Biology subjects

Chen, L.

Publications and source records attributed to Chen, L..

At least 649 records · Page 36Linked to original sources

bioRxiv2022

Identification of ASPDH as a novel NAADP-binding protein

NAADP is a signaling molecule that can induce calcium release from intracellular acidic stores. However, proteins that bind to NAADP are understudied. Here, we identify ASPDH as an NAADP-binding protein through biochemical purification from pig livers. ITC experiment using the recombinantly expressed protein shows a 1:1 binding stoichiometry and a Kd of 455 nM between NAADP and mouse ASPDH. In contrast, recombinantly expressed JPT2 and LSM12, two proteins previously identified as NAADP-receptors, show no binding in ITC experiments.

biochemistry↗

bioRxiv2022

Portrait of a genus: genome sequencing reveals evidence of adaptive variation in Zea

Maize is a globally valuable commodity and one of the most extensively studied genetic model organisms. However, we know surprisingly little about the extent and potential utility of the genetic variation found in the wild relatives of maize. Here, we characterize a high-density genomic variation map from 744 genomes encompassing maize and all wild taxa of the genus Zea, identifying over 70 million single nucleotide polymorphisms (SNPs) and nearly 9 million Insertion/Deletion (InDel) polymorphisms. The variation map reveals evidence of selection within taxa displaying novel adaptations to traits such as waterlogging, perenniality and regrowth. We focus in detail on adaptive alleles in highland teosinte and temperate maize and highlight the key role of flowering time related pathways in highland and high latitude adaptation. To show how this data can identify useful genetic variants, we generated and characterized novel mutant alleles for two flowering time candidate genes. This work provides the most extensive sampling to date of the genetic diversity of the genus Zea, resolving questions on evolution and identifying adaptive variants for direct use in modern breeding.

genetics↗

bioRxiv2022

Genomic Insights of Bruneian Malays

The Malays and their many sub-ethnic groups collectively make up one of the largest population groups in Southeast Asia. However, their genomes, especially those from Brunei, remain very much underrepresented and understudied. Here, we analysed the publicly available WGS and genotyping data of two and 39 Bruneian Malay individuals, respectively. NGS reads from the two individuals were first mapped against the GRCh38 human reference genome and their variants called. Of the total [~]5.28 million short nucleotide variants and indels identified, [~]217K of them were found to be novel; with some predicted to be deleterious and associated with risk factors of common non-communicable diseases in Brunei. Unmapped reads were next mapped against the recently reported novel Chinese and Japanese genomic contigs and de novo assembled. [~]227 Kbp genomic sequences missing in GRCh38 and a partial open reading frame encoding a potential novel small zinc finger protein were successfully discovered. Interestingly, although the Malays in Brunei, Singapore and Malaysia share >83% common variants, principal component and admixture analysis comparing the genetic structure of the local Malays against other Asian population groups suggested that they are genetically closer to some Filipino ethnic groups than the Malays in Malaysia and Singapore. Taken together, our work provides the first comprehensive insight into the genomes of the Bruneian Malay population.

genomics↗

bioRxiv2022

Ensemble-specific deficit in neuronal intrinsic excitability in aged mice

With the prevalence of age-related cognitive deficits on the rise, it is essential to identify cellular and circuit alterations that contribute to age-related memory impairment. Increased intrinsic neuronal excitability after learning is important for memory consolidation, and changes to this process could underlie memory impairment in old age. Some studies find age-related deficits in hippocampal neuronal excitability that correlate with memory impairment but others do not, possibly due to selective changes only in activated neural ensembles. Thus, we tagged CA1 neurons activated during learning and recorded their intrinsic excitability 5 hours or 7 days post-training. Adult mice exhibited increased neuronal excitability 5 hours after learning, specifically in ensemble (learning-activated) CA1 neurons. As expected, ensemble excitability returned to baseline 7 days post-training. In aged mice, there was no ensemble-specific excitability increase after learning, which was associated with impaired hippocampal memory performance. These results suggest that CA1 may be susceptible to age-related impairments in post-learning ensemble excitability and underscore the need to selectively measure ensemble-specific changes in the brain.

neuroscience↗

bioRxiv2022

Within-host genotypic and phenotypic diversity of contemporaneous carbapenem-resistant Klebsiella pneumoniae from blood cultures of patients with bacteremia

Carbapenem-resistant Klebsiella pneumoniae (CRKP) are major pathogens globally. It is unknown whether bloodstream infections (BSIs) by CRKP and other bacteria are commonly caused by single organisms or mixed microbial populations. We hypothesized that contemporaneous CRKP from blood cultures of individual patients are genetically and phenotypically distinct. We determined short-read whole genome sequences of 10 strains from single colonies from CRKP-positive blood cultures in each of 6 patients (Illumina HiSeq). All strains were sequence type (ST)-258 K. pneumoniae that were unique by core genome single nucleotide polymorphism phylogeny, antibiotic resistance and virulence genes, capsular polysaccharide (CPS) gene mutations, and/or plasmid loss. Strains from each of 3 patients that differed in antibiotic resistance, virulence and/or CPS gene content underwent long-read sequencing for genome completion (Oxford Nanopore), and were tested for phenotypes in vitro and pathogenicity during mouse BSIs. Genetically distinct strains within individual patients exhibited significant differences in carbapenem, beta-lactam/beta-lactamase inhibitor and other antibiotic responses, CPS production, mucoviscosity, and susceptibility to serum killing. In 2 patients, strains differed significantly in their ability to infect organs and cause mortality in mice. In conclusion, we identified genotypic and phenotypic variant ST258 K. pneumoniae strains from blood cultures of individual patients, which were not detected by the clinical laboratory at time of BSI diagnosis. The data support a new paradigm of CRKP population diversity during BSIs. If validated for other BSIs, within-host bacterial diversity may have profound implications for medical, microbiology laboratory and infection prevention practices, and for understanding emergence of antibiotic resistance and pathogenesis. IMPORTANCEIn processing positive microbiologic cultures, standard clinical laboratory practice is to test a single bacterial strain from each morphologically distinct colony. We performed comprehensive whole genome sequence analyses on 10 carbapenem-resistant Klebsiella pneumoniae (CRKP) strains from positive blood cultures from each of 6 patients. Our findings that all strains were genetically unique and that genetic variants manifested differences in phenotypes like antibiotic responsiveness and virulence suggest that CRKP bloodstream infections may be commonly caused by mixed bacterial populations. Results raise questions about laboratory protocols and treatment decisions that are directed against a single strain. The observation that pan-genome analyses revealed inter-strain differences that were not evident by studying core genomes has important implications for investigating nosocomial outbreaks and transmission. Data also suggest a model of pathogenesis of CRKP infections, in which environmental pressures in vivo may select for outgrowth of variants that manifest antibiotic resistance, tolerance or specific virulence attributes.

microbiology↗

bioRxiv2022

DeepPHiC: Predicting promoter-centered chromatin interactions using a novel deep learning approach

MotivationPromoter-centered chromatin interactions, which include promoter-enhancer and promoter-promoter interactions, are important to decipher gene regulation and disease mechanisms. The development of next generation sequencing technologies such as promoter capture Hi-C (pcHi-C) leads to the discovery of promoter-centered chromatin interactions. However, pcHi-C experiments are expensive and thus may be unavailable for tissues or cell types of interest. In addition, these experiments may be underpowered due to insufficient sequencing depth or various artifacts, which results in a limited finding of interactions. ResultsTo overcome these challenges, we develop a supervised multi-modal deep learning model, which utilizes a comprehensive set of features including genomic sequence, epigenetic signal and anchor distance to predict tissue/cell type-specific genome-wide promoter-enhancer and promoter-promoter interactions. We further extend the deep learning model in a multi-task learning and a transfer learning framework. We demonstrate that the proposed approach outperforms state-of-the-art deep learning methods and is robust to the inclusion of anchor distance as a feature. In addition, we find that the proposed approach can achieve comparable prediction performance using biologically relevant tissues/cell types compared to using all tissues/cell types especially for predicting promoter-enhancer interactions. Availabilityhttps://github.com/lichen-lab/DeepPHiC

bioinformatics↗

bioRxiv2022

Ratiometric NAD+ sensors reveal subcellular NAD+ modulators

Mapping NAD+ dynamics in live cells and human are essential for translating NAD+ interventions into effective therapies. Yet genetically encoded NAD+ sensors with better specificity and pH-resistance are still needed for cost-effective monitoring of subcellular and clinical NAD+. We introduce multicolor, resonance energy transfer-based NAD+ sensors that cover nano- to milli-molar concentration ranges for clinical NAD+ measurement and subcellular NAD+ visualization. The sensors captured the blood NAD+ increase induced by NMN supplementation and revealed the distinct subcellular effects of NAD+ supplements and modulators. Then, the sensors enabled high-throughput screenings for mitochondrial and nucleic NAD+ modulators and identified -GPC, a cognition-related metabolite, induces NAD+ redistribution from mitochondria to nucleus relative to the total adenine nucleotides, which was confirmed by NAD+ FRET microscopy.

bioengineering↗

bioRxiv2022

Photoperiod and vernalization alleles and their combinations greatly affected phenological and agronomic traits in bread wheat under autumn and spring sowing conditions

The responses to temperature (vernalization) and day length (photoperiod) in wheat (Triticum aestivum L.) are entirely defined by allelic differences at the respective VRN-1 and PPD-1 loci. These two physiological factor greatly affect wheat phenology. Using molecular markers, a panel of 61 diverse bread wheat varieties were genotyped for VRN-1 and PPD-1 alleles, and the traits were evaluated under autumn and spring sowing conditions to investigate the impact of the alleles and their combinations on phenological stage, morphological traits, and yield traits. The results revealed that the earlier heading (HD), and flowering (FD) conferred by insensitive alleles for VRN-1 and PPD-1 genes was consistent regardless of the variation under the two sowing conditions. The effects of a single dominant allele of VRN-1 were similar, with an additive effects when combined. The effects of a single PPD-1 insensitive allele differed, with Ppd-D1a stronger than Ppd-B1a, and Ppd-A1a was the least, with an additive effects when combined. Secondly, varieties sown under spring condition recorded a larger flag leaf area when compared to those sown under autumn condition regardless of the single and combined influence at the VRN-1 and PPD-1 loci. When compared to other genes, PPD-D1 had a consistent and significant effect on flag leaf area, spike length, and plant height across both sowing conditions. Furthermore, the sensitive allele Ppd-D1b had larger biomass across both sowing conditions, but this did not translate into increased yield because the Ppd-D1a insensitive allele recorded higher yield. The impact of PPD-D1 gene on grain yield was evident across both sowing conditions. Overall, according to the findings of this study, VRN-1 and PPD-1 genes impact in modulating phenology stages, morphological, spike and yield traits was dependent on the sowing conditions and also on genetic background with the spring and insensitive alleles poised to be favored for selection.

plant biology↗

bioRxiv2022

ISG15 Monomer Promotes IFNα-mediated Antiviral Activity against Pseudorabies Virus by Facilitating phosphorylation of STAT1/STAT2

Pseudorabies virus (PRV), which presently lacks both an antiviral drug and a viable therapeutic option, is a major viral pathogen that poses a danger to the pig industry worldwide. Interferon-stimulated gene 15 (ISG15) is strongly upregulated during viral infections and has been reported to have proviral or antiviral properties, depending on the virus and host species. Our previous studies demonstrated ISG15 was remarkably upregulated during PRV infection, and the overexpression of ISG15 inhibited PRV replication. Nevertheless, the exact mechanism through which ISG15 influences PRV replication poorly understood unclear. Here, we demonstrate that ISG15 accumulation induced by PRV infection requires viral gene expression and viral growth. Conjugation inhibition assays showed that ISG15 imposes its antiviral effects via unconjugated (free) ISG15 and affects the viral release. In addition, we found ISG15 promoted IFN-mediated antiviral activity against PRV by facilitating the phosphorylation of STAT1 and STAT2, along with an increase of ISGF3-induced ISRE promoter activity. Furthermore, we evaluated the role of ISG15 in host defense to control PRV infection by using ISG15-/- mice model. When challenged with PRV, ISG15-/- mice exhibited increased morbidity and mortality, as well as viral load compared to WT mice. Pathological examination revealed increased lesions, mononuclear cellular infiltration and neuronal death in the brains of ISG15-/- mice, along with the upregulation of the cytokines. Our findings establish the importance of ISG15 in IFN-induced antiviral response and in the control of PRV infection.

molecular biology↗

bioRxiv2022

Retinoic acid-gated BDNF synthesis in neuronal dendrites drives presynaptic homeostatic plasticity

Homeostatic synaptic plasticity is a non-Hebbian synaptic mechanism that adjusts synaptic strength to maintain network stability while achieving optimal information processing. Among the molecular mediators shown to regulate this form of plasticity, synaptic signaling through retinoic acid (RA) and its receptor, RAR, has been shown to be critically involved in the homeostatic adjustment of synaptic transmission in both hippocampus and sensory cortices. In this study, we explore the molecular mechanism through which postsynaptic RA and RAR regulates presynaptic neurotransmitter release during prolonged synaptic inactivity at excitatory synapses. We show that RAR binds to a subset of dendritically sorted brain-derived neurotrophic factor (BDNF) mRNA splice isoforms and represses their translation. The RA-mediated translational de-repression of postsynaptic BDNF results in the retrograde activation of presynaptic Tropomyosin receptor kinase B (TrkB) receptors, facilitating presynaptic homeostatic compensation through enhanced presynaptic release. Together, our study illustrates a RA-mediated retrograde synaptic signaling pathway through which postsynaptic protein synthesis during synaptic inactivity drives compensatory changes at presynaptic site.

neuroscience↗

bioRxiv2022

Structural insights into the high selectivity of the anti-diabetic drug mitiglinide

Mitiglinide is a highly selective fast-acting anti-diabetic drug that inhibits pancreatic KATP channels to induce insulin secretion. However, how mitiglinide binds KATP channels remains unknown. Here, we show the cryo-EM structure of the SUR1 subunit in complex with mitiglinide. The structure reveals that mitiglinide binds inside the common insulin secretagogue-binding site in the transmembrane domain of SUR1, locking SUR1 in a NBD-separated inward-facing conformation. The detailed structural analysis uncovers the molecular basis of the high selectivity of mitiglinide.

biophysics↗

bioRxiv2022

Single cell RNA-seq identifies inflammation-induced loss of CFTR-expressing airway ionocytes in non-eosinophilic asthma

Asthma is the most common chronic airways disease worldwide and the severe treatment resistant subtype of asthma is responsible for the majority of disease burden. Asthma is heterogeneous in nature and can be classified according to airway infiltrates as eosinophilic or non-eosinophilic (sometimes referred to as Type 2 low), which is further divided into paucigranulocytic (low levels of granulocytes), or neutrophilic asthma characterized by elevated neutrophils, and mixed Type 1 and Type 17 cytokines in airway tissue, sputum, and bronchoalveolar lavage. Severe non-eosinophilic asthma currently has fewer effective treatment options and many of these patients fail to qualify for newer biologic monoclonal therapies. The cystic fibrosis transmembrane conductance regulator (CFTR) is a key protein whose function is dysregulated in multiple respiratory diseases including cystic fibrosis and chronic obstructive pulmonary disease (COPD) and has proven a valuable therapeutic target. Using human bronchial epithelial cells (hBECs) isolated differentiated at air-liquid interface we demonstrated a reduced function of the CFTR in non-eosinophilic asthma. Characterization of the cell and molecular differences in airway epithelial cells between severe asthma subtypes using single cell RNA-sequencing (scRNAseq) revealed that airway epithelial cells from non-eosinophilic asthma, and in particular neutrophilic asthma patients, fail to differentiate into CFTR-expressing ionocytes compared with eosinophilic asthma or healthy donors. We identified a novel ionocyte transcriptional signature, which was present in both bronchial and tracheal airway epithelial samples indicating conserved anatomical gene regulation. Using protein markers and immunofluorescent quantification loss of ionocytes was confirmed in non-eosinophilic asthma hBECs. Similarly, ioncytes were also diminished in the airways of a murine model of neutrophilic-dominant but not eosinophilic allergen asthma models. Furthermore, treatment of hBECs from healthy donors with a neutrophilic asthma-like inflammatory cytokine mixture, but not IL-13, led to loss of ionocytes primarily due to IFN-{gamma}. Inflammation-induced loss of CFTR-expressing ionocytes in airway cells from non-eosinophilic asthma may represent a key feature of disease pathogenesis and a novel drug target for this difficult-to-treat disease.

cell biology↗

bioRxiv2022

SMURF: embedding single-cell RNA-seq data with matrix factorization preserving selfconsistency

The advance of single-cell RNA-sequencing (scRNA-seq) sheds light on cell-specific transcriptomic studies of cell developments, complex diseases, and cancers. Nevertheless, scRNA-seq techniques suffer from "dropout" events, and imputation tools are proposed to address the sparsity. Here, rather than imputation, we propose a tool, SMURF, to embed cells and genes into their latent space vectors utilizing matrix factorization with a mixture of Poisson-Gamma divergent as objective while preserving self-consistency. As for wet lab dataset evaluation, SMURF exhibited feasible cell subpopulation discovery efficacy with the latent vectors on all the eight-cell line mixtures. Furthermore, SMURF can embed the cell latent vectors into a 1D-oval and recover the time course of the cell cycle. SMURF can also serve as an imputation tool, the in silico data assessment shows that SMURF paraded the most robust gene expression recovery power with low root mean square error and high Pearson correlation. Moreover, SMURF recovers the gene distribution for the WM989 Drop-seq data. SMURF is available at https://github.com/deepomicslab/SMURF.

bioinformatics↗

bioRxiv2022

Scalable production of recombinant three-finger proteins: from inclusion bodies to high quality molecular probes

We introduce a working pipeline for expression, purification and validation of disulfide-bond rich three-finger proteins using E. coli as the expression host. With this pipeline, we have successfully obtained highly purified and bioactive recombinant -Bungarotoxin, k-Bungarotoxin, Hannalgesin, Mambalgin-1, -Cobratoxin, MT, Slurp1, Pate B etc. Milligrams to hundreds of milligrams of recombinant three finger proteins were obtained within weeks in the lab. The recombinant proteins showed specificity in binding assay and six of them were crystallized and structurally validated using X-ray diffraction protein crystallography. As many three finger proteins have attractive therapeutic or research interests and due to the extremely high quality of the recombinant three finger proteins we obtained, our method provides a competitive alternative to either their native counterparts or chemically synthetic ones, and should facilitate related research and applications.

biochemistry↗

bioRxiv2022

DeepPerVar: a multimodal deep learning framework for functional interpretation of genetic variants in personal genome

MotivationUnderstanding the functional consequence of genetic variants, especially the noncoding ones, is important but particularly challenging. Genome-wide association studies or quantitative trait locus analyses may be subject to limited statistical power and linkage disequilibrium, and thus are less optimal to pinpoint the causal variants. Moreover, most existing machine learning approaches, which exploit the functional annotations to interpret and prioritize putative causal variants, cannot accommodate the heterogeneity of personal genetic variations and traits in a population study, targeting a specific disease. ResultsBy leveraging paired whole genome sequencing data and epigenetic functional assays in a population study, we propose a multi-modal deep learning framework to predict genome-wide quantitative epigenetic signals by considering both personal genetic variations and traits. The proposed approach can further evaluate the functional consequence of noncoding variants on an individual level by quantifying the allelic difference of predicted epigenetic signals. By applying the approach to the ROSMAP cohort studying Alzheimers disease (AD), we demonstrate that the proposed approach can accurately predict quantitative genome-wide epigenetic signals and in key genomic regions of AD causal genes, learn canonical motifs reported to regulate gene expression of AD causal genes, improve the partitioning heritability analysis, and prioritize putative causal variants in a GWAS risk locus. Finally, we release the proposed deep learning model as a stand-alone Python toolkit and a web server. Availabilityhttps://github.com/lichen-lab/DeepPerVar

bioinformatics↗

bioRxiv2022

K2P channel TWK-40 Regulates a Rhythmic Behavior in C. elegans

Leak potassium (K+) currents, conducted by two-pore domain K+ (K2P) channels, are critical for the stabilization of the membrane potential. The effect of K2P channels on motor rhythm remains enigmatic. We show here that the K2P TWK-40 regulates the rhythmic defecation motor program (DMP) in Caenorhabditis elegans. Disrupting TWK-40 suppresses the expulsion defects of nlp-40 and aex-2 mutants. By contrast, a gain-of-function (gf) mutant of twk-40 significantly reduces the expulsion frequency per DMP cycle. In situ whole-cell patch clamping demonstrates that TWK-40 forms an outward current that hyperpolarize the resting membrane potential of DVB neuron. In addition, TWK-40 substantially contributes to the rhythmic activity of DVB. Specifically, DVB Ca2+ oscillations exhibit obvious defects in twk-40 mutants. Expression of TWK-40(gf) in DVB recapitulates the expulsion deficiency of the twk-40(gf) mutant, and inhibits DVB Ca2+ oscillations in both wild-type and twk-40(lf) animals. Moreover, DVB innervated enteric muscles also exhibit rhythmic Ca2+ defects. Taken together, these results demonstrate that TWK-40 is an essential neuronal inhibitor of DMP, thus revealing a cellular mechanism linking leak K2P channels with rhythmic motor activity.

neuroscience↗

bioRxiv2022

A descending interneuron with depolarized resting membrane potential controls C. elegans motor states

Continuity of behaviors requires animals to make smooth transitions between successive and mutually exclusive behavioral states. Neural principles that govern these transitions are not well understood. C. elegans spontaneously switch between two opposite motor states, forward and backward movement, a phenomenon long thought to reflect the reciprocal inhibition between two interneurons that separately gate the forward and backward motor circuits, AVB and AVA. Combining experimental data and mathematical modeling, we report that spontaneous forward and backward locomotion and their corresponding motor circuits are not separately controlled. AVA and AVB are neither functionally equivalent nor strictly reciprocally inhibitory. Instead, while AVA phasically inhibits the forward promoting interneuron AVB at a fast timescale, it maintains a tonic, extrasynaptic excitation on AVB over the longer timescale. AVAs depolarized spontaneous membrane potential is necessary for this tonic excitation. We propose a new, master neuron model for locomotion. AVA, with tonic and phasic activity of opposite polarities on different time scales, acts as a master neuron to break the symmetry between the underlying forward and backward motor circuits. This offers a parsimonious solution for sustained locomotion consisted of mutually exclusive motor states. TeaserA tonically active C. elegans premotor interneuron functions as the master neuron that underlies continuous modulation of forward and backward movement to ensure smooth transitions between the two opposing motor states.

neuroscience↗

bioRxiv2022

Studying meiosis in mitosis: Activating the meiosis-specific Red1-Hop1-Mek1 complex in mitotic budding yeast cells

In mitosis, sister chromatids are preferred repair templates for homologous recombination, whereas in meiosis interhomolog-based repair is promoted. How this switch, which is a defining event in sexual reproduction, is accomplished remains poorly understood. In budding yeast, a meiosis-specific complex consisting of Red1, Hop1 and Mek1 (RHMc) enforces meiotic interhomolog bias, potentially through inhibition of intersister-based repair. The current data points to a linear assembly governing RHMc formation: the HORMA protein Hop1 associates with Red1 via a closure-motif-HORMA domain interaction, and Mek1 kinase is recruited through phospho-mediated interactions with Hop1. Here, via expression in mitotic cells we autonomously establish the RHM complex. In vivo analysis complemented with in vitro biochemical reconstitution shows that Mek1 associates with Red1, in a manner that might resemble binding of other kinases with scaffolding activators. The NH2-terminus of Red1 contributes to Hop1 binding, suggesting cooperative binding between Red1 and the HORMA domain of Hop1, beyond closure motif-based interactions. Meiotic activation of Mek1 kinase is dictated by complex formation and upstream DNA break-dependent signaling. We find Mek1 can be activated under DNA damaging conditions in mitotically dividing cells, where activation depends on upstream Mec1 kinase function and RHMc integrity. We perform a structure-function analysis of RHMc formation and Mek1 activation. Finally, we show that activation of Mek1 in mitosis leads to rad51{Delta}-like DNA break sensitivity, providing evidence for the model that RHMc instates meiotic interhomolog-based repair by inhibiting mitotic homologous recombination. Our analysis enables querying downstream effects of RHMc action on DNA repair. Because aberrant re-expression of homologs of Red1 and Hop1 leads to DNA repair defects in human cancer, our system can be used to study roles of these genes during tumorigenesis.

genetics↗