title: A phase separated active subnuclear compartment safeguards repressive chromatin domains [TSA-seq]

city: Zurich

first: Raffaella

last: Santoro

address: Winterthurerstrasse 190

department: Dep. of Molecular Mechanisms  of Disease

institute: University of Zurich

summary: The nucleus is composed of membrane-less subnuclear compartments, containing intrinsically disordered proteins and RNAs that phase separate (PS) and contribute to specific nuclear reactions. However, whether and how different subnuclear compartments can intercommunicate remains elusive. Here we identified nuclear bodies with PS features composed of BAZ2A, a factor associating with active chromatin. BAZ2A-bodies depend on active transcription, RNAs, and the non-disordered BAZ2A RNA-binding TAM domain. Although BAZ2A and H3K27me3 occupancies anticorrelate in the linear genome, in the nuclear space BAZ2A-bodies contact H3K27me3-bodies. Disruption of BAZ2A-bodies promotes BAZ2A invasion into H3K27me3-domains, causing H3K27me3-bodies loss, H3K27me3 decrease, and gene upregulation. BAZ2A-bodies formation is negatively regulated by the nuclear-speckles associated lncRNA Malat1 that interacts with BAZ2A and mediates BAZ2A association to chromatin at nuclear speckles, which do not contact BAZ2A-bodies. The results unravel how active compartments protect repressive compartments using PS mechanisms that are promoted or impaired according to the type of RNA interactions with RNA-binding proteins.

overall_design: TSAseq in mouse ESCs.

first: Luigi

last: Lerra

first: Martina

last: Panatta

first: Dominik

last: Bär

first: Isabella

last: Zanini

first: Jennifer

middle: Y

last: Tan

first: Agnese

last: Pisano

first: Celia

last: Baroux

first: Vikram

middle: G

last: Panse

first: Ana

middle: C

last: Marques


title: A phase separated active subnuclear compartment safeguards repressive chromatin domains [RNA-seq]

status: Public on Apr 09 2024

country: Switzerland

type: Expression profiling by high throughput sequencing

overall_design: RNAseq in mouse ESCs.


title: Freezing responses during threat memory retrieval reflect trait-like anxiety endophenotypes in inbred mice

city: Mainz

first: Anna

last: Wierczeiko

address: Hanns-Dieter-Hüsch-Weg 15

department: Institute for Human Genetics

country: Germany

institute: University Medical Center, Johannes Gutenberg University Mainz

summary: Trait anxiety is one of the major contributors to susceptibility to stress-induced and anxiety disorders in humans. Although vulnerability to stress-induced pathologies is clearly highly individual, animal models still largely fail to account for it.  Using the threat imminence continuum theory, we designed and validated an experimental pipeline in mice centred around auditory aversive conditioning. Harnessing the naturally occurring variability of defensive freezing responses combined with a model-based clustering strategy enabled us to operationalize trait anxiety. We identified sustained freezing during prolonged retrieval sessions as a behavioural marker of an anxiety endophenotype in both males and females. RNA-sequencing of key defence circuit brain regions revealed massive differences in transcriptomes of phasic and sustained responders, correlating with transcriptomic signatures of psychiatric disorders such as PTSD and schizophrenia. We provide compelling evidence that trait anxiety in inbred mice can be leveraged to develop translationally relevant preclinical models to investigate mechanisms of stress susceptibility.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for different fear conditioning phenotypes in four brain regions of wild-type mice

first: Irina

last: Kovlyagina

first: Hristo

last: Todorov

first: Eric

last: Jacobi

first: Margaryta

last: Tevosian

first: Jakob

last: von Engelhardt

first: Susanne

last: Gerber


title: A phase separated active subnuclear compartment safeguards repressive chromatin domains [ChIP-seq]

first: Raffaella

last: Santoro

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: ChIPseq in mouse ESCs.


title: A phase separated active subnuclear compartment safeguards repressive chromatin domains [iCLIP]

overall_design: iCLIP in mouse ESCs.

first: Luigi

last: Lerra


title: Genome-wide transcriptomes of adult mouse cardiomyocytes (CMs) with ectopic expression of reprogramming factors

city: SINGAPORE

first: Yike

last: Zhu

state: Singapore

address: 14 Medical Dr, Singapore, MD6 - Centre for Translational Medicine

country: Singapore

institute: National University of Singapore

summary: We carried out bulk RNA sequencing of normal adult mouse CMs (Control-CMs) and adult mouse CMs with ectopic overexpression of Yamanaka factors (Oct4, Sox2, Klf4, Myc, OSKM-CMs), based on a hypothesis that OSKM would induce at least partial CM dedifferentiation in vivo. More than 2,000 genes were differentially expressed between Control- and OSKM-CMs, which were enriched for gene ontology (GO) terms related to multiple key aspects of CM dedifferentiation. This study provides a genome-wide transcriptional profile of dedifferentiating CMs induced by OSKM.

overall_design: Bulk RNA sequencing of normal adult mouse CMs (Control-CMs, n = 2) and adult mouse CMs with AAV9-mediated ectopic overexpression of OSKM (OSKM-CMs, n = 3)

first: Wilson

first: Roger

last: Foo


title: Identification of an RNA-binding region in 18S dimethyladenosine methyltransferase DIMT1 essential for proliferation of acute myeloid leukemia cells

city: philadelphia

first: Fange

last: Liu

state: PA

address: 415 curie blvd, Clinical Research Building Rm 350

country: USA

institute: UPENN

summary: Aberrant assembly and function of ribosomes are implicated in cancers, including acute myeloid leukemia (AML). Here, we find that 18S rRNA methyltransferase DIMT1 is essential for AML proliferation through a non-catalytic function. We demonstrate that a positively charged cleft on DIMT1 plays an important role in RNA binding. Mutations at the identified cleft (5mutA-DIMT1) significantly weakens binding of DIMT1 to rRNA in vitro and in cells. Strikingly, the RNA-binding deficient 5mutA-DIMT1 is predominantly located in the nucleoplasm, in contrast with the primarily nucleolar localization of wild-type DIMT1. Furthermore, we demonstrate that rRNA binding is required for DIMT1 to undergo liquid-liquid phase separation in vitro, which likely promotes DIMT1’s nucleolar localization and its role in rRNA processing in cells. Importantly, re-expression of wild-type but not 5mutA-DIMT1 supports AML proliferation in competition-based cell proliferation assays. These results highlight the importance of targeting the catalytically independent RNA-binding activity of DIMT1 for therapy of AML.

overall_design: Ribosome profiling followed by high-throughput sequencing (ribo-seq) in MOLM13C cells with DIMT1 or Rosa (a negative control) depletion.

first: Kathy

middle: F

first: Julian

last: Stoute

first: Yulia

last: Gonskikh

first: Hui

last: Shen


title: Disruption of TGF-β signaling pathway is required to mediate effective killing of hepatocellular carcinoma by human iPSC-derived NK cells

status: Public on Apr 08 2024

city: San Diego

first: Dan

middle: S

last: Kaufman

state: California

address: 2880 Torrey Pines Scenic Dr

department: School of Medicine

institute: University of California San Diego

summary: Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. Transforming growth factor beta (TGF-β) is highly expressed in the liver tumor microenvironment and is known to inhibit immune cell activity. Here, we used human iPSCs to produce natural killer (NK) cells engineered to mediate improved anti-HCC activity. Specifically, we produced iPSC-NK cells with either knock out TGF-β receptor 2 (TGFBR2-KO) or expression of a dominant negative (DN) form of the TGF-β receptor 2 (TGFBR2-DN) combined with CARs that target either GPC3 or AFP. The TGFBR2-KO and TGFBR2-DN iPSC-NK cells are resistant to TGF-β inhibition and improved anti-HCC activity. However, expression of anti-HCC CARs on iPSC-NK cells did not lead to effective anti-HCC activity unless there was also inhibition of TGF-b activity. Our findings demonstrate that TGF-β signaling blockade is required for effective NK cell function against HCC and potentially other malignancies which express high levels of TGF-β.

overall_design: The WT, TGFBR2-KO and TGFBR2-DN iPSC deived NK cells are differentiated and expanded were cultured in standard and memory like conditions. The libraries were prepared for RNA samples using the Illumina Stranded mRNA Library Prep Kit, targeting mRNA via poly-A selection for strand-specific cDNA synthesis. The libraries were sequenced using Illumina Novaseq X+ in the IGM Genomics Center at UCSD.

first: Jaya

middle: L

last: Thangaraj

first: Michael

last: Coffey

first: Edith

last: Lopez


title: Twig age-dependent negative correlation between Taxol accumulation and endophytic fungal assembles provide insights into screening efficient Taxol-related fungal elicitors

city: Hangzhou

first: Chenjia

state: Zhejiang

address: No.58,Haishu Rd, Cangqian, Yuhang District

department: College of Life and Environmental Sciences

country: China

institute: Hangzhou Normal University

summary: We identified two effective fungal elicitors, L01 (Guignardia) and J02 (Diaporthe).  The two fungal elicitors might activate Taxol biosynthesis by regulating the MYC2a-controlled JA signaling pathway.

overall_design: Changes in taxol content in twig of Taxus mairei under fungal elicitors J2 and L1 infection compared with control.

first: Kailin

last: Hou

first: Xueshuang

last: Liang

first: Chenliang

last: Yu


title: Selective molecular inhibition of the HDAC6 ZnF-UBP binding domain impairs multiple myeloma cell proliferation

city: Geneva

first: Rafael

last: Rincon Perez

state: Geneva

address: Rue Michel Servet 1, 1221, Genève

institute: Université de Genève

summary: Histone deacetylase 6 (HDAC6) is overexpressed in multiple myeloma (MM) patients and may be involved in the acquisition of resistance to conventional treatments. Here, we propose a two-residue mutation impairing the HDAC6 ZnF-UBP domain. HDAC6 knockout and ZnF-UBP mutant RPMI 8226 MM cell lines were generated using CRISPR-Cas9. MM cells harboring this mutation showed impaired aggresome formation, cell growth and a dysregulated gene expression profile in a more pronounced manner than the HDAC6 knockout cells.

overall_design: To investigate the role of the HDAC6 ZnF-UBP domain in MM pathogenesis, we established two RPMI 8226 cell lines: one with a knock-out of HDAC6 (KO), and another one with a two-residue mutation in the HDAC6 ZnF-UBP domain (R1155A, Y1155A; RY) preventing its ubiquitin-binding capacity. Both cell lines were obtained by CRISPR-Cas9 edition. We then performed gene expression profiling analysis using data obtained by bulk RNA-seq of the three cell lines (RPMI 8226 WT, HDAC6 KO and HDAC6 RY).

last: Rincón

first: Isabel

last: Coira

first: Muriel

last: Cuendet


title: Effect of Activin A and Gpnmb on gene expression in NAFLD/NASH liver in C57BL/6J mice and gene expression change in C57BL/6J mice fed on sugar water-NAFLD/NASH diet compared with standard chow diet

city: Boston

first: Huan

state: Massachusetts

address: 185 Cambridge Street

department: CVRC

institute: Massachusetts General Hospital

summary:
To investigate the mechanism of hepatic Activin A and Gpnmb in NAFLD/NASH, we studied C57BL/6J mice on a FPC NASH diet and sugar water for 16 weeks, compared with standard chow diet, and used adeno-associated viral vectors with a liver-specific thyroxine binding globulin (TBG) promoter to express Activin A or GFP (control), or AAV8-H1-shRNA to knockdown of Gpnmb or scramble control in NAFLD/NASH liver.
We then performed gene expression profiling analysis using data obtained from RNA-seq of 3 different livers in each group.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for liver expressed AAV-TBG-GFP (Control) and AAV-TBG-Activin A, AAV8-shRNA SC and AAV8-shRNA Gpnmb on sugar water-FPC NASH diet, as well as liver expressed AAV-TBG-GFP (Control) on standard chow diet.

first: Anthony

last: Rosenzweig


title: Intratracheal instillation of quaternary ammonium compounds in rats

city: Jeongeup

first: Yoon

last: Cho

state: Jeollabuk-do

address: 30, Baekhak 1-gil

country: South Korea

institute: Korea Institute of Toxiology

summary: There is a lack of comprehensive toxicity studies of QACs associated with pulmonary toxicity. Therefore, we aimed to elucidate the potential pulmonary toxic effects of QACs based on toxicogenomic approach. We conducted transcriptome analysis using RNA sequencing to identify alterations in gene expression associated with QACs exposure.

overall_design: The SD rats were administered to a single intratracheal instillation (ITI) of each QACs at varying concentrations. Within this range of concentrations, bronchoalveolar lavage fluid (BALF) analysis showed increases in total cell count, lactate dehydrogenase (LDH), and total protein levels in response to QACs exposure. Based on these results, transcriptome analysis using quantSeq 3` mRNA sequencing was conducted on lung tissues from SD rats intratracheally administered with four QACs with a 2.0-fold change cutoff and p < 0.05 (n=4/group) to assess the toxicogenomic responses.

first: Min-Seok

last: Kim


title: Effects of cold stress changes on subchondral bone and cartilage of tibial  knee joint in rats and molecule related to hypothermia and arthritis

city: Xi’an

first: Chunsheng

last: Wang

address: No. 157, West Fifth Road

institute: Second Affiliated Hospital of Xi’an Jiaotong University

summary: Forty eight-week-old male SD-rats were randomly divided into four groups:  control group (4 weeks), hypothermia group (4 weeks), control group (8 weeks) and hypothermia group (8 weeks). The hypothermia group was fed at 4±2℃, and the normal group was fed at 25±2℃. After 4 weeks and 8 weeks, Micro-CT and saffron solid green staining were performed to observe the conditions of subchondral bone and cartilage. At 4 weeks, rat subchondral bone tissue was cut for second generation high-throughput sequencing, differential gene screening and gene enrichment analysis, and PCR validation.

overall_design: To find out which molecules play a role in hypothermia and arthritis,  we performed high-throughput sequencing of subchondral bone  in rats at low and normal temperatures, followed by PCR validation

first: Zhuo

last: Huang

first: Yutao

last: Chen

first: Zeyu

last: Liu

first: Xiaohui


title: Comparative transcriptomic analysis of circulating endothelial cells in sickle cell stroke

city: Campinas

first: Mônica

last: Barbosa de Melo

state: Sao Paulo

address: Avenue Candido Rondon, 400. Cidade Universitária

department: Center for Molecular Biology and Genetic Engineering - CBMEG

country: Brazil

institute: Campinas University

summary: Ischemic Stroke (IS) is one of the most impairing complications of sickle cell anemia (SCA), responsible for 20% of mortality in patients. Rheological alterations, adhesive properties of sickle reticulocytes, leukocyte adhesion, inflammation and endothelial dysfunction are related to the vasculopathy observed prior to ischemic events. The role of the vascular endothelium in this complex cascade of mechanisms is emphasized, as well as in the process of ischemia-induced repair and neovascularization. The aim of the present study was to perform a comparative transcriptomic analysis of endothelial colony-forming cells (ECFCs) from SCA patients with and without IS. Next, to gain further insights of the biological relevance of differentially expressed genes (DEGs), functional enrichment analysis, protein-protein interaction network (PPI) construction and in silico prediction of regulatory factors were performed. Among the 2,469 DEGs, genes related to cell proliferation (AKT1, E2F1, CDCA5, EGFL7), migration (AKT1, HRAS), angiogenesis (AKT1, EGFL7) and defense response pathways (HRAS, IRF3, TGFB1), important endothelial cell molecular mechanisms in post ischemia repair were identified. Despite the severity of IS in SCA, widely accepted molecular targets are still lacking, especially related to stroke outcome. Thus, these exploratory results may contribute to a better understanding of the role of endothelial cells in SCA ischemic stroke recovery, and promote new insights to further studies on therapeutic strategies for this severe complication.

overall_design: RNA-Seq was performed from RNA samples extracted from endothelial colony-forming cells (ECFCs) obtained from eight sickle cell anemia (SCA) patients, divided into case group: four individuals with ischemic stroke (IS), and control group: four individuals without the occurrence of IS.

first: Júlia

middle: N

last: de Castro

first: Sueli

middle: M

last: Costa

first: Ana

last: Camargo

first: Mirta

middle: T

last: Ito

first: Bruno

middle: B

last: de Souza

first: Victor

middle: H

last: Bertozzo

first: Thiago

middle: A

last: Rodrigues

first: Carolina

last: Lanaro

first: Dulciéia

last: Albuquerque

first: Roberta

last: Saez

first: Sara

last: Saad

first: Margareth

last: Ozelo

first: Fernando

last: Cendes

last: de Melo


title: Pseudomonas aeruginosa lasR QS network promotes phage infection by regulating the biosynthesis of LPS and type IV pili  by regulating the biosynthesis of LPS and type IV pili

city: wenzhou

first: Changrui

last: Qian

state: zhejiang

address: wenzhou medical university

institute: wenzhou medical university

summary:
Purpose: The purpose of this study was to investigate the effect of quorum sensing on phage infection.
Methods: We constructed the lasR gene knockout strain of Pseudomonas aeruginosa PAO1  and performed transcriptome sequencing.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for wild-type PAO1 strain,  lasR  gene knockout strain (PAO1ΔlasR).

first: Yan

first: Tieli

last: Zhou


title: Macrophage histone lactylation during ischemia induced-muscle regeneration [CUT&Tag]

city: Schwerzenbach

first: Adhideb

last: Ghosh

address: Schorenstrasse 16

department: Health Sciences and Technology

institute: ETH Zurich

summary: After injury, muscles require fully functional macrophages to recover completely. During muscle regeneration, macrophages transition from a pro-inflammatory to a pro-restorative phenotype ensures the proper regeneration. Lactate is a crucial molecule for muscle-residing macrophage polarisation. Histone lactylation, a direct derivative of lactate, has been implicated in macrophage polarisation in vitro .  In this work, we show for the first time that macrophages recruited to the muscle after ischemic injury modify their histone lactylome between 2 and 4 days post injury. Absolute histone lactylation levels increase significantly. Although subtly, also the genomic enrichment of H3K18la changes from 2 to 4 days post injury and this correlates with gene expression changes. Interestingly, we find that H3K18la genomic enrichment changes from day 2 to day 4 post injury are predictive for gene expression changes later in time, from day 4 to day 7, rather than being a reflection of past gene expression changes from day 1 to day 2. Our results suggest that histone lactylation dynamics are functionally important for the resolving action of macrophages during muscle regeneration.

overall_design: C57BL6J wild type males (Charles River) from 8 to 12 weeks old were used in this study. Animals were housed in a normal light cycle with access to food and water ad libitum. Hind-limb ischemia experiments were performed as described in Zhang et al., 2020. Briefly, mice were anesthetized with isoflurane. The hind limb was shaved, and the skin was incised. The proximal end of the femoral artery and the distal portion of the saphenous artery were ligated. The artery and all side-branches were dissected to be freed and the femoral artery and attached side-branches were excised. Procedures were approved by the ethics committee (number ZH050-2021). To label circulating CD45+ cells, mice were anaesthetised with a combination of ketamine and xylazine and i.v. injected with 3 µg of PE anti-mouse CD45 antibody (Biolegend [30-F11], 103106). Five minutes later, mice were sacrificed and muscle was processed for isolating macrophages.

first: Eva

last: Galle

first: Thibaut

last: Desgeorges

first: Katrien

last: De Bock

first: Ferdinand

last: von Meyenn


title: IKKβ Inhibition Attenuates Epithelial Mesenchymal Transition of Human Stem Cell-Derived Retinal Pigment Epithelium

city: Dallas

first: Srinivasa R.

last: Sripathi

state: Texas

address: 9600 N Central Expressway Suite 200

institute: Retina Foundation of the Southwest

summary: Epithelial-mesenchymal transition (EMT), which is well known for its role in embryonic development, malignant transformation, and tumor progression, has also been implicated in a variety of retinal diseases, including proliferative vitreoretinopathy (PVR), age-related macular degeneration (AMD), and diabetic retinopathy. EMT of the retinal pigment epithelium (RPE), although important in the pathogenesis of these retinal conditions, is not well understood at the molecular level. We and others have shown that a variety of molecules, including the co-treatment of human stem cell-derived RPE monolayer cultures with transforming growth factor beta (TGF–β) and the inflammatory cytokine tumor necrosis factor alpha (TNF–α), can induce RPE–EMT; however, small molecule inhibitors of RPE–EMT have been less well studied. Here, we demonstrate that BAY651942, a small molecule inhibitor of nuclear factor kapa-B kinase subunit beta (IKKβ) that selectively targets NF-κB signaling, can modulate TGF–β/TNF–α-induced RPE–EMT. Next, we performed RNA-seq studies on BAY651942 treated hRPE monolayers to dissect altered biological pathways and signaling events. Further, we validated the effect of IKKβ inhibition on RPE–EMT-associated factors using a second IKKβ inhibitor, BMS345541, with RPE monolayers derived from an independent stem cell line. Our data highlights the fact that pharmacological inhibition of RPE–EMT restores RPE identity and may provide a promising approach for treating retinal diseases that involve RPE dedifferentiation and EMT.

overall_design: We compared the transcriptomic profiles of human stem cell-derived RPE monolayer cultures co-treated with TGF–β/TNF–α and a small molecule (BAY651942) inhibitor of nuclear factor kapa-B kinase subunit beta (IKKβ)

first: Srinivsa

middle: R

first: Ming-Wen

last: Hu

first: Ravi

last: Chakra Turaga

first: Rebekah

last: Mikeasky

first: Ganesh

last: Satyanarayana

first: Jie

last: Cheng

first: Yukan

last: Duan

first: Julien

last: Maruotti

first: Karl

middle: J

last: Wahlin

first: Cynthia

last: Berlinicke

first: Jiang

first: Noriko

last: Esumi

first: Donald

last: Zack


title: Sendai virus is robust and consistent in delivering genes into human pancreatic cancer cells.

city: PORTLAND

first: JUNGSUN

last: KIM

state: OR

address: KCRB 5001.51, 2720 SW Moody Ave

institute: Oregon Health & Science University

summary: Pancreatic ductal adenocarcinoma (PDAC) is highly intratumorally heterogeneous and has several subtypes. Effective gene delivery to all PDAC cells is essential for modulating gene expression and identifying potential gene-based therapeutic targets in PDAC. Most current gene delivery systems for pancreatic cells are optimized for islet or acinar cells. Lentiviral vectors are the current main gene delivery vectors for PDAC, but their transduction efficiencies vary depending on pancreatic cell type, and are especially poor for the classical subtype of PDAC cells from both primary tumors and cell lines. Herein, we systemically compare transduction efficiencies of glycoprotein G of vesicular stomatitis virus (VSV-G)-pseudotyped lentiviral and Sendai viral vectors in human normal pancreatic ductal and PDAC cells. We find that the Sendai viral vector gives the most robust gene delivery efficiency regardless of PDAC cell type. Therefore, we propose using Sendai viral vectors for transducing ectopic genes into PDAC cells.

overall_design: To compare transduction efficiency of lentiviral and Sendai virus vectors in different sybtypes of human pancreatic ductal adenocarcinoma (PDAC) primary cell culture and PDAC cell lines we established primary cell cultures with patient-derived xenograft (PDX) PDAC (ST-7599, ST-14490  and ST-7270). To determine subtypes of  established  primary PDAC cell cultures (ST-7599, ST-14490, and ST-7270)  and PDAC cell lines (Panc-1, CFPAC-1) based on  previosly published PDAC subtypes gene expression signatures we conducted RNA-Seq and performed hierarchical and consensus clustering with normalized gene expression.

first: Dmytro

last: Grygoryev

first: Taelor

last: Ekstrom

first: Elise

last: Manalo

first: Jason

last: Link

first: Dove

last: Keith

first: Brittany

last: Allen-Petersen

first: Brett

last: Sheppard

first: Terry

last: Morgan

first: Rosalie

last: Sears

first: Jungsun

last: Kim


title: Macrophage histone lactylation during ischemia induced-muscle regeneration [RNA-Seq]

first: Adhideb

last: Ghosh

first: Eva

last: Galle

first: Thibaut

last: Desgeorges

first: Katrien

last: De Bock

first: Ferdinand

last: von Meyenn


title: siqRNA-seq is a spike-in-independent technique for quantitative mapping of mRNA landscape

city: Shenzhen

first: Zhenzhen

last: Wang

address: 97 Buxin Road

institute: Chinese Academy of Agricultural Sciences

summary: Normalization of RNA sequencing (RNA-seq) data for gene expression comparison is essential to ensure accurate gene expression quantification. It has been argued that samples with large differences in global expression level cannot be properly normalized without spike-in control RNAs, however, spike-in controls are expensive and not yet widely used. Here, we presented a spike-in independent quantitative RNA sequencing (siqRNA-seq) method, which uses reads from genome DNA as an internal reference to quantify gene expression level. We showed that siqRNA-seq profiles gene expression as traditional RNA-seq, but allows to identify different expression genes between samples with distinct mRNA content. We also showed siqRNA-seq enable us to assess the copy number of mRNA per cell without counting cells and adding spike-ins. Thus, siqRNA-seq provides a convenient and versatile means to quantitatively profile the mRNA landscape in cells.

overall_design: To develop a spike-in-independent method for gene expression quantification, we designed siqRNA-seq that utilize genome DNA (gDNA) as internal reference for normalization .      As we known, each cell has a set of genomic DNA, so we considered that it can be taken as an internal reference but with large molecular weight.       To that end, whole nucleic acids were extracted from samples to construct two libraries in parallel for sequencing in siqRNA-seq, mRNA library and mRNA&gDNA library , respectively.       Genome DNA was removed by digested with DNase I in mRNA library while retained in mRNA&gDNA library as internal reference.       mRNA was reverse-transcribed with oligo(dT) as the primer to synthesis the first strand of the complementary DNA (cDNA).       To reduce bias and simplify our pipeline, fragmented cDNA without second strand synthesis and gDNA were denatured by heat following library preparation using a high-efficient ssDNA ligation technique, Adaptase from Accel-NGS 1S Plus DNA Library Kit (Swift Accel-NGS).       Adaptase is a commercial enzyme mixture with very high ligation efficiency and low bias for single-stranded DNA (ssDNA) library construction with strand-specific information, which we have used to develop ssDRIP-seq for R-loop profiling and DEtail-seq for DNA break detection .       Compared to traditional RNA-seq, mRNA library in siqRNA-seq was directly prepared from single-stranded cDNA, therefore we called this method as ssRNA-seq hereafter.

first: Kehan

last: Tao

first: Changbin

last: Sun

first: Wei

last: Xu


title: Macrophage histone lactylation during ischemia induced-muscle regeneration

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

overall_design: Refer to individual Series


title: Exploration of the transcriptional regulation network of the suppression of ILTV infection by PP1 and PP2 in chickenLMH cells

city: Xi'an

first: Hai

last: Li

address: No.76 Yanta West Road

department: Department of Pathogenic Microbiology and Immunology

institute: Xi'an Jiaotong University

summary: To investigate the transcriptional regulation network of thesuppression of ILTV infection by PP1 and PP2 in chickenLMH cells, we treated LMH cells with PP1 or PP2 upon ILTV infection. We then performed gene expression profiling analysis using data obtained from RNA-seq of LMH cells.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for uninfected LMH cells and infected LMH cells upon different treatments (PP1, and PP2). Four biological replicates for each condition have been done.

first: Shengwang

last: Liu


title: Construction of Danio rerio Asymmetrical Maps (DreAM)

city: Hang Zhou

first: Tao

last: cheng

state: Zhe Jiang

address: 886 Yuhangtang Road

department: Institute of genetics

institute: Zhejiang University

summary: Purpose: Construction of 3D zebrafish spatial transcriptomics data for studying the establishment of AP axis. Methods:  We performed serial bulk RNA-seq data of zebrafish embryo at three development points. Using the published spatial transcriptomics data as references, we implemented Palette to infer spatial gene expression from bulk RNA-seq data and constructed 3D embryonic spatial transcriptomics. The constructed 3D transcriptomics data was then projected on zebrafish embryo images with 3D coordinates, establishing a spatial gene expression atlas named Danio rerio Asymmetrical Maps (DreAM). Results: DreAM provides a powerful platform for visualizing gene expression patterns on zebrafish morphology and investigating spatial cell-cell interactions. Conclusions: Our work used DreAM to explore the establishment of anteroposterior (AP) axis, and identified multiple morphogen gradients that played essential roles in determining cell AP positions. Finally, we difined a hox score, and comprehensively demonstrated the spatial collinearity of Hox genes at single-cell resolution during development.

overall_design: Bulk RNA-seq was performed on the serial cryosections taken along the left-right axis of zebrafish embryos at 10 hpf, 12 hpf and 16 hpf.

first: Yang

last: Dong

last: Cheng

first: Xiang


title: Rnmt and Ram regulate the activated CD4 T cell transcriptome

city: Glasgow

first: Alison

last: Galloway

address: Garscube Estate, Switchback Road

country: United Kingdom

institute: Beatson CRUK

summary: We investigated the role of RNMT and its cofactor RAM in T cells using conditional knockout mouse models. We demonstrate that RNMT and RAM have broadly overlapping roles in CD4 T cell activation.

overall_design: Sequencing of rRNA-depleted RNA from 20 hour activated CD4 T cells from Rnmt fl/fl CD4cre (Rnmt cKO), Rnmt fl/fl (Rnmt control), Ram fl/fl CD4cre (Ram cKO) and Ram fl/fl (Ram control) mice.

first: Victoria

last: Cowling


title: Propionate stimulates MUC2 expression through metabolism of β-oxidation-like pathway that boosts mitochondrial respiration and resultant induction of HIF-2α activation

city: seoul

first: seongho

last: ma

address: Anamro 145

institute: Korea university

summary: Microbiota-derived short chain fatty acids (SCFAs) are one of the hottest issues for its various bioactivities in the gut, immune system extending to brain or other organs through several axises. Though many intensive studies have been implementing til recent, some parts among a plenty of actions occurred in the gut solely are unrevealed so as their mechanism. Propionate, one of the representative SCFAs with butyrate is basically known for its inhibiting activity of histone deacetylation (HDAC) and stimulation of mucin expression in mucosae. However, the mechanism of the stimulating action needs to be elucidated in more detail and understandable way. Here, we firstly investigated the effect of propionate on expression of MUC2, one of the main subtype mucin, in goblet cell line LS 174T and suggest a mechanism of the action along the order of events, linked to metabolic shift. Propionate at the concentration of 1 mM to 4 mM induced the expression of MUC2 gradually in the transcriptional- and protein level at 24 h. At the initial parts of treatment of propionate, the odd SCFA was metabolized along vitamin B12-independent β-oxidation-like pathway and the metabolism led cells to boost mitochondrial respiration until 18 h after treatment. However, another possible metabolism pathway, vitamin B12-dependent carboxylation pathway was not involved to the resulted action. At the latter parts, the master regulator of hypoxia, Hypoxia-induced factors (HIFs) were fluctuated respectively and among them, activated HIF-2α was identified to regulate the expression level of MUC2. In summary, propionate might induce β-oxidation-like pathway at the initial part so that mitochondrial respiration is upregulated. In an effort of LS 174T to adapt to naturally formed hypoxia by response to propionate, each activity of HIF subtypes was regulated in a way of entangled to each other and activated HIF-2α stimulated MUC2 expression.

overall_design: 2 samples in triplicate, total 6 samples; negative control and propionate 4 mM treated LS 174T.

first: Seongho

last: Ma

first: Jiah

last: Yeom

first: Young

last: Lim


title: CLCF1/NF-κB Signaling Pathway Regulates Macrophage Efferocytosis to Ameliorate Neural Damage and Cognitive Dysfunction After CO Poisoning.

status: Public on Apr 07 2024

city: Jinan

first: Xudong

last: Zhou

address: 4655 University Road, University Science and Technology Park, Changqing District, Jinan City, Shandong Province, China

department: First Clinical College

institute: Shandong University of Traditional Chinese Medicine

summary: Severe carbon monoxide (CO) poisoning can cause structural damage to the nervous system, leading to long-term cognitive dysfunction in patients. Correctly terminating the inflammatory response caused by neuronal damage is a prerequisite for tissue repair. Macrophages can clear the cell corpses/fragments caused by brain injury through efferocytosis, and produce cytokines to coordinate the immune response, promoting neuronal repair and regeneration. However, in the microenvironment of the nervous system affected by CO poisoning, the function of macrophages is inhibited. Our research found that CLCF1 can regulate the secretion of cytokines such as TNF-α, IL-1β, and IL-10 through the NF-κB signaling pathway, thereby affecting neural cell repair and regeneration. Simultaneously, CLCF1 can regulate the efferocytosis function of macrophages, thus controlling the degree of inflammation and assisting in the repair of the damaged nervous system. In experiments, it was observed that targeting the regulation of macrophage CLCF1 expression led to improvements in memory, learning, and motor abilities in rats poisoned with CO.

overall_design: Rats were used to create an acute carbon monoxide (CO) poisoning model, following the method described by Li et al.[1]. The rats were placed in a sealed experimental chamber and initially inhaled CO gas with a concentration of 1000 ppm for 40 minutes; this was followed by inhalation of CO gas at a concentration of 3000 ppm for 20 minutes. The model was considered successful for acute severe CO poisoning if the rats exhibited symptoms of impaired consciousness accompanied by high carboxyhemoglobin (HbCO) concentrations (>60%). Brain tissue cell suspensions were extracted from three CO-poisoned rats and three normal control (NC) group rats for single-cell sequencing. Gel beads containing barcode sequences were mixed with the samples and combined with oil surfactants to form Gel Bead-In-Emulsions (GEMs). The GEMs were collected into a reservoir for constructing 10x single-cell transcriptome libraries. An Illumina NovaSeq PE150 sequencing strategy was employed, targeting 50,000-100,000 reads per cell, corresponding to 15-30M data points per cell. Each experimental group was repeated three times to verify the reliability of the results. The raw data were mapped to the Rnor_6.0 rat reference genome using CellRanger (10x Genomics).

first: Huiping

last: Song

last: han

first: Qin


title: α7 Nicotinic Acetylcholine Receptor Regulates Macrophage-Mediated Neuroinflammation in Dry Eye Disease

city: shanghai

first: xujiao

last: zhou

address: fenyang road 83

institute: Eye & ENT Hospital, Fudan University

summary: Patients with dry eye disease (DED) often exhibit neurological abnormalities and may even suffer from neuropathic pain and pain-related anxiety or depression. However, addressing nerve abnormalities in DED remains a formidable challenge, as current therapies fail to halt disease progression. Our study found that activating α-7 nicotinic acetylcholine receptor (α7nAChR), a pivotal regulator in the anti-inflammatory pathway connecting the nervous and immune systems, effectively restores corneal epithelium integrity and enhances nerve sensitivity in DED, pointing to its promising therapeutic potential. Furthermore, we have revealed that α7nAChR stimulates genes involved in immune-mediated inflammatory progression and neuroregulation, inhibits the expression of transient receptor potential vanilloid-1 (TRPV1), reinstates corneal nerve density, and alleviates anxiety-like behaviors associated with severe DED by downregulating the proportion of CD86+ M1 macrophages (pro-inflammatory phenotypes). In summary, our findings underscore the activation of α7nAChR as a pioneering therapeutic approach for preserving corneal nerves balance and controlling inflammation in DED.

overall_design: To investigate the α7 Nicotinic Acetylcholine Receptor in the regulation of dry eye disease


title: Polymer-mediated oligo delivery enables construction of spatially encoded 3D cultures for analysis by single-cell RNA sequencing

city: Crawley

first: Jessica

last: King

state: WA

address: 35 Stirling Hwy

department: School of Molecular Sciences

country: Australia

institute: University of Western Australia

summary: Spatial transcriptomics has been widely used to capture gene expression profiles, which are realised as a two-dimensional (2D) projection of RNA captured from tissue sections. Three-dimensional (3D) cultures such as spheroids and organoids are highly promising alternatives to overly simplistic and homogeneous 2D cell culture models, but existing spatial transcriptomic platforms do not have sufficient resolution and RNA capture efficiency for robust analysis of 3D cultures. We present a transfection-based method for fluorescent DNA barcoding of cell populations, and the subsequent construction of spheroidal cellular architectures using barcoded cells in a layer-by-layer approach. For the first time, changes in gene expression throughout this 3D culture architecture are interrogated using multiplex single-cell RNA sequencing in which DNA barcodes encode the spatial positioning of cells. We show that transfection with fluorophore-conjugated barcode oligonucleotides enables both imaging and sequencing at single-cell resolution, providing spatial maps of gene expression and drug response. Additionally, we show that fluorescently-tagged DNA barcodes support correlative imaging studies such as quantitative microelastography (QME) to capture information about mechanical heterogeneity in 3D cultures, also with spatial resolution. The ability to create customised, spatially encoded cellular assemblies is a general approach that can resolve spatial differences in gene expression in 3D cell culture models.

overall_design: NOT PROVIDED; REQUESTED

first: Alireza

last: Mowla

first: Sebastian

middle: E

last: Amos

last: Kretzmann

first: Marck

last: Norret

first: Somayra

last: Mamsa

first: Ulrich

last: Kadolsky

first: Alka

last: Saxena

first: Brendan

last: Kennedy

first: Yu

last: Choi

first: K

last: Iyer

first: Nicole

last: Smith

first: Cameron

middle: W

last: Evans


title: APOE-Ɛ4 astrocytes exhibit deficits in endocytic uptake that contribute to the delayed maturation of neuronal network activity and alter neuronal tau uptake in co-cultures of human iPSC-derived brain cells

city: Cambridge

first: Robin

last: Kleiman

state: MA

address: 225 Binney Street

institute: Biogen

summary: To explore the mechanistic basis of ApoE Ɛ4 vs ApoE Ɛ3 protein expression on endocytic pathways responsible for tau uptake in neurons and astrocytes and the maturation of neuronal networks, we have developed genotype matched co-cultures of iPSC derived astrocytes and neurons derived from isogenic triads of iPSC lines generated by the ADAPTED consortium (Schmid et al., 2019, 2020) .  We show that isogenic iPSC derived APOE-E4 expressing astrocytes take up less extracellular tau than APOE-E3 or APOE null astrocytes, while isogenic neurons in monoculture take up equivalent amounts of tau primarily through macropinocytosis. Co-culture of neurons with genotype matched astrocytes increases the general uptake capacity of neurons by increasing the dependence of neuronal endocytosis on dynamin mediated pathways. Co-culture also enhances the emergence of spontaneous neuronal activity; however, the emergence of synchronous network activity is impaired by the expression of APOE-E4 genotype. We performed RNA-Seq on the astrocytes, neurons and co-cultures to understand the molecular pathway changes associated with different ApoE genotypes.

overall_design: RNAseq profile of APOE E3E3, E4E4 and KO iPSC lines and its derived astrocytes, neurons and co-cultures

first: Laura

last: Blaha

first: Guoqiang

last: Zhang


title: Mucociliary epidermis RNA-seq data from controls during early patterning (st. 14-20)

city: Freiburg

first: Peter

last: Walentek

state: BW

address: Breisacherstr. 113

department: Internal Medicine IV & IMITATE

institute: University Freiburg Medical Center

summary: This study was conducted to investigate the temporal expression of patterning genes in the Xenopus mucociliary epidermis.

overall_design: Unmanipulated embryos were used to generate animal cap explants at embryonic stage 8. Explants were grown into mucociliary organoids and collected at embryonic stages 14, 16,and 20 for total RNA extraction.

first: Magdalena

last: Brislinger-Engelhardt


title: Lung airway expression

city: Chapel Hill

first: Scott

middle: H.

last: Randell

state: North Carolina

address: CB 7248, Room 1117 Marsico Hall, 125 Mason Farm Rd.

institute: UNC Chapel Hill

summary: This study seeks to characterize airway epithelial gene expression across small, medium and large airways in the normal human lung.

overall_design: Lung tissue samples were harvested from donor lungs with no history of chronic lung disease and unused for transplant.

first: Matthew

middle: D

last: Wilkerson

first: David

last: Hayes

first: Michele

last: Hayward

last: Soloway

first: Ashley

last: Salazar


title: A cell-free pipeline for recreating methylation patterns  radically enhances DNA transformation in bacteria

city: Wuerzburg

first: Chase

last: Beisel

address: Josef-Schneider-Str. 2 / Bau D15

institute: Helmholtz Institute for RNA-based Infection Research

summary: The bacterial world offers diverse strains for understanding medical and environmental processes and for engineering synthetic-biology chasses. However, genetically manipulating these strains has faced a long-standing bottleneck: how to efficiently transform DNA. Here we report IMPRINT, a generalized, rapid and scalable approach to overcome DNA restriction, a prominent barrier to transformation. IMPRINT utilizes cell-free systems to express DNA methyltransferases from the bacterial host’s restriction-modification systems. The expressed methyltransferases then methylate DNA in vitro to match the host DNA’s methylation pattern, circumventing restriction and enhancing transformation. Unlike established approaches, IMPRINT can be completed in under one day, readily accommodates all methyltransferase types, and avoids methylation-induced cytotoxicity. With IMPRINT, we efficiently multiplex DNA methylation and maximize plasmid transformation in gram-negative and gram-positive bacteria. We also developed a high-throughput pipeline that identifies the most consequential methyltransferases in one transformation. Overall, IMPRINT can vastly enhance DNA transformation, enabling use of increasingly sophisticated genetic manipulation tools across the bacterial world.

sample_organism:
Escherichia coli
Bifidobacterium breve

platform_organism:
Escherichia coli
Bifidobacterium breve

overall_design: To efficiently transform DNA, we sought to harness TXTL as a distinct means to overcome DNA restriction. TXTL recapitulates transcription and translation in a lysate or solution of purified components, allowing the functional expression of RNA and protein from any bacterium in minutes to hours without the need for cell culturing or protein purification29. We envisioned a TXTL-based pipeline in which DNA MTases identified in a host bacterium (e.g. through bioinformatics or listed on the REBASE database30) are expressed and combined with DNA in a methylation reaction, and the resulting DNA is purified and transformed into the host. DNA lacking any methylation (e.g. PCR product, plasmid DNA extracted from a MTase-free strain) serves as the starting point to immediately circumvent Type IV R-M systems that cleave methylated DNA26. We call the resulting pipeline IMPRINT (Imitating Methylation Patterns Rapidly IN TXTL) (Fig. 1a). With IMPRINT, methylation by multiple MTases, including Type I MTases that also require expressing a specificity protein, is straightforward and completed in under a day. Any cytotoxicity concerns are also minimized due to the absence of replicating cells for plasmid methylation.

first: Justin

last: Vento

first: Deniz

last: Durmusoglu

first: Sean

last: Sullivan

first: Fani

last: Ttofali

first: John

last: van Schaik

first: Yanying

last: Yu

first: Lars

last: Barquist

first: Nathan

last: Crook


title: Loss of CRY2 promotes regenerative myogenesis by enhancing PAX7 expression and satellite cell proliferation

city: Suzhou

first: Ting

last: Xue

state: Jiangsu

address: 199 Ren Ai Road, Suzhou 215123, China

department: Hematology Center, Cyrus Tang Medical Institute

institute: Soochow University

summary: Skeletal muscle has remarkable capacity to regenerate upon injury due to the presence of satellite cells. The maintenance and function of satellite cells are regulated by circadian clock. Cryptocrhome 2 (CRY2) is a key component of the circadian clock and its role in skeletal muscle regeneration remains controversial. Here, we report that CRY2 is down-regulated during muscle regeneration. Using the satellite cell specific CRY2 knockout mice (CRY2scko), we show that deletion of CRY2 enhances muscle regeneration. Single myofiber analysis showed that deletion of CRY2 enhances satellite cell self-renewal. In the absence of CRY2, the ERK1/2 and JNK1/2 signaling pathways become activated, which phosphorylates the transcription factor ETS1, which in turn binds to the promoter of PAX7 to induce its transcription. CRY2 deficient myoblasts survived better in ischemic muscle. Deletion of CRY2 also alleviated myopathy in mdx mice. Therefore, CRY2 plays an essential role in regulating satellite cell function and skeletal muscle regeneration.

overall_design: Total RNA from 8-week old CRY2flox mouse and CRY2scko mouse.

first: Yao-Hua

last: Song


title: RNA-seq analysis CARM1 inhibitor Ellagic acid effect on MDA-MB-231 cells

city: beijing

first: yan

last: wang

address: 17 panjiayuan nanli, chaoyang district, Beijing, China

country: Zimbabwe

institute: National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences

summary: Ellagic acid is a kind of polyphenol lactone, which is widely found in various fruits and nuts. It’s the natural inhibitor of CARM1. Ellagic acid was reported to inhibit breast cancer growth and metastasis. But the mechanism of Ellagic acid has not been investivaged clearly in triple-negative breast cancer (TNBC). Our results indicate that direct inhibition of CDK4 by Ellagic acid limits breast cancer proliferation and metastasis. Our research has determined the molecular basis of Ellagic acid effect in TNBC, which may provide new ideas and drugs for cancer therapy.

overall_design: Ellagic acid or control was added into MDA-MB-231 cells (n=3 per group) for 24 hours. Total RNA was extracted using Trizol reagent (Invitrogen, USA). Agilent 2100 Bioanalyzer (Agilent Technologies, USA) was used to evaluate the RNA quality.

first: Dandan

last: Feng

first: Yan

last: Wang


title: ALOX15B controls macrophage cholesterol homeostasis via lipid peroxidation, ERK1/2 and SREBP2

city: Frankfurt

first: Yvonne

last: Benatzy

address: Theodor-Stern-Kai 7, building 74

department: Institute of Biochemistry I

institute: Goethe-University Frankfurt

summary: Macrophage cholesterol homeostasis is crucial for health and disease and has been linked to the lipid-peroxidizing enzyme arachidonate 15-lipoxygenase type B (ALOX15B), albeit molecular mechanisms remain obscure. We performed global transcriptome and immunofluorescence analysis in ALOX15B-silenced primary human macrophages and observed a reduction of nuclear sterol regulatory element-binding protein (SREBP) 2, the master transcription factor of cellular cholesterol biosynthesis. Consequently, SREBP2-target gene expression was reduced as were the sterol biosynthetic intermediates desmosterol and lathosterol as well as 25- and 27-hydroxycholesterol. Mechanistically, suppression of ALOX15B reduced lipid peroxidation in primary human macrophages and thereby attenuated activation of mitogen-activated protein kinase ERK1/2, which lowered SREBP2 abundance and activity. Low nuclear SREBP2 rendered both, ALOX15B-silenced and ERK1/2-inhibited macrophages refractory to SREBP2 activation upon blocking the NPC intracellular cholesterol transporter 1. These studies suggest a regulatory mechanism controlling macrophage cholesterol homeostasis based on ALOX15B-mediated lipid peroxidation and concomitant ERK1/2 activation.

overall_design: To investigate the biological function of ALOX15B, differentiated primary human macrophages were transfected with ALOX15B-specific or control siRNA.

first: Ryan

last: Snodgrass

first: Bernhard

last: Brüne


title: Transcriptome-based analysis reveals functional differences in Dendrobium officinale from different growing regions and with different quality levels

city: Beijing

first: Honglei

last: Dang

address: 18 Life Science Parkway, Changping District

institute: National Engineering Research Center for Beijing Biochip Technology

summary: Dendrobium officinale is a common and expensive traditional Chinese medicine used as a medicinal agent and food that has immunoregulatory and anti-inflammatory effects. Methods to authenticate D. officinale are lacking. Although chemical profiles of D. officinale were published, transcriptomic profiling is lacking. Here, we collected D. officinale from two regions (Yunnan and Miandian) and the analogue D. compactum. The Yunnan samples had three different quality levels. All samples were subjected to genome-wide biological response fingerprinting (BioReF) in RAW264.7 cells using RNA sequencing.

overall_design: The analysis uses the comparison between the LPS treatment group and the DMSO control group as the model group, and the experimental samples treated with extracts of  Dendrobium officinale are comparison with the LPS group , genes expression changes were determined by RNA-Seq to determine Dendrobium officinale responsive genes.

first: Hong

first: Lan

last: Xie

first: Xiao

last: Zhang


title: Transcriptome analysis revealed that Arabidopsis model plant invokes the activation of heat shock proteins and ER stress response against cesium stress

status: Public on Apr 06 2024

city: Anseong-si

first: Dasom

last: Choi

state: Gyeonggi-do

address: 4726, Seodong-daero

institute: Chung-Ang University

summary: We investigated the root growth of several knockout mutants of heat shock protein family genes and found that heat stress response was compromised in these mutants compared to wild type plants. It suggested that heat shock protein genes including heat shock protein genes including HSP17s, HSP23s, HSP101, and HSFA2 proteins are deployed upon exposure to Cs for plant stress tolerance. Our study provided novel insights into the molecular events occurring in Cs-stressed plants.

overall_design: Performed a comparative transcriptomic analysis between control and Cs-treated plants via RNA-seq.

first: Dae

middle: K

last: Ko

first: Dong-Hwan

last: Kim


title: RNA-seq in gWAT from diabetic mice treated with canagliflozin

city: Bunkyo-ku

first: Goro

last: Katsuumi

state: Tokyo

address: Hongo 2-1-1

country: Japan

institute: Juntendo University

summary: We found canagliflozin shows elimination of senescent cells from organs in age-related pathology mouse model including diabetes. To clarify detailed change by canagliflozin treatement, we conducted RNA-seq analysis in gonadal white adipose tissue(gWAT).

overall_design: gWAT was harvested from high-fat-diet induced diabetec mouse with or without 7-days canagliflozin treatment (0.03% w/w mixed in food)

first: Masahide

last: Seki

first: Tohru

last: Minamino


title: Factors with Dual Roles as Activators and Repressors Regulate Intermediate Expression Levels

city: Haidian District

first: Jiawei

last: Yu

state: Beijing

address: Tsinghua  University

department: School of Pharmaceutical Sciences

institute: Tsinghua University

summary: Precise control of gene expression levels is essential for normal cell functions, yet how they are defined and tightly maintained, particularly at intermediate levels, remains elusive. Here, using a series of new sequencing, imaging, and functional assays, we uncover a class of transcription factors, referred to as dual TFs, which reduce high expression but increase low expression to achieve stable intermediate levels. Dual TFs directly exert activating and repressing functions via condensate-forming domains that compartmentalize core transcriptional unit selectively. Clinically relevant mutations in these domains, which are linked to a range of developmental disorders, impair condensate selectivity and dual TF activity. These results collectively address a fundamental question in expression regulation and demonstrate the potential of dual TFs as powerful effectors for engineering controlled expression levels.

overall_design: ACC-seq to capture differential chromatin accessibility features in response to modulations of condensate occupancy of mESC (R1 strain)


title: VSMC phenotypic transition

first: li

institute: State Key Laboratory of Cardiovascular Disease, Fuwai Hospital

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

overall_design: Refer to individual Series


title: Super cChIP-seq, a robust and universal epigenetic profiling method using 10 cells

city: Shanghai

first: Xiaodong

last: Zhao

address: 800 Dongchuan RD

institute: Shanghai Jiao Tong University

type: Methylation profiling by high throughput sequencing

summary: ChIP-seq is the standard technique to characterize the genome-wide epigenomic modifications, which requires millions of cells as starting material. Although some methods have recently reported to be suitable for epigenomic profiling with a small number of cells, there are still no methods capable of profiling both histone modifications and DNA methylation with extremely low input. Here, we developed a new method by supplementing carrier materials of chemically modified epigenetic markers and dUPT-containing DNA fragments during ChIP procedures. Using this strategy, we generated high-quality epigenomic profiles of both histone modifications (super cChIP-seq) and DNA methylation (super cMeDIP-seq) with as few as 10 cells. Thus, we provide a robust and straightforward universal approach for epigenomic profiling starting with extremely low input.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Extremely-low input ChIP-seq. Fragmentated chromatin DNA and carrier are enriched with antibody, and followed by library construction.

first: C

last: Hu

first: J

last: Wu

first: Y

last: Kang

first: Q

first: W

last: Du

first: Z

last: Shao

first: X


title: Hyperoxia impairs iPSC derived endothelial cells and drives an atherosclerosis-like transcriptional phenotype

status: Public on Apr 05 2024

city: Atlanta

first: Katherine

last: Owsiany

state: GA

address: 1365 Clifton Rd NE

institute: Emory University

summary: Induced pluripotent stem cells (iPSCs) directed to endothelial identity (iPSC-ECs) lose expression of key identity markers under standard in vitro conditions, limiting their clinical applications. We examined iPSC-ECs at late passage (>2 weeks) under hyperoxic (21%)conditions  with single cell RNA sequencing

overall_design: Y6 pluripotent stem cells were directed to endothelial identity by treatment with CHIR99021, a GSK3 inhibitor, as well as BMP4 for 3 days, followed by VEGFa and forskolin treatment for 2 days according to the protocol described by Cowen et al 2014, Nature Cell Biology DOI 10.1038/ncb3205. Then, cells were purified using CD144 magnetic beads, and were subsequently cultured for two weeks at atmospheric oxygen level (21%) in a conventional cell culture incubator before harvest and analysis by scRNAseq


title: miRNA changes in the serum of sarcopenia patients

first: Ling

last: Wang

address: 899 Pinghai Road

institute: Department of General Medicine, The First Affiliated Hospital of Soochow University

summary: To identify putative miRNAs in the serum of  in sarcopenia patients, we carried out a microarray analysis.

overall_design: The RNA was collected from the serum of  in sarcopenia patients and further applied for microarray analysis.


title: Translational Insights from Cell-type variation across Amygdala Subnuclei in Rhesus Monkeys and Humans

city: Davis

first: Andrew

last: Fox

state: CA

address: 1 Shields Ave

institute: University of California, Davis Davis, CA

summary: Theories of amygdala function are central to our understanding of psychiatric and neurodevelopmental disorders. However, our limited knowledge of the molecular and cellular composition of the amygdala impedes translational research aimed at developing new treatments and interventions. Here, we use single-nucleus RNA-sequencing from multiple amygdala subnuclei in both humans (n=3, Male) and rhesus macaques (n=3, Male) to begin to bridge the gap between preclinical rodent models and human disorders. Our results reveal substantial heterogeneity between regions, even when restricted to inhibitory or excitatory neurons. Consistent with prior work, our data highlight the complexities of individual marker genes for uniquely targeting specific cell-types. Cross-species analyses suggest the rhesus monkey model to be well-suited to understanding the human amygdala, but also identify limitations. For example, we identified a cell-cluster in the ventral lateral nucleus (vLA) of the amygdala that is enriched in humans, relative to rhesus macaques. Additionally, we identify specific cell-clusters with relative enrichment of disorder-related genes. These analyses point to the human-enriched vLa cell-cluster as relevant to ASD, potentially highlighting a vulnerability to neurodevelopmental disorders that has emerged in recent primate evolution. Further, we identified a cluster of cells expressing markers for intercalated cells (ITCs) that is enriched for genes reported in human genome-wide association studies of neuroticism, anxiety disorders, and depressive disorders. Together, these data shed light on the composition of the amygdala and identify specific cell-types that can be prioritized in basic science research to better understand human psychopathology and guide the development of potential treatments

sample_organism:
Macaca mulatta
Homo sapiens

platform_organism:
Macaca mulatta
Homo sapiens

overall_design: Subjects include n=3 male rhesus macaques (Macaca Mulatta; age: 3.5-4 years) and n=3 male human donors (Homo Sapiens; Age: 15-19 years, postmortem interval, mean: 23.5 hrs, range: 8-32 hrs). To understand heterogeneity across the primate amygdala, tissue samples were collected from the corresponding areas of the dorsal amygdala Ce-region and the ventral portions of the La-region (vLa) in both primate species. Rhesus tissue was obtained from the California National Primate Research Center. All study procedures were performed in accordance with the guidelines set forth by the University of California Davis Animal Care and Use Committee (IACUC). Human tissue was obtained from the Brain Endowment for Mental Health (BEMH, University of California Davis). All experiments were approved by the Institutional Review Board (IRB) and the IACUC at the University of California, Davis. Under the guidance of expert primate neuroanatomists (CS; AF; JF), 1.5mm tissue biopsy punches at a depth of 2 to 3 mm were used to retrieve samples from Ce and vLA, including embedded ITCs. Single nuclei suspensions were made following minor modifications to 10x genomics demonstrated protocol: Nuclei Isolation from Cell Suspensions and Tissue for Single Cell RNA Sequencing Libraries were prepared by the UC Davis Genomics Core and sequenced on a NovaSeq S4 300 150 bp pair-ended reads (PE150) which targeted a total of 13,149 nuclei for Ce and 8,245 nuclei for vLa across all three Rhesus subjects with 6,973 nuclei for Ce and 9,289 nuclei for vLa across all three Human subjects. Resulting in a total of 67,312 reads per nuclei for Rhesus and 52,378 reads per nuclei for Human

first: Shawn

last: Kamboj

first: Erin

last: Carlson

first: Bradley

middle: P

last: Ander

first: Kari

last: Hanson

first: Karl

last: Murray

first: Julie

last: Fudge

first: Melissa

last: Bauman

first: Cynthia

last: Schumann


title: Tumoral acidosis promotes adipose tissue depletion by fostering adipocyte lipolysis

city: Brussels

first: Camille

last: Lefevre

state: Belgium

address: Avenue Emmanuelle Mounier 73 (bte B1.73.11)

department: Louvain Drug Research Institute (LDRI)

country: Belgium

institute: UClouvain

summary: Adaptation to acidosis has been reported to induce a shift towards fatty acid metabolism. We observed that conditioned medium of HCT, FaDu and SiHa adapted to acidosis (pH6.5) induced higher lipolysis of primary subcutaneous and mesenteric adipocytes compared to cancer cells maintained at pH7.4. In order to identify the prolipolytic factors induced by acidosis, we examined the differences between cancer cells adapted to pH6.5 and those maintained at pH7.4 by RNA sequencing. Cancer cells adapted to pH6.5 showed profound alterations in their gene expression compared with cancer cells maintained at pH7.4. Here, we focused on predicted secreted genes. We determined that 84 altered genes predicted to be secreted proteins overlapped between the 3 cancer cell lines, 24 of which were overexpressed. The role of these overexpressed genes was determined in vitro. We studied their role in inducing adipocyte lipolysis and in the depletion of adipose tissue observed in cancer cachexia.

overall_design: Human colon HCT116, hypopharyngeal FaDu and cervix SiHa cancer cell lines  were  exposed to medium with pH adjusted to 7.4 or 6.5. pH6.5-exposed cancer cells were maintained at pH6.5 for a minimum of 1 month before.

first: Laure

last: Bindels

first: Morgane

last: Thibaut


title: Transcriptomic profiling of the role of Ctla4 in the innate immune system

city: London

first: Nick

last: Powell

address: Du Cane Road

department: Department of Metabolism, Digestion and Reproduction

institute: Imperial College London

summary: The maintenance of intestinal homeostasis is a fundamental process critical for organismal integrity. Sitting at the interface of the gut microbiome and mucosal immunity, adaptive and innate lymphoid populations regulate the balance between commensal micro-organisms and pathogens. Checkpoint inhibitors (CPI), particularly those targeting the CTLA-4 pathway, disrupt this fine balance and can lead to inflammatory bowel disease (IBD) and immune checkpoint colitis (CPI-C). Here, we show that CTLA-4 is expressed by innate lymphoid cells (ILC) and that its expression is regulated by ILC subset-specific cytokine cues in a microbiota-dependent manner. Genetic deletion or antibody blockade of CTLA-4 demonstrates that this pathway plays a key role in intestinal homeostasis and is conserved in human IBD and CPI-induced colitis (CPI-C). We propose that this population of CTLA-4-positive ILC may serve as an important target for the treatment of idiopathic and iatrogenic intestinal inflammation.

overall_design: Comparative analysis of transcriptomic profiles from distal colon segments of Rag1-/- mice compared to Rag1-/- x Ctla4-/-

first: Jon

last: Lo

first: Domenico

last: Cozzetto


title: Acidovorax temperans polarizes T17 cells and skews neutrophil maturation to promote lung adenocarcinoma development

city: Bethesda

first: Josh

last: Stone

state: MD

address: 9000 Rockville Pike

department: CCR

institute: National Cancer Institue

summary: Dysbiosis, or changes within the microbiome, is a common feature of solid tumors, however whether this dysbiosis directly contributes to tumor development is largely unknown. We previously characterized the human lung cancer microbiome and identified the Gram-negative Acidovorax temperans as enriched in tumors and associated with smoking status and TP53 mutations. To determine if A. temperans exposure could contribute to the development of lung cancer, we investigated its effect in a genetically engineered mouse model of lung adenocarcinoma driven by mutant Kras and Tp53. In comparison to control mice and those instilled with a commensal species Lactobacillus gasseri, we found that repeated A. temperans exposure accelerates tumor development and burden through infiltration of proinflammatory cells in the lungs. Comparing immune cell infiltrates at two timepoints revealed increased proinflammatory cells immediately following A. temperans instillation while later the number of T cells, especially IL-17+ cells, was increased. These data indicate a clear role for microbiota-induced inflammation as a key mechanism in the development of lung cancer, demonstrating that dysbiosis contributes to tumor growth.

overall_design: Acidovorax teemperans, dysbiotic in human lungs, was repeatedly instilled into the lungs of mice bearing K-ras/p53 mtuations, to accelerate lung adenocarcinoma development. Lungs were harvested and RNA was extracted from tumors for sequencing.

first: Joshua

first: Mohammed

last: Khan

first: Curtis

last: Harris


title: Effect of erythropoietin on the gene expression of Xenopus tropicalis peripheral blood cells.

city: Shinjuku

first: Kazuki

last: Omata

address: Wakamatsucho, 2-2

department: Graduate school of advanced science and engineering

institute: Waseda University

summary: The regulatory system of erythropoiesis through erythropoietin (EPO) and its receptor (EPOR) is essential for vertebrates' life. In humans, EPO affects the erythroid progenitors and stimulates proliferation and differentiation. The EPO-EPOR axis is mainly mediated by the JAK2-STAT5 pathway. After terminal maturation, erythrocytes lost EPOR expression; however, erythrocytes of amphibian Xenopus tropicalis maintain EPOR expression even after their terminal maturation. Because erythrocytes of vertebrates except for adult mammals are nucleated, we hypothesized that EPO can alter its transcriptional state. To explore the effects of EPO on Xenopus mature erythrocytes, we performed RNA-Seq analysis on EPO-stimulated Xenopus peripheral blood cells. The EPO-stimulated group showed increased expression of direct target genes of STAT such as cish, socs3, and socs1 indicating a functional signal transduction system. Furthermore, we observed several EPO-responsive genes that were not reported in mammalian erythroid progenitors. These findings suggest the functional difference between enucleated erythrocytes in adult mammals and nucleated erythrocytes in fetal mammals and non-mammalian vertebrates.

overall_design: Xenopus peripheral blood was corrected from the heart. Recombinant Xenopus tropicalis was EPO expressed in E. coli. The control group was cultured in 7/9 x IMDM with 10% fetal calf serum. EPO added group was cultured in the same condition supplemented with 1 ng/mL EPO. Fresh blood cells before culture and cells cultured for 1.5 or 4.0 hours were harvested and their total RNA was extracted by the acid guanidine phenol -chloroform method. Three biological replicates were made for each experimental group. We then performed gene expression profiling analysis using the data obtained from 3' RNA-Seq analysis.

first: Makoto

last: Kashima

first: Hiromi

last: Hirata

first: Takashi

last: Kato


title: Single cell transcriptomics reveals dysregulated immnue homeostasis in different stages in HPV-induced cutaneous squamous cell carcinoma

city: changsha

first: Liqing

last: Ding

address: xiangya street

institute: Central South University

summary: Cutaneous squamous cell carcinoma (cSCC) is a common skin cancer in humans. HPV persistence in the epidermis and excessive wart or squamous is associated with cancer development. Various viral warts can be found on the skin infected with HPV, like condyloma acuminatum and bowenoid papulosis (BP). At present, it is believed that BP is a potential malignant squamous cell carcinoma in situ. In this study, we reported a very rare case of HPV-positive patient with concurrence of BP and cSCC. This patient progressed at a very fast rate and transferred to death quickly. To reveal the cell microenvironment changes from normal skin (NS) to BP and cSCC after HPV infection, we performed scRNA-seq on four pathological lesions skin (NS, BP, paracancerous squamous cell carcinoma (pc-cSCC) and cSCC) from this patient. Among immune cells, CD52+ macrophages were only detected in pc-cSCC and cSCC tissue, and two clusters of T cells, CD8+ Tex cells and CD4+ Tregs as well as matrix cells like MMP1+, MMP11+ fibroblasts were increased in pc-cSCC and cSCC tissue. Widely interactions were also found among specific T cells/macrophages and keratinocytes, which might be involve in the cSCC progress. Targeting these cells or their interactions may be beneficial for the diagnosis and treatment of cSCC.

overall_design:
We diagnosed and followed up on a very rare HPV-induced cSCC patient who progressed at a very fast rate and transferred to death quickly. We performed single-cell RNA sequencing (scRNA-seq) of 11,379 cells from the skin tissues of this patient with four different skin statuses after HPV infection. Immunofluorescence experiments were used for validation.

>>>Raw data are not available due to patient privacy concerns.<<<


title: B cell directed CAR-T cell therapy results in activation of CD8+ cytotoxic CAR-negative bystander T cells in both non-human primates and patients

city: Boston

first: Jim

last: Kaminski

address: 1 Blackfan Circle

institute: Boston Children's Hospital

summary: There is growing appreciation for the emergence of CARneg bystander T cells after CAR-T cell infusion. However, their phenotypic and transcriptomic hallmarks and mechanisms of activation remain uncertain. We performed single-cell RNA-Seq (scRNA-Seq) on non-human primate (NHP) and patient-derived T cells to interrogate CARneg T cells following B cell targeted CAR-T cell therapy. In a NHP model, we observed a distinct population of activated CD8+ CARneg T cells emerging during CAR-T cell expansion. These bystander CD8+ CARneg T cells exhibited a unique transcriptional signature with upregulation of NK-cell markers (KIR3DL2, CD160, KLRD1), chemokines and chemokine receptors (CCL5, XCL1, CCR9), and downregulation of naive T cell-associated genes (SELL, CD28). A transcriptionally similar population was identified in patients following Tisangelecleucel infusion. Mechanistic studies revealed that IL-2 and IL-15 exposure induced bystander-like CD8+ T cells. These T cells efficiently killed leukemic cells through a TCR-independent mechanism. Together, these data identify bystander CD8+ T cells as a novel mechanism by which CAR-T cell infusion can induce further anti-leukemic activity, measurable in both NHP and in patients.

overall_design: scRNA-Seq of peripheral blood T cells obtained from NHPs undergoing CAR-T treatment

first: Ryan

last: Fleming

first: Ulrike

last: Gerdemann


title: Effect of depletion of GPRC5C from smooth muscle cells (SMC) on gene expression in mice

city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

institute: MPI for heart and lung research

summary: To investigate the consequence of Gprc5c knocking out in SMC on mRNA transcriptioinal level, EGFP-expressing smooth muscle cells were sorted by FACS  from skeletal muscle vasculature of tamoxifen-induced control and iSM-G5C-KOs carrying the mTmG reporter strain.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for SMC from skeletal muscle vasculature Control vs iSM-G5C-Kos

first: Tianpeng

last: Wang

last: Guenther

first: Nina

last: Wettschureck


title: Transcriptome Analysis Reveals Gene Expression Changes in a mouse Model of Hyperoxia-induced acute lung injury

first: hu

last: xiaolin

state: Shanghai

address: south congqing road 227#

institute: Shanghai Jiaotong University School of Medicine

summary: Oxygen therapy is essential for cure critically patients in ICU, but hyperoxia can cause damage to many organs, including the lungs, leading to Hyperoxia Acute Lung Injury (HALI), a mild type of acute respiratory distress syndrome  (ARDS). However, the comprehensive pathogenesis and regulation mechanisms underlying the development of HALI remain unclear.  In this study, we conducted a mouse model with hyperoxia treatment and sequenced the gene expression pattern of HALI-mice and relative control (RNA-seq).

overall_design: Comparative gene expression profiling analysis of RNA-seq data for HALI and wild type mouse

first: Qin

last: Song

first: Wang

last: Xiaoqin

first: Wei

last: Chunyan

first: Hu

last: Xiaolin


title: Single-cell sequencing of muose thymic epithelial cell organoids

city: Utrecht

first: Sangho

last: Lim

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

summary: Mouse thymic epithelial cell organoids, cultured in (1) expansion medium, (2) differentiation medium, or (3) differentiation medium with Rank ligand and retinoic acid (DM+RR), were FACS sorted into plates to follow the SORT-seq protocol (Muraro et al., 2016).

overall_design: Mouse thymic epithelial cell organoids cultured in (1) expansion medium or (2) differentiation medium were collected and DAPI-negative viable cells were FACS-sorted and analyzed using scRNA-seq. Mouse thymic epithelial cell oganoid, which is CRISPR knock-in line to have Aire-P2A-tdTomato and Foxn1-P2A-Clover, cultured in (DM+RR) were analyzed by using scRNA-seq. In brief, DAPI-negative & tdTomato-positive cells or DAPI-negative & Clover-positive cells, or DAPI-negative without Clover and tdTomato werer FACS sorted for analysis.

first: Sam

last: Willemsen

first: Gijs

last: van Son

first: Ni-Luh

last: Eka Yanti

first: Amanda

last: Rolf

first: Jeroen

last: Korving

first: Harry

last: Begthel

first: Hans

last: Clevers


title: Intervertebral disc degeneration

city: La Jolla

first: Padma

last: Natarajan

address: 10550 N.Torrey Pines Road

department: CCBB

institute: The Scripps Research Institute


title: Shared and compartment-specific processes in nucleus pulposus and annulus fibrosus during intervertebral disc degeneration

summary: Elucidating how cell populations promote onset and progression of intervertebral disc degeneration (IDD) has the potential to enable more precise therapeutic targeting of cells and mechanisms. Single cell RNA-sequencing (scRNA-seq) was performed on surgically separated annulus fibrosus (AF) (19,978; 26,983 cells) and nucleus pulposus (NP) (20,884; 24,489 cells) from healthy and diseased human intervertebral discs (IVD). In both tissue types, we observed depletion of cell subsets involved in maintenance of healthy IVD, specifically the immature cell subsets – fibroblast progenitors and stem cells – indicative of an impairment of normal tissue self-renewal. We also identified tissue-specific changes. In NP, several fibrotic populations were increased in degenerated IVD, indicating tissue-remodeling. In degenerated AF, we identified a novel disease-associated subset, which is a stem cell-derived abnormally differentiated chondrocytic population and expresses disease-promoting genes. It was associated with pathogenic biological processes and the main gene regulatory networks includedthrombospondinsignaling and FOXO1 transcription factor. Our data reveal new insights of both shared and tissue-specific changes in specific cell populations in AF and NP during IVD degeneration. These identified mechanisms and molecules are novel and more precise targets for IDD prevention and treatment.

overall_design: Single cell level analysis of annulus fibrosus and nucleus pulposus tissues from human diseased (n=10, n=8) intervertebral discs.

first: Hannah

last: Swahn

first: Jasmin

last: Mertens

first: Merissa

last: Olmer

first: Kevin

last: Myers

first: Tony

last: Mondala

first: Padmaja

first: Steven

last: Head

first: Oscar

last: Alvarez-Garcia

first: Martin

last: Lotz


title: The cellular landscape of the healthy human intervertebral disc: identification of new immature cell populations and their relationship with mature populations at single cell resolution

summary: For this study, we applied scRNA-seq to cells from surgically separated annulus fibrosus and nucleus pulposus tissues to identify A) thecellular landscapes of healthytissues, and B) characterize in detail the composition andgene regulatory networksof immature cell populations.

overall_design: Single cell level analysis of annulus fibrosus and nucleus pulposus tissues from human healthy (n=3, n=3) intervertebral discs (IVDs).


title: Gene expression profiling of colon biopsies from ulcerative colitis patients and healthy volunteers

first: Nick

last: Powell

summary: Genome-wide transcriptional profiling of colonic biopsies endoscopically acquired from the rectosigmoid area of healthy donors and UC patients.

overall_design: Biopsies were sampled from 6 healthy donors who lacked completely mucosal inflammation both histologically and endoscopically and from 16 UC patients with evidence of active disease.

first: Jonathan

last: Digby-Bell

first: Domenico

last: Cozzetto


title: Effects of re-expressed RASSF1A  on gene expression in rhabdoid tumor of the kidney cells.

city: Setagaya-ku

first: NOBUTAKA

last: KIYOKAWA

department: Pediatric Hematology & Oncology Research

institute: National Research Institute for Child Health & Development

summary:
Several groups reported the correlation between hypermethylation of RASSF1A and aggressiveness or a poor outcome in various pediatric tumors, including Wilms tumor. In addition, it is known that the overexpression of RASSF1A promotes apoptotic cell cycle arrest and reduces the tumorigenicity. In an attempt to identify epigenetic targets related to the biological features of rhabdoid tumor of the kidney (RTK), we analyzed the methylation level of the RASSF1A gene in RTK. RASSF1A promoter region in RTK is hypermethylated and the gene and protein expression levels were low. To examine the effect of re-expression of RASSF1A in RTK cells from the viewpoint of gene expression, we performed micro array analysis.
We observed neither clear apoptosis induction nor cell-cycle arrest in RASSF1A-expressed RTK cell lines. Furthermore, the geneexpression profiling induced by RASSF1A expression in RTK cell line is curious, and CDK6 expression was significantly increased while CDKN2A expression was significantly decreased after RASSF1A expression. The changes in the gene expression profile that we observed in RTK cells after RASSF1A expression may not be causative events involving the suppression of cell proliferation, but rather antagonizingevents reflecting cellular responses against RASSF1A expression in RTK cells that are deficient in SMARCB1.

overall_design: RTK cell were infected with a retro-viral vector for RASSF1A re-expression or an empty retro-viral vector as control. The RNAs isolated from these cells were used for hybridization on Affymetrix microarrays.

first: Nobutaka

last: Kiyokawa

first: Hitomi

last: Ueno-Yokohata

first: Hajime

last: Okita


title: HCT116 colorectal cancer cells treated with 2.5 mM Metformin

city: Delhi

first: Muzaffer

middle: Ahmed

last: Bhat

state: Delhi

address: ANSARI NAGAR NEW DELHI 110029

department: Physiology

country: India

institute: AIIMS

summary: Metformin is a drug used in the treatment of type 2 diabetes mellitus. Various studies have elucidated its anticancer properties. In this study, the effect of metformin on the differentiation and tumor microenvironment of colorectal cancer cells (CRC) was evaluated. For our study, we have used HCT116 colorectal cancer cell line and treated the cells with Metformin. Maximum tolerable non-toxic dose of metformin on HCT116 cells was determined by MTT assay. Cells were treated with 2.5 mM Metformin for 2 weeks. Analysis of apoptosis was done by flow cytometry using Annexin V / PI. CSC population was determined by flow cytometry using CSC markers CD44 and CD166. Metformin's ability to induce differentiation in CSC was assessed by analyzing Cytokeratin 20 (CK20) by flow cytometry and CDX1 (transcription factor for CK20), by RT-QPCR. Expression of Ki67 (proliferation marker) was done by RT-QPCR. RNA was isolated from 2.5 mM Metformin-treated and untreated cells populations. Microarray of untreated and 2.5 mM Metformin-treated RNA was done to study the whole genome transcriptomic changes.

overall_design: 8x60k whole genome expression arrays for comparing transcriptome of HCT116 colorectal cancer cells (n=4) with HCT116 cells treated with 2.5 mM Metformin (n=4).

first: Amar

last: Preet Kaur

first: Aditi

last: Bhattacharya

last: Ahmed Bhat

first: Sudip

last: Sen


title: Sleep Deprivation Impairs Vascular Function through Reduced Endothelial Oxidant Stress Clearance in Women

city: NY

first: Riddhi

last: Shah

state: NY

address: 630W 168th Street

department: Medicine

institute: CUIMC

summary:
Introduction: Sleep deprivation is associated with increased cardiovascular risk, which is more pronounced in women than men; however, causal evidence is lacking and the underlying mechanisms are unclear. We used randomized crossover design of prolonged sleep deprivation that mimics “life-like conditions” and endothelial cells (ECs) harvested from healthy women to investigate directly whether sleep deprivation impairs endothelial function and to identify underlying mechanisms.
Methods: Healthy women with normal habitual sleep (7-9 h/day) were randomly allocated to 6-weeks of adequate sleep (7-9 h/day) or sleep deprivation (1.5 h less than habitual sleep) in a crossover design. Sleep duration was monitored objectively by actigraphy. EC harvesting and brachial artery flow-mediated dilation (FMD) were performed at baseline and the end of adequate sleep and sleep deprivation period.
Results: Twenty-eight healthy women (mean [SE] age 35±13 years; BMI 25±3 kg/m2) participated. Compared with adequate sleep, sleep deprivation reduced FMD (mean±SE 8.65±0.48 vs. 7.35±0.39, p=0.02) and increased EC inflammation (nuclear factor-κB nuclear fluorescence area mean±SE 1.36±0.24 vs. 2.04±0.38 μm2, p=0.03 and mRNA expression of vascular cell adhesion molecule-1 mean±SE 1.00±0.19 vs. 2.31±0.72, p=0.04) compared with adequate sleep. Sleep deprivation increased EC oxidative stress by 67% compared with adequate sleep (redox sensitive fluorogenic probe fluorescence intensity, p=0.002) without upregulating antioxidant response. Using RNA-seq and a predicted protein-protein interaction algorithm, we identified reduced expression of serum response factor, a transcription factor that primes cortical response to sleep deprivation, and its endothelial target Defective in Cullin Neddylation-1 Domain Containing 3 as novel mediators of impaired nuclear factor (erythroid-derived 2)-like 2-mediated antioxidant responses in ECs in sleep deprivation.
Conclusion: Sleep deprivation impairs clearance of endothelial oxidant stress accumulated during wakefulness leading to endothelial dysfunction and potentially increased cardiovascular risk in women. Trial Registration Number: ClinicalTrials.gov NCT02835261

overall_design: We analysed endothelial cells harvested from women in a randomized crossover design of prolonged sleep deprivation, which mimics "life-like" condition, to observe any changes in gene expression after  sleep deprivation compared to adequate sleep.

first: Vikas

last: Malik

first: Sanja

last: Jelic


title: Arthritogenic SKG T cells have a transcriptional program of activation and a repertoire pruned by superantigen

city: San Francisco

first: Yang

last: Sun

address: 513 Parnassus Ave

department: Rheumatology

institute: UCSF

summary:
Purpose: The goals of this study were to study the transcriptome and TCR repertoire of naive CD4 T cells before the induction of arthritis in the SKG and wild-type (WT) mouse with eGFP-Nur77 as a reporter of antigen stimulation (SKGNur and WTNur).
Methods: Bulk RNA sequencing was used to generate the mRNA profiles for the top 10% and bottom 10% of GFP expressing cells from three SKGNur and three WTNur BALB/c mice resulting in 12 samples overall (3 WT Low, 3 WT High, 3 SKG Low, 3 SKG High) using the Truseq Stranded mRNA kit followed by sequencing on a HiSeq 2500 sequencer. The sequence reads that passed quality filters were aligned with STAR and then analyzed with DESeq2.
Results: We mapped about 38,000,000 million reads per sample to the mouse genome (mm10). The differential expression pairwise comparisons (WT Low v WT High, WT Low v SKG Low, SKG Low v SKG High, and WT High v SKG High) generated a collection of 991 differentially expressed genes with log2FC >1 and Benjamini-Hochberg adjusted p value < 0.05. Hierarchical clustering of differentially expressed genes uncovered six modules of gene expression which highlighted heterogeneity within the naive CD4 T cell compartment and transcriptional signatures associated with the SKG High samples.
Methods: Single cell RNA gene expression and TCR sequencing libraries were generated for the top 10% and bottom 10% of GFP expressing cells from two SKGNur and two WTNur BALB/c mice resulting in 8 samples overall (2 WT Low, 2 WT High, 2 SKG Low, 2 SKG High) using the 5' 10x single cell 5"+V(D)J v1 kit and were sequenced on a NovaSeq 6000 sequencer. The reads were aligned using cellranger and analyzed using a combination of single cell tools including scanpy and scvelo.
Results: We mapped about 785,000,000 sequence reads and about 379,000,000 sequence reads per sample for the gene expression and TCR libraries to the mouse genome (build mm10 with the addition of the eGFP transcript) and TCR reference (vdj GRCm38 v 3.1.0), respectively, using cellranger. We identified 31,054 transcripts across 101,869 cells and recovered paired TRA and TRB chains from more than 80% of cells. After filtering cells and genes by our quality control metrics, we identified 1119 highly variable genes which were used for PCA analysis to create a nearest neighbors graph for leiden clustering and UMAP projection. We identified 13 clusters that we collapsed into 9 cell sub-types. Within the cluster (T.4_Nr4a1) exclusively expressing our marker gene of interest, Nr4a1, we found that SKG High cells upregulated TCR signaling genes despite their impaired impaired TCR signaling ability due to a Zap70 mutation.  Within the T.4Nr4a1 cluster we found two states of acute to chronic TCR signaling, marked by Egr2 and Tnfrsf9, and we used trajectory inference to uncover a continuum of cell states with these acute to chronic TCR signaling states as the endpoints. Interestingly, SKG High cells in the T.4 Nr4a1 cluster seemed arrested in the earlier more acute TCR signaling state suggesting they are unable to appropriately fully upregulate the chronic TCR signaling and more anergic cell state. We also uncovered TRBV restriction within the SKG High samples and specifically within the SKG High cells within the T.4 Nr4a1 cluster that seems to be driven by a failure of negative selection by endogenously expressed super antigens from MMTVs present in the BALB/c mouse. Thus the naive CD4 T cell compartment within the SKG High cells are poised to enter into a reactive immune response via both a unique transcriptomic and TCR repertoire which may underlie the mechanism of high arthritogenic potential of these cells.

overall_design: Bulk RNA seq and single cell RNA and TCR sequencing of naïve CD4 T cells before arthritis induction in Nur77-eGFP SKG and wild-type mice

first: Judith

last: Ashouri

first: Elizabeth

last: McCarthy

first: Steven

last: Yu

first: Noah

last: Perlmutter

first: Charles

last: Lin

first: Joe

last: DeRisi

first: Chun

last: Ye

first: Arthur

last: Weiss


title: Gag proteins encoded by endogenous retroviruses are required for zebrafish development

status: Public on Apr 04 2024

city: Ithaca

first: Jonathan

last: Wells

address: 216 Biotechnology Building

institute: Cornell University

summary: Transposable elements (TEs) make up the bulk of eukaryotic genomes and examples abound of TE-derived sequences repurposed for organismal function. The process by which TEs become coopted remains obscure because most cases involve ancient, transpositionally inactive elements. Reports of active TEs serving beneficial functions are scarce and often contentious due to difficulties in manipulating repetitive sequences. Here we show that recently active TEs in zebrafish encode products critical for embryonic development. Knockdown and rescue experiments demonstrate that the endogenous retrovirus family BHIKHARI-1 (Bik-1) encodes a Gag protein essential for mesoderm development. Mechanistically, Bik-1 Gag associates with the cell membrane and its ectopic expression in chicken embryos alters cell migration. Similarly, depletion of BHIKHARI-2 Gag, a relative of Bik-1, causes defects in neural crest development in zebrafish. We propose an “addiction” model to explain how active TEs can be integrated into conserved developmental processes.

overall_design: RNA-seq of BHIKHARI endogenous retrovirus LNA-based knockdown and scrambled controls in developing zebrafish embryos.

first: Chang

last: Ni-Chen

first: Wells

last: Jonathan


title: FXR Agonism with Bile Acid Mimetic Reduces Pre-Clinical Triple-Negative Breast Cancer Burden

city: Memphis

first: Liza

last: Makowski

state: Tennessee

address: 19 South Manassas St

department: Medicine- Heme Onc

institute: UTHSC

summary: Breast cancer (BC) is the most common malignancy and the leading cause of cancer mortality in women. It is possible that signals arising from the gut microbiome are protective in BC. We used common drugs designed to turn on pathways to mimic one major signal coming from gut microbes, namely bile acids. Taking advantage of a drug called Ocaliva, which is commercially available for another disease, we repurposed this drug to test its anti-cancer potential. Ocaliva worked on a specific protein target called FXR to reduce tumor growth and kill cancer cells. In contrast, a similar drug that targets a different protein called TGR5 was not effective in killing cancer cells. Taken together, these findings suggest that using Ocaliva or other bile acid signals derived from the gut microbiome to target the protein FXR could be an important new therapeutic strategy for individuals with aggressive BC.

overall_design: RNA -Seq data from bulk tumor (orthotopically injected with MDAMB231 Human Triple Negative Breast Cancer Cells) in NSG immunocompetent mice, treated with vehicle (40% DMSO and& 60% polyethylene glycol (PEG), N = 8), OCA (30 mg/kg "mpk", N = 7), or chemotherapy paclitaxel (“Ptax”, 10 mg/kg "mpk", N = 6), grown for 14 days, excised and Snap frozen.

first: Sydney

middle: C

last: Joseph

first: Samson

last: Simon

first: Margaret

middle: S

last: Bohm

first: Minjeong

last: Kim

first: Madeline

last: Pye

first: Boston

last: Simmons

first: Dillon

middle: G

last: Graves

first: Stacey

last: Thomas-Gooch

first: Ubaid

last: Tanveer

first: Jeremiah

last: Holt

first: Suriyan

last: Ponnusamy

first: Laura

last: Sipe

first: D

last: Hayes

first: Katherine

last: Cook

first: Ramesh

last: Narayanan

first: Joseph

middle: F

last: Pierre


title: A single infusion of engineered long-lived and multifunctional T cells confers durable remission of asthma in mice [scRNA-Seq]

city: Beijing

first: Gang

last: Jin

address: Zhongguancun North street

department: Institute for Immunology

institute: Tsinghua University

summary: The majority of common chronic human diseases remain incurable, impacting a significant portion of the population and necessitating lifelong treatments that impose a substantial burden on health, the economy, and society. Asthma, the most prevalent respiratory disease, exemplifies this challenge, affecting over 300 million people and causing more than 250,000 deaths annually. Here, we demonstrate the achievement of long-term remission of type 2-high asthma through a single infusion of engineered CAR T cells. By utilizing IL-5 as the targeting domain and depleting BCOR and ZC3H12A, we engineer long-lived CAR T cells designed to eradicate IL-5R+ eosinophils, termed Immortal-like and Functional IL-5 CAR T (5TIF) cells. Furthermore, we enhance 5TIF cells by engineering them to secrete an IL-4 mutein that blocks the signaling of both IL-4 and IL-13, two inflammatory cytokines driving asthma pathology, resulting in the creation of 5TIF4 cells. In multiple asthma models, a single infusion of 5TIF4 cells in fully immunocompetent mice, without any conditioning regimen, leads to long-term depletion of pathological eosinophils and blockade of IL-4/IL-13 actions. This results in sustained repression of type 2 inflammation and alleviation of asthmatic symptoms. Additionally, 5TIF4 cells can be induced in human T cells in NSG mice. These findings demonstrate that asthma, a prevalent and incurable disease, can undergo durable remission through a single infusion of engineered CAR T cells. This breakthrough paves the way for the potential functional cure of chronic noncancerous diseases using long-lived CAR T cells.

overall_design:
A single-cell RNA sequencing (scRNA-seq) assay was conducted to comprehensively investigate the phenotype of human 5TIF4 cells.
One month after transfer, GFP+ human 5TIF4 cells from recipient mice were sorted by flow cytometry. And the single cell suspensions were directly loaded on a microfluidic chip (Singleron GEXSCOPETM Single Cell RNA-seq Kit, Singleron Biotechnologies) and processed for scRNA-seq library preparation according to manufacturer’s protocol. The ultimate constructed and purified library with the target recovery of ~ 13,000 single cells was sequenced on Illumina novaseq 6000.

first: Min

last: Peng


title: A single infusion of engineered long-lived and multifunctional T cells confers durable remission of asthma in mice [RNA-Seq]

overall_design:
In order to study the molecular mechanisms of unconstained expasion and long-term persistence of 5TIF in mice and IL-4 mutein's influence on 5TIF cell phenotypes, we performed bulk-RNA seq assay for Endogenous CD8 T cell from PBS group, 5TIF cells and 5TIF4 cells.
One month after cell transfer, CD8+ Thy1.1+ 5TIF, 5TIF4 cells or endogenous CD8+ T cells were sorted by flow cytometry with purity > 95% from spleen of recipient mice. RNA samples were isolated and purified using TIANGEN RNAprep Pure Cell/Bacteria Kit, then shipped to BGI for library preparation and RNA sequencing on a DNBseq™.