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Rao, A.

Publications and source records attributed to Rao, A..

At least 91 records · Page 5Linked to original sources
bioRxiv2020

A Structured Professional Development Curriculum for Postdoctoral Fellows Leads to Recognized Knowledge Growth

Postdoctoral training enables research independence and professional readiness. National reports have emphasized professional development as a critical component of this training period. In response, many institutions are establishing transferable skills training workshops for postdocs; however, the lack of structured programs and an absence of methods to assess outcomes beyond participant satisfaction surveys are critical gaps in postdoctoral training. To address these shortcomings, we took the approach of structured programming and developed a method for controlled assessment of outcomes. Our program You3 (You, Your Team, Your Project), co-designed by postdoctoral fellows, focused on a structured array of management and leadership skills agnostic of ultimate career path(s). We then measured outcomes in a controlled manner, by systematically comparing perceived knowledge and growth of participants with non-participants as the control group. You3 participants discern greater growth, independent of number of years in training, in competencies overall compared to the control group. This growth was shown by multiple criteria including self-reporting and associative analysis. Correspondingly, You3 participants reported greater knowledge in 75% of the modules when compared to controls. These data indicate that structured learning, where postdocs commit to a curriculum via a cohort-structure, leads to positive outcomes and provides a framework for programs to assess outcomes in a rigorous manner.

scientific communication and education↗
bioRxiv2020

Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma

In melanoma, transcriptional profiling has revealed multiple co-existing cell states, including proliferative versus invasive sub-populations that have been posited to represent a "go or grow" tradeoff. Both of these populations are maintained in tumors, but how they physically interact to promote metastasis is unknown. We demonstrate that these subpopulations form spatially structured heterotypic clusters that cooperate in the seeding of metastasis. We unexpectedly found that INV cells were tightly adherent to each other, and formed clusters with a rim of PRO cells. Intravital imaging demonstrated cooperation between these populations, in which the INV cells facilitated the spread of less metastatic PRO cells. We identified the TFAP2 neural crest transcription factor as a master regulator of both clustering and the PRO/INV states. Our data suggest a framework for the co-existence of these two divergent cell populations, in which differing cell states form heterotypic clusters that promote metastasis via cell-cell cooperation.

cancer biology↗
bioRxiv2020

Role of Anti-SARS-CoV-2 antibodies in different cohorts: Can they provide clues for appropriate patient triaging?

The emergence of coronavirus disease 2019 (COVID-19) has become a major global health crisis. Currently, diagnosis is based on molecular techniques, which detect the viral nucleic acids when present at detectable levels. The serum IgG response against SARS-CoV-2 was examined by using an ELISA-based assay. Serum samples, along with nasopharyngeal specimens were collected from various cohorts and analyzed by ELISA and rRT-PCR, respectively. A total of 167 serum samples were tested for serum IgG antibodies against SARS-CoV-2 in outpatient cohorts, 15 (8.9%) were positive by rRT-PCR and the remaining 152 (91%) were negative. We used these data to generate two different assay cutoffs for serum IgG assay and investigated percent concordance with rRT-PCR test results. The emergency department data revealed, out of 151 nasopharyngeal swabs, 4 (2.6%) were positive by rRT-PCR and 18 (11.9%) were positive for serum IgG assay. Among the 18 patients that were positive for serum IgG, 13 (72.2%) exhibited 1-3 symptoms of COVID-19 and 5 (27.7%) patients did not present with any COVID-19 related symptoms, per CDC criteria. All 4 (100%) patients that were positive by rRT-PCR had symptoms of COVID-19 disease. A longitudinal study from the inpatient population suggested there was a sharp increase in the serum IgG titers in 5 patients, a moderate increase in 1 patient and a plateau in 3 patients. Sero-prevalence of COVID-19 disease in pre-procedure patients was 5.5%. Our findings suggest serological tests can be used for appropriate patient triaging when performed as an adjunct to existing molecular testing.

microbiology↗
bioRxiv2020

RAS-inhibiting biologics identify and probe druggable pockets including an SII-α3 allosteric site.

RAS mutations are the most common oncogenic drivers across human cancers, but there remains a paucity of clinically-validated pharmacological inhibitors of RAS, as druggable pockets have proven difficult to identify. We have identified two RAS-binding Affimer proteins, K3 and K6, that inhibit nucleotide exchange and downstream signalling pathways with distinct isoform and mutant profiles. Affimer K6 is the first biologic to bind in the SI/SII pocket, whilst Affimer K3 is the first non-covalent inhibitor of the SII region, revealing a novel RAS conformer with a large, druggable SII/3 pocket. This work demonstrates the potential of using biologics with small interface surfaces to select novel druggable conformations in conjunction with pharmacophore identification for hard-to-drug proteins.

molecular biology↗
bioRxiv2020

Chromosome scale reference genome of Cluster bean (Cyamopsis tetragonoloba (L.) Taub.)

Clusterbean (Cyamopsis tetragonoloba (L.) Taub.), also known as Guar is a widely cultivated dryland legume of Western India and parts of Africa. Apart from being a vegetable crop, it is also an abundant source of a natural hetero-polysaccharide called guar gum or galactomannan which is widely used in cosmetics, pharmaceuticals, food processing, shale gas drilling etc. Here, for the first time we are reporting a chromosome-scale reference genome assembly of clusterbean, from a high galactomannan containing popular guar cultivar, RGC-936, by combining sequenced reads from Illumina, 10x Chromium and Oxford Nanopore technologies. The initial assembly of 1580 scaffolds with an N50 value of 7.12 Mbp was generated. Then, the final genome assembly was obtained by anchoring these scaffolds to a high density SNP map. Finally, a genome assembly of 550.31 Mbp was obtained in 7 pseudomolecules corresponding to 7 chromosomes with a very high N50 of 78.27 Mbp. We finally predicted 34,680 protein-coding genes in the guar genome. The high-quality chromosome-scale cluster bean genome assembly will facilitate understanding of the molecular basis of galactomannan biosynthesis and aid in genomics-assisted breeding of superior cultivars.

genomics↗
bioRxiv2020

Mobilization of tissue-resident memory CD4+ T lymphocytes and their contribution to a systemic secondary immune reaction

While it is generally accepted that tissue-resident memory T lymphocytes protect host tissues from secondary immune challenges, it is unclear whether, and if so, how they contribute to systemic secondary immune responses. Here we show that in human individuals with an established immune memory to measles, mumps and rubella viruses, when challenged with the measles-mumps-rubella (MMR) vaccine again, tissue-resident memory CD4+ T cells are mobilized into the blood within 16 to 48 hours after vaccination. These cells then leave the blood again, and apparently contribute to the systemic secondary immune reaction, as is evident from the representation of mobilized T cell receptor V{beta} clonotypes among newly generated circulating memory T lymphocytes, from day 7 onwards. Mobilization of the tissue-resident memory T cells is cognate, in that memory T lymphocytes recognizing other antigens, e.g. tetanus toxin, are not mobilized, unless they cross-react with the vaccine. These data originally demonstrate the essential contribution of tissue-resident memory T cells to secondary systemic immune responses, confirming that immunological memories to systemic pathogens are maintained (also) by tissue-resident memory T cells. In practical terms, the present work defines day 1 to 2 after antigenic challenge as a time window to assess the entire immunological T cell memory for a certain pathogen, including mobilized tissue-resident memory T cells, and its correlates of effectivity. Capsule summaryThe study demonstrates the rapid and cognate mobilization of tissue-resident memory CD4+ T cells into the blood upon antigenic rechallenge, and their contribution to secondary systemic immune responses.

immunology↗
bioRxiv2019

Determination of secretory granule maturation times in pancreatic islet beta-cells by serial block face scanning electron microscopy

It is shown how serial block-face electron microscopy (SBEM) of insulin-secreting beta cells in wild-type mouse pancreatic islets of Langerhans can be used to determine maturation times of secretory granules. Although SBEM captures the beta cell structure at a snapshot in time, the observed ultrastructure can be considered representative of a dynamic equilibrium state of the cells since the pancreatic islets are maintained in culture in approximate homeostasis. It is found that 7.2{+/-}1.2% ({+/-}st. dev.) of the beta cell volume is composed of secretory granule dense-cores exhibiting angular shapes surrounded by wide (typically {gtrsim}100 nm) electron-lucent halos. These organelles are identified as mature granules that store insulin for regulated release through the plasma membrane, with a release time of 96{+/-}12 hours, as previously obtained from pulsed 35S-radiolabeling of cysteine and methionine. Analysis of beta cell 3D volumes reveals a subpopulation of secretory organelles without electron-lucent halos, identified as immature secretory granules. Another subpopulation of secretory granules is found with thin (typically [lsim]30 nm) electron-lucent halos, which are attributed to immature granules that are transforming from proinsulin to insulin by action of prohormone convertases. From the volume ratio of proinsulin in the immature granules to insulin in the mature granules, we estimate that the newly formed immature granules remain in morphologically-defined immature states for an average time of 135{+/-}14 minutes, and the immature transforming granules for an average time of 130{+/-}17 minutes.

biophysics↗
bioRxiv2019

Impact of admixture and ancestry on eQTL analysis and GWAS colocalization in GTEx

BackgroundPopulation structure among study subjects may confound genetic association studies, and lack of proper correction can lead to spurious findings. The Genotype-Tissue Expression (GTEx) project largely contains individuals of European ancestry, but the final release (v8) also includes up to 15% of individuals of non-European ancestry. Assessing ancestry-based adjustments in GTEx provides an opportunity to improve portability of this research across populations and to further measure the impact of population structure on GWAS colocalization.\n\nResultsHere, we identify a subset of 117 individuals in GTEx (v8) with a high degree of population admixture and estimate genome-wide local ancestry. We perform genome-wide cis-eQTL mapping using admixed samples in six tissues, adjusted by either global or local ancestry. Consistent with previous work, we observe improved power with local ancestry adjustment. At loci where the two adjustments produce different lead variants, we observe only 0.8% of tests with GWAS colocalization posterior probabilities that change by 10% or more. Notably, both adjustments produce similar numbers of significant colocalizations. Finally, we identify a small subset of GTEx v8 eQTL-associated variants highly correlated with local ancestry (R2 > 0.7), providing a resource to enhance functional follow-up.\n\nConclusionsWe provide a local ancestry map for admixed individuals in the final GTEx release and describe the impact of ancestry and admixture on gene expression, eQTLs, and GWAS colocalization. While the majority of results are concordant between local and global ancestry-based adjustments, we identify distinct advantages and disadvantages to each approach.

genetics↗
bioRxiv2019

Widespread dose-dependent effects of RNA expression and splicing on complex diseases and traits

The resources generated by the GTEx consortium offer unprecedented opportunities to advance our understanding of the biology of human diseases. Here, we present an in-depth examination of the phenotypic consequences of transcriptome regulation and a blueprint for the functional interpretation of genome-wide association study-discovered loci. Across a broad set of complex traits and diseases, we demonstrate widespread dose-dependent effects of RNA expression and splicing. We develop a data-driven framework to benchmark methods that prioritize causal genes and find no single approach outperforms the combination of multiple approaches. Using colocalization and association approaches that take into account the observed allelic heterogeneity of gene expression, we propose potential target genes for 47% (2,519 out of 5,385) of the GWAS loci examined. Our results demonstrate the translational relevance of the GTEx resources and highlight the need to increase their resolution and breadth to further our understanding of the genotype-phenotype link.

genetics↗
bioRxiv2019

Multispectral Imaging Differentiates Unique Macrophage Profiles in Patients with Distinct Chronic Liver Diseases

Intrahepatic macrophages influence the composition of the microenvironment, host immune response to liver injury, and development of fibrosis. Compared to stellate cells, the role of intrahepatic macrophages in the development of fibrosis remains ill defined. Multispectral imaging allows detection of multiple markers in situ in human formalin-fixed, paraffin-embedded tissue. This cutting-edge technology is ideal for analyzing human liver tissues since it allows spectral unmixing of fluorophore signals, subtraction of auto-fluorescence, and preserves architecture and the in vivo hepatic milieu. We analyzed resident Kupffer cells (CD68+), monocyte-derived macrophages (Mac387+), pro-fibrogenic macrophages (CD163+), and co-expression of pro-inflammatory (CD14) and anti-inflammatory (CD16) markers in liver biopsies from patients with hepatitis C virus (HCV) and different stages of fibrosis. Liver biopsies with advanced fibrosis showed increased accumulation of CD163+, MAC387+ and CD68+ macrophages in the portal tracts when compared to those with minimal fibrosis. Imaging software generated t-distributed stochastic neighbor embedding (t-SNE) plots and phenotype matrices that facilitated comparison of macrophage profiles. These included monocyte-derived (CD68+/Mac387+) and pro-fibrotic/anti-inflammatory (CD163+/CD16+) phenotypes. We established that the utility of this platform could be extended to liver biopsies from patients with other chronic liver diseases including nonalcoholic steatohepatitis and autoimmune hepatitis. Each disease exhibited a unique profile after spectral imaging analysis and this platform holds the potential to identify patients predisposed to progressive liver disease based on the macrophage composition. In summary, spectral imaging is a powerful tool that enables analysis of macrophage profiles in different types of chronic liver diseases and has potential to change the manner in which we evaluate liver biopsies leading to more personalized treatment strategies.

pathology↗
bioRxiv2019

Long non-coding RNA gene regulation and trait associations across human tissues

Long non-coding RNA (lncRNA) genes are known to have diverse impacts on gene regulation. However, it is still a major challenge to distinguish functional lncRNAs from those that are byproducts of surrounding transcriptional activity. To systematically identify hallmarks of biological function, we used the GTEx v8 data to profile the expression, regulation, network relationships and trait associations of lncRNA genes across 49 tissues encompassing 87 distinct traits. In addition to revealing widespread differences in regulatory patterns between lncRNA and protein-coding genes, we identified novel disease-associated lncRNAs, such as C6orf3 for psoriasis and LINC01475/RP11-129J12.1 for ulcerative colitis. This work provides a comprehensive resource to interrogate lncRNA genes of interest and annotate cell type and human trait relevance.\n\nOne Sentence SummarylncRNA genes have distinctive regulatory patterns and unique trait associations compared to protein-coding genes.

genetics↗
bioRxiv2019

DDAP: docking domain affinity and biosynthetic pathway prediction tool for type I polyketide synthases

SummaryDDAP is a tool for predicting the biosynthetic pathways of the products of type I modular polyketide synthase (PKS) with the focus on providing a more accurate prediction of the ordering of proteins and substrates in the pathway. In this study, the module docking domain (DD) affinity prediction performance on a hold-out testing data set reached AUC = 0.88; the MRR of pathway prediction reached 0.67. DDAP has advantages compared to previous informatics tools in several aspects: (i) it does not rely on large databases, making it a high efficiency tool, (ii) the predicted DD affinity is represented by a probability (0 to 1), which is more intuitive than raw scores, (iii) its performance is competitive compared to the current popular rule-based algorithm. To the best of our knowledge, DDAP is so far the first machine learning based algorithm for type I PKS pathway prediction. We also established the first database of type I modular PKSs, featuring a comprehensive annotation of available docking domains information in bacterial biosynthetic pathways.\n\nAvailability and implementationThe DDAP database is available at https://tylii.github.io/ddap. The prediction algorithm DDAP is freely available on GitHub (https://github.com/tylii/ddap) and released under the MIT license.\n\nContactukarvind@umich.edu

bioinformatics↗
bioRxiv2019

Heterogeneous disease-propagating stem cells in juvenile myelomonocytic leukemia

Juvenile Myelomonocytic Leukemia (JMML) is a poor prognosis childhood leukemia usually caused by germline or somatic RAS-activating mutations. The cellular hierarchy in JMML is poorly characterized, including the identity of leukemia stem cells (LSCs). FACS and single-cell RNA-sequencing reveal marked heterogeneity of JMML hematopoietic stem/progenitor cells (HSPCs), including an aberrant Lin-CD34+CD38-CD90+CD45RA+ population. Single-cell HSPC index-sorting and clonogenic assays show that (1) all somatic mutations can be backtracked to the phenotypic HSC compartment with RAS-activating mutations as a \"first hit\", (2) mutations are acquired with both linear and branching patterns of clonal evolution and (3) mutant HSPCs are present after allogeneic HSC transplant before molecular/clinical evidence of relapse. Stem cell assays reveal inter-patient heterogeneity of JMML-LSCs which are present in, but not confined to, the phenotypic HSC compartment. RNA-sequencing of JMML-LSCs reveals upregulation of stem cell and fetal genes (HLF, MEIS1, CNN3, VNN2, HMGA2) and candidate therapeutic targets/biomarkers (MTOR, SLC2A1, CD96) paving the way for LSC-directed disease monitoring and therapy in this disease.

cancer biology↗
bioRxiv2019

Mapping interactions of microbial metabolites and human receptors

Despite evidence linking the human microbiome to health and disease, the mechanistic details of how the microbiota affects human physiology remain largely unknown. Metabolites encoded by bacteria are expected to play an integral role in the microbiotas effect on its human host. Assigning function to these metabolites is therefore critical to determining the molecular underpinnings of the host-microbe relationship and ultimately developing microbiota inspired therapies. Here we use large-scale functional screening of small molecules produced by individual members of a simplified human microbiota to identify bacterial metabolites that agonize G-protein coupled receptors (GPCR). This analysis revealed a complex network of metabolite host receptor interactions and guided our identification of multiple microbiota derived agonists of GPCRs associated with diverse biological functions within the nervous and immune systems, among others. Collectively, the metabolite-receptor pairs we uncovered indicate that diverse aspects of human health are potentially modulated by structurally simple metabolites arising from primary bacterial metabolism.\n\nStatement of SignificanceBacteria residing within the human body have been shown to influence human health. It is likely that physiological responses to the human microbiota are mediated by the collection of small molecules encoded within these bacteria. In this study we use direct functional screening of small molecules produced by individual members of a simplified human microbiota to identify new G protein coupled receptor-metabolite interactions that seek to explain the molecular underpinnings of the microbiotas influence on its human host.

microbiology↗
bioRxiv2019

Adaptive substitutions underlying cardiac glycoside insensitivity in insects exhibit epistasis in vivo.

Predicting how species will respond to selection pressures requires understanding the factors that constrain their evolution. We use genome engineering of Drosophila to investigate constraints on the repeated evolution of unrelated herbivorous insects to toxic cardiac glycosides, which primarily occurs via a small subset of possible functionally-relevant substitutions to Na+,K+-ATPase. Surprisingly, we find that frequently observed adaptive substitutions at two sites, 111 and 122, are lethal when homozygous and adult heterozygotes exhibit dominant neural dysfunction. We identify a phylogenetically correlated substitution, A119S, that partially ameliorates the deleterious effects of substitutions at 111 and 122. Despite contributing little to cardiac glycoside-insensitivity in vitro, A119S, like substitutions at 111 and 122, substantially increases adult survivorship upon cardiac glycoside exposure. Our results demonstrate the importance of epistasis in constraining adaptive paths. Moreover, by revealing distinct effects of substitutions in vitro and in vivo, our results underscore the importance of evaluating the fitness of adaptive substitutions and their interactions in whole organisms.

evolutionary biology↗
bioRxiv2019

MeCP2 nuclear dynamics in live neurons results from low and high affinity chromatin interactions

Loss of function mutations in Methyl-CpG-binding Protein 2 (MeCP2) cause the severe neurological disorder Rett Syndrome. MeCP2 is a highly abundant nuclear protein particularly enriched in neurons. Although biochemical and genomic analyses of MeCP2-DNA interaction and genomic distribution demonstrate that MeCP2 binding on chromatin is dependent on DNA modification state, the dynamic behavior of individual MeCP2 proteins in live neurons has not been explored. Here we use live-cell single-molecule imaging to assess the detailed kinetic features of MeCP2 in distinct sub-nuclear regions at high spatial and temporal resolution. Surprisingly, we found that, in granule cell nuclei, MeCP2 has unique diffusion and chromatin binding kinetics that are distinct from highly mobile sequence-specific transcription factors (TF) and immobile histone proteins. Approximately, half of MeCP2 is bound to DNA in a transiently stable mode that is similar to TF binding to their cognate sites. The binding of meCP2 to DNA requires its methyl-binding domain (MBD) and is sensitive to the levels of both DNA methylation and hydroxymethylation. However, when not stably bound, MeCP2 moves slowly in the nucleus most closely resembling histone H1.0. The rate of MeCP2 diffusion in compact, granule cell nuclei is determined by weak, transient DNA interactions mediated primarily by the MBD and three AT-hook domains located in the C-terminal portion of the protein. Both the fraction of stably bound MeCP2 and its rate of diffusion depend on the level of chromatin compaction and neuronal cell type. Our data reveal new features of MeCP2 that dictate its dynamic behavior in neuronal nuclei and suggest that the limited nuclear diffusion of MeCP2 in live neurons may contribute to its local impact on chromatin structure and gene expression.

molecular biology↗
bioRxiv2019

Loss of TET proteins in regulatory T cells unleashes effector function

TET enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and other oxidized methylcytosines, mediating DNA demethylation and serving as new epigenetic marks. Here we examine the role of TET proteins in Foxp3+ regulatory T (Treg) cells, a distinct lineage of CD4+ T cells that prevent autoimmunity and maintain immune homeostasis. We report that Tet2/3fl/flFoxp3Cre mice that lack Tet2 and Tet3 specifically in Treg cells develop splenomegaly, leukocyte infiltration into tissues and inflammatory disease. Treg cells from these mice showed altered expression of Treg signature genes, with upregulation of genes involved in cell cycle regulation, DNA damage repair and cancer. In littermate mice with more severe inflammation, both CD4+ Foxp3+ and CD4+ Foxp3- cells showed strong skewing towards Tfh and/or Th17 phenotypes. Notably, the presence of wild type Treg cells in mixed bone marrow chimeras and Tet2/3fl/flFoxp3WT/Cre heterozygous female mice did not suppress the aberrant phenotype and function of Tet2/3fl/flFoxp3Cre Treg cells. Fate-mapping experiments indicated that Treg cells from Tet2/3fl/flFoxp3Cre mice were more prone to lose Foxp3 expression; moreover, transfer of total CD4+ T cells isolated from Tet2/3fl/flFoxp3Cre mice could elicit inflammatory disease in fully immunocompetent mice. Together, these data indicate that Tet2 and Tet3 are guardians of Treg cell stability and immune homeostasis in mice.

immunology↗