Biology subjects

Chen, K.

Publications and source records attributed to Chen, K..

At least 379 records · Page 21Linked to original sources

bioRxiv2020

Targeting uncoupling of copy number and gene expression in cancers

The high degree of aneuploidy in cancer is likely tolerated via extensive uncoupling of copy number (CN) and mRNA expression (UCNE) of deleterious genes located in copy number aberrations (CNAs). To test the extent and role of UCNE in cancer, we performed integrative analysis of multiomics data across The Cancer Genome Atlas (TCGA), encompassing [~] 5000 individual tumors. We found many genes having UCNE, the degree of which are associated with increased oncogenic signaling, proliferation and immune-suppression. The occurrence of UCNE appears to be orchestrated by complex epigenetic and regulatory changes, with transcription factors (TFs) playing a prominent role. To further dissect the regulatory mechanisms, we developed a systems-biological approach to identify candidate TFs, which upon perturbation can offset UCNE and reduce tumor fitness. Applying our approach on TCGA data, we identified 20 putative targets, 45% of which were validated by independent sources. Among them are IRF1, which plays a prominent role in anti-tumor immunity and response to immune checkpoint therapy, ETS1, TRIM21 and GATA3, which are associated with anti-tumor immunity, tumor proliferation and metastasis. Together, our study indicates that UCNE is likely an important mechanism in cancer development that can be exploited therapeutically.

cancer biology↗

bioRxiv2020

A robust and versatile nanobody platform for drug delivery

Therapeutic and diagnostic efficacies of numerous small biomolecules and chemical compounds are hampered by the short half-lives. Here we report the development of a repertoire of diverse, high-affinity albumin-nanobodies (NbHSA) to facilitate drug delivery. By integrating biophysics, and hybrid structural biology, we have systematically characterized the NbHSA for albumin binding, mapped the epitopes, and resolved the architecture of a tetrameric Nb-albumin complex. We employed quantitative proteomics for accurate, multiplex Nb pharmacokinetic analysis. Using a humanized albumin mouse model, we found that the NbHSA has outstanding pharmacokinetics; the most stable NbHSA has a 771-fold T1/2 improvement compared with a control Nb. Interestingly, the pharmacokinetics of NbHSA is related to their biophysical and structural properties. To demonstrate the utility of NbHSA, we developed a highly stable NbHSA-hIL-2 cytokine conjugate "Duraleukin" and confirmed its improved anticancer properties than hIL-2 alone. We envision that this high-quality Nb resource will advance research into novel biotherapeutics.

bioengineering↗

bioRxiv2020

Probing nanoscale diffusional heterogeneities in cellular membranes through multidimensional single-molecule and super-resolution microscopy

Diffusion properties notably determine the behavior of biomembranes. Here we report the concurrent nanoscale fine-mapping of membrane topography, diffusivity, and packing order in live mammalian cells through a synergy of single-molecule and super-resolution methods. By identifying a bright, lipophilic fluorescence turn-on probe that enables sustained single-molecule imaging of cellular membranes under stroboscopic excitation, we accumulate the positions and transient displacements of >106 probe molecules to achieve super-resolution topography and diffusivity mapping. We thus determine a trend that the membrane diffusivity drops with increased lipid packing order when comparing the endoplasmic reticulum (ER) membrane, plasma membrane, and nanodomains induced by cholera toxin B. Utilizing our nanoscale mapping capability, we further unveil reduced diffusivity in the ER membrane at ER-plasma membrane contact sites. By next integrating spectrally resolved single-molecule imaging, we show this localized diffusion slowdown is not due to altered lipid packing order, but may instead be attributed to local protein crowding. Our integrated multidimensional single-molecule approach thus unveils and differentiates between nanoscale diffusional heterogeneities of different origins in live-cell membranes.

biophysics↗

bioRxiv2020

MACMIC Reveals Dual Role of CTCF in Epigenetic Regulation of Cell Identity Genes

Numerous studies of relationship between epigenomic features have focused on their strong correlation across the genome, likely because such relationship can be easily identified by many established methods for correlation analysis. However, two features with little correlation may still colocalize at many genomic sites to implement important functions. There is no bioinformatic tool for researchers to specifically identify such feature pair. Here, we develop a method to identify feature pair in which two features have maximal colocalization but minimal correlation (MACMIC) across the genome. By MACMIC analysis of 3,385 feature pairs in 15 cell types, we reveal a dual role of CTCF in epigenetic regulation of cell identity genes. Although super-enhancers are associated with activation of target genes, only a subset of super-enhancers colocalized with CTCF regulate cell identity genes. At super-enhancers colocalized with CTCF, the CTCF is required for the active marker H3K27ac in cell type requiring the activation, and also required for the repressive marker H3K27me3 in other cell types requiring the repression. Our work demonstrates the biological utility of the MACMIC analysis and reveals a key role for CTCF in epigenetic regulation of cell identity.

bioinformatics↗

bioRxiv2020

Amitosis as a strategy of cell division - Insight from the proliferation of Tetrahymena thermophila macronucleus

Cell division is a necessity of life which can be either mitotic or amitotic. While both are fundamental, amitosis is sometimes considered a relic of little importance in biology. Nevertheless, eukaryotes often have polyploid cells, including cancer cells, which may divide amitotically. To understand how amitosis ensures the completion of cell division, we turn to the macronuclei of ciliates. The grand scheme governing the proliferation of the macronuclei of ciliate cells, which involves chromosomal replication and the amitosis, is currently unknown. Using a novel model that encompasses a wide range of mechanisms together with experimental data of the composition of mating types at different stages derived from a single karyonide of Tetrahymena thermophila, we show that the chromosomal replication of the macronucleus has a strong head-start effect, with only about five copies of chromosomes replicated at a time and persistent reuse of the chromosomes involved in the early replication. Furthermore the fission of a fully grown macronucleus is non-random, with a strong tendency to push chromosomes and their replications to the same daughter cell. Similar strategies may exist for other Tetrahymena species or ciliates, and have implications to the amitosis of polyploid cells of multicellular organisms.

genetics↗

bioRxiv2020

Requirement of the antimicrobial peptide CRAMP for macrophages to eliminate phagocytosed E. coli through an autophagy pathway

Host-derived antimicrobial peptides play an important role in the defense against extracellular bacterial infections. However, the capacity of antimicrobial peptides derived from macrophages as potential antibacterial effectors against intracellular pathogens remains unknown. In this study, we report that normal (wild type, WT) mouse macrophages increased their expression of the cathelicidin-related antimicrobial peptide (CRAMP) after infection by viable E. coli or stimulation with inactivated E. coli and its product LPS, a process involving activation of NF-{kappa}B followed by protease-dependent conversion of CRAMP from an inactive precursor to an active form. The active CRAMP was required by WT macrophages to eliminate phagocytosed E. coli, with participation of autophagy-related proteins ATG5, LC3-II, and LAMP-1 as well as conjugation of the bacteria with p62. The autophagy-mediated elimination of E. coli was impaired in CRAMP-/- macrophages resulting in retention of intracellular bacteria and fragmentation of macrophages. These results indicate CRAMP as a critical component in autophagy-mediated clearance of intracellular E. coli by macrophages.

immunology↗

bioRxiv2020

Resolving Cargo-motor-track Interactions in Living Cells with Bifocal Parallax Single Particle Tracking

Resolving coordinated biomolecular interactions in living cellular environments is vital for understanding the mechanisms of molecular nanomachines. The conventional approach relies on localizing and tracking target biomolecules and/or subcellular organelles labeled with imaging probes. However, it is challenging to gain information on rotational dynamics, which can be more indicative of the work done by molecular motors and their dynamic binding status. Herein, a bifocal parallax single particle tracking method using half-plane point spread functions has been developed to resolve the full-range azimuth angle (0-360°), polar angle, and 3D displacement in real time under complex living cell conditions. Using this method, quantitative rotational and translational motion of the cargo in a 3D cell cytoskeleton was obtained. Not only well-known active intracellular transport and free diffusion were observed but new interactions, tight attachment and tethered rotation, were discovered for better interpretation of the dynamics of cargo-motor-track interactions at various types of microtubule intersections.Significance Statement Translation and rotational motion of cargo during pauses at the microtubule intersections in living cells were revealed by high-accuracy three-dimensional single particle rotational tracking. The current study demonstrates the potential of studying coordinated interactions in living cellular environments by resolving characteristic rotational motions.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2020

Single cell analysis of FOXP3 deficiencies in humans and mice unmasks intrinsic and extrinsic CD4+ T cell perturbations

ABSTRACTFOXP3 deficiency in humans with IPEX syndrome and mice results in fatal systemic autoimmunity by altering regulatory T cell (Treg) physiology, but actual cellular and molecular mechanisms of disease are unclear, part because Treg surface markers may be unreliable in disease states. We used deep profiling by flow cytometry, population and single-cell RNAseq to analyze Tregs and conventional (Tconv) CD4+ T lymphocytes in cohorts of IPEX patients with a range of genetic lesions, and in Foxp3-deficient mice. In all patients and mice, heterogeneous Treg-like cells with an active FOXP3 locus were observed, some differing very little from normal Tregs, others more distant. Tconv showed no widespread activation or Th bias. The dominant mark was a monomorphic signature equally affecting all CD4+ T cells, unexpectedly dampening tumor-Treg and cytokine-signaling modules. In mixed bone marrow chimeras, WT Tregs exerted dominant suppression, normalizing the states of mutant Treg and Tconv, extinguishing the disease signature, and revealing a small gene cluster truly regulated, cell-intrinsically, by FOXP3. These results suggest a two-step pathogenesis model, with therapeutic implications: limited downregulation of a few core Treg genes de-represses a systemic mediator(s), which imprints the disease signature on all T cells, and further dampens Treg function.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗

bioRxiv2020

Network-based cancer gene relationship prediction method reveals perturbations in the cancer gene network

The perturbations of protein-protein interactions (PPIs) were found to be the main cause of cancer. Previous gene relationship prediction methods which were trained with non-disease gene interaction data were not compatible to map the PPI network in cancer. Therefore, we established a novel cancer specific PPI prediction method dubbed NECARE, which was based on relational graph convolutional network (R-GCN) with knowledge-based features. It achieved the best performance with a Matthews correlation coefficient (MCC) = 0.84{+/-}0.03 and an F1 = 91{+/-}2% comparing with other methods. With NECARE, we mapped the cancer interactome atlas and revealed that the perturbations of PPIs were enriched on 1362 genes, which were named cancer hub genes. Those genes were found to over-represent with mutations occurring at protein-macromolecules binding interfaces. Furthermore, over 42% of cancer treatment-related genes belonged to hub genes, which were significantly related to the prognosis of 32 types of cancers. Finally, by coimmunoprecipitation, we confirmed that the NECARE prediction method was highly reliable with a 90% accuracy. Overall, we provided the novel network-based protein-protein interaction prediction method and mapped the perturbation of cancer interactome. NECARE is available at: https://github.com/JiajunQiu/NECARE.

bioinformatics↗

bioRxiv2020

Spatially distributed representation of taste quality in the gustatory insular cortex of awake behaving mice

Visual, auditory and somatosensory cortices are topographically organized, with neurons responding to similar sensory features clustering in adjacent portions of the cortex. Such topography has not been observed in the piriform cortex, whose responses to odorants are sparsely distributed across the cortex. The spatial organization of taste responses in the gustatory insular cortex (GC) is currently debated, with conflicting evidence from anesthetized rodents pointing to alternative and mutually exclusive models. Here, we rely on calcium imaging to determine how taste and task-related variables are represented in the superficial layers of GC of alert, licking mice. Our data show that the various stimuli evoke sparse responses from a combination of broadly and narrowly tuned neurons. Analysis of the distribution of responses over multiple spatial scales demonstrates that taste representations are distributed across the cortex, with no sign of spatial clustering or topography. Altogether, data presented here support the idea that the representation of taste qualities in GC of alert mice is sparse and distributed, analogous to the representation of odorants in piriform cortex.

neuroscience↗

bioRxiv2020

Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM

The recent outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its rapid international spread pose a global health emergency. The trimeric spike (S) glycoprotein interacts with its receptor human ACE2 to mediate viral entry into host-cells. Here we present cryo-EM structures of an uncharacterized tightly closed SARS-CoV-2 S-trimer and the ACE2-bound-S-trimer at 2.7-Å and 3.8-Å-resolution, respectively. The tightly closed S-trimer with inactivated fusion peptide may represent the ground prefusion state. ACE2 binding to the up receptor-binding domain (RBD) within S-trimer triggers continuous swing-motions of ACE2-RBD, resulting in conformational dynamics of S1 subunits. Noteworthy, SARS-CoV-2 S-trimer appears much more sensitive to ACE2-receptor than SARS-CoV S-trimer in terms of receptor-triggered transformation from the closed prefusion state to the fusion-prone open state, potentially contributing to the superior infectivity of SARS-CoV-2. We defined the RBD T470-T478 loop and residue Y505 as viral determinants for specific recognition of SARS-CoV-2 RBD by ACE2, and provided structural basis of the spike D614G-mutation induced enhanced infectivity. Our findings offer a thorough picture on the mechanism of ACE2-induced conformational transitions of S-trimer from ground prefusion state towards postfusion state, thereby providing important information for development of vaccines and therapeutics aimed to block receptor binding.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2020

Ab initio Spillover Compensation in CyTOF Data

Signal intensity measured in a mass cytometry (CyTOF) channel can often be affected by the neighboring channels due to technological limitations. Such signal artifacts are known as spillover effects and can substantially limit the accuracy of cell population clustering. Current approaches reduce these effects by using additional beads for normalization purposes known as single-stained controls. While effective in compensating for spillover effects, incorporating single-stained controls can be costly and require customized panel design. This is especially evident when executing large-scale immune profiling studies. We present a novel statistical method, named CytoSpill that independently quantifies and compensates the spillover effects in CyTOF data without requiring the use of single-stained controls. Our method utilizes knowledge-guided modeling and statistical techniques, such as finite mixture modeling and sequential quadratic programming, to achieve optimal error correction. We evaluated our method using five publicly available CyTOF datasets obtained from human peripheral blood mononuclear cells (PBMCs), C57BL/6J mouse bone marrow, healthy human bone marrow, chronic lymphocytic leukemia patient, and healthy human cord blood samples. In the PBMCs with known ground truth, our method achieved comparable results to experiments that incorporated single-stained controls. In datasets without ground-truth, our method not only reduced spillover on likely affected markers, but also led to the discovery of potentially novel subpopulations expressing functionally meaningful, cluster-specific markers. CytoSpill (developed in R) will greatly enhance the execution of large-scale cellular profiling of tumor immune microenvironment, development of novel immunotherapy, and the discovery of immune-specific biomarkers. The implementation of our method can be found at https://github.com/KChen-lab/CytoSpill.git.

bioinformatics↗

bioRxiv2020

Designed peptides as potential fusion inhibitors against SARA-CoV-2 coronavirus infection

Inspired by fusion-inhibitory peptides from heptad repeat 1 (HR1) and heptad repeat 2 (HR2) domains from human immuno-deficiency virus type 1 (HIV-1) envelope glycoprotein gp41 and severe acute respiratory syndrome-coronavirus (SARS-CoV) based on viral fusogenic mechanism in the present work, we provided a similar approach to design the synthesized peptides against the entry into host cells of SARA-CoV-2 virus that causes 2019 novel coronavirus disease (COVID-19). These peptides derived from HR1 and HR2 of SARA-CoV-2 spike protein were further tested for their interaction and potential fusion possibility through circular dichroism spectrum. Here we used the peptide COVID-2019-HR1P1 (40 amino acids) as the target, which was derived from HR1 of SARA-CoV-2 spike protein, while the designed peptides including COVID-2019-HR2P1 (37 amino acids), COVID-2019-HR2P2 (32 amino acids) and others derived from HR2 of SARA-CoV-2 were tested for their binding to COVID-2019-HR1P1. Interestingly, results showed that both COVID-2019-HR2P1 and COVID-2019-HR2P2 can form the complex with COVID-2019-HR1P1, respectively. This implied that these designed peptides could play an important role in blocking SARA-CoV-2 entry into mammalian host cells via viral fusogenic mechanism, and thus could be used for preventing SARA-CoV-2 infection.

microbiology↗

bioRxiv2020

An emerging role of Prevotella histicola on estrogen-deficiency induced bone loss through the gut microbiota-bone axis

Gut microbiota (GM)-bone axis has emerged as a crucial mediator in the maintenance of bone homeostasis. Estrogen-deficiency induced bone loss is closely associated with an altered GM. However, the underlying mechanisms remain not fully understood. To this end, feces samples collected from the postmenopausal patients with osteoporosis (PMO) and with normal bone mass (PMN) were examined by 16s rRNA gene sequencing. We found that GM in PMO group was featured by a significant decreased proportion of genus Prevotella in comparison with that in the PMN group. Next, Prevotella histicola (P.histicola), a typical specie of Prevotella, was orally given to the mice following the ovariectomy (OVX) procedure and it significantly prevented OVX induced bone loss. Mechanistically, the protective effects of P.histicola on bone mass were found to be associated with the inhibition of osteoclastic resorption by attenuating osteoclastogenic cytokines expression and ameliorating gut permeability. Thus, P.histicola may prevent estrogen-deficiency induced bone loss through the GM-bone axis.

microbiology↗

bioRxiv2020

Sensei: How many samples to tell evolution in single-cell studies?

1AO_SCPLOWBSTRACTC_SCPLOWCellular heterogeneity underlies cancer evolution and metastasis. Advances in single-cell technologies such as single-cell RNA sequencing and mass cytometry have enabled interrogation of cell type-specific expression profiles and abundance across heterogeneous cancer samples obtained from clinical trials and preclinical studies. However, challenges remain in determining sample sizes needed for ascertaining changes in cell type abundances in a controlled study. To address this statistical challenge, we have developed a new approach, named Sensei, to determine the number of samples and the number of cells that are required to ascertain such changes between two groups of samples in single-cell studies. Sensei expands the t-test and models the cell abundances using a beta-binomial distribution. We evaluate the mathematical accuracy of Sensei and provide practical guidelines on over 20 cell types in over 30 cancer types based on knowledge acquired from the cancer cell atlas (TCGA) and prior single-cell studies. We provide a web application to enable user-friendly study design via https://kchen-lab.github.io/sensei/table_beta.html.

bioinformatics↗

bioRxiv2020

Reconstruction of Fur pan-regulon uncovers the complexity and diversity of transcriptional regulation in E. coli

Regulons for many transcription factors have been elucidated in model strains leading to an understanding of their role in producing physiological states. Comparative analysis of a regulon and its target genes between different strains of the same species is lacking. Ferric uptake regulator (Fur), involved in iron homeostasis, is one of the most conserved TFs, and is present in a wide range of bacteria. Using ChIP-exo experiments, we performed a comprehensive study of Fur binding sites in nine Escherichia coli strains with different lifestyles. 79 of the 431 target genes (18%) found belong to Fur core regulon, comprising genes involved in ion transport and metabolism, energy production and conversion, and amino acid metabolism and transport. 179 of the target genes (42%) comprise the accessory regulon, most of which were related to cell wall structure and biogenesis, and virulence factor pathways. The remaining target genes (173 or 40%) were in the unique regulon, with gene functions that were largely unknown. Furthermore, deletion of the fur gene led to distinct phenotypes in growth, motility, antibiotic resistance, and the change of siderophore production. These results provide a more complete understanding of how Fur regulates a set of target genes with surprising variation in closely related bacteria.

systems biology↗

bioRxiv2020

Single-cell regulatory landscape and disease vulnerability map of adult Macaque cortex

Single cell approaches have increased our knowledge about the cell type composition of the non-human primate (NHP), but a detailed characterization of area-specific regulatory features remains outstanding. We generated single-cell chromatin accessibility (single-cell ATAC) and transcriptomic data of 358,237 cells from prefrontal cortex (PFC), primary motor cortex (M1) and primary visual cortex (V1) of adult cynomolgus monkey brain, and integrated this dataset with Stereo-seq (Spatio-Temporal Enhanced REsolution Omics-sequencing) of the corresponding cortical areas to assign topographic information to molecular states. We identified area-specific chromatin accessible sites and their targeted genes, including the cell type-specific transcriptional regulatory network associated with excitatory neurons heterogeneity. We reveal calcium ion transport and axon guidance genes related to specialized functions of PFC and M1, identified the similarities and differences between adult macaque and human oligodendrocyte trajectories, and mapped the genetic variants and gene perturbations of human diseases to NHP cortical cells. This resource establishes a transcriptomic and chromatin accessibility combinatory regulatory landscape at a single-cell and spatially resolved resolution in NHP cortex.

neuroscience↗

bioRxiv2020

DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

Dengue virus (DENV) is a mosquito-borne pathogen that causes a spectrum of diseases including life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Vascular leakage is a common clinical crisis in DHF/DSS patients which is closely associated with increased endothelial permeability. The presence of vascular leakage causes hypotension, circulatory failure or disseminated intravascular coagulation as the disease progresses, which can lead to death under such conditions. However, the mechanisms by which DENV infection caused the vascular leakage are not fully understood. This study reveals a distinct mechanism by which DENV induces endothelial permeability and vascular leakage in human endothelial cells and mice tissues. We initially show that DENV2 promotes the matrix metalloproteinase-9 (MMP-9) expression and secretion in DHF patient serum, peripheral blood mononuclear cells (PBMCs) and macrophages, and further reveal that DENV non-structural protein 1 (NS1) induces MMP-9 expression through activating the nuclear factor {kappa}B (NF-{kappa}B) signaling pathway. Additionally, NS1 inhibits TIMP-1 expression to facilitates the MMP-9 enzymatic activity which alters the adhesion and tight junctions and vascular leakage in human endothelial cells and mouse tissues. Moreover, NS1 recruits MMP-9 to interact with {beta}-catenin and Zona occludins protein-1/2 to degrade the important adhesion and tight junction proteins, thereby inducing endothelial hyperpermeability and vascular leakage in human endothelial cells and mouse tissues. Thus, we reveal that DENV NS1 and MMP-9 cooperatively induce vascular leakage by impairing endothelial cell adhesion and tight junction, and suggest that MMP-9 may serve as a potential target for the treatment of hypovolemia in DSS/DHF patients. Author SummaryDENV is the most common mosquito-transmitted viral pathogen in humans. In general, DENV-infected patients are either asymptomatic or have flu-like symptoms with fever and rash. However, in severe cases of DENV infection, the disease may progress to dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), the leading causes of morbidity and mortality in school-age children in tropical and subtropical regions. DENV-induced vascular leakage is characterized by enhanced vascular permeability without morphological damage to the capillary endothelium. We found that a distinct mechanism of DENV NS1 and MMP-9 cooperatively induce vascular leakage is the main reason leading to death in severe dengue patients. Also, NS1 recruits MMP-9 to degrade {beta}-catenin, ZO-1, ZO-2 to intervene endothelial hyperpermeability in human endothelial cells and mouse vascular. Finally, we reveal that DENV activating NF-{kappa}B signaling pathway induces MMP-9 expression, in patients, mice, PBMC and macrophages though the viral NS1 protein. This study would provide new in signs into the pathogenesis caused by DENV infection, and suggest that MMP-9 may acts as a drug target for the prevention and treatment of DENV-associated diseases.

microbiology↗