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Chen, C.

Publications and source records attributed to Chen, C..

At least 415 records · Page 23Linked to original sources
bioRxiv2023

Improving AlphaFold2-based Protein Tertiary Structure Prediction with MULTICOM in CASP15

Since CASP14, AlphaFold2 has become the standard method for protein tertiary structure prediction. One remaining challenge in the field is to further improve the accuracy of AlphaFold2-based protein structure prediction. To address this challenge, we developed a new version of the MULTICOM system to sample diverse multiple sequence alignments (MSAs) and structural templates to improve the input for AlphaFold2 to generate structural models. The models are then ranked by both the pairwise model similarity and AlphaFold2 self-reported model quality score. The top ranked models are further refined by a novel structure alignment-based refinement method powered by Foldseek. Moreover, for a monomer target that is a subunit of a protein assembly (complex), MULTICOM integrates tertiary and quaternary structure prediction together to account for tertiary structural changes induced by protein-protein interaction in the assembly. The MULTICOM system participated in the tertiary structure prediction in the 15th Critical Assessment of Techniques for Protein Structure Prediction (CASP15) in 2022 as server and human predictors. Our best server predictor (MULTICOM_refine) ranked 3rd among 47 CASP15 server predictors and our best human predictor (MULTICOM) ranked 7th among all 132 human and server predictors. The average GDT-TS score and TM-score of the first structural models that MULTICOM_refine predicted for 94 CASP15 domains are [~]0.80 and [~]0.92, 9.6% and 8.2% and higher than [~]0.73 and 0.85 of the standard AlphaFold2 predictor respectively. The results demonstrate that our approach can significantly improve the accuracy of the AlphaFold2-based protein tertiary structure prediction. The source code of MULTICOM is available at: https://github.com/BioinfoMachineLearning/MULTICOM3.

bioinformatics↗
bioRxiv2023

Conformational dynamics of the μ-opioid receptor determine ligand intrinsic efficacy

The -opioid receptor (OR) is an important target for pain management and the molecular understanding of drug action will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance (DEER) and single-molecule fluorescence resonance energy transfer (smFRET), how ligand-specific conformational changes of the OR translate into a broad range of intrinsic efficacies at the transducer level. We identify several cytoplasmic receptor conformations interconverting on different timescales, including a pre-activated receptor conformation which is capable of G protein binding, and a fully activated conformation which dramatically lowers GDP affinity within the ternary complex. Interaction of {beta}-arrestin-1 with the OR core binding site appears less specific and occurs with much lower affinity than binding of G protein Gi. One-Sentence SummaryLigand-dependent conformational dynamics of the -opioid receptor determine downstream signaling efficacy.

biochemistry↗
bioRxiv2023

Metabolic Reprogramming via targeting ACOD1 promotes polarization and anti-tumor activity of human CAR-iMACs in solid tumors

The pro-inflammatory state of macrophages is crucial in conferring its role in combating tumor cells. That state is closely associated with metabolic reprogramming. Here we identified key metabolic genes regulating macrophage pro-inflammatory activation in a pooled metabolic gene knockout CRISPR screen. We found that KEAP1 and ACOD1 are strong regulators of the pro-inflammatory state, and therefore developed human ACOD1 knockout macrophages with our induced pluripotent stem cell-derived CAR-macrophage (CAR-iMAC) platform. The engineered iMACs showed stronger and more persistent polarization toward the pro-inflammatory state, more ROS production, and more potent phagocytosis and cytotoxic functions against cancer cells in vitro. Upon transplantation to ovarian or pancreatic cancer mouse models, ACOD1 depleted CAR-iMACs exhibited enhanced capacity in repressing tumors in vivo and prolonged the lifespan of mice. In addition, combining ACOD1-depleted CAR-iMACs with immune check point inhibitors (ICIs), such as the anti-CD47 antibody or anti-PD1 antibody resulted in stronger tumor suppressing effect. Mechanistically, the depletion of ACOD1 reduced the immunometabolite itaconate, allowing KEAP1 to prevent NRF2 from entering the nucleus to activate the anti-inflammatory program. This study demonstrates that ACOD1 is a new myeloid target for cancer immunotherapy and metabolically engineered human iPSC-derived CAR-iMACs exhibit enhanced polarization and anti-tumor functions in adoptive cell transfer therapies.

immunology↗
bioRxiv2023

Functional analysis of Salix purpurea genes support roles for ARR17 and GATA15 as master regulators of sex determination

The Salicaceae family is of growing interest in the study of dioecy in plants because the sex determination region (SDR) has been shown to be highly dynamic, with differing locations and heterogametic systems between species. Without the ability to transform and regenerate Salix in tissue culture, previous studies investigating the mechanisms regulating sex in the genus Salix have been limited to genome resequencing and differential gene expression, which are mostly descriptive in nature, and functional validation of candidate sex determination genes has not yet been conducted. Here we used Arabidopsis to functionally characterize a suite of previously identified candidate genes involved in sex determination and sex dimorphism in the bioenergy shrub willow Salix purpurea. Six candidate master regulator genes for sex determination were heterologously expressed in Arabidopsis, followed by floral proteome analysis. In addition, 11 transcription factors with predicted roles in mediating sex dimorphism downstream of the SDR were tested using DAP-Seq in both male and female S. purpurea DNA. The results of this study provide further evidence to support models for the roles of ARR17 and GATA15 as master regulator genes of sex determination in S. purpurea, contributing to a regulatory system that is notably different from that of its sister genus Populus. Evidence was also obtained for the roles of two transcription factors, an AP2/ERF family gene and a homeodomain-like transcription factor, in downstream regulation of sex dimorphism.

plant biology↗
bioRxiv2023

Integrin beta4 promotes DNA damage drug resistance in triple-negative breast cancer via TNFAIP2/IQGAP1/Rac1

Anti-tumor drug resistance is a challenge for human triple-negative breast cancer treatment. Our previous work demonstrated that TNFAIP2 activates RAC1 to promote triple-negative breast cancer cell proliferation and migration. However, the mechanism by which TNFAIP2 activates RAC1 is unknown. In this study, we found that TNFAIP2 interacts with IQGAP1 and Integrin {beta}4. Integrin {beta}4 activates RAC1 through TNFAIP2 and IQGAP1 and confers DNA damage-related drug resistance in triple-negative breast cancer. These results indicate that the Integrin {beta}4/TNFAIP2/IQGAP1/RAC1 axis provides potential therapeutic targets to overcome DNA damage-related drug resistance in triple-negative breast cancer.

cancer biology↗
bioRxiv2023

MdMYB44-like positively regulates salt and drought tolerance via the MdPYL8-MdPP2CA module in apple

Abscisic acid (ABA) is involved in salt and drought stress responses, but the underlying molecular mechanism remains unclear. Here, we demonstrated that the overexpression of MdMYB44-like, an R2R3-MYB transcription factor (TF), significantly increases the salt and drought tolerance of transgenic apple and Arabidopsis. MdMYB44-like inhibits the transcription of MdPP2CA, which encodes a type 2C protein phosphatase that acts as a negative regulator in ABA response, thereby enhancing ABA signaling-mediated salt and drought tolerance. Furthermore, we found that MdMYB44-like and MdPYL8, an ABA receptor, form a protein complex that further enhances the transcriptional inhibition of the MdPP2CA promoter by MdMYB44-like. Significantly, we discovered that MdPP2CA can interfere with the physical association between MdMYB44-like and MdPYL8 in the presence of ABA, partially blocking the inhibitory effect of the MdMYB44-like-MdPYL8 complex on the MdPP2CA promoter. Thus, MdMYB44-like, MdPYL8, and MdPP2CA form a regulatory loop that tightly controls ABA signaling homeostasis under salt and drought stress. Our data revealed a previously unidentified mechanism by which MdMYB44-like precisely modulates ABA-mediated salt and drought tolerance in apple through the MdPYL8-MdPP2CA module.

plant biology↗
bioRxiv2023

Analytical Comparability to Evaluate Impact of Manufacturing Changes of ARX788, an Anti-HER2 ADC in Late-Stage Clinical Development

ARX788 is an anti-HER2 antibody drug conjugate (ADC) developed using Ambrx proprietary Engineered Precision Biologics technology. The manufacturing process of ARX788 has been optimized during the course of early to late-phase clinical development. A comprehensive evaluation of side-by-side comparability between pre- and post-change process for ARX788 drug substance and drug product from a quality perspective was conducted based on ICH Q5E guidelines consisting of batch release assays, physicochemical and biophysical characterization, biological characterization, and forced degradation studies. All results have demonstrated a high degree of similarity between the pre- and post-change ARX788 drug substance batches and drug product lots, demonstrating that the process manufacturing changes did not impact product quality.

cancer biology↗
bioRxiv2023

Phase Separation and Ageing of Glycine-Rich Protein from Tick Adhesive

Hard ticks feed on their host for multiple days. To ensure firm attachment, they secrete a protein-rich saliva that eventually forms a solid cement cone. The underlying mechanism of this liquid-to-solid transition is not yet understood. This study focuses on the phase transitions of a disordered glycine-rich protein (GRP) that is prominent in tick saliva. We show that GRP undergoes liquid-liquid phase separation via simple coacervation to form biomolecular condensates in salty environments. Cation-pi and pi-pi interactions near the C-terminus promote coacervation while a negatively charged N-terminus prolongs its onset through electrostatic repulsion. Interestingly, GRP condensates exhibit ageing and undergo liquid-to-gel transition to form viscoelastic networks as well as solid-like condensates. Lastly, we provide evidence for protein-rich condensates in natural tick saliva. Our findings provide a starting point to gain insights into the bioadhesion of ticks, develop novel tick control strategies, and towards biomedical applications such as tissue sealants.

biochemistry↗
bioRxiv2023

Supervised learning of high-confidence phenotypic subpopulations from single-cell data

Accurately identifying phenotype-relevant cell subsets from heterogeneous cell populations is crucial for delineating the underlying mechanisms driving biological or clinical phenotypes. Here, by deploying a learning with rejection strategy, we developed a novel supervised learning framework called PENCIL to identify subpopulations associated with categorical or continuous phenotypes from single-cell data. By embedding a feature selection function into this flexible framework, for the first time, we were able to select informative features and identify cell subpopulations simultaneously, which enables the accurate identification of phenotypic subpopulations otherwise missed by methods incapable of concurrent gene selection. Furthermore, the regression mode of PENCIL presents a novel ability for supervised phenotypic trajectory learning of subpopulations from single-cell data. We conducted comprehensive simulations to evaluate PENCILs versatility in simultaneous gene selection, subpopulation identification and phenotypic trajectory prediction. PENCIL is fast and scalable to analyze 1 million cells within 1 hour. Using the classification mode, PENCIL detected T-cell subpopulations associated with melanoma immunotherapy outcomes. Moreover, when applied to scRNA-seq of a mantle cell lymphoma patient with drug treatment across multiple time points, the regression mode of PENCIL revealed a transcriptional treatment response trajectory. Collectively, our work introduces a scalable and flexible infrastructure to accurately identify phenotype-associated subpopulations from single-cell data.

bioinformatics↗
bioRxiv2023

Evaluating performance and applications of sample-wise cell deconvolution methods on human brain transcriptomic data

Sample-wise deconvolution methods have been developed to estimate cell-type proportions and gene expressions in bulk-tissue samples. However, the performance of these methods and their biological applications has not been evaluated, particularly on human brain transcriptomic data. Here, nine deconvolution methods were evaluated with sample-matched data from bulk-tissue RNAseq, single-cell/nuclei (sc/sn) RNAseq, and immunohistochemistry. A total of 1,130,767 nuclei/cells from 149 adult postmortem brains and 72 organoid samples were used. The results showed the best performance of dtangle for estimating cell proportions and bMIND for estimating sample-wise cell-type gene expression. For eight brain cell types, 25,273 cell-type eQTLs were identified with deconvoluted expressions (decon-eQTLs). The results showed that decon-eQTLs explained more schizophrenia GWAS heritability than bulk-tissue or single-cell eQTLs alone. Differential gene expression associated with multiple phenotypes were also examined using the deconvoluted data. Our findings, which were replicated in bulk-tissue RNAseq and sc/snRNAseq data, provided new insights into the biological applications of deconvoluted data.

genomics↗
bioRxiv2023

Development of monoclonal antibody-based blocking ELISA for detecting SARS-CoV-2 exposure in animals

The global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a significant threat to public health. Besides humans, SARS-CoV-2 can infect several animal species. Highly sensitive and specific diagnostic reagents and assays are urgently needed for rapid detection and implementation of strategies for prevention and control of the infection in animals. In this study, we initially developed a panel of monoclonal antibodies (mAbs) against SARS-CoV-2 nucleocapsid (N) protein. To detect SARS-CoV-2 antibodies in a broad spectrum of animal species, a mAb-based bELISA was developed. Test validation using a set of animal serum samples with known infection status obtained an optimal percentage of inhibition (PI) cut-off value of 17.6% with diagnostic sensitivity of 97.8% and diagnostic specificity of 98.9%. The assay demonstrates high repeatability as determined by a low coefficient of variation (7.23%, 6.95%, and 5.15%) between-runs, within-run, and within-plate, respectively. Testing of samples collected over time from experimentally infected cats showed that the bELISA was able to detect seroconversion as early as 7 days post-infection. Subsequently, the bELISA was applied for testing pet animals with COVID-19-like symptoms and specific antibody responses were detected in two dogs. The panel of mAbs generated in this study provides a valuable tool for SARS-CoV-2 diagnostics and research. The mAb-based bELISA provides a serological test in aid of COVID-19 surveillance in animals. IMPORTANCEAntibody tests are commonly used as a diagnostic tool for detecting host immune response following infection. Serology (antibody) tests complement nucleic acid assays by providing a history of virus exposure, no matter symptoms developed from infection or the infection was asymptomatic. Serology tests for COVID-19 are in high demand, especially when the vaccines become available. They are important to determine the prevalence of the viral infection in a population and identify individuals who have been infected or vaccinated. ELISA is a simple and practically reliable serological test, which allows high-throughput implementation in surveillance studies. Several COVID-19 ELISA kits are available. However, they are mostly designed for human samples and species-specific secondary antibody is required for indirect ELISA format. This paper describes the development of an all species applicable monoclonal antibody (mAb)-based blocking ELISA to facilitate the detection and surveillance of COVID-19 in animals.

microbiology↗
bioRxiv2023

Graph theory analysis reveals an assortative pain network vulnerable to attacks

The neural substrate of pain experience has been described as a dense network of connected brain regions. However, the connectivity pattern of these brain regions remains elusive, precluding a deeper understanding of how pain emerges from the structural connectivity. Here, we use graph theory to systematically characterize the architecture of a comprehensive pain network, including both cortical and subcortical brain areas. This structural brain network consists of 49 nodes denoting pain-related brain areas, linked by edges representing their relative incoming and outgoing axonal projection strengths. Sixty-three percent of brain areas in this structural pain network share reciprocal connections, reflecting a dense network. The clustering coefficient, a measurement of the probability that adjacent nodes are connected, indicates that brain areas in the pain network tend to cluster together. Community detection, the process of discovering cohesive groups in complex networks, successfully reveals two known subnetworks that specifically mediate the sensory and affective components of pain, respectively. Assortativity analysis, which evaluates the tendency of nodes to connect with other nodes with similar features, indicates that the pain network is assortative. Finally, robustness, the resistance of a complex network to failures and perturbations, indicates that the pain network displays a high degree of error tolerance (local failure rarely affects the global information carried by the network) but is vulnerable to attacks (selective removal of hub nodes critically changes network connectivity). Taken together, graph theory analysis unveils an assortative structural pain network in the brain processing nociceptive information, and the vulnerability of this network to attack opens up the possibility of alleviating pain by targeting the most connected brain areas in the network.

neuroscience↗
bioRxiv2023

Mucus production, host-microbiome interactions, hormone sensitivity, and innate immune responses modeled in human endo- and ecto-cervix chips

Modulation of mucus production by the human endo- and ecto-cervical epithelium by steroid hormones and associated interactions with commensal microbiome play a central role in the physiology and pathophysiology of the female reproductive tract. However, most of our knowledge about these interactions is based on results from animal studies or in vitro models that fail to faithfully mimic the mucosal environment of the human cervix. Here we describe microfluidic organ-on-a-chip (Organ Chip) models of the human cervical mucosa that recreate the cervical epithelial-stromal interface with a functional epithelial barrier and produce abundant mucus that has compositional, biophysical, and hormone-responsive properties similar to the living cervix. Application of continuous fluid flow to chips lined with primary human cervical epithelial cells from a commercial source that contained a mixture of primary human ecto- and endo-cervical epithelial cells promoted preferential expression of the ecto-cervical phenotype, whereas use of periodic flow including periods of stasis induced endo-cervical specialization. When the periodic flow Cervix Chips were co-cultured with living Lactobacillus crispatus and Gardnerella vaginalis bacterial communities to respectively mimic the effects of human host interactions with optimal (healthy) or non-optimal (dysbiotic) microbiome associated with an ascending infection in the female reproductive tract, significant differences in tissue innate immune responses, barrier function, cell viability and protein profile, and mucus composition were detected reminiscent of those observed in vivo. Thus, these Organ Chip models of human cervix provide a physiologically relevant experimental in vitro model to study cervical mucus physiology and its role in human host-microbiome interactions as well as a potential preclinical testbed for development of therapeutic interventions to enhance womens health.

bioengineering↗
bioRxiv2023

Epoxyeicosatrienoic acids and sEH inhibition prevent cardiac dysfunction in CVB3-induced myocarditis by positively regulating type I interferon signaling

Myocarditis is a challenging inflammatory disease of the heart and better understanding of its etiology is needed to develop specific drug therapies. Epoxyeicosatrienoic acids (EETs), active molecules synthesized by cytochrome P450 enzymes from arachidonic acids and hydrolyzed to less active dihydroxyeicosatrienoic acids by soluble epoxide hydrolase (sEH), have been attributed anti-inflammatory activity. Here, we investigated whether EETs have immunomodulatory activity and exert protective effects on coxsackie B3 virus (CVB3)-induced myocarditis. Viral infection altered eicosanoid epoxide and diol levels in both patients with myocarditis and, in the murine heart, and correlated with the increased expression and activity of the sEH after CVB3 infection. Administration of a sEH inhibitor prevented CVB3-induced cardiac dysfunction and inflammatory infiltration. Importantly, EET/sEH inhibitor treatment attenuated vial infection/ improved viral resistance by activating type I interferon (IFN) signaling. At the molecular level, EETs enhanced the interaction between glycogen synthase kinase 3{beta} (GSK3{beta}) and TANK-binding kinase 1 (TBK1) to promote IFN-{beta} production. Our findings revealed that EETs and sEH inhibitors prevent the progress of CVB3-induced myocarditis, particularly by promoting viral resistance by increasing IFN production.

immunology↗
bioRxiv2023

UPF3A is a ubiquitously expressed NMD factor among mouse tissues

Nonsense-mediated mRNA decay (NMD), an important post-transcriptional regulatory mechanism in gene expression, is actively involved in a series of cellular and physiological processes, thus controlling cell fate and tissue homeostasis. Defects in NMD cause human diseases such as neurodevelopmental disorders, tumorigenesis and autoimmunity. UPF3 (Up- frameshift protein 3), first identified in the bakers yeast, is a core NMD factor. UPF3A and UPF3B, the two UPF3 paralogs emerging in vertebrates, have either activating or suppressing roles in NMD. Previous studies found that UPF3B protein is ubiquitously expressed in almost all mammalian organs, while UPF3A protein is hardly detectable in most of mammalian tissues, except in the testis. One hypothesis explaining this phenomena is the functional antognism between UPF3A and UPF3B in NMD. Thus, UPF3B competitively binds to UPF2 with higher affinity than UPF3A, which finally destabilizes UPF3A protein. In the present study, we quantitatively evaluated the expression of UPF3A and UPF3B in nine major tissues and reproductive organs of wild type male and female mice. Our study confirmed that UPF3A has the highest expression in male germlines. To our surprise, we found in most tissues, including brain and thymus, the protein level of UPF3A is comparable with that of UPF3B. In spleen and lung, UPF3A is higher than UPF3B. These findings are further supported by publicly available gene expression data. Thus, our study demonstrated that UPF3A protein is ubiquitously expressed in mouse tissues, and may play important roles in the homeostasis of multiple mammalian tissues.

cell biology↗
bioRxiv2023

Radioprotective drug screening in a salivary gland tissue chip.

Ionizing radiation damage to the salivary glands during head and neck cancer treatment often causes a permanent loss of secretory function. Due to the resulting decrease in saliva production, patients experience difficulty with eating, speaking, and swallowing and are predisposed to oral infections and tooth decay. While the radioprotective drug amifostine is approved to prevent radiation-induced hyposalivation, it has intolerable side effects that limit its use and motivate research into discovering alternatives. To address this issue, we have developed a salivary gland mimetic (SGm) tissue chip platform for use in high-content drug discovery. Here, we report on the development and validation of in-chip assays to quantify reduced glutathione and cellular senescence ({beta}-galactosidase) as measures of radiation damage and protection using WR-1065, the active form of amifostine. Following validation, we next tested our assays using other reported radioprotective drugs including Edaravone, Tempol, N-acetylcysteine, Rapamycin, Ex-Rad, and Palifermin. The validated assays were then used to screen a library of FDA-approved compounds for radioprotection. We screened 438 compounds, obtained 25 hits that were further tested for EC50 values and downselected using information from the PubChem database. Lead compounds were identified that are being tested in preclinical models.

bioengineering↗
bioRxiv2023

Homeostatic control of an iron repressor in a GI tract resident

The transition metal iron plays a crucial role in living cells. However, high level of iron is potentially toxic through the production of reactive oxygen species (ROS), serving as a deterrent to the commensal fungus Candida albicans for colonization in the iron-rich gastrointestinal (GI) tract. We observe that the mutant lacking an iron-responsive transcription factor Hap43 is hyper-fit for colonization in murine gut. We demonstrate that high iron specifically triggers multiple post-translational modifications (PTMs) and proteasomal degradation of Hap43, a vital process guaranteeing the precision of intestinal ROS detoxification. Reduced levels of Hap43 lead to de-repression of antioxidant genes and therefore alleviate the deleterious ROS derived from iron metabolism. Our data reveal that Hap43 functions as a negative regulator for oxidative stress-adaptation of C. albicans to gut colonization and thereby provide a new insight into understanding the interplay between iron homeostasis and fungal commensalism. ImportanceIron homeostasis is critical for creatures. Candida albicans is one of the major commensals in the GI tract where is iron-replete environment. Transcriptional factor Hap43 was believed to repress iron utilizations genes in iron-depleted conditions for decades. However, the mystery in iron-replete conditions of Hap43 has never been uncovered. We discovered that reduced levels of Hap43 via phosphorylation-dependent nuclear export, followed by proteosome-mediated protein degradation, leads to de-repression of downstream antioxidant genes and promote its colonization in GI tract. We propose that C. albicans has a strict detoxification process to ensure its survival, which has important implications for understanding how the fungi survives in the mammalian host.

microbiology↗