IU Indianapolis ScholarWorks :: Browsing by Subject "RNA-seq"

Browsing by Subject "RNA-seq"

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Aspergillus versicolor Inhalation Triggers Neuroimmune, Glial, and Neuropeptide Transcriptional Changes
(Sage, 2021) Ladd, Thatcher B.; Johnson, James A., Jr.; Mumaw, Christen L.; Greve, Hendrik J.; Xuei, Xiaoling; Simpson, Ed; Barnes, Mark A.; Green, Brett J.; Croston, Tara L.; Ahmed, Chandrama; Lemons, Angela; Beezhold, Donald H.; Block, Michelle L.; Medical and Molecular Genetics, School of Medicine
Increasing evidence associates indoor fungal exposure with deleterious central nervous system (CNS) health, such as cognitive and emotional deficits in children and adults, but the specific mechanisms by which it might impact the brain are poorly understood. Mice were exposed to filtered air, heat-inactivated Aspergillus versicolor (3 × 105 spores), or viable A. versicolor (3 × 105 spores) via nose-only inhalation exposure 2 times per week for 1, 2, or 4 weeks. Analysis of cortex, midbrain, olfactory bulb, and cerebellum tissue from mice exposed to viable A. versicolor spores for 1, 2, and 4 weeks revealed significantly elevated pro-inflammatory (Tnf and Il1b) and glial activity (Gdnf and Cxc3r1) gene expression in several brain regions when compared to filtered air control, with the most consistent and pronounced neuroimmune response 48H following the 4-week exposure in the midbrain and frontal lobe. Bulk RNA-seq analysis of the midbrain tissue confirmed that 4 weeks of A. versicolor exposure resulted in significant transcriptional enrichment of several biological pathways compared to the filtered air control, including neuroinflammation, glial cell activation, and regulation of postsynaptic organization. Upregulation of Drd1, Penk, and Pdyn mRNA expression was confirmed in the 4-week A. versicolor exposed midbrain tissue, highlighting that gene expression important for neurotransmission was affected by repeated A. versicolor inhalation exposure. Taken together, these findings indicate that the brain can detect and respond to A. versicolor inhalation exposure with changes in neuroimmune and neurotransmission gene expression, providing much needed insight into how inhaled fungal exposures can affect CNS responses and regulate neuroimmune homeostasis.
BERMUDA: a novel deep transfer learning method for single-cell RNA sequencing batch correction reveals hidden high-resolution cellular subtypes
(BioMed Central, 2019-08-12) Wang, Tongxin; Johnson, Travis S.; Shao, Wei; Lu, Zixiao; Helm, Bryan R.; Zhang, Jie; Huang, Kun; Medical and Molecular Genetics, School of Medicine
To fully utilize the power of single-cell RNA sequencing (scRNA-seq) technologies for identifying cell lineages and bona fide transcriptional signals, it is necessary to combine data from multiple experiments. We present BERMUDA (Batch Effect ReMoval Using Deep Autoencoders), a novel transfer-learning-based method for batch effect correction in scRNA-seq data. BERMUDA effectively combines different batches of scRNA-seq data with vastly different cell population compositions and amplifies biological signals by transferring information among batches. We demonstrate that BERMUDA outperforms existing methods for removing batch effects and distinguishing cell types in multiple simulated and real scRNA-seq datasets.
Characterizing the heterogeneity of triple-negative breast cancers using microdissected normal ductal epithelium and RNA-sequencing
(Springer, 2014-01) Radovich, Milan; Clare, Susan E.; Atale, Rutuja; Pardo, Ivanesa; Hancock, Bradley A.; Solzak, Jeffrey P.; Kassem, Nawal; Mathieson, Theresa; V. Storniolo, Anna Maria; Rufenbarger, Connie; Lillemoe, Heather A.; Blosser, Rachel J.; Choi, Mi Ran; Sauder, Candice A.; Doxey, Diane; Henry, Jill E.; Hilligoss, Eric E.; Sakarya, Onur; Hyland, Fiona C.; Hickenbotham, Matthew; Zhu, Jin; Glasscock, Jarret; Badve, Sunil; Ivan, Mircea; Liu, Yunlong; Sledge, George W.; Schneider, Bryan P.; Department of Surgery, IU School of Medicine
Triple-negative breast cancers (TNBCs) are a heterogeneous set of tumors defined by an absence of actionable therapeutic targets (ER, PR, and HER-2). Microdissected normal ductal epithelium from healthy volunteers represents a novel comparator to reveal insights into TNBC heterogeneity and to inform drug development. Using RNA-sequencing data from our institution and The Cancer Genome Atlas (TCGA) we compared the transcriptomes of 94 TNBCs, 20 microdissected normal breast tissues from healthy volunteers from the Susan G. Komen for the Cure Tissue Bank, and 10 histologically normal tissues adjacent to tumor. Pathway analysis comparing TNBCs to optimized normal controls of microdissected normal epithelium versus classic controls composed of adjacent normal tissue revealed distinct molecular signatures. Differential gene expression of TNBC compared with normal comparators demonstrated important findings for TNBC-specific clinical trials testing targeted agents; lack of over-expression for negative studies and over-expression in studies with drug activity. Next, by comparing each individual TNBC to the set of microdissected normals, we demonstrate that TNBC heterogeneity is attributable to transcriptional chaos, is associated with non-silent DNA mutational load, and explains transcriptional heterogeneity in addition to known molecular subtypes. Finally, chaos analysis identified 146 core genes dysregulated in >90 % of TNBCs revealing an over-expressed central network. In conclusion, use of microdissected normal ductal epithelium from healthy volunteers enables an optimized approach for studying TNBC and uncovers biological heterogeneity mediated by transcriptional chaos.
Coxiella burnetii Virulent Phase I and Avirulent Phase II Variants Differentially Manipulate Autophagy Pathway in Neutrophils
(American Society for Microbiology, 2022) Kumaresan, Venkatesh; Wang, Juexin; Zhang, Wendy; Zhang, Yan; Xu, Dong; Zhang, Guoquan; Medical and Molecular Genetics, School of Medicine
Coxiella burnetii is an obligate intracellular Gram-negative bacterium that causes Q fever in humans. The virulent C. burnetii Nine Mile phase I (NMI) strain causes disease in animal models, while the avirulent NM phase II (NMII) strain does not. In this study, we found that NMI infection induces severe splenomegaly and bacterial burden in the spleen in BALB/c mice, while NMII infection does not. A significantly higher number of CD11b+ Ly6G+ neutrophils accumulated in the liver, lung, and spleen of NMI-infected mice than in NMII-infected mice. Thus, neutrophil accumulation correlates with NMI and NMII infection-induced inflammatory responses. In vitro studies also demonstrated that although NMII exhibited a higher infection rate than NMI in mouse bone marrow neutrophils (BMNs), NMI-infected BMNs survived longer than NMII-infected BMNs. These results suggest that the differential interactions of NMI and NMII with neutrophils may be related to their ability to cause disease in animals. To understand the molecular mechanism underlying the differential interactions of NMI and NMII with neutrophils, global transcriptomic gene expressions were compared between NMI- and NMII-infected BMNs by RNA sequencing (RNA-seq) analysis. Interestingly, several genes involved in autophagy-related pathways, particularly membrane trafficking and lipid metabolism, are upregulated in NMII-infected BMNs but downregulated in NMI-infected BMNs. Immunofluorescence and immunoblot analyses indicate that compared to NMI-infected BMNs, vacuoles in NMII-infected-BMNs exhibit increased autophagic flux along with phosphatidylserine translocation in the cell membrane. Similar to neutrophils, NMII activated LC3-mediated autophagy in human macrophages. These findings suggest that the differential manipulation of autophagy of NMI and NMII may relate to their pathogenesis.
Estrogen-Dependent Upregulation of Adcyap1r1 Expression in Nucleus Accumbens Is Associated With Genetic Predisposition of Sex-Specific QTL for Alcohol Consumption on Rat Chromosome 4
(Frontiers, 2018-12-04) Spence, John Paul; Reiter, Jill L.; Qiu, Bin; Gu, Hao; Garcia, Dawn K.; Zhang, Lingling; Graves, Tamara; Williams, Kent E.; Bice, Paula J.; Zou, Yi; Lai, Zhao; Yong, Weidong; Liang, Tiebing; Medicine, School of Medicine
Humans show sex differences related to alcohol use disorders (AUD). Animal model research has the potential to provide important insight into how sex differences affect alcohol consumption, particularly because female animals frequently drink more than males. In previous work, inbred strains of the selectively bred alcohol-preferring (P) and non-preferring (NP) rat lines revealed a highly significant quantitative trait locus (QTL) on rat chromosome 4, with a logarithm of the odds score of 9.2 for alcohol consumption. Recently, interval-specific congenic strains (ISCS) were developed by backcrossing the congenic P.NP line to inbred P (iP) rats to further refine the chromosome 4 QTL region. Two ISCS sub-strains, ISCS-A and ISCS-B, were obtained with a narrowed QTL, where the smallest region of overlap consisted of 8.9 Mb in ISCS-B. Interestingly, we found that females from both ISCS lines consumed significantly less alcohol than female iP controls (p < 0.05), while no differences in alcohol consumption were observed between male ISCS and iP controls. RNA-sequencing was performed on the nucleus accumbens of alcohol-naïve female ISCS-B and iP rats, which revealed differentially expressed genes (DEG) with greater than 2-fold change and that were functionally relevant to behavior. These DEGs included down-regulation of Oxt, Asb4, Gabre, Gabrq, Chat, Slc5a7, Slc18a8, Slc10a4, and Ngfr, and up-regulation of Ttr, Msln, Mpzl2, Wnt6, Slc17a7, Aldh1a2, and Gstm2. Pathway analysis identified significant alterations in gene networks controlling nervous system development and function, as well as cell signaling, GABA and serotonin receptor signaling and G-protein coupled receptor signaling. In addition, β-estradiol was identified as the most significant upstream regulator. The expression levels of estrogen-responsive genes that mapped to the QTL interval and have been previously associated with alcohol consumption were measured using RT-qPCR. We found that expression of the Adcyap1r1 gene, encoding the pituitary adenylate cyclase-activating polypeptide type 1 (PAC1) receptor, was upregulated in female ISCS-B compared to female iP controls, while no differences were exhibited in males. In addition, sequence variants in the Adcyap1r1 promoter region showed a differential response to estrogen stimulation in vitro. These findings demonstrate that rat chromosome 4 QTL contains genetic variants that respond to estrogen and are associated with female alcohol consumption.
Experimental competition induces immediate and lasting effects on the neurogenome in free-living female birds
(National Academy of Sciences, 2021-03-30) Bentz, Alexandra B.; George, Elizabeth M.; Wolf, Sarah E.; Rusch, Douglas B.; Podicheti, Ram; Buechlein, Aaron; Nephew, Kenneth P.; Rosvall, Kimberly A.; Biology, School of Science
Periods of social instability can elicit adaptive phenotypic plasticity to promote success in future competition. However, the underlying molecular mechanisms have primarily been studied in captive and laboratory-reared animals, leaving uncertainty as to how natural competition among free-living animals affects gene activity. Here, we experimentally generated social competition among wild, cavity-nesting female birds (tree swallows, Tachycineta bicolor). After territorial settlement, we reduced the availability of key breeding resources (i.e., nest boxes), generating heightened competition; within 24 h we reversed the manipulation, causing aggressive interactions to subside. We sampled females during the peak of competition and 48 h after it ended, along with date-matched controls. We measured transcriptomic and epigenomic responses to competition in two socially relevant brain regions (hypothalamus and ventromedial telencephalon). Gene network analyses suggest that processes related to energy mobilization and aggression (e.g., dopamine synthesis) were up-regulated during competition, the latter of which persisted 2 d after competition had ended. Cellular maintenance processes were also down-regulated after competition. Competition additionally altered methylation patterns, particularly in pathways related to hormonal signaling, suggesting those genes were transcriptionally poised to respond to future competition. Thus, experimental competition among free-living animals shifts gene expression in ways that may facilitate the demands of competition at the expense of self-maintenance. Further, some of these effects persisted after competition ended, demonstrating the potential for epigenetic biological embedding of the social environment in ways that may prime individuals for success in future social instability.
Global Release of Translational Repression Across Plasmodium’s Host-to-Vector Transmission Event
(bioRxiv, 2024-03-16) Rios, Kelly T.; McGee, James P.; Sebastian, Aswathy; Moritz, Robert L.; Feric, Marina; Absalon, Sabrina; Swearingen, Kristian E.; Lindner, Scott E.; Pharmacology and Toxicology, School of Medicine
Malaria parasites must be able to respond quickly to changes in their environment, including during their transmission between mammalian hosts and mosquito vectors. Therefore, before transmission, female gametocytes proactively produce and translationally repress mRNAs that encode essential proteins that the zygote requires to establish a new infection. This essential regulatory control requires the orthologues of DDX6 (DOZI), LSM14a (CITH), and ALBA proteins to form a translationally repressive complex in female gametocytes that associates with many of the affected mRNAs. However, while the release of translational repression of individual mRNAs has been documented, the details of the global release of translational repression have not. Moreover, the changes in spatial arrangement and composition of the DOZI/CITH/ALBA complex that contribute to translational control are also not known. Therefore, we have conducted the first quantitative, comparative transcriptomics and DIA-MS proteomics of Plasmodium parasites across the host-to-vector transmission event to document the global release of translational repression. Using female gametocytes and zygotes of P. yoelii, we found that nearly 200 transcripts are released for translation soon after fertilization, including those with essential functions for the zygote. However, we also observed that some transcripts remain repressed beyond this point. In addition, we have used TurboID-based proximity proteomics to interrogate the spatial and compositional changes in the DOZI/CITH/ALBA complex across this transmission event. Consistent with recent models of translational control, proteins that associate with either the 5' or 3' end of mRNAs are in close proximity to one another during translational repression in female gametocytes and then dissociate upon release of repression in zygotes. This observation is cross-validated for several protein colocalizations in female gametocytes via ultrastructure expansion microscopy and structured illumination microscopy. Moreover, DOZI exchanges its interaction from NOT1-G in female gametocytes to the canonical NOT1 in zygotes, providing a model for a trigger for the release of mRNAs from DOZI. Finally, unenriched phosphoproteomics revealed the modification of key translational control proteins in the zygote. Together, these data provide a model for the essential translational control mechanisms used by malaria parasites to promote their efficient transmission from their mammalian host to their mosquito vector.
Isolation of mouse brain-infiltrating leukocytes for single cell profiling of epitopes and transcriptomes
(Elsevier, 2021-05-13) Guldner, Ian H.; Golomb, Samantha M.; Wang, Qingfei; Wang, Emilia; Zhang, Siyuan; Medicine, School of Medicine
High dimensional compositional and transcriptional profiling of heterogeneous brain-infiltrating leukocytes can lead to novel biological and therapeutic discoveries. High-quality single-cell leukocyte preparations are a prerequisite for optimal single cell profiling. Here, we describe a protocol for epitope and RNA-preserving dissociation of adult mouse brains and subsequent leukocyte purification and staining, which is adaptable to homeostatic and pathogenic brains. Leukocyte preparation following this protocol permits exquisite single-cell surface protein and RNA profiling in applications including CyTOF and CITE-seq. For complete details on the use and execution of this protocol, please refer to Guldner et al. (2020) and Golomb et al. (2020).
IsoTree: A New Framework for De novo Transcriptome Assembly from RNA-seq Reads
(IEEE, 2018-02) Zhao, Jin; Feng, Haodi; Zhu, Daming; Zhang, Chi; Xu, Ying; Medical and Molecular Genetics, School of Medicine
High-throughput sequencing of mRNA has made the deep and efficient probing of transcriptome more affordable. However, the vast amounts of short RNA-seq reads make de novo transcriptome assembly an algorithmic challenge. In this work, we present IsoTree, a novel framework for transcripts reconstruction in the absence of reference genomes. Unlike most of de novo assembly methods that build de Bruijn graph or splicing graph by connecting $k-mers$ which are sets of overlapping substrings generated from reads, IsoTree constructs splicing graph by connecting reads directly. For each splicing graph, IsoTree applies an iterative scheme of mixed integer linear program to build a prefix tree, called isoform tree. Each path from the root node of the isoform tree to a leaf node represents a plausible transcript candidate which will be pruned based on the information of paired-end reads. Experiments showed that in most cases IsoTree performs better than other leading transcriptome assembly programs. IsoTree is available at https://github.com/Jane110111107/IsoTree.
Knocking out Fkbp51 decreases CCl4-induced liver injury through enhancement of mitochondrial function and Parkin activity
(Springer Nature, 2024-01-02) Qiu, Bin; Zhong, Zhaohui; Dou, Longyu; Xu, Yuxue; Zou, Yi; Weldon, Korri; Wang, Jun; Zhang, Lingling; Liu, Ming; Williams, Kent E.; Spence, John Paul; Bell, Richard L.; Lai, Zhao; Yong, Weidong; Liang, Tiebing; Medicine, School of Medicine
Background and aims: Previously, we found that FK506 binding protein 51 (Fkbp51) knockout (KO) mice resist high fat diet-induced fatty liver and alcohol-induced liver injury. The aim of this research is to identify the mechanism of Fkbp51 in liver injury. Methods: Carbon tetrachloride (CCl4)-induced liver injury was compared between Fkbp51 KO and wild type (WT) mice. Step-wise and in-depth analyses were applied, including liver histology, biochemistry, RNA-Seq, mitochondrial respiration, electron microscopy, and molecular assessments. The selective FKBP51 inhibitor (SAFit2) was tested as a potential treatment to ameliorate liver injury. Results: Fkbp51 knockout mice exhibited protection against liver injury, as evidenced by liver histology, reduced fibrosis-associated markers and lower serum liver enzyme levels. RNA-seq identified differentially expressed genes and involved pathways, such as fibrogenesis, inflammation, mitochondria, and oxidative metabolism pathways and predicted the interaction of FKBP51, Parkin, and HSP90. Cellular studies supported co-localization of Parkin and FKBP51 in the mitochondrial network, and Parkin was shown to be expressed higher in the liver of KO mice at baseline and after liver injury relative to WT. Further functional analysis identified that KO mice exhibited increased ATP production and enhanced mitochondrial respiration. KO mice have increased mitochondrial size, increased autophagy/mitophagy and mitochondrial-derived vesicles (MDV), and reduced reactive oxygen species (ROS) production, which supports enhancement of mitochondrial quality control (MQC). Application of SAFit2, an FKBP51 inhibitor, reduced the effects of CCl4-induced liver injury and was associated with increased Parkin, pAKT, and ATP production. Conclusions: Downregulation of FKBP51 represents a promising therapeutic target for liver disease treatment.

Browsing by Subject "RNA-seq"

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