Biology subjects

Wu, C.

Publications and source records attributed to Wu, C..

At least 307 records · Page 17Linked to original sources

bioRxiv2020

Global impact of phosphorylation on protein endurance

Post-translational modifications such as phosphorylation can have profound effects on the physicochemical and biological properties of proteins. However, high-throughput and systematic approaches have not yet been developed to assess the effects of specific modification types and sites on protein lifetime, which represents a key parameter for understanding signaling rewiring and drug development. Here we describe a proteomic method, DeltaSILAC, to quantify the impact of site-specific phosphorylation on the endurance of thousands of proteins in live cells. Being configured on the reproducible data-independent acquisition mass spectrometry (DIA-MS), the pulse labeling approach using stable isotope-labeled amino acids in cells (SILAC), together with a novel peptide-level matching strategy, this multiplexed assay revealed the global delaying effect of phosphorylation on protein turnover in growing cancer cells. Further, we identified local sequence and structural features in proximity to the phosphorylated sites that could be associated with protein endurance alterations. We found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness and evolutionarily conserved. DeltaSILAC provides a generalizable approach for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology, which is highly complementary to existing methods. Finally, DeltaSILAC is widely applicable to diverse post-translational modification types and different cell systems.

systems biology↗

bioRxiv2020

Reprogramming the endogenous type III-A CRISPR-Cas system for genome editing, RNA interference and CRISPRi screening in Mycobacterium tuberculosis

Mycobacterium tuberculosis (M.tb) causes the current leading infectious disease. Examination of the functional genomics of M.tb and development of drugs and vaccines are hampered by the complicated and time-consuming genetic manipulation techniques for M.tb. Here, we reprogrammed M.tb endogenous type III-A CRISPR-Cas10 system for simple and efficient gene editing, RNA interference and screening via simple delivery of a plasmid harboring a mini-CRISPR array, thereby avoiding the introduction of exogenous proteins and minimizing proteotoxicity. We demonstrated that M.tb genes were efficiently and specifically knocked-in/out by this system, which was confirmed by whole-genome sequencing. This system was further employed for single and simultaneous multiple-gene RNA interference. Moreover, we successfully applied this system for genome-wide CRISPR interference screening to identify the in-vitro and intracellular growth-regulating genes. This system can be extensively used to explore the functional genomics of M.tb and facilitate the development of new anti-Mycobacterial drugs and vaccines. SummaryTuberculosis caused by Mycobacterium tuberculosis (M.tb) is the current leading infectious disease affecting more than ten million people annually. To dissect the functional genomics and understand its virulence, persistence, and antibiotics resistance, a powerful genome editing tool and high-throughput screening methods are desperately wanted. Our study developed an efficient and a robust tool for genome editing and RNA interference in M.tb using its endogenous CRISPR cas10 system. Moreover, the system has been successfully applied for genome-wide CRISPR interference screening. This tool could be employed to explore the functional genomics of M.tb and facilitate the development of anti-M.tb drugs and vaccines.

microbiology↗

bioRxiv2020

Deep neural networks identify context-specific determinants of transcription factor binding affinity

Transcription factors (TFs) bind DNA by recognizing highly specific DNA sequence motifs, typically of length 6-12bp. A TF motif can occur tens of thousands of times in the human genome, but only a small fraction of those sites are actually bound. Despite the availability of genome-wide TF binding maps for hundreds of TFs, predicting whether a given motif occurrence is bound and identifying the influential context features remain challenging. Here we present a machine learning framework leveraging existing convolutional neural network architectures and state of the art model interpretation techniques to identify, visualize, and interpret context features most important for determining binding activity for a particular TF. We apply our framework to predict binding at motifs for 38 TFs in a lymphoblastoid cell line and achieve superior classification performance compared to existing frameworks. We compute importance scores for context regions at single base pair resolution and uncover known and novel determinants of TF binding. Finally, we demonstrate that important context bases are under increased purifying selection compared to nearby bases and are enriched in disease-associated variants identified by genome-wide association studies.

genomics↗

bioRxiv2020

The CEPR2-mediated phosphorylation of NRT1.2 mainly regulates ABA import in Arabidopsis

Abscisic acid (ABA) transport plays important role in systematic plant responses to environmental factors. Here, we showed that C-terminally encoded peptide receptor 2 (CEPR2) directly interacted with the ABA transporter NRT1.2. Using transgenic seedlings, we demonstrated that NRT1.2 positively regulated the ABA response, and that CEPR2 acted on NRT1.2 epistatically and negatively. Under normal conditions, CEPR2 phosphorylated NRT1.2 at least at serine 292 to promote the degradation of NRT1.2. However, ABA and serine 292 nonphospharylation strongly inhibited the degradation of NRT1.2, indicating that ABA-inhibited the phosphorylation of NRT1.2. Transport assays in yeast and Xenopus oocytes showed that nonphosphorylated NRT1.2 had high levels of ABA-import activity, but phosphorylated NRT1.2 did not import ABA. Analyses of complement nrt1.2 mutants by mimicking nonphospharylated and phospharylated NRT1.2 confirmed that nonphospharylated NRT1.2S292A had high stability and ABA import activity in planta. Further experiments indicated that NRT1.2 was degraded via the 26S proteasome and vacuolar degradation pathways. UBC32, UBC33, and UBC34 interacted with, and mediated the ubiquitination of NRT1.2. UBC32, UBC33, and UBC34 acted on NRT1.2 epistatically and negatively in planta. Thus, our results suggested the existence of novel plant mechanisms regulating NRT1.2 stability and ABA import activity in response to environmental conditions.

molecular biology↗

bioRxiv2020

Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction

The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription of noncoding RNAs prior to premature termination, in addition to transcription of mRNAs, are responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.

molecular biology↗

bioRxiv2020

Large-scale Quantification of Vertebrate Biodiversity in Ailaoshan Nature Reserve from Leech iDNA

1Protected areas are central to meeting biodiversity conservation goals, but measuring their effectiveness is challenging. We address this challenge by using DNA from leech-ingested bloodmeals to estimate vertebrate occupancies across the 677 km2 Ailaoshan reserve in Yunnan, China. 163 park rangers collected 30,468 leeches from 172 patrol areas. We identified 86 vertebrate species, including amphibians, mammals, birds, and squamates. Multi-species occupancy modelling showed that species richness increased with elevation and distance to reserve edge, including the distributions of most of the large mammals (e.g. sambar, black bear, serow, tufted deer). The exceptions were the three domestic mammal species (cows, sheep, goats) and muntjak deer, which were more common at lower elevations. eDNA-estimated vertebrate occupancies are Granular, Repeatable, Auditable, Direct, Efficient, and Simple-to-understand measures that can be used to assess conservation effectiveness and thus to improve the contributions that protected areas make to achieving global biodiversity goals.

ecology↗

bioRxiv2020

Glutamate and γ-aminobutyric acid differentially modulate glymphatic clearance of amyloid β through pulsation- and aquaporin-4 dependent mechanisms

The glymphatic system contributes to a large proportion of brain waste clearance, including removal of amyloid {beta} (A{beta}). We have demonstrated that glutamate and {gamma}-aminobutyric acid (GABA) influence glymphatic clearance through distinct mechanisms whereby GABA exerts modulatory effects in an aquaporin-4 (AQP4)-dependent manner while the actions of glutamate are pulsation-dependent. The efficacy of GABA and glutamate in alleviating A{beta} in APP-PS1 and Angiotensin-II (Ang-II) induced hypertension mouse models was further evaluated. Notably, increasing GABA or inhibiting glutamate levels led to reduced binding of A{beta} to pre-labeled plaques to similar extents in APP-PS1 mice while GABA appeared more efficient in A{beta} clearance in hypertensive animals than the glutamate inhibitor. Our findings support the modulation of neurotransmitters that influence the glymphatic pathway via distinct mechanisms as a potentially effective therapeutic strategy for clearance of A{beta} deposits from the brain.

neuroscience↗

bioRxiv2020

The role of HMGB1 in invasive Candida albicans infection

BackgroundHigh mobility group box 1 (HMGB1) is an important "late" inflammatory mediator in bacterial sepsis. Ethyl pyruvate (EP), an inhibitor of HMGB1, can prevent bacterial sepsis by decreasing HMGB1 levels. However, the role of HMGB1 in fungal sepsis is still unclear. Therefore, we investigated the role of HMGB1 and EP in invasive C. albicans infection. MethodsWe measured serum HMGB1 levels in patients with sepsis with C. albicans infection and without fungal infection, and control subjects. We collected clinical indices to estimate correlations between HMGB1 levels and disease severity. Furthermore, we experimentally stimulated mice with C. albicans and C. albicans + EP. Then, we examined HMGB1 levels from serum and tissue, investigated serum levels of tumor necrosis factor (TNF-) and interleukin 6 (IL-6), determined pathological changes in tissues, and assessed mortality. ResultsSerum HMGB1 levels in patients with severe sepsis with C. albicans infection were elevated. Increased HMGB1 levels were correlated with procalcitonin (PCT), C-reactive protein (CRP), 1,3-{beta}-D-Glucan (BDG) and C. albicans sepsis severity. HMGB1 levels in serum and tissues were significantly increased within seven days after mice were infected with C. albicans. The administration of EP inhibited HMGB1 levels, decreased tissue damage, increased survival rates and inhibited the release of TNF- and IL-6. ConclusionsHMGB1 levels were significantly increased in invasive C. albicans infections. EP prevented C. albicans lethality by decreasing HMGB1 expression and release. HMGB1 may provide an effective diagnostic and therapeutic target for invasive C. albicans infections.

microbiology↗

bioRxiv2020

RIP1 kinase activity promotes steatohepatitis through mediating cell death and inflammation in macrophages

Hepatocyte cell death and liver inflammation have been well recognized as central characteristics of nonalcoholic steatohepatitis (NASH), however, the underlying molecular basis remains elusive. The kinase receptor-interacting protein 1 (RIP1) is a key regulator of apoptosis, necroptosis and inflammation, we thus hypothesized that the kinase activity of RIP1 may be involved in the pathogenesis of NASH. Wild-type and RIP1 kinase-dead (Rip1K45A/K45A) mice were fed with methionine-and choline-deficient diet (MCD) or high-fat diet (HFD) to establish distinct NASH models. In both models, compared to WT mice, Rip1K45A/K45A mice exhibited significantly less liver injury, less steatosis, decreased inflammation, and less cell death in liver tissue. Moreover, hepatic fibrosis as characterized by Sirius Red staining, expression of -SMA and other fibrosis markers, were significantly alleviated in Rip1K45A/K45A mice than WT controls. Furthermore, using bone marrow transplantation to create chimeric mice, we found that it is the RIP1 kinase in hematopoietic-derived macrophages contributing mostly to the disease progression in NASH. Results from in vitro studies were in agreement with the in vivo data, demonstrating that RIP1 kinase was required for inflammasome activation and cell death induced by saturated fatty acid (palmitic acid) in bone marrow-derived macrophages (BMDMs). At last, we also found that the phosphorylation and expression of RIP1 was obviously increased in patients with NAFLD or NASH, but not in healthy controls. In summary, our results indicate that RIP1 kinase is activated during the pathogenesis of steatohepatitis, and consequently induces inflammation and cell death in macrophages, contributing to the disease progression. Our study suggests that macrophage RIP1 kinase represents a specific and potential target for the treatment of NASH.

immunology↗

bioRxiv2019

The Two Amino Acids Proximate to the Renin Cleavage Site of Human Angiotensinogen Do Not Affect Angiotensin II-mediated Functions in Mice

ObjectiveRenin cleavage of angiotensinogen (AGT) has species specificity. Since the residues at positions 11 and 12 of AGT are different between human and mouse AGT, we determined whether these two residues in AGT affect renin cleavage and angiotensin II-mediated functions using an adeno-associated viral (AAV) approach for manipulating AGT in vivo. Approach and ResultsHepatocyte-specific AGT deficient (hepAGT-/-) mice in an LDL receptor -/- background were infected with AAVs containing a null insert, human AGT, or mouse AGT expressing the same residues of the human protein at positions 11 and 12 [mouse AGT (L11V;Y12I)]. Expression of human AGT in hepAGT-/- mice led to high plasma human AGT concentrations without changes in plasma mouse endogenous AGT, plasma renin concentrations, blood pressure, or atherosclerosis. This is consistent with human AGT not being cleaved by mouse renin. To determine whether the residues at positions 11 and 12 in human AGT lead to the inability of mouse renin to cleave human AGT, hepAGT-/- mice were injected with AAV encoding mouse AGT (L11V;Y12I). Expression of mouse AGT (L11V;Y12I) resulted in increased plasma mouse AGT concentrations, reduced renin concentrations, and increased renal AngII concentrations that were comparable to their concentrations in hepAGT+/+ mice. This mouse AGT variant increased blood pressure and atherosclerosis in hepAGT-/- mice to the magnitude of hepAGT+/+ mice. ConclusionReplacement of L11 and Y12 to V11 and I12, respectively, in mouse AGT does not affect renin cleavage and AngII-mediated functions in mice. HIGHLIGHTSO_LIHuman AGT is not cleaved by mouse renin and does not change AngII-mediated functions in hepatocyte-specific AGT -/- mice. C_LIO_LIReplacement of the N-terminal amino acids at 11 and 12 positions from mouse to human AGT does not affect renin cleavage and AngII-mediated functions in hepatocyte-specific AGT -/- mice. C_LI

pathology↗

bioRxiv2019

AnthOligo: Automating the design of oligonucleotides for capture/enrichment technologies

SummaryA number of methods have been devised to address the need for targeted genomic resequencing. One of these methods, Region-specific extraction (RSE) of DNA is characterized by the capture of long DNA fragments (15-20 kb) by magnetic beads, after enzymatic extension of oligonucleotides hybridized to selected genomic regions. Facilitating the selection of the most optimal capture oligos targeting a region of interest, satisfying the properties of temperature (Tm) and entropy ({Delta}G), while minimizing the formation of primer dimers in a pooled experiment is therefore necessary. Manual design and selection of oligos becomes an extremely arduous task complicated by factors such as length of the target region and number of targeted regions. Here we describe, AnthOligo, a web-based application developed to optimally automate the process of generation of oligo sequences to be used for the targeting and capturing the continuum of large and complex genomic regions. Apart from generating oligos for RSE, this program may have wider applications in the design of customizable internal oligos to be used as baits for gene panel analysis or even probes for large-scale comparative genomic hybridization (CGH) array processes. Implementation and AvailabilityThe application written in Java8 and run on Tomcat9 is a lightweight Java Spring MVC framework that provides the user with a simple interface to upload an input file in BED format and customize parameters for each task. A Redis-like MapReduce framework is implemented to run sub-tasks in parallel to optimize time and system resources alongside a task-queuing system that runs submitted jobs as a server-side background daemon. The task of probe design in AnthOligo commences when a user uploads an input file and concludes with the generation of a result-set containing an optimal set of region-specific oligos. AnthOligo is currently available as a public web application with URL: http://antholigo.chop.edu.

bioinformatics↗

bioRxiv2019

Phen2Gene: Rapid Phenotype-Driven Gene Prioritization for Rare Diseases

Human Phenotype Ontology (HPO) terms are increasingly used in diagnostic settings to aid in the characterization of patient phenotypes. The HPO annotation database is updated frequently and can provide detailed phenotype knowledge on various human diseases, and many HPO terms are now mapped to candidate causal genes with binary relationships. To further improve the genetic diagnosis of rare diseases, we incorporated these HPO annotations, gene-disease databases, and gene-gene databases in a probabilistic model to build a novel HPO-driven gene prioritization tool, Phen2Gene. Phen2Gene accesses a database built upon this information called the HPO2Gene Knowledgebase (H2GKB), which provides weighted and ranked gene lists for every HPO term. Phen2Gene is then able to access the H2GKB for patient-specific lists of HPO terms or PhenoPackets descriptions supported by GA4GH (http://phenopackets.org/), calculate a prioritized gene list based on a probabilistic model, and output gene-disease relationships with great accuracy. Phen2Gene outperforms existing gene prioritization tools in speed, and acts as a real-time phenotype driven gene prioritization tool to aid the clinical diagnosis of rare undiagnosed diseases. In addition to a command line tool released under the MIT license (https://github.com/WGLab/Phen2Gene), we also developed a web server and web service (https://phen2gene.wglab.org/) for running the tool via web interface or RESTful API queries. Finally, we have curated a large amount of benchmarking data for phenotype-to-gene tools involving 197 patients across 76 scientific articles and 85 patients de-identified HPO term data from CHOP.

bioinformatics↗

bioRxiv2019

Quantitative analysis of 1300-nm three-photon calcium imaging in the mouse brain

1300-nm three-photon calcium imaging has emerged as a useful technique to allow calcium imaging in deep brain regions. Application to large-scale neural activity imaging entails a careful balance between recording fidelity and tissue heating. We calculated and experimentally verified the excitation pulse energy to achieve the minimum photon count required for the detection of calcium transients in GCaMP6s-expressing neurons for 920-nm two-photon and 1320-nm three-photon excitation, respectively. Brain tissue heating by continuous three-photon imaging was simulated with Monte Carlo method and experimentally validated with immunohistochemistry. We observed increased immunoreactivity with 150 mW excitation power at 1.0- and 1.2-mm imaging depths. Based on the data, we explained how three-photon excitation achieves better calcium imaging fidelity than two-photon excitation in the deep brain and quantified the imaging depth where three-photon microscopy should be applied. Our analysis presents a translatable model for the optimization of three-photon calcium imaging based on experimentally tractable parameters.

neuroscience↗

bioRxiv2019

CDK13 Mutations Drive Melanoma via Accumulation of Prematurely Terminated Transcripts

Transcriptional Cyclin Dependent Kinases modulate RNA Polymerase II function to impact gene expression. Here, we show that CDK13 is mutated in 4% of patient melanomas and mutation or downregulation is associated with poor overall survival. Mutant CDK13 lacks kinase activity and overexpression in zebrafish leads to accelerated melanoma. CDK13 mutant fish and human melanomas accumulate prematurely terminated RNAs that are translated into truncated proteins. CDK13 binds to and regulates the phosphorylation of ZC3H14, a member of the PolyA eXosome Targeting (PAXT) RNA degradation complex. ZC3H14 phosphorylation recruits the PAXT complex to degrade prematurely terminated polyadenylated transcripts in the nucleus. In the presence of mutant CDK13, ZC3H14 phosphorylation is compromised and consequently fails to recruit the PAXT complex, leading to truncated transcript stabilization. This work establishes a role for CDK13 and the PAXT nuclear RNA degradation complex in cancer and has prognostic significance for melanoma patients with mutated or downregulated CDK13.

biophysics↗

bioRxiv2019

LIMD1 phase separation contributes to cellular mechanics and durotaxis by regulating focal adhesion dynamics in response to force

The mechanical environement affects cell morphology, differentiation and motility. The ability of cells to follow gradients of extracellular matrix stiffness-durotaxis has been implicated in development, fibrosis, and cancer. Cells sense and respond to extra-cellular mechanical cues through cell-matrix adhesions. Interestingly, the maturation of focal adhesions (FAs) is reciprocally force-dependent. How biomechanical cues dictate the status of cell motility and how FAs coordinate force sensing and self-organization remain enigmatic. LIMD1, a member of the LIM domain proteins, localizes to the FAs and has been reported to negatively regulate the Hippo-YAP pathway in response to tension. Here we identify the force sensitive recruitment of LIMD1 to the FAs. We discover that LIMD1 regulates cell spreading, maintains FA dynamics and cellular force, and is critical for durotaxis. Intriguingly, LIMD1 selectively recruits late but not early FA proteins through phase separation at the FAs under force. We suggest a model in which localization of LIMD1 to the FAs, triggered by mechanical force, serves as a phase separation hub for assembling and organizing late FA proteins, allowing for effective FA maturation and efficient cellular mechano-transduction.

cell biology↗

bioRxiv2019

Increasing vitrification temperature improves the cryo-electron microscopy reconstruction

At the initial stage of the cryo-electron microcopy (cryo-EM) samples irradiated by electrons, the cryo-EM samples suffer from a rapid \"burst\" phase (first 3~4 e-/[A]2) of beam induced motion (BIM) which is too fast to be corrected by existing motion correction software, and lowers the quality of the initial frames. Therefore, these least radiation damaged, but ruined frames are commonly excluded or down-weighted during data processing, which reduces the undamaged signals in the reconstruction and decreases the reconstruction resolution by weakening the averaging power. Here, we show that increasing the freezing temperature of cryo-EM samples suppresses the BIM in this phase. The quality of initial frames is partially recovered after BIM correction and is better than that of subsequent frames in certain frames. Incorporating these initial frames into the reconstruction increases the resolution, at an equivalent of ~60% extra data. Moreover, these frames are least radiation damaged, thus preserves the high quality cryo-EM density of radiation sensitive residues. Such density is usually damaged or very weak in the canonical 3D reconstruction. In addition, we found that a different kind of radiation damage neglected previously occurs in the per-frame reconstruction after the exposure of 2.5 e-/[A]2. Such radiation damage distorts the density of atoms. The deformation can be avoided by only including the frames from the first 2.5 e-/[A]2 into the reconstruction. Overall, the high temperature freezing not only provides extra undamaged signal to the reconstruction, but also increases the resolution of the reconstruction.

biophysics↗

bioRxiv2019

Microsatellite instability as a tool to diagnose genome instability in CHO cell culture

As bioprocess intensification has increased over the last 30 years, yields from mammalian cell processes have increased from 10s of milligrams to over 10s of grams per liter. Most of these gains in productivity have been due to increasing cell numbers in the bioreactors, and with those increases in cell numbers, strategies have been developed to minimize metabolite waste accumulation, such as lactate and ammonia. Unfortunately, cell growth cannot occur without some waste metabolite accumulation, as central metabolism is required to produce the biopharmaceutical. Inevitably, metabolic waste accumulation leads to decline and termination of the culture. While it is understood that the accumulation of these unwanted compounds imparts a less than optimal culture environment, little is known about the genotoxic properties and the influence of these compounds on global genome instability. In this study, we examined the effects on Chinese hamster ovary (CHO) cells genome sequences and physiology due to exposure to elevated ammonia levels. We identified genome-wide de novo mutations, in addition to variants in functional regions of certain genes involved in the mismatch repair (MMR) pathway, such as DNA2, BRCA1 and RAD52, which led to loss-of-function and eventual genome instability. Additionally, we characterized the presence of microsatellites against the most recent Chinese Hamster genome assembly and discovered certain loci are not replicated faithfully in the presence of elevated ammonia, which represents microsatellite instability (MSI). Furthermore, we found 124 candidate loci that may be suitable biomarkers to gauge genome stability in CHO cultures.

genomics↗

bioRxiv2019

Wikidata as a FAIR knowledge graph for the life sciences

Wikidata is a community-maintained knowledge base that epitomizes the FAIR principles of Findability, Accessibility, Interoperability, and Reusability. Here, we describe the breadth and depth of biomedical knowledge contained within Wikidata, assembled from primary knowledge repositories on genomics, proteomics, genetic variants, pathways, chemical compounds, and diseases. We built a collection of open-source tools that simplify the addition and synchronization of Wikidata with source databases. We furthermore demonstrate several use cases of how the continuously updated, crowd-contributed knowledge in Wikidata can be mined. These use cases cover a diverse cross section of biomedical analyses, from crowdsourced curation of biomedical ontologies, to phenotype-based diagnosis of disease, to drug repurposing.

bioinformatics↗