Biology subjects

Sinha, A.

Publications and source records attributed to Sinha, A..

At least 91 records · Page 5Linked to original sources

bioRxiv2022

Transcriptional circuitry of NKX2-1 and SOX1 defines an unrecognized lineage subtype of small cell lung cancer

RationaleThe current molecular classification of small cell lung cancer (SCLC) based on expression of four lineage transcription factors still leaves its major subtype SCLC-A as a heterogeneous group, necessitating more precise characterization of lineage subclasses. ObjectivesTo refine the current SCLC classification with epigenomic profiles and to identify features of the re-defined SCLC subtypes. MethodsWe performed unsupervised clustering of epigenomic profiles on 25 SCLC cell lines. Functional significance was evaluated by cell growth, apoptosis and xenograft using CRISPR-Cas9-mediated deletion. The specific cistromic profiles by ChIP-seq and its functional transcriptional partners using co-immunoprecipitation followed by mass spectrometry were determined. Rb1fl/flTrp53fl/fl and Rb1fl/flNkx2-1fl/fl mouse models were engineered to explore the function of Nkx2-1 in tumor initiation and differentiation. H3K27ac profiles were analyzed to reveal 6 human SCLC specimen and 20 mice tumors epigenomic landscapes. Measurements and Main ResultsWe identified an epigenomic subclusters of the major SCLC-A subtype, named SCLC-A and SCLC-A{sigma}. SCLC-A was characterized by the presence of a super-enhancer at the NKX2-1 locus, which was observed in human SCLC specimens and a murine SCLC model. We found NKX2-1, a dual lung and neural lineage factor, is uniquely relevant in SCLC-A. We further found maintenance of this neural identity in SCLC-A is mediated by collaborative transcriptional activity with another neuronal transcriptional factor SOX1. ConclusionsWe comprehensively describe an additional epigenomic heterogeneity of the major SCLC-A subtype, and define SCLC-A subtype by the core regulatory circuitry representing NKX2-1 and SOX1 super-enhancers and their functional collaborations to maintain neuronal linage state.

molecular biology↗

bioRxiv2022

Generating an in vitro gut model with physiologically relevant biophysical mucus properties

IntroductionGastrointestinal (GI) in vitro models have received lasting attention as an effective tool to model drug and nutrient absorption, study GI diseases, and design new drug delivery vehicles. A complete model of the GI epithelium should at a minimum include the two key functional components of the GI tract: mucus and the underlying epithelium. Mucus plays a key role in protecting and lubricating the GI tract, poses a barrier to orally administered therapies and pathogens, and serves as the microenvironment for the GI microbiome. These functions are reliant on the biophysical material properties of the mucus produced, including viscosity and pore size. MethodsIn this study, we generated in vitro models containing Caco-2 enterocyte-like cells and HT29-MTX goblet-like cells and determined the effects of coculture and mucus layer on epithelial permeability and biophysical properties of mucus using multiple particle tracking (MPT). ResultsWe found that mucus height increased as the amount of HT29-MTX goblet-like cells increased. Additionally, we found that increasing the amount of HT29-MTX goblet-like cells within culture corresponded to an increase in mucus pore size and mucus microviscosity, measured using MPT. When compared to ex vivo mucus samples from mice and pigs, we found that a 90:10 ratio of Caco-2:HT29-MTX coculture displayed similar mucus pore size to porcine jejunum and that the mucus produced from 90:10 and 80:20 ratios of cells shared mechanical properties to porcine jejunum and ileum mucus. ConclusionsGI coculture models are valuable tools in simulating the mucus barrier and can be utilized for a variety of applications including the study of GI diseases, food absorption, or therapeutic development. BiographyDr. Maisel joined the University of Maryland in January 2019 having done interdisciplinary training in nanotechnology, mucosal immunology, lymphatic immunology, and immunoengineering. She completed her PhD in Biomedical Engineering at the Johns Hopkins University in 2014 after which she was an NIH postdoctoral fellow at the University of Chicago in Molecular Engineering and Immunology. The Maisel Lab uses in vitro modeling, nanotechnology, and immunoengineering approaches to study and develop treatments for diseases at mucosal surfaces. They are interested in designing nanoparticles to take advantage of and study the interface between biological barriers, particularly the lymphatics, interstitial tissue, and mucosal surfaces, and nanoparticles. Dr. Maisel has won a number of awards, including NSF GRFP and NIH F32 fellowships as a trainee, the American Lung Association Dalsemer Award, LAM Foundation Career Development Award, an NSF CAREER Award, and an NIH NIGMS Maximizing Investigator Research Award. Her work has led to numerous high-impact publications, particularly in the field of drug delivery and mucosal immunoengineering, and several patents.

bioengineering↗

bioRxiv2021

miR-409-3p represses Cited2 at the evolutionary emergence of the callosal and corticospinal projections

Callosal projection neurons are a broad population of interhemispheric projection neurons that extend an axon across the corpus callosum to connect the two cerebral hemispheres. The corticospinal tract, comprised of the axons of corticospinal projection neurons, is unique to mammals, and its full extension to the lumbar segments that control walking is, like the corpus callosum, unique to placental mammals. The emergence of these two distinct axonal tracts is thought to underpin the evolutionary expansion of complex motor and cognitive abilities. The molecular mechanisms regulating the divergence of corticospinal and callosal projection neurons are incompletely understood. Our recent work identifies a genomic cluster of microRNAs (12qF1/Mirg) unique to placental mammals. These clustered miRNAs are specifically expressed by corticospinal vs. callosal projection neurons during the molecular refinement of corticospinal vs. callosal projection neuron fate (1). One of these, miR-409-3p, can convert layer V callosal into corticospinal projection neurons, acting in part through repression of the callosal-expressed transcriptional regulator Lmo4. This conversion is partial, however, suggesting that miR-409-3p represses multiple callosal projection neuron control genes in order to specify corticospinal projection neurons. One potential additional target of miR-409-3p repression is the callosal-expressed transcriptional co-activator Cited2. Cited2 interacts genetically with Lmo4, and Lmo4 can partially functionally compensate for Cited2 in thymus development(2). Further, Cited2 and Lmo4 function as opposing molecular controls over specific areal identity within superficial layer callosal projection neurons of the somatosensory and motor cortices, respectively (3). Cited2 is highly expressed by callosal, relative to corticospinal, projection neurons from the earliest stages of neurogenesis. Cited2 is necessary for the expansion of intermediate progenitor cells (IPCs) in the subventricular zone (SVZ), and the resulting generation of superficial layer callosal projection neurons. Here we show that miR-409-3p and Cited2 interact in IPCs and in corticospinal vs. deep layer callosal projection neuron development. miR-409-3p represses the Cited2 3UTR in luciferase assays. Mirg, which encodes miR-409-3p, and Cited2, are reciprocally expressed in IPCs at e15.5 by qPCR. Furthermore, miR-409-3p gain-of-function results in a phenocopy of established Cited2 loss-of-function in IPCs. Later on, miR-409-3p and Cited2 exert opposing effects on the adoption of corticospinal vs. callosal projection neuron subtype identity. Taken together, our work suggests that miR-409-3p, and possibly other 12qF1 miRNAs, represses Cited2 in IPCs to limit their proliferation, and in developing corticospinal and deep layer callosal projection neurons to favor corticospinal fate.

neuroscience↗

bioRxiv2021

Demographic changes of a tropical understory bird in naturally patchy montane habitats in southern India

The occurrence, density and survival of a species often depend on various aspects of the habitat that it occupies including patch size and disturbance. The demography of most threatened tropical species largely remain unstudied but could provide valuable information about their biology and insights for their conservation. Our study examined the effect of patch size and disturbance on different demographic parameters of an understory, threatened, endemic bird, the white-bellied shortwing in the tropical biodiversity hotspot of the Western Ghats in India. We sampled eight plots on a sky-island using mist-nets for four years in a Robust design mark-recapture framework. Based on model selection using AIC values, the model with survival as a function of disturbance fits the data better than models with abundance or with these parameters modeled as functions of forest patch size. Shortwing density and sex ratio were not different across forest patch sizes or differing disturbance regimes. However, the survival rate of the species significantly decreased with increasing disturbance but was not related to forest patch size. Our study is possibly the first to provide critical baseline information on the demography of a tropical understory species from this region.

ecology↗

bioRxiv2021

Transplantation of bacteriophages from ulcerative colitis patients shifts the gut bacteriome and exacerbates severity of DSS-colitis

Inflammatory bowel diseases (IBDs) including Crohns disease (CD) and ulcerative colitis (UC) are characterized by chronic and debilitating gut inflammation. Altered bacterial communities of the intestine are strongly associated with IBD initiation and progression. The gut virome, which is primarily composed of bacterial viruses (bacteriophages, phages) is thought to be an important factor regulating and shaping microbial communities in the gut. While alterations in the gut virome have been observed in IBD patients, the contribution of these viruses to alterations in the bacterial community and heightened inflammatory responses associated with IBD patients remains largely unknown. Here, we performed in vivo microbial cross-infection experiments to follow the effects of fecal virus-like particles (VLPs) isolated from UC patients and healthy controls on bacterial diversity and severity of experimental colitis in human microbiota-associated (HMA) mice. Shotgun metagenomics confirmed that several phages were transferred to HMA mice, resulting in treatment-specific alterations in the gut virome. VLPs from healthy and UC patients also shifted gut bacterial diversity of these mice, an effect that was amplified during experimental colitis. VLPs isolated from UC patients specifically altered the relative abundance of several bacterial taxa previously implicated in IBD progression. Additionally, UC VLP administration heightened colitis severity in HMA mice, as indicated by shortened colon length and increased pro-inflammatory cytokine production. Importantly, this effect was dependent on intact VLPs. Our findings build on recent literature indicating that phages are dynamic regulators of bacterial communities in the gut and implicate the intestinal virome in modulating intestinal inflammation and disease.

microbiology↗

bioRxiv2021

Genomes of Wolbachia endosymbionts from the human filarial parasites Mansonella perstans and Mansonella ozzardi

The intracellular endosymbiotic proteobacteria Wolbachia have evolved across the phyla nematoda and arthropoda. In Wolbachia phylogeny, supergroup F is the only clade known so far with members from both arthropod and filarial nematode hosts and therefore can provide unique insights into their evolution and biology. In this study, 4 new supergroup F Wolbachia genomes have been assembled using a metagenomic assembly and binning approach, wMoz and wMpe from the human filarial parasites Mansonella ozzardi and Mansonella perstans, and wOcae and wMoviF from the blue mason bee Osmia caerulescens and the sheep ked Melophagus ovinus respectively. A comprehensive phylogenomic analysis revealed two independent origins of filarial Wolbachia in supergroup F, most likely from ancestral arthropod hosts. The analysis also reveals that the switch from arthropod to filarial host is accompanied by a convergent pseudogenization and loss of the bacterioferritin gene, a phenomenon found to be shared by all filarial Wolbachia, even those outside supergroup F. These observations indicate that differences in heme metabolism might be a key feature distinguishing filarial and arthropod Wolbachia. The new genomes provide a valuable resource for further studies on symbiosis, evolution, and the discovery of new antibiotics to treat mansonellosis. Significance statementWolbachia are bacterial endosymbionts of medically important parasitic filarial nematodes and arthropods. The evolutionary history and biological roles of Wolbachia in these different hosts are not well understood. The supergroup F in Wolbachia phylogeny harbors members from both filarial and arthropod hosts, providing an unparalleled opportunity for genomic comparisons to uncover distinguishing characteristics. This study provides new genomes from filarial and arthropod Wolbachia from this unique supergroup. Their phylogenomic analysis reveals multiple, independent transfers of Wolbachia from arthropod to filariae. Remarkably, these transfers were associated with a convergent loss of the bacterioferritin gene, a key regulator of heme metabolism. Heme supplementation is considered a critical component of Wolbachia - filaria symbiosis. We demonstrate bacterioferritin loss is a novel feature exclusive to filarial Wolbachia.

genomics↗

bioRxiv2021

Bone Marrow-Derived AXL Tyrosine Kinase Promotes Mitogenic Crosstalk and Cardiac Allograft Vasculopathy

Cardiac Allograft Vasculopathy (CAV) is a leading contributor to late transplant rejection. Although implicated, the mechanisms by which bone marrow-derived cells promote CAV remain unclear. Emerging evidence implicates the cell surface receptor tyrosine kinase AXL to be elevated in rejecting human allografts. AXL protein is found on multiple cell types, including bone marrow-derived myeloid cells. The causal role of AXL from this compartment and during transplant is largely unknown. This is important because AXL is a key regulator of myeloid inflammation. Utilizing experimental chimeras deficient in the bone marrow-derived Axl gene, we report that Axl antagonizes cardiac allograft survival and promotes CAV. Flow cytometric and histologic analyses of Axl-deficient transplant recipients revealed reductions in both allograft immune cell accumulation and vascular intimal thickness. Co-culture experiments designed to identify cell-intrinsic functions of Axl uncovered complementary cell-proliferative pathways by which Axl promotes CAV-associated inflammation. Specifically, Axl-deficient myeloid cells were less efficient at increasing the replication of both antigen-specific T cells and vascular smooth muscle cells (VSMCs), the latter a key hallmark of CAV. For the latter, we discovered that Axl-was required to amass the VSMC mitogen Platelet-Derived Growth Factor. Taken together, our studies reveal a new role for myeloid Axl in the progression of CAV and mitogenic crosstalk. Inhibition of AXL-protein, in combination with current standards of care, is a candidate strategy to prolong cardiac allograft survival.

immunology↗

bioRxiv2021

H3K27me3 mediated KRT14 upregulation promotes TNBC peritoneal metastasis

Triple Negative Breast Cancer (TNBC) is known to have poor prognosis and adverse clinical outcome among all breast cancer subtypes due to the absence of available targeted therapy for it. Emerging literature indicates that epigenetic reprogramming is now appreciated as a driving force for TNBC pathophysiology. High expression of epigenetic modulator EZH2 (Enhancer of zeste homolog 2) has been shown to correlate with TNBC poor prognosis but the contribution of EZH2 catalytic (H3K27me3) versus non-catalytic EZH2 (NC-EZH2) function in TNBC growth and progression remains elusive. In the process of dissecting the impact of H3K27me3 versus NC-EZH2 function in TNBC pathogenesis, we reveal that selective hyperactivation of H3K27me3 over NC-EZH2 not only promotes TNBC metastasis but also alters the metastatic landscape of TNBC. Using extensive in- vivo live animal imaging, we present conclusive evidence that peritoneal metastasis, particularly splenic metastasis of TNBC is governed by H3K27me3. Transcriptome analyses of hyperactive H3K27me3 cells lead us to discover Cytokeratin-14 (KRT14) as a new target of H3K27me3. Unlike classical H3K27me3 mediated suppression of gene expression, here; we observe that H3K27me3 enhances KRT14 transcription by attenuating the binding of transcriptional repressor Sp1 to its promoter. Further, loss of KRT14 significantly reduces TNBC migration, invasion and splenic metastasis. Finally, genetic ablation of EZH2 or pharmacological inhibition of EZH2 catalytic function by FDA approved drug tazemetostat (EPZ6438) robustly inhibits TNBC peritoneal metastasis. Altogether, our preclinical findings posit a rational insight for the clinical development of H3K27me3 inhibitor like tazemetostat as a targeted therapy against TNBC.

cancer biology↗

bioRxiv2021

Immunogenic Potential of DNA Vaccine candidate, ZyCoV-D against SARS-CoV-2 in Animal Models

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), initially originated in China in year 2019 and spread rapidly across the globe within 5 months, causing over 96 million cases of infection and over 2 million deaths. Huge efforts were undertaken to bring the COVID-19 vaccines in clinical development, so that it can be made available at the earliest, if found to be efficacious in the trials. We developed a candidate vaccine ZyCoV-D comprising of a DNA plasmid vector carrying the gene encoding the spike protein (S) of the SARS-CoV-2 virus. The S protein of the virus includes the receptor binding domain (RBD), responsible for binding to the human angiotensin converting enzyme (ACE-2) receptor. The DNA plasmid construct was transformed into E. coli cells for large scale production. The immunogenicity potential of the plasmid DNA has been evaluated in mice, guinea pig, and rabbit models by intradermal route at 25, 100 and 500g dose. Based on the animal studies proof-of-concept has been established and preclinical toxicology (PCT) studies were conducted in rat and rabbit model. Preliminary animal study demonstrates that the candidate DNA vaccine induces antibody response including neutralizing antibodies against SARS-CoV-2 and also provided Th-1 response as evidenced by elevated IFN-{gamma} levels.

immunology↗

bioRxiv2021

Transient response of basal ganglia network in healthy and low-dopamine state

The basal ganglia (BG) are crucial for a variety of motor and cognitive functions. Changes induced by persistent low-dopamine (e.g. in Parkinsons disease), result in aberrant changes in steady-state population activity ({beta}-band oscillations) and transient response of the BG. Typically, brief cortical stimulation results in a triphasic response in the substantia nigra pars reticulata (SNr, output of the BG). The properties of the triphasic responses are shaped by dopamine levels. While it is relatively well understood how changes in BG result in aberrant steady state activity, it is not clear which BG interactions are crucial for the aberrant transient responses in the BG. Moreover, it is also not clear whether the same or different mechanisms underlie the aberrant changes in steady-state activity and aberrant transient response. Here we used numerical simulations of a network model of BG to identify the key factors that determine the shape of the transient responses. We show that an aberrant transient response of the SNr in low-dopamine state, involves changes in both, the direct pathway and the recurrent interactions within the globus pallidus externa (GPe) and between GPe and sub-thalamic nucleus. We found that the connections from D2-type spiny projection neurons to GPe are most crucial in shaping the transient response and by restoring them to their healthy level, we could restore the shape of transient response even in low-dopamine state. Finally, we show that the changes in BG that result in aberrant transient response are also sufficient to generate pathological oscillatory activity. Significance statementTo understand how changes induced by low-dopamine (e.g. in Parkinsons disease, PD) affect basal ganglia (BG) function, we need to identify the factors that determine the shape of BG responses to brief cortical stimuli. We show that transient response of the BG is also affected by recurrent interactions within the subnuclei of the BG, and not just feedforward pathways. We found that input and local connectivity within the globus pallidus externa are crucial for shaping the transient response. We also show that the same network changes may underlie both, pathological {beta}-band oscillations and aberrant transient responses. Our results highlight the importance of the recurrent connectivity within the BG and provide a coherent view of emergence of pathological activity in PD.

neuroscience↗

bioRxiv2020

Identification of Covalent Modifications Regulating Immune Signaling Complex Composition and Phenotype

Cells signal through rearrangements of protein communities governed by covalent modifications and reversible interactions of distinct sets of proteins. A method that identifies those post-transcriptional modifications regulating signaling complex composition and functional phenotypes in one experimental setup would facilitate an efficient identification of novel molecular signaling checkpoints. Here we devised Modifications, Interactions and Phenotypes by Affinity Purification Mass Spectrometry (MIP-APMS), comprising the streamlined cloning and transduction of tagged proteins into functionalized reporter cells as well as affinity chromatography, followed by MS-based quantification. We report the time-resolved interplay of more than 50 previously undescribed modification and hundreds of protein-protein interactions of 19 immune protein complexes in monocytes. Validation of interdependecies between covalent, reversible and functional protein complex regulations by knock-out or site-specific mutation, revealed isgylation and phosphorylation of TRAF2 as well as ARHGEF18 interaction in Toll-like receptor 2 signaling. Moreover, we identify distinct mechanisms of action for small molecule inhibitors of p-38 (MAPK14). Our method provides a fast and cost-effective pipeline for the molecular interrogation of protein communities in diverse biological systems and primary cells. HighlightsO_LIExperimental framework to reveal dynamic signaling checkpoints in primary cells C_LIO_LIIdentification of crosstalk between protein modifications and interactions in signaling complexes C_LIO_LIDiscovery of TRAF2 isgylation, phosphorylation and ARHGEF18 interaction in monocytes C_LIO_LIDifferential drug mode of action for p-38 (MAPK14) kinase inhibitors C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/384750v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@1e833d3org.highwire.dtl.DTLVardef@1a2805forg.highwire.dtl.DTLVardef@199e1eaorg.highwire.dtl.DTLVardef@1becf99_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2020

Rub1/NEDD8, a ubiquitin-like modifier, is also a ubiquitin modifier

Of all ubiquitin-like small protein modifiers, Rub1/NEDD8 is the closest kin of ubiquitin in sequence and in structure. Despite their profound similarities, prevalence of ubiquitin and of Rub1 is starkly different: targets of ubiquitin modification reach into the thousands, whereas unique targets of Rub1/NEDD8 appear limited to one family of proteins, Cullins. This distinction is likely due to dedicated E1 activating enzymes that select either one or the other and relay the modifier until it is covalently attached to a target. To convert typical neddylation targets for modification by ubiquitin, and vice versa, we designed reciprocal substitutions at position 72 of Rub1 and of ubiquitin to render them substrates for activation by their non-cognate E1 activating enzymes. We found that this single amino acid is sufficient to distinguish between Ub and Rub1 in living cells, and determine their targets. Thus, modification of Cullins by UbR72T could compensate for loss of Rub1, even as it maintained its ability to polymerize and direct conjugates for degradation. Conversely, Rub1T72R activated by ubiquitin-activating enzyme entered into the ubiquitination cascade, however was not efficiently polymerized, essentially capping polyubiquitin chains. Upon shortage of free ubiquitin under stress, even native Rub1 spilled-over into the ubiquitinome suppressing polyubiquitination. By contrast, the need to maintain monomeric modifications on unique targets is a likely explanation for why the Rub1-activating enzyme strictly discriminates against ubiquitin. Swapping Rub1 and ubiquitin signals uncovered a reason for maintaining two separate pathways across eukaryotic kingdom.

biochemistry↗

bioRxiv2020

HIF2α is a Direct Regulator of Neutrophil Motility

Orchestrated recruitment of neutrophils to inflamed tissue is essential during initiation of inflammation. Inflamed areas are usually hypoxic, and adaptation to reduced oxygen pressure is typically mediated by hypoxia pathway proteins. However, it is still unclear how these factors influence the migration of neutrophils to and at the site of inflammation either during their transmigration through the blood-endothelial cell barrier, or their motility in the interstitial space. Here, we reveal that activation of the Hypoxia Inducible Factor-2 (HIF2) due to deficiency of HIF-prolyl hydroxylase domain protein-2 (PHD2) boosts neutrophil migration specifically through highly confined microenvironments. In vivo, the increased migratory capacity of PHD2-deficient neutrophils resulted in massive tissue accumulation in models of acute local inflammation. Using systematic RNAseq analyses and mechanistic approaches, we identified RhoA, a cytoskeleton organizer, as the central downstream factor that mediates HIF2-dependent neutrophil motility. Thus, we propose that the here identified novel PHD2-HIF2-RhoA axis is vital to the initial stages of inflammation as it promotes neutrophil movement through highly confined tissue landscapes.

immunology↗

bioRxiv2020

A bipartite element with allele-specific functions safeguards DNA methylation imprints at the Dlk1-Dio3 locus

Dysregulation of imprinted gene loci also referred to as loss of imprinting (LOI) can result in severe developmental defects and other diseases, but the molecular mechanisms that ensure imprint stability remain incompletely understood. Here, we dissect the functional components of the imprinting control region of the essential Dlk1-Dio3 locus (called IG-DMR) and the mechanism by which they ensure imprinting maintenance. Using pluripotent stem cells carrying an allele-specific reporter system, we demonstrate that the IG-DMR consists of two antagonistic regulatory elements: a paternally methylated CpG-island that prevents the activity of Tet dioxygenases and a maternally unmethylated regulatory element, which serves as a non-canonical enhancer and maintains expression of the maternal Gtl2 lncRNA by precluding de novo DNA methyltransferase function. Targeted genetic or epigenetic editing of these elements leads to LOI with either bi-paternal or bi-maternal expression patterns and respective allelic changes in DNA methylation and 3D chromatin topology of the entire Dlk1-Dio3 locus. Although the targeted repression of either IG-DMR or Gtl2 promoter is sufficient to cause LOI, the stability of LOI phenotype depends on the IG-DMR status, suggesting a functional hierarchy. These findings establish the IG-DMR as a novel type of bipartite control element and provide mechanistic insights into the control of Dlk1-Dio3 imprinting by allele-specific restriction of the DNA (de)methylation machinery. HIGHLIGHTSO_LIThe IG-DMR is a bipartite element with distinct allele-specific functions C_LIO_LIA non-canonical enhancer within the IG-DMR prevents DNA methyltransferase activity C_LIO_LITargeted epigenome editing allows induction of specific imprinting phenotypes C_LIO_LICRISPRi reveals a functional hierarchy between DMRs that dictates imprint stability C_LI

developmental biology↗

bioRxiv2020

Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems

AbstractMethods for highly multiplexed RNA imaging are limited in spatial resolution, and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to mouse brain, yielding readout of thousands of genes, including splice variants and novel transcripts. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in neurons of the mouse hippocampus, revealing patterns across multiple cell types; layer-specific cell types across mouse visual cortex; and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus ExSeq enables highly multiplexed mapping of RNAs, from nanoscale to system scale. One Sentence SummaryIn situ sequencing of physically expanded specimens enables multiplexed mapping of RNAs at nanoscale, subcellular resolution.

genomics↗

bioRxiv2020

Computational front end to diverse therapeutic phage cocktails

New therapies are necessary to combat increasingly antibiotic-resistant bacterial pathogens. We have developed a technology platform of computational, molecular biology, and microbiology tools which together enable on-demand production of phages that target virtually any given bacterial isolate. Two complementary computational tools that identify and precisely map prophages and other integrative genetic elements (IGEs) in bacterial genomes are used to identify prophage-laden bacteria that are close relatives of the target strain. Phage genomes are engineered to disable lysogeny, through use of long amplicon PCR and Gibson assembly. Finally, the engineered phage genomes are introduced into host bacteria for phage production. As an initial demonstration, we used this approach to produce a phage cocktail against the opportunistic pathogen Pseudomonas aeruginosa PAO1. Two prophage-laden P. aeruginosa strains closely related to PAO1 were identified, ATCC 39324 and ATCC 27853. Deep sequencing revealed that mitomycin C treatment of these strains induced seven phages that grow on P. aeruginosa PAO1. The most diverse five of these were engineered for non-lysogeny by deleting the integrase gene (int), which is readily identifiable and typically conveniently located at one end of the prophage. The {Delta}int phages, individually and in cocktails, showed killing of P. aeruginosa PAO1 in vitro as well as in a waxworm (Galleria mellonella) model of infection. SIGNIFICANCE STATEMENTThe antibiotic-resistance crisis in medicine and agriculture has led to renewed interest in phage therapy as an alternative means of treating infection. However, conventional methods for isolating pathogen-specific phage are slow, labor-intensive, and frequently unsuccessful. We have demonstrated that prophages carried by near-neighbor bacteria can serve as starting material for production of engineered phages that kill the target pathogen. Our approach and technology platform offer new opportunity for rapid development of phage therapies against most, if not all, bacterial pathogens, a foundational advance for use of phage in treating infectious disease.

microbiology↗

bioRxiv2020

Genome Sequences of Burkholderia thailandensis strains E421, E426, and DW503

We present the draft genome sequences of three Burkholderia thailandensis strains: E421, E426, and DW503. E421 consists of 90 contigs of 6,639,935bp and 67.73% GC content. E426 consists of 106 contigs of 6,587,853bp and 67.73% GC content. DW503 consists of 102 contigs of 6,458,767bp and 67.64% GC content.

genomics↗

bioRxiv2019

Integrative genomic and epigenomic analyses identify a distinct role of c-Myc and L-Myc for lineage determination in small cell lung cancer

Comprehensive genomic analyses of small cell lung cancer (SCLC), the most aggressive form of lung cancer, have revealed near universal loss of tumor suppressors (RB1 and TP53) and frequent genomic amplification of all three MYC family members. The amplification of each Myc family member is mutually exclusive; hence it had been long suggested that they are functionally equivalent. However, their expression has more recently been associated with specific neuroendocrine markers and distinct histopathology. In this study, we explored a novel role of c-Myc and L-Myc as lineage determining factors contributing to SCLC molecular subtypes and histology. Integrated analyses of a gene regulatory network generated from mRNA expression of primary SCLC tumor and chromatin state profiling of SCLC cell lines showed that Myc family members impart distinct transcriptional programs associated with lineage state; wherein the L-Myc signature was enriched for neuronal pathways while the c-Myc signature was enriched for Notch signaling and epithelial-to-mesenchymal transition. We investigated the functional redundancy and distinction of c-Myc and L-Myc, and noted the insufficiency of L-Myc to induce lineage switch in contrast to the potential of c-Myc to induce trans-differentiation. c-Myc rewires the Myc-accessible landscape and activates neuron al repressor, Rest to mediate transition from ASCL1-SCLC to NeuroD1-SCLC characterized by distinct LCNEC-like histopathology. Collectively, our findings reveal a previously undescribed role of historically defined general oncogenes, c-Myc and L-Myc, for regulating lineage plasticity across molecular subtypes as well as histological subclasses.

cancer biology↗