title: Chromatin Conformation Dynamics are essential to Control the Transcription of INK4/ARF locus

city: Memphis

first: Chunliang

state: TN

address: 262 Danny Thomas Place

institute: St.Jude Children's Research Hospital

summary: The INK4/ARF (CDKN2A/B) locus has been identified as a hotspot for susceptibility to genetic changes associated with a vast variety of cancers and aging related diseases including coronary artery disease and type II diabetes. Using chromatin Capture-C technologies, we discovered a distal enhancer upstream INK4/ARF looped to CDKN2A, ARF and CDKN2B in human fibroblast cells IMR90 and cancer cell lines HCT116 and SEM, but absent in human fibroblast cell-derived induced pluripotent stem cells, which maintained an epigenetically silenced INK4/ARF locus. Bi-allelic deletion of the ~1.4 Kb core enhancer sequence by CRISPR/Cas9 technology in human HCT116 cells abrogated the long-range chromatin interaction, thereafter disrupting gene expression of these tumor suppressor genes. Moreover, cross-species conservation of the distal enhancer elements between human and mouse was observed.

overall_design: Using combinational strategies including histone modifier chromatin immunoprecipitation, chromatin conformation capture, and CRISPR/Cas9 genome editing tools, we identified a distal genomic segment containing cis-acting elements required for transcriptional regulation of INK4/ARF. This series includes the Capture-C samples.

first: Shaela

last: Wright

first: Judith

last: Hyle

first: Yang

last: Zhang


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

status: Public on Jul 31 2024

city: Boston

first: Merrick

middle: Pierson

last: Smela

state: MA

address: 3 Blackfan Circle

country: USA

institute: Harvard University

type: Expression profiling by high throughput sequencing

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

overall_design: RNA sequencing of human induced oogonia-like cells (generated using different combinations of transcription factors) and induced pluripotent stem cells

middle: D

last: Pierson Smela

first: Christian

middle: C

last: Kramme

first: Patrick

last: Fortuna

first: Bennet

last: Wolf

first: Shrey

last: Goel

first: Jessica

last: Adams

first: Carl

last: Ma

first: Sergiy

last: Velychko

first: Ursula

last: Widocki

first: Venkata

last: Kavirayuni

first: Tianlai

last: Chen

first: Sophia

last: Vincoff

first: Edward

last: Dong

first: Richie

last: Kohman

first: Mutsumi

last: Kobayashi

first: Toshi

last: Shioda

first: George

middle: M

last: Church

first: Pranam

last: Chatterjee


title: Chromatin conformation dynamics are essential to control the transcription of INK4/ARF locus

status: Public on Aug 01 2024

first: Chunliang

last: Li

overall_design: Using combinatihttps://www.ncbi.nlm.nih.gov/geo/submission/update/?acc=GSE119947#onal strategies including histone modifier chromatin immunoprecipitation, chromatin conformation capture, and CRISPR/Cas9 genome editing tools, we identified a distal genomic segment containing cis-acting elements required for transcriptional regulation of INK4/ARF.

first: Shaela

last: Wright

first: Judith

last: Hyle

first: Yang

last: Zhang


title: Human arterial endothelial cells from pluripotent stem cells for in vitro hematopoiesis and lymphopoiesis

city: Melbourne

first: Jacky

middle: Yisheng

state: Victoria

address: 50 Flemington Rd

department: Cellular Biology

country: Australia

institute: MCRI

summary: Arterial endothelial cells (AECs) are the founder cell for intraembryonic haematopoiesis. Here, we report a method for the efficient generation of human haemogenic DLL4+ AECs from pluripotent stem cells (PSC). Time-series single cell RNA-sequencing revealed the dynamic evolution of haematopoiesis and lymphopoiesis from endothelial stage, generating cell types with counterparts present in early human embryos. Our study describes an efficient method for producing lymphoid cells and provides insights into their endothelial and haematopoietic ontogeny, paving a new path to investigations of the human blood system.

overall_design: This dataset includes a time-series single cell RNA-sequencing (scRNA-seq) analysis of arterial-hemogenic cultures at day 6, 12, 15, 19, and 25 (haematopoietic cells only on day 25), encompassing period from the generation of haemogenic endothelium to lymphoid commitment.

first: Shicheng

last: Sun

middle: Y

first: Andrew

middle: G

last: Elefanty

first: Edouard

last: Stanley


title: Transcriptional and methylation outcomes of didehydro-Cortistatin A use in HIV-1 infected CD4+T cells

city: Jupiter

first: Susana

middle: T

last: Valente

state: FL

address: 130 Scripps Way

department: Immunology and Microbiology

institute: The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology

summary: Ongoing viral transcription from the reservoir of long-lived CD4+ T cells containing integrated HIV-1 DNA presents a barrier to cure and associates with poorer health outcomes for people living with HIV, including chronic immune activation and inflammation. We previously reported that didehydro-Cortistatin A (dCA), an HIV-1 Tat inhibitor, blocks HIV-1 transcription. We sought to extend this work and examine the impact of dCA on host immune CD4+ T cell transcriptional and epigenetic states. Here, we performed a comprehensive analysis of genome-wide transcriptomic and DNA methylation profiles upon long-term dCA-treatment of primary human memory CD4+ T cells. dCA prompted specific transcriptional and DNA methylation changes in cell cycle, histone, interferon-response and T cell lineage transcription factor genes, through inhibition of both HIV-1 and Mediator kinases. These alterations establish a tolerogenic Treg/Th2 phenotype, reducing viral gene expression and mitigating inflammation in primary CD4+T cells during HIV-1 infection. Additionally, dCA suppresses expression of lineage-defining transcription factors for Th17 and Th1 cells, critical HIV-1 targets and reservoirs. dCA’s benefits thus extend beyond viral transcription inhibition, modulating immune cell landscape to limit HIV-1 acquisition and inflammatory environment linked to HIV-infection.

overall_design: A comparative RNA-seq analysis of primary memory CD4+ T cells treated with ART or ART + 50 nm didehydro-Cortistatin A for 5 weeks. Memory CD4+ T cells in the Kessing Study were isolated from people living with HIV and expanded and treated in vitro. Memory CD4+ T cells in the Mori study were isolated from people not living with HIV and expanded, treated and infected with HIV in vitro (or left uninfected). We compared cells treated with dCA versus those untreated and sought to distinguish the effects of dCA in HIV-infected cells versus the effects of dCA in uninfected cells.

first: Luisa

middle: P

last: Mori

first: Michael

middle: J

last: Corley

last: McAuley

first: Alina

last: Pang

first: Thomas

last: Venables

first: Lishomwa

last: Ndhlovu

first: Matthew

middle: E

last: Pipkin


title: scRNA for 8wks trabecular bone

city: Pok Fu Lam

first: Peikai

last: Chen

address: Sassoon Road 5

department: Faculty of Medicine

country: Hong Kong

institute: The University of Hong Kong

summary: Col10a1-Cre;Rosa26-tdTomato;Mmp14F, Col1-2.3-GFP

overall_design: trabecular bone cells were extracted

first: Nelson

last: Chu

first: Kathryn

middle: S

last: Cheah


title: Genome-wide localization profile (ChIP-seq) and differential expression profile upon RNAi treatment (RNA-seq) of L(3)mbt and its interactors in ovarian somatic cells[OSCs] in Drosophila

city: Yayoi, Bunkyo-ku

first: Hitomi

last: Yamamoto-Matsuda

state: Tokyo

department: Dept. Biol. Sci.

country: Japan

institute: the University of Tokyo

type: Genome binding/occupancy profiling by high throughput sequencing

summary: L(3)mbt regulate the transcription of germ-specific genes in somatic cells but the detailed mechanism remains unclear. We performed ChIP-seq of L(3)mbt and a newly identified interactor in OSCs to elucidate the genome-wide localization. Then we performed RNA-seq of OSCs before and after RNAi treatment of L(3)mbt and its interactors and performed differential expression analysis.

overall_design: ChIP-seq of OSCs and RNA-seq of OSCs before and after RNAi treatment of L(3)mbt and its interactors were performed in duplicate.

first: Mikiko

last: Siomi


title: Alectinib Induces Sinus Bradycardia through Inhibiting Cacna1d Expression, Mediating Electro-Dysfunction in Sinus Node

city: Jinan

first: Guoxuan

last: Liu

address: qingdao road 6699

country: China

institute: Shandong First Medical University & Shandong Academy of Medical Sciences

summary: Alectinib is the first-line therapy for anaplastic lymphoma kinase rearranged non-small cell lung cancer. Sinus node bradycardia as a major adverse cardiac event still widely impact patient’s quality of life. However, the underlying mechanism of alectinib induce sinus bradycardia (AISB) remains elusive. The aim of this study was to reveal the pathogenesis of the AISB in a rat model including the electrophysiology alterations and the molecular basis

overall_design: We generated the alectinib induced sinus node bradycardia rat model and the vehicle control. The difference of RNA profile in sinus node between the two groups were analysed.

first: Fan

last: Diao

first: Qiang

last: Zheng

first: Donghai


title: Single-cell RNA sequencing reveals the CRTAC1+ population actively contributes to the pathogenesis of spinal ligament degeneration by SPP1+ macrophage

city: Shanghai

first: Yulong

last: Tang

state: --- Select One ---

address: Songhu Road 2005

institute: Fudan University

summary: Degenerative spinal stenosis is a chronic disease that affects the spinal ligaments and associated bones, resulting in back pain and disorders of the limbs among the elderly population. There are few preventive strategies for such ligament degeneration. We here aimed to establish a comprehensive transcriptomic atlas of ligament tissues to identify high-priority targets for pharmaceutical treatment of ligament degeneration. Here, single-cell RNA sequencing was performed on six degenerative ligaments and three traumatic ligaments to understand tissue heterogeneity.

overall_design: Tissue samples, including bone (spinous process, facet, and lamina) and ligaments (ligamentum flavum, interspinous ligament, and supraspinal ligament) from Homo Sapiens were obtained during spinal canal decompression surgery. They were then sorted by Fluorescence-activated cell sorting using BD FACS Aria III (BD Biosciences) into CD45+ and CD45-, followed by applying CD45+ and CD45- cells at 3:7 ratio to the 10X Genomics system.

first: Yuexin

last: Yu

first: Qing

last: Zhang

first: Jiucun

last: Wang

first: Jing


title: RNA-seq after chmA knockdown in PhiKZ-infected  PAO1 cells

city: Würzburg

first: Jörg

last: Vogel

address: Josef-Schneider-Straße 2

department: Institute for Molecular Infection Biology

country: Germany

institute: Würzburg University

summary: ChmA forms the phage nucleus in PhiKZ-infected PAO1 cells, we aimed to illuminate the effects on the transcriptome upon knockdown (KD) of ChmA translation through anti-sense-based technology. We identified phage transcripts that are affected in level upon KD of ChmA and clustering allowed to group these together that likely are expressed at a similar stage and dependency in the phage replication cycle.

sample_organism:
Pseudomonas aeruginosa
Pseudomonas phage phiKZ

overall_design: PAO1 cells were pretreated with anti-sense-based molecules against the start codon and ribosome binding site of the chmA transcript. Cells were infected with PhiKZ and at defined time-points post-infection, cells were harvested. RNA was extracted from cells and sequenced.

first: Milan

last: Gerovac


title: Single-cell profiling of prurigo nodularis demonstrates immune-stromal crosstalk driving profibrotic responses and reversal with nemolizumab

city: Los Angeles

first: Feiyang

last: Ma

state: CA

address: 610, Charles Young Dr East, TLSB 3000C

department: Molecular Biology Institute

institute: UCLA

summary: Prurigo nodularis (PN) is a chronic neuroimmune skin disease characterized by bilaterally distributed pruritic hyperkeratotic nodules on extremities and trunk. Neuroimmune dysregulation and chronic scratching are believed to both induce and maintain the characteristic lesions. This study sought to provide a comprehensive view of the molecular pathogenesis of PN at the single-cell level to identify and outline key pathologic processes and the cell types involved. Features that distinguish PN skin from the skin of patients with atopic dermatitis were of particular interest. We further aimed to determine the impact of the IL31RA antagonist, nemolizumab, and its specificity at the single-cell level.

overall_design: Single cell RNA sequencing on lesional and nonlesional skin cells from Prurigo nodularis patients

first: Johann

last: Gudjonsson


title: RNA-seq after knockdown of PhiKZ genes in PhiKZ-infected Pseudomonas aeruginosa PAO1 cells

summary: We aimed to illuminate the effects on the transcriptome upon translation knockdown (KD) of core genes and other characterised genes or early expressed genes in PHIKZ through anti-sense-based technology. We identified phage transcripts that are affected in level upon KD and clustering allowed to group these together that likely are expressed at a similar stage and dependency in the phage replication cycle. The read coverage can point to alterations in transcripts and mode of action for several candidates that were knocked down.

overall_design: PAO1 cells were pretreated with anti-sense-based molecules against the start codon and ribosome binding site of PHIKZ transcripts. Cells were infected with PhiKZ and at defined time-points post-infection, cells were harvested. RNA was extracted from cells and sequenced.


title: Single-Cell RNA-Sequencing facilitates the effectiveness analysis of ATO and DAC co-treatment in PBMCs from PANDA-T0 trial

first: Huaxin

last: Song

address: Shanghai Jiao Tong University School of Medicine

summary: The goals of this study is to reveal the transcriptome of structural p53 mutations rescued by ATO and DAC in AML/MDS patients.

overall_design: The PBMCs were collected from pre-treatment and post-treatment patient #1 and #3, both responders in the trial and in matched isogenic cell assays.

first: Min

last: Lu

first: Yuting

last: Dai

first: Xinjie


title: GTestimate: Improving relative gene expression estimation in scRNA-seq using the Good-Turing estimator

city: Vienna

first: Martin

last: Fahrenberger

state: Vienna

address: Dr.-Bohr-Gasse 9

department: Structural and Computational Biology

country: Austria

institute: Max Perutz Labs

summary: Single-cell RNA-seq suffers from unwanted technical variation between cells, caused by its complex experiments and shallow sequencing depths. We present GTestimate a new normalization method based on the Good-Turing estimator, which improves upon conventional methods by accounting for unobserved genes. We validate GTestimate using new ultra-deep sequencing data, generated via a novel cell targeted PCR-amplification approach, and show substantial improvements in cell-cell distance estimation and downstream results.

overall_design:
A human cerebral-organoid scRNA-seq library was split into two aliquotes and sequenced; once as a ‘typical’ run and once after cell targeted PCR-amplification (cta-seq). This second run enabled a ‘ultra-deep’ sequencing depth for a small set of targeted cells.

***************************************************************
Submitter statement concerning raw data: Raw sequencing data could not be included in this submission due to patient privacy concerns. Raw-data will be made available through controlled access at the European Genome-Phenome Archive at a later date.
***************************************************************

first: Christopher

last: Esk

first: Arndt

last: von Haeseler


title: Characterization of HBV and co-infection with HDV and HIV through spatial transcriptomics

city: London

first: Dimitra

last: Peppa

address: Pond Street

country: United Kingdom

institute: University College London

summary: Background and aims: The intrahepatic processes associated with chronic hepatitis B (CHB), especially in the context of HDV and HIV co-infection, require a better understanding. Spatial transcriptomics can provide new insights into the complex intrahepatic biological processes, guiding new personalized treatments. Our aim is to evaluate this method to characterize the hepatic transcriptional landscape, cellular composition and biological pathways in liver biopsy samples from patients infected with HBV and HDV or HIV co-infection.   Method: The GeoMx nanostring Digital Spatial Profiling (DSP) platform was employed to assess expression of HBsAg and CD45 in formalin fixed paraffin embedded (FFPE) biopsies from three treatment-naïve patients with chronic HBV and HDV or HIV co-infection. The GeoMx whole transcriptome human atlas assay quantified the expression of genes enriched in specific regions of interest (ROIs). Cell type proportions within ROIs were deconvoluted using a training matrix from the human liver cell atlas. A weighted gene correlation network analysis (WGCNA) evaluated transcriptomic signatures across sampled regions.  Results: We identified spatially discrete transcriptomic signatures and distinct biological pathways that associate with HBV infection/disease status and immune responses. Shared features including cytotoxicity and B cell receptor signaling were consistent across patients, suggesting common elements alongside individual traits. HDV/HBV co-infection exhibited upregulated genes linked to apoptosis and immune cell recruitment, whereas HIV/HBV co-infection featured genes related to interferon response regulation. Varied cellular characteristics and immune cell populations, with an abundance of T cells in the HDV/HBV sample, were observed within analyzed regions. Transcriptional differences in hepatocyte function suggest disrupted metabolic processes in HDV/HBV co-infection potentially impacting disease progression.     Conclusion: This proof-of-principle study shows the value of this platform in investigating the complex immune landscape highlighting relevant host pathways to disease pathogenesis.

overall_design:
Archived formalin-fixed paraffin-embedded (FFPE) liver biopsies from three patients with chronic HBV (PT1), one with HDV/HBV co-infection (PT2) and one patient with HIV/HBV (PT3) co-infection were analyzed in this study. Participants were recruited at The Royal London Hospital, Barts Health NHS Trust and samples were obtained during routine diagnostic procedures following written informed consent. Slides were submitted for Digital Spatial Profiling (DSP) and whole transcriptome sequencing, according to the manufacturer’s recommendations for GeoMx-NGS RNA BOND RX slide preparation (manual no. MAN-10131-02). The panel of morphology markers was designed to include HBsAg ((Polyclonal) 488 1:200 from Bioss #bs-1557G-A488), CD45 (D9M8I) 594 1:100 from Cell Signaling Technology #13917BF, CD3 (clone UMAB54; Origene #UM500048CF) and a DNA dye (Syto83dye; Invitrogen). Deparaffinization, rehydration, heat-induced epitope retrieval (for 20 minutes at 100°C), and enzymatic digestion (1 μg/mL proteinase K for 15 minutes at 37°C) were carried on the Leica BOND-RX. Tissues were incubated with 10% neutral buffered formalin for 5 minutes and for 5 minutes with NBF Stop buffer. The tissue sections were hybridized with the oligonucleotide probe mix (whole Transcriptome Atlas) overnight, then blocked and incubated with the four fluorescently labelled markers for 1 hour. Tissue sections were loaded into the GeoMx platform and scanned for an immunofluorescent signal.

*** FASTQ raw data files have been requested. ***


title: Molecular mechanisms of Target organ damage in youth with primary hypertension [bulk RNA-Seq 1]

city: Cincinnati

first: Kwangmin

last: Choi

state: Ohio

address: 333 Burnet Avenue

department: Experimental Hematology & Cancer Biology

institute: Cincinnati Children's Hospital Medical Center

summary: High blood pressure or Primary Hypertension is associated with target organ damage (TOD) in both adults and youth, where TOD predicts the adverse cardiovscular (CV) events. The mechanisms involved in blood pressure mediated TOD are limitedly understood in onset of  TOD. To understand this,  first using RNA sequencing, we identified candidate differentially expressed genes  in circulating PBMCs of study participants with and without hypertension and elevated  left ventrivlar mass index . Next, through miRNA and whole genome bisulphite sequencing of circulating PBMCs we determined the epigenetic regulators of TOD. Also, using LCMS, serum proteomics, the key circulatory protein involved in advancing CV- TOD events was identified. In conclusion,  multiomics approach was utilized to define the circulatory regulators involved in BP mediated CV-TOD.

overall_design: The design and methods of the SHIP AHOY (Study of High Blood Pressure in Pediatrics, Adult Hypertension Onset in Youth) project have been discussed elsewhere  (Mendizabal B, et al., 2018, Hypertension . 2018 Sep;72(3):625-631.). Briefly, in the current cross-sectional study, youth aged 11 to less than 19 years with BP levels (N=397) across the BP distribution, were enrolled at five clinical sites (Cincinnati, Houston, Philadelphia, Rochester, and Seattle) from 2015 to 2017 from pediatric specialty clinics (four nephrology clinics, and one joint nephrology and cardiology clinic). Efforts were made to achieve body mass index (BMI) balance across BP groups by periodically checking differences in BMI percentile among groups and actively recruiting normotensive obese controls. Out of 397 subjects, 132 adolescents of both sexes and all races with normal (Cut off: SBP <80th%), and high- systolic blood pressure (Cut off: SBP >90th%), and then stratified by with or without TOD, like Low LVMI (Left ventricular mass index) (Cut off: <38.6 gm/m2.7), and High LVMI (Cut off: >38.6 gm/m2.7), were selected in the present study cohort. Urine and Blood samples (for PBNCs and serum) were collected to investigate genetic, epigenetic, and proteomics changes that influence the development of hypertensive TOD in youth.  We excluded youth with symptomatic severe hypertension, on antihypertensive or lipid-lowering medication in the past 6 months, with diabetes mellitus (type 1 or 2), kidney disease, or other chronic medical conditions. All study participants and their parents provided written informed consent and assent according to local investigational review board requirements.

first: Tammy

last: Brady

first: Kalyani

last: Ananthamohan

first: Mohammed

last: Arif

first: Stephen

last: Daniels

first: Bonita

last: Falkner

first: Michael

last: Ferguson

first: Joseph

last: Flynn

first: Coral

last: Hanevold

last: Hooper

first: Julie

last: Inglefinger

first: Marc

last: Lande

first: Lisa

last: Martin

first: Meyer

last: SK

first: Mark

last: Mitsnefes

first: Bernard

last: Rosner

first: Joshua

middle: A

last: Samuels

first: Gina

last: Kuffel

last: Zilliox

first: Richard

last: Becker

first: Elaine

last: Urbina

first: Sakthivel

last: Sadayappan


title: Chrom-seq Identifies RNAs at Chromatin Marks [ChIP-Seq]

city: Xi'an

first: Wei

last: Sun

state: Shaanxi

address: No. 229 Taibai North Road, Beilin District

department: College of Life Sciences and Medicine

institute: Northwest University

summary: Chromatin marks are associated with transcriptional regulatory activities. Here, We developed a method termed Chrom-seq to efficiently capture RNAs associated with various chromatin marks in living cells. Chrom-seq jointly applies highly specific chromatin-mark reader with APEX2 which catalyzes the oxidation of biotin-aniline to label the adjacent RNAs for isolation by streptavidin-coated beads. Using the readers of mCBX7/dPC, mCBX1 and mTAF3, we detected RNA species significantly associated with H3K27me3, H3K9me3 and H3K4me3, respectively. Chrom-seq provides an antibody-free approach to systematically map RNAs at chromatin marks with potential regulatory roles in different epigenetic events.

overall_design: To validate the function of lncRNA identified by chrom-seq, antisense oligonucleotides (ASOs) including ASOSETD5-AS1, ASOLINC00641 and ASO LINC00662 were transfected into HEK293T cells grown on the 6-well plates, respectively. ASO_NC was used for the negative control. Next, ChIP-seq of H3K9me3 and H3K27me3 was performed in cells depleted of these lncRNAs using antisense oligos (ASO).

first: Ligang

last: Fan

first: Yitong

last: Lyu

first: Furong

last: Ju

first: Wenju

first: Jie

last: Chen

first: Haiqian

last: Ma

first: Shifei

last: Yang

first: Xiaomin

last: Zhou

last: Wu

first: Wenkai

last: Yi

first: Erfei

first: Rodrigo

last: Villaseñor

first: Tuncay

last: Baubec

first: Jian

last: Yan


title: Chrom-seq Identifies RNAs at Chromatin Marks [Chrom_and_RNA_seq]

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Chrom-seq leverages highly specific reader protein to bring the engineered ascorbate peroxidase APEX2 close to the modified chromatin. Upon exposure to H2O2, APEX2 catalyzes oxidation of surrounding biotin-aniline (Btn-An) which then reacts with guanosines in adjacent RNA molecules to form a covalent adduct, facilitating subsequent purification of tagged RNAs with streptavidin-conjugated beads for identification by sequencing.


title: Keratin 17- and PKCα-dependent transient amplification of neutrophil influx after repeated stress to the skin

city: Ann Arbor

first: Michigan

middle: CDB

last: Bioinformatics

state: MI

address: 109 Zina Pitcher Pl

department: Cell & Developmental Biology

institute: University of Michigan

summary: Neutrophils contribute to the pathogenesis of chronic inflammatory skin diseases. Little is known about the source and identity of the signals mediating their recruitment in inflamed skin. We used the phorbol ester TPA and UVB, alone or in combination, to induce sterile inflammation in mouse skin and assess whether keratinocyte-derived signals impact neutrophil recruitment. A single TPA treatment results in a neutrophil influx in the dermis that peaks at 12h and resolves within 24h. A second TPA treatment or a UVB challenge, when applied at 24h but not 48h later, accelerates, amplifies, and prolongs neutrophil infiltration. This transient amplification response (TAR) is mediated by local signals in inflamed skin, can be recapitulated in ex vivo culture, and involves the K17-dependent sustainment of protein kinase Cα (PKCα) activity and release of neutrophil chemoattractants by stressed keratinocytes. We show that K17 binds RACK1, a scaffold essential for PKCα activity. Finally, analyses of RNAseq data reveal the presence of a transcriptomic signature consistent with TAR and PKCα activation in chronic inflammatory skin diseases. These findings uncover a novel, transient, and keratin-dependent mechanism that amplifies neutrophil recruitment to the skin under stress, with direct implications for inflammatory skin disorders.

overall_design: We performed bulk RNAseq analysis of WT and Krt17 null mouse ear tissue obtained at 6h after dual TPA or dual Acetnone treatments with 24h interval to investigate TAR-related changes in gene expression

first: Yang

last: Xu

first: Erez

last: Cohen

first: Craig

middle: N

last: Johnson

first: Carole

last: Parent

first: Pierre

last: Coulombe


title: Chrom-seq Identifies RNAs at Chromatin Marks [Chrom-seq]

first: Wei

last: Sun

first: Ligang

last: Fan

first: Yitong

last: Lyu

first: Furong

last: Shifei


title: Interferon Gamma signaling triggers dysplastic alveolar remodeling during respiratory viral infection

first: Guilin

last: Yang

address: Yueyang road 320

institute: Center for Excellence in Molecular Cell Science

summary: A single-cell transcriptional analysis was performed on lung epithelial cells isolated from influenza PR8 infected mice at 14 days post injury. The goal is to investigate the potential signaling pathway that regulates dysplastic KRT5 cells expansion. The left lung lobe from influenza infected mice were dissociated to single cells and subjected to fluorescence activated cell sorting (FACS) to select all Epcam+ cells. We found that the regulation of actin cytoskeleton, focal adhesion, Hippo signaling pathway are highly activated in dysplastic KRT5+ cells, suggesting that these signaling pathway may play a role in the expansion of dysplastic KRT5 cells in influenza infected lungs.

overall_design: Influenza PR8 infected 14 days post injury wild type mice left lung lobe were dissociated to single cells and subjected to Magnetic bead sorting (MACS) to remove CD31+ and CD45+ cells. The rest of cells were subjected to fluorescence activated cell sorting (FACS) to select all Epcam+CD31-CD45-cells. The sorted cells were using for scRNA-seq with a 10x Genomics platform.

first: Xiuyu

last: Lin

first: Pengfei

last: Sui


title: Genome-wide Mapping of Topoisomerase Binding Sites Suggests Topoisomerase 3a (TOP3A) as a Reader of Transcription-Replication Conflicts (TRC)

city: BESTHESDA

first: Hongliang

last: Zhang

state: MD

address: 37 Convent Dr

department: DTB

institute: NCI

summary: Both transcription and replication can take place simultaneously on the same DNA template, potentially leading to transcription-replication conflicts (TRCs) and topological problems. Here we asked which topoisomerase(s) is/are the best candidate(s) for sensing TRC. Genome-wide topoisomerase binding sites were mapped in parallel for all the nuclear topoisomerases (TOP1, TOP2A, TOP2B, TOP3A and TOP3B). To increase the signal to noise ratio (SNR), we used ectopic expression of those topoisomerases in H293 cells followed by a modified CUT&Tag method. Although each topoisomerase showed distinct binding patterns, all topoisomerase binding signals positively correlated with gene transcription. TOP3A binding signals were suppressed by DNA replication inhibition. This was also observed but to a lesser extent for TOP2A and TOP2B. Hence, we propose the involvement of TOP3A in sensing both head-on TRCs (HO-TRCs) and codirectional TRCs (CD-TRCs). In which case, the TOP3A signals appear concentrated within the promoters and first 20 kb regions of the 5’ -end of genes, suggesting the prevalence of TRCs and the recruitment of TOP3A in the 5’-regions of transcribed and replicated genes.

overall_design: We transfected HEK293 cells with FLAG-tagged topoisomerase constructs. For TOP1, we constructed the TOP1-T718A/N722H double-mutant; For TOP2a, we constructed the TOP2A-D48N mutant; For TOP2b, we constructed the TOP2B-D64N mutant; For TOP3a, we used the TOP3A-R364W mutant; For TOP3b, we used the TOP3B-R338W mutant. We carried out the modified CUT&Tag protocol without fixation on all 5 human nuclear topoisomerases with and without aphidicolin treatment. We also tested 3 forms of TOP3A (wild-type, inactive-form, and self-trapping-form) following the same protocol.

first: Zhang

last: Hongliang

first: Sun

last: Yilun

first: Saha

last: Sourav

last: Liton Kumar

first: Pongor

last: Lorinc S.

first: Dhall

last: Anjali

first: Pommier

last: Yves


title: MYC family amplification dictates sensitivity to BET bromodomain protein inhibitor Mivebresib (ABBV-075) in small cell lung cancer

city: North Chicago

first: Josh

last: Plotnik

state: IL

address: 1 North Waukegan Rd, AP10/210A

institute: AbbVie Inc.

summary: Small-cell lung cancer (SCLC) accounts for nearly 15% of all lung cancers. Although patients respond to first line therapy readily, rapid relapse is inevitable with few treatment options in the second line setting. Here, we describe SCLC cell lines harboring amplification of MYC and MYCN, but  not MYCL1 nor non-amplified MYC cell lines, exhibit superior sensitivity to treatment with the pan-BET bromodomain protein inhibitor Mivebresib (ABBV-075). Silencing MYC and MYCN partially rescued SCLC cell lines harboring these respective amplifications from the anti-proliferative effects of mivebresib. Furthermore, the activity of mivebresib in SCLC cell lines occurs primarily through BRD2, rather than BRD4. Further characterization of genome-wide binding of MYC, MYCN, and MYCL1 uncovered unique enhancer and  epigenetic preferences. Our study suggests that chromatin landscapes could establish cell states with unique gene expression programs, conveying sensitivity to epigenetic inhibitors such as mivebresib.

overall_design: Chromatin-Immunoprecipitation DNA sequencing (ChIP-seq) was performed using H3K27ac, MYC, MYCL, and MYCN antibodies in SCLC cell lines. ATAC-seq was also performed in SCLC cell lines.

first: Joshua

first: Zha

last: Zheng

first: Weiguo

last: Feng

first: Irene

last: Lee

first: Erin

last: Murphy

first: Tyler

last: Mansfield

first: Mark

last: Domanus

first: Xin

last: Lu

first: Tamar

last: Uziel

first: Lloyd

last: Lam


title: Single-cell RNA-sequencing of mouse ChP tissue and CSF infiltrate following intracerebroventricular injection of LPS

first: Peter

last: Lotfy

address: 300 Longwood Ave

institute: Boston Children's Hospital

summary: scRNA-seq of mouse ChP and CSF following ICV administration of LPS.

overall_design: Mice treated with ICV PBS or LPS for 24-72 hr were euthanized, then their CSF and ChP were harvested and dissociated into single cells. These cells were stained with TotalSeq-B hashing antibodies, then pooled and sequenced for 10X 3' v3.1 scRNA-seq.

first: Huixin

last: Xu

first: Maria

last: Lehtinen

first: Jose

last: Ordovas-Montanes


title: Premature aging in aneuploid yeast is influenced by aneuploidy-induced defects in Ribosome Quality Control [RiboSeq]

city: Madison

first: Audrey

last: Gasch

state: WI

address: 425G Henry mall

department: Genetics

institute: University of Wisconsin

summary: Premature aging is a hallmark of Down syndrome, caused by trisomy of human chromosome 21;  but the reason is unclear and difficult to study in humans.  We used an aneuploid model in wild yeast to show that chromosome amplification disrupts nutrient-induced cell-cycle arrest, quiescence entry, and healthy aging, across genetic backgrounds and amplified chromosomes.  We discovered that these defects are due in part to aneuploidy-induced dysfunction in Ribosome Quality Control (RQC).  Aneuploids entering quiescence display aberrant ribosome profiles, accumulate RQC intermediates, and harbor an increased load of protein aggregates. Although they have normal proteasome capacity, aneuploids show signs of ubiquitin dysregulation, which impacts cyclin abundance to disrupt arrest.  Remarkably, inducing ribosome stalling in euploids produces similar aberrations, while up-regulating limiting RQC subunits or proteins in ubiquitin metabolism alleviates many of the aneuploid defects.  Our results raise major implications for other aneuploidy disorders including Down syndrome.

overall_design: Euploid and select aneuploid Saccharomyces cerevisiae strains (haploids with one additional copy of chromosome XII) were cultured in rich media and allowed to exhaust glucose and enter stationary phase. Samples for ribosome profiling were harvested at log-phase and 4 days after start of experiment. Each strain and timepoint was measured in biological duplicate.

first: Leah

last: Escalante


title: Regulation of blood-brain barrier by astrocyte through mitochondrial transfer II

first: Junjie

last: Gao

address: 600 Yishan Road

institute: Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine

summary: In our study, astrocytes were proven to be capable of delivering mitochondria to endothelial cells, and thus regulate the integrity of blood-brain barrier. We utilized RNA seq to investigate the effect of astrocyte-derived mitochondria on endothelial cells.

overall_design: Mitochondria were isolated from primary mice astrocytes and were then transplanted to cultured bEnd.3 endothelial cells in vitro. After 24h of co-incubation, bEnd.3 endothelial cells transplanted with or without astrocyte-derived mitochondria were collected for high-throughput RNA seq.

first: Delin

last: Liu


title: Regulation of blood-brain barrier by astrocyte through mitochondrial transfer

summary: The subset of astrocytes that express Dmp1 were considered to wrap around blood vessels. We used scRNA-seq to investigate the mobility of mitochondria from astrocytesDMP1.

overall_design: Brain single cells with mito-COX8Dendra2 fluorescence from Dmp1Cre-COX8Dendra2 mice were enriched by Fluorescence-activated cell sorting (FACS) according to the presence of Dendra2 signal and analyzed using scRNAseq.


title: Improved characterization of single-cell RNA-seq libraries with paired-end avidity sequencing

status: Public on Jul 30 2024

city: Aurora

first: Austin

last: Gillen

state: CO

address: 12801 East 17th Avenue

institute: University of Colorado Anschutz Medical Campus

summary: Prevailing poly(dT)-primed 3’ single-cell RNA-seq protocols generate barcoded cDNA fragments containing the reverse transcriptase priming site, which is expected to be the poly(A) tail or a genomic adenine homopolymer. Direct sequencing across this priming site was historically difficult because of DNA sequencing errors induced by the homopolymeric primer at the ‘barcode’ end. Here, we evaluate the capability of “avidity base chemistry” DNA sequencing from Element Biosciences to sequence through this homopolymer accurately, and the impact of the additional cDNA sequence on read alignment and precise quantification of polyadenylation site usage. We find that the Element Aviti instrument sequences through the thymine homopolymer into the subsequent cDNA sequence without detectable loss of accuracy.

overall_design: Previously published AML CITE-seq gene expression libraries were resequenced on the Element Biosciences Aviti platform to assess homopolymer performance in the context of mRNA 3' end mapping.

first: John

last: Chamberlin

first: Aaron

middle: R

last: Quinlan


title: Characterizing the Transcriptome of Turkey Hemorrhagic Enteritis Virus

city: Provo

first: Abraham

last: Quaye

state: UT

address: 4007 Life Sciences Building

department: Microbiology & Molecular Biology

institute: Brigham Young University

summary: Characterizing the splice map of turkey hemorrhagic enteritis virus (THEV) is an essential step that would allow studies of individual genes mediating its immunosuppressive functions. We used an RNA-sequencing experiment to characterize the transcriptome of THEV for the first time, providing key insight into the THEV gene expression and mRNA structures. Researchers previously annotated the genome of THEV as encoding 23 open reading frames (ORFs). In this work we identified 29 spliced transcripts all of which consisted of novel exons although some exons matched some previously annotated ORFs. The three annotated splice junctions were also corroborated by our data. We performed PCR amplification of THEV cDNA, cloned the PCR products, and used Sanger sequencing to validate all identified splice junctions. During validation we identified five additional unique transcripts, a subset of which were further validated by 3' rapid amplification of cDNA ends (3' RACE) experiments. Thus, we report that the genome of THEV contains 34 transcripts with the coding capacity for all annotated ORFs. However, we found six of the previously annotated ORFs (ORF1, E3, 33K, ORF8, IVa2, and protease) to be truncated ORFs on the basis of the identification of an in-frame upstream start codon or the detection of additional coding exons. We also identified three of the annotated ORFs with longer or shorter isoforms, and seven novel unannotated ORFs that could potentially be translated; although it is beyond the scope of this manuscript to investigate whether they are translated. Similar to other adenoviruses (AdVs), THEV also produces multiple distinctly spliced transcripts that code for the same proteins across its genome. Our data show that all THEV transcripts are spliced and organized into five transcription units under the control of their cognate promoters like other AdVs.

overall_design:
To understand the genetic basis by which THEV causes immunosuppression (IS) in infected turkeys, we performed an RNA-seq experiment to characterize the transcriptome (structure and splicing patterns) of its genes to set the stage for further experimentation with specific viral genes that may mediate IS.
A THEV vaccine was used to infect a turkey cell line (MDTC-RP19) and harvested at 4-, 12-, 24-, and 72-hpi in triplicates. RNA-seq was done for polyA-tailed mRNA
Reads mapping to THEV's genome were filtered out as new BAM files. The mapped reads were assembled into transcripts using StringTie. StringTie set to expression estimation mode was used to calculate FPKM scores for all transcripts after which Ballgown in R was used to perform the statistical analysis on the transcript expression levels

first: Brian

last: Poole

first: Brett

last: Pickett

first: Joel

last: Griffitts

first: Bradford

middle: K

last: Berges


title: Molecular mechanisms of Target organ damage in youth with primary hypertension [smallRNA-Seq 1]

first: Kwangmin

last: Choi

type: Non-coding RNA profiling by high throughput sequencing

overall_design: The design and methods of the SHIP AHOY (Study of High Blood Pressure in Pediatrics, Adult Hypertension Onset in Youth) project have been discussed elsewhere (Mendizabal B, et al., 2018, Hypertension . 2018 Sep;72(3):625-631.).  Briefly, in the current cross-sectional study, youth aged 11 to less than 19 years with BP levels (N=397) across the BP distribution, were enrolled at five clinical sites (Cincinnati, Houston, Philadelphia, Rochester, and Seattle) from 2015 to 2017 from pediatric specialty clinics (four nephrology clinics, and one joint nephrology and cardiology clinic). Efforts were made to achieve body mass index (BMI) balance across BP groups by periodically checking differences in BMI percentile among groups and actively recruiting normotensive obese controls. Out of 397 subjects, 132 adolescents of both sexes and all races with normal (Cut off: SBP <80th%), and high- systolic blood pressure (Cut off: SBP >90th%), and then stratified by with or without TOD, like Low LVMI (Left ventricular mass index) (Cut off: <38.6 gm/m2.7), and High LVMI (Cut off: >38.6 gm/m2.7), were selected in the present study cohort. Urine and Blood samples (for PBNCs and serum) were collected to investigate genetic, epigenetic, and proteomics changes that influence the development of hypertensive TOD in youth.  We excluded youth with symptomatic severe hypertension, on antihypertensive or lipid-lowering medication in the past 6 months, with diabetes mellitus (type 1 or 2), kidney disease, or other chronic medical conditions. All study participants and their parents provided written informed consent and assent according to local investigational review board requirements.

first: Tammy

last: Brady

first: Kalyani

last: Ananthamohan

first: Mohammed

last: Arif

first: Stephen

last: Daniels

first: Bonita

last: Falkner

first: Michael

last: Ferguson

first: Joseph

last: Flynn

first: Coral

last: Hanevold

last: Hooper

first: Julie

last: Inglefinger

first: Marc

last: Lande

first: Lisa

last: Martin

first: Meyer

last: SK

first: Mark

last: Mitsnefes

first: Bernard

last: Rosner

first: Joshua

last: Samuels

first: Gina

last: Kuffel

last: Zilliox

first: Richard

last: Becker

first: Elaine

last: Urbina

first: Sakthivel

last: Sadayappan


title: Chrom-seq Identifies RNAs at Chromatin Marks

first: Wei

last: Sun

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

overall_design: Refer to individual Series


title: A Microphysiological HHT-on-a-Chip Platform Recapitulates Vascular Lesions of Human Patients

city: Irvine

first: Christopher

last: Hatch

address: 2400 Biological Sciences III

institute: University of California, Irvine

summary: Hereditary Hemorrhagic Telangiectasia (HHT) is a rare congenital disease in which fragile vascular malformations focally develop in multiple organs. These can be small (telangiectasias) or large (arteriovenous malformations, AVMs) and can compromise tissue perfusion. Critically, they are prone to rupture leading to frequent and uncontrolled bleeding. As a result, HHT patients experience several debilitating clinical manifestations that reduce patient quality-of-life. Most HHT patients are heterozygous for loss-of-function mutations affecting signaling through the Alk1 pathway; yet, the pathogenesis of vascular malformation (VM) in HHT patients remains unclear and there are still few treatment options and no cure. Due to the complex, three-dimensional, and multicellular nature of vascular lesions in HHT, they are traditionally studied in intact genetically-modified mouse and zebrafish models, and studies in these animals have yielded important insights into HHT disease progression. However, intact animal studies can be time-consuming and expensive, and may be difficult to translate to human patients. In this study, we take advantage of our recently developed Vascularized Micro-Organ platform to develop an in vitro fully-humanized HHT-on-a-chip microphysiological model (HHT-VMO) that recapitulates vascular lesions of HHT patients. Using a tunable IPTG-inducible ACVRL1 (Alk1)-knockdown approach, we control the timing and extent of endogenous Alk1 expression in primary human endothelial cells seeded into the HHT-VMO device. We report that Alk1-deficient EC reliably form networks with aberrant morphology that includes lesions reminiscent of both patient telangiectasias and AVMs. HHT-VMO lesions are dependent upon the timing of Alk1 knockdown and their formation is blocked by the Vascular Endothelial Growth Factor Receptor (VEGFR) inhibitor pazopanib. Lastly, we find that AVM-like lesions that form in the HHT-VMO are comprised of mixed Alk1-intact and Alk1-deficient EC suggesting cell non-autonomous effects. Taken together, we report development of a novel HHT-on-a-chip model that appears to faithfully reproduce HHT patient lesions. In the future, we hope to use this model to better understand HHT disease biology and to perform drug screening studies to identify a cure for HHT.

overall_design: IPTG inducible shALK1 HUVECs and normal human lung fibroblasts were loaded into the vascularized mirco-organ, a 3D microphysiological system. At Day 0, 3 mm IPTG were added to cell culture medium in the experimental group to knockdown ALK1/ACVRL1 expression. Vessels were allowed to grow until day 10, when devices were harvested and cells sent to sequencing.

first: Jennifer

last: Fang

first: Jillian

last: Andrejecsk

first: William

last: van Trigt

first: Damie

last: Juat

first: Yu-His

last: Chen

first: Satomi

last: Matsumoto

first: Abe

last: Lee

last: Hughes


title: Molecular mechanisms of Target organ damage in youth with primary hypertension

first: Kwangmin

last: Choi

type: Methylation profiling by high throughput sequencing


title: Single Nucleus Transcriptomics Identifies Separate Classes of UCP1 and Futile Cycle Adipocytes [human snRNA-seq]

city: Schwerzenbach

first: Adhideb

last: Ghosh

address: Schorenstrasse 16

department: Health Sciences and Technology

country: Switzerland

institute: ETH Zurich

summary: Adipose tissue can recruit catabolic adipocytes which utilize chemical energy to dissipate heat. This process occurs either by uncoupled respiration through uncoupling protein 1 (UCP1) or by utilizing ATP-dependent futile cycles (FCs). However, it remains unclear how these pathways coexist since both processes rely on the mitochondrial membrane potential. Utilizing single-nucleus RNA-sequencing to deconvolute the heterogeneity of subcutaneous adipose tissue in mice and humans, we identify at least 2 distinct subpopulations of beige adipocytes: FC-beige and UCP1-beige adipocytes. Importantly, we demonstrate that the FC-utilizing beige subpopulation is metabolically highly active and utilizes FCs to dissipate energy thus contributing to thermogenesis independent of UCP1. Furthermore, FC-beige adipocytes are important drivers of systemic energy homeostasis and linked to glucose metabolism and obesity resistance in humans. Taken together, our findings identify a noncanonical thermogenic adipocyte subpopulation, which could be an important regulator of energy homeostasis in mammals.

overall_design: 10x single nuclei RNA seq of human subcutanous neck adipose tissue

first: Tongtong

last: Wang

first: Christian

last: Wolfrum


title: A replisome-associated parental histone H3-H4 tetramer chaperone required for faithful epigenetic inheritance

status: Public on Jul 29 2024

city: Boston

first: Danesh

last: Moazed

address: 240 Longwood Avenue, LHRRB 517

institute: Harvard Medical School/HHMI

summary: Faithful transfer of parental histones to newly replicated daughter DNA strands is critical for inheritance of epigenetic states. Although replication proteins that facilitate parental histone transfer have been identified, how intact histone H3-H4 tetramers travel relatively large distances from the front to the back of the replication fork remains unknown. Here, we use AlphaFold-Multimer structural predictions combined with biochemical and genetic approaches to identify the Mrc1/CLASPIN subunit of the replisome as a histone chaperone. Mrc1 contains a conserved histone binding domain that forms a brace around the H3-H4 tetramer mimicking nucleosomal DNA and H2A-H2B histones, is required for heterochromatin inheritance, and promotes parental histone recycling during replication. We further identify binding sites for the FACT histone chaperone in Swi1/TIMELESS and DNA polymerase α that are required for heterochromatin inheritance. We propose that Mrc1, in concert with FACT acting as a mobile co-chaperone, coordinates the distribution of parental histones to newly replicated DNA.

overall_design: eSPAN (enrichment and sequencing of protein-associated nascent DNA) experiments to detect the strand bias of the distribution of parental histone surrogate H3K4me3 at the replication origins in S. pombe

first: Yu

last: Juntao

first: Fang

last: Yimeng

first: Hua

last: Xu

first: Zhang

last: Zhiguo

first: Jia

last: Songtao

first: Moazed

last: Danesh


title: Stem cell secretome-treatment improves whole-body metabolism, reduces adiposity and promotes skeletal muscle function in aged mice

city: Salt Lake City

first: Micah

last: Drummond

institute: University of Utah

summary: Aging coincides with the progressive loss of muscle mass and strength, increased adiposity, and diminished physical function. Accordingly, interventions aimed at improving muscle, metabolic, and/or physical health are of interest to mitigate the adverse effects of aging. In this study, we tested a stem-cell secretome product which contains a host of growth, cytoskeletal remodeling, and immunomodulatory factors. We examined the effects of 4 weeks of bi-weekly unilateral intramuscular secretome injections (quadriceps) in ambulatory aged male C57Bl/6 mice (22-23 months) compared to saline injected aged-matched controls. After 4 weeks of treatment, secretome delivery substantially increased whole body lean mass and decreased fat mass corresponding to higher fiber cross sectional area and smaller adipocyte size, respectively. Secretome-treated mice also displayed improved whole-body grip strength and rotarod performance and had higher energy expenditure and physical activity levels compared to control mice. Furthermore, secretome-treated mice had greater skeletal muscle Pax7+ cell abundance, capillary density, and collagen turnover. Finally, the secretome product reduced lipid content and lipid droplet size in adipocytes in vitro. These data suggest intramuscular treatment secretome improves whole-body metabolism, physical function, and remodels skeletal muscle and adipose tissue in aged mice.

overall_design: Comparative gene expression of RNA-seq data following an acute injection of saline (control) and stem cell secretome product into the quadriceps of old (26-28 months) mice, n=10 per group.

first: Zachary

last: Fennel


title: DNA sequence context and the chromatin landscape differentiate sequence-specific transcription factor binding in the human malaria parasite, Plasmodium falciparum

city: University Park

first: Shaun

last: Mahony

state: PA

address: 404 South Frear Bldg

department: Biochemistry & Molecular Biology

institute: Penn State University

summary: Global transcription of the malaria parasite, Plasmodium falciparum, is finely regulated, yet the genome encodes for a limited number of sequence-specific transcription factors (TFs) to coordinate this pattern. A subset of these TFs bind overlapping DNA motifs (i.e., CACACA and GTGCAC); however, mechanisms of binding site selection and redundancy have not yet been investigated in P. falciparum. Therefore, we integrated a variety of approaches from new and published work such as high-throughput in vitro and in vivo binding assays, DNA shape predictions, epigenetic post translational modification mapping, and genome-wide chromatin accessibility mapping to comprehensively interrogate the impact of DNA sequence context and the chromatin landscape on TF binding in P. falciparum. We show that sequence context of the CACACA-binding TFs does not greatly impact binding site selection, while chromatin accessibility, epigenetic patterns, and co-factor recruitment differentiates in vivo binding. In contrast, the GTGCAC-binding TFs do prefer significantly different sequence contexts and DNA shape profiles both in vitro and in vivo, in concert with chromatin dynamics. Our results demonstrate that TFs select binding sites based on unique combinations of sequence context, DNA topology, epigenetic patterns, and chromatin accessibility, thereby contributing to the complex gene regulatory mechanisms governing malaria pathogenesis.

overall_design: Three biological replicates of chromatin immunoprecipitation followed by sequencing (ChIP-seq) was used to identify the genome-wide binding sites of sequence-specific transcription factor, AP2-LT (gene ID: PF3D7_0802100) in a 3D7 strain, Plasmodium falciparum parasite line with a C-terminal 3xHA tag.

first: Victoria

last: Bonnell

first: Yuning

last: Zhang

first: Alan

last: Brown

first: John

last: Horton

first: Tsu-Pei

last: Chiu

first: Remo

last: Rohs

first: Raluca

last: Gordan

first: Manuel

last: Llinas


title: Regulation of blood-brain barrier by astrocyte through mitochondrial transfer.

city: Shanghai

first: Junjie

last: Gao

summary: Previous data revealed the role of Mfn2 in mediating mitochondrial transfer from a subset of astrocytes that express Dmp1 to cerebrovascular endothelial cells. We used scRNA-seq to investigate the role of Mfn2-mediated mitochondrial transfer in endothelial cell functions.

overall_design: Mfn2 was conditionally depleted in astrocytesDMP1 by establishing Dmp1Cre-Mfn2flox/flox transgenic mice with Dmp1Cre and Mfn2flox/flox mouse lineages. Next, 3 wild type (WT) mouse brains and 3 age-matched Dmp1Cre-Mfn2flox/flox mouse brains were digested into single cells by standard protocols and mixed into one sample, respectively. Mixed single cells from WT mouse brains and Dmp1Cre-Mfn2flox/flox mouse brains were processed for further scRNA sequencing.

first: Delin

last: Liu


title: Single Nucleus Transcriptomics Identifies Separate Classes of UCP1 and Futile Cycle Adipocytes [mouse snRNA-Seq Ucp1KO]

first: Adhideb

last: Ghosh

overall_design: isolate cold exposure UCP1 KO mice inguinal adipocytes nuclei for 10x single nuclei RNA seq

first: Tongtong

last: Wang

first: Christian

last: Wolfrum


title: Glial-enriched stem-cell 3D model resembling the cellular landscape of the human brain mimics the glial-immune neurodegenerative phenotypes of multiple sclerosis.

status: Public on Jul 26 2024

city: Milan

first: Edoardo

last: Pedrini

state: Lombardy

address: Via Olgettina 60

department: Division of Neuroscience

country: Italy

institute: Hospital San Raffaele

summary: The role of central nervous system (CNS) glia in sustaining self-autonomous inflammation and driving clinical progression in multiple sclerosis is attracting increasing scientific interest. Here, we applied a single transcription factor (SOX10)-based protocol for accelerating oligodendrocyte differentiation from human induced pluripotent stem cell (hiPSC)-derived neural precursor cells to produce three dimensional, multilineage organoids integrating submillimetric self-organizing forebrain organoids (consisting of neurons, astrocytes, oligodendrocyte precursors cells, and myelinating oligodendrocytes). To achieve an immunocompetent organotypic model, hiPSC-derived microglia was also incorporated. Within an 8-week time frame, organoids reproducibly generated a rich diversity of mature cell types, with single-cell transcriptional profiles similar to the human adult brain. This cellular system is able to respond to complex inflammatory stimuli and to properly mimic macroglia-microglia neurodegenerative phenotypes and crosstalk, as seen in chronic active multiple sclerosis. The results obtained pave the way for the implementation of this novel 3D model in the identification of druggable targets for inflammatory neurodegeneration as drug screening platform.

overall_design: For each condition we pooled 30 organoids. We compared WT vs SOX10 induced clones and organoids exposed or not to inflammatory CSF. We performed droplet-based scRNA-seq, libraries were prepared using the Chromium Single Cell 3ʹ v3, according to the manufacturer's protocol (10x Genomics) without modifications, targeting 5,000 cells per sample. Using the Illumina Novaseq platform, all samples were sequenced at 50,000 reads per cells

first: Francesca

last: Fagiani

first: Stefano

last: Taverna

first: Elena

last: Brambilla

first: Valentina

last: Murtaj

first: Paola

last: Podini

last: Ruffini

first: Erica

last: Butti

first: Roberta

last: Magliozzi

first: Lena

last: Smirnova

first: Tanja

last: Kuhlmann

first: Angelo

last: Quattrini

first: Daniel

last: Reich

first: Gianvito

last: Martino

last: Panina-Bordignon

first: Martina

last: Absinta


title: PPARg T166 phosphorylation-mediated lipid synthesis sustains the reparative function of macrophages during tissue repair [CUT&RUN]

city: Nanjing

first: Yuxin

last: Wang

address: 163 Xianlin Road

institute: Nanjing University

summary: Macrophages exhibit a reparative phenotype that supports tissue repair and remodeling in response to tissue injury. However, the metabolic requirements that support this process have remained incompletely understood. Here, we showed that posttranslational modification (PTM) of peroxisome proliferator-activated receptor g (PPARg) regulated lipid synthesis in response to wound microenvironmental cues and that metabolic rewiring orchestrated the function of reparative macrophages. In injured tissues, repair signaling attenuated macrophage PPARg threonine 166 (T166) phosphorylation, which induced a partially active PPARg program with increased binding activity to the regulator regions of lipid synthesis-associated genes, thereby activating lipogenesis. The accumulated lipids served as signaling molecules, triggering signal transducer and activator of transcription 3 (STAT3)-mediated growth factor expression, and supporting the synthesis of phospholipids for the expansion of the endoplasmic reticulum (ER), which is required for the secretion of proteins. Genetic or pharmacological inhibition of PPARg T166 phosphorylation promoted the reparative function of macrophages and facilitated tissue regeneration. In summary, we identified that PPARg T166-regulated lipid biosynthesis was essential for the anabolic demands of the activation and function of macrophages and provided a rationale for therapeutic targeting of tissue repair.

overall_design: CUT&RUN-seq for STAT3 in bone marrow-derived macrophages (BMDMs).

first: Shiman

last: Zuo


title: Transcriptome analysis of high-risk cattle supplemented with inorganic or organic Co, Cu, Mn, and Zn

city: Canyon

first: Matthew

middle: Adam

last: Scott

state: TX

address: 3201 Russell Long Blvd

department: Large Animal Clinical Sciences

institute: Texas A&M University

summary: This objective of this study was to identify gene expression and biological mechanisms related to trace mineral metabolism, central to inflammatory mediation and disease development, through the evaluation of the whole blood transcriptome in post‐weaned cattle receiving different mineral treatments. Here, 56 high‐risk cross-bred beef cattle (~250 kg) were assigned to receive ad libitum access to supplements containing 1 of 3 treatments over a 60-day period: 1) sulfate sources of Cu, Co, Mn, and Zn in feed (INR; custom blend; n= 40); 2) organic complexed source of Cu, Mn, Co, and Zn in feed (AAC; Availa4; n = 40); or 3) organic complexed source of Cu, Mn, Co, and Zn in feed and administration of a drench product [ProFusion] upon arrival (COMBO; n = 40). Whole blood RNA from 26 cattle (~4 calves/pen) were matched and utilized to assess the host transcriptome at days 0, 13, 28, 45, and 60 for the assessment of host transcriptome responses over time.

overall_design: Whole blood from 26 randomly selected and matched individuals per treatment group (n=3;INR, AAC, COMBO) were used for gene expression profiling and differential analysis across five time points (Day 0, 13, 28, 45, 60).

first: Kelsey

last: Harvey

first: Brandi

last: Karisch

first: Amelia

last: Woolums


title: PPARg T166 phosphorylation-mediated lipid synthesis sustains the reparative function of macrophages during tissue repair [RNA-seq]

overall_design:
mRNA profiling of bone marrow-derived macrophages (BMDMs) from PPARg WT, T166A mutant mice.
mRNA profiling of skin wound (6 dpi) macrophages from PPARg WT, T166A mutant mice.


title: Self-Renewing Presomitic Mesoderm Progenitor Lines Reprogrammed from human urine cells [Reprogramming ATAC-seq]

city: Hangzhou

first: Xingnan

last: Huang

address: No.18 Shilongshan Road

institute: Westlake University

summary: Vertebrae formation is the defining feature of all vertebrates1,2. Yet, each vertebrate species appears to have a unique timing mechanism for forming somites along the vertebral column3-5. Human vertebrate formation remains poorly studied due to technical and ethical limitations. Here we report the generation of self-renewing stem cells with characteristic presomitic mesoderm (PSM) features by reprogramming epithelial cells isolated from human urine. These induced expandable presomitic mesoderm progenitor cells (UiPSM) proliferated extensively for more than 30 passages in chemically defined conditions, robustly producing 1040 UiPSM cells. UiPSM established presomitic mesodermal transcription profile, not detected pluripotency, ectoderm and endoderm related genes. UiPSM developed into presomitic mesodermal lineage cells, such as skeletal muscle cells(skm), osteoblast and chrondroblast cells in vivo and vitro, when transplanted UiPSM derived human skm cells in muscle injury model, the skm cells can survive in vivo and contribute to muscle regeneration up to one month. Thus, UiPSM is a powerful system to study human somite development and provide strategies for regenerative medicine in musculoskeletal system

overall_design: Sequencing the collected the cell samples in the reprogramming proocess including Day0,Day3,Day6, Day9


title: RNA sequencing of norepinephrine- or vehicle-treated mouse colon cancer organoids

city: New York

first: Hiroki

last: Kobayashi

state: NY

address: Irving Cancer Research Center, Room 901, 1130 St. Nicholas Avenue

department: Division of Digestive & Liver Diseases

institute: Columbia University

summary: To profile transcriptomic alterations induced by catecholamine stimulation, we treated ApcΔ/Δ, KrasG12D/Δ, Trp53Δ/Δ  mouse colon cancer organoids with norepinephrine or vehicle and then performed RNA sequencing.

overall_design: RNA-seq profiles of  ApcΔ/Δ, KrasG12D/Δ, Trp53Δ/Δ  mouse colon cancer organoids treated with norepinephrine (10 μM) or vehicle for 48 hours. Four independent replicates each treatment group were used.

first: Timothy


title: PPARg T166 phosphorylation-mediated lipid synthesis sustains the reparative function of macrophages during tissue repair


title: Self-Renewing Presomitic Mesoderm Progenitor Lines Reprogrammed from human urine cells [xxx scRNA-seq]

overall_design: Building library and sequencing process of producing cells that have been transplanted into mouse -- skeletal muscle cells Day 30 (overexpression of MYOD), Day 60 and urine cells


title: PPARg T166 phosphorylation-mediated lipid synthesis sustains the reparative function of macrophages during tissue repair [ChIP-seq]

overall_design: ChIP-seq for PPARg in PPARg WT, T166A bone marrow-derived macrophages (BMDMs).


title: Self-Renewing Presomitic Mesoderm Progenitor Lines Reprogrammed from human urine cells [induced cell type bulk RNA-seq]

overall_design: Building library and sequencing process of producing Skeletal Muscle cell (SKM) Day30, Day60, Osteoblast, Chondroblast, Overexpression MYOD Skeletal Muscle cell (SKM) Day15, Day30


title: Single Nucleus Transcriptomics Identifies Separate Classes of UCP1 and Futile Cycle Adipocytes [mouse snRNA-seq]

first: Adhideb

last: Ghosh

overall_design: adipo_cre X nuclei_Red mice were housed at room temperature, cold exposure 4 days and cold exposure 7 days. Mice adipocytes nuclei were isolated by FACS. Isolated nuclei were performed 10X single nuclei sequencing

first: Tongtong

first: Christian

last: Wolfrum


title: Single Nucleus Transcriptomics Identifies Separate Classes of UCP1 and Futile Cycle Adipocytes

overall_design: Refer to individual Series


title: Spatial analysis of murine lungs exposed to fibrogenic stress

status: Public on Jul 24 2024

first: Alessandro

last: Venosa

department: Pharmacology and Toxicology

summary: Acute inflammatory exacerbations (AIEs) represent immune-driven deteriorations of many chronic lung conditions, including COPD, asthma, and pulmonary fibrosis (PF). The first line of therapy is represented by broad-spectrum immunomodulation. To better study immune cell heterogeneity during AIE-PF, this work leverages a clinically relevant model of inflammatory exacerbations triggered by mutation in the alveolar epithelial type 2 cell Surfactant Protein-C gene [SP-CI73T]. SP-CI73T mice received corn oil (controls) or tamoxifen (175 mg/kg, orally) to excise a neomycin cassette placed on the surfacant protein C gene locus, hence inducing the expression of the mutant transcript. We have previously shown that a complex inflammatory response arises by 14 days post tamoxifen administration. Paraffin sections of murine lungs were therefore collected in controls and 14 days post induction. Immunofluorescence for DNA, CD45, CD68 was performed to identify macrophages (CD45+CD68+) and non-immune ("other", DNA+CD45-) cells within  healthy, peri-injured and injured regions of the lung. A total of 12 regions (680um x 700um in size) were selected and macrophages and "other" cells isolated. RNA sequencing was then performed. Principal component and pathway analysis revealed spatial heterogeneity with an hyperactive peri-injured compartment (extracellular matrix remodeling, innate and adaptive immunity, senescence). Cells isolated from injured areas of the lung were relatively quiescent. These results illustrate regional heterogeneity in immune and non-immune cells of the lung during fibrogenic stress.

overall_design: Paraffin-embedded sections of SP-C mutant lungs (one oil treated control and one 14 day injured) were assessed through spatial analysis of CD45+CD68+ macrophages and DNA+CD45- non-immune ("other") cells.

first: Jessica

last: Noll


title: The Impact of SIV-Induced Immunodeficiency on SARS-CoV-2 Disease, Viral Dynamics, and Antiviral Immune Response in a Nonhuman Primate Model of Coinfection

status: Public on Jul 23 2024

city: Covington

first: Alexandra

last: Melton

address: 18703 Three Rivers Road

institute: Tulane National Primate Research Center

summary: Abstract: The effects of immunodeficiency associated with chronic HIV infection on COVID-19 disease and viral persistence have not been directly addressed in a controlled setting. In this pilot study, we exposed two pigtail macaques (PTMs) chronically infected with SIVmac239, exhibiting from very low to no CD4 T cells across all compartments, to SARS-CoV-2. We monitored the disease progression, viral replication and evolution, and compared these outcomes with SIV-naïve PTMs infected with SARS-CoV-2. No overt signs of COVID-19 disease were observed in either animal, and the SARS-CoV-2 viral kinetics and evolution in the SIVmac239 PTMs were indistinguishable from those in the SIV-naïve PTMs in all sampled mucosal sites. However, the single-cell RNA sequencing of bronchoalveolar lavage cells revealed an infiltration of functionally inert monocytes after SARS-CoV-2 infection. Critically, neither of the SIV-infected PTMs mounted detectable anti-SARS-CoV-2 T-cell responses nor anti-SARS-CoV-2 binding or neutralizing antibodies. Thus, HIV-induced immunodeficiency alone may not be sufficient to drive the emergence of novel viral variants but may remove the ability of infected individuals to mount adaptive immune responses against SARS-CoV-2.

overall_design:
Single-Cell RNA Sequencing (scRNAseq) of BAL Cells
For single-cell sequencing of bronchoalveolar lavage (BAL) cells, we collected samples before SARS-CoV-2 inoculation and on days 2, 7, 21, and 28 post-challenge. BAL samples were centrifuged at room temperature for 5 min at 1800 rpm, and the resulting cell pellets were resuspended in DMEM supplemented with 10% FBS and 1% Anti-Anti. We used the Parse Biosciences cell fixation kit, following the manufacturer’s instructions for PBMCs, to fix the cells (Parse Biosciences, Seattle, WA, USA). Specifically, we fixed 1 million cells per animal/timepoint in a 15 mL falcon tube. The fixed cells were stored at −20 °C until all samples were collected.
To enable multiplexing of samples, the Parse Single-Cell whole transcriptome kit, which utilizes a combinatorial barcoding approach (Evercode WT, Parse Biosciences, USA), was employed. This allowed us to barcode and multiplex 10 samples collected from the coinfected animals across five timepoints. For analysis of the processed cells, we conducted two separate runs: the first run included approximately 15,000 cells, while the second run consisted of approximately 42,000 cells. The sublibraries from each run were pooled and sequenced on an Illumina NextSeq 2000 platform, yielding an average depth of 27,165 reads per cell for the first batch and 29,088 reads per cell for the second.

first: Lori

last: Rowe

first: Toni

last: Penney

first: Clara

last: Krzykwa

first: Kelly

last: Goff

first: Sarah

last: Scheuermann

first: Hunter

last: Williams

first: Nadia

last: Golden

first: Kristyn

last: Moore Green

first: Brandon

last: Smith

first: Kasi

last: Russell-Lodrigue

first: Jason

last: Dufour

first: Lara

last: Doyle-Meyers

first: Faith

last: Schiro

first: Pyone

last: Aye

first: Jeffery

middle: D

last: Lifson

last: Beddingfield

first: Robert

middle: V

last: Blair

first: Rudolf

last: Bohm

first: Jay

last: Kolls

last: Rappaport

first: James

last: Hoxie

first: Nicholas

last: Maness


title: Transcriptomic profile of IEC4.1 cells treated with an siRNA to lncRNA NR_126553 (Nostril) in response to IFN-gamma protein (IFNγ)

status: Public on Jul 22 2024

city: Chicago

first: Aiyu

last: Gong

address: 1735 W. Harrison St

department: MPI

institute: Rush University

summary: This study is to investigate the potential impact of an lncRNA NR_126553 (Nostril) in murine intestinal epithelial cells (IEC4.1 cells) in response to  IFN-gamma protein (IFNγ) stimulation.  NR_126553 (Nostril) was knocked down by using a pool of gene specific siRNA (SiNostril). Cells treated with scramble non-specific siRNA were used as control (siNegative control). After siRNA transfection 24h, cells were treated with or without mouse IFNγ (1ng/ml for 4hrs). Then total RNA was collected for sequencing via DNBSEQ platform to obtain a comprehensive view of the transcriptome.

overall_design: IEC 4.1 cells were transfected with the siRNAs (or SiNegative control) for 24 hours to allow gene knockdown or other desired effects. Four groups were included in this set up, Group A: Non-specific scrambled siRNA control treatment. Group B: Non-specific scrambled siRNA control treatment + IFNγ stimulation. Group C: Double-strand RNA encoding siNR_126553 (SiNostril) treatment. Group D: Double-strand RNA encoding siNR_126553 ( (SiNostril) +IFNγ stimulation.  Total RNAs were prepared with the RNeasy Mini kit (Qiagen) according to the manufacturer’s instruction.

first: Xianming

last: Chen


title: The OxyR regulon in the entomopathogenic bacterium Xenorhabdus

city: Montpellier

first: Anne

last: Lanois

address: 75 rue du Truel CC54

country: France

institute: INRA

summary: Xenorhabdus nematophila is a Gram-negative bacterium, mutually associated with the soil nematode Steinernema carpocapsae and this nematobacterial complex is parasitic for a broad spectrum of insects. The transcriptional regulator OxyR is widely conserved in bacteria, but the OxyR regulon can vary significantly between species. OxyR activates the transcription of a set of genes that influence cellular defense against oxidative stress. It is also involved in the virulence of several bacterial pathogens. The aim of this study was to identify the X. nematophila OxyR regulon and investigate its role in the bacterial life cycle. An oxyR-mutant was constructed in X. nematophila and phenotypically characterized in vitro and in vivo after reassociation with its nematode partner. OxyR plays a major role during the X. nematophila resistance to oxidative stress in vitro. Transcriptome analysis allowed the identification of 59 genes differentially regulated in the oxyR mutant compared to the parental strain. In vivo, the oxyR mutant was able to reassociate with the nematode as efficiently as the control strain. These nematobacterial complexes harboring the oxyR mutant symbiont were able to rapidly kill the insect larvae in less than 48h after infestation, suggesting that factors other than OxyR could also allow X. nematophila to cope with oxidative stress encountered during this phase of infection in insect. The significant increased number of offspring of the nematobacterial complex when reassociated with the X. nematophila oxyR mutant compared to the control strain, revealed a potential role of OxyR during this symbiotic stage of the bacterial life-cycle.

overall_design:
Three independent RNA samples, each corresponding to a pool of total RNA extracted independently from 3 biological replicates cultures of:
F1 WT grown in LB  or LB supplemented with paraquat (10mM) (=oxydative stress) harvested in exponential phase (OD= 0.3)
OxyR mutant grown in LB or LB supplemented with paraquat (10mM) (=oxydative stress) harvested in exponential phase (OD= 0.3)
Deep sequencing, performed using Illumina HiSeq 2000

first: Victoria

last: Bientz

first: Nadège

last: Ginibre

first: Sylvie

last: Pagès

first: Jean-Claude

last: Ogier

first: Simon

last: George

first: Stéphanie

last: Rialle

first: Julien

last: Brillard


title: Human-Induced Pluripotent Stem Cell (iPSC)-Derived  GABAergic Neuron Differentiation in Bipolar Disorder

city: Ann Arbor

first: Michigan

last: Bioinformatics

address: 109 Zina Pitcher Pl

institute: University of Michigan

summary: Bipolar disorder (BP) is a recurring psychiatric condition characterized by alternating episodes of low energy (depressions) followed by manias (high energy). Cortical network activity produced by GABAergic interneurons may be critical in maintaining the balance in excitatory/inhibitory activity in the brain during development. Initially, GABAergic signaling is excitatory; with maturation, these cells undergo a functional switch that converts GABAA channels from depolarizing (excitatory) to hyperpolarizing (inhibitory), which is controlled by the intracellular concentration of two chloride transporters. The earliest, NKCC1, promotes chloride entry into the cell and depolarization, while the second (KCC2) stimulates movement of chloride from the neuron, hyperpolarizing it. Perturbations in the timing or expression of NKCC1/KCC2 may affect essential morphogenetic events including cell proliferation, migration, synaptogenesis and plasticity, and thereby the structure and function of the cortex. We derived induced pluripotent stem cells (iPSC) from BP patients and undiagnosed control (C) individuals, then modified a differentiation protocol to form GABAergic interneurons, harvesting cells at sequential stages of differentiation. qRT-PCR and RNA sequencing indicated that after six weeks of differentiation, controls transiently expressed high levels of NKCC1.

overall_design: skin biopsis were taken from patients diagnosed with bipolar I disorder (BP) who carry the rs1006737 risk allele(AA) in CACNA1C and from control (C) patients with no psychiatric diagnosis.  The rs1006737 risk allele in three BP iPSC were targeted for correction via CRISPR-Cas9- mediated editing and repair (BP-C). After 6 weeks of differentiation of GABAergic nuerons from iPSC, total RNA was extracted from 4 controls(C), 4 bipolar(BP), and 3 BP-Corrected samples. Two independent biological differentiations were performed per sample.

first: Craig

last: Johnson

first: Daniel

last: Schill

first: Sue

last: O'Shea


title: Tomato RS2Z35 and RS2Z36

city: Frankfurt am Main

first: Remus

middle: Richard Elrik

last: Rosenkranz

address: Max-von-Laue-Str. 9

institute: Goethe University Frankfurt

summary:
Assessment of RS2Z protein function under heat stress in Solanum Lycopersicum via RNASeq and iCLIP2.

This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series.


title: Effect of RS2Z35 and RS2Z36 single and double knockout on gene expression and splicing during control and heat stress conditions in tomato (Solanum Lycopersicum) leaf tissue

summary:
To investigate the role of Serine/Arginine-rich proteins RS2Z35 and RS2Z36 in tomato heat stress response and thermotolerance, we established single and double knockouts of the respective splicing factor(s) in tomato
We then performed gene expression and splicing analysis using data obtained from RNA-seq of wildtype, single knockout and double knockout plants under control and heat stress conditions (1 h 40°C)

overall_design: Differential gene expression analysis of RNA-seq data for RS2Z protein knockout plants compared to WT, under control and heat stress conditions. 3 biological replicates each

first: Sotirios

last: Fragkostefanakis


title: CD4+ CAR-T cell exhaustion associated with early relapse of multiple myeloma after BCMA CAR-T cell therapy I

city: Seattle

first: Lawrence

last: Fong

state: Washington

address: 1100 Fairview Ave N

department: Immunotherapy Integrated Research Center, Translational Science and Therapeutics Division

institute: Fred Hutch Cancer Center

summary: Multiple myeloma is characterized by frequent clinical relapses following conventional therapy. Recently, chimeric antigen receptor T (CAR-T) cells targeting B-cell maturation antigen (BCMA) has been established as a treatment for patients with relapsed or refractory disease. However, while >70% of patients initially respond to this treatment, clinical relapse and disease progression occurs in most cases. Recent studies showed persistent expression of BCMA at the time of relapse, indicating that immune intrinsic mechanisms may contribute to this resistance. While there were no pre-existing T cell features associated with clinical outcomes, we found that patients with a durable response to CAR-T cell treatment had greater persistence of their CAR-T cells compared to patients with transient clinical responses.  They also possessed a significantly higher proportion of CD8+ T effector memory cells. In contrast, patients with short-lived responses to treatment have increased frequencies of cytotoxic CD4+ CAR-T cells. These cells expand in vivo early after infusion but express exhaustion markers (HAVCR2 and TIGIT) and remain polyclonal. Finally, we demonstrate that non-classical monocytes are enriched in the myeloma niche and may induce CAR-T cell dysfunction through mechanisms that include TGFβ. These findings shed new light on the role of cytotoxic CD4+ T cells in disease progression after CAR-T cell therapy.

overall_design: Bone marrow aspirates and peripheral blood samples were obtained from multiple myeloma patients pre- and post- anti-BCMA CAR T treatment and from healthy donors undergoing hip replacement surgery. Multiple myeloma patients were enrolled in clinical trials NCT02658929, NCT03274219 and NCT03430011. Bone marrow and PBMC samples were harvested prior to CAR T infusion and at 14days, 30days, 90days, 180days, 270days and 360days post infusion for analysis when available.

first: Guy

last: Ledergor

first: Zenghua

last: Fan

first: Kai

last: Wu

first: Elizabeth

last: McCarthy

first: Axel

last: Hyrenius Wittsten

first: Alec

last: Starzinski

first: Hewitt

last: Chang

first: Serena

last: Kwek

first: Alexander

last: Cheung

first: Sophia

last: Bylsma

first: Erik

last: Hansen

first: Jeffrey

last: Wolf

first: Sandy

middle: W

last: Wong

first: Nina

last: Shah

first: Kole

last: Roybal

first: Thomas

last: Martin

first: Jimmie

last: Ye


title: Neurological Cockayne Syndrome Results from R-Loops Induced by Stalled RNA Polymerase II during Transcription Elongation

city: La Jolla

first: Alon

last: Goren

address: 9500 Gilman Drive

institute: University of California San Diego

summary: Mutations in the Cockayne Syndrome group B (CSB) gene cause severe neurodevelopmental defects and premature aging. As a member of the SWI/SNF family of chromatin remodelers, CSB is best known for its role in transcription-coupled  nucleotide excision (TC-NER), but this function neither explains the major disease phenotype nor offers any clue about the selective vulnerability in neurons. Pursuing Cockayne Syndrome-associated genome instability, we uncover an intrinsic mechanism by which elongating RNA polymerase II (RNAPII) undergoes transient pausing at internal T-runs where CSB is required to push RNAPII forward. Consequently, CSB deficiency retards RNAPII elongation in these regions, and when coupled upstream G-rich sequences, such functional defects are further amplified to induce genome instability via augmented R-loop formation. As such R-loop prone motifs are proportionally represented in long genes that predominately function in neurons, this mechanism provides critical insights into selective neuronal vulnerability. Moreover, because of divergent intronic sequences between mice and humans, this mechanism also explains why mice deficient for CSB do not develop severe neurological abnormalities as seen in humans, suggesting that the manifestation of Cockayne Syndrome phenotype results from progressive genome evolution in mammals.

overall_design: To examine the genome instability induced by CSB-deficiency, we performed R-ChIP, PolII ChIP-seq, PRO-seq and chromatin associated RNA-seq under siNC and siCSB condition. Besides, We also performed the CSB ChIP-seq to identify the CSB binding site. All data have two replicates and controls.

first: Xuan

last: Zhang

first: Xiang-Dong

last: Fu


title: A genome wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6⍺

city: Cambridge

first: Adriana

last: Ordonez

state: Cambridgeshare

address: Keith Peters Building, Biomedical Campus, Hills Rd

department: Biochemistry

institute: Cambridge Institute for medical Research

summary: Activating transcription factor 6 alpha (ATF6⍺) is one of the three endoplasmic reticulum (ER) transmembrane stress sensors that mediate the unfolded protein response (UPR). Despite its significant involvement in long-term ER stress adaption, regulation of ATF6⍺ signalling is still poorly understood, possibly because its activation involves Golgi and nucleus trafficking. Here, we have generated a dual CHO-K1 ATF6⍺/IRE1⍺ reporter cell line to perform an unbiased genome-wide CRISPR/Cas9 mutagenesis screen, in the presence and absence of ER stress, to systematically profile genetic factors that specifically contribute to ATF6⍺ signalling. Anticipated and new candidate genes that regulate ATF6⍺ activation were discovered. Among these, calreticulin (CRT), a key ER luminal chaperone, emerged as a selective repressor molecule of ATF6⍺ signalling. Cells lacking CRT constitutively activated a BiP::sfGFP ATF6⍺-dependent reporter, had higher BiP levels and an increased rate of trafficking and processing of ATF6⍺. Purified CRT interacts with the luminal domain of ATF6⍺ in vitro and the two proteins co-immunoprecipitated from cell lysates. CRT depletion exposed a negative feedback loop implicating ATF6⍺ in repressing IRE1⍺ activity basally and overexpression of CRT reversed phenotype. Our data indicate that CRT, in addition to its known role as a chaperone, also serves as an ER repressor of ATF6⍺ to maintain selective regulation of the UPR.

overall_design: Examination of genomic DNA, pooled from sorted cells in different bins (based on ATF6::sfGFP and XBP1s::mCherry reporter signals) at different stages of the phenotypic enrichment process and from unsorted control cells, was subjected to high-throughput sequencing and MAGeCK bioinformatics analysis. In total 16 samples were examinated.

first: Heather

last: Harding

first: David

last: Ron


title: Expansion of Tumor-Infiltrating CD8+ T Cell Clonotypes Occurs in the Spleen in Response to Immune Checkpoint Blockade

status: Public on Jul 19 2024

city: CAMBRIDGE

first: Duncan

middle: Matthew

last: Morgan

state: MA

address: 500 Main St

department: Koch Institute

institute: MIT

summary: Immune checkpoint blockade (ICB) enhances tumor-reactive T cell responses against cancer, leading to long-term tumor control and survival in a fraction of patients. Given the increasingly recognized complexity of CD8+ T cell differentiation that occurs in response to chronic antigen stimulation, it remains unclear precisely which T cell differentiation states are critical for the response to ICB, as well as the anatomic sites at which ICB-mediated reinvigoration of these T cells occurs. We used paired single-cell RNA and T cell receptor (TCR) sequencing to profile endogenous, tumor-reactive CD8+ T cells isolated from tumors, tumor-draining lymph nodes, and spleens of mice treated with ICB. We identified an intermediate-exhausted population of T cells in the spleen which underwent the greatest expansion in response to ICB and gave rise to the majority of tumor-infiltrating clonotypes. Increasing concentrations of systemic antigen perturbed the differentiation of this phenotype towards an exhausted_KLR state, while a lack of cross-presentation of tumor-derived antigen blunted differentiation in the spleen. We identified an analogous population of exhausted_KLR CD8+ T cells in matched human tumor and blood samples that exhibited diminished tumor-trafficking ability. Collectively, our data demonstrate the critical role of antigen density within the spleen in coordinating the differentiation and expansion of tumor-infiltrating T cell clonotypes in response to ICB.

overall_design: Single-cell whole transcriptome and TCR profiling was performed on endogeneous, SIY-reactive cells isolated from the tumor, lymph node, and spleen of untreated mice and mice undergoing treatment with checkpiont blockade immunotherapy.

middle: M

first: Brendan

middle: L

last: Horton

first: V

last: Bhandakar

first: R

last: Van

first: T

last: Dinter

first: M

last: Zagorulya

first: J

middle: C

last: Love

first: S

last: Spranger


title: Cancer-associated snaR-A noncoding RNA interacts with core splicing machinery and disrupts processing of mRNA subpopulations

city: Urbana

first: Sihang

last: Zhou

address: 601 S Goodwin Ave

institute: University of Illinois Urbana-Champaign

summary: RNA polymerase III (Pol III) activity in cancer is linked to the production of small noncoding (nc)RNAs that are otherwise silent in most tissues. snaR-A (small NF90-associated RNA isoform A) - a hominid-specific ncRNA shown to enhance cell proliferation, migration, and invasion - is a cancer-emergent Pol III product that remains largely uncharacterized despite promoting growth phenotypes. Here, we applied a combination of genomic and biochemical approaches to predict the biogenesis and subsequent protein interactions of snaR-A to better understand its role as a putative driver of cancer progression. By profiling the chromatin landscapes across a multitude of primary tumor types, we show that predicted snaR-A upregulation is broadly linked with unfavorable outcomes among cancer patients. At the molecular level, we unexpectedly discover widespread interactions between snaR-A and mRNA splicing factors, including SF3B2 - a core component of the U2 small nuclear ribonucleoprotein (snRNP). We find that SF3B2 levels are sensitive to high snaR-A abundance, and that depletion of snaR-A alone is sufficient to decrease intron retention levels across subpopulations of mRNA enriched for U2 snRNP occupancy. snaR-A sensitive genes are characterized by high GC content, close spatial proximity to nuclear bodies concentrated in pre-mRNA splicing factors, and functionally enriched for proteins involved in deacetylation and autophagy. We highlight examples of splicing resolution and increased protein levels following snaR-A depletion for a wide-ranging set of factors, suggesting snaR-A-driven splicing defects may have far-reaching effects that re-shape the cellular proteome. These findings help clarify the molecular activities and consequences of snaR-A express- ion in cancer, and altogether establish a novel mechanism through which Pol III overactivity may promote tumorigenesis.

overall_design: To investigate the effect of snaR-A on splicing of mRNAs, we performed snaR-A knockdown using three different siRNAs targeting snaR-A in HEK293T cells and deep sequenced the samples. The experiment included three biological replicates for both the scramble knockdown and the snaR-A knockdown. Raw mRNA signal counts were extracted.

first: Kevin

last: Van Bortle


title: Identifications of vernalization regulatory elements by epigenomic approaches in winter wheat (ATAC-Seq)

city: Beijing

first: Zefu

last: Lu

state: Haidian

address: No.12 Zhongguancun South St.,Haidian District, Beijing, P.R.China

institute: Institute of Crop Sciences

summary:
Epigenetic modification plays a key role in the vernalization process and maintains gene expression states after low temperature. In this study, the Chinese main cultivar Aikang58 was used to construct the chromatin accessibility maps, expression profile and histone modification (including H3K27me3, H3K27ac, H3K36me3 and H3K4me3) maps of winter wheat before and after vernalization.  We provided a crucial data resource for deep understanding of epigenome in vernalization. What’s more, important core regulatory elements and corresponding transcription factors were discovered, and the complex regulatory network of wheat vernalization was analyzed.
ATAC-seq of leaf, axillary bud and shoot apex before and after vernalization


title: Identifications of vernalization regulatory elements by epigenomic approaches in winter wheat (ChIP-Seq)

summary: Epigenetic modification plays a key role in the vernalization process and maintains gene expression states after low temperature. In this study, the Chinese main cultivar Aikang58 was used to construct the chromatin accessibility maps, expression profile and histone modification (including H3K27me3, H3K27ac, H3K36me3 and H3K4me3) maps of winter wheat before and after vernalization.  We provided a crucial data resource for deep understanding of epigenome in vernalization. What’s more, important core regulatory elements and corresponding transcription factors were discovered, and the complex regulatory network of wheat vernalization was analyzed.

overall_design: ChIP-seq of leaf and vernalized leaf, including H3K27me3, H3K27ac, H3K36me3 and H3K4me3


title: Scar matrix drives Piezo1 mediated stromal inflammation leading to placenta accreta spectrum

city: Farmington

first: Yasir

last: Suhail

state: Connecticut

address: 263 Farmington Avenue

department: BME

institute: UCHC

summary: Scar tissue formation is a hallmark of wound repair in adults and can chronically affect tissue architecture and function. To understand the general phenomena, we sought to explore scar-driven imbalance in tissue homeostasis caused by a common, and standardized surgical procedure, the uterine scar due to cesarean surgery. Deep uterine scar is associated with a rapidly increasing condition in pregnant women, placenta accreta spectrum (PAS), characterized by aggressive trophoblast invasion into the uterus, frequently necessitating hysterectomy at parturition. We created a model of uterine scar, recapitulating PAS-like invasive phenotype, showing that scar matrix activates mechanosensitive ion channel, Piezo1, through glycolysis-fueled cellular contraction. Piezo1 activation increases intracellular calcium activity and Protein kinase C activation, leading to NF-κB nuclear translocation, and MafG stabilization. This inflammatory transformation of decidua leads to production of IL-8 and G-CSF, chemotactically recruiting invading trophoblasts towards scar, initiating PAS. Our study demonstrates aberrant mechanics of scar disturbs stroma-epithelia homeostasis in placentation, with implications in cancer dissemination.

overall_design: We asked if the aberrant mechanical signals present at the scar transform decidual fibroblasts to be more invasable to EVT invasion, we created a scar-like model of decidua (Scar) and its physiological counterpart (Physio). To gain insight into the underlying mechanisms contributing to the aberrant EVT invasion, we isolated the RNA from dESFs on Physio and Scar for sequencing and then performed gene expression profiling analysis.

first: Wenqiang

last: Du

first: Kshitiz

last: Gupta


title: TMEFF1 is a neuron-specific restriction factor for herpes simplex virus

city: Aarhus

first: Søren

middle: Riis

last: Paludan

address: Høegh-Guldbergs Gade 10

department: Department of Biomedicine

country: Denmark

institute: Aarhus University

summary: The brain is highly sensitive to damage caused by infection and inflammation. Herpes simplex virus-1 (HSV-1) is a neurotropic virus and the cause of herpes simplex encephalitis. It is unknown whether neuron-specific antiviral factors control virus replication to prevent infection and excessive inflammatory responses, hence protecting the brain. Using genome-wide CRISPR screening for HSV-1 restriction factors, we identified TMEFF1, which is expressed specifically in CNS neurons and not regulated by type I interferon, as the best-known innate antiviral system controlling virus infections. Depletion of TMEFF1 in stem-cell-derived human neurons led to elevated viral replication and neuronal death upon HSV-1 infection. TMEFF1 blocked the HSV-1 replication cycle at the level of viral entry through interactions with Nectin-1 and non-muscle myosin heavy chain IIA/B, which are core proteins in virus-cell binding and virus-cell fusion, respectively. Importantly, Tmeff1-/- mice exhibited increased susceptibility to HSV-1 infection in the brain but not in the periphery. Within the brain, elevated viral load was observed specifically in neurons. Our study identifies TMEFF1 as a neuron-specific restriction factor essential for prevention of HSV-1 replication in the CNS.

overall_design: Cas9+ SK-N-SH 614 cells were transduced with lentiviral preparations of GeCKO v2 CRISPR library (library part A for screen 1 and library part B for screen 2). Subsequently, the library transduced cell populations were infected with HSV-1-K26GFP at an MOI of 0.25, 17 h after infection cells with high GFP expression was sorted by FACS. Analysis was performed by comparing the GFPhigh sample with the non-sorted control.

first: Paludan

last: Riis Søren