title: Disease-relevant upregulation of P2Y1 receptor in astrocytes enhances neuronal excitability via IGFBP2

city: Chuo

first: Eiji

last: Shigetomi

state: Yamanashi

address: 1110 Shimokato

institute: University of Yamanashi

summary: Reactive astrocytes play a pivotal role in the pathogenesis of neurological diseases; however, their functional phenotype and the downstream molecules by which they modify disease pathogenesis remain unclear. Here, we genetically increase P2Y1 receptor (P2Y1R) expression, which is upregulated in reactive astrocytes in several neurological diseases, in astrocytes of male mice to explore its function and the downstream molecule. This astrocyte-specific P2Y1R overexpression causes neuronal hyperexcitability by increasing both astrocytic and neuronal Ca2+ signals. We identify insulin-like growth factor-binding protein 2 (IGFBP2) as a downstream molecule of P2Y1R in astrocytes; IGFBP2 acts as an excitatory signal to cause neuronal excitation. In neurological disease models of epilepsy and stroke, reactive astrocytes upregulate P2Y1R and increase IGFBP2. The present findings identify a mechanism underlying astrocyte-driven neuronal hyperexcitability, which is likely to be shared by several neurological disorders, providing insights that might be relevant for intervention in diverse neurological disorders.

overall_design: Hippocampal astrocytes were isolated from astrocyte-specific P2Y1 receptor overexpression mice or their littermate control using MACS with anti-mouse astrocyte cell surface antigen-2 (ACSA2)-coated microbeads for RNA collection. 11-week-old male mice were used. Three mice were used for each sample.

first: Schuichi

last: Koizumi


title: Epithelial cells maintain memory of prior infection with Streptococcus pneumoniae through di-methylation of histone H3

status: Public on May 17 2024

city: Paris

first: Claudia

last: Chica

address: 25-28 Rue du Dr Roux

country: France

institute: Institut Pasteur

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Epithelial cells maintain memory of prior infection with Streptococcus pneumoniae through di-methylation of histone H3 [Affymetrix]

summary:
Epithelial cells are the first point of contact for bacteria entering the respiratory tract.  Streptococcus pneumoniae is an obligatory human pathobiont of the nasal mucosa, carried asymptomatically but also the cause of severe pneumoniae. The role of the epithelium in maintaining homeostatic interactions or mounting an inflammatory response to invasive S. pneumoniae is currently poorly understood. However, studies have shown that chromatin modifications, at the histone level, induced by bacterial pathogens interfere with the host transcriptional program and promote infection. In this study, we demonstrate that S. pneumoniae actively induces di-methylation of histone H3 on lysine 4 (H3K4me2), which persists for at least 9 days upon clearance of bacteria with antibiotics. We show that infection establishes a unique epigenetic program affecting the transcriptional response of epithelial cells, rendering them more permissive upon secondary infection. Our results establish H3K4me2 as a unique modification induced by infection, distinct from H3K4me3, which localizes to enhancer regions genome-wide. Therefore, this study reveals evidence that bacterial infection leaves a memory in epithelial cells after bacterial clearance, in an epigenomic mark, thereby altering cellular responses for subsequent infections.
Transcriptome analysis aimed at testing whether cells respond differently in the primary (1°) and secondary (2°)  infection. Differential gene expression and gene set enrichment analysis performed.

overall_design: Microarrays are used to measure gene expression along an infection time course: uninfected (UI), primary infection (1°) and post-infection (PI), 3 replicates per condition.

first: Christine

last: Chevalier

first: Adrien

last: Pain

first: Mélanie

last: Hamon


title: Epithelial cells maintain memory of prior infection with Streptococcus pneumoniae through di-methylation of histone H3 [ChIPseq]

summary: Epithelial cells are the first point of contact for bacteria entering the respiratory tract.  Streptococcus pneumoniae is an obligatory human pathobiont of the nasal mucosa, carried asymptomatically but also the cause of severe pneumoniae. The role of the epithelium in maintaining homeostatic interactions or mounting an inflammatory response to invasive S. pneumoniae is currently poorly understood. However, studies have shown that chromatin modifications, at the histone level, induced by bacterial pathogens interfere with the host transcriptional program and promote infection. In this study, we demonstrate that S. pneumoniae actively induces di-methylation of histone H3 on lysine 4 (H3K4me2), which persists for at least 9 days upon clearance of bacteria with antibiotics. We show that infection establishes a unique epigenetic program affecting the transcriptional response of epithelial cells, rendering them more permissive upon secondary infection. Our results establish H3K4me2 as a unique modification induced by infection, distinct from H3K4me3, which localizes to enhancer regions genome-wide. Therefore, this study reveals evidence that bacterial infection leaves a memory in epithelial cells after bacterial clearance, in an epigenomic mark, thereby altering cellular responses for subsequent infections.

overall_design: Chromatin immunoprecipitation and sequencing (ChIP-seq) for histone modifications H3K4me2 and H3K4me3 in A549 cells along an infection time course: uninfected (UI), primary infection (1°) and post-infection (PI), 2 replicates per condition.


title: Transcription factor shapes chromosomal conformation and regulates gene expression in bacterial adaptation [RNA-Seq]

city: Chengdu

first: Mao

last: Chen

address: Section 4-13, Renmin Rd. South

country: China

institute: Biogas Institute of Ministry of Agriculture and Rural Affairs

type: Expression profiling by high throughput sequencing

summary:
Genomic mutations allow bacteria to adapt rapidly to adverse stress environments. The three-dimensional conformation of the genome also may plays an important role in transcriptional regulation and environmental adaptation. Here, using chromosome conformation capture, we investigate the high-order architecture of the Zymomonas mobilis chromosome in response to genomic mutant and ambient stimuli (acetic acid and furfural, derived from lignocellulosic hydrolysate). We find that genomic mutation only influences the local chromosome contacts, whereas stress of acetic acid and furfural restrict the long-range contacts and change the chromosome organization at domain scales significantly. Further deciphering the domain feature unveils the important transcription factors, Ferric uptake regulation (Fur) proteins, which act as nucleoid-associated proteins to promote long-range (> 200 kb) chromosomal communications and regulate the expression of genes involved in stress response. Our work suggests that ubiquitous transcription factors in prokaryotes mediate chromosome organization and regulate stress-resistance genes in bacterial adaptation.
RNA-seq analysis to reveal the regulation process of Fur proteins.

overall_design: The gene expression levels of different strains were compared under different growth conditions

first: Mingxiong

last: He

first: Bo

last: Wu

first: Samina

last: Shabbir


title: Transcription factor shapes chromosomal conformation and regulates gene expression in bacterial adaptation [ChIP-Seq]

summary:
Genomic mutations allow bacteria to adapt rapidly to adverse stress environments. The three-dimensional conformation of the genome also may play an important role in transcriptional regulation and environmental adaptation. Here, using chromosome conformation capture, we investigate the high-order architecture of the Zymomonas mobilis chromosome in response to genomic mutant and ambient stimuli (acetic acid and furfural, derived from lignocellulosic hydrolysate). We find that genomic mutation only influences the local chromosome contacts, whereas stress of acetic acid and furfural restrict the long-range contacts and change the chromosome organization at domain scales significantly. Further deciphering the domain feature unveils the important transcription factors, Ferric uptake regulation (Fur) proteins, which act as nucleoid-associated proteins to promote long-range (> 200 kb) chromosomal communications and regulate the expression of genes involved in stress response. Our work suggests that ubiquitous transcription factors in prokaryotes mediate chromosome organization and regulate stress-resistance genes in bacterial adaptation.
ChIP-seq analysis of Fur proteins binding in the genome of ZM532.

overall_design: Binding sites of Fur and Zur in strain Zymomonas mobilis ZM532 under RM condition


title: Epigenetic regulation by dynamic RNA G-quadruplex folding and unfolding (ChIP-Seq)

city: Boston

first: yongwoo

last: lee

state: MA

address: 185 Cambridge St

department: Mol bio

country: USA

institute: MASSACHUSETTS GENERAL HOSPITAL

summary: Whether RNA G-quadruplexes (rG4) form extensively in vivo and what roles they play remain actively debated. Among their proposed functions is recognition of Polycomb repressive complex 2 (PRC2), but how the interaction results in epigenetic regulation is not understood. Here we demonstrate that rG4s form dynamically during ES cell differentiation and require ATRX’s helicase function to unwind competing secondary structures. Mutating ATRX causes rG4 depletion on a transcriptome-wide basis and dramatically increases gene expression. We identify and mutate rG4s within Xist RNA and mechanistically separate PRC2’s recruitment versus catalysis. Surprisingly, although rG4s recruit PRC2, unfolding the rG4 structure causes PRC2 hyperactivation, entrapment of PRC2 in the S1 chromosomal compartment, and loss of gene silencing. Thus, we link dynamic rG4 folding and unfolding to PRC2 recruitment, trans-compartmental Xist migration, regulated activation of PRC2, and whole-chromosome gene silencing.

overall_design: Modified existing rG4-seq and RT-Stop protocols were performed and generated an “in vitro” and “in vivo” pipeline, based on differential sensitivity of various RNA structures to dimethyl sulfate (DMS), reverse transcriptase (RT), and the monovalent cations Li+ versus K+.

first: Yong

middle: W

last: Lee

first: Uri

last: Weissbein

first: Roy

last: Blum

first: Jeannie

middle: T


title: Transcription factor shapes chromosomal conformation and regulates gene expression in bacterial adaptation [Hi-C]

summary:
Genomic mutations allow bacteria to adapt rapidly to adverse stress environments. The three-dimensional conformation of the genome also may plays an important role in transcriptional regulation and environmental adaptation. Here, using chromosome conformation capture, we investigate the high-order architecture of the Zymomonas mobilis chromosome in response to genomic mutant and ambient stimuli (acetic acid and furfural, derived from lignocellulosic hydrolysate). We find that genomic mutation only influences the local chromosome contacts, whereas stress of acetic acid and furfural restrict the long-range contacts and change the chromosome organization at domain scales significantly. Further deciphering the domain feature unveils the important transcription factors, Ferric uptake regulation (Fur) proteins, which act as nucleoid-associated proteins to promote long-range (> 200 kb) chromosomal communications and regulate the expression of genes involved in stress response. Our work suggests that ubiquitous transcription factors in prokaryotes mediate chromosome organization and regulate stress-resistance genes in bacterial adaptation.
Hi-C analysis revealed the three-dimensional conformation of Zymomonas mobilis.

overall_design: The chromosomal conformation analysis of wild-type strain (ZM4-RM), genomic mutation strain (ZM532-RM), genomic mutation strain under environmental stress (ZM532-AF), and strains with the deletion of Fur proteins (zur and zurfur).

first: Weiting

last: Wang


title: Microenvironment Shapes Small Cell Lung Cancer Neuroendocrine States and Presents Therapeutic Opportunities [DMS273 SCLC cell lines]

status: Public on May 16 2024

city: Bethesda

first: Anish

last: Thomas

state: MARYLAND

address: Center for Cancer Research, National Cancer Institute, Building 10 Room 4-5330

department: National Cancer Institute

institute: National Institutes of Health

summary: Small-cell lung cancer (SCLC) is the most fatal form of lung cancer. Intra-tumoral heterogeneity, marked by neuroendocrine (NE) and non-neuroendocrine (non-NE) cell states, defines SCLC, but the drivers of SCLC plasticity are poorly understood. To map the landscape of SCLC tumor microenvironment (TME), we apply spatially resolved transcriptomics and quantitative mass spectrometry-based proteomics to metastatic SCLC tumors obtained via rapid autopsy. The phenotype and overall composition of non-malignant cells in the tumor microenvironment (TME) exhibits substantial variability, closely mirroring the tumor phenotype, suggesting TME-driven reprogramming of NE cell states. We identify cancer-associated fibroblasts (CAF) as a crucial element of SCLC TME heterogeneity, contributing to immune exclusion, and predicting exceptionally poor prognosis. Together, our work provides a comprehensive map of SCLC tumor and TME ecosystems, emphasizing their pivotal role in SCLCs adaptable nature, opening possibilities for re-programming the intercellular communications that shape SCLC tumor states.

overall_design: DMS273 SCLC cell lines treated with Vehicle (control) and Erdafitinib (Pan-FGFR inhibitor) at 33.33 micromolar concentration for 5 days followed by RNA-seq analysis

first: Desai

last: Parth

first: Thomas

last: Anish

first: Kumar

last: Rajesh


title: Epigenetic regulation by dynamic RNA G-quadruplex folding and unfolding (RNA-Seq)


title: Unveiling the Immune Dynamics of Neisseria Persistent Oral Colonization

city: Baltimore

first: Suvarna

last: Nadendla

state: MD

address: 670 W.Baltimore Street

department: Institute for Genome Sciences

institute: University of Maryland School of Medicine

summary: Commensal bacteria are crucial in maintaining host physiological homeostasis, immune system development, and protection against pathogens. Despite their significance, the factors influencing persistent bacterial colonization and their impact on the host still need to be fully understood. Animal models have served as valuable tools to investigate these interactions, but most have limitations. The bacterial genus Neisseria, which includes both commensal and pathogenic species, has been studied from a pathogenicity to humans’ perspective, but lacks models that study immune responses in the context of long-term persistence. Neisseria musculi, a recently described natural commensal of mice, offers a unique opportunity to study long-term host-commensal interactions. In this study, for the first time we have used this model to study the transcriptional, phenotypic, and functional dynamics of immune cell signatures in the mucosal and systemic tissue of mice in response to Neisseria musculi colonization.  We found key genes and pathways vital for immune homeostasis in palate tissue, validated by flow cytometry of immune cells from lung, blood and spleen. This study offers a novel avenue for advancing our understanding of host-bacteria dynamics and may provide a platform for developing efficacious interventions against mucosal persistence by pathogenic Neisseria.

overall_design: Investigation of immune responses to Neisseria musculi colonization in a novel AJ mouse model. Hard palate samples were harvested 5 or 33 days post inoculation or mock inoculation, and used for total RNA extraction and RNAseq.

first: Mario

last: Alles

first: Manuja

last: Gunasena

first: Tauqir

last: Zia

first: Adonis

last: D’Mello

first: Saroj

last: Bhattarai

first: Will

last: Mulhern

first: Luke

last: Terry

first: Trenton

last: Scherger

first: Saranga

last: Wijeratne

first: Sachleen

last: Singh

first: Asela

middle: J

first: Dhanuja

last: Kasturiratna

first: Hervé

last: Tettelin

first: Nathan

last: Weyand

first: Namal

middle: P

last: Liyanage


title: Increased ONECUT2 induced by Helicobacter pylori promotes gastric cancer cell stemness via AKT-related pathway

city: Fuzhou

first: Sheng-Ze

middle: long

last: Wu

state: None Selected

address: NO.29,XINQUAN ROAD,FUZHOU 350001 FUJIAN PROVINCE,CHINA.

institute: Concord Hospital of the Fujian Medical University

summary: Background: Helicobacter pylori (HP) infection may initiate and promote progression of gastric carcinogenesis. ONECUT2 shows promise for tumor diagnosis, prognosis, and treatment. This study explored ONECUT2's role and specific mechanism underlying HP infection-associated gastric carcinogenesis to suggest a basis for targeting ONECUT2 as a therapeutic strategy for gastric cancer (GC). Methods: Public data, single-cell RNA sequencing, spatial transcriptome analysis, RNA sequencing, GC tissue specimens, and clinical survival data were analyzed. Human GC organoids, HP infection, 3D sphere-forming, and extreme-dilution nude mouse models were constructed. Western blotting, qPCR, immunohistochemical staining, dual-luciferase reporter assays, phosphokinase microarray, and immunofluorescence were used. Results: Multidimensional data supported an association between ONECUT2, HP infection, and GC pathogenesis. HP infection upregulated ONECUT2 transcriptional activity via NFκB. In vitro and in vivo experiments demonstrated that ONECUT2 increases stemness in GC cells. ONECUT2 was also shown to inhibit PPP2R4 transcription, resulting in reduced PP2A activity, which in turn increased AKT/β-catenin phosphorylation. AKT/β-catenin phosphorylation facilitates β-catenin translocation to the nucleus, initiating transcription of downstream stemness-associated genes in GC cell. HP infection could upregulate the phosphorylation of AKT and β-catenin triggered by ONECUT2 downregulation via induction of ONECUT2. Clinical survival analysis indicated that high ONECUT2 expression might be an indicator of poor prognosis in GC. Conclusion: This study highlights a critical role played by ONECUT2 in promoting HP infection-associated GC by enhancing cell stemness through the PPP2R4/AKT/β-catenin signaling pathway. These findings suggest promising therapeutic strategies and potential therapeutic targets for GC treatment. Keywords: Helicobacter pylori; gastric cancer; prognosis; tumor stemness; ONECUT2

overall_design: To investigate the high ONECUT2 mRNA levels in human GC tissue and to investigate ONECUT2 oncogenic mechanisms.Our analysis revealed a significant association between ONECUT2 and PI3K/AKT and other cancer-related pathways by performing RNA-seq analysis.

first: Mi

last: Lin

first: Ru-hong

last: Tu

first: Sheng-ze

first: Qing

last: Zhong


title: Epigenetic regulation by dynamic RNA G-quadruplex folding and unfolding (CHART-Seq)

first: yongwoo

last: lee

first: Yong

last: Lee

first: Uri

last: Weissbein

first: Roy

last: Blum

first: Jeannie


title: Suppressing Aberrant Hedgehog Pathway and Overcoming Resistance to Smoothened Antagonists via Targeting Super-enhancer-driven Transcriptional Dependencies [ChIP-seq]

city: Shanghai

first: Yi

last: Sui

address: South Chongqing Road

institute: Shanghai Jiao Tong University School of Medicine

summary: Aberrant activation of Hedgehog (Hh) signaling pathway plays important roles in both oncogenesis and targeted therapy of many cancers. The clinical application of FDA-approved Hh-targeted Smoothened inhibitor (SMOi) drugs is hindered due to the emergence of various primary or acquired drug resistance, indicating the need of novel anti-Hh therapies. Our previous studies demonstrate that epigenetic/transcriptional targeted therapies represent a promising direction for anti-Hh drug development. In this study, we identified CDK9 and CDK12, two transcription elongation regulators, as novel therapeutic targets for antagonizing the aberrant Hh pathway and overcoming SMOi resistance. CDK9 inhibition and CDK12 inhibition exhibited similarly potent anti-Hh activities when treating various SMOi responsive or resistant Hh-driven tumor models as previously reported BET inhibition or CDK7 inhibition. We also utilized SHH-subtype medulloblastoma (SHH-MB) as the representative Hh-driven cancer model to perform Super-enhancer (SE) analysis and elucidate the crucial roles of SE in Hh-driven oncogenesis and above-mentioned anti-Hh epigenetic/transcriptional targeted therapies. Furthermore, we identified IRS1, encoding a critical component and cytoplasmic adaptor protein of the IGF pathway, as an oncogenic Hh-driven SE target gene and effective therapeutic target of multiple Hh-driven tumor models, including the SMOi-resistant ones. Collectively, our study demonstrates that the SE-driven transcriptional dependencies represent promising therapeutic vulnerabilities for suppressing the aberrant Hh pathway and overcoming the SMOi resistance. As CDK9 inhibitor and IRS inhibitor drugs have already entered human clinical trials for cancer treatment, our study provides comprehensive preclinical support for expanding their trials to Hh-driven cancers in near future.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: We utilize mouse SHH-MB models SmoWT-MB and SMB56 as representative Hh-driven tumor models to investigate the potential roles of SE in Hh-driven oncogenesis and epigenetic/transcriptional targeted anti-Hh therapy. To do so, we generated H3K27ac ChIP-seq data of SmoWT-MB and SMB56 to profile their active enhancer landscape. The samples were collected under two different conditions, in vitro cultured (SmoWT-1 and SMB56-1) or freshly separated from subcutaneous tumor tissues (SmoWT-2 and SMB56-2). Also, we performed similar SE profiling in two SHH-MB patient-derived xenograft (PDX) models SHH-MB_PDX1 and SHH-MB_PDX2. Meanwhile, we performed anti-HA ChIP-seq analysis of SmoWT-MB cells stably expressing an active form of HA-tagged human GLI2 without the N-terminal region (HA-GLI2-DeltaN) to investigate direct Hh target gene in mouse SHH-MB.

first: Teng

last: Wang

first: Chaochen

first: Yujie

last: Tang


title: Suppressing Aberrant Hedgehog Pathway and Overcoming Resistance to Smoothened Antagonists via Targeting Super-enhancer-driven Transcriptional Dependencies [RNA-seq]

overall_design: mouse SHH-MB models SmoWT-MB and SMB56 were treated with SMO, BET, CDK7, CDK9, and CDK12 inhibitors with DMSO-treated controls. SHH-MB patient-derived xenograft (PDX) models SHH-MB_PDX1 were treated with BET, CDK7, CDK9, and CDK12 inhibitors with DMSO-treated controls. P60 cerebellum, P7 cerebellum, and SMOi-resistant model SmoWT-MB-GR cells were also collected. We then performed RNA-seq analysis to identify the gene expression profiling of the above samples.


title: The Protective Role of Integrin α4β7 and Amphiregulin-Expressing Innate Lymphoid Cells in Lupus Nephritis

city: Seoul

first: Hye Young

last: Kim

address: 103 Daehak-ro, Jongro-gu

country: South Korea

institute: Seoul National University

summary: Type-2 innate lymphoid cells (ILC2s) have emerged as key immune-response regulators in renal-inflammatory diseases such as lupus nephritis. However, the adhesion and migration of ILC2s, including in the homeostatic and diseased kidney, are poorly understood. By integrating transcriptomic profiling, flow cytometry, live-cell imaging, and in-vivo models, we showed that renal ILCs are retained in the homeostatic kidney by the adherence of their cell-surface integrin α4β7 to VCAM-1, E-cadherin or fibronectin on structural kidney cells. When cultured on these ligands, ILC2s demonstrated remarkable migration. Knocking down integrin-α4β7 expression reduced ILC2 Areg production. In lupus nephritis, TLR7/9 may downregulate ILC2 expression of integrin-α4β7, thereby both reducing Areg expression and promoting ILC2 egress. Areg loss may promote the proinflammatory-cytokine secretion by T cells. IL-33 upregulated ILC2 integrin-α4β7 and Areg expression. Notably, IL-33 treatment enhanced survival in lupus nephritis by mitigating kidney inflammation. Thus, ILC2 adhesion may be a therapeutic target for autoimmune kidney diseases.

overall_design: To investigate the role of ILCs in lupus nephritis, we used pooledkidneys from old or young MRL-lpr mice. Renal immune cells, particularly innate lymphoid cells, were further enriched using density-gradient centrifugation and an ILC enrichment kit to ensure that the scRNA-seq analysis detected a low frequency cell population of ILCs in the oldMRL-lpr kidney.

first: Seungwon

last: Ryu

first: Hye

middle: Y


title: Heterochromatic 3D genome organization is directed by HP1a and H3K9-dependent and independent mechanisms.

status: Public on May 24 2024

city: Chapel Hill

first: Alexis

last: Stutzman

state: NC

address: 250 Bell Tower Drive

institute: University of North Carolina at Chapel Hill


title: TFE3 fusions escape from controlling of mTOR signaling pathway and accumulate in the nucleus promoting genes expression in Xp11.2 translocation renal cell carcinomas.

status: Public on May 15 2024

city: Nanjing

last: Chen

address: 22 Hankou Road

department: Medical school

institute: Nanjing Unversity

summary: ChIP analysis demonstrated that TFE3 fusions could bind to the promoters of the target genes as a wild-type TFE3 protein.

overall_design: Chip-seq for TFE3 in UOK120 cells and UOK109 cells

first: Xiaoqin

last: Yin

first: Dongmei

last: Li

first: Weidong

last: Gan


title: A fluorescent perilipin 2 knock-in mouse model reveals a high abundance of lipid droplets in the developing and adult brain

city: Lausanne

first: Ludovic

last: Telley

address: Department of Biomedical Sciences University of Lausanne Rue du Bugnon 9

country: Switzerland

institute: UNIL

summary: Lipid droplets (LDs) are dynamic lipid storage organelles. They are tightly linked to metabolism and can exert protective functions, making them important players in health and disease. Most LD studies in vivo rely on staining methods, providing only a snapshot. We therefore developed a LD-reporter mouse by endogenously labelling the LD coat protein perilipin 2 (PLIN2) with tdTomato, enabling staining-free fluorescent LD visualisation in living and fixed tissues and cells. Here we validate this model under standard and high-fat diet conditions and demonstrate that LDs are highly abundant in various cell types in the healthy brain, including neurons, astrocytes, ependymal cells, neural stem/progenitor cells and microglia. Furthermore, we also show that LDs are abundant during brain development and can be visualized using live-imaging of embryonic slices. Taken together, our tdTom-Plin2 mouse serves as a novel tool to study LDs and their dynamics under both physiological and diseased conditions in all tissues expressing Plin2.

overall_design: Single cell RNA sequencing (scRNA-seq) on sorted TdTom-Plin2 positive and negative

first: Sofia

last: Madsen

first: Ana

middle: C

last: Delgado

first: Christelle

last: Cadilhac

first: Vanille

last: Maillard

first: Fabrice

last: Battiston

first: Carla

middle: M

last: Igelbüscher

first: Simon

last: De Neck

first: Elia

last: Magrinelli

first: Denis

last: Jabaudon

first: Marlen

last: Knobloch


title: Suppressing Aberrant Hedgehog Pathway and Overcoming Resistance to Smoothened Antagonists via Targeting Super-enhancer-driven Transcriptional Dependencies

first: Yi

last: Sui


title: Helicobacter pylori-induced angiopoietin-like 4 promotes gastric bacterial colonization and gastritis

city: Chongqing

first: yuan

last: zhuang

address: Gaotanyan Street

institute: Third Military Medical University

summary: Helicobacter pylori (H. pylori) infection is characterized as progressive processes of bacterial persistence and chronic gastritis with features of infiltration of mononuclear cells more than granulocytes in gastric mucosa. Angiopoietin-like 4 (ANGPTL4) is considered a double-edged sword in inflammation-associated diseases, but its function and clinical relevance in H. pylori-associated pathology is unknown. Here we demonstrate both pro-colonization and pro-inflammation roles of ANGPTL4 in H. pylori infection. Increased ANGPTL4 in the infected gastric mucosa was produced from gastric epithelial cells (GECs) synergistically induced by H. pylori and IL-17A in a cagA-dependent manner. Human gastric ANGPTL4 correlated with H. pylori colonization and the severity of gastritis, and mouse ANGPTL4 from non-bone marrow-derived cells promoted bacteria colonization and inflammation. Importantly, H. pylori colonization and inflammation were attenuated in Il17a-/-, Angptl4-/-, Il17a-/-Angptl4-/- mice. Mechanistically, ANGPTL4 bound to integrin αV (ITGAV) on GECs to suppress CXCL1 production by inhibiting ERK, leading to decreased gastric influx of neutrophils, thereby promoting H. pylori colonization; ANGPTL4 also bound to ITGAV on monocytes to promote CCL5 production by activating PI3K-AKT-NF-κB, resulting in increased gastric influx of regulatory CD4+ T cells (Tregs) via CCL5-CCR4-dependent migration. In turn, ANGPTL4 induced Treg proliferation through binding to ITGAV to activate PI3K-AKT-NF-κB, promoting H. pylori-associated gastritis. Overall, we propose a model in which ANGPTL4 collectively ensures H. pylori persistence and promotes gastritis. Efforts to inhibit ANGPTL4-associated pathway may prove valuable strategies in treating H. pylori infection.

overall_design: To investigate the function of ANGPTL4 in gastric epithelial cells, AGS cells were stimulated with ANGPTL4, cANGPTL4 or nANGPTL4 (1 μg/ml) for 24 h

first: Rui

last: Xie

last: You

first: Wan-Yan

first: Peng

last: Zhu

first: Pan

last: Wang

first: Yi-Pin

last: Lv

first: Geng-Yu

last: Yue

first: Xiao-Lin

last: Xu

first: Jiang-Bo

last: Wu

first: Jing-Yu

first: Si-Xu

last: Liu

first: Mu-Han

last: Lü

first: Sheng-Qian

last: Yang

first: Ping

last: Cheng

first: Fang-Yuan

last: Mao

first: Yong-Sheng

last: Teng

first: Liu-Sheng

last: Peng

first: Jin-Yu

last: Zhang

first: Ya-Ling

last: Liao

first: Shi-Ming

first: Yong-Liang

last: Zhao

first: Weisan

first: Quan-Ming

last: Zou

first: Yuan

last: Zhuang


title: Defined conditions control the morphological dualism of rat primitive extraembryonic endoderm stem cells

city: Ansan

first: Young Gyu

last: Chai

state: Gyeonggi-do

address: 55 Hanyangdaehak-ro

department: Molecualr & Life Science

institute: Hanyang Univ.

summary: We report two chemically defined conditions that differ by the addition of the cytokine Lif and the β-catenin-stabilizing drug Chir99021 and enable permanent self-renewal as mesenchymal and epithelial morphotypes, respectively. The morphotypes are interconvertible and differentiate equipotently. Surprisingly, the proliferation of both morphotypes requires Lif/Gp130/Stat3 signaling (autocrine in the absence of added Lif) and non-canonical Wnt signaling (autocrine). Additionally, the epithelial version requires β-catenin for proliferation and morphology. Interestingly, the mesenchymal cells also express key epithelial markers, but those are improperly structured and/or not functional, indicating a primed state. The results provide an improved platform for studying the proliferation and plasticity of the early extraembryonic endoderm, which occurs in mesenchymal and epithelial forms in vivo.

overall_design: mRNA profiles of rat pXEN cells routinely cultured in T,LPE and CPE conditions were generated by deep sequencing, in triplicate, on an Illumina HiSeq 2500

first: Bert

last: Binas

first: Young

middle: G


title: Gene expression alterations in primary choroid plexus epithelial cells infected with JCPyV

city: Providence

first: Sheila

last: Haley

state: RI

address: 70 Ship Street

department: MCB

institute: Brown University

summary: The human polyomavirus, JCPyV, establishes a lifelong persistent infection in the peripheral organs of a majority of the human population worldwide. Patients who are immunocompromised due to underlying infections, cancer, or to immunomodulatory treatments for autoimmune disease are at risk for developing progressive multifocal leukoencephalopathy (PML) when the virus invades the CNS and infects macroglial cells in the brain parenchyma. It is not yet known how the virus enters the CNS to cause disease. The blood-choroid plexus barrier is a potential site of virus invasion as the cells that make up this barrier are known to be infected with virus both in vivo and in vitro. To understand the effects of virus infection on these cells we challenged primary choroid plexus epithelial cells with JCPyV and profiled changes in host gene expression. We found that JC infection induced the expression of proinflammatory chemokines and downregulated junctional proteins essential for maintaining blood-CSF and blood-brain barrier function. These data contribute to our understanding of how virus infection of the choroid plexus can modulate the host cell response to neuroinvasive pathogens.

overall_design: Gene expression profiling analysis of primary human choroid plexus epithelial cells comparing in triplicate cells infected with human polyomavirus JCPyV with control uninfected cells, 7 days post infection. 3 replicates per condition, 6 overall total samples.

middle: A

first: Walter

last: Atwood

first: Christoph

last: Schorl


title: Modeling the Blood-Brain Barrier Formation and Cerebral Cavernous Malformations in human PSCs- and primary tissue-derived organoids

city: Cincinnati

first: Ziyuan

last: Guo

address: 3333 Burnet Ave

institute: CCHMC

summary: The human blood-brain barrier (hBBB) is a highly specialized structure that regulates passage across blood and CNS compartments. Despite its critical physiological role, there are no reliable in vitro models that can mimic hBBB development and function. Here, we constructed hBBB assembloids from brain and blood vessel organoids derived from human pluripotent stem cells. We validated the acquisition of BBB-specific molecular, cellular, transcriptomic, and functional characteristics and uncovered an extensive neuro-vascular crosstalk with a spatial pattern within hBBB assembloids. When we used patient-derived hBBB assembloids to model cerebral cavernous malformations (CCMs), we found that these assembloids recapitulated the cavernoma anatomy and BBB breakdown observed in patients. Upon comparison of phenotypes and transcriptome between patient-derived hBBB assembloids and primary human cavernomas tissues, we uncovered CCM-related molecular and cellular alterations. Taken together, we report hBBB assembloids that mimic core properties of the human BBB and identify a potentially underlying cause of CCMs.

overall_design: BBB assembloids was prepared by combining a H9 ESCs derived cerebral organoids, where the astrocytic differentiation is promotated by adding LIF with 15%FBS, with a blood vessel organoid from H9 ESCs indurced mesoderm. Both organoids were developed separately and embedded in a matrigel droplet.  We then repurposed the used Illumina Nextseq 2000 sequencing flow cells to a spatial barcoded matrix for RNA hybirdizations. By appling the HIFI-Slide protocol on the assembloid fresh frozen sections for RNA capture and library preparations, we sequencing the extracted library to confirm the cell type and gene expression co-localizations of the BBB assembloids.

first: Lan

last: Dao

first: Zhen

last: You

first: Lu

last: Lu

first: Tianyang

last: Xu

first: Avijite

middle: K

last: Sarkar

first: Hui

last: Zhu

first: Miao

last: Liu

first: Riccardo

last: Calandrelli

first: George

last: Yoshida

first: Pei

last: Lin

first: Yifei

last: Miao

first: Sarah

last: Mierke

first: Srijan

last: Kalva

first: Mingxia

last: Gu

first: Sudhakar

last: Vadivelu

first: Sheng

last: Zhong

first: L

middle: F

last: Huang


title: PKD1 mutant clones within cirrhotic livers inhibit steatohepatitis without promoting cancer [Western Diet + CCl4]

city: Dallas

first: Hao

state: TX

address: 6000 Harry Hines Blvd.

department: Children’s Research Institute

institute: University of Texas Southwestern Medical Center

summary: Somatic mutations in non-malignant tissues are selected for because they confer increased clonal fitness, however, it is uncertain if these clones can benefit organ health. Here, ultra-deep targeted sequencing of 150 liver samples from 30 chronic liver disease patients revealed recurrent somatic mutations. PKD1 mutations were observed in 30% of patients, whereas they were only detected in 1.3% of hepatocellular carcinomas (HCCs). To interrogate tumor suppressor functionality, we perturbed PKD1 in two HCC cell lines and six in vivo models, in some cases showing that PKD1 loss protected against HCC, but in most cases showing no impact. However, Pkd1 haploinsufficiency accelerated regeneration after partial hepatectomy. We tested Pkd1 in fatty liver disease, showing that Pkd1 loss was protective against steatosis and glucose intolerance. Mechanistically, Pkd1 loss selectively increased mTOR signaling without SREBP activation. In summary, PKD1 mutations in cirrhotic livers exert adaptive functionality without increasing transformation risk.

overall_design: Pkd1 WT, Het, and Homo litternate mice on "Western Diet + CCl4"NASH model for 3 months


title: An optimized workflow for transcriptomic analysis from archival paraformaldehyde-fixed retinal tissues collected by laser capture microdissection

city: Philadelphia

first: Raghavi

last: Sudharsan

address: 3900 Delancey Street

institute: University of Pennsylvania

summary: RNA sequencing (RNA-seq) coupled with laser capture microdissection (LCM) is a powerful tool for transcriptomic analysis in unfixed fresh-frozen tissues. Fixation of ocular tissues for immunohistochemistry commonly involves the use of paraformaldehyde (PFA) followed by embedding in Optimal Cutting Temperature (OCT) medium for long-term cryopreservation. However, the quality of RNA derived from such archival PFA-fixed/OCT-embedded samples is often compromised, limiting its suitability for transcriptomic studies. In this study, we aimed to develop a methodology to extract high-quality RNA from PFA-fixed canine eyes by utilizing LCM to isolate retinal tissue. We demonstrate the efficacy of an optimized LCM and RNA purification protocol for transcriptomic profiling of PFA-fixed retinal specimens. We compared four pairs of canine retinal tissues, where one eye was subjected to PFA-fixation prior to OCT embedding, while the contralateral eye was embedded fresh frozen (FF) in OCT without fixation. Since the mRNA obtained from PFA-fixed retinas were contaminated with genomic DNA, we employed two rounds of DNase I treatment to obtain RNA suitable for RNA-seq. Notably, the quality of sequencing reads and gene sets identified from both PFA-fixed and FF tissues were nearly identical. In summary, our study introduces an optimized workflow for transcriptomic profiling from PFA-fixed archival retina. This refined methodology paves the way for improved transcriptomic analysis of preserved ocular tissue, bridging the gap between optimal sample preservation and high-quality RNA data acquisition.

overall_design: To compare the transcriptomic profile between fresh frozen and PFA-fixed canine retinal tissues, bulk RNA sequencing was performed with four biological replicates.

first: William

last: Beltran

first: Kei

last: Takahashi


title: The pleiotropic phenotype of FlbA of Aspergillus niger is explained in part by the activity of seven of its downstream-regulated transcription factors.

city: Utrecht

first: Peter Jan

last: Vonk

state: Utrecht

address: Padualaan 8

department: Biology

country: Netherlands

institute: Utrecht University

summary: Aspergillus niger fulfills an important role in nature in nutrient cycling and is a main cell factory for the industrial production of metabolites and enzymes. Inactivation of flbA in A. niger results in thinner cell walls, increased cell lysis, abolished sporulation, and an increased secretome complexity. A total of 36 transcription factor genes are differentially expressed in ∆flbA. Here, seven of these genes (abaA, aslA, aslB, azf1, htfA, nosA, and srbA) were inactivated. Inactivation of each of the genes affected sporulation. Moreover, inactivation of each of the genes impacted protein secretion with the exception of abaA. The impact on secretion was strongest in the case of ΔaslA and ΔaslB that showed increased pepsin, cellulase, and amylase activity. Biomass formed on agar plates was reduced when either abaA, aslA, nosA, or srbA were inactivated. In contrast, biomass of ∆aslA and ∆aslB was higher in liquid shaken cultures. The ∆aslA and ∆htfA strains showed reduced resistance to H2O2, while ∆abaA showed increased resistance to cell wall perturbing agents. By contrast, AslA, AslB, HtfA, Azf1, NosA, and SrbA were shown to be important for cell wall integrity. Together, the seven genes downstream of FlbA impact biomass formation, sporulation, protein secretion, and stress resistance in A. niger, and thereby explain part of the pleiotropic phenotype of the ∆flbA strain.

overall_design: Total RNA of A. niger was isolated from 16 h TM-G and 4 h MM-X liquid shaking cultures. Mycelium of biological triplicates of liquid shaken cultures of A. niger strains MA234.1, ΔaslA, and ΔaslB were homogenized in a Tissue Lyzer II (Qiagen) under liquid nitrogen. Total RNA was isolated using TRIzol reagent (Invitrogen, www.thermofisher.com). The RNA was purified using the Rnase Plant Mini Kit (Qiagen) and quantified with a Nanodrop ND-1000 (Thermo Scientific, www.thermofisher.com). Sequencing of RNA was performed with Illumina NextSeq2000 2x50bp paired-end technology (Utrecht Sequencing Facility; useq.nl).

first: Xiaoyi

last: Chen

first: Juan

last: Torres

first: Peter

first: J

last: Damen

first: Karli

middle: R

last: Reiding

first: Lugones

last: Luis

first: Han

last: Wösten


title: Platelet-derived microvesicles isolated from type-2 diabetes mellitus patients harbour an altered miRNA signature and drive MDA-MB-231 triple-negative breast cancer cell invasion

city: Leeds

first: Jim

last: Boyne

address: Leeds Beckett University

department: Biomedical Science

country: United Kingdom

institute: Leeds Beckett University

type: Non-coding RNA profiling by high throughput sequencing

summary: The underlying causes of breast cancer are diverse, however, there is a striking association between type 2 diabetes (T2DM) and poor patient outcomes.  Platelets become activated in both T2DM and breast cancer patients and have been implicated in tumorigenesis through a multitude of pathways. Here transcriptomic analysis of T2DM-patient derived PMVs revealed an altered micro-RNA (miRNA) signature compared with normoglycaemic control patients. Interestingly, interrogation of these data identifies a shift towards an oncogenic signature in T2DM-derived PMVs, with increased levels of several miRNA implicated in breast cancer progression and poor prognosis. Functional studies demonstrate that PMVs isolated from T2DM patient blood are internalised by triple-negative breast cancer (TNBC) cells in vitro, and that co-incubation with T2DM-patient derived PMVs led to significantly increased TNBC cell invasion compared with PMVs from healthy volunteers. Together, these data suggest that circulating PMVs in T2DM patients may contribute to the progression of TNBC.

overall_design:
To investigate the role of T2DM-derived PMV miRNA signature on the risk of breast cancer progression in diabetic patients, we extracted PMV RNA from healthy young adult volunteers (n=8) and T2DM patients with HbA1C>50 (n=4).
We then prepared small RNA-Seq libraries using NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (NEB, UK) and analysed the samples using via NextSeq 500.
Differential  miRNA expression analysis was performed between diabetic (D) and non-diabetic (C) samples.

first: Chinedu

last: Anene


title: yHSC and oHSC scRNAseq with oHSC AThi ATlo Index Sorting. Assessing the transcriptional changes associated with autophagy engagement in old hematopoietic stem cells at single cell resolution. [scRNA-Seq]

city: New York

first: Paul

middle: Vincent

last: Dellorusso

state: NY

address: 701 West 168th Street, 724 Hammer Health Sciences Center

department: Genetics and Development

institute: Columbia University Irving Medical Center

summary: Index sorted SMART-Seq2 sequencing to map AThi oHSC vs ATlo oHSC based on GFP-LC3 marker levels. Between yHSC and oHSC the largest transcriptional differences observed in the G0/G1 cell cycle phase cluster. Within oHSCs, AThi oHSCs were almost exclusively observed in the G0/G1 cluster, whereas ATlo oHSCs were spread across the activation continuum, with cells still in the G0/G1 cluster found more proximal to S cluster cells.

overall_design: 96 HSCs were sorted into individual wells recording information on surface markers and GFP-LC3 level.

first: Emmanuelle

last: Passegue

first: Fernando

last: Jose Calero Nieto

first: Xiaonan

last: Wang


title: PDX1+ cell budding morphogenesis in a stem cell-derived islet spheroid system

city: Vancouver

first: Howard

last: Cen

state: BC

address: 2350 Health Sciences Mall

country: Canada

institute: University of British Columbia

summary: Remarkable advancements in protocol development have been achieved to manufacture insulin-secreting islets from human pluripotent stem cells. Distinct from current approaches, we devised a tunable strategy to generate islet spheroids enriched for major islet cell types by incorporating PDX1+ cell budding morphogenesis into the differentiation process of staged transcriptional programming. In this process that appears to mimic normal islet morphogenesis, the differentiating islet spheroids self-organize with endocrine cells that are intermingled or arranged in a core-mantle architecture, accompanied with functional heterogeneity. Through in vitro modelling of human pancreas development, we illustrate the importance of PDX1 in eliciting cell budding morphogenesis, and the requirement for EphB3/4 signaling. We show how RFX6 deficiency affects pancreatic patterning and uncover an expected role of RFX6 in early pancreas morphology. The tunable differentiation system and stem cell-derived islet models described in this work may facilitate addressing fundamental questions in islet biology and probing human pancreas diseases.

overall_design: Intact buds and bodies of stage 6 spheroids from 4 independent differentiation were enzymatically separated and sequenced.

first: Jia

last: Zhao

first: Timothy

last: Kieffer


title: CPEB3 Maintains Developmental Competence of the Oocyte

city: Gainesville

first: Zongliang

last: Jiang

state: FL

address: 2033 Mowry Rd

department: Animal Sciences

institute: University of Florida

summary: Mammalian oocyte development depends on the temporally controlled translation of maternal transcripts, particularly in the coordination of meiotic and early embryonic development when transcription has ceased. The translation of mRNA is regulated by various RNA-binding proteins. We show that the absence of cytoplasmic polyadenylation element-binding protein 3 (CPEB3) neg-atively affects female reproductive fitness. CPEB3-depleted oocytes undergo meiosis normally but experience early embryonic arrest due to a disrupted transcriptome, leading to aberrant protein expression and the subsequent failure of embryonic transcription initiation. We found that CPEB3 stabilizes a subset of mRNAs with a significantly longer 3’UTR that is enriched in its distal region with cytoplasmic polyadenylation elements. Overall, our results suggest that CPEB3 is an im-portant maternal factor that regulates the stability and translation of a subclass of mRNAs that are essential for the initiation of embryonic transcription and thus for embryonic development.

overall_design: MII oocytes and 2-cell embryos from CPEB3 HET and CPEB3 KO  were collected for RNA-seq analysis.

first: Andrej

last: Susor


title: Phage SEP1 hijacks S. epidermidis stationary cells metabolism to replicate

city: Braga

first: Maria Daniela

last: Silva

address: Campus de Gualtar,

department: Centre of Biological Engineering

country: Portugal

institute: University of Minho

summary: In nature, bacteria often survive in a stationary state, with low metabolic activity. Phages use the metabolic machinery of the host cell to replicate and, therefore, their efficacy against non-dividing cells is usually limited. Nevertheless, it was previously shown that the Staphylococcus epidermidis phage SEP1 has the remarkable capacity to actively replicate in stationary-phase cells, reducing their numbers. Here, we studied for the first time the transcriptomic profiles of both exponential and stationary cells infected with SEP1 phage, using RNA-seq, to have a better understanding of this rare phenomenon. We showed that SEP1 successfully takes over the transcriptional apparatus of both exponential and stationary cells. Infection was, however, delayed in stationary cells, with genes within the gp142-gp154 module putatively implicated in host takeover. S. epidermidis responded to SEP1 infection by upregulating three genes involved in a DNA modification system, with this being observed already 5 min after infection in exponential cells and later in stationary cells. In stationary cells, a significant number of genes involved in translation and RNA metabolic and biosynthetic processes were upregulated after 15 and 30 min of SEP1 infection in comparison with the uninfected control, showing that SEP1 activates metabolic and biosynthetic pathways necessary to its successful replication.

overall_design: Exponential and stationary cultures of S. epidermidis 9142 were infected with SEP1 phage. RNA was extracted from samples collected shortly before (0 min) and at 5, 15, and 30 min post-infection and the transcriptomes analyzed by RNA-seq. Trimmed reads were aligned to both bacterial and phage genomes. Gene expression was normalized by calculating Reads per Kilobase per Million Mapped Reads (RPKM). Statistically significant alterations between different time-points (0 min vs 5, 15, and 30 min post-infection) and cell state (exponential vs stationary cells) were identified.

first: Maria

middle: D

first: Graca

last: Pinto

first: Angela

last: Franca

first: Joana

last: Azeredo

first: Luís

last: Melo


title: Salivary Gland Tissue Recombination Can Modify Cell Fate [scRNA-seq]

city: Bethesda

first: Rei

last: Sekiguchi

state: Maryland

address: 30 Convent Drive

department: CBS

institute: NIDCR

summary: Although mesenchyme is essential for inducing the epithelium of ectodermal organs, its precise role in organ-specific epithelial fate determination remains poorly understood. To elucidate roles of tissue interactions in cellular differentiation, we performed single cell RNA sequencing and imaging analyses of recombined tissues in which embryonic mouse salivary gland mesenchyme and epithelium were switched ex vivo. We found partial induction of molecules that define gland-specific acinar and myoepithelial cells in recombined salivary epithelium. Parotid epithelium (serous gland) recombined with submandibular mesenchyme (mixed serous-mucous, but predominantly mucous gland) began to express mucous acinar genes not intrinsic to the parotid gland. While myoepithelial cells do not normally line parotid acini, newly induced myoepithelial cells densely populated recombined parotid acini. However, mucous acinar and myoepithelial markers continued to be expressed in submandibular epithelial cells recombined with parotid mesenchyme. Consequently, some epithelial cells appeared to be plastic, such that their fate could still be altered in response to mesenchymal signaling, whereas other epithelial cells appeared to be already committed to a specific fate. We also discovered evidence for bidirectional induction: transcriptional changes were observed not only in the epithelium but also in the mesenchyme after heterotypic tissue recombination. For example, parotid epithelium induced the expression of muscle-related genes in submandibular fibroblasts that began to mimic parotid fibroblast gene expression. These studies provide the first comprehensive unbiased molecular characterization of tissue recombination approaches exploring the regulation of cell fate.

overall_design:
E13 submandibular and parotid glands (scRNA-seq). E13 salivary glands cultured for 6 days: Control (with or without Dispase treatment) and experimental (mesenchymal switching)
(Contributor) NIDCD/NIDCR Genomics and Computational Biology Core

first: Daniel

last: Martin

first: Shaohe

first: Andrew

last: Doyle

first: Kenneth

last: Yamada


title: Salivary Gland Tissue Recombination Can Modify Cell Fate

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

overall_design: Refer to individual Series


title: H3K27ac Profiling in LIRIL2R-Deficient and LIRIL2R-Sufficient Induced Regulatory T Cells

city: Turku

first: Riitta

last: Lahesmaa

address: Tykistökatu 6A

department: Turku Bioscience

country: Finland

institute: University of Turku

summary: Factors that determine the epigenetic landscape of regulatory T cells (Tregs), which is critical for their function, remain poorly understood. Long intergenic noncoding RNAs (LincRNA)s are important for shaping and maintaining the epigenetic landscape in different cell types. We identified a novel uncharacterised lincRNA, which we named LincRNA regulator of IL2RA (LIRIL2R), that is upregulated during early differentiation of human Tregs. RNA sequencing (RNA-seq) and data independent (DIA) proteomic analysis of LIRIL2R-deficient Tregs, with genome-wide chromatin occupancy analysis, identified IL2RA as direct LIRIL2R target. This nuclear lincRNA directly binds upstream of IL2RA locus and regulates its transcription by facilitating the accessibility of the locus. LIRIL2R-deficient Tregs had reduced expression of Treg-related genes, including FOXP3, IL2RA, STAT5, CTLA4 and PDCD1, as well as reduced suppressive capacity. Modulating LIRIL2R expression may hold promise for better therapeutic strategies against autoimmunity and cancer.

overall_design: CD4 T cells were isolated from human umbilical cord blood.  Cells were treated with either control non targeting (NT) antisense loked nucleic acid oligos (LNAs) or two LNAs (LNA1 and LNA2) targeting LIRIL2R. The cells were rested for 24 h followed by differentiation under iTreg conditions for 48 hours and CUT&Tag-seq was performed using H3K27ac antibody or control IgG antibody.

first: Syed

middle: B

last: Andrabi

first: Omid

last: Rasool

first: Ubaid

middle: U

last: Kalim

first: Sini

last: Junttila

first: Laura

middle: L

last: Elo


title: Salivary Gland Tissue Recombination Can Modify Cell Fate [bulk RNA-seq]

overall_design: E13 submandibular and parotid glands (bulk RNAseq). E13 salivary glands were cultured for 6 days and dissected.

last: Wang


title: H3K4me3 Profiling in Induced Regulatory T Cells

overall_design: CD4 T cells were isolated from human umbilical cord blood.  The cells were differentiated under iTreg conditions or control Tho conditions for 72 hours and CUT&Tag-seq was performed using H3K4me3 antibody or control IgG antibody.

first: Ritta


title: yHSC and oHSC 10X scRNAseq under ad lib, fasted, or fast refeed conditions to evaluate transcriptional impact of transient autophagy engagement in hematopoietic stem cells.

first: Paul

last: Dellorusso

summary: We observed that 24hr of fasting followed by a 24hr refeeding period improved the regenerative capacity of hematopoietic stem cells isolated from old mice, which was associated with an increase in glycolytic capacity. We sought to evaluate transcriptional changes in yHSC and oHSC compartment following different mouse feeding paradigms at single cell resolution. In yHSCs, AL and F/R groups showed overlapping transcriptional signatures with a distinct pattern for the fasted group. In contrast, in oHSCs, AL and fasted groups showed a strong overlap with a distinct transcriptional signature for the F/R group, reflective of differences in metabolic set points with age.

overall_design: Three young and three old mice were analyzied (one per feeding condition - Ad lib, 24hr Fast, 24hr Fast 24hr Refeed)

first: Emmanuelle

last: Passegue


title: AThi oHSC ATlo oHSC WES - Identifying clonal differences or differences in mutation burden between autophagy engaging and non-engaging hematopoietic stem cells during mouse aging.

summary: We performed paired WES from DNA isolated from AThi and ATlo hematopoietic stem cells (FACS sorted on the basis of GFP marker intensity, 33% GFPlo as ATHi, 33% GFPhi as ATlo) using tail DNA as an internal control to identify somatic mutations.  Few mutations were called at a variant allele frequency of ≥ 5% in this bulk sequencing approach, in line with recent results obtained with more sensitive methods. We also observed a high degree of shared mutations between AThi and ATlo oHSCs within each biological replicate, and no conserved mutations across biological replicates. The majority of called mutations were intronic or synonymous single nucleotide variants with limited expected consequences for their encoded proteins. Taken together, these results indicate that differing autophagy levels in oHSCs are not explained by clonal divergence or differential mutagenic burden, nor do they point to mutagenic burden as a driver of oHSC dysfunction in 24-month-old mice. They also do not provide evidence for clonal evolution between AThi and ATlo oHSC subsets.

overall_design: A total of 6 paired AThi/ATlo oHSC biological replicates were sequenced alongside tail DNA from each old Gfp-Lc3 mouse to establish baseline germline mutations.


title: yHSC AThi oHSC ATlo oHSC ATAC-Seq - Differences in Chromatin Accessibility in Autophagy engaging vs non-autophagy engaging old hematopoeitic stem cells (HSC) comapred to young HSC.

summary: Experiment was designed to assess differences in chromatin accessibility across the three indicated HSC populations of interest. By ATAC-seq, we observed large differences in chromatin accessibility between young and old HSCs. However, we found that the chromatin accessibility landscape of AThi and ATlo oHSCs was largely conserved, with no statistically different peaks between the two oHSC subsets and predominantly shared peaks differentially accessible between oHSC subsets and yHSC. Both oHSC subsets had a unidirectional increase in peak accessibility across greater than four thousand statistically significant loci compared to yHSCs, indicating epigenetic de-repression with aging. Pathway analysis of ATAC-seq peaks differing between oHSCs and yHSCs revealed epigenetic poising towards engagement of inflammatory responses, cell stress, and altered metabolic programs in both AThi and ATlo oHSC subsets, with some of the most differentially accessible peaks located in the promoters of genes involved in lipid metabolism. These results suggest that cellular responses associated with oHSC dysfunction are, to some extent, embedded in their epigenetic state.

overall_design: Paired AThi/ATlo oHSC biological replicates and 5 yHSC replicates were generated over 2 isolation and transposition days for ATAC-seq.


title: yHSC and oHSC scRNAseq with oHSC AThi ATlo Index Sorting. Assessing the transcriptional changes associated with autophagy engagement in old hematopoietic stem cells at single cell resolution.

summary: 10X genomics single cell analysis of yHSC vs oHSC from Gfplc3/Gfplc3 mice with index sorted SMART-Seq2 sequencing to map AThi oHSC vs ATlo oHSC based on GFP-LC3 marker levels. 10X Genomics data harmonized by nearest neighbor integration and uniform manifold approximation and projection (UMAP) representation distinguished yHSC from oHSC, with the largest transcriptional differences observed in the G0/G1 cell cycle phase cluster. Within oHSCs, AThi oHSCs were almost exclusively observed in the G0/G1 cluster, whereas ATlo oHSCs were spread across the activation continuum, with cells still in the G0/G1 cluster found more proximal to S cluster cells.

overall_design: For 10X genomics, two biological replicates each of yHSC and oHSC were preprared using 30,000 cells per sample. For SmartSeq2, 96 HSCs from the same samples were sorted into individual wells recording information on surface markers and GFP-LC3 level.


title: Genome-Wide Binding Analysis of LIRIL2R in Induced Regulatory T Cells

overall_design: CD4 T cells were isolated from human umbilical cord blood.The cells were differentiated under iTreg conditions for 48 hours and ChIRP-seq was performed.

first: Mohd

last: Khan

first: Meraj

middle: H


title: yHSC AThi oHSC ATlo oHSC Bulk RNA-seq - Understanding transcriptional changes between autophagy engaging and non-engaging hematopoietic stem cells during mouse aging.

summary: We sought to understand the transcriptional profile of autophagy engaging vs non-engaging old hematopoietic stem cells (HSC) referenced against young HSCs. Bulk RNA-seq analyses indicated a large transcriptional divergence between the two oHSC subsets, with pathway analyses of differentially expressed genes (DEG) demonstrating enrichment in inflammatory signaling in AThi oHSCs and in oxidative metabolism signaling in ATlo oHSCs. Ingenuity Pathway Analysis (IPA) inferred that several inflammation-coupled pro-autophagy regulators including p53, Bnip3l, Nupr1, and Nlrp3[22,23], in addition to quiescence enforcing checkpoints FoxO3a, Rb1 and Cdkn1a, were activated in AThi oHSCs.

overall_design: RNA from HSCs of two young (8-12wks) and three old GFP-LC3 (>24mo) mice was processed for bulk RNA-sequencing. For the old HSC biological replicates, the 33% GFPlo (AThi) and 33% GFPhi (ATlo) HSCs were isolated by FACS prior to RNA isolation.


title: Chromatin Accessibility Analysis of LIRIL2R-Deficient and LIRIL2R-Sufficient Induced Regulatory T Cells

overall_design: CD4 T cells were isolated from human umbilical cord blood. Cells were treated with either contrl non targeting (NT) antisense loked nucleic acid oligos (LNAs) or LNAs targeting LIRIL2R. The cells were differentiated under iTreg conditions for 24/72 hours and ATAC-seq was performed.


title: MATRIN3 deficiency leads to cGAS-STING activation (NanoString)

first: Stefan

last: Muljo

address: 10 Center Drive

institute: NIH, NIAID

summary: To investigate the role of Matrin 3 (MATR3) in innate immune response, we analyzed and compared MATR3 KO clones versus wildtype HAP1 cells.

overall_design: Comparative gene expression profiling analysis of Nanostring digital count data between MATR3 KO versus wildtype HAP1 cells.

first: Zohir

last: Islam

first: Bryan

last: Chim

first: Amir

last: Foroushani


title: MATRIN3 deficiency leads to cGAS-STING activation (Illumina RNA-Seq)

summary: To investigate the role of Matrin 3 (MATR3) in the innate immune response we analyzed and compared MATR3 KO Clones versus wildtype HAP1 cells.

overall_design: Comparative gene expression profiling analysis of MATR3 KO versus wildtype HAP1 cells using Illumina RNA-sequencing.


title: Gene Expression Analysis of LIRIL2R-Deficient and LIRIL2R-Sufficient Induced Regulatory T Cells

overall_design: CD4 T cells were isolated from human umbilical cord blood. Cells were treated with either contrl non targeting (NT) antisense loked nucleic acid oligos (LNAs) or LNAs targeting LIRIL2R. The cells were differentiated under iTreg conditions for 24/48/72 hours and RNA-seq was performed.

first: Senthil

last: Palani


title: Autophagy engaging and non-engaging hematopoietic stem cells


title: Developmentally regulated alternate 3’ end cleavage of nascent transcripts controls dynamic changes in protein expression in an adult stem cell lineage

status: Public on May 14 2024

city: Stanford

first: Cameron

middle: W

last: Berry

state: California

address: 279 W. Campus Drive, B300

department: Developmental Biology

institute: Stanford University

summary:
To determine the extent of alternative 3' end cleavage during Drosophila spermatogenesis, we performed 3' end sequencing of Drosophila testes from flies at different time points post heat shock (pHS) that were mutant for bam and also had a heat shock inducible transgene expressing Bam.
We then performed Polysome profiling of testes at 24, 48 and 72 hours PHS and created 3' end sequencing libraries from the free, 40S, 60s, 80S, 2-3 ribosomes, and 4+ ribosomes fractions for each dataset

overall_design: 3' end sequencing of testes from flies (genotype: bam-/-; hsBam) 0, 16, 24, 32, 48, 72 hours PHS and 3' end sequencing of Polysome Fractions (free, 40S, 60S, 80S, 2-3ribosomes, 4+ ribosomes) of testes from flies 24, 48 and 72 hours PHS

first: Berry

last: Cameron

first: Fuller

last: Margaret


title: MATRIN3 deficiency leads to cGAS-STING activation (MinION RNA-Seq)

summary: To investigate the role of Matrin 3 (MATR3) in innate immune response we analyzed and compared MATR3 KO Clones versus wildtype HAP1 cells.

overall_design: Comparative gene expression profiling analysis of MATR3 KO versus wildtype HAP1 cells using Oxford Nanopore long-read RNA-sequencing.


title: The landscape of RNA-chromatin interaction reveals small non coding RNAs as essential mediators of leukemia maintenance [ChIP-seq]

city: Stuttgart

first: Haiyang

last: Yun

address: Auerbachstraße 112

country: Germany

institute: Robert Bosch Center for Tumor Diseases

summary: RNA constitutes a large fraction of chromatin. Spatial distribution and functional relevance of most of RNA interactions within the chromatin remain unknown. We established a landscape analysis of RNA-chromatin interactions in human acute myeloid leukemia (AML). In total more than 50 million interactions were captured in an AML cell line. Protein-coding mRNAs and long non-coding RNAs (lncRNAs) exhibited a substantial number of interactions with chromatin in cis suggesting transcriptional activity. In contrast, small nucleolar RNAs (snoRNAs) and small nuclear RNAs (snRNAs) associated with chromatin predominantly in trans suggesting chromatin specific functions. Of note, snoRNA-chromatin interaction is associated with chromatin modification and is independent of the classical snoRNA-RNP complexes. Two non-canonical C/D box snoRNAs, namely SNORD118 and SNORD3A, displayed high frequency of trans-association with chromatin. The transcription of SNORD118 and SNORD3A was increased upon leukemia transformation and enriched in leukemia stem cells, but decreased during myeloid differentiation. Suppression of SNORD118 and SNORD3A impaired leukemia cell proliferation and colony forming capacity in AML cell lines and in primary AML blast samples. Notably, this effect was leukemia specific with minimal impact on healthy CD34+ hematopoietic stem and progenitor cells (HSPCs). These findings highlight the functional importance of chromatin-associated RNAs overall and in particular of SNORD118 and SNORD3A in maintaining leukemia propagation.

overall_design: Chromatin immunoprecipitation coupled with next generation sequencing (ChIP-seq) for Fibrillarin occupancy on chromatin and histone modifications (including H3K27ac and H3K9me3) in three acute myeloid leukemia cell lines – MV4-11, OCI-AML2 and Kasumi-1, transduced with lentiviral shRNA control or targeting Fibrillarin.

first: Christian

last: Rohde

first: Carsten

last: Müller-Tidow


title: CRISPR Screening Uncovers a Long-Range Enhancer for ONECUT1 in Pancreatic Differentiation and Links a Diabetes Risk Variant

first: Samuel

middle: Joseph

last: Kaplan

state: New York

address: 430 E 67th St

department: Developmental Biology Program

institute: Sloan Kettering Institute

overall_design: Refer to individual Series


title: The landscape of RNA-chromatin interaction reveals small non coding RNAs as essential mediators of leukemia maintenance [ATAC-Seq]

overall_design: Chromatin accessibility was profiled by performing ATAC-seq in MV4-11 cells transduced with lentiviral shRNA control or targeting SNORD118.


title: The landscape of RNA-chromatin interaction reveals small non coding RNAs as essential mediators of leukemia maintenance [iMARGI]

overall_design: Genome-wide profiling of RNA-chromatin interaction in MV4-11 cells by running in situ mapping of RNA–genome interactome (iMARGI).


title: The landscape of RNA-chromatin interaction reveals small non coding RNAs as essential mediators of leukemia maintenance [ChIRP]

overall_design: Chromatin isolation by RNA purification (ChIRP) coupled with next generation sequencing for U1, U3 and U8 occupancy on chromatin in MV4-11 cells.


title: Male autism spectrum disorder is linked to brain aromatase disruption by prenatal BPA in multimodal investigations and 10HDA ameliorates the related mouse phenotype (10HDA)

city: Melbourne

first: Wah Chin

last: Boon

state: Victoria

address: 30 Royal Parade Parkville

country: Australia

institute: The Florey Institute of Neuroscience and Mental Health

summary: Primary cortical neurons were obtained from male Cyp19-EGFP mouse embryos at gestational day 15.5. Embryos were genotyped for SRY to determine sex, and only male embryos were used. Cells were seeded in 24-well plates containing 12 mm glass coverslips, coated with 100 µg/mL poly D-lysine to a density of ~0.45 x 106 cells/well and incubated in a humidified CO2 incubator (5% CO2, 37°C). Primary cortical from Cyp19-EGFP mouse embryos were obtained on GD 15.5. Neuronal cell cultures were treated with vehicle or 1mM 10HDA (Matreya, PA ,USA)

overall_design: To investigate the effect of 10HDA on RNA expression in cortical primarly cell culture (obtained from Cyp19-EGFP mice at gestational day 15.5), we treated primary cortical neurons obtained from mice with either vehicle (DSMO) or 1mM 10HDA.


title: Detection of the tRNA-derived fragments in human pancreatic islets of control individuals

city: Lausanne

first: Cecile

last: Jacovetti

address: Rue du Bugnon 9

department: Fundamental Neurosciences

institute: University of Lausanne

summary: Cytosolic and mitochondrial fragments that arise from the cleavage of tRNAs and mt-tRNAs, respectively, have been identified as potential novel regulators of cellular functions. In this study we identified hundreds of fragments in the pancreatic islets of healthy human individuals. We were able to annotate 3593 tRFs (16-55 base pairs) present in the islets of healthy human individuals (we considered a minimum of 5 cpm values in each of the 3 samples). Interestingly, the vast majority of the tRFs whose levels are altered in pancreatic islets of prediabetic NOD mice and in extracellular vesicles released by activated CD4+ T lymphocytes, are conserved in humans and were detected in human pancreatic islets. This suggests a possible conservation of the findings of this study in a human context.

overall_design: In order to characterize the expression profile of tRFs (tRNA-derived fragments) in  human islets, we isolated total RNA from pancreatic islets of control healthy individuals.

first: Flora

last: Brozzi

first: Cristina

last: Cosentino

first: Véronique

last: Menoud

first: Kejing

last: Wu

first: Mustafa

last: Bayazit

first: Baroj

last: Abdulkarim

last: Iseli

first: Nicolas

last: Guex

first: Claudiane

last: Guay

first: Romano

last: Regazzi


title: Tn-seq of Staphylococcus aureus exposed to Calprotectin

city: Nashville

first: Eric

middle: P

last: Skaar

state: Tennessee

address: 1161 21st Avenue South

department: Pathology, Microbiology, and Immunology

institute: Vanderbilt University Medical Center

summary: The host protein calprotectin inhibits the growth of a variety of bacterial pathogens through metal sequestration in a process known as 'nutritional immunity'. Staphylococcus aureus growth is inhibited by calprotectin in vitro and calprotectin is localized in vivo to staphylococcal abscesses during infection. However, the staphylococcal adaptations that provide defense against nutritional immunity and the role of metal-responsive regulators are not fully characterized. In this work, we define the transcriptional response of S. aureus and the role of the metal-responsive regulators, Zur, Fur, and MntR, in response to metal limitation by calprotectin exposure. Additionally, we identified genes affecting the fitness of S. aureus during metal limitation through a Transposon sequencing (Tn-seq) approach. Loss of function mutations in clpP, which encodes a proteolytic subunit of the ATP-dependent Clp protease, demonstrate reduced fitness of S. aureus to the presence of calprotectin. ClpP contributes to pathogenesis in vivo in a calprotectin-dependent manner. These studies establish a critical role for ClpP to combat metal limitation by calprotectin and reveal the genes required for S. aureus to outcompete the host for metals.

overall_design: This study sought to uncover genes that affect the fitness of S. aureus when the bacteria experience calprotectin-mediated metal starvation by performing a genome-wide Tn-seq screen on bacteria grown in the presence of WTCP or ∆s1CP

first: Valeria

last: Reyes Ruiz

first: Jeffrey

last: Freiberg

first: Andy

last: Weiss

first: Erin

last: Green

first: Mary-Elizabeth

last: Jobson

first: Emily

last: Felton

first: Lindsey

middle: N

last: Shaw

first: Walter

middle: J

last: Chazin


title: The effect of mug174 deletion or red1 deletion mutation on gene expression in the fission yeast Schizosaccharomyces pombe

city: Shanghai

first: Tomoyasu

last: Sugiyama

address: 393 Middle Huaxia Road

institute: ShanghaiTech University

summary: Our characterization of Mug174 using genetic and biochemical approaches revealed that Mug174 is the Coilin (an integral component of the Cajal body in multicellular organisms) ortholog in the fission yeast S. pombe. To investigate the Mug174 role in shaping transcriptome, we delete the mug174 gene and performed RNA-seq. We found that intron and exon-intron junction reads were elevated in mug174∆, indicating that pre-mRNA splicing is compromised in the absence of Mug174. Red1 is not a splicing factor, but it promotes the degradation of unspliced mRNAs. By comparing the RNA-seq data from mug174∆ and red1∆, we found that Mug174 and Red1 share the target introns. Our analyses are consistent with the previous finding that the Cajal body is required for proper pre-mRNA splicing, thereby supporting our idea that Mug174 is indeed the ortholog of Coilin.

overall_design:
We constructed mug174∆ or red1∆ cells using the standard gene replacement method in a parental wild-type S. pombe strain.
We then isolated total RNA from vegetative wild-type, mug174∆, and red1∆ cells twice (two biological replicates) and performed gene expresssion profiling analyses using data obtained from two independent RNA-seq of the three S.pombe strains.

first: Xiaoling

last: DENG

first: Attila

last: Horváth

first: Tamas

last: Fischer

last: SUGIYAMA


title: The coordinated adaptation of Staphylococcus aureus to calprotectin dependent metal sequestration [RNA-Seq]

overall_design: To study the response of S. aureus to nutrient metal limitation by treatment with CP, a global transcriptomic analysis by RNA-seq was performed on a wild-type (WT) S. aureus strain, or metal-sensing regulator mutants (Fur, Zur, MntR) grown in media with either WT calprotectin (WTCP) which restricts bacteria from Zn, Fe, Mn, Cu, and Ni, or a  His6 site mutant calprotectin (∆s1CP) that only restricts Zn and Cu. A vehicle control using CP buffer and TSB was used.

middle: M


title: Human trophectoderm becomes multi-layered by internalization at the polar region

city: Edinburgh

first: Lawrence

middle: E

last: Bates

address: Crewe Road

department: Human Genetics Unit

institute: University of Edinburgh

summary: The human blastocyst is composed of outer trophectoderm and an inner cell mass, which segregates into epiblast and hypoblast. scRNA sequencing of human blastocyst cells was performed to identify subpopulations following immunosurgery to remove outer cells.

overall_design: Donated human embryos from IVF clinics were subjected to immunosurgery and surviving cells were analysed by single cell sequencing

first: Elena

last: Corujo-Simon

first: Stephen

last: Clark


title: Effect of lactic acid bacterium 2004 on transcription profile in wildtype animals and daf-16 mutants.

city: Kobe

first: Masaharu

last: Uno

address: 2-2-3, Minatojima-minamimachi, Chuo-ku

department: BDR

country: Japan

institute: RIKEN

summary: Examine the effect of lactic bacterium 2004 supplementation on expression profile in wld type animals.

overall_design: Total RNA was isolated using TRIzol reagent (Invitrogen) from C. elegans Bristol strain N2 cultured in S basal medium with E. coli OP50 or L. paracasei subsp. paracasei 2004 for three days from three days after hatching. RNA-seq library preparation and RNA sequencing were performed by Macrogen Inc.

first: Saya

last: Kishimoto

first: Masanori

last: Nono

first: Eisuke

last: Nishida


title: T cell mediated microglia activation triggers myelin pathology in a model of amyloidosis [MERFISH]

city: Munich

first: Peter

last: Androvic

state: Bavaria

address: Feodor-Lynen-Strasse 17

institute: LMU Hospital, Institute for Stroke and Dementia Research

summary: Age-related myelin damage induces inflammatory responses, yet its involvement in Alzheimer's disease remains uncertain, despite age being a major risk factor. Using a mouse model of Alzheimer's disease, we found that amyloidosis itself triggers age-related oligodendrocyte and myelin damage. Mechanistically, CD8+ T cells promote the progressive accumulation of abnormally interferon-activated microglia that display myelin-damaging activity. Thus, our data suggest that immune responses against myelinating oligodendrocytes may contribute to neurodegenerative diseases with amyloidosis.

overall_design: Brain sections were collected from mouse model of amyloidosis and analyzed by MERFISH

first: Ozgun

last: Gokce


title: Non-classical CD45RBlo memory B-cells are the majority of circulating antigen-specific B-cells following mRNA vaccination and COVID-19 infection.

city: Suita

first: Daisuke

last: Motooka

state: Osaka

address: 3-1, Yamadaoka

institute: NGS core facility, Research Institute for Microbial Diseases, Osaka University

summary: Resting memory B-cells can be divided into classical and non-classical groups based on differential expression of markers such as CD27 and CD11c, while activated memory B-cells express a combination of markers, making their ontogeny hard to determine. Here by longitudinal analysis of COVID-19, bacterial sepsis, and BNT162b2 mRNA vaccine recipients by mass cytometry and CITE-seq we describe a three-branch structure of resting B-cell memory consisting of “classical” CD45RB⁺ memory and two branches of CD45RB^lo memory further defined by expression of CD23 and CD11c respectively. Stable differences in CD45RB upon activation allowed tracking of activated B-cells and plasmablasts derived from CD45RB⁺ classical and CD45RB^lo non-classical memory B-cells. In both COVID-19 patients and mRNA vaccination, CD45RB^lo B-cells formed the majority of SARS-CoV2 specific memory B-cells while CD45RB⁺ memory was most strongly activated by bacterial Sepsis. These results suggest that diverse non-classical CD45RB^lo memory B-cells consisting of branches of CD11c⁺Tbet⁺ and CD23⁺ fractions form a critical part of responses to viral infection and vaccination.

overall_design: Frozen human PBMCs from COVID-19 patients, Sepsis patients or mRNA vaccinated individuals were defrosted then hashtagged with TotalSeqC hashtags. Hashtagged samples were then mixed and stained with Flourecently labelled antibodies, surface TotalSeqC antibodies and SARS-CoV2 spike protein-Strepaaviding tetramers. IgD negative B-cells were FACS sorted as IgD-CD19+CD14-CD4-.

first: James

last: Wing


title: Inflammatory Alterations to Renal Lymphatic Endothelial Cell Gene Expression in Mouse Models of Hypertension

city: Bryan

first: Brett

last: Mitchell

address: 8441 Riverside Pkwy

department: Medical Physiology

institute: Texas A&M University

summary: To evaluate the specific changes that renal LECs undergo in hypertension (HTN), we performed single-cell RNA sequencing. Using the angiotensin II-induced and salt-sensitive mouse models of HTN, we isolated renal CD31+ and podoplanin+ cells from hypertensive and normotensive mice. Sequencing of these cells revealed three distinct cell types (LECs, myeloid immune cells, and an unusual third population we dubbed multipotent nephron-associated support cells) with unique expression profiles.

overall_design: Mice underwent the angiotensin-II and salt sensitive models of hypertension and had their kidneys removed and aggregated per group (A2 HTN, A2 control, SS HTN, SS control). Kidneys were digested and turned to a single-cell suspension, followed by the isolation of CD31+ and podoplanin+ cells by magnetic separation. We sought to investigate the effects of hypertension on CD31+/podoplanin+ renal lymphatic endothelial cells and to investigate model-specific differences.

first: Justin

middle: G

last: McDermott

first: Bethany

last: Goodlett

first: Heidi

middle: A

last: Creed

first: Shobana

last: Navaneethabalakrishnan

first: Joseph

last: Rutkowski


title: The tumor suppressor SMAD4 acts through WNT signaling in progression from benign lesions to oral squamous cell carcinoma

first: Anna

last: Mean

address: 1161 21st Ave S

department: Department of Surgery

summary: SMAD4 is a tumor suppressor mutated or silenced in multiple cancers including oral squamous cell carcinoma (OSCC). Human clinical samples and cell lines, mouse models and organoid culture were used to investigate the role that SMAD4 plays in progression from benign disease to invasive OSCC. Human OSCC lost detectable SMAD4 protein within tumor epithelium in 24% of cases and this loss correlated with worse progression free survival independent of other major clinical and pathological features. In a mouse model, Kras mutation induced benign papillomas while additional loss of Smad4 expression resulted in rapid development of invasive carcinoma. Examining regulatory pathways in organoids from SMAD4+ and SMAD4- mouse tumors, we found that either loss of SMAD4 or inhibition of TGFb signaling upregulated the WNT pathway and altered the extracellular matrix. The gene signature of the mouse tumor organoids lacking SMAD4 was highly similar to the gene signature of human head and neck squamous cell carcinoma. In summary, this work has uncovered novel mechanisms by which SMAD4 acts as a tumor suppressor in OSCC.

overall_design: In this study, we measured the whole transcriptome gene expression of 12 mouse mouse organoid samples with or without SMAD4 expression and treated with or without SB431542.

first: Jing

last: Yang

middle: S

last: Lewis

first: Jinghuan

last: Zi

first: Thomas

last: Andl

first: Ethan

last: Lee

first: Claudia

first: Qi

last: Liu

first: Robert

last: Beauchamp

last: Means


title: Investigation of the Mechanism of Action by Comprehensive Transcriptome Analysis

city: Minoh

first: Kazuhide

last: Nakamura

address: 5-1-35, Saito-aokita,

department: Department of Medical Innovations, Osaka Research Center for Drug Discovery

institute: Otsuka Pharmaceutical Co.,Ltd.

summary: To elucidate the SLFN12 RNase effector function, we used whole-genome microarray expression profiling as a discovery platform to identify genes that potentially drive anticancer efficacy upon OPB-171775 treatment. The compound was treated for 8 or 24 hours in control PFSK1 cells and siSLFN12 knockdown PFSK1 cells.  Gene set enrichment analysis (GSEA) revealed a significant enrichment of gene sets associated with ribosomal function and the endoplasmic reticulum (ER) stress response in OPB-treated cells compared to control cells.

overall_design: PFSK1 cells were treated with siControl and siSLFN12 for 48 hours. Cells were then treated with OPB-171775 or DMSO control for 8 or 24 hours. Three independent experiments were performed at each time point (8 or 24 hours).

first: Takashi

last: Watanabe

first: Yuki

last: Kuniyoshi


title: Tagln+ progenitors contribute to the development and maintenance of nucleus pulposus

city: Pok Fu Lam

first: Peikai

last: Chen

address: Sassoon Road 5

department: Faculty of Medicine

country: Hong Kong

institute: The University of Hong Kong

summary: Failure of intervertebral disc components, e.g. the nucleus pulposus causes intervertebral disc disease and associated low-back pain. Despite the high prevalence of disc disease, the changes in intervertebral disc cells and their regenerative potential with ageing and degeneration are not fully elucidated. Understanding the cell lineage, cell differentiation and maintenance of nucleus pulposus may have therapeutic application for the regeneration of degenerative disc, with significant impact for healthy ageing. Here we found that TAGLN expressing cells are present in human healthy nucleus pulposus, but diminish in degenerative disc. By lineage analyses in mice, we found cells in the nucleus pulposus are derived from a peripherally located population of notochord-derived Tagln expressing cells (PeriNP cells). The PeriNP cells are proliferative and can differentiate into the inner part of the nucleus pulposus. The Tagln+ cells and descendants diminish during aging and puncture induced disc degeneration. The maintenance and differentiation of PeriNP cells is partially regulated by Smad4 dependent signaling. Removal of Smad4 by nucleus pulposus specific Cre (Foxa2mNE-Cre), results in decreased Tagln+ cells and abnormal disc morphology, leading to disc degeneration. Our findings propose that the PeriNP Tagln expressing cells are a pool of notochord-derived progenitors that are important for maintenance of the nucleus pulposus and provide insights for regenerative therapy against intervertebral disc degeneration.

overall_design: The Tagln-CreERt2;R26tdTomato mice were activated with tamoxifen at P5 stage, and IVDs from lumbar region (L1 to L6 levels) and tail region (proximal T5 to T9 levels) were harvested at P10 and 8-week stages. Muscles and blood vessels were removed as much as possible and blood cells were harshly washed away with HBSS (Gibco). Remaining IVD tissues were cut into small fragments and digested with 0.25% Dispase (Worthington) and 0.25% type II collagenase (Worthington) in HBSS (5ml in total) at 37ºC with gentle shaking for 1 hour. Digestion was filtered with a 40µm cell strainer. The cells washed with HBSS twice and sent for sequencing using Chromium single cell platform (10X Inc.) at the Centre for PanorOmic Sciences (CPOS, HKU).

first: Tan

last: Zhijia

first: Kathryn

last: Cheah


title: Microenvironment Shapes Small Cell Lung Cancer Neuroendocrine States and Presents Therapeutic Opportunities

status: Public on May 13 2024

first: Anish

last: Thomas

state: MARYLAND

address: Center for Cancer Research, National Cancer Institute, Building 10 Room 4-5330

department: National Cancer Institute

institute: National Institutes of Health

summary: Small-cell lung cancer (SCLC) is the most fatal form of lung cancer. Intra-tumoral heterogeneity, marked by neuroendocrine (NE) and non-neuroendocrine (non-NE) cell states, defines SCLC, but the drivers of SCLC plasticity are poorly understood. To map the landscape of SCLC tumor microenvironment (TME), we apply spatially resolved transcriptomics and quantitative mass spectrometry-based proteomics to metastatic SCLC tumors obtained via rapid autopsy. The phenotype and overall composition of non-malignant cells in the tumor microenvironment (TME) exhibits substantial variability, closely mirroring the tumor phenotype, suggesting TME-driven reprogramming of NE cell states. We identify cancer-associated fibroblasts (CAF) as a crucial element of SCLC TME heterogeneity, contributing to immune exclusion, and predicting exceptionally poor prognosis. Together, our work provides a comprehensive map of SCLC tumor and TME ecosystems, emphasizing their pivotal role in SCLCs adaptable nature, opening possibilities for re-programming the intercellular communications that shape SCLC tumor states.

overall_design: Rapid autopsy derived patient tumor tissues were subjected to RNA-seq

first: Desai

last: Parth

last: Anish

first: Kumar

last: Rajesh


title: ILC2 development requires GATA3-related super-enhancers after lineage commitment

city: Chiba-shi

first: Arifumi

last: Iwata

state: Chiba

address: 1-8-1 Inohana, Chuou-ku

department: Department of Allergy and Clinical Immunology

institute: Chiba University

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

Group 2 innate lymphoid cells (ILC2) are a subset of innate lymphocytes that produce type 2 cytokines, including IL-4, IL-5, and IL-13. GATA3 being a critical transcription factor for ILC2 development at multiple stages. However, when and how GATA3 is induced to the levels required for ILC2 development remains elusive. Herein, we identified ILC2-specific tandem GATA3-related super-enhancers (G3SE) that induce high GATA3 in ILC2-committed precursors. G3SE-deficient mice exhibit ILC2 loss in the BM, lung, liver, and small intestine, a slight ILC1 increase, and no difference in ILC3, LTi, NK cells, nor Th2 cells. Single-cell RNA-sequencing revealed that GATA3 induction in late ILC2-committed precursors depends on G3SEs and is required for the transition to mature ILC2. Cnot6l, part of the CCR4-Not deadenylase complex, is a possible GATA3 target during ILC2 development. Our findings indicate a novel regulatory mechanism for GATA3 expression during ILC2 development.

overall_design: Refer to individual Series


title: Selective CAR-T cell mediated B cell depletion suppresses interferon signature in SLE [Bcells]

city: Berlin

first: Artur

last: Wilhelm

address: Virchowweg 11

department: Rheumatology and clinical immunology

institute: Charite Berlin

summary: Applying advanced molecular profiling together with highly specific targeted therapies offers the possibility to better dissect the mechanisms underlying immune mediated inflammatory diseases such as systemic lupus erythematosus (SLE) in humans. Here we apply a combination of single cell RNA sequencing and T/B cell repertoire analysis to perform an in-depth characterization of molecular changes in the immune-signature upon CD19 CAR T cell-mediated depletion of B cells in SLE patients. The resulting datasets do not only confirm a selective CAR T cell-mediated reset of the B cell response, but simultaneously reveal consequent changes in the transcriptional signature of monocyte and T cell subsets that respond with a profound reduction in type 1 interferon signaling. Our current data thus provide evidence for a causal relationship between the B cell response and the increased interferon signature observed in SLE and additionally demonstrate the usefulness of combining targeted therapies and novel analytic approaches to decipher molecular mechanisms of immune-mediated inflammatory diseases in humans.

overall_design: We generated single cell sequencing data of isolated B cells from SLE patients prior to and after anti CD19 CAR-T cell therapy

first: David

last: Chambers

first: Fabian

last: Müller

first: Aline

last: Bozec

first: Ricardo

last: Grieshaber-Bouyer

last: Winkler

first: Dimitrios

last: Mougiakakos

first: Andreas

last: Mackensen

first: Georg

last: Schett

first: Gerhard

last: Krönke


title: Biochemical properties of chromatin domains define genome compartmentalization [III]

city: Milan

first: Chiara

last: Lanzuolo

address: Via Francesco Sforza 35

country: Italy

institute: CNR and Istituto Nazionale Genetica Molecolare

summary: Chromatin three-dimensional (3D) organization inside the cell nucleus determines the separation of euchromatin and heterochromatin domains. Their segregation results in the definition of active and inactive chromatin compartments, whereby the local concentration of associated proteins, RNA and DNA results in the formation of distinct subnuclear structures. Thus, chromatin domains spatially confined in a specific 3D nuclear compartment are expected to share similar epigenetic features and biochemical properties, in terms of accessibility and solubility. Based on this rationale, we developed the 4f-SAMMY-seq to map euchromatin and heterochromatin based on their accessibility and solubility, starting from as little as 10,000 cells. Adopting a tailored bioinformatic data analysis approach we reconstruct also their 3D segregation in active and inactive chromatin compartments and sub-compartments, thus recapitulating the characteristic properties of distinct chromatin states. A key novelty of the new method is the capability to map both the linear segmentation of open and closed chromatin domains, as well as their 3D compartmentalization in one single experiment.

overall_design: SAMMY-seq analysis on mouse C2C12 myoblasts cell line using the 4fraction protocol (4f-SAMMY-seq), two replicates on 2M cells

first: Federica

last: Lucini

first: Cristiano

last: Petrini

first: Elisa

last: Salviato

first: Koustav

last: Pal

first: Valentina

last: Rosti

first: Francesca

last: Gorini

first: Philina

last: Santarelli

first: Roberto

last: Quadri

first: Giovanni

last: Lembo

first: Giulia

last: Graziano

first: Emanuele

last: Di Patrizio Soldateschi

first: Ilario

last: Tagliaferri

first: Eva

last: Pinatel

first: Endre

last: Sebastyén

first: Luca

last: Rotta

first: Francesco

last: Gentile

last: Vaira

last: Ferrari


title: Osr2 functions as a biomechanical checkpoint to aggravate CD8+ T cell exhaustion in tumor

city: Xiamen

first: Dawang

last: Zhou

state: Fujian

address: Xiang'an south rd, Xiang'an district

department: Life Science

institute: Xiamen University

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


title: Integrated OMICs approach reveals energy metabolism pathway is vital for Salmonella Pullorum survival within the egg white

city: Hangzhou

first: Xiamei

last: Kang

state: Zhejiang

address: No. 388, Yuhangtang Road

institute: Zhejiang University

summary: Purpose: To elucidate the survival strategies in egg white of Salmonella Pullorum, a host-restricted pathogen with vertical transmission capability. Methods: The logarithmic-phase wild-type and Δfim mutant strains were inoculated into LB medium and egg white (final concentration 80%) and statically cultured at 42°C for 6 hours. RNA samples were then extracted for sequencing. Three biological replicates were carried out per sample. RNA sample was sequenced with Illumina HiSeq 2000 System. EdgeR method was used to calculate the differentially expressed genes. qRT–PCR and gene mutation were used to validate RNA-seq result. Results: A total of 12 samples were sequenced. The average clean bases of each sample was 3.63 Gb, and the average comparison rate with the reference genome was more than 90%. Compared with LB broth, there were 1606 differentially expressed genes (FDR ≤0.05 and |Log2Fold change| ≥1) in egg white with 762 genes upregulated and 844 genes downregulated. These accounted for about 35% of the whole genome indicating a large shift in the transcriptional response to egg white. qRT–PCR result of 7 selected genes highly consistent with the RNA-seq results. Five mutants showed decreased survival ability in egg white, suggested that the DEGs from the RNAseq results correlated significantly with the egg white resistance phenotype.Moreover, a significant downregulation of all genes within the fim gene cluster was observed and the RNA-seq analysis of Δfim mutant in the egg white was further conducted. In comparison with wild-type in egg white, the fim mutant showed 36 differentially expressed genes, with most of them associated with energy metabolism pathways.

sample_organism: Salmonella enterica subsp. enterica serovar Pullorum

overall_design: To understand the mechanisms employed by Salmonella Pullorum to withstand the challenging environment in egg white, three biological replicates of wild-type and Δfim mutant incubated in LB broth and egg white (totally 12 samples) were conducted RNA sequncing.

first: Hongli

last: An

first: Baikui

last: Wang

first: Min

last: Yue


title: Biochemical properties of chromatin domains define genome compartmentalization [II]

first: Chiara

last: Lanzuolo

overall_design: SAMMY-seq analysis on primary fibroblast cell lines, using two different protocols with three biological replicates each. For the 4-fractions protocol, we performed also replicates on 50k cells and on 10k cells

first: Federica

last: Lucini

first: Cristiano

last: Petrini

first: Elisa

last: Salviato

first: Koustav

last: Pal

first: Valentina

last: Rosti

last: Gorini

last: Santarelli


title: Auxin-mediated regulation of susceptibility to toxic metabolites, c-di-GMP levels, and phage infection in the rhizobacterium Serratia plymuthica

city: Little Rock

first: Zulema

last: Udaondo

state: AR

address: 501 Jack Stephens Drive

department: Biomedical Informatics

institute: UAMS

summary: To investigate the broader effects of exogenous IAA on gene expression in S. plymuthica A153, we used transcriptome sequencing (RNA-seq) to analyze changes in the global transcriptome of A153 in response to IAA treatment.

overall_design: RNA sequencing was done at the GENYO Research Center (Granada, Spain). Prior to preparation of the RNA library, ribosomal RNAs were removed from the samples using the RiboZeroTM kit (Epicentre, Gram-Negative-Bacteria#MRZGN126) following the manufacturers’ instructions. Subsequently, samples were processed with the TruSeq Stranded Total RNA Library Prep Kit (Illumina) following the provided sample preparation guide. The final library (adapter and index included) was validated using the DNA-specific chip Agilent DNA 1000.

first: Miguel

last: Matilla