Biology subjects

Xie, C.

Publications and source records attributed to Xie, C..

106 records · Page 6Linked to original sources

bioRxiv2020

Development of high-throughput screening assays for profiling snake venom Phospholipase A2 activity after high resolution chromatographic fractionation

Many organisms, ranging from plants to mammals, contain phospholipase A2 enzymes (PLA2s), which catalyze the production of lysophospholipids and fatty acid proinflammatory mediators. PLA2s are also common constituents of animal venoms, including bees, scorpions and snakes, and they cause a wide variety of toxic effects including neuro-, myo-, cyto-, and cardio-toxicity, anticoagulation and edema. The aim of this study was to develop a generic method for profiling enzymatically active PLA2s in snake venoms after chromatographic separation. For this, low-volume high-throughput assays for assessment of enzymatic PLA2 activity were evaluated and optimized. Subsequently, the assays were incorporated into a nanofractionation platform that combines high-resolution fractionation of crude venoms by liquid chromatography (LC) with bioassaying in 384-well plate format, and parallel mass spectrometric (MS) detection for toxin identification. The miniaturized assays developed are based on absorbance or fluorescence detection (respectively, using cresol red or fluorescein as pH indicators) to monitor the pH drop associated with free fatty acid formation by enzymatically active PLA2s. The methodology was demonstrated for assessment of PLA2 activity profiles of venoms from the snake species Bothrops asper, Echis carinatus, Echis coloratus, Echis ocellatus, Oxyuranus scutellatus and Daboia russelii russelii.

biochemistry↗

bioRxiv2020

Identifying strategies to target the metabolic flexibility of tumours.

Plasticity of cancer metabolism can be a major obstacle for efficient targeting of tumour-specific metabolic vulnerabilities. Here, we identify and quantify the compensatory mechanisms following the inhibition of major pathways of central carbon metabolism in c-MYC-induced liver tumours. We find that glutaminase isoform Gls2, expressed in normal liver, compensates for the deletion of Gls1 isoform expressed in tumours. Inhibiting both glutaminases significantly delays tumourigenesis but does not completely block glutamine catabolism through the Krebs cycle. We reveal that glutamine catabolism is then driven by amidotransferases. Consistently, the synergistic effect of glutaminase and amidotransferase inhibitors on proliferation of mouse and human tumour cells is observed in vitro and in vivo. Furthermore, when Gls1 is deleted the Krebs cycle activity and tumour formation can also be significantly affected if glycolysis is co-inhibited (Gls1KO/Hk2KO). Finally, the inhibition of either serine (Psat1KO) or fatty acid (FasnKO) biosynthesis can be compensated by uptake of circulating nutrients. Thus, removing these nutrients from the diet produces synergistic effects on suppression of tumourigenesis. These results highlight the high flexibility of tumour metabolism and demonstrate how targeting compensatory mechanisms can improve a therapeutic outcome.

cancer biology↗

bioRxiv2019

An antibody drug conjugate targeting a GSTA glycosite-signature epitope of mucin1 expressed by non-small cell lung cancer

BackgroundAntibodies targeting abnormally glycosylated proteins have been ineffective in treating cancer. Antibody-drug conjugates are emerging as an efficient option, which allow specific delivery of drugs into tumors. We and others have dissected the abnormally glycosylated tandem repeat region of MUC1 glycoprotein as three site-specific glycosylated neoantigen peptide motifs (PDTR, GSTA, GVTS) for monoclonal antibody binding. MethodsInternalization of monoclonal antibodies was studied by immunofluorescence staining and colocalization with lysosomal markers in live cells. Antibody positivity in tumor and peritumoral tissue samples were studied by immunohistochemistry. The efficacy of anti-MUC1 ADCs were evaluated with various cancer cell lines and mouse tumor xenograft model. ResultsWe describe an anti-MUC1 ADC by conjugating GSTA neoantigen-specific 16A with monomethyl auristatin E (MMAE). 16A-MMAE showed potent antitumoral efficacy with IC50 ranging from 0.2 to 49.4 nM toward multiple types of cancer cells. In vivo, 16A-MMAE showed dose-dependent inhibition of tumor growth in mouse xenograft of NCI-H838 NSCLC cell line, with minimum effective dose at 1 mg/kg. At the dose of 3 mg/kg, 16A-MMAE did not cause significant toxicity in a transgenic mouse expressing human MUC1. ConclusionsThe high antitumoral efficacy of 16A-MMAE suggest that aberrant glycosylated MUC1 neoantigen is a target with high positivity in multiple cancer types for ADC development. Personalized therapy may be achieved by development of glycosite-specific antibody-drug conjugates.

pharmacology and toxicology↗

bioRxiv2019

High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches

Snakebite is a neglected tropical disease that results in a variety of systemic and local pathologies in envenomed victims and is responsible for around 138,000 deaths every year. Many snake venoms cause severe coagulopathy that makes victims vulnerable to suffering life-threating haemorrhage. The mechanisms of action of coagulopathic snake venom toxins are diverse and can result in both anticoagulant and procoagulant effects. However, because snake venoms consist of a mixture of numerous protein and peptide components, high throughput characterizations of specific target bioactives is challenging. In this study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins from a wide diversity of snake species that perturb coagulation. To do so, we used a high-throughput screening approach consisting of a miniaturised plasma coagulation assay in combination with a venom nanofractionation approach. Twenty snake venoms were first separated using reversed-phase liquid chromatography, and a post-column split allowed a small fraction to be analyzed with mass spectrometry, while the larger fraction was collected and dispensed onto 384-well plates before direct analysis using a plasma coagulation assay. Our results demonstrate that many snake venoms simultaneously contain both procoagulant and anticoagulant bioactives that contribute to coagulopathy. In-depth identification analysis from seven medically-important venoms, via mass spectrometry and nanoLC-MS/MS, revealed that phospholipase A2 toxins are frequently identified in anticoagulant venom fractions, while serine protease and metalloproteinase toxins are often associated with procoagulant bioactivities. The nanofractionation and proteomics approach applied herein seems likely to be a valuable tool for the rational development of next-generation snakebite treatments by facilitating the rapid identification and fractionation of coagulopathic toxins, thereby enabling specific targeting of these toxins by new therapeutics such as monoclonal antibodies and small molecule inhibitors.\n\nAuthor summarySnakebite is a neglected tropical disease that results in more than 100,000 deaths every year. Haemotoxicity is one of the most common signs of systemic envenoming observed after snakebite, and many snake venoms cause severe impairment of the blood coagulation that makes victims vulnerable to suffering life-threating hemorrhage. In this study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins from a wide diversity of snake species that interfere with blood coagulation. Twenty snake venoms were screened for their effects on the blood coagulation cascade and based on the initial results and the medical relevance of the species, seven venoms were selected for in-depth analysis of the responsible toxins using advanced identification techniques. Our findings reveal a number of anticoagulant toxins that have not yet been reported before as such. The methodology described herein not only enables the identification of both known and unknown toxins that cause impairment of the blood coagulation, but offers a throughput platform to effectively screen for inhibitory molecules relevant for the development of next generation snakebite treatments.

pharmacology and toxicology↗

bioRxiv2019

H. pylori infection inhibits autophagy to aggravate DNA damage by p62-mediated Rad51 ubiquitination

Helicobacter pylori (H. pylori) infection is the strongest known risk factor for gastric carcinogenesis. DNA damage response (DDR) and autophagy play key roles in tumorigenic transformation. However, it remains unclear how H. pylori infection modulate DNA damage and autophagy. Here we report that H. pylori infection promotes DNA damage via suppression of Rad51 expression through inhibition of autophagy and accumulation of p62 in gastric carcinogenesis. We find that H. pylori infection caused alteration of DDR pathway and autophagy in gastric cells and Mongolian gerbils in a CagA-dependent manner. Moreover, loss of autophagy led to promotion of DNA damage in H. pylori-infected cells. Furthermore, knockdown of autophagic substrate p62 upregulated Rad51 expression, and p62 promoted ubiquitination of Rad51 via the direct interaction of the UBA domain with Rad51. Finally, H. pylori infection was associated with elevated levels of p62 in gastric intestinal metaplasia and decreased levels of Rad51 in dysplasia compared to their H. pylori- counterparts. Our findings provide a novel mechanism into the linkage of H. pylori infection, autophagy, DNA damage and gastric tumorigenesis.

microbiology↗

bioRxiv2019

Interleukin 11 expression causes murine inflammatory bowel disease

Interleukin 11 (IL11) is a profibrotic cytokine, secreted by myofibroblasts and damaged epithelial cells. Smooth muscle cells (SMCs) also secrete IL11 under pathological conditions and express the IL11 receptor. Here we examined the effects of SMC-specific, conditional expression of murine IL11 in a transgenic mouse (Il11SMC). Within days of transgene activation, Il11SMC mice developed loose stools and progressive bleeding and rectal prolapse, which was associated with a 65% mortality by two weeks. The bowel of Il11SMC mice was inflamed, fibrotic and had a thickened wall, which was accompanied by activation of ERK and STAT3. In other organs, including heart, lung, liver, kidney and skin there was a phenotypic spectrum of fibro-inflammation, together with consistent ERK activation. To investigate further the importance of stromal-derived IL11 in the inflammatory bowel phenotype we used a second model with fibroblast-specific expression of IL11, the Il11Fib mouse. This additional model largely phenocopied the Il11SMC bowel phenotype. These data show that IL11 secretion from the stromal niche is sufficient to drive inflammatory bowel disease in mice. Given that IL11 expression in colonic stromal cells predicts anti-TNF therapy failure in patients with ulcerative colitis or Crohns disease, we suggest IL11 as a therapeutic target for inflammatory bowel disease.

pathology↗

bioRxiv2019

Magnetic manipulation of axonal endosome transport in live neurons

Noninvasive control of axonal cargos in live neurons is a challenging prospect that can enable novel research on the mechanisms of axonal cargo transport, cargo-mediated signaling and axonal traffic jams in neurons. However, conventional techniques for force manipulation such as optical traps are limited to a few micron-sized cargos and are not applicable to the small axonal cargos in live neurons. Here, we present a new methodology that permits the external control of axonal endosome transport via tailored magnetic forces. By culturing neurons in a microfluidic device made up of microfabricated magnetic arrays, we can exert 3 - 48 pN forces on retrograde axonal endosomes carrying fluorescent magnetic nanoparticles, 100 - 260 nm in size. The magnetic force counters the forces exerted by molecular motors driving the endosomes and results in a wide range of perturbations on endosome transport in axons. These perturbations, captured by oblique illumination fluorescence imaging, reveal new insights on the collective function of dyneins and the nature of paused and stationary states during retrograde endosome transport in axons. Most notably, we demonstrate controllable capture and release of retrograde endosomes in axons by toggling the external magnetic field. This technical advance has great potential to elucidate the spatiotemporal origins of long-distance endosome signaling pathways as well as the ramifications of axonal traffic jams in neurons.

biophysics↗

bioRxiv2019

Novel immune and stromal subtype classification system of lung adenocarcinoma based on tumor microenvironment

BackgroundTumor microenvironment has complex effects on tumorigenesis and metastasis in lung adenocarcinoma (LUAD). However, there is still a lack of comprehensive understanding of the relationship between immune and non-immune stromal characteristics in tumor microenvironment.\n\nPatients and methodsEight cohort of 1681 lung caner patients were included in this study. The immune and non-immune stromal signatures of tumor microenvironment were identified by eigendecomposition and extraction algorithms. We developed a novel immune and stromal scoring system to quantify anti-tumor immune and promote-metastasis stromal activation, namely PMBT (prognostic model based on tumor microenvironment) as an R package. Tumors were classified into 4 subtypes according to PMBT system. Comprehensive analysis was performed in different subtypes.\n\nResultsThe 4 subgroups had different mutation landscape, molecular, cellular characteristics and prognosis, which validated by 7 data sets containing 1175 patients. 19% patients was characterized by highly active anti-tumor reaction with high production of immunoactive mediators, immunocyte, low fibroblasts infiltration, low TGF-{beta}, VEGFA, collagen and glucose catabolic, low immune checkpoint per T cell, tumor mutation burden, and favourable overall survival (all, P < 0.05) named high-immune and low-stromal activation subgroup (HL). Cellular paracrine network showed both high humoral and cellular immune interaction in HL group. The low-immune and high-stromal activation group (19%) had opposite characteristics with HL group. Moreover, the PMBT system showed the value to predict overall survival and immunotherapy responses (all, P < 0.05).\n\nConclusionsDifferent molecular, cellular characteristics, mutation landscape and prognosis were discovered in the 4 subgroups. Our classification, PMBT system provided novel insight for clinical monitoring and treatment in LUAD.

bioinformatics↗

bioRxiv2019

Complementary roles for ventral pallidum cell types and their projections in relapse

Ventral pallidum (VP) is a key node in the neural circuits controlling relapse to drug seeking but how this role relates to different VP cell types and their projections is poorly understood. Using male rats, we show how different forms of relapse to alcohol-seeking are assembled from VP cell types and their projections to lateral hypothalamus (LH) and ventral tegmental area (VTA). First, we used RNAScope in situ hybridization to characterize activity of different VP cell types during relapse to alcohol-seeking provoked by renewal (context-induced reinstatement). We found that VP Gad1 and parvalbumin (PV), but not vGlut2, neurons show relapse-associated changes in c-Fos expression. Next, we used retrograde tracing, chemogenetic, optogenetic, and electrophysiological approaches to study the roles of VPGad1 and VPPV neurons in relapse. We show that VPGad1 neurons contribute to contextual control over relapse (renewal), but not to relapse during reacquisition, via projections to LH where they converge with ventral striatal inputs onto LHGad1 neurons. This convergence of striatopallidal inputs at the level of individual LHGad1 neurons may be critical to balancing propensity for relapse versus abstinence. In contrast, VPPV neurons contribute to relapse during both renewal and reacquisition via projections to VTA but not LH. These findings show complementary roles for different VP cell types and their projections in relapse. VPGad1 neurons control relapse during renewal via projections to LH whereas VPPV neurons control relapse during both renewal and reacquisition via projections to VTA. Targeting these different pathways may provide tailored interventions for different forms of relapse.

neuroscience↗

bioRxiv2019

Studying the dawn of de novo gene emergence in mice reveals fast integration of new genes into functional networks

The de novo emergence of new transcripts has been well documented through genomic analyses. However, a functional analysis, especially of very young protein-coding genes, is still largely lacking. Here we focus on three loci that have evolved from previously intergenic sequences in the house mouse (Mus musculus) and are not present in its closest relatives. We have obtained knockouts and analyzed their phenotypes, including a deep transcriptomic analysis, based on a dedicated power analysis. We show that the transcriptional networks are significantly disturbed in the knockouts and that all three genes have effects on phenotypes that are related to their expression patterns. This includes behavioral effects, skeletal differences and the regulation of the reproduction cycle in females. Substitution analysis suggests that all three genes have directly obtained an activity, without new adaptive substitutions. Our findings support the hypothesis that de novo genes can quickly adopt functions without extensive adaptation.\n\nImpact statementNew protein-coding genes emerging out of non-coding sequences can become directly functional without signatures of adaptive protein changes

evolutionary biology↗

bioRxiv2018

The permeabilized SecY protein-translocation channel can serve as a nonspecific sugar transporter

As the initial step in carbohydrate catabolism in cells, the substrate-specific transporters via active transport and facilitated diffusion play a decisive role in passage of sugars through the plasma membrane into the cytoplasm. The SecY complex (SecYEG) in bacteria forms a membrane channel responsible for protein translocation. This work demonstrates that weakening the sealability of the SecY channel allowed free diffusion of sugars, including glucose, fructose, mannose, xylose, arabinose, and lactose, into the engineered cells, facilitating its rapid growth on a wide spectrum of monosaccharides and bypassing/reducing stereospecificity, transport saturation, competitive inhibition, and carbon catabolite repression (CCR), which are usually encountered with the specific sugar transporters. The SecY channel is structurally conserved in prokaryotes, thus it may be engineered to serve as a unique and universal transporter for bacteria to passage sugars as demonstrated in Escherichia coli and Clostridium acetobutylicum.

bioengineering↗

bioRxiv2018

Functional connectivity mechanisms subserving automatic emotion regulation by implementation intention

Automatic emotion regulation (AER) has received increasing attention. However, the effortless hypothesis of AER has yet to be directly tested and the neural mechanisms supporting AER remain unclear. Here, we addressed these two issues, by simultaneous recording behavioral assessments and functional MRI (fMRI) signals when thirty-one participants were required to watch neutral and disgust pictures in three experimental conditions: Watching, Goal Intention (GI) and Reappraisal by Implementation Intention (RII). RII decreased negative feelings and bilateral amygdala activity without engendering cognitive efforts, evidenced by the reduced self-reported efforts and less prefrontal engagement during RII compared to Watching and GI. These regulation effects should not be explained by emotional habituation, as further experiment did not show significant emotional habituation for the repetition of the same stimuli. Moreover, RII and GI produced similar functional connectivity (FC) of vACC (ventral anterior cingulate cortex) to left insula and right precuneus, corresponding to the common goal pursuit component of both strategies. Furthermore, RII relative to GI exhibited weaker FC in distributed brain networks subserving effortful control (e.g. inferior-superior parietal lobe FC), memory retrieval (e.g. inferior-middle temporal lobes and lingual-putamen FCs), aversive anticipation and emotion-related motor planning (e.g. putamen-operculum and vACC-supramarginal gyrus FCs). These findings suggest that RII downregulates disgust effortlessly, most likely mediated by the reduced online mobilization of neural systems subserving emotion-related coping.

neuroscience↗

bioRxiv2018

IL-11 is a therapeutic target in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) remains a progressive disease despite best medical management. We previously identified IL-11 as a critical factor for cardiovascular fibrosis and examine here its role in pulmonary fibrosis. IL-11 is consistently upregulated in IPF genomic datasets, which we confirmed by histology. Pulmonary fibroblasts stimulated with IL-11 transform into invasive myofibroblasts whereas fibroblasts from Il11ra deleted mice did not respond to pro-fibrotic stimuli. In the mouse, injection of recombinant Il-11 or fibroblast-specific expression of Il-11 caused pulmonary fibrosis. We then generated a neutralising IL-11 binding antibody that blocks lung fibroblast activation across species. In a mouse model of IPF, anti-IL-11 therapy attenuated lung fibrosis and specifically blocked Erk activation. These data prioritise IL-11 as an accessible drug target in IPF.\n\nOne Sentence SummaryNon-canonical IL-11 signalling is a central hallmark of idiopathic pulmonary fibrosis and represents a novel target for antibody therapies.

molecular biology↗

bioRxiv2017

MAK and CCRK kinases regulate kinesin-2 motility in C. elegans neuronal cilia

Kinesin-2 motors power the anterograde intraflagellar transport (IFT), a highly ordered process that assembles and maintains cilia. It remains elusive how kinesin-2 motors are regulated in vivo. Here we perform forward genetic screen to isolate suppressors that rescue the ciliary defects in the constitutive active mutation of OSM-3-kinesin (G444E) in C. elegans sensory neurons. We identify the C. elegans DYF-5 and DYF-18, which encode the homologs of mammalian male germ cell-associated kinase (MAK) and cell cycle-related kinase (CCRK). Using time-lapse fluorescence microscopy, we show that DYF-5 and DYF-18 are IFT cargo molecules and are enriched at the distal segments of sensory cilia. Mutations of dyf-5 and dyf-18 generate the elongated cilia and ectopic localization of kinesin-II at the ciliary distal segments. Genetic analyses reveal that dyf-5 and dyf-18 are also important for stabilizing the interaction between IFT particle and OSM-3-kinesin. Our data suggest that DYF-5 and DYF-18 act in the same pathway to promote handover between kinesin-II and OSM-3 in sensory cilia.

cell biology↗

bioRxiv2017

Polymodal Allosteric Regulation of Type 1 Serine/Threonine Kinase Receptors via a Conserved Electrostatic Lock

Type 1 Serine/Threonine Kinase Receptors (STKR1) transduce a wide spectrum of biological signals mediated by TGF-{beta} superfamily members. The STKR1 activity is tightly controlled by their regulatory glycine-serine rich (GS) domain adjacent to the kinase domain. Despite decades of studies, it remains unknown how physiological or pathological GS domain modifications are coupled to STKR1 kinase activity. Here, by performing molecular dynamics simulations and free energy calculation of Activin-Like Kinase 2 (ALK2), we found that GS domain phosphorylation, FKBP12 dissociation, and disease mutations all destabilize a D354-R375 salt-bridge, which normally acts as an electrostatic lock to prevent coordination of adenosine triphosphate (ATP) to the catalytic site. We developed a WAFEX-guided principal analysis and unraveled how phosphorylation destabilizes this highly conserved salt-bridge in temporal and physical space. Using current-flow betweenness scores, we identified an allosteric network of residue-residue contacts between the GS domain and the catalytic site that controls the formation and disruption of this salt bridge. Importantly, our novel network analysis approach revealed how certain disease-causing mutations bypass FKBP12-mediated kinase inhibition to produce leaky signaling in the absence of ligand. We further provide experimental evidence that this salt-bridge lock exists in other STKR1s, and acts as a general safety mechanism in STKR1 to prevent pathological leaky signaling. In summary, our study provides a compelling and unifying allosteric activation mechanism in STKR1 kinases that reconciles a large number of experimental studies and sheds light on a novel therapeutic avenue to target disease-related STKR1 mutants.\n\nAUTHOR SUMMARYKinases play central role in essential physiological process and are attractive therapeutic drug targets. One of the important kinase families is Type 1 Serine/Threonine Kinase Receptors (STKR1), which control gene expression in response to extracellular growth factors. The activities of STKR1 are tightly controlled by their regulatory domain, which is distant from the kinase catalytic site. The underlying molecular mechanism is elucidated here. We identified that formation or disruption of a highly conserved charge-charge interaction located near the ATP binding site, mediates the physiological inhibition or activation of STKR1. We find that the stability of this charge-charge interaction is remotely controlled by interactions propagated from the distant regulatory domain. Several disease-causing mutations are located at the regulatory domain. We demonstrate how those mutations bypass these endogenous STKR1 inhibition mechanisms to produce pathological phenotypes. This study provides a general activation mechanism in STKR1 kinases, thus may benefit understanding the molecular mechanism of diseases and drug development.

biophysics↗

bioRxiv2016

The detection of covariation of mRNA levels of large sets of genes across multiple human populations

In this study, we analyse RNA-Seq data from panels of human lymphoblastoid cell lines (LCLs) to identify covariation in the mRNA levels of large numbers of genes. Such large scale covariation may have biological origin or be due to technical variation in analysis (generally referred to as batch effects). We show that batch effects cannot explain this covariation by demonstrating reproducibility across different human populations and across different methods of analysis. This view is also supported by enrichment of single and combinations of transcription factors (TFs) binding to cognate promoter regions, enrichment of genes shown to be sensitive to the knockdown of individual TFs, enrichment of functional pathways, and finally enrichment of protein-protein interactions in proteins encoded by groups of covarying genes. The properties of the groups of covarying genes are therefore most readily explained by the influence of cumulative variations in the effectors of gene expression that act in trans on cognate genes. We suggest that covariation has functional outcomes by showing that covariation of 83 genes involved in the spliceosome pathway accounts for 8-16% of the variation in the alternative splicing patterns of genes expressed in human LCLs.

genetics↗