title: Xenotransplanted Human Small Intestinal Organoids Reveal the Transepithelial Zinc Transport Pathway as a Key Mediator of Intestinal Adaptation in Short Bowel Syndrome

status: Public on Aug 16 2024

city: Baltimore

first: David

middle: J

last: Hackam

address: 1800 Orleans Street

department: Pediatric Surgery

institute: Johns Hopkins University School of Medicine

summary: Intestinal adaptation is crucial to improving outcomes in short bowel syndrome (SBS). We performed scRNA sequencing of human intestinal organoid explanted from mice with experimental SBS to examine the genes and pathways involved in this adaptation.

overall_design: Human intestinal organoids were grown from human induced pluripotent stem cells and implanted into mice with experimental SBS or a control sham surgery. 25-28 days after surgery, the organoids were harvested genes, pathways and cell types were analyzed with scRNA sequencing.

first: Maame

middle: E

last: Sampah

first: Hannah

last: Moore

first: Raheel

last: Ahmad

first: Johannes

last: Duess

first: Peng

last: Lu

first: Carla

last: Lopez

last: Scheese

first: Zachariah

last: Raouf

first: Koichi

last: Tsuboi

last: Ding

first: Connor

last: Caputo

first: Madison

last: McFarland

first: William

middle: B

last: Fulton

first: Sanxia

first: Meghan

first: Thomas

last: Prindle

first: Vered

last: Gazit

first: Deborah

middle: C

last: Rubin

first: Samuel

last: Alaish

first: Chhinder

last: Sodhi


title: RNF20-mediated transcriptional pausing and alternative splicing of pro-angiogenic genes orchestrate vessel growth [06ATAC_siRNF20_HUVEC]

status: Public on Aug 18 2024

city: Mannheim

first: Gergana

last: Dobreva

state: Baden Württemberg

address: Ludolf-Krehl-Str. 7-11

department: Cardiovascular Genomics and Epigenomics

country: Germany

institute: Medical Faculty Mannheim/University of Heidelberg

type: Expression profiling by high throughput sequencing

summary: Signal-responsive gene expression is essential for vascular growth and patterning, yet the mechanisms allowing the integration of signaling inputs and transcriptional activities are largely unknown. Here we show that RNF20, the major E3 ubiquitin ligase of histone H2B, plays a key role during sprouting angiogenesis by mediating Pol II promoter-proximal pausing at pro-angiogenic genes. Furthermore, it orchestrates large-scale mRNA processing events, which change the bioavailability and function of critical pro-angiogenic factors, such as VEGFA. Mechanistically, we show that RNF20 restricts ERG-dependent gene activation through the VEGF-ERK1/2 axis while enabling Notch1 to bind to chromatin, thereby modulating the VEGF-Notch signaling circuits. Since perturbations of RNF20 are associated with cardiovascular malformations in human patients, our work may provide novel therapeutic avenues for diseases caused by vascular dysfunction.

overall_design: HUVEC cells were transfected with siCtr or siRNF20. 60h after transfection, cells were harvested to perform ATAC-seq.

first: Julio

last: Cordero

first: Nalan

last: Tetik


title: Npac regulates pre-mRNA splicing in mouse embryonic stem cells

city: macau

first: YIWEI

last: QIAN

state: Other (Non U.S.)

address: Avenida Wai Long,Taipa,Macau

country: China

institute: macau university of science and technology

summary: As a reader of tri-methylated lysine 36 on histone H3 (H3K36me3), Npac has been shown a significant role in gene transcription elongation. However, its potential implication in RNA splicing remains unknown. Here, we characterized the phenotypes of Npac knockout in mES cells. We discovered that loss of Npac disrupts pluripotency and identity in mESCs. We also found that Npac is associated with many cellular activities including cell proliferation, differentiation, transcription regulation.   Notably, we uncovered that Npac is associated with RNA splicing machinery. Furthermore, we found that Npac regulates alternative splicing through its interaction with the splicing factors including Srsf1. Our research thus highlights the important role of Npac in maintaining ESC identity through regulation of pre-mRNA splicing.

overall_design: To elucidate the cooperative role of Npac in regulating mouse embryonic stem cells (mESCs), we generated E14 cell lines in which specific target genes were knocked out using CRISPR-Cas9 technology. Following this, we performed a comparative gene expression profiling analysis by employing RNA-sequencing. This process enables a comprehensive overview of the RNA-seq data analysis, comparing the gene expression patterns between the wild-type (WT) E14 cells and their knockout (KO) derivatives.


title: Early N-hydroxy-pipecolic acid transcriptional profile

city: Stanford

first: Mary Beth

last: Mudgett

state: CA

address: 371 Jane Stanford Way

department: Biology

country: USA

institute: Stanford University

summary: N-hydroxy-pipecolic acid (NHP) is a mobile metabolite essential for inducing and amplifying systemic acquired resistance (SAR) following pathogen attack. In Arabidopsis thaliana (Arabidopsis), exogenous NHP is sufficient to activate SAR-related immune responses, including salicylic acid (SA) accumulation and changes in global transcriptional profile. Previous studies have tracked these changes 1-2 days after an initial NHP treatment, resulting in little knowledge of the very early phases of NHP signaling and transcriptional responses leading to immunity. Here we show NHP elicits transcriptional changes within minutes of treatment. We report distinct waves of expression over the course of minutes to hours defined by transient induction of jasmonic acid/wound-related responses, a primary induction of WRKY transcription factor expression, and subsequent induction of WRKY-regulated defense genes. The upregulation of WRKYs and the majority of defense-related genes occurred in the sid2-2 mutant, which is unable to accumulate SA upon NHP treatment, suggesting NHP is sufficient to drive the early phase of transcriptional changes in a low SA environment. We also show that WRKY70 is required for the expression of a set of genes defining the secondary transcriptional changes, as well as NHP enhancement of ROS production and SAR.

overall_design: To investigate how the presence of NHP affects transcriptional changes within minutes to hours of treatment, six-well plates containing 10-day-old hydroponically grown Arabidopsis wild type, sid2-2, and fmo1-1 seedlings were treated with mock or NHP. The sid2-2 and fmo1-1 mutants were analyzed to identify transcriptional changes dependent on NHP-induced salicylic acid biosynthesis and endogenous NHP production, respectively. All seedlings from a single well were pooled and collected 15 min, 30 min, 3 h, and 6 h following treatment for a single sample, this was repeated for a total of three samples per condition, n = 3.

first: Jessica

last: Foret

first: Jung-Gun

last: Kim

first: Elizabeth

last: Sattely


title: A regulatory loop involving the cytochrome P450-soluble epoxide hydrolase axis and TGF-b signaling

status: Public on Aug 15 2024

city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

institute: MPI for heart and lung research

summary: Fatty acid metabolites generated by the sequential actions of cytochrome P450 enzymes and the soluble epoxide hydrolase (sEH) regulate inflammation. Here we report that the TGF--induced polarization of macrophages to a pro-resolving phenotype requires the activation of Alk5 and Smad2 to increase sEH expression and activity. Macrophages lacking sEH failed to fully repolarize, were less efficient at phagocytosis, and retained a pro-inflammatory gene expression profile. 11,12-Epoxyeicosatrienoic acid (EET) was one of the fatty acid metabolites altered in sEH-/-  macrophages and was able to reproduce the effect of sEH deletion on gene expression. Importantly, 11,12-EET also attenuated the expression of Alk5 to inhibit the TGF--induced phosphorylation of Smad2 by eliciting the cytosolic translocation of the E3 ligase Smurf2. These results indicate that the expression of sEH is not only controlled by TGF- but that the activity of the enzyme, which keeps 11,12-EET levels low, actually promotes TGF- signaling by preventing the proteolytic degradation of Alk5. Thus, an autocrine loop between the sEH/11,12-EET and TGF-1 determines macrophage function.

overall_design: Monocytes were isolated from bone marrow and then differenciated into macrophages with a combination of M-CSF and GM-CSF for 7 days. M1 polarization was carried out with a combination of IFN-γ and LPS and after 24 hours TGF-β1 was added on top for additional 48 hours.

first: Xiaoming

last: Li

first: Fredy

last: Delgado Lagos

first: Ingrid

last: Fleming


title: RNA sequestration in P-bodies sustains myeloid leukemia [HSPC_RNAseq]

city: Houston

first: Srikanth

last: Kodali

state: TX

address: 1 Baylor Plaza

institute: Baylor College of Medicine

summary: We performed RNA-seq to profile transcriptional changes in hematopoietic stem and progenitor cells following Ddx6 deletion.

overall_design: RNA-seq

first: Yingzhi

last: Cui

first: Bruno

last: Di Stefano


title: Conservation of antiviral defences in the non-vascular plant Marchantia polymorpha

city: Cerdanyola del Vallès

first: Ignacio

last: Rubio-Somoza

state: Barcelona

address: Carrer de la Vall Moronta s/n, Campus UAB - Edifici CRAG

country: Spain

institute: CRAG

summary: Most of our current knowledge about the molecular events ruling plant-virus interaction come from studies focusing on vascular plants. We here characterized the molecular, cellular and physiological events goberning plant-virus interactions in the non-vascular liverwort Marchantia polymorpha.

overall_design: 2 weeks old Marchantia plants grown in sterile conditions were incubated with sap from either WT or TMV infected  tobacco plants. Two weeks after, samples were collected from infected areas (TMV samples) and anatomically equivalent areas from mock plants (Mock samples). RNA was isolated from 4 pools. mRNA enriched libraries were prepared using Illumina TrueSeq kit and following instructions from manufacturer.

first: Eric

last: Ross

first: Tamara

last: Jimenez-Gongora

first: Luis

last: Villar-Martin

first: Víctor

middle: M

last: González-Miguel

first: Denis

last: Kutnjak


title: Characterization of gene profile between ALDH high LNCaP cells and ALDH low LNCaP cells

city: Durham

first: Che-Chia

last: Hsu

state: NC

address: 210 Research Drive, GSRB2, Rm#4014

department: Pathology

institute: Duke University

summary: Acquisition of prostate cancer stem cells (PCSCs) manifested during androgen ablation therapy (ABT) contributes to castration-resistant prostate cancer (CRPC). However, little is known about the specific metabolites critically orchestrating this process. Here, we show t+B13hat IMPA1-derived inositol enriched in PCSCs is a key metabolite crucially maintaining PCSCs for CRPC progression and ABT resistance.

overall_design: For fluorescence-activated cell sorting, cells were labeled with ALDEFLUOR Kit and sorted using MoFlo Astrios Cell Sorter (BECKMAN COULTER) in two ways including highest 10 % ALDH positive cells (ALDHhigh) and the lowest 10% ALDH negative cells (ALDHlow/-), following by cell culture and immunoblotting.


title: RNA sequencing in 22RV1 cells stably expressing shLuc, shIMPA1, shIMPDH2

summary: We investigated gene expession profiles of IMPA1 knockdown and IMPDH2 knockdown in CRPC 22RV1 cells by using RNA-seq

overall_design: Examination of mRNA profiling in control knockdown, IMPA1 knockdown or IMPDH2 knockdown 22RV1 cells


title: METTL14 is a chromatin regulator independent of its RNA N6-methyladenosine methyltransferase activity

city: Shanghai

first: Xiaoyang

last: Dou

address: 320 Yue Yang Road

institute: CAS Center for Excellence in Molecular Cell Science, CAS

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series

first: Lulu

last: Huang

first: Yu

last: Xiao

first: Chuan

last: He

first: Jun

last: Liu


title: Genetic Screen in a Pre-Clinical Model of Sarcoma Development Defines Drivers and Therapeutic Vulnerabilities

city: Sacramento

first: Janai

middle: R.

last: Carr-Ascher

department: Department of Internal Medicine

institute: University of California Davis

summary: High-grade complex karyotype sarcomas are a heterogeneous group of more than seventy tumors that vary in histology, clinical course, and patient demographics. Despite these differences, these high-grade sarcomas are treated similarly with varying outcomes. Pre-clinical models of distinct human sarcoma subtypes would advance insights into the relationships between sarcomas and inform therapeutic decisions. We describe the transformation of human mesenchymal stem cells into multiple subtypes of high-grade sarcoma. Using a pooled genetic screening approach, we identified key drivers and potential modifiers of transformation. YAP1, KRAS, CDK4, and PIK3CA were validated as drivers of four distinct sarcoma subtypes, undifferentiated pleomorphic sarcoma (UPS), myxofibrosarcoma (MFS), leiomyosarcoma (LMS), and osteosarcoma (OS). Histologically and phenotypically these tumors reflect human sarcomas including the pathognomonic complex karyotype and YAP1 amplification. Transcriptome analysis confirmed that these tumors accurately recapitulate human disease. This model is a tool that can be used to begin to understand pathways and mechanisms driving human sarcoma development, the relationship between sarcoma subtypes and to identify and test new therapeutic targets for this aggressive and heterogeneous disease.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for ASC52telo cells and its KO derivatives (TP53, RB1) with or without overexpression of YAP1, KRAS, PIK3CA, CDK4.

middle: R

first: Thomas

middle: G

last: Graeber

first: Jack

last: Freeland


title: Gene expression profiles of human iPSC-derived microglia in monoculture and in co-culture with isogenic iPSC-derived neural cells

city: Cambridge

first: Emile

last: Wogram

state: MA

address: 455 Main St

institute: Whitehead Institute for Biomedical Research

summary: The goal of the study was to developed a new method for the rapid isolation of pure and intact phagosomes from human pluripotent stem cell-derived microglia under various, highly defined experimental conditions.

overall_design: Here, human pluripotent stem cell-derived microglia were either cultured in monocultures, or together with isogenic, human pluripotent stem cell-rerived neural cells

first: George

middle: W

last: Bell


title: Serotonin deficiency from constitutive SKN-1 activation drives pathogen apathy

city: Los Angeles

first: Sean

middle: Patrick

last: Curran

address: 3715 McClintock Ave #350

department: Geronotology

institute: University of Southern California

summary: When an organism encounters a pathogen, the host innate immune system is activated to defend against pathogen colonization and the toxic xenobiotics produced.  C. elegans employ multiple defense systems to ensure survival when exposed to Pseudomonas aeruginosa (PA14) including activation of the cytoprotective transcription factor SKN-1/NRF2. Although wildtype C. elegans quickly learn to avoid pathogens, here we describe a peculiar apathy to PA14 in animals with constitutive activation of SKN-1, whereby animals choose not to leave while continuing to feed on the pathogen even when a non-pathogenic food option is available. Although lacking the urgency to escape the infectious environment, animals with constitutive SKN-1 activity are not oblivious to the presence of the pathogen and display a typical intestinal distension from PA14 colonization and eventual demise, but surprisingly, fail to learn to avoid pathogen with training.  SKN-1 activation, specifically in neurons and intestinal tissues, orchestrates a unique transcriptional program which leads to defects in serotonin signaling from both neurons and non-neuronal tissues that drives the pathogen apathy behavior and pleiotropic responses to selective serotonin reuptake inhibitors (SSRIs). Taken together, our work reveals new insights into how animals perceive environmental pathogens and subsequently alter behavior and cellular programs to promote survival.

overall_design: To investigate the effect of exposure to Pseudomonas aeruginosa (PA14) in wildtype and skn-1gf(lax188) C elegans. E. coli OP50 was used as the control exposure. Worms were raised on OP50 and then either moved to a plate with OP50 or PA14 for 4 hours before collection at L4 stage.

first: Tripti

last: Nair

first: Nicole

middle: L

last: Stuhr

first: Brandy

last: Weathers

first: James

middle: D

last: Nhan

middle: P


title: MBL-1/Muscleblind regulates neuronal differentiation and controls the splicing of a terminal selector in Caenorhabditis elegans

status: Public on Aug 14 2024

city: HK

first: Chaogu

last: Zheng

address: The University of Hong Kong, Pokfulam Road

department: School of Biological Science

country: Hong Kong

institute: University of Hong Kong

summary: The muscleblind family of mRNA splicing regulators is conserved across species and regulate the development of muscles and the nervous system. However, how Muscleblind proteins regulate neuronal fate specification and neurite morphogenesis at the single-neuron level is not well understood. In this study, we found that the C. elegans Muscleblind/MBL-1 promotes axonal growth in the touch receptor neurons (TRNs) by regulating microtubule stability and polarity. Transcriptomic analysis identified dozens of MBL-1-controlled splicing events in genes related to neuronal differentiation or microtubule functions. Among the MBL- 1 targets, the LIM-domain transcription factor mec-3 is the terminal selector for the TRN fate and induces the expression of many TRN terminal differentiation genes. MBL-1 promotes the splicing of the mec-3 long isoform, which are essential for TRN fate specification, and inhibits the short isoforms that have much weaker activities in activating downstream genes. MBL-1 promotes mec-3 splicing through three “YGCU(U/G)Y” motifs located downstream of the included exon, which is similar to the mechanisms used by mammalian Muscleblind and suggests a deeply conserved context-dependency of the splicing regulation. Interestingly, the expression of mbl-1 in the TRNs is dependent on the mec-3 long isoform, indicating a positive feedback loop between the splicing regulator and the terminal selector. Finally, through a forward genetic screen, we found that MBL-1 promotes neurite growth partly by inhibiting the DLK-1/p38 MAPK pathway. In summary, our study provides mechanistic understanding of the role of Muscleblind in regulating cell fate specification and neuronal morphogenesis.

overall_design: To investigate the molecular mechanism of Muscleblind in regulating neuronal differentiation in TRNs

first: Ho

last: Lee

first: Hui

middle: Y

last: Lim

first: Haoming

first: Chun

last: Lau


title: Cell-intrinsic regulation of phagocyte function by interferon lambda during pulmonary viral, bacterial super-infection

city: Pittsburgh

first: John

last: Alcorn

state: Pennsylvania

address: 4401 Penn Avenue

department: Pediatrics

institute: University of Pittsburgh

summary: Influenza infections result in a significant number of severe illnesses annually, many of which are complicated by secondary bacterial super-infection. Primary influenza infection has been shown to increase susceptibility to secondary methicillin-resistant Staphylococcus aureus (MRSA) infection by altering the host immune response, leading to significant immunopathology. Type III interferons (IFNs), or IFNλs, have gained traction as potential antiviral therapeutics due to their restriction of viral replication without damaging inflammation. The role of IFNλ in regulating epithelial biology in super-infection has recently been established; however, the impact of IFNλ on immune cells is less defined. In this study, we infected wild-type and IFNLR1-/- mice with influenza A/PR/8/34 followed by S. aureus USA300. We demonstrated that global IFNLR1-/- mice have enhanced bacterial clearance through increased uptake by phagocytes, which was shown to be cell-intrinsic specifically in myeloid cells in mixed bone marrow chimeras. We also showed that depletion of IFNLR1 on CX3CR1 expressing myeloid immune cells, but not neutrophils, was sufficient to significantly reduce bacterial burden compared to mice with intact IFNLR1. These findings provide insight into how IFNλ in an influenza-infected lung impedes bacterial clearance during super-infection and show a direct cell intrinsic role for IFNλ signaling on myeloid cells.

overall_design: To investigate global trancsriptome changes in myeloid cells lacking IFNLR1, we treated inducible CX3CR1-Cre-eYFP-IFNLR1fl/fl mice with Tamoxifen or corn oil control before infection with 900 PFU influenza PR8 strain. On Day 6 of infection, mice were sacrificed and whole lungs were processed into single cell suspension before eYFP+ cells were sorted out. We isolated RNA from sorted eYFP+ cells from 9 total mice (5 TAM treated and 4 corn oil controls) for RNA sequencing.

first: Alcorn

last: John

first: Antos

last: Danielle


title: Therapeutic Delivery of CCL2 Modulates Immune Response and Restores Host-microbe Homeostasis

city: Buffalo

first: Lu

last: Li

state: NY

address: 3435 Main Street

institute: University at Buffalo

summary: The oral cavity is an easily accessible environment that allows for protective interventions aiming at modulating the immune response to control disease processes driven by a breakdown of host- microbe homeostasis. Periodontal disease (PD) is a prevalent condition in which quantitative and qualitative changes of the oral microbiota (dysbiosis) trigger non-resolving chronic inflammation, progressive bone loss and ultimately tooth loss. Here, we demonstrate the therapeutic benefit of local sustained delivery of the myeloid-recruiting chemokine CCL2 in murine ligature-induced PD using clinically relevant models as a preventive, interventional or reparative therapy. Local delivery of CCL2 into the periodontium inhibited bone loss and accelerated bone gain that could be ascribed to reduced osteoclasts numbers. CCL2 treatment upregulated M2-macrophage and downregulated pro-inflammatory and pro-osteoclastic markers. Furthermore, single cell RNA sequencing indicated that CCL2 therapy reversed disease-associated transcriptomic profiles of murine gingival macrophages via inhibiting TREM-1 signaling in classically activated macrophages and inducing PKA signaling in infiltrating macrophages. Finally, 16S rRNA sequencing showed mitigation of microbial dysbiosis in the periodontium that correlated with a reduction in microbial load in CCL2- treated mice. This study reveals a novel protective effect of CCL2 local delivery in PD as a model for chronic inflammatory diseases caused by a disturbance in host-microbe homeostasis.

overall_design: PD was induced  for 7 days in 6-8 weeks Balb/C mice using silk ligatures around the second maxillary molar.  Polymer (PLGA) microparticles (MPs) loaded with CCL2 chemokine, IL-4 (positive control) or blank MPs were locally delivered at the same time of ligature placement. Healthy mice without PD induction or mice with PD induction only (untreated) were used as controls.  At the end of the disease induction period, the gingival tissues around the ligated molar  in each treatment group were dissected and enzymatically digested to obtain a single cell suspension.  The CD45 positive cells were isolated from the cell suspesions using column based magnetic cell isolation and magnetic CD45 microbeads.  The viability of the isolated CD45+ cells was assessed using an auto cell counter before proceeding to the Sc-RNA Seq workflow.

first: Mostafa

last: Shehabeldin

first: Jin

last: Gao

first: Yejin

last: Cho

first: Rong

last: Chong

first: Tracy

last: Tabib

first: Matthew

last: Smardz

first: Sarah

last: Gaffen

first: Patricia

middle: I

last: Diaz

first: Robert

last: Lafyatis

first: Steven

last: Little

first: Charles

last: Sfeir


title: Transcriptome Dynamics in Mouse Amygdala under Acute and Chronic Stress Revealed by Thiol-labeled RNA Sequencing

first: Yang

last: Yang

address: 393 Middle Huaxia Road, Pudong, Shanghai

institute: ShanghaiTech University

summary: Both acute and chronic stress have significant impact on brain functions. The amygdala is essential in mediating stress responses, but how its transcriptomic dynamics change under stress remains elusive. To overcome the difficulties in detecting subtle stress-induced changes by evaluating total RNA using classic RNA sequencing, we conducted thiol-labeled RNA sequencing (SLAM-seq). We injected 4-thiouridine (4sU) into mouse amygdala followed by SLAM-seq to detect nascent mRNA induced by acute and chronic restraint stress, and found that SLAM-seq could label actively transcribed genes in the major neuronal and glial subtypes. Using SLAM-seq, we found that chronic stress led to higher turnover of a group of genes associated with myelination, and this finding is confirmed by immunostaining which showed increased myelination in the chronically stressed amygdala. Additionally, genes detected by SLAM-seq and RNA-seq only partially overlapped, with SLAM-seq particularly sensitive to transcriptional changes in genes with high basal transcription levels. Thus, by applying SLAM-seq in vivo, we obtained a rich dataset of nascently transcribed genes in the amygdala under stress, and revealed distinct transcriptional dynamics associated with acute and chronic stress.

overall_design: To investigate transcriptomic dynamics of amygdala under stress, we established an in vivo SLAM-seq protocol by injecting 4-thiouridine (4sU) or DMSO into mouse amygdala, and subjected mice to two stress conditions. We then performed SLAM-seq analysis (T-C conversion rates for all genes detected) and gene expression profiling analysis using data obtained from RNA-seq of 2 different stress conditions and 1 no-stress control condition.

first: Dan

last: Zhao

last: Zhang


title: Spatial transcriptomic analysis of human dorsoal root ganglia neurons [Xenium]

city: Philadelphia

first: Huasheng

last: Yu

state: PENNSYLVANIA

address: 3610 Hamilton Walk

department: Neuroscience

institute: University of Pennsylvania

summary: Xenium platform was used for the spatial transcriptomic analysis of human DRG neurons, 100 marker genes were selected as the customized probe panel and hybridized to fresh frozen hDRG sections. Manual segmentation of each neuron soma was performed, based on expressions of pan-neuronal marker gene PGP9.5, satellite glia cell marker FAB7B, and the corresponding H.E. staining. In total, 1340 neurons were identified (excluding 75 region-of-interest with poor or unclear neuronal soma morphology in H & E staining) and clustered into 16 groups. The 16 clusters were assigned as different cell types based on marker genes expression.

overall_design: In the study presented here, four dorsoal root ganglia tissues from two healthy donors were used for Xenium spatial transcriptomics analysis, A hundred gene panel (including 87 neuronal genes from our single-soma sequencing dataset and 13 non-neuronal cell marker genes) were selected to perform spatial transcriptomics. The spatial distribution of these genes in neurons and non-neuronal cells was successfully profiled and quantified.

first: Wenqin

last: Luo


title: Dysregulation of zebrin-II cell subtypes in the cerebellum is a shared feature across polyglutamine ataxia mouse models and patients

city: Irvine

first: Albert

last: La Spada

address: 419 S. Circle View Dr.

department: Neurology

institute: University of California Irvine

summary: Spinocerebellar ataxia type 7 (SCA7) is a genetic neurodegenerative disorder caused by a CAG-polyglutamine repeat expansion. Purkinje cells (PCs) are central to the pathology of ataxias, but their low abundance in the cerebellum underrepresents their transcriptomes in sequencing assays. To address this issue, we developed a PC enrichment protocol and sequenced individual nuclei from mice and patients with SCA7. Single-nucleus RNA sequencing in SCA7-266Q mice revealed dysregulation of cell identity genes affecting glia and PCs. Specifically, genes marking zebrin-II PC subtypes accounted for the highest proportion of DEGs in symptomatic SCA7-266Q mice. These transcriptomic changes in SCA7-266Q mice were associated with increased numbers of inhibitory synapses as quantified by immunohistochemistry and reduced spiking of PCs in acute brain slices. Dysregulation of zebrin-II cell subtypes was the predominant signal in PCs of SCA7-266Q mice and was associated with the loss of zebrin-II striping in the cerebellum at motor symptom onset. We furthermore demonstrated zebrin-II stripe degradation in additional mouse models of polyglutamine ataxia and observed decreased zebrin-II expression in the cerebella of patients with SCA7. Our results suggest that a breakdown of zebrin subtype regulation is a shared pathological feature of polyglutamine ataxias.

overall_design:
Nuclei from cerebellum were extracted and enriched for Purkinje cells from 2 male and 2 female mice of WT C57BL/6 and SCA7-266Q C57BL/6. Nuclei from each animal were labeled with MULTI-seq CMO barcodes and sorted into a common tube as input to 10X Genomics 3' snRNA-seq v3.1. Experiment performed at 5-week and 8.5-week timepoints.
Please note that, due to MULTI-seq multiplexing structure,  snRNA-seq fastq files contain 8 biosamples, which can only be demultiplexed with paired MULTIseq-fastq files.

first: Luke

last: Bartelt

first: Craig

last: Lowe


title: NGS5522 - hMDMS response to IFNg and TNFa

city: South San Francisco

first: Sandra

last: Melo

address: 1 DNA Way

institute: Genentech, Inc.

summary: human primay macrophages (hMDMs) were unstimulated,  stimulated with IFNg or costimulated with IFNg and TNFa  for 18h


title: NGS5439 - BMDMs cytokine response

summary: mouse primary BMDMs were stimulated with IFNg, IFNg + hypoxia, IFNg + IL1b, IFNg + LPS, IFNg +TNFa for 18h


title: NGS5523 - ATAC-seq WTAP and ZC3H13 KOs in hMDMS

summary: human primary macropahges (hMDMs) were genetically perturbed for WTAP and ZC3H13. ATAC-seq was performed 18h post IFNg (2ng/ml) or TNFa (10ng/ml) stimulation


title: Acral Melanoma microRNA Ratio Classification

city: Salt Lake City

first: Dekker

last: Deacon

state: Utah

address: 2000 Circle of Hope Dr #1950

department: Dermatology

institute: University of Utah

type: Non-coding RNA profiling by high throughput sequencing

summary: We performed microRNA sequencing of primary human FFPE Acral Melanoma (AM), Cutaneous Melanoma (CM), Acral Nevi (AN), and Cutaneous Nevi (CN). We found that previously identified ratios of microRNAs, particularly miR-21-5p and miR-211-5p, were able to accurately classify benign and malignant melanocytic neoplasia, both in non-acral cutaneous melanomas and nevi (CM vs CN), as well as matched acral melanoma and nevi (AM vs AN). Receiver operating characteristic area under the curve (AUC) of Ensemble models trained using these microRNA ratios demonstrated AUCs of 0.88-0.90 across these melanoma subtypes, suggesting the potential utility of these ratios as a biomarker of malignancy in melanocytic neoplasia.

first: Chris

last: Stubben

first: Eleonora

last: Marcacci

first: Caroline

last: Stone

first: Michael

last: Birdsall

first: Ken

last: Boucher

first: Doug

last: Grossman

last: Judson-Torres

first: Scott

last: Florell


title: NGS5413 - METTL3 and METTL14 KOs in hMDMs

summary: human primay macrophages (hMDMs) were genetically perturbed for METTL3 and stimulated with IFNg for 18h

overall_design: human primay macrophages (hMDMs) were genetically perturbed for METTL3 across 5 individual healthy donors and stimulated with IFNg for 18h, total RNA was extracted and sequenced


title: Astrocyte allocation during brain development is controlled by Tcf4-mediated fate restriction

first: Yunli

last: Xie

address: 131 Dong`an Road

department: Institutes of Brain Science

institute: Fudan University

summary: Astrocytes within specific brain regions contribute uniquely to regional circuits for higher-order brain function through interactions with local neurons. The regional diversification of astrocytes is dictated by their embryonic origin, yet the mechanisms governing their regional allocation remain unknown. Here we show that allocation of astrocytes to specific brain regions requires the transcription factor 4 (Tcf4) mediated fate restriction during brain development. Loss of Tcf4 in ventral telencephalic neural progenitors alters the fate of oligodendrocyte precursors to transient intermediate astrocyte precursor cells, resulting in mislocated astrocytes in the dorsal neocortex. These ectopic astrocytes originated from the ventral telencephalon engage with neurons and acquire features reminiscent of local neocortical astrocytes. Furthermore, Tcf4 functions as a suppressor of astrocyte fate during differentiation of oligodendrocyte precursors, thereby restricting the fate to oligodendrocyte lineage. Our study reveals that fate restriction governs regional astrocyte allocation, contributing to astrocyte diversification across brain regions.

overall_design: For all expresiments, cells from the Nkx2.1 lineage in the neocortex from control and Tcf4 cKO mice  were collected by FACS sorting. After cell variablity checking,  single cell sequencing library were prepared using 10x Genomics Chromium Single Cell 3`v3 assays, or ATACseq and cut and tag were performed.

first: Yandong

last: Li


title: CRISPR-Enabled Profiling of Enhancer RNA-Producing Super-Enhancers in Triple-Negative Breast Cancer Identifies Drivers of Tumor Progression [RNA-Seq]

city: Chapel Hill

first: Hector

last: Franco

address: 120 Mason Farm Rd

department: Genetics

institute: UNC Lineberger Cancer Center

summary: Triple-negative breast cancer (TNBC) is the most therapeutically recalcitrant form of breast cancer. Pioneering genomic studies have identified the repertoire of expressed genes in the distinct molecular subtypes of breast cancer and have been instrumental in providing diagnostic, prognostic, and therapeutic guidance. Yet breast cancer remains the second leading cause of cancer mortality in women. This is due, in part, to the paucity of targeted therapies for triple-negative breast cancer (TNBC). We posit that a systematic analysis of regulatory elements that extend beyond protein coding genes will lead to novel avenues for therapeutic intervention. To this end, we analyzed the regulatory mechanisms of TNBC-specific transcriptional enhancers together with their non-coding enhancer RNA (eRNA) transcripts. We systematically probed the functions of the top 30 eRNA-producing super-enhancers using a high-throughput CRISPR-interference (dCas9-KRAB) assay coupled to RNA-seq that enabled unbiased detection of target genes genome-wide. Utilizing Hi-C, we constructed 3D chromatin interaction maps to annotate direct target genes for each super-enhancer and highlight their proclivity for genes that portend worse clinical outcomes in TNBC patients.  To illustrate the utility of this dataset, we selected a novel super-enhancer controlling the nearby PODXL gene and demonstrate that deletion of the super-enhancer (via Cas9 deletion) or specific degradation of its enhancer RNAs (via Cas13 RNA cleavage) leads to profound effects on target gene expression, cell proliferation, and migration. Furthermore, complete knockout of the PODXL super-enhancer leads to a significant decrease in tumor growth and metastatic potential in mouse xenograft experiments. Collectively, this work examines several fundamental questions about how regulatory information encoded into eRNA-producing super-enhancers drives gene expression networks that underlie the biology of triple-negative breast cancer.

overall_design: We used dCas9-KRAB expressing triple-negative breast cancer  (TNBC) cells (MDA-MB-436) to perform a CRISPR interference screen that epigenetically repressed each of 30 TNBC-specific super-enhancers individually per sample condition. This was followed by high-throughput RNA-sequencing to determine the downstream gene expression changes associated with epigenetic perturbation.

last: Lewis


title: Induction of senescence upon loss of the Ash2l subunit of histone H3K4 methyltransferase complexes (ChIP-Seq)

city: Aachen

first: Ivan

middle: G.

last: Costa

state: NRW

address: Pauwelsstr. 19

department: RWTH Aachen Medical Faculty

institute: Institute for Computational Genomics

summary: Post-translational modifications of core histones participate in controlling the expression of genes. Methylation of lysine 4 of histone H3 (H3K4) is associated with open chromatin and gene transcription. This histone mark is catalyzed by type 2 lysine methyltransferase (KMT2) complexes. In mammals, these consist of one of 6 different KMT2 enzymes, each associated with 4 core subunits, WDR5, RBBP5, ASH2L and DPY30, which are necessary for catalytic activity. The knockout of Ash2l is embryonically lethal in mice as well as in adult animals when hepatocytes or hematopoietic cells are targeted. To expand on the mechanistic understanding of Ash2l, we have used mouse embryo fibroblasts (MEFs). The knockout of Ash2l resulted in the downregulation of H3K4me3 at a large number of promoters, preferentially those associated with CpG islands, accompanied with broad repression of gene expression. The consequence in MEFs was induction of senescence concomitant with a set of down-regulated signature genes. Thus, although the loss of Ash2l in MEFs has broad and complex consequences on the transcriptional program, the biological consequences were distinct, culminating in senescence.

overall_design: Two independently generated mouse embryo fibroblast lines (from two different litters) with floxed exon 4 of Ash2l (KO1 and KO2). Two additional lines  with wt Ash2l (WT1 and WT2). All express Cre-ER and were immortalized using shArf. Cells were treated  -/+ 4-hydroxytamoxifen for 5 days.ChIP was performed with antibodies for H3K4me1 and H3K4me3.

first: Agnieszka

last: Bochyńska

first: Chao-Chung

last: Kuo

first: Alexander

middle: T

last: Stenzel

first: Roksaneh

last: Sayadi Boroujeni

first: Weili

last: Liang

first: Bhanu

last: Natarajan

first: Benjamin

middle: A

last: Garcia

first: Juliane

last: Lüscher-Firzlaff

first: Bernhard

last: Lüscher


title: Neoself-antigens are the primary target for autoreactive T cells in human lupus

status: Public on Aug 13 2024

city: Suita

first: Daisuke

last: Motooka

state: Osaka

address: 3-1, Yamadaoka

country: Japan

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

summary: Major histocompatibility complex class II (MHC-II) is the most significant genetic risk factor for systemic lupus erythematosus (SLE), but the nature of the self-antigens that trigger autoimmunity remains unclear. Unusual self-antigens, termed neoself-antigens, are presented on MHC-II in the absence of the invariant chain essential for peptide presentation. Here, we demonstrate that neoself-antigens are the primary target for autoreactive T cells clonally expanded in SLE. When neoself-antigen presentation was induced by deleting the invariant chain in adult mice, neoself-reactive T cells were clonally expanded, leading to the development of lupus-like disease. Furthermore, we found that neoself-reactive CD4+ T cells were significantly expanded in SLE patients. A high frequency of Epstein-Barr virus reactivation is a risk factor for SLE. Neoself-reactive lupus T cells were activated by Epstein-Barr-virus-reactivated cells through downregulation of the invariant chain. Together, our findings imply that neoself-antigen presentation by MHC-II plays a crucial role in the pathogenesis of SLE.

overall_design:
We performed scRNA sequencing to understand the effect of MHC-class II dysfunction on autoimmunity.
details of the HTO antibodies
HTO: barcode sequence: hashtagged sample (condition/treatment)
TotalSeq-C0301 anti-mouse Hashtag 1 Antibody: ACCCACCAGTAAGAC: mouse splenic CD4 T cells isolated by magnetic beads, replicate 1
TotalSeq-C0304 anti-mouse Hashtag 4 Antibody: AAAGCATTCTTCACG: mouse splenic CD4 T cells isolated by magnetic beads, replicate 2
TotalSeq-C0306 anti-mouse Hashtag 6 Antibody: TATGCTGCCACGGTA: mouse splenic CD4 T cells isolated by magnetic beads, replicate 3

first: Shunsuke

last: Mori

last: Okuzaki

first: Hisashi

last: Arase


title: Dynamic upregulation of retinoic acid RA signal in the early postnatal murine heart promotes cardiomyocyte cell cycle exit and maturation

city: Showa-ku, Nagoya

first: Shingo

last: Narita

state: Aichi

address: 65 Tsurumai-cho

department: Cardiology

institute: Nagoya University Graduate School of Medicine

summary: Retinoic acid (RA), the major active metabolite of vitamin A, is essential for organogenesis, homeostasis, and tissue morphogenesis. Despite the importance of RA signaling in embryo heart development, little is known about its function in the early postnatal period. To analyze the function of RA signaling during perinatal heart development, we investigated the mRNA expression levels of multiple retinoid metabolic genes at various perinatal stages using RNA-seq analysis.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for each stage of ventricles from P3, P6, P12 and P24 mice.

first: Yusuke

last: Fujikawa

first: Katsuhiro

last: Kato

first: Kazumasa

last: Unno


title: Dynamic upregulation of retinoic acid RA signal in the early postnatal murine heart promotes cardiomyocyte cell cycle exit and maturation [PRVCMs_Cont-ATRA]

summary: Retinoic acid (RA), the major active metabolite of vitamin A, is essential for organogenesis, homeostasis, and tissue morphogenesis. Despite the importance of RA signaling in embryo heart development, little is known about its function in the early postnatal period. For a comprehensive transcriptional evaluation of the effect of RA signaling on neonatal cardiomyocyte cell cycle arrest and maturation, we performed RNA-seq analysis of cultured perinatal cardiomyocytes treated with all-trans retinoic acid (ATRA) in vitro.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for culture prenatal rat ventricular cardiomyocytes (PRVCMs) treated with 10 µM ATRA or Dimethyl sulfoxide (DMSO) as vehicle for 48 h.


title: Localization of HIRA in PML bodies is tightly linked SASP expression

city: La Jolla

first: Xue

last: Lei

address: 10901 N Torrey Pines Rd

institute: SBP medical discovery institute

summary: Cellular senescence, a stable proliferation arrest caused by a range of cellular stresses, is a bona fide cause of cell and tissue aging. As well as proliferation arrest, cell senescence is associated with a potent pro-inflammatory phenotype, the senescence-associated secretory phenotype (SASP). Given that SASP is regulated at various levels, gaining a comprehensive understanding of its regulation is crucial. This understanding can pave the way for potential therapeutic approaches aimed at modulating SASP, which could promote healthy aging and alleviate the burden of senescence-associated diseases and degeneration. We show here that both the Promyelocytic Leukemia (PML) protein and HIRA histone chaperone are required for SASP expression in senescent cells. PML protein is the key organizer of PML nuclear bodies, nuclear features up to 1mM in diameter, containing many proteins and previously implicated in diverse cellular processes, including control of cell senescence and cellular intrinsic anti-viral immunity. HIRA is a histone chaperone best known for its ability to incorporate histone variant H3.3 into nuclear chromatin in a DNA replication-independent manner, including in non-proliferating senescent cells. HIRA localizes to PML nuclear bodies in senescent cells. We show that both HIRA and PML are required for activation of NF-kB and SASP. We found that HIRA regulates cytoplasmic NF-kB signaling in senescent cells through CCF-cGAS-STING-TBK1 pathway and interaction with autophagy cargo receptor Sequestosome-1 (p62/SQSTM1).

overall_design: mRNA profiling in senescent fibroblasts after ectopic expression of a mutant form of HIRA known as delHIRA(520-1017)

first: Nirmalya

last: Dasgupta

first: Christina

last: Shi

first: Peter

last: Adams


title: ZC3H13 and WTAP KO in hMDMs multi stimuli(NGS5421)

first: Sandra

last: Melo

summary: Here, we revealed an immunosuppressive role of m6A mediated by ZC3H13 and WTAP on TNF-driven inflammatory phenotype in human macrophages. In the following experiment we would like to investigate if the negative regulatory role of m6A is consistent across multiple stimuli using innate ligands (LPS, R848 and polyIC). We generated ZC3H13 KOs, WTAP KOs and NTC (WT) in monocyte-derived macrophages from 4 individual human donors and stimulated cells over night with LPS (5ng/ml) , R848 (50ng/ml) , polyIC (50ng/ml) and unstimulated cells as control. This yielded a total of 48 total samples.


title: mRNA  sequence of renal cortex from Type 2 diabetic (db/db)mice and non-diabetic (db/m) mice

city: Changzhou

first: Chao

last: Tu

state: Jiangsu

address: 185 Juqian Road

institute: The Third Affiliated Hospital of Soochow University

summary: We investigated the gene expression profiles of mRNA isolated from renal cortex in aged, 17 week old  db/db mice and db/m mice.

overall_design: In order to investigate the consequences of hyperglycemia on the pathogenesis and progression of diabetic nephropathy from 4 diabetic and 4 non-diabetic mice. Renal cortex were isolated and RNA purified for RNA-Seq analysis on the Illumina Novaseq 6000. The goal of the project was to generate comprehensive list of mRNA genes differentially regulated between the two conditions in order to identify novel targets for further study.

first: CHAO

last: TU

first: LAN

last: WEI

first: QIANRU

last: TAO


title: Rapid degradation of DHX36 revealing its transcriptional role by interacting with G-quadruplex

city: Wuhan

first: Ziang

last: Lu

address: Wuhan University, No.299, Bayi Road, Wuchang District

institute: Wuhan University

summary: More evident supports G-quadruplex are involved in transcription. Recent research revealed that DHX36 preferentially resolves G-quadruplex (G4) structure over the canonical G4, raising the possibility that DHX36 could bind and resolve G4 structures to regulate gene transcription. However, the hypothesis has yet been validated systematically in vivo.In this study, we investigated the role of DHX36 interacting with G4s in transcription. Firstly, we performed CUT&TAG to map the binding sites in chromatin of MCF7. Next, we utilized nascent RNA-seq and AID protein degradation system to identify the direct gene targets by transcriptional regulation of DHX36 and found these sites are enriched with G4 structure. We picked three G4 sequences in oncogene from these sites and found DHX36 binds and resolves these G4 structures in vitro, suggesting DHX36 may be recruited to these promoter G4 sites and regulate gene transcription by resolving G4 structure.

overall_design: Cleavage Under Target & Tagmentation (CUT&TAG) for DHX36 in MCF7 cells, and nascent RNA-seq acrylonitrile-mediated uridine-to-cytidine conversion (AMUC-seq) with or without depletion of DHX36 by Auxin Inducible Degron (AID) assay.

first: Jinglei

last: Xu

first: Yuqi

last: Chen

first: Xiang

last: Zhou


first: Daisuke

last: Motooka

overall_design: We performed scRNA sequencing to understand the effect of MHC-class II dysfunction on autoimmunity.

first: Shunsuke

last: Mori

last: Okuzaki

first: Hisashi

last: Arase


title: Sorafenib induces cachexia by impeding transcriptional signaling of the SET1/MLL complex on muscle-specific genes

status: Public on Aug 12 2024

city: Hannover

first: Bushra

last: Khan

state: Lower Saxony

address: Carl-Neuberg-Straße 1

department: Institute of Molecular and Cell Physiology

institute: Hannover Medical School

summary: Adverse effects of chemotherapies can outweigh the benefits in cancer patients. Various chemotherapeutics are linked to muscle wasting or cachexia, drastically reducing the survival rate of cancer patients. Insights into the molecular basis of chemotherapy-induced cachexia is an unmet need to improve treatment strategies. Here, we demonstrated that Sorafenib- tyrosine kinase inhibitor class of chemotherapeutic agents- induced cachexia. Sorafenib-treated muscle cells show sarcomere disarray, impaired sarcoplasmic reticulum-mediated calcium handling, loss of contractile ability, and reduced mitochondrial oxidative respiratory capacity. To identify the molecular mechanisms underlying Sorafenib-induced cachexia, we performed whole-genome transcriptome profiling or RNA sequencing of muscle cells in response to Sorafenib treatment, compared to DMSO-treated (control) cells. We identified a total of 3030 genes that were downregulated and 2128 genes that were upregulated in Sorafenib-treated cells, as compared to the control. Gene ontology analyses revealed that Sorafenib led to transcriptional downregulation of genes coding for proteins of the sarcomere, sarcoplasmic reticulum, and proteins responsible for muscle contraction, sarcomere organization, and calcium ion handling. Additionally, genes associated with mitochondrial oxidative phosphorylation or electron transport chain were significantly downregulated. In summary, Sorafenib deregulates the transcriptional processes of protein-coding genes associated with sarcomere assembly, calcium ion homeostasis, and mitochondrial oxidative respiration, ultimately leading to impaired muscle function.

overall_design: To investigate the effects of Sorafenib treatment on gene expression in muscle cells, we treated murine C2C12-derived myotubes/muscle cells wiht 20 µM Sorafenib for 24 h. Muscle cells treated with 0.1 % DMSO for 24 h were used as the control. After 24 h treatment, total RNA was isolated from the cells. Three independent experiments were performed to generate three samples of DMSO (control)- and Sorafenib-treated muscle cells (N = 3 for both conditions). Global gene expression of Sorafenib-treated cells was comapred to the control (DMSO-treated) cells.

first: Arnab

last: Nayak

first: Oliver

last: Dittrich-Breiholz


title: Spatially exploring RNA biology in archival formalin-fixed paraffin-embedded tissues

city: New Haven

first: Rong

last: Fan

state: Connecticut

address: 55 Prospect St.

department: Biomedical Engineering

institute: Yale University

summary: The capability to spatially explore RNA biology in formalin-fixed paraffin-embedded (FFPE) tissues holds transformative potential for histopathology research. Here, we present pathology-compatible deterministic barcoding in tissue (Patho-DBiT) by combining in situ polyadenylation and computational innovation for spatial whole transcriptome sequencing, tailored to probe the diverse RNA species in clinically archived FFPE samples. It permits spatial co-profiling of gene expression and RNA processing, unveiling region-specific splicing isoforms, and high-sensitivity transcriptomic mapping of clinical tumor FFPE tissues stored for five years. Furthermore, genome-wide single nucleotide RNA variants can be captured to distinguish malignant subclones from non-malignant cells in human lymphomas. Patho-DBiT also maps microRNA regulatory networks and RNA splicing dynamics, decoding their roles in spatial tumorigenesis. Cellular-level Patho-DBiT dissects the spatiotemporal cellular dynamics driving tumor clonal architecture and progression. Patho-DBiT stands poised as a valuable platform to unravel rich RNA biology in FFPE tissues to aid in clinical pathology evaluation.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Patho-DBiT initiates with tissue section deparaffinization and heat-induced crosslink reversal. After tissue permeabilization, enzymatic in situ polyadenylation enables detection of various RNAs, followed by complementary DNA (cDNA) strand synthesis. Spatial barcoding is then achieved using microfluidic chips featuring 50 microchannels. These channels sequentially deliver horizontal and perpendicular barcodes, creating a unique barcode array. Post-imaging, the tissue undergoes digestion to extract barcoded cDNA to perform template switch, PCR amplification, rRNA removal, library preparation, and sequencing.

first: Zhiliang

last: Bai


title: CRISPR Screening of Transcribed Super-Enhancers Identifies Drivers of Triple-Negative Breast Cancer Progression

first: Hector

last: Franco

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

overall_design: Refer to individual Series


title: Baseline Immune State and T cell Clonal Kinetics are Associated with Response to CAR-T Therapy in Large B-cell Lymphoma

city: New York

first: Isabella

last: Grabski

address: 101 Avenue of the Americas

institute: New York Genome Center

summary: Engineered cellular therapy with CD19-targeting chimeric antigen receptor T-cells (CAR-T) has revolutionized outcomes for patients with relapsed/refractory Large B-Cell Lymphoma (LBCL), but the cellular and molecular features associated with response remain largely unresolved. We analyzed serial peripheral blood samples ranging from day of apheresis (day -28/baseline) to 28 days after CAR-T infusion from 50 patients with LBCL treated with axicabtagene ciloleucel (axi-cel) by integrating single cell RNA and TCR sequencing (scRNA-seq/scTCR-seq), flow cytometry, and mass cytometry (CyTOF) to characterize features associated with response to CAR-T. Pretreatment patient characteristics associated with response included presence of B cells and increased lymphocyte-to-monocyte ratio (ALC/AMC). Infusion products from responders were enriched for clonally expanded, highly activated CD8+ T cells. We expanded these observations to 99 patients from the ZUMA-1 cohort and identified a subset of patients with elevated baseline B cells, 80% of whom were complete responders. We integrated B cell proportion and ALC/AMC into a two-factor predictive model and applied this model to the ZUMA-1 cohort. Estimated progression free survival (PFS) at 1 year in patients meeting one or both criteria was 65% versus 31% for patients meeting neither criterion. Our results suggest that patients’ immunologic state at baseline affects likelihood of response to CAR-T through both modulation of the T cell apheresis product composition and promoting a more favorable circulating immune compartment prior to therapy. These baseline immunologic features, measured readily in the clinical setting prior to CAR-T, can be applied to predict response to therapy.

overall_design:
PBMC samples were collected at day of apheresis (~day -28) and at days +7 and +14 post-treatment. Infusion products were collected by rinsing the infusion bag. Samples were analyzed with CITE-seq and scTCR-seq.

***Submitters state that raw data is available through dbGaP (phs002922.v2)***


title: Interleukin-11 causes alveolar type 2 cell dysfunction and prevents alveolar regeneration

city: Singapore

first: Benjamin

last: Ng

address: 8 College Road

country: Singapore

institute: Duke-National University of Singapore Medical School


title: Host-microbe interactions and changes during intestinal epithelial cell death-dependent dysbiosis

city: Gent

first: Christian

last: Maueroeder

state: Flanders

address: Technologiepark-Zwijnaarde

department: IRC

country: Belgium

institute: VIB


title: E. coli transcription in response to apoptotic metabolites [MeMix]

summary: E. coli was grown in the presence or absence of critical metabolites derived from apoptotic mammalian epithelial cells.

overall_design: E. coli was grown in media (control), the presence of 6 apoptotic metabolites (MM6), or the presence of 3 apoptotic metabolites (MM3). The transcriptional profile was assessed during mid log growth phase, prior to the onset of any growth phenotypes across conditions. E. coli was grown aerobically in vitro.

first: C

middle: J

last: Anderson

first: J

last: Burgoa Cardas

first: L

last: Boeckaerts

first: K

middle: S

last: Ravichandran


title: E. coli response to apoptotic epithelial cells [Doxo]

summary: The goal was to determine the chemotherapy-induced, mammalian cell death-dependent transcriptional response in a human commensal strain of E. coli. Murine intestinal epithelial cells were used to induce chemotherapy-driven cell death, and a human commensal strain of E. coli was used as the 'recipient' bacteria.

overall_design: Mammalian epithelial cells were induced to die via apoptosis using the chemotherapeutic doxorubicin. Following death, cell free supernatants were used as growth medium for E. coli. Transcriptional profiles were compared to media controls and cell free supernatants where mammalian cell death had been inhibited using the pan caspase inhibitor QVD.


title: Interleukin-11 causes alveolar type 2 cell dysfunction and prevents alveolar regeneration [RNA-seq]

summary: Following lung injury, alveolar regeneration is characterized by the transformation of alveolar type 2 (AT2) cells, via a transitional KRT8+ state, into alveolar type 1 (AT1) cells. In lung disease, dysfunctional intermediate cells accumulate, AT1 cells are diminished and fibrosis occurs. Using single cell RNA sequencing datasets of human interstitial lung disease, we found that interleukin-11 (IL11) is specifically expressed in aberrant KRT8 expressing KRT5-/KRT17+ and basaloid cells. Stimulation of AT2 cells with IL11 or TGFβ1 caused EMT, induced KRT8+ and stalled AT1 differentiation, with TGFβ1 effects being IL11 dependent. In bleomycin injured mouse lung, IL11 was increased in AT2-derived KRT8+ cells and deletion of Il11ra1 in lineage labeled AT2 cells reduced KRT8+ expression, enhanced AT1 differentiation and promoted alveolar regeneration, which was replicated in therapeutic studies using anti-IL11. These data show that IL11 maintains AT2 cells in a dysfunctional transitional state, impairs AT1 differentiation and blocks alveolar regeneration across species.

overall_design: Differential gene expression analysis of RNA-seq data for human pulmonary alveolar epithelial cells (HPAEpiC ) with or without TGFβ1 or IL11 stimulation

first: Kevin

last: Huang

first: Chee

last: Pua

first: Sivakumar

last: Viswanathan

first: Wei-Wen

last: Lim

first: Fathima

middle: F

last: Kuthubudeen

first: Yu-Ning

last: Liu

first: An

last: Hii

last: George

first: Anissa

last: Widjaja

first: Enrico

last: Petretto

first: Stuart

last: Cook


title: Interleukin-11 causes alveolar type 2 cell dysfunction and prevents alveolar regeneration [scRNA-Seq]

last: Ng

summary: Following lung injury, alveolar regeneration is characterized by the transformation of alveolar type 2 (AT2) cells, via a transitional KRT8+ state, into alveolar type 1 (AT1) cells. In lung disease, dysfunctional intermediate cells accumulate, AT2 and AT1 cells are diminished and fibrosis occurs. Using single cell RNA sequencing (scRNA-seq) datasets of human interstitial lung disease, we found that Interleukin-11 (IL11) is specifically expressed in aberrant KRT8 expressing KRT5-/KRT17+ epithelial cells and basaloid cells. Stimulation of AT2 cells and distal airway epithelial cells with IL11 or TGFβ1 caused epithelial-to-mesenchymal transition (EMT), induced extracellular matrix (ECM) production, increased KRT8 expression and stalled AT2-to-AT1 differentiation, with TGFβ1 effects being partially IL11 dependent. In bleomycin injured mouse lung, IL11 was increased in AT2-derived Krt8+ transitional cells and deletion of Il11ra1 in AT2 lineage cells prevented the accumulation of Krt8+ transitional cells, enhanced AT1 differentiation and promoted alveolar regeneration, which was replicated in therapeutic studies using anti-IL11 antibodies. scRNA-seq analysis of lung epithelial cells from mice with deletion of Il11ra1 in AT2 lineage cells further identified the importance of IL11 signaling for the potentiation and polarization of a disease-causing, ECM producing KRT8+ transitional cells that contributes to pathological lung remodeling. Overall, our data show that IL11 maintains damaged AT2 cells in a transitional state, impairs reparative AT1 differentiation and impairs endogenous alveolar regeneration to cause fibrotic lung disease.

overall_design: Mouse lung epithelial single cell suspensions of uninjured or bleomycin injured Sftpc-Cre;Il11ra1fl/fl or Il11ra1+/+ mice were enriched by FACS sorting for live CD45- CD31- EpCAM+ cells. Single cell capturing was performed using the BD Rhapsody™ Express Single-Cell Analysis System (BD Biosciences). Libraries were generated using BD Rhapsody Whole Transcriptome Analysis (WTA)


title: CRISPR-Enabled Profiling of Enhancer RNA-Producing Super-Enhancers in Triple-Negative Breast Cancer Identifies Drivers of Tumor Progression [Hi-C]

first: Hector

last: Franco

summary: Triple-negative breast cancer (TNBC) is the most therapeutically recalcitrant form of breast cancer. Pioneering genomic studies have identified the repertoire of expressed genes in the distinct molecular subtypes of breast cancer and have been instrumental in providing diagnostic, prognostic, and therapeutic guidance. Yet breast cancer remains the second leading cause of cancer mortality in women. This is due, in part, to the paucity of targeted therapies for triple-negative breast cancer (TNBC). We posit that a systematic analysis of regulatory elements that extend beyond protein coding genes will lead to novel avenues for therapeutic intervention. To this end, we analyzed the regulatory mechanisms of TNBC-specific transcriptional enhancers together with their non-coding enhancer RNA (eRNA) transcripts. We systematically probed the functions of the top 30 eRNA-producing super-enhancers using a high-throughput CRISPR-interference (dCas9-KRAB) assay coupled to RNA-seq that enabled unbiased detection of target genes genome-wide. Utilizing Hi-C, we constructed 3D chromatin interaction maps to annotate direct target genes for each super-enhancer and highlight their proclivity for genes that portend worse clinical outcomes in TNBC patients.  To illustrate the utility of this dataset, we selected a novel super-enhancer controlling the nearby PODXL gene and demonstrate that deletion of the super-enhancer (via Cas9 deletion) or specific degradation of its enhancer RNAs (via Cas13 RNA cleavage) leads to profound effects on target gene expression, cell proliferation, and migration. Furthermore, complete knockout of the PODXL super-enhancer leads to a significant decrease in tumor growth and metastatic potential in mouse xenograft experiments. Collectively, this work examines several fundamental questions about how regulatory information encoded into eRNA-producing super-enhancers drives gene expression networks that underlie the biology of triple-negative breast cancer.

overall_design: We identified several super-enhancers and their target genes through a CRISPR interference-based screen linked with RNA-seq. Using these results, we identified direct contact relationships and confirmed this using a Hi-C seq experiment to confirm physical interaction of these candidates in wildtype cells.

first: Michael

last: Lewis


title: Effect of OPA1 loss on expression of genes associated with cellular susceptibility to ferroptosis

city: Bronx

first: Felix

last: Liang

state: New York

address: 1300 Morris Park Ave, Forchheimer G01

institute: Albert Einstein College of Medicine

summary: OPA1 is a dynamin-related GTPase that plays a critial role in governing mitochondrial fusion, cristae structure, and energetics. Here we found that OPA1 functions as a postive regulator of a nonapoptotic form of cell death termed ferroptosis. Although we determined that the OPA1 GTPase activity is required to drive ferroptosis, the fusogenic activity of OPA1 is dispensable for ferroptosis execution. Mechanistically, our studies show that OPA1 promotes ferroptosis through distinct mechanisms involving the generation of mitochondrial reactive oxygen species and suppression of the integrated stress response.

overall_design: To investigate the effect of OPA1 loss on the transcriptome, we subjected Opa1-/- MEFs and Opa1-/- MEFs reconstituted with OPA1 to RNA sequencing. Comparative gene expression profiling analysis was performed on data from RNA seq.


title: The profound effects of solid tumour-induced B cell dysregulation [PyMT-N tumor]

city: Houston

first: Xiaoxin

last: Hao

address: 1 Baylor Plaza-Alkek N1130

department: Lester and Sue Smith Breast Center

institute: Baylor College of Medicine

summary: Solid tumours evade immunosurveillance through a plethora of mechanisms. It remains elusive how the development and functions of B cells vary in different tumour contexts. Here, we observed distinct patterns of B cell abnormality in triple-negative breast cancer and termed these patterns “tumour-induced B cell abnormality” type (TiBA). TiBA results from disrupted early B cell development in the bone marrow, which is associated with abnormal myelopoiesis. This study highlights the heterogeneity of tumour-induced systemic changes on B cells and lays foundation for the precision treatment of patients with different B cell abnormalities.

overall_design: We performed scRNA-seq on the TiBA-2 tumor (PyMT-N)-infiltrating immune cells.

first: Xiang

last: Zhang


title: Central regulation of feeding and body weight by ciliary GPR75

city: Dallas

first: Zhao

state: Texas

address: 5323 Harry Hines Blvd.

department: Center for the Genetics of Host Defense

institute: The University of Texas Southwestern Medical Center

summary: Variants of the G protein-coupled receptor 75 (GPR75) are associated with lower BMI in large-scale human exome sequencing studies. However, how GPR75 regulates body weight remains poorly understood. Using random germline mutagenesis in mice, we identified a missense allele (Thinner) of Gpr75 that resulted in a lean phenotype and verified the decreased body weight and fat weight in Gpr75 knockout (Gpr75–/–) mice. Gpr75–/– mice displayed reduced food intake under a high-fat diet (HFD), and pair-feeding normalized their body weight. The endogenous GPR75 protein was exclusively expressed in the brains of 3xFlag tagged Gpr75 knock-in (3xFlag-Gpr75) mice, with consistent expression across different brain regions. GPR75 interacted with Gαq to activate various signaling pathways after HFD feeding. Additionally, GPR75 was localized in the primary cilia of hypothalamic cells, whereas the Thinner mutation (L144P) and human GPR75 variants with lower BMI failed to localize in the cilia. Loss of GPR75 selectively inhibited weight gain in HFD-fed mice but failed to suppress the development of obesity in Leptin ob mice and Adenylate cyclase 3 (Adcy3) mutant mice on a chow diet. Our data reveal that GPR75 is a ciliary protein expressed in the brain and plays an important role in regulating food intake.

overall_design: To gain insights into the role of GPR75 in the central regulation of feeding, we performed a transcriptomics analysis (RNA-seq) of the hypothalamus from wild-type (WT) and Gpr75-/- (Gpr75 KO) mice under a chow diet or 2 weeks of high-fat diet (HFD) since 6 weeks of age. There are four groups in total (chow_wt, chow_75ko, hfd_wt, hfd_75ko) with three age-matched male mice were used in each group, resulting a total of 12 samples.

first: Yiao

last: Jiang

first: Yu

last: Xun


title: The interplay between RNA sequestration in P-bodies and chromatin architecture sustains myeloid leukemia [CUT&RUN]

status: Public on Aug 11 2024

city: Badalona

first: Laureano

last: Tomás-Daza

address: Ctra de Can Ruti, Camí de les Escoles s/n

institute: Josep Carreras Leukaemia Research Institute

summary: To investigate chromatin remodeling in AML cells following loss of P-bodies, we knocked down DDX6 in MOLM-13 cells.

overall_design: CUT&RUN of histones modifications H3K27ac, H3K4me1 and IgG of MOLM-13 cells

first: Ainoa

last: Riverola

first: Biola

middle: M

last: Javierre

first: José

last: Sardina

first: Anna

middle: V

last: López


title: Histone chaperone HIRA and PML are required for SASP expression but not for proliferation arrest

first: Xue

last: Lei

state: CA

summary: Cellular senescence, a stable proliferation arrest caused by a range of cellular stresses, is a bona fide cause of cell and tissue aging. As well as proliferation arrest, cell senescence is associated with a potent pro-inflammatory phenotype, the senescence-associated secretory phenotype (SASP). Given that SASP is regulated at various levels, gaining a comprehensive understanding of its regulation is crucial. This understanding can pave the way for potential therapeutic approaches aimed at modulating SASP, which could promote healthy aging and alleviate the burden of senescence-associated diseases and degeneration. We show here that both the Promyelocytic Leukemia (PML) protein and HIRA histone chaperone are required for SASP expression in senescent cells. PML protein is the key organizer of PML nuclear bodies, nuclear features up to 1mM in diameter, containing many proteins and previously implicated in diverse cellular processes, including control of cell senescence and cellular intrinsic anti-viral immunity. HIRA is a histone chaperone best known for its ability to incorporate histone variant H3.3 into nuclear chromatin in a DNA replication-independent manner, including in non-proliferating senescent cells. HIRA localizes to PML nuclear bodies in senescent cells. We show that both HIRA and PML are required for activation of NF-kB and SASP. We found that HIRA regulates cytoplasmic NF-kB signaling in senescent cells through CCF-cGAS-STING-TBK1 pathway and interaction with autophagy cargo receptor Sequestosome-1 (p62/SQSTM1).

overall_design: mRNA profiling in senescent fibroblasts after knocking-down HIRA and PML

first: Nirmalya

last: Dasgupta

first: Christina

last: Shi

first: Peter

last: Adams


title: Effect of misregulation of mitochondrial 6mA on gene expression in C. elegans

city: Brisbane

first: Steven

last: Zuryn

state: Queensland

address: QBI Building 79, University of Queensland

department: Queensland Brain Institute

country: Australia

institute: The University of Queensland

summary: In virtually all eukaryotes, the mitochondrial genome (mitochondrial DNA, mtDNA) encodes proteins necessary for oxidative phosphorylation (OXPHOS) and the RNA machinery required for their synthesis inside the mitochondria. Appropriate regulation of mtDNA copy number and expression is essential for ensuring the correct stoichiometric formation of OXPHOS complexes assembled from both nuclear- and mtDNA-encoded subunits. The mechanisms of mtDNA regulation are not completely understood but are essential to organismal viability and lifespan. Here, using multiple approaches, we identify the presence of N6-methylation of adenosine (6mA) on the mtDNA of diverse animal and plant species. Importantly, we also demonstrate that this modification is regulated in C. elegans by the DNA methyltransferase DAMT-1, and DNA demethylase ALKB-1, which localize to mitochondria. Misregulation of mtDNA 6mA through targeted overexpression of these enzymatic activities inappropriately alters mtDNA copy number and transcript levels, impairing OXPHOS function and producing increased oxidative stress, as well as a shortened lifespan. Compounding defects in mtDNA regulation, reductions in mtDNA 6mA methylation promote the propagation of a deleterious mitochondrial genome across generations. Together, these results reveal that mtDNA 6mA is highly conserved among eukaryotes and regulates lifespan by influencing mtDNA copy number, expression, and heritable mutation levels in vivo.

overall_design:
To investigate the effects of 6mA methylation of the mtDNA, we generated transgenic worms overexpressing mtDNA 6mA modifiers, establishing 6mA hypo- and hypermethylation strains.
We extracted total RNA from mtDNA 6mA hypo- and hypermethylation animals in a glp-4(bn2) background that do not develop a germline.
Comparative gene expression analysis of mRNA-seq data for mtDNA 6mA hypo- and hypermethylation animals and a corresponding control without altered mtDNA 6mA levels.

first: Anne

last: Hahn

first: Grace

middle: C

last: Hung

first: Arnaud

last: Ahier

first: Chuan-Yang

last: Dai

first: Ina

last: Kirmes

first: Brian

last: Forde

first: Daniel

last: Campbell

first: Rachel

last: Lee

first: Josiah

last: Sucic

first: Tessa

last: Onraet


title: Blockade of LAG-3 and PD-1 leads to co-expression of cytotoxic and exhaustion gene modules in CD8+ T cells to promote antitumor immunity

city: Pittsburgh

first: Anthony

middle: Richard

last: Cillo

state: PA

address: The Assembly, Room 4070C Bay 10, 5051 Centre Ave

department: Immunology

institute: University of Pittsburgh

summary: Relatlimab (rela; anti-LAG-3) plus nivolumab (nivo; anti-PD-1) is safe and effective for treatment of advanced melanoma. We designed a trial (NCT03743766) where advanced melanoma patients received rela, nivo, or rela+nivo to interrogate the immunologic mechanisms of rela+nivo. Analysis of biospecimens from this ongoing trial demonstrated that rela+nivo led to enhanced capacity for CD8+ T cell receptor signaling and altered CD8+ T cell differentiation, leading to heightened cytotoxicity despite the retention of an exhaustion profile. Co-expression of cytotoxic and exhaustion signatures was driven by PRDM1, BATF, ETV7, and TOX. Effector function was upregulated in clonally expanded CD8+ T cells that emerged after rela+nivo. A rela+nivo intratumoral CD8+ T cell signature was associated with a favorable prognosis. This intratumoral rela+nivo signature was validated in peripheral blood as an elevated frequency of CD38+TIM3+CD8+ T cells. Overall, we demonstrated that cytotoxicity can be enhanced despite the retention of exhaustion signatures, which will inform future therapeutic strategies.

overall_design: Peripheral blood and tumor biopsies were collected from patients with advanced melanoma prior to and 4 weeks following treatment with either relatlimab (anti-LAG3) alone, nivolumab (anti-PD1) alone, or the combination of relalimab+nivolumab. Follow-up blood and tumor specimens were collected when available at either 12 or 16 weeks post-treatment.

middle: R

first: John

last: Kirkwood

first: Tullia

last: Bruno

first: Dario

last: Vignali


title: Single nucleus RNA sequencing analysis of hypothalamus ARC neurons

status: Public on Aug 10 2024

first: Han

last: Tan

address: 1230 York Avenue

department: Molecular Genetics

institute: Rockefeller University

summary: We performed snRNA-seq analysis on hypothalamus ARC tissues dissected from adult mice to analyze the diversity of ARC neurons.

overall_design: Nuclei were isolated from micro-dissected hypothalamus ARC tissues, stained with a fluorescent antibody to NeuN, a specific marker of neuronal nuclei, and subsequently subjected to fluorescent activated cell sorting followed by single-cell sequencing using the 10X single-cell analysis platform

first: Jeffrey

last: Friedman


title: Single-cell RNA-seq of the human tonsil and blood lymphocytes

city: Stanford

first: Menglan

last: Xiang

address: 450 Serra Mall

institute: Stanford University

summary: Tissue-selective chemotactic mechanisms direct lymphocytes to epithelial surfaces of the body to establish local immune environments, to regulate immune responses to food antigens and commensal organisms, and to protect the body from pathogens. Chemoattractants for small intestines, colon, and skin are known, but chemotropic mechanisms selective for respiratory tract and other non-intestinal mucosal tissues (NIMT) remain poorly understood. Here we leveraged diverse omics datasets to identify GPR25 as a lymphocyte receptor for CXCL17, a chemoattractant cytokine whose expression by epithelial cells of airways, upper gastrointestinal and squamous mucosae unifies the NIMT and distinguishes them from intestinal mucosae. Analyses of single cell transcriptomic data shows that GPR25 is induced on innate lymphocytes prior to emigration to the periphery, and is imprinted in secondary lymphoid tissues on activated B and T cells responding to immune challenge. GPR25 characterizes B and T tissue resident memory and regulatory T lymphocytes in NIMT and lungs in human and we show that it mediates lymphocyte homing to barrier epithelia of the the respiratory tract, stomach and biliary and genitourinary tracts in mouse models. GPR25 is also expressed by T cells in cerebrospinal fluid and CXCL17 by neurons, suggesting a link to CNS immune regulation. We reveal widespread imprinting and expression of GPR25 on regulatory T cells, suggesting a mechanistic link to population genetic evidence that GPR25 is protective in autoimmunity. Our results define a GPR25-CXCL17 chemoaffinity axis with the potential to integrate immunity and tolerance at non-intestinal mucosae and the CNS.

overall_design: Activated T and B cells from tonsil and total T cells from blood were isolated and enriched by FACS. Single-cell profiling was performed using the 5' 10x Genomics workflow.

first: Kevin

last: Brulois

first: Borja

last: Ocón

first: Manali

last: Kunte

first: Nicole

last: Lazarus

first: Eugene

last: Butcher


title: Defining heritability, plasticity, and transition dynamics of cellular phenotypes in somatic evolution

first: Joshua

middle: Seth

last: Schiffman

summary: Single-cell sequencing has revealed cell state heterogeneity across diverse healthy and malignant tissues. However, the extent these cell states are plastic or heritable remains largely unknown. To address this, we introduce PATH (Phylogenetic Analysis of Trait Heritability), a framework to quantify cell state heritability versus plasticity and infer cell state transition and proliferation dynamics from single-cell lineage tracing data. Here, we used PATH to measure phylogenetic correlations on single-cell phylogenies derived from gliomaspheres to reveal the somatic evolutionary relationships between Neural Progenitor-like (NPC), Oligodendrocyte Progenitor-like (OPC), Astrocyte-like (AC), and Mesenchymal-like (MES) cancer cell states.

overall_design: Single-cell RNA sequencing and lineage tracing using molecular recording in gliomasphere.

first: Yilin

last: Fan

first: Toshiro

last: Hara

first: Mario

last: Suvà

first: Dan-Avi

last: Landau


title: Ptk2 ablation in a Trp53fl/fl, Ptenfl/fl, Rb1fl/fl mouse brain tumor model

city: Toronto

first: Graham

last: MacLeod

state: ON

address: 144 College Street

department: Pharmaceutical Sciences

country: Canada

institute: University of Toronto

summary: Ptk2 was identified as a targetable genetic vulnerability in glioblastoma (GBM) cells. Our objective was to test the effect of Ptk2 deletion on survival and tumor phenotype in a mouse model of GBM. At P0, mice carrying Trp53fl/fl (Trp53tm1Brn, JAX #008462), Ptenfl/fl (Ptentm1Hwu/J, JAX #006440), Rb1fl/fl and R26-LSL-Cas9-GFP (JAX #026175) alleles were subjected to stereotactic injection to deliver lentiviral particles expressing Cre and either Ptk2-targeting or control gRNAs to the NSCs residing in the lateral subventricular zone. This injection initiates a GBM tumor in the mice. At time of sacrifice, tumor tissue was harvested and RNA was extracted for sequencing.

overall_design: RNA was harvested from 3 Ptk2-gRNA tumors and 3 control-gRNA (TIGRE) tumors. Gene expression profiling and differential expression analysis were conducted.

first: Shahan

last: Haider

first: Stephane

last: Angers

first: Daniel

last: Schramek


title: Set7: ERCC bulk short-read RNA-seq by Illumina NextSeq platform, additional samples

status: Public on Aug 19 2024

first: Shuchang

last: Liu

address: 203 Lothrop Street

summary: DNA sequencing is a critical tool in modern biology. Over the last two decades, it has been revolutionized by the advent of massively parallel sequencing, leading to significant advances in genome and transcriptome sequencing of various organisms. Nevertheless, challenges such as accuracy, lack of competitive options and prohibitive costs associated with high throughput parallel short-read sequencing persist. Here, we conduct a comparative analysis between the Element Biosciences AVITI platform and its counterpart the Illumina NextSeq platform, testing DNA and RNA short-read sequencing, as well as synthetic long-read sequencing for single cells and RNA transcripts.

overall_design: Bulk short-read RNA-seq on ERCC samples (Mix 1) by Illumina NextSeq platform; with three batches (b1, b2 and b3) and three repeats each

first: Silvia

first: Caroline

last: Obert

first: Yan-Ping

last: Yu

first: Junhua

last: Zhao

first: Bao-Guo

last: Ren

first: Kelly

last: Blease

first: Benjamin

last: Krajacich

first: Wenjia

last: Wang

first: Kyle

last: Metcalfe

first: Mat

last: Smith

first: Tuval

last: Ben-Yehezkel

first: Jian-Hua

last: Luo


title: Low Molecular Weight Cyclin E Confers a Vulnerability to PKMYT1 Inhibition in Triple-Negative Breast Cancer

status: Public on Aug 09 2024

first: Khandan

last: Keyomarsi

state: TX

address: 6565 MD Anderson Blvd. Unit 1052

department: Experimental Radiation Oncology

institute: MD Anderson Cancer Center

summary: Cyclin E is a regulatory subunit of CDK2 that mediates S phase entry and progression. Cleavage of full-length cyclin E (FL-cycE) to low molecular weight isoforms (LMW-E) dramatically alters the substrate specificity, promoting G1/S cell cycle transition and accelerating mitotic exit. Approximately 70% of triple-negative breast cancers (TNBC) express LMW-E, which correlates with poor prognosis. PKMYT1 also plays an important role in mitosis by inhibiting CDK1 to block premature mitotic entry, suggesting it could be a therapeutic target in TNBC expressing LMW-E. Here, analysis of TNBC patient tumor samples revealed that co-expression of LMW-E and PKMYT1-catalyzed CDK1 phosphorylation predicted poor response to neoadjuvant chemotherapy. Compared to FL-cycE, LMW-E specifically upregulated PKMYT1 expression and protein stability, elevating CDK1 phosphorylation. Inhibiting PKMYT1 with the selective inhibitor RP-6306 (lunresertib) elicited LMW-E dependent antitumor effects, accelerating premature mitotic entry, inhibiting replication fork restart, and enhancing DNA damage, chromosomal breaks, apoptosis, and replication stress. Importantly, TNBC cell line xenografts expressing LMW-E showed greater sensitivity to RP-6306 than tumors with empty vector or FL-cycE. Furthermore, RP-6306 exerted tumor suppressive effects in LMW-E transgenic murine mammary tumors and LMW-E-high TNBC patient-derived xenografts but not in the LMW-E null models examined in parallel. Lastly, transcriptomic and immune profiling demonstrated that RP-6306 treatment induced interferon responses and T-cell infiltration in the LMW-E-high tumor microenvironment, enhancing the antitumor immune response. These findings highlight the LMW-E/PKMYT1/CDK1 regulatory axis as a promising therapeutic target in TNBC, providing the rationale for further clinical development of PKMYT1 inhibitors in this aggressive breast cancer subtype.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for cyclin E transgenic mouse tumors treated with PKMYT inhibitor different for different time periods. Also, comparing gene expression profiles of cyclin E transgenic mice tumors as compared to PyMT tumors..

first: Mi

last: Li

first: Amriti

last: Lulla

first: Y

first: Spyros

last: Tsavachidis

first: Fuchenchu

first: Cansu

last: Karakas

first: Tuyen

last: Nguyen

last: Bui

first: Marc

last: Pina

first: Mei-Kuang

last: Chen

first: Sofia

last: Mastoraki

first: Asha

last: Multani

first: Natlaie

last: Fowlkes

first: Ayse

last: Sahin

first: Gary

last: Marshall

last: Hunt

last: Keyomasi


title: Targeting axonal guidance dependencies in glioblastoma with ROBO1 CAR T cells

first: Jason

last: Moffat

state: Ontario

address: 686 Bay Street

department: Genetics and Genome Biology

institute: Hospital for Sick Children

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

overall_design: Refer to individual Series


title: scRNAseq analysis of peritoneal leukocytes in mice with metastatic ovarian cancer

first: Juan

last: Cubillos-Ruiz

state: NY

address: 1300 York Ave.

institute: Weill Cornell Medicine

summary: NOT PROVIDED; REQUESTED

overall_design: CD45+ viable leukocytes were sorted from the peritoneal cavity of mice bearing ID8-Defb29/Vegf-A ovarian cancer for 25 days and cells were used for scRNAseq analysis

first: Chang-Suk

last: Chae

first: Camilla

last: Salvagno

first: Sung-Min

last: Hwang


title: Antagonism between H3K27me3 and genome-lamina association drives atypical spatial genome organization in the totipotent embryo [CELseq]

city: Utrecht

first: Jop

last: Kind

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

summary: In this study, we profiled genome-Nuclear Lamina (NL) contacts during the first stages of mouse embryonic development. We discovered a remarkable cell-to-cell variability in NL-contacts at the 2-cell stage, which is particularly strong on the paternal allele. We additionally obtained single-cell profiles for H3K27me3, H3K9me3 and DNA accessibility at this stage, but did not observe the same large-scale variability. The variability in NL-contacts did not appear to affect the trascription of underlying genes. Comparing NL-contact profiles with diverse histone modification profiles showed that large regions of typical NL-contacts are lost and instead are inriched for H3K27me3 during early development. To investigate the relationship between H3K27me3 and NL association, we used a conditional EED KO mouse model, which results in an absence of H3K27me3 during oocyte development and the early embryo. Profiling NL-contacts at the 2-cell stage in this syste revealed that regions enriched with H3K27me3 in WT regain NL association in the EED maternal KO. In addition, the loss of H3K27me3 resulted in more similar NL association profiles on the maternal and paternal allele. Together, these results suggest that H3K27me3 antagonizes NL association and that the non-canonical broad H3K27me3 domains present in the early embryo may be responsible for the early-embryo specific loss of NL associations in these regions.

overall_design: Profiling Nuclear Lamina (NL) contacts, H3K27me3, H3K9me3 and accessibility in mouse preimplantation embryos using scDamID and scDam&T-seq.

first: Isabel

last: Geirreiro

first: Franka

last: Rang

first: Yumiko

last: Kitazawa

first: Femke

last: Groenveld

first: Ramada

last: van Beek

first: Silke

last: Lochs

first: Ellen

last: Boele

first: Antoine

middle: H

last: Peters


title: RAS-mutant AML LSCs originate from GMPs and drive clinical resistance to BH3 mimetics [CMPs and GMPs scRNA-Seq]

first: BiNGS

last: Core

address: 1470 Madison Ave

institute: Icahn School of Medicine at Mount Sinai

summary: Cancer driver mutations often show distinct temporal acquisition patterns, but the biological basis for this, if any, remains unknown. RAS mutations occur invariably late in the course of acute myeloid leukemia (AML), upon progression or relapsed/refractory disease1-6. Here, by employing synthetic leukemogenesis in human cells, we first show that RAS mutations are obligatory late events that need to succeed earlier cooperating mutations. We provide the mechanistic explanation for this in a requirement for mutant RAS to specifically transform committed progenitors of the myelomonocytic lineage (granulocyte-monocyte progenitors, GMPs) harboring previously acquired driver mutations, revealing that advanced leukemic clones originate from a different cell type than more ancestral clones. Furthermore, we demonstrate that RAS-mutant leukemia stem cells (LSCs) give rise to monocytic disease, as frequently observed in patients with poor responses to treatment with the BCL2 inhibitor drug Venetoclax (VEN). We show that this is because RAS-mutant LSCs, in contrast to RAS-WT LSCs, have altered BCL2 family gene expression profiles and are resistant to VEN, driving clinical resistance and relapse with monocytic features. Our findings demonstrate that a specific genetic driver by imposing a specific LSC target cell restriction shapes the non-genetic cellular hierarchy of AML and critically impacts therapeutic outcomes in patients.

overall_design: Single-cell RNA-sequencing was performed with the Chromium 10x Genomics 3’ protocol (v3.0) on genetically engineered human cord blood FACS-sorted CMPs and GMPs

first: Eirini

last: Papapetrou


title: Transcriptomic analysis unveils bona fide molecular signatures of microglia under conditions of homeostasis and viral encephalitis

first: Ulrich

last: Kalinke

address: Feodor-Lynen-Str. 7

department: Institute for Experimental Infection Research

institute: Twincore, Centre for Experimental and Clinical Infection Research

summary: Microglia provide a front-line defense against neuroinvasive viral infection, however, determination of their bona fide transcriptional profiles under conditions of health and disease is challenging. Here, we used a combination of experimental tools to delineate the overall transcriptional landscape of microglia during viral infection. By exploiting the ribosomal tagging approach, we developed the concept of enrichment of relevant marker genes by comparing immunoprecipitated RNA with total RNA. Enriched transcripts corresponding to genes expressed in target cells were instrumental in defining bona fide signatures of microglia. With this approach, we generated a comprehensive and accurate transcriptome of microglia at the in situ environment under conditions of health and virus infection. These unified microglial signatures may serve as a benchmark to retrospectively assess ex vivo artefacts from available atlases. Leveraging the microglial translatome, we found enrichment of genes implicated in T-cell activation and cytokine production during the course of VSV infection. These data linked microglia with T-cell re-stimulation and further underscored that microglia shape anti-viral T-cell responses in the brain. Collectively, this study faithfully defines the transcriptional landscape of microglia in steady state and during viral encephalitis and highlights cellular interactions between microglia and T cells that contribute to the control of virus dissemination.

overall_design: Mixed gender CX3CR1-CreERT2+/- Rpl22wt/HA mice aged 6 weeks were subcutaneously injected with 4 mg of tamoxifen for two consecutive days. 8 weeks after initial tamoxifen injection, mice weeks were intranasally instilled with 10 µL of VSV. Controls animals received same volume of PBS. Six days post infection the animals were sacrificed by cervical dislocation prior to brain harvesting. Thereafter, brain was homogenized and lysate used to pull down HA-tagged ribosomes and to isolate RNA from “input” and RiboTag-IP fractions for bulk RNA-sequencing.

first: Felix

last: Mulenge

first: Olivia

middle: L

last: Gern

first: Andreas

last: Pavlou


title: RAS-mutant AML stem cells acquire resistance to BH3 mimetics through altered BCL-2 family expression

summary: Venetoclax (VEN) has transformed the therapy of acute myeloid leukemia (AML), but resistance and relapse are a major challenge. Monocytic differentiation was proposed as a cause of VEN resistance, but clinical and laboratory evidence is conflicting. Here we harness AML patientderived induced pluripotent stem cells (iPSCs) and Genotyping of Transcriptomes (GoT) to interrogate how mutational status and differentiation stage affect VEN sensitivity independently of one another. Findings in primary and iPSC-derived AML stem cells (LSCs) and monocytic blasts, together with clinical trial data, reveal that monocytic blasts are resistant to VEN, in contrast to LSCs, which express high levels of BCL2 and are sensitive, and that it is the latter, but not the former, that determines the clinical outcome. Crucially, N/KRAS-mutant LSCs produce more monocytic blasts, downregulate BCL2 and are resistant to VEN, driving clinical resistance or relapse. We thus provide a unifying mechanistic model of VEN resistance in AML.

overall_design: Single-cell RNA-sequencing was performed with the Chromium 10x Genomics 3’ protocol (v3.0) on hCD45+ cells from and AML-iPSC-derived xenografts sorted using Magnetic Activated Cell Sorting.

first: Saul

last: Carcamo

first: Dan

last: Hasson


title: The interplay between RNA sequestration in P-bodies and chromatin architecture sustains myeloid leukemia

first: Laureano

last: Tomás-Daza

state: Barcelona

overall_design: Low input capture Hi-C of MOLM-13 cells

first: A

last: Planas-Riverola

first: B

last: Javierre

first: L

last: Lopez-Rubio

first: J

last: Sardina


title: RAS-mutant AML LSCs originate from GMPs and drive clinical resistance to BH3 mimetics [scRNA-Seq]

overall_design: Single-cell RNA-sequencing was performed with the Chromium 10x Genomics 3’ protocol (v3.0) on genetically engineered human cord blood HSPCs


title: Piezo2 promotes resistance to radiation by suppressing CD8+ T cell-mediated antitumor immunity via IRF-1/IL-15 signaling

city: Shanghai

first: Wenwen

last: Li

address: Room 1004, West #5 Building, 280 South Chongqing Road, Shanghai 200025, China

institute: Shanghai Institute of Immunology; Department of Immunology and Microbiology, Shanghai Jiao Tong University School of Medicine


title: RAS-mutant AML LSCs originate from GMPs and drive clinical resistance to BH3 mimetics


title: SIRPα-CD47 axis regulates ILC2 effector function and alleviates airway hyperreactivity via modulating energy metabolism.

city: Los Angeles

first: Yoshihiro

last: Sakano

address: 1450 Biggy street

department: Molecular Microbiology & Immunology

institute: Unversity of Southern California

summary: Type-2 innate lymphoid cells (ILC2) are part of a growing family of innate lymphocytes known for their crucial role in both the development and exacerbation of allergic asthma. In this study, we aim to elucidate the critical role of the suppressor molecule signal regulatory protein alpha (SIRPα), which interacts with CD47, in controlling ILC2-mediated airway hyperreactivity (AHR). Our data indicate that activated ILC2s upregulate the expression of SIRPα, and the interaction between SIRPα and CD47 effectively suppresses both ILC2 proliferation and effector function. To evaluate the efficacy of SIRPα in regulating ILC2-mediated AHR, we utilized SIRPα and CD47 deficient mice in a murine model of allergen-induced AHR. Our findings suggest that the absence of SIRPα leads to the overactivation of ILC2s. Conversely, engagement with SIRPα reduces ILC2 cytokine production and effectively regulates ILC2-dependent AHR. Furthermore, the SIRPα-CD47 axis modulates mitochondrial metabolism through the JAK/STAT and ERK/MAPK signaling pathways, thereby regulating NF-κB activity and the production of type two cytokines. Additionally, our studies on human cells have revealed that SIRPα is inducible and expressed on human ILC2s, and administration of a SIRPα agonist effectively suppresses the effector function and cytokine production of ILC2s. Moreover, administering human CD47-Fc to humanized ILC2 mice effectively alleviated AHR and lung inflammation. These findings highlight the promising therapeutic potential of targeting the SIRPα-CD47 axis in the treatment of ILC2-dependent allergic asthma.

overall_design: mRNA profiles of mouse lung ILC2s from WT and SIRP KO mice were generated by deep sequencing, in duplicates, using a NextSeq 500.


title: Olig1/2 Coordinately Control Oligodendrocyte Versus Interneuron Fate Acquisition in Normal Brain and H3.3G34R/V-mutant Gliomas

city: SHANGHAI

first: YU

last: TIAN

state: SHANGHAI

address: 131 DONGAN ROAD

institute: FUDAN UNIVERSITY

summary: The co-opting of the developmental gliogenesis program is a hallmark characteristic of glioblastoma (GBM). The bHLH transcription factors Olig1/2 are co-expressed in tri-potential intermediate progenitors (tri-IPCs) and GBM cells, yet their functions in these cells remain largely unknown. Here, we demonstrate that Olig1/2 coordinately promote the generation of oligodendrocytes while repressing the generation of olfactory bulb interneurons in tri-IPCs and GBM. Mechanistically, Olig1/2 directly bind to multiple enhancers, such as hs687, DSG (downstream from Gsx2) and a putative enhancer to regulate the expression of Gsx2, thereby regulating the fate determination process of tri-IPCs during gliogenesis. Furthermore, we provide compelling evidence that human histone H3.3G34R/V-mutation tumors, a subtype of pediatric high-grade gliomas, originate from cortical-derived tri-IPCs. Altogether, our findings suggest that the molecular mechanism of Olig1/2 in gliogenesis and gliomagenesis are conserved, which will deepen our understanding of the gaps between normal development and tumorigenesis.

overall_design: ScRNA-seq were performed on FACS sorted tdT+ cells from the P1 Olig1/2f/f;IS-reporter mice cortex which were electroporated of pCAG-Cre plasmid at E15.5.Emx1-CRE;Olig1/2f/f mice cortex were dissected out at P7 and immediately submerged in fresh ice-cold Hanks balanced salt solution. The cortex was then cut into pieces and dissociated into a single-cell suspension using a Papain Cell Dissociation Kit according to the manufacturer’s instructions.

first: Yu

last: Tian

first: Ziwu

last: Wang

first: Feihong

last: Yang

first: Wen

last: Zhang

first: Jialin

first: Lin

first: Tongye

last: Fu

first: Wenhui

last: Zheng

first: Zhejun

last: Xu

first: Yan

last: You

first: Xiaosu

first: Jiangang

last: Song

first: Yunli

last: Xie

first: Zhengang

first: Zhuangzhi


title: RAS-mutant AML LSCs originate from GMPs and drive clinical resistance to BH3 mimetics [RNA-Seq]

first: BiNGS

last: Core

overall_design: Synthetic leukemogenesis models in human iPSC-HSPCs and primary CB HSPCs

first: Eirini

last: Papapetrou