Biology subjects

Du, J.

Publications and source records attributed to Du, J..

16 recordsLinked to original sources

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

Induction of Sertoli cells from human fibroblasts by NR5A1 and GATA4

Sertoli cells are essential nurse cells in the testis that regulate the process of spermatogenesis and establish the immune-privileged environment of the blood-testis-barrier (BTB). The induction of human Sertoli cells from fibroblasts could provide cellular sources for fertility and transplantation treatments. Here, we report the in vitro reprogramming of human fibroblasts to Sertoli cells and characterize these human induced Sertoli cells (hiSCs). Initially, five transcriptional factors (NR5A1, GATA4, WT1, SOX9 and DMRT1) and a gene reporter carrying the AMH promoter were utilized to obtain the hiSCs. We further reduce the number of reprogramming factors to two, i.e., NR5A1 and GATA4, and show that these hiSCs have transcriptome profiles that are similar to those of primary human Sertoli cells. Consistent with the known cellular properties of Sertoli cells, hiSCs attract endothelial cells and exhibit high number of lipid droplets in the cytoplasm. More importantly, hiSCs can sustain the viability of spermatogonia cells harvested from mouse seminiferous tubules. In addition, hiSCs suppress the production of IL-2 and proliferation of human T lymphocytes. When hiSCs were cotransplanted with human embryonic kidney cells, these xenotransplanted human cells survived longer in mice with normal immune systems. hiSCs also allow us to determine a gene associated with Sertoli-only syndrome (SCO), CX43, is indeed important in regulating the maturation of Sertoli cells.

developmental 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

Crystal Structure of Human Nocturnin Catalytic Domain

Nocturnin (NOCT) helps the circadian clock to adjust metabolism according to day and night activity. NOCT is upregulated in early evening and it has been proposed to serve as a deadenylase for metabolic enzyme mRNAs. We present a 2.7-[A] crystal structure of the catalytic domain of human NOCT. Our structure shows that NOCT has a close overall similarity to CCR4 deadenylase family members, PDE12 and CNOT6L, and to a DNA repair enzyme TDP2. All the key catalytic residues present in PDE12, CNOT6L and TDP2 are conserved in NOCT and have the same conformations. However, we observe substantial differences in the surface properties of NOCT, an unexpectedly narrow active site pocket, and conserved structural elements in the vicinity of the catalytic center, which are unique to NOCT and absent in the deadenylases PDE12/CNOT6L. Our work thus reveals the structure of an intriguing circadian protein and suggests that NOCT has considerable differences from the related deadenylases, which may point to a unique cellular function of this enzyme.

biophysics↗

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

Isolation and characterization of antagonistic bacteria with the potential for biocontrol of soil-borne wheat diseases

Bacillus amyloliquefaciens subsp. plantarum XH-9 is a plant-beneficial rhizobacterium that shows good antagonistic potential against phytopathogens by releasing diffusible and volatile antibiotics, and secreting hydrolytic enzymes. Furthermore, the XH-9 strain possesses important plant growth-promoting characteristics, including nitrogen fixation (7.92 {+/-} 1.05 mg/g), phosphate solubilization (58.67 {+/-} 4.20 g/L), potassium solubilization (10.07 {+/-} 1.26 g/mL), and the presence of siderophores (4.92 {+/-} 0.46 g/mL), indole-3-acetic acid (IAA) (7.76 {+/-} 0.51 g/mL) and 1-aminocyclopropane-1-carboxylic acid deaminase (ACC-deaminase) (4.67 {+/-} 1.21 nmol/[mg*h]). Moreover, the XH-9 strain showed good capacities for wheat, corn, and chili root colonization, which are critical prerequisites for controlling soil-borne diseases as a bio-control agent. Real-time quantitative polymerase chain reaction experiments showed that the amount of Fusarium oxysporum DNA associated with the XH-9 strain after treatment significantly decreased compared with control group. Accordingly, wheat plants inoculated with the XH-9 strain showed significant increases in the plant shoot heights (14.20%), root lengths (32.25%), dry biomass levels (11.93%), and fresh biomass levels (16.28%) relative to the un-inoculated plants. The results obtained in this study suggest that the XH-9 strain has potential as plant-growth promoter and biocontrol agent when applied in local arable land to prevent damage caused by F. oxysporum and other phytopathogens.\n\nImportancePlant diseases, particularly soilborne pathogens, play a significant role in the destruction of agricultural resources. Although these diseases can be controlled to some extent with crop and fungicides, while these measures increase the cost of production, promote resistance, and lead to environmental contamination, so they are being phased out. Plant growth-promoting rhizobacteria are an alternative to chemical pesticides that can play a key role in crop production by means of siderophore and indole-3-acetic acid production, antagonism to soilborne root pathogens, phosphate and potassium solubilization, and nitrogen fixation. These rhizobacteria can also promote a beneficial change in the microorganism community by significantly reducing its pathogenic fungi component. Their use is fully in accord with the principles of sustainability.

microbiology↗

bioRxiv2018

Gene2Vec: Distributed Representation of Genes Based on Co-Expression

Existing functional description of genes are categorical, discrete, and mostly through manual process. In this work, we explore the idea of gene embedding, distributed representation of genes, in the spirit of word embedding. From a pure data-driven fashion, we trained a 300 dimension vector representation of all human genes, using gene co-expression patterns in 984 data sets from the GEO databases. These vectors capture functional relatedness of genes in terms of recovering known pathways - the average inner product (similarity) of genes within a pathway is 1.68X greater than that of random genes. Using t-SNE, we produced a gene co-expression map that shows local concentrations of tissue specific genes. We also illustrated the usefulness of the embedded gene vectors, laden with rich information on gene co-expression patterns, in tasks such as gene-gene interaction prediction. Overall, we believe that this distributed representation of genes may be useful for more bioinformatics applications.

bioinformatics↗

bioRxiv2018

Structure of the human lipid-sensitive cation channel TRPC3

The TRPC channels are crucially involved in store-operated calcium entry and calcium homeostasis, and they are thus implicated in human diseases such as neurodegenerative disease, cardiac hypertrophy, and spinocerebellar ataxia. We present structure of the full-length human TRPC3, a lipid-gated TRPC member, in a lipid-occupied, closed state at 3.3 Angstrom. TRPC3 has an acorn-like shape with four elbow-like membrane reentrant helices prior to the first transmembrane helix. The TRP helix is perpendicular to, and thus disengaged from, the pore-lining S6, suggesting a different gating mechanism. The third transmembrane helix S3 is remarkably long, resulting in a windmill-like transmembrane domain, and constituting an extracellular domain that may serve as a sensor of external stimuli. We identified two lipid binding sites, one being sandwiched between the pre-S1 elbow and the S4-S5 linker, and the other being close to the ion-conducting pore, where the conserved LWF motif of the TRPC family is located.

biophysics↗

bioRxiv2018

A comprehensive automated pipeline for human microbiome sampling, 16S rRNA gene sequencing and bioinformatics processing

The advent of affordable high-throughput DNA sequencing has opened up a golden age of studies in the human microbiome. In order to understand the role of the human microbiota, standardized methods for large-scale, population-level studies are needed to avoid underpowered or poorly designed studies. The biggest bottlenecks to population-level microbiomics are sample collection, storage and DNA extraction. Here, we describe a flexible automated approach to process intestinal biopsies, fecal samples and vaginal swabs from sample collection to OTU table. We have evaluated storage conditions, DNA extraction methods, PCR strategies and bioinformatic pipelines for these three sample types, and present here a set of guidelines and best practices for each of these steps.

microbiology↗

bioRxiv2017

Three classes of response elements for human PRC2 and MLL1/2-trithorax complexes

Polycomb group (PcG) and trithorax group (TrxG) proteins are essential for maintaining epigenetic memory in both embryonic stem cells and differentiated cells. To date, how they are localized to hundreds of specific target genes within a vertebrate genome had remained elusive. Here, by focusing on short cis-acting DNA elements of single functions, we discovered, for the first time, to our knowledge, three classes of response elements in human genome: PcG response elements (PREs), MLL1/2-TrxG response elements (TREs) and PcG/TrxG response elements (P/TREs). We further demonstrated that, in contrast to their proposed roles in recruiting PcG proteins to PREs, YY1 and CpG islands are specifically enriched in TREs and P/TREs, but not in PREs. The three classes of response elements as unraveled in this study open new doors for a deeper understanding of PcG and TrxG mechanisms in vertebrates.

biochemistry↗

bioRxiv2017

The functional and genetic associations of neuroimaging data: a toolbox

Advances in neuroimaging and sequencing techniques provide an unprecedented opportunity to map the function of brain regions and to identify the roots of psychiatric diseases. However, the results generated by most neuroimaging studies, i.e., activated clusters/regions or functional connectivities between brain regions, frequently cannot be conveniently and systematically interpreted, rendering the biological meaning unclear. We describe a Brain Annotation Toolbox (BAT), a toolbox that helps to generate functional and genetic annotations for neuroimaging results. The toolbox can take data from brain regions identified with an atlas, or from brain regions identified as activated in tasks, or from functional connectivity links or networks of links. Then, the voxel-level functional description from the Neurosynth database and the gene expression profile from the Allen Brain Atlas are used to generate functional and genetic knowledge for such region-level data. Parametric (Fishers exact test) or non-parametric (permutation test) statistical tests are adopted to identify significantly related functional descriptors and genes for the neuroimaging results. The validity of the approach is demonstrated by showing that the functional and genetic annotations for specific brain regions are consistent with each other; and further the region by region functional similarity network and gene co-expression networks are highly correlated for many major brain atlases. One application of BAT is to help provide functional and genetic annotations for the newly discovered regions with unknown functions, e.g., the 97 new regions identified in the Human Connectome Project. Importantly too, this toolbox can help understand differences between patients with psychiatric disorders and controls, and this is demonstrated using data for schizophrenia and autism, for which the functional and genetic annotations for the neuroimaging data differences between patients and controls are consistent with each other and help with the interpretation of the differences.

neuroscience↗

bioRxiv2017

Whole exome sequencing study of colorectal cancer in Chinese population reveals novel prevalently mutated genes and decreased mutation frequency of APC and Wnt signaling in lymph node positive cancer

Colorectal cancer is the fifth prevalent cancer in China. Nevertheless, a large-scale characterization of Chinese colorectal cancer mutation spectrum has not been carried out. In this study, we have performed whole exome-sequencing analysis of 98 patients tumor samples with matched pairs of normal colon tissues using Illumina and Complete Genomics high-throughput sequencing platforms. Canonical CRC somatic gene mutations with high prevalence (>10%) have been verified, including TP53, APC, KRAS, SMAD4, FBXW7 and PIK3CA. PEG3 is identified as a novel frequently mutated gene (10.6%). APC and Wnt signaling exhibit significantly lower mutation frequencies than those in TCGA data. Analysis with clinical characteristics indicates that APC gene and Wnt signaling display lower mutation rate in lymph node positive cancer than negative ones, which are not observed in TCGA data. APC gene and Wnt signaling are considered as the key molecule and pathway for colorectal cancer initiation, and these findings greatly undermine their importance in tumor progression for Chinese patients. Taken together, the application of next-generation sequencing has led to the determination of novel somatic mutations and alternative disease mechanisms in colorectal cancer progression, which may be useful for understanding disease mechanism and personalizing treatment for Chinese patients.

genomics↗

bioRxiv2017

Gene family information facilitates variant interpretation and identification of disease-associated genes

Differentiating risk-conferring from benign missense variants, and therefore optimal calculation of gene-variant burden, represent a major challenge in particular for rare and genetic heterogeneous disorders. While orthologous gene conservation is commonly employed in variant annotation, approximately 80% of known disease-associated genes are paralogs and belong to gene families. It has not been thoroughly investigated how gene family information can be utilized for disease gene discovery and variant interpretation. We developed a paralog conservation score to empirically evaluate whether paralog conserved or nonconserved sites of in-human paralogs are important for protein function. Using this score, we demonstrate that disease-associated missense variants are significantly enriched at paralog conserved sites across all disease groups and disease inheritance models tested. Next, we assessed whether gene family information could assist in discovering novel disease-associated genes. We subsequently developed a gene family de novo enrichment framework that identified 43 exome-wide enriched gene families including 98 de novo variant carrying genes in more than 10k neurodevelopmental disorder patients. 33 gene family enriched genes represent novel candidate genes which are brain expressed and variant constrained in neurodevelopmental disorders.

genetics↗

bioRxiv2017

Biochemical Adaptations Of The Retina And Retinal Pigment Epithelium Support A Metabolic Ecosystem In The Vertebrate Eye

Here we report multiple lines of evidence for a comprehensive model for retinal energy metabolism. Metabolic flux, locations of key enzymes and our finding that glucose enters the neural retina almost entirely through photoreceptors support a conceptually new model for retinal metabolism. In this model, glucose from the choroidal blood supply passes through the retinal pigment epithelium to the retina where photoreceptors convert it to lactate. Photoreceptors then export the lactate as fuel for the retinal pigment epithelium and for neighboring Muller glial cells. A key feature of this model is that aerobic glycolysis in photoreceptors produces lactate to suppress glycolysis in the neighboring retinal pigment epithelium. That enhances the flow of glucose to the retina by minimizing consumption of glucose within the retinal pigment epithelium. This framework for metabolic relationships in retina provides new insights into the underlying causes of retinal disease, age-related vision loss and metabolism-based therapies.

biochemistry↗

bioRxiv2017

Rapid Whole Brain Imaging Of Neural Activities In Freely Behaving Larval Zebrafish

The internal brain dynamics that link sensation and action are arguably better studied during natural animal behaviors. Here we report on a novel volume imaging and 3D tracking technique that monitors whole brain neural activity in freely swimming larval zebrafish (Danio rerio). We demonstrated the capability of our system through functional imaging of neural activity during visually evoked and prey capture behaviors in larval zebrafish.

neuroscience↗

bioRxiv2017

Estimation of immune cell content in tumour tissue using single-cell RNA-seq data

As interactions between the immune system and tumour cells are governed by a complex network of cell-cell interactions, knowing the specific immune cell composition of a solid tumour may be essential to predict a patients response to immunotherapy. Here, we analyse in depth how to derive the cellular composition of a solid tumour from bulk gene expression data by mathematical deconvolution, using indication- and cell type-specific reference gene expression profiles (RGEPs) from tumour-derived single-cell RNA sequencing data. We demonstrate that tumour-derived RGEPs are essential for the successful deconvolution and that RGEPs from peripheral blood are insufficient. We distinguish nine major cell types as well as three T cell subtypes. As the ratios of CD4+, CD8+ and regulatory T cells have been shown to predict overall survival, we extended our analysis to include the estimation of prognostic ratios that may enable the application in a clinical setting. Using the tumour derived RGEPs, we can estimate, for the first time, the content of cancer associated fibroblasts, endothelial cells and the malignant cells in a patient sample by a deconvolution approach. In addition, improved tumour cell gene expression profiles can be obtained by this method by computationally removing contamination from non-malignant cells. Given the difficulty around sample preparation and storage to obtain high quality single-cell RNA-seq data in the clinical context, the presented method represents a computational solution to derive the cellular composition of a tissue sample.

bioinformatics↗