Biology subjects

Li, B.

Publications and source records attributed to Li, B..

At least 415 records · Page 23Linked to original sources

bioRxiv2022

Characterization and identification of a novel daidzein reductase involved in (S)-equol biosynthesis in Clostridium C1

(S)-equol is an isoflavone with high estrogen-like activity and no toxic effects in the human body, and is only produced by some gut bacteria in vivo. It plays an important role in maintaining individual health, however, the dearth of resources associated with (S)-equol-producing bacteria has seriously restricted the production and application of (S)-equol. We report here a novel functional gene C1-07020 that was identified from a chick (S)-equol-producing bacterium (Clostridium C1). We found that recombinant protein of C1-07020 possessed similar function to daidzein reductase (DZNR), which can convert daidzein (DZN) into R/S-dihydrodaidzein (R/S-DHD). Interestingly, C1-07020 can reverse convert (R/S)-DHD (DHD oxidases) into DZN even without cofactors or anaerobic conditions. Additionally, high concentrations of (S)-equol can directly promote DHD oxidase but inhibit DZNR activity. Molecular docking and site-directed mutagenesis revealed that the amino acid Arg 75 was the active site of DHD oxidases. Subsequently, an engineered E. coli strain based on C1-07020 was constructed and showed higher yield of (S)-equol than the engineered bacteria from our previous work. Metagenomics analysis and PCR detection surprisingly revealed that C1-07020 and related bacteria may be prevalent in the gut of humans and animals and their (S)-equol production state may cause differed between (S)-equol producer and non-producer. Overall, a novel DZNR from Clostridium C1 was found and identified in this study, and its bidirectional enzyme activities and wide distribution in the gut of humans and animals provide alternative strategies for revealing the individual regulatory mechanisms of (S)-equol-producing bacteria. Importance(S)-equol is a final product of DZN that metabolized by some enteric bacteria. Although (S)-equol played very important roles in maintaining human health, larger differences in equol production varied between different populations. Here, a novel DZNR gene C1-07020, which related to (S)-equol production, was reported. The bidirectional enzyme functions and wide distribution of C1-07020 in human and animal gut provided additional insights into the metabolic regulation of (S)-equol. Additionally, C1-07020 can be used for improving the production of (S)-equol in vitro.

microbiology↗

bioRxiv2022

CRISPR-based targeted haplotype-resolved assemblies of a megabase region

Constructing high-quality haplotype-resolved genome assemblies has substantially improved the ability to detect and characterize genetic variants. A targeted approach providing readily access to the rich information from haplotype-resolved genome assemblies will be appealing to groups of basic researchers and medical scientists focused on specific genomic regions. Here, using the 4.5 megabase, notoriously difficult-to-assemble major histocompatibility complex (MHC) region as an example, we demonstrated an approach to construct haplotype-resolved de novo assemblies of targeted genomic regions with the CRISPR-based enrichment. Compared to the results from haplotype-resolved genome assemblies, our targeted approach achieved comparable completeness and accuracy with greatly reduced computing complexity, sequencing cost, as well as the amount of starting materials. Moreover, using the targeted assembled personal haplotypes as the reference both improves the quantification accuracy for sequencing data and enables allele-specific functional genomics analyses. Given its highly efficient use of resources, our approach can greatly facilitate population genetic studies of targeted regions, and may pave a new way to elucidate the molecular mechanisms in disease etiology.

genomics↗

bioRxiv2022

Membrane-mediated interactions between hinge-like particles

Adsorption of nanoparticles on a membrane can give rise to interactions between particles, mediated by membrane deformations, that play an important role in self-assembly and membrane remodeling. Previous theoretical and experimental research has focused on nanoparticles with fixed shapes, such as spherical, rod-like, and curved nanoparticles. Recently, hinge-like DNA origami nanostructures have been designed with tunable mechanical properties. Inspired by this, we investigate the equilibrium properties of hinge-like particles adsorbed on an elastic membrane using Monte Carlo and umbrella sampling simulations. The configurations of an isolated particle are influenced by competition between bending energies of the membrane and the particle, which can be controlled by changing adsorption strength and hinge stiffness. When two adsorbed particles interact, they effectively repel one another when the strength of adhesion to the membrane is weak. However, a strong adhesive interaction induces an effective attraction between the particles, which drives their aggregation. The configurations of the aggregate can be tuned by adjusting the hinge stiffness: Tip-to-tip aggregation occurs for flexible hinges, whereas tip-to-middle aggregation also occurs for stiffer hinges. Our results highlight the potential for using the mechanical features of deformable nanoparticles to influence their self-assembly when the particles and membrane mutually influence one another.

biophysics↗

bioRxiv2022

Interactive effect matters: a combination of herbivory degree and the ratio of generalist to specialist better predicts evolution of plant defense

Herbivory degree and the ratio of generalist to specialist herbivores have long been treated as two important but independent factors in shaping the evolution of plant defense. However, this assumption of independency is poorly supported and has resulted in great controversy in explaining the patterns of plant defense. Here we investigated the possible interaction between herbivory degree and generalist-to-specialist ratio using a cost-benefit model of defense evolution in plants. Our results showed that, with increasing generalist herbivore proportion, plant defense investment increases when herbivory degree is low and decreases when herbivory degree is high. These results provide the first theoretical support for the interactive effect of herbivory degree and ratio of generalist/specialist affecting plant defense, which integrate many of the previous results (e.g. latitudinal patterns of plant defense and defense evolution of invasive plants) and put them into a more general theoretical context.

evolutionary biology↗

bioRxiv2022

Highly efficient multiplex genome editing in dicot plants using improved CRISPR/Cas systems

CRISPR/Cas9-mediated gene editing provides a powerful tool for dissecting gene function and improving important traits in crops. However, there are still persisting challenges to obtain high homozygous/bi-allelic (ho/bi) mutations in dicot plants. Here, we develop an improved CRISPR/Cas9 system harboring a calreticulin-like gene promoter, which can boost targeted mutations in dicots. Additionally, the pDC45_dsg construct, combining a 35Spro-tRNA_sgRNA-EU unit and PCE8pro-controlled Cas9, can achieve more than 80.0% ho/bi mutations at target sites in allotetraploid tobacco. We construct pDC45_Fast system that can simultaneously fulfill gene editing and shorten the life span of T0 generation tobacco and tomato. This study provides new tools for improving targeted gene mutagenesis in dicots, and makes manipulations of genes in Solanum more feasible.

plant biology↗

bioRxiv2022

Drosophila Mechanical Nociceptors Preferentially Sense Localized Poking

Mechanical nociception is an evolutionarily conserved sensory process required for the survival of living organisms. Previous studies have revealed much about the neural circuits and key sensory molecules in mechanical nociception, but the cellular mechanisms adopted by nociceptors in force detection remain elusive. To address this issue, we study the mechanosensation of a fly larval nociceptor (class IV da neurons, c4da) using a customized mechanical device. We find that c4da are sensitive to mN-scale forces and make uniform responses to the forces applied at different dendritic regions. Moreover, c4da showed a greater sensitivity to more localized forces, consistent with them being able to sense the poking of sharp objects, such as wasp ovipositor. Further analysis reveals that high morphological complexity, mechanosensitivity to lateral tension and active signal propagation in the dendrites altogether facilitate the mechanosensitivity and sensory features of c4da. In particular, we discover that Piezo and Ppk1/Ppk26, two key mechanosensory molecules, make differential but additive contributions to the mechanosensation of c4da. In all, our results provide updates into understanding how c4da process mechanical signals at the cellular level and reveal the contributions of key molecules.

neuroscience↗

bioRxiv2022

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

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

molecular biology↗

bioRxiv2021

Prefrontal top-down projections control context-dependent strategy selection

The rules governing behavior often vary with behavioral contexts. As a consequence, an action rewarded in one context may be discouraged in another. Animals and humans are capable of switching between behavioral strategies under different contexts and acting adaptively according to the variable rules, a flexibility that is thought to be mediated by the prefrontal cortex (PFC)1-4. However, how the PFC orchestrates context-dependent switch of strategies remains unclear. Here we show that pathway-specific projection neurons in the medial PFC (mPFC) differentially contribute to context-instructed strategy selection. In a decision-making task in which mice have been trained to flexibly switch between a previously established rule and a newly learned rule in a context-dependent manner, the activity of mPFC neurons projecting to the dorsomedial striatum encodes the contexts, and further represents decision strategies conforming to the old and new rules. Moreover, the activity of these neuron is required for context-instructed strategy selection. In contrast, the activity of mPFC neurons projecting to the ventral midline thalamus does not discriminate between the contexts, and represents the old rule even if mice have adopted the new one; furthermore, these neurons act to prevent the strategy switch under the new rule. Our results suggest that the mPFC[->]striatum pathway promotes flexible strategy selection guided by contexts, whereas the mPFC[->]thalamus pathway favors fixed strategy selection by preserving old rules. Balanced activity between the two pathways may be critical for adaptive behaviors.

neuroscience↗

bioRxiv2021

Salubrinal induces fetal hemoglobin expression via the stress-signaling pathway in human sickle erythroid progenitors and sickle cell disease mice

Sickle cell disease (SCD) is an inherited blood disorder caused by a mutation in the HBB gene leading to hemoglobin S production and polymerization under hypoxia conditions leading to vaso-occlusion, chronic hemolysis, and progressive organ damage. This disease affects [~]100,000 people in the United States and millions worldwide. An effective therapy for SCD is fetal hemoglobin (HbF) induction by pharmacologic agents such as hydroxyurea, the only Food and Drug Administration-approved drug for this purpose. Therefore, the goal of our study was to determine whether salubrinal (SAL), a selective protein phosphatase 1 inhibitor, induces HbF expression through the stress-signaling pathway by activation of p-eIF2 and ATF4 trans-activation in the {gamma}-globin gene promoter. Sickle erythroid progenitors treated with 24{micro}M SAL increased F-cells levels 1.4-fold (p=0.021) and produced an 80% decrease in reactive oxygen species. Western blot analysis showed SAL enhanced HbF protein by 1.6-fold (p=0.0441), along with dose-dependent increases of p-eIF2 and ATF4 levels. Subsequent treatment of SCD mice by a single intraperitoneal injection of SAL (5mg/kg) produced peak plasma concentrations at 6 hours. Chronic treatments of SCD mice with SAL mediated a 2.3-fold increase in F-cells (p=0.0013) and decreased sickle erythrocytes supporting in vivo HbF induction.

molecular biology↗

bioRxiv2021

Isolation and characterization of fucosylated extracellular vesicles based on a novel GlyExo-Capture technique

Owing to their diagnostic and therapeutic potential, extracellular vesicles (EVs) derived from tumour cells have recently garnered great interest. The presence of different glycosylation sites at the EV surface supports the need for efficient glycosylated EV isolation. Here, we developed a GlyExo-Capture technique for robustly capturing fucosylated EVs from sera and cell supernatants. Lens culinaris lectin (LCA)-immobilized magnetic complexes were found to capture approximately 60% of the total EVs from HepG2 cells. The capture efficiency was reduced to less than 40% in nontumorigenic MIHA cells. Notably, the cellular uptake pattern of highly fucosylated EVs was markedly different from that of EVs with low fucosylation. The unearthing of enriched fucosylated EV miRNA cargos by next-generation deep sequencing (NGS) revealed 75 differentially expressed miRNAs (DEMs) in hepatocellular carcinoma (HCC). Among them, a 4-miRNA panel was chosen and yielded an area under the ROC curve (AUC) of 0.86 and 0.84 for the detection of HCC from non-HCC controls in testing samples and independent validation samples, respectively. The 4-miRNA signature was independent of alpha-fetoprotein (AFP), and a combined model with AFP yielded an increased AUC of 0.92. In conclusion, we developed a high-throughput method for capturing fucosylated EVs efficiently and shed light on the use of fucosylated EVs as potential sources of miRNAs for cancer biomarker detection.

cancer biology↗

bioRxiv2021

Mutations in the spike RBD of SARS-CoV-2 omicron variant may increase infectivity without dramatically altering the efficacy of current multi-dosage vaccinations

With the continuous evolution of SARS-CoV-2, variants of concern (VOCs) and their mutations are a focus of rapid assessment. Vital mutations in the VOC are found in spike protein, particularly in the receptor binding domain (RBD), which directly interacts with ACE2 on the host cell membrane, a key determinant of the binding affinity and cell entry. With the reporting of the most recent VOC, omicron, we performed amino acid sequence alignment of the omicron spike protein with that of the wild type and other VOCs. Although it shares several conserved mutations with other variants, we found that omicron has a large number of unique mutations. We applied the Hopp-Woods scale to calculate the hydrophilicity scores of the amino acid stretches of the RBD and the entire spike protein, and found 3 new hydrophilic regions in the RBD of omicron, implying exposure to water, with the potential to bind proteins such as ACE2 increasing transmissibility and infectivity. However, careful analysis reveals that most of the exposed domains of spike protein can serve as antigenic epitopes for generating B cell and T cell-mediated immune responses. This suggests that in the collection of polyclonal antibodies to various epitopes generated after multiple doses of vaccination, some can likely still bind to the omicron spike protein and the RBD to prevent severe clinical disease. In summary, while the omicron variant might result in more infectivity, it can still bind to a reasonable repertoire of antibodies generated by multiple doses of current vaccines likely preventing severe disease. Effective vaccines may not universally prevent opportunistic infections but can prevent the sequelae of severe disease, as observed for the delta variant. This might still be the case with the omicron variant, albeit, with increased frequency of infection.

biochemistry↗

bioRxiv2021

Scaling between DNA and cell size governs bacterial growth homeostasis and resource allocation

Bacteria maintain a stable cell size and a certain DNA content through proliferation as described by classic growth laws. How cells behave when this inherent scaling is broken, however, has rarely been interrogated. Here we engineered Escherichia coli cells with extremely low DNA contents using a tunable synthetic tool CRISPRori that temporarily inhibited chromosome replication initiation. A detailed mechanistic model coupling DNA replication, cell growth, and division revealed a fundamental DNA-centric growth law, which was validated by two observations. First, lineage dynamics were robust to large CRISPRori perturbations with division cycles rapidly restoring through a timer mechanism rather than the adder rule. Second, cellular growth transitioned into a linear regime at low DNA-cytoplasm ratios. Experiments and theory showed that in this regime, cellular resource was redirected to plasmid-borne gene expression. Together with the ability of CRISPRori to bi-directionally modulate plasmid copy numbers, these findings suggest a novel strategy for bio-production enhancement.

systems biology↗

bioRxiv2021

An active learning framework improves tumor variant interpretation

For precision medicine to reach its full potential for treatment of cancer and other diseases, protein variant effect prediction tools are needed that characterize variants of unknown significance (VUS) in a patients genome with respect to their likelihood to influence treatment response and outcomes. However, the performance of most variant prediction tools is limited by the difficulty of acquiring sufficient training and validation data. To overcome these limitations, we applied an iterative active learning approach starting from available biochemical, evolutionary, and functional annotations. The potential of active learning to improve variant interpretation was first demonstrated by applying it to synthetic and deep mutational scanning (DMS) datasets for four cancer-relevant proteins. We then probed its utility to guide interpretation and functional validation of tumor VUS in a potential biomarker for cancer therapy sensitivity, the nucleotide excision repair (NER) protein Xeroderma Pigmentosum Complementation Group A (XPA). A quantitative high-throughput cell-based NER activity assay, fluorescence-based multiplex flow-cytometric host cell reactivation (FM-HCR), was used to validate XPA VUS selected by the active learning strategy. In all cases, selecting VUS for validation by active learning yielded an improvement in performance over traditional learning. These analyses suggest that active learning is well-suited to significantly improve interpretation of VUS and cancer patient genomes.

cancer biology↗

bioRxiv2021

Transcription factor induction of vascular blood stem cell niches in vivo

The hematopoietic niche is a supportive microenvironment comprised of distinct cell types, including specialized vascular endothelial cells that directly interact with hematopoietic stem and progenitor cells (HSPCs). The molecular factors that specify niche endothelial cells and orchestrate HSPC homeostasis remain largely unknown. Using multi-dimensional gene expression and chromatin accessibility analyses, we define a conserved gene expression signature and cis-regulatory landscape unique to sinusoidal endothelial cells in the HSPC niche. Using enhancer mutagenesis and transcription factor overexpression, we elucidate a transcriptional code involving members of the Ets, Sox and Nuclear Hormone Receptor families that is sufficient to induce ectopic niche endothelial cells that associate with mesenchymal stromal cells and support the recruitment, maintenance and division of HSPCs in vivo. These studies set forth an approach for generating synthetic HSPC niches, in vitro or in vivo, and for effective therapies to modulate the endogenous niche.

developmental biology↗

bioRxiv2021

JAK-STAT Signaling Enables Lineage Plasticity-driven AR Targeted Therapy Resistance

Emerging evidence indicates that various cancers can gain resistance to targeted therapies by acquiring lineage plasticity. Although various genomic and transcriptomic aberrations correlate with lineage plasticity-driven resistance, the molecular mechanisms of acquiring lineage plasticity have not been fully elucidated. Through integrated transcriptomic and single cell RNA-Seq (scRNA-Seq) analysis of more than 80,000 cells, we reveal for the first time that the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling is a crucial executor in promoting lineage plasticity-driven AR targeted therapy resistance in prostate cancer. Ectopic activation of JAK-STAT signaling is specifically required for the AR targeted therapy resistance of subclones expressing multilineage, stem-like and epithelial-to-mesenchymal transition (EMT) lineage transcriptional programs and represents a potential therapeutic target for overcoming AR targeted therapy resistance. One-Sentence SummaryJAK-STAT signaling is a crucial executor in promoting lineage plasticity-driven AR therapy resistance in prostate cancer.

cancer biology↗

bioRxiv2021

Biology-inspired data-driven quality control for scientific discovery in single-cell transcriptomics

Quality control (QC) of cells, a critical step in single-cell RNA sequencing data analysis, has largely relied on arbitrarily fixed data-agnostic thresholds on QC metrics such as gene complexity and fraction of reads mapping to mitochondrial genes. The few existing data-driven approaches perform QC at the level of samples or studies without accounting for biological variation in the commonly used QC criteria. We demonstrate that the QC metrics vary both at the tissue and cell state level across technologies, study conditions, and species. We propose data-driven QC (ddqc), an unsupervised adaptive quality control framework that performs flexible and data-driven quality control at the level of cell states while retaining critical biological insights and improved power for downstream analysis. On applying ddqc to 6,228,212 cells and 835 mouse and human samples, we retain a median of 39.7% more cells when compared to conventional data-agnostic QC filters. With ddqc, we recover biologically meaningful trends in gene complexity and ribosomal expression among cell-types enabling exploration of cell states with minimal transcriptional diversity or maximum ribosomal protein expression. Moreover, ddqc allows us to retain cell-types often lost by conventional QC such as metabolically active parenchymal cells, and specialized cells such as neutrophils or gastric chief cells. Taken together, our work proposes a revised paradigm to quality filtering best practices - iterative QC, providing a data-driven quality control framework compatible with observed biological diversity.

bioinformatics↗

bioRxiv2021

Lactate Increases Stemness of CD8+ T Cells to Augment Anti-Tumor Immunity

Nutrients and metabolites play important roles in immune functions. Recent studies show lactate instead of glucose can serve as a primary carbon fuel source for most tissues. The role of lactate in tumor immunity is not well understood with immune suppressive functions reported for lactic acid, the conjugate acid form of lactate. In this study, we report lactate increases the stemness of CD8+ T cells and augments anti-tumor immunity. Subcutaneous administration of lactate but not glucose shows CD8+ T cell-dependent tumor growth inhibition. Single cell transcriptomics analysis revealed lactate treatment increased a subpopulation of stem-like TCF-1-expressing CD8+ T cells, which is further validated by ex vivo culture of CD8+ T cells from mouse splenocytes and human peripheral blood mononuclear cells. The inhibition of histone deacetylase activity by lactate increased acetylation in the histone H3K27 site at the Tcf7 super enhancer locus and increased the gene expression of Tcf7. Adoptive transfer of CD8+ T cells pretreated with lactate in vitro showed potent tumor growth inhibition in vivo. Our results elucidate the immune protective role of lactate in anti-tumor immunity without the masking effect of acid. These results may have broad implications for T cell therapy and the understanding of lactate in immune metabolism.

immunology↗

bioRxiv2021

Microscopic Quantification of Oxygen Consumption across Cortical Layers

The cerebral cortex is organized in cortical layers that differ in their cellular density, composition, and wiring. Cortical laminar architecture is also readily revealed by staining for cytochrome oxidase - the last enzyme in the respiratory electron transport chain located in the inner mitochondrial membrane. It has been hypothesized that a high-density band of cytochrome oxidase in cortical layer IV reflects higher oxygen consumption under baseline (unstimulated) conditions. Here, we tested the above hypothesis using direct measurements of the partial pressure of O2 (pO2) in cortical tissue by means of 2-photon phosphorescence lifetime microscopy (2PLM). We revisited our previously developed method for extraction of the cerebral metabolic rate of O2 (CMRO2) based on 2-photon pO2 measurements around diving arterioles and applied this method to estimate baseline CMRO2 in awake mice across cortical layers. To our surprise, our results revealed a decrease in baseline CMRO2 from layer I to layer IV. This decrease of CMRO2 with cortical depth was paralleled by an increase in tissue oxygenation. Higher baseline oxygenation and cytochrome density in layer IV may serve as an O2 reserve during surges of neuronal activity or certain metabolically active brain states rather than baseline energy needs. Our study provides the first quantification of microscopically resolved CMRO2 across cortical layers as a step towards informed interpretation and modeling of cortical-layer-specific Blood Oxygen Level Dependent (BOLD) functional Magnetic Resonance Imaging (fMRI) signals.

neuroscience↗