Biology subjects

Zhang, T.

Publications and source records attributed to Zhang, T..

32 records · Page 2Linked to original sources

bioRxiv2018

Programmable single and multiplex base-editing in Bombyx mori using RNA-guided cytidine deaminases

Standard genome editing tools (ZFN, TALEN and CRISPR/Cas9) edited genome depending on DNA double strand breaks (DSBs). A series of new CRISPR tools that convert cytidine to thymine (C to T) without the requirement for DNA double-strand breaks were developed recently, which have changed this status and have been quickly applied in a variety of organisms. Here, we demonstrate that CRISPR/Cas9-dependent base editor (BE3) converts C to T with a high frequency in the invertebrate Bombyx mori silkworm. Using BE3 as a knock-out tool, we inactivated exogenous and endogenous genes through base-editing-induced nonsense mutations with an efficiency of up to 66.2%. Furthermore, genome-scale analysis showed that 96.5% of B. mori genes have one or more targetable sites being knocked out by BE3 with a median of 11 sites per gene. The editing window of BE3 reached up to 13 bases (from C1 to C13 in the range of gRNA) in B. mori. Notably, up to 14 bases were substituted simultaneously in a single DNA molecule, with a low indel frequency of 0.6%, when 32 gRNAs were co-transfected. Collectively, our data show for the first time that RNA-guided cytidine deaminases are capable of programmable single and multiplex base-editing in an invertebrate model.

genetics↗

bioRxiv2018

Genome analysis and data sharing informs timing of molecular events in pancreatic neuroendocrine tumour

Neuroendocrine tumours (NETs) are rare, slow growing cancers that present in a diversity of tissues. To understand molecular underpinnings of gastrointestinal (GINET) and pancreatic NETs (PNETs), we profiled 45 tumours combining exome, RNA, and shallow whole genome sequencing, as well as fluorescent in situ hybridization. In addition to expected somatic mutations and copy number alterations, we found that PNETs contained a highly consistent copy neutral loss-of-heterozygosity (CN-LOH) profile affecting over half of the genome; a greater percentage than any cancer analyzed to date. Our data indicates that onset of extreme autozygosity may be progressive, associated with metastasis, and initially triggered by the loss of DAXX/ATRX, and subsequent biallelic loss of MEN1. We confirmed this molecular timing model using targeted clinical sequencing data from an additional 43 NETs made available by the AACR GENIE project. Against this background of CN-LOH, several chromosomal regions consistently retained heterozygosity, suggesting selection for crucial allele-specific components specific to PNET progression and potential new therapeutic targets.\n\nStatement of significanceWe have discovered that pancreatic neuroendocrine tumours contain a characteristic pattern of copy neutral loss-of-heterozygosity affecting the majority of the genome following mutations of MEN1 and ATRX/DAXX. Against this background of loss-of-heterozygosity, specific genomic regions are consistently retained and may therefore contain vulnerable therapeutic targets for pancreatic neuroendocrine tumours.

cancer biology↗

bioRxiv2017

Cell-type specific eQTL of primary melanocytes facilitates identification of melanoma susceptibility genes

Most expression quantitative trait loci (eQTL) studies to date have been performed in heterogeneous tissues as opposed to specific cell types. To better understand the cell-type specific regulatory landscape of human melanocytes, which give rise to melanoma but account for <5% of typical human skin biopsies, we performed an eQTL analysis in primary melanocyte cultures from 106 newborn males. We identified 597,335 cis-eQTL SNPs prior to LD-pruning and 4,997 eGenes (FDR<0.05), which are higher numbers than in any GTEx tissue type with a similar sample size. Melanocyte eQTLs differed considerably from those identified in the 44 GTEx tissues, including skin. Over a third of melanocyte eGenes, including key genes in melanin synthesis pathways, were not observed to be eGenes in two types of GTEx skin tissues or TCGA melanoma samples. The melanocyte dataset also identified cell-type specific trans-eQTLs with a pigmentation-associated SNP for four genes, likely through its cis-regulation of IRF4, encoding a transcription factor implicated in human pigmentation phenotypes. Melanocyte eQTLs are enriched in cis-regulatory signatures found in melanocytes as well as melanoma-associated variants identified through genome-wide association studies (GWAS). Co-localization of melanoma GWAS variants and eQTLs from melanocyte and skin eQTL datasets identified candidate melanoma susceptibility genes for six known GWAS loci including unique genes identified by the melanocyte dataset. Further, a transcriptome-wide association study using published melanoma GWAS data uncovered four new loci, where imputed expression levels of five genes (ZFP90, HEBP1, MSC, CBWD1, and RP11-383H13.1) were associated with melanoma at genome-wide significant P-values. Our data highlight the utility of lineage-specific eQTL resources for annotating GWAS findings and present a robust database for genomic research of melanoma risk and melanocyte biology.

genetics↗

bioRxiv2017

Real-Time Tracking of Selective Auditory Attention from M/EEG: A Bayesian Filtering Approach

Humans are able to identify and track a target speaker amid a cacophony of acoustic interference, which is often referred to as the cocktail party phenomenon. Results from several decades of studying this phenomenon have culminated in recent years in various promising attempts to decode the attentional state of a listener in a competing-speaker environment from non-invasive neuroimaging recordings such as magnetoencephalography (MEG) and electroencephalography (EEG). To this end, most existing approaches compute correlation-based measures by either regressing the features of each speech stream to the M/EEG channels (the decoding approach) or vice versa (the encoding approach). These procedures operate in an offline fashion, i.e., require the entire duration of the experiment and multiple trials to provide robust results. Therefore, they cannot be used in emerging applications such as smart hearing aid devices, where a single trial must be used in real-time to decode the attentional state. In this paper, we close this gap by developing an algorithmic pipeline for real-time decoding of the attentional state. Our proposed framework consists of three main modules: 1) Real-time and robust estimation of encoding or decoding coefficients, achieved by sparse adaptive filtering, 2) Extracting reliable markers of the attentional state, and thereby generalizing the widely-used correlation-based measures thereof, and 3) Devising a near real-time state-space estimator that translates the noisy and variable attention markers to robust and reliable estimates of the attentional state with minimal delay. Our proposed algorithms integrate various techniques including forgetting factor-based adaptive filtering, {ell}1-regularization, forward-backward splitting algorithms, fixed-lag smoothing, and Expectation Maximization. We validate the performance of our proposed framework using comprehensive simulations as well as application to experimentally acquired M/EEG data. Our results reveal that the proposed real-time algorithms perform nearly as accurate as the existing state-of-the-art offline techniques, while providing a significant degree of adaptivity, statistical robustness, and computational savings.

neuroscience↗

bioRxiv2017

AQMM: Enabling Absolute Quantification of Metagenome and Metatranscriptome

Metatranscriptome has become increasingly important along with the application of next generation sequencing in the studies of microbial functional gene activity in environmental samples. However, the quantification of target active gene is hindered by the current relative quantification methods, especially when tracking the sharp environmental change. Great needs are here for an easy-to-perform method to obtain the absolute quantification. By borrowing information from the parallel metagenome, an absolute quantification method for both metagenomic and metatranscriptomic data to per gene/cell/volume/gram level was developed. The effectiveness of AQMM was validated by simulated experiments and was demonstrated with a real experimental design of comparing activated sludge with and without foaming. Our method provides a novel bioinformatic approach to fast and accurately conduct absolute quantification of metagenome and metatranscriptome in environmental samples. The AQMM can be accessed from https://github.com/biofuture/aqmm.

bioinformatics↗

bioRxiv2017

Deficit in parietal memory network underlies auditory hallucination: a longitudinal study

Auditory hallucination is a prominent and common symptom in schizophrenia. Previous neuroimaging studies have yielded mixed results of its brain network deficits. We proposed a novel hypothesis that parietal memory network, centered at the precuneus, plays a critical role in auditory hallucination. This network is adjacent and partially overlaps with the default mode network, and has been associated with brain function of familiarity labelling in memory processing. Using a longitudinal design and a large cohort of first-episode, drug-naive schizophrenia patients, we examined this hypothesis and further investigated whether the functional connectivity patterns of the parietal memory network can serve as a neuroimaging marker for auditory hallucination and help to predict future treatment effects. Resting-state scans from 59 first-episode drug-naive schizophrenic patients (27 with and 32 without hallucination) and 53 healthy control subjects were acquired at the baseline test, and 56 of them were scanned again after two months. Functional connectivity strength within the parietal memory network and between this network and memory hubs was across the three groups at baseline and follow-up scans. Results showed that decreased functional connectivity strength within the parietal memory network was specific to the auditory hallucination group (p = 0.009, compare to the healthy subjects; p = 0.029, compare to the patients without hallucination), with the precuneus representing the largest group difference. The intra-network connectivity strength of the precuneus negatively correlated with the severity of hallucination at the baseline scan (r = -0.437, p = 0.029), and it was significantly increased after two-month medication (p = 0.039). Logistic regression analysis and crossvalidation test demonstrated that the functional connectivity strength of the precuneus and precuneus-hippocampus connectivity could differentiate patients with or without auditory hallucination with a sensitivity of 0.750 and a specificity of 0.708. Moreover, crossvalidation test showed that these imaging features at the baseline scan well predicted the extents of positive symptom improvement in the hallucination group after the two-month medication (R2 = 0.433, p = 0.022). Our results provide evidence for a critical role of the parietal memory network underlying auditory hallucination, and further propose a novel neuroimaging marker for identifying patients, accessing severity, and prognosis of treatment effect for auditory hallucination.\n\nAbbreviations

neuroscience↗

bioRxiv2017

Fundamentals of injection molding for microfluidic cell-based assays

Microscale cell-based assays have demonstrated unique capabilities in reproducing important cellular behaviors for diagnostics and basic biological research. As these assays move beyond the prototyping stage and into biological and clinical research environments, there is a need to produce microscale culture platforms more rapidly, cost-effectively, and reproducibly. Rapid injection molding is poised to meet this need as it enables some of the benefits of traditional high volume injection molding at a fraction of the cost. However, rapid injection molding has limitations due to the material and methods used for mold fabrication. Here, we characterize advantages and limitations of rapid injection molding for microfluidic device fabrication through measurement of key features for cell culture applications including channel geometry, feature consistency, floor thickness, and surface polishing. We demonstrate phase contrast and fluorescence imaging of cells grown in rapid injection molded devices and provide design recommendations to successfully utilize rapid injection molding methods for microscale cell-based assay development in academic laboratory settings.

bioengineering↗

bioRxiv2017

DNA 5-Hydroxymethylcytosines from Cell-free Circulating DNA as Diagnostic Biomarkers for Human Cancers

DNA modifications such as 5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC) are epigenetic marks known to affect global gene expression in mammals(1, 2). Given their prevalence in the human genome, close correlation with gene expression, and high chemical stability, these DNA epigenetic marks could serve as ideal biomarkers for cancer diagnosis. Taking advantage of a highly sensitive and selective chemical labeling technology(3), we report here genome-wide 5hmC profiling in circulating cell-free DNA (cfDNA) and in genomic DNA of paired tumor/adjacent tissues collected from a cohort of 90 healthy individuals and 260 patients recently diagnosed with colorectal, gastric, pancreatic, liver, or thyroid cancer. 5hmC was mainly distributed in transcriptionally active regions coincident with open chromatin and permissive histone modifications. Robust cancer-associated 5hmC signatures in cfDNA were identified with specificity for different cancers. 5hmC-based biomarkers of circulating cfDNA demonstrated highly accurate predictive value for patients with colorectal and gastric cancers versus healthy controls, superior to conventional biomarkers, and comparable to 5hmC biomarkers from tissue biopsies. This new strategy could lead to the development of effective blood-based, minimally-invasive cancer diagnosis and prognosis approaches.

cancer biology↗

bioRxiv2017

The pomegranate (Punica granatum L.) genome provides insights into fruit quality and ovule developmental biology

Pomegranate (Punica granatum L.) with an uncertain taxonomic status has an ancient cultivation history, and has become an emerging fruit due to its attractive features such as the bright red appearance and the high abundance of medicinally valuable ellagitannin-based compounds in its peel and aril. However, the absence of genomic resources has restricted further elucidating genetics and evolution of these interesting traits. Here we report a 274-Mb high-quality draft pomegranate genome sequence, which covers approximately 81.5% of the estimated 336 Mb genome, consists of 2,177 scaffolds with an N50 size of 1.7 Mb, and contains 30,903 genes. Phylogenomic analysis supported that pomegranate belongs to the Lythraceae family rather than the monogeneric Punicaceae family, and comparative analyses showed that pomegranate and Eucalyptus grandis shares the paleotetraploidy event. Integrated genomic and transcriptomic analyses provided insights into the molecular mechanisms underlying the biosynthesis of ellagitannin-based compounds, the color formation in both peels and arils during pomegranate fruit development, and the unique ovule development processes that are characteristic of pomegranate. This genome sequence represents the first reference in Lythraceae, providing an important resource to expand our understanding of some unique biological processes and to facilitate both comparative biology studies and crop breeding.

genomics↗

bioRxiv2017

Discovery and reporting of clinically-relevant germline variants in advanced cancer patients assessed using whole-exome sequencing

PurposeIn precision cancer care, WES-based analysis of tumor-normal samples helps reveal somatic alterations but can also identify cancer-associated germline variants important for disease surveillance, treatment choice and cancer prevention. WES can also identify germline secondary findings impacting risk of cardiac, neurodegenerative or metabolic diseases. In patients with advanced cancer, the frequency of reportable secondary findings encountered with WES is not well defined.\n\nMethodsTo address this question, we analyzed a cohort of 343 patients with advanced, metastatic cancer for whom we have performed tumor and germline WES interrogating more than 21,000 genes using a CLIA/CLEP approved assay.\n\nResults17% of patients in our cohort have one or more reportable germline variants, including patients with pathogenic variants in the BRCA1 and BRCA2 genes. The frequency of non-cancer clinically relevant germline variants (8.8%) was within the range of two control non-cancer cohorts (11.0% and 6.5%). The frequency of variants in cancer-associated genes was significantly higher (p<0.0005) in our advanced cancer cohort (8.2%) compared to control cohorts (2.7% and 3.8%). More than 50% of patients with reportable germline cancer variants had a family history of cancer.\n\nConclusionthese results stress the importance of returning germline results found during somatic genomic tumor testing.

genomics↗

bioRxiv2017

BMP signaling orchestrates a transcriptional network to control the fate of mesenchymal stem cells (MSCs)

Signaling pathways are used reiteratively in different developmental processes yet produce distinct cell fates through activating specific downstream transcription factors. In this study, we used tooth root development as a model to investigate how the BMP signaling pathway regulates specific downstream transcriptional complexes to direct the fate determination of multipotent mesenchymal stem cells (MSCs). We first identified the MSC population supporting mouse molar root growth as Gli1+ cells. Using a Gli1-mediated transgenic animal model, our results provide the first in vivo evidence that BMP signaling activity is required for the odontogenic differentiation of MSCs. Specifically, we identified transcription factors that are downstream of BMP signaling and are expressed in a spatially restricted pattern consistent with their potential involvement in determining distinct cellular identities within the dental mesenchyme. Finally, we found that overactivation of one key transcription factor, Klf4, associated with the odontogenic region, promotes odontogenic differentiation of MSCs. Collectively, our results demonstrate the functional significance of BMP signaling in regulating the fate of MSCs during root development and shed light on how BMP signaling can achieve functional specificity in regulating diverse organ development.\n\nSummary StatementBMP signaling activity is required for the lineage commitment of MSCs and transcription factors downstream of BMP signaling may determine distinct cellular identities within the dental mesenchyme.

developmental biology↗

bioRxiv2017

Efficient and flexible strategies for gene cloning and vector construction using overlap PCR

Gene cloning and vector construction are basic technologies in modern molecular biology for gene functional study. Here, we present flexible and efficient strategies for gene cloning and vector construction using overlap PCR. We firstly cloned the open reading frames (ORFs) of the porcine MSTN, chicken OVA and human -glucosidase genes by overlap PCR-based assembling of their exons, which could be amplified with genomic DNAs as the templates without RNA extraction and RT-PCR reaction. Secondly, we generated additionally three designed functional cassettes by overlap PCR-based assembling of different DNA elements, which facilitated the construction their expression vectors greatly. Moreover, we further developed an interesting overlap-circled PCR method for fast plasmid vector construction without any cutting and ligating procedure. These advanced applications of overlap PCR provide useful alternative tools for gene cloning and vector construction.

molecular biology↗

bioRxiv2017

Gene expression profiling reveals U1 snRNA regulates cancer gene expression

U1 small nuclear RNA (U1 snRNA), as one of the most abundant noncoding RNA in eukaryotic cells plays an important role in splicing of pre-mRNAs. Compared to other studies which have focused on the primary function of U1 snRNA and the neurodegenerative diseases caused by the abnormalities of U1 snRNA, this study is to investigate how the U1 snRNA over-expression affects the expression of genes on a genome-wide scale. In this study, we built a model of U1 snRNA over-expression in a rat cell line. By comparing the gene expression profiles of U1 snRNA over-expressed cells with those of their controls using the microarray experiments, 916 genes or loci were identified significantly differentially expressed. These 595 up-regulated genes and 321 down-regulated genes were further analyzed using the annotations from the GO terms and the KEGG database. As a result, three of 12 enriched pathways are well-known cancer pathways, while nine of them were associated to cancers in previous studies. The further analysis of 73 genes involved in 12 pathways suggests that U1 snRNA regulates cancer gene expression. The microarray data with ID GSE84304 is available in the NCBI GEO database.

cancer biology↗

bioRxiv2016

EthSEQ: ethnicity annotation from whole exome sequencing data

Whole exome sequencing (WES) is widely utilized both in translational cancer genomics studies and in the setting of precision medicine. Stratification of individual's ethnicity is fundamental for the correct interpretation of personal genomic variation impact. We implemented EthSEQ to provide reliable and rapid ethnicity annotation from whole exome sequencing individual's data and validated it on 1,000 Genome Project and TCGA data demonstrating high precision (>99%). EthSEQ can be integrated into any WES based processing pipeline and exploits multi-core capabilities. Source code, manual and other data is available at http://demichelislab.unitn.it/EthSEQ.

bioinformatics↗