Biology subjects

Wang, D.

Publications and source records attributed to Wang, D..

At least 739 records · Page 41Linked to original sources

bioRxiv2019

Single cell RNA sequencing reveals cellular diversity of trisomy 21 retina

Retina is a crucial tissue for the capturing and processing of light stimulus. Characterization of the retina at single cell level is essential for the understanding of its biological functions. A variety of abnormalities in terms of morphology and function were reported in T21 retina. To evaluate the effects of chromosome aneuploidy on retina development, we characterized single cell transcriptional profiles of a T21 fetus and performed comprehensive bioinformatic analyses. Our data revealed the diversity and heterogeneity of cellular compositions in T21 retina. In total, we identified seven major cell types, and detected several subtypes within each cell type, followed by the detection of corresponding molecular markers including previously reported ones and a series of novel markers. Our analyses identified extensive communication networks between distinct cellular types, among which a few ligand-receptor interactions were associated with the development of retina and immunoregulatory interactions. Taken together, our data provided the first single cell transcriptome profile for human T21 retina which facilitates our understanding on the dosage effects of chromosome 21 on the development of retina.

cell biology↗

bioRxiv2019

Extension of the in vivo haploid induction system from diploid maize to hexaploid wheat

Doubled haploid breeding technology has been one of the most important techniques for accelerating crop breeding. In compare to in vivo haploid induction in maize, which is efficient and background independent, wheat haploid production by interspecific hybridization pollinated with maize is influenced by genetic background and requires rescue of young embryos. Here, we analyzed the homologues of maize haploid induction gene MTL/ZmPLA1/NLD in several crop species systematically, the homologues are highly conserved in sorghum, millet and wheat etc. Since wheat is a very important polyploidy crop, as a proof of concept, we demonstrated that the in vivo haploid induction method could be extended from diploid maize to hexaploid wheat by knocking out the wheat homologues (TaPLAs). Result showed that double knock-out mutation could trigger wheat haploid induction at ~ 2%-3%, accompanied by 30% - 60% seed setting rate. The performance of haploid wheat individual showed shorter plant, narrower leaves and male sterile. Our results also revealed that knockout of TaPLA-A and TaPLA-D do not affect pollen viability. This study not only confirmed the function of the induction gene and explored a new approach for haploid production in wheat, but also provided an example that the in vivo haploid induction could be applied in more crop species with different ploidy levels. Furthermore, by combining with gene editing, it would be a fast and powerful platform for traits improvement in polyploidy crops breeding.

plant biology↗

bioRxiv2019

Motto: Representing motifs in consensus sequences with minimum information loss

Sequence analysis frequently requires intuitive understanding and convenient representation of motifs. Typically, motifs are represented as position weight matrices (PWMs) and visualized using sequence logos. However, in many scenarios, representing motifs by wildcard-style consensus sequences is compact and sufficient for interpreting the motif information and search for motif match. Based on mutual information theory and Jenson-Shannon Divergence, we propose a mathematical framework to minimize the information loss in converting PWMs to consensus sequences. We name this representation as sequence Motto and have implemented an efficient algorithm with flexible options for converting motif PWMs into Motto from nucleotides, amino acids, and customized alphabets. Here we show that this representation provides a simple and efficient way to identify the binding sites of 1156 common TFs in the human genome. The effectiveness of the method was benchmarked by comparing sequence matches found by Motto with PWM scanning results found by FIMO. On average, our method achieves 0.81 area under the precision-recall curve, significantly (p-value < 0.01) outperforming all existing methods, including maximal positional weight, Douglas and minimal mean square error. We believe this representation provides a distilled summary of a motif, as well as the statistical justification.\n\nAVAILABILITYMotto is freely available at http://wanglab.ucsd.edu/star/motto.

bioinformatics↗

bioRxiv2019

Noda-like RNA viruses infecting Caenorhabditis nematodes: sympatry, diversity and reassortment

Three RNA viruses related to nodaviruses were previously described to naturally infect the nematode Caenorhabditis elegans and its relative C. briggsae. Here we report on a collection of over 50 viral variants from wild-caught Caenorhabditis. We describe the discovery of a new related virus, the M[e]lnik virus, infecting C. briggsae, which similarly infects intestinal cells. In France, a frequent pattern of co-infection of C. briggsae by the Santeuil virus and Le Blanc virus was observed at the level of an individual nematode and even a single cell. We do not find evidence of reassortment between the RNA1 and RNA2 molecules of Santeuil and Le Blanc viruses. However, by studying patterns of evolution of each virus, reassortments of RNA1 and RNA2 among variants of each virus were identified. We develop assays to test the relative potency and competitive ability of the viral variants and detect an interaction between host genotype and Santeuil virus genotype, such that the result of the competition depends on the host strain.\n\nImportanceThe roundworm Caenorhabditis elegans is a laboratory model organism in biology. We study natural populations of this small animal and its relative C. briggsae and the viruses that infect them. We previously discovered three RNA viruses related to nodaviruses and here describe a fourth one, called the Melnik virus. These viruses have a genome composed of two RNA molecules. We find that two viruses may infect the same animal and the same cell. The two RNA molecules may be exchanged between variants of a given viral species. We study the diversity of each viral species and devise an assay of their competitive ability. Using this assay, we show that the outcome of the competition also depends on the host.

microbiology↗

bioRxiv2019

Function and clinical relevance of RHAMM isoforms in pancreatic tumor progression

The receptor for hyaluronic acid-mediated motility (RHAMM) is upregulated in various cancers. We previously screened genes upregulated in human hepatocellular carcinomas for their metastatic function in a mouse model of pancreatic neuroendocrine tumor (PNET) and identified that human RHAMMB promoted liver metastasis. It was unknown whether RHAMMB is upregulated in pancreatic cancer or contributes to its progression. In this study, we found that RHAMM protein was frequently upregulated in human PNETs. We investigated alternative splicing isoforms, RHAMMA and RHAMMB, by RNA-Seq analysis of primary PNETs and liver metastases. RHAMMB, but not RHAMMA, was significantly upregulated in liver metastases. RHAMMB was crucial for in vivo metastatic capacity of mouse and human PNETs. RHAMMA, carrying an extra 15-amino acid-stretch, did not promote metastasis in spontaneous and experimental metastasis mouse models. Moreover, RHAMMB was substantially higher than RHAMMA in pancreatic ductal adenocarcinoma (PDAC). RHAMMB, but not RHAMMA, correlated with both higher EGFR expression and poorer survival of PDAC patients. Knockdown of EGFR abolished RHAMMB-driven PNET metastasis. Altogether, our findings suggest a clinically relevant function of RHAMMB, but not RHAMMA, in promoting PNET metastasis in part through EGFR signaling. RHAMMB can thus serve as a prognostic factor for pancreatic cancer.

cancer biology↗

bioRxiv2019

Clinical long-read sequencing of the human mitochondrial genome for mitochondrial disease diagnostics

PurposeLong-read, third generation, sequencing technologies have the potential to improve current state of the art diagnostic strategies. In order to determine if long-read sequencing technologies are suitable for the diagnosis of mitochondrial disorders due to mitochondrial DNA (mtDNA) variants, particularly large deletions, we compared the performance of Oxford Nanopore Technologies (ONT) MinION to current diagnostic methods.\n\nMethodsWe sequenced mtDNA from nine patients with mtDNA deletion disorders and three normal controls with both ONT MinION and Illumina MiSeq. We applied a computational pipeline to estimate the positions of mtDNA deletions in patients, and subsequently validated the breakpoints using Sanger sequencing.\n\nResultsWe were able to detect mtDNA deletions with a MinION workflow, successfully calling the disease causing event in all cases. Sequencing coverage was in most cases significantly more (p=0.03, Wilcoxon test) uniform with MinION than with MiSeq and subsequent correction of MinION reads improved breakpoint accuracy and reduced false positives. Although heteroplasmic single nucleotide variants are detectable, the high number of false positives and false negatives precludes their use in diagnostics at this time.\n\nConclusionThe MinION is becoming an increasingly attractive diagnostic tool due to the reducing cost, increasing accuracy, and the speed at which data can be obtained.

genomics↗

bioRxiv2019

Oligotyping and Genome-Resolved Metagenomics Reveal Distinct Candidatus Accumulibacter Communities in Full-Scale Side-Stream versus Conventional Enhanced Biological Phosphorus Removal (EBPR) Configurations

Candidatus Accumulibacter phosphatis (CAP) and its sub-clades-level diversity has been associated and implicated in successful phosphorus removal performance in enhanced biological phosphorus removal (EBPR). Development of high-throughput untargeted methods to characterize clades of CAP in EBPR communities can enable a better understanding of Accumulibacter ecology at a higher-resolution beyond OTU-level in wastewater resource recovery facilities (WRRFs). In this study, for the first time, using integrated 16S rRNA gene sequencing, oligotyping and genome-resolved metagenomics, we were able to reveal clade-level differences in Accumulibacter communities and associate the differences with two different full-scale EBPR configurations. The results led to the identification and characterization of a distinct and dominant Accumulibacter oligotype - Oligotype 2 (belonging to Clade IIC) and its matching MAG (RC14) associated with side-stream EBPR configuration. We are also able to extract MAGs belonging to CAP clades IIB (RCAB4-2) and II (RC18) which did not have representative genomes before. This study demonstrates and validates the use of a high-throughput approach of oligotyping analysis of 16S rRNA gene sequences to elucidate CAP clade-level diversity. We also show the existence of a previously uncharacterized diversity of CAP clades in full-scale EBPR communities through extraction of MAGs, for the first time from full-scale facilities.

microbiology↗

bioRxiv2019

Infection of Plasmodiophora brassicae changes the fungal endophyte community of tumourous stem mustard roots as revealed by high-throughput sequencing and culture-dependent methods

Diverse fungal endophytes live in plants and are shaped by some abiotic and biotic stresses. Plant disease as particular biotic stress possibly gives an impact on the communities of fungal endophytes. In this study, clubroot disease caused by an obligate biotroph protist, Plasmodiophora brassicae, was considered to analyze its influence on the fungal endophyte community using an internal transcribed spacer (ITS) through high-throughput sequencing and culture-dependent methods. The results show that the diversity of the endophyte community in the healthy roots was much higher than the clubroots. Ascomycota was the dominant group of endophytes (including Phoma, Mortierella, Penicillium, etc.) in the healthy roots while P. brassicae was the dominant taxon in the clubroots. Hierarchical clustering, principal component analysis (PCA) and principal coordinates analysis (PCoA) indicated significant differences between the endophyte communities in the healthy roots and clubroots. Linear discriminant analysis effect size (LefSe) analysis showed that the dominant genera could be regarded as potential biomarkers. The endophyte community in the healthy roots had a more complex network compared with the clubroots. Also, many plant pathogenic Fusarium were isolated from the clubroots by the culture-dependent method. The outcome of this study illustrates that P. brassicae infection may change the fungal endophyte community associated with the roots of tumourous stem mustard and facilitates the entry of soil pathogen into the roots.

microbiology↗

bioRxiv2019

The role of genetic constraints and social environment in explaining female extra-pair mating

Why females of socially monogamous species copulate with males other than their partner has been a long-standing, unresolved puzzle. We previously reported that female promiscuity appears to be a genetic corollary of male promiscuity (intersexual pleiotropy hypothesis). Here we put this earlier finding to a critical test using the same population of zebra finches Taeniopygia guttata. After three generations of artificial selection on male courtship rate, a correlate of extra-pair mating, we assess whether female promiscuity changed by indirect selection and we re-examine the crucial genetic correlations. Our new analyses with substantially increased statistical power clearly reject the hypothesis that male and female promiscuity are genetically homologous traits. Our study highlights that individual females show low repeatability in extra-pair mating behavior across different social environments. This emphasizes the potential importance of pair bond strength and the availability of favored extra-pair males as factors explaining variation in patterns of female promiscuity.

evolutionary biology↗

bioRxiv2019

A multi-omic cohort as a reference point for promoting a healthy human gut microbiome

More than a decade of gut microbiome studies have a common goal for human health. As most of the disease studies sample the elderly or the middle-aged, a reference cohort for young individuals has been lacking. It is also not clear what other omics data need to be measured to better understand the gut microbiome. Here we present high-depth metagenomic shotgun sequencing data for the fecal microbiome together with other omics data in a cohort of 2,183 adults, and observe a number of vitamins, hormones, amino acids and trace elements to correlate with the gut microbiome and cluster with T cell receptors. Associations with physical fitness, sleeping habits and dairy consumption are identified in this large multi-omic cohort. Many of the associations are validated in an additional cohort of 1,404 individuals. Our comprehensive data are poised to advise future study designs to better understand and manage our gut microbiome both in population and in mechanistic investigations.

microbiology↗

bioRxiv2019

3.1A structure of yeast RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion solved using streptavidin affinity grids

Despite significant advances in all aspects of single particle cryo-electron microscopy (cryo-EM), specimen preparation still remains a challenge. During sample preparation, macromolecules interact with the air-water interface, which often leads to detrimental effects such as denaturation or adoption of preferred orientations, ultimately hindering structure determination. Randomly biotinylating the protein of interest and then tethering it to a cryo-EM grid coated with two-dimensional crystals of streptavidin (acting as an affinity surface) can prevent the protein from interacting with the air-water interface. Recently, this approach was successfully used to solve a high-resolution structure of a test sample, a bacterial ribosome. However, the general applicability of this method to samples where interaction with the air-water interface is problematic remains to be determined. Here we report a 3.1[A] structure of a RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion (Pol II EC(CPD)) solved using streptavidin grids. Our previous attempt to solve this structure using conventional sample preparation methods resulted in a poor quality cryo-EM map due to Pol II EC(CPD)s adopting a strong preferred orientation. Imaging the same sample on streptavidin grids led to a high-resolution structure with little anisotropy, showing that streptavidin affinity grids could be used as a general strategy to address challenges posed by interaction with the air-water interface.

molecular biology↗

bioRxiv2019

The protein repertoire in early vertebrate embryogenesis

We present an unprecedentedly comprehensive characterization of protein dynamics across early development in Xenopus laevis, available immediately via a convenient Web portal. This resource allows interrogation of the protein expression data in conjunction with other data modalities such as genome wide mRNA expression. This study provides detailed data for absolute levels of [~]14K unique Xenopus proteins representing homologues of [~]9K unique human genes - a rich resource for developmental biologists. The purpose of this manuscript is limited to presenting and releasing the data browser.\n\nHighlightsO_LIRelative protein expression from stage IV oocyte, blastula, gastrula, neurula, and early organogenesis\nC_LIO_LIBiological triplicates with confidence intervals on protein expression reflect certainty in dynamic patterns\nC_LIO_LIConvenient time-series Web-browser integrated with the multi-media Xenbase portal\nC_LIO_LIGene-symbol search and multi-gene protein/mRNA juxtaposition capabilities\nC_LI

developmental biology↗

bioRxiv2019

MicroRNA Overexpression screen reveals a noncanonical role of CDK2 in regulating neutrophil migration

Neutrophil migration is essential for inflammatory responses to kill pathogens, however it also causes tissue injury. To discover novel therapeutic targets that modulate neutrophil migration, we performed a neutrophil-specific microRNA overexpression screen in zebrafish, and identified eight microRNAs as potent suppressors of neutrophil migration. Among those, miR-199 decreases neutrophil chemotaxis in zebrafish and human neutrophil-like cells. Intriguingly, in terminally differentiated neutrophils, miR-199 alters the cell cycle-related pathways and directly suppresses cyclin-dependent kinase 2 (cdk2), whose known activity is restricted to cell cycle progression and cell differentiation. Inhibiting CDK2, but not DNA replication, disrupts cell polarity and chemotaxis of zebrafish neutrophils. Chemotaxis of primary human neutrophils are also reduced by CDK2 inhibition. Furthermore, miR-199 overexpression or CDK2 inhibition significantly improves the outcome of lethal systemic inflammation challenges in zebrafish. Together, our results reveal previously unknown functions of miR-199 and CDK2 in regulating neutrophil migration and provide new directions in alleviating systemic inflammation.\n\nOne Sentence SummarymiR-199 directly suppresses cdk2 expression, neutrophil chemotaxis and systemic inflammation.

immunology↗

bioRxiv2019

Production of nonnatural straight chain amino acid 6-aminocaproate via an artificial recursive carbon-chain-extension cycle

Bioplastics produced from microbial source are promising green alternatives to traditional petrochemical-derived plastics. Nonnatural straight-chain amino acids, especially 5-aminovalerate, 6-aminocaproate and 7-aminoheptanoate are potential monomers for the synthesis of polymeric bioplastics as their primary amine and carboxylic acid are ideal functional groups for polymerization. Previous pathways for 5-aminovalerate and 6-aminocaproate biosynthesis in microorganisms are derived from L-lysine catabolism and citric acid cycle, respectively. Here, we show the construction of an artificial iterative carbon-chain-extension cycle in Escherichia coli for simultaneous production of a series of nonnatural amino acids with varying chain length. Overexpression of L-lysine -oxidase in E. coli yields 2-keto-6-aminocaproate as a non-native substrate for the artificial iterative carbon-chain-extension cycle. The chain-extended -ketoacid is subsequently decarboxylated and oxidized by an -ketoacid decarboxylase and an aldehyde dehydrogenase, respectively, to yield the nonnatural straight-chain amino acid products. The engineered system demonstrated simultaneous in vitro production of 99.16 mg/L of 5-aminovalerate, 46.96 mg/L of 6-aminocaproate and 4.78 mg/L of 7-aminoheptanoate after 8 hours of enzyme catalysis starting from 2-keto-6-aminocaproate as the substrate. Furthermore, simultaneous production of 2.15 g/L of 5-aminovalerate, 24.12 mg/L of 6-aminocaproate and 4.74 mg/L of 7-aminoheptanoate was achieved in engineered E. coli. This work illustrates a promising metabolic-engineering strategy to access other medium-chain organic acids with -NH2,-SCH3, -SOCH3, -SH, -COOH, -COH, or -OH functional groups through carbon-chain-elongation chemistry.

synthetic biology↗

bioRxiv2019

Cancer gene therapy by NF-κB-activated cancer cell-specific expression of CRISPR/Cas9 targeting to telomere

NF-{kappa}B has been a luring target for cancer therapy due to its over activation in all tumors. In this study, we showed that a gene therapy named as NF-{kappa}B-activated gene expression (Nage) could be used to induce cancer cell death in vitro and in vivo by utilizing the NF-{kappa}B activity in cancer cells; however, it had no effect on normal cells. In this gene therapy, we constructed a NF-{kappa}B-specific promoter by fusing a NF-{kappa}B decoy sequence to a minimal promoter, which could be bound by the intracellular over activated NF-{kappa}B and thus activate the expression of downstream effector gene in a NF-{kappa}B-specific manner. In this study, we firstly demonstrated the cancer cell-specific activation of NF-{kappa}B. We then demonstrated the cancer cell specificity of Nage vector expression by introducing a Nage vector that could express a reporter gene ZsGreen in various cell lines. We next demonstrated that a Nage vector that could express CRISPR/Cas9 protein and a telomere-targeting sgRNA could be used to specifically induce death of cancer cells. We finally showed that the Cas9/sgRNA Nage vector packaged into the adeno-associated virus (AAV) could be used to inhibit the growth of xenografted tumors in mouse by intravenously injecting recombinant AAV.

cancer biology↗

bioRxiv2019

Cellular structure image classification with small targeted training samples

MotivationCell shapes provide crucial biology information on complex tissues. Different cell types often have distinct cell shapes, and collective shape changes usually indicate morphogenetic events and mechanisms. The identification and detection of collective cell shape changes in an extensive collection of 3D time-lapse images of complex tissues is an important step in assaying such mechanisms but is a tedious and time-consuming task. Machine learning provides new opportunities to automatically detect cell shape changes. However, it is challenging to generate sufficient training samples for pattern identification through deep learning because of a limited amount of images and annotations. ResultWe present a deep learning approach with minimal well-annotated training samples and apply it to identify multicellular rosettes from 3D live images of the Caenorhabditis elegans embryo with fluorescently labelled cell membranes. Our strategy is to combine two approaches, namely, feature transfer and generative adversarial networks (GANs), to boost image classification with small training samples. Specifically, we use a GAN framework and conduct an unsupervised training to capture the general characteristics of cell membrane images with 11,250 unlabelled images. We then transfer the structure of the GAN discriminator into a new Alex-style neural network for further learning with several dozen labelled samples. Our experiments showed that with 10-15 well-labelled rosette images and 30-40 randomly selected non-rosette images our approach can identify rosettes with over 80% accuracy and capture over 90% of the model accuracy achieved with a training dataset that is five times larger. We also established a public benchmark dataset for rosette detection. This GAN-based transfer approach can be applied to study other cellular structures with minimal training samples. Contactdwang7@utk.edu, baoz@mskcc.org

bioinformatics↗

bioRxiv2019

Synapse-specific Opioid Modulation of Thalamo-cortico-striatal Circuits

The medial thalamus (MThal), anterior cingulate cortex (ACC) and striatum play important roles in affective-motivational pain processing and reward learning. Opioids affect both pain and reward through uncharacterized modulation of this circuitry. This study examined opioid actions on glutamate transmission between these brain regions in mouse. Mu-opioid receptor (MOR) agonists potently inhibited MThal inputs without affecting ACC inputs to individual striatal medium spiny neurons (MSNs). MOR activation also inhibited MThal inputs to the pyramidal neurons in the ACC. In contrast, delta-opioid receptor (DOR) agonists disinhibited ACC pyramidal neuron responses to MThal inputs by suppressing local feed-forward GABA signaling from parvalbumin-positive interneurons. As a result, DOR activation in the ACC facilitated poly-synaptic (thalamo-cortico-striatal) excitation of MSNs by MThal inputs. These results suggest that opioid effects on pain and reward may be shaped by the relative selectivity of opioid drugs to the specific circuit components.

neuroscience↗

bioRxiv2019

Gene activation by a CRISPR-assistant trans enhancer

Gene activation is essential to the basic biological research and biomedicine. Therefore, various gene activators such as activation domain-ZNF, TALE and CRISPR proteins have been developed for this end, in which the CRISPR protein dead Cas9 (dCas9) is now most widely used. However, the current gene activators are still limited by their inefficient gene activation activity. In this study, we developed a new strategy, CRISPR-assistant trans enhancer, for activating gene expression in high efficiency by combining dCas9-VP64/sgRNA with a widely used strong enhancer, the CMV enhancer. In this strategy, a trans CMV enhancer DNA was recruited to target gene by dCas9-VP64/sgRNA via annealing between 3 end of sgRNA and CMV enhancer. The trans enhancer activates gene transcription as the natural looped cis enhancer. The trans enhancer could activate both exogenous reporter gene and variant endogenous genes in various cells, with much higher activation efficiency than the current dCas9 activators.

biochemistry↗