Biology subjects

Shen, C.

Publications and source records attributed to Shen, C..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

A unique peptide recognition mechanism by the human relaxin family peptide receptor 4 (RXFP4)

Members of the insulin superfamily regulate a variety of biological processes through two types of target-specific but structurally conserved peptides, insulin/insulin-like growth factors and relaxin/insulin-like peptides. The latter bind to the human relaxin family peptide receptors (RXFPs), which are class A G protein-coupled receptors (GPCRs), to exert pleiotropic actions. Here, we report three cryo-electron microscopy structures of RXFP4-Gi protein complexes in the presence of the endogenous ligand insulin-like peptide 5 (INSL5) or one of the two small molecule agonists, compound 4 and DC591053, both were discovered through medicinal chemistry efforts. The B chain of INSL5 adopts a single -helix that penetrates into the orthostatic pocket, while the A chain sits above the orthosteric pocket to interact with the extracellular surface of RXFP4, revealing a unique peptide-binding mode previously unknown. Together with mutagenesis and functional analyses, the key determinants responsible for the peptidomimetic agonism and subtype selectivity were identified. DC591053 selectively mimicked the action of INSL5 at RXFP4 whereas compound 4 activated both RXFP3 and RXFP4. Comparison of peptide binding modes within the insulin superfamily displayed diverse interaction mechanisms distinct to each type of the peptides. Our findings not only provide valuable insights into ligand recognition and subtype selectivity among class A GPCRs, but also expand the knowledge of signaling mechanisms in the insulin superfamily.

molecular biology↗

bioRxiv2022

eMAFFTadd: scaling MAFFT-linsi-add to large datasets

Multiple sequence alignment is essential for many biological downstream analyses, but accurate alignment of large datasets, especially those exhibiting high rates of evolution or sequence length heterogeneity, is still unsolved. We present SALMA, a new multiple sequence alignment that provides high accuracy and scalability, even for datasets exhibiting high rates of evolution and great sequence length heterogeneity that arises from evolutionary processes. Like some prior methods (e.g., UPP, WITCH, and MAFFT-sparsecore), SALMA operates in two distinct stages: the first stage computes a "backbone alignment" for a subset of the sequences, and the second stage adds the remaining sequences into the backbone alignment. The main novelty in SALMA is how it adds the remaining ("query") sequences into the backbone alignment. For this step, which we refer to as SALMA-add, we use divide-and-conquer to scale MAFFT-linsi--add to enable it to add sequences into large backbone alignments. We show that SALMA-add has an advantage over other sequence-adding techniques for many realistic conditions and can scale to very large datasets with high accuracy (hundreds of thousands of sequences). We also show that SALMA is one of the most accurate compared to standard alignment methods. Our open source software for SALMA is available at https://github.com/c5shen/SALMA.

bioinformatics↗

bioRxiv2022

Nitric oxide is not responsible for the initial sensory-induced neurovascular coupling response in mouse cortex.

Neurovascular coupling ensures that changes in neural activity are accompanied by localised changes in cerebral blood flow. While much is known about the involvement of excitatory neurons in neurovascular coupling, the role of inhibitory interneurons is unresolved. While nNOS-expressing interneurons have been shown to be capable of eliciting vasodilation, the role of nitric oxide in functional hyperemia remains a matter of debate. Therefore in the present study we applied a combination of optogenetic and pharmacological approaches, 2-dimensional optical imaging spectroscopy, and electrophysiology to investigate the role of nitric oxide in neurovascular coupling responses evoked by nNOS-expressing interneurons and whisker stimulation in mouse sensory cortex. The haemodynamic response evoked by nNOS-expressing interneurons was significantly altered in the presence of the NOS inhibitor LNAME, revealing a large initial 20-HETE-dependent vasoconstriction. In contrast, the haemodynamic response induced by sensory stimulation was largely unchanged by LNAME. Our results suggest that while nitric oxide plays a key role in neurovascular responses evoked by nNOS-expressing interneurons it does not mediate the initial sensory-induced neurovascular coupling response in mouse cortex. Thus, our results call into question the involvement of nNOS-expressing interneurons and nitric oxide in sensory-evoked functional hyperemia.

neuroscience↗

bioRxiv2022

UPP2: Fast and Accurate Alignment Estimation of Datasets with Fragmentary Sequences

MotivationMultiple sequence alignment (MSA) is a basic step in many bioinformatics pipelines. However, achieving highly accurate alignments on large datasets, especially those with sequence length heterogeneity, is a challenging task. UPP (Ultra-large multiple sequence alignment using Phylogeny-aware Profiles) is a method for MSA estimation that builds an ensemble of Hidden Markov Models (eHMM) to represent an estimated alignment on the full length sequences in the input, and then adds the remaining sequences into the alignment using selected HMMs in the ensemble. Although UPP provides good accuracy, it is computationally intensive on large datasets. ResultsWe present UPP2, a direct improvement on UPP. The main advance is a fast technique for selecting HMMs in the ensemble that allows us to achieve the same accuracy as UPP but with greatly reduced runtime. We show UPP2 produces more accurate alignments compared to leading MSA methods on datasets exhibiting substantial sequence length heterogeneity, and is among the most accurate otherwise. Availabilityhttps://github.com/gillichu/sepp Contactwarnow@illinois.edu

bioinformatics↗

bioRxiv2022

SAI: Fast and automated quantification of stomatal parameters on microscope images

Using microscopy to investigate stomatal behaviour is a common technique in plant physiology research. Manual inspection and measurement of stomatal features is a low throughput process in terms of time and human effort, which relies on expert knowledge to identify and measure stomata accurately. This process represents a significant bottleneck in research pipelines, adding significant researcher time to any project that requires it. To alleviate this, we introduce StomaAI (SAI): a reliable and user-friendly tool that measures stomata of the model plant Arabidopsis (dicot) and the crop plant barley (monocot grass) via the application of deep computer vision. We evaluated the reliability of predicted measurements: SAI is capable of producing measurements consistent with human experts and successfully reproduced conclusions of published datasets. Hence, SAI boosts the number of images that biologists can evaluate in a fraction of the time so is capable of obtaining more accurate and representative results.

plant biology↗

bioRxiv2022

Close relatives of MERS-CoV in bats use ACE2 as their functional receptors

Middle East Respiratory Syndrome coronavirus (MERS-CoV) and several bat coronaviruses employ Dipeptidyl peptidase-4 (DPP4) as their functional receptors1-4. However, the receptor for NeoCoV, the closest MERS-CoV relative yet discovered in bats, remains enigmatic5. In this study, we unexpectedly found that NeoCoV and its close relative, PDF-2180-CoV, can efficiently use some types of bat Angiotensin-converting enzyme 2 (ACE2) and, less favorably, human ACE2 for entry. The two viruses use their spikes S1 subunit carboxyl-terminal domains (S1-CTD) for high-affinity and species-specific ACE2 binding. Cryo-electron microscopy analysis revealed a novel coronavirus-ACE2 binding interface and a protein-glycan interaction, distinct from other known ACE2-using viruses. We identified a molecular determinant close to the viral binding interface that restricts human ACE2 from supporting NeoCoV infection, especially around residue Asp338. Conversely, NeoCoV efficiently infects human ACE2 expressing cells after a T510F mutation on the receptor-binding motif (RBM). Notably, the infection could not be cross-neutralized by antibodies targeting SARS-CoV-2 or MERS-CoV. Our study demonstrates the first case of ACE2 usage in MERS-related viruses, shedding light on a potential bio-safety threat of the human emergence of an ACE2 using "MERS-CoV-2" with both high fatality and transmission rate.

microbiology↗

bioRxiv2021

Robust and Efficient Assessment of Potency (REAP): A Quantitative Tool for Dose-response Curve Estimation

The median-effect equation has been widely used to describe the dose-response relationship and identify compounds that activate or inhibit specific disease targets in contemporary drug discovery. However, the experimental data often contain extreme responses, which may significantly impair the estimation accuracy and impede valid quantitative assessment in the standard estimation procedure. To improve the quantitative estimation of the dose-response relationship, we introduce a novel approach based on robust beta regression. Substantive simulation studies under various scenarios demonstrate solid evidence that the proposed approach consistently provides robust estimation for the median-effect equation, particularly when there are extreme outcome observations. Moreover, simulation studies illustrate that the proposed approach also provides a narrower confidence interval, suggesting a higher power in statistical testing. Finally, to efficiently and conveniently perform common lab data analyses, we develop a freely accessible web-based analytic tool to facilitate the quantitative implementation of the proposed approach for the scientific community.

pharmacology and toxicology↗

bioRxiv2021

Full-length NLRP3 forms oligomeric cages to mediate NLRP3 sensing and activation

The nucleotide-binding domain and leucine-rich-repeat (LRR) containing protein 3 with a pyrin domain (NLRP3) is emerging to be a critical intracellular inflammasome sensor of membrane integrity and a highly important clinical target against chronic inflammation. Here we report that the endogenous, stimulus-responsive form of full-length NLRP3 is a 12-16 mer double ring cage held together by LRR-LRR interactions with the pyrin domains shielded within the assembly to avoid premature activation. Surprisingly, this NLRP3 form is predominantly membrane localized, which is consistent with previously noted localization of NLRP3 at various membrane organelles. Structure-guided mutagenesis reveals that trans-Golgi network dispersion into vesicles, an early event observed for all NLRP3 activating stimuli, requires the double ring cages of NLRP3. Double ring-defective NLRP3 mutants further abolish inflammasome punctum formation, caspase-1 processing and cell death. Thus, unlike other inflammasome sensors that are monomeric when inactive, our data uncover a unique NLRP3 oligomer on membrane that is poised to sense diverse signals to induce inflammasome activation.

immunology↗

bioRxiv2021

From Text-based Genome, Population Variations, and Transcriptome Datafiles to SQLite Database and Web Application: A Bioinformatical Study on Alfalfa

In this study, a web database application with the Flask framework was developed to implement three types of queries and visualize the results over a bioinformatical dataset from Alfalfa (Medicago sativa). A backend SQLite database was constructed from genome FASTA, population variations, transcriptome, and annotation files with extensions ".fasta", ".gff", "vcf", ".annotate", etc. Further, a supplementary command-line-based Java application was also developed for faster access to the database without direct SQL programming. Overall, Python, Java, and HTML were the main programming languages used in this application. Those scripts and the development procedures are valuable for bioinformaticians to build online databases from similar raw datasets of other species.

bioinformatics↗

bioRxiv2021

Pre-existing chromatin accessibility and gene expression differences among naïve CD4+ T cells influence effector potential

CD4+ T cells have a remarkable potential to differentiate into diverse effector lineages following activation. Here, we probed the heterogeneity present among naive CD4+ T cells before encountering their cognate antigen to ask whether their effector potential is modulated by pre-existing transcriptional and epigenetic differences. Using single-cell RNA sequencing, we showed that key drivers of variability are genes involved in T cell receptor (TCR) signaling. Using CD5 expression as a read-out of the strength of tonic TCR interactions with self-peptide MHC, and sorting on the ends of this self-reactivity spectrum, we find that pre-existing transcriptional differences among naive CD4+ T cells impact follicular helper cell (TFH) versus non-TFH effector lineage choice. Moreover, our data implicate TCR signal strength during thymic development in establishing differences in naive CD4 T cell chromatin landscapes that ultimately shape their effector potential.

immunology↗

bioRxiv2021

Screening of HLA-A restricted T cell epitopes of SARS-CoV-2 and induction of CD8+ T cell responses in HLA-A transgenic mice

While SARS-CoV-2-specific T cells have been characterized to play essential roles in host immune protection in COVID-19 patients, few researches focus on the functional validation of T cell epitopes and development of vaccines inducing specific T cell responses. In this study, 120 CD8+ T cell epitopes from E, M, N, S and RdRp proteins were validated. Among them, 110 epitopes have not been reported previously; 110, 15, 6, 14 and 12 epitopes were highly homologous with SARS-CoV, OC43, NL63, HKU1, and 229E, respectively; 4 epitopes from S protein displayed one amino acid distinct from the current variants of SARS-CoV-2. Thirty-one epitopes restricted by HLA-A2 molecule were used to generate peptide cocktail vaccines in combination with Poly(I:C), R848 or polylactic-co-glycolic acid nanoparticles, which elicited robust specific CD8+ T cell responses in wild-type and HLA-A2/DR1 transgenic mice. Seven of the 31 epitopes were found to be cross-presented by HLA-A2 and H-2K/Db molecules. Unlike previous researches, this study established a modified cell co-culture system of DC-peptide-PBL using healthy donors PBMCs to validate the CD8+ T cell epitope on-silicon predicted; provided a library of CD8+ T cell epitopes restricted by a series of high-frequency HLA-A allotypes which covering broad Asian populations; identified the HLA-A cross-restrictions of these CD8+ T cell epitopes using competitive binding experiments with HMy2.CIR cell lines expressing indicated HLA-A molecules; and initially confirmed the in vivo feasibility of 9 or 10-mer peptide cocktail vaccines of SARS-CoV2. These data will facilitate the development of vaccines inducing antiviral CD8+ T cell responses.

immunology↗

bioRxiv2021

Molecular Response to PARP1 Inhibition in Ovarian Cancer Cells as Determined by Mass Spectrometry Based Proteomics

Poly (ADP)-ribose polymerase (PARP) inhibitors have entered routine clinical practice for the treatment of high-grade serous ovarian cancer (HGSOC), yet the molecular mechanisms underlying treatment response to PARP1 inhibition (PARP1i) are not fully understood. Here, we used unbiased mass spectrometry based proteomics with data-driven protein network analysis to systematically characterize how HGSOC cells respond to PARP1i treatment. We found that PARP1i leads to pronounced proteomic changes in a diverse set of cellular processes in HGSOC cancer cells, consistent with transcript changes in an independent perturbation dataset. We interpret decreases in the levels of the pro-proliferative transcription factors SP1 and {beta}-catenin and in growth factor signaling as reflecting the anti-proliferative effect of PARP1i; and the strong activation of pro-survival processes NF-{varkappa}B signaling and lipid metabolism as PARPi-induced adaptive resistance mechanisms. Based on these observations, we nominate several protein targets for therapeutic inhibition in combination with PARP1i. When tested experimentally, the combination of PARPi with an inhibitor of fatty acid synthase (TVB-2640) has a 3-fold synergistic effect and is therefore of particular pre-clinical interest. Our study improves the current understanding of PARP1 function, highlights the potential that the anti-tumor efficacy of PARP1i may not only rely on DNA damage repair mechanisms and informs on the rational design of PARP1i combination therapies in ovarian cancer.

cancer biology↗

bioRxiv2020

STAT3 inhibits Myocardin induced cardiac hypertrophy

BackgroundIn order to explore the molecular mechanism of cardiomyocyte-dependent myocardial gene expression and cardiomyocyte differentiation in cardiac hypertrophy, and to provide new insights for cardiac hypertrophy. MethodsCardiac myocytes were isolated from day 1-3 Sprague-Dawley rat pups. Real time quantitative PCR, western blot and immunocytochemistry Assay were used to detect the expression and localization of related genes. CO-IP was used to detect direct protein interactions between Myocardin and STAT3. Luciferase reporter assay and chromatin immunoprecipitation were used to detect the binding of Myocardin to the promoter of a downstream target gene. Microinjection of zebrafish embryos was used to examine the effects of STAT3 and Myocardin interactions on cardiac development in vivo ResultsThe N-terminus of STAT3 directly binds to the basic domain of myocardin and inhibits the transcriptional activity of Myocardin-mediated cardiac-specific genes ANF and -actinin, thereby inhibiting their expression, and further inhibit myocardin-mediated cardiac hypertrophy in vivo. ConclusionsIn summary, our report states that signal transduction and transcriptional activation factor 3 (STAT3) are inhibitors of the major cardiac hypertrophic transcription factor Myocardiin, which is required for cardiomyocyte differentiation. The STAT3-cardiacin interaction identified nuclear hormone receptor-mediated and cardiac-specific gene-regulated convergence sites and suggested a possible mechanism for cardioprotective effects.

cell biology↗

bioRxiv2020

Co-infection of influenza A virus enhances SARS-CoV-2 infectivity

The upcoming flu season in the northern hemisphere merging with the current COVID-19 pandemic raises a potentially severe threat to public health. Through experimental co-infection of IAV with either pseudotyped or SARS-CoV-2 live virus, we found that IAV pre-infection significantly promoted the infectivity of SARS-CoV-2 in a broad range of cell types. Remarkably, increased SARS-CoV-2 viral load and more severe lung damage were observed in mice co-infected with IAV in vivo. Moreover, such enhancement of SARS-CoV-2 infectivity was not seen with several other viruses probably due to a unique IAV segment as an inducer to elevate ACE2 expression. This study illustrates that IAV has a special nature to aggravate SARS-CoV-2 infection, and prevention of IAV is of great significance during the COVID-19 pandemic.

microbiology↗

bioRxiv2020

Robust and scalable manifold learning via landmark diffusion for long-term medical signal processing

Motivated by analyzing long-term physiological time series, we design a robust and scalable spectral embedding algorithm, coined the algorithm RObust and Scalable Embedding via LANdmark Diffusion (ROSE-LAND). The key is designing a diffusion process on the dataset, where the diffusion is forced to interchange on a small subset called the landmark set. In addition to demonstrating its application to spectral clustering and image segmentation, the algorithm is applied to study the long-term arterial blood pressure waveform dynamics during a liver transplant operation lasting for 12 hours long.

bioinformatics↗

bioRxiv2020

Cryo-EM structures of inactive and Gi-coupled GABAB heterodimer

Metabotropic GABAB G protein-coupled receptor functions as a mandatory heterodimer of GB1 and GB2 subunits and mediates inhibitory neurotransmission in the central nervous system. Each subunit is composed of the extracellular Venus flytrap (VFT) domain and transmembrane (TM) domain. Here we present cryo-EM structures of human full-length heterodimeric GABAB receptor in the antagonist-bound inactive state and in the active state complexed with agonist and positive allosteric modulator in the presence of Gi1 protein at a resolution range of 2.8-3.0 [A]. Cryo-EM analysis of the activated-GABAB-Gi1 complex revealed that Gi1 couples to the activated receptor primarily in three major conformations, one via GB1 TM and two via GB2 TM, respectively. Our structures reveal that agonist binding stabilizes the closure of GB1 VFT, which in turn triggers a rearrangement of TM interfaces between two subunits from TM3-TM5/TM3-TM5 in the inactive state to TM6/TM6 in the active state and finally induces the opening of intracellular loop 3 and synergistically shifting of TM3, 4 and 5 helices in GB2 TM domain to accommodate the 5-helix of Gi1. These results provide a structural framework for understanding class C GPCR activation and a rational template for allosteric modulator design targeting dimeric interface of GABAB receptor.

molecular biology↗

bioRxiv2020

An Experimental Validation of the Computer Fluid Dynamics of Normal Human Nasal Airflow Using a Precise 3-D Printing Model

Computer Fluid Dynamics (CFD) is a popular method for studying airflow of nasal cavities. However, the data of CFD studies has rarely been validated through experiments. To test the accuracy of CFD computation, we studied the consistency of the air pressure of nasal cavities in the CFD and the experiment. A proportional resin model of a normal human subjects nasal cavities was created by a 3-d printer with a precision value of 0.1mm. The pressure of 63 check points in the nasal cavities in different breathing states was measured. The experimental data was compared with the data obtained by CFD simulation. At the flow rates of 180 ml s-1 and 560 ml s-1, the pressure in all check points remained highly consistent with the CFD data. At 1100 ml s-1 flow rate, there was a significant deviation in the posterior segment of the nasal cavity during exhalation. The method used in this study to measure the pressure in the nasal cavities can be used in experimental validation of CFD data. The computational methods and the boundary conditions used in this study resulted in a high agreement between the results of the CFD simulation and the experiment. Author SummaryIn the contemporary era, Computer fluid dynamics (CFD) is the mainstream method for studying air flow. Due to the complex anatomical structure of the nasal cavity, the CFD results of the nasal flow have rarely been experimentally verified. This study provides a method to verify the methods and results of nasal CFD. We printed an accurate model of a normal persons nasal cavity with a high-precision 3D printer. In this nasal cavity model, we set 63 small holes to detect the air pressure of the places we concerned. Three different nasal flow quantity are used to represent different breathing conditions: high (1100 ml s-1), medium (560 ml s-1), and low (180 ml s-1). In medium and low nasal flow quantities, our CFD results are in good agreement with the experimental pressure values. On this basis, we analyzed the characteristics of nasal airflow in normal people. The method used in this study to measure the pressure in the nasal cavities can be used in experimental measurements of the partial resistance of the nasal cavity. With proper modification, it can be applied to the clinical practice for nasal resistance, giving more help for the design of the operation plan.

biophysics↗

bioRxiv2020

Viral Architecture of SARS-CoV-2 with Post-Fusion Spike Revealed by Cryo-EM

Since December 2019, the outbreak of Coronavirus Disease 2019 (COVID-19) spread from Wuhan, China to the world, it has caused more than 87,000 diagnosed cases and more than 3,000 deaths globally. To fight against COVID-19, we carried out research for the near native SARS-CoV-2 and report here our preliminary results obtained. The pathogen of the COVID-19, the native SARS-CoV-2, was isolated, amplified and purified in a BSL-3 laboratory. The whole viral architecture of SARS-CoV-2 was examined by transmission electron microscopy (both negative staining and cryo-EM). We observed that the virion particles are roughly spherical or moderately pleiomorphic. Spikes have nail-like shape towards outside with a long body embedded in the envelope. The morphology of virion observed in our result indicates that the S protein of SARS-CoV-2 is in post-fusion state, with S1 disassociated. This state revealed by cryo-EM first time could provide an important information for the identification and relevant clinical research of this new coronavirus.

microbiology↗