Search bioRxiv⌕ Search

Biology subjects

Lu, J.

Publications and source records attributed to Lu, J..

At least 379 records · Page 21Linked to original sources

High-Speed Automatic Characterization of Rare Events in Flow Cytometric Data

A new computational framework for FLow cytometric Analysis of Rare Events (FLARE) has been developed specifically for fast and automatic identification of rare cell populations in very large samples generated by platforms like multi-parametric flow cytometry. Using a hierarchical Bayesian model and information-sharing via parallel computation, FLARE rapidly explores the high-dimensional marker-space to detect highly rare populations that are consistent across multiple samples. Further it can focus within specified regions of interest in marker-space to detect subpopulations with desired precision.

bioinformatics↗

A Polybasic Domain in aPKC Mediates Par-6-Dependent Control of Plasma Membrane Targeting and Kinase Activity

Mechanisms coupling the atypical PKC (aPKC) kinase activity to its subcellular localization are essential for cell polarization. Unlike other members of the PKC family, aPKC has no well-defined plasma membrane (PM) or calcium binding domains, leading to the assumption that its subcellular localization relies exclusively on protein-protein interactions. Here we show that in both Drosophila and mammalian cells the pseudosubstrate region (PSr) of aPKC acts as a polybasic domain capable of targeting aPKC to the PM via electrostatic binding to PM PI4P and PI(4,5)P2. However, physical interaction between aPKC and Par-6 is required for the PM-targeting of aPKC, likely by allosterically exposing the PSr to bind PM. Binding of Par-6 also inhibits aPKC kinase activity and such inhibition can be relieved through Par-6 interaction with apical polarity protein Crumbs. Our data suggest a potential mechanism in which allosteric regulation of polybasic PSr by Par-6 couples the control of both aPKC subcellular localization and spatial activation of its kinase activity. eTOC SummaryDong et al. discover that the pseudo-substrate region (PSr) in aPKC is a polybasic domain capable of electrostatically targeting aPKC to plasma membrane. Allosteric regulation of PSr by Par-6 couples the control of both aPKC subcellular localization and spatial activation of kinase activity.

cell biology↗

Causal Network Inference from Gene Transcriptional Time Series Response to Glucocorticoids

Gene regulatory network inference is essential to uncover complex relationships among gene pathways and inform downstream experiments, ultimately paving the way for regulatory network re-engineering. Network inference from transcriptional time series data requires accurate, interpretable, and efficient determination of causal relationships among thousands of genes. Here, we develop Bootstrap Elastic net regression from Time Series (BETS), a statistical framework based on Granger causality for the recovery of a directed gene network from transcriptional time series data. BETS uses elastic net regression and stability selection from bootstrapped samples to infer causal relationships among genes. BETS is highly parallelized, enabling efficient analysis of large transcriptional data sets. We show competitive accuracy on a community benchmark, the DREAM4 100-gene network inference challenge, where BETS is one of the fastest among methods of similar performance but additionally infers whether the causal effects are activating or inhibitory. We apply BETS to transcriptional time series data of 2, 768 differentially-expressed genes from A549 cells exposed to glucocorticoids over a period of 12 hours. We identify a network of 2, 768 genes and 31, 945 directed edges (FDR [≤] 0.2). We validate inferred causal network edges using two external data sources: overexpression experiments on the same glucocorticoid system, and genetic variants associated with inferred edges in primary lung tissue in the Genotype-Tissue Expression (GTEx) v6 project. BETS is freely available as an open source software package at https://github.com/lujonathanh/BETS.

bioinformatics↗

Selection of a Malignant Subpopulation from a Colorectal Cancer Cell Line

Colorectal cancer (CRC) is a leading cause of death from cancer worldwide. Thus, there is an emerging need for new experimental models that allow identification and validation of biomarkers for CRC-specific progression. In this study, we propose a repeated sphere-forming assay as a strategy to select a malignant subpopulation from a CRC line, HCT116. We validated our assay by confirming that three canonical stemness markers, Nanog, Oct4, and Lgr5, were up-regulated in the sphere state at every generation of the selection assay. The resulting line, after eight rounds of selection, exhibited an increased sphere-forming capacity in vitro and tumorgenicity in vivo. Furthermore, dipeptidase 1 (DPEP1) was identified as the major differentially expressed gene in the selected clone, and depletion of DPEP1 suppressed the elevated sphere-forming capacity in vitro and tumorgenicity in vivo. Overall, we have established an experimental strategy for the isolation of a malignant subpopulation from a CRC cell line. Results from our model also suggested that DPEP1 can serve as a promising prognostic biomarker for CRC.

cancer biology↗

MicroRNA Overexpression screen reveals a noncanonical role of CDK2 in regulating neutrophil migration

Neutrophil migration is essential for inflammatory responses to kill pathogens, however it also causes tissue injury. To discover novel therapeutic targets that modulate neutrophil migration, we performed a neutrophil-specific microRNA overexpression screen in zebrafish, and identified eight microRNAs as potent suppressors of neutrophil migration. Among those, miR-199 decreases neutrophil chemotaxis in zebrafish and human neutrophil-like cells. Intriguingly, in terminally differentiated neutrophils, miR-199 alters the cell cycle-related pathways and directly suppresses cyclin-dependent kinase 2 (cdk2), whose known activity is restricted to cell cycle progression and cell differentiation. Inhibiting CDK2, but not DNA replication, disrupts cell polarity and chemotaxis of zebrafish neutrophils. Chemotaxis of primary human neutrophils are also reduced by CDK2 inhibition. Furthermore, miR-199 overexpression or CDK2 inhibition significantly improves the outcome of lethal systemic inflammation challenges in zebrafish. Together, our results reveal previously unknown functions of miR-199 and CDK2 in regulating neutrophil migration and provide new directions in alleviating systemic inflammation.\n\nOne Sentence SummarymiR-199 directly suppresses cdk2 expression, neutrophil chemotaxis and systemic inflammation.

immunology↗

Noroviruses subvert the core stress granule component G3BP1 to promote viral VPg-dependent translation.

Knowledge of the host factors required for norovirus replication has been hindered by the challenges associated with culturing human noroviruses. We have combined proteomic analysis of the viral translation and replication complexes with a CRISPR screen, to identify host factors required for norovirus infection. The core stress granule component G3BP1 was identified as a host factor essential for efficient human and murine norovirus infection, demonstrating a conserved function across the Norovirus genus. Furthermore, we show that G3BP1 functions in the novel paradigm of viral VPg-dependent translation initiation, contributing to the assembly of translation complexes on the VPg-linked viral positive sense RNA genome by facilitating 40S recruitment. Our data suggest that G3BP1 functions by providing viral RNA a competitive advantage over capped cellular RNAs, uncovering a novel function for G3BP1 in the life cycle of positive sense RNA viruses and identifying the first host factor with pan-norovirus pro-viral activity.

microbiology↗

EPIC: MHC-I epitope prediction integrating mass spectrometry derived motifs and tissue-specific expression profiles

BackgroundAccurate prediction of epitopes presented by human leukocyte antigen (HLA) is crucial for personalized cancer immunotherapies targeting T cell epitopes. Mass spectrometry (MS) profiling of eluted HLA ligands, which provides high-throughput measurements of HLA associated peptides in vivo, can be used to faithfully model the presentation of epitopes on the cell surface. In addition, gene expression profiles measured by RNA-seq data in a specific cell/tissue type can significantly improve the performance of epitope presentation prediction. However, although large amount of high-quality MS data of HLA-bound peptides is being generated in recent years, few provide matching RNA-seq data, which makes incorporating gene expression into epitope prediction difficult. MethodsWe collected publicly available HLA peptidome and matching RNA-seq data of 34 cell lines derived from various sources. We built position score specific matrixes (PSSMs) for 21 HLA-I alleles based on these MS data, then used logistic regression (LR) to model the relationship among PSSM score, gene expression and peptide length to predict whether a peptide could be presented in each of the cell line. We further built a universal LR model, termed Epitope Presentation Integrated Prediction (EPIP), based on more than 180,000 unique HLA ligands collected from public sources and ~3,000 HLA ligands generated by ourselves, to predict epitope presentation for 66 common HLA-I alleles. ResultsWhen evaluating EPIP on large, independent HLA eluted ligand datasets, it performed substantially better than other popular methods, including MixMHCpred (v2.0), NetMHCpan (v4.0), and MHCflurry (v1.2.2), with an average 0.1% positive predictive value (PPV) of 52.01%, compared to 37.24%, 36.96%, 24.90% and 23.76% achieved by MixMHCpred, NetMHCpan-4.0 (EL), NetMHCpan-4.0 (BA) and MHCflurry, respectively. It is also comparable to EDGE, a recent deep learning-based model that is not publicly available, on predicting epitope presentation and selecting immunogenic cancer neoantigens. However, the simplicity and flexibility of EPIP makes it easier to be applied in diverse situations, and we demonstrated this by generating MS data for the HCC4006 cell line and adding the support of HLA-A*33:03 to EPIP. EPIP is publicly available as a web tool < http://epip.genomics.cn/>. Conclusionswe have developed an easy to use, publicly available epitope prediction tool, EPIP, that incorporates information from both MS and RNA-seq data, and demonstrated its superior performance over existing public methods.

bioinformatics↗

EpiAlignment: alignment with both DNA sequence and epigenomic data

Comparative epigenomics, by subjecting both epigenome and genome to interspecies comparison, has become a powerful approach to reveal regulatory features of the genome. Thus elucidated regulatory features surpassed the information derived from comparison of genomic sequences alone. Here, we present EpiAlignment, a web-based tool to align genomic regions with both DNA sequence and epigenomic data. EpiAlignment takes DNA sequence and epigenomic profiles derived by ChIP-seq, DNase-seq, or ATAC-seq from two species as input data, and outputs the best semi-global alignments. These alignments are based on EpiAlignment scores, computed by a dynamic programming algorithm that accounts for both sequence alignment and epigenome similarity. For timely response, the EpiAlignment web server automatically initiates up to 140 computing threads depending on the size of user input data. For users convenience, we have pre-compiled the comparable human and mouse epigenome datasets in matched cell types and tissues from the Roadmap Epigenomics and ENCODE consortia. Users can either upload their own data or select pre-compiled datasets as inputs for EpiAlignment analyses. Results are presented in graphical and tabular formats where the entries can be interactively expanded to visualize additional features of these aligned regions. EpiAlignment is available at https://epialign.ucsd.edu/.

bioinformatics↗

Rapid Detection of Burkholderia pseudomallei with a Lateral Flow Recombinase Polymerase Amplification Assay

Melioidosis is a severe infectious disease caused by gram-negative, facultative intracellular pathogen Burkholderia pseudomallei (B. pseudomallei). Although cases are increasing reported from other parts of the world, it is an illness of tropical and subtropical climates primarily found in southeast Asia and northern Australia. Because of a 40% mortality rate, this life-threatening disease poses a public health risk in endemic area. Early detection of B. pseudomallei infection benefits greatly to implement effective treatment timely, which is vital for prognosis of a melioidosis patient. In this study, a novel isothermal recombinase polymerase amplification combined with lateral flow dipstick (LF-RPA) assay was established for rapid detection of B.pseudomallei. A set of probe and primers targeting orf2 gene of B. pseudomallei were generated and parameters for the LF-RPA assay were optimized. Result can be easy visualized in 30 minutes with the limit of detection (LoD) as low as 20 femtogram (ca. 25.6 copies) of B. pseudomallei genomic DNA. The assay is highly specific as no cross amplification was observed with 35 non-B. pseudomallei pathogens. Isolates (N=19) from patients of Hainan province of China were retrospectively confirmed by the newly developed method. LoD for B. pseudomallei spiked soil and blood samples were 2.1x103 CFU/g and 4.2x103 CFU/ml respectively. Sensitivity of the LF-RPA assay was comparable to TaqMan Real-Time PCR, however, the LF-RPA assay exhibited a better tolerant to inhibitors in blood than the later. Our results showed that the LF-RPA assay is an alternative to existing PCR-based methods for detection of B. pseudomallei with a potentiality of early accurate diagnosis of melioidosis at point of care or in-field use.

microbiology↗

Olfactory Co-Stimulation Influences Intranasal Somatosensory Perception

Olfactory sensitivity is influenced by intranasal trigeminal sensation. For instance, sniffing is central to how humans and animals perceive odorants. Here, we investigated the influence of olfactory co-stimulation on the perception of intranasal somatosensory stimulation. In this study, twenty-two healthy human subjects, with normal olfactory function, performed a localization task for weak air-puff stimuli, in the presence or absence of a pure odorant, phenyl ethyl alcohol (PEA; rose odor). Visual cues were used to inform participants to briefly hold their breath while weak, poorly localizable, air-puffs and/or PEA were delivered to either nostril. Although PEA alone could not be localized, when accompanied by a weak air-puff in the ipsilateral nostril, localization accuracy significantly improved, relative to presentation of the air-puff without the odorant. The enhancement of localization was absent when the air-puff and PEA were presented to opposite nostril. Since ipsilateral but not contralateral co-stimulation with PEA increased the accuracy of weak air-puff localization, the results argue against a non-specific alerting effect of PEA. These findings suggest an interaction between the olfactory and somatosensory trigeminal systems.

neuroscience↗

Multiplexed, sequential secretion analysis of the same single cells reveals distinct effector response dynamics dependent on the initial basal state

The effector response of immune cells dictated by an array of secreted proteins is a highly dynamic process, requiring sequential measurement of all relevant proteins from single cells. Herein we show a microchip-based, 10-plexed, sequential secretion assay on the same single cells and at the scale of ~5000 single cells measured simultaneously over 4 time points. It was applied to investigating the time course of single human macrophage response to Toll-like receptor 4 (TLR4) ligand lipopolysaccharide and revealed four distinct activation modes for different proteins in single cells. In particular, we observed that secreted factors regulated by transcription factor NFO_SCPLOWKC_SCPLOWB (e.g., TNF and CCL2) predominantly show on-off mode over off-on mode. The dynamics of all proteins combined classified the cells into two major activation states, which were found to be dependent on the basal state of each cell. Single-cell RNA-Seq was performed on the same samples at the matched time points and further demonstrated at the transcriptional level the existence of two major activation states, which are enriched for translation vs inflammatory programs, respectively. These results showed a cell-intrinsic heterogeneous response in phenotypically homogeneous cell population. This work demonstrated the longitudinal tracking of protein secretion signature in thousands of single cells at multiple time points, providing dynamic information to better understand how individual immune cells react to pathogenic challenges over time and how they together constitute a population response.

bioengineering↗

A fluorescent reporter enables instantaneous measurement of cell cycle speed in live cells

Cell proliferation changes concomitantly with fate transitions during reprogramming, differentiation, regeneration, and oncogenesis. Methods to resolve cell cycle length heterogeneity in real-time are currently lacking. Here, we describe a genetically encoded fluorescent reporter that captures live cell cycle speed using a single measurement. This reporter is based on the color-changing Fluorescent Timer (FT) protein, which emits blue fluorescence when newly synthesized before maturing into a red fluorescent protein. We generated a mouse strain expressing an H2B-FT fusion reporter from a universally active locus, and demonstrate that faster-cycling cells can be distinguished from slower-cycling ones based on the intracellular fluorescence ratio between the FTs blue and red states. Using this reporter, we reveal the native cell cycle speed distributions of fresh hematopoietic cells, and demonstrate its utility in analyzing cell proliferation in solid tissues. This system is broadly applicable for dissecting functional heterogeneity associated with cell cycle dynamics in complex tissues.

cell biology↗

Norovirus infection results in assembly of virus-specific G3BP1 granules and evasion of eIF2α signaling.

During viral infection, the accumulation of RNA replication intermediates or viral proteins imposes major stress on the host cell. In response, cellular stress pathways can rapidly impose defence mechanisms by shutting off the protein synthesis machinery, which viruses depend on, and triggering the accumulation of mRNAs into stress granules to limit the use of energy and nutrients. Because this threatens viral gene expression, viruses need to evade these pathways to propagate. Human norovirus is responsible for gastroenteritis outbreaks worldwide. Previously we showed that murine norovirus (MNV) regulates the activity of eukaryotic initiation factors (eIFs). Here we examined how MNV interacts with the eIF2a signaling axis controlling translation and stress granules accumulation. We show that while MNV infection represses host cell translation, it results in the assembly of virus-specific granules rather than stress granules. Further mechanistic analyses revealed that eIF2a signaling is uncoupled from translational stalling. Moreover the interaction of the RNA-binding protein G3BP1 with viral factors together with a redistribution of its cellular interacting partners could explain norovirus evasion of stress granules assembly. These results identify novel strategies by which norovirus ensure efficient replication propagation by manipulating the host stress response.

microbiology↗

Molecular mechanism of the Orai channel activation

The Orai channel is characterized by voltage independence, low conductance and high Ca2+ selectivity and plays an important role in Ca2+ influx through the plasma membrane. How the channel is activated and promotes Ca2+ permeation are not well understood. Here, we report the crystal structure and cryo-electron microscopy reconstruction of a Drosophila melanogaster Orai mutant (P288L) channel that is constitutively active according to electrophysiology. The open state of the Orai channel showed a hexameric assembly in which six TM1 helices in the center form the ion-conducting pore, and six TM4 helices in the periphery form extended long helices. Orai channel activation requires conformational transduction from TM4 to TM1 and eventually causes the basic section of TM1 to twist outward. The wider pore on the cytosolic side aggregates anions to increase the potential gradient across the membrane and thus facilitate Ca2+ permeation. The open-state structure of the Orai channel offers insights into channel assembly, channel activation and Ca2+ permeation.

biochemistry↗

Transcriptional heterogeneity in cancer-associated regulatory T cells is predictive of survival.

Regulatory T cells (Tregs) are a population of T cells that exert a suppressive effect on a variety of immune cells and non-immune cells. The suppressive effects of Tregs are detrimental to anti-tumor immunity. Recent investigations into cancer-associated Tregs have identified common expression patterns for tumor-infiltration, however the functional heterogeneity in tumor-infiltrating (TI) Treg is largely unknown. We performed single-cell sequencing on immune cells derived from renal clear cell carcinoma (ccRCC) patients, isolating 160 peripheral-blood (PB) Tregs and 574 TI Tregs. We identified distinct transcriptional TI Treg cell fates, with a suppressive subset expressing CD177. We demonstrate CD177+ TI-Tregs have preferential suppressive effects in vivo and ex vivo. Gene signatures derived the CD177+ Treg subset had superior ability to predict survival in ccRCC and seven other cancer types. Further investigation into the development and regulation of TI-Treg heterogeneity will be vital to the application of tumor immunotherapies that possess minimal side effects.

cancer biology↗

Bile acid metabolites control Th17 and Treg cell differentiation

Bile acids are abundantly present in the mammalian gut, where they undergo bacteria-mediated transformation, generating a large pool of bioactive molecules. While they have been shown to affect host metabolism, cancer progression and innate immunity, it is unknown whether bile acids affect the function of adaptive immune cells such as T cells expressing IL-17a (Th17 cells) and regulatory T cells (Tregs) that mediate inflammatory and anti-inflammatory responses, respectively. By screening a small-molecule library primarily composed of bile acid metabolites, we identified two distinct derivatives of lithocholic acid (LCA), 3-oxoLCA and isoalloLCA, as specific regulators of Th17 and Treg cells. While 3-oxoLCA inhibited Th17 cell differentiation by directly binding to its key transcription factor ROR{gamma}t (retinoid-related orphan receptor {gamma} t), isoalloLCA enhanced differentiation of Tregs through mitochondrial-dependent metabolic changes, leading to an increased expression of Foxp3. IsoalloLCA-dependent Treg enhancement required an intronic Foxp3 enhancer, the conserved noncoding sequence 3 (CNS3), which acts as an epigenetic switch that confers a poised state to the Foxp3 promoter. Lastly, oral administration of 3-oxoLCA and isoalloLCA to mice led to reduced Th17 and increased Treg cell differentiation in the intestinal lamina propria. Altogether, our data suggest novel mechanisms by which bile acid metabolites control host immune responses by directly modulating the Th17 and Treg balance.

immunology↗

Identifying Emerging Phenomenon in Plant Long Temporal Phenotyping Experiments

The rapid improvement of phenotyping capability, accuracy, and throughput have greatly increased the volume and diversity of phenomics data. A remaining challenge is an efficient way to identify phenotypic patterns to improve our understanding of the quantitative variation of complex phenotypes, and to attribute gene functions. To address this challenge, we developed a new algorithm to identify emerging phenomena from large-scale temporal plant phenotyping experiments. An emerging phenomenon is defined as a group of genotypes who exhibit a coherent phenotype pattern during a relatively short time. Emerging phenomena are highly transient and diverse, and are dependent in complex ways on both environmental conditions and development. Identifying emerging phenomena may help biologists to examine potential relationships among phenotypes and genotypes in a genetically diverse population and to associate such relationships with the change of environments or development. We present an emerging phenomenon identification tool called Temporal Emerging Phenomenon Finder (TEP-Finder). Using large-scale longitudinal phenomics data as input, TEP-Finder first encodes the complicated phenotypic patterns into a dynamic phenotype network. Then, emerging phenomena in different temporal scales are identified from dynamic phenotype network using a maximal clique based approach. Meanwhile, a directed acyclic network of emerging phenomena is composed to model the relationships among the emerging phenomena. The experiment that compares TEP-Finder with two state-of-art algorithms shows that the emerging phenomena identified by TEP-Finder are more functionally specific, robust, and biologically significant. The source code, manual, and sample data of TEP-Finder are all available at: http://phenomics.uky.edu/TEP-Finder/.

bioinformatics↗

HiAlc Klebsiella pneumonia, one of potential chief culprits of non-alcoholic fatty liver disease: through generation of endogenous ethanol

Non-alcoholic fatty liver disease (NAFLD), a prelude of cirrhosis and hepatocellular carcinoma, is the most common chronic liver disease worldwide. NAFLD has been considerated to be associated with the composition of gut microbiota. However, causal relationship between change of gut microbiome and NAFLD remains unclear. Here we show that Klebsiella pneumoniae was significantly associated with NAFLD through inducing generation of endogenous ethanol. A strain of high alcohol-producing Klebsiella pneumoniae (HiAlc Kpn) was initially isolated from fecal samples of patient with non-alcoholic steatohepatitis (NASH) accompanied with auto-brewery syndrome (ABS). Gavage of HiAlc Kpn was capable of inducing murine model of fatty liver disease (FLD) in which had typical pathological changes of hepatic steatosis and similar liver gene expression profiles to those of alcohol intake in mice. Data derived from germ-free mice by gnotobiotic gavage further demonstrated that the HiAlc Kpn is the major cause of the changes in FLD mice. Furthermore, using proteomic and metabolitic analysis, we found that HiAlc Kpn induced generation of endogenous alcohol through the 2,3-butanediol fermentation pathway. More interestingly, the blood alcohol concentration was elevated in FLD mice induced by HiAlc Kpn after glucose intake. Clinical analysis showed that HiAlc Kpn were observed in up to 60% of patients with NAFLD. Our results suggested that HiAlc Kpn make important contribution to NAFLD, possibly through generation of the endogenous alcohol. Thus, targeting these bacteria might provide a novel therapeutic for clinical treatment of NAFLD.\n\nIn BriefFatty liver disease induced by high alcohol-producing Klebsiella pneumoniae\n\nCompeting Financial Interest StatementThe authors declare no conflicts of interest.

microbiology↗