Biology subjects

Chen, X.

Publications and source records attributed to Chen, X..

At least 1,369 records · Page 76Linked to original sources

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

Maternal vitamin D deficiency increases the risk of obesity in male mice offspring by affecting the immune response

Recently, many epidemiological and animal studies have indicated that obesity have their origin in the early stages of life including the inappropriate balance of some nutrients, the objective of this study is to determine the risk of obesity in male mice offspring as a consequence of maternal VD deficiency-mediated disordering of the immune response. Four-week-old C57BL/6J female mice were fed VD-deficient or normal reproductive diets during pregnancy and lactation. Their male offspring were weighted and euthanized after being fed control and high-fat diets (HFD) for 16 weeks starting at the weaning. The serum was collected for biochemical analyses. Epididymal (eWAT) and inguinal (iWAT) white adipose tissues were excised for histological examination, immunohistochemistry, gene expressions of inflammatory factors, and for determining the proportions of immune cells by flow cytometry. Insufficient maternal VD intake exacerbated the development of obesity both in non-obese and obese male offspring as evidenced by larger adipose cells and abnormal glucose and lipid metabolisms. Also, the expression of proinflammatory cytokine genes was increased and that of anti-inflammatory cytokines was decreased in maternal VD-deficient groups in the eWAT and/or iWAT. This was accompanied by higher levels of TNF- or/and INF-{beta}, and lower levels of IL-4 and IL-10. Insufficient maternal VD intake was also observed to induce a shift in the profiles of immune cells in the eWAT and/or iWAT, resulting in increased percentages of M1 macrophage, ATDCs, and CD4+ and CD8+ T cells, but caused a significant decrease in the percentage of M2 macrophages, both in non-obese and obese male offspring. All these changes in the immune cell profile were more obvious in the eWAT than in the iWAT. These results indicated that insufficient maternal VD intake promoted the development of obesity in male offspring by modulating the immune cell populations and causing a polarization in the adipose depots. ImportanceEvidence in this study has indicated that insufficient maternal VD intake promotes the development of obesity in the male offspring by modulating the recruitment of immune cell populations and their polarization as well as the expression and secretion of proinflammatory adipokines in the adipose depots in a weight-independent manner, which is more obvious in eWAT than that in the iWAT.

immunology↗

bioRxiv2020

Potentiating the anti-tumor response of tumor infiltrated T cells by NAD+ supplementation

Tumor immunotherapies have provided clinical benefits, yet great potential remains for optimizing therapeutic effects. Here, we show that low NAD+ levels restrict the function of tumor infiltrating T lymphocytes (TILs). TILs harvested from human ovarian tumor tissues showed decreased NAD+ levels compared with T cells from paired peripheral blood samples. The combination of whole-genome CRISPR and large-scale metabolic inhibitor screens implicated the NAD+ biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT) is required for T cell activation. Further isotopic labeling and LC-MS studies confirmed that NAD+ depletion suppressed mitochondrial energy biosynthesis in T cells. Excitingly, NAD+ supplementation significantly enhanced the tumor cell-killing efficacy of CAR-T cells ex vivo, and extended animal survive in both adoptive CAR-T model and immune checkpoint blockade treatment models in vivo. This study demonstrates an over-the-counter nutrient supplement NAD+ could robustly boost the efficacy of T cell-based immunotherapy and provides insights into the cellular basis of T cell metabolic reprogramming in treating cancers. One Sentence SummaryNAD+ supplementation during cancer immunotherapies significantly enhances T cell activation and tumor killing capacity.

immunology↗

bioRxiv2020

Click-ExM enables expansion microscopy for all biomolecules

Expansion microscopy (ExM) allows super-resolution imaging on conventional fluorescence microscopes, but has been limited to proteins and nucleic acids. Here we develop click-ExM, which integrates click-labeling into ExM to enable a "one-stop-shop" method for nanoscale imaging of various types of biomolecules. Using 18 clickable labels for click-ExM imaging of DNA, RNA, proteins, lipids, glycans and small molecules, we demonstrate its universality, compatibility with signal-amplification techniques, and broad applications in cellular imaging.

bioengineering↗

bioRxiv2020

Gli3 utilizes Hand2 to synergistically regulate tissue-specific transcriptional networks

Despite a common understanding that Gli TFs are utilized to reiterate a Hh morphogen gradient, genetic analyses suggest craniofacial development does not completely fit this paradigm. We demonstrated that rather than being driven by a Hh threshold, robust Gli3 transcriptional activity during skeletal and glossal development required interaction with the bHLH TF Hand2. Not only did genetic and expression data support a co-factorial relationship, but genomic analysis further revealed that Gli3 and Hand2 were enriched at regulatory elements for genes essential for mandibular patterning and development. Interestingly, motif analysis at sites co-occupied by Gli3 and Hand2 uncovered mandibular-specific, low-affinity, divergent Gli binding motifs (dGBMs). Functional validation revealed these dGBMs conveyed synergistic activation of Gli targets essential for mandibular patterning and development. In summary, this work elucidates a novel, sequence-dependent mechanism for Gli transcriptional activity within the craniofacial complex that is independent of a graded Hh signal.

developmental biology↗

bioRxiv2020

Surgery and Radioactive Iodine Therapeutic Strategy for Patients Greater than 60 Years of Age with Differentiated Thyroid Cancer

PurposeThe purpose of the current study was to determine whether older patients with differentiated thyroid cancer (DTC) who received surgical treatment had a better cause-specific survival (CSS) than patients who were recommended surgery, but declined, and whether patients who underwent post-operative RAI-131 therapy had an impact on CSS based on TNM staging and number of lymph node metastases for all total or near-total thyroidectomy patients. Patients and MethodsAll DTC patients information were obtained from the SEER*Stat 8.3.6 program, and only patients [≥] 60 years or older were considered. The patients were divided into two groups (underwent surgery and surgery recommend, but not performed). Furthermore, patients were grouped as follows: T4; N1b; M1; T1-3N0-1a; specific number of lymph node metastases; and total or near-total thyroidectomy. ResultsThe 120-month cause-specific survival (CSS) rate of females and males showed a gradual declining trend from 60-64 to [≥]80 years of age in the group that underwent surgery. The CSS rate of females and males showed a marked downward and irregular trend with an increase in age in the recommended, but no surgery group. Univariate analysis indicated that the surgery group had a higher 120-month CSS in females in most stages and males, compared with the no surgery group. RAI-131 therapy was associated with an improved 80-month CSS in T4/N1b/M1 females (P<0.0183) and males (P<0.0011). There was no CSS difference in females or males between the T1-3N0 and T1-3N1a patients. There was no statistical difference between the two subgroups. ConclusionsSurgical treatment should be recommended for elderly DTC patients because surgery can lead to a better CSS. High-risk patients achieve a higher benefit-to-risk ratio with RAI-131 therapy. To avoid the adverse effects associated with RAI-131 therapy, a multidisciplinary discussion should be arranged for intermediate- and low-risk patients.

cancer biology↗

bioRxiv2020

Excessive Polyamine Generation in Keratinocytes Promotes Self-RNA Endosomal Sensing by Dendritic Cells in Psoriasis

The mechanisms underlying tissue-specific chronic inflammation are elusive. Here we report that mice lacking Protein Phosphatase 6 in keratinocytes are predisposed to psoriasis-like skin inflammation, with an inordinate urea cycle and enhanced oxidative phosphorylation that supports hyperproliferation. This phenotype is mediated by increased Arginase-1 production resulting from CCAAT/enhancer-binding protein beta activation. Single-cell RNA-seq of the psoriatic epidermis revealed that the rate-limiting enzyme for Arginine biosynthesis, Argininosuccinate synthetase 1, maintains the Arginine pool, which is indispensable for immune responses. Accumulated polyamines branched from the urea cycle promote endosomal Tlr7-dependent self-RNA sensing by myeloid dendritic cells. This process is achieved with the assistance of an RNA-binding peptide that originates from the heterogeneous nuclear ribonucleoprotein A1, a probable autoantigen in psoriasis. Finally, targeting urea cycle wiring with an arginase inhibitor markedly improved skin inflammation in murine and non-human primate models of psoriasis. Our findings suggest that urea cycle alteration and excessive polyamine production by psoriatic keratinocytes promote self-RNA sensing by dendritic cells, which links the hyperproliferation of stationary cells with innate-immune activation in an auto-inflammatory condition.

immunology↗

bioRxiv2020

Super-enhancer switching drives a burst in germline gene expression at the mitosis-to-meiosis transition

The testis has the most diverse and complex transcriptome of all organs due to bursts in expression of thousands of germline-specific genes. Much of this unique gene expression takes place when mitotic germ cells differentiate and enter into meiotic prophase. Here, we demonstrate that the genome-wide reorganization of super-enhancers (SEs) drives bursts of germline genes after the mitosis-to-meiosis transition. At the mitosis-to-meiosis transition, mitotic SEs dissolve while meiotic SEs are established. Meiotic SEs are associated with the activation of key germline genes, defining the cellular identity of germ cells. This SE switching is regulated by the establishment of meiotic SEs via A-MYB (MYBL1), a key transcription factor for germline genes, and by the resolution of mitotic SEs via SCML2, a germline-specific Polycomb protein required for spermatogenesis-specific gene expression. Prior to the entry into meiosis, meiotic SEs are preprogrammed in mitotic spermatogonia, serving to direct the unidirectional differentiation of spermatogenesis. We identify key regulatory factors for both mitotic and meiotic enhancers, revealing a molecular logic for the concurrent activation of mitotic enhancers and suppression of meiotic enhancers in the somatic and/or mitotically proliferating phase.

developmental biology↗

bioRxiv2020

Reprogramming the endogenous type III-A CRISPR-Cas system for genome editing, RNA interference and CRISPRi screening in Mycobacterium tuberculosis

Mycobacterium tuberculosis (M.tb) causes the current leading infectious disease. Examination of the functional genomics of M.tb and development of drugs and vaccines are hampered by the complicated and time-consuming genetic manipulation techniques for M.tb. Here, we reprogrammed M.tb endogenous type III-A CRISPR-Cas10 system for simple and efficient gene editing, RNA interference and screening via simple delivery of a plasmid harboring a mini-CRISPR array, thereby avoiding the introduction of exogenous proteins and minimizing proteotoxicity. We demonstrated that M.tb genes were efficiently and specifically knocked-in/out by this system, which was confirmed by whole-genome sequencing. This system was further employed for single and simultaneous multiple-gene RNA interference. Moreover, we successfully applied this system for genome-wide CRISPR interference screening to identify the in-vitro and intracellular growth-regulating genes. This system can be extensively used to explore the functional genomics of M.tb and facilitate the development of new anti-Mycobacterial drugs and vaccines. SummaryTuberculosis caused by Mycobacterium tuberculosis (M.tb) is the current leading infectious disease affecting more than ten million people annually. To dissect the functional genomics and understand its virulence, persistence, and antibiotics resistance, a powerful genome editing tool and high-throughput screening methods are desperately wanted. Our study developed an efficient and a robust tool for genome editing and RNA interference in M.tb using its endogenous CRISPR cas10 system. Moreover, the system has been successfully applied for genome-wide CRISPR interference screening. This tool could be employed to explore the functional genomics of M.tb and facilitate the development of anti-M.tb drugs and vaccines.

microbiology↗

bioRxiv2020

Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites

The outbreak of coronavirus disease (COVID-19) in China caused by SARS-CoV-2 virus continually lead to worldwide human infections and deaths. It is currently no specific viral protein targeted therapeutics yet. Viral nucleocapsid protein is a potential antiviral drug target, serving multiple critical functions during the viral life cycle. However, the structural information of SARS-CoV-2 nucleocapsid protein is yet to be clear. Herein, we have determined the 2.7 [A] crystal structure of the N-terminal RNA binding domain of SARS-CoV-2 nucleocapsid protein. Although overall structure is similar with other reported coronavirus nucleocapsid protein N-terminal domain, the surface electrostatic potential characteristics between them are distinct. Further comparison with mild virus type HCoV-OC43 equivalent domain demonstrates a unique potential RNA binding pocket alongside the {beta}-sheet core. Complemented by in vitro binding studies, our data provide several atomic resolution features of SARS-CoV-2 nucleocapsid protein N-terminal domain, guiding the design of novel antiviral agents specific targeting to SARS-CoV-2.

molecular biology↗

bioRxiv2020

A novel bat coronavirus reveals natural insertions at the S1/S2 cleavage site of the Spike protein and a possible recombinant origin of HCoV-19

The unprecedented epidemic of pneumonia caused by a novel coronavirus, HCoV-19, in China and beyond has caused public health concern at a global scale. Although bats are regarded as the most likely natural hosts for HCoV-191,2, the origins of the virus remain unclear. Here, we report a novel bat-derived coronavirus, denoted RmYN02, identified from a metagenomics analysis of samples from 227 bats collected from Yunnan Province in China between May and October, 2019. RmYN02 shared 93.3% nucleotide identity with HCoV-19 at the scale of the complete virus genome and 97.2% identity in the 1ab gene in which it was the closest relative of HCoV-19. In contrast, RmYN02 showed low sequence identity (61.3%) to HCoV-19 in the receptor binding domain (RBD) and might not bind to angiotensin-converting enzyme 2 (ACE2). Critically, however, and in a similar manner to HCoV-19, RmYN02 was characterized by the insertion of multiple amino acids at the junction site of the S1 and S2 subunits of the Spike (S) protein. This provides strong evidence that such insertion events can occur in nature. Together, these data suggest that HCoV-19 originated from multiple naturally occurring recombination events among those viruses present in bats and other wildlife species.

microbiology↗

bioRxiv2020

Sapphire Nanopores for Low-Noise DNA Sensing

Silicon-supported (SiS) solid-state nanopores have broad applications in single-molecule biosensing and diagnostics, but their high capacitive noise has seriously limited both their sensing accuracy and recording speed. Nanopores on insulating glass have demonstrated reduced capacitance and noise, but it remains challenging to bulk-etch amorphous glass to create membranes reproducibly and uniformly. Here a new approach is reported to form triangular sapphire-supported (SaS) nanopore membranes by batch-processing-compatible anisotropic wet etching of sapphire, with membrane dimension demonstrated from ~200 μm to 5 μm. A SaS nanopore in 68 μm-wide silicon nitride membrane has 130 times smaller capacitance (10 pF) compared to a SiS nanopore (~4 μm SiN membrane, ~1.3 nF), despite a 100 times larger membrane. It has a current noise of 18 pA over 100 kHz bandwidth, much smaller than that from our SiS nanopore (46 pA) and comparable with the best reported low-noise nanopores. Further, the SaS nanopore displays a higher signal-to-noise ratio (SNR, 21 versus 11 for SiS nanopore) in DNA sensing, although the SNR can be further improved using thinner membranes and smaller pores. The SaS nanopore presents a simple platform in both fabrication and structure that is particularly suitable for low-noise and high-speed molecular diagnostics.Competing Interest StatementThe authors have declared no competing interest.View Full Text

bioengineering↗

bioRxiv2020

Metabolites alleviate staphylococcal bloodstream infection in a NO-dependent manner via arginase inhibition

Staphylococcus aureus is a notorious bacterial pathogen that often causes soft tissue and bloodstream infections and invariably garners resistance mechanisms against new antibiotics. Host innate immunity modulated by metabolites has been proved as a powerful strategy against bacterial infections. However, few studies focus on the application of this strategy against S. aureus infection. Here, we identified four metabolite biomarkers, L-proline, L-isoleucine, L-leucine, and L-valine (PILV), by a metabolomics study. In animal models of S. aureus bloodstream infection, exogenous administration of each metabolite or PILV shows an anti-infective effect, while PILV treatment has higher protection than individual metabolite treatment. Each metabolite targets nitric oxide (NO) to kill S. aureus via arginase inhibition, and PILV exhibits additive inhibition of arginase activity that causes further killing. This suppression also contributes to the metabolite-mediated phagocytic killing of S. aureus in human blood. Our finding demonstrates the metabolite-mediated innate immunity as a therapeutic intervention for S. aureus infection.

microbiology↗

bioRxiv2020

Reprogramming progressive cells display low CAG promoter activity

There is wide variability in the propensity of somatic cells to reprogram into pluripotency in response to the Yamanaka factors. How to segregate these variability to enrich for cells of specific traits that reprogram efficiently remains challenging. Here we report that the variability in reprogramming propensity is associated with the activity of the MKL1/SRF transcription factor and concurs with small cell size as well as rapid cell cycle. Reprogramming progressive cells can be prospectively identified by their low activity of a widely used synthetic promoter, CAG. CAGlow cells arise and expand during cell cycle acceleration in the early reprogramming culture of both mouse and human fibroblasts. Our work illustrate a molecular scenario underlying the distinct reprogramming propensities and demonstrate a convenient practical approach for their enrichment.

cell biology↗

bioRxiv2020

Representing Organic Matter Thermodynamics in Biogeochemical Reactions via Substrate-Explicit Modeling

Predictive biogeochemical modeling requires data-model integration that enables explicit representation of the sophisticated roles of microbial processes that transform substrates. Data from high-resolution organic matter (OM) characterization are increasingly available and can serve as a critical resource for this purpose, but their incorporation into biogeochemical models is often prohibited due to an over-simplified description of reaction networks. To fill this gap, we proposed a new concept of biogeochemical modeling--termed substrate-explicit modeling--that enables parameterizing OM-specific oxidative degradation pathways and reaction rates based on the thermodynamic properties of OM pools. The resulting kinetic models are characterized by only two parameters regardless of the complexity of OM profiles, which can greatly facilitate the integration with reactive transport models for ecosystem simulations by alleviating the difficulty in parameter identification. For every detected organic molecule in a given sample, our approach provides a systematic way to formulate reaction kinetics from chemical formula, which enables the evaluation of the impact of OM character on biogeochemical processes across conditions. In a case study of two sites with distinct OM thermodynamics, our method not only predicted oxidative degradation to be primarily driven by thermodynamic efficiency of OM consistent with experimental rate measurements, but also revealed previously unknown critically important aspects of biogeochemical reactions, including their condition-specific response to carbon and/or oxygen limitations. Lastly, we showed that the proposed substrate-explicit modeling approach can be synergistically combined with enzyme-explicit approach to provide improved predictions. This result led us to present integrative biogeochemical modeling as a unifying framework that can ideally describe the dynamic interplay among microbes, enzymes, and substrates to address advanced questions and hypotheses in future studies. Altogether, the new modeling concept we propose in this work provides a foundational platform for unprecedented predictions of biogeochemical and ecosystem dynamics through enhanced integration with diverse experimental data and extant modeling approaches.

systems biology↗

bioRxiv2020

Mechanical Heterogeneity in the Bone Microenvironment as Characterised by Atomic Force Microscopy

Bones are structurally heterogeneous organs with diverse functions that undergo mechanical stimuli across multiple length scales. Mechanical characterisation of the bone microenvironment is important for understanding how bones function in health and disease. Here we describe the mechanical architecture of cortical bone, the growth plate, metaphysis and marrow in fresh murine bones, probed using atomic force microscopy in physiological buffer. Both elastic and viscoelastic properties are found to be highly heterogeneous with moduli ranging over 3 to 5 orders of magnitude, both within and across regions. All regions include extremely soft areas, with moduli of a few Pascal and viscosities as low as tens Pa*s. Aging impacts the viscoelasticity of the bone marrow strongly but has limited effect on the other regions studied. Our approach provides the opportunity to explore the mechanical properties of complex tissues at the length scale relevant to cellular processes and how these impact on aging and disease. SIGNIFICANCEThe mechanical properties of biological materials at cellular scale are involved in guiding cell fate. However, there is a critical gap in our knowledge of such properties in complex tissues. The physiochemical environment surrounding the cells in in-vitro studies differs significantly from that found in vivo. Existing mechanical characterisation of real tissues are largely limited to properties at larger scales, structurally simple (e.g. epithelial monolayers) or non-intact (e.g. through fixation) tissues. In this paper, we address this critical gap and present the micro-mechanical properties of the relatively intact bone microenvironment. The measured Youngs moduli and viscosity provide a sound guidance in bioengineering designs. The striking heterogeneity at supracellular scale reveals the potential contribution of the mechanical properties in guiding cell behaviour.

biophysics↗

bioRxiv2020

Cellular heterogeneity-adjusted clonal methylation (CHALM) provides better prediction of gene expression

Promoter DNA methylation is a well-established mechanism of transcription repression, but its global correlation with gene expression is surprisingly weak. This weak correlation is due in part to the failure of current methylation quantification methods to consider the heterogeneity among sequenced bulk cells. Here, we introduce Cell Heterogeneity-Adjusted cLonal Methylation (CHALM) as a novel methylation quantification method. CHALM better explains the functional consequences of DNA methylation, such as its correlations with gene expression and H3K4me3. When applied to different methylation datasets, the CHALM method has uniquely detected differentially methylated genes that exhibit distinct biological functions supporting underlying mechanisms.

bioinformatics↗

bioRxiv2020

Mixed Culture of Bacterial Cell for Large Scale DNA Storage

DNA emerged as novel material for mass data storage, the serious problem human society is facing. Taking advantage of current synthesis capacity, massive oligo pool demonstrated its high-potential in data storage in test tube. Herein, mixed culture of bacterial cells carrying mass oligo pool that was assembled in a high copy plasmid was presented as a stable material for large scale data storage. Living cells data storage was fabricated by a multiple-steps process, assembly, transformation and mixed culture. The underlying principle was explored by deep bioinformatic analysis. Although homology assembly showed sequence context dependent bias but the massive digital information oligos in mixed culture were constant over multiple successive passaging. In pushing the limitation, over ten thousand distinct oligos, totally 2304 Kbps encoding 445 KB digital data including texts and images, were stored in bacterial cell, the largest archival data storage in living cell reported so far. The mixed culture of living cell data storage opens up a new approach to simply bridge the in vitro and in vivo storage system with combined advantage of both storage capability and economical information propagation.

molecular biology↗