title: Nuclear receptor Nur77 regulates immunomechanics of macrophages

city: Amsterdam

first: Claudia

last: van Tiel

address: Meibergdreef 15

department: Medical Biochemistry

country: Netherlands

institute: Amsterdam UMC

summary: Macrophages exhibit remarkable plasticity in their morphology and mechanics, enabling them to adapt and execute essential inflammatory functions, such as navigating through inflamed tissue and pathogen engulfment. However, the precise regulatory mechanisms governing these dynamic changes in macrophage mechanics during inflammation remain poorly understood. Although the involvement of nuclear receptor Nur77 in macrophage inflammation has been extensively investigated, its potential correlation with cellular mechanics has not been investigated. In this study, we employ cytoskeletal imaging, single-cell acoustic force spectroscopy (AFS), migration and phagocytosis experiments, and RNA-sequencing analysis on bone marrow-derived macrophages (BMDMs) to compare wild-type (WT) and Nur77-deficient (Nur77-KO) cells. Our findings reveal that Nur77-KO BMDMs exhibit changes to their actin networks compared to WT BMDMs, which is associated with a stiffer and more rigid phenotype. Subsequent in-vitro experiments validated our observations, showcasing that Nur77 deficiency leads to enhanced migration, reduced adhesion, and increased phagocytic activity. The transcriptomics data confirmed altered mechanics-related pathways in Nur77-deficient macrophage that are accompanied by a robust pro-inflammatory phenotype. Utilizing previously obtained ChIP-data, we revealed that Nur77 directly targets differentially expressed genes associated with cellular mechanics. In conclusion, while Nur77 is recognized for its role in reducing inflammation of macrophages by inhibiting the expression of pro-inflammatory genes, our study identifies a novel regulatory mechanism where Nur77 governs macrophage inflammation through the modulation of expression of genes involved in cellular mechanics.

overall_design: Comparison of gene expression in murine macrophages, derived from Nur77-deficient mice and wild-type mice, stimulated with 100 ng/ml LPS for 0, 4 and 18 hours. (n=4 per group for WT cells, n=3 per group for Nur77-deficient cells)

first: Sanne

middle: C

last: Lith

first: Tom

last: Evers

first: Beatriz

last: Marton-Freire

first: Alireza

last: Mashaghi

first: Carlie

last: de Vries


title: Transcriptomic and proteomic spatial profiling of tertiary lymphoid aggregates in head and neck cancer reveal spatial tissue dynamics and profiles associated with immunotherapy response [protein]

status: Public on Aug 02 2024

city: Brisbane

first: Habib

last: Sadeghirad

state: Queensland

address: 37 Kent St, Woolloongabba, 37 Kent St, Woolloongabba

country: Australia

institute: The University of Queensland

summary:
DSP protein profiling using the nanostring immuno-oncology kit was performed on head and neck tumor tissue samples
De proteins were identified in tumour and stromal compartments

overall_design: DE was performed by immunotherapy outcomes

first: Arutha

last: Kulasinghe


title: Effects of Palovarotene on Ext1-deficient and wild type cultured mouse chondrocytes

city: Baltimore

first: Carrie

last: McCracken

state: MD

address: 670 W. Baltimore St

department: Institute for Genome Sciences

country: USA

institute: Institute of Genome

type: Expression profiling by high throughput sequencing

summary: Palovarotene, the agonist of the retionic acid nuclear receptor gamma has been shonw to inhibit osteochondroma formation in the mouse osteochondroma models. To understand the biological cation of Palovarotene, the transcriptome analysis was performed using 3D chondrocyte cultures Compaison of Palovarotene-treated groups with vehicle control groups demonstrated the gene lists and biological pathway modulated by Palovarotene-activated retionic acid receptor signaling pathways.

overall_design: The epiphyseal chondrocytes are isolated from proxiimal tibia and deistal femur growth plate  from the Ext1-mutant mice (the compound mice of the AggrecanCreER and Ext1e2neofloxed mice) and the control mice (Ext1e2neofloxed mice) at postnatal 10 days of age after tamoxifen injections at postnatal 5 and 7 days of ages.  Th isolated cells were embeded in alginate beads and cultured in high glucose DMEM containing 10 % FBS. The cultures were then treated with 300 nM Palovaroten or the same volume of 100% ethanol for 2 days. The cells were collected from the alginate beads and subjected to the bulk RNAseq. There are 4 goups (Aggrecan;Ext1e2nelf/e2neof with vehicel or Palovarotene and Ext1e2neof/2eneof with vehicel or Palovarotene)

first: Motomi

last: Enonmoto-Iwamoto

first: Kimberly

last: Wilson


title: Gene expression profiles of N-propargylglycine (N-PPG) treatment on wildtype and Huntington's mice

city: Stanford

first: Madeleine

last: Scott

state: CA

address: 291 Campus Dr

institute: Stanford Medicine

summary:
To investigate if N-PPG has neuroprotective effects on Huntington and Wildtype mice
Gene profiling of whole brian tissue was processed after treatment of mice with N-PPG

overall_design: Comparative gene expression profiling analysis of RNA-seq data for N-PPG treated and untreated Wildtype and Huntingtons mice

middle: K

first: Gary

first: Christopher

last: Benz


title: Expression profile and gap-junctional transfer of microRNAs in the bovine cumulus-oocyte complex

city: Ghent

first: Yannick

last: Gansemans

address: Ottergemsesteenweg 460

department: Pharmaceutical Sciences

country: Belgium

institute: Ghent University

type: Non-coding RNA profiling by high throughput sequencing

summary: MicroRNAs (miRNA) are important regulators of oocyte maturation, playing a key role in modulating gene expression both in a temporal- and spatial-specific manner. These small non-coding RNAs are involved in important processes during oocyte maturation, acting as messengers between the oocyte and its surrounding cumulus cells. Despite its significance, the bidirectional communication mechanism is still unknown. To test miRNA communication between oocyte and surrounding cumulus cells through the gap junctions the gap junctions were either blocked with carbenoxolone or not. MiRNA sequencing of oocytes at 1, 6, and 22 hours of in vitro maturation was then performed. Among the differentially expressed miRNAs, bta-miR-21-5p, a regulator of cumulus cell viability and oocyte maturation, was the only previously known miRNA. Furthermore, by labeling a bta-miR-21-5p mimic with FAM, crossing of this miRNA through the gap junctions within the cumulus-oocyte complex could be visualized and internalization in the oocyte was confirmed by RT-qPCR. In conclusion, this study provides, for the first time, evidence that miRNA communication within the bovine cumulus-oocyte complex is enabled through the gap junctional network.

overall_design: To investigate the miRNA communication in the bovine COC through the gap-junctional network, we blocked the gap-junctions using carbenoxolone. Together with three control groups (normal maturation conditions, denuded state maturation, denuded stat maturation with COCs in co-culture) oocytes were collected after 1, 6, and 22 hours of maturation for miRNAseq on each sample in triplicate.

first: Rani

last: Six

first: Luc

last: Peelman

first: Filip

last: Van Nieuwerburgh


title: mRNA expression profiles in productively HIV-1 infected and bystander primary human CD4+ T-cells

city: Montreal

first: Eric

middle: A

last: Cohen

state: Quebec

address: 110 Ave des Pins Ouest

country: Canada

institute: IRCM

summary: CD4+ T-cells are the main target of HIV-1 and several host factors can positively or negatively modulate HIV-1 infection of these cells. MiRNAs aresmall regulatory RNAs that are involved in the regulation of basic cellular functions. They are also increasingly recognized as host factors regulatingHIV-1 infection, replication and persistence. In order to identify miRNAs involved in HIV-1 infection of CD4+ T-cells, we performed globaltranscriptomic analyses of productively infected and HIV-1 exposed but non infected bystander CD4+ T-cells and compared their MIRNA profiles with uninfected cells. Theseanalyses were performed in CD4+T-cells isolated from 3 different healthy blood donors. Both miRNA and mRNA expression profiles were compared. Our results show that bystander and uninfected CD4+ T-cells do not display important differences in their miRNA expression profiles even though their respectivemRNA expression profiles were markedly different. In contrast, both mRNA and miRNA expressionprofiles from productively infected CD4+ T-cells were significantly different from those of uninfected cells. Overall, these results suggest that HIV-1infection impacts the miRNA expression profile of primary CD4+ T-cells.

overall_design: Human CD4+ T-cells from 3 different healthy blood donors were isolated from peripheral blood mononuclear cells (PBMCs) and activated for 3 days in the presence of anti-CD3/anti-CD28 and IL-2 in RPMI+10% fetal bovine serum (FBS). Activated T-cells were infected with GFP-marked HIV-1 (strain NL4.3-ADA-IRES-GFP) for 48h at a multiplicity of infection (MOI)=2. Productively infected (GFP+) and bystander cells (GFP-) were sorted by FACS and their total RNA extracted using the RNeasy kit from QIAGEN.

first: Nicolas

last: Bellini

first: Robert

last: Lodge


title: miRNA expression profiles in productively HIV-1 infected and bystander primary human CD4+ T-cells


title: Productively HIV-1 infected and bystander primary human CD4+ T-cells

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

overall_design: Refer to individual Series


title: Mucosal Single-Cell Profiling of Crohn's-Like Disease of the Pouch Reveals Unique Pathogenesis and Therapeutic Targets

city: Saint Louis

first: Khai

last: Nguyen

state: MO

address: 425 South Euclid Ave, 8107, 8th floor BJCIH,

department: Pathology & Immunology

institute: Washington University in St. Louis

summary: The pathophysiology of Crohn’s-like disease of the pouch (CDP) that develops after restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC) is unknown. We examined mucosal cells from patients with and without CDP using single cell analyses.

overall_design:
Endoscopic samples were collected from the pouch body and pre-pouch ileum (refer to as “pouch/ileum”) of 50 patients with an IPAA. Single-cell RNA sequencing (scRNA-seq) was performed on pouch/ileal tissues of patients with normal pouch/ileum and CDP. Mass cytometry was performed on mucosal immune cells from patients with normal pouch/ileum, CDP, pouchitis, as well as those with familial adenomatous polyposis (FAP) after pouch formation. Findings were independently validated using immunohistochemistry.

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Submitter states that missing raw files are due to patient privacy concerns.
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first: Siyan

last: Cao

first: Marco

last: Colonna


title: Prognostic implication of blood immune cell composition in Metastatic Castration-Resistant Prostate Cancer (mCRPC)

city: Madrid

first: Enrique

last: Perez Navarro

state: Madrid

address: Av. de Córdoba

department: Oncology Department

country: Spain

institute: Hospital 12 de Octubre

summary: The prognosis of patients with metastatic castration-resistant prostate cancer (mCRPC) is variable. Several blood-related prognostic factors have been reported, including transcriptional profiling of whole blood and neutrophil to lymphocyte ratio. We aimed to address the contribution of distinct whole blood immune cell components to the prognosis of these patients.  Subjects/Patients and Methods:  Pre-treatment whole blood samples from chemotherapy-naïve mCRPC patients were prospectively collected before treatment with enzalutamide. The study consisted of a training cohort including 98 patients treated with enzalutamide in a phase 2 biomarker clinical trial (NCT02288936)

overall_design: The study involved serial whole-blood sample collection from participants using PAX-Gene RNA, with samples stored at -80ºC. Collection times included before treatment, at 12 weeks during treatment, and at tumor progression or end of treatment. The focus here is on samples taken prior to treatment. For RNA extraction and microarray analysis, blood samples in PAXgene RNA tubes underwent isolation and purification as per the manufacturer's protocol. RNA quality was assessed using NanoDrop 2000 and Bioanalyzer 2100, with only RNA samples having an RNA integration number (RIN) of seven or above being used. Complementary DNA (cDNA) was synthesized from these RNA samples and prepared for hybridization. Hybridization used the GeneChip Human Transcriptome Array HTA 2.0, incubating with single-strand DNA (ssDNA) for 16 hours. Post-hybridization, non-specific probes were washed away, and the array was scanned with the GeneChip Scanner 3000.


title: MORC2 regulates RBM39-mediated CDK5RAP2 alternative splicing to promote EMT and metastasis in colon cancer

city: shenyang

first: Yuxin

last: He

address: 77 Puhe Road, Shenbei New District

country: China

institute: China Medical University


title: MORC2 regulates RBM39-mediated CDK5RAP2 alternative splicing to promote EMT and metastasis in colon cancer [shRBM39 mRNA-seq]

summary: To investigate the role RBM39 plays in alternative splicing, we generated SW480 cell lines in which RBM39 was knocked down by shRNA.We then performed RNA-seq data for alternative splicing gene analysis.

overall_design: Comparative  for alternative splicing gene analysis gene expression profiling of RNA-seq data from control (shCtrl) and knockdown RBM39 (shRBM39) groups in SW480 cells.

first: Yangguang

last: Shao

first: Feng

last: Li


title: MORC2 regulates RBM39-mediated CDK5RAP2 alternative splicing to promote EMT and metastasis in colon cancer [shMORC2 mRNA-seq]

summary: To investigate the role MORC2 plays in alternative splicing, we generated SW480 cell lines in which MORC2 was knocked down by shRNA.We then performed RNA-seq data for alternative splicing gene analysis.

overall_design: Comparativefor alternative splicing gene analysis gene expression profiling of RNA-seq data from control (shCtrl) and knockdown MORC2 (shMORC2) groups in SW480 cells.


title: MORC2 regulates RBM39-mediated CDK5RAP2 alternative splicing to promote EMT and metastasis in colon cancer [RBM39 RIP-seq]

summary: RNA immunoprecipitation sequencing (RIP-seq) for the detection of RBM39 protein-binding RNA in SW480 cells.

overall_design: Differential gene analysis of RIP-seq data was compared between SW480 cell control group (Input) and RBM39 immunoprecipitation group (IP).


title: histone H3K18 acetylation for syncytialization of human placental trophoblast stem cells

city: BEIJING

first: xin

last: yu

state: Beijing

address: DATUN ROAD

institute: Institute of Zoology, Chinese Academy of Sciences

summary: CUT&Tag seq of H3K18ac in TS, STB-D1, STB-D4, STB-D4 treated with oxamate, STB-D4 treated with oxamate and acetate

overall_design: this submission is bigwig file for every sample

first: Xin

last: Yu


title: histone HK16 acetylation for syncytialization of human placental trophoblast stem cells

type: Genome binding/occupancy profiling by high throughput sequencing

summary: CUT&Tag seq of HK16ac in TS(trophoblast stem cells), STB-D1(TS cells culture in STB medium for syncytialization one day), STB-D4(TS cells culture in STB medium for syncytialization 4 day), STB-D4 treated with oxamate(TS cells culture in STB medium for syncytialization 4 day and treated with oxamate), STB-D4 treated with oxamate and acetate(TS cells culture in STB medium for syncytialization 4 day and treated with oxamate and acetate). we explored the relationship between glycolysis and syncytializaion.

overall_design: Five samples with Cut&Tag HK16ac


title: histone H3K27 acetylation for syncytialization of human placental trophoblast stem cells

summary: CUT&Tag seq of H3K27ac in TS, STB-D1, STB-D4, STB-D4 treated with oxamate, STB-D4 treated with oxamate and acetate


title: histone H3K9 acetylation for syncytialization of human placental trophoblast stem cells

summary: CUT&Tag seq of H3K9ac in TS, STB-D1, STB-D4, STB-D4 treated with oxamate, STB-D4 treated with oxamate and acetate


title: Time-series proteomic analysis of body fluid extracellular vesicles in amyotrophic lateral sclerosis

city: Kanagawa

first: Satoru

last: Morimoto

address: 3－25－10, Tonomachi, Kawasaki-ku, Kawasaki-shi

department: Keio Regenerative Medicine Research Center (KRM)

country: Japan

institute: Keio University

summary: Extracellular vesicles (EVs) hold the potential for elucidating the pathogenesis and serving as biomarkers of amyotrophic lateral sclerosis (ALS). Notably, the comparative and longitudinal alterations in the protein profiles of EVs in serum (sEVs) and CSF (cEVs) of sporadic ALS (SALS) patients remain unexplored. This study sought to reveal such changes by collecting serum and CSF at fixed intervals from 10 controls and 20 SALS patients participating in the Ropinirole Hydrochloride Remedy for Amyotrophic Lateral Sclerosis trial. We also aimed to reveal longitudinal changes with disease progression and the effects of ropinirole hydrochloride (ROPI, a dopamine D2 receptor agonist identified as an anti-ALS drug candidate using induced pluripotent stem cell drug discovery) on protein profiles of EVs. Comprehensive proteomic analyses of EVs extracted from these samples were conducted using liquid chromatography-mass spectrometry to trace longitudinal shifts linked to disease progression and the influence of ROPI on EV protein profiles. The findings revealed notable disparities but high congruity in sEV and cEV protein profiles concerning disease status, time, and ROPI administration. Moreover, the sEV and cEV protein profiles converged over time in SALS patients, at least in part, suggestive of a loosening of the blood–brain barrier. In SALS patients, both sEVs and cEVs presented elevated inflammation-related protein levels but reduced levels of proteins associated with the unfolded protein response. These results mirrored the longitudinal changes after disease onset and correlated with the revised ALS Functional Rating Scale at sampling time, suggesting a link to the onset and progression of SALS. ROPI appeared to counteract these changes, attenuating inflammation-related protein levels and boosting those tied to the unfolded protein response in SALS, suggesting an anti-ALS effect on EV protein profiles. Reverse translational research using induced pluripotent stem cell-derived astrocytes indicated that these changes may partly reflect the DRD2-dependent neuroinflammatory inhibitory effects of ROPI. Baseline levels of OGN in sEVs and FRMPD1 in cEVs correlated with subsequent disease progression, indicating their potential as prognostic biomarkers for SALS. Machine learning-driven biomarker searches and diagnostic classification yielded an accuracy of 90.4% for sEVs and 80.3% for cEVs. Despite the limited sample size, this study is the first to report time-series proteomic alterations in sEVs and cEVs from SALS patients, offering comprehensive insights into SALS pathogenesis, ROPI-induced changes, and potential prognostic and diagnostic biomarkers.

overall_design: Astrocytes were differentiated from induced pluripotent stem cells derived from healthy control and performed total RNA-sequencing under the conditions of cell culture with and without ropinirole hydrochloride.

first: Chris

last: Kato

first: Koji

last: Ueda

first: Shinichi

last: Takahashi

first: Shiho

last: Nakamura

first: Fumiko

last: Ozawa

first: Daisuke

last: Ito

first: Yugaku

last: Dàte

first: Kensuke

last: Okada

first: Naoki

last: Kobayashi

first: Jin

last: Nakahara

first: Hideyuki

last: Okano


title: Dual Gene Expression Analysis Identifies Factors Associated with Staphylococcus aureus Virulence in Diabetic Mice

city: Livermore

first: Kunal

last: Poorey

address: 7011 East Avenue MS9292

institute: Sandia National Labs

summary: Staphylococcus aureus is a major human pathogen of the skin. The global burden of diabetes is high, with S. aureus being a major complication of diabetic wound infections. We investigated how the diabetic environment influences S. aureus skin infection and observed an increased susceptibility to infection in mouse models of both type I and type II diabetes. A dual gene expression approach was taken to investigate transcriptional alterations in both the host and bacterium after infection. While analysis of the host response revealed only minor changes between infected control and diabetic mice, we observed that S. aureus isolated from diabetic mice had significant increases in the levels of genes associated with translation and posttranslational modification and chaperones and reductions in the levels of genes associated with amino acid transport and metabolism. One family of genes upregulated in S. aureus isolated from diabetic lesions encoded the Clp proteases, associated with the misfolded protein response. The Clp proteases were found to be partially glucose regulated as well as influencing the hemolytic activity of S. aureus Strains lacking the Clp proteases ClpX, ClpC, and ClpP were significantly attenuated in our animal model of skin infection, with significant reductions observed in dermonecrosis and bacterial burden. In particular, mutations in clpP and clpX were significantly attenuated and remained attenuated in both normal and diabetic mice. Our data suggest that the diabetic environment also causes changes to occur in invading pathogens, and one of these virulence determinants is the Clp protease system.

overall_design: RNA was extracted with control or diabetic mice by punch biopsy that had been infected subcutaneously with Staphylococcus aureus

first: Dane

last: Parker

first: Annette

last: LaBauve

first: Robert

last: Meagher


title: Regulatory role of Gnao1 in neuronal differentitation

city: Fukuoka

first: Yasunari

last: Sakai

address: 3-1-1 Maidashi, Higashi-ku

department: Department of Pediatrics

institute: Kyushu University

summary: Pathogenic variations in GNAO1 are associated with developmental and epileptic encephalopathy (DEE). To understand molecular mechanisms underlying GNAO1-associated DEE, we performed a transcriptomic analysis (RNA sequencing) on Neuro2a cells after siRNA-mediated silencing of Gnao1.

overall_design: Comparative analysis of Neuro2a cell lines treated with siRNA to Gnao1 (n = 3) and those with control siRNA (n = 3). Bulk-prepared RNA was used for RNA sequencing. Both RSEM and rMATS algorithms were used.

first: Ryoji

last: Taira

first: Satoshi

last: Akamine


title: Evaluation of the effect of maternal immune activation and juvenile distress on hypothalamus of 60 day old pigs

city: Urbana

first: Sandra

last: Rodriguez Zas

state: IL

address: 1207 W. Gregory Dr.

institute: University of Illinois

summary: Female and male pigs were studied. Half of the pigs were exposed to immune activation from virally-infected mothers, and half were not exposed. At 60 days of age about one third of the pigs were given metabolic distress (fasting), one third of the pigs were given immune distress (poly(I:C)), and one third of the pigs were given Saline (control).

overall_design: RNA samples were obtained at 60 days of age. Hypothalamus RNA samples were subjected to paired-end RNA sequencing

middle: R

last: Zas


title: Rapid CRISPR/Cas12a-based dual-crRNA library screening in high fidelity reveals novel genetic interactions during hepatocarcinogenesis [target-seq]

city: Wuhan city

first: Qian

last: Chen

state: Hubei province

address: No.299 bayi road, wuchang district

department: College of Life Sciences

institute: Wuhan University

summary: CRISPR/Cas12a-based combinational screening has shown remarkable potential in identifying genetic interactions. Here, we described an innovative method for combinational genetic screen with rapid constructing of dual-crRNA library by gene splicing through overlap extension PCR (SOE PCR) and the adoption of CeCas12a, which was identified previously by us with strict PAM recognition and low off- targeting, to guarantee the fidelity and efficiency. The custom, pooled SOE crRNA array (SOCA) library for double knockout screen could be conveniently constructed in lab for widespread use and CeCas12a mediated high fidelity screen display good performances even under negative selection screen. By designing an SOCA dual-crRNA library which covered the most of kinase and metabolism-associated gene targets of FDA- approved drugs that were implicated in hepatocellular carcinoma (HCC) tumorigenesis, novel cross talks between the two gene sets were negatively selected out to synergistically inhibit HCC cell growth in vitro and in vivo and also validated by virtual double knockdown screening based on TCGA databases. Thus through our rapid, efficient and high fidelity double knockout screening system, it is very promising to systemically dig genetic interactions between multiple gene sets or synergistic combinations of FDA-approved drugs for clinical translational medicine in the future.

overall_design: The main objectives of this study were (i) to determine whether SOE PCR could successfully construct a double knockout library with complete coverage and (ii) to demonstrate the efficiency and accuracy of SOCA library-based combinational genetic screening. We performed T7E1 assay, targeted deep sequencing and GUIDE-seq to identify the library candidate crRNA with high cleavage activity and Cas12a nucleases with high editing efficiency and low off-target rate and ensured the efficiency and accuracy of screening results. We constructed primer pool by SOE PCR to amplify core fragments (DR-SpacerX-DR-Overlaps-DR- SpacerY-DR), and obtain an entire SOE crRNA array (SOCA) library using infusion cloning. After that, using the SOCA library, we performed in vitro negative screening in the murine HCC cell line (Hepa1-6 cell) and analyzed the correlation between two biological replicates and between two Cas12a nucleases. For in vivo screening, we transplanted target cells into 5-6 nu/nu mice per group and harvested tumors at different time of tumor growth, and then we extracted the genome of the tumor cells and analyzed the crRNA abundance. We performed in vivo and in vitro assays to verify the synergistic interaction of the novel identified combinations and the accuracy of our library screening.

first: Lei

last: Yin


title: Rapid CRISPR/Cas12a-based dual-crRNA library screening in high fidelity reveals novel genetic interactions during hepatocarcinogenesis [guide-seq]


title: Rapid CRISPR/Cas12a-based dual-crRNA library screening in high fidelity reveals novel genetic interactions during hepatocarcinogenesis

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Rapid CRISPR/Cas12a-based dual-crRNA library screening in high fidelity reveals novel genetic interactions during hepatocarcinogenesis [library seq]


title: Rapid CRISPR/Cas12a-based dual-crRNA library screening in high fidelity reveals novel genetic interactions during hepatocarcinogenesis [RNA-Seq]


title: Aging- and Alcohol-Associated Spatial Transcriptomic Signature in Mouse Acute Pancreatitis

city: Houston

first: Wenbo

address: 7000 Fannin Street

institute: The University of Texas Health Science Center at Houston

summary: The rapidly aging population has an increased alcohol use, resulting in increased risk and mortality of alcohol-associated acute pancreatitis in the elderly population. We established an aging- and alcohol-associated acute pancreatitis mouse model and applied spatial transcriptomics to profile heterogenous tissue composition in the mouse pancreas and the associated gene expression. By comparing with the young cohorts, our analysis has revealed the key differentially expressed genes in functional tissue compartments for understanding the mechanisms and developing targeted therapeutic strategies.

overall_design: Pancreatic formalin-fixed paraffin-embedded tissue sections from four C57BL/6 male mice (2 young and 2 aging) with acute pancreatitis induced by alcohol liquid diet feeding and cerulein injection were analyzed using spatial transcriptomics.

first: Rachel

last: Tindall

first: Yuntao

last: Yang

first: Yanna

last: Cao

first: Jim

last: Zheng

first: Tien

last: Ko


title: OT-II T cells derived from uninfected or N.b. infected hosts transferred into congenic recipient post immunization

city: New Haven

first: Carla

last: Rothlin

state: CT

address: 300 Cedar Street

institute: Yale University

summary: The goal was to determine changes in transcription in OT-II T cells post activation if pre-conditioned in a helminth-infected host

overall_design: 7 biological replicates were anaylzed in total. 4 samples of OT-II T cells derived from uninfected hosts and 3 samples of OT-II T cells derived from N.b. infected hosts.

first: Alexandre

middle: P

last: Meli

first: Sourav

last: Ghosh

middle: V


title: Histone 3 lysine 9 methylation balance controls transient site-specific DNA amplification and genomic rearrangements of Mixed Lineage Leukemia (MLL)

city: Boston

first: Kashish

last: Chetal

state: MA

address: 55 Fruit St

department: Molecular biology

institute: MGH

summary: DNA amplifications and rearrangements are observed in cancer. In infant and adult leukemia as well as therapy-induced leukemia, MLL/KMT2A amplifications and translocations are prevalent. To date, the exact molecular contributor(s) to these alterations are unclear. In this study, we demonstrate that histone H3 K9 mono- and di-methylation (H3K9me1/2) balance at the MLL/KMT2A locus is regulating the amplifications and rearrangements. This balance is controlled by the cross-talk between lysine demethylase KDM3B and methyltransferase G9a/EHMT2. KDM3B loss or chemical suppression increases H3K9me1/2 and reduced CTCF occupancy in KMT2A, and in turn, promotes amplification and rearrangements. Reducing CTCF levels was also sufficient to generate these alterations. Furthermore, Doxorubicin (Dox), which promotes MLL/KMT2A amplifications and rearrangements, suppresses KDM3B and CTCF protein levels. By suppressing G9a or overexpressing KDM3B, the Dox induced MLL/KMT2A alterations are rescued. Therefore, MLL/KMT2A induced amplifications and rearrangements are controlled by distinct epigenetic enzymes that are druggable, which has clinical implications.

overall_design: Chromatin Immunoprecipitation DNA-Sequencing (ChIP-seq) for histone modifications H3K9me1, me2, and me3 and CTCF binding sites in RPE cells

first: Johnathan

last: Whetstine

first: Reuben

last: Duttweiler

first: Zachary

last: Gray

first: Ruslan

last: Sadreyev


title: RNA Binding protein KPNA2 promotes the progress of gastric cancer by regulating alternative splicing of related genes

city: Lanzhou

first: Yuping

last: Wang

address: the first hospital of lanzhou university, Lanzhou, Gansu

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


title: RNA Binding protein KPNA2 promotes the progress of gastric cancer by regulating alternative splicing of related genes [RNA-seq]

institute: the first hospital of lanzhou university

summary: RNA binding proteins (RBPs) took a critical part in genome regulation. kpna2, as an essential member of the RBP family, was found to be highly expressed and associated with lymph node metastasis in gastric cancer (GC). Transcriptional sequencing data were used to find that KPNA2 primarily regulated alternative 5' splice site (A5SS), alternative 3'splice site (A3SS), exon skipping (ES) and cassette exon. Vitally, KPNA2 was involved in the transcriptional regulation of cancer-related genes. Therefore, we screened potential targets in AGS cells with differential expression and alternative splicing (AS) levels regulated by KPNA2, further identifying that KPNA2 was engaged in biological processes concerning immune response, cell proliferation and apoptosis in GC through transcriptional regulation. Besides, the binding sites of KPNA2 to spliceosomes were identified using improved RNA immunoprecipitation sequencing techniques, and KPNA2 was preferentially bound to the intron region. Notably, KPNA2 regulated the A3SS AS mode of WDR62, which may increase the malignancy of GC. Finally, we also discovered that AS of immune-related molecules can be regulated by KPNA2. Overall, for the first time, our results demonstrated that KPNA2 served as an oncogenic splicing factor in GC regulating AS and differential expression of GC-related genes and may represent a potential target for GC therap

overall_design: transcrptional analysis of KPNA2 knockdown and control in AGS cells


title: Transcriptional profiling of wounds in WT mice and Cthrc1-/- mice

city: Beijing

first: Sha

last: Huang

address: 51 Fu Cheng Road

institute: PLA General Hospital

summary: Collagen triple helix repeat containing 1 (CTHRC1) has been reported to be associated with tissue repair in response to injury. To investigate the role of CTHRC1 in the skin wound repair, we  used next-generation sequencing (NGS) to clarify the changes of transcriptome in the mice skin wounds after Cthrc1 deletion.

overall_design: Using NGS, we explored the differences in gene expression in dermal tissues of  wild type (WT) and Cthrc1-/- murine model of paired equivalent-sized wounds. Samples were examined at day 0 and day 7, and represented by 3 biological replicates.

first: Xianlan

last: Duan

first: Xingyu

last: Yuan


title: B-cell transcription factor PAX5 functions as a tumor suppressor by regulating the cell cycle in primary effusion lymphoma

city: Kumamoto

first: Seiji

last: Okada

address: 2-2-1 Honjo, Chuo-ku

institute: Kumamoto University

summary: Primary effusion lymphoma (PEL) is an aggressive B-cell lymphoma arising from Kaposi sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8) infection. PEL shows invasive behavior, causing body cavity effusions without apparent solid tumors. The typical prognosis and clinical course of patients with PEL are poor and potentially lethal. Clarification of the pathogenic mechanisms of PEL is urgently needed in order to develop novel therapies. PEL cells generally lack B-cell surface markers such as CD19, and we therefore hypothesized that the B-cell transcription factor, PAX5, would be down-regulated in PEL. PAX5 is expressed from the pro-B to the mature B-cell stage and is indispensable for the differentiation of B-cells. PAX5 was silenced in PEL cells via methylation of the PAX5 promoter region. Up-regulation of PAX5 induced several genes coding for B-cell surface marker mRNA, but not protein level. PAX5 inhibited cell growth via G1 cell cycle arrest. PAX5 bound to RB and increased its protein expression. RB/E2F-regulated genes were significantly down-regulated in microarray analysis and PCR experiments. To clarify the in vivo function of PAX5, we analyzed the restoration of PAX5 in a PEL xenograft model using severely immunodeficient, NOD/SCID/Jak3-deficient (NOJ) mice. The ascites volume and organ invasions were significantly suppressed by PAX5 restoration. Reduction of PAX5 has a critical role in the oncogenesis of PEL, and PAX5 is a tumor suppressor in PEL. Targeting PAX5 could represent a novel therapeutic approach for patients with PEL.

overall_design: We generated BCBL-1 cells that conditionally express PAX5 using a tet-on system. We compared control and PU.1-up-regulated BCBL-1 cells with Dox using microarrays

first: Hiroki

last: Goto


title: DNA methylation profiling of immune cells from tuberculosis-exposed individuals overlaps with BCG-induced epigenetic changes and correlates with the emergence of anti-mycobacterial ‘corralling cells’

city: Linköping

first: Jyotirmoy

last: Das

address: Sandbäcksgatan 7

department: Medical Microbiology

country: Sweden

institute: Linkoping University

type: Methylation profiling by genome tiling array

summary: The mechanism of protection of the only approved tuberculosis (TB) vaccine, Bacillus Calmette Guérin (BCG) is poorly understood. In recent years, epigenetic modifications induced by BCG have been demonstrated to reflect a state of trained immunity. The concept of trained immunity is now explored as a potential prevention strategy for a variety of infections, including Covid-19. Studies on human TB immunity are dominated by those using peripheral blood as surrogate markers for immunity. Here, we instead studied the lung compartment by obtaining induced sputum from subjects included in a TB contact tracing. CD3- and HLA-DR-positive cells were isolated from the collected sputum and DNA methylome analyses performed.  Unsupervised cluster analysis revealed that DNA methylomes of cells from TB-exposed individuals and controls appeared as separate clusters, and the numerous genes that were differentially methylated were functionally connected. The enriched pathways were strongly correlated to previously reported epigenetic changes in immune cells exposed to the BCG-vaccine in human and animal studies. We further demonstrated that similar pathways were epigenetically modified in human macrophages exposed to BCG in vitro. Finally, we found evidence of an M. tuberculosis-triggered emergence of a non-macrophage cell population from BCG-exposed macrophage cultures. These cells did not phagocytose M. tuberculosis, but instead ‘corralled’ the bacteria into focal points, resulting in limitation of bacterial growth. Our study demonstrates that similar epigenetic changes are induced by M. tuberculosis and BCG and suggest that the modifications promote transformation of macrophages (or an unknown progenitor) to establish a yet undescribed cellular defense mechanism which we term ‘corralling’, based on the metaphorical resemblance to sheepdog herding.

overall_design: 10 sample DNAs were isolated from the HLA-DR, CD3 and PBMC cells and 4 sample DNAs were isolated from HLA-DR and CD3 using AllPrep DNA/RNA Mini Kit (Qiagen, Germany). DNA concentration was measured by Qubit fluorometer. DNA samples were bisulphite converted, amplified, fragmented and hybridized to an Illumina Infinium Human Methylation 450K Bead Chip and scanned.

first: Isabelle

last: Pehrson

first: Clara

last: Braian

first: Lovisa

last: Karlsson

first: Nina

last: Idh

first: Eva

last: Danielsson

first: Jakob

last: Paues

first: Maria

last: Lerm


title: RNA Binding protein KPNA2 promotes the progress of gastric cancer by regulating alternative splicing of related genes [RIP-seq]

overall_design: RIP-seq analysis of KPNA2 IP versus input in AGS cells


title: Role of the STAR proteins in early mammalian development [Ribosome profiling]

city: Barcelona

first: Elias

last: Bechara

address: Dr Aiguader 88

institute: CRG

summary: RNA binding proteins (RBPs), are emerging as important master regulators of the biology of stem cells in both pluripotency and differentiation. The Signal transduction and activation of RNA (STAR) is family of proteins shown to be involved in mammalian development. We characterized the role of two members of the family, Sam68 and Quaking (QKI), in pluripotency and differentiation of mouse embryonic stem cells (mESCs) by making use of CRISPR-Cas9 generated knock-out (KO) cell lines along with RNA-sequencing, ribosome profiling and RNA crosslinking followed by immunoprecipitation (iCLIP2). Our results indicate that both proteins are important in the regulation of mESCs cell cycle and self-renewal and are indispensable for proper cardiomyocyte differentiation by regulating expression, alternative splicing and translation of the cardiogenic  factors GATA4. This work highlights the importance of the RBPs during the differentiation of mESCs.

overall_design: 6 samples in biological triplicate: a wild type E14 cell line of reference and KO lines (Sam68 or Qauking) of the same genetic background. Each line was analyzed in 2 time points: day 0 and day 10 of Embryoid body differentiation

first: Alessandro

last: Dasti

last: Antonelli


title: Role of the STAR proteins in early mammalian development

overall_design: Refer to individual Series


title: Role of the STAR proteins in early mammalian development [iCLIP]

overall_design: 6 samples in biological triplicate:  wild type E14 cell line precipitated with antibody against either Sam68, QKI or using IgG as negative control


title: Rapid Human Oogonia-like Cell Specification via Combinatorial Transcription Factor-Directed Differentiation [barcode]

status: Public on Aug 01 2024

first: Merrick

middle: Pierson

last: Smela

address: 3 Blackfan Circle

institute: Harvard University

summary: The generation of germline cell types from human induced pluripotent stem cells (hiPSCs) represents a key milestone toward in vitro gametogenesis, which has the potential to transform reproductive modeling and medicine. Methods to recapitulate advanced germline cell specification in vitro have relied on extensive, long term culture methods, the most notable of which is a four-month culture protocol employing xenogeneic reconstituted ovaries with mouse embryonic ovarian somatic cells. Recently, transcription factor (TF)-based methods have demonstrated the feasibility of exogenous factor expression to directly differentiate hiPSCs into cell types of interest, including various ovarian cell types. The protocols leveraged in these studies, however, utilize more local methods of factor selection, such as basic differential gene expression analysis, and lower-throughput screening strategies via iterative testing of a small set of TFs. In this work, we integrate our recently-described graph theory pipeline and highly-parallelized screening protocols to globally identify and screen 46 oogenesis-regulating TFs for their role in human germline formation. We identify ZNF281, LHX8, SOHLH1, ZGLP1, and ANHX whose combinatorial overexpression drives DDX4+ induced oogonia-like cell (iOLC) formation from hiPSCs. In contrast to previous methods, our protocol employs a simple four-day, feeder-free monolayer culture condition. We additionally demonstrate a method of post-isolation, feeder-free expansion of DDX4+ iOLCs that shows retained cell identity in vitro. We additionally identify DLX5, HHEX, and FIGLA whose individual overexpression enhances hPGCLC formation from hiPSCs. We characterize these TF-based iOLCs and hPGCLCs via gene and protein expression analyses and demonstrate their broad similarity to in vivo and in vitro-derived oogonia and primordial germ cells. Together, these results identify new regulatory factors that enhance in vitro human germ cell specification and further establish unique computational and experimental tools for human in vitro oogenesis research.

overall_design: DNA methylation sequencing of human induced oogonia-like cells, induced pluripotent stem cells, and primordial germ cell-like cells

middle: D

last: Pierson Smela

first: Christian

last: Kramme

first: Patrick

last: Fortuna

first: Bennet

last: Wolf

first: Shrey

last: Goel

first: Jessica

last: Adams

first: Carl

last: Ma

first: Sergiy

last: Velychko

first: Ursula

last: Widocki

first: Venkata

last: Kavirayuni

first: Tianlai

last: Chen

first: Sophia

last: Vincoff

first: Edward

last: Dong

first: Richie

last: Kohman

first: Mutsumi

last: Kobayashi

first: Toshi

last: Shioda

first: George

middle: M

last: Church

first: Pranam

last: Chatterjee


title: DeepRNA-Reg: A Deep-Learning Based Approach for Comparative Analysis of CLIP Experiments

city: San Francisco

first: K. Mark

last: Ansel

address: 513 Parnassus Ave. UCSF Box 0414

department: Microbiology & Immunology

institute: UCSF

summary: DeepRNA-Reg employs advances in deep learning to enable high-fidelity comparative analysis of paired datasets of high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). In a HITS-CLIP experimental paradigm where Ago2 activity is precisely perturbed via gene knock-out of a microRNA cluster, DeepRNA-Reg offers a superior prediction set than the current best prescription for differential HITS-CLIP; furthermore, DeepRNA-Reg predictions adhere better to the ground-truth of the RNA primary and secondary structural motifs that enable miRNA-mediated targeting of RNA. DeepRNA-Reg uncovered novel mediators in the mechanism of microRNA-mediated restraint of type-2 immunity in T-Helper 2 cells. In a comparative analysis, DeepRNA-Reg predictions show greater translatability across distinct biological milieux, offering prediction sets with wide applicability for investigators.

overall_design: To investigate the ability of DeepRNA-Reg to identify differentially protein-bound loci between a pair of HITS-CLIP experiments, we  performed Ago2 HITS CLIP on mouse Th2 cells with the Mirc11 and Mirc22 clusters (miRNAs 23, 24, and 27) knocked-out and Th2 cells with a WT genotype.

first: Harshaan

last: Sekhon

first: Heather

middle: H

last: Pua

first: Eric

middle: J

last: Wigton

first: Robin

last: Kageyama

first: K


title: A CLIC1 network coordinates matrix stiffness and the Warburg effect to promote tumor growth in pancreatic cancer. Zheng et al.

city: Shanghai

first: De-Jun

last: Liu

state: Shanghai

address: Pujian Road 160

department: Department of Biliary-Pancreatic Surgery

institute: Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University

summary: Pancreatic ductal adenocarcinoma (PDAC) features substantial matrix stiffening and reprogrammed glucose metabolism, particularly the Warburg effect. However, the complex interplay between these traits and their impact on tumor advancement remains inadequately explored. Here, we integrated clinical, cellular, and bioinformatics approaches to explore the connection between matrix stiffness and the Warburg effect in PDAC, identifying CLIC1 as a key mediator. Elevated CLIC1 expression, induced by matrix stiffness through Wnt/β-catenin/TCF4 signaling, signifies poorer prognostic outcomes in PDAC. Functionally, CLIC1 serves as a catalyst for glycolytic metabolism, propelling tumor proliferation. Mechanistically, CLIC1 fortifies HIF1α stability by curbing hydroxylation via ROS. Collectively, PDAC cells elevate CLIC1 levels in a matrix stiffness-responsive manner, bolstering the Warburg effect to drive tumor growth via ROS/HIF1α signaling. Our insights highlight opportunities for targeted therapies that concurrently address matrix properties and metabolic rewiring, with CLIC1 emerging as a promising intervention point.

overall_design: Comparative gene expression profiling analysis via RNA-seq in CLIC1-knockdown vs. control PATU8988 cells (n = 3 each).

first: Jia-Hao

last: Zheng

first: Yu-Heng

last: Zhu

first: Jian

last: Yang

first: Pei-Xuan

last: Ji

first: Rui-Kang

last: Zhao

first: Zong-Hao

last: Duan

first: Hong-Fei

last: Yao

first: Qin-Yuan

last: Jia

first: Yi-Fan

last: Yin

first: Li-Peng

last: Hu

first: Qing

last: Li

first: Shu-Heng

last: Jiang

first: Yan-Miao

last: Huo

first: Wei

first: Yong-Wei

last: Sun


title: RNA-sequencing assay to identify changed gene expression  in lung adenocarcinoma (LUAD) cells before and after osimertinib resistance

city: Tianjin

first: Jing

last: Zhou

address: Huanhuxi Street

department: Department of Thoracic Oncology

institute: Tianjin Medical University Cancer Institute & Hospital

summary: The H1975 osimertinib resistant (H1975 OR) cell lines were successfully established by subjecting parental H1975 cells to a gradually increasing concentration of osimertinib up to 1μM for a duration exceeding 2 months. In order to investigate the molecular alterations associated with osimertinib resistance, we analyzed the transcriptional profiling of parental H1975 cells and H1975 OR cells by RNA sequencing.

overall_design: Three biological replicates for each group H1975 and H1975 OR were subjected to RNA-sequencing analysis respectively, among which H1975 acted as the reference sample for H1975 OR.

first: Richeng

first: Xinyue

last: Wang


title: The JNK and Hippo pathways control epithelial integrity and prevent tumour initiation by regulating an overlapping transcriptome [RNA-Seq]

city: Melbourne

first: Kieran

last: Harvey

state: Victoria

address: 305 Grattan Street

department: Research

institute: Peter MacCallum Cancer Centre

summary: Epithelial organs maintain their integrity and prevent tumour initiation by actively removing defective cells, such as those that have lost apicobasal polarity. Here, we identify how transcription factors of two key signalling pathways – Jun-N-terminal kinase (JNK) and Hippo – regulate epithelial integrity by controlling transcription of an overlapping set of target genes. Targeted DamID experiments reveal that in proliferating cells of the Drosophila melanogaster eye, the AP-1 transcription factor Jun, and the Hippo pathway transcription regulators Yorkie and Scalloped bind to a common suite of target genes that regulate organ growth. In defective neoplastic cells, AP-1 transcription factors repress transcription of growth genes together with the CtBP co-repressor. If gene repression by AP-1/CtBP fails, neoplastic tumour growth ensues, driven by Yorkie/Scalloped. Thus, AP-1/CtBP eliminates defective cells and prevents tumour initiation by acting in parallel to Yorkie/Scalloped to repress expression of a shared transcriptome. These findings shed new light on the maintenance of epithelial integrity and tumour suppression.

overall_design: RNAseq of third instar larval eye-antennal discs for the following mutant genotypes: fosKM, CtBP87De10, scrib1, scrib1 fosKM, CtBP87De-10 scrib1 and FRT82B control

first: Katrina

last: Arissa Mitchell

first: Joseph

last: Vissers

first: Jonathan

last: Pojer

first: Elliot

last: Brooks

first: Abdul

last: Jabbar Saiful Hilmi

first: Anthony

middle: T

last: Papenfuss

first: Jan

last: Schröder

last: Forrest Harvey


title: The JNK and Hippo pathways control epithelial integrity and prevent tumour initiation by regulating an overlapping transcriptome [DamID-seq]

overall_design: Targeted DamID sequencing of Dam control, Yki-Dam, Dam-Sd and Dam-Jun fusions in Drosophila early third instar larval eye-antennal discs (3 replicates each)

last: Mitchell

middle: F


title: Disentangling the mutational effects on protein stability and interaction of human MLH1

city: Copenhagen

first: Sven

last: Larsen-Ledet

state: N

address: Ole Maaloes Vej 5

country: Denmark

institute: University of Copenhagen

summary: Missense mutations can have diverse effects on proteins, depending on their location within the protein and the specific amino acid substitution. Mutations in the DNA mismatch repair gene MLH1 are associated with Lynch syndrome, yet the underlying mechanism of most disease-causing mutations remain elusive. To address this gap, we aim to disentangle the mutational effects on two essential  properties for MLH1 function: protein stability and protein-protein interaction. We systematically examine the cellular abundance and interaction with PMS2 of 4839 (94%) MLH1 variants in the C-terminal domain. Our combined data shows that most MLH1 variants lose interaction with PMS2 due to reduced cellular abundance. However, substitutions to charged residues in the canonical interface lead to reduced interaction with PMS2. Unexpectedly, we also identify a distal region in the C-terminal domain of MLH1 where substitutions cause both decreased and increased binding with PMS2. Our data successfully distinguish benign from pathogenic MLH1 variants and correlate with thermodynamic stability predictions and evolutionary conservation. This work provides mechanistic insights into variant consequences and may help interpret MLH1 variants.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: The C-terminal domain of human MLH1 was divided into seven tiles. In each tile, we generated site saturation mutagenesis libraries comprising all possible missense variants and cloned them into pDONR221. The libraries were subsequently cloned into pDEST-DHFR-PCA for the abundance assay and pDEST22 for the interaction assay and transformed into the wild-type BY4741 yeast strain and the MaV203 yeast strain, respectively. For the abundance assay, cells were plated on medium without uracil and with (selection) or without (control) methotrexate. For the interaction assay, cells were plated on medium with (control) or without (selection) histidine. After selection, cells were harvested and plasmids were isolated to prepare amplicons that was sequenced using the Illumina NextSeq 550 System. Based on the relative change in frequency of variants between the control and selection conditions compared to synonymous wild-type variants, we calcuated an abundance and interaction score for each variant.

first: Amelie

last: Stein


title: Elucidating the transcriptomic response of adult hypothalamic neuron cells to cannabidiol (CBD) exposure

city: Kraków

first: Artur

last: Gurgul

address: Rędzina 1C

department: Center for Experimental and Innovative Medicine

country: Poland

institute: University of Agriculture in Krakow

summary: Cannabidiol (CBD) actions in the brain are largely complex and not fully understood. One important brain structure that is a target for CBD, and for which health-beneficial CBD consequences can be seen is the hypothalamus. Given the fundamental role of gene expression in the hypothalamus's regulatory functions, this study undertook the analysis of changes arising in hypothalamic cells’ transcriptomes following various CBD treatments. For this purpose, we used a hypothalamic cellular model, namely we employ adult-derived mHypoA-2/12 mouse cell lines. In the course of the study, the neural cells were treated with different CBD doses (ranging from 0.325 to 3 µM) and vehicle as a control for 6 and 24 hours. The experiment setup allowed us to evaluate both the dosage and time-dependent effect of CBD on hypothalamic cells, particularly on their viability, apoptosis, and transcriptome profile.

overall_design: The adult mouse hypothalamic cell lines mHypoA-2/12 (Clu177) were purchased from Cedarlane (Canada) and cultured in recommended conditions. After culture stabilization, the cells were supplemented with CBD (0.325, 0.75, 1.5 and 3 µM) and cultured either for 6 hours (n = 3 for each concentration) or for 24 hours (n = 3 for each concentration). The vehicle control was 0.1% DMSO in the DMEM with GlutaMAX. When treatment time passed, cells were trypsinized, transferred to centrifuge tubes, and used for RNA isolation. Purified RNA was used for 3’mRNA-Seq to evaluate changes that arise in the transcriptome of the hypothalamic cells following different CBD treatments.

first: Igor

last: Jasielczuk

first: Tomasz

last: Szmatoła

first: Ewelina

last: Semik-Gurgul

first: Mirosław

last: Kucharski

first: Karolina

last: Mizera-Szpilka

first: Ewa

last: Ocłoń


title: Rapid Human Oogonia-like Cell Specification via Combinatorial Transcription Factor-Directed Differentiation [BiSulfite-seq]

first: Merrick

last: Smela

type: Methylation profiling by high throughput sequencing

last: Pierson Smela

first: Christian

last: Kramme

first: Patrick

last: Fortuna

first: Bennet

last: Wolf

first: Shrey

last: Goel

first: Jessica

last: Adams

first: Carl

last: Ma

first: Sergiy

last: Velychko

first: Ursula

last: Widocki

first: Venkata

last: Kavirayuni

first: Tianlai

last: Chen

first: Sophia

last: Vincoff

first: Edward

last: Dong

first: Richie

last: Kohman

first: Mutsumi

last: Kobayashi

first: Toshi

last: Shioda

first: George

last: Church

first: Pranam

last: Chatterjee


title: Keratinocyte glucocorticoids enable local immune resilience to preserve skin homeostasis.

city: Konstanz

first: Rebekka

last: Lambrecht

address: Universitaetsstrasse 10

country: Germany

institute: University of Konstanz

summary: Endogenous glucocorticoids (GCs) are pivotal in controlling inflammation. Keratinocyte-derived GCs contribute to local skin homeostasis as deletion of the GC-producing enzyme 11β-hydroxylase (Cyp11b1) in keratinocytes exacerbated skin inflammation. Since local tamoxifen-induced knockout (KO) induction may contribute to skin irritation, we implemented intraperitoneal injections to induce a systemic skin GC depletion preventing experimental skin irritation in order to reveal the importance of skin GC in steady-state. Both, local and systemic skin GC deficiency models exhibited reduced skin GC levels and increased migration of skin antigen-presenting cells to draining lymph nodes. However, systemic skin GC ablation did not result in pronounced skin inflammation as seen in local model. Interestingly, systemic skin GC deficiency elevated systemic inflammatory markers and provoked adrenal GC synthesis. RNA sequencing of keratinocytes revealed distinct gene expression patterns between local and systemic KOs. Local skin GC ablation showed a stronger inflammatory and apoptotic response, while systemic skin GC deficiency triggered several compensatory regulatory pathways, mitigating extensive skin inflammation. These findings underscore the critical role of local GCs in skin immune resilience against minor skin irritations and highlight the interplay between skin and adrenal GC levels.

overall_design: To investigate the effect of systemic skin GC deficiency and its difference to local skin GC deficiency, K14-CreERTam x Cyp11b1-L2/L2 mice (conditional knockout, cKO) and Cre-negative Cyp11b1-L2/L2 littermate control mice (floxed control, ctrl_fl) were treated with tamoxifen for five consecutive days, either by intra-peritoneal (i.p.) injection (systemic), or by topical application (local) to induce knockout of Cyp11b1 in keratinocytes and thereby ablate skin GC synthesis. For systemic administration, mice were daily i.p. injected with tamoxifen in oil-ethanol solution, for local application, back hair was gently dry-shaved, and dorsal and ear skin was treated daily with tamoxifen in ethanol. On day six, keratinocytes were isolated from ear and dorsal skin and enriched by flow cytometry sorting. Total RNA of sorted keratinocytes were isolated and gene expression was analyzed by bulk RNA sequencing. Five mice per experimental group (ctrl_fl local, cKO local, ctrl_fl systemic, cKO systemic) were used.

first: Truong

middle: S

last: Phan

first: Verena

last: Merk

first: Alice

last: Wiedmann

first: Pascale

last: Zwicky

first: Burkhard

last: Becher

first: Thomas

last: Brunner


title: Epigenome-wide association study of Prostate Cancer in African American Men identified differentially methylated genes

city: Tampa

first: Anders

last: Berglund

state: Florida

address: 12902 Magnolia Drive

department: Department of Biostatistics and Bioinformatics

institute: Moffitt Cancer Center

summary: Men with African ancestry have the highest incidence and mortality rates of prostate cancer (PCa) worldwide. This study aimed to identify differentially methylated genes between tumor vs. adjacent normal and aggressive vs. indolent PCa in 121 African American patients. Epigenome-wide DNA methylation patterns in tumor DNA were assessed using the human Illumina Methylation EPIC V1 array. Around 5,139 differentially methylated CpG-sites (q<0.01, lΔβl>0.2) were identified when comparing normal vs. tumor, with an overall trend of hypermethylation in prostate tumors.  Multiple representatives differentially methylated regions (DMRs), including immune-related genes, such as CD40, Galectin3, OX40L, and STING, were detected in prostate tumors when compared to adjacent normal tissues. Based on an epigenetic clock model, we observed that tumors' total number of stem cell divisions and the stem cell division rate were significantly higher than adjacent normal tissues. Regarding aggressiveness, 2,061 differentially methylated CpG-sites (q<0.05, lΔβl>0.05) were identified when the grade group (GG)1 was compared with GG4/5. Among these 2,061 CpG sites, 155 probes were consistently significant in more than one comparison. Among these genes, several immune system genes, such as COL18A1, S100A2, ITGA4, HLA-C, and ADCYAP1, have previously been linked to tumor progression in PCa.  Several differentially methylated genes involved in immune-oncologic pathways associated with disease risk or aggressiveness were identified. In addition, 261 African American-specific differentially methylated genes related to the risk of PCa were identified. These results can shed light on potential mechanisms contributing to PCa disparities in the African American population.

overall_design: The Illumina Methylation EPIC V1 BeadChip was used for measuring methylation levels using genomic DNA samples from formalin-fixed paraffin-embedded prostate tumor (n=120) and adjacent normal  tissue samples (n=118).

first: Kosj

last: Yamoah

first: Steven

last: Eschrich

first: Rana

last: Falahat

first: James

last: Mulé

first: Sungjune

last: Kim

first: Jaime

last: Matta

first: Julie

last: Dutil

first: Gilberto

last: Ruiz-Deya

first: Carmen

middle: O

last: Sanchez

first: Liang

last: Wang

first: Hyun

last: Park

first: Hirendra

middle: N

last: Banerjee

first: Tamara

last: Lotan

first: Kathryn

last: Barry

first: Ryan

last: Putney

first: Seung

first: Clement

last: Gwede

first: Jacob

last: Kresovich

first: Youngchul

first: Hui-Yi

last: Lin

first: Jasreman

last: Dhillon

first: Ratna

last: Chakrabarti

first: Jong

middle: Y


title: Gene expression 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 RNA-seq comparing the CD19+ B cells and CD138+ plasma cells at different time points during the organoid culture.  Two stimulation conditions were used including influenza antigen alone or in combination with the TLR7 agonist R848.  Finally, two B cell sources were compared to udnerstand if tissue of origin impacted organoid based B cell differentiation.

overall_design: RNA-seq of 2-3 replicates of each sample group.

first: Christopher

first: Ankur

last: Singh


title: The effect of Stat3 loss on inflammation and endometriosis

city: Columbia

first: Jae-Wook

last: Jeong

address: 1030 Hitt Street

department: Obstetrics, Gynecology and Women's Health

institute: University of Missouri

summary:
Aberrant STAT3 activation is a major contributor to endometriosis.
We used microarrays to detail the mechanism underlying Stat3 loss.

overall_design: Uteri of Stat3 f/f and Stat3 d/d mice were selected at 3.5 dpc for RNA extraction and hybridization on Affymetrix microarrays.

first: Tae

last: Kim


title: Molecular mechanisms of PD-1 immune checkpoint inhibitors in mouse mammary tumours

city: Dunedin

first: Sarah

last: Barber

address: 362 Leith Street

department: Biochemistry

country: New Zealand

institute: University of Otago

summary: Understanding the molecular actions of anti-PD-1  immune checkpoint inhibitors in ER+ mouse mammary tumours is crucial to aid in understanding of how this treatment can be used in ER+ breast cancer. Here, we demonstrate that treatment of mouse mammary tumours with anti-PD-1 alone results in limited genome wide transcriptional changes. Treatment with the endocrine therapy, letrozole, results in large genome wide transcriptional changes with predominant influence on oestrogen responsive genes and genes involved in cell cycle related processes. Further, we report that treatment with a combination of both letrozole and anti-PD-1 results again produced large genome wide transcriptional changes, all of which were observed upon letrozole treatment alone.

overall_design: To investigate the transcriptional effects of anti-PD-1 and letrozole treatments, we transcriptionally profiled SSM3 ER+ breast cancer tumours in immunocompetent 129/SvEv mice treated with either letrozole alone, anti-PD-1 alone, letrozole + anti-PD-1 or a vehicle control. RNA-seq was conducted on RNA was extracted from five biological replicates of each treatment.

middle: L

first: Anita

last: Dunbier


title: LPS-induced TIR domain complex acetylation activated TLR4/MAL/MyD88 signal pathway in sepsis [scRNA-Seq]

city: Hangzhou

first: Facai

last: Zhang

state: Zhejiang

address: 158 Shangtang Road

institute: Zhejiang Provincial People‘s Hospital

summary: Among the diseases caused by Toll-like receptor 4 (TLR4) abnormal activation by bacterial endotoxin, sepsis is the most dangerous one. The reprogramming of macrophages plays a crucial role in orchestrating the pathogenesis of sepsis. However, the precise mechanism underlying TLR4 activation in macrophages remained incompletely understood. Our studies revealed that upon lipopolysaccharide (LPS) stimulation, CREB-binding protein (CBP)  was recruited to the TLR4 signalosome complex and resulted in pronounced acetylation in the TIR domains of TLR4, Myeloid differentiation factor 88 (MyD88) and MyD88 adapter-like (MAL), which significantly enhanced the activation of the NF-κB signaling pathway and polarization of M1 macrophages. In sepsis patients, significantly elevated TLR4-TIR acetylation was detected in CD16+ monocytes combined with elevated expression of M1 macrophage markers and production of pro-inflammatory cytokines. In contrast, histone deacetylase 1 (HDAC1) served as a key deacetylase in the deacetylation of the TIR domain complex. The inhibition of HDAC1 accelerated sepsis-associated syndromes, while the inhibition of CBP alleviated this process. Overall, our findings highlighted the crucial role of TIR domain complex acetylation in the regulation of inflammatory immune response and suggested that the reversible acetylation of the complex emerged as a promising therapeutic target for M1 macrophages during the progression of sepsis.

overall_design: The purified monocytes from sepsis were labeled with TLR4-acK and FITC-conjugated secondary antibody, then positively stained cells were enriched through the BD FACSAria™ III Cell Sorter.

first: Xue

last: Li

first: Xiangrong


title: LPS-induced TIR domain complex acetylation activated TLR4/MAL/MyD88 signal pathway in sepsis [RNA-Seq]

overall_design: WT, TLR4-K730R and TLR4-K810R mouse peritoneal macrophages were obtained as previously described and subjected to treatment with LPS (100 ng/mL) for 6 hours. Subsequently, total RNA from the cells was isolated using the RNeasy Mini Kit (QIAGEN, Valencia, CA, USA), following the manufacturer’s instructions. The RNA-seq analysis was performed by GENEWIZ, Inc., utilizing an Illumina sequencing platform.


title: Primary cilia are required for cell-type determination and angiogenesis in the pituitary development

city: Chiba

first: SAISHU

last: YOSHIDA

address: 2-2-1 Miyama, Funabashi-shi

department: Department of Biomolecular Science,

institute: Toho University

summary: To understand the molecular mechanisms underlying developmental abnormalities of the pituitary in  Dyrk2-/- mice, we performed comprehensive whole-genome RNA sequencing of the pituitaries of wild-type and Dyrk2-/- mice at E12.5

overall_design: We isolated Total RNA from the pituitaries of wild-type and Dyrk2-/- mice at E12.5

first: Saishu

last: Yoshida

first: Yousuke

last: Tsuneoka

first: Takehiro

last: Tsukada

first: Takashi

last: Nakakura

first: Akira

last: Kawamura

first: Wataru

last: Kai

first: Kiyotsugu


title: Characterization of gammadelta T cells infiltrating human colon adenocarcinomas

city: Goteborg

first: Marianne

last: Quiding Jarbrink

address: Box 435

institute: University of Gothenburg

summary: gd T cell infiltration into tumours usually correlates with improved patient outcome, but both tumour-promoting and tumoricidal effects of γδ T cells have been documented. Human γδ T cells can be divided into functionally distinct subsets based on T cell receptor Vd usage. Still, the contribution of these different subsets to tumour immunity remains elusive. Here, we provide a detailed gd T cell profiling in colon tumours, comprising mRNA quantification using the Nanostring platform, in combination with mass and flow cytometry and TCR sequencing. δ chain usage in both the macroscopically unaffected colon mucosa and tumours varied considerably between patients, with substantial fractions of Vδ1, Vδ2, and non-Vd1Vd2 cells. Nanostring analyses of flow cytometry sorted Vd1, Vd2 and non-Vd1Vd2 cells showed a large variation in gd T cell subsets between individual tumours, and we suggest that individual gd T cell composition in colon tumours may contribute to the balance between favourable and adverse immune responses, and thereby also patient outcome.

overall_design: Vd1, Vd2, and non-Vd1Vd2 cells isolated from tumors originating from 4 different patients were analysed with Nanostring nCounter human immunology v2 panel.

first: William

last: Rodin

middle: Q

last: Järbrink


title: Effect of overexpession of Stonewall (Stwl) on gene expression in germline stem cell (GSC)-like cells compared to the control

city: Zurich

first: Madhav

last: Jagannathan

address: Otto Stern-Weg 3

department: Biology

country: Switzerland

institute: ETH Zurich

summary: The association of genomic loci to the nuclear periphery is proposed to facilitate cell type-specific gene repression and influence cell fate decisions. However, the interplay between gene position and expression remains incompletely understood, in part because the proteins that position genomic loci at the nuclear periphery remain unidentified. Here, we used an Oligopaint-based HiDRO screen targeting ∼1000 genes to discover novel regulators of nuclear architecture in Drosophila cells.We identified the heterochromatin-associated protein Stonewall (Stwl) as a factor promoting perinuclear chromatin positioning. In female germline stem cells (GSCs), Stwl binds and positions chromatin loci, including GSC differentiation genes, at the nuclear periphery. Strikingly, Stwl-dependent perinuclear positioning is associated with transcriptional repression, highlighting a likely mechanism for Stwl’s known role in GSC maintenance and ovary homeostasis. Thus, our study identifies perinuclear anchors in Drosophila and demonstrates the importance of gene repression at the nuclear periphery for cell fate.

overall_design:
To investigate the role of Stonewall on gene expression in GSCs, we enriched for GSC-like cells using genetic tools.
We overexpressed Stwl in GSC-like cells and performed differential gene expression analysis using four replicates for each condition
The two conditions include Control  (Control_Stwl_Replicate (1-4)) or Stonewall overexpression (Test_StwlEY_Replicate (1-4))

first: Ana

last: Sintosva

first: Ankita

last: Chavan


title: Bidirectional enhancer RNAs regulate different MYB promoters

first: Yucheng

last: Wang

address: Huchenghuan Rd

department: College of Fisheries and Life Science

institute: Shanghai Ocean University

summary:
MYB is a pivotal oncogenic driver in leukemia and is overexpressed through various mechanisms. Transcriptional regulation of MYB is complex with an alternative MYB promoter (TSS2) located in intron 1.

Here, we identified two bidirectional enhancer RNAs transcribed from the -34 kb enhancer of MYB. These two eRNAs both upregulate MYB transcription, and promote proliferation and migration in leukemia cells. To further elucidate how eRNAs regulate MYB expression, we analyzed MYB differential exon usage in RNASeq data with the DEXSeq package after overexpression of eRNAs in K562 cells. We found that bidirectional enhancer RNAs regulate different MYB promoters.

Transcription initiating from TSS2 produces N-terminally truncated MYB protein lacking the first 20 amino acids. To analyze the genes and pathways affected by both MYB isoforms, RNA-seq was performed after MYB or N-terminally truncated MYB was overexpressed in K562 cells. We found that N-terminally truncated MYB exhibits different activity in K562 cells, where it not only activates different primary targets, but also different downstream pathways altogether.

overall_design:
The MYB 34kb upstream enhancer encodes two eRNAs. To investigate the function two eRNAs in the regulation of differential exon usage, we overexpressed two eRNAs in K562 cells, respectively. We then performed DEXSeq analysis using data obtained from RNA-seq.

To investigate the genes affected by both MYB isoforms, we overexpressed MYB or N-terminally truncated MYB in K562 cells. Comparative gene expression profiling analysis of RNA-seq data for K562 overexpressing two MYB isoforms, respectively.

first: Xiang

first: Mengjia

first: Junfang

first: Bingshe

last: Han


title: Comparison of skin spatial transciptomics before and after IL-1-targeted biological treatment in Pityriasis rubra pilaris

city: Cambridge

first: Eloi

last: Schmauch

state: Massachusetts

address: 32 Vassar St

department: Computer Science and Artificial Intelligence Laboratory

institute: Massachusetts Institute of Technology

summary: Pityriasis rubra pilaris (PRP) is a rare inflammatory skin disease with a poorly understood pathogenesis. Through a molecularly-driven precision medicine approach and an extensive mechanistic pathway analysis we identified IL-1β as a key mediator, orchestrating an NF-κB-mediated IL-1β-CCL20 axis. Treatment with the IL-1-antagonists anakinra and canakinumab in patients resulted in rapid clinical improvement. Here we show by spatial profiling that in skin samples after treatment the molecular signature of the PRP was reversed: positive DEGs before treatment showed significant negative enrichment after treatment; the same accounts for GSEA analysis of selected pathways of interest, such as TNF-alpha signaling via NF-kB; predicted activation scores of IPA annotations for disease, pathways, and upstream regulator exhibited a significant negative correlation between active PRP and after treatment; and finally IPA mechanistic network analysis showed that gene regulation arising from IL1B was reversed with treatment.

overall_design: Skin biopsies from active PRP lesions were taken from two patients before and at week 8 of anakinra treatment. The biopsies were processed using the GeoMx Human Whole Transcriptome Atlas (WTA). This technique utilizes mRNA-specific in situ hybridization probes linked to distinct DNA barcodes. Upon selection of a "region of interest" (ROI), these barcodes are detached using UV cleavage. Subsequently, the extracted RNA undergoes sequencing on an Illumina system, generating a transcriptome for each designated ROI. Specific ROIs targeted were the epidermis (Pan-CK), CD3+ and CD68+ cells, but due to low overall counts in ROIs, the samples were all pooled together, to increase statistical power. This resulted in 24 technical replicates for patient 1 (13 pre-treatment, 11 during treatment) and 26 for patient 2 (13 pre-treatment, 13 during treatment).

first: Antonios

last: Kolios


title: Pathways for macrophage uptake of cell-free circular RNAs

city: Stanford

first: Laura

last: Amaya

address: 269 Campus Drive

institute: Stanford University

summary: Circular RNAs (CircRNAs) are stable RNAs present in cell-free RNA, comprising cellular debris and pathogen genomes. As a prerequisite for the development of efficient circRNA-based therapies, we investigated the phenomenon and mechanism of cellular uptake and intracellular fate of circRNAs. Human myeloid cells and B cells selectively internalize extracellular circRNAs. Macrophage uptake of circRNA is rapid, energy-dependent, and saturable. CircRNA uptake can lead to translation of encoded sequences and antigen presentation. The route of internalization influences immune activation after circRNA uptake, with distinct gene expression programs depending on the mechanism involved. Genome-scale CRISPR screens and chemical inhibitor studies nominate macrophage scavenger receptor MSR1, toll-like receptors, and mTOR signaling as key regulators of receptor-mediated phagocytosis of circRNAs, a dominant pathway to internalize circRNAs in parallel to macropinocytosis. These results suggest that cell-free circRNA serves as an "eat me" signal and a danger-associated molecular pattern, indicating orderly pathways of recognition and disposal.

overall_design:
To investigate the mechanisms of naked circRNA uptake vs. lipid-mediated transfection, we compared the transcriptome profile of cells after 24 hrs of these two conditions by RNA-seq, in RAW264 cells and MutuDC cells. We also compared uptake of circRNA and ntranslatable circRNA, referered as circSTOP in J774 cells.
We deployed a pooled CRISPR screening approach to systematically investigate the essential requirements for circRNA uptake in J774 cells, comparing bound and unbound cells when incubated with IONP-circRNA, IONP-mRNA and IONP-only control

first: Howard

last: Chang


title: The SAGA acetyltransferase module is required for the maintenance of MAF and MYC oncogenic gene expression programs in multiple myeloma [RNA-seq]

city: Houston

first: Yue

last: Lu

state: TX

address: 1881 East Road

department: Epigenetics & Molecular Carcinogenesis

institute: The University of Texas MD Anderson Cancer Center

summary: Despite recent advances in therapeutic treatments, multiple myeloma (MM) remains an incurable malignancy. Epigenetic factors contribute to the initiation, progression, relapse, and clonal heterogeneity in MM, but our knowledge on epigenetic mechanisms underlying MM development is far from complete. The SAGA complex serves as a coactivator in transcription and catalyzes acetylation and deubiquitylation. Analyses of datasets in the Cancer Dependency Map Project revealed many SAGA components are selective dependencies in MM. To define SAGA-specific functions, we focused on ADA2B, the only subunit in the lysine acetyltransferase (KAT) module that specifically functions in SAGA. Integration of RNA-seq, ATAC-seq, and CUT&RUN results identified pathways directly regulated by ADA2B include MTORC1 signaling, MYC, E2F, and MM-specific MAF oncogenic programs. We discovered that ADA2B is recruited to MAF and MYC gene targets, and that MAF shares a majority of its targets with MYC in MM cells. Furthermore, we found the SANT domain of ADA2B is required for interaction with both GCN5 and PCAF acetyltransferases, incorporation into SAGA, and ADA2B protein stability. Our findings uncover previously unknown SAGA KAT module-dependent mechanisms controlling MM cell growth, revealing a vulnerability that might be exploited for future development of MM therapy.

overall_design: Total RNA sequencing was performed on MM.1S cells harboring either shADA2B or shNT treated with Doxycycline for 3 or 5 days

first: Ying-Jiun

last: Chen

first: Bhaskara

last: Govinal Badiger

first: Kevin

last: Lin

first: Sharon

last: Dent


title: The SAGA acetyltransferase module is required for the maintenance of MAF and MYC oncogenic gene expression programs in multiple myeloma [ATAC-seq]

overall_design: ATAC-sequencing was performed on MM.1S cells harboring either shADA2B or shNT treated with Doxycycline for 3 or 5 days


title: The SAGA acetyltransferase module is required for the maintenance of MAF and MYC oncogenic gene expression programs in multiple myeloma [CUT&RUN]

overall_design: CUT&RUN was performed on MM.1S cells harboring either FLAG-3xHA-ADA2B or FLAG-3xHA using HA, H3K9ac, or IgG antibodies.


title: ACSS2 and ACLY link nutrient-dependent chromatin accessibility to regulation of CD8 T cell effector responses

city: Grand Rapids

first: Russell

last: Jones

state: Michigan

address: 333 Bostwick Ave. NE

institute: Van Andel Institute

summary: Coordination of cellular metabolism is essential for optimal T cell responses. Here, we identify cytosolic acetyl-CoA production as an essential metabolic node for CD8 T cell function in vivo. We show that acetyl-CoA derived from mitochondrial citrate via the enzyme ATP citrate lyase (Acly) is required for CD8 T cell responses to infection. However, ablation of Acly triggers an alternative, acetate-dependent pathway for acetyl-CoA production in T cells mediated by acyl-CoA synthetase short chain family member 2 (Acss2). Mechanistically, acetate fuels both the TCA cycle and cytosolic acetyl-CoA production, impacting T cell effector responses, acetate-dependent histone acetylation, and effector gene expression by altering chromatin accessibility. When Acly is functional, Acss2 is not required, suggesting acetate is not an obligate metabolic substrate for CD8 T cell function. However, deletion of Acly renders CD8 T cells dependent on acetate (via Acss2) to maintain acetyl-CoA production and effector function.  Thus, together Acly and Acss2 coordinate cytosolic acetyl-CoA production in CD8 T cells to maintain chromatin accessibility and T cell effector function.

overall_design: CD8 T cells isolated from wild type (Acly f/f Cd4Cre-) or Acly KO (Acly f/f Cd4Cre+) mice were activated in VIM media with plate bound CD3/CD28 and IL-2 for 48 hours in the presence or absence of Acss2 inhibitor or acetate. At 48 hours post activation ATAC-sequencing was performed.

first: McLane

last: Watson

first: Irem

last: Kaymak

first: Brandon

last: Oswald

first: Dominic

middle: G

last: Roy

first: Shixin

last: Ma

first: Benjamin

last: Johnson

first: Lisa

last: DeCamp

first: Batsirai

last: Mabvakure

first: Katarzyna

last: Luda

first: Eric

first: Kin

last: Lau

first: Zhen

last: Fu

first: Brejnev

last: Muhire

first: Susan

last: Kitchen-Goosen

first: Alexandra

last: VanderArk

first: Michael

last: Dahabieh

first: Bozena

last: Samborska

first: Matthew

last: Vos

first: Hui

last: Shen

first: Zi

last: Fan

first: Thomas

last: Roddy

first: Gillian

middle: A

last: Kingsbury

first: Cristovão

last: Sousa

first: Connie

last: Krawczyk

first: Kelsey

last: Williams

first: Ryan

last: Sheldon

last: Kaech


title: Perinatal Brain Injury Triggers Niche-Specific Changes to Cellular Biogeography [MERFISH]

city: Boston

first: Brian

last: Kalish

address: 220 Longwood Ave

department: Neurobiology

institute: Boston Children's Hospital

summary: Preterm infants are at risk for brain injury and long term neurodevelopmental impairment due, in part, to white matter injury following chronic hypoxia exposure. However, the precise molecular mechanisms by which perinatal hypoxia disrupts early neurodevelopment are poorly understood. Here, we constructed a brain-wide map of the regenerative response to newborn brain injury using high resolution imaging-based spatial transcriptomics (MERFISH) to analyze over 1.3 million cells in a mouse model of chronic neonatal hypoxia. We also developed a new method for inferring condition-associated differences in cell type spatial proximity, enabling the identification of niche-specific changes in cellular architecture. We observed significant hypoxia-associated changes in region-specific cell states, cell type composition, and spatial organization. Our findings suggest that perinatal hypoxia disrupts oligodendrocyte formation and crosstalk signaling with other cell types in their niche. Importantly, our analysis of spatially-informed gene expression patterns revealed specific mechanisms of reparative neurogenesis and gliogenesis, and nominated pathways that may impede circuit rewiring following perinatal hypoxia. Altogether, our work provides a comprehensive description of the brain-wide response to newborn brain injury and identifies candidate signaling pathways for functional interrogation.

overall_design: Multiplexed error-robust fluorescence in situ hybridization (MERFISH) was performed on postnatal day 21 (P21) mice from two conditions: 1) mice that were exposed to 21% oxygen during development (normoxia), 2) mice that were exposed to 10% oxygen (hypoxia) between P3-P11. Coronal sections of the entire brain were used for analysis.

first: Nareh

last: Tahmasian

first: Min

last: Feng

first: Keon

last: Arbabi

first: Bianca

last: Rusu

first: Wuxinhao

last: Cao

first: Bharti

last: Kukreja

first: Shreejoy

last: Tripathy


title: Combination of Eribulin and Anlotinib Exerts Synergistic Cytotoxicity in Retroperitoneal Liposarcoma by Inducing Endoplasmic Reticulum Stress

city: Beijing

first: Jianhui

last: Wu

address: No.52 Fucheng Road, Haidian District

department: Department of Hepato-Pancreato-Biliary Surgery

institute: Peking University Cancer Hospital & Institute

summary: Primary retroperitoneal liposarcoma (RLPS) is a rare heterogeneous tumor occurring within retroperitoneal space, and its overall survival did not improve much in the past few decades. Based on a small-sample clinical practice at our center, patients with RLPS can greatly benefit from combination of anlotinib and eribulin. In this study, we investigated combinational effect of anlotinib and eribulin on RLPS. Through transcriptome analysis, we uncovered and validated that synergistic effect mainly induced by endoplasmic reticulum stress (ERS) pathway both in vitro and in vivo.

overall_design: To elucidate the underlying mechanisms by which combination of anlotinib and eribulin inhibited RLPS cells, RNA-seq was conducted to evaluate transcriptome changes in RLPS cells after being treated with optimal concentration which could elicit maximum synergy effect. 93T449, 94T778 and sw872 cell lines were used in this study.

first: Shuquan

last: Li

first: Hao

last: Yu

first: Yifan

first: Xiuyun

last: Tian

first: Chunyi

last: Hao


title: Transcriptome-wide mapping of RNA:protein interactions of Arid5a in mouse P19 cells.

city: Frankfurt am Main

first: Michaela

last: Müller-McNicoll

address: Max-von-Laue-Str. 13

institute: Goethe University Frankfurt

summary: We performed Arid5a iCLIP2 in P19 cells transiently expressing GFP-tagged Arid5a.

overall_design: 3 biological replicates were subjected to the iCLIP2 procedure with UV crosslinking at 254 nm. For immunoprecipitation, an anti-GFP antibody was used. No UV treatment served as negative control.

first: Anke

last: Busch


title: Metabolic effects of cesarean section induced gut microbial dysbiosis in high-fat diet induced obese mice

city: Frederiksberg C

first: Camilla

middle: Hartmann Friis

last: Hansen

address: Ridebanevej 9, 2nd floor

department: Faculty of Health Sciences

institute: University of Copenhagen

summary: The aim of this study was to investigate the causative effect of CS induced dysbiosis on obesity and insulin resistance in a high-fat diet induced obese (DIO) mouse model. Male germ-free BALB/c mice were humanized by fecal microbiota transfer using samples from children born by CS or VD and fed HFD for 16 weeks. Adipose tissue was sampled for RNAseq at study termination.

overall_design: Male mice were either inoculated with GM from vaginally born infants (HM_VD) or cesarian section born infants (HM_CS) anf then subjectesd to 16 week HFD feeding

first: Caroline

last: Mentzel


title: Alloreactive memory CD4 T cells promote transplant rejection by engaging DCs to induce innate inflammation and CD8 T cell priming

city: Cincinnati

first: Irene

last: Saha

state: Ohio

address: 3333 Burnet Avenue

department: Immunobiology

institute: Cincinnati Children's Hospital and Medical center

summary:
Alloreactive memory t cells have been implicated as central drivers of transplant rejection. In our study we find that alloreactive memory T cells can interact with and engage Dendritic cells(DCs)
Alloantigen-specific memory T cells were generated in vitro by co-culturing naïve CD4T cells from BALB/c mice with splenic DCs derived from B6 mice( by negatively sorted to remove T,B,NK and macrophages) for 5 days and then rested with IL2 for 2 days. Syngeneic memory T cells were similarly derived from B6 mice and polarized in vitro. Alloreactive or syngeneic memory T cells were then co-cultured for  3 hours with B6 splenic DCs. CD11b+CD11c+ cells were sorted away from T cells  after 3 hours and lysed for RNA seq analysis.

overall_design: mRNA profiles of B6 splenic DCs stimulated with syngeneic or allogeneic effector memory T cells for 3 hours by Illumina sequencing in triplicate.

first: Chandrashekhar

last: Pasare

first: Amanpreet

last: Singh Chawla

first: Ana

last: Paula B N Oliviera

first: Eileen

middle: E

last: Elfers

first: Kathrynne

last: Warrick

first: Hannah

last: Meibers

first: Thomas

last: Hagan

first: Jonathan

last: Katz

first: Viral

last: Jain


title: Biomechanical basis of intramyocardial hydrogel injection treatment for ischemic cardiomyopathy [snRNA-seq]

first: Li

last: Shen

state: Zhejiang Province

address: 866 Yuhangtang Road

institute: Life Sciences Institute, Zhejiang University

summary: Adverse mechanical cues promote pathological remodeling of ischemic left ventricle, which leads to heart failure and mortality in ischemic cardiomyopathy (ICM) patients. Intramyocardial injection of hydrogels reduces the mechanical stress in left ventricular (LV) wall, thus preserves cardiac function and geometry. The therapeutic concept has been tested in clinical trials and presented exciting potential. However, the theory of intramyocardial hydrogel injection still needs to be improved as the molecular biological connection between the mechanical effect and therapeutic outcomes is weak, and it is difficult to clearly separate the contribution of hydrogel mechanical support from physiological reaction to hydrogel chemistry and combinational surgical procedures in previous clinical translation studies. In this study, we transendocardially injected alginate hydrogel via a percutaneous coronary intervention in 10 ICM patients with end-stage heart failure. Thirty days after injection, cardiac function was improved, and LV size was reduced. Stress in the myocardium decreased as evaluated by finite element analysis. Similar mechanical and functional effects by hydrogel injections were observed in rat myocardial infarction (MI) model. Bulk and single-cell RNA sequencing revealed restoration in transcription levels of mechanosensing, cardiac contraction genes and other important pathways, which was mainly attributed to changes in cardiomyocytes. Such effects were not observed in rats receiving diluted hydrogel, confirming that mechanical effect instead of material chemistry is the main contributor. The resemblance in transcriptomic profile and the effect of hydrogel injection on mechanical stress and remodeling of LV wall between ICM patients and diseased rats indicated the applicability of the major mechanistic conclusions in rat model to human.

overall_design: The rats were subjected to left anterior descending (LAD) coronary artery ligation (MI) along with Sham controls (Sham), and soft hydrogel (MI-soft) and stiff hydrogel (MI-stiff) treatment respectively, followed by heart tissue collection 28 days post MI to mimic the MI/R injury in AMI patients.The heart infarct zones (MI, MI-soft, MI-stiff and Sham) were isolated to single-nucleus transcriptome analysis. Collected nuclei for each sample were loaded onto 10X Chrominum platform. We obtained 69,608 cells and 20,748 genes. Five major cell types were identified, which included cardiomyocytes (CMs), endothelial cells (ECs), fibroblasts (FBs), macrophages (MACs) and smooth muscle cells (SMCs).

first: Yunwen

last: Lu

first: Fei

last: Huang

first: Yun

last: Gao


title: Biomechanical basis of intramyocardial hydrogel injection treatment for ischemic cardiomyopathy

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

overall_design: Refer to individual Series


title: BRAF V600E in Pediatric Low-Grade Gliomas: Exploring the Tumor Microenvironment and Immune Cell Infiltration dynamics in vivo.

city: Columbus

first: Alessandro

last: Canella

state: OH

address: 700 Children’s Drive

department: Institute for Genomic Medicine

institute: Nationwide Children's Hospital

summary: We established a RCAS-BRAF V600E murine model and we analyzed the tumor infiltrating immune cells at the late stage of the tumor progression.

overall_design: Immune cells from the TME were isolated at day 3 post-treatment, and processed for Total RNA extraction and Total RNA sequencing.

first: Matthew

last: Nazzaro

first: Lakshmi

middle: P

last: Venkata

first: Diana

last: Thomas

first: Justin

last: Lyberger

first: Katherine

last: Miller

first: Gregory

last: Behbehani

first: Claudia

middle: K

last: Petritsch

first: Prajwal

last: Rajappa


title: Biomechanical basis of intramyocardial hydrogel injection treatment for ischemic cardiomyopathy [bulk RNA-seq]

overall_design: The rats were subjected to left anterior descending (LAD) coronary artery ligation (MI) along with Sham controls (Sham), and soft hydrogel (MI-soft) and stiff hydrogel (MI-stiff) treatment respectively. Each group contains four replicates.


title: The unique proteasomal processing of viral ORF1 drives HEV-induced liver fibrosis

summary: Hepatitis E virus (HEV) is a globally prevalent pathogen that causes 20 million infections and 60,000 fatalities annually, endangering particularly pregnant women and immunosuppressed individuals. Liver cirrhosis, which results from advanced fibrosis, is the primary symptom and leading mortality cause in chronic hepatitis E patients. However, the causation and process of liver fibrosis triggered by chronic HEV infection remain poorly understood. Here, we unexpectedly discovered that the viral multiple-domain replicase (ORF1) undergoes unique ubiquitin-proteasomal processing in HEV replicon hepatocytes, HEV-infected gerbil livers, and HEV-infected patient livers, which follows a CHIP-mediated K48 ubiquitination and produces the HEV-Derived Smad Activator (HDSA). Lacking putative helicase and RNA polymerase domains, this enriched viral polypeptide in hepatocytes and gerbil livers is non-HSP90-bound, stable, and exhibits exclusively nuclear localization. Surprisingly, HDSA markedly potentiates the fibrogenic TGF-β/Smad pathway in livers by facilitating promoter binding and coactivator recruitment of SMAD3, leading to profound liver fibrotic symptoms and damage. Thus, we have identified the first viral protein derived from the unique proteasomal processing of the host, defined its notable role in liver fibrosis, and highlighted the nature of complex host-HEV interactions that drives HEV pathogenesis.

overall_design: Bulk RNA sequencing performed with human HEK293 cell line treated with Vector-SB, Vector-TGFb and HDSA-TGFb respectively.

last: Zhang

first: Yinghao

last: Pan


title: RNA-Seq of human cutaneous squamous cell carcinoma cell lines and normal human epidermal keratinocytes.

city: Turku

first: Liisa

last: Nissinen

address: Medisiina D, 5th floor; Kiinamyllynkatu 10

department: Dermatology

country: Finland

institute: University of Turku

summary: The incidence of keratinocyte-derived skin cancer, cutaneous squamous cell carcinoma (cSCC) is increasing worldwide making it the second most common metastatic skin cancer. In this project we used total RNA sequencing to characterize gene expression profiles in normal human epidermal keratinocytes (NHEKs) and cSCC cell lines.

overall_design: Total RNAs from negative NHEKs (n=4) and cutaneous UT-SCC cell lines (n=8) were extracted. The samples were sequenced using Illumina sequencing.

first: Veli-Matti

last: Kähäri


title: Transcriptomic analysis reveals regulation of adipogenesis via long non-coding RNA, alternative splicing, and alternative polyadenylation

first: Claire

last: Moore

address: 150 Harrison Ave

institute: Tufts University

summary: Obesity is characterized by dysregulated adipogenesis leading to increased number and/or size of adipocytes. Understanding the molecular mechanisms governing regulation of adipogenesis is therefore key to designing therapeutic interventions against obesity. In our study, we analyzed 3’-end sequencing data generated from sequencing of human preadipocytes and adipocytes, as well as data available in publicly available databases, to propose mechanisms of molecular regulation of adipogenesis. We discovered lncRNAs that have not been previously characterized but may be key regulators of both brown and white adipogenesis. We also demonstrated possible mechanisms of direct or indirect regulation of adipogenesis through alternative splicing. Finally, we show that usage of alternative polyadenylation sites of key adipogenesis genes leads to isoform diversity, which can have significant biological consequences on differentiation efficiency. Therefore, our research reveals potential therapeutic avenues for obesity through manipulation of long non-coding RNA levels, alternative splicing as well as the usage of alternative polyadenylation sites.

overall_design: To investigate changes in gene expression and alternative polyadenylation during white adipogenesis of primary human preadipocytes (Day 0) to mature adipocytes (Day 14).

first: Salwa

middle: M

last: Mostafa

first: Luyang

last: Wang