Biology subjects

Zhou, J.

Publications and source records attributed to Zhou, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2018

Base pair editing of goat embryos: nonsense codon introgression into FGF5 to improve cashmere yield

The ability to alter single bases without DNA double strand breaks provides a potential solution for multiplex editing of livestock genomes for quantitative traits. Here, we report using a single base editing system, Base Editor 3 (BE3), to induce nonsense codons (C-to-T transitions) at four target sites in caprine FGF5. All five progenies produced from microinjected single-cell embryos had alleles with a targeted nonsense mutation and yielded expected phenotypes. The effectiveness of BE3 to make single base changes varied considerably based on sgRNA design. Also, the rate of mosaicism differed between animals, target sites, and tissue type. PCR amplicon and whole genome resequencing analyses for off-target changes caused by BE3 were low at a genome-wide scale. This study provides first evidence of base editing in livestock, thus presenting a potentially better method to introgress complex human disease alleles into large animal models and provide genetic improvement of complex health and production traits in a single generation.

genetics↗

bioRxiv2018

New genetic signals for lung function highlight pathways and pleiotropy, and chronic obstructive pulmonary disease associations across multiple ancestries

Reduced lung function predicts mortality and is key to the diagnosis of COPD. In a genome-wide association study in 400,102 individuals of European ancestry, we define 279 lung function signals, one-half of which are new. In combination these variants strongly predict COPD in deeply-phenotyped patient populations. Furthermore, the combined effect of these variants showed generalisability across smokers and never-smokers, and across ancestral groups. We highlight biological pathways, known and potential drug targets for COPD and, in phenome-wide association studies, autoimmune-related and other pleiotropic effects of lung function associated variants. This new genetic evidence has potential to improve future preventive and therapeutic strategies for COPD.

genomics↗

bioRxiv2018

Next-Generation Sequencing Could be a Promising Diagnostic Approach for Pathogen Detection: Pathogenic Analysis of Pediatric Bacterial Meningitis by Next-Generation Sequencing Technology Directly from Cerebrospinal Fluid Specimens

BackgroundBacterial meningitis remains one of the major challenges in infectious diseases, leading to sequel in many cases. A prompt diagnosis of the causative microorganism is critical to significantly improve outcome of bacterial meningitis. Although various targeted tests for cerebrospinal fluid (CSF) samples are available, it is a big problem for the identification of etiology of bacterial meningitis.\n\nMethodsHere we describe the use of unbiased sequence analyses by next-generation sequencing (NGS) technology for the identification of infectious microorganisms from CSF samples of pediatric bacterial meningitis patients in the Department of Infectious Diseases from Beijing Childrens Hospital.\n\nResultsIn total, we had 99 bacterial meningitis patients in our study, 55 (55.6%) of these were etiologically confirmed by clinical microbiology methods. Combined with NGS, 68 cases (68.7%) were etiologically confirmed. The main pathogens identified in this study were Streptococcus pneumoniae (n=29), group B streptococcus (n=15), Staphylococcus aureus (n=7), Escherichia coli (n=7). In addition, two cases with cytomegalovirus infection and one with Taenia saginata asiatica were confirmed by NGS.\n\nConclusionsNGS could be a promising alternative diagnostic approach for critically ill patients suffering from bacterial meningitis in pediatric population.\n\nSummaryWe conducted the study for the identification of microorganisms by next-generation sequencing directly from CSF samples of pediatric bacterial meningitis patients. And the study showed that NGS could be a promising alternative diagnostic approach for bacterial meningitis in pediatric population.

microbiology↗

bioRxiv2018

Selection for protein stability enriches for epistatic interactions

A now classical argument for the marginal thermodynamic stability of proteins explains the distribution of observed protein stabilities as a consequence of an entropic pull in protein sequence space. In particular, most sequences that are sufficiently stable to fold will have stabilities near the folding threshold. Here we extend this argument to consider its predictions for epistatic interactions for the effects of mutations on the free energy of folding. Although there is abundant evidence to indicate that the effects of mutations on the free energy of folding are nearly additive and conserved over evolutionary time, we show that these observations are compatible with the hypothesis that a non-additive contribution to the folding free energy is essential for observed proteins to maintain their native structure. In particular through both simulations and analytical results, we show that even very small departures from additivity are sufficient to drive this effect.

evolutionary biology↗

bioRxiv2018

A genetically-encoded fluorescent sensor enables rapid and specific detection of dopamine in flies, fish, and mice

Dopamine (DA) is a central monoamine neurotransmitter involved in many physiological and pathological processes. A longstanding yet largely unmet goal is to measure DA changes reliably and specifically with high spatiotemporal precision, particularly in animals executing complex behaviors. Here we report the development of novel genetically-encoded GPCR-Activation-Based-DA (GRABDA) sensors that enable these measurements. In response to extracellular DA rises, GRABDA sensors exhibit large fluorescence increases ({Delta}F/F0[~]90%) with sub-second kinetics, nanomolar to sub-micromolar affinities, and excellent molecular specificity. Importantly, GRABDA sensors can resolve a single-electrical-stimulus evoked DA release in mouse brain slices, and detect endogenous DA release in the intact brains of flies, fish, and mice. In freely-behaving mice, GRABDA sensors readily report optogenetically-elicited nigrostriatal DA release and depict dynamic mesoaccumbens DA changes during Pavlovian conditioning or during sexual behaviors. Thus, GRABDA sensors enable spatiotemporal precise measurements of DA dynamics in a variety of model organisms while exhibiting complex behaviors.

neuroscience↗

bioRxiv2018

Long-read sequencing identified a causal structural variant in an exome-negative case and enabled preimplantation genetic diagnosis

For a proportion of individuals judged clinically to have a recessive Mendelian disease, only one pathogenic variant can be found from clinical whole exome sequencing (WES), posing a challenge to genetic diagnosis and genetic counseling. Here we describe a case study, where WES identified only one pathogenic variant for an individual suspected to have glycogen storage disease type Ia (GSD-Ia), which is an autosomal recessive disease caused by bi-allelic mutations in the G6PC gene. Through Nanopore long-read whole-genome sequencing, we identified a 7kb deletion covering two exons on the other allele, suggesting that complex structural variants (SVs) may explain a fraction of cases when the second pathogenic allele is missing from WES on recessive diseases. Both breakpoints of the deletion are within Alu elements, and we designed Sanger sequencing and quantitative PCR assays based on the breakpoints for preimplantation genetic diagnosis (PGD) for the family planning on another child. Four embryos were obtained after in vitro fertilization (IVF), and an embryo without deletion in G6PC was transplanted after PGD and was confirmed by prenatal diagnosis, postnatal diagnosis, and subsequent lack of disease symptoms after birth. In summary, we present one of the first examples of using long-read sequencing to identify causal yet complex SVs in exome-negative patients, which subsequently enabled successful personalized PGD.

genetics↗

bioRxiv2018

Whole-genome deep learning analysis reveals causal role of noncoding mutations in autism

We address the challenge of detecting the contribution of noncoding mutations to disease with a deep-learning-based framework that predicts specific regulatory effects and deleterious disease impact of genetic variants. Applying this framework to 1,790 Autism Spectrum Disorder (ASD) simplex families reveals autism disease causality of noncoding mutations by demonstrating that ASD probands harbor transcriptional (TRDs) and post-transcriptional (RRDs) regulation-disrupting mutations of significantly higher functional impact than unaffected siblings. Importantly, we detect this significant noncoding contribution at each level, transcriptional and post-transcriptional, independently and after multiple hypothesis correction. Further analysis suggests involvement of noncoding mutations in synaptic transmission and neuronal development, and reveals a convergent genetic landscape of coding and noncoding (TRD and RRD) de novo mutations in ASD. We demonstrate that sequences carrying prioritized proband de novo mutations possess transcriptional regulatory activity and drive expression differentially, and highlight a link between noncoding mutations and IQ heterogeneity in ASD probands. Our predictive genomics framework illuminates the role of noncoding mutations in ASD, prioritizes high impact transcriptional and post-transcriptional regulatory mutations for further study, and is broadly applicable to complex human diseases.

genomics↗

bioRxiv2018

Structural survey of HIV-1-neutralizing antibodies targeting Env trimer delineates epitope categories and suggests vaccine templates

HIV-1 broadly neutralizing antibodies are desired for their therapeutic potential and as templates for vaccine design. Such antibodies target the HIV-1-envelope (Env) trimer, which is shielded from immune recognition by extraordinary glycosylation and sequence variability. Recognition by broadly neutralizing antibodies thus provides insight into how antibody can bypass these immune-evasion mechanisms. Remarkably, antibodies neutralizing >25% of HIV-1 strains have now been identified that recognize all major exposed surfaces of the prefusion-closed Env trimer. Here we analyzed all 206 broadly neutralizing antibody-HIV-1 Env complexes in the PDB with resolution suitable to define their interaction chemistries. These segregated into 20 antibody classes based on ontogeny and recognition, and into 6 epitope categories (V1V2, glycan-V3, CD4-binding site, silent face center, fusion peptide, and subunit interface) based on recognized Env residues. We measured antibody neutralization on a 208-isolate panel and analyzed features of paratope and B cell ontogeny. The number of protruding loops, CDR H3 length, and level of somatic hypermutation for broadly HIV-1 neutralizing antibodies were significantly higher than for a comparison set of non-HIV-1 antibodies. For epitope, the number of independent sequence segments was higher (P < 0.0001), as well as the glycan component surface area (P = 0.0005). Based on B cell ontogeny, paratope, and breadth, the CD4-binding site antibody IOMA appeared to be a promising candidate for lineage-based vaccine design. In terms of epitope-based vaccine design, antibody VRC34.01 had few epitope segments, low epitope-glycan content, and high epitope-conformational variability, which may explain why VRC34.01-based design is yielding promising vaccine results.

bioinformatics↗

bioRxiv2018

The Biological Evaluation of Fusidic Acid and Its Hydrogenation Derivative as Antimicrobial and Anti-inflammatory Agents

Fusidic acid (WU-FA-00) is the only commercially available antimicrobial from the fusidane family that has a narrow spectrum of activity against Gram-positive bacteria. Herein, the hydrogenation derivative (WU-FA-01) of fusidic acid was prepared, and both compounds were examined against a panel of six bacterial strains. In addition, their anti-inflammation properties were evaluated using a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema model. The results of the antimicrobial assay revealed that both WU-FA-00 and WU-FA-01 displayed a high level of antimicrobial activity against Gram-positive strains. Moreover, killing kinetic studies were performed, and the results were in accordance with the MIC and MBC results. We also demonstrated that the topical application of WU-FA-00 and WU-FA-01 effectively decreased TPA-induced ear edema in a dose-dependent manner. This inhibitory effect was associated with the inhibition of TPA-induced up-regulation of pro-inflammation cytokines IL-1{beta}, TNF- and COX-2. WU-FA-01 significantly suppressed the expression levels of p65, I{kappa}B-, and p-I{kappa}B- in the TPA-induced mouse ear model. Overall, our results showed that WU-FA-00 and WU-FA-01 not only had effective antimicrobial activities in vitro, especially to the Gram-positive bacteria, but also possessed strong anti-inflammatory effects in vivo. These results provide a scientific basis for developing fusidic acid derivatives as antimicrobial and anti-inflammatory agents.

pharmacology and toxicology↗

bioRxiv2018

Destabilization of Ran C-terminus promotes GTP loading and occurs in multiple Ran cancer mutations

Ran (Ras-related nuclear protein) plays several important roles in nucleo-cytoplasmic transport, mitotic spindle formation, nuclear envelope/nuclear pore complex assembly, and other diverse functions in the cytoplasm, as well as in cellular transformation when activated. Unlike other Ras superfamily proteins, Ran contains an auto-inhibitory C-terminal tail, which packs against its G domain and bias Ran towards binding GDP over GTP. The biological importance of this C-terminal tail is not well understood. By disrupting the interaction between the C-terminus and the G domain, we were able to generate Ran mutants that are innately active and potently bind to RanBP1 (Ran Binding Protein 1), nuclear export factor CRM1 and nuclear import factor KPNB1. In contrast to previously reported activated Ran mutants, the C-terminus destabilized mutants are hydrolysis competent in cells, support nuclear transport, and do not form nuclear rim staining. Crystal structures show that one of these C-terminal mutations slightly changes its mode of binding to RanBP1. Finally, a high percentage of Ran C-terminus mutations from cancer patients were found to be destabilizing and hyperactivating, suggesting that Ran C-destabilization might be an unprecedented cellular transformation mechanism in affected cancers. This study also highlights a new drug design strategy towards treating patients with hyperactivated Ras proteins including K-Ras.

biochemistry↗

bioRxiv2018

Distinct RanBP1 nuclear export and cargo dissociation mechanisms between fungi and animals

Ran binding protein 1 (RanBP1), the primary effector of nuclear GTPase Ran, is a cytoplasmic-enriched and nuclear-cytoplasmic shuttling protein, playing important roles in nuclear transport through preventing RanGTP from being trapped with karyopherin proteins and dissociating cargoes from nuclear export factor CRM1. Much of what we know about RanBP1 is learned from fungi. Here we show that animal RanBP1 has distinct cargo dissociation and nuclear export mechanisms. In contrast to CRM1-RanGTP sequestration mechanism of cargo dissociation in fungi, animal RanBP1 solely sequesters RanGTP from nuclear export complexes. In fungi, RanBP1, CRM1 and RanGTP form a 1:1:1 nuclear export complex; in contrast, animal RanBP1, CRM1 and RanGTP form a 1:1:2 nuclear export complex. The key feature for the two mechanistic changes from fungi to animals is the loss of affinity between RanBP1-RanGTP and CRM1, since residues mediating their interaction in fungi are not conserved in animals. The biological significances of these different mechanisms in fungi and animals are also studied and discussed. Our study illustrates how orthologous proteins may play conserved functions through distinct routes, and may provide directions for design of antifungal medicines.

biochemistry↗

bioRxiv2018

Robust single-cell DNA methylome profiling with snmC-seq2

Single-cell DNA methylome profiling has enabled the study of epigenomic heterogeneity in complex tissues and during cellular reprogramming. However, broader applications of the method have been impeded by the modest quality of sequencing libraries. Here we report snmC-seq2, which provides improved read mapping, reduced artifactual reads, enhanced throughput, as well as increased library complexity and coverage uniformity compared to snmC-seq. snmC-seq2 is an efficient strategy suited for large scale single-cell epigenomic studies.

genomics↗

bioRxiv2018

A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 nucleases in rice

Targeting specificity has been an essential issue for applying genome editing systems in functional genomics, precise medicine and plant breeding. Understanding the scope of off-target mutations in Cas9 or Cpf1-edited crops is critical for research and regulation. In plants, only limited studies had used whole-genome sequencing (WGS) to test off-target effects of Cas9. However, the cause of numerous discovered mutations is still controversial. Furthermore, WGS based off-target analysis of Cpf1 has not been reported in any higher organism to date. Here, we conducted a WGS analysis of 34 plants edited by Cas9 and 15 plants edited by Cpf1 in T0 and T1 generations along with 20 diverse control plants in rice, a major food crop with a genome size of ~380 Mb. The sequencing depth ranged from 45X to 105X with reads mapping rate above 96%. Our results clearly show that most mutations in edited plants were created by tissue culture process, which caused ~102 to 148 single nucleotide variations (SNVs) and ~32 to 83 insertions/deletions (indels) per plant. Among 12 Cas9 single guide RNAs (sgRNAs) and 3 Cpf1 CRISPR RNAs (crRNAs) assessed by WGS, only one Cas9 sgRNA resulted in off-target mutations in T0 lines at sites predicted by computer programs. Moreover, we cannot find evidence for bona fide off-target mutations due to continued expression of Cas9 or Cpf1 with guide RNAs in T1 generation. Taken together, our comprehensive and rigorous analysis of WGS big data across multiple sample types suggests both Cas9 and Cpf1 nucleases are very specific in generating targeted DNA modifications and off-targeting can be avoided by designing guide RNAs with high specificity.

molecular biology↗

bioRxiv2018

Development of a Cx46 targeting strategy for cancer stem cells

Gap junction-mediated cell-cell communication enables tumor cells to synchronize the execution of complex processes. Despite the connexin family of gap junction proteins being considered tumor suppressors, we previously found that glioblastoma cancer stem cells (CSCs) express higher levels of Cx46 compared to non-stem tumor cells, and this was necessary and sufficient for CSC maintenance. To develop a Cx46 targeting strategy, we utilized point mutants to disrupt specific functions of Cx46 and found that gap junction coupling was the critical function of Cx46 for CSCs. Based on this finding, we screened a clinically relevant library of small molecules and identified clofazimine as an inhibitor of Cx46-specific cell-cell communication. Clofazimine attenuated proliferation, self-renewal, and tumor growth and synergized with temozolomide to induce apoptosis. These data suggest that combining clofazimine with standard-of-care therapies may target glioblastoma CSCs. Furthermore, these results demonstrate the importance of targeting cell-cell communication as an anti-cancer therapy.

cancer biology↗

bioRxiv2018

Single-molecule optical mapping enables accurate molecular diagnosis of facioscapulohumeral muscular dystrophy (FSHD)

Facioscapulohumeral Muscular Dystrophy (FSHD) is a common adult muscular dystrophy in which the muscles of the face, shoulder blades and upper arms are among the most affected. FSHD is the only disease in which \"junk\" DNA is reactivated to cause disease, and the only known repeat array-related disease where fewer repeats cause disease. More than 95% of FSHD cases are associated with copy number loss of a 3.3kb tandem repeat (D4Z4 repeat) at the subtelomeric chromosomal region 4q35, of which the pathogenic allele contains less than 10 repeats and has a specific genomic configuration called 4qA. Currently, genetic diagnosis of FSHD requires pulsed-field gel electrophoresis followed by Southern blot, which is labor-intensive, semi-quantitative and requires long turnaround time. Here, we developed a novel approach for genetic diagnosis of FSHD, by leveraging Bionano Saphyr single-molecule optical mapping platform. Using a bioinformatics pipeline developed for this assay, we found that the method gives direct quantitative measurement of repeat numbers, can differentiate 4q35 and the highly paralogous 10q26 regions, can determine the 4qA/4qB allelic configuration, and can quantitate levels of post-zygotic mosaicism. We evaluated this approach on 5 patients (including two with post-zygotic mosaicism) and 2 patients (including one with post-zygotic mosaicism) from two separate cohorts, and had complete concordance with Southern blots, but with improved quantification of repeat numbers resolved between haplotypes. We concluded that single-molecule optical mapping is a viable approach for molecular diagnosis of FSHD and may be applied in clinical diagnostic settings once more validations are performed.

genomics↗

bioRxiv2018

BAP1 Loss Predicts Therapeutic Vulnerability in Malignant Peritoneal Mesothelioma

BackgroundMalignant Peritoneal Mesothelioma (PeM) is a rare but frequently fatal cancer that originates from the peritoneal lining of the abdomen. Standard treatment of PeM is limited to cytoreductive surgery and/or chemotherapy, and no effective targeted therapies for PeM yet exist. In the search for novel therapeutic target candidates in PeM, we performed a comprehensive integrative multi-omics analysis of 19 treatment-naive PeM tumors.\n\nResultsThe analysis identified PeM tumors with BAP1 loss to form a distinct molecular subtype characterized by distinct expression patterns of genes involved in chromatin remodeling, DNA repair pathway, and immune checkpoint receptor activation. This PeM subtype could potentially benefit from immune checkpoint, PARP, or HDAC inhibition therapies.\n\nConclusionsOur findings uncover BAP1 as a trackable prognostic and predictive biomarker, and refine PeM disease classification. This integrated molecular characterization provides a comprehensive foundation for developing PeM precision medicine.

cancer biology↗

bioRxiv2018

PKD2 influence uric acid levels and gout risk by interacting with ABCG2

BackgroundUric acid is the final product of purine metabolism and elevated serum urate levels can cause gout. Conflicting results were reported for the effect of PKD2 on serum urate levels and gout risk. Therefore, our study attempted to state the important role of PKD2 in influencing the pathogenesis of gout.\n\nMethodSNPs in PKD2 (rs2725215 and rs2728121) and ABCG2 (rs2231137 and rs1481012) were tested in approximately 5,000 Chinese individuals.\n\nResultsTwo epistatic interactions between loci in PKD2 (rs2728121) and ABCG2 (rs1481012 and rs2231137) showed distinct contributions to uric acid levels with P int values of 0.018 and 0.004, respectively, and the associations varies by gender and BMI. The SNP pair of rs2728121 and rs1481012 justly played roles in uric acid in females (P int = 0.006), while the other pair did in males (P int = 0.017). Regarding BMI, the former SNP pair merely contributed in overweigh subjects (P int = 0.022) and the latter one did in both normal and overweigh individuals (P int = 0.013 and 0.047, respectively). Furthermore, the latter SNP pair was also associated with gout pathology (P int = 0.001), especially in males (P int = 0.001). Finally, functional analysis showed potential epistatic interactions in those genes region and PKD2 mRNA expression had a positive correlation with ABCG2s (r = 0.743, P = 5.83e-06).\n\nConclusionOur study for the first time identified that epistatic interactions between PKD2 and ABCG2 influenced serum urate concentrations and gout risk, and PKD2 might affect the pathogenesis from elevated serum urate to hyperuricemia to gout by modifying ABCG2.

genetics↗

bioRxiv2018

Single-cell analysis of progenitor cell dynamics and lineage specification of the human fetal kidney

The mammalian kidney develops through repetitive and reciprocal interactions between the ureteric bud and the metanephric mesenchyme to give rise to the entire collecting system and the nephrons, respectively. Most of our knowledge of the developmental regulators driving this process has been gained from the study of gene expression and functional genetics in mice and other animal models. In order to shed light on human kidney development, we have used singlecell transcriptomics to characterize gene expression in different cell population, and to study individual cell dynamics and lineage trajectories during development. Single cell transcriptome analyses of 3,865 cells identified 17 clusters of specific cell types as defined by their gene expression profile, including markers of ureteric bud tip- and metanephric mesenchyme-specific progenitors, as well as their intermediate and differentiated lineages including the mature collecting ducts, the renal vesicle and comma- and s-shaped bodies, immature and mature podocytes, proximal tubules, loops of Henle and distal tubules. Other lineages identified include mesangium and cortical and medullary interstitium, endothelial and immune cells as well as hematopoietic cells. Novel markers for these cell types were revealed in the analysis as well as components of key signaling pathways driving renal development in animal models. Altogether, we provide a comprehensive and dynamic gene expression array of the human developing kidney at the single-cell level.

developmental biology↗