title: Roles of H2A.Z histone variant isoforms during enterocyte-like differentiation process (ChIP-Seq)

city: TOULOUSE

last: Trouche

address: Université Toulouse III Paul Sabatier - Bât 4R4, 118 route de Narbonne

institute: Centre for Integrative Biology (CBI) of Toulouse MCD

summary: The involvement of the histone variant H2A.Z and its isoforms in the regulation of gene expression is an increasing exciting field considering the impact of such regulations in physio-pathology. Indeed, we and other recently showed that H2A.Z.1 and H2A.Z.2 isoforms exert cooperative or antagonistic transcriptional regulations on subsets of genes involved in key processes, such as proliferation, senescence or several organ functions. In this work, we analyze the relative role of both H2A.Z isoforms on parameters of the intestinal epithelial homeostasis. We observed that the amount of H2A.Z.1 and H2A.Z.2 at TSS and gene bodies are highly correlated and that the two H2A.Z isoforms can replace each other when depleted. We highlighted the role of their respective deposition onto chromatin by specific incorporators in some discrete isoform-specific contribution to the differentiation process. We have also uncovered an unexpected link between H2A.Z isoforms occupancy at gene bodies and the propensity of genes to be induced in enterocyte differentiation.

overall_design: Caco-2/15 cells is a useful model to study the intestinal enterocyte-type differentiation process, since it spontaneously acquired lineage-specific differentiation features upon confluence (Van Beers et al., 1995). To decipher the contribution of each H2A.Z isoform to gene expression pattern modifications that occur during intestinal epithelial differentiation, we first generated Caco-2/15 cell -based models in which we were able to heterozygously introduce a Flag tag into the 3’ regions of H2AFZ or H2AFV genes, by CRISPR-Cas9 genome editing, to bypass the lack of isoform-specific antibody. These models allowed us to follow, during the differentiation process, patterns of each isoform expression and incorporation into chromatin.  Using these models, we performed ChIP-seq and RNA-seq experiments for cell cultures in subconfluent (Sc) or 7-days post-confluent (Pc) state, allowing us to highlight H2A.Z isoforms enrichment and regulated genes subsets during differentiation.

first: Jérémie

last: Rispal

first: Virginie

last: Jouffret

first: Fabrice

last: Escaffit


title: RNA helicase DDX10 is Required for Pluripotency of Stem Cells by Regulating 18S rRNA Processing (CLIP-Seq)

status: Public on Oct 15 2024

city: guangzhou

first: gongcheng

last: Hu

state: guangdong

address: 190 Kai Yuan Avenue, Science Park, Guangzhou, China

country: China

institute: Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences

summary: Ribosomes, as a protein synthesis machine, are required for stem cells to maintain self-renewal. Here, we find that DEAD-box RNA helicase DDX10 is necessary for cellular pluripotency acquisition in somatic cell reprogramming, and in mouse embryonic stem cells (mESCs), DDX10 degradation disrupts cellular homeostasis, leads to cell cycle arrest in G1 phase and markedly inhibits cell proliferation. DDX10 is localized in dense fiber component (DFC) and granular component (GC), mainly binds to 45S ribosomal RNA (rRNA) and participates in regulating ribosome biogenesis. Specifically, DDX10 degradation prevents the release of U3 snoRNA from pre-rRNA, and disrupts pre-rRNA processing and maturation of 18S rRNA, leading to impaired ribosomal small subunit production. Together, this study reveals that DDX10 functions as an important regulator of ribosome biogenesis, and is essential for the survival, induction and maintenance of pluripotent stem cells.

overall_design: CLIP-seq experiments were performed in mESCs with stable expression of Flag-tagged DDX10.

first: Gongcheng

first: Hongjie

last: Yao


title: Roles of H2A.Z histone variant isoforms during enterocyte-like differentiation process (RNA-Seq)

first: Didier

country: France

type: Expression profiling by high throughput sequencing


title: Cross-Talk between Haematopoietic Cells and Stromal cells Subsets Drives the Remodelling of the Bone Marrow Microenvironment in Myeloproliferative Neoplasms

city: Oxford

first: Antonio

last: Rodriguez-Romera

address: John Radcliffe Hospital, Headley Way

department: Radcliffe Department of Medicine

country: United Kingdom

institute: University of Oxford

summary: In a proportion of patients with myeloproliferative neoplasms (MPNs), severe remodelling of the bone marrow (BM) microenvironment develops, with myelofibrosis (MF) and osteosclerosis leading to cytopenias and poor survival. Mesenchymal stromal cells (MSC) have been implicated in MF pathogenesis (Leimkühler, Cell Stem Cell, 2021), including nestin+ (Arranz, Nature, 2014), Gli1+ (Schneider, Cell Stem Cell, 2017) and leptin-receptor+ MSCs (Decker, Nat Cell Bio, 2017). However, little is known about the cellular subtypes and transcriptional states of fibroblast subtypes in MF, and the key receptor-ligand (R-L) interactions that lead to their activation.  Our aim was to build a fully comprehensive atlas of MF BM and clarify the pathological interactions between MPN cells and their stromal environment.

overall_design: We used 10x genomics platform to profile single-cell transcriptome of total momonuclear cells, hematopoietic stem/progenitor cells, CD41+ cells and stromal cells from the murine MPL W515L model of myelofibrosis


title: Impact of HSC-derived immune training on granulocyte/macrophage progenitor gene expression

city: Aurora

first: Andrew

last: Goodspeed

state: CO

address: 12801 E 17th Ave, Mail Stop 8303

country: USA

institute: University of Colorado Anschutz Medical Campus

summary: Trained immunity occurs when inflammatory stimulation reprograms myeloid cells to have increased effector functions upon secondary stimulation.  Interestingly, the secondary stimulation can occur well beyond the lifetime of an individual myeloid cell, thus memory of immune training must reside in the hematopoietic system. After showing that long-term hematopoietic stem cells (HSCLT) from mice treated with pristane for eight weeks generate trained macrophages, we sought to uncover mechanisms underlying the heritable trained immunity program in myeloid cells. To do this we transplanted HSCLT from pristane mice into congenic recipients, and after 18 weeks of engraftment sorted out donor derived granulocyte monocyte progenitors (GMP). We then analyzed the chromatin landscape and transcriptome using low input chromatin accessibility and transcriptomics sequencing (LiCAT-seq) which allows for the co-generation of RNA-seq and ATAC-seq libraries from the same cellular pool. With this approach we found a unique epigenetic phenotype of reduced chromatin accessibility at metabolic genes in GMP from pristane HSCLT, which resulted in reduced transcription of metabolic genes, leading to reduced metabolism in mature macrophages.

overall_design: Mice were treated ± pristane for eight weeks, then HSCLT were sorted out and transplanted into lethally irradiated congenic recipients. After 18 weeks of engraftment donor derived CD45.2+ GMP were sorted for LiCAT-seq analysis.

first: Taylor

middle: S

last: Mills

first: Eric

middle: M

last: Pietras


title: RNA helicase DDX10 is Required for Pluripotency of Stem Cells by Regulating 18S rRNA Processing (RNA-Seq)

overall_design: Gene expression profiling of auxin-induced degradation of DDX10 were performed at different time points (0 h, 2 h, 4 h, 6 h,12 h, 1 day, 2 days and  auxin 2 days followed by withdrawal 2 days).


title: Successful eculizumab-based desensitization in ABO-incompatible living donor kidney transplantation and its molecular phenotypes

city: Seoul

first: Minsun

last: Jung

address: 50-1 Yonsei-ro

country: South Korea

institute: Yonsei University College of Medicine

summary:
Background: Plasmapheresis/rituximab-based desensitization therapy has successfully reduced anti-ABO antibody levels and suppressed antibody-mediated rejection (AMR) in ABO-incompatible (ABOi) kidney transplantation (KT). However, high titers of anti-ABO antibodies in some patients are refractory to standard desensitization, leading to loss of KT opportunities or AMR.
Methods: Eculizumab-based desensitization was used to rescue high-titer ABOi KT patients refractory to plasmapheresis/rituximab-based desensitization.
Results: The initial titers of anti-ABO IgG antibodies in the two patients were 1:512 and >1:1024; the final pre-transplant titers after desensitization were 1:128 and 1:64. Both patients received eculizumab from the day of KT to two or four weeks post-KT and maintained stable renal function up to one-year post-transplantation without overt infectious complications, despite early episodes of suspicious AMR or borderline T cell-mediated rejection. Molecular phenotype analysis of allograft biopsies using the Banff Human Organ Transplant gene panel revealed that gene expression patterns in the ABOi KT with eculizumab group overlapped with those in the ABOi KT with AMR group more than in the ABOi KT without AMR group, except for complement pathway-related gene expression. Anti-ABO antibody titers decreased to low levels 1–3 months post-transplant in the eculizumab group in parallel with decreasing anti-B-specific B cells at this time point.
Conclusions: Short-term eculizumab-based desensitization therapy is promising for rescuing ABOi KT recipients with unacceptably high anti-ABO antibody titers refractory to plasmapheresis-based desensitization therapy.

overall_design: We analyzed immunological gene expression in kidney allograft biopsies using the B-HOT panel (NanoString, Seattle, WA, USA). We compared the transcriptomic profiles of 12 biopsies obtained from case 1 (two biopsies) and 2 (one biopsy) in the eculizumab group (n=3) and control groups, including AMR1 (n=3), NR (n=3), and AMR2 (n=3).

first: Jaeseok

last: Yang


title: Impact of autoimmune inflammation on HSPC and monocyte gene expression

overall_design: Mice were treated +/- pristane for four weeks then SLAM, MPPG/M, GMP, and BM Mon were sorted out for RNA-seq analysis


title: Autoimmune inflammation triggers an HSC-derived trained immunity program in myeloid cells

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: 5' droplet-based single cell RNA sequencing of human gastrointestinal organoids and Intestinal Metaplasia (IM) organoids [scRNA-seq]

city: HongKong

first: Suetyi

last: Leung

address: Queen Mary Hospital, Pokfulam Road

department: Department of Pathology

institute: The University of Hong Kong

summary: Using 5' droplet-based single cell sequencing, we profiled single cells dervied from human gastric, intestinal and IM organoids.

overall_design:
Normal gastric, intestinal and IM organoids were cultured in standard gastric/intestinal organoid medium or specified culture conditions to achieve differentiated state. Samples were then FACS-sorted to obtain single cells and were subjected to sequencing.
***Please note that raw data is not provided due to subject privacy concerns.***

first: SuetYi


title: Effect of HS-mediated telomere disruption on Nalm6 cells

status: Public on Oct 14 2024

city: Shenzhen

first: Kun

last: Sun

state: Guangdong

address: Rm A1921, Guangming International Innovation Center,

institute: Shenzhen Bay Laboratory

summary: Nalm6 tumour cells were engineered with focused ultrasound (FUS)-controllable CRISPR (heat-inducible CRISPR) with telomere-targeting gRNA or control non-targeting (NT) gRNA, and either treated with heat shock (HS) or no HS (control, CT). We observed that a relatively short duration of HS (10 min) significantly inhibited the proliferation of the cells engineered with telomere FUS-CRISPR, but not that of the cells with NT FUS-CRISPR, suggesting that telomere disruption rather than hyperthermia itself suppressed cell growth. Bulk RNA-seq further revealed that FUS-CRISPR-mediated telomere disruption led to the upregulation of multiple genes associated with the stress response p53 signaling pathway and apoptotic process and the TNF family in the engineered cells to trigger cell cycle arrest.

overall_design: Nalm6 cells engineered with telomere-targeting or NT FUS-CRISPR were subjected to 10 min HS or no treatment (CT). Total RNA was collected at 24, 48, and 96 h after HS using the RNA microprep kit (Zymo Research, R1050) and sent for bulk RNA-seq (Novogene).

first: Yiqian

last: Wu

first: Yingxiao

last: Wang


title: DNA methylation array analysis of human gastrointestinal organoids and Intestinal Metaplasia (IM) organoids

type: Methylation profiling by genome tiling array

summary: Using DNA methylation array, we profiled epigentic changes of human gastric, intestinal and IM organoids.

overall_design: Normal gastric, intestinal and IM organoids were cultured in standard gastric/intestinal organoid medium.


title: Lack of TYK2 Signaling Enhances Host Resistance to Candida albicans Skin Infection

city: Vienna

first: Birgit

last: Strobl

address: Veterinärplatz 1

country: Austria

institute: Veterinary University Vienna

summary: Candida albicans is the most common human fungal pathogen, causing diseases ranging from local to life-threating systemic infections. Tyrosine kinase 2 (TYK2), a crucial mediator in several cytokine signaling pathways, has been associated with protective functions in various microbial infections. However, its specific contribution in the immune response to fungal infections has remained elusive. In this study, we show that mice lacking TYK2 or its enzymatic activity exhibit enhanced resistance to C. albicans skin infections, limiting fungal spread and accelerating wound healing. Impaired TYK2-signaling prompted the formation of a distinctive layer of necrotic neutrophils around the fungal pathogens. Transcriptomic analysis revealed TYK2's pivotal role in regulating interferon-inducible genes in neutrophils, thereby impacting their antifungal capacity during infection. Furthermore, we show that TYK2-dependent interferon-gamma (IFNg) production contributes to fungal dissemination from the skin to the kidneys. Our study uncovers a hitherto unrecognized detrimental role of TYK2 in cutaneous C. albicans infections.

overall_design: To better understand the impact of TYK2 on the functionality of skin-infiltrating neutrophils, we performed RNA-sequencing of sorted neutrophils. To obtain sufficient and equal numbers of viable neutrophils, we isolated myeloid cells (CD45+CD3-CD19-NK1.1-) from infected skin at day 1 p.i from WT, Tyk2-/- (full body TYK2 knockout) and Tyk2-K923E (kinase-inactive TYK2) mice. The sorted population consisted of around 80% neutrophils with more than 80% viability.

first: Sara

last: Miranda

first: Hrönn

last: Kjartansdottir


title: Lipid nanoparticles as adjuvant of Norovirus VLP vaccine augment cellular and humoral immune responses in a TLR 9- and type I IFN-dependent pathway

city: Tianjin

first: Weiqian

last: Dai

address: Xinxing Sub-district, Heping District

institute: Tianjin Medical University

summary: Norovirus (NoV) virus-like particles (VLPs) adjuvanted with aluminum hydroxide (Alum) are common vaccine candidates in clinical studies. Alum adjuvants usually inefficiently assist recombinant proteins to induce cellular immune responses. Thus, novel adjuvants are required to develop NoV vaccines that could induce both efficient humoral and robust cellular immune responses. Lipid nanoparticles (LNPs) are well-known mRNA delivery vehicles. Increasing evidence suggests that LNPs may have intrinsic adjuvant activity and can be used as adjuvants for recombinant protein vaccines; however, the underlying mechanism remains poorly understood. In this study, we compared the adjuvant effect of LNPs and Alum for a bivalent GI.1/GII.4 NoV VLP vaccine. Compared with Alum, LNP-adjuvanted vaccines induced earlier production of binding, blocking and neutralizing antibodies and promoted a more balanced IgG2a/IgG1 ratio. It is crucial that LNP-adjuvanted vaccines induced stronger Th1-type cytokine-producing CD4+ T cell and CD8+ T cell responses than Alum. The adjuvant activity of LNPs depended on the ionizable lipid components. Mechanistically, LNPs activated innate immune responses in a type I IFN-dependent manner and were partially dependent on Toll-like receptor (TLR) 9, thus affecting the adaptive immune responses of the vaccine. This conclusion was supported by RNA-seq analysis and in vitro cell experiments and by the deeply blunted T cell responses in IFNαR1−/− mice immunized with LNP-adjuvanted vaccines. This study not only identified LNPs as a high quality adjuvant for NoV VLP vaccines, but also clarified the underlying mechanism of LNPs as a potent immunostimulatory component for improving protein subunit vaccines.

overall_design:
To explore the underlying mechanism of LNPs in initiating an innate immune response, which is required for generating an effective adaptive immune response.
Female BABL/c mice were randomly divided into seven groups (n = 4/group), as follows: (1) Naive, (2) Alum, (3) High LNP, (4) Low LNP + GI.1/GII.4, (5) High LNP + GI.1/GII.4, (6) Alum + GI.1/GII.4, and (7) GI.1/GII.4. Mice were immunized i.m. in both hind limbs. After 24 h, the mouse draining lymph nodes (dLNs), including popliteal, inguinal, and iliac LNs were collected for RNA-seq (n = 4/group).

first: Man

last: Xing

first: Lingjin

first: Lihui

last: Lv

first: Xiang

first: Yihan

first: Xueyang

last: Pang

first: Yingying

last: Guo

first: Jiling

last: Ren

first: Dongming

last: Zhou


title: CDK4 Inactivation Hinders Cell Death and Metabolism Remodeling Mitochondria-ER contacts in Triple-Negative Breast cancer cells

city: Lausanne

first: Dorian

middle: Vincent

last: ZIEGLER

state: Vaud

address: Quartier UNIL-Sorge - Batiment Gnopode

department: Center for Integrative Genomics (CIG)

country: Switzerland

institute: University of Lausanne (UNIL)

summary: The shift in the energetic demands of proliferating cells during tumorigenesis requires extensive crosstalk between the cell cycle and metabolism. Beyond their role in cell proliferation, cell cycle regulators also modulate intracellular metabolism in normal tissues. However, in the context of cancer, where CDK4 is upregulated or stabilized, the metabolic role of CDK4 is barely understood. Here, using both genetic and pharmacological approaches, we sought to determine the metabolic role of CDK4 in triple-negative breast cancer (TNBC) cells. Unexpectedly, the deletion of CDK4 only modestly reduced TNBC cell proliferation and did not hinder tumor formation in vivo. Furthermore, proapoptotic stimuli failed to induce appropriate cell death in TNBC cells upon CDK4 depletion or long-term CDK4/6 inhibitor treatment. Mechanistically, CDK4 enhances mitochondria-endoplasmic reticulum contact (MERC) formation, thereby promoting mitochondrial fission and ER-mitochondrial calcium signaling.  Phosphoproteomic analysis also revealed a role for CDK4 in regulating PKA activity at MERCs to sustain ER-mitochondrial calcium signaling. Such CDK4-mediated mitochondrial calcium signaling is required for the metabolic flexibility of TNBC cells. Taken together, these results demonstrate that CDK4 inhibition leads to cell death resistance by inhibiting mitochondrial apoptosis and functions through attenuated MERCs formation and ER-mitochondrial calcium signaling in TNBC. Overall, this study provides new insights into the mechanisms of TNBC resistance to CDK4/6i therapy and paves the way to explore potential synergistic therapeutic targeting of MERCs-associated metabolic shifts.

overall_design:
To investigate the effect of CDK4 on transcriptome of triple-negative breast cancer cells MDA-MB-231, we generated a specific CRISPR-Cas9 CDK4-KO cell line model
We then performed gene expression profiling analysis in normal/basal conditions with 4 different biological replicates.

middle: V

last: Ziegler

first: Lucia

last: Leal-Esteban

first: Luis

last: Fajas


title: Transcriptome and proteome profiling reveals TREM2-dependent and -independent glial response and metabolic perturbation in an Alzheimer’s mouse model

city: Columbus

first: Jie

last: Gao

state: OH

address: 460 W. 12th Ave

department: The Department of Neuroscience

institute: The Ohio State University

summary: Elucidating the intricate molecular mechanisms of Alzheimer's disease (AD) requires a multidimensional analysis incorporating various omics data. In this study, we employed transcriptome and proteome profiling of AppNL-G-F, a human APP knock-in model of amyloidosis, at the early and mid-stages of amyloid-beta (Aβ) pathology, to delineate the impacts of Aβ deposition on brain cells. By contrasting AppNL-G-F mice with TREM2 (Triggering receptor expressed on myeloid cells 2) knockout models, our study further investigates the role of TREM2, a well-known AD risk gene, in influencing microglial responses to Aβ pathology. Our results highlight microglial activation as a central feature of Aβ pathology, characterized by the significant upregulation of microglia-specific genes related to immune responses such as complement system and antigen presentation, and catabolic pathways such as phagosome formation and lysosome biogenesis. The absence of TREM2 markedly diminishes the induction of these genes, impairs Aβ clearance, and exacerbates dystrophic neurite formation. Importantly, TREM2 is required for the microglial engagement with Aβ plaques and the formation of compact Aβ plaque cores. Furthermore, this study reveals substantial disruptions in energy metabolism and protein synthesis, signaling a shift from anabolism to catabolism in response to Aβ deposition. This metabolic alteration, coupled with a decrease in synaptic protein abundance, occurs independently of TREM2, suggesting the direct effects of Aβ deposition on synaptic integrity and plasticity. In summary, our findings demonstrate significant microglial activation and metabolic disruption following Aβ deposition, offering mechanistic insights into Aβ pathology and highlighting the potential of targeting these pathways in AD therapy.

overall_design: We performed transcriptome and proteome profiling of single human APP knock-in AppNL-G-F mice at the onset and middle stages of Aβ pathology to comprehensively investigate the pathway alterations and molecular mechanisms associated with Aβ deposition. AppNL-G-F knock-in mice were selected as they eliminate artifacts related to APP overexpression, such as the mis-localization of APP and the accumulation of APP fragments including CTF-β (C-terminal fragment of APP) and AICD (APP intracellular domain). Furthermore, we also profiled AppNL-G-F, TREM2 knockout mice to examine how the absence of TREM2 affects the responses of microglia and other glial cells to Aβ deposition.

first: Da

last: Lin


title: Dual roles for a tick protein disulfide isomerase during the life cycle of the Lyme disease agent [I. scapularis]

city: Hangzhou

first: Xiaotian

last: Tang

state: Zhejiang

address: Agro-Bio-Environ Building, Zhejiang University

department: Entomology

institute: Zhejiang University

summary: To determine whether the influences of Ixodes scapularis protein disulfide isomerase A6 (IsPDIA6) on Borrelia burgdorferi colonization is the result of physiological responses, we performed RNA-sequencing (RNA-seq) to compare the transcriptomes of control (GFP) and IsPDIA6-silenced ticks.

overall_design: We compared the transcriptome of GFP (control) and IsPDIA6-silenced ticks upon Borrelia burgdorferi infection.

first: Erol

last: Fikrig


title: Dual roles for a tick protein disulfide isomerase during the life cycle of the Lyme disease agent

summary: To understand the immunomodulation roles of Ixodes scapularis protein disulfide isomerase A6 (IsPDIA6), we utilized RNA sequencing (RNA-seq) to investigate the effect of IsPDIA6 on the murine transcriptome.

overall_design: We compared the transcriptome of murine splenocytes stimulated by the Lyme disease agent Borrelia burgdorferi, with or without IsPDIA6, for 6 hours.


title: RNA tertiary structure and conformational dynamics revealed by BASH MaP

city: New York

first: Samie

last: Jaffrey

state: New York

address: 1300 York Ave

department: Pharmacology

institute: Weill Cornell Medicine

summary: The functional effects of an RNA can arise from complex three-dimensional folds known as tertiary structures. However, predicting the tertiary structure of an RNA and whether an RNA adopts distinct tertiary conformations remains challenging. To address this, we developed BASH MaP, a single-molecule dimethyl sulfate (DMS) footprinting method and DAGGER, a computational pipeline, to identify alternative tertiary structures adopted by different molecules of RNA. BASH MaP utilizes potassium borohydride to reveal the chemical accessibility of the N7 position of guanosine, a key mediator of tertiary structures. We used BASH MaP to identify diverse conformational states and dynamics of RNA G-quadruplexes, an important RNA tertiary motif, in vitro and in cells. BASH MaP and DAGGER analysis of the fluorogenic aptamer Spinach reveals that it adopts alternative tertiary conformations which determine its fluorescence states. BASH MaP thus provides an approach for structural analysis of RNA by revealing previously undetectable tertiary structures.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: To determine the structural features which impact N7G DMS reactivity, we performed DMS MaP and BASH MaP on in vitro transcribed RNAs and cellular mRNAs.

first: Maxim

last: Oleynikov


title: Genome-wide binding of FRUITFULL complexes (FUL-SOC1, FUL-AG-SEP3,  FUL, FUL-AG, FUL-SEP3) determined by DNA-affinity purification sequencing (DAP-seq)

city: Wageningen

first: Kai

last: Thoris

address: Droevendaalsesteeg 1

country: Netherlands

institute: WUR

summary: With this experiment, our objective is to identify the binding sites of MADS-domain protein complexes and determine if they exhibit binding to different targets or with differing affinities, which could be a mechanism to acquire target gene specificity. Specifically, we aim to investigate this phenomenon for the tetramers FUL-SOC1 and FUL-AG-SEP3, as they are enriched in different tissues, thus likely regulating distinct functions. FUL-SOC1 is enriched in the inflorescence meristem, whereas the FUL-AG-SEP3 complex is enriched in the siliques. Using the DAP-seq protocol, we established their genome-wide binding profiles and included other FUL complexes (FUL, FUL-AG, and FUL-SEP3) as controls.

overall_design: We use an in vitro genome-wide DNA binding assay, known as DAP-seq, to determine the genome-wide binding patterns of FUL-SOC1, FUL-AG-SEP3, FUL, FUL-AG, and FUL-SEP3.

first: Marian

last: Bemer

first: Gerco

last: Angenent


title: single cell transcriptome analysis of regulated transgenic GATA1 expression as a gene therapy for Diamond-Blackfan anemia

city: Boston

first: Alexis

last: Caulier

state: Massachusetts

address: 1 Blackfan street

department: Oncology/Hematology

institute: Boston Children's Hospital

summary: We analyzed the transcriptional effects of transgenic expression of our erythroid-specific codon-optimized GATA1 lentiviral vector in  differentiating red blood cells of patients diagnosed with Diamond-Blackfan anemia.

overall_design: CD34+ cells from DBA patients BCH-002 and BCH-008 were treated with hG1E-GATA1 (transgene) or HMD-GFP (control) and cultured for 10 days in erythroid differentiation media prior to collection and immediate processing for scRNA sequencing with 10x RNA 3’ V3 kit (10x Genomics)

first: Richard

last: Voit

first: Vijay

last: Sankaran


title: ZBTB7A is a modulator of KDM5-driven transcriptional networks in basal breast cancer  (ChIP-Seq)

first: Kornelia

last: Polyak

state: MA

address: 450 Brookline Ave

department: Medical Oncology

institute: Dana-Farber Cancer Institute

summary: We previously identified KDM5B, encoding a histone H3 lysine 4 (H3K4) demethylase, as an oncogene in estrogen receptor positive (ER+) breast cancer driving endocrine resistance. Here we describe that KDM5A is frequently amplified and overexpressed in basal breast tumors and is associated with chemotherapy resistance. Using CRISPR knockout viability screens -/+  KDM5 inhibition (KDM5i), we found that deletion of the transcription factor ZBTB7A and core SAGA complex increased sensitivity to KDM5i, whereas knockout of RHO-GTPases led to resistance. Integrated ChIP-seq and RNA-seq analyses revealed colocalization of ZBTB7A and KDM5s at promoters with high H3K4me3 signal and dependence of KDM5A binding on ZBTB7A. ZBTB7A knockout had a pleiotropic effect on transcriptional responses to KDM5i, in which it modulates the KDM5i-induced innate immune signaling and NF-kB-regulated genes. ZBTB7A knockout and KDM5i cooperate to alter cell states with KDM5i decreasing basal-like and ZBTB7A knockout inducing mesenchymal-like gene expression patterns. Our work furthers our understanding of KDM5-mediated gene regulation in breast cancer and identifies key pathways that mediate sensitivity to KDM5 inhibition

overall_design: Since our CRISPR screen identified ZBTB7A as a modulator of C70-sensitivity, we aimed to investigate how ZBTB7A and KDM5A/B interact to regulate H3K4me3 and gene expression. We generated ZBTB7A, KDM5A, and KDM5B knockout cell lines in SM149. As a WT control, cells expressing the ROSA26-targeting gRNA were generated (i.e., ROSA26_KO). The effect of each knockout and treatment (-/+ 10 µM C70 for 7 days) on chromatin binding patterns for ZBTB7A, KDM5A, KDM5B, and H3K4me3 were examined.

first: Benedetto

last: DiCiaccio


title: Transcriptional and Cellular Response of hiPSC-derived Microglia-Neural Progenitor Co-Cultures Exposed to IL-6

city: London

first: Amalie

middle: Charlotte Maria

last: Couch

address: 5 Cutcombe Road

department: Basic and Clinical Neuroscience

institute: King's College London

summary: Elevated interleukin (IL-)6 levels during prenatal development have been linked to increased risk for neurodevelopmental disorders (NDD) in the offspring, but the mechanism remains unclear. Human-induced pluripotent stem cell (hiPSC) models offer a valuable tool to study the effects of IL-6 on features relevant for human neurodevelopment in vitro. We previously reported that hiPSC-derived microglia-like cells (MGLs) respond to IL-6, but neural progenitor cells (NPCs) in monoculture do not.  Therefore, we investigated whether co-culturing hiPSC-derived MGLs with NPCs would trigger a cellular response to IL-6 stimulation via secreted factors from the MGLs. Using N=4 donor lines without psychiatric diagnosis, we first confirmed that NPCs can respond to IL-6 through trans-signalling when recombinant IL-6Ra is present, and that this response is dose-dependent. MGLs secreted soluble IL-6R, but at lower levels than found in vivo and below that needed to activate trans-signalling in NPCs. Whilst transcriptomic and secretome analysis confirmed that MGLs undergo substantial transcriptomic changes after IL-6 exposure and subsequently secrete a cytokine milieu, NPCs in co-culture with MGLs exhibited a minimal transcriptional response. Furthermore, there were no significant cell fate-acquisition changes when differentiated into post-mitotic cultures, nor alterations in synaptic densities in mature neurons. These findings highlight the need to investigate if trans-IL-6 signalling to NPCs is a relevant disease mechanism linking prenatal IL-6 exposure to increased risk for psychiatric disorders. Moreover, our findings underscore the importance of establishing more complex in vitro human models with diverse cell types, which may show cell-specific responses to microglia-released cytokines to fully understand how IL-6 exposure may influence human neurodevelopment.

overall_design: To characterize the response of human induced pluripotent stem cell (hiPSC-)derived microglia-like cells (MGL) and neural progenitor cells (NPC) in co-culture, we exposed N=4 donor lines (M3_CTR_36S, 127_CTM_01, 069_CTF_01 and 014_CTM_02) on day 14 of MGL and 18 of NPC differentiation to 24h of IL-6 stimulation (100ng/ml) and carried out bulk-RNAseq analysis on each cell type to profile their transcriptomic response.

first: Anthony

middle: C

last: Vernon


title: HKU Gastric Intestinal Metaplasia study: Single-cell RNA sequencing, transcriptome and methylation profiling

first: Suetyi

last: Leung


title: RNA sequencing of human gastrointestinal organoids and Intestinal Metaplasia (IM) organoids

summary: Using RNA sequencing, we profile transcriptome dervied from human normal gastric, intestinal and IM organoids.

overall_design:
Normal gastric, intestinal and IM organoids were cultured in standard gastric/intestinal organoid medium, respectively. Five IM organoids were cultured in vitro for over 6 months.
**Please note that raw data is not provided due to subject privacy concerns**

first: SuetYi


title: MCRS1 sensitizes pancreatic cancer to T cell immunity

city: Shanghai

first: Xiaofei

last: Yu

address: 2005 Songhu Rd

institute: Fudan University


title: MCRS1 sensitizes pancreatic cancer to T cell immunity [ChIP-seq]

summary: Pancreatic cancer is one of the deadliest cancers in humans and is yet refractory to immunotherapy. Identifying factors that can sensitize pancreatic cancer to immune cells may thus hold the key to improving immunotherapy efficacy. Herein, by pooled CRISPR activation screening, we find that the transcriptional regulator Mcrs1 (Microspherule protein 1) is pivotal to the immune sensitivity of pancreatic cancer cells, with elevated Mcrs1 expression inducing enhanced immune responses and tumor suppression in mice.

overall_design: Mcrs1 ChIP-Seq was performed with the mouse pancreatic cancer cell line, HT, to identify genomic regions bound by MCRS1.

first: Jin

last: Zheyu

first: Yi

last: Han

first: Jiang

last: Kaitao

first: Yu

last: Xiaofei


title: Impact of HSC-derived immune training on granulocyte/macrophage progenitor chromatin landscape

first: Andrew

last: Goodspeed

summary: Trained immunity occurs when inflammatory stimulation reprograms myeloid cells to have increased effector functions upon secondary stimulation.  Interestingly, the secondary stimulation can occur well beyond the lifetime of an individual myeloid cell, thus memory of immune training must reside in the hematopoietic system. After showing that long-term hematopoietic stem cells (HSCLT) from mice treated with pristane for eight weeks generate trained macrophages, we sought to uncover mechanisms underlying the heritable trained immunity program in myeloid cells. To do this we transplanted HSCLT from pristane mice into congenic recipients, and after 18 weeks of engraftment sorted out donor derived granulocyte monocyte progenitors (GMP). We then analyzed the chromatin landscape and transcriptome using low input chromatin accessibility and transcriptomics sequencing (LiCAT-seq) which allows for the co-generation of RNA-seq and ATAC-seq libraries from the same cellular pool. With this approach we found a unique epigenetic phenotype of reduced chromatin accessibility at metabolic genes in GMP from pristane HSCLT, which resulted in reduced transcription of metabolic genes, leading to reduced metabolism in mature macrophages.

overall_design: Mice were treated ± pristane for eight weeks, then HSCLT were sorted out and transplanted into lethally irradiated congenic recipients. After 18 weeks of engraftment donor derived CD45.2+ GMP were sorted for LiCAT-seq analysis.

first: Taylor

last: Mills

first: Etienne

last: Danis

first: Eric

last: Pietras


title: MCRS1 sensitizes pancreatic cancer to T cell immunity [cellline RNA]

overall_design: The mouse pancreatic cancer cell line HT was transduced with a mouse Mcrs1-encoding lentivirus to generate a Mcrs1-overexpressing cell line (Mcrs1ox).Transcriptome profiling was performed for the Mcrs1ox and vector control cells to identify Mcrs1-regulated genes.


title: Centralized control of dynamic gene regulatory circuits governs human T cell rest and activation

city: San Francisco

first: Alexander

last: Marson

state: CA

address: 1650 Owens St

institute: UCSF Gladstone Institutes

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

overall_design: Refer to individual Series


title: Set2 and H3K36 regulate the Drosophila male X chromosome in a context-specific manner (L1)

city: Chapel Hill

first: Greg

last: Matera

state: NC

address: 3352 Genome Sciences Building

institute: UNC Chapel Hill

summary: Current models of MSL spreading in the context of Drosophila dosage compensation (DC) focus on interactions of MSL3 (male-specific lethal 3) with histone marks; especially Set2-dependent H3 lysine-36 trimethylation (H3K36me3). However, previous studies investigating the role of H3K36me3 in DC do not fully account for: other targets of Set2, redundancy between canonical H3.2 and replication-dependent H3.3, and X chromosome effects that are not sex-specific.To differentiate amongst these possibilities and to assess whether Set2 and/or H3K36 is essential for DC, we use RNA-Seq in WL3 brain (male and female) and L1 larvae (mixed sex) to compare Set2, H3.2K36R, H3.3K36R, and combined H3K36R mutant genotypes. In brains, we observe heterogeneous regulation of chrX genes by these factors in both males and females. These effects are not clearly linked to MSL3 binding or H4K16ac occupancy, but in some groups of genes, are clearly stronger in males. At L1, we also observe heterogeneous gene regulation distinct from an H4K16R control. Simultaneous mutation of both H3.2K36 and H3.3K36 results in an increase in expression in genes most strongly reduced in H4K16R mutants. Overall, the data are inconsistent with the prevailing model wherein H3K36me3 is essential for spreading the MSL complex to genes along the male X. Rather, we propose that Set2 and H3K36 support DC indirectly, via processes that are utilized by MSL, but common to both sexes.

overall_design: 1) mRNA-seq WL3 brain, both sexes, Set2, H3.2K36R, H3.3K36R mutant genotypes and controls matched for genetic background 2) Total RNA-seq L1 larvae, mixed sex, Set2, combined H3K36R, controls matched for genetic background, and H4K16R control. Note: yw, rep1 was dropped from the analysis due to a library construction error.

first: Harmony

last: Salzler


title: Interpretable machine learning uncovers epithelial transcriptional rewiring and a role for Gelsolin in COPD

city: Pittsburgh

first: Justin

last: Sui

state: PA

address: 200 Lothrop Street

institute: University of Pittsburgh School of Medicine

summary: Transcriptomic analyses have advanced the understanding of complex disease pathophysiology including chronic obstructive pulmonary disease (COPD). However, identifying relevant biologic causative factors has been limited by the integration of high dimensionality data. COPD is characterized by lung destruction and inflammation with smoke exposure being a major risk factor. To define previously unknown biological mechanisms in COPD, we utilized unsupervised and supervised interpretable machine learning analyses of single cell-RNA sequencing data from the gold standard mouse smoke exposure model to identify significant latent factors (context-specific co-expression modules) impacting pathophysiology. The machine learning transcriptomic signatures coupled to protein networks uncovered a reduction in network complexity and new biological alterations in actin-associated gelsolin (GSN), which was transcriptionally linked to disease state. GSN was altered in airway epithelial cells in the mouse model and in human COPD. GSN was increased in plasma from COPD patients, and smoke exposure resulted in enhanced GSN release from airway cells from COPD patients. This method provides insights into rewiring of transcriptional networks that are associated with COPD pathogenesis and provide a translational analytical platform for other diseases.

overall_design:
C57BL/6J mice were obtained from Jackson Laboratories (female mice at 10-12 weeks of age, n = 3-4 per group) and subjected to the smoke of 4 unfiltered cigarettes per day (lot# 3R4F; University of Kentucky), 5 days a week for a duration of 6 months, using a smoking apparatus that delivers targeted cigarette smoke to single mice isolated in individual chambers. The controls in each group were exposed to room-air alone. Whole lung single cell suspensions were created and single cell RNA seq was performed.

***************************************************************
we do not have the raw FastQ files, as the sequencing and CellRanger alignment was done a few years ago by a different team
***************************************************************

first: Sui

last: Justin

first: Kliment

last: Corrine


title: ZBTB7A is a modulator of KDM5-driven transcriptional networks in basal breast cancer

first: Kornelia

last: Polyak


title: Set2 and H3K36 regulate the Drosophila male X chromosome in a context-specific manner (WL3 brain)

overall_design: 1) mRNA-seq WL3 brain, both sexes, Set2, H3.2K36R, H3.3K36R mutant genotypes and controls matched for genetic background 2) Total RNA-seq L1 larvae, mixed sex, Set2, combined H3K36R, controls matched for genetic background, and H4K16R control.


title: Short-term gene expression changes induced by malformin C in NCH421k patient-derived glioblastoma stem-like cells

city: Heidelberg

first: Emma

last: Phillips

address: In Neuenheimer Feld 580

department: Brain Tumor Translational Targets

country: Germany

institute: DKFZ

summary: Malformin C was identified in a fungal extract screen to selectively induce cell death in glioblastoma stem-like cells (GSCs). Gene expression changes were investigated at early time points before cell death was induced in order to gain insight into the mode of action of malformin C in GSCs.

overall_design: Total RNA extracted from NCH421k GSCs incubated with 50 nM malformin C for 2, 4 and 6 hours, or DMSO for 6 hours. 3 biological replicates for each condition were performed.


title: MCRS1 sensitizes pancreatic cancer to T cell immunity [ATAC-seq]

first: Xiaofei

last: Yu

overall_design: The chromatin accessibilty was profiled by ATAC-Seq in control, Mcrs1ox and Mcrs1ox cells deficient in YY1 (Mcrs1oxYy1ko).


title: Embryonic macrophages support endocrine commitment during human pancreatic differentiation

city: Toronto

first: Adriana

last: Migliorini

state: Ontario

address: 101 College St

department: McEwen Stem Cell Institute

country: Canada

institute: University Health Network

summary: Organogenesis is a complex process that relies on a dynamic interplay between extrinsic factors originating from the microenvironment and tissue-specific intrinsic factors. For pancreatic endocrine cells, the local niche consists of acinar and ductal cells as well as neuronal, immune, endothelial, and stromal cells. Hematopoietic cells have been detected in human pancreas as early as 6 post-conception weeks, but whether they play a role during human endocrinogenesis remains unknown. To investigate this, we performed single nuclei RNA sequencing of the second trimester human pancreas and identified a wide range of hematopoietic cells, including two distinct subsets of tissue-resident macrophages. Leveraging this discovery, we developed a co-culture system of human embryonic stem cell-derived endocrine-macrophage organoids to model their interaction in vitro. Here, we show that macrophages support the differentiation and viability of endocrine cells in vitro and enhance tissue engraftment, highlighting their potential role in tissue engineering strategies for diabetes.

overall_design: Human fetal pancreatic samples at 14, 16 and 18 post conception weeks (PCW) were subjected to cells nuclei isolation for snRNAseq using the 10X Genomics platform. Cells nuclei isolation was performed according to standard protocols. Fluorescent Activated Cell Sorting was used to enrich for intact nuclei.

first: Sabrina

last: Ge

first: Michael

middle: H

last: Atkins

first: Amanda

last: Oakie

first: Rangarajan

last: Sambathkumar

first: Gregory

last: Kent

first: Haiyang

last: Huang

first: Angel

last: Sing

first: Conan

last: Chua

first: Adam

middle: J

last: Gehring

first: Gordon

last: Keller

first: Faiyaz

last: Notta

first: Maria

last: Nostro


title: Adenosine uptake through equilibrative nucleoside transporter-1 suppresses antitumor immunity by pyrimidine starvation

status: Public on Oct 13 2024

city: Montreal

first: John

last: Stagg

state: QC

address: 900 St-Denis

institute: CRCHUM

summary: Immunosuppression by adenosine is an important cancer immune checkpoint. Extracellular adenosine signals through specific receptors and can be transported across the cell membrane through nucleoside transporters. While adenosine receptors are well-known to regulate tumor immunity, the impact of adenosine transporters remains unexplored. In this study, we investigated the effect on tumor immunity of equilibrative nucleoside transporter-1 (ENT1), the major regulator of extracellular adenosine concentrations. Using selective inhibitors and gene targeting approaches, we demonstrated that blocking or deleting host ENT1 significantly enhanced CD8+ T cell-dependent anti-tumor responses. Tumors inoculated into ENT1-deficient mice showed increased infiltration of effector CD8+ T cells with an enhanced cytotoxic transcriptomic profile and significant upregulation of granzyme B, IFN-g, IL-2, TNF-a and CXCL10. ENT1-deficiency was further associated with decreased tumor-infiltrating T regulatory cells and CD206high macrophages, and suppressed CCL17 production. ENT1-deficiency notably potentiated the therapeutic activity of PD-1 blockade. T cells upregulated ENT1 upon activation, and blocking ENT1 enhanced their function when co-cultured with cognate antigen/HLA-matched melanoma cells. Mechanistically, ENT1-mediated adenosine uptake inhibited the activity of phosphoribosylpyrophosphate synthetase (PRPS) in activated T cells, thereby suppressing production of uridine 5′-monophosphate (UMP) and its derivatives required for DNA and RNA synthesis. In summary, our study identified ENT1-mediated adenosine uptake as an important and previously unappreciated mechanism of adenosine-mediated immunosuppression via pyrimidine starvation that can be targeted to enhance antitumor T cell responses.

overall_design: MCA205 tumors were implanted in C57Bl/6 WT and ENT1-deficient mice and CD45+ CD8+ T cells were isolated after 14 days by FACS-based cell sorting. At least 30 000 purified CD8+ T cells per condition (4 ENT1KO samples and 3 WT samples) were stored at -80°C in trizol stabilizing solution and submitted for Illumina next-generation sequencing to the IRIC Genomics Platform.

first: David

last: Allard

first: Jeanne

last: Cormery

first: Salma

last: Bricha

first: Camille

last: Fuselier

first: Farnoosh

last: Abbas Aghababazadeh

first: Lucie

last: Giraud

last: Skora

first: Benjamin

last: Haibe-Kains


title: A p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR/Cas9 gene editing in Hematopoietic Stem and Progenitor Cells

city: Milan

first: Raffaella

last: Di Micco

address: Via Olgettina, 60

country: Italy

institute: San Raffaele Telethon Institute for Gene Therapy

summary: Ex vivo activation is a prerequisite to reaching adequate levels of gene editing by homology-driven repair (HDR) for hematopoietic stem and progenitor cell (HSPC)-based clinical applications. Here, we show that shortening culture time mitigates the p53-mediated DNA damage response to CRISPR/Cas9-induced DNA double-strand breaks, enhancing the reconstitution capacity of edited HSPCs. However, this results in lower HDR efficiency, rendering ex vivo culture necessary yet detrimental. Mechanistically, ex vivo activation triggers a multi-step process initiated by p38 MAPK phosphorylation, which generates mitogenic ROS, promoting fast cell cycle progression and subsequent proliferation-induced DNA damage. Thus, p38 inhibition before gene editing delays G1/S transition and expands transcriptionally-defined HSCs, ultimately endowing edited cells with superior multi-lineage differentiation, persistence throughout serial transplantation, enhanced polyclonal repertoire, and better-preserved genome integrity. Our data identify proliferative stress as a driver of HSPC dysfunction with fundamental implications for designing more effective and safer gene correction strategies for clinical applications.

overall_design: Droplet-based single-cell RNA sequencing (scRNA-seq) of non edited, HS Cas9, HS+AAV6(GFP-/GFP+) edited cells treated with p38i treatment across HSPC subsets. To enhance the representation of the more primitive HSCs, we sorted a pool of HSC-enriched cells (defined as CD34+CD133+CD90+CD45RA-) and mixed them with the bulk sample population upon hashtag marking.

first: Lucrezia

last: della Volpe

first: Roberta

last: Vacca

first: Federico

last: Midena

first: Teresa

last: Tavella

first: Ivan

last: Merelli


title: Transcriptional profiles of microglia-like progeny cells derived from Cx3cr1 haplo-insufficient HSPCs and wild-tyoe HSPCs

first: Danilo

last: Pellin

address: 300 Longwood Ave

institute: Boston Childrens Hospital

summary: Favoring the engraftment and maturation of the engineered HSPCs in the central nervous system could allow enhancing further the therapeutic potential of the transplantation of engineered HSPCs for treating neurometabolic diseases. We propose a gene addition strategy involving Cx3cr1, a microglia chemokine receptor regulating microglia ontogeny and function. Here we characterize the transcriptional profile of microglia-like cell generated by Cx3cr1 haplo-insufficient and WT HSPC

overall_design: Microglia-like cell generated by Cx3cr1 haplo-insufficient and WT HSPC were isolated from the CNS of conditioned mice


title: Gene expression profiles of interactions between colorectal cancer-associated fibroblasts and myeloid cells in 2D co-cultures and in novel next generation triple cultures containing colorectal cancer patient-derived organoids

city: Vienna

first: Matthias

last: Farlik

state: Vienna

address: Währinger Gürtel 18-20

institute: Medical University of Vienna

summary: In this study colorectal cancer (CRC) patient-derived cancer-associated fibroblasts (CAFs) and primary monocytes or macrophages were co-cultured in 2D set-up and single-cell transcriptomics was employed to analyze the phenotypic and functional changes of the myeloid cells and CAFs upon their interaction. The results showed that CAFs instruct monocytes to gain a tumor-associated macropages (TAM)-like cells phenotype. In the next step, novel next generation tumor organoid cultures of myelid cells, CAFs and patient-derived CRC organoids (PDOs) were utilized to mimic the tumor microenvironment and study the phenotypic changes of cells under chemotherapy and oncolytic virus treatments. The sequencing data revealed that both treatments triggered distinct phenotypic changes and identified immunomodulatory effects of chemotherapy in TAM-like cells. Oncolytic virus treatment led to strong upregulation of Type I and II interferon signaling, while both therapies activated multiple cell-cell communication pathways, highlighting key molecular interactions relevant to the tumor microenvironment. This co-culture model provided insights into TAM-like responses, aiding precision cancer treatment evaluations.

overall_design:
Co-cultures of CAFs and myeloid cells and single cultures (controls) were performed for 24h and followed by scRNA-seq. Triple cultures were established for 3 days, followed by the 24h treatment with chemotherapy (20 𝝁M oxaliplatin + 0.5 mM 5-fluorouracil), oncolytic virus  (H1N1-NS1116) or mock (control). Single cells were stained with DAPI and live cells were sort purified and used for scRNA-seq.

***************************************************************
Submitter states that missing raw files are due to patient privacy concerns.
***************************************************************

first: Julijan

last: Kabiljo

first: Anna

last: Theophil

first: Jakob

last: Homola

first: Annalena

middle: F

last: Renner

first: Nathalie

last: Stürzenbecher

first: Daphni

last: Ammon

first: Rebecca

last: Zirnbauer

first: Simone

last: Stang

first: Loan

last: Tran

first: Johannes

last: Laengle

first: Askin

last: Kulu

last: Chen

first: Markus

last: Fabits

first: Velina

last: Atanasova

first: Oliver

last: Pusch

first: Wolfgang

last: Weninger

first: Henning

last: Walczak

first: Dietmar

last: Herndler-Brandstetter

first: Gerda

last: Egger

first: Helmut

last: Dolznig

last: Kusienicka

first: Michael

last: Bergmann


title: The tRNA methyltransferase Mettl1 governs ketogenesis through translational regulation and drives metabolic reprogramming in cardiomyocyte maturation

city: Guangzhou

first: Tailai

last: Du

address: No.58, Zhongshan 2nd road

department: The First Affiliated Hospital

institute: Sun Yat-sen University

summary: Following birth, the heart undergoes intricate transitions in response to the altered environment, including shifts in energy metabolism and cytoarchitecture, such as the switch from glucose to lipid utilization and the transformation of fetal-to-adult sarcomeric gene isoforms, all aimed at achieving functional maturation. These adaptations facilitate efficient energy production and cardiac contraction, enabling the heart to effectively pump blood throughout the body. Although the early postnatal period is widely recognized as a critical phase for cardiomyocyte maturation, the precise mechanisms initiating and orchestrating this process remain elusive. Using in vivo and in vitro models incorporated with multi-omic analyses, we here show that Mettl1 is a critical regulator in postnatal cardiomyocyte maturation. We demonstrate that Mettl1 is required for the proper ketogenesis in cardiomyocyte maturation via regulating Hmgcs2 translation. Loss of Mettl1 leads to aberrant metabolic reprogramming and subsequent cardiomyocyte immaturation through the deficiency in lysine β-hydroxybutyrylation of TCA cycle-related proteins.

sample_organism:
Mus musculus
Rattus norvegicus

platform_organism:
Mus musculus
Rattus norvegicus

overall_design: RNA-seq, Ribo-seq, and TRAC-seq were performed to identify differentially expressed genes, differentially translated genes and tRNA m7G methylome in 4-week-old Mettl1-cKO and control mouse hearts. RNA-seq was performed to identify differentially expressed genes in Mettl1-null and WT H9C2 cardiomyocytes.

first: Yanchuang

last: Han

first: Zhan-Peng

last: Huang


title: ZBTB7A is a modulator of KDM5-driven transcriptional networks in basal breast cancer  (scRNA-seq)

first: Kornelia

last: Polyak

summary: We previously identified KDM5B, encoding a histone H3 lysine 4 (H3K4) demethylase, as an oncogene in estrogen receptor positive (ER+) breast cancer driving endocrine resistance. Here we describe that KDM5A is frequently amplified and overexpressed in basal breast tumors and is associated with chemotherapy resistance. Using CRISPR knockout viability screens -/+  KDM5 inhibition (KDM5i), we found that deletion of the transcription factor ZBTB7A and core SAGA complex increased sensitivity to KDM5i, whereas knockout of RHO-GTPases led to resistance. Integrated ChIP-seq and RNA-seq analyses revealed colocalization of ZBTB7A and KDM5s at promoters with high H3K4me3 signal and dependence of KDM5A binding on ZBTB7A. ZBTB7A knockout had a pleiotropic effect on transcriptional responses to KDM5i, in which it modulates the KDM5i-induced innate immune signaling and NF-kB-regulated genes.

overall_design: To investigate resistance to the KDM5 inhibitor C70, we generated a C70-resistant cell line through prolonged passaging in 10 µM C70 (i.e., SUM149CR). The SUM149CR derivative and parental SUM149 cells were compared via RNA-seq  (2 or 7 days reatment with -/+ 10 µM C70) and scRNA-seq (7 days treatment with -/+ 10 µM C70).   Since our CRISPR screen identified ZBTB7A as a modulator of C70-sensitivity, we aimed to test the effect of ZBTB7A knockout on transcriptional response to C70. We developed ZBTB7A knockouts across multiple cell lines: SUM149, MDA-MB-436, MCF7, and T-47D. As a WT control, cells expressing the ROSA26-targeting gRNA (i.e., ROSA26-KOs) were generated. All cell lines were treated for 7 days -/+ 10 µM C70 and the effect of ZBTB7A-KO on transcriptional response was assessed.

first: Benedetto

last: DiCiaccio


title: MCRS1 sensitizes pancreatic cancer to T cell immunity [Tumor]

first: Xiaofei

last: Yu

overall_design: Mcrs1ox and control cells were subcutaneously injected into the flanks of mice and tumor tissues were collected 4 weeks later.  Transcriptome profiling of  control and Mcrs1ox the dissected tumors were performed to identify MCRS1-regulated genes in vivo.

first: Jiang

last: Kaitao

first: Li

last: Xue

first: Jin

last: Zheyu

first: Yu

last: Xiaofei


title: Metformin targets mitochondrial complex I to lower blood glucose levels

status: Public on Oct 12 2024

city: Chicago

first: Rogan

middle: A

last: Grant

state: IL

address: 303 E Superior St, Simpson Querrey Biomedical Research Center 5-522

department: Pulmonary and Critical Care Medicine

institute: Northwestern University

summary: Metformin is among the most prescribed anti-diabetic drugs, but the primary molecular mechanism by which metformin lowers blood glucose levels is unknown.  Previous studies have proposed numerous mechanisms by which acute metformin lowers blood glucose, including the inhibition of mitochondrial complex I of the electron transport chain (ETC).  Here, we used transgenic mice that globally express the Saccharomyces cerevisiae protein NDI1 to determine whether the glucose lowering effect of acute oral administration of metformin requires inhibition of mitochondrial complex I of the ETC in vivo.  NDI1 is a yeast NADH dehydrogenase enzyme that complements the loss of mammalian mitochondrial complex I electron transport function and is insensitive to pharmacologic mitochondrial complex I inhibitors including metformin.  We demonstrate that NDI1 expression attenuates metformin’s ability to lower blood glucose levels under standard chow and high-fat diet conditions.  Our results indicate that acute oral administration of metformin targets mitochondrial complex I to lower blood glucose.

overall_design: The objective of this study was to use NDI1 in vivo to determine whether metformin mediates its blood glucose lowering effect through inhibition of mitochondrial complex I. RNA-seq was performed on bulk liver samples from wild-type and NDI1+ mice to confirm NDI1 expression in the liver.

first: Reczek

middle: R

last: Colleen

first: Chakrabarty

middle: P

last: Ram

first: D'Alessandro

middle: B

last: Karis

first: Sebo

middle: L

last: Zachary

first: Grant

last: Rogan

first: Gao

last: Peng

first: Budinger

last: GR Scott

first: Chandel

last: Navdeep


title: CD4+ T cell-derived IL21 regulates stem cell fate in acute myeloid leukemia by activation of p38-MAPK signaling

city: Bern

first: Carsten

last: Riether

address: Murtenstrasse 35

department: DBMR

institute: University Bern

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: ZBTB7A is a modulator of KDM5-driven transcriptional networks in basal breast cancer  (RNA-Seq)

first: Kornelia

last: Polyak

summary: We previously identified KDM5B, encoding a histone H3 lysine 4 (H3K4) demethylase, as an oncogene in estrogen receptor positive (ER+) breast cancer driving endocrine resistance. Here we describe that KDM5A is frequently amplified and overexpressed in basal breast tumors and is associated with chemotherapy resistance. Using CRISPR knockout viability screens -/+  KDM5 inhibition (KDM5i), we found that deletion of the transcription factor ZBTB7A and core SAGA complex increased sensitivity to KDM5i, whereas knockout of RHO-GTPases led to resistance. Integrated ChIP-seq and RNA-seq analyses revealed colocalization of ZBTB7A and KDM5s at promoters with high H3K4me3 signal and dependence of KDM5A binding on ZBTB7A. ZBTB7A knockout had a pleiotropic effect on transcriptional responses to KDM5i, in which it modulates the KDM5i-induced innate immune signaling and NF-kB-regulated genes. ZBTB7A knockout and KDM5i cooperate to alter cell states with KDM5i decreasing basal-like and ZBTB7A knockout inducing mesenchymal-like gene expression patterns. Our work furthers our understanding of KDM5-mediated gene regulation in breast cancer and identifies key pathways that mediate sensitivity to KDM5 inhibition

overall_design: To investigate resistance to the KDM5 inhibitor C70, we generated a C70-resistant cell line through prolonged passaging in 10 µM C70 (i.e., SUM149CR). The SUM149CR derivative and parental SUM149 cells were treated for 2 or 7 days -/+ 10 µM C70 and compared via RNA-seq.   Since our CRISPR screen identified ZBTB7A as a modulator of C70-sensitivity, we aimed to test the effect of ZBTB7A knockout on transcriptional response to C70. We developed ZBTB7A knockouts across multiple cell lines: SUM149, MDA-MB-436, MCF7, and T-47D. As a WT control, cells expressing the ROSA26-targeting gRNA (i.e., ROSA26-KOs) were generated. All cell lines were treated for 7 days -/+ 10 µM C70 and the effect of ZBTB7A-KO on transcriptional response was assessed.


title: CD4+ T cell-derived IL21 regulates stem cell fate in acute myeloid leukemia by activation of p38-MAPK signaling - IL21R KO experiment

summary: Self-renewal programs in leukemia stem cells (LSCs) predict poor prognosis in acute myeloid leukemia (AML) patients. We identified CD4+ T cell-derived interleukin (IL) 21 as an important negative regulator of self-renewal of murine and human LSCs, but not hematopoietic stem cells. IL21/IL21R signaling favored asymmetric cell division and differentiation in LSCs through accumulation of reactive oxygen species (ROS) and activation of p38-MAPK signaling, resulting in reduced LSCs number and significantly prolonged survival in murine AML models. In human AML, serum IL21 at diagnosis was identified as an independent positive prognostic biomarker for outcome and correlated with better survival and higher complete remission rate in patients that underwent high-dose chemotherapy. IL21 inhibited primary AML LSCs function in vitro by activating ROS and p38-MAPK signaling and this effect was enhanced by cytarabine treatment. Consequently, promoting IL21/IL21R signaling on LSCs may be a novel approach to decrease stemness and increase differentiation in AML.

overall_design: Transcriptomic profiles of FACS-purified L-GMPs derived from bone marrow of IL21R+/- (WT) or IL21-/- (KO) AML mice were assessed in biological replicates using Illumina Nova 6000.

first: Viviana

last: Rubino


title: CD4+ T cell-derived IL21 regulates stem cell fate in acute myeloid leukemia by activation of p38-MAPK signaling - IL21KO experiment

overall_design: Transcriptomic profiles of FACS-purified L-GMPs derived from bone marrow of BL/6 (WT) or IL21-/- (KO) AML mice were assessed in biological replicates using Illumina Nova 6000.


title: Thyroidal transcriptomic profiles of pathoadaptive responses to congenital hypothyroidism in XB130 knockout mic

first: Aaron

last: Wong

address: 101 College Street

department: TGHRI

summary: Congenital hypothyroidism is a genetic condition in which the thyroid gland fails to produce sufficient thyroid hormone (TH), resulting in metabolic dysfunction and growth retardation. Xb130-/- mice exhibit perturbations of thyrocyte cytoskeleton and polarity, develop postnatal transient growth retardation due to congenital hypothyroidism and leading to multinodular goiter in elderly. To determine the underlying mechanisms, we performed transcriptomic analyses on thyroid glands of mice at three age points – week 2 (W2, before visible growth retardation), W4 (at the nadir of growth), and W12 (immediately before full growth recovery. We compared gene expression between Xb130+/+ and Xb130-/- mice.

overall_design: This study compared gene expression of of Xb130+/+ and Xb130-/- mice at 3 ages, 2 weeks, 4 weeks, and 3 months.

first: Junichi

last: Sugihara

first: Hae-Ra

last: Cho

first: Yingchun

last: Wang

first: Ricardo

last: Zamel

first: Mingyao

last: Liu


title: Promotion of TLR7-MyD88-dependent Inflammation and Autoimmunity through Stem-loop Changes in Lnc-Atg16l1

city: Beijing

first: Zongheng

last: Yang

address: Dongdansantiao 5th

institute: Peking Union Medical College, Chinese Academy of Medical Sciences

summary: In this study, we employed a combination of Endosome RNA-seq and iCLIP-seq technologies to identify long non-coding RNA associated with TLR7 and Myd88 in mouse HA-mTLR7 stable overexpression and wild type RAW264.7 cells. RNA-seqs of wild type and Lnc-Atg16l1 RAW264.7 cells were applied to function study. We performed icSHAPE-seq to investigate RNA secondary structure of Lnc-Atg16l1 in RAW264.7 cells with or without stumulatied by R837, as well as in spleen cells from BXSB.Yaa and BXSB.B6 mice.

overall_design: RNAs from endosome in mouse HA-mTLR7 stable overexpression RAW264.7 cells  was analyzed using illumine Novaseq 6000. RNAs from RAW264.7 cells with or without R837 stimulating was analyzed using illumine Novaseq 6000. TLR7 activated iCLIP were performed using anti-HA antibody in RAW264.7 cells stably expressing HA-mTLR7. TLR7 noactivated iCLIP were performed using anti-TLR7 antibody in RAW264.7 cells. Two iCLIP of Myd88 were performed using anti-Myd88 antibody in RAW264.7 cells with or without R837 stimulating. icSHAPE experiments were performed through click reaction in RAW264.7 cells with or without R837 stimulating and spleen cells from BXSB.Yaa and BXSB.B6 mice. All RNAs of iCLIP and icSHAPE were analyzed using illumima Hiseq 2500.

first: Shuchen

last: Ji

first: Lun

first: Shuo

first: Bingjing

first: Yuanwu

last: Ma

first: Xuetao

last: Cao


title: CD4+ T cell-derived IL21 regulates stem cell fate in acute myeloid leukemia by activation of p38-MAPK signaling - Human AMK

overall_design: Transcriptomic profiles of FACS-purified CD34+ AML LSCs of 4 patients cultured in presence and absence of recombinant human IL21 were assessed in biological replicates using Illumina Nova 6000.


title: Type 2 innate immunity promotes the development of pulmonary fibrosis in Hermansky-Pudlak syndrome

status: Public on Oct 11 2024

city: Providence

first: Yang

last: Zhou

state: Rhode Island

address: 171 Meeting St, BMC 624

department: Molecular Microbiology and Immunology

institute: Brown University

summary: Hermansky-Pudlak syndrome (HPS), particularly in types 1 and 4, is characterized by progressive pulmonary fibrosis, a major cause of morbidity and mortality. However, the precise mechanisms driving pulmonary fibrosis in HPS are not fully elucidated. Our previous studies have suggested that CHI3L1-driven fibroproliferation may be a significant factor in HPS-associated fibrosis. This study aimed to explore the role of CHI3L1-CRTH2 interaction on ILC2s and explored the potential contribution of ILC2-fibroblast crosstalk in the development of pulmonary fibrosis in HPS. We identified ILC2s in lung tissues from idiopathic pulmonary fibrosis (IPF) and HPS patients. Our findings suggest that ILC2s may directly stimulate the proliferation and differentiation of primary lung fibroblasts partially through Areg-EGFR-dependent mechanisms. Additionally, specific overexpression of CHI3L1 in the ILC2 population using the IL-7Rcre driver, which was associated with increased fibroproliferation, indicates that ILC2-mediated, CRTH2-dependent mechanisms might contribute to optimal CHI3L1-induced fibroproliferative repair in HPS-associated pulmonary fibrosis.

overall_design: Using bleomycin-challenged wild type (WT) and Hps1−/− mice we observed that ILC2s were recruited and appeared to contributed to fibrosis development in the Hps1−/− mice, with CRTH2 playing a notable role in ILC2 accumulation. We sorted ILC2s, profiled fibrosis-related genes and mediators, and conducted co-culture experiments with primary lung ILC2s and fibroblasts.

first: Parand

last: Sorkhdini

first: Kiran

last: Klubock-Shukla

first: Selena

last: Seth

first: Dongquin

first: Alina

last: Xiaoyu Yang

first: Carmelissa

last: Norbrun

first: Wendy

last: Introne

first: Bernadette

last: Gochuico


title: Targeted Manipulation of Cellular RNA m6A Methylation at the Single-Base Level

city: Hangzhou

first: Jianzhao

address: 866 Yuhangtang Rd, Hangzhou, Zhejiang Province, P.R. China

institute: Zhejiang University

summary: We engineered m6A methyltransferase with dCas13-M14 to achieve site-specific editing of m6A. The resultant m6A editors can be programmed with a guide RNA, allowing functional comparison of single site methylation in different mRNA regions.

overall_design: Examination of m6A methylation patterns after targeted methylation of YTHDF2-1551, SPEN-3525 and GAPDH-782

first: Minsong

last: Gao

first: Shichen

last: Su

first: Jie


title: Expansion of peripheral T cell clonotypes predict response in patients with small cell neuroendocrine bladder and prostate cancers treated with chemo-immunotherapy in a phase I clinical trial [bulkRNA-Seq]

city: Los Angeles

first: Yiqian

last: Gu

address: 5000D Terasaki Life Sciences Building

institute: University of California Los Angeles

summary: To explore the impact of immunotherapy, we report a single institution, dual cohort phase Ib study (NCT 03582475) of platinum-based chemotherapy in combination with PD-1 antagonist pembrolizumab to evaluate safety and preliminary efficacy in patients with small cell bladder and small cell/neuroendocrine prostate cancers. To assess the immunologic basis for responses, single cell gene expression and T cell receptor (TCR) sequencing of serial peripheral blood samples revealed a diverse T cell repertoire with expansion of large clonotypes correlating with response.

overall_design: Total tumor RNA sequencing of formalin-fixed paraffin embedded pre-treatment tumor tissue were performed by next-generation sequencing using the Tempus xR assay (Tempus Labs, Chicago, IL), to investigate the cellulor composition and enriched genes.

first: Ann

last: Ly

first: Arnold

middle: I

last: Chin


title: MCRS1 sensitizes pancreatic cancer to T cell immunity [scRNA]

first: Xiaofei

last: Yu

address: 2005 Songhu Rd

institute: Fudan University

summary: Pancreatic cancer is one of the deadliest cancers in humans and is yet refractory to immunotherapy. Identifying factors that can sensitize pancreatic cancer to immune cells may thus hold the key to improving immunotherapy efficacy. Herein, by pooled CRISPR activation screening, we find that the transcriptional regulator Mcrs1 (Microspherule protein 1) is pivotal to the immune sensitivity of pancreatic cancer cells, with elevated Mcrs1 expression inducing enhanced immune responses and tumor suppression in mice.

overall_design: The tumor-infiltrating lymphocytes in Mcrs1ox or control tumors were isolated with collangenase digestion, followed by centrifugation in a discontinuous Percoll density gradient. TCRb+ CD90.2+ cells were sorted with a FACSAria Fusion Flow Cytometer, with dead cells excluded as DAPI+. Sorted cells were then stained with the Biolegend TotalSeq Hashtags for single cell RNA-seq and TCR-seq.

first: Li

last: Xue

first: Jiang

last: Kaitao

first: Jin

last: Zheyu

first: Yu

last: Xiaofei


title: Methionine cycle dysregulation mediates REDD1 overexpression-induced muscle atrophy in cancer cachexia

city: Nanjing

first: Zhiqiang

last: Huang

state: Jiangsu

address: Hankou 22

department: Medical School

institute: Nanjing University

summary: The essential amino acid methionine plays a pivotal role in one-carbon metabolism, facilitating the production of S-adenosylmethionine (SAMe), a critical supplier for DNA methylation. Here we find the disruption of methionine metabolism by rapid SAMe depletion in skeletal muscle in cancer cachexia, leading to endoplasmic reticulum (ER) stress and the overexpression of regulated in development and DNA damage responses (REDD1). Targeting the DNA methylation process via DNA methyltransferases (DNMTs) and REDD1 knockout can alleviate cancer cachexia-induced skeletal muscle atrophy. Methionine supplementation maintains the DNA methylation of DNA damage-inducible transcript 4 (Ddit4) by DNMT3A, thereby inhibiting activating transcription factor 4 (ATF4)-mediated Ddit4 transcription. Our study suggests that methionine or SAMe supplementation can effectively reverse muscle atrophy in cancer cachexia, providing valuable mechanistic insights and a promising therapeutic strategy for clinical application.

overall_design: Multi-omics studies to explore the transcriptional regulatory mechanisms of muscle tissue in the cachexia model.

first: Kai

last: Lin

first: Hongwei

last: Wang


title: Endogenous self-peptides guard CNS immune privilege [TCR-seq]

city: St. Louis

first: Jonathan

last: Kipnis

state: MO

address: 4515 McKinley Ave

institute: Washington University in St. Louis

summary: The central nervous system (CNS), despite the presence of strategically positioned anatomical barriers designed to protect it, is not entirely isolated from the immune system1,2. In fact, it remains physically connected to and can be influenced by the peripheral immune system. How the CNS retains such responsiveness while maintaining an immunologically unique status remains an outstanding conundrum. In searching for molecular cues that derive from the CNS and allow its direct communication with the immune system, we discovered a repertoire of CNS-derived endogenous regulatory self-peptides presented on major histocompatibility complex (MHC) II molecules at the CNS borders. During homeostasis, a preponderance of these regulatory self-peptides were found to be bound to MHC II molecules throughout the path of lymphatic drainage from the brain to its surrounding meninges and its draining cervical lymph nodes. With neuroinflammatory disease, however, the presentation of regulatory self-peptides diminished. Upon boosting the presentation of these regulatory self-peptides, a population of suppressor CD4+ T cells could be expanded, controlling CNS autoimmunity in a CTLA-4 and TGF dependent manner. This unexpected discovery of CNS-derived autoimmune self-peptides may be the molecular key adapting the CNS to maintain continuous dialogue with the immune system while balancing overt autoreactivity. This sheds new light on how we conceptually think about and therapeutically target neuroinflammatory and neurodegenerative diseases.

overall_design: Lymph nodes were immunized with either MBP peptides alone or MOG and MBP peptides together. The inguinal lymph nodes are then harvested using MBP-specific MHC II tetramers. Both tetramer positive and negative populations were collected; thus resulting in 4 samples in total.

first: Min Woo

last: Kim

first: Wenqing

first: Cheryl

last: Lichti

first: Xingxing

first: Taitea

last: Dykstra

first: Jay

last: Cao

first: Igor

last: Smirnov

first: Pavle

last: Boskovic

first: Denis

last: Kleverov

first: Andrea

last: Salvador

first: Antoine

last: Drieu

first: Kyungdeok

first: Susan

last: Blackburn

first: Claire

last: Crewe

first: Maxim

middle: N

last: Artyomov

first: Emil

last: Unanue


title: RNA helicase DDX10 is Required for Pluripotency of Stem Cells by Regulating 18S rRNA Processing

city: guangzhou

first: gongcheng

last: Hu

state: guangdong

address: 190 Kai Yuan Avenue, Science Park, Guangzhou, China

institute: Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences

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

overall_design: Refer to individual Series


title: Mapping Ischemia Reperfusion Inflammatory Responses in Human Lung Transplantation at Single-Cell Resolution

first: Aaron

last: Wong

summary: Ischemia reperfusion (IR) is an unavoidable step of organ transplantation. IR-induced injury constrains the number of donor lungs used for transplant. Here we performed longitudinal single-cell RNA sequencing (scRNA-seq) from human lungs of six individuals who underwent lung transplantation. Lung biopsies were collected after cold preservation and 2-hour reperfusion for each individual resulting in the profiling of 108,613 cells in total.

overall_design:
Here we performed longitudinal single-cell RNA sequencing (scRNA-seq) from human lungs of six individuals who underwent lung transplantation. Lung biopsies were collected after cold preservation and 2-hour reperfusion for each individual.
Submitter states that raw data will be uploaded to the European Genome-phenome Archive (EGA) to protect patient privacy.

first: Jonathan

last: Yeung

first: Shaf

last: Keshavjee

first: Mingyao

last: Liu


title: Decoding the interplay between m6A modification and stress granule stability by live-cell imaging

city: Shenzhen

first: Qin

last: Peng

address: Gaoke Innovation Center

institute: Shenzhen Bay Laboratory

summary: We present a spatiotemporal m6A imaging system (SMIS) that can monitor the m6A modification and the translation of mRNAs with high specificity and sensitivity in a single live cell.  The SMIS system contains a m6A-modified reporter mRNA and fluorescence resonance energy transfer (FRET) biosensors.  To quantify the methylation viariation achieved at m6A sites in the reporter mRNA after STM2457 treatment, a classical METTL3 inhibitor,  we performed m6ACE-seq to measure the relative m6A level with single-base-resolusion.

overall_design: HeLa cells ovexpressing SMIS-WT system, namely SMIS-WT cells,  were preteated with DMSO or STM2457 for 3 hours and incubated with DMSO or STM2457 (60μM) and DOX (1μg/mL) for 12 hours. Then, the cells was harvested and lyzed for total RNA extraction and mRNA purification. The purified mRNA was further handled according to the protocol of m6ACE-seq that is previously published on nature communication (PMID: 31822664).

first: Yi

last: Zhang

first: Wee Siong

last: Goh

first: Qianqian

last: Li


title: Immunotherapies that repolarize macrophages and regulatory T cells enhance the effect of chemotherapy in high-grade serous ovarian cancer

city: London

first: Samar

last: Elorbany

address: Barbican

department: Tumour Microenvironment

institute: Queen Mary University of London

summary:
We conducted single-cell RNA sequencing (scRNAseq) of tumor-infiltrating immune cells in high-grade serous ovarian cancer (HGSOC) omental biopsies to identify potential targets that could enhance response to neo-adjuvant chemotherapy (NACT). Analysis of 64,097 cells identified NACT-induced overexpression of stabilin-1 (clever-1) on macrophages and FOXP3 in Tregs and these were validated at the protein level. STAB1 inhibition in vitro induced anti-tumor macrophages. FOXP3 anti-sense oligonucleotide (FOXP3-ASO), repolarised Tregs to an effector T cell phenotype.
ScRNAseq on 69,781 cells from an HGSOC syngeneic mouse model recapitulated the patients’ data. Combining chemotherapy with anti-stabilin1 antibody and/or Foxp3-ASO significantly increased survival of mice with established peritoneal disease in two HGSOC syngeneic models and progression-free survival in a third model. Long-term survivors (300 days+) were resistant to tumor rechallenge. Anti-stabilin1 antibody enriched the tumors with CXCL9+ macrophages and Foxp3-ASO increased TBET cell infiltration. Our results suggest targeting these molecules may improve chemotherapy response in patients.

overall_design: RNA sequencing on whole omental tumors from control and treated mice, 3-4 replicates per treatment arm. The included arms are control (n=3), carboplatin and paclitaxel treated (n=3), Anti-stab1 antibody treated (n=3), carboplatin and paclitaxel plus anti-stab1 antibody (n=3), Foxp3-ASO treated (n=3), carboplatin and paclitaxel plus Foxp3-ASO(n=4), carboplatin and paclitaxel plus anti-stab1 antibody and Foxp3-ASO(n=4), Foxp3-ASO plus anti-stab1 antibody (n=3)

first: Chiara

last: Berlato

first: Larissa

last: Carnevalli

first: Eleni

last: Maniati

first: Jun

last: Wang

first: Simon

middle: T

last: Barry

first: Ranjit

last: Manchanda

first: Julia

last: Kzhyshkowska

first: Frances

last: Balkwill


title: Endogenous self-peptides guard CNS immune privilege

last: Kipnis

overall_design: C57BL/6J male mice were immunized either with MOG35-55 or MOG35-55 along with MBP160-175 (n = 3 per group) and 10 days later cells from the draining (inguinal) lymph nodes were harvested. Cells were isolated as described in “single cell isolations”, and sorted to capture those negative for DAPI (live cells), Thy1.2 and TCRß to capture both CD4+ and CD8+ T cells for single-cell sequencing.

first: Min Woo

last: Kim

first: Wenqing

last: Gao

first: Cheryl

last: Lichti

first: Xingxing

last: Gu

first: Taitea

last: Dykstra

first: Jay

last: Cao

first: Igor

last: Smirnov

first: Pavle

last: Boskovic

first: Denis

last: Kleverov

first: Andrea

last: Salvador

first: Antoine

last: Drieu

first: Kyungdeok

first: Susan

last: Blackburn

first: Claire

last: Crewe

first: Maxim

last: Artyomov

first: Emil

last: Unanue


title: The transcriptomic differences between NRP2-high expressing and NRP2-low expressing cells in BT549

city: Worcester

first: Yong-Xu

last: Wang

address: 364 Plantation Street

department: MCCB

institute: UMass Chan Medical School

summary: NRP2 is a critical protein that has been shown to play a role in several cancer stem cell properties such as tumorigencity, self-renewal, and therapy resistance. We wanted to explore the differences in signaling for cells that express NRP2 on their cell surface compared to cells that do not express it on the cell surface.

overall_design: We used flow cytometry to sort the populations with BT549 that express NRP2 (NRP2-high) and those that do not (NRP2-low) and then proceeded with RNA-sequencing

first: Ayush

last: Kumar

first: Hira

last: Lal Goel


title: Transcriptomic differences between radioresistant 4T1 and parental 4T1

first: Rui

last: Li

address: 364 Plantation Street, LRB

institute: University of Massachusetts Medical School

summary: A radioresistant 4T1 cell line, a mouse TNBC cell lines, was developed by giving a total dose of 50Gy (2gyx5, 4Gyx3, 6Gyx3, and 10Gyx1) to the parental cell line. The trancriptomic changes that occur include enhanced antioxidant response, increase in DNA repair pathways, and invasion/metastasis features.

overall_design: RNA-seq of 4T1 radioresistant vs parental cell lines

first: Arthur

last: Mercurio


title: Proximal tubule cell maturation rate and function are controlled by PPARα signaling in kidney organoids.

city: Kobe

first: Yoshiki

last: Sahara

state: Hyogo

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

department: Center for Biosystems Dynamics Research (BDR)

country: Japan

institute: RIKEN

summary: Kidney organoids derived from human iPSCs have cell heterogeneity of maturation. We used single-cell RNA sequencing (scRNAs-seq) to extract the factor related to proximal tubule maturation. Using the kidney organoids cultured with the factors that accelerate the maturation speed of proximal tubules extracted by the aforementioned method, we examined whether the maturity of the proximal tubules increases within the same induction period.

overall_design: Kidney organoids at day 15, 19, 23, 27 and 31 were used for analysis to extract the factor lerated to proximal tubule maturation. To confirm the proximal tubule maturation, kidney organoids were treated with vehicle control and PPARA agonists from day 15 to day 23 and sampled on day 23.

first: Chie

last: Fukui

first: Yuki

last: Kuniyoshi

first: Minoru

last: Takasato


title: A Defective Splicing Machinery Promotes Senescence through MDM4 Alternative Splicing

city: Sherbrooke

first: Mathieu

last: Deschênes

state: Québec

address: 3201, rue Jean-Mignault

department: Microbiologie et Infectiologie

institute: Université de Sherbrooke

summary: Defects in the splicing machinery have been implicated in various diseases, including cancer. We identified a general reduction in the expression spliceosome components and splicing regulators in human cell lines undergoing replicative, stress-induced and telomere uncapping-induced senescence. Supporting the notion that defective splicing contributes to senescence, we showed that the splicing inhibitors herboxidiene and pladienolide B induce senescence both in normal and cancer cell lines. Furthermore, the individual depletion of several spliceosome components also promoted senescence. We noted an alternative splicing shift in MDM4, from the full-length MDM4-FL variant to MDM4-S during replicative and stress-induced senescence. This shift was replicated by splicing inhibition and the depletion of individual spliceosome components. Importantly, decreasing the level of MDM4-FL promoted senescence, while cell survival was improved both by reducing the level of MDM4-FL and by overexpressing MDM4-S. Overall, our work establishes that defects in the splicing machinery alter the alternative splicing of MDM4 to promote senescence.

overall_design:
The BJ cell line was cultured until reaching 63 population doubling. At this point they were identified as undergoing replicative senescence using common senescence markers.
Replicative senescent BJ cells and their young counterpart were then plated in triplicate, 24h before RNA extraction. After dosing and QC, only the best 2 of the 3, for each cell population, were kept for poly-A RNA sequencing (2 experimental samples in duplicate (4 samples total)).

last: Durand

first: Marc-Alexandre

last: Olivier

first: Alicia

last: Pellerin-Viger

first: Francis

last: Rodier

first: Benoit

last: Chabot


title: MHC-II peptidome of the CNS reveals endogenous guardian peptides

first: Jonathan

last: Kipnis

summary: The central nervous system (CNS), despite the presence of strategically positioned anatomical barriers designed to protect it, is not entirely isolated from the immune system. In fact, it remains physically connected to and can be influenced by the peripheral immune system. How the CNS retains such responsiveness while maintaining “immune privilege” remains an outstanding conundrum. In searching for molecular cues that derive from the CNS and allow its direct communication with the immune system, we discovered a repertoire of CNS-derived endogenous guardian peptides presented on major histocompatibility complex (MHC) II molecules at the CNS borders. During homeostasis, a preponderance of these guardian peptides were found to be bound to MHC II molecules throughout the path of lymphatic drainage from the brain to its surrounding meninges and its draining cervical lymph nodes. With neuroinflammatory disease, however, the presentation of guardian peptides was diminished. Fascinatingly, boosting the presence of these guardian peptides reinforced a population of suppressor CD4+ T cells and significantly reduced CNS autoimmune disease. This unexpected discovery of CNS-derived autoimmune guardian peptides may be the molecular key adapting the CNS to receive information and to maintain continuous dialogue with the immune system while balancing overt autoreactivity. This sheds new light on how we conceptually think about and therapeutically target neuroinflammatory and neurodegenerative diseases.

overall_design: Mice were immunized with MOG(35-55) or MOG(35-55) and MBP(160-175) and then 10 days after immunization cells from the draining (inguinal) lymph nodes were harvested. Cells were sorted based on DAPI (live cells) as well as Thy1.2 and TCRb to get T cells from which we get both CD4 and CD8. 3 male C57BL/6J mice were used for each group.

first: Min

middle: K

last: Woo

first: Taitea

last: Dykstra


title: ATLAS-seq: a microfluidic single-cell TCR screen for antigen-reactive TCRs

city: Philadelphia

first: Siwei

last: Luo

state: Pennsylvania

address: 3401 Civic Center Blvd.

institute: Children’s Hospital of Philadelphia

summary: Discovering antigen-reactive T cell receptors (TCRs) is central to developing effective engineered T cell immunotherapies. However, the conventional technologies for isolating antigen-reactive TCRs (i.e., major histocompatibility complex (MHC) multimer staining) focus on high-affinity interactions between the TCR and MHC-antigen complex, and may fail to identify TCRs with high efficacy for activating T cells. Here, we develop a microfluidic single-cell screening method for antigen-reactive T cells named ATLAS-seq (Aptamer-based T Lymphocyte Activity Screening and SEQuencing). This technology isolates and characterizes activated T cells via an aptamer-based fluorescent molecular sensor, which monitors the cytotoxic cytokine IFNγ secretion from single T cells upon antigen stimulation, followed by single-cell RNA and single-cell TCR sequencing. We use ATLAS-seq to screen TCRs reactive to cytomegalovirus (CMV) or prostate specific antigen (PSA) from peripheral blood mononuclear cells (PBMCs). ATLAS-seq identifies distinct TCR clonotype populations with higher T cell activation levels compared to TCRs recovered by MHC multimer staining. Select TCR clonotypes from ATLAS-seq are more efficient in target cell killing than those from MHC multimer staining. Collectively, ATLAS-seq provides an efficient and broadly applicable technology to screen antigen-reactive TCRs for engineered T cell immunotherapy.

overall_design: Briefly, we used the microfluidic chip to generate droplets co-encapsulating aptamer decorated single CD8+ T cell and antigen-loaded aAPCs. Generated droplets were collected and incubated for 2 days to allow T-cell activation by antigen-loaded aAPCs. An activated T-cell containing cognate TCR for the target antigen pMHC may release IFNγ molecules into the same droplet which, in turn, bind to the IFNγ specific aptamer beacons on the same T-cell surface and emit Cy3 fluorescence signal. The droplet emulsions were then broken to release aptamer labelled CD8+ T cells for fluorescent sorting using flow cytometry. CD8+Cy3high T cells were then collected and subject to single cell TCR and RNA sequencing (scTCR-seq and scRNA-seq) prepared by 10X Genomics single-cell sequencing system.

first: Amber

last: Notaro

first: Lan

last: Lin


title: Immunotherapies that repolarize macrophages and regulatory T cells enhance the effect of chemotherapy in high-grade serous ovarian cancer (Mouse)

first: Samar

last: Elorbany

overall_design: Single cell RNA sequencing on whole omental tumours from HGS2 mouse model. Tumours were harvested from three control mice and 3 mice one week after the third dose of carboplatin and paclitaxel.

first: Chiara

last: Berlato

first: Larissa

middle: S

last: Carnevalli

first: Eleni

last: Maniati

first: Jun

last: Wang

first: Simon

last: Barry

first: Ranjit

last: Manchanda

first: Julia

last: Kzhyshkowska

first: Frances

last: Balkwill


title: Perturbation-Specific Transcriptional Mapping for unbiased target elucidation of antibiotics

city: Boston

first: Keith

last: Romano

address: 185 Cambridge St.

department: Molecular Biology

institute: Massachusetts General Hospital

summary: We developed an unbiased strategy for MOA prediction, called Perturbation-Specific Transcriptional Mapping (PerSpecTM), in which large-throughput expression profiling of wildtype or hypomorphic mutants, depleted for essential targets, enables a computational strategy to address this challenge. We applied PerSpecTM to perform reference-based MOA prediction based on the principle that similar perturbations, whether small molecule or genetic, will elicit similar transcriptional responses. Using this approach, we elucidated the MOAs of three new molecules with activity against Pseudomonas aeruginosa by mapping their expression profiles to those of a reference set of antimicrobial compounds with known MOAs. We also show that transcriptional responses to small molecule inhibition maps to those resulting from genetic depletion of essential targets by CRISPRi by PerSpecTM, demonstrating proof-of-concept that correlations between expression profiles of small molecule and genetic perturbations can facilitate MOA prediction when no chemical entities exist to serve as a reference. Empowered by PerSpecTM, this work lays the foundation for an unbiased, readily scalable, systematic reference-based strategy for MOA elucidation that could transform antibiotic discovery efforts.

overall_design: We obtained RNAseq data from Pseudomonas aeurginosa (PA14) under various drug treatments. The majority of the data were taken at 90 minutes after drug exposure, but a subset of the data include 30, 60, 90, and 120 minute time points. There are at least three replicates per condition. RNAseq data from CRISPRi strains are also included, at 270 minutes after arabiniose induction.

first: Josephine

last: Bagnall

first: Thulasi

last: Warrier

first: Jaryd

last: Sullivan

first: Kristina

last: Ferrara

first: Marek

last: Orzechowski

first: Phuong

last: Nguyen

first: Kyra

last: Raines

last: Livny

first: Noam

last: Shoresh

first: Deborah

last: Hung


title: Taste Receptor Type 1 Member 3 Mediates Cognitive Impairment in Diet-Induced Obese Mice

city: Seoul

first: Keon-Hee

last: Lee

address: 1, Gwanak-ro, Gwanak-gu, Seoul, Republic of Korea

department: Food and Nutrition

institute: Seoul National University

summary:
To investigate whether taste receptor type 1 member 3 (TAS1R3) might serve as a key modulator of cognitive impairment led by WD-induced obesity, male Tas1r3-wild type (WT) and knockout (KO) mice were randomized to receive either a normal diet (ND) or western diet (WD) for 18 weeks.
We performed gene expression profiling analysis using data obtained from RNA-seq of hippocampus of two WT mice fed a WD (WT-WD) and two KO-WD mice.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for hippocampus of WT-WD and KO-WD mice.

first: Woo-Jeong

last: Shon

first: Dong-Mi

last: Shin


title: Unraveling potential gene biomarkers for dengue infection through RNA sequencing

status: Public on Oct 10 2024

city: Shah Alam

first: Jeyanthi

last: Suppiah

address: Jalan Setia Murni U13/52, Setia Alam

department: Virology Unit

country: Malaysia

institute: Institute for Medical Research, National Institutes of Health Malaysia

summary: This pilot study, aimed to profile the host transcriptome as a potential strategy for identifying specific biomarkers for dengue prediction and detection. High-throughput RNA-sequencing (RNA-seq) was employed to generate host transcriptome profiles in 16 dengue patients and 10 healthy controls. Differentially expressed genes (DEGs) were identified in patients with severe dengue and those with dengue with warning signs compared to healthy individuals. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to elucidate the functions of upregulated and downregulated genes. Compared to healthy controls, 6466 genes were significantly differentially expressed (P < 0.05) in the dengue with warning signs group and 3082 genes in the severe dengue group, with over half being upregulated. The major KEGG pathways implicated included transport and catabolism (14.4%-16.3%), signal transduction (6.6%-7.3%), global and overview maps (6.7%-7.1%), viral diseases (4.6%-4.8%), and the immune system (4.4%-4.6%). Several genes exhibited consistent and significant upregulation across all dengue patients, regardless of severity: Interferon alpha inducible protein 27 (IFI27), Potassium Channel Tetramerisation Domain Containing 14 (KCTD14), Syndecan 1 (SDC1), DCC netrin 1 receptor (DCC), Ubiquitin C-terminal hydrolase L1 (UCHL1), Marginal zone B and B1 cell specific protein (MZB1), Nestin (NES), C-C motif chemokine ligand 2 (CCL2), TNF receptor superfamily member 17 (TNFSF17), and TNF receptor superfamily member 13B (TNFRSF13B). Further analysis revealed potential biomarkers for severe dengue prediction, including TNF superfamily member 15 (TNFSF15), Plasminogen Activator Inhibitor-2 (SERPINB2), motif chemokine ligand 7 (CCL7), aconitate decarboxylase 1 (ACOD1), Metallothionein 1G (MT1G) and Myosin Light Chain Kinase (MYLK2), which were expressed 3.5 times, 2.9 times, 2.3 times, 2.1 times, 1.7 times, and 1.4 times greater, respectively, than dengue patients exhibiting warning signs. The identification of these host biomarkers through RNA-sequencing holds promising implications and potential to augment existing dengue detection algorithms, contributing significantly to improved diagnostic and prognostic capabilities.

overall_design: Seven milliliters (7ml) of human whole blood were collected in sodium heparin vacutainer.Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples using Ficoll-Paque density gradient centrifugation. Total RNA was extracted using the innuPREP RNA mini kit 2.0 (Analytical Jenna). Enrichment of mRNA was performed using Dynabeads mrRNA Purification Kit (Invitrogen) and library preparation was carried out using MGIEasy RNA Library Prep Set (MGI). After library assessment, sequencing was performed on the DNBSEQ-G400 platform (MGI).