Biology subjects

Zhu, J.

Publications and source records attributed to Zhu, J..

At least 451 records · Page 25Linked to original sources

bioRxiv2021

Nicotinic acid mononucleotide is an allosteric SARM1 inhibitor promoting axonal protection

SARM1 is an inducible NAD+ hydrolase that is the central executioner of pathological axon loss. Recently, we elucidated the molecular mechanism of SARM1 activation, demonstrating that SARM1 is a metabolic sensor regulated by the levels of NAD+ and its precursor, nicotinamide mononucleotide (NMN), via their competitive binding to an allosteric site within the SARM1 N-terminal ARM domain. In healthy neurons with abundant NAD+, binding of NAD+ blocks access of NMN to this allosteric site. However, with injury or disease the levels of the NAD+ biosynthetic enzyme NMNAT2 drop, increasing the NMN/ NAD+ ratio and thereby promoting NMN binding to the SARM1 allosteric site, which in turn induces a conformational change activating the SARM1 NAD+ hydrolase. Hence, NAD+ metabolites both regulate the activation of SARM1 and, in turn, are regulated by the SARM1 NAD+ hydrolase. This dual upstream and downstream role for NAD+ metabolites in SARM1 function has hindered mechanistic understanding of axoprotective mechanisms that manipulate the NAD+ metabolome. Here we reevaluate two methods that potently block axon degeneration via modulation of NAD+ related metabolites, 1) the administration of the NMN biosynthesis inhibitor FK866 in conjunction with the NAD+ precursor nicotinic acid riboside (NaR) and 2) the neuronal expression of the bacterial enzyme NMN deamidase. We find that these approaches not only lead to a decrease in the levels of the SARM1 activator NMN, but also an increase in the levels of the NAD+ precursor nicotinic acid mononucleotide (NaMN). We show that NaMN inhibits SARM1 activation, and demonstrate that this NaMN-mediated inhibition is important for the long-term axon protection induced by these treatments. Analysis of the NaMN-ARM domain co-crystal structure shows that NaMN competes with NMN for binding to the SARM1 allosteric site and promotes the open, autoinhibited configuration of SARM1 ARM domain. Together, these results demonstrate that the SARM1 allosteric pocket can bind a diverse set of metabolites including NMN, NAD+, and NaMN to monitor cellular NAD+ homeostasis and regulate SARM1 NAD+ hydrolase activity. The relative promiscuity of the allosteric site may enable the development of potent pharmacological inhibitors of SARM1 activation for the treatment of neurodegenerative disorders. HighlightsO_LINaMN binds to SARM1 N-terminal allosteric site and inhibits SARM1 NAD+ hydrolase C_LIO_LINaMN inhibits SARM1 activation by stabilizing its open, inactive structure C_LIO_LINMN deamidase promotes strong axonal protection by reducing NMN and increasing NaMN C_LI

neuroscience↗

bioRxiv2021

Neurotoxins subvert the allosteric activation mechanism of SARM1 to induce neuronal loss

SARM1 is an inducible TIR-domain NAD+ hydrolase that mediates pathological axon degeneration. SARM1 is activated by an increased ratio of NMN to NAD+, which competes for binding to an allosteric activating site. When NMN binds, the TIR domain is released from autoinhibition, activating its NAD+ hydrolase activity. The discovery of this allosteric activating site led us to hypothesize that other NAD+-related metabolites might also activate SARM1. Here we show that the nicotinamide analogue 3-acetylpyridine (3-AP), first identified as a neurotoxin in the 1940s, is converted to 3-APMN which activates SARM1 and induces SARM1-dependent NAD+ depletion, axon degeneration and neuronal death. Systemic treatment with 3-AP causes rapid SARM1-dependent death, while local application to peripheral nerve induces SARM1-dependent axon degeneration. We also identify a related pyridine derivative, 2-aminopyridine, as another SARM1-dependent neurotoxin. These findings identify SARM1 as a candidate mediator of environmental neurotoxicity, and furthermore, suggest that SARM1 agonists could be developed into selective agents for neurolytic therapy.

neuroscience↗

bioRxiv2021

VCP protects neurons from proteopathic seeding

Uptake and spread of proteopathic seeds, such as S, Tau, and TDP-43, contribute to neurodegeneration. The cellular machinery necessary for this process is poorly understood. Using a genome-wide CRISPR-Cas9 screen, we identified Valosin Containing Protein (VCP) as a suppressor of S seeding. Dominant mutations in VCP cause multisystem proteinopathy (MSP) with muscle and neuronal degeneration. VCP inhibition or disease mutations increase S seeding in cells and neurons. This is not associated with an increase in seed uptake and is similar to treatment with the lysosomal damaging agent, LLoME. Intrastriatal injection of S seeds into VCP disease mice enhances seeding efficiency compared with controls. This is not specific to S since VCP inhibition or disease mutations increased TDP-43 seeding in neurons. These data support that VCP protects against proteopathic spread of pathogenic aggregates. The spread of distinct aggregate species may dictate pleiotropic phenotypes and pathologies in VCP associated MSP.

neuroscience↗

bioRxiv2021

Critical Assessment of Metagenome Interpretation - the second round of challenges

Evaluating metagenomic software is key for optimizing metagenome interpretation and focus of the community-driven initiative for the Critical Assessment of Metagenome Interpretation (CAMI). In its second challenge, CAMI engaged the community to assess their methods on realistic and complex metagenomic datasets with long and short reads, created from [~]1,700 novel and known microbial genomes, as well as [~]600 novel plasmids and viruses. Altogether 5,002 results by 76 program versions were analyzed, representing a 22x increase in results. Substantial improvements were seen in metagenome assembly, some due to using long-read data. The presence of related strains still was challenging for assembly and genome binning, as was assembly quality for the latter. Taxon profilers demonstrated a marked maturation, with taxon profilers and binners excelling at higher bacterial taxonomic ranks, but underperforming for viruses and archaea. Assessment of clinical pathogen detection techniques revealed a need to improve reproducibility. Analysis of program runtimes and memory usage identified highly efficient programs, including some top performers with other metrics. The CAMI II results identify current challenges, but also guide researchers in selecting methods for specific analyses.

bioinformatics↗

bioRxiv2021

Polybasic patches in both C2 domains of Synaptotagmin-1 are required for evoked neurotransmitter release

Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release. It is composed of a single-pass transmembrane domain linked to two tandem C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is not well understood. Calcium binding to C2B, but not to C2A, is critical for synchronous release and C2B additionally binds the SNARE complex. The C2A domain is also required for Syt1 function, but it is not clear why. Here we asked what critical feature of C2A may be responsible for its functional role, and compared this to the analogous feature in C2B. We focused on highly conserved poly-lysine patches located on the sides of C2A (K189-192) and C2B (K324-327). We tested effects of charge-neutralization mutations in either region (Syt1K189-192A and Syt1K326-327A) side-by-side to determine their relative contributions to Syt1 function in cultured cortical mouse neurons and in single-molecule experiments. Combining electrophysiological recordings and optical tweezers measurements to probe dynamic single C2 domain-membrane interactions, we show that both C2A and C2B polybasic patches contribute to membrane binding, and both are required for evoked release. The readily releasable vesicle pool or spontaneous release were not affected, so both patches are specifically required for synchronization of release. We suggest these patches contribute to cooperative binding to membranes, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release. Significance StatementSynaptotagmin-1 is a vesicular calcium sensor required for synchronous neurotransmitter release. Its tandem cytosolic C2 domains (C2A and C2B) bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. How calcium-binding to Synaptotagmin-1 leads to release and the relative contributions of the two C2 domains is not clear: unlike C2B, calcium-binding to C2A is not critical for evoked release, yet both domains are needed for Syt1 function. Combining electrophysiological recordings from cultured neurons and optical tweezers measurements that probe single C2 domain-membrane interactions, we show that conserved polybasic regions in both domains contribute to membrane binding cooperatively, and both are required for evoked release, likely by increasing the overall affinity of Synaptotagmin-1 for negatively charged membranes.

neuroscience↗

bioRxiv2021

The Genome Sequence Archive Family: Towards Explosive Data Growth and Diverse Data Types

The Genome Sequence Archive (GSA) is a data repository for archiving raw sequence data, which provides data storing and sharing services for worldwide scientific communities. Considering explosive data growth with diverse data types, here we present the GSA family by expanding into a set of resources for raw data archive with different purposes, namely, GSA (https://ngdc.cncb.ac.cn/gsa/), GSA for Human (GSA-Human, https://ngdc.cncb.ac.cn/gsa-human/), and Open Archive for Miscellaneous Data (OMIX, https://ngdc.cncb.ac.cn/omix/). Compared with the 2017 version, GSA has been significantly updated in data model, online functionalities, and web interfaces. GSA-Human, as a new partner of GSA, is a data repository specialized in human genetics-related data with controlled access and security. OMIX, as a critical complement to the two resources mentioned above, is an open archive for miscellaneous data. Together, all these resources form a family of resources dedicated to archiving explosive data with diverse types, accept data submissions from all over the world and provide free open access to all publicly available data in support of worldwide research activities.

bioinformatics↗

bioRxiv2021

ICER controls HIV-1 infection and replication in elite controllers

A rare subset of HIV-infected individuals, termed elite controllers (ECs), can maintain long-term control over HIV replication in the absence of antiretroviral therapy (ART). To elucidate the biological mechanism of resistance to HIV replication at the molecular and cellular levels, we performed RNA sequencing and identified alternative splicing variants from ECs, HIV-infected individuals undergoing ART, ART-naive HIV-infected individuals, and healthy controls. Differential gene expression patterns that are specific to ECs and may influence HIV resistance were identified, including alternative RNA splicing and exon usage variants of the CREM/ICER gene (cAMP-responsive element modulator/inducible cAMP early repressors). The knockout and knockdown of specific ICER exons that were found to be upregulated in ECs resulted in significantly increased HIV infection in CD4+ T cell line and primary CD4+ T cells. Overexpression of ICER isoforms decreased HIV infection in primary CD4+ T cells. Furthermore, ICER regulated HIV-1 LTR promoter activity in a Tat-dependent manner. Together, these results suggest that ICER is an HIV host factor that may contribute to HIV resistance of ECs. These findings will help elucidate the mechanisms of HIV control by ECs and may yield a new approach for treatment of HIV.

immunology↗

bioRxiv2021

High-density transposon mutagenesis in Mycobacterium abscessus identifies an essential penicillin-binding lipo-protein (PBP-lipo) involved in septal peptidoglycan synthesis and antibiotic sensitivity

Mycobacterium abscessus (Mab) is a rapidly growing non-tuberculous mycobacterium (NTM) that causes a wide range of infections. Treatment of Mab infections is difficult because the bacterium is intrinsically resistant to many classes of antibiotics. Developing new and effective treatments against Mab requires a better understanding of the unique vulnerabilities that can be targeted for future drug development. To achieve this, we identified essential genes in Mab by conducting transposon-sequencing (TnSeq) on the reference Mab strain ATCC 19977. We generated [~]51,000 unique transposon mutants and used this high-density library to identify 362 essential genes for in vitro growth. To investigate species-specific vulnerabilities in Mab, we further characterized MAB_3167c, a predicted penicillin-binding-lipoprotein (PBP-lipo) that is essential in Mab and non-essential in Mycobacterium tuberculosis (Mtb). We found that PBP-lipo primarily localizes to the subpolar region and later to the septum as cells prepare to divide. Depletion of Mab PBP-lipo causes cells to elongate, develop ectopic branches, and form multiple septa. Knockdown of PBP-lipo along with PbpB, DacB1, and a carboxypeptidase, MAB_0519 lead to synergistic growth arrest. In contrast, these genetic interactions were absent in the Mtb model organism, Mycobacterium smegmatis, indicating that the PBP-lipo homologs in the two species exist in distinct genetic networks. Finally, repressing PBP-lipo sensitized the reference strain and 11 Mab clinical isolates to several classes of antibiotics, including the {beta}-lactams, ampicillin and amoxicillin by greater than 128-fold. Altogether, this study presents PBP-lipo as a key enzyme to study Mab specific processes in cell wall synthesis and importantly positions PBP-lipo as an attractive drug target to treat Mab infections.

microbiology↗

bioRxiv2021

Voxelated Bioprinting of Mechanically Robust Multiscale Porous Scaffolds for Pancreatic Islets

Three-dimensional (3D) bioprinting additively assembles bio-inks to manufacture tissue-mimicking biological constructs, but with the typical building blocks limited to one-dimensional filaments. Here, we develop a technique for the digital assembly of spherical particles (DASP), which are effectively zero-dimensional voxels - the basic unit of 3D structures. We show that DASP enables on-demand generation, deposition, and assembly of viscoelastic bio-ink droplets. We establish a phase diagram that outlines the viscoelasticity of bio-inks required for printing spherical particles of good fidelity. Moreover, we develop a strategy for engineering bio-inks with independently controllable viscoelasticity and mesh size. Using DASP, we create mechanically robust, multiscale porous scaffolds composed of interconnected yet distinguishable hydrogel particles. Finally, we demonstrate the application of the scaffolds in encapsulating human pancreatic islets for responsive insulin release. Together with the knowledge of bio-ink design, DASP might be used to engineer highly heterogeneous yet tightly organized tissue constructs for therapeutic applications. Table of ContentsA three-dimensional bioprinting technology is developed to enable on-demand generation, deposition, and assembly of viscoelastic bio-ink droplets in a biofriendly environment. A strategy is developed to independently control elasticity, viscosity, and mesh size of bio-inks. The technique allows for easy manufacturing mechanically robust multiscale porous scaffolds, which can be used to encapsulate human pancreatic islets for sustained responsive insulin release. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/449587v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@1c0f2eaorg.highwire.dtl.DTLVardef@8bf924org.highwire.dtl.DTLVardef@191bd3aorg.highwire.dtl.DTLVardef@11e5ce1_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

In planta genome editing with CRISPR/Cas9 ribonucleoproteins

In planta genome editing represents an attractive approach to engineering crops/varieties that are recalcitrant to culture-based transformation methods. Here, we report the direct delivery of CRISPR/Cas9 ribonucleoproteins into the shoot apical meristem using in planta particle bombardment and introduction of a semidwarf1 (sd1)-orthologous mutation into wheat. The triple knockout tasd1 mutant of an elite wheat variety reduced culm length by 10% without a reduction in yield.

plant biology↗

bioRxiv2021

vWCluster: A Network Based Clustering of Multi-omics Breast Cancer Data Based on Vector-Valued Optimal Transport

In this paper, we present a network-based clustering method based on the vector-valued Wasserstein distance derived from optimal mass transport (OMT) theory. This distance allows for the natural integration of multi-layer representations of data in a given network from which one derives clusters via a hierarchical clustering approach. In this study, we applied the methodology, called vector Wasserstein clustering (vW-cluster), to multi-omics data from the two largest breast cancer studies. The resultant clusters showed significantly different survival rates in Kaplan-Meier analysis in both datasets. CIBERSORT scores were compared among the identified clusters. Out of the 22 CIBERSORT immune cell types, 9 were commonly significantly different in both datasets, suggesting the difference of tumor immune microenvironment in the cluster. vWCluster can aggregate multi-omics data represented as a vectorial form in a network with multiple layers, taking into account the concordant effect of heterogeneous data, and further identify subtypes of tumors with different survival rates.

bioinformatics↗

bioRxiv2021

African swine fever virus structural protein p17 inhibits cGAS-STING signaling pathway through interacting with STING

African swine fever (ASF) is highly contagious, causes high mortality in domestic and feral swine, and has a significant economic impact on the global swine industry due to the lack of a vaccine or an effective treatment. African swine fever virus (ASFV) encodes more than 150 polypeptides, which may have intricate and delicate interactions with the host for the benefit of the virus to evade the hosts defenses. However, currently, there is still a lack of information regarding the roles of the viral proteins in host cells. Here, our data demonstrated that the p17, encoded by D117L gene could suppress porcine cGAS-STING signaling pathway, exhibiting the inhibitions of TBK1 and IRF3 phosphorylations, downstream promoter activities, cellular mRNA transcriptions and ISG56 induction, and antiviral responses. Further, we found that p17 was located in endoplasmic reticulum (ER) and Golgi apparatus, and interacted with STING, perturbing it in the recruitment of TBK1 and IKK{varepsilon}. Additionally, it appeared that the transmembrane domain (amino acids 39-59) of p17 could be required for interacting with STING and inhibiting cGAS-STING pathway. Taken together, p17 could inhibit the cGAS-STING pathway through its interaction with STING and interference with STING in the recruitment of TBK1 and IKK{varepsilon}. ImportanceAfrican swine fever (ASF) is a highly contagious disease in domestic and feral swine, posing significant economic impacts on the global swine industry, and the pathogen ASFV is a large icosahedral DNA virus. The innate immune cGAS-STING DNA sensing pathway plays a critical role in sensing invading ASFV and triggering antiviral responses. However, there is still a lack of information regarding the molecular mechanisms of ASFV evasion of the cGAS-STING pathway. We have analyzed the effects of whole genomic open reading frames (ORFs) of ASFV China 2018/1 on the activation of cGAS-STING pathway, and found that p17 was able to inhibit cGAS-STING mediated type I IFN production by targeting STING, altering its capacity to recruit both TBK1 and IKK{varepsilon}. Findings presented here will expand our knowledge on the molecular mechanisms by which ASFV counteracts the antiviral innate immunity and provide deep insights into ASF pathogenesis.

immunology↗

bioRxiv2021

The African swine fever virus protease pS273R inhibits DNA sensing cGAS-STING pathway by targeting IKKε

African swine fever virus (ASFV), a large and complex cytoplasmic double-stranded DNA virus, has developed multiple strategies to evade the antiviral innate immune responses. Cytosolic DNA arising from invading ASFV is mainly detected by the cyclic GMP-AMP synthase (cGAS) and then triggers a series of innate immune responses to prevent virus invasion. However, the immune escape mechanism of ASFV remains to be fully clarified. The pS273R of ASFV is a member of the SUMO-1-specific protease family and is crucial for valid virus replication. In this study, we identified pS273R as a suppressor of cGAS-STING pathway mediated type I interferon (IFN) production by ASFV genomic open reading frame screening. The pS273R was further confirmed as an inhibitor of IFN production as well as its downstream antiviral genes in cGAS-STING pathway. Mechanistically, pS273R greatly decreased the cGAS-STING signaling by targeting IKK{varepsilon} but not TBK1 and pS273R was found to disturb the interaction between IKK{varepsilon} and STING through its interaction with IKK{varepsilon}. Further, mutational analyses revealed that pS273R antagonized the cGAS-STING pathway by enzyme catalytic activity, which may affect the IKK{varepsilon} sumoylation state required for the interaction with STING. In summary, our results revealed for the first time that pS273R acts as an obvious negative regulator of cGAS-STING pathway by targeting IKK{varepsilon} via its enzymatic activity, which shows a new immune evasion mechanism of ASFV. ImportanceAfrican swine fever (ASF) is a devastating disease for domestic pigs and wild boar and the pathogen ASFV is a cytoplasmic double-stranded DNA virus. The innate immune cGAS-STING-IFN signaling pathway exerts a critical role in sensing ASFV infection. However, the functions of half ASFV encoded 150 plus proteins are still unknown and the evasion against the cGAS-STING pathway is not resolved. In our study, via ASFV genomic open reading frame (ORF) screening, we found that 29 ASFV proteins could inhibit cGAS-STING signaling pathway, with pS273R showing the most obvious inhibitory effect. Surprisingly, pS273R was found to antagonize the cGAS-STING signaling by targeting IKK{varepsilon}. Moreover, the pS273R enzyme activity is required for its ability to inhibit the cGAS-STING pathway. Our findings deepen the understanding of the immune evasion mechanism of ASFV, which will provide a support for the development of safe and effective ASFV vaccines.

immunology↗

bioRxiv2021

Multi-omics consensus ensemble refines the classification of muscle-invasive bladder cancer with stratified prognosis, tumour microenvironment and distinct sensitivity to frontline therapies

The molecular classification of muscle-invasive bladder cancer (MIBC) based on transcriptomic signatures has been extensively studied. The complementary nature of information provided by different molecular profiles motivated us to refine MIBC classification by aggregating multi-omics data. We generated a consensus ensemble through ten multi-omics integrative clustering approaches on 396 MIBCs from TCGA. A total of 701 MIBCs from different sequencing technologies were used for external validation. Associations between subtypes and prognosis, molecular profiles, the tumour microenvironment, and potential response to frontline therapies were further analyzed. Nearest template prediction and random forest classification were used to develop a predictive signature/classifier for MIBC refinement. We identified four integrative consensus subtypes of MIBC, which were further designated basal-inflamed, basal-noninflamed, luminal-excluded and luminal-desert by immune profiling. Of note, the refinement of basal-like MIBC classification adds to the literature by identifying a basal-noninflamed MIBC subtype presenting with a significantly poor outcome and a global immune-cold phenotype, which might be triggered by Chr4 deletion and high activation of the oncogenic NRF2 pathway. In contrast, basal-inflamed MIBC showed high immunocyte infiltration and high expression of potential targets for immunotherapy. Using an external metastatic MIBC cohort in which patients received anti-PD-L1 treatment, we suggested that basal-inflamed MIBC had a higher likelihood of responding to immunotherapy than other MIBCs. The R package "refineMIBC" was offered as a research tool to refine MIBC from a single-sample perspective (https://github.com/xlucpu/refineMIBC). This consensus ensemble refines the intrinsic MIBC subtypes, which provides a blueprint for the clinical development of rational targeted and immunotherapeutic strategies.

cancer biology↗

bioRxiv2021

Identification of ubiquitin variants that inhibit the E2 ubiquitin conjugating enzyme, Ube2k

Transfer of ubiquitin to substrate proteins regulates most processes in eukaryotic cells. E2 enzymes are a central component of the ubiquitin machinery, and generally determine the type of ubiquitin signal generated and thus the ultimate fate of substrate proteins. The E2, Ube2k, specifically builds degradative ubiquitin chains on diverse substrates. Here we have identified protein-based reagents, called ubiquitin variants (UbVs), that bind tightly and specifically to Ube2k. Crystal structures reveal that the UbVs bind to the E2 enzyme at a hydrophobic cleft that is distinct from the active site and previously identified ubiquitin binding sites. We demonstrate that the UbVs are potent inhibitors of Ube2k and block both ubiquitin charging of the E2 enzyme, and E3-catalysed ubiquitin transfer. The binding site of the UbVs suggests they directly clash with the ubiquitin activating enzyme, while potentially disrupting interactions with E3 ligases via allosteric effects. Our data reveal the first protein-based inhibitors of Ube2k and unveil a hydrophobic groove that could be an effective target for inhibiting Ube2k and other E2 enzymes.

biochemistry↗

bioRxiv2021

SARS-CoV-2 Nsp14 activates NF-κB signaling and induces IL-8 upregulation

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to NF-{kappa}B activation and induction of pro-inflammatory cytokines, though the underlying mechanism for this activation is not fully understood. Our results reveal that the SARS-CoV-2 Nsp14 protein contributes to the viral activation of NF-{kappa}B signaling. Nsp14 caused the nuclear translocation of NF-{kappa}B p65. Nsp14 induced the upregulation of IL-6 and IL-8, which also occurred in SARS-CoV-2 infected cells. IL-8 upregulation was further confirmed in lung tissue samples from COVID-19 patients. A previous proteomic screen identified the putative interaction of Nsp14 with host Inosine-5-monophosphate dehydrogenase 2 (IMPDH2) protein, which is known to regulate NF-{kappa}B signaling. We confirmed the Nsp14-IMPDH2 protein interaction and found that IMPDH2 knockdown or chemical inhibition using ribavirin (RIB) and mycophenolic acid (MPA) abolishes Nsp14-mediated NF-{kappa}B activation and cytokine induction. Furthermore, IMDPH2 inhibitors (RIB, MPA) efficiently blocked SARS-CoV-2 infection, indicating that IMDPH2, and possibly NF-{kappa}B signaling, is beneficial to viral replication. Overall, our results identify a novel role of SARS-CoV-2 Nsp14 in causing the activation of NF-{kappa}B.

microbiology↗

bioRxiv2021

Effect of water temperature on the behavior of Neptunea cumingii and the histology, immune enzyme activity, and transcriptome of its gills and kidneys

The behavior of the marine snail Neptunea cumingii cultured at different temperatures (0, 4, 8, 12, 16 (control), 20, 24, and 28{degrees}C) and the histology, immune enzyme activity, and transcriptome of its gills and kidneys were studied using ecological and molecular methods. At 0{degrees}C, most of the snails shrank in size and did not eat during the first 6 h. At 28{degrees}C, snails also did not eat, death began to occur at 24 h. when the temperature was 8 {degrees}C and 12 {degrees}C, there was no significant difference in the mean feed intake of N. cumingii (P>0.05). And it was higher than the average conch intake under other temperature conditions. However, the histology of the gills and kidneys differed among test temperatures. At 0{degrees}C, the morphology of the gill pieces was difficult to judge. At 24{degrees}C, edema was present in the small gills, and at 28{degrees}C the small gills were severely deformed, the distance between the gill capillaries and the surrounding area was enlarged, and the gill tissue was severely damaged. Temperature increase or decrease from 16{degrees}C caused the columnar cells of the kidney to become shorter and more numerous. The total antioxidant capacity (T-AOC), catalase (CAT) and superoxide dismutase activities (SOD) of the gill and kidney differed significantly among the temperature conditions (p < 0.05). Transcriptome results showed that temperature change resulted in up-regulation of 2339 genes and down-regulation of 2058 genes in the gills, and in up-regulation of 2300 genes and down-regulation of 2060 genes in the kidneys of N. cumingii. Furthermore, the DEGs were subject to GO and KEGG enrichment analysis, and which showed that most of the DEGs in gill were involved in protein folding, defolding, translation, ribosome, and most of the DEGs in kidney were involved in DNA recombination, nuclear euchromatin, RNA-directed DNA polymerase activity. Finally, the results from this study show that N. cumingii prefers the temperature range was 8 to 16, temperature increase or decrease from that range, many aspects of N. cumingii biology could be significantly affected.

physiology↗

bioRxiv2021

Functional dissection of human mitotic proteins using CRISPR-Cas9 tiling screens

Kinetochores are large protein complexes that assemble at the centromere and bind to mitotic spindle microtubules to ensure accurate chromosome segregation. Like most protein-coding genes, the full multifunctional nature of kinetochore factors remains uncharacterized due to the limited experimental tools for unbiased dissection of human protein sequences. We developed a method that leverages CRISPR-Cas9 induced mutations to identify key functional regions within protein sequences required for cellular outgrowth. Our analysis of 48 human mitotic genes revealed hundreds of regions required for cell proliferation, including known domains and uncharacterized ones. We validated the essential nature for 15 of these regions, including amino acids 387-402 of Mad1, which identified an unknown domain that contributes to Mad1 kinetochore localization and chromosome segregation fidelity. Altogether, we demonstrate that CRISPR-Cas9-based tiling mutagenesis identifies key functional domains in protein-coding genes de novo, which elucidates separation of function mutants and allows functional annotation across the human proteome.

cell biology↗