title: Gliocidin is a nicotinamide mimetic prodrug that targets glioblastoma

first: Mungunsarnai

last: Ganbold

state: NYC

address: 417 East 68st

department: Brain Tumor Center

institute: MSKCC

summary: Glioblastoma is incurable and in urgent need of improved therapeutics. We identify a small compound, Gliocidin, that kills glioblastoma cells while sparing nontumor replicative cells. Gliocidin activity targets a de novo purine synthesis vulnerability in glioblastoma through indirect inhibition of inosine monophosphate dehydrogenase 2 (IMPDH2). IMPDH2 blockade reduces intracellular guanine nucleotide levels causing nucleotide imbalance, replication stress, and tumor cell death. Gliocidin is a prodrug anabolized into its tumoricidal metabolite, Gliocidin-adenine dinucleotide (GAD), by the enzyme nicotinamide nucleotide adenylyltransferase (NMNAT1) of the NAD+ salvage pathway. The cryo-EM structure of GAD together with IMPDH2 demonstrates its entry, deformation, and blockade of the NAD+ pocket. In vivo, Gliocidin penetrates the blood brain barrier and extends orthotopic murine glioblastoma mouse survival. The DNA alkylating agent, temozolomide, induces Nmnat1 expression causing synergistic tumor killing and additional survival benefit in orthotopic patient-derived xenograft models. This study brings Gliocidin to light as a prodrug with potential to improve the survival of glioblastoma patients.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: HTS and MEF were treated with DMSO or Gliocidin (1μM) for 12 hours and harvested for bulk RNA-seq. PDX-161010-1 were orthotopically injected in athymic nude mice. At day 61 post injection, mice were treated with vehicle, Gliocidin (50 mg/kg, I.P., two times a day, total of 5 days), Temozolomide (50 mg/kg/day, I.P., total of 6 days) or combo (Gliocidin + Temozolomide). Tumors were harvested at day 72 post injection and prepared for single cell sequencing.

first: Yu-Jung

last: Chen

first: Luis

last: Parada


title: Transcriptome Analysis of  Responses to Saline-Alkali Stress in Two Different Genotypes of foxtail millet (Setaria italica L.)at the Seedling Stage

status: Public on Oct 10 2024

city: Qinhuangxdao

first: Yucui

last: Han

address: No. 360, West Section, Hebei Street

country: China

institute: Hebei Normal University Of Science and Technology

type: Expression profiling by high throughput sequencing

summary: We present here a transcriptome dataset of millet seedling leaves based on RNA-seq technology. The purpose of this study was to mine the salt and alkali tolerance genes of millet and further explore the mechanism of salt and alkali tolerance of millet. We selected 18 representative samples and conducted in-depth sequencing using the latest sequencing platform to ensure the accuracy and reliability of the data.

overall_design: RNA-seq data of saline-tolerant variety JK3 and saline-sensitive variety B175 were collected from a total of 18 samples at 3 time points, with 3 repeats at each time point.

first: Xiaohu

last: Lin

first: Huimin

last: Wang

first: Yanan

last: Yang


title: Extracellular Nicotinamide Phosphoribosyltransferase (eNAMPT) Drives Abnormal Pericyte-Rich  Vasculature in Triple-Negative Breast Cancer

status: Public on Oct 28 2024

city: Novara

first: Grolla

middle: A

last: Ambra

address: Bovio street, 6

department: Department of Pharmaceutical Sciences

country: Italy

institute: University of Eastern Piedmont

summary:
Tumour angiogenesis supports malignant cells with oxygen and nutrients to promote invasion and metastasis. A number of cytokines released in situ participate in the recruitment of endothelial cells and pericytes to trigger the formation of novel blood vessels, which are often abnormal, leaky, and disorganized.
Nicotinamide phosphoribosyltransferase is a key intracellular enzyme involved in NAD metabolism and is up regulated in many cancers to meet bioenergetic demands. Yet, the same protein is also secreted extracellularly (eNAMPT), where it acts as a pro-inflammatory cytokine. High plasma eNAMPT levels have been reported in breast cancer patients and correlate with aggressiveness and prognosis.
We now report that in a triple-negative breast cancer model, enriching the tumour microenvironment with eNAMPT leads to abundant angiogenesis and increased metastatization. Atypically, the eNAMPT-mediated pro-angiogenic effect is mainly directed to NG2+ pericytes. Indeed, eNAMPT acts as chemoattractant for pericytes and coordinates vessel-like tube formation, in synergism with the classical factor PDGF-BB.
Stimulation of pericytes by eNAMPT leads to a pro-inflammatory activation, characterized by the overexpression of key chemokines (CXCL8, CXCL1, CCL2) and VCAM1, via NF-kB signalling.
All these effects were ablated by the use of C269, an anti-eNAMPT neutralizing antibody, suggesting that this might represent a novel anti-angiogenic pharmacological approach for triple-negative breast cancer.

overall_design: To investigate the mechanim by which eNAMPT regulates pericytes we performed RNA sequencing on immortalized pericytes (Celther POLSKA, CL 05008-CLTH) exposed to recombinant human eNAMPT  [500 ng/mL] (Peprotech, Cat. 130-09), recombinant human PDGF-BB [20 ng/mL] (Peprotech, Cat. 100-14B) or the combination of both. 4x105 cells were plated in 6-well plate and treated for 4 hours. The experimental setting provides 5 replicates for each conditions using Illumina NextSeq 500.

first: Marianna

last: Moro

first: Ambra

last: Grolla

first: Giorgia

last: Colombo

first: Armando

last: Genazzani


title: Dose-dependent changes in gonadal white adipose tissue in TCDD-elicited steatohepatitis

status: Public on Oct 29 2024

city: East Lansing

first: Tim

last: Zacharewski

state: Michigan

address: 1129 Farm Ln

department: Biochemistry and Molecular Biology, Integrative Toxicological Sciences

country: USA

institute: Michigan State University

summary: Bulk RNA-sequencing of 27 C57BL/6 gonadal white adipose tissue samples where ovariectomized females were gavaged 4 days for 92 days with 0.01 to 30 µg/kg TCDD or sesame oil vehicle (n = 3) per dose.

overall_design: Following 4 days acclimation (PND 28), animals (n = 3) were orally gavaged with 0.1 ml sesame oil or 0.01, 0.03, 0.1, 0.3, 1, 3, 10, or 30 microg per kg TCDD (Dow Chemical Company, Midland, MI) every fourth day for 92 days for a total of 23 exposures. Prior to termination animals were fasted for 6 h. Blood was collected by submandibular vein puncture prior to cervical dislocation, centrifuged, and stored at -80 degrees C. Liver and gonadal white adipose tissue (gWAT) were excised, weighed, and frozen at -80 degrees C.

first: Rance

last: Nault

first: Kelly

last: Fader

first: Dustin

last: Ammendolia

first: Peter

last: Dornbos

first: Dave

last: Potter

first: Bonnie

last: Sharratt

first: Kazuyoshi

last: Kumagai

first: Jack

middle: R

last: Harkema

first: Sophia

middle: Y

last: Lunt

first: Timothy


title: LSK immunophenotype hematopoietic stem cells after irradiation at day 2

status: Public on Oct 07 2024

city: 重庆Chongqing

first: Zeyu

last: Fu

state: 重庆Chongqing

address: 高滩岩正街

department: College of Preventive Medicine

institute: AMU

summary: To investigate the impact of radiation damage on hematopoietic stem cell fate decision, we conducted a longitudinal study, collecting single-cell data on days 2 post-radiation exposure. This approach enabled us to observe changes in the subpopulations of hematopoietic stem cells at the LSK level in mice over time. Additionally, we assessed alterations in their fate determination and lineage differentiation tendencies throughout the recovery process.

overall_design: To perform 10X Genomics single-cell sequencing on mouse bone marrow LSK cells, begin by euthanizing the mouse following ethical guidelines and extracting the femurs and tibias. Flush the bone marrow using a syringe with buffer PBS with 2% FBS, to obtain a single-cell suspension. Filter this suspension through a 40 µm cell strainer to remove debris and clumps. Next, enrich the LSK (Lineage^-, Sca-1^+, c-Kit^+) cells using fluorescence-activated cell sorting (FACS). Count the cells and assess viability using trypan blue staining. Adjust the concentration of LSK cells to the required density 700-1200 cells/µL, for optimal performance in the 10X Genomics Chromium Controller. Load the single-cell suspension, reagents, and gel beads into the appropriate wells of a 10X Genomics Single Cell 3' Chip and run the chip in the Chromium Controller to encapsulate single cells into nanoliter-scale Gel Bead-In-EMulsions (GEMs). Perform reverse transcription within the GEMs to generate barcoded cDNA from the mRNA of individual

first: Shilei

last: Chen

first: Song

last: Wang

first: Xinhai

first: Junping


title: A mitochondrial long-chain fatty acid oxidation defect in a mouse leads to dysregulation of plasma long-chain acylcarnitines, dysregulation of plasma amino acids, and an increased reliance on glucocorticoid signaling to maintain euglycemia during fasting

status: Public on Oct 26 2024

city: New York

first: Sander

middle: M

last: Houten

state: NY

address: 1425 Madison Avenue, Box 1498

department: Genetics and Genomic Sciences

institute: Icahn School of Medicine at Mount Sinai

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: single cell transcriptomic analysis of embyronic hearts with fibroblast ablation

city: Pittsburgh

first: guang

last: li

state: PA

address: 530 45th St. 8116 Rangos Research Center

institute: university of pittsburgh

summary: Control and ablated embryonic hearts from the Pdgfra-CreER; Rosa-DTA mice were isolated, dissociated, and multiplexed using the MULTI-seq approach, profiled with the 10X scRNA-seq platform and sequenced with Illumina NovaSeq X.

overall_design: ScRNA-seq analysis of wild type and DTA ablated mouse heart cells at two stages.

first: Yiting

last: Deng

first: Haoting

last: He

first: Guang

last: Li


title: Transcriptome RNA Sequencing Data Set of Differential Gene Expression in Salmonella enterica serovar Typhimurium 14028S within Acanthamoeba castellanii, and under Oxidative and Starvation Stress Conditions

city: Orenburg

first: Alexander

middle: S

last: Balkin

address: 11 Pionerskaya st.

country: Russia

institute: Institute for Cellular and Intracellular Symbiosis

summary: Here we present high-resolution transcriptomic data on Salmonella gene expression under optimal, starvation and hydrogen peroxide conditions, and after 8 and 15 hours of co-culture with A. Castellanii.

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. ATCC 14028

overall_design: To assess changes in global Salmonella gene expression during long-term infection of amoebae, we performed RNA sequencing of total Salmonella RNA within A. castellanii at late stages of infection (8 h and 15 h) and also compared three free-living cultures grown under different conditions, including rich medium (LB), poor medium (PAS), and exposure to hydrogen peroxide.

first: Yuri

last: Gogolev


title: Genome-wide in vivo dynamics of cohesin-mediated loop extrusion and its role in transcription activation [SLAM-seq]

city: La Jolla

first: Jesse

last: Dixon

state: CA

address: 10010 N. Torrey Pines Rd.

institute: Salk Institute for Biological Studies

summary: The organization of the genome in three-dimensional space is highly dynamic, yet how these dynamics are regulated and the role they play in genome function is poorly understood. Here, we utilized acute depletion of NIPBL to characterize the role of cohesin-mediated loop extrusion in vivo. Using this approach, we find that many chromatin loops are rapidly diminished upon loss of NIBPL, consistent with recent single locus imaging studies showing that chromatin loops are transient. However, we also identified a subset of cohesin-dependent chromatin loops that are anchored in repressive chromatin and may be “long-lived”, given that they require NIPBL for their establishment upon mitotic exit, but are persistent when NIPBL is depleted from interphase cells. In addition to the reformation of 3D genome structures, mitotic exit coincides with widespread transcriptional activation. We found that NIPBL is essential for establishing the expression of lineage-defining genes during the M-G1 transition but has diminished impact in the steady-state maintenance of their expression. At genes sensitive to its depletion, NIPBL supports a unique local genome organization defined by greater spatial proximity to nearby enhancers and weaker transcription start site insulation of genomic contacts. Overall, we show that NIPBL-mediated loop extrusion is critical to genome organization and transcriptional regulation in vivo.

overall_design: Role of NIPBL in post-mitotic transcriptional reactivation, chromatin association of cohesin, and 3D genome organization.

first: Tessa

last: Popay


title: Genome-wide in vivo dynamics of cohesin-mediated loop extrusion and its role in transcription activation [ChIP-seq]

type: Genome binding/occupancy profiling by high throughput sequencing


title: Regulatory network analysis of Dclk1 gene expression reveals a tuft cell-ILC2 axis that inhibits pancreatic tumor progression [RNA-seq]

status: Public on Oct 08 2024

first: Giovanni

last: Valenti

address: 1130 St Nicholas Ave

institute: Columbia University

summary: Dclk1 expression defines a rare population of cells in normal pancreas whose frequency is increased at early stages pancreatic tumors. The identity and the precise roles of Dclk1 expressing cells in pancreas are matter of debate, although the concept of their involvement in a number of key functions, including regeneration and neoplasia, has emerged. We employed a novel Dclk1 reporter mouse model and single cell RNAseq analysis to define Dclk1 expressing cells in normal pancreas and pancreatic neoplasia. In normal pancreas, Dclk1 epithelial expression identifies subsets of ductal, islet and acinar cells. In pancreatic neoplasia, Dclk1 expression identifies five epithelial cell populations, among which acinar-to-ductal metaplasia (ADM)-like cells and tuft-like cells represent the main ones. These cells play opposing roles in pancreatic neoplasia, with Dclk1+ ADM-like cells sustaining tumor growth and Dclk1+ tuft-like cells restraining tumor progression. The differentiation of Kras mutant acinar cells into Dclk1+ tuft-like cells requires the activation of SPIB and is further supported by a cellular loop involving cancer group 2 innate lymphoid cells (ILC2) and activated fibroblasts (CAFs) that provide IL13 and IL33, respectively. In turn, Dclk1+ tuft-like cells release angiotensinogen that play protective roles against pancreatic neoplasia. Overall, our study provides novel insights on the biology of Dclk1+ cells in normal pancreas and also unveils a protective axis against pancreatic neoplasia involving Dclk1+ tuft-like cells, ILC2 and CAFs, which ultimately results in angiotensinogen release.

overall_design:
In order to dissect the identity of Dclk1 cells in normal pancreas and early pancreatic neoplasia we performed a scRNA-seq analysis. Specifically, we used a droplet-based scRNA-seq to profile Dclk1 cells isolated from Dclk1-DTR-ZSGreen, Mist1-Kras-TdTomato-Dclk1-DTR-ZSGreen mice at 2 weeks after KRAS activation  and Mist1-Kras-TdTomato-Dclk1-DTR-ZSGreen mice 16 weeks after KRAS activation.
To better identify the genetic programs controlled by SPIB in pancreatic neoplasia, we performed a RNA-seq analysis of pancreatic organoids grown from single cells obtained from normal murine pancreas, in which we overexpressed SPIB as well as mutant KRAS.

first: Pasquale

last: Laise

middle: C


title: 3P-seq analysis of APP, BACE1, MAPT, and SNCA mRNAs in undifferentiated SH-SY5Ycells

status: Public on Oct 24 2024

city: Worcester

first: Sarah

middle: Marie

last: Davis

state: Massachusetts

address: 368 Plantation Street

department: RNA Therapeutics Institute

institute: University of Massachusetts Chan Medical School

summary: To determine expressed 3' end isoforms for APP, BACE1, MAPT, and SNCA mRNAs in undifferentiated SH-SY5Y cells

overall_design: Ran 3P-seq on undifferentiated SH-SY5Y cell lysate

first: Samuel

last: Hildebrand


title: Liver mRNA expression in 25 mice with or without genetic defects of mitochondrial fatty acid oxidation (Acadl and Acadvl knockout) using RNA sequencing

summary:
full title: A mitochondrial long-chain fatty acid oxidation defect in a mouse model leads to dysregulation of plasma long-chain acylcarnitines, dysregulation of plasma amino acids, and an increased reliance on glucocorticoid signaling to maintain euglycemia during fasting. [liver]
The liver is a major source of energy substrates during metabolic stress: fasting, prolonged exercise, febrile illness. Fasting-induced hypoglycemia is a characteristic feature of FAO disorders including very long chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD). However, the pathophysiological mechanisms that underlie the diversity of clinical presentation of FAO dysfunction are not known. Here, we investigated the transcriptional response in liver tissue to the FAO defect in a model of VLCADD: the long-chain acyl-CoA dehydrogenase (LCAD) knockout (KO) mouse. We found that differentially expressed genes from the liver were associated with molecular networks annotated for fatty acid oxidation and cholesterol biosynthesis from population-based networks.

overall_design: We analyzed liver mRNA expression in 25 mice with or without genetic defects of mitochondrial fatty acid oxidation (Acadl and Acadvl knockout) using RNA sequencing.

first: Carmen

last: Argmann

first: Aaron

last: Bender


title: Genome-wide in vivo dynamics of cohesin-mediated loop extrusion and its role in transcription activation [Hi-C]


title: Human single cell RNA-sequencing reveals a targetable CD8+ exhausted T cell population that maintains murine low-grade glioma growth

status: Public on Oct 23 2024

city: Saint Louis

first: David

last: Gutmann

address: 660 S. Euclid Ave

institute: Washington University in St. Louis School of Medicine

summary: Immune cell infiltration is a common feature of solid cancers, where T cells typically function as cytotoxic effectors to limit tumor growth, prompting therapies that activate this antineoplastic property (immune checkpoint inhibition; ICI). Unfortunately, ICI treatments have been largely ineffective for high-grade brain tumors (gliomas; HGGs). Leveraging several single-cell RNA sequencing datasets, we report greater CD8+ exhausted T cells in human pediatric low-grade gliomas (LGGs) relative to adult and pediatric HGGs. Using several preclinical mouse LGG models (Nf1-OPG mice), we showed that these PD1+/TIGIT+ CD8+ terminally exhausted T cells are restricted to the tumor tissue, where they express paracrine factors necessary for OPG growth. Importantly, ICI treatments with anti-PD1 and anti-TIGIT antibodies attenuate Nf1-OPG tumor proliferation through suppression of cytokine-mediated immune axis support, rather than by T cell-mediated cytotoxicity. Collectively, these findings establish a previously unrecognized function for CD8+ exhausted T cells as specialized regulators of LGG maintenance.

overall_design: All experiments were performed under active Animal Studies Committee protocols at Washington University School of Medicine (Washington University in St Louis Institutional Animal Care and Use Committee). Mice harboring a neomycin cassette insertion in exon 31 (Nf1+/-) with GFAP-Cre-mediated Nf1 loss in neuroglial progenitors (Nf1flox/neo; GFAP-Cre, Nf1-OPG mice) (Bajenaru et al., 2003). Nf1+/−, WT or Nf1flox/flox mice were employed as controls. De-identified human PA samples (4 female sporadic PAs and 1 female NF1-PA; clinical information in Supplemental Table 3) were obtained under an approved Human Studies Protocol as dissociated frozen tumor single cells from the St. Louis Children’s Hospital Pediatric Tumor Bank.

first: Rasha

last: Barakat

first: Jit

last: Chatterjee

first: Rui

last: Mu

first: Dahlia

last: Qi

first: Xingxing

last: Gu

first: Igor

last: Smirnov

first: Olivia

last: Cobb

first: Karen

last: Gao

first: Angelica

last: Barnes

first: Jonathan

last: Kipnis

middle: H


title: Skeletal muscle mRNA expression in 25 mice with or without genetic defects of mitochondrial fatty acid oxidation (Acadl and Acadvl knockout) using RNA sequencing.

first: Sander

last: Houten

summary:
A mitochondrial long-chain fatty acid oxidation defect leads to dysregulation of plasma long-chain acyl carnitines, dysregulation of plasma amino acids, and an increased reliance on glucocorticoid signaling to maintain euglycemia during fasting. [muscle]
Skeletal muscle tissue relies on products of fatty acid oxidation (FAO) during conditions of metabolic stress: fasting, prolonged exercise, febrile illness. Fasting-induced hypoglycemia and rhabdomyolysis are characteristic features of FAO disorders including very long chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD). However, the pathophysiological mechanisms that underlie the connection between FAO dysfunction and skeletal muscle dysfunction are not known. Here, we investigated the  transcriptional response in skeletal muscle tissue (gastrocnemius) to the FAO defect in a model of VLCADD: the long-chain acyl-CoA dehydrogenase (LCAD) knockout (KO) mouse. We found that differentially expressed genes in the muscle were associated with molecular networks annotated for the cellular response to starvation from population-based models. To validate the association between the starvation response and FAO, we pharmacologically inhibited both glucocorticoid signaling and FAO in a model of fasting and observed that mice depleted in both pathways lost less weight during fasting and became hypoglycemic. These findings implicate glucocorticoid signaling as a candidate modifier of the cellular response to starvation in muscle tissue in the context of FAO disorders including VLCADD.

overall_design: We analyzed skeletal muscle mRNA expression in 25 mice with or without genetic defects of mitochondrial fatty acid oxidation (Acadl and Acadvl knockout) using RNA sequencing.


title: Bulk RNA-sequencing of kidneys from male C57BL/6 mice subacutely exposed to TCDD.

status: Public on Oct 22 2024

first: Tim

last: Zacharewski

summary: Bulk RNA-sequencing of 40 C57BL/6NCrl mouse-right kidney samples where males were gavaged every 4 days for 28 days with 0.03 to 30 µg/kg TCDD or sesame oil vehicle (n=5 per dose). Gavaging and tissue collection occurred between ZT0-3.

overall_design: Beginning on post-natal day (PND) 30, male C57BL/6NCrl mice were repeatedly gavaged every 4 days for 28 days with some dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (0.03 - 30 µg/kg TCDD) or sesame oil vehicle (n = 5 per dose) and sacrificed on PND 58 with CO2. Gavaging and tissue collection always occurred between ZT0 and ZT3. Tissues were flash frozen in liquid nitrogen and stored at -80°C. For total RNA extraction using trizol, 100 mg was used per kidney sample. Only samples which passed Nanodrop and RIN quality control were used. 150 bp paired-end RNA reads were sequenced using a NovoSeq 6000.

first: Warren

last: Sink

first: Rance

last: Nault


title: Investigation of genetic dependencies in atypical teratoid rhabdoid tumors (ATRTs) [I]

status: Public on Oct 01 2024

city: Tübingen

first: Daniel

middle: J.

last: Merk

address: Otfried-Müller-Str. 27

country: Germany

institute: Hertie-Institute for Clinical Brain Research

summary: CRISPR/Cas9 knockout screens were performed to identify genetic dependencies of ATRT cell lines.

overall_design: Gene expression profiling of naive human ATRT cell lines

middle: J

first: Ghazaleh

last: Tabatabai


title: Biomarker discovery in paired mid luteal endometrial biopsies

status: Public on Oct 21 2024

city: Coventry

first: Jan

last: Brosens

address: University Hospital Coventry and Warwickshire

department: Warwick Medical School

country: United Kingdom

institute: University of Warwick

summary: Cell state determination is apparent within the endometrium during the mid-luteal window of implantation. Using previously identified biomarkers of cell states (SCARA5 and DIO2) we performed bulk RNA sequencing on 6 pairs of endometrial biopsies characterised by either normal biomarker expression in both biopsies, or abnormal biomarker expression in one biopsy (low SCARA5 and high DIO2) and normal expression in a second biopsy. We demonstrate that “abnormal cycles” are associated with a distinct gene expression profile when compared to “normal cycles” and have utilised this dataset for biomarker discovery.

overall_design: Endometrial mRNA profiles of 12 paired midluteal biopsies were generated by deep sequencing.

first: Joanne

last: Muter

first: Pavle

last: Vrljicak

first: Sascha

last: Ott


title: Identification of essential transporter genes in the colorectal carcinoma cell model HCT116 by focused CRISPR/Cas9 KO screens.

status: Public on Sep 25 2024

city: Vienna

first: Giulio

last: Superti-Furga

address: Lazarettgasse 14

country: Austria

institute: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences

summary: Transporters such as solute carriers (SLCs) are required to transport substrates such as nutrients or metals across cellular membranes. We here aimed to indentify which transporters are required for the proliferation of HCT116 WT and KO cells (SLC16A1, SLC16A3, SLC16A13, SLC25A28, SLC25A37 and SLC6A8 KOs).

sample_organism:
Homo sapiens
blank sample

platform_organism:
Homo sapiens
blank sample

overall_design: Pooled CRISPR/Cas9 KO screen in standard media with proliferation readout.

first: Gernot

last: Wolf

first: Svenja

last: Onstein

first: Esther

last: Uijttewaal


title: Myocardial transcriptional, and proteomic landscapes across the pre-menopausal, peri-menopasual, and menopasual continuum

status: Public on Sep 30 2024

city: Tucson

first: Marissa

middle: Anne

last: Lopez-Pier

state: AZ

address: 1656 E. Mabel St.

department: Physiology

institute: The University of Arizona

summary: More women than men have died from heart disease over the last 20-25 years, with morbidity and mortality rising in women after menopause supported to be result of decreased circulating estrogen. Current literature lacks an understanding of the cellular and molecular mechanisms that capture the shift in susceptibility to cardiovascular disease (CVD) during the menopausal transition from pre- to peri- to menopause. Therefore, through use of a chemical model (4-vinylcyclohexene diepoxide; VCD) of ovarian failure, which allows us to preserve the peri-menopause state, we performed transcriptomic and proteomic analysis on post-mortem heart tissue. In general, pre-, peri-, and menopausal hearts clustered more closely (most similar) within each experimental group and perimenopausal hearts clustered more closely with premenopausal hearts than menopausal hearts (least similar). Both proteomes and transcriptomes showed similar trends in genes associated with atherothrombosis, contractility, and impaired nuclear signaling between pre-, peri-, and menopausal hearts. From this, we emphasized a menopause and angiotensin II (AngII) effect on AMP-activated protein kinase (AMPK) signaling and histone deacetylase (HDAC) activity where we found both an estrogen- and pathologic-dependent increase in phosphorylation of AMPK and decrease in class I HDAC activity. Additionally, we found a menopause-dependent decrease in class IIa HDAC activity and increase in class IIb activity. These findings suggest unique changes in pre-, peri-, and menopausal mice indicative of metabolic reprogramming and adverse cardiac remodeling with loss of estrogen.

overall_design:
To investigate the menopasual pathogenic heart and the progression of disease from high estrogen production to low estrogen production, buy utilizing the VCD model in mice.
performed RNA seq profiling on 12 hearts , 4 for each group premenopausal , perimenopausal, and menopausal hearts ( LV and RV).
comparative RNA seq profiling for comparing each group to one another using one-way anova anlaysis.

first: Vito

last: Marino

first: John

last: Konhilas


title: Spatially defined multicellular functional units in colorectal cancer revealed from single cell and spatial transcriptomics

status: Public on Oct 18 2024

city: Cambridge

first: Simon

last: Mages

state: MA

address: 415 Main St.

institute: Broad Institute


title: Spatially defined multicellular functional units in colorectal cancer revealed from single cell and spatial transcriptomics - scRNAseq v2

summary: While advances in single cell genomics have helped to chart the cellular components of tumor ecosystems, it has been more challenging to characterize their specific spatial organization and functional interactions. Here, we combine single cell RNA-seq, spatial transcriptomics by Slide-seq, and in situ multiplex RNA analysis, to create a detailed spatial map of healthy and dysplastic colon cellular ecosystems and their association with disease progression. We profiled inducible genetic CRC mouse models that recapitulate key features of human CRC, assigned cell types and epithelial expression programs to spatial tissue locations in tumors, and computationally used them to identify the regional features spanning different cells in the same spatial niche. We find that tumors were organized in cellular neighborhoods, each with a distinct composition of cell subtypes, expression programs, and local cellular interactions. Comparing to scRNA-seq and Slide-seq data from human CRC, we find that both cell composition and layout features were conserved between the species, with mouse neighborhoods correlating with malignancy and clinical outcome in human patient tumors, highlighting the relevance of our findings to human disease. Our work offers a comprehensive framework that is applicable across various tissues, tumors, and disease conditions, with tools for the extrapolation of findings from experimental mouse models to human diseases.

overall_design: Single-cell suspensions from healthy colon or dysplastic lesions were processed to generate single cell suspensions. All cell suspensions were counted to achieve a uniform concentration of 7,000 cells per microliter for capture by 10x Chromium controller following the manufacturer protocol for the v2 3’ kit (10X Genomics, Pleasanton, CA).

first: Inbal

last: Avraham-Davidi

first: Leah

last: Caplan

first: Danielle

last: Dionne

first: Toni

last: Delorey


title: Genome-wide in vivo dynamics of cohesin-mediated loop extrusion and its role in transcription activation

first: Jesse

last: Dixon


title: Investigation of genetic dependencies in atypical teratoid rhabdoid tumors (ATRTs) [III]

first: Daniel

last: Merk

type: Methylation profiling by genome tiling array

summary:
CRISPR/Cas9 knockout screens were performed to identify genetic dependencies of ATRT cell lines.
This submission covers methylation analyses of human ATRT cell lines as assessed by the Illumina Infinium HumanMethylation850 BeadChip array.

overall_design: Bisulphite converted DNA from 7 samples were hybridized to the Illumina Infinium HumanMethylation850 Beadchip

first: Ghazaleh

last: Tabatabai


title: TNF Drives Aberrant BMP Signaling to Induce Endothelial and Mesenchymal Dysregulation in a Transgenic Model of Pulmonary Hypertension

status: Public on Oct 11 2024

city: Rochester

first: Benjamin

last: Korman

address: 601 Elmwood Avenue, Box 695

institute: AIR

summary: The pathobiology of pulmonary hypertension (PH) is complex and multiple cell types (endothelial, smooth muscle, fibroblast, pericyte) contribute to disease pathogenesis. However, the specific molecular crosstalk between endothelial and mesenchymal cells to promote PH is poorly characterized.  We previously demonstrated that tumor necrosis factor alpha transgenic (TNF-Tg) mice have a severe PH phenotype.

overall_design: Pulmonary endothelial and mesenchymal cells were isolated from WT and TNF-Tg mice at 8,14, and 20 weeks of age and underwent single-cell RNA sequencing.


title: An eRNA Transcription Checkpoint For Diverse Signal-dependent Enhancer Activation Programs

status: Public on Sep 17 2024

city: SAN DIEGO

first: Lishuan

last: Wang

state: California

address: 9500 Gilman Drive

institute: UCSD

summary: While many or most signal and ligand-dependent pathways function by directing binding of specific transcription factors to large cohorts of regulatory enhancers, the possible “universal” strategies underling activation of these lorge enhancer programs by diverse signals remains poorly understood. Here, we report that ligands, exemplified by estrogen-17-b (E2), cause the recruitment of DNA-PKcs-phosphorylated KAP1 to estrogen receptor a (ERa)-bound enhancers, inhibiting its E3 SUMO ligase activity for the CDK9 subunit of P-TEFb, which permits formation of active P-TEFb required for eRNA elongation overcoming this checkpoint and permitting enhancer activation. We find that most, or all, signal and ligand enhancer activation programs similarly require the loss of SUMOligase function by phoshorylation of KAP1 to prevent Pol II pausing at regulated enhancers. This is based on activation of P-TEFb consequent to deSUMOylation of CDK9, permitting eRNA synthesis. Overcoming this checkpoint appears to be a widely, if not universally, used strategy for signal-dependent enhancer activation because similar events occur on AR and NF-kB-regulated enhancers and even following depolarization acutely-activated neuronal enhancers. This study reveals an unexpected strategy required to overcome a checkpoint common to most or all signal/ligand-regulated enhancers critical to activate broad programs of biologically-important regulatory enhancers that implement the instructions of the endocrine system.

overall_design: For hormone treatments, cells were incubated at 37°C and 5% CO2 for at least 3 days in phenol red-free DMEM (GIBCO/Invitrogen) supplemented with 5% charcoal dextran-stripped FBS (GIBCO/Invitrogen). 17-β-Estradiol (E2; Steraloids, Inc.) was added to a final concentration of 100 nM. For knock-down of KAP1 genes, the shRNA were employed.


title: Non-Canonical PRC1.1 is required for the activity of Menin-MLL1 Inhibitors in NUP98-Rearranged AML [RNA-seq]

status: Public on Sep 16 2024

city: Boston

first: Scott

last: Armstrong

address: 450 Brookline Ave

department: pediatric oncology

institute: Dana Farber Cancer Institute

summary: Translocations involving the Nucleoporin 98 (NUP98) gene predict a poor prognosis for patients with acute myeloid leukemia. These translocations generate NUP98-fusion proteins which interact with the mixed-lineage leukemia (MLL1/KMT2A) chromatin modifying enzyme and its binding partner Menin. The mechanisms by which the NUP98-fusion proteins maintain gene expression remain unclear. We demonstrate that the NUP98-fusion-Menin-KMT2A complex is essential for maintenance of H3K27Ac and active transcription at key loci such as Meis1 and the Hox cluster, among others. However, eviction of the complex from chromatin is insufficient to completely silence gene expression. Accumulation of the PRC1.1 complex and deposition of H2aK119Ub and H3K27Me3 is required to transition to a transcriptionally repressive chromatin state. Furthermore, we demonstrate that loss of PRC1.1 function leads to resistance to small molecule Menin inhibitors. Therefore, a critical function of the NUP98-fusion-Menin-KMT2A complex is to antagonize repressive chromatin complexes. These same mechanisms are critical for efficacy of newly developed therapeutics.

overall_design: NUP98-rearranged murine leukemia cell lines were engineered to constitutively express CRISPR-Cas9 and sgRNA targeting individual components of PRC1.1 (Bcor, Kdm2b, Rnf2, or Pcgf1) or the sgluciferase control. Vectors containing sgRNA also contained an RFP fluorescent marker. Cell lines were established by sorting RFP+ cells, and CRISPR editing was confirmed by Sanger sequencing with the Synthego ICE algorithm. RNA-seq libraries were constructed from RNA extracted from established cell lines at steady state.

first: Emily

middle: B

last: Heikamp

first: Charles

last: Hatton

first: Yanhe

last: Wen


title: Regulatory network analysis of Dclk1 gene expression reveals a tuft cell-ILC2 axis that inhibits pancreatic tumor progression [scRNA-seq]

first: Giovanni

last: Valenti

first: Pasquale

last: Laise

first: Timothy


title: Non-Canonical PRC1.1 is required for the activity of Menin-MLL1 Inhibitors in NUP98-Rearranged AML [SLAM-Seq]

overall_design: NUP98-HOXA9 and NUP98-JARID1A murine leukemia cells expressing FKBP-tagged versions of the fusion protein and treated with 500 nM dTAG-13 for 4 hours to detect changes in nascent transcription upon fusion protein degradation.

first: Charlie


title: Non-Canonical PRC1.1 is required for the activity of Menin-MLL1 Inhibitors in NUP98-Rearranged AML

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: mRNA sequencing after BMSC treatment of BDL-induced liver fibrosis

status: Public on Sep 13 2024

city: Taiyuan

first: Tingjuan

last: Huang

address: NO.56 Road Jiefang

institute: Shanxi Medical University

summary: Cholestatic liver disease (CLD) represents a severe pathological condition that can advance to cirrhosis and hepatocellular carcinoma. The activation of hepatic stellate cells (HSCs) is integral to the pathogenesis of CLD. Previous research has indicated that bone marrow mesenchymal stem cells (BMSCs) may offer therapeutic benefits for liver diseases. Nonetheless, the specific therapeutic efficacy and underlying mechanisms of BMSCs in the context of CLD remain inadequately understood. In this study, we aimed to elucidate the therapeutic efficacy and potential therapeutic genes of BMSCs transplantation in a murine model of cholangioligation-induced cholestatic liver fibrosis (CLF).

overall_design: The CLF model was prepared by bile duct ligation (BDL). The mice in the BDL+BMSC group were transplanted with one million BMSCs through the portal vein before abdominal closure after ligation of the common bile duct, and the samples were collected 4 weeks later.


title: Non-Canonical PRC1.1 is required for the activity of Menin-MLL1 Inhibitors in NUP98-Rearranged AML [CRISPR-Seq]

overall_design: NUP98-HOXA9 murine leukemia cells expressing the CRISPR-Cas9 enzyme were transduced with a lentiviral sgRNA library targeting 616 epigenetic regulators (CRISPR Epi-Screen).  Following transduction and antibiotic selection, cells were treated with DMSO or 200 nM VTP50469 for 14 days.


title: Investigation of genetic dependencies in atypical teratoid rhabdoid tumors


title: Investigation of genetic dependencies in atypical teratoid rhabdoid tumors (ATRTs) [II]

overall_design: Gene expression profiling of human ATRT cell lines treated with the CDK4/6 inhibitor abemaciclib


title: Discovery of chromosomal inversions in the mosquito Aedes aegypti

status: Public on Sep 06 2024

city: Blacksburg

first: Maria

last: Sharakhova

state: VIRGINIA

address: 360 West Campus Drive

department: Entomology

institute: Virginia polytechnic institute and state university

summary: Chromosomal inversions play a fundamental role in evolution and have been shown to be responsible for the epidemiologically important traits in malaria mosquitoes. However, they have never been characterized in the major vector of arboviruses Aedes aegypti because of the poor structure of its polytene chromosomes. In this study, we applied a Hi-C proximity ligation approach to identify chromosomal inversions in 23 recently collected strains of Ae. aegypti from its worldwide distribution, two old laboratory colonies, and Ae. mascarensis.

sample_organism:
Aedes aegypti
Aedes mascarensis

platform_organism:
Aedes aegypti
Aedes mascarensis

overall_design: To invetigate chromosomal inversions in Ae. aegypti, we applied Hi-C to identify chromosomal inversions in 25 strains of Ae. aegypti from its world-wide distribution and Ae. mascarensis


title: Purification of mitochondria from skeletal muscle tissue for transcriptomic analyses reveals localization of nuclear-encoded non-coding RNAs [Mitoplasts]

status: Public on Sep 11 2024

city: Parkville

first: Adam

middle: James

last: Trewin

state: Victoria

address: Royal Pde

department: Anatomy and Physiology

country: Australia

institute: University of Melbourne

summary: Mitochondria are central to cellular function, particularly in metabolically active tissues such as skeletal muscle. Nuclear-encoded RNAs typically localise within the nucleus and cytosol but a small population may also translocate to subcellular compartments such as mitochondria. We aimed to investigate the nuclear-encoded RNAs that localise within the mitochondria of skeletal muscle cells and tissue. Intact mitochondria were isolated via immunoprecipitation (IP) followed by enzymatic treatments (RNase-A and proteinase-K)  to remove transcripts located exterior to mitochondria, making it amenable for high-throughput transcriptomic sequencing. Whole-transcriptome RNA sequencing of enzymatically-purified mitochondria isolated by IP from skeletal muscle tissue showed a striking similarity in the degree of purity compared to mitoplast preparations which lack an outer mitochondrial membrane. In summary, we describe a novel, powerful sequencing approach applicable to animal and human tissues and cells that can facilitate the discovery of nuclear-encoded RNA transcripts localised within skeletal muscle mitochondria.

overall_design: Samples are ribo-depleted libraries construced from the total RNA of various preparations of mitochondria isolated from the gastrocnemius skeletal muscle (SM) tissue of a wistar kyoto rat. Mitochondrial preparations were crude mitochondria isolated by differential centrifugation, immunoprecipitated isolated mitochondria or mitoplasts derived from the latter. All preparations underwent RNase-A treatment, with or without proteinase-K treatment.

first: Jessica

middle: L

last: Silver

first: Stella

last: Loke

first: Larry

last: Croft

first: Mark

last: Ziemann

first: Megan

last: Soria

first: Hayley

last: Dillon

first: Søren

last: Nielsen

first: Séverine

last: Lamon

first: Glenn

middle: D

last: Wadley


title: Identification of genes regulated by knockdown of EWS-FLI1 in Ewing sarcoma cells.

city: Iowa City

first: Emma

last: Croushore

state: Iowa

address: 25 S Grand Ave

department: Pediatric Hematology/Oncology

institute: University of Iowa

summary: EWS-FLI1 is the driver oncogene in >85% of Ewing sarcoma tumors. We used siRNA to knock down EWS-FLI1 for 48h hours in two Ewing sarcoma cell lines (EW8, TC71) to identify downstream genes and pathways affected by loss of EWS-FLI1 activity. RNAseq analysis was performed after 48 hours of treatment with a nontargeting siControl or siEWS-FLI1.

overall_design: We performed RNA-seq in Ewing sarcoma cell lines with 48h siRNA-mediated knockdown of EWS-FLI1 or siControl.

middle: E

first: David

last: Gordon


title: Changes in the astrocyte translatome induced by developmental ethanol mediate dysregulation of neuronal development: role of the extracellular matrix and relevance to Fetal Alcohol Spectrum Disorders

city: Portland

first: Marina

last: Guizzetti

state: OR

address: 3710 SW US Veterans Hospital Rd

department: Behavioral Neuroscience

institute: Oregon Health & Science University; Portland VA Research Service

summary:
Background: Fetal Alcohol Spectrum Disorders (FASD) cause life-long cognitive and behavioral impairment and are highly prevalent across the world. The effects of developmental ethanol exposure on astrocyte functions in vivo are poorly understood. To fill this gap, we assessed changes in the astrocyte translatome following in vivo developmental ethanol exposure and mechanistically linked some of these changes to altered neuronal development.
Methods: The translating RNA from hippocampal astrocytes of neonatal Aldh1l1-EGFP-Rpl10a mice exposed to ethanol was isolated by Translating Ribosomal Affinity Purification and sequenced; neuronal dendritic morphology was assessed by Golgi-Cox staining and morphometric analysis; chondroitin sulfate glycosaminoglycan (CS-GAG) disaccharides were measured by Liquid Chromatography/Mass Spectrometry; neurite outgrowth was assessed in in vitro hippocampal neurons incubated in the presence of astrocyte-conditioned medium prepared from control and Chpf2-silenced astrocytes.
Results: Translatome results suggested that ethanol alters mechanisms of astrocyte-neuron interactions involved in neuronal development. Moreover, ethanol increased dendritic arborization in hippocampal pyramidal neurons, inhibited the astrocyte translation of enzymes involved in the biosynthesis of inhibitory CS-GAGs, and decreased the levels of disaccharides forming CS-GAGs in vivo. Lastly, the silencing of CS-GAG biosynthetic enzyme Chpf2 in astrocyte cultures increased neurite branching of hippocampal neuron in vitro.
Conclusions: Translatome analysis revealed novel effects of developmental ethanol exposure on astrocytes and provided mechanistic insights into altered brain development possibly relevant to the pathophysiology of FASD. We also identified a novel mechanism by which ethanol-induced reduction of astrocyte-produced inhibitory extracellular matrix components CS-GAGs increases dendritic arborization.

overall_design: Aldh1l1-EGFP/RPL10a mice were exposed to ethanol vapor or air for 4 hours a day from postnatal day (PD) 2 to 7. On PD7, immediately after removal from the chambers, hippocampus samples were collected. From each hippocampus sample, input total homogenate RNA was collected as well as astrocyte-specific RNA via the translating ribosome affinity purification (TRAP) procedure. Both input and TRAP fractions were sequenced.

first: Joel

middle: G

last: Hashimoto

first: Nicholas

last: Margolies

first: Xiaolu

last: Zhang

first: Joshua

last: Karpf

first: Brett

last: Davis

first: Fuming

first: Robert

last: Linhardt

first: Lucia

last: Carbone


title: Targeted single cell expression profiling identifies integrators of sleep and metabolic state

status: Public on Sep 27 2024

city: Stony Brook

first: Meng-Fu

middle: Maxwell

last: Shih

address: 100 Nicolls Road

department: Anesthesiology

institute: Stony Brook University

summary: The fruit fly, Drosophila melanogaster, suppresses sleep in response to acute food deprivation, presumably to forage for food.  This process is dependent on a single pair of neurons, termed the Lateral Horn Leucokinin (LHLK) neurons, that secrete the neuropeptide Leucokinin.  These neurons signal to insulin producing cells and suppress sleep under periods of starvation. Here, we use single-cell sequencing of LHLK neurons to examine the transcriptional responses to starvation.

overall_design: LHLK neurons were genetically labeled by GFP expression (LK-GAL4>UAS-GFP). Later after behavioral purturbation, the targeted LHLK neurons were harvested on a custom rig, followed by CEL-Seq-based single cell RNA sequencing protocol. CEL-Seq data of LHLK neurons from female flies subjected to 24-hr starvation were compared to the control or fed group to identify differential expression in response to starvation.

first: Jiwei

first: Elizabeth

last: Brown

first: Josh

last: Dubnau

first: Alex

last: Keene


title: Data Mining of IFNgamma-induced macrophage and Atherosclerotic Plaque Transcriptomes predicts a macrophage-specific role of STAT1 in human atherosclerosis [BMDM]

status: Public on Sep 09 2024

city: Poznan

first: Hans

last: Bluyssen

address: Uniwersytetu Poznańskiego 6

department: Laboratory of Human Molecular Genetics

country: Poland

institute: Adam Mickiewicz University

summary: Atherosclerosis is a chronic inflammatory disease of the blood vessels, characterized by atherosclerotic lesions. Early vascular injury initiates excessive inflammatory and immune responses, communicated by blood leukocytes and cells from the vasculature. Key factors contributing to early stages of atherosclerosis and plaque development include the pro-inflammatory cytokine IFNγ, which is vital for both innate and adaptive immunity and is also expressed at high levels in atherosclerotic lesions. Recent discoveries provide novel evidence to suggest that IFNγ-activated STAT1 orchestrates a platform of cell-type common and specific inflammatory responses in Vascular Smooth Muscle Cells (VSMC) and macrophages (MΦ) that are instrumental in the onset and progression of atherosclerotic plaque formation. Therefore, we compared genome-wide transcriptional responses of mouse VSMCs and bone marrow-derived macrophages (BMDMs) in time-dependent IFNγ response using RNA-seq. We identified genes upregulated both in a common and cell-type-specific manner. Next, comparative analysis with RNAseq data from aortic plaques from HFD-treated ApoEKO mice identified 225 differentially expressed genes with a BMDM-specific character. These included many known IFNγ target genes containing putative GAS and ISRE sites in their promoters. Likewise, using a scRNAseq data set obtained from human coronary atherosclerotic plaques, identified 98 pro-inflammatory and pro-atherogenic genes that were characterized as BMDM-specific and exclusively expressed in foamy, inflammatory, monocytes and tissue-resident BMDM subtypes. Finally, we identified a 25 BMDM-specific gene subset commonly expressed in mouse and human plaques, over-representing STAT1-binding sites and predominantly associated with Leukocyte Chemotaxis and Migration.  These results provide strong evidence for a BMDM-specific role of STAT1 in coronary artery plaque development and identify a BMDM-specific STAT1-dependent pro-inflammatory gene signature that could serve to monitor and diagnose plaque progression in human atherosclerosis.

overall_design: RNA-seq samples were derived from Bone Marrow Derived Macrophages in 3 replicates in untreated or mIFN gamma-treated at 0,5h, 2h, 4h, 8h and 24h.


title: LSK immunophenotype hematopoietic stem cells in Dapp1 knockout mice

status: Public on Sep 18 2024

city: 重庆Chongqing

first: Zeyu

last: Fu

address: 高滩岩正街

institute: AMU

summary: To investigate the impact of perturbing key differentiation driver genes on hematopoietic stem cell fate decision, we employed a Dapp1 gene knockout approach. This allowed us to observe changes in the subpopulations of hematopoietic stem cells at the LSK level in mice. Additionally, we assessed alterations in their fate decision and lineage differentiation tendencies

overall_design:
We conducted an in-depth single-cell RNA sequencing study using the 10X Genomics platform to analyze mouse bone marrow LSK (Lin^⁻Sca-1^⁺c-Kit^⁺) cells. Starting with ethically euthanized mice, we extracted femurs and tibias to harvest the bone marrow. The marrow cells were flushed out using a syringe with PBS containing 2% FBS to create a single-cell suspension. This suspension was filtered through a 40 µm cell strainer to eliminate debris and cell clumps, ensuring a pure population of cells for analysis. To isolate our target LSK cells, we employed fluorescence-activated cell sorting (FACS), selecting for Lineage-negative, Sca-1-positive, and c-Kit-positive cells. We assessed the viability of these cells using trypan blue staining and adjusted the concentration to approximately 1,000 cells/µL, optimizing conditions for the 10X Genomics Chromium Controller. We then loaded the purified single-cell suspension, along with the necessary reagents and gel beads, into a 10X Genomics Single Cell 3' Chip. Running the chip in the Chromium Controller, we encapsulated individual cells into Gel Bead-In-Emulsions (GEMs), enabling the barcoding of mRNA from each cell during reverse transcription within the GEMs. Following emulsion breakage, we purified the barcoded cDNA and performed PCR amplification. The amplified cDNA was evaluated for quality and quantity using an Agilent Bioanalyzer to ensure downstream sequencing success. Constructing sequencing libraries from the high-quality cDNA, we followed the 10X Genomics protocol meticulously. These libraries were then sequenced on an Illumina NovaSeq platform, adhering to the recommended read lengths and depths to achieve comprehensive single-cell transcriptional coverage.
The sequencing data generated were processed using the 10X Genomics Cell Ranger pipeline, which aligned reads and produced a gene expression matrix for each cell. For data analysis, we utilized various bioinformatics tools, prominently featuring our novel scRL (single-cell Reinforcement Learning) framework. This advanced analytical approach allowed us to identify distinct cell populations and gene expression patterns with high precision. Most critically, employing scRL enabled us to obtain quantitative indicators of the strength of cell lineage differentiation fate decisions—a level of insight not achievable with existing methods.

first: Song

last: Wang

first: Shilei

last: Chen

first: Junping


title: Device-free isolation of photoreceptor cells from patient iPSC-derived retinal organoids

status: Public on Sep 02 2024

first: Nathaniel

middle: Kevin

last: Mullin

state: IA

address: 375 Newton Road

summary:
Purpose: Autologous cell replacement shows great promise for the treatment of inherited retinal degeneration. While mature differentiation protocols exist to produce retinal organoids from patient-derived stem cells, not all cell types present in these organoids are desirable for transplant. To increase the potency of future cell therapies, methods for isolating photoreceptors from dissociated retinal organoids are needed. In this work, we show how partial dissociation can be used to exploit the spatial organization of retinal organoids to produce highly pure photoreceptor populations without the use of specialized sorting devices or reagents such as xenobiotic antibodies.
Methods: Retinal organoids were generated as we have described previously. For flow cytometry experiments, organoids were dissociated using papain for 30 minutes or for 90 minutes followed by trituration. For scRNAseq experiments, liberated cells were collected from papain-dissociating organoids at 20-minute intervals. At the conclusion of the experiment, remaining tissue was fully dissociated. A control 60-minute full dissociation was performed in parallel. Photoreceptor purity was assessed via flow cytometry and scRNAseq.
Results: Photoreceptors desired for transplant are typically found in the outer layers of retinal organoids, suggesting that partial dissociation could selectively release these cells. Flow cytometry results indicating an increase in purity of CD133+ cells from 21.1% to 91.7% when cells were partially dissociated as compared to full dissociation. A time dependent release of photoreceptors, with a maximum purity of 96% CRX+ cells at 40 minutes as compared to 66% pure for traditional dissociation.
Conclusions: By timing the dissociation of retinal organoids, highly pure photoreceptor cell populations suitable for cell replacement therapies can be obtained under cGMP without the use of sorting reagents or equipment.

overall_design: Human retinal organoids were subjected to various partial dissociation conditions and profiled using 3' scRNAseq.

first: Nicholas

last: Stone

first: Laura

middle: R

last: Bohrer

middle: K

first: Budd

middle: A

last: Tucker


title: Effects of neobractatin and trametinib on gene expression in pancreatic cancer cells

status: Public on Sep 01 2024

city: Shanghai

first: Jiaqi

last: Tan

state: Shanghai

address: Cailun road 1200

institute: Shanghai University of Traditional Chinese Medicine

summary: Pancreatic ductal adenocarcinoma carcinoma (PDAC) is a highly lethal disease and ranks as the seventh leading cause of cancer-related deaths worldwide. Neobractatin (NBT), a natural compound derived from Garcinia bracteate, has shown great antitumor effects and has been identified as a regulator of pyroptosis in esophageal cancer cells. The effectiveness of combining NBT with the common targeted drug trametinib in inhibiting PDAC and its underlying mechanisms remain unclear. In this study, we aimed to explore the therapeutic potential of combining NBT with trametinib in PDAC.

overall_design: To investigate the synergistic effects of NBT and trametinib in regulating anti-tumor effects, MIA PaCa-2 cells were treated with NBT (1 μM) or trametinib (5 μM) or in a combination for 12 h. We then performed gene expression profiling analysis using data obtained from RNA-seq of 4 different cells. Comparative gene expression profiling analysis of RNA-seg data for MIA PaCa-2 cells.


title: An eRNA Transcription Checkpoint For Diverse Signal-dependent Enhancer Activation Programs

first: Lishuan


title: DLX4 regulates the invasiveness of Rheumatoid Arthritis Fibroblast-Like Synoviocytes and a cancer transcriptomic signature

status: Public on Sep 05 2024

city: New York

first: Teresina

last: Laragione

address: 1468 Madison Ave

department: Medicine

institute: Icahn School of Medicine at Mount Sinai

summary: Background: The fibroblast-like synoviocyte (FLS) has a central role in rheumatoid arthritis (RA) pathogenesis and its invasive behavior strongly correlates with disease severity and joint damage. Yet, the regulation of FLS invasiveness is incompletely understood. Distal-less homeobox 4 (DLX4) is a transcription factor implicated in cancer cell invasion and metastasis, and we considered that it might also be involved in the regulation of FLS phenotypes.  Results: siRNA knockdown of DLX4 significantly reduced RA FLS invasiveness (P=0.028) and migration in the scratch/wound healing assay (P=0.008). RNA sequencing analyses revealed that DLX4 knockdown significantly affected processes and pathways implicated in diverse types of cancer and in cancer biology and cell invasion, including cell cycle, DNA replication, transcription, p53 signal transduction, RHO GTPase signaling, regulation of cytokinesis, as well as response to oxygen levels and to oxidative stress. HMOX1 and SOD2 were among the genes with the most significantly increased expression, while CPNE3 and HADHA were among those with the most significantly decreased expression in FLS knocked down for DLX4 compared with controls.  Conclusion: We describe a new role for DLX4 in the regulation of RA FLS behaviors relevant to disease pathogenesis and joint damage, and identify a new transcriptomic signature regulated by this gene. These findings raise the possibility that DLX4, or one of its target genes and pathways may become a target for treatment

overall_design: To investigate the role of DLX4 in rheumatoid arthritis siRNA was used to knockdown DLX4 in RA FLS, compared with a control siRNA. Cells were then studied in invasion, migration, proliferation and adhesion assays, and RNA was used for RNA sequencing and pathway analyses.

first: Carolyn

last: Harris

first: Percio

last: Gulko


title: Non-Canonical PRC1.1 is required for the activity of Menin-MLL1 Inhibitors in NUP98-Rearranged AML [PRO-Seq]

first: Scott

last: Armstrong

overall_design: NUP98-HOXA9 murine leukemia cells expressing FKBP-tagged versions of the fusion protein, the CRISPR-Cas9 enzyme, and sgRNA targeting luciferase or Pcgf1 were treated with 500 nM dTAG-13 for 1 hour, then evaluated by PRO-seq to detect changes in nascent transcription upon fusion protein degradation.

first: Emily

last: Heikamp

first: Charlie

last: Hatton

first: Yanhe

last: Wen


title: CCL2-mediated Endothelial Injury Drives Cardiac Dysfunction in Long COVID [RNAseq_washOut_COVID]

status: Public on Aug 31 2024

city: Palo Alto

first: Nazish

last: Sayed

address: 240 Pasteur Dr

institute: Stanford University

summary: Chronic endotheliitis and various cardiovascular co-morbidities are more likely to develop in patients who are recovering from a post-acute SARS-CoV-2 infection. Despite a growing body of clinical data suggesting that the endothelium could be the cause of both cardiac injury and the multi-organ damage found in COVID-19 patients, there is no clear link between endothelial (EC) dysfunction and increased cardiac risk during long COVID. Here, we studied long COVID-19-associated endotheliitis and its implications on cardiac dysfunction. Thrombotic vascular tissues from long COVID patients were harvested and profiled to identify the different mechanisms of viral-induced EC pathogenesis. Human induced pluripotent stem cell (iPSC)–derived ECs were leveraged to model endotheliitis in-a-dish after exposure to SARS-CoV-2, which showed similar EC dysfunction and upregulation of specific cytokines such as CCL2 and IL6, as seen in the primary ECs of long COVID patients. 3D fabricated cardiac organoids generated from iPSC-ECs and iPSC-derived cardiomyocytes (iPSC-CMs) were utilized to understand the pathological influence of endotheliitis on cardiac dysfunction. Notably, cardiac dysfunction was observed only in cardiac organoids that were fabricated with both iPSC-CMs and iPSC-ECs after exposure to SARS-CoV-2. Simultaneous profiling of chromatin accessibility and gene expression dynamics via integration of ATAC-seq and RNA-seq at a single cell resolution revealed CCL2 as the prime cytokine responsible for the non-endothelial “phenotype switching” and the impending cardiac dysfunction in cardiac organoids. This was further validated by high-throughput proteomics that showed CCL2 to be released only by cardiac organoids that were fabricated with iPSC-CMs and iPSC-ECs after SARS-CoV-2 infection. Lastly, disease modeling of the cardiac organoids as well as exposure of human ACE2 transgenic mice to SARS-CoV-2 spike proteins uncovered a putative mechanism for the cardiac dysfunction involving posttranslational modification of cardiac proteins driven by oxidative stress and inflammation. These results suggest that EC-released cytokines can contribute to the pathogenesis of long COVID-associated cardiac dysfunction, and thus a thorough clinical profiling of vascular health could help identify early signs of heart disease in COVID-19 patients.

overall_design: Gene expression profiling analysis of RNA-seq data for cardiac organoids fabricated with iPSC-ECs and iPSC-CMs.

first: Sadhana

last: Gaddam


title: Data Mining of IFNgamma-induced macrophage and Atherosclerotic Plaque Transcriptomes predicts a macrophage-specific role of STAT1 in human atherosclerosis [VSMC]

first: Hans

last: Bluyssen

overall_design: RNA-seq samples were derived from Vascular Smooth Muscle Cells in 3 replicates in untreated or mIFN gamma-treated at 0,5h, 2h, 4h, 8h and 24h.


title: Dishevelled-1 regulates global transcriptomic changes in MDA-MB-231 triple negative breast cancer cells

status: Public on Aug 30 2024

city: Chapel Hill

first: Dalia

last: Martinez-Marin

state: NC

address: 450 West Dr

department: Dept of Pharmacology

institute: University of North Carolina

overall_design: Refer to individual Series


title: Dishevelled-1 regulates global transcriptomic changes in MDA-MB-231 triple negative breast cancer cells (ChIP-Seq).

summary: Dishevelled (DVL) is a crucial component of the Wnt-signaling pathway and is vital for multiple physiological processes. Previously thought to have a primarily cytoplasmic role, the discovery of DVL translocation to the nucleus reframed how DVL is viewed functionally. Despite our advances in elucidating DVL nuclear function, further investigation is needed to determine its transcriptomic and epigenetic regulatory abilities. We show here that modification of DVL1 expression globally affects the transcriptomic landscape. Additionally, analysis of DVL1 ChIP-seq allowed us to map global binding sites and reveal the extensive reach of DVL1 binding sites. Integration of RNA-Sequencing and ChIP-Sequencing further revealed DVL1 transcription factor binding partners to regulate gene expression. These findings provide insight to nuclear DVL1 regulation of gene transcription.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for DVL1 in triple-negative breast cancer cell line MDA-MB-231.

first: Kevin

last: Pruitt

first: Monica

last: Sharma


title: Non-Canonical PRC1.1 is required for the activity of Menin-MLL1 Inhibitors in NUP98-Rearranged AML [ChIP-Seq]

overall_design: Chromatin immunoprecipitation DNA Sequencing (ChIP-Seq) for FKBP-tagged NUP98-fusion proteins, activating and repressive histone modifications, and PRC1.1 Complex members in NUP98-rearranged murine leukemia cells treated with dTAG-13


title: Data Mining of IFNgamma-induced macrophage and Atherosclerotic Plaque Transcriptomes predicts a macrophage-specific role of STAT1 in human atherosclerosis


title: Gene expression profile at single cell level of aortic endothelial cells (ECs) from endothelial-specific Epas1 knockout mice (Epas1fl/fl, Cdh5Cre/+) and controls (Epas1fl/fl, Cdh5+/+)

status: Public on Aug 27 2024

city: London

first: Paul

last: Evans

address: Charterhouse square

institute: QMUL

summary: Single cell RNA sequencing (scRNA-seq) was used to analyse the diversity of ECs in the mouse aorta, as well as the effect of endothelial-specific Epas1 deletion on aortic endothelial cells.

overall_design: The aorta from Epas1 EC-KO mice and  Epas1 EC-WT mice was dissected and digested. Single endothelial cells were isolated by Fluorescence-activated cell sorting (FACS), selecting CD45-, CD31+ and Topro3- cells. Cells were analyzed using scRNAseq.

first: Mark

last: Dunning

first: Blanca

last: Tardajos Ayllón


title: Genomic and cellular responses to aspirin treatment in colonic organoids from African- and European-Americans

city: Chicago

first: Sonia

middle: S

last: Kupfer

state: IL

department: Human Genetics

institute: University of Chicago

summary: Aspirin (ASA) is a proven chemoprotective agent for sporadic and hereditary colorectal cancer (CRC), though mechanisms underlying these effects are incompletely understood. Human-derived epithelial organoids are an ideal system to study host-environment interactions in the colon across individuals. Here, colonic organoids from a diverse cohort of African-Americans (AA) and European-Americans (EA) were used to profile genomic and cellular ASAresponses.

overall_design: Human colonic organoids derived from 67 healthy individuals (33 AA and 34 EA) were cultured, differentiated and treated with 3mM ASA or vehicle control (DMSO) for 24 hours(h). Gene expression was measured using RNA-sequencing, and differentially responsive genes were analyzed by condition and population as well as for gene set enrichment. Top differentially responsive genes were assessed by time (3h, 6h & 24h) and dose (0.5mM and 3mM) using qPCR in independent organoid lines. eQTL mapping was performed to identify genetic variants associated with condition-specific responses. Proliferation, apoptosis and necrosis assays were performed in organoids, and apoptosis gene expression measured at several ASA doses and time points.

first: Hina

last: Usman

first: David

last: Witonsky

first: Margaret

middle: C

last: Bielski

first: Kristi

middle: M

last: Lawrence

first: Bharathi

last: Laxman


title: CCL2-mediated Endothelial Injury Drives Cardiac Dysfunction in Long COVID [LiveOrganoids]

address: 240 Pasteur Drive, 3500 BMI

overall_design: Bulk was conduced on iPSC-EC and iPSC-CM organoids treated with COVID pseudovirus and mock. scRNA-seq was conducted on human arteries that were collected from Long COVID patients that had undergone below-knee amputation. Similarly, scRNA-seq was conducted on arteries collected from NON-COVID patients that suffered from peripherial artery disease (PAD) and undergone below-knee amputation due to complete occlusion of their arteries.

last: Wu


title: Synchronization of growth and self-organizing digit pattern by WNT signaling [ChIPseq]

city: Lausanne

first: Lucille

last: Lopez-Delisle

address: Station 19

country: Switzerland

institute: EPFL

summary: Molecular analysis of the spatio-temporal dynamics of destabilization and self-reorganization underscore the crucial role of WNT and BMP signaling modulators as part of the Turing system.

overall_design: b-Catenin and H3K27Ac ChIP-seq embryonic forelimb buds 6 hours after DMSO or C59 injection at E10.5.

first: Jonas

last: Malkmus

first: Alexandre

last: Mayran

first: Aimée

last: Zuniga

first: Rolf

last: Zeller

first: Rushikesh

last: Sheth


title: Characteristics of Age-Associated Changes in the Whole Transcriptome of Human Peripheral Blood Leukocytes and Establishment of an Aging Clock

city: Beijing

first: xin

last: gao

address: dongcheng street

department: The Key Laboratory of Geriatrics, Beijing Institute of Geriatrics

institute: Beijing Hospital

summary: In the post-pandemic era, there are limited methods for identifying accelerated aging through peripheral blood transcriptomics. Therefore, we conducted whole-transcriptome sequencing on 35 healthy individuals (22-88 years old). Analyzing mRNA, lncRNA, and miRNA expression, we constructed a ceRNA network and employed a random forest algorithm to develop an aging clock based on 10 genes, validated by pseudo-time analysis and RT-PCR. Utilizing the OEP001041 dataset (277 healthy individuals, aged 17-75) and datasets comprising patients with infectious diseases (n = 1558), we validated the aging clock's ability to categorize the aging rates among different individuals, uncovering associations between aging rates and infectious diseases. Further investigations revealed that infections accelerate aging by elevating inflammation and oxidative stress. Importantly, the aging clock demonstrated alterations post-infection, showcasing its potential in assessing the aging rate after patient recovery. This study uncovers potential factors contributing to accelerated aging and promotes the development of healthy aging.

overall_design: We recruited 35 healthy individuals (22-88 years old), referred to as the "BJH cohort," from April 2022 to October 2022 from residential areas in Beijing and the Beijing Hospital Health Examination Center. The individuals were categorized into three age groups: Young (n=11), with an average age of 27.5 (±2.54); Middle (n=10), with an average age of 60.4 (±2.22); and Old (n=14), with an average age of 80.5 (±4.57). Individuals participating in the study had to meet the following criteria: not suffering from major diseases that severely impact physiological functions, such as cancer, stroke, blood disorders, or severe immune abnormalities. Each volunteer underwent a health examination, and 5 mL of peripheral blood was collected. For each blood sample, one portion was collected in a tube containing EDTA for routine clinical blood testing and RNA extraction, while another portion was collected in an additive-free dry vacuum tube for biochemical analysis.

first: Xin

last: Gao

first: Jian-Ping

last: Cai


title: Dishevelled-1 regulates global transcriptomic changes in MDA-MB-231 triple negative breast cancer cells (RNA-Seq).

first: Dalia

last: Martinez-Marin

overall_design: To investigate the role of DVL1 in the regulation of gene transcription in triple-negative breast cancer, we estabilished MDA-MB-231 cell lines that express HA-DVL1.

first: Kevin

last: Pruitt

first: Monica

last: Sharma


title: CCL2-mediated Endothelial Injury Drives Cardiac Dysfunction in Long COVID [tissue]

first: Nazish

last: Sayed

overall_design: Bulk was conducted on long COVID, healthy, and PAD tissues. scRNA-seq was conducted on human arteries that were collected from Long COVID patients that had undergone below-knee amputation. Similarly, scRNA-seq was conducted on arteries collected from NON-COVID patients that suffered from peripherial artery disease (PAD) and undergone below-knee amputation due to complete occlusion of their arteries.


title: Respiratory syncytial virus (RSV) enhances translation of virus-resembling AU-rich host transcripts

status: Public on Sep 03 2024

city: Toronto

first: Kyra

last: Kerkhofs

state: ON

address: 4700 Keele Street

country: Canada

institute: York University

summary: Viral infection often triggers eukaryotic initiator factor 2α (eIF2α) phosphorylation, leading to global 5’-cap-dependent translation inhibition. RSV encodes messenger RNAs (mRNAs) mimicking 5’-cap structures of host mRNAs and thus inhibition of cap-dependent translation initiation would likely also reduce viral translation. We confirmed that RSV limits widespread translation initiation inhibition and unexpectedly found that the fraction of ribosomes within polysomes increases during infection, indicating higher ribosome loading on mRNAs during infection. We found that AU-rich host transcripts that are less efficiently translated under normal conditions become more efficient at recruiting ribosomes, similar to RSV transcripts. Viral transcripts are transcribed in cytoplasmic inclusion bodies, where the viral AU-rich binding protein M2-1 has been shown to bind viral transcripts and shuttle them into the cytoplasm. We further demonstrated that M2-1 is found on polysomes, and that M2-1 might deliver host AU-rich transcripts for translation.

overall_design: To determine which viral and host transcripts are associated with polysomes during RSV infection, total and  polysome-associated RNA from mock- and RSV-infected cells was collected, Trizol extracted and sequenced after poly(A) (A+) enrichment. RNA-sequencing samples contain biological triplicates for (1) mock-infected total RNA, (2) RSV-infected total RNA, (3) mock-infected polysome-associated RNA, and (4) RSV-infected polysome-associated RNA.

first: Nicholas

last: Guydosh

first: Mark

last: Bayfield


title: Synchronization of growth and self-organizing digit pattern by WNT signaling [RNAseq]

overall_design: RNA-seq on embryonic forelimb buds at 1, 6, 12, 18, 24 hours after DMSO or C59 injection at either E10.5 or E11.5, respectivley.


title: Oligonucleotide-directed proximity-interactome mapping (O-MAP): A unified method for discovering RNA-interacting proteins, transcripts and genomic loci in situ [Xist OMAP RIP]

status: Public on Aug 22 2024

city: Seattle

first: Evan

middle: Erik

last: Kania

state: WA

address: 1959 NE Pacific Street, HSB K554, Box 357750

department: Pharmacology

institute: University of Washington

summary: Throughout biology, RNA molecules form complex networks of molecular interactions that are central to their function, but remain challenging to investigate. Here, we introduce Oligonucleotide-mediated proximity-interactome MAPping (O-MAP), a straightforward method for elucidating the biomolecules near an RNA of interest, within its native cellular context. O-MAP uses programmable oligonucleotide probes to deliver proximity-biotinylating enzymes to a target RNA, enabling nearby molecules to be enriched by streptavidin pulldown. O-MAP induces exceptionally precise RNA-localized in situ biotinylation, and unlike alternative methods it enables straightforward optimization of its targeting accuracy. Using the 47S pre-ribosomal RNA and long noncoding RNA Xist as models, we develop O-MAP workflows for unbiased discovery of RNA-proximal proteins, transcripts, and genomic loci. This revealed unexpected co-compartmentalization of Xist and other chromatin-regulatory RNAs and enabled systematic characterization of nucleolar-chromatin interactions across multiple cell lines. O-MAP is portable to cultured cells, organoids, and tissues, and to RNAs of various lengths, abundances, and sequence composition. And, O-MAP requires no genetic manipulation and uses exclusively off-the-shelf parts. We therefore anticipate its application to a broad array of RNA phenomena.

overall_design: Oligonucleotide-directed proximity-interactome mapping (O-MAP) followed by streptavidin enrichment of biotinylated material and isolation of DNA or RNA for sequencing. O-MAP was performed in HeLa, 8988T, ASPC-1, Panc3.27, and SUIT2 cell lines against the internal transcribed spacer 1 of the 47S pre-ribosomal RNA.  O-MAP was performed in Patski cells against the Xist transcript.

first: Ashley

middle: F

last: Tsue

first: Diana

middle: Q

last: Lei

first: Rose

last: Fields

first: Christopher

last: McGann

first: Elliot

last: Hershberg

first: Maryanne

last: Kihiu

first: Shao-En

last: Ong

first: Sita

last: Kugel

first: Brian

last: Beliveau

first: Devin

last: Schweppe

first: David

last: Shechner


title: ACSS2 alleviates alcoholic liver disease via modulating hepcidin-mediated systemic iron homeostasis and hepatic ferroptosis

status: Public on Aug 28 2024

city: Hefei

first: Mengyao Wang

middle: Mengyao

last: Wang

state: None Selected

address: Hefei University of Technology

institute: hefei university of technology

summary: Alcoholic liver disease (ALD) is one of the most prevalent types of liver disease associated with high morbidity and mortality, caused by hepatotoxicity originating from alcohol metabolism, but current therapeutic drugs are still limited. The role of ACSS2 as an enzyme that metabolizes acetate in ALD remains unknown. Our study aimed to investigate the function of ACSS2 and its potential mechanism in ALD progression.

overall_design: To explore the function of ACSS2 in alcoholic liver disease, hepatocyte-specific knockout mice were generated and the liver transcriptome analysis was performed

first: Mengyao


title: CCL2-mediated Endothelial Injury Drives Cardiac Dysfunction in Long COVID [RNA-seq]

first: Nazish

last: Sayed

first: Sadhana

last: Gaddam


title: Oligonucleotide-directed proximity-interactome mapping (O-MAP): A unified method for discovering RNA-interacting proteins, transcripts and genomic loci in situ [Xist OMAP ChIP]

middle: J


title: CCL2-mediated Endothelial Injury Drives Cardiac Dysfunction in Long COVID

summary: This SuperSeries is composed of the SubSeries listed below.


title: CCL2-mediated Endothelial Injury Drives Cardiac Dysfunction in Long COVID [Pseudo_Mock_Organoids]

last: Wu


title: Synchronization of growth and self-organizing digit pattern by WNT signaling

first: Lucille

last: Lopez-Delisle


title: Oligonucleotide-directed proximity-interactome mapping (O-MAP): A unified method for discovering RNA-interacting proteins, transcripts and genomic loci in situ [ITS OMAP RIP]

first: Evan

last: Kania


title: ERH regulates type II interferon immune signaling through post-transcriptional regulation of JAK2 mRNA [4]

city: Vienna

first: Gijs

middle: A.

last: Versteeg

address: Dr. -Bohrgasse 9

department: Department of Microbiology

institute: Max Perutz Labs, University of Vienna

summary: Type II interferon (IFNγ) signaling is essential for innate immunity and critical for effective immunological checkpoint blockade in cancer immunotherapy. Genetic screen identification of post-transcriptional regulators of this pathway has been challenging since such factors are often essential for cell viability. Here, we utilize our inducible CRISPR/Cas9 approach to screen for key post-transcriptional regulators of IFNγ signaling, and in this way identify ERH and the ERH-associated splicing and RNA export factors MAGOH, SRSF1, and ALYREF. Loss of these factors impairs post-transcriptional mRNA maturation of JAK2, a crucial kinase for IFNγ signaling, resulting in abrogated JAK2 protein levels and diminished IFNγ signaling. Further analysis highlights a critical role for ERH in preventing intron retention in AU-rich regions in specific transcripts, such as JAK2. This regulation is markedly different from previously described retention of GC-rich introns. Overall, these findings reveal that post-transcriptional JAK2 processing is a critical rate-limiting step for the IFNγ-driven innate immune response.

overall_design: To investigate post-transcriptional mRNA regulation of ERH, we lentivirally transduced human RKO-iCas9 cells with sgRNA expression vectors targeting AAVS1 (negative control) or ERH. The targeted genes were knocked out by five days of doxycycline-induced Cas9 expression. We then treated cells with DMSO (control) or the translational inhibitor cyclohexamide (CHX) for 4 hours to determine if translationally coupled processes such as nonsense mediated decay (NMD) had a role in altered mRNA expression. Finally, we utilized subcellular fractionation and PolyA-enriched RNA-Seq to separately identify nuclear and cytoplasmic RNA abundance changes (analyzed by DESeq2) and mis-splicing events resulting in intron retention (analyzed by IRFinder-S and DESeq2).

first: Adrian

last: Soderholm

first: Niko

last: Popitsch


title: ERH regulates type II interferon immune signaling through post-transcriptional regulation of JAK2 mRNA  [1]

overall_design: To investigate how ERH affected the expression of immune-stimulated mRNAs in human cells, we lentivirally transduced human RKO-iCas9 cells with sgRNA expression vectors targeting AAVS1 (negative control), or ERH. The targeted genes were knocked out by five days of doxycycline-induced Cas9 expression. We then treated cells with IFNγ, IL-1beta, or MilliQ water (non-treated control) for 4 hours, after which we harvested total RNA with Trizol and performed 3’ mRNA sequencing (Quant-Seq).


title: ERH regulates type II interferon immune signaling through post-transcriptional regulation of JAK2 mRNA [3]

overall_design: To investigate how ERH and potential cooperating post-transcriptional regulatory factors affected the nuclear and cytoplasmic mRNA landscape, we lentivirally transduced human RKO-iCas9 cells with sgRNA expression vectors targeting AAVS1 (negative control), ERH, ALYREF, MAGOH, SRSF1, or POLDIP3. The targeted genes were knocked out by by five days of doxycycline-induced Cas9 expression. We then utilized subcellular fractionation and 3’ mRNA sequencing (Quant-Seq) to separately identify nuclear and cytoplasmic RNA abundance changes.


title: CCL2-mediated Endothelial Injury Drives Cardiac Dysfunction in Long COVID [Single Cell Multiome ATAC + Gene Expression]

overall_design: Gene expression and open chromatin profiling analysis of single nuclei RNA-seq and single nuclei ATAC-seq of cardiac organoids fabricated with iPSC-ECs and iPSC-CMs and exposed to SARS-CoV-2.


title: The TLR8 agonist Selgantolimod regulates Kupffer cell differentiation status and indirectly impairs HBV entry into hepatocytes via an IL-6-dependent mechanism (PHH) [Liver]

status: Public on Aug 10 2024

city: Lyon

first: Xavier

last: Grand

address: 46 allée d'Italie

country: France

institute: INSERM U1052, UCBL1, ENS de Lyon

summary: Background & Aims: In spite of universal vaccination programs, HBV remains a global burden due to the limited therapeutic options available. Therefore, achieving the goal of HBV cure will require a continuous effort to develop novel molecules and combination therapies. In this context, the Toll-like receptor 8 (TLR8) agonist selgantolimod (SLGN) has shown promising results during its clinical evaluation. Although the effect of SLGN has been explored in the peripheral immune compartment, little is known regarding its intrahepatic response. Therefore, we characterized the transcriptomic changes and intercellular communication events associated with SLGN in the liver microenvironment. Approach & Results: The analysis of single-cell RNA sequencing (scRNA-seq) data allowed us to identify that hepatic TLR8 expression takes place in the myeloid compartment. Therefore, we stablished a method for the isolation of human Kupffer cells (KCs). We found that in vitro treatment of KCs with SLGN induces the upregulation of monocyte markers (e.g., EREG, S100A12) and the downregulation of genes associated with the KC identity (e.g., SPIC, FOLR2). Moreover, treatment of hepatocytes with SLGN-conditioned media (CM) led to the downregulation of NTCP and impaired HBV entry. Finally, co-treatment with SLGN-CM and an IL-6-inhibitory antibody identified this cytokine as mediating the reduced HBV entry. Conclusions: Our results suggest that in addition to its previously described therapeutic activity against HBV, SLGN also presents a prophylactic effect. Furthermore, our characterization of SLGN sheds light into the transcriptional programs regulating KC activation and underscores the importance of considering cell states when annotating cell populations based on scRNA-seq data.

overall_design: RNA-seq profiles from primary human hepatocytes (PHH) exposed (72 h) to selgantolimod-conditioned media (SLGN-CM, n=3) or Mock-CM (n=3) produced in KCs.

first: Armando

last: Andres

first: Roca

last: Suarez


title: The TLR8 agonist Selgantolimod regulates Kupffer cell differentiation status and indirectly impairs HBV entry into hepatocytes via an IL-6-dependent mechanism (PHH) [Kupffer cells]

overall_design: RNA-seq profiles from human Kupffer cells (KCs) exposed (24 h) to selgantolimod (SLGN, 150 nM, n=3) or DMSO (n=3).


title: Paradoxical regulation of IGF2 in promoting lipid metabolism in adipose tissues

status: Public on Aug 08 2024

city: Hangzhou

first: Xihua

last: Lin

state: Zhejiang

address: 3 East Qingchun Road

department: Department of Endocrinology and Metabolism

institute: Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University

summary: Background and aims: Metabolic diseases, including diabetes, obesity, and dyslipidemia, are significant public health concerns worldwide. The insulin-like growth factor 2 (IGF2) gene have been implicated in various physiological processes, but its specific role in lipid metabolism remains unclear. We aim to elucidate the role of IGF2 in regulating lipid metabolism in adipose tissues and its association with metabolic syndrome (MetS).  Methods: The research employed a multidisciplinary approach to investigate the role of IGF2 in lipid metabolism. We investigated the correlation between genetic variations within the IGF2 gene and metabolic parameters. We conduct a cross-sectional human study to evaluate relationships between varying IGF2 serum concentrations and the incidence of MetS. Additionally, manipulation of IGF2 expression levels in mouse and cell models via overexpression and knockdown to assess impacts on lipid metabolism in adipose tissue, specifically lipid accumulation, insulin resistance, and the balance between lipogenesis and lipolysis. Furthermore, the study employs metabolomics techniques to scrutinize the broader metabolic profiles in adipose tissues in response to IGF2 modulation.  Results: Multiple SNP loci in the IGF2 gene were significantly associated with BMI, HbA1c, and diabetes. Insufficient or excessive expression of IGF2 was identified as a risk factor for hyperlipidemia, low HDL-c, and central obesity in MetS. We observed that IGF2 was mainly concentrated in adipose tissues and adipocytes. Enhanced IGF2 expression stimulated adipogenesis and lipid accumulation, whereas IGF2 knockdown hindered lipolysis, exacerbating ectopic lipid accumulation and insulin resistance. There is a substantial enlargement of pancreatic tissue and heightened insulin generation in mice deficient in IGF2. Activation of the PI3K/Akt pathway through IGF1R in IGF2 excess or INSR in conditions of IGF2 scarcity, along with inhibition of AMPK, implies a common downstream process that favors lipid accumulation and metabolic reprogramming in adipocytes.  Conclusions: Our study demonstrated that upregulation of IGF2 enhanced adipogenesis and lipogenesis, while knockdown of IGF2 inhibited lipolysis, which resulting in accelerating lipid accumulation through PI3K/Akt-AMPK pathway.

overall_design: Global transcriptome profiling was performed by RNA-Seq using epididymal white adipose tissue of Ad-IGF2 (n=2, 2 replicate sequencing)  or Ad-GFP mice (n=3, 2 replicate sequencing) and IGF2-RNAi  (n=2, 2 replicate sequencing) or NC-RNAi mice (n=3, 2 replicate sequencing). RNA was extracted, sequenced, and analyzed by a custom service provided by Novogene using an Illumina NovaSeq 6000. The bioinformatics analysis was conducted utilizing the OmicStudio tools available at the following website: https://www.omicstudio.cn/tool. The differential expression cut-off threshold was determined relied on |log2fold change (log2FC) | > 1 and p-value < 0.05.

first: Caiping

last: Zhou


title: Oligonucleotide-directed proximity-interactome mapping (O-MAP): A unified method for discovering RNA-interacting proteins, transcripts and genomic loci in situ

sample_organism:
Homo sapiens
Mus musculus x Mus spretus

platform_organism:
Homo sapiens
Mus musculus x Mus spretus

overall_design: Refer to individual Series


title: Thr4 phosphorylation on RNA Pol II occurs at early transcription regulating 3’-end processing

status: Public on Aug 01 2024

city: Austin

first: Svetlana

last: Panina

state: TX

address: 101 East 21st St.

department: Molecular Biosciences

institute: The University of Texas at Austin

summary: RNA polymerase II relies on a repetitive sequence domain (YSPTSPS) within its largest subunit to orchestrate transcription. While phosphorylation on Ser2/Ser5 of the C-terminal heptad repeats is well-established, Thr4’s role remains enigmatic. Paradoxically, Thr4 phosphorylation was only detected after transcription end sites despite functionally implicated in pausing, elongation, termination, and mRNA processing. Our investigation revealed that Thr4 phosphorylation detection was obstructed by flanking Ser5 phosphorylation at the onset of transcription, which can be removed selectively. Subsequent proteomic analyses identified many proteins recruited to transcription via Thr4 phosphorylation, which previously were attributed to Ser2. Importantly, loss of Thr4 phosphorylation greatly reduces Ser2 phosphorylation, revealing a crosstalk between the two marks. Finally, the function analysis of the Thr4 phosphorylation highlighted its role in alternative 3’-end processing within pro-proliferative genes. Our findings unveil the true genomic location of this evolutionarily conserved phosphorylation mark and prompt a reassessment of functional assignments of the CTD.

overall_design:
ChIP-Sequencing of pThr4 CTD Pol II, pSer2 CTD Pol II, and HA-RPRD1B, two biological replicates per sample (HEK293 cells).

***Please note that the records have been updated with three additional samples on Aug 6, 2024.****

first: Rosamaria

middle: Y

last: Moreno

middle: B

first: Seema

last: Irani

first: Haley

last: Hardtke

first: Renee

last: Stephenson

first: Brendan

last: Floyd

first: Edward

last: Marcotte

first: Qian

last: Zhang

first: Jessie


title: Transcriptome profiles of etiolated cotyledons exposed to ABA during seedling deetiolation.

city: Cerdanyola del Vallès

first: Guiomar

last: Martín

state: Barcelona

address: Vall Moronta

country: Spain

institute: Centre for Research in Agricultural Genomics

summary: RNA-seq experiment of dark-grown cotyledons and cotyledons exposed to light for three hours in the absence or presence of absicic acid (ABA).

overall_design: Transcriptome profiles of cotyledons from seedlings grown for 3 days in the dark and then exposed to white light  (40 µmol·m-2·s-1) for 3 hours in the presence or absence of ABA (100 µM). Triplicate biological samples were analyzed.

first: Paula

last: Duque


title: Oligonucleotide-directed proximity-interactome mapping (O-MAP): A unified method for discovering RNA-interacting proteins, transcripts and genomic loci in situ [ITS OMAP ChIP]

first: Evan

last: Kania

middle: E

first: Ashley

last: Tsue

first: Diana

last: Lei

first: Rose

last: Fields

first: Christopher

last: McGann

first: Elliot

last: Hershberg

first: Maryanne

last: Kihiu

first: Shao-En

last: Ong

first: Sita

last: Kugel

first: Brian

last: Beliveau

first: Devin

last: Schweppe

first: David

last: Shechner


title: Methylglyoxal activates the NRF2 pathway and impairs  immune responses controlling the  growth control of Mycobacterium tuberculosis (Mtb) in macrophages.

status: Public on Jul 20 2024

city: Solna

first: Martin

last: Rottenberg

address: Solna vägen 9

country: Sweden

institute: Karolinska Institutet

summary: We compared the transcriptome  profile of M. tuberculosis-infected murine bone marrow derived macrophages (BMM) treated or not with 200 uM methylglyoxal (MGO) and uninfected controls as described below.   We found 3606 differentially expressed genes (DEGs) between uninfected and M. tuberculosis-infected BMM, being approximately half of these upregulated . 1699 DEGs were determined by comparing Mtb-MGO  vs M. tuberculosis BMM, with 2/3 of these DEGs downregulated. Genes in the glyoxalate, glutathione and selenocompound metabolic KEEG pathways were increased comparing Mtb-MGO vs M. tuberculosis BMM. Instead genes involved in type I IFN responses, cytokine receptor and chemokine signalling gene ontology clusters (GO) were increased after infection with Mtb as compared to uninfected controls and decreased in Mtb-MGO  vs M. tuberculosis  BMM.

overall_design: RNA seq was performed in triplicate independent cultures of BMM treated or not with 200 uM MGO, infected 4 h after treatment with M. tuberculosis  at a MOI of 5:1. RNA was extracted 4h after Mtb infection from uninfected BMM controls (Control), Mtb infected BMM (Mtb), and MGO-treated, Mtb-infected BMM  (Mtb-MGO).

first: Hanxiong

last: Li

first: Ruining

last: Liu


title: Single-cell RNA sequencing for heart failure heart of mice

status: Public on Aug 15 2024

city: Wenzhou

first: Guang

last: Liang

address: Wenzhou medical university of higher education park chashan town

department: School of Pharmaceutical Sciences

institute: Wenzhou Medical University

summary: Heart failure (HF) is the ultimate outcome of most cardiovascular disorders with high prevalence and poor prognosis. The transverse aortic constriction (TAC) method is currently the most widely utilized approach for investigating HF induced by left ventricle pressure overload. We explore potential genes with significant changes in HF through single-cell sequencing.

overall_design: Samples were prepared from uninjured mice, as well as from mice which underwent TAC operation 6 weeks to model pressure overload injury. Hearts were dissociated into single cells. We combined single-cell suspensions from 3-5 hearts into one sample.

first: Jibo

last: Han

first: Jinfu

last: Qian


title: ERH regulates type II interferon immune signaling through post-transcriptional regulation of JAK2 mRNA [2]

first: Gijs

last: Versteeg

overall_design: To investigate how ERH affected the expression of immune-stimulated mRNAs in murine cells, we lentivirally transduced mouse RAW264.7-iCas9 cells with sgRNA expression vectors targeting ROSA (negative control) or ERH. The targeted genes were knocked out by by five days of doxycycline-induced Cas9 expression. We then treated cells with IFNγ, IL-1beta, or MilliQ water (non-treated control) for 4 hours, after which we harvested total RNA with Trizol and performed 3’ mRNA sequencing (Quant-Seq).

first: Adrian

last: Soderholm

first: Niko

last: Popitsch


title: scRNA-seq time course of B cell differentiation in organoid cultures

city: Atlanta

first: Chris

last: Scharer

state: GA

address: 1510 Clifton Rd, Suite 3086A

department: Microbiology and Immunology

institute: Emory University

summary: To understand how different stimulation conditions impacted the maturation of B cells and formation of plasma cells in an ex vivo organoid culture, we preformed 10X single-cell RNA-seq comparing the CD19+ B cells and CD138+ plasma cells at different time points during the organoid culture.  Three stimulation conditions were used including H1N1 influenza antigen alone, R848, H1N1 in combination with the TLR7 agonist R848.  Additionally, a final set of organoid cultures were stimulated with H1N1 in combination with the TLR7 agonist R848 and at days 16 and 24 inhibitors to BCL6 and EZH2 were added.\

overall_design: scRNA-seq of 4 time points across two antigen stimulation conditions.

first: Christopher

middle: D