Biology subjects

Zou, J.

Publications and source records attributed to Zou, J..

At least 199 records · Page 11Linked to original sources

bioRxiv2019

IL-22 induced cell extrusion and IL-18-induced pyroptosis prevent and cure rotavirus infection

Administration of bacterial flagellin elicits production of TLR5-mediated IL-22 and NLRC4-mediated IL-18 that act in concert to cure and prevent rotavirus (RV) infection. This study investigated the mechanism by which these cytokines act to impede this virus. Although IL-18 and IL-22 induce each others expression, we found that IL-18 and IL-22 both impeded RV independently of each other and did so by distinct mechanisms, in both cases via activation of their cognate receptors in intestinal epithelial cells (IEC). IL-22 drove IEC proliferation and migration toward villus tips, which resulted in increased extrusion of highly differentiated IEC that serve as the site of RV replication. In contrast, IL-18 induced pyroptotic death of RV-infected IEC thus directly interrupting the RV replication cycle, resulting in spewing of incompetent virus into the intestinal lumen and causing a rapid drop in levels of RV-infected IEC. Together, these actions resulted in rapid and complete expulsion of RV, even in hosts with severely compromised immune systems. These results suggest that IL-18/22 might be a means of treating viral infections that preferentially target short-lived epithelial cells.

immunology↗

bioRxiv2019

Architecture of the Mto1/2 microtubule nucleation complex

Proteins that contain a Centrosomin Motif 1 (CM1) domain are key regulators of{gamma} -tubulin complex-dependent microtubule nucleation, but how they are organized in higher-order structures is largely unknown. Mto1[bonsai], a truncated functional version of the Schizosaccharomyces pombe CM1 protein Mto1, interacts with Mto2 to form an Mto1/2[bonsai] complex in vivo. Here we show that recombinant Mto1/2[bonsai] forms higher-order multimers in vitro and that Mto2 alone can also multimerize. We demonstrate that Mto2 multimerization involves two separate homodimerization domains, the near N-terminal domain (NND) and the twin-cysteine domain (TCD). The TCD crystal structure reveals a stable homodimer with a novel dimerization interface. While the NND homodimer is intrinsically less stable, using crosslinking mass spectrometry we show that within Mto1/2[bonsai] complexes, it can be reinforced by additional cooperative interactions involving both Mto2 and Mto1[bonsai]. We propose a model for Mto1/2[bonsai] complex architecture that is supported by functional analysis of mutants in vivo.

cell biology↗

bioRxiv2019

Structural Basis for CAL1-Mediated Centromere Maintenance

Centromeres are microtubule attachment sites on chromosomes defined by the enrichment of CENP-A-containing nucleosomes. To preserve centromere identity, CENP-A must be escorted to centromeres by a CENP-A-specific chaperone for deposition. Despite this essential requirement, many eukaryotes differ in the composition of players involved in centromere maintenance highlighting the plasticity of this process. In humans, CENP-A recognition and centromere targeting is achieved by HJURP and the Mis18 complex, respectively. Here, using crystal structures, we show how Drosophila CAL1, an evolutionarily distinct CENP-A chaperone, targets CENP-A to the centromere receptor CENP-C without the requirement of the Mis18 complex: while the N-terminal CAL1 fragment (CAL11-160) wraps around CENP-A/H4 through multiple physical contacts, the C-terminal CAL1 fragment (CAL1893-914) directly binds CENP-C cupin dimer. Our work shows CAL1, though divergent at the primary structure, employs evolutionarily conserved and adaptive structural principles to recognise CENP-A/H4 and CENP-C providing insights into the minimalistic principles underlying centromere maintenance.

biochemistry↗

bioRxiv2019

Robust generation of erythroid and multilineage hematopoietic progenitors from human iPSCs using a scalable monolayer culture system

One of the most promising objectives of clinical hematology is to derive engraftable autologous hematopoietic stem cells (HSCs) from human induced pluripotent stem cells (iPSCs). Progress in translating iPSC technologies to the clinic relies on the availability of scalable differentiation methodologies. In this study, human iPSCs were differentiated for 21 days using STEMdiff, a monolayer-based approach for hematopoietic differentiation of human iPSCs that requires no replating, co-culture or embryoid body formation. Both monolayer and suspension cells were functionally characterized throughout differentiation. In the supernatant fraction, an early transient population of primitive CD235a+ erythroid cells first emerged, followed by hematopoietic progenitors with multilineage differentiation activity in vitro but no long-term engraftment potential in vivo. In later stages of differentiation, a nearly exclusive production of definitive erythroid progenitors was observed. In the adherent monolayer, we identified a prevalent population of mesenchymal stromal cells and limited arterial vascular endothelium (VE), suggesting that the cellular constitution of the monolayer may be inadequate to support the generation of HSCs with durable repopulating potential. Quantitative modulation of WNT/{beta}-catenin and activin/nodal/TGF{beta} signaling pathways with CHIR/SB molecules during differentiation enhanced formation of arterial VE, definitive multilineage and erythroid progenitors, but was insufficient to orchestrate the generation of engrafting HSCs. Overall, STEMdiff provides a clinically-relevant and readily adaptable platform for the generation of erythroid and multilineage hematopoietic progenitors from human pluripotent stem cells.\n\nHighlightsO_LIRobust, scalable and clinically-relevant monolayer-based culture system for hematopoietic differentiation of human iPSCs.\nC_LIO_LISuccessive emergence of primitive erythroid cells, definitive multilineage HSPCs and erythroid progenitors in the culture supernatant.\nC_LIO_LIAbundant mesenchymal cells and limited arterial vascular endothelium in the culture monolayer.\nC_LIO_LICHIR/SB molecules increase arterial vascular endothelium formation, suppress primitive hematopoiesis and promote definitive multilineage and erythroid progenitors.\nC_LI

developmental biology↗

bioRxiv2019

Cross-linked RNA Secondary Structure Analysis using Network Techniques

Next generation sequencing and biochemical cross-linking methods have been combined into powerful tools to probe RNA secondary structure. One such method, known as PARIS, has been used to produce near base-pair maps of long-range and alternative RNA structures in living cells. However, the procedure for generating these maps typically relies on laborious manual analysis. We developed an automated method for producing RNA secondary structure maps using network analysis techniques. We produced an analysis pipeline, dubbed cross-linked RNA secondary structure analysis using network techniques (CRSSANT), which automates the grouping of gapped RNA sequencing reads produced using the PARIS assay, and tests the validity of secondary structures implied by the groups. We validated the clusters and secondary structures produced by CRSSANT using manually-produced grouping maps and known secondary structures. We implemented CRSSANT in Python using the network analysis package NetworkX and RNA folding software package ViennaRNA. CRSSANT is fast and efficient, and is available as Python source code at https://github.com/ihwang/CRSSANT.

bioinformatics↗

bioRxiv2019

Direct Nucleosome Binding of Borealin Secures Chromosome Association and Function of the Chromosomal Passenger complex

Chromosome association of the Chromosomal Passenger Complex (CPC; consisting of Borealin, Survivin, INCENP and the Aurora B kinase) is essential to achieve error-free chromosome segregation during cell division. Hence, understanding the mechanisms driving the chromosome association of the CPC is of paramount importance. Here using a multifaceted approach, we show that the CPC binds nucleosomes through a multivalent interaction predominantly involving Borealin. Strikingly, Survivin, previously suggested to target the CPC to centromeres [1-3] failed to bind nucleosomes on its own and requires Borealin and INCENP for its binding. Disrupting Borealin-nucleosome interactions excluded the CPC from chromosomes and caused chromosome congression defects. We also show that Borealin-mediated chromosome association of the CPC is critical for Haspin- and Bub1-mediated centromere enrichment of the CPC and works upstream of the latter. Our work thus establishes Borealin as a master regulator determining the chromosome association and function of the CPC.

cell biology↗

bioRxiv2019

Folate deficiency facilitates coordination of KDM6A with p53 in response to DNA damage

Folate contributes to the accumulation of DNA strand breaks (DSBs) in the genome. Kdm6a plays a critical role in early embryogenesis but it is unknown whether Kdm6a is involved in the DNA damage response under folate deficiency. Here, we established a low folate environment for mouse embryonic cells using a folate antagonist (methotrexate, MTX). We found increased enrichment of DSBs in Kdm6a in MTX-treated cells, resulting in reduced Kdm6a expression. MTX treatment enriched KDM6A in the nonhomologous end-joining (NHEJ) repair pathway and controlled the expression of the Ku heterodimer (Ku70/80) and XRCC4. The activation of NHEJ repair pathway-associated genes under folate deficiency relied on the specific interaction between KDM6A and p53. p53 silencing increased Ku heterodimer expression. In addition, in a neural tube defect (NTD) mouse model and low folate neural tube defect human brain samples, KDM6A levels were also decreased and accompanied by over-expression of Ku80. Our findings highlight how alterations in folate levels affect KDM6A with respect to DNA breakage and DNA repair, offering a new insight into the molecular function of KDM6A.\n\nSummary statementOur study demonstrates for the first time that in mammals, Kdm6a and p53 have synergistic effects during DNA fragmentation repair.

bioinformatics↗

bioRxiv2018

Nationwide population genetic screening improves outcomes of newborn screening for hearing loss in China

PurposeConcurrent newborn hearing and genetic screening has been reported, but its benefits have not been statistically proven due to limited sample sizes and outcome data. To fill this gap, we analyzed outcomes of a large number of newborns with genetic screening results.\n\nMethodsNewborns in China were screened for 20 hearing-loss-related genetic variants from 2012-2017. Genetic results were categorized as positive, at-risk, inconclusive, or negative. Hearing screening results, risk factors, and up-to-date hearing status were followed-up via phone interviews.\n\nResultsWe completed genetic screening on one million newborns and followed up 12,778. We found that a positive genetic result significantly indicated a higher positive predictive value of the initial hearing screening (60% vs. 5.0%, P<0.001) and a lower rate of loss-to-follow-up (5% vs. 22%, P<0.001) than an inconclusive one. Importantly, 42% of subjects in the positive group with reported or presymptomatic hearing loss were \"missed\" by conventional hearing screening. Furthermore, genetic screening identified 0.23% of subjects predisposed to preventable ototoxicity.\n\nConclusionOur results demonstrate that limited genetic screening identified additional cases, reduced loss-to-follow-up, and informed families of ototoxicity risks, providing convincing evidence to support integrating genetic screening into universal newborn hearing screening programs.

genetics↗

bioRxiv2018

DR-SIP: Protocols for Higher Order Structure Modeling with Distance Restraints- and Cyclic Symmetry-Imposed Packing

MotivationQuaternary structure determination for proteins is difficult especially for transmembrane proteins. Even if the monomeric constituents of complexes have been experimentally resolved, computational prediction of quaternary structures is a challenging task particularly for higher order complexes. It is essential to have a reliable computational protocol to predict quaternary structures of both transmembrane and soluble proteins leveraging experimentally determined distance restraints and/or cyclic symmetry (Cn symmetry) found in most homo-oligomeric transmembrane proteins.\n\nResultsWe survey 115 X-ray crystallographically solved structures of homo-oligomeric transmembrane proteins (HoTPs) to discover that 90% of them are Cn symmetric. Given the prevalence of Cn symmetric HoTPs and the benefits of incorporating geometry restraints in aiding quaternary structure determination, we introduce two new filters, the distance-restraints (DR) filter and the Symmetry-Imposed Packing (SIP) filter which takes advantage of the statistically derived tilt angle cutoff and the Cn symmetry of HoTPs without prior knowledge of the number (\"n\") of monomers. Using only the geometrical filter, SIP, near-native poses of the 115 HoTPs can be correctly identified in the top-5 for 52% of all cases, or 49% among the HoTPs having an n >2 (~60% of the dataset), while ZDOCK alone returns 41% and 24%, respectively. Applying only SIP to three HoTPs with distance restraints, the near-native poses for two HoTPs are ranked 1st and the other 7th among 54,000 possible decoys. With both filters, the two remain 1st while the other improved to 2nd. While a soluble system with distance restraints is recovered at the 1st-ranked pose by applying only DR.\n\nAvailability and Implementationhttps://github.com/capslockwizard/drsip\n\nSupplementary informationSupplementary methods and results are available.

bioinformatics↗

bioRxiv2018

AdaFDR: a Fast, Powerful and Covariate-Adaptive Approach to Multiple Hypothesis Testing

Multiple hypothesis testing is an essential component of modern data science. Its goal is to maximize the number of discoveries while controlling the fraction of false discoveries. In many settings, in addition to the p-value, additional information/covariates for each hypothesis are available. For example, in eQTL studies, each hypothesis tests the correlation between a variant and the expression of a gene. We also have additional covariates such as the location, conservation and chromatin status of the variant, which could inform how likely the association is to be due to noise. However, popular multiple hypothesis testing approaches, such as Benjamini-Hochberg procedure (BH) and independent hypothesis weighting (IHW), either ignore these covariates or assume the covariate to be univariate. We introduce AdaFDR, a fast and flexible method that adaptively learns the optimal p-value threshold from covariates to significantly improve detection power. On eQTL analysis of the GTEx data, AdaFDR discovers 32% and 27% more associations than BH and IHW, respectively, at the same false discovery rate. We prove that AdaFDR controls false discovery proportion, and show that it makes substantially more discoveries while controlling FDR in extensive experiments. AdaFDR is computationally efficient and can process more than 100 million hypotheses within an hour and allows multi-dimensional covariates with both numeric and categorical values. It also provides exploratory plots for the user to interpret how each covariate affects the significance of hypotheses, making it broadly useful across many applications.

bioinformatics↗

bioRxiv2018

Inherited defects in natural killer cells shape tumor immune microenvironment, clinical outcome and immunotherapy response

Tumor immune microenvironment (TIME) plays an important role in metastasis and immunotherapy. However, it has been not much known how to classify TIMEs and how TIMEs are genetically regulated. Here we showed that tumors were classified into TIME-rich, -intermediate and -poor subtypes which had significant differences in clinical outcomes, abundances of tumor-infiltrating lymphocytes (TILs), the degree of key immune programs activation, and immunotherapy response across 13 common cancer types (n= [~]6,000). Furthermore, TIME-intermediate/-poor patients had significantly more inherited genetic defects (i.e., functional germline variants) in natural killer (NK) cells, antigen processing and presentation (APP) and Wnt signaling pathways than TIME-rich patients, and so did cancer patients than non-cancer individuals (n=4,500). These results suggested that individuals who had more inherited defects in NK cells, APP and Wnt pathways had a higher risk of developing cancers. Moreover, in the 13 common cancers the number of inheritably defected genes of NK cells was significantly negative-correlated with patients survival, TILs abundance in TIMEs and immunotherapy response, suggesting that inherited defects in NK cells alone were sufficient to shape TILs recruitment, clinical outcome, and immunotherapy response, highlighting that NK cell activation was required in the 13 cancer types to drive the recruitment of immune troops into TIMEs. Thus, we proposed that cancer was a disease of NK cell inherited deficiencies. These results had implications in identifying of high-risk individuals based on germline genomes, implementing precision cancer prevention by adoptive transfer of healthy NK cells, and improving existing immunotherapies by combining of adoptive NK cell transfer (i.e., converting TIME-intermediate/-poor tumors into TIME-rich tumors) and anti-PD-1 or CAR-T therapy.\n\nContactEW (edwin.wang@ucalgary.ca)

cancer biology↗

bioRxiv2018

A genetically encoded fluorescent sensor for rapid and specific in vivo detection ofnorepinephrine

Norepinephrine (NE) and epinephrine (Epi), two key biogenic monoamine neurotransmitters, are involved in a wide range of physiological processes. However, their precise dynamics and regulation remain poorly characterized, in part due to limitations of available techniques for measuring these molecules in vivo. Here, we developed a family of GPCR Activation-Based NE/Epi (GRABNE) sensors with a 230% peak {Delta}F/F0 response to NE, good photostability, nanomolar-to-micromolar sensitivities, sub-second rapid kinetics, high specificity to NE vs. dopamine. Viral- or transgenic- mediated expression of GRABNE sensors were able to detect electrical-stimulation evoked NE release in the locus coeruleus (LC) of mouse brain slices, looming-evoked NE release in the midbrain of live zebrafish, as well as optogenetically and behaviorally triggered NE release in the LC and hypothalamus of freely moving mice. Thus, GRABNE sensors are a robust tool for rapid and specific monitoring of in vivo NE/Epi transmission in both physiological and pathological processes.

neuroscience↗

bioRxiv2018

Genome-wide selection footprints and deleterious variations in young Asian allotetraploid rapeseed

Brassica napus (AACC, 2n=38), is an important oilseed crop grown worldwide. However, little is known about the population evolution of this species, the genomic difference between its major genetic clusters, such as European and Asian rapeseed, and impacts of historical large-sale introgression events in this young tetraploid. In this study, we reported the de novo assembly of the genome sequences of an Asian rapeseed (B. napus), Ningyou 7 and its four progenitors and carried out de novo assembly-based comparison, pedigree and population analysis with other available genomic data from diverse European and Asian cultivars. Our results showed that Asian rapeseed originally derived from European rapeseed, but it had subsequently significantly diverged, with rapid genome differentiation after intensive local breeding selection. The first historical introgression of B. rapa dramatically broadened the allelic pool of Asian B. napus, but decreased their deleterious variations. The secondary historical introgression of European rapeseed (canola-quality) has reshaped Asian rapeseed into two groups, accompanied by an increase in genetic load. This study demonstrates distinctive genomic footprints by recent intra- and inter-species introgression events for local adaptation, and provide novel insights for understanding the rapid genome evolution of a young allopolyploid crop.

genomics↗

bioRxiv2018

Systematic characterization of genome editing in primary T cells reveals proximal genomic insertions and enables machine learning prediction of CRISPR-Cas9 DNA repair outcomes

The Streptococcus pyogenes Cas9 (SpCas9) nuclease has become a ubiquitous genome editing tool due to its ability to target almost any location in DNA and create a double-stranded break1,2. After DNA cleavage, the break is fixed with endogenous DNA repair machinery, either by non-templated mechanisms (e.g. non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ)), or homology directed repair (HDR) using a complementary template sequence3,4. Previous work has shown that the distribution of repair outcomes within a cell population is non-random and dependent on the targeted sequence, and only recent efforts have begun to investigate this further5-11. However, no systematic work to date has been validated in primary human cells5,7. Here, we report DNA repair outcomes from 1,521 unique genomic locations edited with SpCas9 ribonucleoprotein complexes (RNPs) in primary human CD4+ T cells isolated from multiple healthy blood donors. We used targeted deep sequencing to measure the frequency distribution of repair outcomes for each guide RNA and discovered distinct features that drive individual repair outcomes after SpCas9 cleavage. Predictive features were combined into a new machine learning model, CRISPR Repair OUTcome (SPROUT), that predicts the length and probability of nucleotide insertions and deletions with R2 greater than 0.5. Surprisingly, we also observed large insertions at more than 90% of targeted loci, albeit at a low frequency. The inserted sequences aligned to diverse regions in the genome, and are enriched for sequences that are physically proximal to the break site due to chromatin interactions. This suggests a new mechanism where sequences from three-dimensionally neighboring regions of the genome can be inserted during DNA repair after Cas9-induced DNA breaks. Together, these findings provide powerful new predictive tools for Cas9-dependent genome editing and reveal new outcomes that can result from genome editing in primary T cells.

cell biology↗

bioRxiv2018

eTumorRisk, an algorithm predicts cancer risk based on comutated gene networks in an individual’s germline genome

Early cancer detection has potentials to reduce cancer burden. A prior identification of the high-risk population of cancer will facilitate cancer early detection. Traditionally, cancer predisposition genes such as BRCA1/2 have been used for identifying high-risk population of developing breast and ovarian cancers. However, such high-risk genes have only a few. Moreover, the complexity of cancer hints multiple genes involved but also prevents from identifying such predictors for predicting high-risk subpopulation. Therefore, we asked if the germline genomes could be used to identify high-risk cancer population. So far, none of such predictive models has been developed. Here, by analyzing of the germline genomes of 3,090 cancer patients representing 12 common cancer types and 25,701 non-cancer individuals, we discovered significantly differential co-mutated gene pairs between cancer and non-cancer groups, and even between cancer types. Based on these findings, we developed a network-based algorithm, eTumorRisk, which enables to predict individuals cancer risk of six genetic-dominant cancers including breast, colon, brain, leukemia, ovarian and endometrial cancers with the prediction accuracies of 74.1-91.7% and have 1-3 false-negatives out of the validating samples (n=14,701). The eTumorRisk which has a very low false-negative rate might be useful in screening of general population for identifying high-risk cancer population.

bioinformatics↗

bioRxiv2018

Modeling Spatial Correlation of Transcripts With Application to Developing Pancreas

Recently high-throughput image-based transcriptomic methods were developed and enabled researchers to spatially resolve gene expression variation at the molecular level for the first time. In this work, we develop a general analysis tool to quantitatively study the spatial correlations of gene expression in fixed tissue sections. As an illustration, we analyze the spatial distribution of single mRNA molecules measured by in situ sequencing on human fetal pancreas at three developmental time points 80, 87 and 117 days post-fertilization. We develop a density profile-based method to capture the spatial relationship between gene expression and other morphological features of the tissue sample such as position of nuclei and endocrine cells of the pancreas. In addition, we build a statistical model to characterize correlations in the spatial distribution of the expression level among different genes. This model enables us to infer the inhibitory and clustering effects throughout different time points. Our analysis framework is applicable to a wide variety of spatially-resolved transcriptomic data to derive biological insights.

bioinformatics↗

bioRxiv2018

Auditory and Language Contributions to Neural Encoding of Speech Features in Noisy Environments

Recognizing speech in noisy environments is a challenging task that involves both auditory and language mechanisms. Previous studies have demonstrated noise-robust neural tracking of the speech envelope, i.e., fluctuations in sound intensity, in human auditory cortex, which provides a plausible neural basis for noise-robust speech recognition. The current study aims at teasing apart auditory and language contributions to noise-robust envelope tracking by comparing 2 groups of listeners, i.e., native listeners of the testing language and foreign listeners who do not understand the testing language. In the experiment, speech is mixed with spectrally matched stationary noise at 4 intensity levels and the neural responses are recorded using electroencephalography (EEG). When the noise intensity increases, an increase in neural response gain is observed for both groups of listeners, demonstrating auditory gain control mechanisms. Language comprehension creates no overall boost in the response gain or the envelope-tracking precision but instead modulates the spatial and temporal profiles of envelope-tracking activity. Based on the spatio-temporal dynamics of envelope-tracking activity, the 2 groups of listeners and the 4 levels of noise intensity can be jointly decoded by a linear classifier. All together, the results show that without feedback from language processing, auditory mechanisms such as gain control can lead to a noise-robust speech representation. High-level language processing, however, further modulates the spatial-temporal profiles of the neural representation of the speech envelope.

neuroscience↗

bioRxiv2018

Reliable Multiplex Sequencing with Rare Index Mis-Assignment on DNB-Based NGS Platform

BackgroundMassively-parallel-sequencing, coupled with sample multiplexing, has made genetic tests broadly affordable. However, intractable index mis-assignments (commonly exceeds 1%) were repeatedly reported on some widely used sequencing platforms.\n\nResultsHere, we investigated this quality issue on BGI sequencers using three library preparation methods: whole genome sequencing (WGS) with PCR, PCR-free WGS, and two-step targeted PCR. BGIs sequencers utilize a unique DNB technology which uses rolling circle replication for DNA-nanoball preparation; this linear amplification is PCR free and can avoid error accumulation. We demonstrated that single index mis-assignment from free indexed oligos occurs at a rate of one in 36 million reads, suggesting virtually no index hopping during DNB creation and arraying. Furthermore, the DNB-based NGS libraries have achieved an unprecedentedly low sample-to-sample mis-assignment rate of 0.0001% to 0.0004% under recommended procedures.\n\nConclusionsSingle indexing with DNB technology provides a simple but effective method for sensitive genetic assays with large sample numbers.

genomics↗