Biology subjects

Wu, J.

Publications and source records attributed to Wu, J..

At least 667 records · Page 37Linked to original sources

bioRxiv2021

Cryo-EM structures of human TMEM120A and TMEM120B

TMEM120A (Transmembrane protein 120A) was recently identified as a mechanical pain sensing ion channel named as TACAN, while its homologue TMEM120B has no mechanosensing property1. Here, we report the cryo-EM structures of both human TMEM120A and TMEM120B. The two structures share the same dimeric assembly, mediated by extensive interactions through the transmembrane domain (TMD) and the N-terminal coiled coil domain (CCD). However, the nearly identical structures cannot provide clues for the difference in mechanosensing between TMEM120A and TMEM120B. Although TMEM120A could mediate conducting currents in a bilayer system, it does not mediate mechanical-induced currents in a heterologous expression system, suggesting TMEM120A is unlikely a mechanosensing channel. Instead, the TMDs of TMEM120A and TMEM120B resemble the structure of a fatty acid elongase, ELOVL7, indicating their potential role of an enzyme in lipid metabolism.

biophysics↗

bioRxiv2021

Spatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis

Multiplexed mRNA profiling in the spatial context provides important new information enabling basic research and clinical applications. Unfortunately, most existing spatial transcriptomics methods are limited due to either low multiplexing or assay complexity. Here, we introduce a new spatialomics technology, termed Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA), that integrates in situ labeling of mRNA and protein markers in cells or tissues with combinatorial fluorescence spectral and lifetime encoded probes, spectral and time-resolved fluorescence imaging, and machine learning-based target decoding. This technology is the first application combining the biophotonic techniques, Spectral and Fluorescence Lifetime Imaging and Microscopy (FLIM), to the field of spatial transcriptomics. By integrating the time dimension with conventional spectrum-based measurements, MOSAICA enables direct and highly-multiplexed in situ spatial biomarker profiling in a single round of staining and imaging while providing error correction removal of background autofluorescence. We demonstrate mRNA encoding using combinatorial spectral and lifetime labeling and target decoding and quantification using a phasor-based image segmentation and machine learning clustering technique. We then showcase MOSAICAs multiplexing scalability in detecting 10-plex targets in fixed colorectal cancer cells using combinatorial labeling of only five fluorophores with facile error-correction and removal of autofluorescent moieties. MOSAICAs analysis is strongly correlated with sequencing data (Pearsons r = 0.9) and was further benchmarked using RNAscopeand LGC Stellaris. We further apply MOSAICA for multiplexed analysis of clinical melanoma Formalin-Fixed Paraffin-Embedded (FFPE) tissues that have a high degree of tissue scattering and intrinsic autofluorescence, demonstrating the robustness of the approach. We then demonstrate simultaneous co-detection of protein and mRNA in colorectal cancer cells. MOSAICA represents a simple, versatile, and scalable tool for targeted spatial transcriptomics analysis that can find broad utility in constructing human cell atlases, elucidating biological and disease processes in the spatial context, and serving as companion diagnostics for stratified patient care.

bioengineering↗

bioRxiv2021

Single-molecule, full-length transcript isoform sequencing reveals disease mutation-associated RNA isoforms in cardiomyocytes

Alternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we established an experimental and computational pipeline that performs de novo transcript annotation and accurately quantifies transcript isoforms from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generated a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms (http://steinmetzlab.embl.de/iBrowser/). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 (RBM20) mutations associated with aggressive dilated cardiomyopathy (DCM), we identified 121 differentially expressed transcript isoforms in 107 cardiac genes. Our approach enables quantitative dissection of complex transcript architecture instead of mere identification of inclusion or exclusion of individual exons, as exemplified by novel IMMT isoforms. Thereby we achieve a path to direct differential expression testing independent of an existing annotation of transcript isoforms as the functional unit, instead of genes or exons, providing more immediate biological interpretation and higher resolution transcriptome comparisons.

genetics↗

bioRxiv2021

SARS-CoV-2 spike P681R mutation enhances and accelerates viral fusion

During the current SARS-CoV-2 pandemic, a variety of mutations have been accumulated in the viral genome, and currently, four variants of concerns (VOCs) are considered as the hazardous SARS-CoV-2 variants to the human society1. The newly emerging VOC, the B.1.617.2/Delta variant, closely associates with a huge COVID-19 surge in India in Spring 20212. However, its virological property remains unclear. Here, we show that the B.1.617.2/Delta variant is highly fusogenic, and notably, more pathogenic than prototypic SARS-CoV-2 in infected hamsters. The P681R mutation in the spike protein, which is highly conserved in this lineage, facilitates the spike protein cleavage and enhances viral fusogenicity. Moreover, we demonstrate that the P681R-bearing virus exhibits higher pathogenicity than the parental virus. Our data suggest that the P681R mutation is a hallmark that characterizes the virological phenotype of the B.1.617.2/Delta variant and is closely associated with enhanced pathogenicity.

microbiology↗

bioRxiv2021

Combining Multi-Dimensional Molecular Fingerprints to Predict hERG Cardiotoxicity of Compounds

At present, drug toxicity has become a critical problem with heavy medical and economic burdens. acLQTS (acquired Long QT Syndrome) is acquired cardiac ion channel disease caused by drugs blocking the hERG channel. Therefore, it is necessary to avoid cardiotoxicity in the drug design and computer models have been widely used to fix this plight. In this study, we present a molecular fingerprint based on the molecular dynamic simulation and uses it combined with other molecular fingerprints (multi-dimensional molecular fingerprints) to predict hERG cardiotoxicity of compounds. 203 compounds with hERG inhibitory activity (pIC50) were retrieved from a previous study and predicting models were established using four machine learning algorithms based on the single and multi-dimensional molecular fingerprints. Results showed that MDFP has the potential to be an alternative to traditional molecular fingerprints and the combination of MDFP and traditional molecular fingerprints can achieve higher prediction accuracy. Meanwhile, the accuracy of the best model, which was generated by consensus of four algorithms with multi-dimensional molecular fingerprints, was 0.694 (RMSE) in the test dataset. Besides, the number of hydrogen bonds from MDFP has been determined as a critical factor in the predicting models, followed by rgyr and sasa. Our findings provide a new sight of MDFP and multi-dimensional molecular fingerprints in building models of hERG cardiotoxicity prediction.

pharmacology and toxicology↗

bioRxiv2021

SE(3)-Equivariant Energy-based Models for End-to-End Protein Folding

Accurate prediction of protein structures is critical for understanding the biological function of proteins. Nevertheless, most structure optimization methods are built upon pre-defined statistical energy functions, which may be sub-optimal in formulating the conformation space. In this paper, we propose an end-to-end approach for protein structure optimization, powered by SE(3)-equivariant energy-based models. The conformation space is characterized by a SE(3)-equivariant graph neural network, with substantial modifications to embed the protein-specific domain knowledge. Furthermore, we introduce continuously-annealed Langevin dynamics as a novel sampling algorithm, and demonstrate that such process converges to native protein structures with theoretical guarantees. Extensive experiments indicate that SE(3)-Fold achieves comparable structure optimization accuracy, compared against state-of-the-art baselines, with over 1-2 orders of magnitude speed-up.

bioinformatics↗

bioRxiv2021

Strain-Programmable Patch for Diabetic Wound Healing

Chronic wounds with impaired healing capability such as diabetic foot ulcers (DFU) are devastating complications in diabetic patients, inflicting rapidly growing clinical and economic burdens in aging societies. Despite recent advances in therapeutic approaches, limited benefits of the existing solutions highlight the critical need for novel therapeutic solutions for diabetic wound healing. Here we propose a strain-programmable patch capable of rapid robust adhesion on and programmable mechanical contraction of wet wounded tissues over days to offer a new therapeutic platform for diabetic wounds. The strain-programmable patch, consisting of a dried bioadhesive layer and a pre-stretched elastomer backing, implements a hydration-based shape-memory mechanism to achieve both uniaxial and biaxial contractions and stress remodeling of wet wounds in a programmable manner. We develop theoretical and numerical models to rationally guide the strain-programming and mechanical modulation of wounds. In vivo rodent and ex vivo human skin culture models validate the programmability and efficacy of the proposed platform and identify mechanisms of action for accelerated diabetic wound healing. One Sentence SummaryA strain-programmable bioadhesive patch is developed for accelerated closure and healing of wounds in diabetic mice and human skin.

bioengineering↗

bioRxiv2021

Immunological profiling of COVID-19 patients with pulmonary sequelae

Cellular immunity may be involved in organ damage and rehabilitation in patients with coronavirus disease 2019 (COVID-19). We aimed to delineate immunological features of COVID-19 patients with pulmonary sequelae (PS) one year after discharge. 50 COVID-19 survivors were recruited and classified according to radiological characteristics: 24 patients with PS and 26 patients without PS. Phenotypic and functional characteristics of immune cells were evaluated by multiparametric flow cytometry. Patients with PS had an increased proportion of natural killer (NK) cells and lower percentage of B cells compared to patients without PS. Phenotypic and functional features of T cells in patients with PS were predominated by the accumulation of CD4+ T cells secreting IL-17A, short-lived effector-like CD8+ T cells (CD27-CD62L-) and senescent T cells with excessive secretion of granzyme-B/perforin/IFN-{gamma}. NK cells were characterized by the excessive secretion of granzyme-B and perforin and the downregulation of NKP30 and NKP46; highly activated NKT and {gamma}{delta} T cells exhibited NKP30 and TIM-3 upregulation and NKB1 downregulation in patients with PS. However, immunosuppressive cells were comparable between the two groups. The interrelation of immune cells in COVID-19 was intrinsically identified, whereby T cells secreting IL-2, IL-4 and IL-17A were enriched among CD28+ and CD57-cells and cells secreting perforin/granzyme-B/IFN-{gamma}/TNF- expressed markers of terminal differentiation. CD57+NK cells, CD4+perforin+ T cells and CD8+CD27+CD62L+ T cells were identified as the independent predictors for residual lesions. Overall, our findings unveil the profound imbalance of immune landscape that may correlate with organ damage and rehabilitation in COVID-19. IMPORTANCEA considerable proportion of COVID-19 survivors have residual lung lesions, such as ground glass opacity and fiber streak shadow. To determine the relationship between host immunity and residual lung lesions, we performed an extensive analysis of immune responses in convalescent patients with COVID-19 one year after discharge. We found significant differences in immunological characteristics between patients with pulmonary sequelae and patients without pulmonary sequelae one year after discharge. Our study highlights the profound imbalance of immune landscape in the COVID-19 patients with pulmonary sequelae, characterized by the robust activation of cytotoxic T cells, NK cells and {gamma}{delta} T cells as well as the deficiencies of immunosuppressive cells. Importantly, CD57+NK cells, CD4+perforin+ T cells and CD8+CD27+CD62L+ T cells were identified as the independent predictors for residual lesions.

immunology↗

bioRxiv2021

LRRC23 is a conserved component of the radial spoke that is necessary for sperm motility and male fertility in mice

Cilia and flagella are ancient structures that achieve controlled motor functions through the coordinated interaction of structures including dynein arms, radial spokes (RSs), microtubules, and the dynein regulatory complex (DRC). RSs facilitate the beating motion of these organelles by mediating signal transduction between dyneins and a central pair (CP) of singlet microtubules. RS complex isolation from Chlamydomonas axonemes enabled the detection of 23 different proteins (RSP1-23), with the roles of RSP13, RSP15, RSP18, RSP19, and RSP21 remained poorly understood. Herein, we show that Lrrc23 is an evolutionarily conserved testis-enriched gene encoding an RSP15 homolog in mice. Through immunoelectron microscopy, we demonstrate that LRRC23 localizes to the RS complex within murine sperm flagella. We further found that LRRC23 was able to interact with RSHP9 and RSPH3A/B. The knockout of Lrrc23 resulted in RS disorganization and impaired motility in murine spermatozoa, whereas the ciliary beating was unaffected by the loss of this protein. Spermatozoa lacking LRRC23 were unable to efficiently pass through the uterotubal junction and exhibited defective zona penetration. Together these data indicate that LRRC23 is a key regulator underpinning the integrity of RS complex within the flagella of mammalian spermatozoa, whereas it is dispensable in cilia. Author summary

cell biology↗

bioRxiv2021

Oligodendrocyte precursor cells prune axons in the mouse neocortex

Neurons in the developing brain undergo extensive structural refinement as nascent circuits adopt their mature form1. This transformation is facilitated by the engulfment and degradation of excess axonal branches and inappropriate synapses by surrounding glial cells, including microglia and astrocytes2,3. However, the small size of phagocytic organelles and the complex, highly ramified morphology of glia has made it difficult to determine the contribution of these and other glial cell types to this process. Here, we used large scale, serial electron microscopy (ssEM) with computational volume segmentation to reconstruct the complete 3D morphologies of distinct glial types in the mouse visual cortex. Unexpectedly, we discovered that the fine processes of oligodendrocyte precursor cells (OPCs), a population of abundant, highly dynamic glial progenitors4, frequently surrounded terminal axon branches and included numerous phagolysosomes (PLs) containing fragments of axons and presynaptic terminals. Single- nucleus RNA sequencing indicated that cortical OPCs express key phagocytic genes, as well as neuronal transcripts, consistent with active axonal engulfment. PLs were ten times more abundant in OPCs than in microglia in P36 mice, and declined with age and lineage progression, suggesting that OPCs contribute very substantially to refinement of neuronal circuits during later phases of cortical development.

neuroscience↗

bioRxiv2021

Comprehensive Analysis of Spatial Architecture in Primary Liver Cancer

Heterogeneity is the major challenge for cancer prevention and therapy. Here, we firstly constructed high-resolution spatial transcriptomes of primary liver cancers (PLCs) containing 84,823 spots within 21 tissues from 7 patients. The sequential comparison of spatial tumor microenvironment (TME) characteristics from non-tumor to leading-edge to tumor regions revealed that the tumor capsule potentially affects intratumor spatial cluster continuity, transcriptome diversity and immune cell infiltration. Locally, we found that the bidirectional ligand-receptor interactions at the 100 m wide cluster-cluster boundary contribute to maintaining intratumor architecture. Our study provides novel insights for diverse tumor ecosystem of PLCs and has potential benefits for cancer intervention.

cancer biology↗

bioRxiv2021

Broad neutralization against SARS-CoV-2 variants induced by a modified B.1.351 protein-based COVID-19 vaccine candidate

Beginning in late 2020, the emergence and spread of multiple variant SARS-CoV-2 strains harboring mutations which may enable immune escape necessitates the rapid evaluation of second generation COVID-19 vaccines, with the goal of inducing optimized immune responses that are broadly protective. Here we demonstrate in a mouse immunogenicity study that two doses of a modified B.1.351 spike (S)-Trimer vaccine (B.1.351 S-Trimer) candidate can induce strong humoral immune responses that can broadly neutralize both the original SARS-CoV-2 strain (Wuhan-Hu-1) and Variants of Concern (VOCs), including the UK variant (B.1.1.7), South African variant (B.1.351) and Brazil variant (P.1). Furthermore, while immunization with two doses (prime-boost) of Prototype S-Trimer vaccine (based on the original SARS-CoV-2 strain) induced lower levels of cross-reactive neutralization against the B.1.351 variant, a third dose (booster) administered with either Prototype S-Trimer or B.1.351 S-Trimer was able to increase neutralizing antibody titers against B.1.351 to levels comparable to neutralizing antibody titers against the original strain elicited by two doses of Prototype S-Trimer.

immunology↗

bioRxiv2021

CD147 antibody specifically and effectively inhibits infection and cytokine storm of SARS-CoV-2 variants

SARS-CoV-2 and its variants are raging worldwide. Unfortunately, the global vaccination is not efficient enough to attain a vaccine-based herd-immunity and yet no special and effective drug is developed to contain the spread of the disease. Previously we have identified CD147 as a novel receptor for SARS-CoV-2 infection. Here, we demonstrated that CD147 antibody effectively inhibits infection and cytokine storm caused by SARS-CoV-2 variants. In CD147KO VeroE6 cells, infections of SARS-CoV-2, its variants (B.1.1.7, B.1.351) and pseudovirus mutants (B.1.1.7, B.1.351, B.1.525, B.1.526 (S477N), B.1.526 (E484K), P.1, P.2, B.1.617.1, B.1.617.2) were decreased. Meanwhile, CD147 antibody effectively blocked the entry of variants and pseudomutants in VeroE6 cells, and inhibited the expression of cytokines. A model of SARS-CoV-2-infected hCD147 transgenic mice was constructed, which recapitulated the features of exudative diffuse alveolar damage and dynamic immune responses of COVID-19. CD147 antibody could effectively clear the virus and alveolar exudation, resolving the pneumonia. We found the elevated level of cyclophilin A (CyPA) in plasma of severe/critical cases, and identified CyPA as the most important proinflammatory intermediate causing cytokine storm. Mechanistically, spike protein of SARS-CoV-2 bound to CD147 and initiated the JAK-STAT pathway, which induced expression of CyPA. CyPA reciprocally bound to CD147, triggered MAPK pathway and consequently mediated the expression of cytokine and chemokine. In conclusion, CD147 is a critical target for SARS-CoV-2 variants and CD147 antibody is a promising drug to control the new wave of COVID-19 epidemic.

immunology↗

bioRxiv2021

Long-term stress and trait anxiety alter brain network balance in dynamic decisions during working memory

Long-term stress has a profound impact on the human brain and cognition, and trait anxiety influences stress-induced adaptive and maladaptive effects. However, the neurocognitive mechanisms underlying long-term stress and trait anxiety interactions remain elusive. Here we investigated how long-term stress and trait anxiety interact to affect dynamic decisions during working-memory (WM) by altering functional brain network balance. In comparison to controls, male participants under long-term stress experienced higher psychological distress and exhibited faster evidence accumulation but had a lower decision-threshold during WM. This corresponded with hyper-activation in the anterior insula, less WM-related deactivation in the default-mode network, and stronger default-mode network decoupling with the frontoparietal network. Critically, high trait anxiety under long-term stress led to slower evidence accumulation through higher WM-related frontoparietal activity, and increased decoupling between the default-mode and frontoparietal networks. Our findings provide neurocognitive evidence for long-term stress and trait anxiety interactions on executive functions with (mal)adaptive changes.

neuroscience↗

bioRxiv2021

The Impact of a Dynamic Working Environment on Human Gut Microbiota

Gut microbiota dysbiosis is associated with a variety of diseases, such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), metabolic diseases, allergic diseases, neurodevelopmental disorders and cancer. The human gut microbiota can be influenced by a variety of factors, including geography, dietary habits, living environment, age and altered lifestyle etc. This study was conducted to explore the gut microbiota compositions in officials who are in a stable working environment and train drivers who are in a dynamic working environment. Microbiota communities in the feces of 80 officials and 88 train drivers were analyzed using Illumina MiSeq sequencing targeting the V3-V4 region of 16S ribosomal RNA (rRNA) gene and ITS1 region of fungi. There were significant differences between the two groups in diversity and richness of gut microbiota, while the microbial community compositions of the two groups were similar. The relationship between gut microbiota and clinical characteristics was investigated. We found that more bacteria and fungi were positively correlated with clinical characteristics. Functional prediction analysis of the gut microbiota between the two groups by PICRUSt2 revealed significant differences between the official group and the train driver group. Elucidating these differences of the microbiome between the two groups will provide a foundation understanding of the impact of a dynamic environment on gut microbiota.

microbiology↗

bioRxiv2021

No observable guide-RNA-independent off-target mutation induced by prime editor

Prime editor (PE) has been recently developed to induce efficient and precise ontarget editing, whereas its guide RNA (gRNA)-independent off-target effects remain unknown. Here, we used whole-genome and whole-transcriptome sequencing to determine gRNA-independent off-target mutations in cells expanded from single colonies, in which PE generated precise editing at on-target sites. We found that PE triggered no observable gRNA-independent off-target mutation genome-wide or transcriptome-wide in transfected human cells, highlighting its high specificity.

biochemistry↗

bioRxiv2021

Development of a mini-replicon-based reverse-genetics system for rice stripe tenuivirus

Negative-stranded RNA (NSR) viruses include both animal- and plant-infecting viruses that often cause serious diseases in human and livestock, and in agronomic crops. Rice stripe tenuivirus (RSV), a plant NSR virus with four negative-stranded/ambisense RNA segments, is one of the most destructive rice pathogens in many Asian countries. Due to the lack of a reliable reverse-genetics technology, molecular studies of RSV gene functions and its interaction with host plants are severely hampered. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV gene functional analysis in Nicotiana benthamiana. We first developed a mini-replicon system expressing RSV genomic RNA3 eGFP reporter (MR3(-)eGFP), a nucleocapsid (NP), and a codon usage optimized RNA-dependent RNA polymerase (RdRpopt), respectively. Using this mini-replicon system we determined that RSV NP and RdRpopt are indispensable for the eGFP expression from MR3(-)eGFP. The expression of eGFP from MR3(-)eGFP can be significantly enhanced in the presence of NSs and P19-HcPro-{gamma}b. In addition, NSvc4, the movement protein of RSV, facilitated eGFP trafficking between cells. We also developed an antigenomic RNA3-based replicon in N. benthamiana. However, we found that the RSV NS3 coding sequence acts as a cis-element to regulate viral RNA expression. Finally, we made mini-replicons representing all four RSV genomic RNAs. This is the first mini-replicon-based reverse-genetics system for monocot-infecting tenuivirus. We believe that this mini-replicon system described here will allow the studies of RSV replication, transcription, cell-to-cell movement and host machinery underpinning RSV infection in plants. IMPORTANCEPlant-infecting segmented negative-stranded RNA (NSR) viruses are grouped into 3 genera: Orthotospovirus, Tenuivirus and Emaravirus. The reverse-genetics systems have been established for members in the genera Orthotospovirus and Emaravirus, respectively. However, there is still no reverse-genetics system available for Tenuivirus. Rice stripe virus (RSV) is a monocot-infecting tenuivirus with four negative-stranded/ambisense RNA segments. It is one of the most destructive rice pathogens and causes significant damages to rice industry in Asian countries. Due to the lack of a reliable reverse-genetics system, molecular characterizations of RSV gene functions and the host machinery underpinning RSV infection in plants are extremely difficult. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV in Nicotiana benthamiana. This is the first mini-replicon-based reverse-genetics system for tenuivirus. We consider that this system will provide researchers a new working platform to elucidate the molecular mechanisms dictating segmented tenuivirus infections in plant.

pathology↗

bioRxiv2021

Twelve-month specific IgG response to SARS-CoV-2 receptor-binding domain among COVID-19 convalescent plasma donors in Wuhan

To investigate the duration of humoral immune response in convalescent coronavirus disease 2019 (COVID-19) patients, we conduct a 12-month longitudinal study through collecting a total of 1,782 plasma samples from 869 convalescent plasma donors in Wuhan, China and test specific antibody responses. The results show that positive rate of IgG antibody against receptor-binding domain of spike protein (RBD-IgG) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the COVID-19 convalescent plasma donors exceeded 70% for 12 months post diagnosis. The level of RBD-IgG decreases with time, with the titer stabilizing at 64.3% of the initial level by the 9th month. Moreover, male plasma donors produce more RBD-IgG than female, and age of the patients positively correlates with the RBD-IgG titer. A strong positive correlation between RBD-IgG and neutralizing antibody titers is also identified. These results facilitate our understanding of SARS-CoV-2-induced immune memory to promote vaccine and therapy development.

immunology↗