Biology subjects

Rajan, A.

Publications and source records attributed to Rajan, A..

51 records · Page 3Linked to original sources

bioRxiv2021

Fat Body Phospholipid State Dictates Hunger Driven Feeding Behavior

Diet-induced obesity (DIO) leads to dysfunctional feeding behavior. However, the precise molecular nodes underlying diet-induced dysregulation of satiety sensing and feeding motivation are poorly understood. The fruit fly is a simple genetic model system yet displays significant evolutionary conservation to mammalian nutrient sensing and energy balance. Using a longitudinal high sugar regime in Drosophila, we sought to address how lipid alteration in fat cells alters feeding behavior. We find that prolonged exposure to HSD degrades the hunger-driven feeding (HDF) response. Lipidomics analysis reveals that longitudinal exposure to HSD significantly alters whole body phospholipid profiles. By performing a systematic screen for phospholipid enzymes, we identify Pect as a critical regulator of hunger-driven feeding. Pect is a rate-limiting enzyme in the phosphatidylethanolamine (PE) biosynthesis pathway and the fly ortholog of human PCYT2. We show that disrupting Pect only in the fat body causes insulin-resistant phenotypes and a loss of hunger-driven feeding. Excitingly, we find that overexpression of Pect restores HSD-induced loss of hunger-driven feeding response. Strikingly human studies have noted a correlation between PCYT2/Pect levels and clinical obesity. Now, our unbiased studies in Drosophila provide specific genetic evidence for Pect in maintaining nutrient sensing during DIO. Our study provides novel insights into the role of phospholipids in interorgan communication of nutrient status. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/472854v4_ufigA1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@1ecf3eorg.highwire.dtl.DTLVardef@b800f2org.highwire.dtl.DTLVardef@461a7eorg.highwire.dtl.DTLVardef@75651c_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2021

Fatquant: An automated image analysis tool for identification of fat cells in heterogeneous hematoxylin and eosin tissue sections.

Fatty infiltration in pancreas leading to steatosis is a major risk factor in pancreas transplantation. Hematoxylin and eosin (H and E) is one of the common histological staining techniques that provides information on the tissue cytoarchitecture. Adipose (fat) cells accumulation in pancreas has been shown to impact beta cell survival, its endocrine function and pancreatic steatosis and can cause non-alcoholic fatty pancreas disease (NAFPD). The current automated tools (E.g. Adiposoft) available for fat analysis are suited for white adipose tissue which is homogeneous and easier to segment unlike heterogeneous tissues such as pancreas where fat cells continue to play critical physiopathological functions. The currently, available pancreas segmentation tool focuses on endocrine islet segmentation based on cell nuclei detection for diagnosis of pancretic cancer. In the current study, we present a fat quantifying tool, Fatquant, which identifies fat cells in heterogeneous H and E tissue sections with reference to diameter of fat cell. Using histological images of pancreas from a publicly available database, we observed an intersection over union of 0.797 to 0.966 for manual versus fatquant based machine analysis. Author SummaryWe have developed an automated tool, Fatquant, for identification of fat cells based on its diameter in complex hematoxylin and eosin tissue sections such as pancreas which can aid the pathologist for diagnosis of fatty pancreas and related metabolic conditions. Fatquant is unique as current fat automated tools (adiposoft, adipocount) works well for homogeneous white adipose tissue but not for other tissue samples. The currently available pancreas analysis tool are mostly suited for segmentation of endocrine {beta}-cell based on cell nuclei detection, extracting colour features and cannot estimate fat cell infiltration in pancreas. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/472341v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@bfaa73org.highwire.dtl.DTLVardef@14f918aorg.highwire.dtl.DTLVardef@dc02e0org.highwire.dtl.DTLVardef@40d92f_HPS_FORMAT_FIGEXP M_FIG C_FIG Currently available fat quantification tools like adiposoft can analyze homogenous adipose tissue (left) with intersection over union (IoU) of 0.935 and 0.954 with adiposoft and fatquant, respectively. While in heterogenous tissue (e.g. pancreas on right) which contains adipose (fat cells), acinar cells, adiposoft fails to detect fat cells with IoU=0 while fatquant had IoU=0.797.

bioinformatics↗

bioRxiv2021

The human nose organoid respiratory virus model: an ex-vivo human challenge model to study RSV and SARS-CoV-2 pathogenesis and evaluate therapeutics

There is an unmet need for pre-clinical models to understand the pathogenesis of human respiratory viruses; and predict responsiveness to immunotherapies. Airway organoids can serve as an ex-vivo human airway model to study respiratory viral pathogenesis; however, they rely on invasive techniques to obtain patient samples. Here, we report a non-invasive technique to generate human nose organoids (HNOs) as an alternate to biopsy derived organoids. We made air liquid interface (ALI) cultures from HNOs and assessed infection with two major human respiratory viruses, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Infected HNO-ALI cultures recapitulate aspects of RSV and SARS-CoV-2 infection, including viral shedding, ciliary damage, innate immune responses, and mucus hyper-secretion. Next, we evaluated the feasibility of the HNO-ALI respiratory virus model system to test the efficacy of palivizumab to prevent RSV infection. Palivizumab was administered in the basolateral compartment (circulation) while viral infection occurred in the apical ciliated cells (airways), simulating the events in infants. In our model, palivizumab effectively prevented RSV infection in a concentration dependent manner. Thus, the HNO-ALI model can serve as an alternate to lung organoids to study respiratory viruses and testing therapeutics.

microbiology↗

bioRxiv2021

Multiple RSV strains infecting HEp-2 and A549 cells reveal cell line-dependent differences in resistance to RSV infection.

Respiratory syncytial virus (RSV) is a leading cause of pediatric acute respiratory infection worldwide. There are currently no approved vaccines or antivirals to combat RSV disease. A few transformed cell lines and two historic strains have been extensively used to study RSV. Here we report a thorough molecular and cell biological characterization of HEp-2 and A549 cells infected with four strains of RSV representing both major subgroups as well as historic and more contemporaneous genotypes -- [RSV/A/Tracy (GA1), RSV/A/Ontario (ON), RSV/B/18537 (GB1), RSV/B/Buenos Aires (BA)] -- via measurements of viral replication kinetics and viral gene expression, immunofluorescence-based imaging of gross cellular morphology and cell-associated RSV, and measurements of host response including transcriptional changes and levels of secreted cytokines and growth factors. Our findings strongly suggest 1) the existence of a conserved difference in gene expression between RSV subgroups A and B; 2) the A549 cell line is a more stringent and natural host of replicating RSV than the HEp-2 cell line; and 3) consistent with previous studies, determining the full effects of viral genetic variation in RSV pathogenesis requires model systems as tractable as transformed cell lines but better representative of the human host. IMPORTANCEInfection with respiratory syncytial virus (RSV) early in life is essentially guaranteed and can lead to severe disease. In vitro data from two historic RSV/A strains and two cell lines, HEp-2 and A549, constitute most of our knowledge; but RSV contains ample variation from two evolving subgroups (A and B) showing recent convergent evolution. Here we measure viral action and host response in HEp-2 and A549 cells infected with four RSV strains from both subgroups and representing both historic and more contemporaneous strains. We discover a subgroup-dependent difference in viral gene expression and find A549 cells are more potently antiviral and more sensitive, albeit subtly, to viral variation. Our findings reveal important differences between RSV subgroups and two widely used cell lines and provide baseline data for experiments with model systems better representative of natural RSV infection.

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

Drivers of Transcriptional Variance in Human Intestinal Epithelial Organoids

Background & AimsHuman intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the intestinal epithelium. Here, we explored the effect on the transcriptome of common variations in culture methods, including extracellular matrix substrate, format, tissue segment, differentiation status, and patient heterogeneity. MethodsRNA-sequencing datasets from 251 experiments performed on 35 human enteroid and colonoid lines from 28 patients were aggregated from several groups in the Texas Medical Center. DESeq2 and Gene Set Enrichment Analysis (GSEA) was used to identify differentially expressed genes and enriched of pathways. ResultsPERMANOVA, Pearson correlations, and dendrogram analysis of all data indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3D, transwell, and monolayer) had the largest effect (7,271-1,305 differentially expressed genes-DEGs); segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect (5,977-420 DEGs), and patient heterogeneity and specific experimental manipulations (e.g., pathogen infection) had the smallest effect. GSEA identified hundreds of pathways that varied between culture methods, such as IL1 cytokine signaling enriched in transwell vs. monolayer cultures, and cholesterol biosynthesis genes enriched in Matrigel vs. collagen cultures. ConclusionsSurprisingly large differences in organoid transcriptome were driven by variations in culture methods such as format and substrate, whereas experimental manipulations such as infection had modest effects. These results show that common variations in culture conditions can have large effects on intestinal organoids and should be accounted for when designing experiments and comparing results between laboratories. Our data constitute the largest RNA-seq dataset interrogating human intestinal organoids.

molecular biology↗

bioRxiv2021

Atg8 licenses adipokine nuclear exit

Adipokines released from the adipocytes function as a systemic adipometer; they impinge on neural circuits to signal nutrient status. On starvation, adipokines must be retained to signal energy deficit; else, it significantly reduces starvation survival. But how fat cells retain adipokines is unclear. Here, we demonstrate that Atg8, a cell-intrinsic autophagy factor, regulates the starvation-induced acute retention of the Leptin Drosophila ortholog Upd2. We show that on starvation, as a direct consequence of Atg8s lipidation, Upd2 accumulates in the nucleus. We illustrate that Upd2s nuclear retention is critical to fat mobilization and increased starvation resilience. Furthermore, nuclear Upd2 promotes the expression of a secreted innate immune gene signature. This hints at an unanticipated connection between adipokine nuclear retention and increased innate immunity. In conclusion, we propose that, during starvation, Atg8s role is not just limited to autophagy but is critical for withholding adipokines in the nucleus to promote starvation resilience. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=178 HEIGHT=200 SRC="FIGDIR/small/428913v5_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1826425org.highwire.dtl.DTLVardef@17cca52org.highwire.dtl.DTLVardef@1c70ac7org.highwire.dtl.DTLVardef@94a024_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIIn fed state Upd2 requires Atg8 for nuclear exit and cytosolic localization. C_LIO_LIAtg8s lipidation on starvation results in Upd2s nuclear accumulation. C_LIO_LIUpd2 nuclear retention on starvation increases fat mobilization and post-starvation hunger. C_LIO_LIOn starvation Upd2 nuclear retention increases expression of a secreted innate immune signature. C_LI

physiology↗

bioRxiv2020

Oligonucleotide Capture Sequencing of the SARS-CoV-2 Genome and Subgenomic Fragments from COVID-19 Individuals

The newly emerged and rapidly spreading SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). To facilitate a deeper understanding of the viral biology we developed a capture sequencing methodology to generate SARS-CoV-2 genomic and transcriptome sequences from infected patients. We utilized an oligonucleotide probe-set representing the full-length genome to obtain both genomic and transcriptome (subgenomic open reading frames [ORFs]) sequences from 45 SARS-CoV-2 clinical samples with varying viral titers. For samples with higher viral loads (cycle threshold value under 33, based on the CDC qPCR assay) complete genomes were generated. Analysis of junction reads revealed regions of differential transcriptional activity and provided evidence of expression of ORF10. Heterogeneous allelic frequencies along the 20kb ORF1ab gene suggested the presence of a defective interfering viral RNA species subpopulation in one sample. The associated workflow is straightforward, and hybridization-based capture offers an effective and scalable approach for sequencing SARS-CoV-2 from patient samples.

genomics↗

bioRxiv2020

Role of Inferior Frontal Junction (IFJ) in the Control of Feature vs Spatial Attention

Feature-based attention refers to preferential selection and processing of items and objects based on their non-spatial attributes such as color or shape. While it is intuitively an easier form of attention to relate to in our day to day lives, the neural mechanisms of feature-based attention are not well understood. Studies have long implicated the dorsal attention network as a key control system for voluntary spatial, feature and object-based attention. Recent studies have expanded on this model by focusing on the inferior frontal junction (IFJ), a region in the pre-frontal cortex to be the source of feature attention control, but not spatial attention control. However, the extent to which IFJ contributes to spatial attention remains a topic of debate. We investigated the role of IFJ in the control of feature versus spatial attention in a cued visual spatial (attend left or right) and feature attention (attend red or green) task using fMRI. Analyzing single-trial cue-evoked fMRI responses using univariate GLM and multi-voxel pattern analysis (MVPA), we observed the following. First, the univariate BOLD activation responses yielded no significant differences between feature and spatial cues. Second, MVPA analysis showed above chance level decoding in classifying feature attention (attend-red vs. attend-green) in both the left and right IFJ, whereas during spatial attention (attend-left vs. attend-right) decoding was at chance. Third, while the cue-evoked decoding accuracy was significant for both left and right IFJ during feature attention, target stimulus-evoked neural responses were not different. Importantly, only the connectivity patterns from the right IFJ was predictive of target-evoked activity in visual cortex (V4); this was true for both left and right V4. Finally, the strength of this connectivity between right IFJ and V4 (bilaterally) was found to be predictive of behavioral performance. These results support a model where the right IFJ plays a crucial role in top down control of feature but not spatial attention.

neuroscience↗

bioRxiv2020

Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals

microbiology↗

bioRxiv2020

Targeting of Mammalian Glycans Enhances Phage Predation in the Gastrointestinal Tract

The human mucosal surface consists of a eukaryotic epithelium, a prokaryotic microbiota, and a carbohydrate-rich interface that separates them. Bacteriophage parasitize the prokaryotes but are not known to associate with eukaryotic cells. In the gastrointestinal tract, the interaction of these two domains influences the health of the host, especially colonization with invasive pathobionts. Antibiotics may be used but they also kill protective commensals and lack the physio-chemical properties to be specifically and optimally active in this complex milieu. Here, we report a novel phage whose lytic cycle is enhanced in intestinal environments. The enhanced activity is encoded in its tail fiber gene, whose protein product binds human heparan sulfated proteoglycans and localizes the phage to the epithelial cell surface, thereby positioning it near its bacterial host, a type of locational targeting mechanism. This finding offers the prospect of developing epithelial-targeting phage to selectively remove invasive pathobiont species from mucosal surfaces. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/212829v2_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@13219f6org.highwire.dtl.DTLVardef@469d7borg.highwire.dtl.DTLVardef@d31e0org.highwire.dtl.DTLVardef@458e14_HPS_FORMAT_FIGEXP M_FIG C_FIG Model showing (1) mucins from the intestinal mucus layer inhibit phage infection, (2) phage ES17 can bind to mucin and utilize other intestinal glycans as a receptor to infect and kill mucus-coated bacteria, and (3) phages like ES17 can be utilized to coat the intestinal epithelium by binding heparan sulfate glycans to protect from invasive pathogen infection.

microbiology↗

bioRxiv2020

The structure of enteric human adenovirus 41 - a leading cause of diarrhoea in children

Human adenovirus (HAdV) types F40 and F41 are a prominent cause of diarrhea and diarrhea-associated mortality in young children worldwide. These enteric HAdVs differ strikingly in tissue tropism and pathogenicity from respiratory and ocular adenoviruses, but the structural basis for this divergence has been unknown. Here we present the first structure of an enteric HAdV - HAdV-F41 - determined by cryo-EM to a resolution of 3.8[A]. The structure reveals extensive alterations to the virion exterior as compared to non-enteric HAdVs, including a unique arrangement of capsid protein IX. The structure also provides new insights into conserved aspects of HAdV architecture such as a proposed location of protein V, which links the viral DNA to the capsid, and assembly-induced conformational changes in the penton base protein. Our findings provide the structural basis for adaptation to a fundamentally different tissue tropism of enteric HAdVs.

microbiology↗

bioRxiv2019

Adipokines set neural tone by regulating synapse number

Energy sensing neural circuits decide to expend or conserve resources by integrating tonic steady-state energy store information with phasic signals for hunger and food intake. Tonic signals, in the form of adipose tissue-derived adipokines, set the baseline level of energy-sensing neuron activity, providing context for interpretation of phasic messages. However, the mechanism by which tonic adipokine information establishes baseline neuronal function is unclear. Here we show that Upd2, a Drosophila Leptin ortholog, regulates actin-based synapse reorganization by reducing inhibitory synaptic contacts, thereby providing a permissive neural tone for insulin release under conditions of nutrient surplus. Unexpectedly, Insulin acts on the same upstream inhibitory neurons to conversely increase synapse number, hence re-instating negative tone. Our results suggest that two surplus-sensing hormonal systems, Leptin/Upd2 and Insulin, converge on a neuronal circuit with opposing outcomes that establish tonic, energy-store-dependent neuron activity. HighlightsO_LIThe adipokine Upd2 regulates number of inhibitory synaptic contacts on Insulin neurons. C_LIO_LIUpd2 activates an actin-regulating complex of Arouser, Basigin, and Gelsolin in target neurons. C_LIO_LIArouser, Basigin, and Gelsolin reduce the extent of inhibitory contact on Insulin neurons. C_LIO_LIInsulin resets negative tone by increasing the number of synaptic contacts made by its own upstream inhibitory neurons. C_LI

physiology↗

bioRxiv2019

An Interscholastic Network to Generate LexA Enhancer Trap Lines in Drosophila

Binary expression systems like the LexA-LexAop system provide a powerful experimental tool kit to study gene and tissue function in developmental biology, neurobiology and physiology. However, the number of well-defined LexA enhancer trap insertions remains limited. In this study, we present the molecular characterization and initial tissue expression analysis of nearly 100 novel StanEx LexA enhancer traps, derived from the StonEx1 index line. This includes 76 insertions into novel, distinct gene loci not previously associated with enhancer traps or targeted LexA constructs. Additionally, our studies revealed evidence for selective transposase-dependent replacement of a previously-undetected KP element on chromosome III within the StanEx1 genetic background during hybrid dysgenesis, suggesting a molecular basis for the over-representation of LexA insertions at the NK7.1 locus in our screen. Production and characterization of novel fly lines were performed by students and teachers in experiment-based genetics classes within a geographically diverse network of public and independent high schools. Thus, unique partnerships between secondary schools and university-based programs have produced and characterized novel genetic and molecular resources in Drosophila for open-source distribution, and provide paradigms for development of science education through experience-based pedagogy.

genetics↗

bioRxiv2018

Integrated proteogenomic analysis of metastatic thoracic tumors identifies APOBEC mutagenesis and copy number alterations as drivers of proteogenomic tumor evolution and heterogeneity

Elucidation of the proteogenomic evolution of metastatic tumors may offer insight into the poor prognosis of patients harboring metastatic disease. We performed whole-exome and transcriptome sequencing, copy number alterations (CNA) and mass spectrometry-based quantitative proteomics of 37 lung adenocarcinoma (LUAD) and thymic carcinoma (TC) metastases obtained by rapid autopsy and found evidence of patient-specific, multi-dimensional heterogeneity. Extreme mutational heterogeneity was evident in a subset of patients whose tumors showed increased APOBEC-signature mutations and expression of APOBEC3 region transcripts compared to patients with lesser mutational heterogeneity. TP53 mutation status was associated with APOBEC hypermutators in our cohort and in three independent LUAD datasets. In a thymic carcinoma patient, extreme heterogeneity and increased APOBEC3AB expression was associated with a high-risk germline APOBEC3AB variant allele. Patients with CNA occurring late in tumor evolution had corresponding changes in gene expression and protein abundance indicating genomic instability as a mechanism of downstream transcriptomic and proteomic heterogeneity between metastases. Across all tumors, proteomic heterogeneity was greater than copy number and transcriptomic heterogeneity. Enrichment of interferon pathways was evident both in the transcriptome and proteome of the tumors enriched for APOBEC mutagenesis despite a heterogeneous immune microenvironment across metastases suggesting a role for the immune microenvironment in the expression of APOBEC transcripts and generation of mutational heterogeneity. The evolving, heterogeneous nature of LUAD and TC, through APOBEC-mutagenesis and CNA illustrate the challenges facing treatment outcomes.

cancer biology↗