Biology subjects

Wu, P.

Publications and source records attributed to Wu, P..

At least 109 records · Page 6Linked to original sources

bioRxiv2021

SARS-CoV-2 nucleocapsid protein dually regulates innate immune responses

The recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the ongoing global pandemic of COVID-19, may trigger immunosuppression in the early stage and a cytokine storm in the late stage of infection, however, the underlying mechanisms are not well understood. Here we demonstrated that the SARS-CoV-2 nucleocapsid (N) protein dually regulated innate immune responses, i.e., the low-dose N protein suppressed type I interferon (IFN-I) signaling and inflammatory cytokines, whereas high-dose N protein promoted IFN-I signaling and inflammatory cytokines. Mechanistically, the SARS-CoV-2 N protein interacted with the tripartite motif protein 25 (TRIM25), thereby dually regulating the phosphorylation and nuclear translocation of IRF3, STAT1 and STAT2. Additionally, low-dose N protein combined with TRIM25 could suppress retinoic acid-inducible gene I (RIG-I) ubiquitination and activation. Our findings revealed a regulatory mechanism of innate immune responses by the SARS-CoV-2 N protein, which would contribute to understanding the pathogenesis of SARS-CoV-2 and other SARS-like coronaviruses, and development of more effective strategies for controlling COVID-19.

microbiology↗

bioRxiv2021

Discovery of a molecular glue that enhances UPRmt to restore proteostasis via TRKA-GRB2-EVI1-CRLS1 axis

Lowering proteotoxicity is a potentially powerful approach for the treatment of neurological disorders, such as Parkinsons disease. The unfolded protein response (UPR) is a major mechanism that preserves the network maintaining cellular proteostasis. In the present study, we developed the screening strategy to discover compounds that significantly enhanced the activation of mitochondrial UPR (UPRmt) through increasing cardiolipin content. We identified that ginsenoside Rg3 (Rg3) increased cardiolipin depending on cardiolipin synthase 1 (CRLS1) in both worms and in human neural cells. Using LiP-SMap (limited proteolysis-mass spectrometry) strategy, we identified GRB2 (growth factor receptor bound protein 2) as a direct target of Rg3 in human neural cells. Rg3 enhances the binding between GRB2 and TRKA, that transduces signals via phosphrorylation of ERK. We provide bioinformatic and experimental evidence that EVI1, the critical oncogenic transcriptional regulator in leukemia, binds to CRLS1 promoter region and stimulated CRLS1 expression and subsequently increased cardiolipin content in the presence of Rg3. In a Parkinsons disease mouse model, Rg3 restores motor function by protecting nigral dopaminergic neurons dependent on Grb2. Our data recapitulate the TRKA-GRB2-EVI1-CRLS1 axis in maintaining proteostasis in Parkinsons disease via UPRmt.

pharmacology and toxicology↗

bioRxiv2021

L-WNK1 is required for BK channel activation in intercalated cells

BK channels expressed in intercalated cells (ICs) in the aldosterone-sensitive distal nephron (ASDN) mediate flow-induced K+ secretion. In the ASDN of mice and rabbits, IC BK channel expression and activity increase with a high K+ diet. In cell culture, the long isoform of the kinase WNK1 (L-WNK1) increases BK channel expression and activity. Apical L-WNK1 expression is selectively enhanced in ICs in the ASDN of rabbits on a high K+ diet, suggesting that L-WNK1 contributes to BK channel regulation by dietary K+. We examined the role of IC L-WNK1 expression in enhancing BK channel activity in response to a high K+ diet. Mice with an IC-selective deletion of L-WNK1 (IC-L-WNK1-KO) and littermate control mice were placed on a high K+ (5% K+ as KCl) diet for at least 10 days. IC-L-WNK1-KO mice exhibited higher blood K+ concentrations ([K+]) than controls. BK channel-dependent whole-cell currents in ICs from cortical collecting ducts of high K+ fed IC-L-WNK1-KO mice were reduced compared to controls. Six-hour urinary K+ excretion in response a saline load was similar in IC-L-WNK1-KO mice and controls. The observations that IC-L-WNK1-KO mice have higher blood [K+] and reduced IC BK channel currents are consistent with impaired urinary K+ secretion, and suggest that IC L-WNK1 has a role in the renal adaptation to a high K+ diet.

physiology↗

bioRxiv2021

AsmMix: A pipeline for high quality diploid denovo assembly

In this paper, we report a pipeline, AsmMix, which is capable of producing both contiguous and high-quality diploid genomes. The pipeline consists of two steps. In the first step, two sets of assemblies are generated: one is based on co-barcoded reads, which are highly accurate and haplotype-resolved but contain many gaps, the other assembly is based on single-molecule sequencing reads, which is contiguous but error-prone. In the second step, those two sets of assemblies are compared and integrated into a haplotype-resolved assembly with fewer errors. We test our pipeline using a dataset of human genome NA24385, perform variant calling from those assemblies and then compare against GIAB Benchmark. We show that AsmMix pipeline could produce highly contiguous, accurate, and haplotype-resolved assemblies. Especially the assembly mixing process could effectively reduce small-scale errors in the long read assembly.

genomics↗

bioRxiv2020

Non-coding loci without epigenomic signals can be essential for maintaining global chromatin organization and cell viability

The majority of the non-coding regions in the human genome do not harbor any annotated element and are even not marked with any epigenomic signal or protein binding. An understudied aspect of these regions is their possible roles in stabilizing the 3D chromatin organization. To illuminate their "structural importance", we chose to start with the non-coding regions forming many 3D contacts (referred to as hubs) and identified dozens of hubs essential for cell viability. Hi-C and single cell transcriptomic analyses showed that their deletion could significantly alter chromatin organization and impact gene expression located distal in the genome. This study revealed the 3D structural importance of non-coding loci that are not associated with any functional element, providing a new mechanistic understanding of the disease-associated genetic variations (GVs). Furthermore, our analyses also suggested a powerful approach to develop "one-drug-multiple-targets" therapeutics targeting the disease-specific non-coding regions.

genetics↗

bioRxiv2020

Regulatory elements can be essential for maintaining broad chromatin organization and cell viability

Increasing evidence shows that promoters and enhancers could be related to 3D chromatin structure, thus affecting cellular functions. Except for functioning through the canonical chromatin loops formed by promoters and enhancers, their roles in maintaining broad chromatin organization have not been well studied. Here, we focused on the active promoters/enhancers (referred to as hotspots) predicted to form many 3D contacts with other active promoters/enhancers, and identified dozens of loci critical for cell survival. While the essentiality of hotspots is not resulted from their association with essential genes, deletion of an essential hotspot could lead to change of broad chromatin organization and expressions of distal genes. We demonstrated that multiple affected genes that are individually non-essential could have synergistic effects to cause cell death.

bioinformatics↗

bioRxiv2020

Controlling Cellular Arrangements via Stretched Bioprinting

Bioprinting is a common method to replicate geometrical architecture of native tissues. However, it usually fails to modulate cellular arrangements, which is critical for the tissues functionality. To our knowledge, no method has successfully addressed this challenge. Here, we report a method of controlling cellular orientation during the bioprinting process by integrating a stretch process into a modified bioprinting frame. We demonstrate that the cellular orientation is a result of cells sensing and responding to the tensile stress, instead of shear stress or topographical patterns. Moreover, our method shows a potent capability to induce myoblast differentiation, fusion and maturation without the presence of differentiation medium. As a potential clinical application, we demonstrate that aligned myofibers directly printed onto injured muscle in vivo, can not only repair the structure of damaged tissue, but also recover the muscle functionalities effectively. This study shows that the new method can produce tissues with precise control of cellular arrangements and more clinically viable functionalities. Significance StatementDue to no method could reproduce the exact cellular arrangements of native tissues in engineered tissues, tissue engineering facing difficult in fabricating 3D tissues that possess desirable biological and mechanical functionalities for biomedical applications. For the first time, we report a method of controlling cellular orientation during 3D bio-printing process. This method can be used to produce engineered tissues with controlled cellular arrangement with several different cell types. Moreover, this method shows a potent capability of fabricating fully mature and aligned myofibers in vitro in the absence of differentiation medium. As potential clinical applications, with this method, engineered tissues could be directly printed in vivo with high efficacy of tissue repair and function recovery.

cell biology↗

bioRxiv2020

A meta-analysis and experiment assessing phage-based FMDV vaccine

Foot-and-mouth disease (FMD) is a pathological disease caused by the foot- and-mouth disease virus (FMDV), which mainly affects cloven-hoofed animals. This study was conducted to a meta-analysis and experiment on the effect of bacteriophages used in the development of FMDV vaccines. A systematic search was conducted for the collection of the protection effect for the phage-based FMDV vaccine using sensitive search strategies. The extracted data were analyzed using Rev-Man 5.4 software. This experiment used the T7 phage to express the capsid protein VP1 of the OHM-02 strain, and the recombinant VP1 phage was termed OHM-T7. Antibodies and cytokines levels were assessed after immunizing BALB/C mice with OHM-T7. The results showed that a total of 115 articles were retrieved, and 4 of them met the inclusion criteria. There was no heterogeneity with I2 = 0%, 20% or 43%. We used a fixed-effect model for meta-analysis, and the results showed a protective effect on FMDV between the phage group and control group (P<0.01) and between FMDV group and control group (P<0.01). Furthermore, when the phage group was compared to the FMDV group, there was also no significant difference (P>0.05). After successfully obtained the ohm-t7 strain and immunized the mice, it could induce high levels of IFN-{gamma} levels in mice with little effect on IL-4 levels. OHM-T7 could be used to detect antibodies produced by mice immunized with different FMDV antigens and produce high levels of anti-FMD antibodies. In summary, these results showed the potential of phage-based FMDV vaccines in FMDV prevention.

immunology↗

bioRxiv2020

Repopulating Kupffer Cells Originate Directly from Hematopoietic Stem Cells

Kupffer cells (KCs) originate from yolk sac progenitors before birth. Throughout adulthood, they self-maintain independently from the input of circulating monocytes (MOs) at stead state, and are replenished within 2 weeks after having been depleted, but the origin of repopulating KCs in adult remains unclear. The current paradigm dictates that repopulating KCs originate from preexisting KCs or monocytes, but there remains a lack of fate-mapping evidence. In current study, we firstly traced the fate of preexisting KCs and that of monocytic cells with tissue-resident macrophage-specific and monocytic cell-specific fate mapping mouse models, respectively, and found no evidences that repopulating KCs originate from preexisting KCs or MOs. Secondly, we performed genetic lineage tracing to determine the type of progenitor cells involved in response to KC depletion in mice, and found that in response to KC depletion, hematopoietic stem cells (HSCs) proliferated in the bone marrow, mobilized into the blood, adoptively transferred into the liver and differentiated into KCs. Finally, we traced the fate of HSCs in a HSC-specific fate-mapping mouse model, in context of chronic liver inflammation induced by repeated carbon tetrachloride treatment, and confirmed that repopulating KCs originated directly from HSCs. Taken together, these findings provided strong in vivo fate-mapping evidences that repopulating KCs originate directly from Hematopoietic stem cells not from preexisting KCs or from MOs. SignificanceThere is a standing controversy in the field regarding the cellular origin of repopulating macrophages. This paper provides strong in vivo fate-mapping evidences that repopulating KCs originate directly from hematopoietic stem cells not from preexisting KCs or from MOs, which presenting a completely novel understanding of the cellular origin of repopulating Kupffer Cells and shedding light on the divergent roles of KCs in liver homeostasis and diseases.

cell biology↗

bioRxiv2020

A Systematic Review and Meta-analysis of the Protective Effect of FMDV VP1 on Animals

The FMDV VP1 protein has different structures which could decrease or increase the immune response. We undertook a meta-analysis to evaluate the protective effect of VP1 on the FMDV. A systematic search of the PubMed, Embase, CNKI and Wan fang DATA was conducted up to April 2020. Experimental studies involving the VP1 protection effect on FMDV were included. Extracted data were analyzed using Rev-Man 5.3 software. Chi-square tests were used to analyze the heterogeneity among the documents. The fixed-effect model was used for meta-analysis to find the combined effect value and 95% confidence interval. Sensitivity analysis was performed on the differences in the combined values of model effects, and the inverted funnel chart method was used to assess the publication bias of the included literature. A total of 12 articles were included for meta-analysis. The results of showed that VP1 had a protective effect on FMDV [MH = -0.66, 95% CI = (-0.75, -0.56), P < 0.00001]. Sensitivity analysis showed that the results were robust. The funnel graph method showed that the published literature had a small publication bias and met the requirements of this study. It is necessary to study the epitopes of VP1 to produce new vaccines. VP1 could protect animals from FMDV attacks. It is necessary to study the VP1 protein and its epitopes and use it as a new vaccine and diagnostic product.

immunology↗

bioRxiv2020

Modulate Siglec-7 Signaling via in situ Created High-affinity cis-Ligands

Sialic acid-binding immunoglobulin-like lectins, also known as Siglecs, have recently been designated as glyco-immune checkpoints. Through their interactions with sialylated glycan epitopes overexpressed on tumor cells, inhibitory Siglecs on innate and adaptive immune cells modulate signaling cascades to restrain anti-tumor immune responses. However, the mechanisms underlying these processes are just starting to be elucidated. We discover that when human natural killer (NK) cells attack tumor cells, glycan remodeling occurs on the target cells at the immunological synapse. This remodeling occurs through both transfer of sialylated glycans from NK cells to target tumor cells and accelerated de novo synthesis of sialosides on the tumor cell themselves. The functionalization of NK cells with a high-affinity ligand of Siglec-7 leads to multifaceted consequences in modulating Siglec-7-regulated NK-activation. At high levels, the added Siglec-7 ligand suppresses NK-cytotoxicity through the recruitment of Siglec-7, whereas at low levels the same ligand triggers the release of Siglec-7 from the cell surface into the culture medium, preventing Siglec-7-mediated inhibition of NK cytotoxicity. These results suggest that glycan engineering of NK cells may provide a means to boost NK effector functions.

biochemistry↗

bioRxiv2020

An emerging role of Prevotella histicola on estrogen-deficiency induced bone loss through the gut microbiota-bone axis

Gut microbiota (GM)-bone axis has emerged as a crucial mediator in the maintenance of bone homeostasis. Estrogen-deficiency induced bone loss is closely associated with an altered GM. However, the underlying mechanisms remain not fully understood. To this end, feces samples collected from the postmenopausal patients with osteoporosis (PMO) and with normal bone mass (PMN) were examined by 16s rRNA gene sequencing. We found that GM in PMO group was featured by a significant decreased proportion of genus Prevotella in comparison with that in the PMN group. Next, Prevotella histicola (P.histicola), a typical specie of Prevotella, was orally given to the mice following the ovariectomy (OVX) procedure and it significantly prevented OVX induced bone loss. Mechanistically, the protective effects of P.histicola on bone mass were found to be associated with the inhibition of osteoclastic resorption by attenuating osteoclastogenic cytokines expression and ameliorating gut permeability. Thus, P.histicola may prevent estrogen-deficiency induced bone loss through the GM-bone axis.

microbiology↗

bioRxiv2020

Genetic dissection of glutamatergic neuron subpopulations and developmental trajectories in the cerebral cortex

Diverse types of glutamatergic pyramidal neurons (PyNs) mediate the myriad processing streams and output channels of the cerebral cortex, yet all derive from neural progenitors of the embryonic dorsal telencephalon. Here, we establish genetic strategies and tools for dissecting and fate mapping PyN subpopulations based on their developmental and molecular programs. We leverage key transcription factors and effector genes to systematically target the temporal patterning programs in progenitors and differentiation programs in postmitotic neurons. We generated over a dozen temporally inducible mouse Cre and Flp knock-in driver lines to enable combinatorial targeting of major progenitor types and projection classes. Intersectional converter lines confer viral access to specific subsets defined by developmental origin, marker expression, anatomical location and projection targets. These strategies establish an experimental framework for understanding the hierarchical organization and developmental trajectory of PyN subpopulations that assemble cortical processing networks and output channels.

neuroscience↗

bioRxiv2020

Blocking gephyrin phosphorylation or microglia BDNF signaling prevents synapse loss and reduces infarct volume after ischemia

Microglia interact with neurons to facilitate synapse plasticity; however, signal transducers between microglia and neuron remain unknown. Here, using in vitro organotypic hippocampal slice cultures and transient MCAO in genetically-engineered mice in vivo, we report that at 24 h post-ischemia microglia release BDNF to downregulate glutamatergic and GABAergic synapses within the peri-infarct area. Analysis of the CA1 hippocampal formation in vitro shows that proBDNF and mBDNF downregulate glutamatergic dendritic spines and gephyrin scaffold stability through p75NTR and TrkB receptors respectively. Post-MCAO, we report that in the peri- infarct area and in the corresponding contralateral hemisphere similar neuroplasticity occur through microglia activation and gephyrin phosphorylation at Ser268, Ser270 in vivo. Targeted deletion of the Bdnf gene in microglia or GphnS268A/S270A (phospho-null) point-mutations protect against ischemic brain damage, neuroinflamation and synapse downregulation normally seen post-MCAO. Collectively, we report that gephyrin phosphorylation and microglia derived BDNF faciliate synapse plasticity after transient ischemia.

neuroscience↗

bioRxiv2020

Chloroquine Kills Hair Cells in Zebrafish Lateral Line and Murine Cochlear Cultures: Implications for Ototoxicity

Hearing and balance deficits have been reported during and following treatment with the antimalarial drug chloroquine. However, experimental work examining the direct actions of chloroquine on mechanoreceptive hair cells in common experimental models is lacking. This study examines the effects of chloroquine on hair cells using two common experimental models: the zebrafish lateral line and neonatal mouse cochlear cultures. Zebrafish larvae were exposed to varying concentrations of chloroquine phosphate or hydroxychloroquine for 1 hr or 24 hr, and hair cells assessed by antibody staining. A significant, dose-dependent reduction in the number of surviving hair cells was seen across conditions for both exposure periods. Hydroxycholroquine showed similar toxicity. In mouse cochlear cultures, chloroquine damage was specific to outer hair cells in tissue from the cochlear basal turn, consistent with susceptibility to other ototoxic agents. These findings suggest a need for future studies employing hearing and balance monitoring during exposure to chloroquine and related compounds, particularly with interest in these compounds as therapeutics against viral infections including coronavirus.

neuroscience↗

bioRxiv2020

Virus-host interactome and proteomic survey of PMBCs from COVID-19 patients reveal potential virulence factors influencing SARS-CoV-2 pathogenesis

The ongoing coronavirus disease (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is a global public health concern due to relatively easy person-to-person transmission and the current lack of effective antiviral therapy. However, the exact molecular mechanisms of SARS-CoV-2 pathogenesis remain largely unknown. We exploited an integrated proteomics approach to systematically investigate intra-viral and virus-host interactomes for the identification of unrealized SARS-CoV-2 host targets and participation of cellular proteins in the response to viral infection using peripheral blood mononuclear cells (PBMCs) isolated from COVID-19 patients. Using this approach, we elucidated 251 host proteins targeted by SARS-CoV-2 and more than 200 host proteins that are significantly perturbed in COVID-19 derived PBMCs. From the interactome, we further identified that non-structural protein nsp9 and nsp10 interact with NKRF, a NF-[Kcy]B repressor, and may precipitate the strong IL-8/IL-6 mediated chemotaxis of neutrophils and overexuberant host inflammatory response observed in COVID-19 patients. Our integrative study not only presents a systematic examination of SARS-CoV-2-induced perturbation of host targets and cellular networks to reflect disease etiology, but also reveals insights into the mechanisms by which SARS-CoV-2 triggers cytokine storms and represents a powerful resource in the quest for therapeutic intervention.

microbiology↗

bioRxiv2020

Glycoengineering of NK cells with glycan ligands of CD22 and selectins for B-cell lymphoma therapy

CD22, a member of Siglec family of sialic acid binding proteins, has restricted expression on B cells. Antibody-based agents targeting CD22 or CD20 (Rituxan) on B lymphoma and leukemia cells exhibit clinical efficacy for treating these malignancies, but also attack normal B cells leading to immune deficiency. Here, we report a chemoenzymatic glycocalyx editing strategy to introduce high-affinity and specific CD22 ligands onto NK-92MI and cytokine-induced killer (CIK) cells to achieve tumor-specific CD22 targeting. These CD22-ligand modified cells exhibited significantly enhanced tumor cell binding and killing in vitro without harming healthy B cells. For effective lymphoma cell killing in vivo we further functionalized CD22 ligand-modified NK-92MI cells with the E-selectin ligand sialyl Lewis X to promote trafficking to bone marrow. The cells containing the ligands of both CD22 and selectins resulted in the efficient suppression of B lymphoma in a xenograft model. Our results suggest that NK cells modified with glycan ligands to CD22 and selectins promote both targeted killing of B lymphoma cells and improved trafficking to sites where the cancer cells reside, respectively.

bioengineering↗

bioRxiv2020

Detecting Tumor Specific Antigen-Reactive T cells from Tumor Infiltrating Lymphocytes via Interaction Dependent Fucosyl-biotinylation

Re-activation and clonal expansion of tumor specific antigen (TSA)-reactive T cells are critical to the success of checkpoint blockade and adoptive transfer of tumor-infiltrating lymphocyte (TIL) based therapies. There are no reliable markers to specifically identify the repertoire of TSA-reactive T cells due to their heterogeneous composition. Here we introduce FucoID as a general platform to detect endogenous antigen-specific T cells and study their biology. Through this interaction dependent labeling approach, TSA-reactive T cells can be detected and separated from bystander T cells in primary tumor digests based on their cell-surface enzymatic fucosyl-biotinylation. Compared to bystander TILs, TSA-reactive TILs possess a distinct TCR repertoire and unique gene features. Though exhibiting a dysfunctional phenotype, this subset of TILs possesses substantial capabilities of proliferation and tumor specific killing. FucoID features genetic manipulation-free procedures and a quick turnover cycle, and therefore should have the potential of accelerating the pace of personalized cancer treatment. HighlightsInteraction dependent fucosylation enables the detection and isolation of bona fide intratumoral tumor specific antigen-reactive T cells Tumor specific antigen-reactive CD8+ T cells possess capabilities to be expanded and adoptively transferred for tumor control Tumor specific antigen-reactive CD8+ T cells feature oligoclonal expansion and upregulate genes for the steroid biosynthesis and metabolic process Intratumoral bystander CD8+ T cells can be separated into two groups based on PD-1 expression that feature distinct gene modules

immunology↗