Biology subjects

Wu, L.

Publications and source records attributed to Wu, L..

At least 253 records · Page 14Linked to original sources

bioRxiv2021

CDCP: a visualization and analyzing platform for single-cell datasets

Advances in single-cell sequencing technology provide a unique approach to characterize the heterogeneity and distinctive functional states at single-cell resolution, leading to rapid accumulation of large-scale single-cell datasets. A big challenge undertaken by research community especially bench scientists is how to simplify the way of retrieving, processing and analyzing the huge number of datasets. Towards this end, we developed Cell-omics Data Coordinate Platform (CDCP), a platform that aims to share and integrate comprehensive single-cell datasets, and to provide a network analysis toolkit for personalized analysis. CDCP contains single-cell RNA-seq and ATAC-seq datasets of 474 572 cells from 6 459 samples in species covering humans, non-human primate models and other animals. It allows querying and visualization of interested datasets and the expression profile of distinct genes in different cell clusters and cell types. Besides, this platform provides an analysis pipeline for non-bioinformatician experimental scientists to address questions not focused by the submitters of the datasets. In summary, CDCP provides a user-friendly interface for researchers to explore, visualize, analyze, download and submit published single-cell datasets and it will be a valuable resource for investigators to explore the global transcriptome profiling at single-cell level.

bioinformatics↗

bioRxiv2021

Ecological dynamics of the gut microbiome in response to dietary fiber

Dietary fibers are generally thought to benefit intestinal health. Their impacts on the composition and metabolic function of the gut microbiome, however, vary greatly across individuals. Previous research showed that each individuals response to fibers depends on their baseline gut microbiome, but the ecology driving microbiota remodeling during fiber intake remained unclear. Here, we studied the long-term dynamics of gut microbiome and short-chain fatty acids (SCFAs) in isogenic mice with distinct microbiota baselines fed with the fermentable fiber inulin compared to the non-fermentable fiber cellulose. We found that inulin produced generally rapid response followed by gradual stabilization to new equilibria, and those dynamics were baseline-dependent. We parameterized an ecology model from the timeseries data, which revealed a group of bacteria whose growth significantly increases in response to inulin. and whose baseline abundance and interspecies competition explains the baseline-dependence of microbiome density and community composition dynamics. Fecal levels of of SCFAs, such as propionate, is associated with the abundance of inulin responders, yet inter-individual variation of gut microbiome impedes the prediction of SCFAs by machine learning models. Finally, we showed that our methods and major findings are generalizable to dietary resistant starch. This study emphasizes the importance of ecological modeling to understand microbiome responses to dietary changes and the need for personalized interventions.

ecology↗

bioRxiv2021

Ageing impairs the airway epithelium defence response to SARS-CoV-2

Age-dependent differences in the clinical response to SARS-CoV-2 infection is well-documented1-3 however the underlying molecular mechanisms involved are poorly understood. We infected fully differentiated human nasal epithelium cultures derived from healthy children (1-12 years old), young adults (26-34 years old) and older adults (56-62 years old) with SARS-COV-2 to identify age-related cell-intrinsic differences that may influence viral entry, replication and host defence response. We integrated imaging, transcriptomics, proteomics and biochemical assays revealing age-related changes in transcriptional regulation that impact viral replication, effectiveness of host responses and therapeutic drug targets. Viral load was lowest in infected older adult cultures despite the highest expression of SARS-CoV-2 entry and detection factors. We showed this was likely due to lower expression of hijacked host machinery essential for viral replication. Unlike the nasal epithelium of young adults and children, global host response and induction of the interferon signalling was profoundly impaired in older adults, which preferentially expressed proinflammatory cytokines mirroring the "cytokine storm" seen in severe COVID-194,5. In silico screening of our virus-host-drug network identified drug classes with higher efficacy in older adults. Collectively, our data suggests that cellular alterations that occur during ageing impact the ability for the host nasal epithelium to respond to SARS-CoV-2 infection which could guide future therapeutic strategies.

immunology↗

bioRxiv2021

Advancing quality-control for NGS measurement of actionable mutations in circulating tumor DNA

The primary objective of the FDA-led Sequencing and Quality Control Phase 2 (SEQC2) project is to develop standard analysis protocols and quality control metrics for use in DNA testing to enhance scientific research and precision medicine. This study reports a targeted next generation sequencing (NGS) method that enables more accurate detection of actionable mutations in circulating tumor DNA (ctDNA) clinical specimens. This advancement was enabled by designing a synthetic internal standard spike-in for each actionable mutation target, suitable for use in NGS following hybrid-capture enrichment and unique molecular index (UMI) or non-UMI library preparation. When mixed with contrived ctDNA reference samples, internal standards enabled calculation of technical error rate, limit of blank, and limit of detection for each variant at each nucleotide position, in each sample. True positive mutations with variant allele fraction too low for detection by current practice were detected with this method, thereby increasing sensitivity.

genomics↗

bioRxiv2021

piRNA pathway is essential for generating functional oocytes in golden hamster

Piwi-interacting RNAs (piRNAs) are small RNAs predominantly expressed in germ cells that are critical for gametogenesis in various species. However, PIWI-deficient female mice are fertile and mouse oocytes express a panel of small RNAs that do not appear widely representative of mammals, and piRNA function in the oogenesis of other mammals has therefore remained elusive. Recent studies revealed the small RNA and PIWI transcriptional profiles in golden hamster oocytes more closely resemble that of humans than mice. Herein, we generated PIWIL1-, PLD6- and MOV10L1-deficient golden hamsters and found that all female mutants were sterile, with embryos arrested at the two-cell stage. In PIWIL1 mutant oocytes, we observed transposon accumulation and broad transcriptomic dysregulation, while zygotic gene activation was impaired in early embryos. Intriguingly, PIWIL1-piRNAs exhibited a unique, preferential silencing of endogenous retroviruses (ERVs), whereas silencing LINE1s depended on both PIWIL1- and PIWIL3-piRNAs. Moreover, we showed that piRNAs participate in the degradation of maternal mRNAs in MII oocytes and embryos via partially complementary targets. Together, our findings demonstrate that piRNAs are indispensable for generating functional oocytes in golden hamster, and show the informative value of this model for functional and mechanistic investigations of piRNAs, especially those related to female infertility.

developmental biology↗

bioRxiv2021

Genome Size Estimation and Full-length Transcriptome of Sphingonotus tsinlingensis: Genetic Background for the Drought-Adapted Grasshopper

Sphingonotus Fieber, 1852 (Orthoptera: Acrididae) is a species-rich grasshopper genus with ~146 species. All species of this genus prefer dry environments, such as: desert, steppe, sand, and stony benchland. This genomic study aimed to understand the evolution and ecology of these grasshopper species. Here, the genome size of Sphingonotus tsinlingensis was estimated using flow cytometry and the first high-quality full-length transcriptome of this species is presented, which may serve as a reference genetic resource for the drought-adapted grasshopper species of Sphingonotus Fieber. The genome size of Sphingonotus tsinlingensis was ~12.8 Gb. Based on the 146.98 Gb Pacbio isoform sequencing data, 221.47 Mb full-length transcripts were assembled. Among these transcripts, 88,693 non-redundant isoforms were identified with an average length of 2,497 bp and an N50 value of 2,726 bp, which was much longer than the formal grasshopper transcriptome assemblies. A total of 48,502 protein coding sequences were determined, and 37,569 were annotated in public gene function databases. A total of 36,488 simple tandem repeats, 12,765 long non-coding RNAs, and 414 transcription factors were also identified. According to gene functions, 70 heat shock proteins and 61 P450 genes that may correspond to drought adaptation of S. tsinlingensis were identified. The genome of Sphingonotus tsinlingensis is an ultra-large and complex genome. Full-length transcriptome sequencing is an ideal strategy for genomic research. This is the first full-length transcriptome of the genus Sphingonotus. The assembly parameters were better than all known grasshopper transcriptomes. This full-length transcriptome may be used to understand its genetic background and the evolution and ecology of grasshoppers.

genetics↗

bioRxiv2021

Arkadia-SKI/SnoN signaling cascade differentially regulates TGF-β-induced iTreg and Th17 cell differentiation

TGF-{beta} signaling is fundamental for both Th17 and regulatory T cell (Treg) differentiation. However, these cells differ in requirements for downstream signaling components, such as various SMAD effectors, for their differentiation. To further characterize the mechanisms that distinguish TGF-{beta} signaling requirements for Th17 and Treg cell differentiation, we investigated the role of Arkadia (RNF111), a RING type E3 ubiquitin ligase known to enhance TGF-{beta} signaling during development. We find that Arkadia mediates the differentiation of induced-Treg (iTreg) but not Th17 cells both in vitro and in vivo. Inactivation of Arkadia in CD4+ T cells resulted in impairment of Treg cell differentiation in vitro and loss of ROR{gamma}t+FOXP3+ iTreg cells in the intestinal lamina propria in vivo, which increased susceptibility to microbiota-induced mucosal inflammation. Furthermore, genetic ablation of two substrates of Arkadia, the transcriptional co-repressors SKI and SnoN, rescued in vitro and in vivo iTreg cell-differentiation of Arkadia-deficient cells. These results reveal distinct TGF-{beta} signaling modules that govern Th17 and iTreg cell differentiation programs and that could be exploited therapeutically to selectively modulate T cell subsets in pathological settings such as autoimmunity or cancer.

immunology↗

bioRxiv2021

Harnessing the Power of Antibodies to Fight Bone Metastasis

Over the past 20 years, antibody-based therapies have proved to be of great value in cancer treatment. Despite the clinical success of these biopharmaceuticals, reaching targets in the bone micro-environment has proved to be difficult perhaps due to the relatively low vascularization of bone tissue and the presence of physical barriers that impair drug penetration. Here, we have used an innovative bone targeting (BonTarg) technology to generate a first-in-class bone-targeting anti-body. Moreover, we have used two xenograft models to demonstrate the enhanced therapeutic efficacy of this bone-targeting antibody against bone metastases, compared to the efficacy of traditional antibodies. Our strategy involves the use of pClick antibody conjugation technology to chemically couple the bone-targeting moiety bisphosphonate to the human epidermal growth factor receptor 2 (HER2)-specific antibody trastuzumab. Bisphosphonate modification of therapeutic antibodies results in delivery of higher conjugate concentrations to the bone metastatic niche, relative to other tissues. In both HER2-positive and negative xenograft mice models, this strategy provides enhanced inhibition of experimental bone metastases as well as multi-organ secondary metastases that arise from the bone lesions. Specific delivery of therapeutic antibodies to the bone therefore represents a promising strategy for the treatment of bone metastatic cancers and other bone diseases.

cancer biology↗

bioRxiv2021

Enhanced Cas12a multi-gene regulation using a CRISPR array separator

The type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple crRNAs. Here, we report that Cas12a array performance is hypersensitive to the GC content of crRNA spacers, as high-GC spacers can impair activity of the downstream crRNA. We analyzed naturally occurring CRISPR arrays and observed that repeats always contain an AT-rich fragment that separates crRNAs; we term this fragment a CRISPR separator. Inspired by this observation, we designed short, AT-rich synthetic separators (synSeparators) that successfully removed the disruptive effects between crRNAs. We demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously unknown feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.

synthetic biology↗

bioRxiv2021

Large field of view-spatially resolved transcriptomics at nanoscale resolution

Spatially resolved transcriptomic technologies are promising tools to study cell fate decisions in a physical microenvironment, which is fundamental for enhancing our knowledge of mammalian development. However, the imbalance between resolution, transcript capture and field of view of current methodologies precludes their systematic application to analyze relatively large and three-dimensional mid- and late-gestation mammalian embryos. Here, we combined DNA nanoball (DNB) patterned arrays and tissue RNA capture to create SpaTial Enhanced REsolution Omics-sequencing (Stereo-seq). This approach allows transcriptomic profiling of large histological sections with high resolution and sensitivity. We have applied Stereo-seq to study the kinetics and directionality of transcriptional variation in a time course of mouse organogenesis. We used this information to gain insight into the molecular basis of regional specification, neuronal migration and differentiation in the developing brain. Furthermore, we mapped the expression of a panel of developmental disease-related loci on our global transcriptomic maps to define the spatiotemporal windows of tissue vulnerability. Our panoramic atlas constitutes an essential resource to investigate longstanding questions concerning normal and abnormal mammalian development.

cell biology↗

bioRxiv2021

Dependency of LKB1-inactivated lung cancer on aberrant CRTC-CREB activation

Lung cancer with loss-of-function of the LKB1 tumor suppressor is a common aggressive subgroup with no effective therapies. LKB1-deficiency induces constitutive activation of cAMP/CREB-mediated transcription by a family of three CREB-regulated transcription coactivators (CRTC1-3). However, the significance and mechanism of CRTC activation in promoting the aggressive phenotype of LKB1-null cancer remain poorly characterized. Here we observed overlapping CRTC expression patterns and mild growth phenotypes of individual CRTC-knockouts in lung cancer, suggesting functional redundancy of CRTC1-3. We consequently designed a dominant-negative mutant (dnCRTC) to block all three CRTCs to bind and co-activate CREB. Expression of dnCRTC efficiently inhibited the aberrantly activated cAMP/CREB-mediated oncogenic transcriptional program induced by LKB1-deficiency, and specifically blocked the growth of LKB1-inactivated lung cancer. Collectively, this study provides direct proof for an essential role of the CRTC-CREB activation in promoting the malignant phenotypes of LKB1-null lung cancer and proposes the CRTC-CREB interaction interface as a novel therapeutic target.

cancer biology↗

bioRxiv2021

Mutational analysis of DinG-family helicase YoaA: its expression and interaction with replication clamp-loader protein HolC in E. coli

The XP-D/DinG family of DNA helicases contribute to genomic stability in all three domains of life. We investigate here the role of one of these proteins,YoaA, of Escherichia coli. In E. coli,YoaA aids tolerance to the nucleoside azidothymidine (AZT), a DNA replication inhibitor and physically interacts with a subunit of the DNA polymerase III holoenzyme, HolC. We map the residues of YoaA required for HolC interaction to its C-terminus by yeast two-hybrid analysis. We propose that this interaction competes with HolCs interaction with HolD and the rest of the replisome;YoaA indeed inhibits growth when overexpressed, dependent on this interaction region. By gene fusions we show YoaA is repressed by LexA and induced in response to DNA damage as part of the SOS response. Induction of YoaA by AZT is biphasic with an immediate response after treatment and a slower response that peaks in the late log phase of growth. This growth-phase dependent induction by AZT is not blocked by lexA3 (Ind-), which normally negates its self-cleavage, implying another means to induce the DNA damage response that responds to the nutritional state of the cell. We propose that YoaA helicase activity increases access to the 3 nascent strand during replication; consistent with this,YoaA appears to aid removal of potential A-to-T transversion mutations in ndk mutants, which are prone to nucleotide misincorporation. YoaA and its paralog DinG also may initiate template-switching that leads to deletions between tandem repeats in DNA. IMPORTANCEMaintaining genomic stability is crucial for all living organisms. Replication of DNA frequently encounters barriers that must be removed to complete genome duplication. Balancing DNA synthesis with its repair is critical and not entirely understood at a mechanistic level.The YoaA protein, studied here, is required for certain types of DNA repair and interacts in an alternative manner with proteins that catalyze DNA replication. YoaA is part of the well-studied LexA-regulated response to DNA damage, the SOS response. We describe an unusual feature of its regulation that promotes induction after DNA damage as the culture begins to experience starvation. Replication fork repair integrates both DNA damage and nutritional signals. We also show that YoaA affects genomic stability.

genetics↗

bioRxiv2020

Landscapes and dynamic diversifications of B-cell receptor repertoires in COVID-19 patients

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the pandemic of coronavirus disease 2019 (COVID-19). Great international efforts have been put into the development of prophylactic vaccines and neutralizing antibodies. However, the knowledge about the B cell immune response induced by the SARS-CoV-2 virus is still limited. Here, we report a comprehensive characterization of the dynamics of immunoglobin heavy chain (IGH) repertoire in COVID-19 patients. By using next-generation sequencing technology, we examined the temporal changes in the landscape of the patients immunological status, and found dramatic changes in the IGH within the patients immune system after the onset of COVID-19 symptoms. Although different patients have distinct immune responses to SARS-CoV-2 infection, by employing clonotype overlap, lineage expansion and clonotype network analyses, we observed a higher clonotype overlap and substantial lineage expansion of B cell clones during 2-3 weeks of illness, which is of great importance to B-cell immune responses. Meanwhile, for preferences of V gene usage during SARS-CoV-2 infection, IGHV3-74 and IGHV4-34 and IGHV4-39 in COVID-19 patients were more abundant than that of healthy controls. Overall, we present an immunological resource for SARS-CoV-2 that could promote both therapeutic development as well as mechanistic research.

immunology↗

bioRxiv2020

Decidual cell differentiation is evolutionarily derived from fibroblast activation

What the molecular mechanisms underlying the evolutionary origin of novel cell types are is a major unresolved question in biology. The uterine decidual cell is a novel cell type of placental mammals which serves as the interface between maternal and fetal tissues during pregnancy. In this paper, we investigate two models for the nature of the differentiation of decidual cells: first, that it represents a mesenchymal-epithelial transition (MET), and second, that it evolved from wound-induced fibroblast activation (WIFA). Immunocytochemistry and RNA-seq analysis of decidualizing human endometrial fibroblasts cast doubt on the MET hypothesis and instead demonstrate a similarity between decidualization and fibroblast activation, including a central role for TGFB1. Through single-cell RNA-seq, we found a transient myofibroblast-like cell population in the in vitro differentiation trajectory of human decidual cells and found that these cells represent a pre-decidual state approaching the inferred transcriptomic transition to decidual cells. We propose an evolutionary developmental model wherein the decidual cell is a novel cell type not equivalent to the myofibroblast, but the process of decidual differentiation itself evolved as an endometrial-specific modification to fibroblast activation in response to the wound caused by embryo implantation.

evolutionary biology↗

bioRxiv2020

RORα enforces stability of the T-helper-17 cell effector program

T helper 17 (Th17) cells regulate mucosal barrier defenses, but also promote multiple autoinflammatory diseases. Although many molecular determinants of Th17 cell differentiation have been described, the transcriptional programs that sustain Th17 cells in vivo remain obscure. The transcription factor ROR{gamma}t is critical for Th17 cell differentiation, but a distinct role of the closely-related ROR, which is co-expressed in Th17 cells, is not known. Here we demonstrate that, although dispensable for Th17 cell differentiation, ROR governs optimal Th17 responses in peripheral tissues. Thus, the absence of ROR in T cells led to significant reductions in both ROR{gamma}t expression and effector function amongst Th17 cells, due to need for cooperative ROR and ROR{gamma}t binding to a newly-identified Rorc enhancer element that is essential for Th17 lineage maintenance in vivo. Altogether, these data point to a non-redundant role of ROR in Th17 lineage maintenance via reinforcement of the ROR{gamma}t transcriptional program.

immunology↗

bioRxiv2020

Catechol-Containing Compounds are a Broad Class of Amyloid Inhibitors: II. Rosmarinic Acid Potently Detoxifies Amylin Amyloid and Ameliorates Diabetic Pathology in HIP Rats

Protein aggregation is associated with a large number of human protein misfolding diseases, yet FDA-approved drugs are currently not available. Amylin amyloid and plaque depositions in the pancreas are hallmark features of type 2 diabetes. Moreover, these amyloid deposits are implicated in the pathogenesis of diabetic complications such as neurodegeneration. We recently discovered that catechols and redox-related quinones/ anthraquinones represent a broad class of protein aggregation inhibitors. Further screening of a targeted library of natural compounds in complementary medicine that were enriched with catechol-containing compounds identified rosmarinic acid as a potent inhibitor of amylin aggregation (estimated inhibitory concentration IC50 = 200-300 nM). Structure-function relationship analysis of rosmarinic acid showed the additive effects of two catechol-containing components of the RA molecule. We further showed that RA does not reverse fibrillation back to monomeric amylin, but lead to non-toxic, remodeled protein aggregates. Rosmarinic acid has significant ex vivo efficacy in reducing human amylin oligomer levels in HIP rat sera as well as in sera from diabetic patients. In vivo efficacy studies of rosmarinic acid treatment with the diabetic HIP rat model demonstrated significant reduction in amyloid islet deposition and strong mitigation of diabetic pathology. Our work provides new in vitro molecular mechanisms and in vivo efficacy insights for a model nutraceutical agent against type 2 diabetes and other aging-related protein misfolding diseases.

pharmacology and toxicology↗

bioRxiv2020

High-throughput Single-cell CNV Detection Reveals Clonal Evolution During Hepatocellular Carcinoma Recurrence

Single-cell genomics provides substantial resources for dissecting cellular heterogeneity and cancer evolution, but classical DNA amplification-based methods are low-throughput and introduce coverage bias during sample preamplification. We developed a single-cell DNA library preparation method without preamplification in nanolitre scale (scDPN). The method has a throughput of up to 1,800 cells per run for copy number variation (CNV) detection. Also, it has a lower level of amplification bias and noise than the multiple displacement amplification (MDA) method and showed high sensitivity and accuracy based on evaluation in cell lines and tumour tissues. We used this approach to profile the tumour clones in paired primary and relapsed tumour samples of hepatocellular carcinoma (HCC). We identified 3 clonal subpopulations with a multitude of aneuploid alterations across the genome. Furthermore, we observed that a minor clone of the primary tumour containing additional alterations in chromosomes 1q, 10q, and 14q developed into the dominant clone in the recurrent tumour, indicating clonal selection during recurrence in HCC. Overall, this approach provides a comprehensive and scalable solution to understand genome heterogeneity and evolution.

cell biology↗

bioRxiv2020

Dynamic integration and segregation of amygdala subregional functional circuits linking to physiological arousal

The dynamical organization of brain networks is essential to support human cognition and emotion for rapid adaption to ever-changing environment. As the core nodes of emotion-related brain circuitry, the basolateral amygdala (BLA) and centromedial amygdala (CMA) as two major amygdalar nuclei, are recognized to play distinct roles in affective functions and internal states, via their unique connections with cortical and subcortical structures in rodents. However, little is known how the dynamical organization of emotion-related brain circuitry reflects internal autonomic responses in humans. Using resting-state functional magnetic resonance imaging (fMRI) with K-means clustering approach in a total of 79 young healthy individuals (cohort 1: 42; cohort 2: 37), we identified two distinct states of BLA- and CMA-based intrinsic connectivity patterns, with one state (integration) showing generally stronger BLA- and CMA-based intrinsic connectivity with multiple brain networks, while the other (segregation) exhibiting weaker yet dissociable connectivity patterns. In an independent cohort 2 of fMRI data with concurrent recording of skin conductance, we replicated two similar dynamic states and further found higher skin conductance level in the integration than segregation state. Moreover, machine learning-based Elastic-net regression analyses revealed that time-varying BLA and CMA intrinsic connectivity with distinct network configurations yield higher predictive values for spontaneous fluctuations of skin conductance level in the integration than segregation state. Our findings highlight dynamic functional organization of emotion-related amygdala nuclei circuits and networks and its links to spontaneous autonomic arousal in humans.

neuroscience↗