Prediction of folding patterns for intrinsic disordered protein.

The conformation flexibility of natural protein causes both complexity and difficulty to understand the relationship between structure and function. The prediction of intrinsically disordered protein primarily is focusing on to disclose the regions with structural flexibility involving relevant biological functions and various diseases. The order of amino acids in protein sequence determines possible conformations, folding flexibility and biological function. Although many methods provided the information of intrinsically disordered protein (IDP), but the results are mainly limited to determine the locations of regions without knowledge of possible folding conformations. Here, the developed protein folding fingerprint adopted the protein folding variation matrix (PFVM) to reveal all possible folding patterns for the intrinsically disordered protein along its sequence. The PFVM integrally exhibited the intrinsically disordered protein with disordering regions, degree of disorder as well as folding pattern. The advantage of PFVM will not only provide rich information for IDP, but also may promote the study of protein folding problem.

Highly accurate and sensitive diagnostic detection of SARS-CoV-2 by digital PCR.

The outbreak of COVID-19 caused by a novel Coronavirus (termed SARS-CoV-2) has spread to over 210 countries around the world. Currently, reverse transcription quantitative qPCR (RT-qPCR) is used as the gold standard for diagnosis of SARS-CoV-2. However, the sensitivity of RT-qPCR assays of pharyngeal swab samples are reported to vary from 30% to 60%. More accurate and sensitive methods are urgently needed to support the quality assurance of the RT-qPCR or as an alternative diagnostic approach. A reverse transcription digital PCR (RT-dPCR) method was established and evaluated. To explore the feasibility of RT-dPCR in diagnostic of SARS-CoV-2, a total of 196 clinical pharyngeal swab samples from 103 suspected patients, 77 close contacts and 16 supposed convalescents were analyzed by RT-qPCR and then measured by the proposed RT-dPCR. For the 103 fever suspected patients, 19 (19/25) negative and 42 (42/49) equivocal tested by RT-qPCR were positive according to RT-dPCR. The sensitivity of SARS-CoV-2 detection was significantly improved from 28.2% by RT-qPCR to 87.4% by RT-dPCR. For 29 close contacts (confirmed by additional sample and clinical follow up), 16 (16/17) equivocal and 1 negative tested by RT-qPCR were positive according to RT-dPCR, which is implying that the RT-qPCR is missing a lot of asymptomatic patients. The overall sensitivity, specificity and diagnostic accuracy of RT-dPCR were 91%, 100% and 93%, respectively. RT-dPCR is highly accurate method and suitable for detection of pharyngeal swab samples from COVID-19 suspected patients and patients under isolation and observation who may not be exhibiting clinical symptoms.

Overexpression of TAF1L Promotes Cell Proliferation, Migration and Invasion in Esophageal Squamous Cell Carcinoma.

Currently, it reported that TAF1L gene mutation is found in a number of carcinomas, but its pathophysiological function has not been well studied. We focused on investigating expressive levels of TAF1L gene and protein in esophageal squamous cell carcinoma (ESCC) with two tissue microarrays, forty fresh paired ESCC and paracancer samples using immunohistochemistry, real-time PCR or Western blot in this study. Furthermore, we executed TAF1L silence with siRNA in ESCC cell lines to evaluate effects of TAF1L expression on cell proliferation, migration and invasion of ESCC via CCK-8, wound healing and transwell chamber assays. Moreover, key proteins related to ESCC development were also analyzed by Western blot. Results from this study showed that the expression of TAF1L mRNA and protein in ESCC tissues were significantly higher than that in matched paracancer tissues. However, its abnormal expression was not associated with other clinic features, such as the age, gender and pathological grade, except of TNM-N stage. Furthermore, the proliferation, migration and invasion of ESCC cells were inhibited after TAF1L gene silencing. As a consequence, the expression of c-Myc and phosphorylated Akt in esophageal squamous cell line after TAF1L-siRNA treatment were inversely decreased, while p53 was increased significantly, compared those to control group. Taken together, the results from this study suggest that TAF1L gene might be served as an oncogene, and its overexpression could accelerate to the tumorigenesis of ESCC via promoting the malignant cell proliferation and tumor metastasis.

Association between LRP1 C766T polymorphism and Alzheimer's disease susceptibility: a meta-analysis.

Low density lipoprotein receptor-related protein 1 (LRP1) C766T polymorphism (rs1799986) has been extensively investigated for Alzheimer's disease (AD) susceptibility. However, results in different studies have been contradictory. Therefore, we conducted a meta-analysis containing 6455 AD cases and 6304 controls from 26 independent case-control studies to determine whether there was an association between the LRP1 C766T polymorphism and AD susceptibility. The combined analysis showed that there was no significant association between LRP1 C766T polymorphism and AD susceptibility (TT + CT versus CC: OR = 0.920, 95% CI = 0.817-1.037, P = 0.172). In subgroup analysis, significant decreased AD susceptibility was found among Asian population in allele model (T versus C: OR = 0.786, 95% CI = 0.635-0.974, P = 0.028) and dominant model (TT + CT versus CC: OR = 0.800, 95% CI = 0.647-0.990, P = 0.040). Moreover, T allele of LRP1 C766T was statistically associated with late onset of AD (LOAD) (T versus C: OR = 0.858, 95% CI = 0.748-0.985, P = 0.029; TT + CT versus CC: OR = 0.871, 95% CI = 0.763-0.994, P = 0.040). In conclusion, our meta-analysis suggested that LRP1 C766T polymorphism was associated with lower risk of AD in Asian, and could reduce LOAD risk especially. Considering some limitations of our meta-analysis, further large-scale studies should be done to reach a more comprehensive understanding.

Genetic Alterations in Esophageal Tissues From Squamous Dysplasia to Carcinoma.

BACKGROUND & AIMS: Esophageal squamous cell carcinoma (ESCC) is the most common subtype of esophageal cancer. Little is known about the genetic changes that occur in esophageal cells during the development of ESCC. We performed next-generation sequence analyses of esophageal nontumor, intraepithelial neoplasia (IEN), and ESCC tissues from the same patients to track genetic changes during tumor development. METHODS: We performed whole-genome, whole-exome, or targeted sequence analyses of 227 esophageal tissue samples from 70 patients with ESCC undergoing resection at Shantou University Medical College in China from 2012 through 2015 (no patients had received chemotherapy or radiation therapy); we analyzed normal tissues, tissues with simple hyperplasia, dysplastic tissues (IEN), and ESCC tissues collected from different regions of the esophagus at the same time. We also obtained 1191 nontumor esophageal biopsy specimens from the Chaoshan region (a high-risk region for ESCC) of China (a high-risk region for ESCC) and performed immunohistochemical and histologic analyses to detect inflammation. RESULTS: IEN and ESCC tissues had similar mutations and copy number alterations, at similar frequencies; these differed from mutations detected in tissues with simple hyperplasia. IEN tissues had mutations associated with apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like-mediated mutagenesis (a DNA damage mutational signature). Genetic analyses indicated that most ESCCs were formed from early stage IEN clones. Trunk mutations (mutations shared by >10% of paired IEN and ESCC tissues) were in genes that regulate DNA repair and cell apoptosis, proliferation and adhesion. Mutations in TP53 and CDKN2A and copy number alterations in 11q (contains CCND1), 3q (contains SOX2), 2q (contains NFE2L2), and 9p (contains CDKN2A) were considered to be trunk variants; these were dominant mutations detected at high frequencies in clones of paired IEN and ESCC samples. In the esophageal biopsy samples from high-risk individuals (residing in the Chaoshan region), 68.9% had an evidence of chronic inflammation; the level of inflammation was correlated with atypical cell structures and markers of DNA damage. CONCLUSIONS: We analyzed mutations and gene copy number changes in nontumor, IEN, and ESCC samples, collected from 70 patients. IEN and ESCCs each had similar mutations and markers of genomic instability, including apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like. Genomic changes observed in precancerous lesions might be used to identify patients at risk for ESCC.

Bivariate genomic analysis identifies a hidden locus associated with bacteria hypersensitive response in Arabidopsis thaliana.

Multi-phenotype analysis has drawn increasing attention to high-throughput genomic studies, whereas only a few applications have justified the use of multivariate techniques. We applied a recently developed multi-trait analysis method on a small set of bacteria hypersensitive response phenotypes and identified a single novel locus missed by conventional single-trait genome-wide association studies. The detected locus harbors a minor allele that elevates the risk of leaf collapse response to the injection of avrRpm1-modified Pseudomonas syringae (P = 1.66e-08). Candidate gene AT3G32930 with in the detected region and its co-expressed genes showed significantly reduced expression after P. syringae interference. Our results again emphasize that multi-trait analysis should not be neglected in association studies, as the power of specific multi-trait genotype-phenotype maps might only be tractable when jointly considering multiple phenotypes.

Association between Polymorphisms of the AKT1 Gene Promoter and Risk of the Alzheimer's Disease in a Chinese Han Population with Type 2 Diabetes.

AIMS: Alzheimer's disease (AD) is a multifactor disease that has been reported to have a close association with type 2 diabetes (T2D) where the v-akt murine thymoma viral oncogene homolog 1 (AKT1) plays an important role in the protein synthesis pathways and cell apoptosis processes. Evidence has been shown that AKT1 protein may be related to AD risk among patients with T2D. The aim of this study was to analyze the potential association between single nucleotide polymorphisms of AKT1 promoter and the risk of AD among patients with T2D. METHODS: The association between AKT1 polymorphisms and AD risk in patients with T2D was assessed among 574 consecutive unrelated subjects including 112 AD patients with T2D, 231 patients with AD, and 231 healthy controls in a case-control study. The cognitive function of all subjects was assessed using MMSE. Six single nucleotide polymorphisms with minor allele frequency >0.2 (rs2498786, rs74090038, rs2494750, rs2494751, rs5811155, and rs2494752) in AKT1 promoter were analyzed by polymerase chain reaction (PCR), and the concentration of AKT1 protein in serum was tested using enzyme-linked immunosorbent assay (ELISA). RESULTS: Overall, there was statistically significant difference in AKT1 rs2498786 polymorphism. The CC frequency of AKT1 rs2498786 polymorphism in AD with T2D group and AD control group was significantly higher than that in healthy control group (PAD+T2D vs. health < 0.0001, PAD vs. health < 0.0001). However, the difference was not found between AD with T2D group and AD control group. Compared with healthy control group, the plasma levels of AKT1 protein in AD with T2D group (PAD+T2D vs. health < 0.0001) and AD control group (PAD vs. health = 0.0003) decreased significantly. Among genotypes of AKT1 rs2498786 polymorphism, the AKT1 protein level in GG genotype was significantly higher than that in GC genotype (PGG vs. GC < 0.0001) and CC genotype (PGG vs. CC < 0.0001). CONCLUSION: The study suggests that AKT1 rs2498786 polymorphism in insulin signaling pathway may be associated with AD risk and different genotypes may affects levels of protein expression. However, the polymorphism is not shown to be exclusive in AD patients with T2D.

The global landscape of intron retentions in lung adenocarcinoma.

BACKGROUND: The transcriptome complexity in an organism can be achieved by alternative splicing of precursor messenger RNAs. It has been revealed that alternations in mRNA splicing play an important role in a number of diseases including human cancers. METHODS: In this study, we exploited whole transcriptome sequencing data from five lung adenocarcinoma tissues and their matched normal tissues to interrogate intron retention, a less studied alternative splicing form which has profound structural and functional consequence by modifying open reading frame or inserting premature stop codons. RESULTS: Abundant intron retention events were found in both tumor and normal tissues, and 2,340 and 1,422 genes only contain tumor-specific retentions and normal-specific retentions, respectively. Combined with gene expression analysis, we showed that genes with tumor-specific retentions tend to be over-expressed in tumors, and the abundance of intron retention within genes is negatively related with gene expression, indicating the action of nonsense mediated decay. Further functional analysis demonstrated that genes with tumor-specific retentions include known lung cancer driver genes and are found enriched in pathways important in carcinogenesis. CONCLUSIONS: We hypothesize that intron retentions and consequent nonsense mediated decay may collectively counteract the over-expression of genes promoting cancer development. Identification of genes with tumor-specific retentions may also help develop targeted therapies.

Lung cancer risk and genetic variants in East Asians: a meta-analysis.

Inconsistent results are often found regarding the risk of genetic variants in lung cancer association studies. To alleviate these conflicts, we performed a large-scale meta-analysis to evaluate the effect of variants on lung cancer in East Asian population (Han Chinese, Japanese, and Korean). Forty-three genetic variants with data from at least three independent case-control studies were under investigation of which two variants (rs1800734 in hMLH1, rs2273953-rs1801173 bi-marker in P73) were first meta-analyzed in East Asians. We found that three variants in CYP1A1, GSTM1, and XRCC1 showed consistently significant associations with lung cancer in mixed analysis and stratified analysis, and several variants showed diverse effects interacting with different environmental factors in stratified analysis. Our study presents a comprehensive and systematic analysis of lung cancer association studies in East Asians and confirms the effect of three variants in lung cancer risk. Additionally, result from stratified analysis suggests the importance of inclusion of environmental factors, such as smoking and tumor histology, in the analysis.

Whole transcriptome sequencing identifies tumor-specific mutations in human oral squamous cell carcinoma.

BACKGROUND: The accumulation of somatic mutations in genes and molecular pathways is a major factor in the evolution of oral squamous cell carcinoma (OSCC), which sparks studies to identify somatic mutations with clinical potentials. Recently, massively parallel sequencing technique has started to revolutionize biomedical studies, due to the rapid increase in its throughput and drop in cost. Hence sequencing of whole transcriptome (RNA-Seq) becomes a superior approach in cancer studies, which enables the detection of somatic mutations and accurate measurement of gene expression simultaneously. METHODS: We used RNA-Seq data from tumor and matched normal samples to investigate somatic mutation spectrum in OSCC. RESULTS: By applying a sophisticated bioinformatic pipeline, we interrogated two tumor samples and their matched normal tissues and identified 70,472 tumor somatic mutations in protein-coding regions. We further identified 515 significantly mutated genes (SMGs) and 156 tumor-specific disruptive genes (TDGs), with six genes in both sets, including ANKRA2, GTF2H5, STOML1, NUP37, PPP1R26, and TAF1L. Pathway analysis suggested that SMGs were enriched in cell adhesion pathways, which are frequently indicated in tumor development. We also found that SMGs tend to be differentially expressed between tumors and normal tissues, implying a regulatory role of accumulation of genetic aberrations in these genes. CONCLUSIONS: Our finding of known tumor genes proves of the utility of RNA-Seq in mutation screening, and functional analysis of genes detected here would help understand the molecular mechanism of OSCC.

Genome-scale identification of Caenorhabditis elegans regulatory elements by tiling-array mapping of DNase I hypersensitive sites.

BACKGROUND: A major goal of post-genomics research is the integrated analysis of genes, regulatory elements and the chromatin architecture on a genome-wide scale. Mapping DNase I hypersensitive sites within the nuclear chromatin is a powerful and well-established method of identifying regulatory element candidates. RESULTS: Here, we report the first genome-wide analysis of DNase I hypersensitive sites (DHSs) in Caenorhabditis elegans. The data was obtained by hybridizing DNase I-treated and end-captured material from young adult worms to a high-resolution tiling microarray. The data show that C. elegans DHSs were significantly enriched within intergenic regions located 2 kb upstream and downstream of coding genes, and also that a considerable fraction of all DHSs mapped to intergenic positions distant to annotated coding genes. Annotated transcribed loci were generally depleted in DHSs relative to intergenic regions, but DHSs were nonetheless enriched in coding exons and UTRs, whereas introns were significantly depleted in DHSs. Many DHSs appeared to be associated with annotated non-coding RNAs and recently detected transcripts of unknown function. It has been reported that nematode highly conserved non-coding elements were associated with cis-regulatory elements, and we also found that DHSs, particularly distal intergenic DHSs, were significantly enriched in regions that were conserved between the C. elegans and C. briggsae genomes. CONCLUSION: We describe the first genome-wide analysis of C. elegans DHSs, and show that the distribution of DHSs is strongly associated with functional elements in the genome.

Localization of linker histone in chromatosomes by cryo-atomic force microscopy.

Linker histones play a fundamental role in determining higher order chromatin structure as a consequence of their association with nucelosomal DNA. Yet the locations and structural consequences of linker histone binding are still enigmatic. Here, using cryo-atomic force microscopy, we show that the linker histone H5 in native chromatin and in chromatosomes reconstituted on the 5S rDNA template is located at the dyad of the nucleosome core particle, within the "stem" structure. Direct measurement also indicates that the length of free linker DNA between chromatosomes in native chromatin is approximately 30 bp, slightly shorter than that estimated from nuclease digestion assays.

Smooth muscle myosin: regulation and properties.

The relationship of the biochemical states to the mechanical events in contraction of smooth muscle cross-bridges is reviewed. These studies use direct measurements of the kinetics of Pi and ADP release. The rate of release of Pi from thiophosphorylated cycling cross-bridges held isometric was biphasic with turnovers of 1.8 s-1 and 0.3 s-1, reflecting properties and forces directly acting on cross-bridges through mechanisms such as positive strain and inhibition by high-affinity MgADP binding. Fluorescent transients reporting release of an ADP analogue 3'-deac-edaADP were significantly faster in phasic than in tonic smooth muscles. Thiophosphorylation of myosin regulatory light chains (RLCs) increased and positive strain decreased the release rate around twofold. The rates of ADP release from rigor cross-bridges and the steady-state Pi release from cycling isometric cross-bridges are similar, indicating that the ADP-release step or an isomerization preceding it may limit the ATPase rate. Thus ADP release in phasic and tonic smooth muscles is a regulated step with strain- and dephosphorylation-dependence. High affinity of cross-bridges for ADP and slow ADP release prolong the fraction of the duty cycle occupied by strongly bound AM.ADP state(s) and contribute to the high economy of force that is characteristic of smooth muscle. RLC thiophosphorylation led to structural changes in smooth muscle cross-bridges consistent with our findings that thiophosphorylation and strain modulate product release.

Cryo-atomic force microscopy of unphosphorylated and thiophosphorylated single smooth muscle myosin molecules.

The purpose of this study was to determine whether steric blockage of one head by the second head of native two-headed myosin was responsible for the inactivity of nonphosphorylated two-headed myosin compared with the high activity of single-headed myosin, as suggested on the basis of electron microscopy of two-dimensional crystals of heavy meromyosin (Wendt, T., Taylor, D., Messier, T., Trybus, K. M., and Taylor, K. A. (1999) J. Cell Biol. 147, 1385-1390; and Wendt, T., Taylor, D., Trybus, K. M., and Taylor, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4361-4366). Our earlier cryo-atomic force microscopy (cryo-AFM) (Zhang, Y., Shao, Z., Somlyo, A. P., and Somlyo, A. V. (1997) Biophys. J. 72, 1308-1318) indicates that thiophosphorylation of the regulatory light chain increases the separation of the two heads of a single myosin molecule, but the thermodynamic probability of steric hindrance by strong binding between the two heads was not determined. We now report this probability determined by cryo-AFM of single whole myosin molecules shown to have normal low ATPase activity (0.007 s-1). We found that the thermodynamic probability of the relative head positions of nonphosphorylated myosin was approximately equal between separated heads as compared with closely apposed heads (energy difference of 0.24 kT (where k is a Boltzman constant and T is the absolute temperature)), and thiophosphorylation increased the number of molecules having separated heads (energy advantage of -1.2 kT (where k is a Boltzman constant and I is the absolute temperature)). Our results do not support the suggestion that strong binding of one head to the other stabilizes the blocked conformation against thermal fluctuations resulting in steric blockage that can account for the low activity of nonphosphorylated two-headed myosin.

Borg/septin interactions and the assembly of mammalian septin heterodimers, trimers, and filaments.

Septins constitute a family of guanine nucleotide-binding proteins that were first discovered in the yeast Saccharomyces cerevisiae but are also present in many other eukaryotes. In yeast they congregate at the bud neck and are required for cell division. Their function in metazoan cells is uncertain, but they have been implicated in exocytosis and cytokinesis. Septins have been purified from cells as hetero-oligomeric filaments, but their mechanism of assembly is unknown. Further studies have been limited by the difficulty in expressing functional septin proteins in bacteria. We now show that stable, soluble septin heterodimers can be produced by co-expression from bicistronic vectors in bacteria and that the co-expression of three septins results in their assembly into filaments. Pre-assembled dimers and trimers bind guanine nucleotide and show a slow GTPase activity. The assembly of a heterodimer from monomers in vitro is accompanied by GTP hydrolysis. Borg3, a downstream effector of the Cdc42 GTPase, binds specifically to a septin heterodimer composed of Sept6 and Sept7 and to the Sept2/6/7 trimer, but not to septin monomers or to other heterodimers. Septins associate through their C-terminal coiled-coil domains, and Borg3 appears to recognize the interface between these domains in Sept6 and Sept7.