title: Regulation of succinate signaling and immunometabolism in diabetes-associated atherosclerosis by miR-369-3p

first: Mark

middle: W

last: Feinberg

address: 77 Avenue Louis Pasteur

department: Medicine

institute: Brigham and Women's Hospital

summary: Atherosclerosis is accelerated under diabetic conditions in part due to altered macrophage metabolism and function. Identifying critical factors to restore metabolic alterations and promote resolution of inflammation remains an unmet goal. MicroRNAs (miRs) orchestrate multiple signaling events in macrophages and regulate inflammation, yet their therapeutic potential in diabetes-associated atherosclerosis remains poorly understood. miRNA profiling of aortic intimal lesions from Ldlr-/- mice on a high-fat sucrose containing (HFSC) diet for 12 weeks revealed low expression of miR-369-3p. miR-369-3p was also reduced in peripheral blood mononuclear cells (PBMCs) from diabetic patients with coronary artery disease. Cell-type expression profiling showed miR-369-3p enrichment in aortic macrophages. Treatment with oxLDL reduced miR-369-3p expression in-stimulation mouse bone marrow-derived macrophages (BMDMs) downregulated miR-369-3p. Overexpression of miR-369-3p restored the oxLDL-mediated decrease of OXPHOS in BMDMs. Mechanistically, we found that miR-369-3p targeted the succinate receptor (GPR91) to alleviate the oxLDL-induced activation of inflammasome signaling pathways. Therapeutic administration of miR-369-3p in HFSC-fed Ldlr-/- mice reduced GPR91 expression in lesional macrophages and the diabetes-mediated accelerated atherosclerosis, evident by a decrease in plaque size and in the accumulation of pro-inflammatory Ly6Chi macrophages. RNA-seq analyses showed more pro-resolving pathways in plaque macrophages from miR-369-3p treated mice, consistent with an increase in macrophage efferocytosis in lesions. These findings establish a therapeutic role for miR-369-3p in halting diabetes-associated atherosclerosis by regulating GPR91 and macrophage succinate metabolism.

overall_design: To investigate the effects of miR-369-3p mimic in atherosclerotic plaques,  Ldlr-/- mice were placed on high fat high sucrose containing diet (HFSC) for 8 weeks to induce atherosclerosis after which the mice were randomized into two treatment groups : Non-specific mimic (NS)control  and miR-369-3p mimic (369-m). Mice received 2 intravenous injections of 2 nmole NS or 369-m every week for a total of four weeks with the continuation of HFSC diet. At the time of sacrificing, aortas were isolated and aortic plaque macrophages were FACs-sorted. We then performed low input RNA-seq for gene expression analyses in FACs-sorted macrophages from plaques of NS and 369-m injected mice.

first: Shruti

last: Rawal

first: Vinay

last: Randhawa

first: Syed

middle: H

last: Rizvi

first: Madhur

last: Sachan

first: Akm

last: Wara

first: Daniel

last: Pérez-Cremades

first: Robert

last: Weisbrod

first: Naomi

last: Hamburg


title: Amentoflavone induces cell autophagy through promoting phosphorylation of ULK1 in hepatocellular carcinoma

status: Public on Dec 04 2024

city: China Shenzhen

first: Yang

last: Wang

address: Sun Yat-Sen University Shenzhen Campus

country: China

institute: Sun Yat-Sen University

type: Expression profiling by high throughput sequencing

summary:
Purpose: To date, the biological activity of AMF has not been fully investigated. We set out to analyze how AMF regulates gene expression in HepG2 cells.
Conclusions: we performed clustering analysis on the various expressed genes associated with autophagy, and the pathways confirmed in the KEGG and BP analysis

overall_design: Methods: RNA-seq was performed with two repetitions in HepG2 followed by treatment with DMSO and 80 μM Tub B for 48 h.

first: Chenyan

last: Shi


title: Early response to cyclic stretch in human intestinal smooth muscle activates transition to a synthetic, proinflammatory phenotype hypothesized to affect gene expression in nearby cells in the bowel wall

city: Philadelphia

first: Katherine

last: Beigel

state: PA

address: 2716 South Street

department: Department of Biomedical and Health Informatics

country: USA

institute: Children's Hospital of Philadelphia

summary: Background and Aims: Bowel smooth muscle experience mechanical stress constantly during normal function, and unusual mechanical stressors in various disease settings. Here, we test the hypothesis that pathologic mechanical stress could alter transcription to induce smooth muscle phenotypic class switching. Methods: Primary human intestinal smooth muscle cells (HISMCs), seeded on electrospun aligned poly-ε-caprolactone nano-fibrous scaffolds, were subjected to uniaxial 3% cyclic stretch at 1 Hz (loaded) or kept unloaded in culture for 6 hours. Bulk total RNA sequencing, RT-qPCR, and quantitative immunohistochemistry defined loading-induced changes in gene expression. NicheNet predicted how differentially expressed genes might impact HISMCs and other bowel cells. Results: Loading induced differential expression of 2500 genes in HISMCs. Loaded HISMCs had a less contractile phenotype, with increased expression of synthetic SMC genes, proinflammatory cytokines, and altered expression of axon guidance molecules, growth factors and morphogens. Many differentially expressed genes encode secreted ligands that could act cell-autonomously on smooth muscle, but also on other cells in the bowel wall. Discussion: HISMCs demonstrate remarkably rapid phenotypic plasticity in response to mechanical stress that may convert contractile HISMC into proliferative fibroblast-like cells or proinflammatory cells. These mechanical stress-induced changes in HISMC gene expression may be relevant for human bowel disease.

overall_design: To test the hypothesis that mechanical stress could rapidly alter gene expression in visceral SMCs and to gain insight into early changes in SMC phenotype events, we decided to evaluate gene expression in cultured human intestinal smooth muscle cells (HISMCs) after only 6 hours with or without cyclic stretching. Compared to most prior studies, we used a very mild mechanical stress (3%, 1 Hz), which rapidly altered the expression of 2500 genes (genes with log2 fold change > 0.48 or < -0.48, out of 4537 genes with adjusted p-value < 0.05)mRNAin loaded cultured human intestinal smooth muscle compared to unloaded. Compared to unstretched HISMCs, stretched cells had increased expression of genes typically produced in synthetic phenotype SMCs, increased production of many cytokines, chemokines, cytokine receptors, axon guidance molecules, junctional proteins, and altered levels of many signaling molecules predicted to act on nearby cells in the bowel. Cyclic stretching at this frequency also enhanced expression of many genes known to be preferentially expressed in vascular compared to visceral SMCs. Collectively, these data suggest that bowel SMC phenotype, in part, depends on the unique physical forces these cells experience as nutrients move through the bowel, as waste is eliminated, and in response to various types of bowel injury or disease.

first: Sharon

last: Wolfson

first: Sierra

last: Anderson

first: Molly

last: Weiner

first: Deanne

middle: M

last: Taylor

first: Su-Jin

last: Heo

middle: O

last: Heuckeroth

first: Sohaib

middle: K

last: Hashmi


title: CRIPTO’s multifaceted role in driving aggressive prostate cancer unveiled by in vivo, organoid,  and patient data

city: Bern

first: Panagiotis

last: Chouvardas

address: Murtenstrasse 24

country: Switzerland

institute: University of Bern

summary: CRIPTO, (or CR-1 or TDGF1), is a protein that plays an active role in tumor initiation and progression. We have confirmed that increased expression of CRIPTO is associated with clinical and prostate-specific antigen (PSA) progression in human prostate tissues. To further study CRIPTO signaling in vivo, we developed new genetically engineered mouse models (GEMMs) in which we genetically knocked out CRIPTO to specifically investigate its role in castration-resistant Nkx3.1 expressing cells (CARNs), which are targeted for oncogenic transformation in prostate cancer (PCa). The most aggressive stage was modeled using an inducible Cre driver, Pten inactivation, oncogenic Kras activation, and lineage tracing with yellow fluorescence protein (E-YFP). Our findings provide evidence that selective depletion of CRIPTO in epithelial cells in vivo reduces the invasive phenotype, particularly in more advanced tumor stages. Moreover, in vitro experiments with CRIPTO overexpression demonstrated alterations in the physical features of organoids, which correlated with increased tumorigenic activity. Transcriptomic analyses revealed a unique CRIPTO/MYC co-activation signature which was further associated with PSA progression in a human PCa cohort.

overall_design: Transcriptomic analysis of organoids derived from prostates of genetically engineered mice (N, NC, NP, NPC, NPK, NPKC) and transcriptomic analysis of tissues of NPKC mice treated with Alk4Fc.

first: Elisa

middle: R

last: Sousa

first: Simone

last: De Brot

first: Eugenio

last: Zoni

first: Mario

last: Scarpa

first: Andrea

last: Brunello

first: Soheila

last: Zeinali

first: Federico

last: La Manna

first: Allen

middle: A

last: Alexander

first: Irena

last: Klima

first: David

last: Freeman

last: Cristaldi

first: Olivier

middle: T

last: Guenat

first: Boudwejin

last: Kruithof

first: Benjamin

last: Spike

first: Marianna

last: Kruithof-de Julio


title: Distinct use of super-enhancer elements controls cell type-specific CD25 transcription and function

status: Public on Dec 03 2024

city: Bethesda

first: Peng

last: Li

state: MD

address: 9000 Rockville Pike

department: NHLBI

institute: NIH

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The IL-2 receptor α chain (IL-2Rα/CD25) is constitutively expressed on double-negative (DN2/DN3 thymocytes and regulatory T cells (Tregs) but induced by IL-2 on T and natural killer (NK) cells, with Il2ra expression regulated by a STAT5-dependent super-enhancer. We investigated CD25 regulation and function using a series of mice with deletions spanning STAT5-binding elements. Deleting the upstream super-enhancer region mainly affected constitutive CD25 expression on DN2/DN3 thymocytes and Tregs, with these mice developing autoimmune alopecia, whereas deleting an intronic region decreased IL-2-induced CD25 on peripheral T and NK cells. Thus, distinct super-enhancer elements preferentially control constitutive versus inducible expression in a cell type-specific manner. The mediator-1 coactivator colocalized with specific STAT5-binding sites. Moreover, both upstream and intronic regions had extensive chromatin interactions, and deletion of either region altered the super-enhancer structure in mature T cells. These results demonstrate differential functions for distinct super-enhancer elements, thereby indicating previously unknown ways to manipulate CD25 expression in a cell type-specific fashion.

overall_design: ChIP-Seq analysis using various immune cells from wild-type and specific CRISPR-deletion mutants to investigate the Il2ra super-enhancer and mechanisms by which cell-type specific or developmental specific expression of this gene is controlled. HiChIP experiments using CD8+ T cells were performed to reveal H3K27Ac-mediated chromatin interactions among the Il2ra promoter in wile-type and mutants.

first: Rosanne

last: Spolski

first: Vivek

last: Chandra

first: Boyoung

last: Shin

first: Shubham

last: Goel

first: Keiko

last: Sakamoto

first: Chengyu

last: Liu

first: Jangsuk

last: Oh

first: Min

last: Ren

first: Yutaka

last: Enomoto

first: Erin

middle: E

last: West

first: Stephen

last: Christensen

first: Edwin

middle: C

last: Wan

first: Meili

last: Ge

first: Jian-Xin

last: Lin

first: Bingyu

last: Yan

first: Majid

last: Kazemian

first: Zu-Xi

last: Yu

first: Keisuke

last: Nagao

first: Pandurangan

last: Vijayanand

first: Ellen

middle: V

last: Rothenberg

first: Warren

middle: J

last: Leonard


title: Reversibility of long-term changes to pulmonary function post early life respiratory viral infection

status: Public on Dec 05 2024

city: Crawley, Perth

first: Emma

last: de Jong

state: WA

address: 35 Stirling Hwy

country: Australia

institute: University of Western Australia

summary: Respiratory viral infections in early life are associated with the development of lung diseases that can persist into adulthood, but the underlying mechanisms are not well understood.We aimed to characterise virus-induced changes to pulmonary gene networks that are linked to impairement of lung development and function. Male and female BALB/c mice were inoculated at seven days of life with either Influenza A/Puerto Rico/8/34 (H1N1), Influenza A/Memphis/1/71 (H3N2) or phosphate buffered saline (PBS), and randomly sacrificed seven, fourteen and twenty-eight days post inoculation. Bulk RNAseq was performed on whole lung tissue for each sex, experimental group and timepoint.

overall_design: 144 samples in total: 8 samples per sex, per experimental group, per timepoint. 72 samples for males, 72 samples for females. 48 samples for seven days post-inoculation, 48 samples for fourteen days post-inoculation, 48 samples for twenty-eight days post-inoculation. 48 samples for Influenza A/Puerto Rico/8/34, 48 samples for Influenza A/Memphis/1/71, 48 samples for phosphate-buffered saline.

first: Laith

last: Harb

first: Patrick

last: Holt

first: Deborah

last: Strickland

first: Anya

last: Jones

first: Michael

last: Serralha

first: David

last: Martino

first: Alexander

last: Larcombe

first: Anthony

last: Bosco


title: The tumor associated antigen PRAME exhibits dualistic functions that are targetable in diffuse large B cell lymphoma

city: Vancouver BC

first: Christian

last: Steidl

state: British Columbia

address: 675, West 10th Ave

department: Department of Lymphoid Cancer Research

country: Canada

institute: BC Cancer

summary: PRAME is a prominent member of the cancer germline antigen family of proteins, which triggers autologous T-cell mediated immune responses. Integrative genomic analysis in diffuse large B-cell lymphoma (DLBCL) uncovered recurrent, and highly focal deletions of 22q11.22 including the PRAME gene, which were associated with poor outcome. PRAME-deleted tumors showed cytotoxic T-cell immune escape and were associated with cold tumor microenvironments. In addition, PRAME down-modulation was strongly associated with somatic EZH2 Y641 mutations in DLBCL, and PRC2-regulated genes were repressed in isogenic PRAME KO vs. PRAME wt lymphoma cell lines, with PRAME directly interacting with EZH2 as a negative regulator. EZH2 inhibition with EPZ-6438 abrogated these extrinsic and intrinsic effects leading to PRAME and microenvironment restoration in vivo. Our data highlight multiple functions of PRAME during lymphomagenesis, and provide a preclinical rationale for synergistic therapies combining epigenetic re-programming with PRAME-targeted therapies.

overall_design: Isogenic PRAME knock-out (KO) cell lines were created using CRISPR/Cas9 genome editing in DLBCL cell lines SU-DHL-4, Karpas-422 and HBL-1. RNAseq analysis was performed to derive PRAME KO-associated gene signatures.

first: Katsuyoshi

last: Takata

first: Daniel

last: Kwon

first: Lauren

last: Chong


title: Gamma entrainment using sensory stimuli alleviate chemo brain pathology and cognitive impairment induced by chemotherapy agents

city: Cambridge

first: Benjamin

last: James

state: MA

address: 32 Vassar St

institute: Massachusetts Institute of Technology

summary: We are using scRNA-seq to analyze how 40 Hz sensory stimulation can modulate chemo brain pathology in mouse hippocampus.

overall_design: Chemo brain model animals were generated with cisplatin injection and control groups received PBS injections. To test if Gamma ENtrainment Using Sensory stimuli (GENUS), which utilizes 40 Hz auditory pulse and visual LED flicker in this experiment, was given from the first day of cisplatin injection resulting in four experimental groups (PBS No Stim, PBS Stim, Cisplatin No Stim, Cisplatin Stim) . GENUS was given 1 hr every day for 21 days and animals were sacrificed and hippocampi were collected on the 22nd day. Replicate 1 (D20_6407~6410) contains two hippocampi from one animal per sample and replicate 2 and 3 contains four hippocampi from two animals.

first: TaeHyun

last: Kim

first: Li-Huei

last: Tsai


title: The effect of acute and chronic temperature stress in Baikal omul (Coregonus migratorius Georgi)

city: Irkutsk

first: Tuyana

last: Sidorova

address: 3, Ulan-Batorskaya

department: Siberian Branch of the Russian Academy of Sciences

country: Russia

institute: Limnological Institute

summary: In this work, we describe the transcriptional profiles of Baikal omul juveniles after acute and chronic temperature stress exposure. The juveniles were kept for 1.5 months at 9–12 °C, followed by exposure to acute stress (heating to 21 °C for 1 hour) and chronic stress (heating to 21 °C for 24 hours 3 times a week for a month) in the Experimental Freshwater Aquarium Complex for Baikal Hydrobionts at the Limnological Institute (LIN SB RAS). The information on the transcriptional profiles will contribute to further understanding of the mechanisms of adaptation of whitefish to the environment.

overall_design:
The experiment aims to study the effects of acute and chronic stress on aquaculture larvae of Baikal omul (Coregonus migratorius Georgi). The individuals were collected from ponds near the Belaya River (Belsky fish hatchery, Sosnovka village, Irkutsk region, 52 ° 50'05.4"N 103 ° 22'51.8"E). Individuals were kept for 1.5 months in aquaculture at a temperature of 9-12 ° C with subsequent heat treatment: acute stress implied rapid heating to 21 ° C for 1 hour, chronic stress implied heating to 21 ° C for 24 hours 3 times a week.

Grant ID No.: RSF 23-24-00644 ( https://rscf.ru/project/23-24-00644/ )
Grant title: Biomarkers of temperature stress and indicators of the well-being of whitefish in the natural environment and in aquaculture for the further use of these biomarkers for monitoring work.
Funding source: Russian Science Foundation

first: Anastasiya

middle: G

last: Koroleva

first: Evgenia

last: Vakhteeva

last: Epifantsev

first: Sergey

last: Potapov

first: Lyubov

last: Sukhanova

first: Olga

middle: Y

last: Glyzina

first: Yulia

middle: P

last: Sapozhnikova


title: Differential expression of genes associated with acute and chronic temperature stress in peled (Coregonus peled Gmelin)

summary: In this work, we describe the transcriptional profiles of peled juveniles after acute and chronic temperature stress exposure. The juveniles were kept for 1.5 months at 9–12 °C and then exposed to acute stress (heating to 21 °C for 1 hour) and chronic stress (heating to 21 °C for 24 hours 3 times a week for a month) in the Experimental Freshwater Aquarium Complex for Baikal Hydrobionts at the Limnological Institute (LIN SB RAS). The information on the transcriptional profiles will contribute to further understanding of the mechanisms of adaptation of whitefish to the environment.

overall_design:
The experiment aims to study the effects of acute and chronic stress on aquaculture larvae of peled (Coregonus peled Gmelin). The individuals were collected from ponds near the Belaya River (Belsky fish hatchery, Sosnovka village, Irkutsk region, 52 ° 50'05.4"N 103 ° 22'51.8"E). Individuals were kept for 1.5 months in aquaculture at a temperature of 9-12° C with subsequent heat treatment: acute stress meant rapid warming to 21 °C for 1 hour, chronic stress meant warming to 21 °C for 24 hours 3 times a week.

Grant ID No.: RSF 23-24-00644 ( https://rscf.ru/project/23-24-00644/ )
Grant title: Biomarkers of temperature stress and indicators of the well-being of whitefish in the natural environment and in aquaculture for the further use of these biomarkers for monitoring work.
Funding source: Russian Science Foundation


title: Comprehensive transcriptome analysis in human gallbladder

city: Shanghai

first: Shengli

last: Li

address: 650 Xinsongjiang Road

institute: Shanghai Jiao Tong University School of Medicine

type: Non-coding RNA profiling by high throughput sequencing

summary: The complexity of transcriptome in human gallbladder has not been clarified quite clearly so far. Here we collect 20 pairs of gallbladder samples.High-throughput RNA sequecing data was generated for each sample.We characterize the linear and circular transcripts.

overall_design: Illuminate the complexity of gallbladder transcriptome through high-throughput sequencing data.


title: Single-cell sequencing results of peritoneal macrophages from ANKRD22 knockout and control mice

city: Hangzhou

first: Tianhui

last: Pan

state: Zhejiang

address: jiefang Road 88

institute: Zhejiang University

summary: Silencing of Ankrd22 altered the distribution of macrophage subtypes in the tumor microenvironment and attenuated their pro-inflammatory activity

overall_design: Ankrd22 knockout mice were commissioned for construction by Cyagen Biosciences using CRISPR/Cas9 technology. The F4/80+ cell subset, isolated from mouse peritoneal cells through FACS sorting, corresponds to the population of mouse peritoneal macrophages. Single-cell suspension was added to the 10x Chromium chip according to the instructions for the 10x Genomics Single Cell 3 v3 Reagent Kit, with the expectation of capturing 8000 cells. cDNA amplification and library construction were performed according to standard protocols. Libraries were sequenced by LC-BioTechnology on an Illumina NovaSeq 6000 sequencing system at a minimum depth of 20,000 reads per cell.

first: Xiaoying

last: Wang

first: Keqing

last: Yang

first: Bin

first: Rui

first: Yongliang

last: Zhu


title: Spatially-resolved molecular sex differences at single cell resolution in the adult human hypothalamus

city: Baltimore

first: Keri

last: Martinowich

state: Maryland

address: 855 N Wolfe St Suite 300

institute: Lieber Institute for Brain Development

summary: The hypothalamus (HYP) underlies basal endocrine, physiological, and behavioral drives which inherently differ across species and sexes—ranging from growth and puberty to appetite and metabolism to social behavior. Especially in the rodent brain, these sex-differentiated functions correspond to differences in the volume, cell types, and gene expression of distinct HYP subareas. Recent studies have elucidated the transcriptional profile of single cells in the rodent ventromedial hypothalamus (VMH) and arcuate (ARC), which influence appetitive/social behaviors and growth/metabolism, respectively. However, there is a paucity of studies examining the molecular architecture of the adult human HYP, and transcriptional sex differences in the human VMH and ARC have not been characterized in single-cell or spatial contexts. Using 10x Genomics Visium and Xenium platforms, we generated a multimodal, spatially-resolved molecular atlas of human postmortem VMH and ARC from male and female adult donors. Sex-differential gene expression analysis within these regions revealed correlated autosomal expression differences, with stronger overall sex effects in the ARC. We then leveraged the single-cell-resolution of Xenium to localize sex-differential expression to cell types, finding most occur in ESR1- and KISS1-expressing neurons of the ARC. Finally, we investigated VMH and ARC gene expression in relation to genes associated with neuropsychiatric diseases, identifying a striking enrichment of genes upregulated in the male VMH for known and candidate risk genes for autism spectrum disorders. This work provides a multisample view of the human VMH and ARC in young/middle adulthood across both sexes, including data at both transcriptome-wide and single-cell resolutions, the first single-cell transcriptional atlas of molecular sex differences in the adult human VMH and ARC, and highlights potential sex-differentiated roles for VMH and ARC in neurobehavioral disorders.

overall_design: We obtained tissue blocks of postmortem human brain containing hypothalamus from 4 male and 4 female neurotypical adult brain donors. We used the 10x Genomics Visium and Xenium platforms to generate spatial transcriptomics of the tuberal (ventromedial portion) of the hypothalamus to profile VMH and ARC in particular. Genes selected for profiling with Xenium included the 10x human brain 'base panel' and 100 custom-selected genes based on marker, sex DE, and spatial variability analyses of Visium data, along with candidate genes from the literature reported to mark cell types or spatial subdivisions of VMH/ARC.

first: Bernard

last: Mulvey

first: Yi

first: Heena

last: Divecha

first: Svitlana V

last: Bach

first: Kelsey

middle: D

last: Montgomery

first: Sophia

last: Cinquemani

first: Yufeng

last: Du

first: Ryan

last: Miller

first: Joel

last: Kleinman

first: Stephanie

last: Page

first: Thomas M

last: Hyde

first: Stephanie C

last: Hicks

first: Kasper

last: Hansen

first: Kristen

last: Maynard


title: Single cell transcriptomic profiling of human CD19+ B cells enriched for CD5+ Innate-like B cells

city: East Lansing

first: Rance

last: Nault

state: United States

address: 1355 Bogue St. B403

department: Department of Pharmacology and Toxicology

institute: Michigan State University

summary: Aryl hydrocarbon receptor is a ligand-activated transcription factor whose activation can regulate B cell-mediated immune responses. Given the diverse nature of human B cell subsets, scRNA-seq transcriptomic profiling was used to ascertain the heterogeneity of AHR expression in discrete B cell subpopulations. We identified a unique cell cluster enriched in expression of AHR and CD9. Further, these cells were diminished in their expression of canonical B cell-associated genes and enriched with a myeloid cell-specified gene signature. These results were confirmed in vitro as CD14 expressing B cells were readily detectable in human donor PBMC and expressed significantly more AHR protein. Following activation in vitro, CD9+ B cells secreted IgM, IL-10, TGFβ1, and several myeloid-associated pro-inflammatory mediators, specifically CXCL8. These results demonstrate CD14 expression by B cells is readily detectable in healthy human donors and their release of CXCL8 could implicate these cells in inflammatory diseases and autoimmunity.

overall_design: PBMC were isolated by ficoll density gradient from 2 anonymous platelet donor leukapheresis packs naïve B cells were isolated by magnetic assisted cell sorting via negative selection. CD5 expressing B cells were enriched by positive selection for CD5 protein and then added back into a 1:1 ratio with CD5 negative B cells. RNA was extracted and then libraries were prepared using the 10X Genomics Chromium Single Cell 3’ v3 kit and Chromium i7 Multiplex Kit.

first: Lance

last: Blevins

first: Norb

last: Kaminski


title: Macrophage-Specific Lactate Dehydrogenase Expression Modulates Inflammatory Function In Vitro

city: Birmingham

first: Gunars

last: Osis

state: Alabama

address: 1720 2nd Avenue South

institute: University of Alabama at Birmingham

summary: Background:  In acute kidney injury, macrophages play a major role in regulating inflammation. Classically activated macrophages (M1) undergo drastic metabolic reprogramming during their differentiation and upregulate the aerobic glycolysis pathway to fulfill their pro-inflammatory functions. NAD+ regeneration is crucial for the maintenance of glycolysis and the most direct pathway by which this occurs is via the fermentation of pyruvate to lactate, catalyzed by lactate dehydrogenase A (LDHA). Our previous study determined that LDHA is predominantly expressed in the proximal segments of the nephron in the mouse kidney and increases with hypoxia. This study investigates the potential of LDHA as a therapeutic target for inflammation by exploring its role in macrophage function in vitro. Methods:  Bone-marrow-derived macrophages (BMDMs) were isolated from myeloid-specific LDHA knockout mice derived from crossbreeding LysM-Cre transgenic mice and LDHA floxed mice. RNA sequencing and LC-MS/MS metabolomics analyses were used in this study to determine the effect of LDHA deletion on BMDM following stimulation with IFN-γ. Results: LDHA deletion in IFN-γ BMDMs resulted in a significant alteration of the macrophage activation and functional pathways, and change in glycolytic, cytokine, and chemokine gene expression. Metabolite concentrations associated with pro-inflammatory macrophage profiles were diminished while anti-inflammatory-associated ones were increased in LDHA KO BMDMs. Glutamate and amino sugars metabolic pathways were significantly affected by the LDHA deletion. A combined muti-omics analysis highlighted changes in Rap1 signaling, cytokine-cytokine receptor interaction, focal adhesion, and MAPK signaling metabolism pathways. Conclusions:  Deletion of LDHA in macrophages results in a notable reduction in the pro-inflammatory profile and concurrent upregulation of anti-inflammatory pathways. These findings suggest that LDHA could serve as a promising therapeutic target for inflammation, a key contributor to the pathogenesis of acute kidney injury.

overall_design: WT and KO mice for the LDHA gene were utilized to isolate Bone Marrow Derived  Macrophages (BMDMs) which then were cultured in DMEM medium (15% FBS), with 30 ng/mL MCSF. Polarization of cells was performed using recombinant mouse interferon-gamma (IFN-γ) towards M1 state and interleukin 4 (IL-4) towards M2 for 24 hours. RNA was extracted with Trizol reagent. RNA quality was checked using Eukaryote Total RNA Nano assay. Samples were sequenced by Illumina NextSeq500 Next Generation Sequencing (NGS). The resulting .fastq files were analyzed and trimmed through FastQC. Data was processed through the STAR (v2.5) pipeline for alignment using the GRCm38 mouse reference genome. Individual gene read number counting was conducted by HTseq (v. 0.6) software, generating FPKM (Fragments Per Kilobase of transcript per Million mapped reads). DESeq2/edgeR was utilized for differential expression calculation and statistical analysis.

first: Osis

last: Gunars

first: Lu

last: Yan

first: Zmijewska

last: Anna

first: Traylor

last: Amie

first: Thurkral

last: Saakshi

first: Wilson

last: Landon

first: Barnes

last: Stephen

first: George

last: James

first: Agarwal

last: Anupam


title: Sphingosine-1-phosphate receptor 3 activation promotes sociability and regulates the expression of genes associated with anxiolytic-like behavior

city: Newark

first: Jose

middle: Emiliano

last: Castro Vildosola

state: NJ

address: Stanley S. Bergen Jr. Building

institute: Rutgers University

summary: We previously demonstrated that sphingosine-1-phosphate receptor 3 (S1PR3) in the medial prefrontal cortex (mPFC) prevents stress-mediated reductions in sociability. S1PR3 is a ubiquitously expressed G-protein coupled receptor that regulates immune system function, although its regulation of other biological processes is not well understood. Pharmacological activators of S1PR3 might provide important insights for understanding the neural substrates underlying sociability and/or serve as novel, preclinical treatments for social anxiety. Here we show that in mice, systemic injections of an S1PR3-specific agonist, CYM5541, promotes sociability in males and females whereas an S1PR3-specific antagonist, CAY10444, increases amygdala activation and promotes social anxiety-like behavior in females. S1PR3 expression is increased in the mPFC and dentate gyrus of females compared to males. RNA sequencing in the mPFC reveals that S1PR3 activation alters the expression of transcripts related to immune function, neurotransmission, transmembrane ion transport, and intracellular signaling. This work provides evidence that S1PR3 agonists, which have classically been used as immune modulators, might also be used as novel anxiolytics. S1PR3 might be an important hub gene for anxiolytic effects as it reduces inflammatory processes caused by stress and increases transcripts linked to anxiolytic neurotransmission.

overall_design: We used RNA samples extracted from mice hippocampus treated with CYM and a control Vehicle

last: Castro-Vildosola

first: Chris-Ann

last: Bryan

first: Nasira

last: Tajamal

first: Sai

last: Anusha Jonnalagadda

first: Akhila

last: Katsuri

first: Jaqueline

last: Tilly

first: Isabel

last: Garcia

first: Renuka

last: Kumar

first: Nathan

last: Fried

first: Tamara

last: Hala

first: Brian

middle: F

last: Corbett


title: Amphiregulin Upregulated in Esophageal Squamous Cell Carcinoma Cells upon Direct Contact with Cancer-Associated Fibroblasts

city: Kamakura

first: Satoshi

last: Kondo

state: Kanagawa

address: Tebiro 6-10-1

department: New Projects Development Division

country: Japan

institute: Toray Industries,Inc.

summary:
Cancer-associated fibroblasts (CAFs) are a pivotal component of the tumor microenvironment, significantly contributing to the progression of esophageal squamous cell carcinoma (ESCC). Direct co-culture of human bone marrow-derived mesenchymal stem cells (MSCs), one of the origins of CAFs, with ESCC cell line, led to increased amphiregulin (AREG) expression and secretion in the ESCC cells. Consequently, the expression and phosphorylation of the AREG receptor EGFR were upregulated in co-cultured ESCC cells. Moreover, AREG treatment enhanced ESCC cell survival and migration through the EGFR-Erk/p38 signaling pathway.
Immunohistochemical analysis of AREG using human ESCC tissues showed a positive correlation between the intensity of AREG expression in tumor invasive front and the expression level of the CAF marker FAP. Furthermore, bioinformatics database analysis confirmed a significant upregulation of AREG expression in ESCC tissues compared to normal tissues. These findings suggest that AREG is involved in CAF-mediated ESCC progression and could be a novel therapeutic target for ESCC.

overall_design: MSCs and ESCC cells were seeded in the same dish and co-cultured for 4 days. Monocultures of both cell types served as controls. Following co-culture or monoculture, cells were washed with phosphate-buffered saline (FUJIFILM Wako Pure Chemical Corporation) and detached themselves from the dish using trypsin (FUJIFILM Wako Pure Chemical Corporation) treatment. The harvested cells were then mixed with anti-CD326 (epithelial cell adhesion molecule; EpCAM) microbeads (130-061-101; Miltenyi Biotec) and subjected to magnetic separation using the autoMACS Pro Separator (Miltenyi Biotec) to isolate high-purity tumor cells. cDNA microarray analysis was performed on monocultured and co-cultured TE-9 cells.

first: T

last: Nakanishi

first: YI

last: Koma


title: Spatially-resolved molecular sex differences at single cell resolution in the adult human hypothalamus [Xenium]

first: Keri

last: Martinowich

first: Bernard

last: Mulvey

first: Yi

last: Wang

first: Heena

last: Divecha

first: Svitlana

last: Bach

first: Kelsey

last: Montgomery

first: Sophia

last: Cinquemani

first: Yufeng

last: Du

first: Ryan

last: Miller

first: Joel

last: Kleinman

first: Stephanie

last: Page

first: Thomas

last: Hyde

last: Hicks

first: Kasper

last: Hansen

first: Kristen

last: Maynard


title: BRAF activation enhances erythropoiesis and treats anemia

city: Beijing

first: Yuelin

last: Deng

state: Beijing

address: Beijing, China

institute: Peking University

summary: Erythropoiesis is a crucial part in hematopoietic system, while facing disruptions from various diseases conditions. Anemia and blood shortages has become global challenges, with current finite pharmacological options like EPO or glucocorticoids having limitations, especially in genetic anemias like Diamond-Blackfan anemia (DBA), calling for novel candidates in stimulating the erythropoiesis. We designed the primary human hematopoietic stem and progenitor cells (HSPCs) chemicals screening system in erythropoiesis and unexpectedly discovered a BRAF inhibitor, effectively against BRAFV600E mutant cancers, delaying erythroid differentiation while promoting progenitors’ proliferation. Further research demonstrated its efficacy in cytokine-restricted conditions and in samples from erythroid disorder patients. Mechanistically, BRAF inhibitors paradoxically activated the RAF-MEK-ERK/MAPK pathway. Unlike oncogenic BRAFV600E impaired hematopoiesis and erythropoiesis via AP-1 hyperactivation, BRAF inhibitors minimally affected HSPCs self-renewal and differentiation. In vivo studies further exhibited BRAF inhibitors' potential to enhance human hematopoiesis and erythropoiesis in severe immunodeficient mouse models and alleviate anemia symptoms in Rpl11 haploinsufficiency DBA models and other anemia models. This discovery sheds light on the MAPK pathway's role in hematopoiesis, making BRAF inhibitors a novel clinically therapeutic choice in improving hematopoietic reconstitution and the recovery of anemias like DBA.

overall_design: CD34+ cells were resuscitated in HSPCs (CD34+) retention culture medium and differentiated to day10. Cells were electroporated with empty vector or BRAFV600E overexpression plasmid, and treated with DMSO or GDC-0879 small molecule. Samples were collected at 48h after electrotransfection for ATACseq.


title: Transcriptomic and Epigenomic Signatures Distinguish High- and Low-Risk Endotypes for Liver Tumor Development

city: Houston

first: Amrit

last: Koirala

state: TX

address: 1 Baylor Plaza

institute: Baylor College of Medicine

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

overall_design: Refer to individual Series


title: Gene expression of two Ewing sarcoma cell lines (A-673, EW-22), transduced with inducible shRNAs against GLRX3

city: Heidelberg

middle: Georg Philipp

last: Grunewald

address: Im Neuenheimer Feld 280

department: Division of Translational Pediatric Sarcoma Research

country: Germany

institute: German Cancer Research Center (DKFZ)

summary:
This dataset comprises transcriptome data of two Ewing sarcoma cell lines (A-673 and EW-22) after knockdown of the gene GLRX3
10 samples were normalized (log2) with Affymetrix Expression Console Software (v1.4) using SST-RMA algorithm. Gene expression data were annotated at gene level with the Affymetrix library for Clariom D arrays (human).

overall_design: Shunya, Ohmura

first: Endrit

last: Vinca

last: Grünewald


title: RLS-associated MEIS transcription factors control distinct processes in human neural stem cells [RNA-Seq]

city: Neuherberg

first: Juliane

last: Winkelmann

address: Ingolstaedter Landstr. 1

department: Institute of Neurogenomics

institute: Helmholtz-Zentrum München

summary: MEIS1 and MEIS2 encode highly conserved homeodomain transcription factors crucial for developmental processes in a wide range of tissues, including the brain. They can execute redundant functions when co-expressed in the same cell types, but their roles during early stages of neural differentiation have not been systematically compared. By separate knockout and overexpression of MEIS1 and MEIS2 in human neural stem cells, we find they control specific sets of target genes, associated with distinct biological processes. Integration of DNA binding sites with differential transcriptomics implicates MEIS1 to co-regulate gene expression by interaction with transcription factors of the SOX and FOX families. MEIS1 harbors the strongest risk factor for restless legs syndrome (RLS). Our data suggest that MEIS1 can directly regulate the RLS-associated genes NTNG1, MDGA1 and DACH1, constituting new approaches to study the elusive pathomechanism or RLS.

overall_design: To investigate individual target genes of MEIS1 and MEIS2, we performed separate CRISPR-activation an CRISPR/Cas9 knockout of MEIS1 and MEIS2 in cultured human neural stem cells (hNSC). RNA extraction from cryopreserved cells was performed with the RNeasy Kit (Qiagen). RNAs with a RIN value > 7 were selected for mRNA sequencing. The libraries were prepared using the TruSeq stranded mRNA Sample Preparation kit (Illumina), following the kit’s instructions. After a final QC, the libraries were sequenced in paired-end mode (2 × 100 bases) on the Novaseq6000 sequencer (Illumina) with a depth of ≥ 10 Million reads per sample. Reads were aligned to the human genome (hg19) using the STAR (v 2.4.2a) and were counted using HTSeq v0.6.0115. DESeq2 v1.34.0 was used for differential gene expression analyses.

first: Volker

last: Kittke

first: Chen

last: Zhao

first: Daniel

last: Lam

first: Philip

last: Harrer

first: Wojciech

last: Krezel

first: Barbara

last: Schormair

first: Konrad

last: Oexle


title: RLS-associated MEIS transcription factors control distinct processes in human neural stem cells [ChIP-Seq]

overall_design:
Chromatin Immunoprecipitation DNA-sequencing (ChIP-seq) for MEIS1/MEIS2 in human neural stem cells (hNSC) from two replicates. Illumina sequencing libraries were generated using the NEBNext Ultra II Library Prep Kit for Illumina (E7645) with NEBNext Multiplex Oligos for Illumina (Dual Index Set1, E7600) according to the manufacturer’s protocol. Size selection was performed using a Pippin Prep (Sage Science), using a 2% cassette with size window set to 300–750 bp. Samples were sequenced 36 bp paired-end on an Illumina NextSeq550.
Adapters were trimmed from raw reads using cutadapt (v1.16), without quality-based trimming. Reads were mapped to the human genome assembly GRCh37 (hg19) using bowtie2 (v2.3.0) and samtools (v1.2) at a cutoff of mapq > = 30. PCR duplicates were removed with Picard tools (v 2.15.0). Peaks were called against input controls by MACS2 in paired-end mode with a P-value cut-off of 0.01, and were used as input for IDR analysis (v.2.0.3), which was used to determine reproducible peaks between the two replicates.


title: Modulation of UV damage formation and repair in nucleosomes in distinct chromatin states in Drosophila

city: Pullman

first: John

middle: J.

last: Wyrick

address: Biotechnology Life Sciences 241

department: School of Molecular Biosciences

institute: Washington State University

type: Genome variation profiling by high throughput sequencing

summary: Here, we describe a new genome-wide map of UV-induced cyclobutane pyrimidine dimers (CPDs) in Drosophila S2 cells and a naked DNA control using CPD-seq. We used this data to analyze CPD formation in nucleosomes and different chromatin states across the Drosophila genome. We analyzed CPD formation alongside existing excision repair-sequencing (XR-seq) data to compare CPD damage and repair rates in five distinct chromatin types in Drosophila. This analysis revealed that CPD repair varied in different chromatin domains, while CPD formation was largely unaffected. Moreover, we observed distinct patterns of repair activity in nucleosomes in different chromatin types.

overall_design: We used the CPD-seq method to map UV-induced cyclobutane pyrimidine dimers (CPDs) a single nucleotide resolution across the Drosophila genome, immediately after UV-radiation of Drosophila S2 cells (UV 0hr) and in Drosophila genomic DNA that was UV irradiated in vitro (naked DNA control). The no UV control represents unirradated sample.

first: Kathiresan

last: Selvam


title: RNA-Seq analysis of b-catenin over expression in Human liver cancer cells

first: Narendra

last: Wajapeyee

state: AL

address: 720 20th Street South, Kaul 540A

department: Department of Biochemistry and Molecular Genetics

summary: The purpose of this study is to understand the role of b-catenin over expression in cancer cell proliferation

overall_design: Total RNA was extracted from all cells, followed by poly-A selection and RNA-sequencing using Illumina Hi-Seq 2500 platform, with three biological replicates for each transformed sample.

first: Dhana

middle: S

last: Reddy


title: Comparative transcriptome analysis on Arabidopsis thaliana treated by guvermectin, gibberellin acid 3, trans-zeatin, indole-3-acetic acid or epibrassinolide

city: Harbin

first: Lu

last: Bai

address: Changjiang Road 600

institute: Northeast Agricultural University

summary:
Purpose: To compare transcriptomic changes after guvermectin (GV), indole-3-acetic acid (IAA), trans-zeatin (ZT), epibrassinolide (eBL) and gibberellin acid 3 (GA3) treatments in Arabidopsis.
Method: 7-day-old MS liquid cultivated Col-0 seedlings were treated by 10 μM GV, 1 μM IAA, 1 μM ZT, 1 μM eBL or 1 μM GA3. Chemicals were added into MS liquid respectively and whole seedling samples were collected after treatment for 5, 30, 180 minutes. In total, we got control group and 5 treatment groups. Three biological replicates were performed.
Results: In total, 54 samples were used for RNA sequencing analysis. At least 6 G clean bases were generated for each sample. Comparative analysis identified differences between GV and other plant growth regulators in transcriptional level.

overall_design: In this study, transcriptome changes after 10 μM GV, 1 μM IAA, 1 μM ZT, 1 μM eBL or 1 μM GA3 treatment were compared at 5, 30, 180 minutes. Whole seedling samples from control and treated plants were collected for RNA isolation. 18 treatments in triplicate were used in this study.

first: Chongxi

last: Liu


title: Single-Cell Multiomics Profiling Reveals Heterogeneity of Müller Cells in the Oxygen-Induced Retinopathy Model

status: Public on Dec 02 2024

city: Tianjin

first: Xueming

last: Yao

address: No.94 Weijin Road, Nankai District

institute: Nankai University

summary: Retinal neovascularization poses heightened risks of vision loss and blindness. Despite its clinical significance, the molecular mechanisms underlying the pathogenesis of retinal neovascularization remain elusive. This study utilized single-cell multiomics profiling in an oxygen-induced retinopathy (OIR) model to comprehensively investigate the intricate molecular landscape of retinal neovascularization.

overall_design: Multiome-seq: Single nuclei were extracted from retinas from postnatal day 17 (P17) mice under the OIR model.Joint snRNA- and scATAC libraries were prepared using the 10x Genomics platform.

first: Hua

last: Yan


title: Transcriptomic analysis of keloid fibroblasts demonstrates reduced expression of vitamin D binding partner Retinoid-X receptor α: Implications for clinical treatment

city: Crawley

first: Andrew

middle: William

last: Stevenson

state: Western Australia

address: Room 112b, Curnow Building, Hackett Drive Carpark Entrance 2, UWA

department: School of Biomedical Sciences

institute: University of Western Australia

summary: Keloids are benign fibroproliferative tumours resulting from skin damage such as trauma, burns or surgery. Keloids are more prevalent in populations with darkly pigmented skin. Links between skin pigmentation and vitamin D production have been established and some studies indicate involvement of vitamin D signalling in keloid pathology. This study assessed the impact of paricalcitol (a selective vitamin D signalling activator) on fibroblasts derived from keloid and normal skin, to further investigate the role and potential clinical relevance of vitamin D signalling in keloid pathology. Analysis of keloid and normal skin tissue using immunohistochemistry demonstrated a significant reduction of nuclear vitamin D receptor (VDR) in keloid tissue. After treatment with paricalcitol, nuclear VDR was increased in both keloid and normal fibroblasts. RNA sequencing of normal fibroblasts treated with paricalcitol demonstrated significant changes in gene expression, with many upregulated genes identified to have anti-fibrotic effects. However, paricalcitol failed to alter gene expression in Keloid fibroblasts. To investigate this further, we performed RNA sequencing of normal and keloid fibroblasts and found that retinoid-X receptor α (RXRα), a key binding partner of VDR required for downstream transcriptional activation, is significantly downregulated in keloid fibroblasts. Our results indicate that paricalcitol can effectively activate VDR translocation to the nucleus but is unable to effect change at the transcriptional level in keloid fibroblasts, most likely due to the reduced expression of RXRα. This suggests Vitamin D signalling may be aberrant in keloids, and that supplementation with Vitamin D alone would likely be ineffective in restoring signalling. Keywords: Keloid, Vitamin D receptor, Paricalcitol, Retinoid-X receptor α

overall_design: To investigate the role of Vitamin D signalling in keloid pathology, we treated normal and keloid skin fibroblasts with the Vitamin D activator Paricalcitol and compared the changes in gene expression to untreated fibroblasts from the same patients. Additionally, using the same untreated samples used previously, we performed additional gene expression analysis comparing untreated normal fibroblasts to untreated keloid fibroblasts.

first: Zhenjun

last: Deng

first: Shiya

last: Tee

middle: W

first: Nicole

last: Hortin

first: Helen

last: Douglas

first: Phillip

last: Melton

first: Janette

last: Bester

first: Jaco

last: Kotzé

first: Fiona

last: Wood

first: Mark

last: W Fear

first: Lucy

last: Barrett


title: O-GlcNAcylation attenuates ischemia-reperfusion–induced pulmonary epithelial cell ferroptosis via the Nrf2/G6PDH pathway

city: Wuhan

first: Xinyi

last: Li

address: Shuiguo street

department: Department of Anesthesiology

institute: Zhongnan hospital of Wuhan University

summary: Lung ischemia-reperfusion (I/R) injury is a common clinical pathology associated with high mortality. The pathophysiology of lung I/R injury involves ferroptosis and elevated protein O-GlcNAcylation levels, while the effect of O-GlcNAcylation on lung I/R injury remains unclear. This research aimed to explore the effect of O-GlcNAcylation on reducing ferroptosis in pulmonary epithelial cells caused by I/R.  First, we identified O-GlcNAc transferase 1 (Ogt1) as a differentially expressed gene in lung epithelial cells of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) patients, using single-cell sequencing, and Gene Ontology analysis (GO analysis) revealed the enrichment of the ferroptosis process. We found a time-dependent dynamic alteration in lung O-GlcNAcylation during I/R injury. Proteomics analysis identified the differentially expressed proteins enriched in ferroptosis and multiple redox-related pathways based on KEGG annotation. Thus, we generated Ogt1-conditional knockout mice and found that Ogt1 deficiency aggravated ferroptosis, as evidenced by lipid reactive oxygen species (lipid ROS), malondialdehyde (MDA), Fe2+, as well as alterations in critical protein expression glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11). Consistently, we found that elevated O-GlcNAcylation inhibited ferroptosis sensitivity in hypoxia/reoxygenation (H/R) injury-induced TC-1 cells via O-GlcNAcylated NF-E2-related factor-2 (Nrf2). Furthermore, both the chromatin immunoprecipitation (ChIP) assay and the dual-luciferase reporter assay indicated that Nrf2 could bind with translation start site (TSS) of glucose-6-phosphate dehydrogenase (G6PDH) and promote its transcriptional activity. As an important rate-limiting enzyme in the pentose phosphate pathway (PPP), elevated G6PDH provided a mass of nicotinamide adenine dinucleotide phosphate (NADPH) to improve the redox state of glutathione (GSH) and eventually led to ferroptosis resistance. Rescue experiments proved that Nrf2 knockdown or Nrf2-T334A (O-GlcNAcylation site) mutation abolished the protective effect of ferroptosis resistance.  In summary, we revealed that O-GlcNAcylation could protect against I/R lung injury by reducing ferroptosis sensitivity via the Nrf2/G6PDH pathway. Our work will provide a new basis for clinical therapeutic strategies for pulmonary ischemia-reperfusion-induced acute lung injury.

overall_design: The control group consists of normal lung tissue adjacent to tumors from cancer patients, while the treatment group consists of lung tissue from patients with acute lung injury/acute respiratory distress syndrome (ALI/ARDS).

first: Liuqing

last: Yang


title: Human visceral adipose tissue RT-PCR in individuals with obesity

city: Coimbra

first: Daniela

middle: Rosendo

last: Silva

address: Azinhaga de Santa Comba, Celas

country: Portugal

institute: Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine, University of Coimbra

summary: Visceral adiposity is more related with insulin resistance than BMI itself. To study the molecular signature of the subsequent evolution steps of the metabolic sequalae of obesity we collected omental adipose tissue samples from individuals with obesity while undergoing bariatric surgery. Participants (all with obesity) were divided in 4 groups according to glycemic profile: Insulin sensitive/normoglycemic group (IS NG) - OxHOMA2<1 and fasting glycemia < 100 mg/dL (n=17); Insulin resistant/normoglycemic group (IR NG) - OxHOMA2 > 1 and fasting glycemia < 100 mg/dL and HbA1c < 5.7% (n=24); Pre diabetes group (PD) - OxHOMA2 > 1 and fasting glycemia > 100 mg/dL or HbA1c [5.7% - 6.4%] (n=33); Type 2 diabetes group (T2D) - OxHOMA2 > 1 and fasting glycemia > 125 mg/dL or HbA1c > 6.4% (n=18). A panel of 43 genes related to inflammation, angiogenesis, lipid metabolism and redox machinery was evaluated in a BioMark HD Fluidigm system.

overall_design: Samples of visceral adipose tissue were collected from the greater omentum while undergoing metabolic surgery, which was always performed by the same appointed surgeon. Adipose tissue biopsies were collected from the patients and kept in liquid nitrogen during transportation to our facilities, to be stored at -80 ºC. Total RNA was isolated from human visceral adipose tissue biopsies (100 mg of tissue) with a RNeasy Lipid Tissue Mini Kit (Qiagen, Germany). Upon isolation, RNA samples were analyzed by NanoDrop One/One spectrophotometer (ThermoFisher, Waltham, MA, USA) at 260nm to evaluate RNA concentration. Each RNA sample was diluted to the same concentration (25 ng/μL). 1μl of Reverse transcriptase enzyme  [qScript cDNA super mix (Quanta BioSciences)] was added to 4μl of each RNA sample to obtain cDNA. Each cDNA sample was pre-amplified (PA). Briefly, 1.25μl of each cDNA sample was mixed with 0.5μl of a mix of pooled primers (500nM final concentration each), 2.25μl water and 1μl of PreAmp Master Mix enzyme (Fluidigm). Thermal cycling was performed according to the enzyme manufacturer for 12 cycles. The samples were then treated with Exonuclease I (New England Biolabs) to remove unincorporated primers. After Exonuclease treatment the samples were diluted 5x in TE buffer (10mM Tris-HCl, 1mM EDTA). For each sample (2.25μl) a Pre-Mix was prepared with 2.5μl SsoFast Eva Green Supermix (BioRad), 0,25μl of 20x DNA binding dye sample reagent (Fluidigm). 5μl of each sample was pipetted into the respective inlet of a Fluidigm® 96.96 Gene expression integrated fluidic circuit (IFC). For each assay (gene) a mix of 12μl was individually prepared: 6μl of 2x Assay loading reagent (Fluidigm), 5.4μl of TE buffer, 1.2 μl from a stock of 50μM each mixed forward and reverse primers. 5μl of each assay was pipetted into their respective assay inlets on the chip. The assay and sample mixes were loaded with the Load mix (136x) script of the HX controller (HD Biomark). After loading, the chip qRT-PCR was carried out using the BioMark HDTM, accordingly to the cycling parameters recommended by Fluidigm® for 96.96 Gene expression IFC. Data were collected with Data Collection Software and were analyzed using Fluidigm® Real Time PCR Analysis v2.1 software. Genes with melting curves displaying more of one peak (amplification of non-specific products) were not included in the analysis. The data were normalized for the reference gene ATCB and ddCt was calculated.The oligos were designed for human transcripts and were synthesized by Sigma-Aldrich and reconstituted to a final concentration of 100 µM in water.

first: D

last: Rosendo-Silva

first: P

last: Matafome


title: Single Cell Profiling of Cotton Ovules Epidermis During Early Fiber Development [scRNA-Seq]

city: Austin

first: Z Jeffrey

middle: Jeffrey

last: Chen

address: 2506 Speedway NMS 3.122 Stop A500

department: Molecular Biosciences

institute: The University of Texas at Austin

summary: Each cotton seed has approximately 25,000 singular fiber cells, which account for 25-30% epidermal cells. Understanding the basis of fiber cell initiation is critical for cotton yield improvement. However, it is difficult to separate and study fiber and non-fiber cells. Here we developed a reliable single-cell protocol to study transcriptome changes in Upland and Pima cotton and in a naked seed mutant. We analyzed over 40,000 single cells derived predominately from the epidermal layer of ovules during early stages of fiber development.

sample_organism:
Gossypium barbadense
Gossypium hirsutum

platform_organism:
Gossypium barbadense
Gossypium hirsutum

overall_design: Whole ovules of Gossypium were collected and briefly digested using macerozyme and cellulase enzyme mix to release protoplasts from the epidermal surrface, including developing fiber cells. Ovules from Gossypium hirsutum TM1 at 0, 1 and 2 days post-anthesis, the N1N1 fiberless mutant at 0 and 2 DPA and Gossypium barbadense at 2 DPA processed this way and used for 10x single-cell RNAseq.

first: Ryan

last: Kirkbride

first: Zenjian


title: Bulk RNA transcriptomic analysis of logic-gated CAR-T cells targeting a combination of CD33 and CD123 in AML

city: Los Angeles

first: Babak

last: Moghimi

state: CA

address: 4650 sunset blvd

institute: CHLA

summary: We identified a gene set that is differentially expressed in gated versus constitutively expressed CAR-T cells, often pointing to naive and unstimulated genes in gated CAR-T cells, in contrast to constitutive CAR-T cells

overall_design: We performed bulk RNA analysis on T cells isolated from CAR-T co-culture with AML  MOLM13 cell line at 48 hours from 3 different donors

first: Samy

last: Jambon

first: Jianping

last: Sun

first: Shawn

last: Barman

first: Sakunthala

last: Muthugounder

first: Rachel

last: Bito

first: Armita

last: Shadfar

first: Alexandra

last: Kovach

first: Brent

first: Thoppey

last: Manoharan

first: Sorana

last: Morrissy

first: Deepa

last: Bhojwani

first: Alan

last: Wayne

first: Michael

last: Pulsipher

first: Yong-Mi

last: Kim

first: Shahab

last: Asgharzadeh

first: Chintan

last: Parekh


title: Novel Roles of Nrf3-Trim5 Axis in Vascular Smooth Muscle Cell Dysfunctions and Neointimal Hyperplasia

city: shanghai

first: shasha

last: sun

address: No. 1665 Kangjiang Road

institute: Xinhua Hospital affiliated to Shanghai Jiaotong University School of Medicine

summary: Neointimal hyperplasia (NIH), driven by vascular smooth muscle cell (VSMC) dysfunction, is a key factor in vascular diseases like atherosclerosis and restenosis. While Nrf3 is known to regulate VSMC differentiation, its role in NIH remains unclear. Using transcriptomic data,  Nrf3 knockout mice (global), we assessed Nrf3’s impact on VSMC function and NIH. We identified Trim5, a gene linked to coronary artery disease, as a downstream target of Nrf3, which promotes autophagy in VSMCs and injured arteries, enhancing VSMC dysfunction and NIH. Nrf3 overexpression increased VSMC proliferation, migration, and inflammation, while deletion or knockdown had the opposite effects. Nrf3-/- and Nrf3ΔSMC mice showed reduced VSMC accumulation and attenuated NIH after vascular injury.  These findings highlight Nrf3 as a novel modulator of VSMC dysfunction and injury-induced NIH, with potential for therapeutic targeting of the Nrf3-Trim5 axis to treat NIH-related vascular diseases.

overall_design: Arterial injury was induced using femoral artery wire injury and carotid artery ligation model (Nrf3 knockout mice (Nrf3-/-) and  control groups (flox mice). Tissue samples were collected at the seventh day post-injury for RNA extraction. RNA-seq libraries were prepared from total RNA using a standard mRNA enrichment protocol, followed by high-throughput sequencing on an Illumina platform. Paired-end sequencing (150 bp) was performed to generate high-quality transcriptomic data. Differential gene expression analysis was performed to identify genes affected by Nrf3 deletion, focusing on pathways related to VSMC dysfunction and injury-induced neointimal hyperplasia (NIH).


title: BRAF inhibitors promote erythropoiesis and hematopoiesis via paradoxical MAPK activation

first: Yuelin

last: Deng


title: Siglecf Deficiency Prevents Fibrosis After Bleomycin-Induced Acute Lung Injury

city: Denver

first: Marika

last: Orlov

state: Colorado

address: 12700 East 19th Avenue  9C03

department: Medicine, Division of Pulmonary Sciences and Critical Care Medicine

institute: University of Colorado

summary: Airway macrophages (AMs) are critical for resolving inflammation and repairing injured lung tissue, but can also contribute to the development of fibrosis.  AMs were isolated from bronchoalveolar lavage fluid of Siglec-F KO and WT mice prior to and 7, 14, and 21 days after bleomycin challenge (single dose, 2.5 units/kg, IT).  RNA was isolated using the Qiagen micro plus RNA kit and 200 ng of total RNA was used to generate RNA-Seq libraries.  The libraries were run on the NovaSeq 6000 with 80 million pair-end reads per sample.

overall_design: To investigate the role of Siglec-F signaling in airspace macrophages (AMs) and contributions to development of fibrosis, we challenged mice with bleomycin (2.5 units/kg, IT) or saline vehicle.  We performed bulk RNAseq on AMs collected from wild type and Siglec-F deficient mice on days 7, 14, and 21 after bleo exposure to evaluate differentially expressed genes.

first: Sunad

last: Rangarajan

first: Ana M

last: Jaramillo

first: Qihua

last: Ye

first: Naoko

last: Hara

first: Kenny

last: Ngo

first: James C

last: NeeDell

first: Anna Q

last: Harder

first: Fan

last: Jia

first: Brian

last: Vestal

first: Rachel Z

last: Blumhagen

first: Ting H

last: Tu

first: Jazalle

last: McLendon

first: Bradford J

last: Smith

first: David A

last: Schwartz

first: William J

last: Janssen

first: Christopher M

last: Evans


title: Transcriptomic Analysis of Air–Liquid Interface Culture in Human Lung Organoids Reveals Regulators of Epithelial Differentiation

city: Seongnam-si

first: Young-Jae

last: Cho

address: 82, Gumi-ro 173 Beon-gil

country: South Korea

institute: Seoul National University Bundang Hospital

summary: We comprehensively analyzed differentiation effects of the air–liquid interface (ALI) culture method on adult tissue-derived human lung organoids and examined the similarity with in vivo human airway epithelium compared to submerged cultures using single-cell RNA sequencing.

overall_design: 8 samples of epithelial cells differentiated from normal lung organoids derived from 2 patients were dissociated and analyzed using scRNAseq.

first: Jieun

last: Kim

first: Eun-Young

last: Eo

first: Bokyong

first: Heetak

last: Lee

first: Jihoon

first: Bon-Kyoung

last: Koo

first: Hyung-Jun

first: Sukki

first: Jinho


title: Genetic variation in the anaphase promoting complex modulates fertility and chromosome alignment defects in the absence of KIF18A

city: Bar Harbor

first: Laura

last: Reinholdt

state: ME

address: 600 Main Street

institute: The Jackson Laboratory

summary: The kinesin family member 18A (Kif18a) is an essential regulator of microtubule dynamics and chromosome alignment during mitosis. Laboratory mice with a loss of function mutation in Kif18a are are infertile due to mitotic arrest of germ cells, but this phenotype shows variable penetrance depending on genetic background. Kif18a is also necessary for mitotic progression of chromosomally unstable cancer cells, which exhibit mitotic arrest in the absence of KIF18A function. While it is clear that functional dependency on KIF18A varies by cell type and genetic context, the heritable factors that influence this dependency remain unknown. To address this, we took advantage of the variable penetrance observed in different mouse strain backgrounds to screen for genetic loci that modulate germ cell depletion in the absence of KIF18A. We found a significant association between the severity of germ cell depletion and a locus on Chr5 wherein anaphase promoting complex subunits 5 (Anapc5) and 7 (Anapc7) were the top candidate genes. We compared the expression of both genes at key timepoints in gondal development and found that both genes were differentially expressed in sensitive strain backgrounds when compared to resistant strain backgrounds. We also identify a novel retroviral insertion in Anapc7 that may in part explain the observed expression differences. In cell line models, we found that depletion of KIF18A induced mitotic arrest, which was partially rescued by co-depletion of APC7 and exacerbated by co-depletion of APC5. These findings suggest that differential expression and activity of Anapc5 and Anapc7 may influence sensitivity to KIF18A depletion in germ cells and CIN cells, with potential implications for optimizing antineoplastic therapies.

overall_design: We generated RNASeq data from testes and ovaries to evaluate the developmental and tissue specific expression profiles of the genes in the Chr5 QTL. Five biological replicates were collected from each sex (male, female), strain (C57BL/6J, CAST/EiJ, and B6CastF1), developmental timepoint~sex (ovaries at 12.5-13.5 days post coitum, testes at 5 days post partum). These developmental timepoints were chosen because mitotically dividing germ cells are enriched. In GEO metadata strain is designated under "genotype" as B6 (C57BL/6J: RRID:IMSR_JAX:000664), CAST (CAST/EiJ: RRID:IMSR_JAX:000928), and F1 offspring 'B6Cast' from a cross between B6 female x CAST male (C57BL/6J x CAST/EiJ F1).


title: The phosphatase PP1 sustains global transcription by promoting RNA polymerase II pause release [ChIP-seq]

city: Shanghai

first: Aixia

last: Song

address: Dong'an Road

institute: Fudan University

summary: We performed calibrated ChIP-seq, PRO-seq, TT-seq, or RNA-seq to investigate the role of  protein phosphotase PNUTS-PP1 in regulating transcription. ChIP-seq, PRO-seq, TT-seq, or RNA-seq were normalizated by MEF as spike-in.  The reads passed the quality check aligned to the human hg19 and mouse mm10 genome. In this study, samples for each condition were collected in biological duplicates. Cells were treated with dTAG13 for 3,12h, the corresponding control cells were treated with vehicle only (DMSO) at the same vol/vol dilution.

overall_design: Zhenning, Wang

first: Fei

middle: X

last: Chen


title: The phosphatase PP1 sustains global transcription by promoting RNA polymerase II pause release [PRO-seq]

summary: RNA polymerase II progression from initiation to elongation is driven in part by a cascade of protein kinases acting on the core transcription machinery.

overall_design: We performed calibrated ChIP-seq, PRO-seq, TT-seq, or RNA-seq to investigate the role of  protein phosphotase PNUTS-PP1 in regulating transcription. ChIP-seq, PRO-seq, TT-seq, or RNA-seq were normalizated by MEF as spike-in.  The reads passed the quality check aligned to the human hg19 and mouse mm10 genome. In this study, samples for each condition were collected in biological duplicates. Cells were treated with dTAG13 for 3,12h, the corresponding control cells were treated with vehicle only (DMSO) at the same vol/vol dilution.

first: Zhenning

last: Wang


title: Mechanosensing regulates plasmacytoid DCs activation in the skin through NRF2 activation

city: New York

first: David

last: Oliver

state: New York

address: 535 E 70th St

department: Research

institute: Hospital for Special Surgery

summary: Plasmacytoid dendritic cells (pDCs) infiltrate the skin, chronically produce type I interferon (IFN-I) and promote skin lesions and fibrosis in autoimmune patients. However what controls their activation in the skin is unknown. Here, we report that increased stiffness, characteristic of the skin microenvironment, inhibits the production of IFN-I by TLR-activated pDCs. Mechanistically, mechanosensing activates stress pathways including the transcription factor NRF2, which, by triggering the pentose phosphate pathway, rewires glycolysis and reduces pyruvate levels, a product necessary for pDC responses. Modulating NRF2 expression in skin-infiltrating pDCs controlled the IFN-I response, leading to either resolution or the sustained activation of pathologic IFN-I pathway in the skin. Moreover, we observed that CXCL4, a pro-fibrotic chemokine that is elevated in fibrotic skin, was able to overcome stiffness-mediated IFN-I inhibition, allowing chronic IFN-I responses by pDCs in the skin. Furthermore, in the skin of patients with systemic sclerosis (SSc), although NRF2 was induced in skin-infiltrating pDCs as compared to blood pDCs, the IFN-response was maintained. Hence, these data identify a novel regulatory mechanism exerted by the skin microenvironment and identify points of dysregulation of this mechanism in patients with skin inflammation and fibrosis.

overall_design: Purified pDCs from Healthy donors were cultured at different stiffness (low stiffness: 0.2kPa and high stiffness: 50kPa) in the absence or presence of a TLR9 agonist (C274 at 0.075uM)) for 5h. Or  Purified pDCs from HDs (n=3) were cultured in media alone or with the NRF2 activator (KI696) for 1h, followed by the TLR9 agonist (C274 at 0.075uM) for 5h.  RNA was extreacted and preformed RNA-sequencing

first: Vidyanath

last: Chaudhary

first: Bikash

last: Mishra

first: Marie Dominique

last: Ah Kioon

first: Yong

last: Du

first: Lionel B

last: Ivashkiv

first: Mary

last: K. Crow

first: Franck

last: J. Barrat


title: E3 ligase substrate adaptor SPOP fine tunes the UPR of pancreatic beta cells

city: Philadelphia

first: Doris

last: Stoffers

state: Pennsylvania

address: 3400 Civic Center Boulevard, TRC 12-166

institute: University of Pennsylvania

summary: The Cullin-3 E3 ligase adaptor protein, SPOP, targets proteins for ubiquitination and proteasomal degradation. We previously established the β cell transcription factor (TF) and human diabetes gene PDX1 as an SPOP substrate, suggesting a functional role for SPOP in the β cell. Here, we generated a β cell specific Spop deletion mouse strain (SpopβKO) and found that Spop is necessary to prevent aberrant basal insulin secretion and for maintaining glucose- stimulated insulin secretion (GSIS) through impacts on glycolysis and glucose-stimulated calcium flux. Integration of proteomic, TF-regulatory gene network and biochemical analyses identified XBP1 as a functionally important SPOP substrate in pancreatic β cells. Further, loss of SPOP strengthened the IRE1α-XBP1 axis of unfolded protein response (UPR) signaling. ER stress promoted proteasomal degradation of SPOP, supporting a model whereby SPOP fine-tunes XBP1 activation during the UPR. These results position SPOP as a regulator of β cell function and proper UPR activation.

overall_design: Pancreatic islets were isolated from 12-week-old female control (Spopfl/fl) mice and mice with beta cell specific deletion of Spop  (SpopβKO). Mice are on a C57BL/6 background.

first: Alexis

last: Oguh


title: The phosphatase PP1 sustains global transcription by promoting RNA polymerase II pause release [RNA-seq]

summary: RNA polymerase II progression from initiation to elongation is driven in part by a cascade of protein kinases acting on the core transcription machinery. Conversely, the corresponding phosphatases, notably PP2A and PP1—the most abundant serine-threonine phosphatases in cells—are thought to mainly impede polymerase progression, respectively restraining pause release at promoters and polymerase elongation at terminators. Here we reveal an unexpected role of PP1, within the PNUTS-PP1 complex, in sustaining global transcriptional activation. Acute disruption of PNUTS-PP1 leads to severe defects in the release of paused polymerase and subsequent downregulation for the majority of transcribed genes. Mechanistically, PNUTS-PP1 promotes pause release by dephosphorylating multiple substrates, including the 7SK snRNP subunit MEPCE, a known regulator of pause release. PNUTS-PP1 exhibits antagonistic functions compared to INTAC phosphatase, which generally inhibits pause release. Our research thus highlights the opposing roles of PP1 and PP2A in modulating genome-wide transcriptional pausing and gene expression.

overall_design: We performed calibrated ChIP-seq, PRO-seq, TT-seq, or RNA-seq to investigate the role of  protein phosphotase PNUTS-PP1 in regulating transcription. ChIP-seq, PRO-seq, TT-seq, or RNA-seq were normalizated by MEF as spike-in.  The reads passed the quality check were aligned to the human hg19 and mouse mm10 genome. In this study, samples for each condition were collected in biological duplicates. Cells were treated with dTAG13 for 3,12h, the corresponding control cells were treated with vehicle only (DMSO) at the same vol/vol dilution.


title: CROWN-seq profiling of snRNA m6Am modification in pig ST cells upon PRV infection

first: Samie

last: Jaffrey

address: 1300 York Ave

department: Pharmacology

institute: Weill Cornell Medicine

summary: Fat mass and obesity-associated protein (FTO) was discovered as the first RNA demethylase and mediates the demethylation of N6,2′-O-dimethyladenosine (m6Am), a prevalent RNA modification adjacent to the 5’ cap. Despite its significance, the regulation of the m6Am demethylase and the potential impact of virus infection on FTO remain largely unexplored. In this study, we show that the alphaherpesvirus pseudorabies virus (PRV) triggers phosphorylation of FTO and that this phosphorylation is caused by the viral serine/threonine protein kinase UL13.  To test whether the phosphorylated FTO can result in different demethylation on snRNA, we performed CROWN-seq to quantify m6Am stoichiometry in cells.

sample_organism:
Sus scrofa
Suid alphaherpesvirus 1

platform_organism:
Sus scrofa
Suid alphaherpesvirus 1

overall_design: Three replicates were performed on ST cells (mock), ST cells transfected by wild-type PRV, and ST cells transfected by UL13-null PRV

first: Ruth

last: Verhamme

first: Jianheng

last: Liu

first: Luke

last: Nicholson

middle: R

first: Herman

last: Favoreel


title: An ageing bone marrow exacerbates lung fibrosis via a dysregulated regulatory T cell driven axis, fuelling profibrotic macrophage persistence.

city: Vienna

first: Sylvia

last: Knapp

address: Währinger Gürtel 18-20

department: Department of Medicine I

country: Austria

institute: Medical University of Vienna

summary: Pulmonary fibrosis is an incurable disease, which increasingly manifests with advanced age. Yet, how the ageing hematopoietic niche influences immune cell function and fibrosis progression in the lungs remains elusive. Using heterochronic transplant mouse models, we discovered that an aged bone marrow exacerbates lung fibrosis irrespective of lung tissue age. Upon lung injury, there was an increased pulmonary infiltration of monocyte-derived alveolar macrophages (Mo-AMs) from an aged bone marrow. These Mo-AMs displayed an enhanced profibrotic profile and a delayed transition in acquiring a homeostatic tissue-resident phenotype. This impaired Mo-AM differentiation was attributed to diminished numbers of regulatory T cells (Tregs) and the reduced availability of the anti-inflammatory cytokine IL-10 in the lung microenvironment of mice engrafted with aged bone marrow. Mechanistically, we show that Tregs are crucial providers of IL-10 that promote timely Mo-AM maturation and attenuate fibrosis progression. Our findings demonstrate the critical influence of an aged bone marrow on the development of lung fibrosis and identify a Treg-mediated resolutory axis as a potential therapeutic target for accelerated tissue repair.

overall_design: To investigate the impact of hematopoietic ageing on lung immune cells during lung fibrosis, we transplanted young recipient mice with young or aged bone marrow. After reconstitution we challenged them with bleomycin intratracheally and sorted cells from the lungs (five cell types) at D7 and (two cell types) at D14 post challenge to analyse any gene expression/transcriptional differences between the recipient groups.

first: Asma

last: Farhat

first: Florian

last: Deckert

first: Riem

last: Gawish


title: The phosphatase PP1 sustains global transcription by promoting RNA polymerase II pause release [TT-seq]

first: Aixia

last: Song

first: Zhenning

last: Wang

first: Fei

last: Chen


title: Age is an intrinsic driver of inflammatory responses to malaria

city: Brisbane

first: Zuleima

last: Pava Imitola

state: Queensland

address: 300 Herston road

institute: QIMR Berghofer

summary: Age is a critical factor influencing the host immune response and disease pathogenesis. In malaria, the risk of severe disease increases with age in non-immune individuals. Malaria disease is in part driven by inflammation, but the specific cells and mechanisms contributing to age-dependent disease risk are incompletely understood. Here, we assessed inflammatory cytokines in malaria in non-immune children and adults, and the phenotypic, functional and transcriptional differences of innate immune cell responders to malaria parasites in malaria-naïve children and adults. During naturally acquired malaria, age was associated with increased plasma levels of inflammatory chemokines CCL2, CCL3, CXCL8, CXLC9, along with CRP, and IDO, which were associated with clinical symptoms. In malaria naïve individuals, classical monocyte and Vδ2+ δ T cell responses from adults were characterized by increased inflammatory cytokine production, and higher transcriptional activation following stimulation with malaria parasites. Classical monocyte responses in adults were dominated by CCL2 production, while in children the response had increased IL-10 production and enrichment in IL-10 signaling pathways upon parasite stimulation. This heightened inflammatory response in adults was not mitigated by regulatory T cells (Tregs). Taken together, these findings identify cellular mechanisms of age-dependent host responses that play crucial roles in driving inflammatory responses in malaria.

overall_design: PBMCs were co-cultured at 37 °C, 5% CO2 in a 96-well U-bottom plate for 4 hours at a 1:1 cell ratio with 1x106 mature trophozoite stage pRBCs. After incubation classical monocytes and Vδ2 T cells subsets were FACS sorted using the BD FACSAria™ III Cell Sorter. RNA was extracted from isolated cell population lysates using the QIAGEN PicoPureTM RNA isolation kit (Applied Biosystems™, KIT0204), and RNA quality confirmed with the 2200 TapeStation system (G2964AA) by High Sensitivity RNA ScreenTape (5067- 5579). RNA sequencing libraries were constructed using the NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (E6420S) and NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs) (E6440S). The libraries were sequenced using a NextSeq 550 system

first: Jessica

last: Loughland

first: Nicholas

last: Dooley

last: Pava

first: Arya

last: SheelaNair

first: Dean

last: Andrew

first: Peta

last: Tipping

first: Peter

last: Bourke

first: Christian

last: Engwerda

first: Alejandro

last: Lopez

first: Kim

last: Piera

first: Tonia

last: Woodberry

first: Timothy

last: William

first: Bridget

last: Barber

first: Matthew

last: Grigg

middle: M

last: Anstey

first: Gabriela

last: Minigo

first: Michelle

last: Boyle


title: Utilization of Bulk RNA Sequencing for the Evaluation of Keratin Nanomaterials as a Coating for Percutaneous Devices

city: Salt Lake City

first: Sujee

last: Jeyapalina

state: Utah

address: 500 Foothill Dr

institute: Department of Veteran Affairs

summary: Despite advances in the design and protocols for maintaining the skin/device interface around percutaneous devices (PDs), no current strategy ensures the permanent attachment of peri-implant epithelial tissue to the device surface. Based on preliminary data, we hypothesized that PDs coated with keratin nanomaterials could provide a biochemically mediated surface that enhances epidermal cell adhesion and downgrowth. To test this hypothesis, fifteen Yucatan miniature pigs were each implanted with six percutaneous titanium devices, comprising three porous and three smooth devices, both with and without keratin coatings (Kerateine [iKNT] and Keratose [gKOS]). The pigs were sacrificed at 4, 8, and 16 weeks post-implantation. The devices and surrounding tissues were harvested and analyzed using histological and RNA sequencing techniques. Compared to smooth peri-implant tissue, porous peri-implant tissue showed a significant decrease in epithelial downgrowth, fibrous capsule thickness, and infections, alongside a significant upregulation of multiple immune marker genes, including IL12B. At the 16-week period, gKOS-coated surfaces demonstrated a more favorable wound healing response than iKTN-coated devices, with a reduction in granulation tissue area and a significant upregulation of several keratin genes related to differentiation. Among the combinations of surface types and coatings studied, the porous gKOS-coated device produced the most favorable wound healing response.

overall_design: Yucatan minipigs were implanted with six sterile percutaneous deviceses with smooth subdermal disks (1 uncoated control, 1 iKTN-coated, and 1 gKOS-coated) and three additional porous subdermal disks (1 uncoated control, 1 iKTN-coated, and 1 gKOS-coated) under their dorsal skin. Granulation tissue was harvested at the planned necrospy for each pig for RNA sequencing.

first: Andrew

last: Miller

first: James

last: Beck

first: Alexis

last: White

first: Jayant

last: Agarwal

first: Kent

middle: N

last: Bachus

first: Mark

middle: V

last: Dyke


title: A novel mouse model of Prader-Willi Syndrome including the deletion of both Necdin and Magel2 genes

city: Marseille

first: Anne-Marie

last: François-Bellan

address: 27 Bd Jean Moulin

department: Facute de Medecine

country: France

institute: CNRS-AMU

summary: Prader-Willi syndrome (PWS) is a multigenic disorder caused by the loss of seven contiguous paternally expressed genes. Mouse models with inactivation of all PWS genes display 100% lethality within the first postnatal week and have not helped understand the postnatal pathophysiology of this syndrome. Knockout (KO) models for each candidate gene were also generated, but they lack the functional interactions and possible compensatory functions between PWS-related genes. Here, we generated a novel double KO mouse model to explore the effect of a combined deletion of Magel2 and Necdin.

overall_design: Mice were maintained under standard animal housing conditions, with free access to food and water,, and in standard 12-h light/12-h dark cycles (LD). C57BL/6J mice  (WT) and double ko mice (MADIN) were sacrificed either six hour after light on (the DAY group) or 18 hours after light on (the NIGHT group) and were processed for total RNA sequencing

first: Denis

last: Becquet


title: Dimethyl fumarate ameliorates chronic graft-versus-host disease by inhibiting Tfh differentiation via Nrf2

city: Suzhou

first: Lyu

last: Fulian

address: Renai Street

institute: Soochow University

summary: Chronic graft-versus-host disease (cGVHD), characterized by chronic tissue inflammation and fibrosis involving multiple organs, remains a major complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Dimethyl fumarate (DMF) is an anti-inflammatory drug approved for the treatment of multiple sclerosis and psoriasis. We previously reported that DMF effectively inhibits acute GVHD (aGVHD) while preserving the graft-versus-leukemia effect. However, the role of DMF in cGVHD progression remains unknown. Here, we found that DMF administration significantly suppresses follicular helper T cell (Tfh) differentiation, germinal center formation and alleviates disease severity in different murine cGVHD models. Mechanistically, DMF treatment downregulates IL-21 transcription by activation of Nrf2, thus orchestrating Tfh-related gene program both in mice and humans. The inhibitory role of DMF on Tfh cell differentiation was diminished in Nrf2 deficient T cells. Importantly, the therapeutic potential of DMF in clinical cGVHD has been validated in human data whereby DMF effectively reduces IL-21 production and Tfh cell generation in peripheral blood mononuclear cells from active cGVHD patients and further attenuates xenograft GVHD. Collectively, our findings reveal that DMF potently inhibits cGVHD development by repressing Tfh cell differentiation via Nrf2, paving way for the treatment of cGVHD in the clinic.

overall_design: To further elucidate the role of DMF in cGVHD development, we established a scleroderma-like cGVHD model.

first: Fulian

last: Lyu

first: Huanle

last: Gong

first: Xiaojin

last: Wu

first: Xin

last: Liu

first: Yinghao

last: Lu


title: Exposure to particulate air pollution disrupts erythrocyte turnover

city: Louisville

first: Eric

middle: Christian

last: Rouchka

state: Kentucky

address: 522 East Gray Street

department: Biochemistry and Molecular Genetics

institute: University of Louisville

summary: Chronic exposure to ambient particulate matter (PM) increases the risk of cardiovascular disease (CVD). One proposed mechanism linking PM exposure and CVD is the induction of low-grade systemic inflammation that underlies disease progression. However, the intermediaries that relay signals from the proximal sites of inflammation to the periphery remain unknown. We have previously shown that fine PM (PM2.5) alter red blood cell distribution width. Therefore, the overall goal of this study was to elucidate how PM exposure impacts the spleen and splenic macrophages which are primarily responsible for the turnover of aged and damaged red blood cells.

overall_design: Mice were exposed to filtered air or PM2.5 for 30 days and spleens were collected.  In the second experiment, mice were exposed to filtered air following air or PM2.5 for 30 days and macrophages were isolated from spleen.

first: Brian

last: Sansbury

middle: C

first: Richa

last: Singhal


title: Cell-in-cell mediated intercellular communication exacerbates the pro-inflammatory progression in asthma

city: 广州市

first: Jiahao

last: Huang

state: 广东省

address: 海珠区昌岗东路250号

institute: 广州医科大学附属第二医院

summary: Cell-in-cell (CIC) structures have been suggested to mediate intracellular substance transport between cells and have been found widely in inflammatory lung tissue of asthma. The aim of this study was to investigate the significance of CIC structures in inflammatory progress of asthma. CIC structures and related inflammatory pathways were analyzed in asthmatic lung tissue and normal lung tissue of mouse model. In vitro, the activation of inflammatory pathways by CIC mediated intercellular communication was analyzed by RNA-Seq and verified by western blotting and immunofluorescence. Results showed CIC structures of lymphocytes and alveolar epithelial cells in asthmatic lung tissue mediated intercellular substance (such as mitochondria) transfer and promoted pro-inflammation in two phases. At early phase, internal lymphocytes triggered inflammasome dependent pro-inflammation and cell death of itself. Then degraded lymphocytes released cellular contents such as mitochondria inside alveolar epithelial cells, further activated multi-pattern-recognition receptors and NF-kappa Bsignalingpathways of alveolar epithelial cells, thereby amplified pro-inflammatory response in asthma. Our work supplements the mechanism of asthma pro-inflammation progression from the perspective of CIC structure of lymphocytes and alveolar epithelial cells, and provides a new idea for anti-inflammatory therapy of asthma.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for LA4 cells

first: Shan

last: Wang

first: Bowen


title: RNA helicase DDX5 maintains cardiac function by regulating CamkIIδ alternative splicing [RNA-seq]

first: Kangni

last: Jia

state: Shanghai

address: 227 Chongqing South Road, Huangpu District, Shanghai, China

institute: Shanghai Jiaotong University

summary: The cardiomyocyte-specific deletion of Ddx5 in mice resulted in heart failure characterized by diminished cardiac function, enlarged heart chambers, and heightened fibrosis. Proteomic analysis unveiled the involvement of DDX5 in RNA splicing within cardiomyocytes. Our study identified DDX5 as a regulator of the abnormal splicing of CamkIIδ, preventing the generation of CaMKIIδA. This isoform activates LTCC through serine phosphorylation of Cacna1c, disrupting Ca2+ homeostasis. Consistently, DDX5-depleted cardiomyocytes exhibited elevated intracellular Ca2+ transients and increased sarcoplasmic reticulum Ca2+ content.

overall_design: To investigate the role of DDX5 in the heart, we obtained cardiac tissues from 8-week-old DDX5fl/flMYH6Cre+ mice (mCKO), with age-matched DDX5fl/fl mouse cardiac tissues serving as controls (CTR1). Additionally, we collected cardiac tissues from Ddx5flox/floxMyh6MerCreMer mice (iCKO) after 7 days of intraperitoneal tamoxifen injection, with age-matched DDX5fl/fl mouse cardiac tissues injected with the same dose of tamoxifen serving as controls (CTR2). RNA sequencing was performed for each condition, followed by differential gene expression analysis and alternative splicing analysis against their respective controls.

first: Haomai

last: Cheng

first: Wenqi

last: Ma

first: Xiaoxiang

last: Yan


title: BRAF inhibitors enhance erythropoiesis and treat anemia through paradoxical activation of MAPK signaling

city: Bei Jing

first: Hsiang-Ying

last: Lee

state: Bei Jing

address: Yiheyuan Road

institute: Peking University

summary: Erythropoiesis is a crucial process in hematopoiesis, yet it remains highly susceptible to disruption by various diseases, which significantly contribute to the global challenges of anemia and blood shortages. Current treatments like erythropoietin (EPO) or glucocorticoids often fall short, especially for hereditary anemias such as Diamond-Blackfan anemia (DBA). To uncover new erythropoiesis-stimulating agents, we devised a screening system using primary human hematopoietic stem and progenitor cells (HSPCs). We discovered that BRAF inhibitors (BRAFi), commonly used to treat BRAFV600E melanoma, can unexpectedly and effectively promote progenitor cell proliferation by temporarily delaying erythroid differentiation. Notably, these inhibitors exhibited pronounced efficacy even under cytokine-restricted conditions and in patient samples of DBA. Mechanistically, although these BRAFi inhibit the MAPK cascade in BRAFV600E mutant cells, they paradoxically act as amplifiers in wild-type BRAF cells, potently enhancing the cascade. Furthermore, we found that while the oncogenic BRAFV600E mutation disrupts hematopoiesis and erythropoiesis through AP-1 hyperactivation, BRAFi minimally impact HSPC self-renewal and differentiation. In vivo studies have shown that BRAFi can enhance human hematopoiesis and erythropoiesis in severe immunodeficient mouse models and alleviate anemia in the Rpl11 haploinsufficiency DBA model, as well as other relevant anemia models. This discovery underscores the role of the MAPK pathway in hematopoiesis and positions BRAFi as a promising therapeutic option for improving hematopoietic reconstitution and treating anemias, including DBA.

overall_design: We sorted human CD71+ CD235a- (single positive, SP) erythroblasts (erythroid progenitor cells) in the erythropoiesis culture system on day 9(starting with human cord blood derived CD34+ cells). Then culturing the sorted SP erythroblasts for 72h with DMSO(Ctrl) or 2 μM GDC-0879(G2), and each group has two biological replicates. Harvested the cells on day 12 with TRIzol™ and extracted the RNA.

first: Shunkang

first: Yuelin

last: Deng

first: Fan


title: Low-Intensity Pulsed Ultrasound Effects on the Human Blood Brain Barrier

city: CHICAGO

first: Yu

last: Luan

state: Illinois

address: 303 E. Superior Simpson Querrey 7-518

institute: Northwestern University Feinberg School of Medicine

summary: Analysis of human peritumoral brain following a low-intensity pulsed ultrasound (LIPU) mediated opening of the blood brain barrier, to enhance the delivery of systemic chemotherapies (paclitaxel or carboplatin).

overall_design: Single-cell RNA-seq of peritumoral brain biopsies from recurrent glioblastoma patients following LIPU.

first: Andrew

last: Gould

first: Adam

last: Sonabend


title: The phosphatase PP1 sustains global transcription by promoting RNA polymerase II pause release

first: Aixia

last: Song

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

overall_design: Refer to individual Series


title: Multiple Generation Distinct Toxicant Exposures Induce Epigenetic-Transgenerational-Inheritance of Enhanced Pathology and Obesity

city: Pullman

first: Michael

middle: K

last: Skinner

state: WA

address: Abelson 507

department: SBS

institute: WSU

type: Methylation profiling by high throughput sequencing

summary: This study examines the actions of the agricultural fungicide vinclozolin on the F0 generation, followed by jet fuel hydrocarbon mixture exposure of the F1 generation, and then pesticide DDT (dichlorodiphenyltrichloroethane) on the F2 generation gestating females. The subsequent F3 and F4 generations and F5 transgenerational-generation were examined for male sperm epigenetic alterations and pathology in males and females. Significant impacts on the male sperm differential DNA methylation regions (DMRs) were observed. The F3-F5 generations were similar in approximately 50% of the DMRs. The pathology of each generation was assessed in the testis, ovary, kidney, prostate, obesity, and tumors. The pathology used a newly developed Deep Learning (DL), artificial intelligence (AI), based histopathology analysis. Observations demonstrated compounded disease impacts in obesity and metabolic parameters, but other pathologies plateaued with smaller increases at the F5 transgenerational generation. Therefore, the multiple generational exposures, which occurs in human populations, may increase epigenetic impacts and disease susceptibility.

overall_design: The experimental design used three successive generations of gestating female exposures during fetal gonadal sex determination, followed by three generations of no exposure to obtain the F5 generation to reveal the ultimate transgenerational phenotype for pathology and disease. The F0 generation gestating female was exposed to the agricultural fungicide vinclozolin. The second successive exposure of the F1 generation gestating female involved jet fuel (JP8). The third successive exposure of the F2 generation gestating female involved DDT (dichlorodiphenyltrichloroethane). The subsequent F3 and F4 generations and F5 transgenerational-generation were examined for male sperm epigenetic alterations and pathology in males and females. Methylation differences between treatment groups was done using an MeDIP-seq procedure. The control lineage was only taken to the F3 generation. This F3 generation control was then compared with the F3, F4, and F5 treatment lineage sample groups.

first: Eric

middle: E

last: Nilsson

first: Margaux

last: McBirney

first: Sarah

last: De Santos

first: Stephanie

last: King

first: Daniel

last: Beck

first: Colin

last: Greeley

first: Lawrence

middle: B

last: Holder


title: Differential analysis of transcriptome of Trichophyton mentagrophytes treated with Trichoderma

city: hangzhou

first: xiao

middle: chen

last: wen

address: desheng_road NO.298

institute: Zhejiang Academy of Agricultural Sciences

summary: Under the action of Trichophyton 1000 UG / ml, the colony of Trichophyton mentagrophyte was completely inhibited. The spore number and germination rate of Trichophyton mentagrophyte under the action of 100ug / ml and 10ug / ml were significantly lower than those in the control group. Under the action of Trichophyton, the mitochondria in the mycelium of Trichophyton mentagrophyte were cleaved. Under the action of trichomycin, the related genes in mitochondria decreased significantly. This showed that mitochondria were obviously damaged during trichomycin treatment. It is speculated that Trichophyton can cause mitochondrial damage and reduce the efficiency of respiratory chain, but Trichophyton can synthesize enough ATP by regulating related ATP synthase to resist the invasive effect caused by Trichophyton stimulation.

overall_design: SDA drug sensitive plates with Trichoderma concentration of 10 and 100 UG / ml were prepared, respectively. The fungal cake was prepared by the method of fungal cake drilling and cultured on the drug sensitive plate for 6 days. Then collect the colonies and store them in liquid nitrogen. Perform the next step of high-throughput sequencing.

first: Xiao

last: Chenwen

first: Liang

last: Siyuan

first: Wang

last: Jiaoyu

first: Lin

last: Fucheng


title: Gene expression following transverse aortic constriction in mice housed under normoxic or hypoxic conditions

city: Braunschweig

first: Robert

last: Geffers

address: Inhoffenstr. 7

department: Dep. Molecular Bacteriology

institute: HCI - Helmholtz Centre for Infection Research

summary: mRNA expression profiles from C57/Bl6J mouse hearts 3 days after transverse aortic constriction (TAC) or sham surgery. Mice were house under normoxic (21% O2) or hypoxic conditions (12% O2).

overall_design: Mice were subjected to transverse aortic constriction (TAC) and sacrificed 3 days after surgery, mRNA was extracted from the apex, and libraries were prepared for high throughput sequencing.

first: C

last: Riehle

first: R

first: W

last: Hofmann