveness but should also include patient’s comfort and satisfaction. Identifying barriers for adherent autoCPAP use in obese pediatric patients would be an important clinical problem to address in future studies.AutoCPAP use by pediatric patients with OSAS and obesity was associated with a significant reduction in OAHI in this study. The use of autoCPAP appears safe and tolerated in this population. There is also concordance between the optimal pressures obtained from the in-lab PSG PAP titration study to the autoCPAP 95th percentile pressure. Long wait times for PSG evaluation and subsequent PAP titration studies can delay the diagnosis and management of OSAS, which can increase patient risk for associated sequelae. AutoCPAP use can mitigate these issues. Additional research is needed to establish the long-term effectiveness, safety, and tolerability of autoCPAP in children with obesity.


Bio-effects following thermal treatments are a function of the achieved temperature profile in tissue, which can be estimated across tumor volumes with real-time MRI thermometry (MRIT). Here, we report on expansion of a previously developed small-animal microwave hyperthermia system integrated with MRIT for delivering thermal ablation to subcutaneously implanted tumors in mice.ex-vivo tissues were heated to temperatures in the range 47–67 °C with custom-made microwave applicators for validating MRIT with the proton resonance frequency shift method against fiberoptic thermometry. HAC15 tumors implanted in nude mice (n = 6) were ablated in vivo and monitored with MRIT in multiple planes. One day post ablation, animals were euthanized, and excised tumors were processed for viability assessment.Computational models were employed to assess suitability of the 2.45 GHz microwave applicators for delivering ablation to subcutaneous tumor targets in mice. Phantoms and ex-vivo ablations. During in-vivo experiments, tumors with volumes ranging between 5.4–35.9 mm3 (mean 14.2 mm3) were ablated (duration: 103–150 s) to achieve 55 °C at the tumor boundary. Thermal doses ≥240 CEM43 were achieved across 90.7–98.0% of tumor volumes for four cases. Ablations were incomplete for remaining cases, attributed to motion-affected thermometry. Thermal dose-based ablative tumor coverage agreed with viability assessment of excised tumors.Average absolute error between temperatures from fiberoptic sensors and MRIT was 0.6 °C across all in-vivo.We have developed a system for delivering microwave ablation to subcutaneous tumors in small animals under MRIT guidance and demonstrated its performance  Small animal tumor models are common and widely accepted as important tools for the evaluation of various therapeutic approaches –4. Thermvia a percutaneous approach [a ~1.5–2 mm device results in a substantial fraction of the tumor volume affected by mechanical trauma. A limitation of most studies is the lack of information regarding the thermal profiles in tissues, with few studies reporting temperature data measured or anticipated within tumors and surrounding tissue. Knowledge of thermal dose delivered to tumor and adjacent tissue may be especially important for studies investigating combination of ablation with other therapeutic modalities [While there are several studies employing microwave ablation (MWA) in small animal tumor targets, there are few reports describing technical details about the instrumentation used for delivering heating, which would be important for characterizing the power absorption and thermal profiles. Since MWA devices designed for ablation of human tumors typically yield >15 mm long ablation zones , due to approach –22, or iapproach –25. For dalities .et al reported on a broadband planar coaxial microwave applicator [et al designed a 1.2 mm diameter dual slot antenna operating at 8 GHz with the goal of achieving spherical ablation zones for treatment of ~1 cm diameter tumors in mice [Some studies have described the design and characterization of thermal ablation devices specific for use in experimental studies in small animals. Yoon plicator  which wa in mice , with th in mice  and ultr in mice .in vivo small-animal investigations. Similarly, MR thermometry with the proposed sequence was not previously validated for ablative heating at high-fields; since data acquired with this system will be used to quantitatively assess thermal profiles in future studies, we believe it is important to benchmark thermometry accuracy, even in the event that the accuracy is similar to that at lower temperature ranges.Our group has previously developed a system for MRI thermometry (MRIT) guided delivery of microwave-induced hyperthermia to experimental small animals ,31. The ex vivo tissue and 3) to demonstrate use of the system for delivering ablation to subcutaneous HAC15 tumors in nude mice in vivo.The objectives of this work were 1) to expand the previously developed system for MRIT guided hyperthermia for the thermal ablation studies in small animals and characterize thermal profiles, 2) to validate the accuracy of volumetric temperature readings from MRIT at 14.1 T by comparison with temperature data from fiber optic sensors during heating in phantom and via Paravision 6.01 software. Specific details of imaging sequences, which were used to acquire anatomic images of ex vivo and in vivo samples are provided in sections ‘System integration and evaluation in tissue phantoms and ex vivo tissue’ and ‘In Vivo heating procedure’, respectively.The small-animal ablation system is comprised of a custom 2.45 GHz microwave ablation platform integrated with a high-field small-animal MRI scanner. The overall schematic of the system is shown in via ethernet cable to a monitoring and control computer with Thermoguidetm software , where phase images are used to estimate transient temperature profiles with the proton resonance frequency shift (PRFS) technique [ex vivo tissue’. Since the PRFS technique provides estimates of relative change in temperature, rather than absolute temperature, a fiber-optic temperature measurement probe was utilized to measure the core temperature of the small animal prior to heating, and thus serve as the baseline temperature.During microwave heating exposure, acquired magnitude and phase images are transmitted echnique ,35. ImagThe microwave heating platform consists of a solid-state microwave generator , MRI compatible directional microwave applicator, and a power meter  for monitoring transmitted and reflected power. A 4 ft long RG393 coaxial cable, with approximate attenuation ~0.12 dB/ft at 2.45 GHz, was used to connect the generator to the power meter, and 6 ft long RG400 coaxial cable, with approximate attenuation ~0.22 dB/ft at 2.45 GHz (Pasternack RF), was used to connect the power meter to the applicator. Details of utilized MWA applicator are provided in the following section. A peristaltic pump (Masterflex L/S 7015–20) was used to circulate water through the applicator from a temperature-controlled water reservoir.We investigated the feasibility of delivering microwave ablation to subcutaneously implanted experimental tumors in small-animals using a water-cooled microwave applicator previously developed for mild-hyperthermia applications , 31. Theex vivo chicken breast and compared temperature changes from MRIT against fiber optic measurements. Even though the temperature profiles in phantoms and ex vivo tissues are expected to be different from heating profiles in tumor in vivo (due to the lack of blood flow and heterogeneities), these experiments are informative to assess the system integration and MRIT monitoring accuracy ahead of in vivo studies. Transient temperature profiles in phantoms as well as ex vivo samples were measured with the use of the proton resonance frequency shift technique during microwave heating. We used a FLASH protocol, with TR = 30.1 ms, TE = 4 ms, FA = 15°, FOV = 30 × 30 mm2, slice thickness = 1 mm, three axial slices, and in-plane pixel size 0.2344 × 0.2344 mm2.The accuracy of MRIT with the 14.1 T high-field scanner at temperatures in the range 40–44 °C was previously found to be on the order of 0.4 °C . Temperaex vivo chicken breast samples. The sample is loaded from the top  under protocol number 4451. A mouse model of aldosterone producing adenoma was prepared by injecting six million HAC15 cells  subcutaneously in the right hind flank of nude mice . Starting at day seven post-injection, the 2-dimensional tumor size was estimated using calipers. When the tumor size reached 5 mm in one direction, the mice were treated. A total of six nude mice were selected for heating procedures. On the day of experiment, the mice were anesthetized using isoflurane  and maintained under anesthesia for the duration of the treatment.h = 200 W/m2/K and external temperature 20 °C.To evaluate the suitability of the directional microwave applicator for heating of subcutaneous tumors, observed tumor shapes and sizes as illustrated in For the time stepping scheme, we used low order (maximum 2) backward differentiating formula (BDF) with relative tolerance within time domain solver was set to 0.005 to aid the convergence of numerical computations. We used a free tetrahedral mesh with the smallest element size of 0.1 mm around the input port to the applicator, gradually increasing the size to 0.25 mm around the applicator shaft and allowing the element size to be gradually increasing up to 3 mm for space more than 1 cm away from the applicator shaft. These parameters were chosen based on the result-convergence method, where the changes in simulated temperature and electric profiles were monitored for simulations with decreasing mesh size and relative tolerance and the parameters resulting in output changes smaller than 1% of initial differences were employed.ex vivo liver, due to lack of data on HAC15 tumors in literature, and liver being commonly accepted as a surrogate tissue for performance evaluation of ablation devices. Baseline properties of tissue were taken from IT’IS tissue database [The tissue block around the applicator was modeled with dielectric and thermal properties of database , temperadatabase , and temdatabase . The ablvia MRIT.After inducing anesthesia, the mice were placed in an acrylic fixture which allows for positioning of the small animal, temperature sensor, breathing mask, and microwave applicator in a defined relationship within MRI probe, which is to be loaded to the 14.1 T MRI scanner. The microwave applicator was then positioned on the skin of the mouse next to the subcutaneous HAC15 tumor and fixed with the tape such that the tumor was in the direction of the applicator’s heating pattern. A rectal thermocouple probe  was used to capture the core temperature of the mouse just before starting the heating procedure to allow absolute temperature measurements ex vivo samples .Once the MRI probe was loaded in the scanner, initial T2-TurboRARE scans were obtained in the axial, sagittal and coronal view to verify the position of the microwave applicator with respect to the tumor as shown in Prior to the initiation of microwave heating, we selected several points along the tumor boundary as regions of interest (ROI), where temperature was monitored in real time. Four points were assigned along the tumor boundary in each of the 3 slices shown in As can be seen in ex vivo tissue. Heating times were in range of 4–5 min with an average 4.58 min. The update time between subsequent images was 3.86 s based on used sequence parameters. The results of MRIT validation in agar phantom and chicken breast are shown in To validate the MRI thermometry, we performed three heating procedures in agar phantom and another three procedures in ex vivo tissue, peak temperatures of 67 °C were observed, with average mean absolute error (MAE) between fiber-optic thermometry and MRI of 0.86 °C across all samples. During heating phantoms and ex vivo experiments.Simulations predicted that the full coverage of tumors with minimum thermal damage 240 CEM43 is attainable in each tumor case in less than 140 s for 30 W and less than 66 s for 50 W input power. Furthermore, comparison between shape of simulated ablation zone and target subcutaneous tumor shapes from anatomical MRI scans at the time of full tumor ablation yielded average DSC 0.65 ranging from 0.48 to 0.77 for 30 W and average DSC 0.62 with range 0.53–0.72 for 50 W input power. DSC value of 0.7 was previously suggested as an indication of conformity between target shape and thermal contour approximating ablation . The ranex vivo liver to assess general ablation shape. The actual heating profiles might be even more favorable for targeted tumor heating in terms of shape similarity, because of possible contrast in dielectric and thermal properties between tumor and fat, where more microwave energy is deposited in volumes with higher dielectric properties  and heat is preserved in tumor area due to thermal insulation of surrounding fat layer.However, MR images as well as visual assessment after tumor extraction following euthanasia indicated tumors were exclusively surrounded by a layer of fat, which is not a critical structure and can tolerate thermal damage without safety concerns. Moreover, since the target tumor biophysical properties are unknown, simulations were performed in homogenous ex vivo chicken breast heating demonstrated the ability of system to heat tissue in regions up to 12 mm from the surface of the applicator to ablative temperatures . In one case of chicken breast tissue  of the tumor volume  was heated to thermal doses exceeding 240 CEM43. In one experiment temperatures recorded over 250 s did not exceed 50.5 °C. In another experiment, MR thermometry data were unusable because of animal movement leading to substantial motion artifacts. In the five experiments, where usable thermometry data was available, thermal dose-based assessment of ablative coverage was in agreement with viability assessment based on TTC staining.During 5 out of 6 in vivo scenario, for instance the dielectric and thermal properties of this specific tumor type are not known. Nevertheless, the model is instructive for characterizing the approximate thermal profiles in soft tissue and thereby provide complementary information to the temperature profiles acquired during MR thermometry. While we have developed a suitable antenna for ablation of small tumor targets and characterized its performance, it is possible, that other applicator designs with different radiation patterns might also be appropriate for ablation of small tumor targets, and characterization of such devices is warranted ahead of their use in experimental studies.A limitation of the presented system is that it remains susceptible to large errors in temperature estimation due to motion. Presently, motion is managed by appropriately securing the animal within the imaging coil. Future studies may incorporate motion compensation techniques to further improve the robustness of MRI thermometry. We note that the utilized computational model is not a perfect representation of the ex vivo tissue, with an average mean absolute error of 0.86 °C across all samples. The system was further applied for in vivo heating of subcutaneously implanted HAC15 tumors in nude mice, where the ability to monitor temperatures at the tumor boundary allowed us to terminate the heating at a time specific to the geometry of each targeted tumor, and therefore to deliver target specific energy.We have developed a system for delivering microwave ablation to subcutaneous tumors in small animals integrated with MRI for real-time volumetric thermometry. We verified the accuracy of MRI-derived temperature measurements in agar phantom and Supplementary material