Biology subjects

Li, P.

Publications and source records attributed to Li, P..

At least 163 records · Page 9Linked to original sources

bioRxiv2024

Role of Hepatocyte RIPK1 in Maintaining Liver Homeostasis during Metabolic Challenges

As a central hub for metabolism, the liver exhibits strong adaptability to maintain homeostasis in response to food fluctuations throughout evolution. However, the mechanisms governing this resilience remain incompletely understood. In this study, we identified Receptor interacting protein kinase 1 (RIPK1) in hepatocytes as a critical regulator in preserving hepatic homeostasis during metabolic challenges, such as short-term fasting or high-fat dieting. Our results demonstrated that hepatocyte-specific deficiency of RIPK1 sensitized the liver to short-term fasting-induced liver injury and hepatocyte apoptosis in both male and female mice. Despite being a common physiological stressor that typically does not induce liver inflammation, short-term fasting triggered hepatic inflammation and compensatory proliferation in hepatocyte-specific RIPK1-deficient (Ripk1{Delta}hep) mice. Transcriptomic analysis revealed that short-term fasting oriented the hepatic microenvironment into an inflammatory state in Ripk1{Delta}hep mice, with upregulated expression of inflammation and immune cell recruitment-associated genes. Single-cell RNA sequencing further confirmed the altered cellular composition in the liver of Ripk1{Delta}hep mice during fasting, highlighting the increased recruitment of macrophages to the liver. Mechanically, our results indicated that ER stress was involved in fasting-induced liver injury in Ripk1{Delta}hep mice. Overall, our findings revealed the role of RIPK1 in maintaining liver homeostasis during metabolic fluctuations and shed light on the intricate interplay between cell death, inflammation, and metabolism.

immunology↗

bioRxiv2024

High-accuracy crRNA array assembly strategy for multiplex CRISPR

Simultaneous targeting of multiple loci with CRISPR system, a tool known as multiplex CRISPR, offers us more feasibility to manipulate and elucidate the intricate and redundant endogenous networks underlying complex cellular functions. Owing to the versatility of the continuously emerging Cas nucleases and the utilization of CRISPR arrays, multiplex CRISPR has been implemented by numerous in vitro and in vivo studies. However, a streamlined practical strategy for CRISPR array assembly with both convenience and accuracy is still lacking. Here, we present a novel high-accuracy, cost- and time-saving strategy for CRISPR array assembly. Using this strategy, we efficiently accomplished the assembly of up to twelve crRNAs (for AsCas12a) or fifteen crRNAs (for RfxCas13d) in a single reaction. Moreover, we reveal that CRISPR arrays driven by Pol II promoters exhibit a distinct targeting pattern compared to Pol III promoters, which could be exploited for specific distributions of CRISPR intensity. For long CRISPR arrays, we designed a better approach than simply selecting one from U6 or EF1a. Collectively, our study provides a flexible and powerful tool for the convenient implementation of multiplex CRISPR across DNA to RNA, facilitating the dissection of sophisticated cellular networks and the future realization of multi-target gene therapy.

synthetic biology↗

bioRxiv2024

Purging viral latency by a bifunctional HSV-vectored therapeutic vaccine in chronically SIV-infected macaques

The persistence of latent viral reservoirs remains the major obstacle to eradicating human immunodeficiency virus (HIV). We herein found that ICP34.5 can act as an antagonistic factor for the reactivation of HIV latency by herpes simplex virus type I (HSV-1), and thus recombinant HSV-1 with ICP34.5 deletion could more effectively reactivate HIV latency than its wild-type counterpart. Mechanistically, HSV-{Delta}ICP34.5 promoted the phosphorylation of HSF1 by decreasing the recruitment of protein phosphatase 1 (PP1), thus effectively binding to the HIV LTR to reactivate the latent reservoirs. In addition, HSV-{Delta}ICP34.5 enhanced the phosphorylation of IKK/{beta} through the degradation of I{kappa}B, leading to p65 accumulation in the nucleus to elicit NF-{kappa}B pathway-dependent reactivation of HIV latency. Then, we constructed the recombinant HSV-{Delta}ICP34.5 expressing simian immunodeficiency virus (SIV) env, gag, or the fusion antigen sPD1-SIVgag as a therapeutic vaccine, aiming to achieve a functional cure by simultaneously reactivating viral latency and eliciting antigen-specific immune responses. Results showed that these constructs effectively elicited SIV-specific immune responses, reactivated SIV latency, and delayed viral rebound after the interruption of antiretroviral therapy (ART) in chronically SIV-infected rhesus macaques. Collectively, these findings provide insights into the rational design of HSV-vectored therapeutic strategies for pursuing an HIV functional cure.

microbiology↗

bioRxiv2024

High-Resolution Laminar Identification in Macaque Primary Visual Cortex Using Neuropixels Probes

Laminar electrode arrays allow simultaneous recording of activity of many cortical neurons and assignment to layers using current source density (CSD) analyses. Electrode arrays with 100-micron contact spacing have been used to estimate borders between layer 4 versus superficial or deep layers, but in macaque primary visual cortex (V1) there are far more layers, such as 4A which is only 50-100 microns thick. Neuropixels electrode arrays have 20-micron spacing, and thus could potentially discern thinner layers and more precisely identify laminar borders. Here we show that laminar distributions of CSDs lack consistency and the spatial resolution required for thin layers and accurate layer boundaries. To take full advantage of high density Neuropixels arrays, we have developed approaches based on higher resolution electrical signals and analyses, including spike waveforms and spatial spread, unit density, high-frequency action potential (AP) power spectrum, temporal power change, and coherence spectrum, that afford far higher resolution of laminar distinctions, including the ability to precisely detect the borders of even the thinnest layers of V1.

neuroscience↗

bioRxiv2023

A comprehensive alignment-filtering methodology improves phylogeny particularly by filtering overly divergent segments

O_LIAlignment problems may be complicated and diverse and the performance of the existing alignment-filtering tools, particularly their potential effect on phylogenetic reconstruction, have remained debated or limitedly explored. C_LIO_LIIn the present study, we developed a new R package named alignmentFilter to treat the diverse alignment problems, especially masking ambiguously-aligned or overly divergent segments using a newly devised grouping-regrouping algorithms acting on sequence divergence in each of sliding windows throughout alignment. Then we tested and compared the power and accuracy of the prevalent alignment-filtering tools, particularly the effect on phylogeny based on Angiospermae genome-scale data and simulated data. C_LIO_LIThe results indicate the alignment-filtering methods alone may affect the phylogeny decisively, producing (strongly-supported) phylogenetic conflicts. In most cases, alignmentFilter most efficiently improves phylogeny, yielding more congruent phylogeny regardless of phylogeny-reconstruction methods, producing phylogeny more concordant to the priori tree regarding to both topology and branch length in simulation, and most efficiently decreasing the root-to-tip length heterogeneity that is implied to be negatively correlated with topological accuracy. C_LIO_LIThe concepts and algorithms of alignmentFilter are quite distinct from existing alignment-filtering methods. It is not susceptible to specific types of alignment errors that usually baffle other methods and can reach the optimal solution frequently with lowest computation complexity. The biological or statistical sense of the key optional stringency-controlling parameter is more straightforward and the setting and outcome may be more adjustable or predictable. A better and more comprehensive solution of alignment problems in this study will benefit tremendous downstream analyses sourced from alignment and phylogeny. C_LI

bioinformatics↗

bioRxiv2023

Conserved and derived expression patterns and positive selection on dental genes reveal complex evolutionary context of ever-growing rodent molars

Continuously growing teeth are an important innovation in mammalian evolution, yet genetic regulation of continuous growth by stem cells remains incompletely understood. Dental stem cells responsible for tooth crown growth are lost at the onset of tooth root formation. Genetic signaling that initiates this loss is difficult to study with the ever-growing incisor and rooted molars of mice, the most common mammalian dental model species, because signals for root formation overlap with signals that pattern tooth size and shape (i.e., cusp patterns). Different species of voles (Cricetidae, Rodentia, Glires) have evolved rooted and unrooted molars that have similar size and shape, providing alternative models for studying roots. We assembled a de novo genome of Myodes glareolus, a vole with high-crowned, rooted molars, and performed genomic and transcriptomic analyses in a broad phylogenetic context of Glires (rodents and lagomorphs) to assess differential selection and evolution in tooth forming genes. We identified 15 dental genes with changing synteny relationships and six dental genes undergoing positive selection across Glires, two of which were undergoing positive selection in species with unrooted molars, Dspp and Aqp1. Decreased expression of both genes in prairie voles with unrooted molars compared to bank voles supports the presence of positive selection and may underlie differences in root formation. Bulk transcriptomics analyses of embryonic molar development in bank voles also demonstrated conserved patterns of dental gene expression compared to mice, with species-specific variation likely related to developmental timing and morphological differences between mouse and vole molars. Our results support ongoing evolution of dental genes across Glires, revealing the complex evolutionary background of convergent evolution for ever-growing molars.

evolutionary biology↗

bioRxiv2023

Histone marks enable formation of immiscible phase-separated chromatin compartments

Eukaryotic chromatin is organized into compartments for gene expression regulation, but the underlying mechanisms remain unclear. Here, we demonstrate that multivalent H3K27me3 and its reader, CBX7-PRC1 complex, regulate facultative heterochromatin via a phase separation mechanism, similar to constitutive heterochromatin1. Facultative and constitutive heterochromatin represent distinct, coexisting condensates in nuclei. In vitro, H3K27me3- and H3K9me3-marked nucleosomal arrays and their reader complexes can phase separate into immiscible condensates that are analogous to the relationship between facultative and constitutive heterochromatin in vivo. Moreover, overexpression of CBX7-PRC1 causes aberrant chromatin compartmentalization in vivo as demonstrated by H3K9me3 CUT&Tag and up-regulation of genes related to cancer such as acute myeloblastic leukemia (AML). CBX7 inhibitor effectively inhibits cancer cell proliferation possibly through phase separation-mediated compartment reorganization. Our data demonstrated how the specificity of compartmentalization is achieved based on the formation of immiscible phase-separated condensates, and offer novel epigenetic mechanistic insights into tumor development.

biochemistry↗

bioRxiv2023

Cell lineage analysis with somatic mutations reveals late divergence of neuronal cell types and cortical areas in human cerebral cortex

The mammalian cerebral cortex shows functional specialization into regions with distinct neuronal compositions, most strikingly in the human brain, but little is known in about how cellular lineages shape cortical regional variation and neuronal cell types during development. Here, we use somatic single nucleotide variants (sSNVs) to map lineages of neuronal sub-types and cortical regions. Early-occurring sSNVs rarely respect Brodmann area (BA) borders, while late-occurring sSNVs mark neuron-generating clones with modest regional restriction, though descendants often dispersed into neighboring BAs. Nevertheless, in visual cortex, BA17 contains 30-70% more sSNVs compared to the neighboring BA18, with clones across the BA17/18 border distributed asymmetrically and thus displaying different cortex-wide dispersion patterns. Moreover, we find that excitatory neuron-generating clones with modest regional restriction consistently share low-mosaic sSNVs with some inhibitory neurons, suggesting significant co-generation of excitatory and some inhibitory neurons in the dorsal cortex. Our analysis reveals human-specific cortical cell lineage patterns, with both regional inhomogeneities in progenitor proliferation and late divergence of excitatory/inhibitory lineages.

neuroscience↗

bioRxiv2023

An end-to-end approach for protein folding by integrating Cryo-EM maps and sequence evolution

Protein structure modeling is an important but challenging task. Recent breakthroughs in Cryo-EM technology have led to rapid accumulation of Cryo-EM density maps, which facilitate scientists to determine protein structures but it remains time-consuming. Fortunately, artificial intelligence has great potential in automating this process. In this study, we present SMARTFold, a deep learning protein structure prediction model combining sequence alignment features and Cryo-EM density map features. First, using density map, we sample representative points along the predicted high confidence areas of protein backbone. Then we extract geometric features of these points and integrate these features with sequence alignment features in our proposed protein folding model. Extensive experiments confirm that our model performs best on both single-chain and multi-chain benchmark dataset compared with state-of-the-art methods, which makes it a reliable tool for protein atomic structure determination from Cryo-EM maps.

bioinformatics↗

bioRxiv2023

M1 Macrophage-Related Genes Model for NSCLC Immunotherapy Response Prediction

2.Patients diagnosed with non-small cell lung cancer have a limited lifespan and exhibit poor immunotherapy outcomes. M1 macrophages have been found to be essential for anti-tumor immunity. This study aimed to develop an immunotherapy response evaluation model for NSCLC patients based on transcriptional expression. RNA sequencing profiles of 254 advanced-stage NSCLC patients treated with immunotherapy were downloaded from POPLAR and OAK projects. Immune cell infiltration in NSCLC patients has been examined, and thereafter different co-expressed genes were identified. Following that, the impact of M1 macrophage related genes on the prognosis of NSCLC patients was investigated. Six M1 macrophage co-expression genes, namely NKX2-1, CD8A, SFTA3, IL2RB, IDO1, and CXCL9, exhibited a strong association with the prognosis of NSCLC and served as effective predictors for immunotherapy response. A response model was constructed using Cox regression model and Lasso Cox regression analysis. The M1 genes were validated on our previous TD- FOREKNOW NSCLC clinical trial by RT-qPCR. The response model showed excellent immunotherapy response predicting and prognosis evaluating value in advanced stage of NSCLC. The model can effectively predict advanced NSCLC prognosis and aid in identifying patients who could benefit from customized immunotherapy as well as sensitive drugs.

immunology↗

bioRxiv2023

Sophocarpine inhibits TRP channels to produce anti-pruritic and analgesic effects in a mouse model of inflammatory itch and pain

Background and objectiveItch, an unpleasant sensation prompting the urge to scratch, and pain, aimed at detecting potential harm through acute withdrawal or protective behaviors, are increasingly recognized as interconnected phenomena. The co-occurrence of itch and pain symptoms in various diseases impairs therapeutic efficacy and the quality of life. In this study, we investigated the potential antipruritic and analgesic effects of sophocarpine (SC), an active compound of Sophorae Flavesentis Radix, in a murine model of inflammatory itch and pain, and sought to elucidate the underlying mechanisms. MethodThe anti-pruritic and analgesic effects of three doses of SC (60 mg/kg, 30 mg/kg, 10 mg/kg) were tested by analyzing the scratching and wiping behaviors in squaric acid dibutylester (SABDE)-induced allergic contact dermatitis (ACD) mouse model accompany by itch and pain, respectively. Psoriasis area and severity index (PASI) score was used to test the anti-inflammatory effect of SC. The underlying mechanisms were studied by real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting. Additionally, the anti-pruritic and analgesic effects of SC were further tested in mice with intradermal injection of allyl-isothiocyanate (AITC), a TRPA1 agonist, or capsaicin (CAP), a TRPV1 agonist, respectively. The relationships between SC, AITC, CAP and TRPV1, TRPA1 were simulated by molecular docking. ResultsSC treatment significantly decreased scratching bouts and wipes, as well as the PASI score. Administration of SC reduced the mRNA and protein expression of both TRPA1 and TRPV1. Moreover, pretreatment of SC decreased scratching bouts and wipes induced by AITC as well as by CAP. Molecular docking revealed potential competitive binding between SC and AITC on TRPA1, and SC and CAP on TRPV1. ConclusionWe demonstrated that SC has strong anti-pruritic and analgesic effects by targeting the TRPA1 and TRPV1 ion channels, and is a potential competitive inhibitor of TRPA1 and TRPV1. These findings suggest that SC has significant therapeutic potential in the therapy of diseases with inflammatory itch and pain.

pharmacology and toxicology↗

bioRxiv2023

Integrating large-scale meta-GWAS and PigGTEx resources to decipher the genetic basis of complex traits in pig.

Understanding the molecular and cellular mechanisms that underlie complex traits in pigs is crucial for enhancing their genetic improvement program and unleashing their substantial potentials in human biomedicine research. Here, we conducted a meta-GWAS analysis for 232 complex traits with 28.3 million imputed whole-genome sequence variants in 70,328 individuals from 14 pig breeds. We identified a total of 6,878 genomic regions associated with 139 complex traits. By integrating with the Pig Genotype-Tissue Expression (PigGTEx) resource, we systemically explored the biological context and regulatory circuits through which these trait-associated variants act and finally prioritized 16,664 variant-gene-tissue-trait circuits. For instance, rs344053754 regulates the expression of UGT2B31 in the liver by affecting the activity of regulatory elements and ultimately influences litter weight at weaning. Furthermore, we investigated the conservation of genetic and regulatory mechanisms underlying 136 human traits and 232 pig traits. Overall, our multi-breed meta-GWAS in pigs provides invaluable resources and novel insights for understanding the regulatory and evolutionary mechanisms of complex traits in both pigs and humans.

genetics↗

bioRxiv2023

Probing Molecular Diversity and Ultrastructure of Brain Cells with Fluorescent Aptamers

Detergent-free immunolabeling has been proven feasible for correlated light and electron microscopy, but its application is restricted by the availability of suitable affinity reagents. Here we introduce CAptVE, a method using slow off-rate modified aptamers for cell fluorescence labeling on ultrastructurally reconstructable electron micrographs. CAptVE provides labeling for a wide range of biomarkers, offering a pathway to integrate molecular analysis into recent approaches to delineate neural circuits via connectomics.

neuroscience↗

bioRxiv2023

Cellular protein painting for structural and binding sites analysis via lysine reactivity profiling with o-phthalaldehyde

The three-dimensional structure and the molecular interaction of proteins determine their roles in many cellular processes. Chemical protein painting with protein mass spectrometry can identify changes in structural conformations and molecular interactions of proteins including their binding sites. Nevertheless, most current protein painting techniques identified protein targets and binding sites of drugs in vitro using cell lysate or purified protein. Here, we screened 11 membrane-permeable lysine-reactive chemical probes for intracellular covalent labeling of endogenous proteins, which reveals ortho-phthalaldehyde (OPA) as the most reactive probe in intracellular environment. An MS workflow was developed and coupled with a new data analysis strategy termed RAPID (Reactive Amino acid Profiling by Inverse Detection) to enhance detection sensitivity. RAPID-OPA successfully identified structural change induced by allosteric drug TEPP-46 on its target protein PKM2, and was applied to profile conformation change of the proteome occurring in cells during thermal denaturation. Application of RAPID-OPA on cells treated with geldanamycin, selumetinib, and staurosporine successfully revealed their binding sites on target proteins. Thus, RAPID-OPA for cellular protein painting permits the identification of ligand-binding sites and detection of protein structural changes occurring in cells. Significance StatementProtein painting can be used to identify changes in the three-dimensional structure and molecular interaction of proteins that govern many cellular processes but are mostly applied to cell lysate or purified protein. We identified lysine reactive probes for the intracellular labeling of endogenous proteins, and developed an MS procedure with new data analysis strategy termed RAPID-OPA to characterize the intracellular conformation change of the proteome during thermal denaturation, and identified structural change mediated by allosteric regulator TEPP-46 on target protein PKM2. Furthermore, the approach could identify ligand binding sites exemplified by labeling of target proteins in cells treated with geldanamycin, selumetinib and staurosporine. Overall, RAPID-OPA for cellular protein painting enables the detection of protein structural changes happening in cells as well as the identification of ligand-binding sites.

cell biology↗

bioRxiv2023

Understanding the biosynthesis of human IgMs through a combinatorial expression of mutant subunits that affect different assembly steps

Polymeric IgMs are secreted from plasma cells abundantly despite their structural complexity and intricate multimerization steps. To gain new insights into IgMs assembly mechanics that underwrite the high-level secretion, we characterized the biosynthetic process of a natural human IgM, SAM-6, using a recombinant HEK293 cell system. By creating a series of mutant subunits that differentially disrupt specific sets of inter-chain disulfide bonds, we assessed their effects on various aspects of IgM biosynthesis in 48 different mutant subunit combinations. The analysis included the visualization of intracellular biosynthetic events such as steady-state subcellular subunit distribution, secretory trafficking bottlenecks, and the ER-associated Russell body formation by fluorescent microscopy. We also characterized various extracellular events including secreted IgM product quality, secretion output, and the release of various assembly intermediates using biochemical and biophysical assays. In this combinatorial mutagenesis approach, we unexpectedly found that the loss of multiple inter-chain disulfide bonds, including the one between HC and {lambda}LC subunits, was tolerated in polymeric IgM formation and secretion. This finding revealed the vital role of underlying non-covalent protein-protein association not only during the orchestration of initial subunit interactions but also in maintaining the polymeric IgM product integrity during ER quality control steps, secretory pathway trafficking, and secretion. We suggest that the IgM assembly process is inherently robust and has a stopgap that permits the secretion of polymeric IgM even when not all the prescribed inter-chain disulfide bonds are formed. This study holistically presents the requirements and exemptions in polymeric IgM biosynthesis by encompassing the characterization of intracellular and extracellular events and the roles of covalent and non-covalent interactions. These findings can guide antibody engineering strategy when designing IgM-based multivalent modalities.

cell biology↗

bioRxiv2023

PRO-LDM: Protein Sequence Generation with Conditional Latent Diffusion Models

AbstractsDeep learning-driven protein design holds enormous potential despite the complexities in sequences and structures. Recent developments in diffusion models yielded success in structure design, but awaits progress in sequence design and are computationally demanding. Here we present PRO-LDM: an efficient framework combining design fidelity and computational efficiency, utilizing the diffusion model in latent space to design proteins with property tuning. The model employs a joint autoencoder to capture latent variable distributions and generate meaningful embeddings from sequences. PRO-LDM (1) learns representations from biological features in natural proteins at both amino-acid and sequence level; (2) generates native-like new sequences with enhanced diversity; and (3) conditionally designs new proteins with tailored properties or functions. The out-of-distribution design enables sampling notably different sequences by adjusting classifier guidance strength. Our model presents a feasible pathway and an integratable tool to extract physicochemical and evolutionary information embedded within primary sequences, for protein design and optimization.

bioengineering↗

bioRxiv2023

Molecular dynamics analyses of CLDN15 pore size and charge selectivity

Claudin-15 (CLDN15) molecules form channels that directly regulate cation and water transport. In the gastrointestinal tract, this transport indirectly impacts nutrient absorption. However, the mechanisms governing ion transport through these channels remain poorly understood. We addressed this question by building on our previous cell culture studies and all atom molecular dynamic simulation model of CLDN15. By mutating D55 to a bulkier glutamic acid (E) or neutral amino acid asparagine (N), our in vitro measurements showed that the D55E mutation decreased charge selectivity and favored small ion permeability, while the D55N mutation led to reduced charge selectivity without markedly altering size selectivity. By establishing a simplified (reduced) CLDN15 molecular dynamics model that excludes non-essential transmembrane regions, we were able to probe how D55 modified cation dehydration, charge interaction, and permeability. These results provide novel insight into organization of the CLDN15 selectivity filter and suggests that D55 plays a dual role in shaping both electrostatic and steric properties of the pore, but its electrostatic role is more prominent in determining CLDN15 cation permeability. This knowledge can be used toward the development of effective strategies to modulate CLDN15 function. The experimental approach established can be further extended to study the function of other claudin channels. Together, these advancements will help us to modulate tight junctions to promote human health. SUMMARYCell culture and molecular dynamics simulations reveal the role of the CLDN15-D55 residue in ion size and charge selectivity. A reduced CLDN15 model offers novel insights into ion conductance, providing a valuable tool for therapeutic modulation of tight junctions.

physiology↗

bioRxiv2023

Pneumonic plague protection induced by a monophosphoryl lipid A decorated Yersinia outer-membrane-vesicle vaccine

A newly constructed Yersinia pseudotuberculosis mutant (YptbS46) carrying the lpxE insertion and pmrF-J deletion exclusively synthesized an adjuvant form of lipid A, monophosphoryl lipid A (MPLA). Outer membrane vesicles (OMVs) isolated from YptbS46 harboring an lcrV expression plasmid, pSMV13, were designated OMV46-LcrV, which contained MPLA and high amounts of LcrV and displayed low activation of Toll-like receptor 4 (TLR4). Similar to the previous OMV44-LcrV, intramuscular prime-boost immunization with 30 {micro}g of OMV46-LcrV exhibited substantially reduced reactogenicity and conferred complete protection to mice against a high-dose of respiratory Y. pestis challenge. OMV46-LcrV immunization induced robust adaptive responses in both lung mucosal and systemic compartments and orchestrated innate immunity in the lung, which were correlated with rapid bacterial clearance and unremarkable lung damage during Y. pestis challenge. Additionally, OMV46-LcrV immunization conferred long-term protection. Moreover, immunization with reduced doses of OMV46-LcrV exhibited further lower reactogenicity and still provided great protection against pneumonic plague. Our studies strongly demonstrate the feasibility of OMV46-LcrV as a new type of plague vaccine candidate.

immunology↗