The HOX11/TLX1 (hereafter referred to as HOX11) homeobox gene was originally identified at a t translocation breakpoint, a chromosomal abnormality observed in 5-7% of T cell acute lymphoblastic leukemias (T-ALLs). We previously reported a predisposition to aberrant spindle assembly checkpoint arrest and heightened incidences of chromosome missegregation in HOX11-overexpressing B lymphocytes following exposure to spindle poisons. The purpose of the current study was to evaluate cell cycle specific expression of HOX11.Cell cycle specific expression studies revealed a phosphorylated form of HOX11 detectable only in the mitotic fraction of cells after treatment with inhibitors to arrest cells at different stages of the cell cycle. Mutational analyses revealed phosphorylation on threonine-247 (Thr247), a conserved amino acid that defines the HOX11 gene family and is integral for the association with DNA binding elements. The effect of HOX11 phosphorylation on its ability to modulate expression of the downstream target, cyclin B1, was tested. A HOX11 mutant in which Thr247 was substituted with glutamic acid (HOX11 T247E), thereby mimicking a constitutively phosphorylated HOX11 isoform, was unable to bind the cyclin B1 promoter or enhance levels of the cyclin B1 protein. Expression of the wildtype HOX11 was associated with accelerated progression through the G2/M phase of the cell cycle, impaired synchronization in prometaphase and reduced apoptosis whereas expression of the HOX11 T247E mutant restored cell cycle kinetics, the spindle checkpoint and apoptosis.Our results demonstrate that the transcriptional activity of HOX11 is regulated by phosphorylation of Thr247 in a cell cycle-specific manner and that this phosphorylation modulates the expression of the target gene, cyclin B1. Since it is likely that Thr247 phosphorylation regulates DNA binding activity to multiple HOX11 target sequences, it is conceivable that phosphorylation functions to regulate the expression of HOX11 target genes involved in the control of the mitotic spindle checkpoint. Given telopment , consistd TCRγδ- . Dysregud TCRγδ- . Additiocultures . The tracultures -15. Addicultures ,17.HOX11 in inducing a tumorigenic state remain unclear, although it is thought that the transactivation of specific downstream genes by HOX11 is responsible, at least in part, for cellular transformation. One-hybrid experiments have elucidated three regions of the HOX11 oncoprotein which are important for optimal transactivation of reporter constructs: an N-terminal glycine/proline rich region, a C-terminal glutamine-rich region, and the homeodomain [HOX11/TLX gene family [HOX11 consensus recognition motif to TAAGTG [Aldh1) [The molecular mechanisms of action of eodomain . A threoeodomain ,20), as  [Aldh1) ,21.Aldh1 [A comprehensive structure-function analysis of the HOX11 protein revealed that the transcriptional transactivation of genes specified by the Thr247 residue of HOX11 was distinct from its immortalizing function, as assayed by the ability to immortalize hematopoietic precursors and generate IL3-dependent myeloid-like cell lines . MutatioAldh1 , but wasAldh1 . Thus, tHOX11 overexpression conferred an ability to aberrantly bypass G2/M cell cycle checkpoint arrest induced by gamma-irradiation [HOX11 exhibit aberrant expression of genes involved in the regulation of the G1/S cell cycle checkpoint, including E2F, c-Myc, and the cAMP-responsive element binding protein (CREBP) [HOX11 demonstrated elevated expression of the cell cycle checkpoint regulators, NFKB2 and SMARCD3 [HOX11 to promote bypass of mitotic checkpoint arrest, leading to chromosome missegregation and aneuploidy [HOX11 overexpression, and may represent a critical mechanism by which HOX11 elicits lymphoma.Several reports have implicated HOX11 in disruptions of various cell cycle checkpoints. In Jurkat T cells, adiation . High th (CREBP) . An inde SMARCD3 . Our andeuploidy -29. ThisGiven the multiple defects in cell cycle regulation subsequent to HOX11 overexpression in several different cellular models, we investigated the expression of the HOX11 protein at various stages of the cell cycle. Surprisingly, we detected a hyperphosphorylation of the HOX11 oncoprotein on the Thr247 residue, which was specific to mitotic cells. The implications of this post-translational modification with respects to cyclin B1 transactivation and mitotic spindle checkpoint were explored.FLAG-tagged wildtype and truncated HOX11 proteins were generated by subcloning polymerase chain reaction (PCR)-amplified fragments, corresponding to the following HOX11 regions in frame with the sequence encoding a FLAG peptide into the HindIII/NotI restriction sites of the p3XFLAG-CMV-7.1 vector : HOX11Δ260 (amino acids 1-260), HOX11Δ187 (amino acids 1-187) and HOX11Δ106 (amino acids 1-106). HOX11 Thr247Ala, Thr247Glu and Thr241Ala were generated using the QuikChange Site-Directed Mutagenesis Kit  prior to cloning as described above. Luciferase reporter constructs were generated by subcloning PCR-amplified fragments corresponding to the following promoter regions upstream of the luciferase reporter gene into the KpnI/HindIII restriction site of the pGL3 vector : CCNB1L-LUC (contains -4902 to +21 relative to TSS of murine cyclin B1 (CCNB1) gene) and CCNB1S-LUC (contains -3310 to +21 relative to TSS of murine cyclin B1 (CCNB1) gene). All constructs were verified by DNA sequencing.6 cells/ml. Relative levels of the HOX11 protein in the different cell lines is shown in Additional file NIH 3T3 fibroblasts stably expressing the HOX11 oncoprotein were generated by transfection of NIH 3T3 cultures with 3 μg MSCV-HOX11 plasmid constructs using the Polyfect Transfection reagent (QIAGEN), and selecting with 800 μg/ml G418 (Sigma) for 14 days. Media changes supplemented with fresh G418 were performed every two days. Following 14 days of selection, 24 G418-resistant colonies were picked, expanded and tested for HOX11 positivity by Western immunoblotting with a HOX11-specific antibody . HOX11-3T3 fibroblasts were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FCS, 2 mM L-glutamine and 1% penicillin/streptomycin. The T cell leukemia cell lines Jurkat  and ALL-Nocodazole treatment was performed 24 hours after seeding, on typically ~60-80% confluent cultures. Phosphoenriched lysates following nocodazole treatment were generated using the PhosphoCruz Protein Purification System (Santa Cruz) according to manufacturer's protocols. Staurosporine (Sigma) was added in conjunction with nocodazole at concentrations of 0, 20, 40, 60, 80 and 100 nM. A dose-response experiment using staurosporine concentrations from 0.05 μM to 500 μM indicated drug concentrations did not impact cell viability  according to manufacturer's protocols. Immunoblotting analysis was performed on SDS-separated proteins blotted to nitrocellulose membrane, and probed at various dilutions with a primary antibody recognizing HOX11 , FLAG , pSer (1:100), pThr (1:100), PP1 (1:800), PP2A  and β-actin  for 4 h at room temperature, followed by three washes with PBST for 20 min each. Membranes were subsequently incubated with either anti-mouse  or anti-rabbit  antibody conjugated to horseradish peroxidase (HRP) for 1 h at room temperature. The anti-pSer and anti-pThr antibodies were obtained from Anaspec, the anti-FLAG antibody was obtained from Sigma. All other antibodies were obtained from Santa Cruz Biotechnologies .Chromatin immunoprecipitations were performed using the Chromatin Immunoprecipitation Kit (Upstate) and a HOX11-specific antibody, clone 1D7,  according to manufacturer's protocols. Primers used for ChIP PCR analyses were:CYCB1-1F: ACTTCATGCTATCAACCTCA; CYCB1-1R: CCTCTTCTATAGAAGTGCCA;CYCB1-2F: CCGCGTTAGTGTTACTGAAA; CYCB1-2R: CAAAGATCTCTATTGCAACTCT; CYCB1-3F: TGTAACCTCAACAATGAGGA; CYCB1-3R: ACACCATCCTGTGCTCTCTA;CYCB1-4F: CAGTCTCCGGTGTGACATAA; CYCB1-4R: GAGACAGGGTTTCTCTGTGT;CYCB1-5F: AACAAGTTCCAGCCTCCACA; CYCB1-5R: CCCATTACCAAGCTAGAGAG;CYCB1-6F: AGGAACAGCCAGAGCTGTTT; CYCB1-6R: AAATGCCAATGGTCTCCCTG.NIH 3T3 fibroblasts were seeded in 24-well dishes 24 hours prior to transfection. Cells were transfected with 0.15 μg FLAG-HOX11 construct and 0.15 μg promoter-reporter construct using the Polyfect transfection reagent (QIAGEN). Each transfection was performed in triplicate. Luciferase assays were performed using the Luciferase Reagent Kit (Promega), according to manufacturer's protocols. Briefly, cells were lysed by addition of 200 μL CCNR lysis buffer, cleared by centrifugation and 20 μL protein lysate was combined with 100 μL luciferase reagent immediately prior to measurement in the luminometer. For each sample, arbitrary luciferase units were standardized to protein concentrations.Nuclear extracts were derived from Jurkat cell lines stably expressing an empty Flag vector, Flag-HOX11-wt, Flag-HOX11-T247A or Flag-HOX11-T247E. The oligonucleotide probes derived from the CCNB1 promoter and mutant probe were as follows: 5'-TCCATCCCAGTAATAAGTGTTTT-3' and 5'-TCCATCCCAGTAAGGGCCCTTTT-3'. Double-stranded oligonucleotide probes were generated by annealing equimolar quantities of complementary oligonucleotide in 1× annealing buffer . Probes were incubated in 1× binding buffer  with 6 μg nuclear extract in a final volume of 10 μl.The samples were incubated at room temperature for 30 min and resolved by electrophoresis in 6% polyacrylamide nondenaturing gels in 0.5 × TBE at 100 voltage for 30 min. Following electrophoresis, the gels were stained using an EMSA staining kit  and photographed using a 300 nm UV transilluminator.Cells were synchronized at the G1/S checkpoint by culturing for 16 h in 100 μg/ml aphidocolin, released into S phase by feeding with fresh medium containing 10% fetal calf serum for 4 h, arrested at G2/M by culturing with 60 μg/ml genistein for 16 h, or trapped in mitosis by using 400 μg/ml colchicine, 50 nM or 100 nM nocodazole  or 3 μg/ml cytochalasin B (for cytokinesis arrest following telophase) for 16 h. Cell cycle distribution was determined by propidium iodide staining and flow cytometry. For each sample, 20,000 events were recorded.Cells were co-transfected with a pmax GFP-expressing vector (Amaxa) and either an empty vector, a vector expressing a wildtype HOX11 or a vector expressing the HOX11Thr247Glu mutant. After 24 h, cells were synchronized in mitosis by a 17 h exposure to colchicine (40 ng/ml) or (10 ng/ml) and stained with Hoechst 33342 (10 μM) in 2% FCS/PBS in 37°C for 10 min. Mitotic indexes of transfected cells were quantified using immunofluorescent microscopy by scoring the nuclear morphology of 200 GFP-expressing cells, with bright-stained nuclei corresponding to prometaphase arrested cells and dull or diffuse-stained nuclei corresponding to cells that had escaped mitotic arrest. Cells were viewed with an Axiovert 200 M epi-fluorescence microscope using Axiovision Rel. 4.5 software . Each experiment was replicated three times.Cell death was monitored by flow cytometric analysis of cells stained by Annexin V (Biovision ) and propidium iodine (PI) (Invitrogen), as per the manufacturer's protocol. 30,000 cells were analyzed.To assess HOX11 expression levels at various stages of the cell cycle, fibroblast cell lines stably expressing the HOX11 oncoprotein (HOX11-3T3) were treated with various inhibitors to induce cell cycle arrest at different stages. HOX11-3T3 cells were synchronized at the G1/S checkpoint by culturing for 16 h in aphidocolin and released into S phase by feeding with fresh medium for 4 h, arrested at G2/M by culturing with genistein for 16 h, blocked in mitosis by exposure to colchicine or nocodazole for 16 h or arrested during cytokinesis following telophase with cytochalasin B for 16 h. While no dramatic cell cycle-related fluctuations in HOX11 expression levels were detected by immunoblotting, we observed the presence of a gel retarded cross-reacting band when HOX11-3T3 cultures were treated with colchicine or nocodazole Figure , but notThe lower mobility band was detected in a nocodazole dose-dependent manner Figure  and in ain vitro with increasing amounts of alkaline phosphatase for 1 h at 37°C. Western immunoblotting of the entire reaction confirmed the disappearance of the upper HOX11 band upon alkaline phosphatase treatment  diminished HOX11 phosphorylation following nocodazole treatment, indicating that HOX11 was being phosphorylated on threonine-247  with respect to potential HOX11 binding sites. Chromatin immunoprecipitations (ChIPs) were performed to detect direct HOX11 protein association with putative promoter regions up to 5 kb upstream of the transcription start site (TSS) of the CCNB1 locus. Six different regions at intervals of 700-1000 bp upstream of the TSS were analyzed. Whereas there was minimal amplification of any of the six regions in NIH 3T3 cells not expressing HOX11, we detected HOX11-binding to four of six regions upstream of the CCNB1 gene, with the strongest spanning a region of DNA approximately -4500 to -4300 from the TSS  and CCNB1S-LUC (Short), respectively. NIH3T3 cells were cotransfected with a wildtype HOX11 cDNA and either an empty reporter construct or constructs containing CCNB1S-LUC or CCNB1L-LUC and luciferase activity was measured following 24 hours.In addition to association with the promoter, we assessed the ability of HOX11 to transcriptionally transactivate the CCNB1 TSS.Transfection of the empty vector in the absence of HOX11 showed background levels of luciferase activity whereas cotransfection of the CCNB1S-LUC reporter construct with a vector expressing the wild type HOX11 cDNA resulted in a three-fold increase in luciferase activity , demonstrating that the CCNB1 regulatory elements in the presence of HOX11 were active in NIH 3T3 cells. Cotransfection of the CCNB1L-LUC construct with wild type HOX11 significantly enhanced luciferase activity  Figure . Thus, HCCNB1 promoter whereas the HOX11 T247A mutant showed similar association with the CCNB1 promoter as wild type HOX11 , a frequently used technique to partially mimic the negative charge introduced by a phosphate group. This approach has been used to simulate a phosphorylation-induced alteration of DNA binding activity of other homeodomain-containing proteins ,33. West1 Figure . EMSA an1 Figure . In cont+ Jurkat T-ALL cell line [Given the role of HOX11 in T lymphoid diseases, we sought to determine whether this phenomenon could be recapitulated in a T cell system. Using the T-ALL cell line, ALL-SIL, which carries a t translocation concomitant with high levels of HOX11 expression  and the ell line  transfecWe previously reported that dysregulated expression of HOX11 contributed to the development of lymphoma by allowing cells with missegregated chromosomes to aberrantly bypass the spindle checkpoint although it remained unclear whether this effect was dependent on the Thr247 residue ,29. TherOf note, studies assessing cell death by flow cytometry of annexin V and propidium iodide stained cells indicated that Jurkat cells expressing wild-type HOX11 exhibited a reduced frequency of early-stage apoptotic cells relative to mock and HOX11 T247E transfected cells suggesting that HOX11 confers a survival advantage to cells Figure . CombineHOX11 expression [HOX11 gene was constitutively active, we observed a post-translational modification of HOX11 which correlated with increased numbers of mitotic cells following treatment with microtubule destabilizing compounds. Radiolabelling studies using several HOX11 truncation and point mutants revealed that this modification was the result of phosphorylation of the HOX11 protein at Thr247 within the homeodomain.The multiple reports of deregulation of cell cycle checkpoints associated with perturbations in pression ,25,27-29The 60-amino acid homeodomain is a conserved DNA binding protein domain consisting of an N-terminal arm and three α helices that are separated by a loop and a turn. DNA binding of the homeodomain is mediated by the third recognition helix and the N-terminal arm with several conserved amino acids, including Arg3, Arg5, Ile47, Gln50 and Asn51, playing a major role in DNA recognition. Three of the residues, Arg5, Gln50 and Asn51, are conserved in the homeodomains of HOX11 gene family members whereas the two other residues are changed from Arg3 to Lys3 and Ile47 to Thr47. Since both Arg3 and Lys3 have side chains with amine groups this change likely does not affect DNA interactions . In contThe data presented in this report indicated that phosphorylation of HOX11 on Thr247 limited the binding of the HOX11 protein to the promoter region of cyclin B1. Furthermore, HOX11 was phosphorylated only during M phase of the cell cycle resulting in transient inhibition of HOX11 DNA binding activity. Since Thr247 makes specific contact with nucleic acids within the major groove of the DNA double helix, phosphorylation of this amino acid residue places a negative charge close to the phosphates of DNA thereby interfering with DNA binding through electrostatic repulsion. Moreover, replacement of Thr247 with glutamic acid to mimic phosphorylation decreased DNA binding activity. Thus phosphorylation of Thr247 provides a mechanism for cells to inactivate target gene expression as cells enter the M phase and reactivate expression as cells undergo cytokinesis and re-enter the G1 phase of the cell cycle.HOXA9 and HOXA10 homeobox genes are frequently over-expressed in acute myeloid leukemias (AMLs). HOXA9 has been reported to be phosphorylated on serine, threonine and tyrosine residues within its homeodomain with phosphorylation correlating with decreased transforming capability in myeloid and murine bone marrow cells [in vitro and in vivo inhibition of DNA binding activity [Phosphorylation represents a common mechanism in regulating the DNA binding capacity of homeodomain-containing transcription factors including the Drosophilia homeodomain proteins Antennapaedia , Engrailow cells ,53. Two ow cells -49,54. Tow cells  and as aow cells -58. Bothow cells -64. PRH ow cells . In otheow cells ,42,65-67ow cells . Moreoveactivity ,42,66. Tvia Thr247-directed interactions with promoter regions containing the HOX11 consensus recognition sequence TAAGTG, and (2) those regulated through association of promoter regions with HOX11-PBX heterodimers [We observed that the transcriptional activity of the HOX11 oncoprotein was modulated by phosphorylation of the Thr247 residue. Specifically, the ability of HOX11 to modulate levels of cyclin B1 expression was impaired by a form of HOX11 that contained an acid amino acid substitution at Thr247 that mimicked a constitutively phosphorylated HOX11. Moreover, we observed that the Thr247-dependent upregulation of cyclin B1 was associated with dysfunction of the spindle assembly checkpoint. In this respect, our findings are consistent with previous studies in delineating an uncoupling between genes targeted by the Thr247 residue associated with deregulating the spindle assembly checkpoint and HOX11 target genes involved in cell immortalization . These drodimers .Aldh1a1, Wt, c-kit and Vegfc [Aldh1a1 and the oncogenic properties of HOX11 are both dependent on an intact homeodomain [On the basis of the DNA binding activity of the HOX11 homeodomain and its ability to transactivate reporter gene expression through both TATA-containing and TATA-less promoters , it has nd Vegfc ,68-70, hnd Vegfc , Meis [7nd Vegfc  or Groucnd Vegfc  and compnd Vegfc . Howevereodomain ,22 suggeThe physiological relevance of HOX11 phosphorylation was demonstrated with respect to its effects on cyclin B1 expression and spindle assembly checkpoint regulation. Expression of wild type HOX11 increased expression of cyclin B1 and this elevated expression was associated with a reduction in transition time through the G2/M checkpoint, a reduced mitotic index and a reduction in the frequency of early apoptotic cells. In contrast, the threonine-to-glutamic acid substitution mutant resulted in a return to normal levels of cyclin B1 expression, cell cycle kinetics, mitotic index and frequency of early apoptotic cells.Cyclin B1 is the regulatory subunit of cyclin dependent kinase (CDK1). The CDK1/cyclin B1 complex is critical for the control of G2/M transition as it controls the centrosome cycle and the onset of mitosis  but no binding of factors present in Jurkat cells stably expressing the HOX11T247E mutant protein . Mutation of the TAAGTG HOX11 binding site in target oligonucleotides prevented factor binding . The presence of the wild type HOX11 protein and the mutant HOX11T247A protein within the factor complex was confirmed by supershift analysis using an anti-flag antibody. Lane 1: free probe. Lane 2: probe + flag-vector. Lane 3: probe + flag-vector + anti-flag antibody. Lane 4: probe + flag-HOX11-wt. Lane 5: probe + flag-HOX11-wt + anti-flag antibody. Lane 6: probe + flag-HOX11-T247A. Lane 7: probe + flag-HOX11-T247A + anti-flag antibody. Lane 8: probe + flag-HOX11-T247E. Lane 9, probe + flag-HOX11-T247E+ anti-Flag antibody.Click here for fileEnhanced viability of Jurkat cells stably expressing HOX11. Jurkat cells stably expressing an empty flag-vector, flag-HOX11-wt or flag-HOX11-T247E cDNA were subjected to mock Amaxa nucleofection in Solution V using program X-001. Cells were stained with Annexin V and propidium iodide (PI) and analyzed by flow cytometry to assess cell death prior to nucleofection and 2 days post nucleofection. Numbers shown in lower right quadrants indicate percentages of early apoptotic cells. The results are representative of three independent experiments.Click here for file


This formulation is repeated twice in the asymmetric unit as the rubidium cation lies on an inversion centre. Each anion or acid molecule is linked to two rubidium cations, while the rubidium cation has close contacts to four symmetry-equivalent organic ligands, with two different coordination modes towards this cation. In addition, each rubidium cation is coordinated by two water O atoms, raising the coordination number to eight. One of the carboxyl groups of the acid holds its H atom, which forms a hydrogen bond to a coordinated water molecule. The other carboxyl group is deprotonated in half of the ligands and protonated in the other half, taking part in a strong O—H⋯O hydrogen bond disordered over an inversion centre. The stabilization of the crystal structure is further assisted by O—H⋯O and O—H⋯N hydrogen-bonding interactions involving the water molecules and carboxylate O atoms.The structural unit of the title compound, [Rb(C DOI: 10.1107/S1600536809002001/hg2450Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


In the ligands, the imidazole units are protonated and two of the hydroxy O atoms of the phosphonate groups are deprotonated and chelate the MnII, thus forming the neutral molecule of the title compound. The two protonated O atoms within the phosphonate groups of one imhedpH3− ligand act as hydrogen-bond acceptors for a bifurcated hydrogen bond originating from the coordinated water molecule. The phosphonate units of neigboring molecules are connected with their equivalents in neighboring molecules via two types of inversion-symmetric hydrogen-bonding arrangements with four and two strong O—H⋯O hydrogen bonds, respectively. The two interactions connect molecules into infinite chains along [111] and [110], in combination forming a tightly hydrogen-bonded three-dimensional supramolecular network. This network is further stabilized by additional hydrogen bonds between the protonated imidazole units and one of the coordinated P—O O atoms and by additional O—H⋯O hydrogen bonds between the water molecules and the P=O O atoms of neigboring molecules.In the title compound, [Mn(C DOI: 10.1107/S160053680905065X/zl2255Isup2.hklStructure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


The hydrogen bond patterns between mainchain atoms in protein structures not only give rise to regular secondary structures but also satisfy mainchain hydrogen bond potential. However, not all mainchain atoms can be satisfied through hydrogen bond interactions that arise in regular secondary structures; in some locations sidechain-to-mainchain hydrogen bonds are required to provide polar group satisfaction. Buried polar residues that are hydrogen-bonded to mainchain amide atoms tend to be highly conserved within protein families, confirming that mainchain architecture is a critical restraint on the evolution of proteins. We have investigated the stabilizing roles of buried polar sidechains on the backbones of protein structures by performing an analysis of solvent inaccessible residues that are entirely conserved within protein families and superfamilies and hydrogen bonded to an equivalent mainchain atom in each family member.We show that polar and sometimes charged sidechains form hydrogen bonds to mainchain atoms in the cores of proteins in a manner that has been conserved in evolution. Although particular motifs have previously been identified where buried polar residues have conserved roles in stabilizing protein structure, for example in helix capping, we demonstrate that such interactions occur in a range of architectures and highlight those polar amino acid types that fulfil these roles. We show that these buried polar residues often span elements of secondary structure and provide stabilizing interactions of the overall protein architecture.Conservation of buried polar residues and the hydrogen-bond interactions that they form implies an important role for maintaining protein structure, contributing strong restraints on amino acid substitutions during divergent protein evolution. Our analysis sheds light on the important stabilizing roles of these residues in protein architecture and provides further insight into factors influencing the evolution of protein families and superfamilies. As Pauling and Corey realised, satisfaction of hydrogen bonding potential of polypeptide mainchain functions is one of the major factors that give rise to the β-strand and α-helix ,2. TheseHowever, these elegant architectures still leave many mainchain functions unsatisfied in their potential to form hydrogen bonds: an early survey of hydrogen bonding in proteins revealed that ~40% of mainchain atoms do not form hydrogen bonds with other mainchain atoms . In gene(1) Where strands and helices terminate, requiring "capping" -10.(2) Where helices and strands bulge ,12 or be(3) In polyproline or irregular, twisted strands ,16(4) In arches and turns -5,17,18.Water molecules or sidechains can usually satisfy the hydrogen bonding potential of mainchain functions that are at the protein surface in a variety of ways and so the residues are often substituted in evolution. However, in the smaller proportion of functions that must be satisfied from the core of the protein, this is achieved by buried sidechains of polar residues.Analysis of the substitution patterns of amino acids within homologous protein families has revealed that buried polar residues that are hydrogen-bonded to mainchain amide atoms are highly conserved, more so than those polar residues forming hydrogen bonds to mainchain carbonyl atoms or other sidechains ,20. Furtin silico analyses of the stabilizing roles that polar sidechains have on the backbone of protein structures have tended to focus on a particular architectural context [Previous  context ,23,24. B context  identifiIn this report we focus purely on buried polar residues that are entirely conserved within protein families and superfamilies, hydrogen bonding to a mainchain atom in each family member. We hypothesise that such buried sidechain-to-mainchain hydrogen bonds satisfy mainchain hydrogen bonding potential where secondary structures cannot be formed, and in so doing become irreplaceable elements of the overall architecture. In order to test this hypothesis we characterize the nature and tertiary structural context of these conserved and buried polar residues. We show that polar sidechains which bridge to mainchain functions in the cores of proteins have conserved tertiary structural roles in homologues. Like the elements of secondary structure, they are born of the need to satisfy hydrogen bonding but, in achieving this, they become key, conserved structural features of many well-known protein architectures. Some are joists or braces, spanning the helices and strands, while others form truss-like structures that support complex loop structures Figure .In HOMSTRAD , a databFor conserved and buried polar residues making hydrogen bonds to mainchain NH functions in the N-terminal regions of α-helices, cysteine has the highest propensity to form such interactions, followed by negatively charged aspartate, histidine and glutamate  was used to define solvent inaccessible (buried) residues. In order to avoid redundancy, where protein families overlapped, the family with the highest sequence coverage was chosen for the analysis.Protein families containing five or more members were selected from HOMSTRAD where the family alignment contained a conserved, buried polar residue and where the sidechain of the polar residue forms a hydrogen bond to a mainchain atom in each family member. The JOY alignmenHBOND . HBOND identifies all possible hydrogen bonds based on a distance criterion (3.5Å between donor and acceptor).Hydrogen bond partner(s) to the conserved, buried polar residues were identified using the program, PROMOTIF, to identify the structural context of the conserved polar residues and their interaction partners [We used the program, partners . The fol1. α-helices  to N-(N+3) for N-terminal residues and N-3 to N+1 for C-terminal residues (where N is the length of the helix).10 helices2. 33. β-strands - edge strands were distinguished from centre strands by referring to the number of hydrogen bonding partner strands. Strands defined as having >1 hydrogen bonding partner strand were defined as centre and all others as edge.4. β-hairpins5. Coil regionsSEGNO[We also identified polyproline helices using the program SEGNO.x to form hydrogen bonds to mainchain atoms in a particular architectural context Parch was calculated using the following equation:The propensity of a particular residue type narch(x) is the number of residues of type x forming hydrogen bonds to mainchain atoms in a particular architectural context, Nx )  is the total number of residues forming hydrogen bonds to mainchain atoms in a particular architectural context and Ntotal)  Polar residues which are entirely conserved, buried in each family member and forming a hydrogen bond to a mainchain atom group in each family member. These numbers were therefore derived from the 233 alignment positions identified in the 66 families.(ii) All polar residues in the 131 family set, regardless of solvent accessibility and conservation but where the polar residue forms a hydrogen bond to a mainchain atom group.CLW participated in the design of the study, performed the computational experiments, analysed the data and drafted the manuscript. TLB conceived of the study, participated in its design and refined the manuscript. Both authors read and approved the final manuscript.Table of the 131 non-redundant families which were used in the analysis.Click here for fileTable of the families and their members that were used in the analysis.Click here for fileFigures S1 to S6 show the propensity of polar amino acids to form hydrogen bonds to mainchain atoms in the various architectural contexts analysed.Click here for file


To establish the frequency of necrotizing funisitis in congenital syphilis, we conducted a prospective descriptive study of maternal syphilis in Bolivia by testing 1,559 women at delivery with rapid plasma reagin (RPR). We examined umbilical cords of 66 infants whose mothers had positive RPR and fluorescent treponemal antibody absorption tests. Histologic abnormalities were detected in 28 (42%) umbilical cords , and 38 [58%] were normal. Of 22 umbilical cords of infants from mothers without syphilis (controls), only two (9%) showed mild funisitis; the others were normal. Testing umbilical cords by using immunohistochemistry is a research tool that can establish the frequency of funisitis due to Treponema pallidum infection.


MYH3, TNNI2, or TNNT3 have been found in about 50% of cases. These genes encode proteins of the contractile apparatus of fast twitch skeletal muscle fibers. The diagnosis of SHS is based on clinical criteria. Mutation analysis is useful to distinguish SHS from arthrogryposis syndromes with similar features . Prenatal diagnosis by ultrasonography is feasible at 18–24 weeks of gestation. If the family history is positive and the mutation is known in the family, prenatal molecular genetic diagnosis is possible. There is no specific therapy for SHS. However, patients benefit from early intervention with occupational and physical therapy, serial casting, and/or surgery. Life expectancy and cognitive abilities are normal.Sheldon-Hall syndrome (SHS) is a rare multiple congenital contracture syndrome characterized by contractures of the distal joints of the limbs, triangular face, downslanting palpebral fissures, small mouth, and high arched palate. Epidemiological data for the prevalence of SHS are not available, but less than 100 cases have been reported in the literature. Other common clinical features of SHS include prominent nasolabial folds, high arched palate, attached earlobes, mild cervical webbing, short stature, severe camptodactyly, ulnar deviation, and vertical talus and/or talipes equinovarus. Typically, the contractures are most severe at birth and non-progressive. SHS is inherited in an autosomal dominant pattern but about half the cases are sporadic. Mutations in either  Sheldon-Hall syndrome (SHS);Distal arthrogryposis type 2B (DA2B);Freeman-Sheldon syndrome variant;Arthrogryposis multiplex congenita, type II.Sheldon-Hall syndrome  or distal arthrogryposis type 2B (DA2B) is an autosomal dominant disorder characterized by congenital contractures of the distal joints of the limbs without a primary neurological defect. SHS is similar to another distal arthrogryposis called Freeman-Sheldon syndrome (FSS or DA2A), first described by Freeman and Sheldon in 1938, but distinguished as a separate entity in 1997 [The prevalence of arthrogryposis is about 1/3000 [The major clinical features of SHS are summarized in Table Among the facial characteristics of SHS the most common finding is a narrow face that is pointed at the chin giving the face a triangular appearance. High arched palate and micrognatia are also very common. Down slanting palpebral fissures are seen in some individuals. Other facial findings include small mouth, high nasal bridge, prominent nasolabial folds, bulbous nose, malar hypoplasia, facial asymmetry, hypertelorism, and posteriorly angulated ears or some other ear abnormalities . Overalle.g., elbow, shoulder, knee, and hip) also can be affected. Contractures of the upper limbs typically manifest as overlapping fingers, camptodactyly, and ulnar deviation. Talipes equinovarus, valgus abnormalities, overlapping toes, vertical talus, and tarsal fusion are the most common findings in the lower limbs. The severity of contractures can vary substantially between upper and lower limbs, and the left and right sides of the body. An affected individual might have both equinovarus and calcaneovalgus abnormalities in his/her feet. Contractures are most severe at birth and non-progressive. However, flexion contractures can worsen over time in the absence of appropriate intervention .In an isolated case, the presence of congenital contractures is mandatory in order to entertain the diagnosis of SHS. The joints of the hands, wrists, feet, and ankles are most commonly involved although more proximal joints  and troponin T (TNNT3), and embryonic myosin (MYH3) [TNNI2, TNNT3, or MYH3, large deletions of these genes, or mutations in other as of yet unidentified genes.SHS is caused by mutations in the genes that encode fast twitch skeletal muscle isoforms of troponin I (n (MYH3) -7. MutatTNNI2 gene which is located on the short arm of chromosome 11 (11p15.5). Troponins are muscle proteins that are part of the contractile apparatus. To date, 4 different TNNI2 mutations have been reported in DA2B patients: p.R156X, p.R174Q, p.E167del, and p.K175del [TNNI2 cause contractures is unclear but troponin I binds to actin and tropomyosin, and in the absence of Ca2+ prevents muscle contraction. In vitro and ex vivo contractility studies of troponin I mutants suggest that p.R156X and p.R174Q increase muscle contractility via increasing Ca2+ sensitivity [2+ levels.The fast twitch skeletal muscle troponin I is encoded by the .K175del . All of TNNT3 gene, which is near TNNI2 on chromosome 11p15.5, was considered a positional and functional candidate [2+-dependent force generation [The andidate . It encoandidate . This muTNNI2 or TNNT3, giving priority to genes expressed during prenatal or perinatal development. This eventually led to the screening of the gene that encodes embryonic myosin heavy chain (MYH3). Mutations in MYH3 were found in 5 of 12 familial (42%) and 7 of 26 sporadic (27%) cases [MYH3 mutations were predicted to affect the formation and stability of the contractile apparatus. Collectively, mutations in the MYH3, TNNI2, and TNNT3 genes account for about half of all studied cases of SHS.After it became evident that SHS was caused by defects of the contractile apparatus of fast-twitch myofibers, we investigated whether SHS could be caused by mutations in genes that encode other proteins of the contractile apparatus . The genTNNI2, TNNT3, or MYH3 have phenotypes that cannot be distinguished from one another. Even individuals with the same mutation exhibit widely variable clinical findings. Nevertheless, an important caveat is that a relatively small number of cases with any specific mutation have been studied, and the phenotypic information from many of these cases is incomplete. Overall, more cases need to be studied to rigorously evaluate if there is a substantial correspondence between genotype and phenotype in individuals with SHS.Extensive within and between family phenotypic variation is observed in SHS. Both the specific joints affected and the degree to which joints are affected varies. The basis of this variability is unclear but could arise by several different mechanisms or a combination thereof as none are mutually exclusive. First, mutation in different genes could cause different phenotypes. However, none of the phenotypic variability between families appears to correspond to disruption of specific proteins much less specific genotypes. In other words, individuals with a mutation in either MYH3 mutations that cause SHS are distinct from those that cause FSS [ause FSS . Almost The diagnosis of SHS is based on clinical characteristics ,4. The mOccasionally women may note decreased fetal movements, but they might also be normal. The clinical findings of SHS can be detected prenatally by ultrasonography. However, detection may not be possible until 18–24 weeks of gestation. If the family history is positive and the mutation is known in the family, prenatal molecular genetic diagnosis is possible .de novo mutations. The recurrence risk in such families is low but recurrent "de novo" mutations in several families with DA suggest that germline mosaicism does occur in DA.SHS is a monogenic disorder with autosomal dominant inheritance. Affected individuals have a 50% risk of transmitting the disease to their offspring. However, many of the patients reported to date have had e.g., diagnosis is in question). Since the majority of mutations have been found in MYH3, this gene should be screened first. If no mutations are found in MYH3, TNNI2 and TNNT3 genes should also be sequenced.Mutation analysis of the known genes should be offered to affected individuals in certain circumstances , however, SHS does not include trismus of the jaw, which is a unique characteristic of DA7.There is no specific treatment for SHS. The contractures occur early in development and they improve postnatally. Although some spontaneous improvement might be expected, prolonged use of braces and casts, or multiple surgeries to correct the limb contractures are often required. Most patients will benefit from physical therapy, which should start soon after birth.Individuals with SHS have a normal life expectancy. Hearing, vision, speech, and motor development should be checked as appropriate. The majority of the patients will require series of orthodontic treatment as well as prolonged orthopedic management. Surgical procedures can be complicated by difficult intravenous access, laringoscopy, and spinal anesthesia. In addition, there is evidence to suggest that individuals with DA syndromes including SHS are at higher risk for malignant hyperthermia .MYH3, TNNI2, or TNNT3 genes. It needs to be addressed whether these patients have mutations in another gene or mutations in regulatory regions of these genes. With respect to the latter, there are no known families that map to any of the known loci and in whom mutations have not been identified. The molecular mechanism underlying the contractures is not well understood. Some mutations appear to increase contractility, although this has not yet been confirmed in ex vivo studies of myofiber contractility in affected individuals. Finally, the SHS phenotype starts to develop in utero, suggesting that a clear understanding of how muscle development is perturbed in SHS will require studies in animal models.Almost 50% of the patients affected with SHS, do not have mutations in MYH3: embryonic myosin heavy chain; SHS: Sheldon-Hall syndrome; TNNI2: troponin I2; TNNT3: troponin T3.DA: distal arthrogryposis; FSS: Freeman-Sheldon syndrome; MIM: Mendelian Inheritance in Man; Written consent for publication of the clinical picture was obtained from the patient.The authors declare that they have no competing interests.RMT wrote the first draft of the manuscript; both authors revised the manuscript and approved the final version.


Flexible electronics sensors for tactile applications in multi-touch sensing and large scale manufacturing were designed and fabricated. The sensors are based on polyimide substrates, with thixotropy materials used to print organic resistances and a bump on the top polyimide layer. The gap between the bottom electrode layer and the resistance layer provides a buffer distance to reduce erroneous contact during large bending. Experimental results show that the top membrane with a bump protrusion and a resistance layer had a large deflection and a quick sensitive response. The bump and resistance layer provided a concentrated von Mises stress force and inertial force on the top membrane center. When the top membrane had no bump, it had a transient response delay time and took longer to reach steady-state. For printing thick structures of flexible electronics sensors, diffusion effects and dimensional shrinkages can be improved by using a paste material with a high viscosity. Linear algorithm matrixes with Gaussian elimination and control system scanning were used for multi-touch detection. Flexible electronics sensors were printed with a resistance thickness of about 32 μm and a bump thickness of about 0.2 mm. Feasibility studies show that printing technology is appropriate for large scale manufacturing, producing sensors at a low cost. We also use an algorithm matrix and control system scanning to solve the array matrix for multi-touch switch identification in the tactile sensors. Flexible electronics actuators for realizing large scale and low-cost applications have been gradually developed in recent years ,12. EspeWe have designed a gap between the bottom electrode and the resistance layer to eliminate erroneous signals during bending actions. For optimal flexible structure characteristics, a protrusion (bump) on the top membrane is used to enhance the sensing sensitivity response. A high viscosity thixotropic material is used to print the thick bump structures to reduce the diffusive effects and dimensional shrinkage after printing and curing. Mechanical properties were investigated by analysis of the deflection and stress distributions using finite element analysis (FEA). The feasibility of fabricating low-cost printable sensors for applications in flexible electronics is demonstrated.2.2.1.This study used commercial PI films and screen printing technology to design and fabricate a flexible large area electronics sensor. PI films with copper foils are widely used in flexible electronics sensors for flexible printed circuits -28. The 4 for electroless plating of Au, to a thickness of about 5 μm, to avoid the oxidation of the copper foil. Next, a cover layer with lattice patterns was laminated on the bottom film using hot pressing to form post structures (II). The hot-press process was performed at a temperature of 180°C for 20 minutes.First, the electrode patterns on the bottom film with a 12 μm copper foil (I), were defined for column and sensing electrodes using the photolithography method. The bottom film was then put into a solution of CuSO4cp and a glass transition temperature of 190°C, was printed on sensing electrode areas using a screen stencil mask. The organic resistance was synthesized using a phenolic resin, organic solvents, filler, and carbon black. The phenolic resin is a bisphenol A (C15H16O2) type of organic compound with two phenol functional groups. The organic solvents included dimethylformamide (formula C3H7NO) and diethylene glycol monobutyl ether (formula CH3CH2OCH2CH2OCH2CH2OH). Dimethylformamide , is a hydrophilic aprotic solvent of the organic compound material that facilitates chemical reactions by polar mechanisms. Diethylene glycol monobutyl ether [2-(2-butoxyethoxy)ethanol], is the main dispersing agent for the synthetic resin and filler. The filler and carbon black are 25.6 wt% and 12.0 wt%, respectively.The second part of the top film, which contains the row electrodes (i), is similar to that of the bottom substrate, except that it has pass-through holes that were drilled using the punching method. Cu was coated on the side of these holes using a chemical plating to transfer the output signal from the back to the top view of top film. An organic resistance material (ii), Model EPO 4X330, with a viscosity of 8 × 106cp and a glass transition temperature of 150°C was printed on the top film using a screen stencil mask to form bump structures (iii). The bump structures were formed after being cured at a temperature of 150°C for 45 minutes. Finally, both PI films were aligned and assembled to form a flexible electronics sensor using adhesive (III), type Cemedine Super-X No.8008  which was printed on the top of the post surface using screen technology.For organic resistance printing, the stencil mask was a stainless steel sheet, which was made using chemical etching techniques. The printing header was a rubber squeegee of type A with a durometer of 70 and a printed angle of 45°. After top electrode and organic resistance fabrication, the film was turned over. A thixotropic material, type EPO 4X282H, with a viscosity of 102.2.1 to E4 has only an ON/OFF switch function, with R11, R12, R21, and R22 ignored, as shown in the upper left of 1, E2, and E3, then E4 will be misread as also being touched because all column and row electrodes are mutually conductive during the column and row scanning processes. An organic resistance material was printed on the surface of the sensing electrodes and an algorithm matrix and control system scanning were used to solve this problem. The schematic control system for multi-touch switching applications is shown in mnR (m × n array elements with a resistance of R) had a pitch of 5 mm with 25 × 25 elements.In general, in array switch elements for multi-touch use without a resistance layer, it is very difficult to correctly identify which elements are really touched. For example, a 2 × 2 matrix sensor with elements ED, VG, …, VG,” outputs “1,0, …, 0” for all columns, indicating that only column 1 was driven. Then, port B sets the impedances of all rows to “high, low, …, low” and is changed to detect all row data. R11 can be detected using algorithm matrix operating. The equivalent circuit of the algorithm matrix for row 1 is shown in In the multi-touch detection process, the parallel buffer IC outputs serial digital data to columns via port A. Only one column is the high bit, 1, and the others are the low bit, 0. Port B first sets the serial data to all rows. One row is high impedance and the others are low impedance. Then, port B is changed to detect all row data to identify which elements were touched. For instance, the serial digital data of port A, “VSimilarly, the serial digital data of port A outputs “0,1,0, …, 0” for all columns.1n=0AY, can be formed as follows:DV is the driving voltage of the microcontroller, entries 11V to 1nV are the voltage values of row 1, and 11Y to 1nY are the conductance values of the resistance at row 1, with 1n=1/R1nY. To solve the linear equations, Gaussian elimination was performed in row-echelon form to reduce the matrix. Then, the back-substitution method was used to solve the final equations of 1nY for multi-touch identification. The average value of resistance of elements after the voltage scanning and calculations was about 2.8 kΩ. Similarly, rows 2 to m can be scanned using the same methods to detect the signals. It is noteworthy that the microprocessor for scanning one column and solving the algorithm takes about 0.015 seconds, indicating a response frequency of 150 Hz. The sensor array of our study is 25 × 25, so this is not a problem for our system. However, we think that solving bigger matrixes will create some problems. Maybe the scanning array matrixes can be segmented and a sub-microprocessor can be used to solve the algorithm equations.Therefore, the algorithm matrix of row 1, 2.3.f, in the z direction, as shown in dx dy, forces of magnitude Pdx and Pdy act on the sides parallel to the y and x axes, respectively. The forces, F, acting along the z direction due to these forces are:P and δ are the pressure and the deflection at the membrane center, respectively.Consider a square plate for a linear motion equation, simply supported on all edges and subjected to a distributed load, f·dx·dy. Hence, the membrane bending by moments distributed can be obtained for the forced transverse vibration in z direction [The pressure force along the z direction is irection ,31:(5)where mt and 0w are the membrane thickness and width, respectively, and E and ν are the material Young's modulus and Possion's ratio, respectively. C and α are the coefficients, which are 21.62 × (1.41-0.292 × ν) and 1.26 × 10-3 [E = 2.5 GPa, ν = 0.34, mt = 50 μm, and 0w = 5 mm when a pressure of P = 400 MPa is applied on the membrane. The central deflection is 3.59 × 10-4 m.The result of the applied pressure and the membrane deflection is given by:3.3.1.The design and simulation of a flexible electronics sensor for static tactile analysis with a large deflection were carried out using commercial FEA, ANSYS v.10.0. The effects of the membranes without bumps, with bumps, and with bumps and a resistance layer were used to analyze the deflection characteristics after a constant force was applied on the top surface. The membranes for the simulations, with a constant thickness of 50 μm, were square-shaped suspension structures. Bump sizes with width ratios of 0.2 to 1, relative to the membrane width, and with thicknesses of 0.05, 0.2, and 0.5 mm were considered. During the simulation, all elements were defined by eight nodes with three degrees of freedom at each node. A free mesh with tetrahedral-shaped elements was used to simplify the geometry creation.maxδ, increased with increasing applied force, as shown in For boundary condition settings, the displacements in the bottom of the posts were fixed in all degrees of freedom and the constant forces from 0.5 to 10 N were applied on the top membrane. Young's modulus, Poisson's ratio, and the density of the PI film were 2.5 GPa, 0.34, and 1.42 g/ml, respectively. The simulation results show that the maximum deflection of all cases, W and bt, are shown in The effects of width and thickness of various bumps, 3, respectively. The maximum deflections and stresses of the membrane with a bump structure after applying a constant force of 5 N were 0.196 mm and 485 MPa, respectively, as shown in The mechanical characteristics analyses, deflections, and von Mises stress distributions of membranes with bumps and of those with bumps and a resistance structure, are shown in 3.2.2, containing 25 × 25 arrays of sensing pads and bumps, respectively, as shown in The flexible electronics of tactile sensors were successfully fabricated using two PI films. The thicknesses of the top and bottom were 50 and 125 μm. The effective area was 15 × 15 cm3, were printed on the top PI film to provide a concentrated force for enhanced touch sensitivity. Results show that bump shapes had excellent morphological profiles, as shown in The bump structures, 2 × 2 × 0.2 mmp - WiW) / iW × 100 %, where iW and pW are the original width and the printed width, respectively. To analyze the printed characteristics, it is necessary to measure the dimensional diffusion and average thickness. The dimensional diffusion ratio of structure dimensions after printing and curing is presented as  with an accuracy of 0.1 N and a cylinder tip with a radius of 0.5 mm, a 6 axis micro-stage, an LCR meter, and a motor controller with a displacement accuracy of 1 μm.3.3.1.For dynamic response measurement, a trapezoid force with a maximum force of 5 N, parallel to the y-axis in the xy-plane, was applied to the elements. The sensing mechanism of the dynamic response is based on the impedance change of resistance after contact. When a force is applied on a bump and the organic resistance layer is compressed, the sensing electronics of the top and bottom conduct the impedance changes. The equivalent circuit of sensing impedance is similar to a variable resistor. 3.3.2.1 to P5 indicate the measurement points at the center and 4 apexes of the array sensor, respectively. Results show that the impedance values sharply decreased with increasing compression force. The characteristic trend of the resistance material after fabricating was similar at the different measurement points. The characteristic output versus compression forces can approximate to linear logarithm regression. Therefore, for multi-touch switch sensing, the thresholds of the resistance response were determined according to the compression forces.For contact characteristic measurement, an external force, parallel to the y-axis in the xy-plane, was applied at the top center of the membrane. The displacements of the stages were adjusted to test the compression force versus the characteristic output at the operating frequency of 1 kHz, related to the measurement accuracy of sensing material, in ambient conditions, as shown in The effects of temperature variations from 5 to 95°C on different structures for switch scanning are shown in 3.3.3.The multi-touch testing of the flexible electronics sensors is shown in ε is the permittivity of free space, ε = 8.84 × 10-12 F/m, and d is the distance between the two substrates. The gap distance, at the radius of infinite curvature, is originally about 41 μm. The results of the bending tests at different curvature radii versus gap distance are shown in For the bending testing, flexible sensors were simply fixed on different curved surfaces of cylinder molds, with curvature radii of 3, 5, and 7 cm, to test the bending response. During experimental measurements, gap distances after differential bending were calculated using the principle of parallel capacitance, C = (ε ×A)/d. where A is the area of each plate, 4.This study has successfully developed a flexible electronics sensor for multi-touch switch detection using an algorithm matrix and using screen printing technology for large-scale manufacturing. For printing thick structures, materials with higher viscosity can be used to print excellent morphological profiles, which can greatly reduce the dimension effects of diffusion and shrinkage, enhance the printing resolution, and decrease the cohesive force between the stencil mask and the sensor substrate. In addition, printed material on flexible substrates with a high surface roughness had good printed patterns and adhesive force. The gap within sensor structure provided a buffer distance to avoid erroneous contact during bending action. This sensor, with a minimum curvature radius of about 3 cm, is flexible enough to be applied anywhere without being damaged. We believe that the results of this study can be applied to many fields of flexible electronics sensors and that they provide useful information for designing and fabricating flexible large area devices.


About one-third of patients suffering from systemic lupus erythematosus have ocular manifestations. The most common manifestation is keratoconjunctivitis sicca. The most vision threatening are retinal vasculitis and optic neuritis/neuropathy. Prompt diagnosis and treatment of eye disease is paramount as they are often associated with high levels of systemic inflammation and end-organ damage. Initial management with high-dose oral or IV corticosteroids is often necessary. Multiple “steroid-sparing” treatment options exist with the most recently studied being biologic agents. Systemic lupus erythematosus (SLE) is a chronic, autoimmune, connective tissue disorder affecting multiple organ systems often with a relapsing and remitting clinical course. Prevalence, clinical manifestations, and morbidity vary significantly between the developing and industrialized worlds. While SLE is more common in people of African and Asian descent, thrombotic complications are more common in Caucasian patients . The higConcordance rates for SLE among monozygotic and dizygotic twins are 25% and 2%, respectively, suggesting a significant genetic contribution . Major hα-actinin. Two major theories exist on how these autoantibodies cause tissue damage. The first model suggests that anti-double-stranded DNA antibodies bind to circulating nucleosomes to form immune complexes that then get deposited in end-organ capillary beds such as the renal glomerulus and activate immune/inflammatory responses [SLE is a complex disease process demonstrating dysregulation of the immune system at multiple levels. Autoantibodies against double-stranded DNA were first isolated from kidney specimens in patients with lupus nephritis in 1967 [esponses . The souesponses .α, INF-γ, IL-10, and B-lymphocyte stimulator. Mass production of autoantibodies relies on multiple factor, which have each independently been targeted as potential immunotherapy in the treatment of lupus. Important steps include T-cell activation via antigen binding to the T-cell receptor and proper costimulation; T-cell activation of B cells; production of cytokines such as TNF-Medications, hormonal influences, and other factors such as sunlight have all been implicated in disease exacerbation. Drug-induced lupus, most commonly due to procainamide, hydralazine, and quinidine, usually presents with disease involving the skin and joints with renal and CNS manifestations being much more rare . HormonaAccording to the 1982 revised criteria for systemic lupus erythematosus, a diagnosis of SLE can be made by the serial or simultaneous presentation of at least 4 of the following 11 criteria: malar rash, discoid rash, photosensitivity, oral ulcers, nonerosive arthritis, serositis, renal dysfunction, neurological derangements , hematologic disorder , immunologic disorder , and presence of antinuclear antibodies. SLE can affect the periorbita, ocular adnexa, eye, and optic nerve. The most common association is keratoconjunctivitis sicca, while the most visually devastating sequelae occur secondary to optic nerve involvement and retinal vaso-occlusion. Orbital involvement is a rare manifestation of SLE. Vasculitis, myositis, and panniculitis have all been described. Signs and symptoms include proptosis, enophthalmos, orbital pain, blurred vision, chemosis, and restriction of extraocular movements. Orbital vasculitis leads to nonperfusion of the globe and extraocular muscles. This has been shown to cause irreversible vision loss secondary to ischemic injury to the optic nerve as well as elevated intraocular pressure from neovascular glaucoma .Orbital myositis is often initially misdiagnosed as bacterial orbital cellulitis, as it usually presents with significant pain, proptosis, periorbital swelling, and globe restriction. CT and orbital ultrasound are both valuable in demonstrating enlargement of one or multiple extraocular muscles. Creatinine kinase, aldolase, and myoglobin levels are markedly elevated. Inflammation and symptoms typically respond to steroids , 13.Subcutaneous inflammation secondary to SLE was first described by Kaposi in 1883, and the term “lupus erythematosus panniculitis” was coined in 1940 . It is mPeriorbital edema is an uncommon manifestation of systemic and discoid lupus erythematosus with an overall incidence of 4.8% . It is mTypical lesions of discoid lupus erythematosus are slightly raised, scaly, and atrophic. Most commonly, they occur on the head, face, neck, and other sun-exposed areas. Rarely does it affect the eyelids. Histopathologic study shows a hyperkeratotic epithelium with liquefactive degeneration of the basal layer and a dense perivascular/periappendageal lymphocytic infiltration , 26. DiaThe most common ocular manifestation of SLE is keratoconjunctivitis sicca with the majority of patients endorsing at least one dry eye symptom . DrynessAn abundance of proinflammatory markers such as IL-17 , 32 can Episcleritis is generally a benign inflammation of the episclera. Typically occurring in young women, symptoms include a dull ache, red eye, and tearing. Decreased visual acuity and severe pain are uncommon. Systemic associations are extremely rare in adults, and a systemic workup is not necessary. Incidence in adult patients with SLE has been reported at 2.4% . HoweverScleritis is a more painful and potentially a vision-threatening condition that warrants prompt treatment. Anterior scleritis can be nodular or diffuse and presents with a red, painful eye that is tender to touch. The injected deep episcleral vessels give a violaceous due to the sclera, which is best appreciated in natural light . PosteriCorneal epitheliopathy, scarring, ulceration, and filamentary keratitis can all occur secondary to keratoconjunctivitis sicca. More rare corneal complications include peripheral ulcerative keratitis , which cCorneal biomechanical properties differ in SLE. Yazici et al.  used ReiLupus retinopathy is one of the most common vision-threatening complications of systemic lupus erythematosus with an incidence of up to 29% in patients with active systemic disease. A strong correlation exists between presence of retinopathy and CNS disease . The mosRetinal vasculitis, a subset of retinal vasculopathy featuring inflammation of the retinal arterioles or venules, tends to have poorer visual outcomes and present in an acute onset fashion. A large percentage of these patients have concomitant antiphospholipid antibodies including anticardiolipin and lupus anticoagulant. In one study, 77% of patients with SLE and retinal involvement had positive antiphospholipid antibody titers, whereas only 29% of SLE patients without retinal disease had positive titers . HistopaImmunosuppression has been successful in improving the appearance of the retinopathy; however, visual recovery has only been reported in few cases. The permanent loss of visual acuity is likely due to retinal ischemia. Addition of anticoagulation to immunosuppression helps to stabilize retinal disease and prevent further vascular events . Other tLupus choroidopathy with exudative retinal detachments is a rare ocular manifestation with fewer than 40 patients reported in the literature . It is gRecently, ICG imaging has been used to visualize the choroidal circulation in lupus choroidopathy. Studies have shown focal, transient early-phase hypofluorescence followed by late-phase diffuse hyperfluorescence, distortion of the large choroidal vessels, and also focal clusters of choroidal hyperfluorescence in the intermediate phase. Transient early hypofluorescence and late hyperfluorescence are likely secondary to choroidal vascular perfusion delay with subsequent leakage due to an increase in vascular permeability, which are also observed in other vascular and inflammatory diseases. Unique findings include focal areas of hyperfluorescence in the intermediate frames, which may represent ICG staining of immune complexes . Although it is a marker of high disease activity, lupus choroidopathy has been shown to be responsive to corticosteroids and other forms of immunosuppression. Given its associations with CNS and renal disease, the presence of choroidopathy is likely an indication for aggressive, long-term immunosuppression. Optic nerve disease is a rare manifestation of SLE and consists of optic neuritis and ischemic optic neuropathy . PresentThe neuromyelitis optica spectrum disorders (NMOSDs) are characterized by a combination of optic neuritis and transverse myelitis. Few cases have been reported in the literature of the presentation of NMOSD in SLE –68. A reOptic neuropathy in SLE is caused by an ischemic process that affects the small vessels supplying both the optic nerve head and retrobulbar nerve. It usually presents as an acute loss of vision with an altitudinal visual field defect with or without optic disc edema. The disease is most commonly bilateral except in patients with circulating antiphospholipid antibodies. In this subset, a focal thrombotic event in the ciliary vasculature is thought to occur as opposed to a generalized vasculitis . StandarEye movement abnormalities are common in SLE and have been reported in up to 29% of patients . IschemiRetrochiasmal involvement can cause visual hallucinations, visual field defects, nystagmus, and cortical blindness. Few cases of idiopathic intracranial hypertension have been reported. 60% of cases reported in the literature are associated with antiphospholipid antibodies . Treatment options for SLE range from nonsteroidal anti-inflammatory drugs, corticosteroids, antimalarials, immunomodulatory, and biologic agents. Significant ocular involvement—orbital inflammation, scleritis, retinal vasculitis, choroiditis, and optic neuritis—warrants systemic therapy. The goal of treatment is to suppress immune activity, specifically decreasing the level of autoantibodies. Corticosteroids are the mainstay and most effective short-term therapy for SLE . They inSteroid-sparing immunosuppressive agents are used in a large amount of patients secondary to treatment failure or harmful side effects of corticosteroids. Antimalarials such as chloroquine and more commonly hydroxychloroquine are often used. These medications are highly efficacious in curtailing future flares with fewer side effects compared to other immunomodulatory drugs such as alkylating agents. However, ocular effects of these drugs are well known. Irreversible vision loss secondary to a drug-induced maculopathy has been well documented in the literature. Factors associated with high risk of developing maculopathy include greater than 5–7 years of therapy, greater than a cumulative dose of 1000 g of hydroxychloroquine, impairment of liver or kidney function, obesity, age greater than 65, and preexisting retinopathy. The American Academy of Ophthalmology recommends a baseline-dilated eye exam on all patients starting hydroxychloroquine followed by annual exams starting at 5 years after initiating therapy. A Humphrey 10–2 automated visual field test along with multi-focal electroretinogram, spectral domain optical coherence tomography, or fundus autofluorescence should be performed at each of these visits. Discontinuation of the drug should be recommended at the earliest sign of toxicity . UnfortuMethotrexate, azathioprine, mycophenolate mofetil, cyclosporine A, cyclophosphamide, and chlorambucil have all been employed with varying degrees of success. In the past few years, newer drugs, categorized as biological agents, have emerged targeting specific molecules involved in B- and T-cell activation. One of the first to be utilized in SLE was rituximab, a chimeric murine/human anti-CD20 antibody. Multiple studies have shown clinical improvement in refractory patients , 87. RitIn summary, a myriad of ocular manifestations of systemic lupus erythematosus have been described, and in some patients, these findings may be a presenting sign of systemic disease. Moreover, their presence can be a sign or a marker of disease activity. In the cases of choroidopathy and retinopathy, ophthalmic findings can be a poor prognostic systemic risk factor with the potential for both ophthalmic and systemic morbidity. For this reason, treatment typically involves a considered assessment of both the systemic and ophthalmic findings in determining the proper therapy and duration of treatment. Close communication between the consultant ophthalmologist and treating rheumatologist is critical in the effective management of these complex clinical situations.


Tornadoes can cause catastrophic destruction. Here total kinetic energy (TKE) as a metric of destruction is computed from the fraction of the tornado path experiencing various damage levels and a characteristic wind speed for each level. The fraction of the path is obtained from a model developed for the Nuclear Regulatory Commission that combines theory with empirical data. TKE is validated as a useful metric by comparing it to other indexes and loss indicators. Half of all tornadoes have TKE exceeding 62.1 GJ and a quarter have TKE exceeding 383.2 GJ. One percent of the tornadoes have TKE exceeding 31.9 TJ. April has more energy than May with fewer tornadoes; March has more energy than June with half as many tornadoes. September has the least energy but November and December have the fewest tornadoes. Alabama ranks number one in terms of tornado energy with 2.48 PJ over the period 2007–2013. TKE can be used to help better understand the changing nature of tornado activity. On average about 1,000 tornadoes occur each year in the United States; more than in any other country . SimilarStorm Data, which includes all known tornado reports dating back to 1950. Each report in the database gives the tornado touchdown location (latitude and longitude), date, length and width of the damage path, and maximum damage rating on a scale from 0 to 5.Current understanding of tornado destruction comes largely from available data records. The Storm Prediction Center’s (SPC) severe weather database is the most extensive set of historical tornado records in the world. Information in the database is compiled from the National Weather Service’s (NWS) The frequency of tornadoes by damage rating is a way to compare tornado occurrences collectively. For instance, Elsner et al.  show thaBy multiplying the path area by the damage rating and summing over all tornadoes in a given day, Thompson and Vescio  define aThe DPI and TDI are useful for summarizing a collection of tornadoes (tornado days for the DPI and tornado years for the TDI) and for comparing outbreaks. Here interest centers on kinetic energy of individual tornadoes. An estimate of KE is made in Agee and Childs  using thIn this study an estimate of total kinetic energy (TKE) is made for all tornadoes in the SPC database from 2007 when the Enhanced Fujita (EF) scale became operational, through 2013. The method uses the fraction of the tornado path experiencing EF damage and a characteristic wind speed for each EF rating . Since tThe SPC maintains the most readily available tornado database in the world. Tornado reports in the database extend back to 1950. Relevant to this study per-tornado information includes occurrence time, location, damage rating, path width, path length, injuries, fatalities, and property loss. Here all tornadoes between 2007 and 2013 are used. The start year of 2007 coincides with the adoption of the EF Scale by the NWS.−1 and EF4 damage corresponds to wind speeds between 75 and 89 m s−1 .Post storm surveys of the destruction in the wake of a tornado allow engineers to rate the damage on a scale from zero to five. Historically the damage scale was related physically to the tornado wind speed , 12. TodA tornado’s EF rating is assigned based on the greatest damage observed within the tornado’s path . The EF Estimates of tornado energy are not directly available in the database. The goal of this paper is to improve the understanding of tornado climatology through two objectives. The first is to use the available information to compute total kinetic energy of each tornado in the database. The second is to use the estimated kinetic energy as a basis for an alternative tornado climatology involving energy rather than frequency.J is the highest EF rating, vj is the midpoint wind speed for each rating , wj is the corresponding fraction of path area, and m is the tornado mass, which is estimated as air density (1 kg m−3) times the volume . The tornado’s height and the density of air are held constant, thus m is only a function of total path area.The main idea of this paper is to distinguish individual tornadoes based on a metric of destruction that has physical units of energy. Estimates of tornado energy have been made previously following the formula in Schielicke and Névir . In part−1 (7.5 m s−1 above the threshold wind speed consistent with the EF4 midpoint speed relative to its threshold). It is acknowledged that tornado height varies considerably perhaps by as much as a factor of 10 or more, but without other information it is fixed at 1 km.Since there is no upper bound on the EF5 wind speeds, the midpoint wind speed is set at 97 m sSince tornado-specific fractions of path area by EF rating are not available in the SPC database, here the NRC model of the fractions is used instead see . The perThe NRC model combines a Rankine vortex with empirical estimates to determine the fraction of path area associated with each EF rating . Fricker12 joules), five percent have TKE exceeding 5.53 TJ and one percent have TKE exceeding 31.9 TJ. The tornado with the most energy is the Tallulah-Yazoo City-Durant tornado of April 24, 2010 with a TKE of 516.7 TJ. It tracked over 240 km from Louisiana through Mississippi with a width of 2.82 km at its widest. It resulted in ten fatalities and 146 injuries. The most severe damage (EF4) occurred in the Mississippi counties of Yazoo and Holmes.TKE is computed for all 8752 tornadoes in the SPC database over the period 2007–2013. There are many more weak tornadoes (EF0) than strong ones (EF2 and higher) so the values are highly skewed with a median TKE of 62.1 gigajoules (GJ) and a interquartile range between 9.1 and 383.2 GJ. On a logarithmic scale the distribution is symmetric . Ten perJ is the highest EF rating and A is the path area estimated by multiplying the path length by the path width. Path width is provided in the SPC database as the maximum width across the tornado path. Per-tornado TDI is computed following Agee and Childs [vJ is the midpoint wind speed for the highest EF rating (J) and W is the path width. Note that TDI and DPI are metrics that have never previously been computed on a per tornado basis.The method to estimate TKE is validated by comparing resulting values with other indexes of tornado destruction and by correlating TKE with casualties and loss variables. This is done on a per-tornado basis. Per-tornado DPI is computed following Thompson and Vescio  asDPI=Ad Childs  asTDI=.A rank of the top ten tornadoes with the most energy shows the tight relationship between TKE and DPI . The topThe top five days with the most energy match the top five days with the highest DPI while the next five days of high energy match the next five high DPI days but in slightly different order. Seven of the top TDI days are in the top ten days with the most energy but not in the same order with the exception of April 27, 2011, which has the largest TDI, DPI, and TKE values.Another validation of TKE as a useful measure of destruction is available by correlating it with fatalities, injuries, and losses . TKE is In the previous section TKE as a metric of tornado destruction is defined with units of energy. Then it is shown that TKE has the same or greater explanatory power  as earlier indexes of destruction including DPI and TDI. Here variations in TKE are examined. Since energy is extensive, the per-tornado TKE values can be summed and averaged. For instance over the period 2007–2013 the average energy of the nine EF5 tornadoes is just over 100 TJ . The ave15 joules) or four times the energy of the next most energetic day  for the 8752 tornadoes in the SPC database over the period 2007–2013. The main idea is to distinguish individual tornadoes based on a variable of destruction that has physical units of energy. The method uses the fraction of the tornado path experiencing EF damage and the midpoint wind speed for each EF rating. The fraction of the path is obtained from a model developed for the NRC that combines theoretical considerations with empirical data. An earlier study showed estimates of TKE from the model matched well those computed from recent tornado observations.The TKE is validated as a useful metric of destruction by comparing it to other destructive indexes in the literature including the DPI and TDI. Additional validation is demonstrated by correlating it to casualties and loss estimates. TKE is highly correlated with DPI since they are both sensitive to path length and width in the same way (path area). As such TKE offers no statistical advantage over DPI as a single indicator of destruction for individual tornadoes. However, since TKE is based on physical theory and has units of energy it can be more easily understood by scientists outside of meteorology. Moreover, TKE has units of Joules  and can be directly compared with environmental energy variables like CAPE and helicity, which have the same units of J/kg. This is important toward the goal of improving understanding of the relationship between tornadoes and their parent-storm environment. Moreover, TKE can be spatially dis-aggregated. For example, if interest lies in the portion of energy within a county from a tornado passing through several counties, then the county-level TKE can be computed based on the contours by EF rating. In this way maps of TKE by area can be drawn accumulated by month, year, and decades.The per-tornado TKE values are summed and averaged to produce an alternative climatology of tornadoes based on energy rather than frequency. Half of all tornadoes have TKE that exceed 62.1 GJ and a quarter have TKE that exceed 383.2 GJ. Five percent of the tornadoes have TKE that exceed 5.53 TJ. The tornado with the greatest amount of energy is the long-tracked EF4 Tallulah-Yazoo City-Durant tornado of April 24, 2010 with a TKE of 516.7 TJ resulting in ten fatalities and 146 injuries. The single day with the most energy is April 27, 2011 with more than 2.6 PJ from 206 tornadoes.The average energy of the nine EF5 tornadoes is just over 100 TJ and the average energy of the 57 EF4 tornadoes is half of that at just over 50 TJ. The average energy of the EF3 tornadoes is less than half of the EF4 and the average energy of the EF2 tornadoes is much less than half of the EF3. Tornado energy peaks from mid to late spring similar to the peak in tornado frequency. Key new findings are that April has more energy than May with fewer tornadoes; March has more than June with half as many tornadoes. September is the month with the least energy but November and December are months with the fewest tornadoes.Another key finding is that Alabama ranks number one in terms of tornado energy with a total of 2.48 PJ followed by Oklahoma with 1.45 PJ. The next four states in decreasing order are Mississippi, Kansas, Arkansas, and Louisiana. On a per-tornado basis, for those states with more than 100 tornadoes, five of the top six states are in the South. The spatial pattern of TKE at this scale suggests a single tornado threat region encompassing both the traditional alley of the Great Plains and the Dixie alley of the South . The proFinally, recent research shows a future with more days when high wind shear coincides with high values of convective available potential energy  indicati


Current clinical practices used to functionally classify heart failure (HF) are time-consuming, expensive, or require complex calculations. This study aimed to design an inquiry list from the perspective of traditional Chinese medicine (TCM) that could be used in routine clinical practice to resolve these problems.The severity of documented HF in 115 patients was classified according to their performance in maximal exercise tests into New York Heart Association (NYHA) functional classification (FC) II or NYHA FC III. Concomitantly, the patients were assessed using the new TCM inquiry list and two validated quality of life questionnaires, namely, the Short Form 36 (SF-36) generic scale and the Minnesota Living with Heart Failure Questionnaire (MLHFQ). Factor analysis was applied to extract the core factors from the responses to the items in TCM inquiry list; logistic regression analysis was then used to predict the severity of HF according to the extracted factors.The TCM inquiry list showed moderate levels of correlation with the physical and emotional components of the SF-36 and the MLHFQ, and predicted the functional class of HF patients reliably using logistic regression analysis, with a correct prediction rate with 64.3 %. Factor analysis of the TCM inquiry list extracted five core factors, namely, Qi Depression, Heart Qi Vacuity and Blood Stasis, Heart Blood Vacuity, Dual Qi-Blood Vacuity, and Yang Vacuity, from the list, which aligned with the perspective of TCM as it relates to the pattern of HF. The correct prediction rate rose to 70.4 % when Dual Qi-Blood Vacuity was combined with the MLHFQ. The excessive false-negative rate is a problem associated with the TCM inquiry list.The TCM inquiry list is a simple scale and similar to patient-reported subjective measures of quality of life in HF, and may help to classify patients into NYHA FC II or NYHA FC III. Factor 4 addresses dizziness, dizzy vision and general weakness, which are critical parameters that distinguish between NYHA FC II and NYHA FC III. Incorporating these three items into the management of HF may help to classify patients from a functional perspective.The online version of this article (doi:10.1186/s12906-016-1306-7) contains supplementary material, which is available to authorized users. Heart failure (HF) is common and its impact on a patient’s quality of life (QoL) depends on its severity . FindingPhysicians who practice traditional Chinese medicine (TCM) use four diagnostic approaches, i.e., inspection, auscultation and olfaction, interrogation, and palpation to record patients’ information in relation to specific patterns , 4. TCM The aim of this study was to design an inquiry list from the perspective of TCM  that could be used in routine clinical practice to resolve the current problems associated with the FC of HF.The aim of this study was to design a TCM inquiry list that could be used in routine clinical practice to resolve the current problems associated with the FC of HF.This investigation was performed in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Chang Gung Memorial Hospital, Taiwan (103-1515B). All of the subjects provided written informed consent after receiving an explanation of the experimental procedures.A TCM inquiry list was created based on the signs and symptoms of HF found in textbooks and reports. Eligible candidates, who had been classified as NYHA FC II or NYHA FC III based on aerobic capacity during CPET, were enrolled to participate in pre-testing of the TCM inquiry list. These subjects were also assessed using the TCM inquiry list and two validated QoL questionnaires, namely, the SF-36 generic scale and the MLHFQ. The TCM inquiry list was validated using the SF-36 and MLHFQ, which are two accepted tools that measure patient-perceived symptom severity and QoL impairment, and have been used in previous studies of HF , 14. FinAn inquiry list was developed as a disease-specific questionnaire to be used when HF patients visit clinics to see TCM physicians. The TCM inquiry list was designed to be simple and self-explanatory for people without a medical background or medical knowledge. Furthermore, the patients were able to complete the questionnaire themselves with little assistance from medical professionals. Each item on the list allowed the patients to respond in a way that reflected the frequency of their symptoms, as follows: 1 = never, 2 = rarely, 3 = sometimes, 4 = frequently, and 5 = always, and in accordance with the severity of their symptoms, as follows: 1 = absent, 2 = mild, 3 = moderate, 4 = severe, and 5 = catastrophic, to facilitate evaluation of the different levels of severity. Data about all items potentially related to the TCM pattern were collected . The proArrindell et al.  proposed2), carbon dioxide production, peak exercise oxygen consumption (VO2 peak), cardiac output, and changes in tissue oxygenation , and the Δtotal hemoglobin concentration [ΔTHb]) in the left frontal cortex region and the vastus lateralis muscle, using standardized protocols previously described by Fu et al. [The baseline evaluation of each patient included completion of the preliminary TCM inquiry list, an MLHFQ, an SF-36 questionnaire, and a maximal exercise test using metabolic gas analysis , a non-invasive cardiac output monitor , and near infrared spectrometry  to measure the ventilatory and hemodynamic parameters, including minute ventilation, oxygen consumption VO, carbon t-test was used to compare quantitative variables between groups. The chi-squared test and Fisher’s Exact test were used to compare categorical variables between groups. Pearson’s correlation coefficient was used to determine the presence of correlations between scores on the TCM inquiry list and several parameters associated with functional capacity, including QoL and maximal exercise capacity. The inquiry list was investigated by factor analysis to explore possible patterns from the TCM perspective. The ability of the TCM inquiry list to predict HF from a functional perspective was evaluated using logistic regression analysis. Finally, the critical elements needed to distinguish between NYHA FC II and NYHA FC III in a population with HF were determined and possible correlations between these factors and latent physiologic traits were investigated. All of the statistical analyses were performed using IBM®SPSS® version 22.0 software .First, the patients’ anthropometric and exercise performance data were analyzed. An independent n = 66) and NYHA FC III (n = 49) groups according to their performance on the maximal exercise test. Patients in the NYHA FC II group had slight  limitations to their physical activity, and those in the NYHA FC III group had marked (>1.5 to ≤5 MET) limitations to their physical activity. The characteristics of the groups are shown in Table This study enrolled 115 HF patients who were divided into NYHA FC II (2 = 0.967). Both of these parameters demonstrated the stability of the TCM inquiry list.The TCM inquiry list showed a high level of internal consistency . The test-retest-reliability coefficient indicated excellent reliability , a moderate negative correlation with the physical component score (PCS) of the SF-36  and the mental component score (MCS) of the SF-36 , and a mild negative correlation with peak VO2 . These findings were consistent with the original design of the TCM inquiry list in which a higher score indicated greater severity. The rates at which each test correctly predicted NYHA FC II and NYHA FC III are shown in Table 2 achieved a correct prediction rate of 100 %, because patients were assigned to the NYHA FC II group or the FC III group based on their aerobic capacity. Correct prediction rates for the PCS of the SF-36, the MCS of the SF-36, the MLHFQ, and the TCM inquiry list were 63.5, 60.0, 60.0, and 64.3 %, respectively. The TCM inquiry list had the best prediction rate of all of the questionnaires used in this study.Table χ2 = 1363 (P < 0.0001), indicating the presence of significant correlations and reinforcing the relevance of the factor analysis.Exploratory factor analysis was conducted to scrutinize the TCM inquiry list and to identify any critical parameters in this list that might distinguish between NYHA FC II and NYHA FC III patients. A value of 0.864 on the Kaiser-Meyer-Olkin test indicated adequate correlation matrices. The Bartlett sphericity test was significant at Exploratory factor analysis extracted five factors without any limit on the possible number of factors in this analysis, for which five-factor varimax rotation explained 69 % of the total variance Table . All of P = 0.025; Table All of the factors on the TCM inquiry list were included in the regression model for prediction of NYHA FC. Factors 2 and 4 were incorporated into the prediction model, and only factor 4 yielded a statistically significant difference P = 0.02; Table 5P < 0.0001) when 11.5 was used as the cutoff value for this novel parameter, a value that had the greatest diagnostic power to distinguish between NYHA FC II and NYHA FC III  was largest when the PCS of the MLHFQ and the score for factor 4 on the TCM inquiry list were combined. The AUC was 0.740 , Heart Qi Vacuity and Blood Stasis (factor 2), Heart Blood Vacuity (factor 3), Dual Qi-Blood Vacuity factor 4), and Yang Vacuity factor 5), as shown in Table , and Yan, as showWhen the NYHA FC II and NYHA FC III categories were used as binary variables and the five TCM factors were entered into the logistic regression model to predict NYHA FC, only factor 4 was significantly associated with differences between NYHA FC II and NYHA FC III. Hence, factor 4 is the critical parameter from the perspective of TCM that can help to differentiate between NYHA FC II and NYHA FC III in a population with HF.The typical clinical manifestations of HF include dyspnea on exertion, nocturnal dyspnea, and leg swelling, and inquiries about these manifestations often comprise parts of the current questionnaires. Factor 4, which includes Q1 (dizziness), Q2 (dizzy vision), and Q11 , and hints at Dual Qi-Blood Vacuity, which is the key pattern associated with coronary artery disease in TCM , 8, comp2, peak workload, VO2 at the anaerobic threshold, oxygenated hemoglobin levels in muscle at the anaerobic threshold, total cerebral hemoglobin level at peak exercise, and the oxygen uptake efficiency slope, and very weak but significant positive correlations with cerebral and muscle deoxygenated hemoglobin levels during peak exercise. The deoxygenated state of the brain and the low cerebral blood flow during the peak exercise phase may be associated with dizziness and dizzy vision. The deoxygenated state of the muscles during the peak exercise phase may be associated with limb weakness. This indicates that the differences between NYHA FC II and NYHA FC III are associated with the blood supply to the brain and muscles, which is in agreement with previous studies [Table  studies . Cardiol2, the MLHFQ score, and the MCS were also noted. Poor correlations between MLHFQ score and CPET parameters have been described in a previous study [This study has several limitations. Our HF population had similar PCS scores, but lower MLHFQ and worse MCS scores than those reported by other researchers , 20. Weaus study ; howeverus study , 20, 22.us study , 24.Q13 was removed from the factor analysis. That means that there is room for improvement in the choice of items included in this inquiry list. The Q13 item was removed from the exploratory factor analysis because of insufficient loading (<0.5 or less) in all factors, or excessive loading (>0.5 or more) in an excessive number of factors (>2 or more). For our purposes, this implies that either Q13 is not an important or common item or that there are surrogate items for Q13 that can be used instead. Therefore, subjects may choose other items first. Deletion of Q13 in this study did not change the final result. Hence, it was reasonable to delete this item.The correlation coefficients demonstrated relationships between the factors, aerobic capacity, and QoL. We found that the factors in the TCM inquiry list had moderate correlations with the QoL questionnaires, but only weak correlations or no correlations at all with aerobic capacity. This may mean that aerobic capacity did not actually correlate with the TCM inquiry list and the QoL; hence, a non-linear relationship may have existed between QoL and the symptoms associated with the TCM inquiry list. Therefore, further analysis of the data is needed. In addition, the sample size in this study was barely adequate for development of a questionnaire, but should not be considered unsatisfactory . MoreoveThe TCM inquiry list is a simple scale and similar to patient-reported subjective measures of quality of life in HF, and may help to classify patients into NYHA FC II or NYHA FC III. Factor 4 addresses dizziness, dizzy vision and general weakness, which are critical parameters that distinguish between NYHA FC II and NYHA FC III. Incorporating these three items into the management of HF may help to classify patients from a functional perspective.