Biology subjects

Chen, L.

Publications and source records attributed to Chen, L..

At least 1,027 records · Page 57Linked to original sources

bioRxiv2018

Bisulfite-free, Base-resolution, and Quantitative Sequencing of Cytosine Modifications

The deamination of unmodified cytosine to uracil by treatment with bisulfite has for decades been the gold standard for sequencing epigenetic DNA modifications including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). However, this harsh chemical reaction degrades the majority of the DNA and generates sequencing libraries with low complexity. Here, we present a novel bisulfite-free and base-resolution sequencing method, TET Assisted Pic-borane Sequencing (TAPS), for detection of 5mC and 5hmC. TAPS relies on mild reactions, detects modifications directly without affecting unmodified cytosines and can be adopted to detect other cytosine modifications. Compared with bisulfite sequencing, TAPS results in higher mapping rates, more even coverage and lower sequencing costs, enabling higher quality, more comprehensive and cheaper methylome analyses.\n\nOne Sentence SummaryA bisulfite-free and base-resolution method to directly sequence epigenetically modified cytosine.

genomics↗

bioRxiv2018

IL-33 regulates gene expression in intestinal epithelial cells independently of its nuclear localization

IL-33 is a cytokine found in the extracellular space (mature IL-33) or in the cell nucleus (full-length IL-33). Nuclear accumulation of IL-33 has been reported in intestinal epithelial cells (IEC) during intestinal inflammation and cancer, but a functional role for this nuclear form remains unclear. To study the role of nuclear IL-33 in IEC, we generated transgenic mice expressing full-length IL-33 in the intestinal epithelium (Vfl33 mice). Expression of full-length IL-33 in the epithelium resulted in accumulation of IL-33 protein in the nucleus and secretion of IL-33. Over-expression of full-length IL-33 by IEC did not promote gut inflammation, but induced expression of genes in the IEC and lamina propria lymphocytes (LPL) that correlated negatively with genes expressed in inflammatory bowel diseases (IBD). Because the IL-33 receptor ST2 is expressed by IEC, there was the potential that both the mature and full-length forms could mediate this effect. To specifically interrogate the transcriptional role of nuclear IL-33, we intercrossed the Vfl33 mice with ST2-deficient mice. ST2 deficiency completely abrogated the transcriptional effects elicited by IL-33 expression, suggesting that the transcriptional effects of IL-33 on IEC are mediated by its mature, not its nuclear form.\n\nHighlightsO_LIExpression of full-length IL-33 in the epithelium resulted in accumulation of IL-33 protein in the nucleus and secretion of IL-33.\nC_LIO_LIFull-length IL-33 induced differential gene expression in IEC and LPL that was negatively associated with intestinal inflammatory diseases\nC_LIO_LIIL-33 regulated gene expression in IEC via its extracellular (mature) form not via its nuclearform.\nC_LI

immunology↗

bioRxiv2018

Implementation of CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing

In contrast to genome editing that introduces genetic changes at DNA level, disrupting or editing genes transcripts provides a distinctive approach to perturb a genetic system, offering benefits complementary to classic genetic approaches. To develop a new toolset for manipulation of RNA, we first implemented a member of type VI CRISPR systems, Cas13a from Leptotrichia shahii (LshCas13a) in Schizosaccharomyces pombe, an important model organism employed by biologists to study key cellular mechanisms conserved from yeast to humans. While it was shown to knock down targeted endogenous genes transcripts, differently from previous studies in E. coli, no collateral cleavage of other non-specific RNA by activated Cas13a-crRNA complex was detected in fission yeast. Second, we engineered a RNA-editing system by tethering an inactive form of LshCas13a (dCas13) to the catalytic domain of human Adenosine Deaminase Act on RNA 2 (hADAR2d), which was shown to be programmable with crRNA to target messenger RNAs and precisely edit specific nucleotide residues. We optimized the system parameters using a dual-florescence reporter and demonstrated its utility in editing of randomly selected endogenous genes transcripts. Our engineered RNA-editing system enables a new toolset for transcriptomic manipulation that is widely applicable in basic genetic and biotechnological research.

synthetic biology↗

bioRxiv2018

Efficient genome editing of Magnetospirillum magneticum AMB-1 by CRISPR-Cas9 system for analyzing magnetotactic behavior

Magnetotactic bacteria are a diverse group of microorganisms with the ability to use geomagnetic fields for direction sensing. This magnetotactic behavior can help microorganisms move towards favorable habitats for optimal growth and reproduction. Highly efficient genomic editing is very useful for a comprehensive understanding of the magnetotactic mechanism at the molecular level. In this study, we adapted an engineered CRISPR-cas9 system for efficient inactivation of gene in a widely used magnetotactic bacteria model strain, Magnetospirillum magneticum AMB-1. By combining an engineered nuclease-deficient Cas9 and single-guide RNA, a CRISPR interference system was successfully developed to silence amb0994 expression. More importantly, we succeeded in the construction of a single amb0994 gene deletion mutant using CRISPR-Cas9 with approximate 60-fold high efficiency compared to classical homology double-crossing replacement procedure. This mutant synthesized normally the magnetosomes, but reacted quicker and with less time than the wild-type strain to abrupt magnetic field reversals. A dynamics simulation by modeling M. magneticum AMB-1 cell as an ellipsoid showed that the difference of the motions between wild and{Delta} amb0994 is due to flagellar influence. The behavior observation being consistent with dynamics simulation indicated that Amb0994 is involved in the cellular response to magnetic torque change via controlling flagella. Besides the contribution to a better understanding of the magnetotaxis mechanism, this study demonstrates the CRISPR system as a useful genetic toolbox for high-efficiency genome editing in magnetotactic bacteria.

microbiology↗

bioRxiv2018

Establishment of signaling interactions with cellular resolution for every cell cycle of embryogenesis

Intercellular signaling interaction plays a key role in breaking fate symmetry during animal development. Identification of the signaling interaction at cellular resolution is technically challenging, especially in a developing embryo. Here we develop a platform that allows automated inference and validation of signaling interaction for every cell cycle of C. elegans embryogenesis. This is achieved by generation of a systems-level cell contact map that consists of 1,114 highly confident intercellular contacts by modeling analysis and is validated through cell membrane labeling coupled with cell lineage analysis. We apply the map to identify cell pairs between which a Notch signaling interaction takes place. By generating expression patterns for two ligands and two receptors of Notch signaling pathway with cellular resolution using automated expression profiling technique, we are able to refine existing and identify novel Notch interactions during C. elegans embryogenesis. Targeted cell ablation followed by cell lineage analysis demonstrates the roles of signaling interactions over cell division in breaking fate symmetry. We finally develop a website that allows online access to the cell-cell contact map for mapping of other signaling interaction in the community. The platform can be adapted to establish cellular interaction from any other signaling pathways.

cell biology↗

bioRxiv2018

Mathematical modelling of molecular heterogeneity identifies novel markers and subpopulations in complex tumors

Intratumor heterogeneity, as both a major confounding factor and an underexploited information source, is widely implicated as a key driver of drug resistance. While a handful of reports have demonstrated the potential of supervised methods to deconvolute intratumor heterogeneity, these approaches require a priori information on the marker genes or composition of known subpopulations. To address the critical problem of the absence of validated marker genes for many (including novel) subpopulations, we developed convex analysis of mixtures (CAM), a fully unsupervised deconvolution method, for identifying marker genes and subpopulations directly from original mixed molecular expressions.

bioinformatics↗

bioRxiv2018

Ligand binding and conformational changes of SUR1 subunit in pancreatic ATP-sensitive potassium channels

ATP-sensitive potassium channels (KATP) are energy sensors on the plasma membrane. By sensing the intracellular ADP/ATP ratio of {beta}-cells, pancreatic KATP channels control insulin release and regulate metabolism at the whole body level. They are implicated in many metabolic disorders and diseases and are therefore important drug targets. Here, we present three structures of pancreatic KATP channels solved by cryo-electron microscopy (cryo-EM), at resolutions ranging from 4.1 to 4.5 [A]. These structures depict the binding site of the antidiabetic drug glibenclamide, indicate how Kir6.2 N-terminus participates the coupling between the peripheral SUR1 subunit and the central Kir6.2 channel, reveal the binding mode of activating nucleotides, and suggest the mechanism of how Mg-ADP binding on nucleotide binding domains (NBDs) drives a conformational change of the SUR1 subunit.

biophysics↗

bioRxiv2018

Structure of the receptor-activated human TRPC6 ion channel

TRPC6 is a receptor-activated nonselective cation channel that belongs to the family of canonical transient receptor potential (TRPC) channels. It is activated by diacylglycerol, a lipid second messenger. TRPC6 is involved in many physiological processes and implicated in human genetic diseases. Here we present the structure of human TRPC6 homotetramer in complex with a newly identified high affinity inhibitor BTDM solved by single-particle cryo-electron microscopy to 3.8 [A] resolution. The structure shows a two-layer architecture, in which the bell-shaped cytosolic layer holds the transmembrane layer. Extensive inter-subunit interactions of cytosolic domain, including N terminal ankyrin repeats and C terminal coiled-coil, contribute to the tetramer assembly. The high affinity inhibitor BTDM wedges between S5-S6 pore domain and voltage sensor-like domain to inhibit channel opening. Our structure uncovers the molecular architecture of TRPC channels and provides a structural basis for understanding the mechanism of these channels.

biophysics↗

bioRxiv2018

MiR-505-3p, a New Repressor of Puberty Onset in Female Mice

Puberty onset is a complex trait regulated by multiple genetic and environmental factors. In this study, we narrowed a puberty-related QTL on chromosome X in female mice to a 1.7 Mb region and deduced that miR-505-3p was the functional gene.\n\nIn a GT1-7 cell line with stable overexpression of miR-505-3p (pGT1-7), both ribosomes and ribosome biogenesis pathways were affected, and the expression of some puberty-related genes was down-regulated. The amount of mRNA and protein products of the Srsf1 gene decreased by 50 percent, and the expression of puberty-related genes was rescued by the overexpression of Srsf1. With the down regulation of Srsf1 expression through shRNA, the mRNA accumulation of puberty-related genes decreased simultaneously in the GT1-7 cell line. The results of RIP-seq showed that SF2, the protein of the Srsf1 gene, primarily bound ribosome protein (RP) mRNAs in GT1-7 cells.\n\nmiR-505-3p knockout female mice showed earlier vaginal opening, higher serum gonadotrophin levels and higher expression of puberty-related and Srsf1 genes in the hypothalamus than their wild-type littermates. B6 female mice with ectopic expression of miR-505-3p in the hypothalamus showed significant growth retardance and later VO than wild types.\n\nThese results suggest that miR-505-3p may regulate puberty onset via the Srsf1 gene and RP expression, which reveals a new regulatory pathway in mammalian puberty onset involving microRNA, SF2 and ribosome proteins.\n\nAuthor summaryIn this study, we identified miR-505-3p in a puberty-related QTL on the X chromosome as a female puberty onset repressor using a positional cloning strategy.\n\nGT1-7 cell lines stably overexpressing miR-505-3p (pGT1-7) showed Kiss1 and GnRH down-regulation. We also identified Srsf1 as the functional target gene of miR-505-3p in GT1-7 cells. Compared to wild-type mice, miR-505-3p knockout female mice showed puberty onset four days sooner, along with the overexpression of miR-505-3p in the hypothalamus 2 days later. Thus, miR-505-3p is a new repressor of puberty onset in female mice.

genetics↗

bioRxiv2018

Physiological Significance of R-fMRI Indices in Detecting Structural Brain Lesions

Resting-state functional MRI (R-fMRI) research has recently entered the era of \"big data\", however, few studies have provided a rigorous validation of the physiological underpinnings of R-fMRI indices. Although studies have reported that various neuropsychiatric disorders exhibit abnormalities in R-fMRI measures, these \"biomarkers\" have not been validated in differentiating structural lesions (brain tumors) as a concept proof. We enrolled 60 patients with intracranial tumors located in the unilateral cranial cavity and 60 matched normal controls to test whether R-fMRI indices can differentiate tumors, which represents a prerequisite for adapting such indices as biomarkers for neuropsychiatric disorders. Common R-fMRI indices of tumors and their counterpart control regions, which were defined as the contralateral normal areas (for amplitude of low frequency fluctuations (ALFF), fractional ALFF (fALFF), regional homogeneity (ReHo) and degree centrality (DC)) and ipsilateral regions surrounding the tumors (for voxel-mirrored homotopic connectivity (VMHC)), were comprehensively assessed. According to paired t-tests with a Bonferroni correction, only ALFF (both with and without Z-standardization) and VMHC (Fishers r-to-z transformed) could successfully differentiate substantial tumors from their counterpart normal regions in patients. And DC was not able to differentiate tumor from normal unless employed Z-standardization. To validate the lower power in the between-subject design than in the within-subject design, each metric was calculated in a matched control group, and two-sample t-tests were used to compare the patient tumors and the normal controls at the same area. Only ALFF (and that with Z-standardization) along with VMHC succeeded in differentiating significant differences between tumors and the sham tumors areas of normal controls. This study tested the premise of R-fMRI biomarkers for differentiating lesions, and brings a new understanding to physical significance of the Z-standardization.

neuroscience↗

bioRxiv2018

Diet Modifies Colonic Microbiota and CD4+ T cell Repertoire to Trigger Flares in a Novel Model of Colitis Induced by IL-23

A wealth of experimental data points to immunological and environmental factors in the pathogenesis of inflammatory bowel disease (IBD). Here we study the role of IL-23, the microbiome, and the diet in the development of colitis. To promote IL-23 expression in vivo, we generated a mouse model in which IL-23 was conditionally expressed by CX3CR1+ myeloid cells, upon cyclic administration of tamoxifen in a specific diet (diet 2019). IL-23 expression induced an intestinal inflammatory disease that resembled ulcerative colitis in humans with cycles of acute disease and remission. The relapses were caused by the diet switch from the conventional diet used in our facility (diet 5053) to the diet 2019, and were not dependent on tamoxifen after the first cycle. The switch in the diet modified the microbiota, but did not alter the levels of IL-23. Colitis induction depended on the microbiota and required CD4 T lymphocytes. Colitis-inducing CD4+ T cells were found in the mesenteric lymph node and large intestine during remission and were able to trigger disease when transferred to lymphopenic mice, but only upon diet modification. The CD4 TCR repertoire in the diseased recipient Rag-/- mice had reduced diversity associated with the expansion of dominant T cell clones. These findings reveal a critical role for IL-23 in generation of a CD4+ T cell population in mice that is sensitive to a modification of intestinal bacterial flora subsequent to a dietary manipulation. Dietary changes occurring in the context of altered IL-23 expression may contribute to the onset and progression of IBD.

immunology↗

bioRxiv2018

Loss of SDHB reprograms energy metabolisms and inhibits high fat diet induced metabolic syndromes

Mitochondrial respiratory complex II utilizes succinate, key substrate of the Krebs cycle, for oxidative phosphorylation, which is essential for glucose metabolism. Mutations of complex II cause cancers and mitochondrial diseases, raising a critical question of the (patho-)physiological functions. To address the fundamental role of complex II in systemic energy metabolism, we specifically knockout SDHB in mice liver, a key complex II subunit that tethers the catalytic SDHA subunit and transfers the electrons to ubiquinone, and found that SHDB deficiency abolishes the assembly of complex II without affecting other respiration complexes while largely retaining SDHA stability. SHDB ablation reprograms energy metabolism and hyperactivates the glycolysis, Krebs cycle and {beta}-oxidation pathways, leading to catastrophic energy deficit and early death. Strikingly, sucrose supplementation or high fat diet resumes both glucose and lipid metabolism and prevent early death. Also, SDHB deficient mice are completely resistant to high fat diet induced obesity. Our findings reveal that the unanticipated role of complex II orchestrating both lipid and glucose metabolisms, and suggest that SDHB is an ideal therapeutic target for combating obesity.

molecular biology↗

bioRxiv2018

Tetracyclines Modify Translation By Targeting Key Human rRNA Substructures.

Apart from their antimicrobial properties, tetracyclines demonstrate clinically validated effects in the amelioration of pathological inflammation and human cancer. Delineation of the target(s) and mechanism(s) responsible for these effects, however, has remained elusive. Here, employing quantitative mass spectrometry-based proteomics, we identified human 80S ribosomes as targets of the tetracyclines Col-3 and doxycycline. We then developed in cell click selective crosslinking with RNA sequence profiling (icCL-Seq) to map binding sites for these tetracyclines on key human rRNA substructures at nucleotide resolution. Importantly, we found that structurally and phenotypically variant tetracycline analogs could chemically discriminate these rRNA binding sites. We also found that tetracyclines both subtly modify human ribosomal translation and selectively activate the cellular integrated stress response (ISR). Together, the data reveal that targeting of specific rRNA substructures, activation of the ISR, and inhibition of translation are correlated with the anti-proliferative properties of tetracyclines in human cancer cell lines.

cell biology↗

bioRxiv2018

Quantifying Waddington’s epigenetic landscape: a comparison of single-cell potency measures

Over 60 years ago Waddington proposed an epigenetic landscape model of cellular differentiation, whereby cell-fate transitions are modelled as canalization events, with stable cell states occupying the basins or attractor states1, 2. A key ingredient of this landscape is the energy potential, or height3, which correlates with cell-potency. To date, very few explicit biophysical models for estimating single-cell potency have been proposed. Using 9 independent experiments, encompassing over 6,600 high-quality single-cell RNA-Seq profiles, we here demonstrate that single-cell potency can be approximated as the graph entropy of a Markov Chain process on a model signaling network. Our analysis highlights that other proposed single-cell potency measures are not robust, whilst also revealing that integration with orthogonal systems-level information improves potency estimates. Thus, this study provides a foundation for an improved systems-level understanding of single-cell potency, which may have profound implications for the discovery of novel stem-and progenitor cell phenotypes.

bioinformatics↗

bioRxiv2018

Genome-wide identification of CDC34 that stabilizes EGFR and promotes lung carcinogenesis

To systematically identify ubiquitin pathway genes that are critical to lung carcinogenesis, we used a genome-wide silencing method in this study to knockdown 696 genes in non-small cell lung cancer (NSCLC) cells. We identified 31 candidates that were required for cell proliferation in two NSCLC lines, among which the E2 ubiquitin conjugase CDC34 represented the most significant one. CDC34 was elevated in tumor tissues in 67 of 102 (65.7%) NSCLCs, and smokers had higher CDC34 than nonsmokers. The expression of CDC34 was inversely associated with overall survival of the patients. Forced expression of CDC34 promoted, whereas knockdown of CDC34 inhibited lung cancer in vitro and in vivo. CDC34 bound EGFR and competed with E3 ligase c-Cbl to inhibit the polyubiquitination and subsequent degradation of EGFR. In EGFR-L858R and EGFR-T790M/Del(exon 19)-driven lung cancer in mice, knockdown of CDC34 by lentivirus mediated transfection of short hairpin RNA significantly inhibited tumor formation. These results demonstrate that an E2 enzyme is capable of competing with E3 ligase to inhibit ubiquitination and subsequent degradation of oncoprotein substrate, and CDC34 represents an attractive therapeutic target for NSCLCs with or without drug-resistant EGFR mutations.

cancer biology↗

bioRxiv2018

Water stress and CO2 concentration interactions affect carbon isotope signatures of leaf and phloem organic matter

Investigation of {delta}13C of leaf and twig phloem water-soluble organic material (WSOM) is a promising approach for analysis of the effects of environmental factors on plant performance. In this study, orthogonal treatments of three CO2 concentrations (Ca) x five soil water contents (SWC) were conducted using Platycladus orientalis saplings to investigate the interaction of water stress and CO2 concentration on {delta}13C of leaf and twig phloem WSOM. Under the lowest SWC, the {delta}13C of leaf and twig phloem WSOM had the most positive values at any Ca and their values decreased as Ca increased. However, at improved soil water conditions, the greatest values of {delta}13C of leaf and twig phloem WSOM were mostly observed at C600. In addition, a more significant relationship between SWC and {delta}13C of twig phloem WSOM than that between SWC and {delta}13C of leaf WSOM demonstrated that {delta}13C of twig phloem WSOM is a more sensitive indicator of SWC. Twig phloem WSOM was generally 13C-depleted compared with leaf WSOM for potential post-photosynthetic fractionation, and the 13C discrimination from leaves to twig phloem was insensitive to the interaction between SWC and Ca. Clearly, interacting effects play a more important role in photosynthetic fractionation than in post-photosynthetic fractionation.\n\nHighlightThe {delta}13C of leaf and twig phloem WSOM exhibited the most positive values at C400x35%-45% FC.\n\nPost-photosynthetic fractionation from leaf to twig was not be impacted by the interacting effects.

ecology↗

bioRxiv2018

A compromised gsdf signaling leads to gamatogenesis confusion and subfertility in medaka

Summary statementGsdf signals trigger the gamatogenesis, alter the somatic expression of Fsh/Lh receptors and brain type aromatase in medaka brain and gonad.\n\nAbstractGonadal soma-derived factor (gsdf) and anti-Mullerian hormone (amh) are somatic male determinants in several species of teleosts, although the mechanisms by which they trigger the indifferent germ cells into the male pathway remain unknown. This study aimed to decipher the roles of gsdf/amh in directing the sexual fate of germ cells using medaka as a model. Transgenic lines (TgcryG) that restrictively and persistently express a Gsdf-Gfp fusion protein in the lens and the hypothalamus-pituitary-gonad (HPG) axis, were generated under the control of a mouse {gamma}F-crystallin promoter. A high frequency (44.4%) of XX male sex reversals was obtained in TgcryG lines, indicating that signals of gsdf-expressing cells in HPG were enough for the spermatogenesis activation in the genetic females. Furthermore, all TgcryG XY individuals with endogenous gsdf depletion (named Sissy) displayed intersex (100%) with enlarged ovotestis in contrast to a giant ovary developed in XY gsdf deficiency. The heterogeneous expression of gsdf led to the confusion of gamatogenesis and ovotestis development, similar to some hotei (amhr2) mutants, suggests that the signaling balance of gsdf/amh is essential for proper gamatogenesis, maintaining sex steroid production and gonadotropin secretion, which are evolutionarily conserved across phyla.

evolutionary biology↗

bioRxiv2017

Comparative analysis of neutrophil and monocyte epigenomes

Neutrophils and monocytes provide a first line of defense against infections as part of the innate immune system. Here we report the integrated analysis of transcriptomic and epigenetic landscapes for circulating monocytes and neutrophils with the aim to enable downstream interpretation and functional validation of key regulatory elements in health and disease. We collected RNA-seq data, ChIP-seq of six histone modifications and of DNA methylation by bisulfite sequencing at base pair resolution from up to 6 individuals per cell type. Chromatin segmentation analyses suggested that monocytes have a higher number of cell-specific enhancer regions (4-fold) compared to neutrophils. This highly plastic epigenome is likely indicative of the greater differentiation potential of monocytes into macrophages, dendritic cells and osteoclasts. In contrast, most of the neutrophil-specific features tend to be characterized by repressed chromatin, reflective of their status as terminally differentiated cells. Enhancers were the regions where most of differences in DNA methylation between cells were observed, with monocyte-specific enhancers being generally hypomethylated. Monocytes show a substantially higher gene expression levels than neutrophils, in line with epigenomic analysis revealing that gene more active elements in monocytes. Our analyses suggest that the overexpression of c-Myc in monocytes and its binding to monocyte-specific enhancers could be an important contributor to these differences. Altogether, our study provides a comprehensive epigenetic chart of chromatin states in primary human neutrophils and monocytes, thus providing a valuable resource for studying the regulation of the human innate immune system.

genomics↗