Biology subjects

Wei, H.

Publications and source records attributed to Wei, H..

At least 109 records · Page 6Linked to original sources

bioRxiv2020

Genome-wide identification and transcriptional analyses of MATE transporter genes in root tips of wild Cicer spp. under aluminium stress

Chickpea is an economically important legume crop with high nutritional value in human diets. Aluminium-toxicity poses a significant challenge for the yield improvement of this increasingly popular crop in acidic soils. The wild progenitors of chickpea may provide a more diverse gene pool for Al-tolerance in chickpea breeding. However, the genetic basis of Al-tolerance in chickpea and its wild relatives remains largely unknown. Here, we assessed the Al-tolerance of six selected wild Cicer accessions by measuring the root elongation in solution culture under control (0 {micro}M Al3+) and Al-treatment (30 {micro}M Al3+) conditions. Al-treatment significantly reduced the root elongation in all target lines compared to the control condition after 2-days growth. However, the relative reduction of root elongation in different lines varied greatly: 3 lines still retained significant root growth under Al-treatment, whilst another 2 lines displayed no root growth at all. We performed genome-wide identification of multidrug and toxic compound extrusion (MATE) encoding genes in the Cicer genome. A total of 56 annotated MATE genes were identified, which divided into 4 major phylogeny groups (G1-4). Four homologues to lupin LaMATE (> 50% aa identity; named CaMATE1-4) were clustered with previously characterised MATEs related to Al-tolerance in various other plants. qRT-PCR showed that CaMATE2 transcription in root tips was significantly up-regulated upon Al-treatment in all target lines, whilst CaMATE1 was up-regulated in all lines except Bari2_074 and Deste_064, which coincided with the lines displaying no root growth under Al-treatment. Transcriptional profiling in five Cicer tissues revealed that CaMATE1 is specifically transcribed in the root tissue, further supporting its role in Al-detoxification in roots. This first identification of MATE-encoding genes associated with Al-tolerance in Cicer paves the ways for future functional characterization of MATE genes in Cicer spp., and to facilitate future design of gene-specific markers for Al-tolerant line selection in chickpea breeding programs.

plant biology↗

bioRxiv2020

Time-resolved cryoEM using Spotiton

We present an approach for preparing cryoEM grids to study short-lived molecular states. Using piezo electric dispensing, two independent streams of ~50 pL sample drops are deposited within 10 ms of each other onto a nanowire EM grid surface, and the mixing reaction stops when the grid is vitrified in liquid ethane, on the order of ~100 ms later. We demonstrate the utility of this approach for four biological systems where short-lived states are of high interest.

biophysics↗

bioRxiv2020

Tocilizumab treatment in severe COVID-19 patients attenuates the inflammatory storm incited by monocyte centric immune interactions revealed by single-cell analysis

ABSTRACTDespite the current devastation of the COVID-19 pandemic, several recent studies have suggested that the immunosuppressive drug Tocilizumab can powerfully treating inflammatory responses that occur in this disease. Here, by employing single-cell analysis of the immune cell composition of severe-stage COVID-19 patients and these same patients in post Tocilizumab-treatment remission, we have identified a monocyte subpopulation specific to severe disease that contributes to inflammatory storms in COVID-19 patients. Although Tocilizumab treatment attenuated the strong inflammatory immune response, we found that immune cells including plasma B cells and CD8+ T cells still exhibited an intense humoral and cell-mediated anti-virus immune response in COVID-19 patients after Tocilizumab treatment. Thus, in addition to providing a rich, very high-resolution data resource about the immune cell distribution at multiple stages of the COVID-19 disease, our work both helps explain Tocilizumab’s powerful therapeutic effects and defines a large number of potential new drug targets related to inflammatory storms.Competing Interest StatementJingwen Fang is the executive officer of HanGen BiotechView Full Text

genomics↗

bioRxiv2020

Non-opioid analgesia based on Gα signalling bias

The development of therapeutic agonists for G protein-coupled receptors (GPCRs) is hampered by the propensity of GPCRs to couple to multiple intracellular signalling pathways. This promiscuous coupling leads to numerous downstream cellular effects, some of which are therapeutically undesirable. This is especially the case for adenosine A1 receptors (A1Rs) whose clinical potential is undermined by the sedation and cardiorespiratory depression caused by conventional agonists. We have discovered that the A1R-selective agonist, BnOCPA, is a potent and powerful analgesic but does not cause sedation, bradycardia, hypotension or respiratory depression. This unprecedented discrimination between native A1Rs arises from BnOCPAs unique and exquisitely selective activation of Gob among the six Gi/o subtypes, and in the absence of {beta}-arrestin recruitment. BnOCPA thus demonstrates a highly-specific G-selective activation of the native A1R, sheds new light on GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of selective G agonism. Short summaryWe describe the selective activation of an adenosine A1 receptor-mediated intracellular pathway that provides potent analgesia in the absence of sedation or cardiorespiratory depression, paving the way for novel medicines based on the far-reaching concept of selective G agonism.

physiology↗

bioRxiv2020

Comparison of long read methods for sequencing and assembly of a plant genome

Sequencing technologies have advanced to the point where it is possible to generate high accuracy, haplotype resolved, chromosome scale assemblies. Several long read sequencing technologies are available on the market and a growing number of algorithms have been developed over the last years to assemble the reads generated by those technologies. When starting a new genome project, it is therefore challenging to select the most cost-effective sequencing technology as well as the most appropriate software for assembly and polishing. For this reason, it is important to benchmark different approaches applied to the same sample. Here, we report a comparison of three long read sequencing technologies applied to the de novo assembly of a plant genome, Macadamia jansenii. We have generated sequencing data using Pacific Biosciences (Sequel I), Oxford Nanopore Technologies (PromethION) and BGI (single-tube Long Fragment Read) technologies for the same sample. Several assemblers were benchmarked in the assembly of PacBio and Nanopore reads. Results obtained from combining long read technologies or short read and long read technologies are also presented. The assemblies were compared for contiguity, accuracy and completeness as well as sequencing costs and DNA material requirements. Overall, the three long read technologies produced highly contiguous and complete genome assemblies of Macadamia jansenii. At the time of sequencing, the cost associated with each method was significantly different but continuous improvements in technologies have resulted in greater accuracy, increased throughput and reduced costs. We propose updating this comparison regularly with reports on significant iterations of the sequencing technologies.

genomics↗

bioRxiv2020

A comprehensive proteomic SWATH-MS workflow for profiling blood extracellular vesicles: a new avenue for glioma tumour surveillance

There is a real need for biomarkers that can indicate glioma disease burden and inform clinical management, particularly in the recurrent glioblastoma (GBM; grade IV glioma) setting where treatment-associated brain changes can confound current and expensive tumour surveillance methods. In this regard, extracellular vesicles (EVs; 30-1000 nm membranous particles) hold major promise as robust tumour biomarkers. GBM-EVs encapsulate molecules that reflect the identity and molecular state of their cell-of-origin and cross the blood-brain-barrier into the periphery where they are readily accessible. Despite the suitability of circulating-EVs for GBM biomarker discovery, sample complexity has hindered comprehensive quantitative proteomic studies. Here, sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) was used in conjunction with a targeted data extraction strategy to comprehensively profile circulating-EVs isolated from plasma. Plasma-EVs sourced from pre-operative glioma II-IV patients (n=41) and controls (n=11) were sequenced by SWATH-MS, and the identities and absolute quantities of the proteins were extracted by aligning the SWATH-MS data against a custom glioma spectral library comprised of 8662 high confidence protein species. Overall, 4054 plasma-EV proteins were quantified across the cohorts, and putative circulating-EV biomarker proteins identified (adjusted p-value<0.05) included previously reported GBM-EV proteins identified in vitro and in neurosurgical aspirates. Principle component analyses showed that plasma-EV protein profiles clustered according to glioma subtype and WHO-grade, and plasma-EV proteins reflected the extent of glioma aggression. Using SWATH-MS, we describe the most comprehensive proteomic plasma-EV profiles for glioma and highlight the promise of this approach as an accurate and sensitive tumour monitoring method. Objective blood-based measurements of glioma tumour activity will support the implementation of next-generation, patient-centred therapies and are ideal surrogate endpoints for recurrent progression that would allow clinical trial protocols to be more dynamic and adapt to the individual patient and their cancer.

pathology↗

bioRxiv2020

Aberrant pathogenic GM-CSF+ T cells and inflammatory CD14+CD16+ monocytes in severe pulmonary syndrome patients of a new coronavirus

Pathogenic human coronavirus infections, such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV), cause high morbidity and mortality 1,2. Recently, a severe pneumonia-associated respiratory syndrome caused by a new coronavirus was reported at December 2019 (2019-nCoV) in the city Wuhan, Hubei province, China3-5, which was also named as pneumonia-associated respiratory syndrome (PARS)6. Up to 9th of February 2020, at least 37, 251 cases have been reported with 812 fatal cases according to the report from China CDC. However, the immune mechanism that potential orchestrated acute mortality from patients of 2019-nCoV is still unknown. Here we show that after the 2019-nCoV infection, CD4+T lymphocytes are rapidly activated to become pathogenic T helper (Th) 1 cells and generate GM-CSF etc. The cytokines environment induces inflammatory CD14+CD16+ monocytes with high expression of IL-6 and accelerates the inflammation. These aberrant and excessive immune cells may enter the pulmonary circulation in huge numbers and play an immune damaging role to causing lung functional disability and quick mortality. Our results demonstrate that excessive non-effective host immune responses by pathogenic T cells and inflammatory monocytes may associate with severe lung pathology. Therefore, we suggest that monoclonal antibody that targets the GM-CSF or interleukin 6 receptor may potentially curb immunopathology caused by 2019-nCoV and consequently win more time for virus clearance.

immunology↗

bioRxiv2020

The transmembrane serine protease inhibitors are potential antiviral drugs for 2019-nCoV targeting the insertion sequence-induced viral infectivity enhancement

At the end of 2019, the SARS-CoV-2 induces an ongoing outbreak of pneumonia in China1, even more spread than SARS-CoV infection2. The entry of SARS-CoV into host cells mainly depends on the cell receptor (ACE2) recognition and spike protein cleavage-induced cell membrane fusion3,4. The spike protein of SARS-CoV-2 also binds to ACE2 with a similar affinity, whereas its spike protein cleavage remains unclear5,6. Here we show that an insertion sequence in the spike protein of SARS-CoV-2 enhances the cleavage efficiency, and besides pulmonary alveoli, intestinal and esophagus epithelium were also the target tissues of SARS-CoV-2. Compared with SARS-CoV, we found a SPRR insertion in the S1/S2 protease cleavage sites of SARS-CoV-2 spike protein increasing the cleavage efficiency by the protein sequence aligment and furin score calculation. Additionally, the insertion sequence facilitates the formation of an extended loop which was more suitable for protease recognition by the homology modeling and molicular docking. Furthermore, the single-cell transcriptomes identified that ACE2 and TMPRSSs are highly coexpressed in AT2 cells of lung, along with esophageal upper epithelial cells and absorptive enterocytes. Our results provide the bioinformatics evidence for the increased spike protein cleavage of SARS-CoV-2 and indicate its potential target cells.

microbiology↗

bioRxiv2019

Cryo-EM Structures and Regulation of Arabinofuranosyltransferase AftD from Mycobacteria

Mycobacterium tuberculosis causes tuberculosis, a disease that kills over one million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides - arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9 [A] resolution structure of M. abscessus AftD determined by single particle cryo-electron microscopy. AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate binding modules. Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is non-covalently complexed with an acyl carrier protein (ACP). 3.4 and 3.5 [A] structures of a mutant with impaired ACP binding reveal a conformational change that suggests the ACP may regulate AftD function. Using a conditional knock-out constructed in M. smegmatis, mutagenesis experiments confirm the essentiality of the putative active site and the ACP binding for AftD function.

biochemistry↗

bioRxiv2019

Rapid phylogenetic analysis of African swine fever virus from metagenomic sequences

African swine fever virus (ASFV) has devastating impacts on swine health and the world economy. Rapid and accurate phylogenetic analysis of ASFV causing outbreaks is important to reveal diversity and evolutionary of ASFV. Because it is time-consuming and needs biosafety laboratories to isolate ASFV, here we present a new way to perform rapid genome-wide phylogenetic analysis of ASFV using an allele calling based on gene by gene approach directly from genome drafts assembled from metagenomic sequences. Using open-accessed chewBBACA software, 41 publicly available ASFV genomes were analyzed to optimize the parameters and find the alleles. Alleles as many as 94 were found for building the phylogenetic trees, which covered more than 56% of the whole genome. Based on the alleles, current ASFV isolates could be divided into two major clades and a few subclades. Then the method is used to analyze two ASFV genome drafts assembled from two metagenomic sequences of a swine whole blood and a swine spleen tissue collected in Wuhan, China. It shows that the two ASFV genomes showed highest similarity to that of Pig/HLJ/2018 strain and DB/LN/2018 strain, which isolated recently in China. This proved that the ASFV in Wuhan originate from the same source causing the earlier outbreaks in Helongjiang and Liaoning province of China. This method makes it possible to analyze phylogenetic analysis of ASFV draft genomes flexibly without the need of ASFV isolation. Furthermore, because it is based on Allele calling, the ASFV-specific genetic markers found could be translated into clinical diagnostics or can be used broadly to identify conserved putative therapeutic candidates.

bioinformatics↗

bioRxiv2019

Development of a PC12 cell-based assay for in vitro screening of catechol-O-methyltransferase inhibitors

The male rat adrenal pheochromocytoma cell-originated PC12 cell line can synthesize and release catecholamine neurotransmitters, and it has been widely used as a model system in cell biology and toxicology research. Catechol-O-methyltransferase (COMT) is involved in the inactivation of the catecholamine neurotransmitters, and it is particularly important for theregulation of dopamine. In this study, we explored the feasibility of using PC12 cells as an in vitro drug screening platform to compare the activity of multiple COMT inhibitors. Incubation of PC12 cells with tolcapone, a highly potent and selective COMT inhibitor, increased the concentrations of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) while reducing the metabolites 3-methoxytyramine (3-MT) and homovanillic acid (HVA) in the cell culture medium. LIBD-3, a novel, non-nitrocatechol COMT inhibitor produced similar effects compared to tolcapone. LIBD-4, a less potent inhibitor, exhibited the expected right-shift in functional inhibition in the assay. These results match the known in vivo effects of COMT inhibition in rodents. Together, these data support the continued use of PC12 cells as an in vitro screen that bridges cell-free enzyme assays and more costly in vivo assays.

neuroscience↗

bioRxiv2019

Mammalian retromer is an adaptable scaffold for cargo sorting from endosomes

In metazoans, retromer (VPS26/VPS35/VPS29) associates with sorting nexin (SNX) proteins to form coats on endosomal tubules and sort cargo proteins to the trans-Golgi network (TGN) or plasma membrane. This core complex is highly conserved from yeast to humans, but molecular mechanisms of metazoan retromer assembly remain undefined. Here we combine single particle cryo-electron microscopy with biophysical methods to uncover multiple oligomer structures formed by mammalian retromer. Two-dimensional class averages in ice reveal the retromer heterotrimer; dimers of trimers; tetramers of trimers; and flat chains. These species are further supported by biophysical studies in solution. We provide cryo-EM reconstructions of all species, including pseudo-atomic resolution detail for key sub-structures. Multi-body refinement demonstrates how retromer heterotrimers and dimers adopt a range of conformations. Our structures identify a flexible yet highly conserved electrostatic interface in dimers formed by interactions between VPS35 subunits. We generate a structure-based mutant to disrupt this key interface in vitro and introduce equivalent mutations into S. cerevisiae to demonstrate the mutant exhibits a cargo sorting defect. Together, structures and complementary functional data in budding yeast imply a conserved assembly interface across eukaryotes. These data further suggest mammalian retromer acts as an adaptable and plastic scaffold that accommodates interactions with different SNXs to sort multiple cargoes from endosomes their final destinations.

cell biology↗

bioRxiv2019

Landscape and dynamics of the transcriptional regulatory network during natural killer cell differentiation

Natural killer (NK) cells are essential in controlling cancer and infection. However, little is known about the dynamics of the transcriptional regulatory machinery during NK cell differentiation. In this study, we applied assay of transposase accessible chromatin with sequencing (ATAC-seq) technique in a self-developed in vitro NK cell differentiation system. Analysis of ATAC-seq data illustrated two distinct transcription factor (TF) clusters that dynamically regulate NK cell differentiation. Moreover, two TFs from the second cluster, FOSL2 and EGR2, were identified as novel essential TFs that control NK cell maturation and function. Knocking down either of these two TFs significantly impacted NK cell transformation. Finally, we constructed a genome-wide transcriptional regulatory network that provides an understanding of the regulatory dynamics during NK cell differentiation.

bioinformatics↗

bioRxiv2019

Genetic Diversity and Phylogenetic Relationships of Annual and Perennial Glycine Species

We estimated average genetic diversity of two Glycine annual and six perennial species based upon 76 orthologous gene sets and performed phylogenetic analysis, divergence analysis and tests for departure from neutrality of the eight species using 52 orthologous gene sets. In addition, 367 orthologous gene sets were used to estimate the relationships of 11 G. canescens accessions. Among the perennials, G. canescens showed the highest nucleotide diversity and the other perennials except G. tomentella had higher nucleotide diversity than the two annuals. Phylogenetic analysis of the Glycine showed a similar genome grouping with the previous report except G. cyrtoloba and G. stenophita formed a sister clade in the study. Divergence analysis supported the phylogenetic relationships, G. falcata was the most divergent from G. max, followed by G. cyrtoloba, G. syndetika, G. tomentella D3, G. stenophita and G. canescens. Neutrality selection tests within species showed that most genes were subjected to a recent directional selection due to a selective sweep or rapid population expansion. Although most gene sequence had negative and significant Tajimas D, the sequences were homogeneous in the levels of polymorphism and divergence between G. max and other Glycine species based on the HKA test, thus, Glycine perennials may have experienced very similar evolutionary selection as inferred by trans-specific mutation analysis. The greater genetic diversity of most perennial Glycine species and their origins from the warmer and drier climates of Australia suggested the perennials as potential sources of heat and drought resistance that will be of value in the face of climate change.

genetics↗

bioRxiv2019

Loss of heterozygosity of essential genes represents a widespread class of potential cancer vulnerabilities

Alterations in non-driver genes represent an emerging class of potential therapeutic targets in cancer. Hundreds to thousands of non-driver genes undergo loss of heterozygosity (LOH) events per tumor, generating discrete differences between tumor and normal cells. Here we interrogate LOH of polymorphisms in essential genes as a novel class of therapeutic targets. We hypothesized that monoallelic inactivation of the allele retained in tumors can selectively kill cancer cells but not somatic cells, which retain both alleles. We identified 5664 variants in 1278 essential genes that undergo LOH in cancer and evaluated the potential for each to be targeted using allele-specific gene-editing, RNAi, or small-molecule approaches. We further show that allele-specific inactivation of either of two essential genes (PRIM1 and EXOSC8) reduces growth of cells harboring that allele, while cells harboring the non-targeted allele remain intact. We conclude that LOH of essential genes represents a rich class of non-driver cancer vulnerabilities.

cancer biology↗

bioRxiv2019

Novel, non-nitrocatechol COMT inhibitors modulate dopamine neurotransmission in the frontal cortex and improve cognitive flexibility

Cognitive impairment is a primary feature of many neuropsychiatric disorders and there is a need for new therapeutic options. Catechol-O-methyltransferase (COMT) inhibitors modulate cortical dopaminergic function and have been proposed as potential cognitive enhancers. Unfortunately, currently available COMT inhibitors are not good candidates due to either poor blood-brain barrier penetration or severe toxicity. To address the need for safe, brain-penetrant COMT inhibitors, we tested multiple novel COMT inhibitors in a set of preclinical in vivo efficacy assays to determine their viability as potential clinical candidates. We found that multiple COMT inhibitors, exemplified by LIBD-1 and LIBD-3, significantly modulated dopaminergic function measured as decreases in homovanillic acid (HVA) and increases in 3,4-Dihydroxyphenylacetic acid (DOPAC), two dopamine metabolites, in cerebrospinal fluid (CSF) and the frontal cortex. Additionally, we found the LIBD-1 significantly improved cognitive flexibility in a rat attentional set-shifting assay (ASST), an effect previously seen with the COMT inhibitor tolcapone. These results demonstrate that LIBD-1 is a novel COMT inhibitor with promising in vivo activity and the potential to serve as a new therapy for cognitive impairment.

neuroscience↗

bioRxiv2018

In vivo generation of post-infarct mouse cardiac muscle by cardiomyocyte progenitors produced with a reproducible laminin-promoted human stem cell differentiation system

Regeneration of human heart muscle after injury is extremely limited and an unmet clinical need. Despite extensive research, there are no methods for the reproducible generation of clinical quality stem-cell-derived cardiovascular progenitors (CVPs). Here, we identified laminin-221 (LN-221) as the main cardiac laminin, which was produced here as human recombinant protein, and showed that LN-221 promotes differentiation of pluripotent hESCs towards cardiomyocyte lineage and downregulates genes associated with pluripotency and teratoma development. We developed a chemically defined, xeno-free laminin-based cardiomyocyte differentiation protocol to reproducibly generate CVPs that form human muscle in vivo. We assessed the reproducibility of the differentiation protocol using time-course bulk RNA sequencing developed from two different hESC lines. Single-cell RNA sequencing of CVPs derived from two hESC lines further showed high reproducibility and identified three main progenitor subpopulations. These CVPs were transplanted into myocardial infarction mice, where heart function was measured by echocardiogram and human heart muscle bundle formation was identified histologically. This method may provide clinical quality cells for use in regenerative cardiology.

cell biology↗

bioRxiv2018

Reliable Multiplex Sequencing with Rare Index Mis-Assignment on DNB-Based NGS Platform

BackgroundMassively-parallel-sequencing, coupled with sample multiplexing, has made genetic tests broadly affordable. However, intractable index mis-assignments (commonly exceeds 1%) were repeatedly reported on some widely used sequencing platforms.\n\nResultsHere, we investigated this quality issue on BGI sequencers using three library preparation methods: whole genome sequencing (WGS) with PCR, PCR-free WGS, and two-step targeted PCR. BGIs sequencers utilize a unique DNB technology which uses rolling circle replication for DNA-nanoball preparation; this linear amplification is PCR free and can avoid error accumulation. We demonstrated that single index mis-assignment from free indexed oligos occurs at a rate of one in 36 million reads, suggesting virtually no index hopping during DNB creation and arraying. Furthermore, the DNB-based NGS libraries have achieved an unprecedentedly low sample-to-sample mis-assignment rate of 0.0001% to 0.0004% under recommended procedures.\n\nConclusionsSingle indexing with DNB technology provides a simple but effective method for sensitive genetic assays with large sample numbers.

genomics↗