Biology subjects

Kong, W.

Publications and source records attributed to Kong, W..

49 records · Page 3Linked to original sources

bioRxiv2020

Polyamine biosynthesis and eIF5A hypusination are modulated by the DNA tumor virus KSHV and promote KSHV viral infection

Polyamines are critical metabolites involved in various cellular processes and often dysregulated in cancers. Kaposis sarcoma associated Herpesvirus (KSHV) is a defined oncogenic virus belonging to the sub-family of human gamma-herpesviruses. KSHV infection leads to the profound alteration of host metabolic landscape to favor the development of KSHV-associated malignancies. In our studies, we identified that polyamine biosynthesis and eIF5A hypusination are dynamically regulated by KSHV infection likely through the modulation of key enzymes of these pathways, such as ODC1, and that in return these metabolic pathways are required for both KSHV lytic switch from latency and de novo infection. The further analysis unraveled that translation of critical KSHV latent and lytic proteins (LANA, RTA) depends on eIF5A hypusination. We also demonstrated that KSHV infection can be efficiently and specifically suppressed by using inhibitors targeting either polyamine biosynthesis or eIF5A hypusination. Above all, our results illustrated that the dynamic and profound interaction of a DNA tumor virus (KSHV) with host polyamine biosynthesis and eIF5A hypusination metabolic pathways promote viral propagation and oncogenesis, which serve as new therapeutic targets to treat KSHV-associated malignancies.

microbiology↗

bioRxiv2020

Prevailing role of mucosal immunoglobulions and B cells in teleost skin immune responses to bacterial infection

The skin of vertebrates is the outermost organ of the body and serves as the first line of defense against external aggressions. In contrast to mammalian skin, that of teleost fish lacks keratinization and has evolved to operate as a mucosal surface containing a skin-associated lymphoid tissue (SALT). Thus far, IgT representing the prevalent immunoglobulin (Ig) in SALT have only been reported upon infection with a parasite. However, very little is known about the types of B cells and Igs responding to bacterial infection in the teleost skin mucosa, as well as the inductive or effector role of the SALT in such responses. To address these questions, here we analyzed the immune response of trout skin upon infection with one of the most widespread fish skin bacterial pathogens, Flavobacterium columnare. This pathogen induced strong skin innate immune and inflammatory responses at the initial phases of infection. More critically, we found that the skin mucus of fish having survived the infection contained significant IgT-but not IgM- or IgD-specific titers against the bacteria. Moreover, we demonstrate the local proliferation and production of IgT+ B-cells and specific IgT titers respectively within the SALT upon bacterial infection. Thus, our findings represent the first demonstration that IgT is the main Ig isotype induced by the skin mucosa upon bacterial infection, and that because of the large surface of the skin, its SALT probably represents a prominent IgT inductive site in fish.

immunology↗

bioRxiv2020

Capybara: A computational tool to measure cell identity and fate transitions

Transitions in cell identity are fundamental to development, reprogramming, and disease. Single-cell technologies enable the dissection of tissue composition on a cell-by-cell basis in complex biological systems. However, highly-sparse single-cell RNA-seq data poses challenges for cell-type identification algorithms based on bulk RNA-seq. Single-cell analytical tools are also limited, where they require prior biological knowledge and typically classify cells in a discrete, categorical manner. Here, we present a computational tool, Capybara, designed to measure cell identity as a continuum, at single-cell resolution. This approach enables the classification of discrete cell entities but also identifies cells harboring multiple identities, supporting a metric to quantify cell fate transition dynamics. We benchmark the performance of Capybara against other existing classifiers and demonstrate its efficacy to annotate cells and identify critical transitions within a well-characterized differentiation hierarchy, hematopoiesis. Our application of Capybara to a range of reprogramming strategies reveals previously uncharacterized regional patterning and identifies a putative in vivo correlate for an engineered cell type that has, to date, remained undefined. These findings prioritize interventions to increase the efficiency and fidelity of cell engineering strategies, showcasing the utility of Capybara to dissect cell identity and fate transitions. Capybara code and documentation are available at https://github.com/morris-lab/Capybara.

genomics↗

bioRxiv2019

Developing a DeSipher method for conformational change detection of membrane receptor complex at residue resolution

While one dimensional 1H nuclear magnetic resonance (1D 1H-NMR) spectroscopy is one of the most important and convenient method for measuring conformation change in biomacromolecules, characterization of protein dynamics in large membrane protein complexes by 1D 1H-NMR remains challenging, due to the difficulty of spectra assignment, low signal-to-noise ratio (S/N) and the need for large amount of protein. Here we report the site-specific incorporation of 4-trimethylsilyl phenylalanine (TMSiPhe) into proteins, through genetic code expansion in Escherichia coli cells, and the measurement of multiple conformational states in membrane protein complex by 1D 1H-NMR. The unique up-field 1H-NMR chemical shift of TMSiPhe, highly efficient and specific incorporation of TMSiPhe enabled facile assignment of the TMSiPhe 1H-NMR signal, and characterization of multiple conformational state in a 150 kilodalton (kD) membrane protein complex, using only 5 M of protein and 20 min spectra accumulation time. This highly efficient and convenient methods should be broadly applicable for the investigation of dynamic conformation change of protein complexes.

biochemistry↗

bioRxiv2019

A lipid code-dependent phosphoswitch directs PIN-mediated auxin efflux in Arabidopsis development

Directional intercellular transport of the phytohormone auxin mediated by PIN FORMED (PIN) efflux carriers plays essential roles in both coordinating patterning processes and integrating multiple external cues by rapidly redirecting auxin fluxes. Multilevel regulations of PIN activity under internal and external cues are complicated; however, the underlying molecular mechanism remains elusive. Here we demonstrate that 3-Phosphoinositide-Dependent Protein Kinase1 (PDK1), which is conserved in plants and mammals, functions as a molecular hub integrating the upstream lipid signalling and the downstream substrate activity through phosphorylation. Genetic analysis uncovers that loss-of-function Arabidopsis mutant pdk1.1 pdk1.2 exhibits a plethora of abnormalities in organogenesis and growth, due to the defective PIN-dependent auxin transport. Further cellular and biochemical analyses reveal that PDK1 phosphorylates D6 Protein Kinase to facilitate its activity towards PIN proteins. Our studies establish a lipid-dependent phosphorylation cascade connecting membrane composition-based cellular signalling with plant growth and patterning by regulating morphogenetic auxin fluxes.

plant biology↗

bioRxiv2019

Mutational signatures in upper tract urothelial carcinoma define etiologically distinct subtypes with prognostic relevance

Parts of East Asia have a very high upper tract urothelial carcinoma (UTUC) prevalence, and an etiological link between the medicinal use of herbs containing aristolochic acid (AA) and UTUC has been established. The mutational signature of AA, which is characterized by a particular pattern of A:T to T:A transversions, can be detected by genome sequencing. Thus, integrating mutational signatures analysis with clinicopathological data may be a crucial step toward personalized treatment strategies for the disease. Therefore, we performed whole-genome sequencing (WGS) of 90 UTUC patients from China. Mutational signature analysis via nonnegative matrix factorization method defined three etiologically distinct subtypes with prognostic relevance: (i) AA, the typical AA mutational signature characterized by signature 22, had the highest tumor mutation burden, the best clinical outcomes; (ii) Age, an age-related group featured by signatures 1 and 5, had the lowest weighted genome instability index score, the worst clinical outcomes; and (iii) DSB, signature with deficiencies in DNA double strand break-repair featured by signatures 3, the intermediate clinical outcomes. Additionally, the distinct AA subtype was associated with AA exposure, the highest number of predicted neoantigens and heavier lymphocytes infiltrating. Thus, it may be good candidate for immune checkpoint blockade therapy. Notably, we showed AA-mutational signature was also identified in histologically \"normal\" urothelial cells. Thus, non-invasive urine test based on the AA-mutational signature could take advantage of this \"field effect\" to screen individuals at increased risk of recurrence due to exposure to herbal remedies containing the carcinogen AA. Collectively, the findings here may accelerate the development of novel prognostic markers and personalized therapeutic approaches for UTUC patients in China.

genomics↗

bioRxiv2019

Single-Cell Analysis Reveals Regional Reprogramming during Adaptation to Massive Small Bowel Resection in Mice

Background & AimsThe small intestine (SI) displays regionality in nutrient and immunological function. Following SI tissue loss (as occurs in short gut syndrome, or SGS), remaining SI must compensate, or adapt; the capacity of SI epithelium to reprogram its regional identity has not been described. Here, we apply single-cell resolution analyses to characterize molecular changes underpinning adaptation to SGS.\n\nMethodsSingle-cell RNA-sequencing was performed on epithelial cells isolated from distal SI of mice following 50% proximal small bowel resection (SBR) vs. sham surgery. Single-cell profiles were clustered based on transcriptional similarity, reconstructing differentiation events from intestinal stem cells (ISCs) through to mature enterocytes. An unsupervised computational approach to score cell identity was used to quantify changes in regional (proximal vs distal) SI identity, validated using immunofluorescence, immunohistochemistry, qPCR, western blotting, and RNA-FISH.\n\nResultsUniform Manifold Approximation and Projection-based clustering and visualization revealed differentiation trajectories from ISCs to mature enterocytes in sham and SBR. Cell identity scoring demonstrated segregation of enterocytes by regional SI identity: SBR enterocytes assumed more mature proximal identities. This was associated with significant upregulation of lipid metabolism and oxidative stress gene expression, which was validated via orthogonal analyses. Observed upstream transcriptional changes suggest retinoid metabolism and proximal transcription factor Creb3l3 drive proximalization of cell identity in response to SBR.\n\nConclusionsAdaptation to proximal SBR involves regional reprogramming of ileal enterocytes toward a proximal identity. Interventions bolstering the endogenous reprogramming capacity of SI enterocytes--conceivably by engaging the retinoid metabolism pathway--merit further investigation, as they may increase enteral feeding tolerance, and obviate intestinal failure, in SGS.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC=\"FIGDIR/small/615054v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (26K):\norg.highwire.dtl.DTLVardef@156d1b4org.highwire.dtl.DTLVardef@e1a3fcorg.highwire.dtl.DTLVardef@1d519bforg.highwire.dtl.DTLVardef@18d4e9d_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisHere, single-cell RNA sequencing reveals interactions between the retinoid metabolism pathway and regional reprogramming of distal small intestinal epithelium to a proximal identity following proximal small bowel resection. This provides novel insight into physiological adaptation to short gut syndrome.

genomics↗

bioRxiv2019

Molecular changes in Mesembryanthemum crystallinum guard cells underlying the C3 to CAM transition

Crassulacean acid metabolism (CAM) is a specialized type of photosynthesis: stomata close during the day, enhancing water conservation, and open at night, allowing CO2 uptake. Mesembryanthemum crystallinum (common ice plant) is a facultative CAM species that can shift from C3 photosynthesis to CAM under salt or drought stresses. However, the molecular mechanisms underlying the stress induced transition from C3 to CAM remain unknown. Here we determined the transition time from C3 to CAM in M. crystallinum under salt stress. In parallel, single-cell-type transcriptomic profiling by 3-mRNA sequencing was conducted in guard cells to determine the molecular changes in this key cell type during the transition. In total, 495 transcripts showed differential expression between control and salt-treated samples during the transition, including 285 known guard cell genes, seven CAM-related genes, 18 transcription factors, and 185 other genes previously not found to be expressed in guard cells. PEPC1 and PPCK1, which encode key enzymes of CAM photosynthesis, were up-regulated in guard cells after seven days of salt treatment, indicating that guard cells themselves can transition from C3 to CAM. This study provides important information towards introducing CAM stomatal behavior into C3 crops to enhance water use efficiency.\n\nSummary statementWe determined the timing of salt induced transition of common ice plant from C3 to CAM and identified transcriptomic changes during the transition. The data support the notion that guard cells themselves can transition from C3 to CAM.

plant biology↗

bioRxiv2018

Force inference predicts local and tissue-scale stress patterns in epithelia

Morphogenesis relies on the active generation of forces, and the transmission of these forces to surrounding cells and tissues. Hence measuring forces directly in developing embryos is an essential task to study the mechanics of development. Among the experimental techniques that have emerged to measure forces in epithelial tissues, force inference is particularly appealing. Indeed it only requires a snapshot of the tissue, as it relies on the topology and geometry of cell contacts, assuming that forces are balanced at each vertex. However, establishing force inference as a reliable technique requires thorough validation in multiple conditions. Here we performed systematic comparisons of force inference with laser ablation experiments in three distinct Drosophila epithelia. We show that force inference accurately predicts single junction tensions, tension patterns in stereotyped groups of cells, and tissue-scale stress patterns, in wild type and mutant conditions. We emphasize its ability to capture the distribution of forces at different scales from a single image, which gives it a critical advantage over perturbative techniques such as laser ablation. Our results demonstrate that force inference is a reliable and efficient method to quantify the mechanics of epithelial tissues during morphogenesis.

biophysics↗

bioRxiv2018

Mucosal immunoglobulins protect the olfactory organ of teleost fish against parasitic infection

The olfactory organ of vertebrates receives chemical cues present in the air or water and, at the same time, they are exposed to invading pathogens. Nasal-associated lymphoid tissue (NALT), which serves as a mucosal inductive site for humoral immune responses against antigen stimulation, is present in teleosts and mammals. IgT in teleosts is responsible for similar functions to those carried by IgA in mammals. Moreover, teleost NALT is known to contain B-cells and teleost nasal mucus contains immunoglobulins (Igs). Yet, whether nasal B cells and Igs respond to infection remains unknown. We hypothesized that water-borne parasites can invade the nasal cavity of fish and elicit local specific immune responses. To address this hypothesis, we developed a model of bath infection with the Ichthyophthirius multifiliis (Ich) parasite in rainbow trout, Oncorhynchus mykiss, an ancient bony fish, and investigated the nasal adaptive immune response against this parasite. Critically, we found that Ich parasites in water could be reach the nasal cavity and successfully invade the nasal mucosa. Moreover, strong parasite-specific IgT responses were exclusively detected in the nasal mucus, and the accumulation of IgT+ B-cells was noted in the nasal epidermis after Ich infection. Strikingly, local IgT+ B-cell proliferation and parasite-specific IgT generation were found in the trout olfactory organ, providing new evidence that nasal-specific immune responses were induced locally by a parasitic challenge. Overall, our findings suggest that nasal mucosal adaptive immune responses are similar to those reported in other fish mucosal sites and that an antibody system with a dedicated mucosal Ig performs evolutionary conserved functions across vertebrate mucosal surfaces.\n\nAuthor SummaryThe olfactory organ is a vitally important chemosensory organ in vertebrates but it is also continuously stimulated by pathogenic microorganisms in the external environment. In mammals and birds, nasopharynx-associated lymphoid tissue (NALT) is considered the first line of immune defense against inhaled antigens and in bony fish, protecting against water-borne infections. However, although B-cells and immunoglobulins (Igs) have been found in teleost NALT, the defensive mechanisms of parasite-specific immune responses after pathogen challenge in the olfactory organ of teleost fish remain poorly understood. Considering that the NALT of all vertebrates has been subjected to similar evolutionary forces, we hypothesize that mucosal Igs play a critical role in the defense of olfactory systems against parasites. To confirm this hypothesis, we show the local proliferation of IgT+ B-cells and production of pathogen-specific IgT within the nasal mucosa upon parasite infection, indicating that parasite-specific IgT is the main Ig isotype specialized for nasal-adaptive immune responses. From an evolutionary perspective, our findings contribute to expanding our view of nasal immune systems and determining the fate of the host-pathogen interaction.

immunology↗

bioRxiv2018

CellTag Indexing: a genetic barcode-based multiplexing tool for single-cell technologies

Single-cell technologies have seen rapid advancements in recent years, along with new analytical challenges and opportunities. These high-throughput assays increasingly require special consideration in experimental design, sample multiplexing, batch effect removal, and data interpretation. Here, we describe a lentiviral barcode-based multiplexing approach, CellTag Indexing, where we transduce and label samples that can then be pooled together for downstream application and analysis. By introducing predefined genetic barcodes that are transcribed and readily detected, we can reliably read out sample identity via genomic or transcriptomic profiling, permitting the simultaneous assessment of cell grouping and transcriptional state. We validate and demonstrate the utility of CellTag Indexing by sequencing transcriptomes at single-cell resolution using a variety of cell types including mouse pre-B cells, primary mouse embryonic fibroblasts, human HEK293T cells, and mouse induced endoderm progenitors. Furthermore, we establish CellTag Indexing as a valuable tool for multiplexing direct lineage reprogramming perturbation experiments. We present CellTag Indexing as a broadly applicable genetic multiplexing tool that is complementary with existing single-cell RNA-sequencing and multiplexing strategies.

genomics↗

bioRxiv2018

Genotyping by sequencing of 393 Sorghum bicolor BTx623 x IS3620C recombinant inbred lines improves sensitivity and resolution of QTL detection

We describe a genetic map with a total of 381 bins of 616 genotyping by sequencing (GBS)-based SNP markers in a F6-F8 recombinant inbred line (RIL) population of 393 individuals derived from crossing S. bicolor BTx623 to S. bicolor IS3620C, a guinea line substantially diverged from BTx623. Five segregation distorted regions were found with four showing enrichment for S. bicolor alleles, suggesting possible selection during formation of this RIL population. A quantitative trait locus (QTL) study with this number of individuals, tripled relative to prior studies of this cross, provided resources, validated previous findings, and demonstrated improved power to detect plant height and flowering time related QTLs relative to other published studies. An unexpected low correlation between flowering time and plant height permitted us to separate QTLs for each trait and provide evidence against pleiotropy. Ten non-random syntenic regions conferring QTLs for the same trait suggest that those QTLs may represent alleles at genes functioning in the same manner since the 96 million year ago genome duplication that created these syntenic relationships, while syntenic regions conferring QTLs for different trait may suggest sub-functionalization after duplication. Collectively, this study provides resources for marker-assisted breeding, as well as a framework for fine mapping and subsequent cloning of major genes for important traits such as plant height and flowering time in sorghum.

genetics↗

bioRxiv2018

Preclinical Study: Sunitinib-suppressed MiR-452-5p Facilitates Renal Cancer Cell Invasion and Metastasis Through Modulating SMAD4/SMAD7/EMT Signals

Although microRNAs (miRNAs) have been revealed as crucial modulators in tumor metastasis and target therapy, our understanding of their roles in metastatic renal cell carcinoma (mRCC) and Sunitinib treatment is limited. Here, We focused on 2 published microarray data to select out our anchored miRNA which was downregulated after Sunitinib treatment while upregulated in metastasis RCC tissues. Then we discovered that treating with Sunitinib, the targeted receptor tyrosine kinase inhibitor (TKI), inhibited renal cell migration and invasion via attenuating the expression of miR-452-5p. The novel identified miR-452-5p was upregulated and associated with poor prognosis in RCC. Preclinical studies using multiple RCC cells and xenografts model illustrated that miR-452-5p could promote RCC cell migration and invasion in vitro and in vivo. Mechanistically, P65 could directly bind to the miR-452-5p promoter and thus transcriptionally induce miR-452-5p expression, which led to post-transcriptionally abrogate SMAD4 expression, thus inhibition of its downstream signals including SMAD7 and EMT (Epithelial-Mesenchymal Transition) associated genes. Our study presented a road map for targeting this newly identified miR-452-5p and its SMAD4/SMAD7/EMT signals pathway, which imparted a new potential therapeutic strategy for mRCC treatment.

cancer biology↗