Biology subjects

Hu, J.

Publications and source records attributed to Hu, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2018

Genomic Analysis of a Transcriptional Networks Directing Progression of Cell States During MGE development.

BackgroundHomeodomain (HD) transcription factor (TF) NKX2-1 critical for the regional specification of the medial ganglionic eminence (MGE) as well as promoting the GABAergic and cholinergic neuron fates via the induction of TFs such as LHX6 and LHX8. NKX2-1 defines MGE regional identity in large part through transcriptional repression, while specification and maturation of GABAergic and cholinergic fates is mediated in part by transcriptional activation via TFs such as LHX6 and LHX8. Here we analyze the signaling and TF pathways, downstream of NKX2-1, required for GABAergic and cholinergic neuron fate maturation.\n\nMethodsDifferential ChIP-seq analysis was used to identify regulatory elements (REs) where chromatin state was sensitive to change in the Nkx2-1cKO MGE at embryonic day (E) 13.5. TF motifs in the REs were identified using RSAT. CRISPR-mediated genome editing was used to generate enhancer knockouts. Differential gene expression in these knockouts was analyzed through RT-qPCR and in situ hybridization. Functional analysis of motifs within hs623 was analyzed via site directed mutagenesis and reporter assays in primary MGE cultures.\n\nResultsWe identified 4782 activating REs (aREs) and 6391 repressing REs (rREs) in the Nkx2-1 conditional knockout (Nkx2-1cKO) MGE. aREs are associated with basic-Helix-Loop-Helix (bHLH) TFs. Deletion of hs623, an intragenic Tcf12 aRE, caused a reduction of Tcf12 expression in the sub-ventricular zone (SVZ) and mantle zone (MZ) of the MGE. Mutation of LHX, SOX and octamers, within hs623, caused a reduction of hs623 activity in MGE primary cultures.\n\nConclusionsTcf12 expression in the sub-ventricular zone (SVZ) of the MGE is mediated through aRE hs623. The activity of hs623 is dependent on LHX6, SOX and octamers. Thus, maintaining the expression of Tcf12 in the SVZ involves on TF pathways parallel and genetically downstream of NKX2-1.

developmental biology↗

bioRxiv2018

Embedding containerized workflows inside data science notebooks enhances reproducibility

Data science notebooks, such as Jupyter, combine text documentation with dynamically editable and executable code and have become popular for sharing computational methods. We present nbdocker, an extension that integrates Docker software containers into Jupyter notebooks. nbdocker transforms notebooks into autonomous, self-contained, executable and reproducible modules that can document and disseminate complicated data science workflows containing code written in different languages and executables requiring different software environments.

bioinformatics↗

bioRxiv2018

Effects of duplicated mapped read PCR artifacts on RNA-seq differential expression analysis based on qRNA-seq

Best practices to handling duplicated mapped reads in RNA-seq analyses has long been discussed but a gold standard method has yet to be established, as such duplicates could originate from valid biological transcripts or they could be PCR-related artifacts. Here we used the NEXTflex qRNA-SeqTM (aka Molecular Indexing) technology to identify PCR duplicates via the random attachment of unique molecular labels to each cDNA molecule prior to PCR amplification. We found that up to 64.3% of the single end and 19.3% of the mouse paired end duplicates originated from valid biological transcripts rather than PCR artifacts. For single end reads, either removing or retaining all duplicates resulted in a substantial number of false positives (up to 47.0%) and false negatives (up to 12.1%) in the sets of significantly differentially expressed genes. For paired end reads, only the alignment retaining all duplicates resulted in a substantial number of false positives. This is the first effort to evaluate the performance of qRNA-seq using real-world biomedical samples, and we found that PCR duplicate identification provided minor benefits for paired end reads but greatly improved the sensitivity and specificity in the determination of the significantly differentially expressed genes for single end reads.

genomics↗

bioRxiv2018

Myosin filaments reversibly generate large forces in cells

We present high resolution experiments performed on elementary contractile units in cells that challenge our current understanding of molecular motor force generation. The key features are the development of a force per motor considerably larger than forces measured in single molecule experiments, a force increase followed by relaxation controlled by a characteristic displacement rather than by a characteristic force, the observation of steps at half the actin filament period even though a large number of motors are at work in an elementary contractile unit. We propose a generic two-state model of molecular motor collections with hand-over-hand contractions and we find that these unexpected observations are spontaneously emerging features of a collective motor behavior.

biophysics↗

bioRxiv2018

Dynamic Extreme Aneuploidy (DEA) in the vegetable pathogen Phytophthora capsici sheds light on instant evolution and intractability.

Oomycete plant pathogens are notoriously difficult to control, and individual isolates are highly unstable; making routine research challenging. Sequencing reveals extreme aneuploidy for single-spore progeny of the vegetable pathogen Phytophthora capsici; a phenomenon dubbed Dynamic Extreme Aneuploidy (DEA). Although extreme, the aneuploidy appears to be moderately stable. A single sporulating plant lesion may produce an armada of genetically unique individuals and helps explain the rapid increase of advantageous alleles (e.g. drug resistance), mating type switches to allow sex and the widely observed phenomenon, loss of heterozygosity (LOH). Investigation of other oomycetes indicate this phenomenon is not unique to P. capsici.

genomics↗

bioRxiv2018

Principles of Computer Numerical Controlled Machining Applied to Cranial Microsurgery

Over the last decade, a plethora of tools have been developed for neuroscientists to interface with the brain. Implementing these tools requires precise removal of sections of the skull to access the brain. These delicate cranial microsurgical procedures need to be performed on sub-millimeter thick bone without damaging the underlying tissue and therefore, require significant training. Automating some of these procedures would not only enable more precise microsurgical operations, but also democratize use of advanced neurotechnologies. Here, we describe the Craniobot, a cranial microsurgery platform that combines automated skull surface profiling with a computer numerical controlled (CNC) milling machine to perform a variety of cranial microsurgical procedures in mice. The Craniobot utilizes a low force contact sensor to profile the skull surface and uses this information to perform micrometer-scale precise milling operations within minutes. We have used the Craniobot to drill pilot holes to anchor cranial implants, perform skull thinning, and open small to large craniotomies. The Craniobot is built using off-the-shelf components for under $1000 and is controlled using open-source CNC programming software.

bioengineering↗

bioRxiv2018

Loss of Peroxisomal Hydroxypyruvate Reductase Inhibits Triose Phosphate Isomerase but Stimulates Cyclic Photosynthetic Electron Flow and the Glc-6P-Phosphate Shunt

The oxygenation of ribulose 1,5-bisphosphate by Rubisco is the first step in photorespiration and reduces the efficiency of photosynthesis in C3 plants. Our recent data indicates that mutants in photorespiration have increased rates of photosynthetic cyclic electron flow around photosystem I. We investigated mutant lines lacking peroxisomal hydroxypyruvate reductase to determine if there are connections between 2-PG accumulation and cyclic electron flow. We found that 2-PG is a competitive inhibitor of triose phosphate isomerase (TPI), an enzyme in the Calvin-Benson cycle that converts glyceraldehyde 3-phosphate to dihydroxyacetone phosphate. This block in metabolism could be overcome if glyceraldehyde 3-phosphate is exported to the cytosol where the cytosolic triose phosphate isomerase could convert it to dihydroxyacetone phosphate. We found evidence that carbon is reimported as Glc-6P-phosphate forming a cytosolic bypass around the block of stromal TPI. However, this also stimulates a Glc-6P-phosphate shunt, which consumes ATP, which can be compensated by higher rates of cyclic electron flow.\n\nOnce Sentence SummaryTriose phosphate isomerase is inhibited in plants lacking hydroxypyruvate reductase 1 and this is overcome by exporting triose phosphate to the cytosol and importing Glc-6P, which stimulates a Glc-6P-phosphate shunt and cyclic electron flow.

plant biology↗

bioRxiv2018

Cell atlas of human uterus

The human uterus is a highly dynamic tissue that undergoes repeated damage repair and regeneration during the menstrual cycle, which make it ideal model to study tissue regeneration and pathological process. Stem/progenitors were speculated to be involved in the regeneration of endometrial epithelial and pathogenesis of endometriosis. But the identity, microenvironment and regulatory mechanisms of the uterus epithelial stem/progenitors in vivo remain unclear. Here, we dissected the cell heterogeneities of the full-thickness human uterus epithelial cells (11 clusters), stroma cells (6 clusters), endothelial cells (5 clusters), smooth muscle cells (2 clusters), myofibroblasts (2 clusters) and immune cells (6 clusters) from 2735 single cell by single cell RNA-seq. Further analysis identified a unique ciliated epithelial cell cluster showing characteristics of stem/progenitors with properties of epithelial-mesenchymal transition (EMT) that mainly localized in the upper functionalis of the endometrium. Ordering the cell subpopulations along the pseudo-space revealed cell clusters possess cellular states of stress, inflammation and apoptosis in the upper functionalis cellular ecosystem of the endometrium. Connectivity map between the human uterus subpopulations revealed potential inflammatory (cytokines and chemokines) and developmental (WNT, FGF, VEGF) signals within the upper functionalis cellular ecosystem of the endometrium, especially from other epithelial clusters, regulating cell plasticity of the EMT-epithelial clusters. This study reconstructed the heterogeneities, space-specific distribution and connectivity map of human uterus atlas, which would provide insight in the regeneration of uterus endometria and reference for the pathogenesis of uterus.

cell biology↗

bioRxiv2018

High-quality ultrastructural preservation using cryofixation for 3D electron microscopy of genetically labeled tissues

Electron microscopy (EM) offers unparalleled power to study cell substructures at the nanoscale. Cryofixation by high-pressure freezing offers optimal morphological preservation, as it captures cellular structures instantaneously in their near-native states. However, the applicability of cryofixation is limited by its incompatibilities with diaminobenzidine labeling using genetic EM tags and the high-contrast en bloc staining required for serial block-face scanning electron microscopy (SBEM). In addition, it is challenging to perform correlated light and electron microscopy (CLEM) with cryofixed samples. Consequently, these powerful methods cannot be applied to address questions requiring optimal morphological preservation and high temporal resolution. Here we developed an approach that overcomes these limitations; it enables genetically labeled, cryofixed samples to be characterized with SBEM and 3D CLEM. Our approach is broadly applicable, as demonstrated in cultured cells, Drosophila olfactory organ and mouse brain. This optimization exploits the potential of cryofixation, allowing quality ultrastructural preservation for diverse EM applications.

biophysics↗

bioRxiv2018

AMPKi overcomes the paradoxical activation of CRAF driven by RAF inhibitors through blocking the 14-3-3 binding to its carboxyl-terminus

The paradoxical activation of RAF kinase is the predominant challenge in cancer therapies with RAF inhibitors. The inhibitor-bound RAF molecules are able to transactivate their wild-type binding partners. 14-3-3 that binds to the carboxyl-terminus of RAF kinase has been suggested to regulate the dimer-dependent activation of RAF kinase under physiological conditions, though the molecular basis is not clear. In this study, we investigated the role of 14-3-3 in the paradoxical effect of RAF inhibitors. Firstly, we found that the 14-3-3 binding to the carboxyl-terminus of CRAF was essential for its transactivation. Further, we demonstrated that this binding enhanced the dimer affinity of CRAF. Since 14-3-3 binds to the phosphorylated motif, we next investigated and identified AMPK and CRAF itself as two putative kinases that phosphorylate redundantly the 14-3-3 binding motif of CRAF. Among RAF isoforms, CRAF plays a dominant role in the paradoxical effect of RAF inhibitors, and we thus determined whether the combinatory inhibition of AMPK and CRAF would block this effect. Indeed, our data showed that AMPKi not only blocked the RAF inhibitor-driven paradoxical activation of RAF signaling and cellular overgrowth in Ras-mutated cancer cells but also reduced the drug-resistant clones derived from BRAF(V600E)-mutated cancer cells. Finally, we showed that the 14-3-3 binding to the carboxyl-terminus of CRAF was dispensable for its catalytic function in vivo. Together, our study unraveled how 14-3-3 regulates the dimerization-driven RAF activation and identified AMPKi as a potential method to relieve the drug resistance and side effect of RAF inhibitors in cancer therapy.

cancer biology↗

bioRxiv2017

DeepMHC: Deep Convolutional Neural Networks for High-performance peptide-MHC Binding Affinity Prediction

Convolutional neural networks (CNN) have been shown to outperform conventional methods in DNA-protien binding specificity prediction. However, whether we can transfer this success to protien-peptide binding affinity prediction depends on appropriate design of the CNN architectue that calls for thorough understanding how to match the architecture to the problem. Here we propose DeepMHC, a deep convolutional neural network (CNN) based protein-peptide binding prediction algorithm for achieving better performance in MHC-I peptide binding affinity prediction than conventional algorithms. Our model takes only raw binding peptide sequences as input without needing any human-designed features and othe physichochemical or evolutionary information of the amino acids. Our CNN models are shown to be able to learn non-linear relationships among the amino acid positions of the peptides to achieve highly competitive performance on most of the IEDB benchmark datasets with a single model architecture and without using any consensus or composite ensemble classifier models. By systematically exploring the best CNN architecture, we identified critical design considerations in CNN architecture development for peptide-MHC binding prediction.

bioinformatics↗

bioRxiv2017

The Activation of MEK1 by Enhanced Homodimerization Drives Tumorigenesis

Hyperactive RAS/RAF/MEK/ERK signaling has a well-defined role in cancer biology. Aberrant pathway activation occurs mostly upstream of MEK; however, MEK mutations are prevalent in some cancer subsets. Here we show that cancer-related MEK mutants can be classified as those activated by relieving the inhibitory role of helix A, and those with in-frame deletions of {beta}3-C loop, which exhibit differential resistance to MEK inhibitors in vitro and in vivo. The {beta}3-C loop deletions activate MEK1 through enhancing homodimerization that can drive intradimer cross-phosphorylation of activation loop. Further, we demonstrate that MEK1 dimerization is required both for its activation by RAF and for its catalytic activity towards ERK. Our study identifies a novel group of MEK mutants, illustrates some key steps in RAF/MEK/ERK activation, and has important implications for the design of therapies targeting hyperactive RAS/RAF/MEK/ERK signaling in cancers.

cancer biology↗

bioRxiv2017

A Rice Dual-localized Pentatricopeptide Repeat Protein is involved in Organellar RNA Editing with MORFs

Flowering plants engage in diverse RNA editing events in mitochondrion and chloroplast on post-transcriptional process. Although several PPRs and MORFs were identified as RNA editing factors, the underlying mechanism of PPRs and the cooperation among them are still obscure. Here, we identified a rice dual-localized PPR mutant Ospgl1. Loss-of-function of OsPGLl resulted in defect of chloroplast RNA editing at ndhD-878 and mitochondrial RNA editing at ccmFc-543, which can be restored via complementary validation. Despite the synonymous editing on ccmFc-543, loss of editing at ndhD-878 caused failure of conversion from serine to leucine, leading to the dysfunction of chloroplast and defective in photosynthetic complex, further studies demonstrated OsPGL1 directly bound to both two transcripts. The interaction between three MORFs (MORF2/8/9) and OsPGL1 were confirmed in vitro and in vivo, implied OsPGL1 functioned on RNA editing via an editosome. It also suggested MORFs assisted and contributed to the flexible PPR-RNA recognition model during RNA editing through the cooperation with PPRs. These results provide new insight into the relationship between RNA editing and plant development on chloroplast.\n\nHighlightWe firstly characterized a dual-localized PPR protein which is required for RNA editing in mitochondrion and chloroplast simultaneously. OsPGL1 binds to two distinguish target transcripts directly and cooperated with MORFs.

molecular biology↗

bioRxiv2017

Stopping Transformed Growth with Cytoskeletal Proteins: Turning a Devil into an Angel

The major hallmark of cancer cells is uncontrollable growth on soft matrices (transformed growth), which indicates that they have lost the ability to properly sense the rigidity of their surroundings. Recent studies of fibroblasts show that local contractions by cytoskeletal rigidity sensor units block growth on soft surfaces and their depletion causes transformed growth. The contractile system involves many cytoskeletal proteins that must be correctly assembled for proper rigidity sensing. We tested the hypothesis that cancer cells lack rigidity sensing due to their inability to assemble contractile units because of altered cytoskeletal protein levels. In four widely different cancers, there were over ten-fold fewer rigidity-sensing contractions compared with normal fibroblasts. Restoring normal levels of cytoskeletal proteins restored rigidity sensing and rigidity-dependent growth in transformed cells. Most commonly, this involved restoring balanced levels of the tropomyosins 2.1 (often depleted by miR-21) and 3 (often overexpressed). Restored cells could be transformed again by depleting other cytoskeletal proteins including myosin IIA. Thus, the depletion of rigidity sensing modules enables growth on soft surfaces and many different perturbations of cytoskeletal proteins can disrupt rigidity sensing thereby causing transformed growth of cancer cells.

cell biology↗

bioRxiv2017

Lamins organize the global three-dimensional genome from the nuclear periphery

Lamins are structural components of the nuclear lamina (NL) that regulate genome organization and gene expression, but the mechanism remains unclear. Using Hi-C, we show that lamins maintain proper interactions among the topologically associated chromatin domains (TADs) but not their overall architecture. Combining Hi-C with fluorescence in situ hybridization (FISH) and analyses of lamina-associated domains (LADs), we reveal that lamin loss causes expansion or detachment of specific LADs in mouse ES cells. The detached LADs disrupt 3D interactions of both LADs and interior chromatin. 4C and epigenome analyses further demonstrate that lamins maintain the active and repressive chromatin domains among different TADs. By combining these studies with transcriptome analyses, we found a significant correlation between transcription changes and the changes of active and inactive chromatin domain interactions. These findings provide a foundation to further study how the nuclear periphery impacts genome organization and transcription in development and NL-associated diseases.\n\nHighlightsO_LILamin loss does not affect the overall TAD structure but alters TAD-TAD interactions\nC_LIO_LILamin null ES cells exhibit decondensation or detachment of specific LAD regions\nC_LIO_LIExpansion and detachment of LADs can alter genome-wide 3D chromatin interactions\nC_LIO_LIAltered chromatin domain interactions are correlated with altered transcription\nC_LI

genomics↗

bioRxiv2017

Deubiquitylation and stabilization of p21 by USP11 is critical for cell cycle progression and DNA damage responses

p21WAF1/CIP1 is a broad-acting cyclin-dependent kinase inhibitor. Its stability is essential for proper cell cycle progression and cell fate decision. Ubiquitylation by the multiple E3 ubiquitin ligases complex is the major regulatory mechanism of p21, which induces p21 degradation. However, it is unclear whether ubiquitylated p21 can be recycled. In this study, we report USP11 as a deubiquitylase of p21. In the nucleus, USP11 binds to p21, catalyzes the removal of polyubiquitin chains conjugated onto p21 and stabilizes p21 protein. As a result, USP11 reverses p21 polyubiquitylation and degradation mediated by SCFSKP2, CRL4CDT2 and APC/CCDT20 in a cell cycle-independent manner. Loss of USP11 causes the destabilization of p21 and induces the G1/S transition in unperturbed cells. Furthermore, p21 accumulation mediated by DNA damage is completely abolished in cells depleted of USP11, which results in abrogation of the G2 checkpoint and induction of apoptosis. Functionally, USP11-mediated stabilization of p21 inhibits cell proliferation and tumorigenesis in vivo. These findings reveal an important mechanism by which p21 can be stabilized by direct deubiquitylation and pinpoint a crucial role of the USP11-p21 axis in regulating cell cycle progression and DNA damage responses.

cell biology↗

bioRxiv2017

Reproducible Bioconductor Workflows Using Browser-Based Interactive Notebooks And Containers

ObjectiveBioinformatics publications typically include complex software workflows that are difficult to describe in a manuscript. We describe and demonstrate the use of interactive software notebooks to document and distribute bioinformatics research. We provide a user-friendly tool, BiocImageBuilder, to allow users to easily distribute their bioinformatics protocols through interactive notebooks uploaded to either a GitHub repository or a private server.\n\nMaterials and methodsWe present three different interactive Jupyter notebooks using R and Bioconductor workflows to infer differential gene expression, analyze cross-platform datasets and process RNA-seq data. These interactive notebooks are available on GitHub. The analytical results can be viewed in a browser. Most importantly, the software contents can be executed and modified. This is accomplished using Binder, which runs the notebook inside software containers, thus avoiding the need for installation of any software and ensuring reproducibility. All the notebooks were produced using custom files generated by BiocImageBuilder.\n\nResultsBiocImageBuilder facilitates the publication of workflows with a point-and-click user interface. We demonstrate that interactive notebooks can be used to disseminate a wide range of bioinformatics analyses. The use of software containers to mirror the original software environment ensures reproducibility of results. Parameters and code can be dynamically modified, allowing for robust verification of published results and encouraging rapid adoption of new methods.\n\nConclusionGiven the increasing complexity of bioinformatics workflows, we anticipate that these interactive software notebooks will become as ubiquitous and necessary for documenting software methods as traditional laboratory notebooks have been for documenting bench protocols.

bioinformatics↗

bioRxiv2017

Temporal fitness fluctuations in experimental Arabidopsis thaliana populations

Understanding the genetics of lifetime fitness is crucial to understand a species ecological preferences and ultimately predict its ability to cope with novel environmental conditions. Yet, there is a dearth of information regarding the impact of the ecological variance experienced by natural populations on expressed phenotypic and fitness differences. Here, we follow the natural dynamics of experimental A. thaliana populations over 5 successive plantings whose timing was determined by the natural progression of the plants life cycle and disentangle the environmental and genetic factors that drive plant ecological performance at a given locality. We show that, at the intermediate latitude where the experiment was conducted, a given genotype can experience different life cycles across successive seasons. Lifetime fitness across these seasons varied strongly, with a fall planting yielding 36-fold higher fitness compared to a spring planting. In addition, the actual life-stage at which plant overwinter oscillated across years, depending on the timing of the end of the summer season. We observed a rare but severe fitness differential after inadequate early flowering in one of the five planting. Substrate variation played a comparatively minor role, but also contributed to modulate the magnitude of fitness differentials between genotypes. Finally, reciprocal introgressions on chromosome 4 demonstrated that the fitness effect of a specific chromosomal region is strongly contingent on micro-geographic and seasonal fluctuations. Our study contributes to emphasize the extent to which the fitness impact of phenotypic traits and the genes that encode them in the genome can fluctuate. Experiments aiming at dissecting the molecular basis of local adaptation must apprehend the complexity introduced by temporal fluctuations because they massively affect the expression of phenotype and fitness differences.

evolutionary biology↗