Biology subjects

Zhang, L.

Publications and source records attributed to Zhang, L..

At least 1,513 records · Page 84Linked to original sources

bioRxiv2019

Aquila_stLFR: assembly based variant calling package for stLFR and hybrid assembly for linked-reads

MotivationIdentifying structural variants (SVs) is of critical importance in health and disease, however, detecting them remains a scientific and computing challenge. Several linked-read sequencing technologies, including 10X linked-read, TELL-Seq, and single tube long fragment read (stLFR), have been recently developed as cost-effective approaches to reconstruct multi-megabase haplotypes (phase blocks) from sequence data of a single sample. These technologies provide an optimal sequencing platform to characterize SVs, though few computational algorithms can utilize them. Thus, we developed Aquila_stLFR, an approach that resolves SVs through haplotype-based assembly of stLFR linked-reads. ResultsAquila_stLFR first partitions LFRs into two haplotype-specific blocks, by taking advantage of the potential phasing ability of the linked-read itself. Each haplotype is then assembled independently, to achieve a complete diploid assembly to finally reconstruct the genome-wide SVs. We benchmarked Aquila_stLFR on a well-studied sample, NA24385, and showed Aquila_stLFR can detect medium to large size (50bp - 10kb) deletions with a high sensitivity and insertions with a high specificity. AvailabilitySource code and documentation are available on https://github.com/maiziex/Aquila_stLFR. Contactmaizie.zhou@vanderbilt.edu Supplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2019

A cold shock protein from a thermophile bacterium promotes the high-temperature growth of bacteria and fungi through binding to diverse RNA species

High temperatures deleteriously affect cells by damaging cellular structures and changing the behavior of diverse biomolecules, and extensive research about thermophilic microorganisms has elucidated some of the mechanisms that can overcome these effects and allow thriving in high-temperature ecological niches. We here used functional genomics methods to screen out a cold-shock protein (CspL) from a high-productivity lactate producing thermophile strain (Bacillus coagulans strain 2-6) grown at 37{degrees}C and 60{degrees}C. We subsequently made the highly striking finding that transgenic expression of CspL conferred massive increases in high temperature growth of other organisms including E. coli (2.4-fold biomass increase at 45{degrees}C) and the eukaryote S. cerevisiae (a 2.7-fold biomass increase at 34{degrees}C). Pursuing these findings, we used bio-layer interferometry assays to characterize the nucleotide-binding function of CspL in vitro, and used proteomics and RNA-Seq to characterize the global effects of CspL on mRNA transcript accumulation and used RIP-Seq to identify in vivo RNA targets of this nucleotide-binding protein (e.g. rpoE, and rmf, etc.). Finally, we confirmed that a nucleotide-binding-dead variant form of CspL does not have increased growth rates or biomass accumulation effects at high temperatures. Our study thus establishes that CspL can function as a global RNA chaperone.

microbiology↗

bioRxiv2019

The Metabolic Effects of Angiopoietin-like protein 8 (ANGPLT8) are Differentially Regulated by Insulin and Glucose in Adipose Tissue and Liver and are Controlled by AMPK Signaling

ObjectiveThe angiopoietin-like protein (ANGPTL) family represents a promising therapeutic target for dyslipidemia, which is a feature of obesity and type 2 diabetes (T2DM). The aim of the present study was to determine the metabolic role of ANGPTL8 and to investigate its nutritional, hormonal and molecular regulation in key metabolic tissues.\n\nMethodsThe metabolism of ANGPTL8 knockout mice (ANGPTL8-/-) was examined in mice following chow and high-fat diets (HFD). The regulation of ANGPTL8 expression by insulin and glucose was quantified using a combination of in vivo insulin clamp experiments in mice and in vitro experiments in hepatocytes and adipocytes. The role of AMPK signaling was examined, and the transcriptional control of ANGPTL8 was determined using bioinformatic and luciferase reporter approaches.\n\nResultsThe ANGPTL8-/-mice had improved glucose tolerance and displayed reduced fed and fasted plasma triglycerides. However, there was no reduction in steatosis in ANGPTL8-/-mice after the HFD. Insulin acutely activated ANGPTL8 expression in liver and adipose tissue, which was mediated by C/EBP{beta}. Using insulin clamp experiments we observed that glucose further enhanced ANGPTL8 expression in the presence of insulin in adipocytes only. The activation of AMPK signaling potently suppressed the effect of insulin on ANGPTL8 expression in hepatocytes.\n\nConclusionThese data show that ANGPTL8 plays an important metabolic role in mice that may extend beyond triglyceride metabolism. The finding that insulin and glucose have distinct roles in regulating ANGPTL8 expression in liver and adipose tissue may provide important clues about the function of ANGPTL8 in these tissues.

physiology↗

bioRxiv2019

Chemistry of cation hydration and conduction in a skeletal muscle ryanodine receptor

Ryanodine receptors (RyRs) are Ca2+-regulated Ca2+ channels of 2.2-megadalton in muscles and neurons for calcium signaling. How Ca2+ regulates ion conduction in the RyR channels remains elusive. We determined a 2.6-[A] cryo-EM structure of rabbit skeletal muscle RyR1, and used multiscale dynamics simulations to elucidate cation interactions with RyR1. We investigated 21 potential cation-binding sites that may together rationalize biphasic Ca2+ response of RyR1. The selectivity filter captures a cation hydration complex by hydrogen-bonding with both the inner and outer hydration shells of water molecules. Molecular dynamics simulations suggest that adjacent Ca2+ ions moving in concert along ion-permeation pathway are separated by at least two cation-binding sites. Our analysis reveals that RyR1 has been evolved to favor its interactions with two hydration shells of cations.

biochemistry↗

bioRxiv2019

Assessment of human diploid genome assembly with 10x Linked-Reads data

BackgroundProducing cost-effective haplotype-resolved personal genomes remains challenging. 10x Linked-Read sequencing, with its high base quality and long-range information, has been demonstrated to facilitate de novo assembly of human genomes and variant detection. In this study, we investigate in depth how the parameter space of 10x library preparation and sequencing affects assembly quality, on the basis of both simulated and real libraries.\n\nFindingsWe prepared and sequenced eight 10x libraries with a diverse set of parameters from standard cell lines NA12878 and NA24385 and performed whole genome assembly on the data. We also developed the simulator LRTK-SIM to follow the workflow of 10x data generation and produce realistic simulated Linked-Read data sets. We found that assembly quality could be improved by increasing the total sequencing coverage (C) and keeping physical coverage of DNA fragments (CF) or read coverage per fragment (CR) within broad ranges. The optimal physical coverage was between 332X and 823X and assembly quality worsened if it increased to greater than 1,000X for a given C. Long DNA fragments could significantly extend phase blocks, but decreased contig contiguity. The optimal length-weighted fragment length (WFL) was around 50 - 150kb. When broadly optimal parameters were used for library preparation and sequencing, ca. 80% of the genome was assembled in a diploid state.\n\nConclusionThe Linked-Read libraries we generated and the parameter space we identified provide theoretical considerations and practical guidelines for personal genome assemblies based on 10x Linked-Read sequencing.

bioinformatics↗

bioRxiv2019

The PRMT7-dependent methylation of shank2 modulates invasion-proliferation switching during breast cancer metastasis

Invasiveness of cancer cells is associated with proliferation inhibition in multiple types of cancers. Here, we identified the pivotal roles of Arginine methyltransferase PRMT7 in promoting invasion and attenuating proliferation of breast cancer cells. PRMT7 exerted its functions through binding to the scaffold protein shank2 to induce the di-methylation of shank2 at R240. Shank2 R240 methylation exposed ANK domain by disrupting its SPN-ANK domain blockade. Moreover, shank2 R240 methylation rendered recruitment of FAK that elicited the FAK auto-phosphorylation, which consequently augmented the shank2-dependent migration and invasion of breast cancer cells. On the other hand, the shank2 R240 methylation impeded proliferation of breast cancer cells by antagonizing the Ras-Raf binding via tethering the mono-ubiquitinated H-Ras. These findings characterize the PRMT7-dependent shank2 methylation as a key player in mediating reciprocal switching between invasion and proliferation, also point to the value of shank2 R240 methylation as a target for tumour metastasis treatment strategies.

cancer biology↗

bioRxiv2019

Mutant GT198 in angiogenesis as a common origin of human prostate and bladder cancers

Prostate and bladder cancers are common cancers in men. It has been speculated that the high concomitant incidence of the two cancers is due to a potential shared cause underlying both cancers. In this report, we have identified a common cause of human prostate and bladder cancers as the mutant oncoprotein GT198 (PSMC3IP). GT198 is a DNA repair factor and a steroid hormone receptor coactivator. GT198 has been previously shown to be mutated in angiogenic pericyte stem cells in solid tumor microenvironment. GT198 is also a direct protein target of chemo drugs paclitaxel and doxorubicin. Here we show, the GT198 gene is mutated with protein overexpression in tumor stroma of human prostate and bladder cancers. Affected stromal cells include angiogenic blood vessel pericyte stem cells, and vascular smooth muscle cell lineages including myofibroblasts in prostate and smooth muscle cells in bladder. In prostate cancers, GT198+ tumor stromal cells are associated with early stages of cancer with lower Gleason scores. In bladder cancers, the presence of angiogenesis and GT198+ stroma are associated with better progression-free survival in docetaxel-treated patients. Together, our evidence suggests that angiogenic pericyte stem cells are initial lesions producing a mutant stroma carrying GT198 somatic mutations. Subsequently, mutant myofibroblasts promote adenocarcinomas in prostate and mutant smooth muscle cells promote urothelial carcinomas in bladder. Chemo drugs targeting to GT198 is more effective in early stages of cancers with GT198+ stromal cells. This study supports oncoprotein GT198 as a common cause and a drug target in human prostate and bladder cancers.

pathology↗

bioRxiv2019

THOC1 deficiency leads to late-onset nonsyndromic hearing loss through p53-mediated hair cell apoptosis

Apoptosis of cochlear hair cells is a key step towards age-related hearing loss. Although numerous genes have been implicated in the genetic causes of late-onset, progressive hearing loss, few show direct links to the proapoptotic process. By genome-wide linkage analysis and whole exome sequencing, we identified a heterozygous p.L183V variant in THOC1 as the probable cause of the late-onset, progressive, non-syndromic hearing loss in a large dominant family. Thoc1, a member of the conserved multisubunit THO/TREX ribonucleoprotein complex, is highly expressed in mouse and zebrafish hair cells. The Thoc1 mutant zebrafish generated by gRNA-Cas9 system lacks the C-startle response, indicative of the hearing dysfunction. Both Thoc1 mutant and knockdown zebrafish have greatly reduced hair cell numbers, while the latter can be rescued by embryonic microinjection of human wild-type THOC1 mRNA but to significantly lesser degree by the p.L183V mutant mRNA. The Thoc1 deficiency resulted in marked apoptosis in zebrafish hair cells. Consistently, transcriptome sequencing of the mutants showed significantly increased gene expression in the p53-associated signaling pathway. Depletion of p53 or applying the p53 inhibitor Pifithrin- significantly rescued the hair cell loss in the Thoc1 knockdown zebrafish. Our results suggested that THOC1 deficiency lead to late-onset, progressive hearing loss through p53-mediated hair cell apoptosis. This is to our knowledge the first human disease associated with THOC1 mutations and may shed light on the molecular mechanism underlying the age-related hearing loss.\n\nSignificance StatementFor the first time, we found that THOC1 deficiency leads to late-onset nonsyndromic hearing loss. Furthermore, we revealed the hypomorphic THOC1 induced p53-mediated hair cell apoptosis. This is to our knowledge the first human disease associated with THOC1 mutations and may shed light on the molecular mechanism underlying the age-related hearing loss.

genetics↗

bioRxiv2019

Rational design of hepatitis C virus E2 core nanoparticle vaccines

Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are critical for cell entry with E2 being the major target of neutralizing antibodies (NAbs). Here, we present a comprehensive strategy for B cell-based HCV vaccine development through E2 optimization and nanoparticle display. We redesigned variable region 2 in a truncated form (tVR2) on E2 cores derived from genotypes 1a and 6a, resulting in improved stability and antigenicity. Crystal structures of three optimized E2 cores with human cross-genotype NAbs (AR3s) revealed how the modified tVR2 stabilizes E2 without altering key neutralizing epitopes. We then displayed these E2 cores on 24- and 60-meric nanoparticles and achieved high yield, high purity, and enhanced antigenicity. In mice, these nanoparticles elicited more effective NAb responses than soluble E2 cores. Next-generation sequencing (NGS) defined distinct B-cell patterns associated with nanoparticle-induced antibody responses, which cross-neutralized HCV by targeting the conserved neutralizing epitopes on E2.\n\nOne Sentence SummaryAn HCV vaccine strategy is presented that displays redesigned E2 cores on nanoparticles as vaccine candidates for eliciting a broadly neutralizing B-cell response.

microbiology↗

bioRxiv2019

Rapid pacing by circulating traveling waves improves maturation of hiPSC-derived cardiomyocytes in self-organized tissue ring

Directed differentiation methods allow acquisition of high-purity cardiomyocytes (CMs) differentiated from human induced pluripotent stem cells (hiPSCs); however, their immaturity characteristic limits their application for drug screening and regenerative therapy. The rapid electrical pacing of cardiomyocytes have been used for efficiently promoting the maturation of cardiomyocytes, here we describe a simple device in modified culture plate on which hiPSC-derived CMs (hiPSC-CMs) can form three-dimensional self-organized tissue rings (SOTRs). Using calcium imaging, we show that within the ring, traveling waves (TWs) of action potential spontaneously originated and ran robustly at a frequency up to 4 Hz. After 2 weeks, SOTRs with TW training showed matured features including structural organization, increased cardiac-specific gene expression, enhanced Ca2+-handling properties, an increased oxygen-consumption rate, and enhanced contractile force. We subsequently used a mathematical model to interpret the origination, propagation, and long-term behavior of the TWs within the SOTRs. This new idea for spontaneous hiPSC-CM maturation also has potential for pacing the electrical excitable cells such as neuron and retina cells for various applications.

bioengineering↗

bioRxiv2019

Withdraw of prophylactic antimicrobials does not change the pigs’ resistome

The use of antimicrobials in the animal industry has increased the prevalence of antimicrobial resistant commensal bacteria in food products derived from animals, which could be associated with antimicrobial resistance in human pathogens. To reduce the influx of antibiotic resistant bacteria (and genes) to the human microbiota, restrictions on antimicrobials (in food animals) have been implemented in different countries. We investigated the impact of antimicrobial restriction in the frequency of antimicrobial resistant bacteria in pigs. No differences in antimicrobial resistance or antimicrobial resistance genes (richness or abundance) was found when we compared animals fed with and without antibiotics. Fitness costs of antimicrobial resistance in bacteria (in the field) seems to be overestimated.

microbiology↗

bioRxiv2019

IFITMs Inhibit Cell Fusion Mediated by Trophoblast Syncytins

Type I interferon (IFN) induced by virus infections during pregnancy causes placental damage, though the mechanisms and identities of IFN-stimulated genes that are involved remain under investigation. The IFN-induced transmembrane proteins (IFITMs) inhibit virus infections by preventing virus membrane fusion with cells and by inhibiting fusion of infected cells (syncytialization). Fusion of placental trophoblasts via expression of endogenous retroviral fusogens known as Syncytins forms the syncytiotrophoblast, a multinucleated cell structure essential for fetal development. We found that IFN blocks fusion of BeWo human placental trophoblasts. Stably-expressed IFITMs 1, 2, and 3 also blocked fusion of these trophoblasts, while making them more resistant to virus infections. Conversely, stable knockdown of IFITMs in BeWo trophoblasts increased their spontaneous fusion and allowed fusion in the presence of IFN, while also making the cells more susceptible to virus infection. Overall, our data demonstrate that IFITMs are anti-viral and anti-fusogenic in trophoblasts.

immunology↗

bioRxiv2019

Protein interactions of Magnaporthe oryzae protein kinase CK2 and secondary data analysis of a large number of transcriptomes suggest chaperone-like activity is integral to its function

CK2, a serine/threonine (S/T) kinase present in eukaryotic cells is known to have a vast number of substrates. We have recently shown that it localizes to nuclei and at pores between hyphal compartments in M. oryzae. We performed a pulldown-proteomics of M. oryzae CK2 catalytic subunit MoCKa to detect interacting proteins. The MoCKa pulldown was enriched for septa and nucleoli proteins and intrinsically disordered proteins (IDPs) containing a CK2 phosphorylation motif proposed to destabilize and unfold alpha helixes. This points to a function for CK2 phosphorylation and corresponding phosphatase dephosphorylation in the formation of functional protein-protein aggregates and protein-RNA/DNA binding. To test this as widely as possible we used secondary data downloaded from databases from a large range of M. oryzae experiments and also for a relatively closely related plant pathogenic fungus, Fusarium graminearum. We found that CKa expression was strongly positively correlated with S/T phosphatases as well as with disaggregase (HSP104, YDJ1, SSA1) and an autophagy indicating protein (ATG8). The latter points to increased protein aggregate formation at high levels of CKa expression. Our results suggest a general role for CK2 in aggregation and disaggregation of IDPs and their binding to proteins, DNA and RNA interactions.

cell biology↗

bioRxiv2019

Predicting mechanism of action of cellular perturbations with pathway activity signatures

MotivationMisregulation of signaling pathway activity is etiologic for many human diseases, and modulating activity of signaling pathways is often the preferred therapeutic strategy. Understanding the mechanism of action (MOA) of bioactive chemicals in terms of targeted signaling pathways is the essential first step in evaluating their therapeutic potential. Changes in signaling pathway activity are often not reflected in changes in expression of pathway genes which makes MOA inferences from transcriptional signatures a difficult problem. ResultsWe developed a new computational method for implicating pathway targets of bioactive chemicals and other cellular perturbations by integrated analysis of pathway network topology, the LINCS transcriptional signatures of genetic perturbations of pathway genes and the transcriptional signature of the perturbation. Our methodology accurately predicts signaling pathways targeted by the perturbation when current pathway analysis approaches utilizing only a transcriptional signature of the perturbation fail. Availability and ImplementationOpen source R package paslincs is available at https://github.com/uc-bd2k/paslincs.

systems biology↗

bioRxiv2019

A Survey and Systematic Assessment of Computational Methods for Drug Response Prediction

Drug response prediction arises from both basic and clinical research of personalized therapy, as well as drug discovery for cancer and other diseases. With gene expression profiles and other omics data being available for over 1000 cancer cell lines and tissues, different machine learning approaches have been applied to solve drug response prediction problems. These methods appear in a body of literature and have been evaluated on different datasets with only one or two accuracy metrics. We systematically assessed 17 representative methods for drug response prediction, which have been developed in the past five years, on four large public datasets in nine metrics. This study provides insights and lessons for future research into drug response prediction.

bioinformatics↗

bioRxiv2019

Photosynthetic conversion of CO2 to hyaluronic acid by engineered cyanobacteria

Hyaluronic acid (HA), consisting of alternating N-acetylglucosamine and glucuronic acid units, is a natural polymer with diverse cosmetic and medical applications. Currently, HA is produced by overexpressing HA synthases from gram-negative Pasteurella multocida (encoded by pmHAS) or gram-positive Streptococcus equisimilis (encoded by seHasA) in various heterotrophic microbial production platforms. Here we introduced these two different types of HA synthase into the fast-growing cyanobacterium Synechococcus sp. PCC 7002 (Syn7002) to explore the capacity for producing HA in a photosynthetic system. Our results show that both HA synthases enable Syn7002 to produce HA photoautotrophically, but that overexpression of the soluble HA synthase (PmHAS) is less deleterious to cell growth and results in higher production. Genetic disruption of the competing cellulose biosynthetic pathway increased the HA titer by over 5-fold (from 14 mg/L to 80 mg/L) and the relative proportion of HA with molecular mass greater than 2 MDa. Introduction of glmS and glmU, coding for enzymes involved in the biosynthesis of the precursor UDP-N-acetylglucosamine, in combination with partial glycogen depletion, allowed photosynthetic production of 112 mg/L of HA in 5 days, an 8-fold increase in comparison to the initial PmHAS expressing strain. Addition of tuaD and gtaB (coding for genes involved in UDP-glucuronic acid biosynthesis) also improved the HA yield, albeit to a lesser extent. Overall our results have shown that cyanobacteria hold promise for sustainable production of pharmaceutically important polysaccharides from sunlight and CO2.

bioengineering↗

bioRxiv2019

Monoclonal IgM antibodies raised against Candida albicans Hyr1 provide cross-kingdom protection against Gram negative bacteria

Recent years have seen an unprecedented rise in the incidence of multidrug resistant (MDR) Gram negative bacteria (GNB) such as Acinetobacter and Klebsiella species. In view of the shortage of novel drugs in the pipeline, alternative strategies to prevent and treat infections by GNB are urgently needed. Previously, we have reported that the C. albicans hyphal-regulated protein Hyr1 shares striking 3D structural homology with cell surface proteins of A. baumannii; and active or passive vaccination with rHyr1p-N or anti-Hyr1p polyclonal antibody, respectively; protect mice from Acinetobacter infections. Here, we show that monoclonal antibodies (mAb) generated against Hyr1p, bind to the surface of Acinetobacter as well as K. pneumoniae. The anti-Hyr1 mAb also block damage to primary endothelial cells by the bacteria, and protect mice from lethal pulmonary infections mediated by A. baumannii and K. pneumoniae. Our current studies emphasize the potential of harnessing Hyr1p mAb as a cross-kingdom immunotherapeutic strategy against MDR GNB.

immunology↗

bioRxiv2019

Engineering oleaginous yeast Yarrowia lipolytica for violacein production: extraction, quantitative measurement and culture optimization

Violacein is a naturally occurring anticancer therapeutic with deep purple color. Yeast fermentation represents an alternative approach to efficiently manufacturing violacein from inexpensive feedstocks. In this work, we optimized the extraction protocol to improve violacein recovery ratio and purity from yeast culture, including the variations of organic solvents, the choice of mechanical shear stress, incubation time and the use of cell wall-degrading enzymes. We also established the quantitative correlation between HPLC and microplate reader method. We demonstrated that both HPLC and microplate reader are technically equivalent to measure violacein from yeast culture. Furthermore, we optimized the yeast cultivation conditions, including carbon/nitrogen ratio and pH conditions. Our results indicated that ethyl acetate is the best extraction solvent with glass beads grinding the cell pellets, the maximum violacein and deoxyviolacein production was 70.04 mg/L and 5.28 mg/L in shake flasks, respectively. Violacein purity reaches 86.92% at C/N ratio of 60, with addition of 10 g/L CaCO3 to control the media pH. Taken together, the development of efficient extraction protocol, quantitative correlation between HPLC and microplate reader, and the optimization of culture conditions set a new stage for engineering violacein production in Y. lipolytica. This information should be valuable for us to build a renewable and scalable violacein production platform from the novel host oleaginous yeast species.

microbiology↗