Biology subjects

Zhu, Y.

Publications and source records attributed to Zhu, Y..

At least 685 records · Page 38Linked to original sources

bioRxiv2020

SMARCAD1 Mediated Active Replication Fork Stability Maintains Genome Integrity

Stalled fork protection pathway mediated by BRCA1/2 proteins is critical for replication fork stability that has implications in tumorigenesis. However, it is unclear if additional mechanisms are required to maintain replication fork stability. We describe a novel mechanism by which the chromatin remodeler SMARCAD1 stabilizes active replication forks that is essential for resistance towards replication poisons. We find that loss of SMARCAD1 results in toxic enrichment of 53BP1 at replication forks which mediates untimely dissociation of PCNA via the PCNA-unloader, ATAD5. Faster dissociation of PCNA causes frequent fork stalling, inefficient fork restart and accumulation of single-stranded DNA resulting in genome instability. Although, loss of 53BP1 in SMARCAD1 mutants restore PCNA levels, fork restart efficiency, genome stability and tolerance to replication poisons; this requires BRCA1 mediated fork protection. Interestingly, fork protection challenged BRCA1-deficient naive- or PARPi-resistant tumors require SMARCAD1 mediated active fork stabilization to maintain unperturbed fork progression and cellular proliferation.

cell biology↗

bioRxiv2020

CBX1 mutations cause a neurodevelopmental syndrome due to heterochromatin organizational alterations

PurposeThis study aimed to establish variants in CBX1, encoding heterochromatin protein 1{beta} (HP1{beta}), as a cause of a novel syndromic neurodevelopmental disorder. MethodsPatients with CBX1 variants were identified, and clinician researchers were connected using GeneMatcher and physician referrals. Clinical histories were collected from each patient. To investigate the pathogenicity of identified variants, we performed in vitro cellular assays, neurobehavioral and cytological analyses of neuronal cells obtained from newly generated Cbx1 mutant mouse lines. ResultsIn three unrelated individuals with developmental delay, hypotonia, and autistic features, we identified heterozygous de novo variants in CBX1. The identified variants were in the chromodomain, the functional domain of HP1 {beta}, which mediates interactions with chromatin. Cbx1 chromodomain mutant mice displayed increased latency-to-peak response, suggesting the possibility of synaptic delay or myelination deficits. Cytological and chromatin immunoprecipitation experiments confirmed the reduction of mutant HP1{beta} binding to heterochromatin, while HP1{beta} interactome analysis demonstrated that the majority of HP1{beta}-interacting proteins remained unchanged between the wild-type and mutant HP1{beta}. ConclusionThese collective findings confirm the role of CBX1 in developmental disabilities through the disruption of HP1{beta} chromatin binding during neurocognitive development. As HP1{beta} forms homodimers and heterodimers, mutant HP1{beta} likely sequesters wild-type HP1{beta} and other HP1 proteins, exerting dominant-negative effects.

genetics↗

bioRxiv2020

Acute noise causes down-regulation of ECM protein expression in guinea pig cochlea

ObjectiveTo study the differences in cochlea protein expression before and after noise exposure using proteomics to reveal the pathological mechanism of noise-induced hearing loss (NIHL). MethodsA guinea pig NIHL model was established to test the ABR thresholds before and after noise exposure. The proteomics technology was used to study the mechanism of differential protein expression in the cochlea by noise stimulation. ResultsThe average hearing threshold of guinea pigs on the first day after noise exposure was 57.00{+/-}6.78dB SPL, high-frequency hearing loss was more severe than low frequency; the average hearing threshold on the seventh day after noise exposure was 45.83{+/-}6.07dB SPL, and the 4k Hz hearing threshold has the best recovery. The proteomics technology identified 3122 different inner ear proteins, of which six proteins related to the hearing were down-regulation: TenascinC, Collagen type XI alpha two chains, Collagen type II alpha one chain, Thrombospondin 2, Collagen type XI alpha one chain and Ribosomal protein L38, and are enriched in protein absorption, focal adhesion, and extracellular matrix receptor pathways. ConclusionImpulse noise can affect the expression of differential proteins through focal adhesion pathways. This data can provide an experimental basis for the research on the prevention and treatment of NIHL.

immunology↗

bioRxiv2020

Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry

Coronavirus interaction with its viral receptor is a primary genetic determinant of host range and tissue tropism. SARS-CoV-2 utilizes ACE2 as the receptor to enter host cell in a species-specific manner. We and others have previously shown that ACE2 orthologs from New World monkey, koala and mouse cannot interact with SARS-CoV-2 to mediate viral entry, and this defect can be restored by humanization of the restrictive residues in New World monkey ACE2. To better understand the genetic determinants behind the ability of ACE2 orthologs to support viral entry, we compared koala and mouse ACE2 sequences with that of human and identified the key residues in koala and mouse ACE2 that restrict viral receptor activity. Humanization of these critical residues rendered both koala and mouse ACE2 capable of binding the spike protein and facilitating viral entry. The single mutation that allowed for mouse ACE2 to serve as a viral receptor provides a potential avenue for the development of SARS-CoV-2 mouse model.

microbiology↗

bioRxiv2020

Discovery of a Novel Inhibitor of Coronavirus 3CL Protease as a Clinical Candidate for the Potential Treatment of COVID-19

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. The designed phosphate prodrug PF-07304814 is metabolized to PF-00835321 which is a potent inhibitor in vitro of the coronavirus family 3CL pro, with selectivity over human host protease targets. Furthermore, PF-00835231 exhibits potent in vitro antiviral activity against SARS-CoV-2 as a single agent and it is additive/synergistic in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of this compound as a potential COVID-19 treatment.

pharmacology and toxicology↗

bioRxiv2020

Distinct mechanisms for TMPRSS2 expression explain organ-specific inhibition of SARS-CoV-2 infection by enzalutamide

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly become a global public health threat due to the lack of effective drugs or vaccines against SARS-CoV-2. The efficacy of several repurposed drugs has been evaluated in clinical trials. Among these drugs, a relatively new antiandrogen agent, enzalutamide, was proposed because it reduces the expression of transmembrane serine protease 2 (TMPRSS2), a key component mediating SARS-CoV-2-driven entry into host cells, in prostate cancer cells. However, definitive evidence for the therapeutic efficacy of enzalutamide in COVID-19 is lacking. Here, we evaluated the antiviral efficacy of enzalutamide in prostate cancer cells, lung cancer cells, human lung organoids and SARS-CoV-2-infected Ad-ACE2-transduced Tmprss2 knockout (Tmprss2-KO) and wild-type (WT) mice. TMPRSS2 knockout significantly inhibited SARS-CoV-2 infection in vivo. Enzalutamide effectively inhibited SARS-CoV-2 infection in human prostate cancer cells (LNCaP) but not in human lung cancer cells or patient-derived lung organoids. Although Tmprss2 knockout effectively blocked SARS-CoV-2 infection in ACE2-transduced mice, enzalutamide showed no antiviral activity due to the AR independence of TMPRSS2 expression in mouse and human lung epithelial cells. Moreover, we observed distinct AR binding patterns between prostate cells and lung cells and a lack of direct binding of AR to TMPRSS2 in human lung cells. Thus, our findings do not support the postulated protective role of enzalutamide in treating COVID-19.

microbiology↗

bioRxiv2020

Acute unilateral emphysema induced by Pseudomonas aeruginosa in mice

Pulmonary emphysema is the major pathological feature of chronic obstructive pulmonary disease (COPD). Although the pathogenesis of emphysema is still not completely understood, but until now a bacterial cause has not really been considered. Recently, we found that the secretion from Pseudomonas aeruginosa could cause severe lung emphysema in mice rapidly. Since the bacterium is ubiquitous and secrets proteases, we hypothesized that direct P. aeruginosa airway infection would have a similar effect. To address this issue, we applied a unilateral lung injury model. First, we observed the dynamic pathophysiology change of acute emphysema. P. aeruginosa secretion was extracted and instilled intratracheally into the left lungs of C57BL/6 and C3H/HeJ mice, while the right lungs were saved as self-control. Alveolar diameter and lung compliance were measured. Later, we tested the effect of P. aeruginosa inoculation in normal C57BL/6 mice, immunosuppressed C57BL/6 mice, and C3H/HeJ (TLR4 deficient) mice. P. aeruginosa secretion extract caused acute panacinar emphysema and decreased dynamic lung compliance. Different types of emphysema are transformable. However, the P. aeruginosa infection could only elicit emphysema in immunosuppressed C57BL/6 mice and C3H/HeJ mice, indicating that normal immunity is essential to protect the hosts from emphysema. Emphysema induced by P. aeruginosa in mice recapitulates all the main features of human emphysema and COPD. Our finding filled a major gap in COPD pathogenesis. We believe P. aeruginosa is the underlying cause of COPD.

pathology↗

bioRxiv2020

Emotional tagging retroactively promotes memory integration through rapid neural reactivation and reorganization

Neutral events preceding emotional experiences are thought to be better remembered by tagging them as significant to simulate future event predictions. Yet, the neurobiological mechanisms how emotion transforms initially mundane events into strong memories remain unclear. By two behavioral and one fMRI studies with adapted sensory preconditioning paradigm, we show rapid neural reactivation and reorganization underlying emotion-tagged retroactive memory enhancement. Behaviorally, emotional tagging enhanced initial memory for neutral associations across the three studies. Neurally, emotional tagging potentiated reactivation of overlapping neural traces in the hippocampus and stimulus-relevant neocortex. Moreover, it induced large-scale hippocampal-neocortical reorganization supporting such retroactive benefit, as characterized by enhanced hippocampal-neocortical coupling modulated by the amygdala during online processing, and a shift from stimulus-relevant neocortex to transmodal prefrontal-parietal areas during offline post-tagging rest. Together, emotional tagging retroactively promotes associations between past neutral events through stimulating rapid reactivation of overlapping representations and reorganizing related memories into an integrated network.

neuroscience↗

bioRxiv2020

UVA radiation could be a significant contributor to sunlight inactivation of SARS-CoV-2

Past experiments demonstrated SARS-CoV-2 inactivation by simulated sunlight; models have considered exclusively mechanisms involving UVB acting directly on RNA. However, UVA inactivation has been demonstrated for other enveloped RNA viruses, through indirect mechanisms involving the suspension medium. We propose a model combining UVB and UVA inactivation for SARS-CoV-2, which improves predictions by accounting for effects associated with the medium. UVA sensitivities deduced for SARS-CoV-2 are consistent with data for SARS-CoV-1 under UVA only. This analysis calls for experiments to separately assess effects of UVA and UVB in different media, and for including UVA in inactivation models. Lay summaryRecent experiments have demonstrated that SARS-CoV-2 is inactivated by simulated sunlight; however, there are still many unknowns, including the mechanism of action and which part of the light spectrum is principally responsible. Our analysis indicates the need for targeted experiments that can separately assess the effects of UVA and UVB on SARS-CoV-2, and that sunlight inactivation models may need to be expanded to also include the effect of UVA. A first UVA-inclusive model is also proposed here. These findings have implications for how to improve the safety of the built environment, and for the seasonality of COVID-19.

biochemistry↗

bioRxiv2020

Antibody Upstream Sequences Diversity and Its Biological Implications Revealed by Repertoire Sequencing

The sequence upstream of antibody variable region (Antibody Upstream Sequence, or AUS) consists of 5 untranslated region (5 UTR) and two leader regions, L-PART1 and L-PART2. The sequence variations in AUS affect the efficiency of PCR amplification, mRNA translation, and subsequent PCR-based antibody quantification as well as antibody engineering. Despite their importance, the diversity of AUSs has long been neglected. Utilizing the rapid amplification of cDNA ends (5RACE) and high-throughput antibody repertoire sequencing (Rep-Seq) technique, we acquired full-length AUSs for human, rhesus macaque (RM), cynomolgus macaque (CM), mouse, and rat. We designed a bioinformatics pipeline and discovered 2,957 unique AUSs, corresponding to 2,786 and 1,159 unique sequences for 5 UTR and leader, respectively. Comparing with the leader records in the international ImMunoGeneTics (IMGT), while 529 were identical, 313 were with single nucleotide polymorphisms (SNPs), 280 were totally new, and 37 updated the incomplete records. The diversity of AUSs impact on related antibody biology was also probed. Taken together, our findings would facilitate Rep-Seq primer design for capturing antibodies comprehensively and efficiently as well as provide a valuable resource for antibody engineering and the studies of antibody at the molecular level.

bioinformatics↗

bioRxiv2020

Snord67 promotes lymph node metastasis and regulates U6-mediated alternative splicing in breast cancer

Small nucleolar RNAs (snoRNAs) have long been considered "housekeeping genes", important for ribosomal biogenesis and protein synthesis. However, there is increasing evidence that this largely ignored class of non-coding RNAs (ncRNAs) also have wide-ranging, non-canonical functions in diseases, including cancer. SnoRNAs have been shown to have both oncogenic and tumor suppressor roles, yet whether snoRNAs regulate metastasis is unknown. Here we show that expression of certain snoRNAs are enriched in lymph node (LN) metastases in a micro-surgical, immune-competent mouse model of breast cancer. We identify the snoRNA Snord67 as a key regulator of LN metastasis. Knockout of Snord67 resulted in significantly decreased LN tumor growth and subsequent development of distant metastases. This was associated with loss of targeted 2-O-methylation on the small nuclear RNA U6, a component of the spliceosome. RNA sequencing revealed distinct alternative splicing patterns in Snord67 knockout cells. Using rapid autopsy breast cancer cases, we found that matched human primary tumor and LN metastases revealed similar alternatively spliced genes, including several that are known to contribute to cancer. These results demonstrate that Snord67 is critical for growth of LN metastases and subsequent spread to distant metastases, and suggest that snoRNA-guided modifications of the spliceosome represent a previously unappreciated, yet targetable pathway in cancer.

cancer biology↗

bioRxiv2020

The S1/S2 boundary of SARS-CoV-2 spike protein modulates cell entry pathways and transmission

The global spread of SARS-CoV-2 is posing major public health challenges. One unique feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site, the function of which remains uncertain. We found that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site instead utilizes a less efficient endosomal entry pathway. This idea was supported by the identification of a suite of endosomal entry factors specific to Sdel virus by a genome-wide CRISPR-Cas9 screen. A panel of host factors regulating the surface expression of ACE2 was identified for both viruses. Using a hamster model, animal-to-animal transmission with the Sdel virus was almost completely abrogated, unlike with Sfull. These findings highlight the critical role of the S1/S2 boundary of the SARS-CoV-2 spike protein in modulating virus entry and transmission.

microbiology↗

bioRxiv2020

SARS-Cov-2-, HIV-1-, Ebola-neutralizing and anti-PD1 clones are predisposed

Antibody repertoire refers to the totality of the superbly diversified antibodies within an individual to cope with the vast array of possible pathogens. Despite this extreme diversity, antibodies of the same clonotype, namely public clones, have been discovered among individuals. Although some public clones could be explained by antibody convergence, public clones in naive repertoire or virus-neutralizing clones from not infected people were also discovered. All these findings indicated that public clones might not occur by random and they might exert essential functions. However, the frequencies and functions of public clones in a population have never been studied. Here, we integrated 2,449 Rep-seq datasets from 767 donors and discovered 5.07 million public clones - ~10% of the repertoire are public in population. We found 38 therapeutic clones out of 3,390 annotated public clones including anti-PD1 clones in healthy people. Moreover, we also revealed clones neutralizing SARS-CoV-2, Ebola, and HIV-1 viruses in healthy individuals. Our result demonstrated that these clones are predisposed in the human antibody repertoire and may exert critical functions during particular immunological stimuli and consequently benefit the donors. We also implemented RAPID - a Rep-seq Analysis Platform with Integrated Databases, which may serve as a useful tool for others in the field.

immunology↗

bioRxiv2020

Chemical-genetic interactions with the proline analog L-azetidine-2-carboxylic acid in Saccharomyces cerevisiae

Non-proteinogenic amino acids, such as the proline analog L-azetidine-2-carboxylic acid (AZC), are detrimental to cells because they are mis-incorporated into proteins and lead to proteotoxic stress. Our goal was to identify genes that show chemical-genetic interactions with AZC in Saccharomyces cerevisiae and thus also potentially define the pathways cells use to cope with amino acid mis-incorporation. Screening the yeast deletion and temperature sensitive collections, we found 72 alleles with negative synthetic interactions with AZC treatment and 12 alleles that suppress AZC toxicity. Many of the genes with negative synthetic interactions are involved in protein quality control pathways through the proteasome. Genes involved in actin cytoskeleton organization and endocytosis also had negative synthetic interactions with AZC. Related to this, the number of actin patches per cell increases upon AZC treatment. Many of the same cellular processes were identified to have interactions with proteotoxic stress caused by two other amino acid analogs, canavanine and thialysine, or a mistranslating tRNA variant that mis-incorporates serine at proline codons. Alleles that suppressed AZC-induced toxicity functioned through the amino acid sensing TOR pathway or controlled amino acid permeases required for AZC uptake.

genetics↗

bioRxiv2020

Persistent bacterial coinfection of a COVID-19 patient caused by a genetically adapted Pseudomonas aeruginosa chronic colonizer

This study characterized a genetically adapted Pseudomonas aeruginosa small colony variant isolated from a COVID-19 patient who suffered persistent bacterial coinfection and eventually recovered from critical illness. Specification and modification of the isolates discovered at genomic and transcriptomic levels with aligned phenotypic observations indicated that these isolates formed excessive biofilm with elevated quorum sensing systems.

microbiology↗

bioRxiv2020

Limits to the cellular control of sequestered cryptophyte prey in the marine ciliate Mesodinium rubrum

The marine ciliate Mesodinium rubrum is famous for its ability to acquire and exploit chloroplasts and other cell organelles from some cryptophyte algal species. We sequenced genomes and transcriptomes of free-swimming Teleaulax amphioxeia, as well as well-fed and starved M. rubrum in order to understand cellular processes upon sequestration under different prey and light conditions. From its prey, the ciliate acquires the ability to photosynthesize as well as the potential to metabolize several essential compounds including lysine, glycan, and vitamins that elucidate its specific prey dependency. M. rubrum does not express photosynthesis related genes itself, but elicits considerable transcriptional control of the acquired cryptophyte organelles. This control is limited as light dependent transcriptional changes found in free-swimming T. amphioxeia got lost after sequestration. We found strong transcriptional rewiring of the cryptophyte nucleus upon sequestration, where 35% of the T. amphioxeia genes were significantly differentially expressed within well-fed M. rubrum. Qualitatively, 68% of all genes expressed within well-fed M. rubrum originated from T. amphioxeia. Quantitatively, these genes contributed up to 48% to the global transcriptome in well-fed M. rubrum and down to 11% in starved M. rubrum. This tertiary endosymbiosis system functions for several weeks, when deprived of prey. After this point in time, the ciliate dies if not supplied with fresh prey cells. M. rubrum represents one evolutionary way of acquiring photosystems from its algal prey, and might represent a step on the evolutionary way towards a permanent tertiary endosymbiosis.

evolutionary biology↗

bioRxiv2020

A CRISPR-based genome-wide screen for adipogenesis reveals new insights into mitotic expansion and lipogenesis.

In response to excess nutrients, white adipose tissue expands by both generating new adipocytes and by upregulating lipogenesis in existing adipocytes. Here, we performed a genome-wide functional genomics screen to identify regulators of adipogenesis in the mouse 3T3-L1 preadipocyte model. The pooled screening strategy utilized FACS to isolate populations based on lipid content by gating for fluorescence intensity of the lipophilic, green fluorescent BODIPY dye. Additionally, the approach categorized if genes functioned during mitotic expansion or lipogenesis. Cellular mechanisms regulating the rate of protein translation and protein stability were found critical for adipogenesis and lipogenesis. These protein-directed mechanisms were further supported by proteomic analyses, which demonstrated that essential changes in protein abundance driving 3T3-L1 adipogenesis were not driven by transcription. We exemplify this theme by showing that the hypusination pathway, a conserved regulator of translation initiation, is critical to translate adipogenic inducers of mitotic expansion and that the neddylation/ubiquitin pathway modulates insulin sensitivity to regulate lipogenesis.

molecular biology↗

bioRxiv2020

Single-cell screening of SARS-CoV-2 target cells in pets, livestock, poultry and wildlife

A few animals have been suspected to be intermediate hosts of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, a large-scale single-cell screening of SARS-CoV-2 target cells on a wide variety of animals is missing. Here, we constructed the single-cell atlas for 11 representative species in pets, livestock, poultry, and wildlife. Notably, the proportion of SARS-CoV-2 target cells in cat was found considerably higher than other species we investigated and SARS-CoV-2 target cells were detected in multiple cell types of domestic pig, implying the necessity to carefully evaluate the risk of cats during the current COVID-19 pandemic and keep pigs under surveillance for the possibility of becoming intermediate hosts in future coronavirus outbreak. Furthermore, we screened the expression patterns of receptors for 144 viruses, resulting in a comprehensive atlas of virus target cells. Taken together, our work provides a novel and fundamental strategy to screen virus target cells and susceptible species, based on single-cell transcriptomes we generated for domesticated animals and wildlife, which could function as a valuable resource for controlling current pandemics and serve as an early warning system for coping with future infectious disease threats.

cell biology↗