Biology subjects

Jin, T.

Publications and source records attributed to Jin, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Quantitative effects of co-culture on T cell motility and cancer-T cell interactions

One of the primary challenges in current cancer immunotherapy is the insufficient infiltration of cytotoxic T cells into solid tumors. Despite ongoing investigations, the mechanisms restricting T cell infiltration in immune-cold tumors remains elusive, hindered by the intricate tumor microenvironment. Here, we co-cultured mouse cancer cell lines with cancer-specific cytotoxic T cells to study the influence of cancer-T cell interactions on T cell motility, a crucial factor for effective tumor infiltration. By quantifying T cell motility patterns, we found that cancer-specific T cells exhibited extended contact time with cancer-cell clusters and higher directional persistence than non-specific T cells. Computational modelling suggested that T cells with stronger persistence could facilitate efficient searching for cancer clusters. Transcriptomic profiling revealed T cells recognizing cancer cells orchestrate accumulation on cancer cell clusters by activating adhesion proteins on both cancer cells and T cells, thereby fostering prolonged interaction on cancer cells. Furthermore, we observed that there were two distinct subpopulations of cancer cells after co-culturing with cancer-specific T cells: one expressing elevated levels of T-cell attractants and antigen-presentation molecules, while the other expressing immunosuppressive molecules and undergoing epithelial-to-mesenchymal transition. These dynamic insights into the complex interplay of cancer-T cell interactions and their impact on T cell motility hold implications for refining more efficacious cancer immunotherapy strategies.

systems biology↗

bioRxiv2023

Multimodal analysis of neuronal maturation in the developing primate prefrontal cortex

The prefrontal cortex (PFC) is critical for myriad high-cognitive functions and is associated with several neuropsychiatric disorders. Here, using Patch-seq and single-nucleus multiomic analyses, we identified genes and regulatory networks governing the maturation of distinct neuronal populations in the PFC of rhesus macaque. We discovered that specific electrophysiological properties exhibited distinct maturational kinetics and identified key genes underlying these properties. We unveiled that RAPGEF4 is important for the maturation of resting membrane potential and inward sodium current in both macaque and human. We demonstrated that knockdown of CHD8, a high-confidence autism risk gene, in human and macaque organotypic slices led to impaired maturation, via downregulation of key genes, including RAPGEF4. Restoring the expression of RAPGEF4 rescued the proper electrophysiological maturation of CHD8-deficient neurons. Our study revealed regulators of neuronal maturation during a critical period of PFC development in primates and implicated such regulators in molecular processes underlying autism.

neuroscience↗

bioRxiv2023

Hypoxia-induced circSTT3A maintains Breast Cancer Stem Cell formation by Stabilizing PGK1 protein

BackgroundHypoxia is a key feature of tumor microenvironment that can cause fundamental changes in cancer cells, and may also lead to the development of breast cancer stem cells (BCSCs) with self-renewal ability. However, the mechanism of hypoxia in inducing BCSCs is not fully understood. MethodsPerforming RNA sequence and bioinformatics analysis, a hypoxia specific circular RNA (circRNA), named circSTT3A, was identified in hypoxic breast cancer cells and tissues. The clinical significance of circSTT3A was investigated in breast cancer (BC) tissues and tissue array. The loss and acquisition of circSTT3A were carried out in vivo and in vitro to confirm its functional roles in BCSC stemness maintenance. HIF1 droved circSTT3A expression was evaluated by chromatin immunoprecipitation and dual luciferase reporter assays. RNA pull-down, RNA immunoprecipitation, silver staining detection, mass spectrographic analysis, co-immunoprecipitation assays and western blotting were exerted to determine circSTT3A directly binding with HSP70 and PGK1 proteins. CircSTT3A-mediated serine metabolism was determined by UHPLC-QTRAP-MS system and ELISA kit. BC mouse model was used to assess the effects of circSTT3A/Hsp70/PGK1 on tumorigenesis and chemotherapy resistance in vivo. ResultsA novel hypoxia specific circSTT3A was significantly upregulated in clinical breast cancer tissues, and was related to the clinical stage and poor prognosis of BC patients. The hypoxia inducible factor 1 alpha (HIF1)-regulated circSTT3A has remarkable effect on mammosphere formation in breast cancer cells. Our work revealed that circSTT3A directly interacting with nucleotide-binding domain of heat shock protein 70 (HSP70) increases the ability of HSP70 to recruit phosphoglycerate kinase 1 (PGK1) via its substrate-binding domain, which reduces the ubiquitination of PGK1 and increases the stability of PGK1. The enhanced PGK1 catalyzes 1,3-diphosphoglycerate (1, 3-BPG) into 3-phosphoglycerate (3-PG) leading to 3-PG accumulation and increase of serine synthesis, thus to facilitate BCSC enrichment under hypoxic microenvironment. Loss of circSTT3A or PGK1 substantially induces suppression in tumor initiation and tumor growth, which dramatically increases tumor sensitivity to Doxorubicin treatment in mice. ConclusionsHypoxia induced circSTT3A/HSP70/PGK1 axis plays a critical role in maintaining BCSC properties and may be meaningful for treating patients with breast cancer.

cancer biology↗

bioRxiv2022

Genetic and histological correlation between the musk gland and skin of Chinese forest musk deer

Chinese forest musk deer (FMD, Moschus berezovskii) glands have the ability to secrete musk, which plays an important role in attracting females during the breeding season. Sebaceous glands (SGs) are exocrine skin glands associated with hair follicles that continuously release a mixture of sebum, lipids and cellular debris, by holocrine secretion. Both the musk glands and the skin tissues of the FMD contain abundant sebaceous glands, and Sox9, Caveolin1, and Androgen receptor (AR) are all involved in the regulation of sebum secretion by the sebaceous glands. However, there are fewer studies on the correlation between skin and musk glands and the expression of Sox9, Caveolin1, and AR in the musk glands and skin tissue of FMD. To address this gap, we analyzed biochemical data from FMD skin tissues and musk glands using transcriptomic data, hematoxylin-eosin (HE) staining, western blotting (WB), immunohistochemistry (IHC), tissue dissection, and RT-qPCR. Anatomical results show that only adult male FMD had complete glandular part and sachets, while 4-month-old FMD do not have well-developed sachets. Transcriptomic data showed that 88.24% of genes were co-expressed in the skin and musk glands tissues of FMD. The WB, IHC, and RT-qPCR results showed that the genes involved in regulating sebum secretion, Sox9, Caveolin1, and AR were expressed in the skin tissues and musk glands. In summary, skin tissues and musk glands tissue have a strong correlation, and Sox9, Caveolin1, and AR may play important roles in skin tissues and musk glands tissue.

biochemistry↗

bioRxiv2022

DeepGAMI: Deep biologically guided auxiliary learning for multimodal integration and imputation to improve phenotype prediction

Genotype-phenotype association is found in many biological systems, such as brain-related diseases and behavioral traits. Despite the recent improvement in the prediction of phenotypes from genotypes, they can be further improved and explainability of these predictions remains challenging, primarily due to complex underlying molecular and cellular mechanisms. Emerging multimodal data enables studying such mechanisms at different scales from genotype to phenotypes involving intermediate phenotypes like gene expression. However, due to the black-box nature of many machine learning techniques, it is challenging to integrate these multi-modalities and interpret the biological insights in prediction, especially when some modality is missing. Biological knowledge has recently been incorporated into machine learning modeling to help understand the reasoning behind the choices made by these models. To this end, we developed DeepGAMI, an interpretable deep learning model to improve genotype-phenotype prediction from multimodal data. DeepGAMI uses prior biological knowledge to define the neural network architecture. Notably, it embeds an auxiliary-learning layer for cross-modal imputation while training the model from multimodal data. Using this pre-trained layer, we can impute latent features of additional modalities and thus enable predicting phenotypes from a single modality only. Finally, the model uses integrated gradient to prioritize multimodal features and links for phenotypes. We applied DeepGAMI to multiple emerging multimodal datasets: (1) population-level genotype and bulk-tissue gene expression data for predicting schizophrenia, (2) population-level genotype and gene expression data for predicting clinical phenotypes in Alzheimers Disease, (3) gene expression and electrophysiological data of single neuronal cells in the mouse visual cortex, and (4) cell-type gene expression and genotype data for predicting schizophrenia. We found that DeepGAMI outperforms existing state-of-the-art methods and provides a profound understanding of gene regulatory mechanisms from genotype to phenotype, especially at cellular resolution. DeepGAMI is an open-source tool and is available at https://github.com/daifengwanglab/DeepGAMI.

bioinformatics↗

bioRxiv2022

CARD8 negatively regulates NLRP1 inflammasome activation level by interaction with NLRP1

NLRP1 inflammasome functions as canonical cytosolic sensor in response to intracellular infections and is implicated in auto-inflammatory diseases. But the regulation and signal transduction mechanisms of NLRP1 are incompletely understood. Here, we show that the T60 variant of CARD8, but not the canonical T48 isoform, negatively regulates NLRP1 inflammasome activation by directly interacting with the receptor molecule NLRP1 and inhibiting inflammasome assembly. Furthermore, our results suggest that the different ASC preference in three types of inflammasomes, namely ASC-indispensable NLRP1 inflammasome, ASC-dispensable mNLRP1b inflammasome and ASC-independent CARD8 inflammasome, is mainly caused by the CARD domain, not the UPA subdomain. Based on the systematic site-directed mutagenesis and structural analysis, we find that the signal transduction of NLRP1 inflammasome relies on multiple interaction surfaces on its death domain superfamily member CARD domain. Finally, our results partly explain the mechanism of the NLRP1 mutation-derived auto-inflammatory diseases caused by the overactivation of the NLRP1 inflammasome. In conclusion, our study not only reveals how CARD8 downregulates NLRP1 inflammasome activation, but also provides insights into the mechanisms of CARD-containing inflammasome assembly.

immunology↗

bioRxiv2022

Selection and validation of reference genes for quantitative real-time polymerase chain reaction in Serratia ureilytica DW2

Serratia ureilytica DW2 is a highly efficient phosphate-solubilizing bacterium isolated from Codonopsis pilosula rhizosphere soil that can promote the growth of C. pilosula. However, no validated reference genes from the genus Serratia for use in quantitative real-time polymerase chain reaction (RT-qPCR) normalization have been reported. To screen stable reference genes in S. ureilytica DW2, the expression of eight candidate reference genes (16S rRNA, ftsZ, ftsA, mreB, recA, slyD, thiC, and zipA) under different treatment conditions (pH, temperature, culture time, and salt content) was assayed by RT-qPCR. The expression stability of these genes was analyzed with different algorithms (geNorm, NormFinder, and BestKeeper). To verify the reliability of the data, the most stably expressed reference gene was used to quantify expression of the glucose dehydrogenase (gdh) gene under different soluble phosphate levels. The results showed that the zipA and 16S rRNA genes were the most stable reference genes, and the least stable were thiC and recA. The expression of gdh was consistent with the phosphate solubilization ability on plates containing National Botanical Research Institute phosphate (NBRIP) growth medium. Therefore, this study provides a stable and reliable reference gene for Serratia, which is vital for the accurate quantification of functional gene expression in future studies.

microbiology↗

bioRxiv2022

A cocktail containing two synergetic antibodies broadly neutralizes SARS-CoV-2 and its variants including Omicron BA.1 and BA.2

Neutralizing antibodies (NAbs) can prevent and treat infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, continuously emerging variants, such as Omicron, have significantly reduced the potency of most known NAbs. The selection of NAbs with broad neutralizing activities and the identification of conserved critical epitopes are still urgently needed. Here, we identified an extremely potent antibody (55A8) by single B-cell sorting from convalescent SARS-CoV-2-infected patients that recognized the receptor-binding domain (RBD) in the SARS-CoV-2 spike (S) protein. 55A8 could bind to wild-type SARS-CoV-2, Omicron BA.1 and Omicron BA.2 simultaneously with 58G6, a NAb previously identified by our group. Importantly, an antibody cocktail containing 55A8 and 58G6 (2-cocktail) showed synergetic neutralizing activity with a half-maximal inhibitory concentration (IC50) in the picomolar range in vitro and prophylactic efficacy in hamsters challenged with Omicron (BA.1) through intranasal delivery at an extraordinarily low dosage (25 g of each antibody daily) at 3 days post-infection. Structural analysis by cryo-electron microscopy (cryo-EM) revealed that 55A8 is a Class III NAb that recognizes a highly conserved epitope. It could block angiotensin-converting enzyme 2 (ACE2) binding to the RBD in the S protein trimer via steric hindrance. The epitopes in the RBD recognized by 55A8 and 58G6 were found to be different and complementary, which could explain the synergetic mechanism of these two NAbs. Our findings not only provide a potential antibody cocktail for clinical use against infection with current SARS-CoV-2 strains and future variants but also identify critical epitope information for the development of better antiviral agents.

immunology↗

bioRxiv2022

Hetero-bivalent Nanobodies Provide Broad-spectrum Protection against SARS-CoV-2 Variants of Concern including Omicron

Following Delta, Omicron variant triggered a new wave of SARS-CoV-2 infection globally, adaptive evolution of the virus may not stop, the development of broad-spectrum antivirals is still urgent. We previously developed two hetero-bivalent nanobodies with potent neutralization against original WT SARS-CoV-2, termed aRBD-2-5 and aRBD-2-7, by fusing aRBD-2 with aRBD-5 or aRBD-7, respectively. Here, we resolved crystal structures of these nanobodies in complex with RBD, and found the epitope of aRBD-2 differs from that of aRBD-5, aRBD-7. aRBD-2 binds to a conserved epitope which renders its binding activity to all variants of concern (VOCs) including Omicron. Interestingly, although monovalent aRBD-5 and aRBD-7 lost binding to some variants, they effectively improved the overall affinity when transformed into the hetero-bivalent form after being fused with aRBD-2. Consistent with the high binding affinities, aRBD-2-5-Fc and aRBD-2-7-Fc exhibited ultra-potent neutralization to all five VOCs; particularly, aRBD-2-5-Fc neutralized authentic virus of Beta, Delta and Omicron with the IC50of 5.98[~]9.65 ng/mL or 54.3[~]87.6 pM. Importantly, aRBD-2-5-Fc provided in vivo prophylactic protection for mice against WT and mouse-adapted SARS-CoV-2, and provided full protection against Omicron in hamster model when administrated either prophylactically or therapeutically. Taken together, we found a conserved epitope on RBD, and hetero-bivalent nanobodies had increased affinity for VOCs over its monovalent form, and provided potent and broad-spectrum protection both in vitro and in vivo against all tested major variants, and potentially future emerging variants. Our strategy provides a new solution in the development of therapeutic antibodies for COVID-19 caused by newly emergent VOCs.

immunology↗

bioRxiv2022

Single-cell network biology characterizes cell-type gene regulation for drug repurposing and phenotype prediction in Alzheimer's disease

Dysregulation of gene expression in Alzheimers disease (AD) remains elusive, especially at the cell type level. Gene regulatory network, a key molecular mechanism linking transcription factors (TFs) and regulatory elements to govern target gene expression, can change across cell types in the human brain and thus serve as a model for studying gene dysregulation in AD. However, it is still challenging to understand how cell type networks work abnormally under AD. To address this, we integrated single-cell multi-omics data and predicted the gene regulatory networks in AD and control for four major cell types, excitatory and inhibitory neurons, microglia and oligodendrocytes. Importantly, we applied network biology approaches to analyze the changes of network characteristics across these cell types, and between AD and control. For instance, many hub TFs target different genes between AD and control (rewiring). Also, these networks show strong hierarchical structures in which top TFs (master regulators) are largely common across cell types, whereas different TFs operate at the middle levels in some cell types (e.g., microglia). The regulatory logics of enriched network motifs (e.g., feed-forward loops) further uncover cell-type-specific TF-TF cooperativities in gene regulation. The cell type networks are highly modular. Several network modules with cell-type-specific expression changes in AD pathology are enriched with AD-risk genes and putative targets of approved and pending AD drugs, suggesting possible cell-type genomic medicine in AD. Finally, using the cell type gene regulatory networks, we developed machine learning models to classify and prioritize additional AD genes. We found that top prioritized genes predict clinical phenotypes (e.g., cognitive impairment). Overall, this single-cell network biology analysis provides a comprehensive map linking genes, regulatory networks, cell types and drug targets and reveals mechanisms on cell-type gene dyregulation in AD.

bioinformatics↗

bioRxiv2022

S100A4 exerts robust mucosal adjuvant activity for co-administered antigens in mice

The lack of clinically applicable mucosal adjuvants is a major hurdle in designing effective mucosal vaccines. We hereby report that the calcium-binding protein S100A4, which regulates a wide range of biological functions, is a potent mucosal adjuvant in mice for co-administered antigens, including the SARS-CoV-2 spike protein, with comparable or even superior efficacy as cholera toxin but without causing any adverse reactions. Intranasal immunization with recombinant S100A4 elicited antigen-specific antibody and pulmonary cytotoxic T cell responses, and these responses were remarkably sustained for longer than six months. As a self-protein, S100A4 did not stimulate antibody responses against itself, a quality desired of adjuvants. S100A4 prolonged nasal residence of intranasally delivered antigens and promoted migration of antigen-presenting cells. S100A4-pulsed dendritic cells potently activated cognate T cells. Furthermore, S100A4 induced strong germinal center responses revealed by both microscopy and mass spectrometry, a novel technique for measuring germinal center activity. In conclusion, S100A4 may be a promising adjuvant in formulating mucosal vaccines, including vaccines against pathogens that infect via the respiratory tract, such as SARS-CoV-2.

immunology↗

bioRxiv2022

Rsc3K, a SMV resistance gene localized on chromosome 2 in soybean cv. Kefeng No.1, interacts with virulence determinant P3

Soybean mosaic virus (SMV) is one of the most devastating viral pathogens in Glycine max (L.) Merr (soybean). Twenty-two SMV strains (SC1-SC22) isolated in China were identified based on their responses to ten soybean cultivars. By using the F2-derived F3 (F2:3) and recombinant inbred line (RIL) populations of resistant Soybean cultivar (cv.) Kefeng No.1 x susceptible cv. Nannong 1138-2, we localized the gene mediating resistant to SMV-SC3 strain to a 90 kb interval on chromosome 2 in Kefeng No.1. Bean pod mottle virus (BPMV)-induced gene silencing (VIGS) were used to study the gene function of candidate genes in the mapping interval and revealed that an recombinant gene, later named as Rsc3K, caused by internal deletion of a genomic DNA frag ement in Kefeng No.1, is the resistant gene to SMV-SC3. By shuffling genes between avirulent isolate SC3 and avirulent SMV isolate 1129, we found that P3 is the virulence determinant causing resistance on Kefeng No.1. We showed the interaction between Rsc3K and P3 by the yeast two-hybrid (Y2H) and bimolecular fluorescent complementation (BiFC) assays. In conclusion, this study demonstrated that Rsc3K plays a crucial role in resistance of Kefeng No.1 to SMV-SC3 by direct interaction with viral protein P3. HighlightRsc3K resistant to SMV-SC3 by interacting with P3.

molecular biology↗

bioRxiv2021

Single-dose AAV-based vaccine induces a high level of neutralizing antibodies and provides long-term protection against SARS-CoV-2 in rhesus macaques

Coronavirus disease 2019 (COVID-19), which is triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, continues to threaten global public health. Developing a vaccine that only requires single immunization but provides long-term protection for the prevention and control of COVID-19 is important. Here, we developed an adeno-associated virus (AAV)-based vaccine expressing a stable receptor-binding domain (SRBD) protein. The vaccine requires only a single shot but provides effective neutralizing antibodies (NAbs) over 598 days in rhesus macaques (Macaca mulatta). Importantly, our results showed that the NAbs were kept in high level and long lasting against authentic wild-type SARS-CoV-2, Beta, Delta and Omicron variants using plaque reduction neutralization test. Of note, although we detected pre-existing AAV2/9 antibodies before immunization, the vaccine still induced high and effective NAbs against COVID-19 in rhesus macaques. AAV-SRBD immune serum also efficiently inhibited the binding of ACE2 with RBD in the SARS-CoV-2 B.1.1.7 (Alpha), B.1.351 (Beta), P.1/P.2 (Gamma), B.1.617.2 (Delta), B.1.617.1/3(Kappa), and C.37 (Lambda) variants. Thus, these data suggest that the vaccine has great potential to prevent the spread of SARS-CoV-2.

immunology↗

bioRxiv2021

Dual regulation of the actin cytoskeleton by CARMIL-GAP

CARMIL (Capping protein Arp2/3 Myosin I Linker) proteins are multi-domain scaffold proteins that regulate actin dynamics by regulating the activity of Capping Protein (CP). Here we characterize CARMIL-GAP, a Dictyostelium CARMIL isoform that contains a ~130 residue insert that, by homology, is a GTPase activating (GAP) domain for Rho-related GTPases. Consistently, this GAP domain binds Dictyostelium Rac1a and accelerates its rate of GTP hydrolysis. CARMIL-GAP concentrates with F-actin in phagocytic cups and at the leading edge of chemotaxing cells, and CARMIL-GAP null cells exhibit pronounced defects in phagocytosis and chemotactic streaming. Importantly, these defects are fully rescued by expressing GFP-tagged CARMIL-GAP in CARMIL-GAP null cells. Finally, rescue with versions of CARMIL-GAP that lack either GAP activity or the ability to regulate CP show that while both activities contribute significantly to CARMIL-GAP function, the GAP activity plays the bigger role. Together, our results add to the growing evidence that CARMIL proteins influence actin dynamics by regulating signaling molecules as well as CP, and that the continuous cycling of the Rho GTPases nucleotide state is often required to drive Rho-dependent biological processes. SUMMARY STATEMENTThe assembly of actin filaments supports a wide array of fundamental cellular functions, including cell migration and phagocytosis. Actin assembly is controlled by a host of regulatory proteins, with Capping Protein being one of the most important. Capping Protein is in turn regulated by the CARMIL family of proteins. Actin assembly is also controlled by signaling pathways that often converge on Rho-related GTPases like Rac1. These GTPases cycle between an active, GTP-bound state and an inactive, GDP-bound state. Guanine nucleotide exchange factors (GEFs) and guanine nucleotide activating proteins (GAPs) drive Rho-related GTPases to their GTP-bound and GDP-bound states, respectively. Here we characterized a version of CARMIL that contains within it a GAP domain for Rac1. We show that CARMIL-GAP supports the actin-based processes of cell migration and phagocytosis. We also show that while CARMIL-GAPs ability to regulate Capping Protein and the nucleotide state of Rac1 are both important for its cellular functions, its ability to regulate Rac1 via its GAP domain plays the bigger role. Finally, our data support the emerging concept that the continuous cycling of Rho GTPases between their GTP- bound and GDP-bound states is often required to drive Rho-dependent biological processes.

cell biology↗

bioRxiv2021

Recovery of high-qualitied Genomes from a deep-inland Salt Lake Using BASALT

Metagenomic binning enables the in-depth characterization of microorganisms. To improve the resolution and efficiency of metagenomic binning, BASALT (Binning Across a Series of AssembLies Toolkit), a novel binning toolkit was present in this study, which recovers, compares and optimizes metagenomic assembled genomes (MAGs) across a series of assemblies from short-read, long-read or hybrid strategies. BASALT incorporates self-designed algorithms which automates the separation of redundant bins, elongate and refine best bins and improve contiguity. Evaluation using mock communities revealed that BASALT auto-binning obtained up to 51% more number of MAGs with up to 10 times better MAG quality from microbial community at low (132 genomes) and medium (596 genomes) complexity, compared to other binners such as DASTool, VAMB and metaWRAP. Using BASALT, a case-study analysis of a Salt Lake sediment microbial community from northwest arid region of China was performed, resulting in 426 non-redundant MAGs, including 352 and 69 bacterial and archaeal MAGs which could not be assigned to any known species from GTDB (ANI < 95%), respectively. In addition, two Lokiarchaeotal MAGs that belong to superphylum Asgardarchaeota were observed from Salt Lake sediment samples. This is the first time that candidate species from phylum Lokiarchaeota was found in the arid and deep-inland environment, filling the current knowledge gap of earth microbiome. Overall, BASALT is proven to be a robust toolkit for metagenomic binning, and more importantly, expand the Tree of Life.

bioinformatics↗

bioRxiv2021

Potent in vitro Neutralization of SARS-CoV-2 by Hetero-bivalent Alpaca Nanobodies Targeting the Spike Receptor-Binding Domain

Cell entry by SARS-CoV-2 requires the binding between the receptor-binding domain (RBD) of the viral Spike protein and the cellular angiotensin-converting enzyme 2 (ACE2). As such, RBD has become the major target for vaccine development, while RBD-specific antibodies are pursued as therapeutics. Here, we report the development and characterization of SARS-CoV-2 RBD-specific VHH/nanobody (Nb) from immunized alpacas. Seven RBD-specific Nbs with high stability were identified using phage display. They bind to SARS-CoV-2 RBD with affinity KD ranging from 2.6 to 113 nM, and six of them can block RBD-ACE2 interaction. The fusion of the Nbs with IgG1 Fc resulted in homodimers with greatly improved RBD-binding affinities (KD ranging from 72.7 pM to 4.5 nM) and nanomolar RBD-ACE2 blocking abilities. Furthermore, fusion of two Nbs with non-overlapping epitopes resulted in hetero-bivalent Nbs, namely aRBD-2-5 and aRBD-2-7, with significantly higher RBD binding affinities (KD of 59.2 pM and 0.25 nM) and greatly enhanced SARS-CoV-2 neutralizing potency. The 50% neutralization dose (ND50) of aRBD-2-5 and aRBD-2-7 was 1.22 ng/mL ([~]0.043 nM) and 3.18 ng/mL ([~]0.111 nM), respectively. These high-affinity SARS-CoV-2 blocking Nbs could be further developed into therapeutics as well as diagnosis reagents for COVID-19. ImportanceTo date, SARS-CoV-2 has caused tremendous loss of human life and economic output worldwide. Although a few COVID-19 vaccines have been approved in several countries, the development of effective therapeutics including SARS-CoV-2 targeting antibodies remains critical. Due to their small size (13-15 kDa), highly solubility and stability, Nbs are particularly well suited for pulmonary delivery and more amenable to engineer into multi-valent formats, compared to the conventional antibody. Here, we report a serial of new anti-SARS-CoV-2 Nbs isolated from immunized alpaca and two engineered hetero-bivalent Nbs. These potent neutralizing Nbs showed promise as potential therapeutics against COVID-19.

immunology↗

bioRxiv2020

Sequencing of clinical samples reveals that adaptation keeps establishing during H7N9 virus infection in humans

The H7 subtype avian influenza viruses (AIV) have a much longer history and their adaptation through evolution pose continuous threat to humans 1. Since 2013 March, the novel reasserted H7N9 subtype have transmitted to humans through their repeated assertion in the poultry market. Through repeated transmission, H7N9 gradually became the second AIV subtype posing greater public health risk after H5N1 2,3. After infection, how the virus tunes its genome to adapt and evolve in humans remains unknown. Through direct amplification of H7N9 and high throughput (HT) sequencing of full genomes from the swabs and lower respiratory tract samples collected from infected patients in Shenzhen, China, we have analyzed the in vivo H7N9 mutations at the level of whole genomes and have compared with the genomes derived by in vitro cultures. These comparisons and frequency analysis against the H7N9 genomes in the public database, 40 amino acids were identified that play potential roles in virus adaptation during H7N9 infection in humans. Various synonymous mutations were also identified that might be crucial to H7N9 adaptation in humans. The mechanism of these mutations occurred in a single infection are discussed in this study.

microbiology↗

bioRxiv2020

Broad transcriptomic dysregulation across the cerebral cortex in ASD

Classically, psychiatric disorders have been considered to lack defining pathology, but recent work has demonstrated consistent disruption at the molecular level, characterized by transcriptomic and epigenetic alterations.1-3 In ASD, upregulation of microglial, astrocyte, and immune signaling genes, downregulation of specific synaptic genes, and attenuation of regional gene expression differences are observed.1,2,4-6 However, whether these changes are limited to the cortical association areas profiled is unknown. Here, we perform RNA-sequencing (RNA-seq) on 725 brain samples spanning 11 distinct cortical areas in 112 ASD cases and neurotypical controls. We identify substantially more genes and isoforms that differentiate ASD from controls than previously observed. These alterations are pervasive and cortex-wide, but vary in magnitude across regions, roughly showing an anterior to posterior gradient, with the strongest signal in visual cortex, followed by parietal cortex and the temporal lobe. We find a notable enrichment of ASD genetic risk variants among cortex-wide downregulated synaptic plasticity genes and upregulated protein folding gene isoforms. Finally, using snRNA-seq, we determine that regional variation in the magnitude of transcriptomic dysregulation reflects changes in cellular proportion and cell-type-specific gene expression, particularly impacting L3/4 excitatory neurons. These results highlight widespread, genetically-driven neuronal dysfunction as a major component of ASD pathology in the cerebral cortex, extending beyond association cortices to involve primary sensory regions.

neuroscience↗