The Hepatokine Adropin Is Regulated by Estrogen

Abstract Introduction: menopause is associated with weight gain, visceral adiposity and NAFLD. Rodent ovariectomy (OVX) is an accepted model for human menopause. Many OVX studies add high-fat diet or old-age to accentuate deranged phenotype. We have shown OVX alone induced weight gain and changes in liver transcriptome including downregulation of Enho, encoding for the hepatokine adropin (1). Here, we explore changes in VAT cytokine and adipokine genes, hepatic miRNA, and liver triglyceride content induced by OVX, in addition to estrogen’s role in regulation of adropin. Methods: 9-week-old C57BL/6J female mice underwent OVX or sham surgery. Groups of 10 mice were sacrificed at 6- and 12-weeks post-surgery and tissues harvested including mesenteric adipose tissue representing VAT. Liver TG was quantified using Cayman colorimetric assay. In-vitro studies performed in the murine hepatic cell-line, BNL1.ME. Adropin was measured using ELISA. Results: OVX induced adverse inflammatory cytokine & adipokine gene expression in VAT at 6-weeks post-surgery (Il18 1.1 p=0.01, Rares2 2.9, p=0.003, Retn 5.5, p=0.002) and 12-weeks post-surgery (Tnfa 2.3 p<0.001, Cxcl5 1.9 p=0.04). In the liver, OVX induced an increase in TG content at 12 weeks post-surgery (realtive increase vs sham 2.0 p=0.05). Hepatic Enho expression showed a strong inverse coorelation with total body weight gain (r= -0.7 p<0.001) and liver TG content (r=-0.4, p=0.04). In-vitro, estrogen induced an increase in Enho (relative mRNA change vs. growing medium 2.6, p=0.004); though protein level was unchanged, a trend for increased adropin was found in supernatant (relative change vs control 2.2 P=0.09). In-silico analysis of data from OVX mice treated with estrogen showed up-regulation of Enho (relative change vs vehicle, 6 p<0.001). At 6-weeks post-surgery OVX induced changes in hepatic miRNA profile with 48 miRNAs differentially expressed vs SHAM (24 up & 24 down). Integrating data from same sample RNA-SEQ and miRNA-SEQ created a network of differently expressed miRNA with oppositely differently expressed known specific mRNA targets. mIR-29, a known regulator of Enho in liver, was not found to be correlated with Enho expression in this context. Conclusions: OVX alone is sufficient to induce adverse changes in VAT gene expression and liver TG. Hepatic adropin gene expression is regulated by estrogen and its downregulation was strongly correlated to phenotypes relevant to menopause induced metabolic dysfunction, weight gain and increased liver fat. Thus, adropin should be further explored as a novel therapeutic and/or biomarker for menopause induced metabolic dysfunction. (1) Stokar, J., Gurt, I., Cohen-Kfir, E., Yakubovsky, O., Hanna, A., Assayag, E., & Dresner-Pollak, R. (2019). RNA-Seq Analysis of Ovariectomy-Induced Changes in Mouse Liver Reveals New Targets for Menopause-Associated Metabolic Derangement. Journal of the Endocrine Society, 3(Supplement_1), SUN-033.

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Single cell RNA sequencing of calvarial and long bone endocortical cells

10.1101/849224 ◽

2019 ◽

Cited By ~ 1

Author(s):

Ugur M. Ayturk ◽

Joseph P. Scollan ◽

Alexander Vesprey ◽

Christina M. Jacobsen ◽

Paola Divieti Pajevic ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Cultured Cells ◽

Neutralizing Antibody ◽

Long Bone ◽

Appendicular Skeleton ◽

Rna Seq ◽

Isolated Cells

ABSTRACTSingle cell RNA-seq (scRNA-seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether scRNA-seq could be used to detect the effect of environmental and pharmacologic perturbations on osteoblasts. We began with a commonly used in vitro system in which freshly isolated neonatal mouse calvarial cells are expanded and induced to produce a mineralized matrix. We used scRNA-seq to compare the relative cell type abundances and the transcriptomes of freshly isolated cells to those that had been cultured for 12 days in vitro. We observed that the percentage of macrophage-like cells increased from 6% in freshly isolated calvarial cells to 34% in cultured cells. We also found that Bglap transcripts were abundant in freshly isolated osteoblasts but nearly undetectable in the cultured calvarial cells. Thus, scRNA-seq revealed significant differences between heterogeneity of cells in vivo and in vitro. We next performed scRNA-seq on freshly recovered long bone endocortical cells from mice that received either vehicle or Sclerostin-neutralizing antibody for 1 week. Bone anabolism-associated transcripts were also not significantly increased in immature and mature osteoblasts recovered from Sclerostin-neutralizing antibody treated mice; this is likely a consequence of being underpowered to detect modest changes in gene expression, since only 7% of the sequenced endocortical cells were osteoblasts, and a limited portion of their transcriptomes were sampled. We conclude that scRNA-seq can detect changes in cell abundance, identity, and gene expression in skeletally derived cells. In order to detect modest changes in osteoblast gene expression at the single cell level in the appendicular skeleton, larger numbers of osteoblasts from endocortical bone are required.

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Enhancing droplet-based single-nucleus RNA-seq resolution using the semi-supervised machine learning classifier DIEM

10.1101/786285 ◽

2019 ◽

Cited By ~ 4

Author(s):

Marcus Alvarez ◽

Elior Rahmani ◽

Brandon Jew ◽

Kristina M. Garske ◽

Zong Miao ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Cell Types ◽

Supervised Machine Learning ◽

Data Sets ◽

Rna Seq ◽

Novel Approach ◽

Single Nucleus ◽

Downstream Analysis

AbstractSingle-nucleus RNA sequencing (snRNA-seq) measures gene expression in individual nuclei instead of cells, allowing for unbiased cell type characterization in solid tissues. Contrary to single-cell RNA seq (scRNA-seq), we observe that snRNA-seq is commonly subject to contamination by high amounts of extranuclear background RNA, which can lead to identification of spurious cell types in downstream clustering analyses if overlooked. We present a novel approach to remove debris-contaminated droplets in snRNA-seq experiments, called Debris Identification using Expectation Maximization (DIEM). Our likelihood-based approach models the gene expression distribution of debris and cell types, which are estimated using EM. We evaluated DIEM using three snRNA-seq data sets: 1) human differentiating preadipocytes in vitro, 2) fresh mouse brain tissue, and 3) human frozen adipose tissue (AT) from six individuals. All three data sets showed various degrees of extranuclear RNA contamination. We observed that existing methods fail to account for contaminated droplets and led to spurious cell types. When compared to filtering using these state of the art methods, DIEM better removed droplets containing high levels of extranuclear RNA and led to higher quality clusters. Although DIEM was designed for snRNA-seq data, we also successfully applied DIEM to single-cell data. To conclude, our novel method DIEM removes debris-contaminated droplets from single-cell-based data fast and effectively, leading to cleaner downstream analysis. Our code is freely available for use at https://github.com/marcalva/diem.

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Changes in Gene Expression during Drying in a Desiccation-Tolerant Grass Sporobolus stapfianus and a Desiccation-Sensitive Grass Sporobolus pyramidalis

Functional Plant Biology ◽

10.1071/pp96073 ◽

1997 ◽

Vol 24 (5) ◽

pp. 617 ◽

Cited By ~ 6

Author(s):

D.F. Gaff ◽

D. Bartels ◽

J.L. Gaff

Keyword(s):

Gene Expression ◽

Desiccation Tolerance ◽

Transcriptional Level ◽

Novel Proteins ◽

Sporobolus Stapfianus ◽

Induced Changes ◽

First Time ◽

Dimensional Electrophoresis ◽

Water Contents

For the first time in the grasses, a desiccation-tolerant species (Sporobolus stapfianus) was examined for evidence of drought-induced changes in gene transcription. Desiccation tolerance (the ability of this species to recover from a water potential of –540 MPa) is induced in the resurrection grass during the drying process itself. Specific mRNA was compared in extracts of air-dry, drying and fully hydrated leaves by comparisons of the encoded proteins translated in vitro and partitioned by 2- dimensional electrophoresis. Forty-one genes, that were not expressed in hydrated leaves, were transcribed during drying, whereas only 25 novel polypeptides (translated in vitro) were detected; this suggests that gene expression was controlled mainly at the transcriptional level, but possibly also at the translational level. Leaves of S. stapfianus become desiccation tolerant as they dry on intact plants with mechanically undisturbed roots, whereas leaves on plants whose roots have been disturbed die during drying. Complements of mRNA from live S. stapfianus leaves changed markedly from full hydration to 70% RWC and to air-dryness; they also differed markedly from drought-sensitive leaves (on plants with disturbed roots) at 70% RWC and dead air-dry S. stapfianus leaves and from leaves of the desiccation sensitive grass S. pyramidalis at the same water contents. Drought-induced injury could not be attributed to low abundance of mRNA in either species. Five criteria which might be involved in desiccation tolerance were applied to specific in vitro proteins of S. stapfianus; 12 novel proteins correlated with desiccation tolerance in a least four of the five criteria.

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Effect of SMARCB1 deficiency in renal medullary carcinoma (RMC) on genes associated with nucleosome assembly and telomere organization.

Journal of Clinical Oncology ◽

10.1200/jco.2018.36.6_suppl.614 ◽

2018 ◽

Vol 36 (6_suppl) ◽

pp. 614-614 ◽

Cited By ~ 1

Author(s):

Pavlos Msaouel ◽

Gabriel G. Malouf ◽

Xiaoping Su ◽

Hui Yao ◽

Durga N Tripathi ◽

...

Keyword(s):

Gene Expression ◽

Growth Rate ◽

Sickle Cell Trait ◽

Regulation Of Gene Expression ◽

Kidney Tissue ◽

Rna Seq ◽

Nucleosome Assembly ◽

P Values ◽

Go Analysis

614 Background: RMC is a highly aggressive tumor with close to universal fatality despite therapy. It is almost exclusively found in young African-Americans with sickle cell trait, and is characterized by complete loss of expression of SMARCB1, a major chromatin remodeler involved in regulation of gene expression. We investigated the effects of SMARCB1 loss on mutation frequency, gene expression, and cell growth in RMC. Methods: Whole exome sequencing (WES) and RNA sequencing (RNA-seq) were performed in RMC tissues from 15 and 11 patients respectively, each with matched adjacent normal kidney tissue controls. In vitro experiments were performed in a cell line (RMC2C) we established from a patient with RMC. SMARCB1 was conditionally re-expressed using a tetracycline-inducible lentivector. Gene ontology (GO) analysis was performed using DAVID. Results: WES showed that RMC harbors a low number (median of < 25/tumor sample) of non-synonymous exomic single nucleotide variants (SNVs) or small indels. GO analysis revealed that the most significant pathways upregulated in RMC compared with normal tissue were those associated with nucleosome assembly and telomere organization (p values < 0.0001). Re-expression of SMARCB1 at near-endogenous levels suppressed the growth rate of RMC2C cells. Subsequent silencing of SMARCB1 expression restored the growth rate of these cells. RNA-seq of RMC2C cells expressing SMARCB1 demonstrated that the most significant downregulated pathways compared with SMARCB1-negative RMC2C cells were those associated with nucleosome assembly and telomere organization (p values < 0.0001). Conclusions: RMC harbors a remarkably simple genome, as evidenced by our WES analysis. Therefore, consistently detected alterations, such as SMARCB1 loss, are likely to serve as drivers for this disease. Indeed, in vitro restoration of SMARCB1 expression suppressed the growth of RMC cells and repressed genes associated with nucleosome assembly and telomere organization, identifying for the first time a causal link between loss of SMARCB1 and dysregulation of these genes. These results provide the basis for future therapeutic strategies targeting SMARCB1 loss in RMC.

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RNA-seq analyses of blood-induced changes in gene expression in the mosquito vector species, Aedes aegypti

BMC Genomics ◽

10.1186/1471-2164-12-82 ◽

2011 ◽

Vol 12 (1) ◽

Cited By ~ 83

Author(s):

Mariangela Bonizzoni ◽

W Augustine Dunn ◽

Corey L Campbell ◽

Ken E Olson ◽

Michelle T Dimon ◽

...

Keyword(s):

Gene Expression ◽

Aedes Aegypti ◽

Mosquito Vector ◽

Rna Seq ◽

Vector Species ◽

Induced Changes

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Gene Expression Profile of the Human Colorectal Carcinoma LoVo Cells Treated With Sporamin and Thapsigargin

Frontiers in Oncology ◽

10.3389/fonc.2021.621462 ◽

2021 ◽

Vol 11 ◽

Author(s):

Chun Yang ◽

Si-Jia Chen ◽

Bo-Wen Chen ◽

Kai-Wen Zhang ◽

Jing-Jie Zhang ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Profile ◽

Expression Profile ◽

Ipomoea Batatas ◽

Expression Profiles ◽

Rna Seq ◽

Ppi Network ◽

Illumina Platform

Sporamin, a proteinase inhibitor isolated from the sweet potato (Ipomoea batatas), has shown promising anticancer effect against colorectal cancer (CRC) in vitro and in vivo but its mechanisms of action are poorly understood. In the present study, high throughput RNA sequencing (RNA-seq) technology was applied to explore the transcriptomic changes induced by sporamin in the presence of thapsigargin (TG), a non-12-O-tetradecanolphorbol-13-acetate type cancer promoter, in the LoVo human CRC cells. Cellular total RNA was extracted from the cells after they were treated with vehicle (CTL), 1 μM of thapsigargin (TG), or 1 μM of TG plus 30 μM of sporamin (TGSP) for 24 h. The migratory capacity of the cells was determined by wound healing assay. The gene expression profiles of the cells were determined by RNA-seq on an Illumina platform. GO enrichment analysis, KEGG pathway analysis, protein-protein interaction (PPI) network construction, and transcription factors (TF) prediction were all performed based on the differentially expressed genes (DEGs) across groups with a series of bioinformatics tools. Finally, the effect and potential molecular targets of the sporamin at the transcriptome level were evaluated. Sporamin significantly inhibited the migration of cells induced by TG. Among the 17915 genes detected in RNA-seq, 46 DEGs were attributable to the effect of sporamin. RT-PCR experiment validated that the expression of RGPD2, SULT1A3, and BIVM-ERCC5 were up-regulated while NYP4R, FOXN1, PAK6, and CEACAM20 were down-regulated. Sporamin enhanced the mineral absorption pathway, worm longevity regulating pathway, and pyrimidine metabolism pathway. Two TFs (SMIM11A and ATOH8) were down-regulated by sporamin. HMOX1 (up-regulated) and NME1-NME2 (down-regulated) were the main nodes in a PPI network consisting of 16 DEGs that were modulated by sporamin in the presence of TG. Sporamin could favorably alter the gene expression profile of CRC cells, up-regulating the genes that contribute to the homeostasis of intracellular metal ions and the activities of essential enzymes and DNA damage repairment. More studies are warranted to verify its effect on specific genes and delineate the mechanism of action implicated in the process.

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Mesenchymal Stem Cell Therapy Ameliorates Metabolic Dysfunction and Restores Fertility in a PCOS Mouse Model Through Interleukin-10

10.21203/rs.3.rs-444566/v1 ◽

2021 ◽

Author(s):

Rishi Man Chugh ◽

Hang-soo Park ◽

Abdeljabar El Andaloussi ◽

Amro Elsharoud ◽

Sahar Esfandyari ◽

...

Keyword(s):

Gene Expression ◽

Polycystic Ovary ◽

Therapeutic Option ◽

Interleukin 10 ◽

Reproductive Age ◽

Metabolic Dysfunction ◽

Mesenchymal Stem Cell Therapy ◽

Anti Inflammatory

Abstract Background: Polycystic ovary syndrome (PCOS) is the most common endocrine and metabolic disorder in reproductive-age women. Excessive inflammation and elevated androgen production from ovarian theca cells are key features of PCOS. Human bone marrow mesenchymal stem cells (BM-hMSC) and their secreted factors (secretome) exhibit robust anti-inflammatory capabilities in various biological systems. We evaluated the therapeutic efficacy of BM-hMSC and its secretome in both in vitro and in vivo PCOS models.Methods: For in vitro experiment, we treated conditioned media from BM-hMSC to androgen producing H293R cells, and analyzed androgen producing gene expression. For in vivo experiment, BM-hMSC were implanted into Letrozole (LTZ) induced mouse PCOS model. BM-hMSC effect in androgen producing cells or PCOS model mice was assessed by monitoring cell proliferation (immunohistochemistry), steroidogenic gene expression (quantitative real-time polymerase chain reaction [qRT-PCR] and Western blot, animal tissue assay (H&E staining), and fertility by pup delivery.Results: BM-hMSC significantly downregulate steroidogenic gene expression, curb inflammation, and restore fertility in treated PCOS animals. The anti-inflammatory cytokine interleukin-10 (IL-10) played a key role in mediating the effects of BM-hMSC in our PCOS models. We demonstrated that BM-hMSC treatment was improve in metabolic and reproductive markers in our PCOS model and able to restore fertility. Conclusion: Our study demonstrates for the first time the efficacy of intra-ovarian injection of BM-hMSC or its secretome to treat PCOS-related phenotypes, including both metabolic and reproductive dysfunction. This approach may represent a novel therapeutic option for women with PCOS. Our results suggest that BM-hMSC can reverse PCOS-induced inflammation through IL-10 secretion. BM-hMSC might be a novel and robust therapeutic approach for PCOS treatment.

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Analysis of thein plantatranscriptome expressed by the corn pathogenPantoea stewartiisubsp.stewartiivia RNA-Seq

PeerJ ◽

10.7717/peerj.3237 ◽

2017 ◽

Vol 5 ◽

pp. e3237 ◽

Cited By ~ 5

Author(s):

Holly Packard ◽

Alison Kernell Burke ◽

Roderick V. Jensen ◽

Ann M. Stevens

Keyword(s):

Gene Expression ◽

Quorum Sensing ◽

Critical Role ◽

Wilt Disease ◽

Sample Type ◽

Dependent Manner ◽

Rna Seq ◽

In Planta ◽

Data Set

Pantoea stewartiisubsp.stewartiiis a bacterial phytopathogen that causes Stewart’s wilt disease in corn. It uses quorum sensing to regulate expression of some genes involved in virulence in a cell density-dependent manner as the bacterial population grows from small numbers at the initial infection site in the leaf apoplast to high cell numbers in the xylem where it forms a biofilm. There are also other genes important for pathogenesis not under quorum-sensing control such as a Type III secretion system. The purpose of this study was to compare gene expression during anin plantainfection versus either a pre-inoculumin vitroliquid culture or anin vitroagar plate culture to identify genes specifically expressedin plantathat may also be important for colonization and/or virulence. RNA was purified from each sample type to determine the transcriptome via RNA-Seq using Illumina sequencing of cDNA. Fold gene expression changes in thein plantadata set in comparison to the twoin vitrogrown samples were determined and a list of the most differentially expressed genes was generated to elucidate genes important for plant association. Quantitative reverse transcription PCR (qRT-PCR) was used to validate expression patterns for a select subset of genes. Analysis of the transcriptome data via gene ontology revealed that bacterial transporters and systems important for oxidation reduction processes appear to play a critical role forP. stewartiias it colonizes and causes wilt disease in corn plants.

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RNASeq analysis of a Pax3-expressing myoblast clone in-vitro and effect of culture surface stiffness on differentiation.

10.1101/2021.09.05.459022 ◽

2021 ◽

Author(s):

Louise Richardson ◽

Dapeng Wang ◽

Ruth E Hughes ◽

Colin Anfimov Johnson ◽

Michelle Peckham

Keyword(s):

Gene Expression ◽

Satellite Cells ◽

Cell Clone ◽

Rna Seq ◽

Myogenic Regulatory Factors ◽

Culture Surface ◽

Γ Interferon ◽

Cells Cultured ◽

Sarcomeric Genes

Skeletal muscle satellite cells cultured on soft surfaces (12kPa) show improved differentiation than cells cultured on stiff surfaces (approximately 100kPa). To better understand the reasons for this, we performed an RNA Seq analysis for a single satellite cell clone (C1F) derived from the H2kb-tsA58 immortomouse, which differentiates into myotubes under tightly regulated conditions (withdrawal of γ-interferon, 37°C). As expected, the largest change in overall gene expression occurred at day 1, as cells switch from proliferation to differentiation. Surprisingly, further analysis showed that proliferating C1F cells express Pax3 and not Pax7, confirmed by immunostaining, yet their subsequent differentiation into myotubes is normal, and enhanced on softer surfaces, as evidenced by significantly higher expression levels of myogenic regulatory factors, sarcomeric genes, enhanced fusion and improved myofibrillogenesis. Levels of RNA encoding extracellular matrix structural constituents and related genes were consistently upregulated on hard surfaces, suggesting that a consequence of differentiating satellite cells on hard surfaces is that they attempt to manipulate their niche prior to differentiating. This comprehensive RNASeq dataset will be a useful resource for understanding Pax3 expressing cells.

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The Oviductal Extracellular Vesicles’ RNA Cargo Regulates the Bovine Embryonic Transcriptome

International Journal of Molecular Sciences ◽

10.3390/ijms21041303 ◽

2020 ◽

Vol 21 (4) ◽

pp. 1303 ◽

Cited By ~ 7

Author(s):

Stefan Bauersachs ◽

Pascal Mermillod ◽

Carmen Almiñana

Keyword(s):

Gene Expression ◽

Extracellular Vesicles ◽

Sequencing Analysis ◽

Rna Seq ◽

High Throughput Analysis ◽

Positive Effects ◽

Early Embryos ◽

And Control ◽

The Impact

Oviductal extracellular vesicles (oEVs) are emerging as key players in the gamete/embryo–oviduct interactions that contribute to successful pregnancy. Various positive effects of oEVs on gametes and early embryos have been found in vitro. To determine whether these effects are associated with changes of embryonic gene expression, the transcriptomes of embryos supplemented with bovine fresh (FeEVs) or frozen (FoEVs) oEVs during in vitro culture compared to controls without oEVs were analyzed by low-input RNA sequencing. Analysis of RNA-seq data revealed 221 differentially expressed genes (DEGs) between FoEV treatment and control, 67 DEGs for FeEV and FoEV treatments, and minor differences between FeEV treatment and control (28 DEGs). An integrative analysis of mRNAs and miRNAs contained in oEVs obtained in a previous study with embryonic mRNA alterations pointed to direct effects of oEV cargo on embryos (1) by increasing the concentration of delivered transcripts; (2) by translating delivered mRNAs to proteins that regulate embryonic gene expression; and (3) by oEV-derived miRNAs which downregulate embryonic mRNAs or modify gene expression in other ways. Our study provided the first high-throughput analysis of the embryonic transcriptome regulated by oEVs, increasing our knowledge on the impact of oEVs on the embryo and revealing the oEV RNA components that potentially regulate embryonic development.

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