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Huang, C.

Publications and source records attributed to Huang, C..

At least 181 records · Page 10Linked to original sources
bioRxiv2021

Assembly status transition offers an avenue for activity modulation of a supramolecular enzyme

Nature has evolved many supramolecular proteins assembled in certain, sometimes even seemingly oversophisticated, morphological manners. The rationale behind such evolutionary efforts is often poorly understood. Here we provide atomic-resolution insights into how the dynamic building of a structurally complex enzyme with higher-order symmetry offers amenability to intricate allosteric regulation. We have established the functional coupling between enzymatic activity and protein morphological states of glutamine synthetase (GS), an old multi-subunit enzyme essential for cellular nitrogen metabolism. Cryo-EM structure determination of GS in both the catalytically active and inactive assembly states allows us to reveal an unanticipated self-assembly-induced dynamics-driven allosteric paradigm, in which the remote interactions between two subcomplex entities significantly rigidify the otherwise structurally fluctuating active sites, thereby regulating activity. We further show in vivo evidences that how the enzyme morphology transitions could be modulated by cellular factors on demand. Collectively, our data present an example of how assembly status transition offers an avenue for allosteric modulation, and sharpens our mechanistic understanding of allostery, dynamics, cooperativity, and other complex functional and regulatory properties of supramolecular enzymes.

biochemistry↗
bioRxiv2021

MOV10 facilitates messenger RNA decay in an N6-methyladenosine (m6A) dependent manner to maintain the mouse embryonic stem cells state

N6-methyladenosine (m6A) is the most predominant internal mRNA modification in eukaryotes, recognised by its reader proteins (so-called m6A-readers) for regulating subsequent mRNA fates -- splicing, export, localisation, decay, stability, and translation -- to control several biological processes. Although a few m6A-readers have been identified, yet the list is incomplete. Here, we identify a new m6A-reader protein, Moloney leukaemia virus 10 homologue (MOV10), in the m6A pathway. MOV10 recognises m6A-containing mRNAs with a conserved GGm6ACU motif. Mechanistic studies uncover that MOV10 facilitates mRNA decay of its bound m6A-containing mRNAs in an m6A-dependent manner within the cytoplasmic processing bodies (P-bodies). Furthermore, MOV10 decays the Gsk-3{beta} mRNA through m6A that stabilises the {beta}-CATENIN expression of a WNT/{beta}-CATENIN signalling pathway to regulate downstream NANOG expression for maintaining the mouse embryonic stem cells (mESCs) state. Thus, our findings reveal how a newly identified m6A-reader, MOV10 mediates mRNA decay via m6A that impact embryonic stem cell biology.

biochemistry↗
bioRxiv2021

Effects of four antibiotics on the diversity of the intestinal microbiota

Oral antibiotics remain the therapy of choice for severe bacterial infections; however, antibiotic use disrupts the intestinal microbiota, which increases the risk of colonization with intestinal pathogens. Currently, our understanding of antibiotic-mediated disturbances of the microbiota remains at the level of bacterial families or specific species, and little is known about the effect of antibiotics on potentially beneficial and potentially pathogenic bacteria under conditions of gut microbiota dysbiosis. Additionally, it is controversial whether the effects of antibiotics on the gut microbiota are temporary or permanent. In this study, we used 16S rRNA gene sequencing to evaluate the short-term and long-term effects of ampicillin, vancomycin, metronidazole, and neomycin on the murine intestinal microbiota by analyzing changes in the relative numbers of potentially beneficial and potentially pathogenic bacteria. We found that the changes in the intestinal microbiota reflected the antibiotics mechanisms of action and that dysbiosis of the intestinal microbiota led to competition between the different bacterial communities. Thus, destruction of bacteria with beneficial potential increased the abundance of bacteria with pathogenic potential. In addition, we found that these oral antibiotics had long-term negative effects on the intestinal microbiota and promoted the development of antibiotic-resistant bacterial strains. These results indicate that ampicillin, vancomycin, metronidazole, and neomycin have long-term negative effects and can cause irreversible changes in the diversity of the intestinal microbiota and the relative proportions of bacteria with beneficial potential and bacteria with pathogenic potential, thereby increasing the risk of host disease.

microbiology↗
bioRxiv2021

Robust Acquisition of High Resolution Spatial Transcriptomes from Preserved Tissues with Immunofluorescence Based Laser Capture Microdissection

The functioning of tissues is fundamentally dependent upon not only the phenotypes of the constituent cells but also their spatial organization in the tissue. However, acquisition of comprehensive transcriptomes of spatially- and phenotypically-defined cells in situ remains challenging. Here we present a general and robust method based on immunofluorescence-guided laser capture microdissection (immuno-LCM-RNAseq) to acquire finely resolved spatial transcriptomes including isoforms with as few as tens of cells from snap-frozen or RNAlater-treated clinical tissues, circumventing the problem of significant RNA degradation during this time-consuming process. The efficacy of this approach is exemplified by the characterization of differences at the transcript isoform level between the mouse small intestine lacteal cells at the tip versus the main capillary body. With the extensive repertoire of specific antibodies that are presently available, our method provides a powerful means by which spatially resolved cellular states can now be delineated in situ with preserved tissues. Moreover, such high quality spatial transcriptomes defined by immunomarkers can be used to compare with the phenotypes derived from single-cell RNAseq of dissociated cells as well as applied to bead-based spatial transcriptomic approaches that require such information a priori for cell identification.

genomics↗
bioRxiv2021

Cytoplasmic tail truncation of SARS-CoV-2 Spike protein enhances titer of pseudotyped vectors but masks the effect of the D614G mutation

The high pathogenicity of SARS-CoV-2 requires it to be handled under biosafety level 3 conditions. Consequently, Spike protein pseudotyped vectors are a useful tool to study viral entry and its inhibition, with retroviral, lentiviral (LV) and vesicular stomatitis virus (VSV) vectors the most commonly used systems. Methods to increase the titer of such vectors commonly include concentration by ultracentrifugation and truncation of the Spike protein cytoplasmic tail. However, limited studies have examined whether such a modification also impacts the proteins function. Here, we optimized concentration methods for SARS-CoV-2 Spike pseudotyped VSV vectors, finding that tangential flow filtration produced vectors with more consistent titers than ultracentrifugation. We also examined the impact of Spike tail truncation on transduction of various cell types and sensitivity to convalescent serum neutralization. We found that tail truncation increased Spike incorporation into both LV and VSV vectors and resulted in enhanced titers, but had no impact on sensitivity to convalescent serum inhibition. In addition, we analyzed the effect of the D614G mutation, which became a dominant SARS-CoV-2 variant early in the pandemic. Our studies revealed that, similar to the tail truncation, D614G independently increases Spike incorporation and vector titers, but that this effect is masked by also including the cytoplasmic tail truncation. Therefore, the use of full-length Spike protein, combined with tangential flow filtration, is recommended as a method to generate high titer pseudotyped vectors that retain native Spike protein functions. IMPORTANCEPseudotyped viral vectors are useful tools to study the properties of viral fusion proteins, especially those from highly pathogenic viruses. The Spike protein of SARS-CoV-2 has been investigated using pseudotyped lentiviral and VSV vector systems, where truncation of its cytoplasmic tail is commonly used to enhance Spike incorporation into vectors and to increase the titers of the resulting vectors. However, our studies have shown that such effects can also mask the phenotype of the D614G mutation in the ectodomain of the protein, which was a dominant variant early in the COVID-19 pandemic. To better ensure the authenticity of Spike protein phenotypes when using pseudotyped vectors, we therefore recommend using full-length Spike proteins, combined with tangential flow filtration methods of concentration, if higher titer vectors are required.

microbiology↗
bioRxiv2021

Effect of Substrate Stiffness on Human Intestinal Enteroids Infectivity by Enteroaggregative Escherichia coli

Human intestinal enteroids (HIE) models have contributed significantly to our understanding of diarrheal diseases and other intestinal infections, but their routine culture conditions fail to mimic the mechanical environment of the native intestinal wall. Because the mechanical characteristics of the intestine significantly alter how pathogens interact with the intestinal epithelium, we used different concentrations of polyethylene glycol (PEG) to generate soft ([~]2 kPa), medium ([~]10 kPa), and stiff ([~]100 kPa) hydrogel biomaterial scaffolds. The height of HIEs cultured in monolayers atop these hydrogels was 18 {micro}m whereas HIEs grown on rigid tissue culture surfaces (with stiffness in the GPa range) were 10 {micro}m. Substrate stiffness also influenced the amount of enteroaggregative E. coli (EAEC strain 042) adhered to the HIEs. We quantified a striking difference in adherence pattern; on the medium and soft gels, the bacteria formed clusters of >100 and even >1000 on both duodenal and jejunal HIEs (such as would be found in biofilms), but did not on glass slides and stiff hydrogels. All hydrogel cultured HIEs showed significant enrichment for gene and signaling pathways related to epithelial differentiation, cell junctions and adhesions, extracellular matrix, mucins, and cell signaling compared to the HIEs cultured on rigid tissue culture surfaces. Collectively, these results indicate that the HIE monolayers cultured on the hydrogels are primed for a robust engagement with their mechanical environment, and that the soft hydrogels promote the formation of larger EAEC aggregates, likely through an indirect differential effect on mucus. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/448533v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@f9c7b3org.highwire.dtl.DTLVardef@a5d5feorg.highwire.dtl.DTLVardef@184224forg.highwire.dtl.DTLVardef@1e8a28d_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

bioengineering↗
bioRxiv2021

CNS antigen-specific control of early B-lineage development in the meninges

The random V(D)J recombination of immunoglobulins (Ig) loci often creates autoreactive B cell progenitors expressing self-recognized B-cell receptors (BCRs)1, which are eliminated or inactivated through an autoantigen-dependent central tolerance checkpoint to prevent autoimmune reactions2,3, a process thought to be restricted to the bone marrow (BM) in the adult mammals4. Here we report that early developing B cells are also present in the meninges of mice at all ages. Single cell RNA-sequencing (scRNA-seq) analysis revealed a consecutive trajectory of meningeal developing B cells in mice and non-human primates (NHPs). Parabiosis together with lineage tracing of hematopoietic stem cells (HSCs) showed that meningeal developing B cells are continuously replenished from the HSC-derived progenitors via a circulationindependent route. Importantly, autoreactive immature B cells which recognize myelin oligodendrocyte glycoprotein (MOG)5, a central nervous system (CNS)-specific antigen, are eliminated from the meninges but not BM. Furthermore, genetic deletion of MOG restored the self-reactive B cells in the meninges. Thus, we propose that meninges function as a unique reservoir for B cell development, allowing in situ negative selection of CNS-antigen-autoreactive B cells to ensure a local non-self-reactive immune repertoire.

immunology↗
bioRxiv2021

Mechanistic insights into dideoxygenation in gentamicin biosynthesis

Gentamicin is an important aminoglycoside antibiotic used for treatment of infections caused by Gram-negative bacteria. Although most of the biosynthetic pathway of gentamicin has been elucidated, a remaining intriguing question is how the intermediates JI-20A and JI-20B undergo a dideoxygenation to form gentamicin C complex. Here we show that the dideoxygenation process starts with GenP-catalyzed phosphorylation of JI-20A and JI-20Ba. The phosphorylated products are converted to C1a and C2a by concerted actions of two PLP (pyridoxal 5-phosphate)-dependent enzymes: elimination of water and then phosphate by GenB3 and double bond migration by GenB4. Each of these reactions liberates an imine which hydrolyses to a ketone or aldehyde and is then re-aminated by GenB3 using an amino donor. Crystal structures of GenB3 and GenB4 have guided site-directed mutagenesis to reveal crucial residues for the enzymes functions. We propose catalytic mechanisms for GenB3 and GenB4, which shed new light on the already unrivalled catalytic versatility of PLP-dependent enzymes.

microbiology↗
bioRxiv2021

Mesenchymal stem cell subpopulations and their heterogeneity of response to inductions revealed by single-cell RNA-seq

Mesenchymal stem/stromal cells (MSCs) are promising cell source for regenerative medicine and treatment of autoimmune disorders. Comparing MSCs from different tissues at single cell level is fundamental for optimizing clinical applications. Here we analyzed single cell RNA-seq data of MSCs from 4 tissues, namely umbilical cord, bone marrow, synovial tissue and adipose tissue. We identified 3 major cell subpopulations, namely osteo-MSCs, chondro-MSCs, adipo/myo-MSCs, across all MSC samples. MSCs from umbilical cord exhibited the highest immunosuppression, potentially indicating it is the best immune modulator for autoimmune diseases. The differentiation potentials of MSC subpopulations, which are strongly associated with their subtypes and tissue sources, showed pronounced subpopulation differences. We found MSC subpopulations expanded and differentiated when their subtypes consist with induction directions, while the other subpopulations shrank. We identified the genes and transcription factors underlying each induction at single cell level and subpopulation level, providing better targets for improving induction efficiency.

genetics↗
bioRxiv2021

Optimization of AAV6 transduction enhances site-specific genome editing of primary human lymphocytes

Adeno-associated virus serotype 6 (AAV6) is a valuable reagent for genome editing of hematopoietic cells due to its ability to serve as a homology donor template. However, a comprehensive study of AAV6 transduction of hematopoietic cells in culture, with the goal of maximizing ex vivo genome editing, has not been reported. Here, we evaluated how the presence of serum, culture volume, transduction time, and electroporation parameters could influence AAV6 transduction. Based on these results, we identified an optimized protocol for genome editing of human lymphocytes based on a short, highly concentrated AAV6 transduction in the absence of serum, followed by electroporation with a targeted nuclease. In human CD4+ T cells and B cells, this protocol improved editing rates up to 7-fold and 21-fold respectively, when compared to standard AAV6 transduction protocols described in the literature. As a result, editing frequencies could be maintained using 50-100-fold less AAV6, which also reduced cellular toxicity. Our results highlight the important contribution of cell culture conditions for ex vivo genome editing with AAV6 vectors and provide a blueprint for improving AAV6-mediated homology-directed editing of human T and B cells.

molecular biology↗
bioRxiv2021

The Actin Bundling Protein Fascin Promotes Lung Cancer Growth and Metastasis by Enhancing Glycolysis and PFKFB3 Expression

Fascin is a pro-metastasis actin bundling protein upregulated in essentially all the metastatic carcinoma. It is believed that fascin promotes cancer cell migration and invasion by facilitating membrane protrusions such as filopodia and invadopodia. Aerobic glycolysis is a key feature of cancer metabolism that provides critical intermediate metabolites for tumor growth and cell proliferation. Here we report that fascin increase glycolysis in lung cancer to promote tumor growth and metastasis. Fascin promotes glycolytic flux by increasing the expression and activities of phosphofructose kinase 1 and 2 (PFK1 and 2). The glycolytic function of fascin depends on activation of YAP1 through its canonical actin bundling activity. Fascin promotes the binding of YAP1 to a TEAD1/4 binding motif located 30 bp upstream of the PFKFB3 transcription start site to activate its transcription. Our interrogation of the TCGA database suggest that the fascin-YAP1-PFKFB3 circuit is likely conserved across different types of cancer. We further showed that the glycolytic function of fascin is essential for promotion of lung cancer growth and metastasis. Importantly, pharmacological inhibitors of fascin could be used to suppress the YAP1-PFKFB3 signaling and inhibit glycolysis in cancer cell lines, organoid cultures and xenograft metastasis models. Taken together, our data reveal an important glycolytic role of fascin in lung cancer metabolism, and suggest that pharmacological inhibitors of fascin could be used to reprogram cancer metabolism in lung cancer and potentially other cancer with fascin upregulation.

cancer biology↗
bioRxiv2021

Depleting Hypothalamic Somatostatinergic Neurons Recapitulates Diabetic Phenotypes in Brain, Bone Marrow, Adipose, and Retina

Hypothalamic inflammation and sympathetic nervous system hyperactivity are hallmark features of metabolic syndrome and type 2 diabetes. Hypothalamic inflammation may aggravate metabolic and immunologic pathologies due to extensive sympathetic activation of peripheral tissues. Loss of somatostatinergic (SST) neurons may contribute to enhanced hypothalamic inflammation. The present data show that leptin receptor deficient (db/db) mice exhibit reduced hypothalamic somatostatinergic cells, particularly in the periventricular nucleus. We model this finding, using adeno-associated virus (AAV) delivery of diphtheria toxin (DTA) driven by an SST-cre system to deplete these cells in SSTcre/gfp mice (SST-DTA). SST-DTA mice exhibit enhanced hypothalamic c-fos expression and brain inflammation as demonstrated by microglial and astrocytic activation. Bone marrow from SST-DTA mice undergoes skewed hematopoiesis, generating excess granulocyte-monocyte precursors and increased pro-inflammatory (CCR2hi) monocytes. Visceral adipose tissue from DTA-treated animals was resistant to catecholamine induced lipolysis. Finally, SST-DTA mice exhibited a "diabetic retinopathy like" phenotype: reduced visual function by optokinetic response and electroretinogram, as well as increased percentages of retinal monocytes. Importantly, hyperglycemia was not observed in SST-DTA mice. Thus, the isolated reduction in hypothalamic somatostatinergic neurons was able to recapitulate several hallmark features of type 2 diabetes in disease relevant tissues.

neuroscience↗
bioRxiv2021

Reduced neutralization of SARS-CoV-2 variants by convalescent plasma and hyperimmune intravenous immunoglobulins for treatment of COVID-19

Hyperimmune immunoglobulin (hCoV-2IG) preparations generated from SARS-CoV-2 convalescent plasma (CP) are under evaluation in several clinical trials of hospitalized COVID-19 patients. Here we explored the antibody epitope repertoire, antibody binding and virus neutralizing capacity of six hCoV-2IG batches as well as nine convalescent plasma (CP) lots against SARS-CoV-2 and emerging variants of concern (VOC). The Gene-Fragment Phage display library spanning the SARS-CoV-2 spike demonstrated broad recognition of multiple antigenic sites spanning the entire spike including NTD, RBD, S1/S2 cleavage site, S2-fusion peptide and S2-heptad repeat regions. Antibody binding to the immunodominant epitopes was higher for hCoV-2IG than CP, with predominant binding to the fusion peptide. In the pseudovirus neutralization assay (PsVNA) and in the wild-type SARS-CoV-2 PRNT assay, hCoV-2IG lots showed higher titers against the WA-1 strain compared with CP. Neutralization of SARS-CoV-2 VOCs from around the globe were reduced to different levels by hCoV-2IG lots. The most significant loss of neutralizing activity was seen against the B.1.351 (9-fold) followed by P.1 (3.5-fold), with minimal loss of activity against the B.1.17 and B.1.429 ([≤]2-fold). Again, the CP showed more pronounced loss of cross-neutralization against the VOCs compared with hCoV-2IG. Significant reduction of hCoV-2IG binding was observed to the RBD-E484K followed by RBD-N501Y and minimal loss of binding to RBD-K417N compared with unmutated RBD. This study suggests that post-exposure treatment with hCoV-2IG is preferable to CP. In countries with co-circulating SARS-CoV-2 variants, identifying the infecting virus strain could inform optimal treatments, but would likely require administration of higher volumes or repeated infusions of hCOV-2IG or CP, in patients infected with the emerging SARS-CoV-2 variants.

immunology↗
bioRxiv2021

EZH2 Abundance Regulated by UHRF1/UBE2L6/UBR4 Ubiquitin System is the Potential Therapeutic Target to Trigger Pigmented Phenotype in Melanoma

Cellular heterogeneity in cancer is linked to disease progression and therapy response, although the mechanisms regulating distinct cellular states within tumours are not well understood. To address this, we identified melanin pigment content as a major source of phenotypic and functional heterogeneity in melanoma and compared RNAseq data from high (HPC) and low pigmented melanoma cells (LPC), revealing the polycomb repressor complex protein, EZH2, as a master regulator of these states. EZH2 protein, but not RNA expression, was found to be upregulated in LPCs and inversely correlated with melanin in pigmented patient melanomas. Surprisingly, conventional EZH2 methyltransferase inhibitors, GSK126 and EPZ6438, had no effect on LPC survival, clonogenicity and pigmentation, despite fully inhibiting methyltransferase activity. In contrast, EZH2 silencing by siRNA strategy or DZNep, MS1943 that reduces EZH2 protein levels, significantly inhibited cell growth in LPCs by hampering ribosome biogenesis. In addition, decline in EZH2 protein level induces pigmented cell phenotype by inducing melanin biosynthesis. Proteasomal inhibitor, MG132 treatment induced EZH2 protein levels in HPCs prompted us to look for differentially regulated ubiquitin system proteins in HPC vs LPCs. UBE2L6, E2 conjugating enzyme has been shown to be downregulated significantly in LPCs by UHRF1-mediated CpG methylation. Both biochemical assays and animal studies demonstrated that UBE2L6 expression decline, in turn, promotes EZH2 protein stability due to lack of ubiquitination on K381 residue in LPCs. UBR4 cooperates with UBE2L6 to facilitate this ubiquitination process. Targeting UHRF1/UBE2L6/UBR4 axis can be a better treatment option to trigger HPC state in melanoma in which conventional EZH2 inhibitors are ineffective.

cancer biology↗
bioRxiv2021

The component parts of bacteriophage virions accurately defined by a machine-learning approach built on evolutionary features.

Antimicrobial resistance (AMR) continues to evolve as a major threat to human health and new strategies are required for the treatment of AMR infections. Bacteriophages (phages) that kill bacterial pathogens are being identified for use in phage therapies, with the intention to apply these bactericidal viruses directly into the infection sites in bespoke phage cocktails. Despite the great unsampled phage diversity for this purpose, an issue hampering the roll out of phage therapy is the poor quality annotation of many of the phage genomes, particularly for those from infrequently sampled environmental sources. We developed a computational tool called STEP3 to use the "evolutionary features" that can be recognized in genome sequences of diverse phages. These features, when integrated into an ensemble framework, achieved a stable and robust prediction performance when benchmarked against other prediction tools using phages from diverse sources. Validation of the prediction accuracy of STEP3 was conducted with high-resolution mass spectrometry analysis of two novel phages, isolated from a watercourse in the Southern Hemisphere. STEP3 provides a robust computational approach to distinguish specific and universal features in phages to improve the quality of phage cocktails, and is available for use at http://step3.erc.monash.edu/. IMPORTANCEIn response to the global problem of antimicrobial resistance there are moves to use bacteriophages (phages) as therapeutic agents. Selecting which phages will be effective therapeutics relies on interpreting features contributing to shelf-life and applicability to diagnosed infections. However, the protein components of the phage virions that dictate these properties vary so much in sequence that best estimates suggest failure to recognize up to 90% of them. We have utilised this diversity in evolutionary features as an advantage, to apply machine learning for prediction accuracy for diverse components in phage virions. We benchmark this new tool showing the accurate recognition and evaluation of phage components parts using genome sequence data of phages from under-sampled environments, where the richest diversity of phage still lies.

microbiology↗
bioRxiv2021

The cryo-EM structure of the neurofibromin dimer reveals the molecular basis for von Recklinghausen disease

Neurofibromin (NF1) is a tumour suppressor mutated in neurofibromatosis type 1 (von Recklinghausen disease), one of the most common human genetic diseases(1). NF1 regulates cellular growth through suppressing the Rat Sarcoma (RAS) pathway and, accordingly, mutations in this protein drive numerous cancers, including melanoma, ovarian, breast and brain cancer(2, 3). Currently, however, the molecular basis for NF1 function remains to be understood. Here we address this problem and use cryogenic Electron Microscopy (cryo-EM) to determine the structure of fulllength NF1. The 640 kDa NF1 homodimer forms an extraordinary lemniscate ({infty}) shaped molecule that is ~30 nm in length and ~ 10 nm wide. Each NF1 monomer comprises an N-terminal HEAT-repeat domain (N-HEAT), a guanosine triphosphatase activating protein (GAP)-related domain (GRD), a Sec14 homologous and pleckstrin homologous module (SEC-PH), and a C-terminal HEAT domain (C-HEAT). The core NF1 scaffold is formed via a head-to-tail dimer of the N- and C-HEAT domains. This platform, which is responsible for interacting with more than 10 regulatory binding partners, comprises an extraordinary array of over 150 -helices. Analysis of these EM data revealed that the GRD and SEC-PH domain are highly mobile with respect to the core scaffold and could not initially be accurately placed in electron density. Strikingly, however, using 3D variability analysis we were able to identify a significant subpopulation of NF1 particles and determine the complete NF1 structure to 5.6 [A] resolution. These data revealed that the catalytic GRD and lipid binding SEC-PH domain are positioned against the core scaffold in a closed, autoinhibited conformation. We postulate that interaction with the plasma membrane may release the closed conformation in order to promote RAS inactivation. Our structural data further allow us to map the location of disease-associated NF1 variants and provide a long sought-after structural explanation for the extreme susceptibility of the molecule to loss-of-function mutations. Finally, it is suggested that approaches to combat NF1-linked diseases may include release of the autoinhibited state in order to improve NF1 catalytic efficiency.

biochemistry↗
bioRxiv2021

CDSeqR: fast complete deconvolution for gene expression data from bulk tissues

BackgroundBiological tissues consist of heterogenous populations of cells. Because gene expression patterns from bulk tissue samples reflect the contributions from all cells in the tissue, understanding the contribution of individual cell types to the overall gene expression in the tissue is fundamentally important. We recently developed a computational method, CDSeq, that can simultaneously estimate both sample-specific cell-type proportions and cell-type-specific gene expression profiles using only bulk RNA-Seq counts from multiple samples. Here we present an R implementation of CDSeq (CDSeqR) with significant performance improvement over the original implementation in MATLAB and an added new function to aid cell type annotation. The R package would be of interest for the broader R community. ResultWe developed a novel strategy to substantially improve computational efficiency in both speed and memory usage. In addition, we designed and implemented a new function for annotating the CDSeq estimated cell types using single-cell RNA sequencing (scRNA-seq) data. This function allows users to readily interpret and visualize the CDSeq estimated cell types. In addition, this new function further allows the users to annotate CDSeq-estimated cell types using marker genes. We carried out additional validations of the CDSeqR software using synthetic, real cell mixtures, and real bulk RNA-seq data from the Cancer Genome Atlas (TCGA) and The Genotype-Tissue Expression (GTEx) project. ConclusionsThe existing bulk RNA-seq repositories, such as TCGA and GTEx, provide enormous resources for better understanding changes in transcriptomics and human diseases. They are also potentially useful for studying cell-cell interactions in the tissue microenvironment. Bulk level analyses neglect tissue heterogeneity, however, and hinder investigation of a cell-type-specific expression. The CDSeqR package may aid in silico dissection of bulk expression data, enabling researchers to recover cell-type-specific information.

bioinformatics↗