A conjugated acceptor-donor-acceptor (A-?-D-?-A) with the Zn-porphyrin core and the di-cyanovinyl substituted thiophene (A) connected at meso positions denoted as was designed and synthesized. The optical and electrochemical properties of were investigated. This new porphyrin exhibits a broad and intense absorption in the visible and near infrared regions. Bulk-heterojunction (BHJ) solution processed organic solar cells based on this porphyrin, as electron donor material, and PC71BM ([6,6]-phenyl C71 butyric acid methyl ester), as electron acceptor material, were fabricated using THF and a pyridine-THF solvent exhibiting a power conversion efficiency of 3.65% and 5.24%, respectively. The difference in efficiencies is due to the enhancement of the short circuit current Jsc and FF of the solar cell, which is ascribed to a stronger and broader incident photon to current efficiency (IPCE) response and a better balanced charge transport in the device processed with the pyridine-THF solvent.
Abstract Remudy (Registry of Muscular Dystrophy), operated by NCNP (the National Center of Neurology and Psychiatry), Japan, runs two national registries for Dystrophinopathy and GNE myopathy in collaboration with the TREAT-NMD (Translational Research in Europe--Assessment and Treatment of Neuromuscular Disease) alliance. The aim of Remudy is to construct a clinical research infrastructure that accelerates the pace of clinical development research for these rare diseases. We successfully provide data sets for feasibility studies, send out appropriate information about clinical trials to speed up the recruitment of candidates, as well as present the natural history and epidemiology data of these rare diseases using a new 'registry based' research style. Remudy presents a prototype model of the clinical research infrastructure to overcome these rare and incurable diseases.
Dolichol phosphate (Dol-P) serves as a carrier of complex polysaccharides during protein glycosylation. Dol-P is synthesized by the phosphorylation of dolichol or the monodephosphorylation of dolichol pyrophosphate (Dol-PP); however, the enzymes that catalyze these reactions remain unidentified in Arabidopsis thaliana. We performed a genome-wide search for cytidylyltransferase motif-containing proteins in Arabidopsis and found that At3g45040 encodes a protein homologous with Sec59p, a dolichol kinase (DOK) in Saccharomyces cerevisiae. At3g45040, designated AtDOK1, complemented defects in the growth and N-linked glycosylation of the S. cerevisiae sec59 mutant, suggesting that AtDOK1 encodes a functional DOK. To characterize the physiological roles of AtDOK1 in planta, we isolated two independent lines of T-DNA-tagged AtDOK1 mutants, dok1-1 and dok1-2. The heterozygous plants showed developmental defects in male and female gametophytes, including an aberrant pollen structure, low pollen viability, and short siliques. Additionally, the mutations had incomplete penetrance. These results suggest that AtDOK1 is a functional DOK required for reproductive processes in Arabidopsis. This article is protected by copyright. All rights reserved.
Simian hemorrhagic fever virus (SHFV) causes a severe and almost uniformly fatal viral hemorrhagic fever in Asian macaques, but is thought to be nonpathogenic for humans. To date, the SHFV lifecycle is almost completely uncharacterized on the molecular level. Here we describe the first steps of the SHFV lifecycle. Our experiments indicate that SHFV enters target cells by low pH-dependent endocytosis. Dynamin inhibitors, chlorpromazine, methyl-?-cyclodextrin, chloroquine, and concanamycin A dramatically reduced SHFV entry efficiency, whereas the macropinocytosis inhibitors EIPA, blebbistatin, and wortmannin, and the caveolin-mediated endocytosis inhibitors nystatin and filipin III had no effect. Furthermore, overexpression and knock-out study and electron-microscopy results indicate that SHFV entry occurs by a dynamin-dependent clathrin-mediated endocytosis-like pathway. Experiments utilizing latrunculin B, cytochalasin B, and cytochalasin D indicate that SHFV does not hijack the actin polymerization pathway. Treatment of target cells with proteases (proteinase K, papain, ?-chymotrypsin, trypsin) abrogated entry, indicating that the SHFV cell-surface receptor is a protein. Phospholipases A2 and D had no effect on SHFV entry. Finally, treatment of cells with antibodies targeting CD163, a cell surface molecule identified as an entry factor for the SHFV-related porcine reproductive and respiratory syndrome virus, diminished SHFV replication, identifying CD163 as an important SHFV entry component.
2-(2'-Hydroxyphenyl)benzoxazole (HBO) is known for undergoing intramolecular proton transfer in the excited state to result in the emission of its tautomer. A minor long-wavelength absorption band in the range of 370-420 nm has been reported in highly polar solvents such as dimethylsulfoxide (DMSO). Yet the nature of this species has not been entirely clarified. In this work, we provide evidence that this long-wavelength absorption band might have been caused by base or metal salt impurities that are introduced into the spectral sample during solvent transport using glass Pasteur pipettes. The contamination by base or metal salt could be avoided by using borosilicate glass syringes or non-glass pipettes in sample handling. Quantum chemical calculations conclude that solvent-mediated deprotonation is too energetically costly to occur without the aid of a base of an adequate strength. In the presence of such a base, the deprotonation of HBO and its effect on emission are investigated in dichloromethane and DMSO, the latter of which facilitates deprotonation much more readily than the former. Finally, the absorption and emission spectra of HBO in 13 solvents are reported, from which it is concluded that ESIPT is hindered in polar solvents that are also strong hydrogen bond acceptors. This article is protected by copyright. All rights reserved.
The acid-base chemistry that drives catalysis in pyridoxal-5'-phosphate (PLP)-dependent enzymes has been the subject of intense interest and investigation since the initial identification of PLP's role as a coenzyme in this extensive class of enzymes. It was first proposed over 50 years ago that the initial step in the catalytic cycle is facilitated by a protonated Schiff base form of the holoenzyme in which the linking lysine ?-imine nitrogen, which covalently binds the coenzyme, is protonated. Here we provide the first (15)N NMR chemical shift measurements of such a Schiff base linkage in the resting holoenzyme form, the internal aldimine state of tryptophan synthase. Double-resonance experiments confirm the assignment of the Schiff base nitrogen, and additional (13)C, (15)N, and (31)P chemical shift measurements of sites on the PLP coenzyme allow a detailed model of coenzyme protonation states to be established.
The lysine acetylation of proteins is a reversible posttranslational modification that plays a critical regulatory role in both eukaryotes and prokaryotes. Mycobacterium tuberculosis is a facultative intracellular pathogen and the causative agent of tuberculosis. Increasing evidence shows that lysine acetylation may play an important role in the pathogenesis of M. tuberculosis. However, only a few acetylated proteins of M. tuberculosis are known, presenting a major obstacle to understanding the functional roles of reversible lysine acetylation in this pathogen. We performed a global acetylome analysis of M. tuberculosis H37Ra by combining protein/peptide prefractionation, antibody enrichment, and LC-MS/MS. In total, we identified 226 acetylation sites in 137 proteins of M. tuberculosis H37Ra. The identified acetylated proteins were functionally categorized into an interaction map and shown to be involved in various biological processes. Consistent with previous reports, a large proportion of the acetylation sites were present on proteins involved in glycolysis/gluconeogenesis, the citrate cycle, and fatty acid metabolism. A NAD+-dependent deacetylase (MRA_1161) deletion mutant of M. tuberculosis H37Ra was constructed and its characterization showed a different colony morphology, reduced biofilm formation, and increased tolerance of heat stress. Interestingly, lysine acetylation was found, for the first time, to block the immunogenicity of a peptide derived from a known immunogen, HspX, suggesting that lysine acetylation plays a regulatory role in immunogenicity. Our data provide the first global survey of lysine acetylation in M. tuberculosis. The dataset should be an important resource for the functional analysis of lysine acetylation in M. tuberculosis and facilitate the clarification of the entire metabolic networks of this life-threatening pathogen.
Children below the age of six months suffer less often from malaria than older children in sub-Saharan Africa. This observation is commonly attributed to the persistence of foetal haemoglobin (HbF), which is considered not to permit growth of Plasmodium falciparum and therefore providing protection against malaria. Since this concept has recently been challenged, this study evaluated the effect of HbF erythrocytes and maternal plasma on in vitro parasite growth of P. falciparum in Central African Gabon.
Au25(SR)18 has provided fundamental insights into the properties of clusters protected by monolayers of thiolated ligands (SR). Because of its ultrasmall core, 1 nm, Au25(SR)18 displays molecular behavior. We prepared a Au25 cluster capped by n-butanethiolates (SBu), obtained its structure by single-crystal X-ray crystallography, and studied its properties both experimentally and theoretically. Whereas in solution Au25(SBu)18(0) is a paramagnetic molecule, in the crystal it becomes a linear polymer of Au25 clusters connected via single Au-Au bonds and stabilized by proper orientation of clusters and interdigitation of ligands. At low temperature, [Au25(SBu)18(0)]n has a nonmagnetic ground state and can be described as a one-dimensional antiferromagnetic system. These findings provide a breakthrough into the properties and possible solid-state applications of molecular gold nanowires.
Accurate allele frequencies are important for measuring subclonal heterogeneity and clonal evolution. Deep-targeted sequencing data can contain PCR duplicates, inflating perceived read depth. Here we adapted the Illumina TruSeq Custom Amplicon kit to include single molecule tagging (SMT) and show that SMT-identified duplicates arise from PCR. We demonstrate that retention of PCR duplicate reads can imply clonal evolution when none exists, while their removal effectively controls the false positive rate. Additionally, PCR duplicates alter estimates of subclonal heterogeneity in tumor samples. Our method simplifies PCR duplicate identification and emphasizes their removal in studies of tumor heterogeneity and clonal evolution.
For living deep-tissue imaging, the optical window favorable for light penetration is in near-infrared wavelengths, which requires fluorescent proteins with emission spectra in the near-infrared region. Here, we report that a single mutant Ser28His of mNeptune with a near-infrared (?650 nm) emission maxima of 652 nm is found to improve the brightness, photostability, and pH stability when compared with its parental protein mNeptune, while it remains as a monomer, demonstrating that there is still plenty of room to improve the performance of the existing near infrared fluorescence proteins by directed evolution.
Despite 20 years of progress in synthesizing thiolated gold nanoclusters (Au NCs), the knowledge of their growth mechanism still lags behind. Herein the detailed process from reduction of Au(I)-thiolate complex precursors to the eventual evolution of and focusing to the atomically precise Au25 NCs was revealed for the first time by monitoring the time evolution of Au(I) precursor and Au NC intermediate species with ESI-MS. A two-stage, bottom-up formation and growth process was proposed: a fast stage of reduction-growth mechanism, followed by a slow stage of intercluster conversion and focusing. Balanced reactions of formation for each identified NC were suggested, backed by theoretical calculations of the thermodynamic driving force. This work advances one step further toward understanding the mechanism of formation and growth of thiolated Au NCs.
Detection of Bacillus anthracis in the field, whether as a natural infection or as a biothreat remains challenging. Here we have developed a new lateral-flow immunochromatographic assay (LFIA) for B. anthracis spore detection based on the fact that conjugates of B. anthracis spores and super-paramagnetic particles labeled with antibodies will block the pores of chromatographic strips and form retention lines on the strips, instead of the conventionally reported test lines and control lines in classic LFIA. As a result, this new LFIA can simultaneously realize optical, magnetic and naked-eye detection by analyzing signals from the retention lines. As few as 500-700 pure B. anthracis spores can be recognized with CV values less than 8.31% within 5min of chromatography and a total time of 20min. For powdery sample tests, this LFIA can endure interference from 25% (w/v) milk, 10% (w/v) baking soda and 10% (w/v) starch without any sample pre-treatment, and has a corresponding detection limit of 6×10(4) spores/g milk powder, 2×10(5) spores/g starch and 5×10(5) spores/g baking soda. Compared with existing methods, this new approach is very competitive in terms of sensitivity, specificity, cost and ease of operation. This proof-of-concept study can also be extended for detection of many other large-sized analytes.
Purpose:3-Methylcrotonyl-CoA carboxylase deficiency (MCCD) is an autosomal recessive disorder of leucine catabolism that has a highly variable clinical phenotype, ranging from acute metabolic acidosis to nonspecific symptoms such as developmental delay, failure to thrive, hemiparesis, muscular hypotonia, and multiple sclerosis. Implementation of newborn screening for MCCD has resulted in broadening the range of phenotypic expression to include asymptomatic adults. The purpose of this study was to identify factors underlying the varying phenotypes of MCCD.Methods:We performed exome sequencing on DNA from 33 cases and 108 healthy controls. We examined these data for associations between either MCC mutational status, genetic ancestry, or consanguinity and the absence or presence/specificity of clinical symptoms in MCCD cases.Results:We determined that individuals with nonspecific clinical phenotypes are highly inbred compared with cases that are asymptomatic and healthy controls. For 5 of these 10 individuals, we discovered a homozygous damaging mutation in a disease gene that is likely to underlie their nonspecific clinical phenotypes previously attributed to MCCD.Conclusion:Our study shows that nonspecific phenotypes attributed to MCCD are associated with consanguinity and are likely not due to mutations in the MCC enzyme but result from rare homozygous mutations in other disease genes.Genet Med advance online publication 06 November 2014Genetics in Medicine (2014); doi:10.1038/gim.2014.157.
With the initial motivation of optimizing hydrogen storage in beryllium nanocrystals, we have thoroughly and systematically studied the structural, cohesive, and electronic properties of Ben and BenHxn (n = 2-160, x = 0.1-2.4) nanoparticles as a function of both size (n) and hydrogen content (x), using density functional theory with a properly selected meta-hybrid functional and high level coupled cluster CCSD(T) theory for comparison. We have calculated the binding energies of Ben, BenHxn and [BeH2]n nanoparticles for a large range of n values. In the limit n??, we have obtained the experimental binding energy of a Be crystal (3.32 eV) with unexpectedly very good agreement (3.26 ± 0.06 eV), and a predicted value of 7.85 eV ± 0.02 eV for the binding energy of the [BeH2]? infinite system. We also predict that the majority of the lowest energy stoichiometric BenH2n nanoparticles are chains or chain-like structures. The tendency towards chain stabilization of BenHxn nanoparticles increases, as x approaches the stoichiometric value x = 2, leading for large values of n, as n??, to polymeric forms of bulk BeH2, which in the past have been considered as the leading forms of solid BeH2. For such 1-dimensional forms of [BeH2]n we have obtained and verified that the binding energy varies exactly proportionally to n(-1). The extrapolated desorption energy for such polymeric forms of solid BeH2 is found to be 19 ± 3 kJ mol(-1) in juxtaposition to the experimental value of 19 kJ mol(-1) for solid BeH2, suggesting that the difference ?E in cohesive energy between the orthorhombic and polymeric form is very small (?E? 3 kJ mol(-1)). This is in full accord with the early discrepancies in the literature in determining and distinguishing the real crystal structure of solid BeH2.
GNE myopathy is a slowly progressive autosomal recessive myopathy caused by mutations in the GNE (glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase) gene. This study aimed to (1) develop a nationwide patient registry for GNE myopathy in order to facilitate the planning of clinical trials and recruitment of candidates, and (2) gain further insight into the disease for the purpose of improving therapy and care.
Imaging of protein-protein and RNA-protein interactions in vivo, especially in live animals, is still challenging. Here we developed far-red mNeptune-based bimolecular fluorescence complementation (BiFC) and trimolecular fluorescence complementation (TriFC) systems with excitation and emission above 600 nm in the 'tissue optical window' for imaging of protein-protein and RNA-protein interactions in live cells and mice. The far-red mNeptune BiFC was first built by selecting appropriate split mNeptune fragments, and then the mNeptune-TriFC system was built based on the mNeptune-BiFC system. The newly constructed mNeptune BiFC and TriFC systems were verified as useful tools for imaging protein-protein and mRNA-protein interactions, respectively, in live cells and mice. We then used the new mNeptune-TriFC system to investigate the interactions between human polypyrimidine-tract-binding protein (PTB) and HIV-1 mRNA elements as PTB may participate in HIV mRNA processing in HIV activation from latency. An interaction between PTB and the 3'long terminal repeat region of HIV-1 mRNAs was found and imaged in live cells and mice, implying a role for PTB in regulating HIV-1 mRNA processing. The study provides new tools for in vivo imaging of RNA-protein and protein-protein interactions, and adds new insight into the mechanism of HIV-1 mRNA processing.
Developing chimeric lysins with a wide lytic spectrum would be important for treating some infections caused by multiple pathogenic bacteria. In the present work, a novel chimeric lysin (Ply187N-V12C) was constructed by fusing the catalytic domain (Ply187N) of the bacteriophage lysin Ply187 with the cell binding domain (146-314aa, V12C) of the lysin PlyV12. The results showed that the chimeric lysin Ply187N-V12C had not only lytic activity similar to Ply187N against staphylococcal strains but also extended its lytic activity to streptococci and enterococci, such as Streptococcus?dysgalactiae, Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecium and Enterococcus faecalis, which Ply187N could not lyse. Our work demonstrated that generating novel chimeric lysins with an extended lytic spectrum was feasible through fusing a catalytic domain with a cell-binding domain from lysins with lytic spectra across multiple genera.
Proteins can be viewed as small-world networks of amino acid residues connected through noncovalent interactions. Nuclear magnetic resonance chemical shift covariance analyses were used to identify long-range amino acid networks in the ? subunit of tryptophan synthase both for the resting state (in the absence of substrate and product) and for the working state (during catalytic turnover). The amino acid networks observed stretch from the surface of the protein into the active site and are different between the resting and working states. Modification of surface residues on the network alters the structural dynamics of active-site residues over 25 Å away and leads to changes in catalytic rates. These findings demonstrate that amino acid networks, similar to those studied here, are likely important for coordinating structural changes necessary for enzyme function and regulation.
We examined the microRNAs (miRNAs) expressed in chronic lymphocytic leukemia (CLL) and identified miR-150 as the most abundant, but with leukemia cell expression levels that varied among patients. CLL cells that expressed ?-chain-associated protein of 70 kDa (ZAP-70) or that used unmutated immunoglobulin heavy chain variable (IGHV) genes, each had a median expression level of miR-150 that was significantly lower than that of ZAP-70-negative CLL cells or those that used mutated IGHV genes. In samples stratified for expression of miR-150, CLL cells with low-level miR-150 expressed relatively higher levels of forkhead box P1 (FOXP1) and GRB2-associated binding protein 1 (GAB1), genes with 3' untranslated regions having evolutionary-conserved binding sites for miR-150. High-level expression of miR-150 could repress expression of these genes, which encode proteins that enhance B-cell receptor signaling, a putative CLL-growth/survival signal. Also, high-level expression of miR-150 was a significant independent predictor of longer treatment-free survival or overall survival, whereas an inverse association was observed for high-level expression of GAB1 or FOXP1 for overall survival. This study demonstrates that expression of miR-150 can influence the relative expression of GAB1 and FOXP1 and the signaling potential of the B-cell receptor, thereby possibly accounting for the noted association of expression of miR-150 and disease outcome.
Bacteria like Escherichia coli can use propionate as sole carbon and energy source. All pathways for degradation of propionate start with propionyl-CoA. However, pathways of propionyl-CoA synthesis from propionate and their regulation mechanisms have not been carefully examined in E. coli. In this study, roles of the acetyl-CoA synthetase encoding gene acs and the NAD(+)-dependent protein deacetylase encoding gene cobB on propionate utilization in E. coli were investigated. Results from biochemical analysis showed that, reversible acetylation also modulates the propionyl-CoA synthetase activity of Acs. Subsequent genetic analysis revealed that, deletion of acs in E. coli results in blockage of propionate utilization, suggesting that acs is essential for propionate utilization in E. coli. Besides, deletion of cobB in E. coli also results in growth defect, but only under lower concentrations of propionate (5mM and 10mM propionate), suggesting the existence of other propionyl-CoA synthesis pathways. In combination with previous observations, our data implies that, for propionate utilization in E. coli, a primary amount of propionyl-CoA seems to be required, which is synthesized by Acs.
Small RNAs (sRNAs) are loaded into ARGONAUTE (AGO) proteins to induce gene silencing. In plants, the 5'-terminal nucleotide is important for sRNA sorting into different AGOs. Here we show that microRNA (miRNA) duplex structure also contributes to miRNA sorting. Base pairing at the 15th nucleotide of a miRNA duplex is important for miRNA sorting in both Arabidopsis AGO1 and AGO2. AGO2 favours miRNA duplexes with no middle mismatches, whereas AGO1 tolerates, or prefers, duplexes with central mismatches. AGO structure modelling and mutational analyses reveal that the QF-V motif within the conserved PIWI domain contributes to recognition of base pairing at the 15th nucleotide of a duplex, while the DDDE catalytic core of AtAGO2 is important for recognition of the central nucleotides. Finally, we rescued the adaxialized phenotype of ago1-12, which is largely due to miR165 loss-of-function, by changing miR165 duplex structure which we predict redirects it to AGO2.
Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is well established as a key regulatory posttranslational modification used in signal transduction to control cell growth, proliferation, and stress responses. However, little is known about its extent and function in diatoms. Phaeodactylum tricornutum is a unicellular marine diatom that has been used as a model organism for research on diatom molecular biology. Although more than 1000 protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues have been predicted in P. tricornutum, no phosphorylation event has so far been revealed by classical biochemical approaches. Here, we performed a global phosphoproteomic analysis combining protein/peptide fractionation, TiO(2) enrichment, and LC-MS/MS analyses. In total, we identified 264 unique phosphopeptides, including 434 in vivo phosphorylated sites on 245 phosphoproteins. The phosphorylated proteins were implicated in the regulation of diverse biological processes, including signaling, metabolic pathways, and stress responses. Six identified phosphoproteins were further validated by Western blotting using phospho-specific antibodies. The functions of these proteins are discussed in the context of signal transduction networks in P. tricornutum. Our results advance the current understanding of diatom biology and will be useful for elucidating the phosphor-relay signaling networks in this model diatom.
Genotypes generated in next generation sequencing studies contain errors which can significantly impact the power to detect signals in common and rare variant association tests. These genotyping errors are not explicitly filtered by the standard GATK Variant Quality Score Recalibration (VQSR) tool and thus remain a source of errors in whole exome sequencing (WES) projects that follow GATK's recommended best practices. Therefore, additional data filtering methods are required to effectively remove these errors before performing association analyses with complex phenotypes. Here we empirically derive thresholds for genotype and variant filters that, when used in conjunction with the VQSR tool, achieve higher data quality than when using VQSR alone.
New Delhi metallo-?-lactamase (NDM)-producing bacteria are considered potential global health threats. It is necessary to monitor NDM-1 and its variants in clinical isolates in order to understand the NDM-1 epidemic and the impact of its variants on ?-lactam resistance. To reduce the lengthy time needed for cloning and expression of NDM-1 variants, a novel PCR-based in vitro protein expression (PCR-P) method was used to detect blaNDM-1 and its variants coding for carbapenemases with different activities (functional variants). The PCR-P method combined a long-fragment real-time quantitative PCR (LF-qPCR) with in vitro cell-free expression to convert the blaNDM-1 amplicons into NDM for carbapenemase assay. The method could screen for blaNDM-1 within 3 h with a detection limit of 5 copies and identify functional variants within 1 day. Using the PCR-P to analyze 5 recent blaNDM-1 variants, 2 functional variants, blaNDM-4 and blaNDM-5, were revealed. In the initial testing of 23 clinical isolates, the PCR-P assay correctly found 8 isolates containing blaNDM-1. This novel method provides the first integrated approach for rapidly detecting the full-length blaNDM-1 and revealing its functional variants in clinical isolates.
Hepatic insulin resistance is a key contributor to the pathogenesis of obesity and type 2 diabetes (T2D). Paradoxically, the development of insulin resistance in the liver is not universal, but pathway selective, such that insulin fails to suppress gluconeogenesis but promotes lipogenesis, contributing to the hyperglycemia, steatosis, and hypertriglyceridemia that underpin the deteriorating glucose control and microvascular complications in T2D. The molecular basis for the pathway-specific insulin resistance remains unknown. Here we report that oxidative stress accompanying obesity inactivates protein-tyrosine phosphatases (PTPs) in the liver to activate select signaling pathways that exacerbate disease progression. In obese mice, hepatic PTPN2 (TCPTP) inactivation promoted lipogenesis and steatosis and insulin-STAT-5 signaling. The enhanced STAT-5 signaling increased hepatic IGF-1 production, which suppressed central growth hormone release and exacerbated the development of obesity and T2D. Our studies define a mechanism for the development of selective insulin resistance with wide-ranging implications for diseases characterized by oxidative stress.
Exfoliated monolayer graphene flakes were embedded in a polymer matrix and loaded under axial compression. By monitoring the shifts of the 2D Raman phonons of rectangular flakes of various sizes under load, the critical strain to failure was determined. Prior to loading care was taken for the examined area of the flake to be free of residual stresses. The critical strain values for first failure were found to be independent of flake size at a mean value of -0.60% corresponding to a yield stress up to -6 GPa. By combining Euler mechanics with a Winkler approach, we show that unlike buckling in air, the presence of the polymer constraint results in graphene buckling at a fixed value of strain with an estimated wrinkle wavelength of the order of 1-2 nm. These results were compared with DFT computations performed on analogue coronene/PMMA oligomers and a reasonable agreement was obtained.
Blood pressure (BP) is a heritable risk factor for cardiovascular disease. To investigate genetic associations with systolic BP (SBP), diastolic BP (DBP), mean arterial pressure (MAP), and pulse pressure (PP), we genotyped ~50,000 SNPs in up to 87,736 individuals of European ancestry and combined these in a meta-analysis. We replicated findings in an independent set of 68,368 individuals of European ancestry. Our analyses identified 11 previously undescribed associations in independent loci containing 31 genes including PDE1A, HLA-DQB1, CDK6, PRKAG2, VCL, H19, NUCB2, RELA, HOXC@ complex, FBN1, and NFAT5 at the Bonferroni-corrected array-wide significance threshold (p < 6 × 10(-7)) and confirmed 27 previously reported associations. Bioinformatic analysis of the 11 loci provided support for a putative role in hypertension of several genes, such as CDK6 and NUCB2. Analysis of potential pharmacological targets in databases of small molecules showed that ten of the genes are predicted to be a target for small molecules. In summary, we identified previously unknown loci associated with BP. Our findings extend our understanding of genes involved in BP regulation, which may provide new targets for therapeutic intervention or drug response stratification.
Axillary recurrence of breast cancer that involves the brachial neurovascular bundle is uncommon. However, for many patients with such recurrence, forequarter amputation can play a palliative role in relieving excruciating pain and paralysis of the upper limb. Further, for those patients who do not have distant metastasis or other local-regional recurrence, forequarter amputation provides a chance for a cure. Only a few case reports of curative amputations for recurrent breast cancer are present in the literature. Here, we report a case of forequarter amputation for curative treatment of axillary recurrent breast cancer, together with a literature review. To date, we have followed the patient for three years after amputation, during which there has been no evidence of recurrence or metastasis. Although radical resection is feasible, it can be accompanied by surgical wound complications and psychosocial stress. Therefore, an organized multidisciplinary approach is needed to ensure the success of radical resection.
Revealing the processes of ligand-protein associations deepens our understanding of molecular recognition and binding kinetics. Hydrogen bonds (H-bonds) play a crucial role in optimizing ligand-protein interactions and ligand specificity. In addition to the formation of stable H-bonds in the final bound state, the formation of transient H-bonds during binding processes contributes binding kinetics that define a ligand as a fast or slow binder, which also affects drug action. However, the effect of forming the transient H-bonds on the kinetic properties is little understood. Guided by results from coarse-grained Brownian dynamics simulations, we used classical molecular dynamics simulations in an implicit solvent model and accelerated molecular dynamics simulations in explicit waters to show that the position and distribution of the H-bond donor or acceptor of a drug result in switching intermolecular and intramolecular H-bond pairs during ligand recognition processes. We studied two major types of HIV-1 protease ligands: a fast binder, xk263, and a slow binder, ritonavir. The slow association rate in ritonavir can be attributed to increased flexibility of ritonavir, which yields multistep transitions and stepwise entering patterns and the formation and breaking of complex H-bond pairs during the binding process. This model suggests the importance of conversions of spatiotemporal H-bonds during the association of ligands and proteins, which helps in designing inhibitors with preferred binding kinetics.
The National Children's Study (NCS) is a prospective epidemiological study in the USA tasked with identifying a nationally representative sample of 100,000 children, and following them from their gestation until they are 21 years of age. The objective of the study is to measure environmental and genetic influences on growth, development, and health. Determination of the ancestry of these NCS participants is important for assessing the diversity of study participants and for examining the effect of ancestry on various health outcomes.
Protein adsorption on nanoparticles is closely associated with the physicochemical properties of particles, in particular, their surface properties. We synthesized two batches of polyacrylic acid-coated nanoparticles under almost identical conditions except for the heating duration and found differences in the head-group structure of the polyacrylic acid. The structure change was confirmed by NMR and MS. The two batches of particles had varied binding affinities to a selected group of proteins. Computational work confirmed that the head group of the polymer on the surface of a nanoparticle could directly interact with a protein, and small structural changes in the head group were sufficient to result in a significant difference in the free energy of binding. Our results demonstrate that protein adsorption is so sensitive to the surface properties of particles that it can reveal even small variations in the structure of a nanoparticle surface ligand, and should be useful for quick assessment of nanoparticle properties.
Few empirical studies have evaluated the mediating effects of quality of life (QoL) among people living with HIV/AIDS (PLWHA). The purposes of this study were to identify the predictors of QoL and to test the mediating effects of social support on depression and QoL among patients enrolled in an HIV case-management program in Taiwan. A cross-sectional, descriptive correlation design collected data from 108 HIV-infected individuals. Individuals were assessed using the Beck Depression Inventory II, the short version of the World Health Organization Quality of Life Assessment (WHOQOL-BREF), and the Multidimensional Scale of Perceived Social Support between September 2007 and April 2010. After adjusting for sociodemographic characteristics (including age, gender, and mode of transmission) and clinical information (including CD4 count and time since diagnosis with HIV), the study findings showed that QoL was significantly and positively correlated with both social support and the initiation of highly active antiretroviral therapy (HAART), and was negatively correlated with depression and time since diagnosis with HIV. The strongest predictors for QoL were depression followed by the initiation of HAART and social support, with an R(2) of 0.40. Social support partially mediated the relationship between depression and QoL. Health professionals should enhance HIV-infected individuals' social support to alleviate the level of depression and further increase the QoL among PLWHA.
Middle East respiratory syndrome coronavirus (MERS-CoV) is a recently isolated betacoronavirus identified as the etiologic agent of a frequently fatal disease in Western Asia, Middle East respiratory syndrome. Attempts to identify the natural reservoirs of MERS-CoV have focused in part on dromedaries. Bats are also suspected to be reservoirs based on frequent detection of other betacoronaviruses in these mammals. For this study, ten distinct cell lines derived from bats of divergent species were exposed to MERS-CoV. Plaque assays, immunofluorescence assays, and transmission electron microscopy confirmed that six bat cell lines can be productively infected. We found that the susceptibility or resistance of these bat cell lines directly correlates with the presence or absence of cell surface-expressed CD26/DPP4, the functional human receptor for MERS-CoV. Human anti-CD26/DPP4 antibodies inhibited infection of susceptible bat cells in a dose-dependent manner. Overexpression of human CD26/DPP4 receptor conferred MERS-CoV susceptibility to resistant bat cell lines. Finally, sequential passage of MERS-CoV in permissive bat cells established persistent infection with concomitant downregulation of CD26/DPP4 surface expression. Together, these results imply that bats indeed could be among the MERS-CoV host spectrum, and that cellular restriction of MERS-CoV is determined by CD26/DPP4 expression rather than by downstream restriction factors.
Loss of pancreatic beta cells is a feature of type-2 diabetes. High glucose concentrations induce endoplasmic reticulum (ER) and oxidative stress-mediated apoptosis of islet cells in vitro. ER stress, oxidative stress and high glucose concentrations may also activate the NLRP3 inflammasome leading to interleukin (IL)-1? production and caspase-1 dependent pyroptosis. However, whether IL-1? or intrinsic NLRP3 inflammasome activation contributes to beta cell death is controversial. This possibility was examined in mouse islets. Exposure of islets lacking functional NLRP3 or caspase-1 to H2O2, rotenone or thapsigargin induced similar cell death as in wild-type islets. This suggests that oxidative or ER stress do not cause inflammasome-mediated cell death. Similarly, deficiency of NLRP3 inflammasome components did not provide any protection from glucose, ribose or gluco-lipotoxicity. Finally, genetic activation of NLRP3 specifically in beta cells did not increase IL-1? production or cell death, even in response to glucolipotoxicity. Overall, our results show that glucose-, ER stress- or oxidative stress-induced cell death in islet cells is not dependent on intrinsic activation of the NLRP3 inflammasome.
The regulation of a series of cellular events requires specific protein-protein interactions, which are usually mediated by modular domains to precisely select a particular sequence from diverse partners. However, most signaling domains can bind to more than one peptide sequence. How do proteins create promiscuity from precision? Moreover, these complex interactions typically occur at the interface of a well-defined secondary structure, ? helix and ? sheet. However, the molecular recognition primarily controlled by loop architecture is not fully understood. To gain a deep understanding of binding selectivity and promiscuity by the conformation of loops, we chose the forkhead-associated (FHA) domain as our model system. The domain can bind to diverse peptides via various loops but only interact with sequences containing phosphothreonine (pThr). We applied molecular dynamics (MD) simulations for multiple free and bound FHA domains to study the changes in conformations and dynamics. Generally, FHA domains share a similar folding structure whereby the backbone holds the overall geometry and the variety of sidechain atoms of multiple loops creates a binding surface to target a specific partner. FHA domains determine the specificity of pThr by well-organized binding loops, which are rigid to define a phospho recognition site. The broad range of peptide recognition can be attributed to different arrangements of the loop interaction network. The moderate flexibility of the loop conformation can help access or exclude binding partners. Our work provides insights into molecular recognition in terms of binding specificity and promiscuity and helpful clues for further peptide design.
The mechanistic basis for resistance of Mycobacterium tuberculosis to para-aminosalicylic acid (PAS), an important agent in the treatment of multi-drug resistant tuberculosis, has yet to be fully defined. As a substrate analog of the folate precursor para-aminobenzoic acid, PAS is ultimately bioactivated to hydroxydihydrofolate which inhibits dihydrofolate reductase and disrupts operation of folate-dependent metabolic pathways. As a result, mutation of dihydrofolate synthase, an enzyme needed for bioactivation of PAS, causes PAS resistance in M. tuberculosis H37Rv. Here, we demonstrate that various missense mutations within the coding sequence of the dihydropteroate (H2Pte) binding pocket of dihydrofolate synthase (FolC) confer PAS resistance in laboratory isolates of M. tuberculosis and M. bovis. From a panel of 85 multi-drug resistant M. tuberculosis clinical isolates, 5 were found to harbor mutations in folC within the H2Pte binding pocket resulting in PAS resistance. While these alterations in the H2Pte binding pocket resulted in reduced dihydrofolate synthase activity, they also abolished bioactivation of hydroxydihydropteroate to hydroxydihydrofolate. Consistent with this model for abolished bioactivation, introduction of a wild type copy of folC fully restored PAS susceptibility in folC mutant strains. Confirmation of this novel PAS resistance mechanism will be beneficial for development of molecular based diagnostics for M. tuberculosis clinical isolates and for further defining the mode of action of this important tuberculosis drug.
Length of female reproductive lifespan is associated with multiple adverse outcomes, including breast cancer, cardiovascular disease and infertility. The biological processes that govern the timing of the beginning and end of reproductive life are not well understood. Genetic variants are known to contribute to about 50% of the variation in both age at menarche and menopause, but to date the known genes explain <15% of the genetic component. We have used genome-wide association in a bivariate meta-analysis of both traits, to identify genes involved in determining reproductive lifespan. We observed significant genetic correlation between the two traits using Genome-wide Complex Trait Analysis (GCTA). However, we found no robust statistical evidence for individual variants with an effect on both traits. A novel association with age at menopause was detected for a variant rs1800932 in the mismatch repair gene MSH6 (P=1.9x10(-9)), which was also associated with altered expression levels of MSH6 mRNA in multiple tissues. This study contributes to the growing evidence that DNA repair processes play a key role in ovarian ageing and could be an important therapeutic target for infertility.
We examined the relations between maternal depressive symptoms and child internalizing and externalizing problems in a sample of 125 adolescent Latina mothers (primarily Puerto Rican) and their toddlers. We also tested the influence of mother-reported partner child care involvement on child behavior problems and explored mother-reported partner characteristics that related to this involvement. Results suggested that maternal depressive symptoms related to child internalizing and externalizing problems when accounting for contextual risk factors. Importantly, these symptoms mediated the link between life stress and child behavior problems. Mother-reported partner child care interacted with maternal depressive symptoms for internalizing, not externalizing, problems. Specifically, depressive symptoms related less strongly to internalizing problems at higher levels of partner child care than at lower levels. Participants with younger partners, co-residing partners, and in longer romantic relationships reported higher partner child care involvement. Results are discussed considering implications for future research and interventions for mothers, their children, and their partners.
Testing the pyrazinamide (PZA) susceptibility of Mycobacterium tuberculosis isolates is challenging. In a previous paper, we described the development of a rapid colorimetric test for the PZA susceptibility of M. tuberculosis by a PCR-based in vitro-synthesized-pyrazinamidase (PZase) assay. Here, we present an integrated approach to detect M. tuberculosis and PZA susceptibility directly from sputum specimens. M. tuberculosis was detected first, using a novel long-fragment quantitative real-time PCR (LF-qPCR), which amplified a fragment containing the whole pncA gene. Then, the positive amplicons were sequenced to find mutations in the pncA gene. For new mutations not found in the Tuberculosis Drug Resistance Mutation Database (www.tbdreamdb.com), the in vitro PZase assay was used to test the PZA resistance. This approach could detect M. tuberculosis within 3 h with a detection limit of 7.8 copies/reaction and report the PZA susceptibility within 2 days. In an initial testing of 213 sputum specimens, the LF-qPCR found 53 positive samples with 92% sensitivity and 97% specificity compared to the culture test for M. tuberculosis detection. DNA sequencing of the LF-qPCR amplicons revealed that 49 samples were PZA susceptible and 1 was PZA resistant. In the remaining 3 samples, with new pncA mutations, the in vitro PZase assay found that 1 was PZA susceptible and 2 were PZA resistant. This integrated approach provides a rapid, efficient, and relatively low-cost solution for detecting M. tuberculosis and PZA susceptibility without culture.
The treatment of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) is a challenge worldwide. In our search for novel antimicrobial agents against MRSA, we constructed a chimeric lysin (named as ClyH) by fusing the catalytic domain of Ply187 (Pc) with the non-SH3b-like cell wall binding domain of phiNM3 lysin. Herein, the antimicrobial activity of ClyH against MRSA strains in vitro and in vivo was studied. Our results showed that ClyH could kill all of the tested clinical isolates of MRSA with higher efficacy than lysostaphin as well as its parental enzyme. The MICs of ClyH against clinical S. aureus strains were found to be as low as 0.05 to 1.61 mg/liter. In a mouse model, a single intraperitoneal administration of ClyH protected mice from death caused by MRSA, without obvious harmful effects. The present data suggest that ClyH has the potential to be an alternative therapeutic agent for the treatment of infections caused by MRSA.
We evaluated the long-term efficacy of prednisolone (PSL) therapy for prolonging ambulation in Japanese patients with genetically confirmed Duchenne muscular dystrophy (DMD). There were clinical trials have shown a short-term positive effect of high-dose and daily PSL on ambulation, whereas a few study showed a long-term effect. Especially in Japan, "real-life" observation was lacking. We utilized the national registry of muscular dystrophy in Japan for our retrospective study. We compared the age at loss of ambulation (LOA) between patients in PSL group and those in without-PSL group. Out of 791 patients in the Remudy DMD/BMD registry from July 2009 to June 2012, 560 were matched with inclusion criteria. Of the 560, all were genetically confirmed DMD patients, 245 (43.8 %) of whom were treated with PSL and 315 (56.2 %) without PSL. There was no difference between the two groups regarding their mutational profile. The age at LOA was significantly greater (11 month on average) in the PSL group than in the without-PSL group (median, 132 vs. 121 months; p = 0.0002). Although strictly controlled clinical trials have shown that corticosteroid therapies achieved a marked improvement in ambulation, discontinuation of the drug due to intolerable side effects led to exclusion of clinical trial participants, which is considered as unavoidable. In our study, patients were not excluded from the PSL group, even if they discontinued the medication shortly after starting it. The results of our study may provide evidence to formulate recommendations and provide a basis for realistic expectations for PSL treatment of DMD patients in Japan, even there are certain limitations due to the retrospectively captured data in the registry.
Members of the DnaQ superfamily are major 3-5 exonucleases that degrade either only single-stranded DNA (ssDNA) or both ssDNA and double-stranded DNA (dsDNA). However, the mechanism by which dsDNA is recognized and digested remains unclear. Exonuclease X (ExoX) is a distributive DnaQ exonuclease that cleaves both ssDNA and dsDNA substrates. Here, we report the crystal structures of Escherichia coli ExoX in complex with three different dsDNA substrates: 3 overhanging dsDNA, blunt-ended dsDNA and 3 recessed mismatch-containing dsDNA. In these structures, ExoX binds to dsDNA via both a conserved substrate strand-interacting site and a previously uncharacterized complementary strand-interacting motif. When ExoX complexes with blunt-ended dsDNA or 5 overhanging dsDNA, a wedge composed of Leu12 and Gln13 penetrates between the first two base pairs to break the 3 terminal base pair and facilitates precise feeding of the 3 terminus of the substrate strand into the ExoX cleavage active site. Site-directed mutagenesis showed that the complementary strand-binding site and the wedge of ExoX are dsDNA specific. Together with the results of structural comparisons, our data support a mechanism by which normal and mismatched dsDNA are recognized and digested by E. coli ExoX. The crystal structures also provide insight into the structural framework of the different substrate specificities of the DnaQ family members.
PCR product cloning is the foundational technology for almost all fields in the life sciences. Numerous innovative methods have been designed during the past few decades. Enzyme-free cloning is the only one that avoids post-amplification enzymatic treatments, making the technique reliable and cost effective. However, the complementary staggered overhangs used in enzyme-free cloning tend to result in self-ligation of the vector under some circumstances. Here, we describe a "T-type" enzyme-free cloning method: instead of designing the complementary staggered overhangs used in conventional enzyme-free cloning, we create "T-type" overhangs that reduce the possibility of self-ligation and are more convenient for multi-vector cloning. In this study, we systematically optimize "T-type" enzyme-free cloning, compare its cloning background with that in conventional enzyme-free cloning, and demonstrate a promising application of this technique in multi-vector cloning. Our method simplifies post-amplification procedures and greatly reduces cost, offering a competitive option for PCR product cloning.
Duchenne muscular dystrophy (DMD) is an X-linked genetic disease, caused by the absence of the dystrophin protein. Although many novel therapies are under development for DMD, there is currently no cure and affected individuals are often confined to a wheelchair by their teens and die in their twenties/thirties. DMD is a rare disease (prevalence <5/10,000). Even the largest countries do not have enough affected patients to rigorously assess novel therapies, unravel genetic complexities, and determine patient outcomes. TREAT-NMD is a worldwide network for neuromuscular diseases that provides an infrastructure to support the delivery of promising new therapies for patients. The harmonized implementation of national and ultimately global patient registries has been central to the success of TREAT-NMD. For the DMD registries within TREAT-NMD, individual countries have chosen to collect patient information in the form of standardized patient registries to increase the overall patient population on which clinical outcomes and new technologies can be assessed. The registries comprise more than 13,500 patients from 31 different countries. Here, we describe how the TREAT-NMD national patient registries for DMD were established. We look at their continued growth and assess how successful they have been at fostering collaboration between academia, patient organizations, and industry.
African-American (AA) women have earlier menarche on average than women of European ancestry (EA), and earlier menarche is a risk factor for obesity and type 2 diabetes among other chronic diseases. Identification of common genetic variants associated with age at menarche has a potential value in pointing to the genetic pathways underlying chronic disease risk, yet comprehensive genome-wide studies of age at menarche are lacking for AA women. In this study, we tested the genome-wide association of self-reported age at menarche with common single-nucleotide polymorphisms (SNPs) in a total of 18 089 AA women in 15 studies using an additive genetic linear regression model, adjusting for year of birth and population stratification, followed by inverse-variance weighted meta-analysis (Stage 1). Top meta-analysis results were then tested in an independent sample of 2850 women (Stage 2). First, while no SNP passed the pre-specified P < 5 × 10(-8) threshold for significance in Stage 1, suggestive associations were found for variants near FLRT2 and PIK3R1, and conditional analysis identified two independent SNPs (rs339978 and rs980000) in or near RORA, strengthening the support for this suggestive locus identified in EA women. Secondly, an investigation of SNPs in 42 previously identified menarche loci in EA women demonstrated that 25 (60%) of them contained variants significantly associated with menarche in AA women. The findings provide the first evidence of cross-ethnic generalization of menarche loci identified to date, and suggest a number of novel biological links to menarche timing in AA women.
As a typical protein nanostructure, virus-based nanoparticle (VNP) of simian virus 40 (SV40), which is composed of pentamers of the major capsid protein of SV40 (VP1), has been successfully employed in guiding the assembly of different nanoparticles (NPs) into predesigned nanostructures with considerable stability. However, the stabilization mechanism of SV40 VNP remains unclear. Here, the importance of inter-pentamer disulfide bonds between cysteines in the stabilization of quantum dot (QD)-containing VNPs (VNP-QDs) is comprehensively investigated by constructing a series of VP1 mutants of cysteine to serine. Although the presence of a QD core can greatly enhance the assembly and stability of SV40 VNPs, disulfide bonds are vital to stability of VNP-QDs. Cysteine at position 9 (C9) and C104 contribute most of the disulfide bonds and play essential roles in determining the stability of SV40 VNPs as templates to guide assembly of complex nanoarchitectures. These results provide insightful clues to understanding the robustness of SV40 VNPs in organizing suprastructures of inorganic NPs. It is expected that these findings will help guide the future design and construction of protein-based functional nanostructures.
Most psychiatric disorders are moderately to highly heritable. The degree to which genetic variation is unique to individual disorders or shared across disorders is unclear. To examine shared genetic etiology, we use genome-wide genotype data from the Psychiatric Genomics Consortium (PGC) for cases and controls in schizophrenia, bipolar disorder, major depressive disorder, autism spectrum disorders (ASD) and attention-deficit/hyperactivity disorder (ADHD). We apply univariate and bivariate methods for the estimation of genetic variation within and covariation between disorders. SNPs explained 17-29% of the variance in liability. The genetic correlation calculated using common SNPs was high between schizophrenia and bipolar disorder (0.68 ± 0.04 s.e.), moderate between schizophrenia and major depressive disorder (0.43 ± 0.06 s.e.), bipolar disorder and major depressive disorder (0.47 ± 0.06 s.e.), and ADHD and major depressive disorder (0.32 ± 0.07 s.e.), low between schizophrenia and ASD (0.16 ± 0.06 s.e.) and non-significant for other pairs of disorders as well as between psychiatric disorders and the negative control of Crohns disease. This empirical evidence of shared genetic etiology for psychiatric disorders can inform nosology and encourages the investigation of common pathophysiologies for related disorders.
The worldwide emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis threatens to make this disease incurable. Drug resistance mechanisms are only partially understood, and whether the current understanding of the genetic basis of drug resistance in M. tuberculosis is sufficiently comprehensive remains unclear. Here we sequenced and analyzed 161 isolates with a range of drug resistance profiles, discovering 72 new genes, 28 intergenic regions (IGRs), 11 nonsynonymous SNPs and 10 IGR SNPs with strong, consistent associations with drug resistance. On the basis of our examination of the dN/dS ratios of nonsynonymous to synonymous SNPs among the isolates, we suggest that the drug resistance-associated genes identified here likely contain essentially all the nonsynonymous SNPs that have arisen as a result of drug pressure in these isolates and should thus represent a near-complete set of drug resistance-associated genes for these isolates and antibiotics. Our work indicates that the genetic basis of drug resistance is more complex than previously anticipated and provides a strong foundation for elucidating unknown drug resistance mechanisms.
Due to the complicated media, monitoring proteases in real physiological environments is still a big challenge. Bioluminescence resonance energy transfer (BRET) is one of the promising techniques but its application is limited by the susceptibility to buffer composition, which might cause serious errors for the assay. Herein we report a novel combination of BRET pair with humanized Gaussia luciferase (hGluc) and highly bright red fluorescence protein tdTomato for sensitive and robust protease activity determination. As a result, the hGluc/tdTomato BRET pair showed much better tolerance to buffer composition, pH and sample matrices, and wide spectral separation (??:~110 nm). With the protease sensor built with this pair, the detection limit for enterokinase reached 2.1 pM in pure buffer and 3.3 pM in 3% serum. The proposed pair would find broad use in both in vitro and in vivo assays, especially for samples with complicated matrix.
Solving the crystal structure of Cbl(TKB) in complex with a pentapeptide, pYTPEP, revealed that the PEP region adopted a poly-L-proline type II (PPII) helix. An unnatural amino acid termed a proline-templated glutamic acid (ptE) that constrained both the backbone and sidechain to the bound conformation was synthesized and incorporated into the pYTPXP peptide. We estimated imposing structural constraints onto the backbone and sidechain of the peptide and preorganize it to the bound conformation in solution will yield nearly an order of magnitude improvement in activity. NMR studies confirmed that the ptE-containing peptide adopts the PPII conformation, however, competitive binding studies showed an order of magnitude loss of activity. Given the emphasis that is placed on imposing structural constraints, we provide an example to support the contrary. These results point to conformational flexibility at the interface, which have implications in the design of potent Cbl(TKB)-binding peptides.
Blood pressure (BP) is a heritable determinant of risk for cardiovascular disease (CVD). To investigate genetic associations with systolic BP (SBP), diastolic BP (DBP), mean arterial pressure (MAP) and pulse pressure (PP), we genotyped ?50 000 single-nucleotide polymorphisms (SNPs) that capture variation in ?2100 candidate genes for cardiovascular phenotypes in 61 619 individuals of European ancestry from cohort studies in the USA and Europe. We identified novel associations between rs347591 and SBP (chromosome 3p25.3, in an intron of HRH1) and between rs2169137 and DBP (chromosome1q32.1 in an intron of MDM4) and between rs2014408 and SBP (chromosome 11p15 in an intron of SOX6), previously reported to be associated with MAP. We also confirmed 10 previously known loci associated with SBP, DBP, MAP or PP (ADRB1, ATP2B1, SH2B3/ATXN2, CSK, CYP17A1, FURIN, HFE, LSP1, MTHFR, SOX6) at array-wide significance (P < 2.4 × 10(-6)). We then replicated these associations in an independent set of 65 886 individuals of European ancestry. The findings from expression QTL (eQTL) analysis showed associations of SNPs in the MDM4 region with MDM4 expression. We did not find any evidence of association of the two novel SNPs in MDM4 and HRH1 with sequelae of high BP including coronary artery disease (CAD), left ventricular hypertrophy (LVH) or stroke. In summary, we identified two novel loci associated with BP and confirmed multiple previously reported associations. Our findings extend our understanding of genes involved in BP regulation, some of which may eventually provide new targets for therapeutic intervention.
Recent advances in the ability to efficiently characterize tumor genomes is enabling targeted drug development, which requires rigorous biomarker-based patient selection to increase effectiveness. Consequently, representative DNA biomarkers become equally important in pre-clinical studies. However, it is still unclear how well these markers are maintained between the primary tumor and the patient-derived tumor models. Here, we report the comprehensive identification of somatic coding mutations and copy number aberrations in four glioblastoma (GBM) primary tumors and their matched pre-clinical models: serum-free neurospheres, adherent cell cultures, and mouse xenografts. We developed innovative methods to improve the data quality and allow a strict comparison of matched tumor samples. Our analysis identifies known GBM mutations altering PTEN and TP53 genes, and new actionable mutations such as the loss of PIK3R1, and reveals clear patient-to-patient differences. In contrast, for each patient, we do not observe any significant remodeling of the mutational profile between primary to model tumors and the few discrepancies can be attributed to stochastic errors or differences in sample purity. Similarly, we observe ?96% primary-to-model concordance in copy number calls in the high-cellularity samples. In contrast to previous reports based on gene expression profiles, we do not observe significant differences at the DNA level between in vitro compared to in vivo models. This study suggests, at a remarkable resolution, the genome-wide conservation of a patients tumor genetics in various pre-clinical models, and therefore supports their use for the development and testing of personalized targeted therapies.
BACKGROUND: Currently, clinical trials for new therapeutic strategies are being planned for Duchenne and Becker muscular dystrophies (DMD/BMD). However, it is difficult to obtain adequate numbers of patients in clinical trials. As solutions to these problems, patient registries are an important resource worldwide, especially in rare diseases such as DMD/BMD. METHODS: We developed a national registry of Japanese DMD/BMD patients in collaboration with TREAT-NMD. The registry includes male Japanese DMD/BMD patients whose genetic status has been confirmed by genetic analysis. The registry includes patients throughout Japan. RESULTS: As of February 2012, 583 DMD and 105 BMD patients were registered. Most individuals aged less than 20 years. In terms of genetic mutations of registrants of DMD and BMD, deletion of exons was the most frequent (61.4% and 79.0%) followed by point mutations (24.5% and 14.3%) and duplications (13.6% and 4.8%), respectively. 43.6% of DMD are capable of walking, and 76.2% of BMD registrants are able to walk. 41.1% of DMD registrants in the database were treated using steroids. 29.5% of DMD and 23.8% of BMD registrants were prescribed one cardiac medicine at least. 22% of DMD used ventilator support, and non-invasive support was common. Small numbers of DMD and BMD registrants, only 3.9% and 1.0% of them, have received scoliosis surgery. 57 (9.8%) patients were eligible to clinical trial focused on skipping exon 51. CONCLUSIONS: The Remudy has already demonstrated utility in clinical researches and standardization of patients care for DMD/BMD. This new DMD/BMD patient registry facilitates the synchronization of clinical drug development in Japan with that in other countries.
Live cell imaging of mRNA-protein interactions makes it possible to study posttranscriptional processes of cellular and viral gene expression under physiological conditions. In this study, red color mCherry-based trimolecular fluorescence complementation (TriFC) systems were constructed as new tools for visualizing mRNA-protein interaction in living cells using split mCherry fragments and HIV REV-RRE and TAT-TAR peptide-RNA interaction pairs. The new mCherry TriFC systems were successfully used to image RNA-protein interactions such as that between influenza viral protein NS1 and the 5 UTR of influenza viral mRNAs NS, M, and NP. Upon combination of an mCherry TriFC system with a Venus TriFC system, multiple mRNA-protein interactions could be detected simultaneously in the same cells. Then, the new mCherry TriFC system was used for imaging of interactions between influenza A virus mRNAs and some of adapter proteins in cellular TAP nuclear export pathway in live cells. Adapter proteins Aly and UAP56 were found to associate with three kinds of viral mRNAs. Another adapter protein, splicing factor 9G8, only interacted with intron-containing spliced M2 mRNA. Co-immunoprecipitation assays with influenza A virus-infected cells confirmed these interactions. This study provides long-wavelength-spectrum TriFC systems as new tools for visualizing RNA-protein interactions in live cells and help to understand the nuclear export mechanism of influenza A viral mRNAs.
Western breast cancer survivors have an increased risk of osteoporosis and bone fracture. Breast cancer occurs 10 to 20 years earlier in Asian women than in Western women. We investigated if younger Asian women with breast cancer also have increased risk of fracture.
Primary central nervous system lymphomas (PCNSL) have a dramatically increased prevalence among persons living with AIDS and are known to be associated with human Epstein Barr virus (EBV) infection. Previous work suggests that in some cases, co-infection with other viruses may be important for PCNSL pathogenesis. Viral transcription in tumor samples can be measured using next generation transcriptome sequencing. We demonstrate the ability of transcriptome sequencing to identify viruses, characterize viral expression, and identify viral variants by sequencing four archived AIDS-related PCNSL tissue samples and analyzing raw sequencing reads. EBV was detected in all four PCNSL samples and cytomegalovirus (CMV), JC polyomavirus (JCV), and HIV were also discovered, consistent with clinical diagnoses. CMV was found to express three long non-coding RNAs recently reported as expressed during active infection. Single nucleotide variants were observed in each of the viruses observed and three indels were found in CMV. No viruses were found in several control tumor types including 32 diffuse large B-cell lymphoma samples. This study demonstrates the ability of next generation transcriptome sequencing to accurately identify viruses, including DNA viruses, in solid human cancer tissue samples.
Studies involving second malignancies in patients with multiple myeloma are limited for the Asian population. Using data from population-based insurance claims, we assessed the risk of developing secondary malignancies after multiple myeloma, in particular hematologic malignancies. A retrospective cohort study was conducted in 3970 patients with newly diagnosed multiple myeloma from the registry of catastrophic illnesses between 1997 and 2009. A total of 15880 subjects without multiple myeloma were randomly selected as comparisons from the insured population, frequency-matched based on gender, age, and the date of diagnosis. The incidence of secondary malignancies was ascertained through cross-referencing with the National Cancer Registry System. The Cox proportional hazards model was used for analyses. The incidence of multiple myeloma in the insured population increased annually. The overall incidence of secondary malignancy was lower in the multiple myeloma cohort than in the comparison cohort (93.6 vs. 104.5 per 10,000 person-years, IRR = 0.90, 95% CI = 0.78-1.04). The incidence of hematologic malignancies was 11-fold greater for multiple myeloma patients (47.2 vs. 4.09 per 10,000 person-years) with an adjusted HR of 13.0 (95% CI = 7.79-21.6) compared with the comparison cohort. The relative risk of secondary malignancy was also strong for myeloid leukemia (21.2 vs. 1.36 per 10,000 person-years). Gender- and age-specific analysis for secondary hematologic malignancies showed that males and patients with multiple myeloma <60 years of age had a higher risk of secondary malignancy than females and patients with multiple myeloma >60 years of age. In conclusion, patients with multiple myeloma, especially younger patients, are at a high risk of hematologic malignancies.
Viruses encapsulating inorganic nanoparticles are a novel type of nanostructure with applications in biomedicine and biosensors. However, the encapsulation and assembly mechanisms of these hybridized virus-based nanoparticles (VNPs) are still unknown. In this article, it was found that quantum dots (QDs) can induce simian virus 40 (SV40) capsid assembly in dissociation buffer, where viral capsids should be disassembled. The analysis of the transmission electron microscope, dynamic light scattering, sucrose density gradient centrifugation, and cryo-electron microscopy single particle reconstruction experimental results showed that the SV40 major capsid protein 1 (VP1) can be assembled into ?25 nm capsids in the dissociation buffer when QDs are present and that the QDs are encapsulated in the SV40 capsids. Moreover, it was determined that there is a strong affinity between QDs and the SV40 VP1 proteins (KD=2.19E-10 M), which should play an important role in QD encapsulation in the SV40 viral capsids. This study provides a new understanding of the assembly mechanism of SV40 virus-based nanoparticles with QDs, which may help in the design and construction of other similar virus-based nanoparticles.
The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70-90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.
Surface monofunctionalization of protein nanostructures will enable precise topological control over the protein-templated assembly of nanoscale motifs, however, this remains a formidable challenge. Here we demonstrated a novel strategy for this purpose with a protein nanocage, virus-based nanoparticle (VNP) of simian virus 40 as a model system. By simultaneously incorporating a function modality (cysteine) and a purification modality (polyhistidine tag) into the building block (VP1) of VNPs through rational design and genetic engineering, the monofunctionalized cysteine-VNPs are readily obtained through a routine affinity chromatography in virtue of the purification modality of polyhistidine tag, after the coassembly of the functional VP1 and the nonfunctional VP1 at an optimal ratio. This strategy has proved to be highly efficient in constructing monofunctionalized protein nanostructures as highlighted by the monofunctionalized-VNP-guided Au/QD-VNP nanostructures. These nanostructures could be utilized in a wide range of disciplines, including basic biological research, novel nanostructures, and nanodevices fabrication, etc.
A systematic study of cross-linking chemistry of the Au(25)(SR)(18) nanomolecule by dithiols of varying chain length, HS-(CH(2))(n)-SH where n = 2, 3, 4, 5, and 6, is presented here. Monothiolated Au(25) has six [RSAuSRAuSR] staple motifs on its surface, and MALDI mass spectrometry data of the ligand exchanged clusters show that propane (C3) and butane (C4) dithiols have ideal chain lengths for interstaple cross-linking and that up to six C3 or C4 dithiols can be facilely exchanged onto the cluster surface. Propanedithiol predominately exchanges with two monothiols at a time, making cross-linking bridges, while butanedithiol can exchange with either one or two monothiols at a time. The extent of cross-linking can be controlled by the Au(25)(SR)(18) to dithiol ratio, the reaction time of ligand exchange, or the addition of a hydrophobic tail to the dithiol. MALDI MS suggests that during ethane (C2) dithiol exchange, two ethanedithiols become connected by a disulfide bond; this result is supported by density functional theory (DFT) prediction of the optimal chain length for the intrastaple coupling. Both optical absorption spectroscopy and DFT computations show that the electronic structure of the Au(25) nanomolecule retains its main features after exchange of up to eight monothiol ligands.
Bionanoparticles and nanostructures have attracted increasing interest as versatile and promising tools in many applications including biosensing and bioimaging. In this study, to image and detect tumor cells, ferritin cage-based multifunctional hybrid nanostructures were constructed that: (i) displayed both the green fluorescent protein and an Arg-Gly-Asp peptide on the exterior surface of the ferritin cages; and (ii) incorporated ferrimagnetic iron oxide nanoparticles into the ferritin interior cavity. The overall architecture of ferritin cages did not change after being integrated with fusion proteins and ferrimagnetic iron oxide nanoparticles. These multifunctional nanostructures were successfully used as a fluorescent imaging probe and an MRI contrast agent for specifically probing and imaging ?(v)?(3) integrin upregulated tumor cells. The work provides a promising strategy for tumor cell detection by simultaneous fluorescence and MR imaging.
Clinical trials for new therapeutic strategies are now being planned for Duchenne and Becker muscular dystrophies (DMD/BMD); however, many challenges exist in the planning and conduction of a clinical trial for rare diseases. The epidemiological data, total number of patients, natural history, and clinical outcome measures are unclear. Adequate numbers of patients are needed to achieve significant results in clinical trials. As solutions to these problems, patient registries are an important infrastructure worldwide, especially in the case of rare diseases such as DMD/BMD. In Europe, TREAT-NMD, a clinical research network for neuromuscular disorders, developeda global database for dystrophinopathy patients. We developed a national registry of Japanese DMD/BMD patients in collaboration with TREAT-NMD. The database includes clinical and molecular genetic data as well as all required items for the TREAT-NMD global patient registry. As of July 2011, 750 patients were registered in the database. The purpose of this registry is the effective recruitment of eligible patients for clinical trials, and it may also provide timely information to individual patients about upcoming trials. This registry data also provides more detailed knowledge about natural history, epidemiology, and clinical care. In recent years, drug development has become dramatically globalized, and global clinical trials (GCTs) are being conducted in Japan. It is appropriate, particularly with regard to orphan diseases, to include Japanese patients in GCTs to increase evidence for evaluation, because such large-scale trials would be difficult to conduct solely within Japan. GCTs enable the synchronization of clinical drug development in Japan with that in Western countries, minimizing drug approval delays.
Rh is unique in its ability to convert syngas to ethanol with the help of promoters. We performed systematic first-principles computations to examine the catalytic performance of pure and Mn modified Rh(100) surfaces for ethanol formation from syngas. CO dissociation on the surface as well as CO insertion between the chemisorbed CH(3) and the surface are the two key steps. The CO dissociation barrier on the Mn monolayer modified Rh(100) surface is remarkably lowered by ~1.5 eV compared to that on Rh(100). Moreover, the reaction barrier of CO insertion into the chemisorbed CH(3) group on the Mn monolayer modified Rh(100) surface is 0.34 eV lower than that of methane formation. Thus the present work provides new mechanistic insight into the role of Mn promoters in improving Rhs selectivity to convert syngas to ethanol.
Five room temperature ionic liquids based on C-2 substituted imidazolium cations and bis(trifluoromethanesulfonyl)imide (TFSI) anions were synthesized and their physicochemical properties: thermal property, density, viscosity, ionic conductivity, self-diffusion coefficients, and electrochemical stability, were systematically investigated. The temperature dependence of both viscosity and ionic conductivities of these ionic liquids can be described by the Vogel-Fulcher-Tamman (VFT) equation. Compared with the reference, 1-propyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide, the introduction of functional groups at the C-2 position generally increased the viscosity and lowered the ionic conductivity. The introduction of an ether group (-CH(2)OCH(2)CH(2)CH(2)CH(3)) at the C-2 position not only enhanced the reduction stability of the ionic liquids but also exhibited the lowest solid electrolyte interfacial resistance (R(SEI)). In contrast, the introduction of a cyano group (-CN) at the C-2 position not only decreased the reduction stability but also adversely increased the SEI resistance. The effect of the C-2 substitution on the reduction stability was explained by the change in the energy level of the lowest unoccupied molecular orbital. The self-diffusion coefficients (D) of each ion were measured by pulsed field gradient nuclear magnetic resonance (PFG-NMR). The lithium transference number (t(Li)) of 0.5 M LiTFSI/IL solutions calculated from the self-diffusion coefficients was in the range of 0.04 to 0.09.
NADH pyrophosphatase (NudC) catalyses the hydrolysis of NAD(H) to AMP and NMN(H) [nicotinamide mononucleotide (reduced form)]. NudC multiple sequence alignment reveals that homologues from most Mycobacterium tuberculosis isolates, but not other mycobacterial species, have a polymorphism at the highly conserved residue 237. To elucidate the functional significance of this polymorphism, comparative analyses were performed using representative NudC isoforms from M. tuberculosis H37Rv (NudC(Rv)) and M. bovis BCG (NudC(BCG)). Biochemical analysis showed that the P237Q polymorphism prevents dimer formation, and results in a loss of enzymatic activity. Importantly, NudC(BCG) was found to degrade the active forms of isoniazid (INH), INH-NAD and ethionamide (ETH), ETH-NAD. Consequently, overexpression of NudC(BCG) in Mycobacterium smegmatis mc(2)155 and M. bovis BCG resulted in a high level of resistance to both INH and ETH. Further genetic studies showed that deletion of the nudC gene in M. smegmatis mc(2)155 and M. bovis BCG resulted in increased susceptibility to INH and ETH. Moreover, inactivation of NudC in both strains caused a defect in drug tolerance phenotype for both drugs in exposure assays. Taken together, these data suggest that mycobacterial NudC plays an important role in the inactivation of INH and ETH.
We present two Parkinsons disease (PD) patients, who experienced heatstroke. Both patients manifested central nervous system dysfunction with elevated core temperature. Despite adequate lowering of the body temperature, multiorgan-dysfunction syndrome including encephalopathy, rhabdomyolysis, acute renal failure, acute respiratory failure, and disseminated intravascular coagulopathy was noted in one patient, leading to permanent neurologic damage. Because the ensuing multiorgan dysfunction could determine the functional prognosis in heatstroke patients, it is important to provide information about the prevention of heatstroke to patients, who are isolated or are severely disabled in the advanced stages of PD.
Porous carbons of high surface area are promising as cost-effective electrode materials for supercapacitors. Although great attention has been given to the anomalous increase of the capacitance as the pore size approaches the ionic dimensions, there remains a lack of full comprehension of the size dependence of the capacitance in nanopores. Here we predict from a classical density functional theory that the capacitance of an ionic-liquid electrolyte inside a nanopore oscillates with a decaying envelope as the pore size increases. The oscillatory behavior can be attributed to the interference of the overlapping electric double layers (EDLs); namely, the maxima in capacitance appear when superposition of the two EDLs is most constructive. The theoretical prediction agrees well with the experiment when the pore size is less than twice the ionic diameter. Confirmation of the entire oscillatory spectrum invites future experiments with a precise control of the pore size from micro- to mesoscales.
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