Biology subjects

Li, B.

Publications and source records attributed to Li, B..

At least 541 records · Page 30Linked to original sources

bioRxiv2020

Evolutionarily conserved regulation of embryonic fast-twitch skeletal muscle differentiation by Pbx factors

Vertebrate skeletal muscles are composed of both slow-twitch and fast-twitch fiber types. How the differentiation of distinct fiber types is activated during embryogenesis is not well characterized. Skeletal muscle differentiation is initiated by the activity of the myogenic basic helix-loop-helix (bHLH) transcription factors Myf5, Myod1, Myf6, and Myog. Myod1 functions as a muscle master regulatory factor and directly activates muscle differentiation genes, including those specific to both slow and fast muscle fibers. Our previous studies showed that Pbx TALE-class homeodomain proteins bind with Myod1 on the promoter of the zebrafish fast muscle gene mylpfa and are required for proper activation of mylpfa expression and the fast-twitch muscle-specific differentiation program in zebrafish embryos. Pbx proteins have also been shown to bind regulatory regions of muscle differentiation genes in mammalian muscle cells in culture. Here, we use new zebrafish mutant strains to confirm the essential roles of zebrafish Pbx factors in embryonic fast muscle differentiation. Furthermore, we examine the requirements for Pbx genes in mouse embryonic skeletal muscle differentiation, an area that has not been investigated in the mammalian embryo. Removing Pbx1 function from skeletal muscle in Myf5Cre/+;Pbx1fl/fl mouse embryos has minor effects on embryonic muscle development. However, concomitantly deleting Pbx2 function in Myf5Cre/+;Pbx1fl/fl;Pbx2-/- mouse embryos causes delayed activation and reduced expression of fast muscle differentiation genes. In the mouse, Pbx1/Pbx2-dependent fast muscle genes closely match those that have been previously shown to be dependent on murine Six1 and Six4. This work establishes evolutionarily conserved requirements for Pbx factors in embryonic fast muscle differentiation. Our studies are revealing how Pbx homeodomain proteins help direct specific cellular differentiation pathways.

developmental biology↗

bioRxiv2020

The RNA binding protein hnRNP-U is required for physiological hypertrophy of skeletal muscle

Skeletal muscle has the remarkable ability to modulate its mass in response to physiological changes associated with nutritional input, functional utilization, systemic disease, and age. A decreased responsiveness to anabolic stimuli is thought to contribute significantly to the loss of skeletal muscle mass and strength associated with sarcopenia, however the molecular mechanisms precipitating this are unclear. The signal transduction pathways that control the relative balance between anabolic and catabolic processes are tightly regulated at the transcriptional and post-transcriptional levels. Alternative splicing produces multiple protein isoforms from a single gene in a cell-type-specific manner and in response to environmental cues. We show that sustained activation of Akt1 in Hnrnpu deficient mice leads to premature muscle wasting, in part, through impaired autophagy while providing mechanistic insights into the development of anabolic resistance.

cell biology↗

bioRxiv2020

An in vitro vesicle formation assay to analyze protein sorting in the secretory transport pathway

The fidelity of protein transport in the secretory transport pathway relies on the accurate sorting of proteins to their correct destination. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on a specific cargo sorting machinery to be efficiently packaged into vesicles. Here, we used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry analyses of the isolated vesicles revealed novel cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner or that interact with GTP-bound Sar1A on vesicle membranes. Functional analysis indicates that two of them, FAM84B and PRRC1, regulate anterograde trafficking. Comparing control cells with cells depleted of the cargo receptors, SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Moreover, our results indicate that vesicles enriched with a specific cargo protein contain specific transmembrane cargo and SNARE proteins. A SNARE protein, Vti1B, is identified to be in vesicles enriched with a planar cell polarity protein, Frizzled6, and promotes vesicular release of Frizzled6. Our results indicate that the vesicle formation assay in combination with quantitative mass spectrometric analysis is a robust and powerful tool to reveal novel cytosolic and transmembrane proteins that regulate trafficking of a specific cargo protein.

cell biology↗

bioRxiv2020

Thousands of novel unannotated proteins expand the MHC I immunopeptidome in cancer

Tumor epitopes - peptides that are presented on surface-bound MHC I proteins - provide targets for cancer immunotherapy and have been identified extensively in the annotated protein-coding regions of the genome. Motivated by the recent discovery of translated novel unannotated open reading frames (nuORFs) using ribosome profiling (Ribo-seq), we hypothesized that cancer-associated processes could generate nuORFs that can serve as a new source of tumor antigens that harbor somatic mutations or show tumor-specific expression. To identify cancer-specific nuORFs, we generated Ribo-seq profiles for 29 malignant and healthy samples, developed a sensitive analytic approach for hierarchical ORF prediction, and constructed a high-confidence database of translated nuORFs across tissues. Peptides from 3,555 unique translated nuORFs were presented on MHC I, based on analysis of an extensive dataset of MHC I-bound peptides detected by mass spectrometry, with >20-fold more nuORF peptides detected in the MHC I immunopeptidomes compared to whole proteomes. We further detected somatic mutations in nuORFs of cancer samples and identified nuORFs with tumor-specific translation in melanoma, chronic lymphocytic leukemia and glioblastoma. NuORFs thus expand the pool of MHC I-presented, tumor-specific peptides, targetable by immunotherapies.

cancer biology↗

bioRxiv2020

Argonaute catalytic activity is required for maternal mRNA clearance in embryos

Inheritance and clearance of maternal mRNAs are two of the most critical events required for animal early embryonic development. However, the mechanisms regulating this process are still largely unknown. Here, we show that together with maternal mRNAs, C. elegans embryos inherit a complementary pool of small non-coding RNAs capable of triggering the cleavage and removal of hundreds of maternal mRNAs. These antisense small RNAs are loaded into the maternal catalytically-active Argonaute CSR-1 and cleave complementary mRNAs no longer engaged in translation in somatic blastomeres. Induced depletion of CSR-1 specifically during embryonic development leads to embryonic lethality in a slicer-dependent manner and impairs the degradation of CSR-1 embryonic mRNA targets. Given the conservation of Argonaute catalytic activity, we propose that a similar mechanism operates to clear maternal mRNAs during the maternal-to-zygotic transition across species.

developmental biology↗

bioRxiv2020

Two-photon voltage imaging of spontaneous activity from multiple neurons reveals network activity in brain tissue

Recording the electrical activity of multiple neurons simultaneously would greatly facilitate studies on the structure and function of neuronal circuits. Using fluorescent genetically encoded voltage indicators (GEVI) would be especially desirable, as it would allow cell type-selectivity, longitudinal recordings, and further optical manipulations. By expressing the GEVI ASAP3 via in utero electroporation and rapidly imaging neurons in densely labelled tissues via random-access multi-photon microscopy, we achieve voltage recording of multiple neurons in brain slice with single-trial single-voxel resolution. This approach enables monitoring of subthreshold membrane potential changes and action potentials from multiple locations in soma and dendrites for tens of minutes. By optically recording spontaneous electrical activities in somatosensory cortex neurons, we provide evidence for the development of intralaminar horizontal connections in layer 2/3 with greater sensitivity than calcium imaging. Single-trial optical voltage recordings using ASAP3 thus enables the investigation of network connectivity at cellular resolution.

neuroscience↗

bioRxiv2020

Analysis of metagenome-assembled genomes from the mouse gut microbiota reveals distinctive strain-level characteristics

The laboratorial mouse harbors a unique gut microbiota with potential value for human microbiota-associated studies. Mouse gut microbiota has been explored at the genus and species levels, but features rarely been showed at the strain level. The identification of 833,051 and 658,438 nonredundant genes of faeces and gut content samples from the laboratorial C57/BL mice showed over half of these genes were newly found compared to the previous mouse gut microbial gene catalogue. Metagenome-assembled genomes (MAGs) was used to reconstruct 46 nonredundant MAGs belonging to uncultured specieses. These MAGs included members across all phyla in mouse gut (i.e. Firmicutes, Bacteroidetes, Proteobacteria, Deferribacteres, Verrucomicrobia, and Tenericutes) and allowed a strain-level delineating of the mouse gut microbiota. Comparison of MAGs with human gut colonies revealed distinctive genomic and functional characteristics of mouses Bacteroidetes and Firmicutes strains. Genomic characteristics of rare phyla in mouse gut microbiota were demonstrated by MAG approach, including strains of Mucispirillum schaedleri, Parasutterella excrementihominis, Helicobacter typhlonius, and Akkermansia muciniphila. ImportanceThe identification of nonredundant genes suggested the existence of unknown microbes in the mouse gut samples. The metagenome-assembled genomes (MAGs) instantiated the specificity of mouse gut species and revealed an intestinal microbial correlation between mouse and human. The cultivation of faeces and gut contents sample validated the existence of MAGs and estimate their accuracy. Full-length 16S ribosomal RNA gene sequencing enabled taxonomic characterization. This study highlighted a unique ecosystem in the gut of laboratorial mice that obviously differed with the human gut flora at the strain level. The outcomes may be beneficial to researches based on laboratorial mouse models.

microbiology↗

bioRxiv2020

Profiling the microbial community structure and functional diversity of a dam-regulated river undergoing gravel bar restoration

BackgroundRiver restoration efforts are expected to influence and change the diversity and functions of microbial communities following the recovery of habitat characteristics in the river ecosystem. The recreation or restoration of gravel bars in the Trinity River in California aims to rehabilitate the environmental heterogeneity downstream of the dam impounded channel. Here, we profiled the community composition, estimated diversity, and annotated putative metabolic functions of the sediment microbial communities to assess whether the construction and restoration of gravel bars in the Trinity River in California enhanced environmental heterogeneity, with the increase in the microbial beta diversity of these in-channel structures against the free-flowing reach of the main channel with comparison to its undisturbed tributaries. ResultsMicrobial community composition of the free-flowing (i.e., no gravel bars) communities were relatively closer regardless of dam influence, whereas the Trinity River gravel bar and tributaries gravel bar communities were highly dissimilar. Proteobacteria, Bacteroidetes, and Acidobacteria were the highly abundant sediment microbial phyla on most sites, specifically in the Trinity River gravel bar communities. Putative functional annotation of microbial taxa revealed that chemoheterotrophy and aerobic chemoheterotrophy were the most prevalent microbial processes, with the Trinity River gravel bars having relatively higher representations. The considerably large abundance of heterotrophic taxa implies that gravel bars provide suitable areas for heterotrophic microorganisms with metabolic functions contributing to the net respiration in the river. ConclusionsOur results provide supporting evidence on the positive impact of habitat restoration being conducted in the Trinity River with the non-dam influenced, undisturbed tributaries as the basis of comparison. Gravel bar recreation and restoration contributed to the increased microbial biodiversity through the restoration of environmental heterogeneity at the river scale. We provided valuable insights into the potential microbial processes in the sediment that might be contributing to the biogeochemical processes carried out by the microbial communities in the Trinity River. The significant positive correlation between the functional diversity of the identified microbial taxa and beta diversity suggests that differences in the detected metabolic functions were closely related to dissimilarities in community composition.

microbiology↗

bioRxiv2020

Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin

Since the SARS outbreak 18 years ago, a large number of severe acute respiratory syndrome related coronaviruses (SARSr-CoV) have been discovered in their natural reservoir host, bats1-4. Previous studies indicated that some of those bat SARSr-CoVs have the potential to infect humans5-7. Here we report the identification and characterization of a novel coronavirus (nCoV-2019) which caused an epidemic of acute respiratory syndrome in humans, in Wuhan, China. The epidemic, started from December 12th, 2019, has caused 198 laboratory confirmed infections with three fatal cases by January 20th, 2020. Full-length genome sequences were obtained from five patients at the early stage of the outbreak. They are almost identical to each other and share 79.5% sequence identify to SARS-CoV. Furthermore, it was found that nCoV-2019 is 96% identical at the whole genome level to a bat coronavirus. The pairwise protein sequence analysis of seven conserved non-structural proteins show that this virus belongs to the species of SARSr-CoV. The nCoV-2019 virus was then isolated from the bronchoalveolar lavage fluid of a critically ill patient, which can be neutralized by sera from several patients. Importantly, we have confirmed that this novel CoV uses the same cell entry receptor, ACE2, as SARS-CoV.

microbiology↗

bioRxiv2020

Calcium-independent astrocytic lipid release modulates neuronal activity through Kv channels

An accumulating amount of data suggests that Ca2+-dependent gliotransmitter release plays a key role in the modulation of neuronal networks. Here, we tested the hypothesis that in response to agonist exposure, astrocytes release lipid modulators through activation of Ca2+-independent phospholipase A2 (iPLA2) activity. We found that cultured rat astrocytes treated with selective ATP and glutamatergic agonists released arachidonic acid (AA) and/or its derivatives, including the endogenous cannabinoid 2-arachidonoyl-sn-glycerol (2AG) and prostaglandin E2 (PGE2). Surprisingly, the buffering of cytosolic Ca2+ resulted in a sharp increase in agonist-induced lipid release by astrocytes. In addition, the astrocytic release of PGE2 increased miniature excitatory postsynaptic potentials (mEPSPs) by inhibiting the opening of neuronal Kv channels in brain slices. This study provides the first evidence showing that a Ca2+-independent pathway regulates the release of PGE2 from astrocytes and further demonstrates the functional role of astrocytic lipid release in the modulation of synaptic activity. SIGNIFICANCEUntil now, most studies that implicate astrocytes in the modulation of synaptic activity have focused on Ca2+-dependent release of traditional gliotransmitters such as D-serine, ATP, and glutamate. Mobilization of intracellular stores of Ca2+ occurs within a matter of seconds, but this novel Ca2+-independent lipid pathway in astrocytes could occur on a faster time scale and thus play a role in the rapid signaling processes involved in synaptic potentiation, attention, and neurovascular coupling.

neuroscience↗

bioRxiv2019

Effects of Endophytic Fungus MF23 on Dendrobium nobile Lindl. in an artificial primary environment

The quality of Dendrobium nobile Lindl. is related to the endophytic fungus. It had reported that the mycorrhizal fungus MF23 helps to increase the content of dendrobine, but few studies had explained the mechanism of the phenomenon. In previous study, we verified the symbiotic mechanism of the mycorrhizal fungus MF23 with D. nobile on the agar medium. In this study, the research carried on the bark medium, nearly like the natural environment, which had special meaning because of its benefits for the widely application. As a result, it showed a significant effect especially in the later period of the cultivation, suggesting that mycorrhizal fungus MF23 had a promotion for D. nobile in the natural environment, which enabled the application of the technique in the experimental field. It also implied that post-modification enzyme genes might play an important role in stimulating biosynthesis of dendrobine.

genetics↗

bioRxiv2019

The α-synuclein hereditary mutation E46K unlocks a more stable, pathogenic fibril structure

Aggregation of -synuclein is a defining molecular feature of Parkinsons disease, Lewy Body Dementia, and Multiple Systems Atrophy. Hereditary mutations in -synuclein are linked to both Parkinsons disease and Lewy Body Dementia; in particular, patients bearing the E46K disease mutation manifest a clinical picture of parkinsonism and Lewy Body Dementia, and E46K creates more pathogenic fibrils in vitro. Understanding the effect of these hereditary mutations on -synuclein fibril structure is fundamental to -synuclein biology. We therefore determined the cryoEM structure of -synuclein fibrils containing the hereditary E46K mutation. The 2.5 [A] structure reveals a symmetric double protofilament in which the molecules adopt a vastly re-arranged, lower energy fold compared to wild-type fibrils. We propose that the E46K misfolding pathway avoids electrostatic repulsion between K46 and K80, a residue pair which forms the E46-K80 salt-bridge in the wild-type fibril structure. We hypothesize that under our conditions the wild-type fold does not reach this deeper energy well of the E46K fold because the E46-K80 salt bridge diverts -synuclein into a kinetic trap - a shallower, more accessible energy minimum. The E46K mutation apparently unlocks a more stable and pathogenic fibril structure. Significance StatementParkinsons is the second most prevalent neurodegenerative condition, leading to movement disorders, and dementia in some cases. Because of the strong association of this condition with amyloid aggregates of the protein -synuclein, structural understanding of these amyloid aggregates may be the path to eventual therapies. Our study of the structure of a variant -synuclein inherited in families afflicted with a clinical picture of parkinsonism and Lewy Body Dementia supplements recent structures of the wild type structure, and shows how a single residue change can result in a greatly changed structure that may underlie the inherited form of the disease.

biochemistry↗

bioRxiv2019

WEDGE: recovery of gene expression values for sparse single-cell RNA-seq datasets using matrix decomposition

The low capture rate of expressed RNAs from single-cell sequencing technology is one of the major obstacles to downstream functional genomics analyses. Recently, a number of imputation methods have emerged for single-cell transcriptome data, however, recovering missing values in very sparse expression matrices remains a substantial challenge. Here, we propose a new algorithm, WEDGE (WEighted Decomposition of Gene Expression), to impute gene expression matrices by using a biased low-rank matrix decomposition method (bLRMD). WEDGE successfully recovered expression matrices, reproduced the cell-wise and gene-wise correlations, and improved the clustering of cells, performing impressively for applications with multiple cell type datasets with high dropout rates. Overall, this study demonstrates a potent approach for imputing sparse expression matrix data, and our WEDGE algorithm should help many researchers to more profitably explore the biological meanings embedded in their scRNA-seq datasets.

bioinformatics↗

bioRxiv2019

Digital polymerase chain reaction in an array of microfluidic printed droplets

Digital polymerase chain reaction (PCR) is a fast-developed technology, which makes it possible to provide absolute quantitative results. However, this technology has not been widely used in research field or clinical diagnostics. Although digital PCR has been born for two decades, the products on this subject still suffer from either high cost or cumbersome user experience, hence very few labs have the willingness or budget to routinely use such product; On the other hand, the unique sensitivity of dPCR over traditional qPCR shows great potential applications. Here, a cost-effective digital PCR method based on a microfluidic printing system was introduced, trying to overcome those shortcomings. The microfluidic droplet printing technology was utilized in this study to directly generate droplet array containing PCR reaction solution onto the simple glass substrate for the subsequent PCR and imaging, which could be done with any regular flat-panel PCR machine and microscope. The method introduces a new perspective in droplet-based digital PCR in that the droplets generated with this method aligns well in an array without touch with each other, therefore the regular glass and oil could be used without any special surfactant. With simple analysis, the data generated with this method showed reliable quality, which followed the Poisson distribution trend. Compared with other expensive digital PCR methods, this system is more affordable and simpler to integrate, especially for those biological or medical labs which are in need for the digital PCR options but short in budget. Therefore, this method is believed to have the great potential in the future market application.

molecular biology↗

bioRxiv2019

High-throughput reclassification of SCN5A variants

RationalePartial or complete loss of function variants in SCN5A are the most common genetic cause of the arrhythmia disorder Brugada Syndrome (BrS1). However, the pathogenicity of SCN5A variants is often unknown or disputed; 80% of the 1,390 SCN5A missense variants observed in at least one individual to date are variants of uncertain significance (VUS). The designation of VUS is a barrier to the use of sequence data in clinical care. ObjectiveWe selected 83 variants for study: 10 previously studied control variants, 10 suspected benign variants, and 63 suspected Brugada Syndrome-associated variants, selected on the basis of their frequency in the general population and in patients with Brugada Syndrome. We used high-throughput automated patch clamping to study the function of the 83 variants, with the goal of reclassifying variants with functional data. Methods and ResultsTen previously studied variants had functional properties concordant with published manual patch clamp data. All 10 suspected benign variants had wildtype-like function. 22 suspected BrS variants had loss of channel function (<10% normalized peak current) and 23 variants had partial loss of function (10-50% normalized peak current). The 73 previously unstudied variants were initially classified as likely benign (n=2), likely pathogenic (n=11), or VUS (n=60). After the patch clamp studies, 16 variants were benign/likely benign, 47 were pathogenic/likely pathogenic, and only 10 were still VUS. 8/22 loss of function variants were partially rescuable by incubation at lower temperature or pretreatment with a sodium channel blocker. Structural modeling identified likely mechanisms for loss of function including altered thermostability, and disruptions to alpha helices, disulfide bonds, or the permeation pore. ConclusionsHigh-throughput automated patch clamping enabled the reclassification of the majority of tested VUSs in SCN5A.

genetics↗

bioRxiv2019

Improved transcriptome annotation and read-through transcript identification with TIF-Seq2

Eukaryotic transcriptomes are complex involving thousands of overlapping transcripts. The interleaved nature of the transcriptome limits our ability to identify regulatory regions and, in some cases, can lead to misinterpretation of gene expression. To improve the understanding of the overlapping transcriptome, we have developed an optimized method, TIF-Seq2, able to sequence simultaneously the 5 and 3 ends of individual RNA molecules at single-nucleotide resolution. We investigated the transcriptome of a well characterized human cell line (K562) and identify thousands of unannotated transcript isoforms. By focusing on transcripts which are challenging to be investigated with RNA-seq, we accurately defined boundaries of lowly expressed unannotated and read-though transcripts putatively encoding fusion genes. We validated our results by targeted long-read sequencing and standard RNA-Seq for chronic myeloid leukaemia patient samples. Taking the advantage of TIF-Seq2, we explore transcription regulation among the overlapping units and investigate their crosstalk. We show that most overlapping upstream transcripts use poly(A) sites within the first 2 kb of the downstream transcription unit. Our work shows that, by paring the 5 and 3 end of each RNA, TIF-Seq2 can improve the annotation of complex genomes, facilitates accurate assignment of promoters to genes and easily identify transcriptionally fused genes. Key pointsO_LIStudy of TSS-PAS co-occurrence allows dissecting complex overlapping transcription units. C_LIO_LIPartially overlapping transcription units in human commonly use PAS within the first 2Kb. C_LIO_LITIF-Seq2 facilitates the identification of lowly expressed and transcriptionally fused genes. C_LI

genomics↗

bioRxiv2019

Recurrent Neoantigens in Colorectal Cancer as Potential Immunotherapy Targets

This study was aimed to investigate the mutations in colorectal cancer (CRC) for recurrent neoantigen identification. A total of 1,779 samples with whole exome sequencing (WES) data were obtained from 7 published CRC cohorts. Common HLA genotypes were used to predict the probability of neoantigens at high frequency mutants in the dataset. Based on the WES data, we not only obtained the most comprehensive CRC mutation landscape so far, but also found 1550 mutation sites which could be identified in at least 5 or more patients, including KRAS G12D (8%), KRAS G12V (5.8%), PIK3CA E545K (3.5%), PIK3CA H1047R (2.5%) and BMPR2 N583Tfs*44 (2.8%). These mutations can also be recognized by multiple common HLA molecules as potential public neoantigens. Many of these mutations also have high mutation rates in metastatic pan-cancers, suggesting their value as therapeutic targets in different cancer types. Overall, our analysis provides recurrent neoantigens as potential cancer immunotherapy targets.

cancer biology↗

bioRxiv2019

A big-data approach to understanding metabolic rate and response to obesity in laboratory mice

Maintaining a healthy body weight requires an exquisite balance between energy intake and energy expenditure. In humans and in laboratory mice these factors are experimentally measured by powerful and sensitive indirect calorimetry devices. To understand the genetic and environmental factors that contribute to the regulation of body weight, an important first step is to establish the normal range of metabolic values and primary sources contributing to variability in results. Here we examine indirect calorimetry results from two experimental mouse projects, the Mouse Metabolic Phenotyping Centers and International Mouse Phenotyping Consortium to develop insights into large-scale trends in mammalian metabolism. Analysis of nearly 10,000 wildtype mice revealed that the largest experimental variances are consequences of institutional site. This institutional effect on variation eclipsed those of housing temperature, body mass, locomotor activity, sex, or season. We do not find support for the claim that female mice have greater metabolic variation than male mice. An analysis of these factors shows a normal distribution for energy expenditure in the phenotypic analysis of 2,246 knockout strains and establishes a reference for the magnitude of metabolic changes. Using this framework, we examine knockout strains with known metabolic phenotypes. We compare these effects with common environmental challenges including age, and exercise. We further examine the distribution of metabolic phenotypes exhibited by knockout strains of genes corresponding to GWAS obesity susceptibility loci. Based on these findings, we provide suggestions for how best to design and conduct energy balance experiments in rodents, as well as how to analyze and report data from these studies. These recommendations will move us closer to the goal of a centralized physiological repository to foster transparency, rigor and reproducibility in metabolic physiology experimentation.

physiology↗