Biology subjects

Zhang, S.

Publications and source records attributed to Zhang, S..

At least 1,063 records · Page 59Linked to original sources

bioRxiv2020

Dietary S. maltophilia promotes fat storage by enhancing lipogenesis and ER-LD contacts in C. elegans

Dietary and symbiotic bacteria can exert powerful influence on metazoan lipid metabolism. Here, we demonstrate that feeding Caenorhabditis elegans (C. elegans) with the opportunistic pathogenic bacteria Stenotrophomonas maltophilia (S. maltophilia) retards growth and promotes excessive fat storage. Gene expression analysis reveals that dietary S. maltophilia induces a lipogenic transcriptional response that includes the SREBP ortholog SBP-1, and fatty acid desaturases FAT-6 and FAT-7. Live imaging and ultrastructural analysis suggest that excess fat is stored in greatly expanded lipid droplets (LDs), as a result of enhanced endoplasmic reticulum-LD interaction. We also report that loss of function mutations in cyp-35B1 or dpy-9 in C. elegans confers resistance to S. maltophilia. Our work delineates a new model for understanding microbial regulation of metazoan physiology.

cell biology↗

bioRxiv2020

Riemannian geometry and statistical modeling correct for batch effects and control false discoveries in single-cell surface protein count data from CITE-seq

Recent advances in next generation sequencing-based single-cell technologies have allowed high-throughput quantitative detection of cell-surface proteins along with the transcriptome in individual cells, extending our understanding of the heterogeneity of cell populations in diverse tissues that are in different diseased states or under different experimental conditions. Count data of surface proteins from the cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) technology pose new computational challenges, and there is currently a dearth of rigorous mathematical tools for analyzing the data. This work utilizes concepts and ideas from Riemannian geometry to remove batch effects between samples and develops a statistical framework for distinguishing positive signals from background noise. The strengths of these approaches are demonstrated on two independent CITE-seq data sets in mouse and human. Python source code implementing the algorithms is available at https://github.com/jssong-lab/SAGACITE.

bioinformatics↗

bioRxiv2020

Adiposome Targeting and Enzymatic Activity of Lipid Droplet-Specific Proteins

New strategies to decode the specific protein targeting mechanism on lipid droplet (LD) are urgently needed. Using adiposome, the LD binding of perilipin 2 (PLIN2), perilipin 3 (PLIN3), and adipose triglyceride lipase (ATGL) were studied. Scatchard analysis found that the binding of PLIN2 to the adiposome surface was saturable, pointing to a specific membrane binding partner. Phosphatidylinositol (PI) was found to inhibit PLIN2 binding while it did not impede PLIN3. Structural analysis combined with mutagenesis revealed that the 73rd glutamic acid of PLIN2 is significant for the effect of PI on the protein binding. The presence of PI significantly stimulated the activity of ATGL in vitro. The phosphorylation site mutants of ATGL were found reducing the lipase activity in the adiposome system. Our study demonstrates the utility of adiposome as a powerful, manipulatable model system for the characterization of LD binding and enzymatic activity of LD proteins in vitro.

biochemistry↗

bioRxiv2020

Cryo-EM structures of inactive and Gi-coupled GABAB heterodimer

Metabotropic GABAB G protein-coupled receptor functions as a mandatory heterodimer of GB1 and GB2 subunits and mediates inhibitory neurotransmission in the central nervous system. Each subunit is composed of the extracellular Venus flytrap (VFT) domain and transmembrane (TM) domain. Here we present cryo-EM structures of human full-length heterodimeric GABAB receptor in the antagonist-bound inactive state and in the active state complexed with agonist and positive allosteric modulator in the presence of Gi1 protein at a resolution range of 2.8-3.0 [A]. Cryo-EM analysis of the activated-GABAB-Gi1 complex revealed that Gi1 couples to the activated receptor primarily in three major conformations, one via GB1 TM and two via GB2 TM, respectively. Our structures reveal that agonist binding stabilizes the closure of GB1 VFT, which in turn triggers a rearrangement of TM interfaces between two subunits from TM3-TM5/TM3-TM5 in the inactive state to TM6/TM6 in the active state and finally induces the opening of intracellular loop 3 and synergistically shifting of TM3, 4 and 5 helices in GB2 TM domain to accommodate the 5-helix of Gi1. These results provide a structural framework for understanding class C GPCR activation and a rational template for allosteric modulator design targeting dimeric interface of GABAB receptor.

molecular biology↗

bioRxiv2020

A Fast High-Affinity Fluorescent Serotonin Biosensor Engineered from a Tick Lipocalin

The authors have withdrawn their manuscript whilst they perform additional experiments to test some of their conclusions. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. DetailsRegrettably, we, and several other researchers in the corresponding authors lab and not involved in the original study, have been unable to reproduce some of the reported key results under conditions originally described. We recently used the pDisplay-G-GESS-M plasmid to transfect HEK 293T cells and observed that the membrane localization of the biosensor was sensitive to FBS batches. By using an FBS batch from Gibco (Cat# 16000-044 and Lot #2103017RP), we were able to replicate the membrane localization similar to what was reported in Fig. 1e. However, the cells required the addition of several hundred micromolar of serotonin for robust fluorescence increase. These new results suggest that the affinity of G-GESS to serotonin is worse than that claimed in the initial article. We further used liquid chromatography-mass spectrometry (LC-MS) to analyze the serotonin stock solutions identified in our -80{degrees}C lab freezer and observed MS peaks suggesting a glutamate contamination in some of the analyzed samples. In addition, we used polymerase chain reaction (PCR) or reverse transcription-PCR (RT-PCR) to amplify gene fragments from our viral stocks and the sequencing results indicate that the viral stocks were contaminated with a glutamate biosensor. While we are still performing further investigation, in light of the existing findings, all authors agree to retract this paper. In the initial article, the sensor was tested in several cell culture systems, brain slices and behaving mice. We are presently unable to fully explain the observed sensor responses described in the initial article, but it is highly possible that these responses could be caused by the contaminated chemical or viral stocks. We deeply apologize to the scientific community for not detecting these problems sooner.

neuroscience↗

bioRxiv2020

Blocking antibodies against SARS-CoV-2 RBD isolated from a phage display antibody library using a competitive biopanning strategy

The infection of the novel coronavirus SARS-CoV-2 have caused more than 150,000 deaths, but no vaccine or specific therapeutic antibody is currently available. SARS-CoV-2 relies on its spike protein, in particular the receptor binding domain (RBD), to bind human cell receptor angiotensin-converting enzyme 2 (ACE2) for viral entry, and thus targeting RBD holds the promise for preventing SARS-CoV-2 infection. In this work, a competitive biopanning strategy of a phage display antibody library was applied to screen blocking antibodies against RBD. High-affinity antibodies were enriched after the first round using a standard panning process in which RBD-His recombinant protein was immobilized as a bait. At the next two rounds, immobilized ACE2-Fc and free RBD-His proteins were mixed with the enriched phage antibodies. Antibodies binding to RBD at epitopes different from ACE2-binding site were captured by the immobilized ACE2-Fc, forming a "sandwich" complex. Only antibodies competed with ACE2 for recognizing RBD at the same or similar epitopes can bind to the free RBD-His in the supernatant and be subsequently separated by the Ni-NTA magnetic beads. Top 1 lead from the competitive biopanning of a synthetic antibody library, Lib AB1, was produced as the full-length IgG1 format. It was proved to competitively block the binding of RBD to ACE2 protein, and potently inhibit SARS-CoV-2 pseudovirus infection of ACE2-overexpressing Hela cells with IC50 values of 12nM. Nevertheless, top 1 lead from the standard biopanning of Lib AB1, can only bind to RBD in vitro but not have the blocking or neutralization activity. Our strategy can efficiently isolate the blocking antibodies of RBD, and it would speed up the discovery of neutralizing antibodies against SARS-CoV-2.

microbiology↗

bioRxiv2020

Synaptotagmin-7 is a calcium sensor for facilitation at the splanchnic-chromaffin cell synapse

Disturbances that threaten homeostasis elicit activation of the sympathetic nervous system (SNS) and the adrenal medulla. The effectors discharge as a unit to drive global and immediate changes in whole-body physiology. Descending sympathetic information is conveyed to the adrenal medulla via preganglionic splanchnic fibers. These fibers pass into the gland and synapse onto chromaffin cells, which synthesize, store, and secrete catecholamines and vasoactive peptides. While the importance of the sympatho-adrenal branch of the autonomic nervous system has been appreciated for many decades, the mechanisms underlying transmission between presynaptic splanchnic neurons and postsynaptic chromaffin cells have remained obscure. In contrast to chromaffin cells, which have enjoyed sustained attention as a model system for exocytosis, even the Ca2+ sensors that are expressed within splanchnic terminals have not yet been identified. This study shows that a ubiquitous Ca2+-binding protein, synaptotagmin-7 (Syt7), is expressed within the fibers that innervate the adrenal medulla, and that its absence can alter synaptic transmission in the preganglionic terminals of chromaffin cells. The prevailing impact in presynapses that lack Syt7 is a decrease in synaptic strength and neuronal short-term plasticity. Evoked excitatory postsynaptic currents (EPSCs) in Syt7 KO preganglionic terminals are smaller in amplitude than in wild-type synapses stimulated in an identical manner. Splanchnic inputs also display robust short-term presynaptic facilitation, which is compromised in the absence of Syt7. These data reveal, for the first time, a role for any synaptotagmin at the splanchnic-chromaffin cell synapse. They also suggest that Syt7 has actions at synaptic terminals that are conserved across central and peripheral branches of the nervous system.

neuroscience↗

bioRxiv2020

Genomic Architecture of Cells in Tissues (GeACT): Study of Human Mid-gestation Fetus

By circumventing cellular heterogeneity, single cell omics have now been widely utilized for cell typing in human tissues, culminating with the undertaking of human cell atlas aimed at characterizing all human cell types. However, more important are the probing of gene regulatory networks, underlying chromatin architecture and critical transcription factors for each cell type. Here we report the Genomic Architecture of Cells in Tissues (GeACT), a comprehensive genomic data base that collectively address the above needs with the goal of understanding the functional genome in action. GeACT was made possible by our novel single-cell RNA-seq (MALBAC-DT) and ATAC-seq (METATAC) methods of high detectability and precision. We exemplified GeACT by first studying representative organs in human mid-gestation fetus. In particular, correlated gene modules (CGMs) are observed and found to be cell-type-dependent. We linked gene expression profiles to the underlying chromatin states, and found the key transcription factors for representative CGMs. HighlightsO_LIGenomic Architecture of Cells in Tissues (GeACT) data for human mid-gestation fetus C_LIO_LIDetermining correlated gene modules (CGMs) in different cell types by MALBAC-DT C_LIO_LIMeasuring chromatin open regions in single cells with high detectability by METATAC C_LIO_LIIntegrating transcriptomics and chromatin accessibility to reveal key TFs for a CGM C_LI

genomics↗

bioRxiv2020

A phenotype-specific framework for identifying the eye abnormalities causative nonsynonymous-variants

The most important role of variant pathogenicity predictors is to identify the disease-phenotype causative variant in studying monogenic diseases. In the last decade, machine-learning based predictors exhibited a relatively accurate performance for distinguishing the pathogenic variants and contributed a significant role for all disease-spectrums. Yet, few predictors can investigate the phenotypic significance of variants. Here we presented a phenotype-specific framework aimed to directly point out the phenotypic significance of predicted candidates, and showed its advancing performance in eye abnormalities. By training on eye-abnormalities causative variants, our method presented 96.2% accuracy, 96.1% precision, 93.4% recall for pathogenicity identification. Inconsistent with the modeling performance, identifying the single phenotype-causative variant from various sequencing variants is challenging for all predictors. Underlying the phenotype-oriented, our method significantly promoted the precision and reduced the cost for identifying the single causative variant from thousands of candidates. These advances highlight the significance of the phenotype-specific training method for studying disease.

bioinformatics↗

bioRxiv2020

A general strategy to red-shift green fluorescent protein based biosensors

Compared to green fluorescent protein (GFP) based biosensors, red fluorescent protein (RFP) based biosensors are inherently advantageous because of reduced phototoxicity, decreased autofluorescence, and enhanced tissue penetration. However, there is a limited choice of RFP-based biosensors and development of each biosensor requires significant effort. Herein, we describe a general and convenient method which uses the genetically encoded amino acid, 3-aminotyrosine (aY), to convert GFPs and GFP-based biosensors into red.

synthetic biology↗

bioRxiv2020

Identification of a Micropeptide on Lipid Droplet That Mediates Lipid Storage and Insulin Sensitivity

Over the past decade, great progress in sequencing technologies and computational biology has revealed that the majority of the mammalian genome considered to be noncoding is rich in functional elements able to produce proteins. Many RNA molecules, mis-annotated as noncoding, actually harbor small open reading frames that are predicted to code for proteins. Some of those proteins have been verified to play critical roles in multiple biological processes. The lipid droplet (LD) is a unique cellular organelle, conserved from bacteria to humans, and is closely associated with cellular lipid metabolism and metabolic disorders. No noncoding RNA-coded proteins have been identified on LDs. Here, for the first time, we searched the organelle for their presence. After the enrichment of small proteins of LDs isolated from myoblasts, we used mass spectrometry coupled with our lab made protein database to identify LD-associated noncoding RNA-encoded proteins (LDANPs). A total of 15 new proteins were identified. One of them was studied further and termed LDANP1. LDANP1 was localized on LDs by imaging, cell fractionation, and immunogold labeling. Like LD resident proteins, LDANP1 was degraded by the proteasome. Using the CRISPR/Cas9-mediated genome editing technique, the endogenous expression of LDANP1 was validated. The stable expression of LDANP1 suppressed the accumulation of triacylglycerol in oleic acid treated myoblasts and inhibited the rescue of palmitate-inhibited insulin sensitivity by oleic acid. In summary, we report for the first time that translatable, nominally noncoding RNA-derived proteins, which are new and cannot be identified using current research methods, were associated with LDs and that among these, LDANP1 modulated lipid metabolism and insulin sensitivity. The discovery of noncoding RNA-encoded proteins on LDs paves a new way for the research of LDs and lipid metabolism.

cell biology↗

bioRxiv2020

Room-temperature-storable PCR Mixes for SARS-CoV-2 Detection

A novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) emerged in late 2019, causing an outbreak of pneumonia [coronavirus disease 2019 (COVID-19)] in Wuhan, China, which then rapidly spread globally. Although the use of ready-made reaction mixes can enable more rapid PCR-based diagnosis of COVID-19, the need to transport and store these mixes at low temperatures presents challenges to already overburdened logistics networks. Here, we present an optimized freeze-drying procedure that allows SARS-CoV-2 PCR mixes to be transported and stored at ambient temperatures, without loss of activity. Additive-supplemented PCR mixes were freeze-dried. The residual moisture of the freeze-dried PCR mixes was measured by Karl-Fischer titration. We found that freeze-dried PCR mixes with [~]1.2% residual moisture are optimal for storage, transport, and reconstitution. The sensitivity, specificity, and repeatability of the freeze-dried reagents were similar to those of freshly prepared, wet reagents. The freeze-dried mixes retained activity at room temperature (18[~]25{degrees}C) for 28 days, and for 14 and 10 days when stored at 37{degrees}C and 56{degrees}C, respectively. The uptake of this approach will ease logistical challenges faced by transport networks and make more cold storage space available at diagnosis and hospital laboratories. This method can also be applied to the generation of freeze-dried PCR mixes for the detection of other pathogens.

microbiology↗

bioRxiv2020

Structural Basis for the Inhibition of the RNA-Dependent RNA Polymerase from SARS-CoV-2 by Remdesivir

The pandemic of Corona Virus Disease 2019 (COVID-19) caused by SARS-CoV-2 has become a global crisis. The replication of SARS-CoV-2 requires the viral RNA-dependent RNA polymerase (RdRp), a direct target of the antiviral drug, Remdesivir. Here we report the structure of the SARS-CoV-2 RdRp either in the apo form or in complex with a 50-base template-primer RNA and Remdesivir at a resolution range of 2.5-2.8 [A]. The complex structure reveals that the partial double-stranded RNA template is inserted into the central channel of the RdRp where Remdesivir is incorporated into the first replicated base pair and terminates the chain elongation. Our structures provide critical insights into the working mechanism of viral RNA replication and a rational template for drug design to combat the viral infection.

molecular biology↗

bioRxiv2020

Lipid Droplet-Anchored Mitochondria Are More Sensitive to Cold in Brown Adipocytes

Brown adipose tissue (BAT) are specialized for uncoupled heat production through mitochondria fueled majorly from fatty acids (FA) of lipid droplets (LDs). How the interaction between the two organelles contributes the generation of heat remains elusive. Here we report that LD-anchored mitochondria (LDAM) were observed in BAT of mice raised at three different temperatures, 30{degrees}C, 23{degrees}C, and 6{degrees}C. The biochemical analyses including Western blotting and electron transport chain subunits showed that LDAM were functional at given temperatures. Comparative proteomics analysis was conducted and revealed that these LDAM had protein level differences from cytoplasmic mitochondria (CM) at different temperatures. Higher expressions of proteins at low temperature were observed for i) FA {beta}-oxidation in LDAM including FA synthesis, and uncoupling, ii) pseudo-futile cycle in CM, and iii) two shuttle systems; glycerol 3-phosphate in both CM and LDAM, and citrate malate in CM. Together, these results suggest that LDs and LDAM are a preorganized and functional organelle complex that permits the rapid response to cold environment.

cell biology↗

bioRxiv2020

A concept shift in the mechanistic understanding of HIFα-regulated developmental myelination

The developing CNS is exposed to physiological hypoxia, under which hypoxia inducible factor alpha (HIF) is stabilized and plays a crucial role in regulating neural development. The cellular and molecular mechanisms of HIF in developmental myelination remain incompletely understood. Previous concept proposes that HIF regulates CNS developmental myelination by activating the autocrine Wnt/{beta}-catenin signaling in oligodendrocyte progenitor cells (OPCs). Here, by analyzing a battery of genetic mice of both sexes, we presented in vivo evidences supporting an alternative understanding of oligodendroglial HIF-regulated developmental myelination. At the cellular level, we found that HIF was required for developmental myelination by transiently controlling upstream OPC differentiation but not downstream oligodendrocyte maturation and that HIF dysregulation in OPCs but not oligodendrocytes disturbed normal developmental myelination. We demonstrated that HIF played a minor, if any, role in regulating canonical Wnt signaling in the oligodendroglial lineage or in the CNS. At the molecular level, blocking autocrine Wnt signaling did not affect HIF-regulated OPC differentiation and myelination. We further identified HIF-Sox9 regulatory axis as an underlying molecular mechanism in HIF-regulated OPC differentiation. Our findings support a concept shift in our mechanistic understanding of HIF-regulated CNS myelination from the previous Wnt-dependent view to a Wnt-independent one and unveil a previously unappreciated HIF-Sox9 pathway in regulating OPC differentiation.

neuroscience↗

bioRxiv2020

Engineered chemotaxis core signaling units indicate a constrained kinase-off state

Bacterial chemoreceptors, the CheA histidine kinase, and the coupling protein CheW comprise transmembrane molecular arrays with remarkable sensing properties. An unanswered question concerns how receptors turn off CheA kinase activity. Chemoreceptor cytoplasmic regions engineered to assume a trimer-of-receptor-dimers configuration form well-defined complexes with CheA and CheW and promote a kinase-off state. These mimics of core signaling units were assembled to homogeneity and investigated by site-directed spin-labeling with pulse-dipolar ESR spectroscopy (PDS), small-angle x-ray scattering, targeted protein cross-linking, and cryo-electron microscopy. The kinase-off state is especially stable, has relatively low domain mobility and associates the histidine substrate domain P1 and docking domain P2 with the kinase core. Distances measured between spin-labeled ADP molecules bound to the P4 kinase domain provide evidence for a "dipped conformation" that has been previously proposed from molecular dynamics simulations. Taken together, the data provide an experimentally restrained model for the inhibited state of the core-signaling unit and suggest that chemoreceptors indirectly sequester the kinase and substrate domains to limit histidine autophosphorylation.

biochemistry↗

bioRxiv2020

Potent human neutralizing antibodies elicited by SARS-CoV-2 infection

The pandemic caused by emerging coronavirus SARS-CoV-2 presents a serious global public health emergency in urgent need of prophylactic and therapeutic interventions. SARS-CoV-2 cellular entry depends on binding between the viral Spike protein receptor-binding domain (RBD) and the angiotensin converting enzyme 2 (ACE2) target cell receptor. Here, we report on the isolation and characterization of 206 RBD-specific monoclonal antibodies (mAbs) derived from single B cells of eight SARS-CoV-2 infected individuals. These mAbs come from diverse families of antibody heavy and light chains without apparent enrichment for particular families in the repertoire. In samples from one patient selected for further analyses, we found coexistence of germline and germline divergent clones. Both clone types demonstrated impressive binding and neutralizing activity against pseudovirus and live SARS-CoV-2. However, the antibody neutralizing potency is determined by competition with ACE2 receptor for RBD binding. Surprisingly, none of the SARS-CoV-2 antibodies nor the infected plasma cross-reacted with RBDs from either SARS-CoV or MERS-CoV although substantial plasma cross-reactivity to the trimeric Spike proteins from SARS-CoV and MERS-CoV was found. These results suggest that antibody response to RBDs is viral species-specific while that cross-recognition target regions outside the RBD. The specificity and neutralizing characteristics of this plasma cross-reactivity requires further investigation. Nevertheless, the diverse and potent neutralizing antibodies identified here are promising candidates for prophylactic and therapeutic SARS-CoV-2 interventions.

immunology↗

bioRxiv2020

FcRn, but not FcgRs, drives maternal-fetal transplacental transport of human IgGantibodies

The IgG Fc domain has the capacity to interact with diverse types of receptors, including FcRn and Fc{gamma}Rs, which confer pleiotropic biological activities. Whereas FcRn regulates IgG epithelial transport and recycling, Fc effector activities, such as ADCC and phagocytosis are mediated by Fc{gamma}Rs, which upon crosslinking transduce signals that modulate the function of effector leukocytes. Despite the well-defined and non-overlapping functional properties of FcRn and Fc{gamma}Rs, recent studies have suggested that Fc{gamma}Rs mediate transplacental IgG transport, as certain Fc glycoforms were reported to be enriched in fetal circulation. To determine the contribution of Fc{gamma}Rs and FcRn to the maternal-fetal transport of IgG, we characterized the IgG Fc glycosylation in paired maternal-fetal samples from patient cohorts from Uganda and Nicaragua. No differences in IgG1 Fc glycan profiles and minimal differences in IgG2 Fc glycans were noted, whereas the presence or absence of galactose on the Fc glycan of IgG1 did not alter Fc{gamma}RIIIA or FcRn binding, half-life, or their ability to deplete target cells in Fc{gamma}R/FcRn humanized mice. Modeling maternal/fetal transport in Fc{gamma}R/FcRn humanized mice confirmed that only FcRn contributed to transplacental transport of IgG; IgG selectively enhanced for FcRn binding resulted in enhanced accumulation of maternal antibody in the fetus. In contrast, enhancing Fc{gamma}RIIIA binding did not result in enhanced maternal/fetal transport. These results argue against a role for Fc{gamma}Rs in IgG transplacental transport, suggesting Fc engineering of maternally administered antibody to only enhance FcRn binding as a means to improve maternal/fetal transport of IgG. Significance StatementTransport of IgG antibodies from the maternal to the fetal circulation is a key process for neonatal immunity, as neonates cannot sufficiently generate IgG antibodies to reach protective levels during the first months after birth. In humans and other primates, maternal to fetal transport of IgG antibodies is largely mediated through the placental tissue. FcRn has been previously identified as the major driver of IgG transplacental transport. Here we examined whether other receptors, such as Fc{gamma}Rs, also contribute to the maternal-fetal IgG transfer. By characterizing the Fc domain structure of paired maternal-fetal IgG samples and modeling transplacental IgG transport in genetically engineered mouse strains, we determined that FcRn, but not Fc{gamma}Rs, is the major receptor that mediates transplacental IgG transport.

immunology↗