title: RNA sensing induced by chromosome missegregation augments anti-tumor immunity I

overall_design: We invetigated gene expression alteration following pulsed MPS1 treatment in H1944 and H647 cells.


title: The identification of a SARs-CoV2 S1 protein derived peptide with super-antigen-like stimulatory properties on T-cells

status: Public on Nov 12 2024

city: Montreal

first: Christopher

middle: E.

last: Rudd

state: Quebec

address: 5345 Boulevard de l'Assomption

department: Département of Médecine

country: Canada

institute: Université de Montréal

type: Expression profiling by high throughput sequencing

summary: Severe COVID-19 is characterized by a cytokine-mediated acute respiratory distress syndrome resembling toxic shock syndrome. We identified a SARS-CoV-2 S1 spike peptide (P3) with sequence homology to bacterial super-antigens. Computational modelling revealed P3 binding to MHC class I/II and TCR regions that partially overlap with staphylococcal enterotoxin B (SEB) and enterotoxin H (SEH) binding sites. In vitro, P3 potently activated 25-40% of human CD4 and CD8 T cells, accompanied by the increased expression of effector molecules IFNg and granzyme B. In vivo, P3 peptide administration to mice induced an inflammatory response characterized by elevated levels of proinflammatory cytokines, IL1b, IL6 and TNFa. Further, the P3 peptide promoted a more robust in vitro immune response of T-cells from COVID-19 patients with severe disease, as evidenced by increased CD69 and IFNg expression. Consistent with its super-antigenic properties, P3 preferentially also expanded T cells bearing restricted TCR Vα and Vβ chain repertoires in vitro. Overall, the homology of the P3 peptide to several endogenous mammalian proteins suggests the potential for the future discovery of related peptides that may contribute to inflammation and autoimmune disorders in humans.

overall_design: To study the effect of in vitro P3 peptide stimulation on human CD8 TCR clonality with CD8 T cells isolated from healthy donor PBMC.

first: Thai Hien

last: Tu

first: Fatima-Ezzahra

last: Bennani

first: Nasser

last: Masroori

first: Chen

last: Liu

first: Atena

last: Nemati

first: Nicholas

last: Rozza

first: Amichai Meir

last: Grunbaum

first: Richard

last: Kremer

first: Catalin

last: Mihalcioiu

first: Denis-Claude

last: Roy

middle: E


title: RNA sensing induced by chromosome missegregation augments anti-tumor immunity V

city: Tokyo

first: Shunsuke

last: Kitajima

address: 3-8-31 Ariake, Koto

country: Japan

institute: Japanese Foundation for Cancer Research

summary: We demonstrate that dsRNA is significantly accumulated in cancer cells following pharmacologic induction of micronuclei, stimulating MAVS-mediated dsRNA sensing in conjunction with the cGAS/STING pathway. Activation of cytosolic dsRNA sensing co-operates with dsDNA sensing to up-regulate immune cell migration and antigen-presenting machinery. Tracing of dsRNA-sequence identity revealed that dsRNA-forming transcripts are predominantly generated from non-exonic regions, particularly in locations proximal to genes exhibiting high chromatin accessibility. Activation of this pathway by pulsed Monopolar Spindle 1 (MPS1) kinase inhibitor therapy, which potently induces micronuclei formation, thus induces cytoplasmic dsRNA-sensing and promotes anti-tumor immunity mediated by type I interferon signaling and cytotoxic lymphocyte activation in vivo. Taken together, these findings reveal a novel aspect of the dsRNA-sensing pathway in enhancing the anti-tumor efficacy of drugs that promote genomic instability and micronuclei formation, facilitating cancer immunogenicity.

overall_design: We invetigated STING and/or MAVS-dependent gene expression alteration following pulsed MPS1 treatment in H1944 cells.

first: Nobunari

last: Sasaki

first: Mizuki

last: Homme


title: Morphoregulatory ADD3 underlies glioblastoma growth and formation of tumor-tumor connections

city: Milan

first: Nereo

last: Kalebic

address: V.le Rita Levi-Montalcini, 1

country: Italy

institute: Human Technopole

summary: Glioblastoma is a major unmet clinical need characterized by striking inter- and intra-tumoral heterogeneity and a population of glioblastoma stem cells (GSCs), conferring aggressiveness and therapy resistance. GSCs communicate through a network of tumor-tumor connections (TTCs), including nanotubes and microtubes, promoting tumor progression. However, very little is known about the mechanisms underlying TTC formation and overall GSC morphology. As GSCs closely resemble neural progenitor cells during neurodevelopment, we hypothesised that GSCs’ morphological features affect tumour progression. We identified GSC morphology as a new layer of tumoral heterogeneity with important consequences on GSC proliferation. Strikingly, we showed that the neurodevelopmental morphoregulator ADD3, is sufficient and necessary for maintaining proper GSC morphology, TTC abundance and cell cycle progression as well as required for cell survival. Remarkably, both the effects on cell morphology and proliferation depend on the stability of actin cytoskeleton. Hence, cell morphology and its regulators play a key role in tumor progression by mediating cell-cell communication. We thus propose that GSC morphological heterogeneity holds the potential to identify new therapeutic targets and diagnostic markers.

overall_design: Glioblastoma stem cells have been treated with ADD3 overexpression or CRISPR-Cas9 mediated knock-out to understand the role of ADD3 on cell morphology and tumour growth.

first: Carlotta

last: Barelli

first: Raffaele M.

last: Iannuzzi

first: Francesco

last: Iorio


title: In vitro to in vivo extrapolation modeling to facilitate the integration of transcriptomics data into genotoxicity assessment

status: Public on Nov 13 2024

city: Ottawa

first: Matthew

last: Meier

state: Ontario

address: 251 Sir Frederick Banting Driveway

department: Health Canada

institute: Government of Canada

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

overall_design: Refer to individual Series


title: Raw data for: Epigenetic Age and Long-Term Cancer Risk Following a Stroke

city: Barcelona

first: Joan

address: Carrer del Doctor Aiguader, 88

country: Spain

institute: Hospital del Mar Research Institute

type: Methylation profiling by genome tiling array

summary: In this study, we investigated whether stroke patients who later developed cancer exhibited an accelerated epigenetic age compared to those who did not. A total of 648 stroke patients were followed for 15 years, during which 83 individuals were diagnosed with cancer. Biological age (B-age) was estimated from DNA methylation data derived from whole blood samples collected within 24 hours of stroke onset. These samples were analyzed using the 450K or EPIC Illumina Beadchip arrays. We employed several epigenetic clocks to calculate B-age, including the Hannum, Horvath, PhenoAge, ZhangBLUP, ZhangEN, and the mitotic epiTOC clocks.    After adjusting for multiple testing, competing risks, and key confounders, we found that patients who developed cancer were biologically older, as measured by the Hannum clock.    This repository provides the raw idat files and processed DNA methylation values.

overall_design: We studied whether biological age, as obtained from DNA methylation, was associated with incidence of cancer in patients with stroke. To that aim, we followed-up a cohort of 648 patients with stroke within 15 years, of whom 83 developed cancer. DNA was extracted from whole peripheral blood collected in 10mL EDTA tubes during the first 24h after stroke onset. DNA methylation was obtained in two different batches. In one (n=410) we measured DNA methylation via the Human Methylation 450K Beadchip (Illumina, Netherlands, Eindhoven) in two different technical runs, while in the second batch (N=238) we used the Infinium Methylation EPIC Beadchip (Illumina, Netherlands, Eindhoven).

last: Jiménez-Balado

first: Antoni

last: Suárez-Pérez

first: Jordi

last: Jiménez-Conde

first: Ángel

last: Ois


title: Perivascular B cells link intestinal angiogenesis to immunity and to the gut-brain axis during neuroinflammation

city: Epalinges

first: Caroline

last: Pot

address: Chemin des Boveresses 155

department: Department of Clinical Neurosciences

country: Switzerland

institute: Lausanne University Hospital

summary: Disruption of gut barrier function and intestinal immune cell homeostasis are increasingly considered critical players in pathogenesis of extra-intestinal inflammatory diseases, including multiple sclerosis (MS) and its prototypical animal model, theexperimental autoimmune encephalomyelitis (EAE). Breakdown of epithelial barriers increases intestinal permeability and systemic dissemination of microbiota-derived molecules. However,whetherthe gut-vascular barrier (GVB) is altered during EAE has not been reported. Here, we demonstrate that endothelial cell proliferation and vessel permeability increase before EAE clinical onset, leadingto vascular remodelingand expansion of intestinal villi capillary bed during disease symptomatic phasein an antigen-independent manner. Concomitant to onset of angiogenesis observed prior to neurological symptoms, we identify an increase of intestinal perivascular immune cells characterized by the surface markerlymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE-1). LYVE-1+is expressed more frequently on B cells that show high levels of CD73 and have proangiogenic properties. B cell depletion was sufficient tomitigate enteric blood endothelial cell proliferation following immunization for EAE. In conclusion, we propose that altered intestinal vasculature driven by a specialized LYVE-1+B cell subset promotes angiogenesis and that loss of GVB function is implicated in EAE development and autoimmunity.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for small intestinal (jejunum section) B lymphocytes isolated from mice immunized for experimental autoimmune encephalomyelitis (EAE) at 7 days post-immunization (DPI) depending on their expression of the surface marker lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE-1). Intestinal B cells were FACS sorted as viable CD45+ CD11b- F4/80- CD11c- B220+ LYVE-1+ and viable CD45+ CD11b- F4/80- CD11c- B220+ LYVE-1- from 6 mice.

first: Benjamin

last: Peter

first: Jessica

last: Rebeaud

first: Solenne

last: Vigne

first: Valentine

last: Bressoud

first: Nicholas

last: Phillips

first: Florian

last: Ruiz

first: Tatiana

middle: V

last: Petrova

first: Jeremiah

last: Bernier-Latmani


title: Microglia regulate cortical remyelination via ΤNFR1-dependent phenotypic polarization [RNA-Seq]

city: Athens

first: Lesley

last: Probert

state: Attiki

address: Vassilisis Sofias 127

department: Immunology

country: Greece

institute: Hellenic Pasteur Institute

summary: Microglia are strongly implicated in demyelinating neurodegenerative diseases with increasing evidence for roles in protection and healing, but the mechanisms that control CNS remyelination are poorly understood. Here we show that microglia-specific deletion of TNFR1 and pharmacological inhibition of soluble TNF (solTNF) or downstream IL-1R allow maturation of highly activated disease-associated microglia with increased size and myelin phagocytosis capacity that accelerate cortical remyelination and motor recovery. Single cell transcriptomic analysis of cortex at disease onset reveal that solTNF inhibition enhances reparative IL-10-responsive, while preventing damaging IL-1-related signatures of disease-associated microglia. Longitudinal brain transcriptome analysis through disease reveal earlier recovery upon therapeutic loss of microglia TNFR1. Functional relevance of microglia inflammatory polarization pathways for disease is validated in vivo. Furthermore, disease-state microglia producing downstream IL-1/IL-18/NLRP3/CASP1 targets are identified in human demyelinating lesions. Overall, redirecting disease microglia polarization by targeting cytokines is a potential approach for improving CNS repair in demyelinating disorders.

overall_design: Brain transcriptome at hallmark disease timepoints of cuprizone demyelination and remyelination, and the effect of microglia TNFR1 deletion in remyelination, by whole brain RNA-Seq analysis from naïve (CPZ0; N), peak of demyelination (CPZ5; DM), and remyelination (CPZ6+1; RM) TNFR1ff controls (backcrossed onto C57BL/6) , and from remyelination mgTNFR1KO (CPZ6+1; RM-KO) (Cx3cr1-CreER.Tnfr1ff) mice after therapeutic tamoxifen-induced  microglia-specific TNFR1 deletion. (n=7 samples/group)

first: Athena

last: Boutou

first: Ilias

last: Roufagalas

first: Katerina

last: Politopoulou

first: Spyros

last: Tastsoglou

first: Giorgos

last: Skoufos

first: Artemis

middle: G

last: Hatzigeorgiou


title: Stereo-seq on human prefrontal cortex from six AD and six normal samples

city: New Orleans

first: Yun

last: Gong

address: 3720 Clio St

country: USA

institute: Tulane University

summary: Aging significantly elevates the risk for Alzheimer’s disease (AD), contributing to the accumulation of AD pathologies, such as amyloid-β (Aβ), inflammation, and oxidative stress. The human prefrontal cortex (PFC) is highly vulnerable to the impacts of both aging and AD. Unveiling and understanding the molecular alterations in PFC associated with normal aging (NA) and AD is essential for elucidating the mechanisms of AD progression and developing novel therapeutics for this devastating disease. In this study, for the first time, we employed a cutting-edge spatial transcriptome platform, STOmics® SpaTial Enhanced Resolution Omics-sequencing (Stereo-seq), to generate the first comprehensive, subcellular resolution spatial transcriptome atlas of the human PFC from six AD cases at various neuropathological stages and six age, sex, and ethnicity matched controls. Our analyses revealed distinct transcriptional alterations across six neocortex layers, highlighted the AD-associated disruptions in laminar architecture, and identified changes in layer-to-layer interactions as AD progresses. Further, throughout the progression from NA to various stages of AD, we discovered specific genes that were significantly upregulated in neurons experiencing high stress and in nearby non-neuronal cells, compared to cells distant from the source of stress. Notably, the cell-cell interactions between the neurons under the high stress and adjacent glial cells that promote Aβ clearance and neuroprotection were diminished in AD in response to stressors compared to NA. Through cell-type specific gene co-expression analysis, we identified three modules in excitatory and inhibitory neurons associated with neuronal protection, protein dephosphorylation, and negative regulation of Aβ plaque formation. These modules negatively correlated with AD progression, indicating a reduced capacity for toxic substance clearance in AD subject samples. Moreover, we have discovered a novel transcription factor, ZNF460, that regulates all three modules, establishing it as a potential new therapeutic target for AD. Overall, utilizing the latest spatial transcriptome platform, our study developed the first transcriptome-wide atlas with subcellular resolution for assessing the molecular alterations in the human PFC due to AD. This atlas sheds light on the potential mechanisms underlying the progression from NA to AD.

overall_design: Stereo-seq on the BA10 area from six AD and six normal samples


title: Microglia regulate cortical remyelination via ΤNFR1-dependent phenotypic polarization [scRNA-Seq]

overall_design: Single-cell RNA-Sequencing (scRNA-Seq) data from 48,265 nuclei isolated from the cortex of untreated healthy naïve (CPZ0-Naive), and of cuprizone week 3 (demyelination onset, microgliosis, astrocytosis) from prophylactic soluble TNF-inhibitor XPro1595- (CPZ3-XPro1595) and saline-treated (CPZ3-Saline) C57BL/6 mice  (n=15 samples/group)

first: Maya

last: Abouzeid

first: Jeong Hun

last: Ko

first: Michael

middle: R

last: Johnson


title: Highly multiplexed spatial transcriptomics in bacteria

city: Boston

first: Jeffrey

last: Moffitt

state: MA

address: 200 Longwood Ave

department: Program in Cellular and Molecular Medicine

institute: Boston Children's Hospital

summary: Single-cell decisions made in complex environments underlie many bacterial phenomena. Image-based, transcriptomics approaches offer an avenue to study such behaviors, yet these approaches have been hindered by the massive density of bacterial mRNA. To overcome this challenge, we combine 1000-fold volumetric expansion with multiplexed error robust fluorescence in situ hybridization (MERFISH) to create bacterial-MERFISH, a method enabling high-throughput, spatially resolved profiling of thousands of operons within individual bacteria. Using bacterial-MERFISH, we dissect the response of E. coli to carbon starvation, systematically map subcellular RNA organization, and chart the adaptation of B. thetaiotaomicron to micron-scale niches in the mammalian colon. We envision bacterial-MERFISH could prove useful in the study of bacterial single-cell decisions made in diverse, spatially structured, and native environments.

sample_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: To measure and validate the performance of bacterial-MERFISH in Escherichia coli (E. coli), we correlated our MERFISH data with bulk RNA-seq of matching samples. We grew and collected WT E. coli MG1655 at OD600=0.33 in Lennox LB, and in diauxic media (0.025% w/v glucose and 0.025% w/v xylose in MOPS minimal media) at OD600=0.18 in the glucose growth phase, and 0.47 during xylose growth. We extracted total RNA following the RNAsnap protocol (Stead et al. 2012). The total RNA was ribosomally depleted and sequenced as 150bp paired-end reads by the Harvard Medical School Biopolymers Core (diauxic samples) and Azenta (LB sample).

first: Ari

last: Sarfatis

first: Yuanyou

last: Wang

first: Nana

last: Twumasi-Ankrah


title: Single cell RNA sequencing of mutant bone marrow stromal cells lacking HIF2 and controls.

city: Philadelphia

first: Ernestina

last: Schipani

state: PA

address: 310A Stemmler Hall, 3450 Hamilton Walk

department: McKay Orthopaedic Research Laboratory

institute: University of Pennsylvania

summary: Osteoblast lineage cells within adult bone marrow are exposed to varying oxygen levels. Hypoxia-Inducible Factor-1α (HIF1α) and HIF2α are pivotal in the cellular response to hypoxia. Our study explores the effects of targeted HIF2α deletion in mesenchymal progenitors and their descendants. We demonstrate that HIF2α acts as a negative regulator of osteoblastogenesis and bone mass accrual. Therapeutically targeting HIF2α could be beneficial in treating low bone mass conditions, such as those observed in chronic diseases, osteoporosis, or during aging. To elucidate the mechanisms underlying the increased bone mass resulting from HIF2α loss, we performed single-cell RNA sequencing (scRNA-seq) on bone marrow stromal cells from both mutant mice lacking HIF2α in PRX1 lineage cells and control mice.

overall_design: To investigate how the deletion of HIF2 affects the transcriptional profile of bone marrow stromal cells, we conditionally knocked out HIF2 in uncommitted mesenchymal progenitors and their descendants using PRX1-Cre driver, and a mouse line homozygous for a floxed Hif2 allele (HIF2f/f). PRX1-Cre mice express Cre recombinase in mesenchymal cells that give rise to chondrocytes and osteoblasts in long bones and calvaria. The Ai14 reporter was used to recognize Ai14-positive bone marrow stromal cells. PRx1-Cre+/Ai14+/-/HIF2f/+ mice were crossed with HIF2f/f or C57 mice to generate PRx1-Cre+/Ai14+/-/HIF2f/f mutants and PRx1-Cre+/Ai14+/-/HIF-/- control littermates. scRNA-seq was performed on bone marrow stromal cells isolated from the long bones hindlimbs of 12-week-old mice.

first: Giulia

last: Lanzolla

first: Elena

last: Sabini


title: Deciphering the effect of UM171 on human hematopoietic progenitor cell fate through clonal analysis

first: Guy

last: Sauvageau

state: QC

address: 2950, Chemin de Polytechnique

institute: Institute for Research in Immunology and Cancer

summary: Ex vivo expansion of hematopoietic stem cells (HSC) requires the maintenance of a stemness state while cells are proliferating. This can be achieved via exposure to UM171 which leads to the degradation of chromatin modifiers and prevents the loss of key epigenetic marks. However, the chromatin landscape varies across populations within the hematopoietic system and the effect of UM171 on self-renewal and differentiation potential of different hematopoietic progenitor cells is less characterized. To address this, we used the CellTag barcoding approach to track the fate of individual stem and progenitor cells during in vitro expansion. We showed that in addition to its HSC self-renewing property, UM171 specifically modulates cell fate of a precursor common to erythroid, megakaryocytic, and mast cells in favor of self-renewal and a mast-bias differentiation trajectory. This differentiation bias can be driven by pro-inflammatory signaling pathways that are activated downstream of UM171 and resulted in an abundant mast cell population that can be transplanted as part of the graft to populate mice tissues in xenotransplantation studies.

overall_design: CD34+CD38low cord blood cells were isolated by Fluorescence-Activated Cell Sorting (FACS) and transduced with a CellTag library (V1 or V3) for clonal tracking. Cells were replated in a 384-well plate, expanded in the presence of 35nM UM171 (V1) or DMSO (V3), and sampled at two time points for scRNA-seq

first: Patrick

last: Coulombe


title: Deconstructing synthetic inflammasomes in tumors drives diverse antitumor responses

city: Ljubljana

first: Iva

last: Hafner Bratkovič

address: Hajdrihova 19

country: Slovenia

institute: National Institute of Chemistry (Slovenia)

summary: Inflammasomes are cardinal defense machines that utilize cytokine and immunogenic cell death components to engage the immune system against pathogens. On the other hand, inflammasomes have ambiguous and context-dependent roles in cancers that are known to downregulate effectors of immunogenic cell death. In this study, we deconstructed inflammasomes by introducing one or both inflammasome signaling arms in diverse types of tumors regardless of the expression of endogenous inflammasome components. To induce immunogenic cell death, we designed tightly regulated GSDMD variants comprising different pore-forming capabilities and diverse modes of activation.  We show that electroporation of plasmids encoding the pyroptotic component into B16 melanoma tumors leads to tumor regression and complete remission in a quarter of treated animals. The inflammatory cytokines IL-1β and IL-18, along with the T-cell activator IL-12, did not provide protection when produced solely within tumors. However, they significantly boosted anti-tumor immunity when combined with pyroptosis. Careful selection of immunostimulatory molecules is imperative as a combination of IL-1β and IL-18 or the introduction of IFNγ antagonizes the protective action of pyroptosis by upregulation of several immune checkpoints. Additionally, we show that deconstruction of inflammasomes locally, without inducing systemic inflammation, provides protection against distant tumors and proves effective across various tumor types. Deconstructed inflammasomes are thus a powerful, tunable and tumor-agnostic strategy to enhance antitumor responses to even the most resilient types of tumors.

overall_design: 2×10e6 B16F10 Red-FLuc cells were subcutaneously inoculated into the right flank of C57BL/6J OlaHSd mice. When tumors were palpable and reached approximately 50 mm3, determined using the volume formula (width x length x height x π)/6, 20 μg of pcDNA empty vector, mIL-1β, mIL-18, single chain mIL-12 or IFNγ plasmid were injected into the tumor followed by electroporation using Gene Pulser Xcell Total Electroporation System (Bio-Rad Laboratories) with the parameters of Voltage 900, pulse width 100 μs, and pulse number 5. When the combination of mIL-1β and mIL-18 was used, 10 µg of each plasmid was electroporated. Two and six days after the first electroporation, tumors were injected with 20 μg of pcDNA empty vector or NT GSDMD plasmid and electroporated using the same parameters. We collected tumor samples 14 days post B16 inoculation and total RNA was extracted from the fresh tumors using TriPure Isolation Reagent (Roche) according to the manufacturer's instructions. Collected RNA from the animals of the same group was pulled together and sent in triplicates for bulk RNA sequencing.

first: Sara

last: Orehek

first: Špela

last: Malenšek


title: Overexpressing the ClpC AAA+ Unfoldase Prematurely Triggers Secondary Differentiation in Chlamydia trachomatis

city: OMaha

first: Scot

middle: P.

last: Ouellette

state: Nebraska

address: 985900 Nebraska Medical Center

department: Pathology, Microbiology, and Immunology

institute: University of Nebraska Medical Center

summary: Chlamydia is an obligate intracellular bacteria that undergoes a complex biphasic developmental cycle, alternating between the smaller infectious non-dividing elementary body (EB) and the larger non-infectious but dividing reticulate body (RB). Due to the differences between these functionally and morphologically distinct forms, we hypothesize protein degradation is essential to chlamydial differentiation. The bacterial Clp system, consisting of an ATPase unfoldase (e.g., ClpX or ClpC) and a proteolytic component (e.g., ClpP), is critical for the physiology of bacteria through its recognition, and usually degradation, of specific substrates. We observed by transmission electron microscopy that overexpression of wild-type ClpC, but not an ATPase mutant isoform, in Chlamydia increased glycogen accumulation within the vacuolar niche of the bacteria earlier in the developmental cycle than typically observed. This suggested ClpC activity may increase expression of EB-associated genes. Consistent with this, targeted RT-qPCR analyses demonstrated a significant increase in several EB-associated gene transcripts earlier in development. These effects were not observed with overexpression of the ATPase mutant of ClpC, providing strong evidence that the activity of ClpC drives secondary differentiation. By analyzing the global transcriptional response to ClpC overexpression using RNA sequencing, we observed a global shift to earlier expression of canonical late developmental cycle genes and other EB-associated genes. Finally, we directly linked overexpression of ClpC with earlier production of EBs. Conversely, disrupting normal ClpC function with an ATPase mutant caused a delay in secondary differentiation. Overall, these findings provide the first mechanistic insight for initiation of secondary differentiation in Chlamydia.

overall_design: The transformant of clpC overexpression were infected into HeLa cells. At 10 hpi, the constructs were induced or not with 20 nM aTc.  RNA samples were prepared from clpC overexpression-infected cells at 18. 20 μg RNA samples were treated with DNase to remove DNA contamination by using DNA-free Turbo kit (Thermo) according to the manusfacturer’s instruction. Ribosomal RNA was depleted from samples using the MICROBEnrich (Thermo) and MICROBExpress kits (Thermo) following the manufacturer’s instructions. RNA samples were processed for sequencing by the UNMC Genomics Core Facility.

middle: P

first: Aaron

last: Jensen


title: Gene expression changes by Glyceollin I treatment of breast cancer cells

city: Ariake, Koto-ku, Tokyo

first: Tatsuro

last: Yamamoto

department: Division of Cancer Biology

institute: The Cancer Institute of JFCR

summary: We examined how glyceolin I, a phytoalexin from soybeands, affects gene expression in breast cancer cells. mRNA was collected at time points of 0, 6, 12, and 24 hours, and mRNA-seqs were performed.

overall_design: Comparative gene expression profiling analysis of RNA-seq data at 0, 6, 12, and 24 hours with Glyceollin I treatment in MCF7, MCF7-LTED, and MDA-MB-468 cells.

first: Kenji

last: Watanabe

first: Noriko

last: Saitoh


title: High-mobility group box1 peptide ameliorates bronchopulmonary dysplasia (BPD) via suppressing inflammation and fibrosis in a mouse model.

city: Suita

first: Takashi

last: Shimbo

state: Osaka

address: 2-2 Yamadaoka

institute: Osaka university

summary: Previously, we have shown that a synthesized peptide originated from high mobility group box-1 protein (HMGB1) induces a regenerative cascade via activating endogenous mesenchymal stem cells in the body. Here, we have tested whether the HMGB1 peptide can ameliorate BPD related manifestations. In a mouse BPD model established by hyperoxia exposure, three shots of the HMGB1 peptide significantly improved the survival and lung function. Single-cell RNA-seq analysis on the lung showed that an inflammatory signature in macrophages and a fibrotic signature in fibroblasts induced by hyperoxia exposure were significantly suppressed by the HMGB1 peptide. These changes in transcriptome were also confirmed at the protein level. Together, the HMGB1 peptide treatment prevented the progression of BPD by suppressing inflammation and fibrosis. Our data serve as a foundation to develop new effective therapies for BPD.

overall_design: Evaluation of responses to HMGB1 peptide in BPD mouse model at transcriptomic and molecular levels

first: Takeya

last: Hara

first: Tomomi

last: Kitayama

first: Tatsuo

last: Masuda


title: Bacteroides expand the functional versatility of a universal transcription factor and transcribed DNA to program capsule diversity

status: Public on Nov 11 2024

city: Madison

first: Robert

last: Landick

state: WI

address: 1550 Linden Drive

department: Biochemistry

institute: University of Wisconsin-Madison

summary: Human gut Bacteroides species encode numerous (eight or more) tightly regulated capsular polysaccharides (CPS). Specialized paralogs of the universal transcription elongation factor NusG, called UpxY (Y), and an anti-Y UpxZ (Z) are encoded by the first two genes of each CPS operon. The Y-Z regulators combine with promoter inversions to limit CPS transcription to a single operon in most cells. Y enhances transcript elongation whereas Z inhibits noncognate Ys. How Y distinguishes among cognate CPS operons and how Z inhibits only noncognate Ys are unknown. Using in-vivo nascent-RNA sequencing and promoter-less in-vitro transcription (PIVoT), we establish that Y recognizes a paused RNA polymerase via sequences in both the exposed non-template DNA and the upstream duplex DNA. Y association is aided by novel ‘pause-then-escape’ nascent RNA hairpins. Z binds non-cognate Ys to directly inhibit Y association. This Y-Z hierarchical regulatory program allows Bacteroides to create CPS subpopulations for optimal fitness.

overall_design: Examination of nascent transcripts from a filtered culture of Bacteroides fragilis rpoC-3xFLAG

first: Jason

last: Saba

first: Katia

last: Flores

first: Bailey

last: Marshall

first: Michael

middle: D

last: Engstrom

first: Yikai

last: Peng

first: Atharv

middle: S

last: Garje

first: Laurie

last: Comstock


title: AAV capsid prioritization in machine-perfused normal and steatotic human livers

city: San Francisco

first: Simone

last: Kurial

state: CA

address: 35 Medical Center Way

department: Surgery

institute: UCSF

summary: Adeno-associated virus (AAV) vectors generated with five different capsids were intravenously injected into human livers undergoing normothermic machine perfusion (NMP). To assess the tropism of the five AAV vectors in the human livers, single-cell suspensions of hepatocytes and non-parenchymal cells (NPCs) were analyzed by single-cell RNA sequencing (scRNAseq). Differentially expressed genes were identified in AAV vector-transduced hepatocytes and unique cell populations.

overall_design: Hepatocytes and NPCs were released from human livers by enzymatic perfusion or chunk digestion and analyzed using scRNAseq (SeqWell).

middle: N

first: Jae-Jun

last: Kim

first: Holger

last: Willenbring


title: Human subcutaneous and visceral adipocyte atlases uncover classical and non-classical adipocytes and depot-specific patterns

city: Palo Alto

first: Lattice

last: Data Coordination

address: 3165 Porter Drive

department: Genetics

institute: Stanford University

summary: Human adipose depots are functionally distinct. Yet, recent single-nucleus RNA-sequencing (snRNA-seq) analyses largely uncovered overlapping/similar cell-type landscapes. We hypothesized that adipocytes subtypes, differentiation trajectories, and/or intercellular communication patterns could illuminate this depot similarity-difference gap. For this, we performed snRNA-seq of human subcutaneous/visceral adipose tissues (5/10 samples, respectively). Of 27,665 adipocyte nuclei in both depots, the majority were “classical”, namely- enriched in lipid metabolism pathways. However, we also observed “non-classical” adipocyte subtypes, enriched in immune-related, extracellular matrix deposition (fibrosis), vascularization/angiogenesis, or ribosomal processes. Pseudo-temporal analysis showed a developmental trajectory from adipose progenitor cells to classical adipocytes via non-classical adipocytes, suggesting that the classical state stems from loss, rather than gain, of specialized functions. Lastly, intercellular communication routes were consistent with the different inflammatory tone of the two depots. Jointly, these findings provide a high-resolution view into the contribution of cellular composition, differentiation, and intercellular communication patterns to human fat depot differences.

overall_design:
snRNA-seq of visceral fat tissue, subcutaneous adipose tissue tissue from 10 human donors.

UPDATE: [Jan-11-2025] In Sample GSM8619168, the sex, ethnicity, and age of donor 3313 were updated.

first: Esti

last: Yeger-Lotem

first: Assaf

last: Rudich

first: Or

last: Lazarescu

first: Maya

last: Ziv-Agam

first: Yulia

last: Haim

first: Idan

last: Hekselman

first: Juman

last: Jubran

first: Ariel

last: Shneyour

first: Habib

last: Muallem

first: Alon

last: Zemer

first: Marina

last: Rosengarten-Levin

first: Daniel

last: Kitsberg

first: Liron

last: Levin

first: Idit

middle: F

last: Liberty

first: Uri

last: Yoel

first: Oleg

last: Dukhno

first: Miriam

last: Adam

first: Julia

last: Braune

first: Claudia

last: Müller

first: Nora

last: Raulien

first: Martin

last: Gericke

first: Antje

last: Körner

first: Rinki

last: Murphy

first: Matthias

last: Blüher

first: Naomi

last: Habib


title: Gene expression profiling of cellular senescence in mouse articular chondrocytes

city: Seoul

first: Jin-Hong

last: Kim

address: 1 Gwanak-ro, Gwanak-gu

country: South Korea

institute: Seoul National University

summary: mRNA profiles of primary mouse chondrocytes exposed to senescence stress (bleomycin, doxorubicin) or acute inflammation (interleukin-1 beta) were generated by mRNA sequencing. This study provides transcriptome landscape of senescent chondrocytes.

overall_design: To investigate the cellular senescence process of chondrocytes, primary culture of mouse articular chondrocytes underwent treatment with senescence-inducing stimuli—bleomycin (50 μg/ml for 24 h plus incubation in fresh medium for five days) or doxorubicin (50 nM for five days). Interleukin-1 beta treatment as an inflammation-induced acute stress model served as a control.

first: Donghyun

last: Kang

first: Jeeyeon

last: Lee


title: Attenuated kidney oxidative metabolism in young adults with type 1 diabetes

city: Seattle

first: Petter

last: Bjornstad

state: Washington

address: 750 Republican St

department: Division of Metabolism, Endocrinology and Nutrition

institute: University of Washington

summary: In type 1 diabetes (T1D), impaired insulin sensitivity may contribute to the development of diabetic kidney disease (DKD) through alterations in kidney oxidative metabolism. Young adults with T1D and healthy controls underwent single-cell RNA sequencing, and spatial metabolomics to assess this relationship.

overall_design:
Cryostor-preserved kidney biopsies were subjected to single cellular RNA-sequencing.
***Please note that the current study was part of a clinical trial (NCT04074668), and thus raw data are not available due to patient privacy concerns.

first: Ye Ji

last: Choi

first: Gabriel

last: Richard

first: Guanshi

last: Zhang

first: Jeffrey

middle: B

last: Hodgin

first: Dawit

last: Demeke

first: Yingbao

last: Yang

first: Jennifer

middle: A

last: Shaub

first: Ian

middle: M

last: Tamayo

first: Bhupendra

middle: K

last: Gurung

first: Abhijit

last: Naik

first: Viji

last: Nair

first: Carissa

last: Birzneiks

first: Alexis

last: MacDonald

first: Phoom

last: Narongkiatikhun

first: Susan

last: Gross

first: Lynette

last: Driscoll

first: Maureen

last: Flynn

first: Kalie

last: Tommerdahl

first: Kristen

middle: J

last: Nadeau

first: Viral

last: Shah

first: Tim

last: Vigers

first: Janet

last: Snell-Bergeon

first: Jessica

last: Kendrick

first: Daniel

middle: H

last: van Raalte

first: Lu-Ping

last: Li

first: Pottumarthi

last: Prasad

first: Patricia

last: Ladd

first: Bennett

last: Chin

first: David

middle: Z

last: Cherney

first: Phillip

last: McCown

first: Fadhl

last: Alawkaa

first: Edgar

last: Otto

first: Frank

middle: C

last: Brosius

first: Pierre

last: Saulnier

first: Victor

last: Puelles

first: Jesse

last: Goodrich

first: Kelly

last: Street


title: Paediatric Bone marrow Mesenchymal Stem cells support acute myeloid leukaemia cell survival and enhance chemoresistance via contact-independent mechanism

city: GLASGOW

first: Alexander

last: Winton

address: Wolfson Wohl Cancer Research Centre, Bearsden

department: School Of Cancer Science

country: United Kingdom

institute: University Of Glasgow

summary: Pediatric mesenchymal stem cells (pMSCs) within the bone marrow (BM) niche play a crucial role in regulating pediatric acute myeloid leukemia (pAML) survival and therapy resistance. In this study, we isolated pMSCs from pAML bone marrow samples and characterized their phenotype and differentiation potential. We performed coculture experiments with pAML cells in both autologous and allogenic settings, with and without direct contact. RNA-sequencing and multiplex immunoassays revealed that pMSCs promote pAML survival through changes in gene expression, cytokine secretion, and extracellular matrix (ECM) components, specifically enhancing chemoprotection from cytarabine and Gemtuzumab Ozogamicin (GO). Inhibition of the JAK/STAT and ERK pathways using ruxolitinib (RUX) abrogated the pMSC-induced chemoprotection, suggesting a contact-independent mechanism. These findings provide new insights into how the BM microenvironment contributes to therapy resistance in pediatric AML and highlights the potential for targeting pMSC-induced signaling pathways in treatment strategies.

overall_design: Pediatric mesenchymal stem cells (pMSCs) were isolated from pediatric acute myeloid leukemia (pAML) bone marrow samples using the plastic adherence method. The pMSCs were characterized by surface markers (CD45-Lin-CD90+CD105+CD73+) and their ability to differentiate into adipocytes, osteoblasts, and chondrocytes. Coculture experiments were performed by directly co-culturing pAML cells with pMSCs, both with and without cytokine supplementation. Parallel experiments included a transwell system to assess contact-independent effects. RNA sequencing was conducted on pAML cells after coculture, and multiplex immunoassays were used to profile the secretome. Chemoprotection assays involved treatment with cytarabine (Ara-C) and Gemtuzumab Ozogamicin (GO), with apoptosis and cell cycle analyzed. Additionally, the role of JAK/STAT and ERK signaling was assessed using ruxolitinib (RUX) to inhibit these pathways and measure changes in chemoprotection.

first: Alison

last: Laing

first: Ahmed

last: Elmarghany

first: Arwa

last: Alghaith

first: Aya

last: Gouma

first: Cassie

last: Clarke

first: Thomas

last: Stevens

first: Jennifer

last: Cassells

first: Christine

last: Harrison

first: Claire

last: Schwab

first: Brenda

last: Gibson

first: Karen

last: Keeshan


title: Single-cell Transcriptional Atlas of Murine Collecting Lymphatic Vessels

first: Pin-Ji

last: Lei

state: Massachusetts

address: 100 Blossom Street

department: Department of Radiation Oncology

institute: Massachusetts General Hospital

summary: The muscle cells within the wall of collecting lymphatic vessels exhibit tonic and autonomous phasic contractions, which drive active lymph transport to maintain tissue-fluid homeostasis and support immune surveillance. Damage to the lymphatic muscle cells (LMC) disrupts lymphatic function and is related to various diseases. Despite their importance, knowledge of the transcriptional signatures in LMC and how they relate to lymphatic function in normal and diseases contexts is largely missing. In this study, we have generated to date the most comprehensive transcriptional single-cell atlas—including LMC—of collecting lymphatic vessels in mouse dermis at various ages.

overall_design: To characterize the transcriptomes of the cells that make up the walls of collecting lymphatic vessels and the surrounding tissue, we isolated dermal collecting lymphatic vessels that connect the inguinal lymph node and axillary lymph node in 6-week-old (young), 12-month-old (middle-aged), and 18-month-old (aged) C56BL/6J mice from both males and females for 10X Genomics single-cell library construction and subsequent Illumina high throughput sequencing.

first: Katarina

last: Ruscic

first: Timothy

last: Padera


title: Gene expression profiling of inhibiting O-GlcNAcylation on the cellular senescence of mouse articular chondrocytes

first: Jin-Hong

last: Kim

summary: mRNA profiles of primary mouse chondrocytes treated with bleomycin, followed by DMSO or ST045849 were generated by mRNA sequencing. This study provides insights into the role of O-GlcNAcylation in chondrocytes.

overall_design: To investigate the role of O-GlcNAcylation during the cellular senescence process, primary culture of mouse articular chondrocytes underwent treatment with bleomycin (50 μg/ml) for 24 h, followed by treatment with or without ST045849 (20 μM) for five days.

first: Donghyun

last: Kang

first: Jeeyeon

last: Lee


title: Extensive binding of uncharacterized human transcription factors to genomic dark matter [GSE58341 Reanalysis]

city: Toronto

first: Arttu

last: Jolma

address: 160 College Street, Room 1350

department: Donnelly Centre

institute: University of Toronto

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Most of the human genome is thought to be non-functional, and includes large segments often referred to as “dark matter” DNA. The genome also encodes hundreds of putative and poorly characterized transcription factors (TFs). We determined genomic binding locations of 166 uncharacterized human TFs in living cells. Nearly half of them associated strongly with known regulatory regions such as promoters and enhancers, often at conserved motif matches and co-localizing with each other. Surprisingly, the other half often associated with genomic dark matter, at largely unique sites, via intrinsic sequence recognition. Dozens of these, which we term “Dark TFs” mainly bind within regions of closed chromatin. Dark TF binding sites are rarely under purifying selection, and are enriched for transposable elements. Many Dark TFs are KZNFs, which contain the repressive KRAB domain, but many are not, and may represent potential pioneer TFs: based on compiled literature information, the Dark TFs exert diverse functions ranging from early development to tumor suppression. Thus, a large fraction of previously uncharacterized human TFs may have unappreciated activities within the dark matter genome.

relation:
SubSeries of: GSE280248
Reanalysis of: GSM1407626
Reanalysis of: GSM1407630
Reanalysis of: GSM1407648

overall_design:
This entry describes re-analysis of previously existing datasets in located in SRA entry SRP043061. These datasets have been analyzed previously (GSE52523), but they were re-analyzed here to be consistent with the newly generated ChIP-seq datasets. Previous datasets were mapped into hg19 assembly and analyzed with different computational approaches

***************************************************************
The table below lists GEO accessions reused/reanalyzed for this study.
***************************************************************

first: Rozita

last: Razavi

first: Ali

last: Fathi

first: Isaac

last: Yellan

first: Alexander

last: Brechalov

first: Kaitlin

middle: U

last: Laverty

first: Hong

last: Zheng

first: Ally

last: Yang

first: Marjan

last: Barazandeh

first: Chun

last: Hu

first: Ilya

last: Vorontsov

first: Zain

last: Patel

first: Ivan

last: Kulakovskiy

first: Philipp

last: Bucher

first: Hamed

last: Najafabadi

first: Quaid

last: Morris

last: Hughes


title: RNA sequencing of Tau overexpressing human SH-SY5Y cell line revels that Tau protein is able to alter global gene expression and chromatin structure

city: Jena

first: Marco

last: Groth

address: Beutenbergstraße 11

department: Core Facility - Next Generation Sequencing

country: Germany

institute: Leibniz Institute on Aging - FLI

summary: Alzheimer’s disease and tauopathies are neurodegenerative disorders characterized by the alteration of neuronal homeostasis and aggregation of Tau protein. Tau is generally considered a protein involved in cytoskeletal stability and transport whose destabilization leads to neuronal dysfunction and death. Despite the knowledge about canonical roles of Tau, in recent years it has been observed that Tau is a key player in several neuronal functions. We previously described that Tau destabilization and nuclear delocalization alters the expression of glutamatergic genes mediating early neuronal damage. In this paper we propose that Tau is able to deeply affect the neuronal transcriptome and that its availability is associated to gene expression alterations occurring in developing intermediate AD phases. In addition, we identify the epigenetic pathway as strongly altered by Tau during AD progression.

overall_design: Differential gene expression analysis of RNA-seq data performed on SH-SY5Y cells overexpressing Tau 0N4R isoform (SH_tau) or a control vector (SH_ctrl). 6 samples in total: 3 samples SH-SY5Y overexpressing Tau 0N4R; 3 samples SH-SY5Y expressing control vector.

first: Cristina

last: Di Primio

first: Giacomo

last: Siano

first: Martina

last: Varisco

first: Antonino

last: Cattaneo

first: Alessandro

last: Cellerino


title: RNA binding by Periphilin plays an essential role in initiating silencing by the HUSH complex

city: Cambridge

first: Paul

last: Lehner

address: Puddicome Way

department: CITIID

institute: University of Cambridge

summary: The human silencing hub (HUSH) complex is a transcription-dependent, epigenetic repressor complex that provides a genome-wide immunosurveillance system for the recognition and silencing of newly-integrated retroelements. The core HUSH complex of TASOR, MPP8 and Periphilin, represses these retroelements through SETDB1-mediated H3K9me3 deposition and MORC2-dependent chromatin compaction. HUSH-dependent silencing is RNA-mediated, yet no HUSH components encode any RNA-binding domain. Here we used an unbiased approach to identify which HUSH component was able to bind RNA and determine whether RNA-binding was essential for HUSH function. We identify Periphilin as the major RNA-binding component of the HUSH complex and show that Periphilin’s N-terminal domain is essential for both RNA binding and HUSH function. Periphilin binding to RNA was independent of its interaction with TASOR or MPP8, as its N-terminal domain was sufficient for RNA targeting. The artificial tethering of Periphilin, to a HUSH-insensitive, nascent transcript, enabled the HUSH-dependent silencing of the transcript. This tethering of Periphilin allowed the RNA-binding region of Periphilin to be removed such that only its C-terminal domain was required, for oligomerisation and interaction with TASOR. We therefore show that Periphilin is the predominant RNA-binding protein of the HUSH complex and this RNA-binding is essential for HUSH activity.

overall_design: To characterize the endogenous targets of PPHLN1 binding we initially used UV-crosslinked RNA immunoprecipitation sequencing (UV-RIPseq) in 293T PPHLN1 and SETDB1 knockout cells, expressing a panel of HA tagged PPHLN1 constructs

first: Stuart

last: Bloor

first: Niek

last: Wit


title: Blocking tumor-intrinsic MNK1 kinase restricts metabolic adaptation and diminishes liver metastasis

city: Montreal

first: Sonia

last: del Rincón

address: 3755 Chemin de la Cote Ste Catherine

institute: McGill University

summary:
To investigate the functions of tumor cell-intrinsic MNK1 kinase, we used CRISPR/Cas9 to generate two MNK1 knockout 4T1 cell lines  (sg-MKNK1-1 and sg-MKNK1-2).
We then performed gene expression profiling analysis using data obtained from RNA-seq, comparing each sg-MKNK1 4T1 cell line to a wild-type 4T1 control (Cas9CTL).

overall_design: Comparative gene expression profiling analysis of RNA-seq data for wild-type (Cas9CTL) and MNK1 knockout (sg-MKNK1) 4T1 cells

first: Samuel

last: Preston

first: Christophe

last: Gonçalves


title: Antigen experience history directs distinct functional states of CD8+ CAR T cells during the anti-leukemia response

city: Aurora

first: Terry

last: Fry

state: CO

institute: University of of Colorado Denver and Children's Hospital Col

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

overall_design: Refer to individual Series


title: Metastasis of colon cancer requires Dickkopf-2 to generate cancer cells with Paneth cell properties [RNA-seq]

first: Jaechul

last: Lim

state: CT

address: 1 Gwanak-ro, 1 Gwanak-gu

summary: Metastasis is the leading cause of cancer-related mortality. Cancer stem cells contribute to metastasis in the murine colon cancer models, but the underlying mechanisms are unclear. Here we report a Wnt ligand, Dickkopf2 (DKK2) is essential for colorectal cancer stemness. Genetic depletion of Dkk2 in intestinal epithelial or stem cells reduced tumorigenesis as well as expression of the stem cell marker gene Lgr5 in a model of colitis-associated cancer. Mechanistically, DKK2 activates c-Src followed by increased LGR5 expressing stem cells in colorectal cancer through degradation of HNF4α1. Splenic injection of DKK2-deficient cancer organoids into C57BL/6 mice resulted in a significant reduction of liver metastases compared to the control cancer organoids in spite of the presence of oncogenic mutations in Apc, Kras and Tp53 genes. These findings suggest that DKK2 is required for stemness of colorectal cancer cells, which in turn contributes to metastasis.

overall_design: Three replicates from murine colon cancer organoids carrying mutations in Apc, Kras and Tp53 genes and five replicates from Dkk2 knockout colon cancer organoids with identical oncogenic mutations. Four and three replicates of colitis-induced cancer cells from wild type and intestinal stem cell-specific tamoxifen-inducible Dkk2 knockout mice, respectively.

first: Jae-Hun

last: Shin

first: Jungmin

last: Choi

first: Jun-Young

last: Hong

first: Alfred

last: Bothwell


title: Metastasis of colon cancer requires Dickkopf-2 to generate cancer cells with Paneth cell properties

overall_design: Two replicates from normal C57BL/6 murine colonic organoids, colon cancer organoids carrying mutations in Apc, Kras and Tp53 genes and Dkk2 knockout colon cancer organoids with identical oncogenic mutations, respectively.


title: Single Cell RNA Sequencing of Synovial Tissue in Adolescents Undergoing ACL Reconstruction

status: Public on Nov 10 2024

city: Charlotte

first: Sarah

last: Romereim

state: NC

address: 1000 Blythe Blvd, CRC 305

department: Musculoskeletal Institute

institute: Atrium Health

summary: Background: Loss of motion and arthrofibrosis after anterior cruciate ligament reconstruction (ACLR) can be a devastating complication for athletes. The cellular and molecular pathogenesis of arthrofibrosis is poorly understood, limiting prevention and treatment options. Synovial inflammation may contribute to post-ACLR arthrofibrosis. Hypothesis/Purpose: We hypothesized that higher synovial immune cell infiltration and inflammatory/catabolic gene expression patterns at the time of ACLR would correlate with poorer motion-related outcomes. Study Design: Case Series Methods: Patients aged 10-18 undergoing primary ACLR were enrolled in a prospective pilot study, and synovial tissue biopsies were obtained during ACLR. Flow cytometry and single cell RNA-sequencing explored synovial cell types/frequencies and gene expression. Principle component analysis followed by clustering grouped patients into distinct immunophenotypes based on their synovial cell composition. Clinical follow-up with knee range of motion (ROM), need for lysis of adhesions, and patient reported outcome measures were collected and compared between immunophenotypes. Results: Enrolled patients (n = 17) underwent ACLR at a median of 37 days post-injury. Analysis revealed three distinct immunophenotypes. Type 1 comprised patients with the longest time between injury and surgery and the lowest hematopoietic and T cell infiltration. Types 2 and 3 had similar times between injury and surgery, and Type 2 had intermediate while Type 3 had the highest hematopoietic and T cell percentages. Type 3 was associated with worse ROM at 2- and 6-weeks post-op; T cell prevalence and ROM were inversely correlated at those time points. The only patient requiring lysis of adhesions for arthrofibrosis was in Type 3. Conclusion: Synovial immune infiltration after ACL injury shows variability between patients that clusters into three immunophenotypes correlating with early ROM and the risk of arthrofibrosis. T cell recruitment and infiltration was the strongest factor correlated with ROM outcomes and presents an exciting venue for future research on post-ACLR arthrofibrosis.

overall_design: Synovial tissue biopsies were obtained during ACL reconstruction from six patients, enzymatically digested to obtain heterogenous single-cell suspensions, and analyzed by scRNA-seq. The six scRNA-seq samples as well as eleven other patient samples were categorized into three Immunotypes based on flow cytometry quantification of the immune cells present in the synovium tissue. The scRNA-seq output was analyzed for a more comprehensive characterization of the cell types present as well as the relative expression of select inflammatory markers and immunomodulatory genes.

first: Romereim

last: Sarah

first: Jin

last: Guangxu

first: Steuerwald

last: Nury

first: Fearing

last: Bailey

first: Riboh

last: Jonathan


title: Meniscus Gene Expression Profiling: Inner and Outer Zone Tissue Comparison to Cartilage and Passaged Monolayer Meniscus Cells

city: Durham

first: Amy

middle: L

last: McNulty

address: DUMC Box 3093

institute: Duke University School of Medicine

summary: Meniscus injuries are common and while surgical strategies have improved, there is a need for alternative therapeutics to improve long-term outcomes and prevent post-traumatic osteoarthritis. Current research efforts in regenerative therapies and tissue engineering are hindered by a lack of understanding of meniscus cell biology and a poorly defined meniscus cell phenotype. This study utilized bulk RNA-sequencing to identify unique and overlapping transcriptomic profiles in cartilage, inner and outer zone meniscus tissue, and passaged inner and outer zone meniscus cells. The greatest transcriptomic differences were identified when comparing meniscus tissue to monolayer cultured cells (> 4,600 differentially expressed genes (DEGs)) and meniscus tissue to cartilage (> 3,100 DEGs). While zonal differences exist within the meniscus tissue (205 DEGs between inner and outer zone meniscus tissue), meniscus resident cells are more similar to each other than to either cartilage or monolayer meniscus cells. Additionally, we identified and validated LUM and PRRX1 as potential markers for meniscus tissue and ACTA2, FSTL1, SFRP2, and TAGLN as novel markers for meniscus cell dedifferentiation. Our data contribute significantly to the current characterization of meniscus cells and provide an important foundation for future work in meniscus cell biology, regenerative medicine, and tissue engineering.

overall_design: RNA was isolated from porcine inner and outer zone meniscus tissue, cartilage (n=3/tissue type), and passage 3 inner (n=3) and outer zone (n=2) meniscus cells.  RNA-Sequencing was performed and differential expression due to tissue type, zone, and cell passaging/dedifferentiation was assessed.

first: Benjamin

last: Andress


title: Single cell RNA-seq of cochlear tissues from Tmie+/- and Tmie-/- mice, a model for recessive non-syndromic deafness model 6.

city: Baltimore

first: Yijun

last: Xu

state: MD

address: 733 N Broadway

department: Neuroscience

institute: Johns Hopkins University School of Medicine

summary: Deafness is the most common form of sensory impairment in humans and frequently caused by defects in hair cells of the inner ear. Here we demonstrate that in a mouse model for recessive non-syndromic deafness (DFNB6), inactivation of Tmie in hair cells disrupts gene expression in the neurons that innervate them. This includes genes regulating axonal pathfinding and synaptogenesis, two processes that are disrupted in the inner ear of the mutant mice. Similar defects are observed in mouse models for deafness caused by mutations in other genes with primary functions in hair cells. Gene therapy targeting hair cells restores hearing and inner ear circuitry in DFNB6 model mice. We conclude that hair cell function is crucial for the establishment of peripheral auditory circuitry. Treatment modalities for deafness thus need to consider restoration of the function of both hair cells and neurons, even when the primary defect occurs in hair cells.

overall_design: Cochlear tissues (including the spiral ganglion, spiral limbus, inner sulcus, organ of Corti, and outer sulcus) from postnatal day 21 aged (P21) Tmie-/- (KO) mice and heterozygous Tmie+/- (HET) littermate controls were isolated and analyzed by scRNA-seq.

first: Riley

middle: T

last: Bottom

first: Caroline

last: Siebald

first: Ulrich

last: Mueller


title: Fam170a deficiency causes male infertility

city: Nantong

first: Yinchuan

last: Li

state: Jiangsu

address: 16 wenfeng road, Chongchuan

department: Medical School

country: China

institute: Nantong University

summary: During spermiogenesis, nucleus condensation packages genomic DNA into tiny sperm heads by histone-to-protamine chromatin remodeling process, which is essential to male fertility. However, this process involving key events and regulators is still poorly understood. Herein, we report that Fam170a orchestrates the histone-to-protamine chromatin remodeling process by interacting with its associated proteins and mediating its associated gene expression.

overall_design: To decipher the potential role of Fam170a  during spermiogenesis, Fam170a knock-out mice were generated. Testes from wild type mice (WT) and adult Fam170a knock-out mice (KO) were obtained for RNA_seq.

first: Jinmei

last: Cheng


title: LPS stimulation of APOE knock-out iPSC-derived microglia with different ApoE isoforms

city: South San Francisco

first: Thomas

last: Sandmann

email: genomics@dnli.com, sandmann@dnli.com

state: California

address: 161 Oyster Point Blvd

institute: Denali Therapeutics

summary: APOE is the main genetic modifier for late onset Alzheimer’s disease (LOAD). While an APOE2/APOE3/APOE4 allelic series is well established for LOAD risk and neuropathology, molecular mechanisms underlying isoform-dependent risk and relevance of ApoE-associated lipids remain elusive. Here, we studied the effects of LPS stimulation on APOE KO iPSC-derived microglia treated with different ApoE isoforms (ApoE2/E3/E4) pre-complexed with BODIPY-cholesteryl ester (CE) and HDL -/+ recombinant LDLR extracellular domain.

overall_design: LPS-stimulated APOE knock-out (KO) iPSC-derived microglia (iMG) were treated with ApoE isoforms (ApoE2/E3/E4) pre-complexed with BODIPY-CE and HDL -/+ recombinant LDLR ECD for one day prior to RNA harvest. Four different treatments were evaluated: (1) BODIPY-CE/HDL/ApoE2, (2) BODIPY-CE/HDL/ApoE3, (3) BODIPY-CE/HDL/ApoE4, (4) BODIPY-CE/HDL/ApoE4 + LDLR extracellular domain (ECD). Samples were collected from 3 independent experiments. For each of the 12 independent biological samples, two replicate sequencing libraries were prepared and analyzed.

first: Jing

last: Guo

first: Dylan

last: Braun

first: Nicholas

middle: E

last: Propson

first: Connie

last: Ha

first: Gilbert

last: Di Paolo


title: Phosphorylation controls mRNA retention via nuclear speckle cohesion - APEX2-seq

city: Zurich

first: A

last: McIntyre

address: Winterthurerstrasse 190

department: Molecular Life Sciences

institute: University of Zurich

summary: Nuclear speckles are membraneless organelles that associate with active transcription sites and participate in post-transcriptional mRNA processing. During mitosis, nuclear speckles dissolve following phosphorylation of their protein components. Here, we identify PP1 phosphatases as responsible for counteracting kinase-mediated dissolution. Their overexpression increases speckle cohesion and leads to retention of polyadenylated RNA within speckles and the nucleus. By performing APEX2 proximity labeling combined with RNA-sequencing, we characterized the association of specific RNAs with nuclear speckles depending on their cohesion state. We find that many transcripts are preferentially enriched within nuclear speckles compared to the nucleoplasm, particularly chromatin- and nucleus-associated transcripts. While total polyadenylated RNA retention increased with greater nuclear speckle cohesion, the ratios of most mRNA species to each other were constant, indicating non-selective, or proportional, retention. We then explored whether nuclear speckle cohesion changes in response to environmental perturbations associated with changes in kinase or phosphatase activity. We found that cellular responses to heat shock, oxidative stress, and hypoxia include changes to the cohesion of nuclear speckles and mRNA retention. Our results demonstrate that tuning the material properties of nuclear speckles provides a mechanism for the acute control of mRNA localization.

overall_design: Biotinylation was performed using APEX2 tagged to an endogenous nuclear speckle protein gene (SON) or a nuclear localization signal (NLS) in human induced pluripotent stem cell lines (hiPSCs). Four treatments were applied: 1) DMSO control, 2) DYRK3 inhibition with GSK626616, 3) splicing inhibition with Pladienolide-B, and 4) transcriptional inhibition with DRB. Biotinylated RNA was extracted and sequenced for 5 replicates under each treatment and cell line, with 3 replicates of input RNA also sequenced per condition (matched to biotinylated replicates 2, 4, and 5).

first: Adrian

last: Tschan

first: Alexa

first: Katrina

last: Meyer

first: Severin

last: Walser

first: Arpan

last: Rai

first: Keisuke

last: Fujita

first: Lucas

last: Pelkmans


title: NLRP3 Amplifies Inflammatory Signaling in Acute Lung Injury Induced by Cell Wall Extract of Gram-positive Bacteria

city: 合肥市

first: 林贵

last: 顾

state: 安徽省

address: 安徽省合肥市蜀山区梅山路安徽医科大学基础医学院

institute: 安徽医科大学

summary: Transcriptomic analysis suggested that LCWE and LPS both induce Toll/NF-kB and TNF signalings in ALI, and LCWE additionally promotes T cell differentiation.We established a murine model of ALI-induced by LCWE. TLR2 receptors are activated by LCWE and transmitted through the MYD88 signaling pathway, activating NF-kappaB which leads to the production of IL1b and activation of TNFR and NLRP3 signaling pathways.

overall_design: Mice were challenged with LCWE from lung. LPS was used as positive control. Lung tissues were collected for RNA preparation and RNA-seq analysis.

first: Xuanming

last: Shi


title: GWAS and 3D chromatin mapping identifies multicancer risk genes associated with hormone-dependent cancers

city: Brisbane

first: Jonathan

last: Beesley

state: QLD

address: 300 Herston Rd Herston

country: Australia

institute: QIMR Berghofer

summary: Hormone-dependent cancers (HDCs) share several risk factors, suggesting a common aetiology. Using data from genome-wide association studies, we showed spatial clustering of risk variants across four HDCs (breast, endometrial, ovarian and prostate cancers), contrasting with genetically uncorrelated traits. We identified 44 multi-HDC risk regions across the genome, defined as overlapping risk regions for at least two HDCs: two regions contained risk variants for all four HDCs, 13 for three HDCs and 28 for two HDCs. Integrating GWAS data, epigenomic profiling and promoter capture HiC maps from diverse cell line models, we annotated 53 candidate risk genes at 22 multi-HDC risk regions. These targets were enriched for established genes from the COSMIC Cancer Gene Census, but many had no previously reported pleiotropic roles. Additionally, we pinpointed lncRNAs as potential HDC targets and identified risk alleles in several regions that altered transcription factors motifs, suggesting regulatory mechanisms. Known drug targets were over-represented among the candidate multi-HDC risk genes, implying that some may serve as targets for therapeutic development or facilitate the repurposing of existing treatments for HDC. Our comprehensive approach provides a framework for identifying common target genes driving complex traits and enhances understanding of HDC susceptibility.

overall_design: We performed promoter capture Hi-C on tumour and normal cell lines relevant to hormone-dependent cancers. We designed baits to target promoters in regions defined as “multi-Hormone Dependent Cancer Regions”, in which risk signals for >1 cancer have been detected by GWAS.

first: Isela

last: Rivera

first: Juliet

last: French

first: Stacey

last: Edwards


title: Antigen experience history directs distinct functional states of CD8+ CAR T cells during the anti-leukemia response [RNA-seq]

first: Terry

last: Fry

summary: Adoptive transfer of immune cells expressing chimeric antigen receptors (CARs) is an effective therapy for B-lineage malignancies. However, most patients will relapse and this therapeutic has yet to show strong efficacy in other hematologic or solid tumors. One opportunity for improvement lies in the ability to select or generate T cells that have the highest potential for potent anti-tumor responses and T cell persistence. Here, we dissect the biology of CD8+ CAR T cells by controlling whether the T cell has encountered cognate TCR antigen prior to CAR generation. We find that prior antigen experience influences multiple aspects of in vitro and in vivo CAR T cell functionality, boosting effector function and leukemia clearance in the setting of limiting target antigen density. However, this comes at the expense of proliferative capacity, resistance to dysfunction, and clearance of wildtype leukemia in the setting of limiting CAR+ cell dose. Epigenetic and transcriptomic comparisons of these cell populations uncover that modulation of the Runx2 transcription factor differentially impacts CAR T cell functionality depending on prior cell state. Collectively, our data demonstrate that prior antigen experience status determines functional attributes of a CAR T cell, as well as amenability to functional enhancement by transcription factor modulation.

overall_design: To investigate the role of prior T cell antigen experience in responses of CD8+ chimeric antigen receptor (CAR) T cells to leukemia, we adoptively transferred C57BL/6-background Rag2-/- OT-I TCR transgenic CD8+ T cells into C57BL/6-background CD45.1+ hosts, followed by vaccination with ovalbumin, anti-mouse CD40, and PolyI:C to generate memory CD8+ T cells ("memory-derived"), or used naïve C57BL/6-background Rag2-/- OT-I TCR transgenic CD8+ T cells from naïve hosts ("naïve-derived"). We then transduced both populations with an anti-mouse CD19 CAR containing CD28 costimulatory domain and CD3zeta domain. Memory or naive-derived T cells were compared by RNAseq at three timepoints, Pre-CAR Transduction ("PreCAR", Day -5, ex vivo), Post-CAR Transduction ("PostCAR", Day 0, in vitro), and 4 days after infusion into C57BL/6-background Rag1-/- mice bearing the mouse CD19+ murine B-cell acute lymphoblastic leukemia cell line, E2A-PBX1 ("Tumor", Day +4, Ex Vivo).

first: Kole

middle: R

last: DeGolier

first: Etienne

last: Danis

first: James

middle: P

last: Scott-Browne


title: Phosphorylation controls mRNA retention via nuclear speckle cohesion - cellular fractionation data

first: A

last: McIntyre

overall_design: Human induced pluripotent stem cells were fractionated into chromatin, nuclear and cytoplasmic fractions using a protocol adapted from Mayer & Churchman, 2017. Five replicates were collected.

first: Adrian


title: Phosphorylation controls mRNA retention via nuclear speckle cohesion during environmental perturbations


title: Synuclein pathology disrupts mitochondrial function in dopaminergic and cholinergic neurons at-risk in Parkinson’s disease

status: Public on Nov 09 2024

city: Chicago

first: D.

middle: James

last: Surmeier

state: IL

address: 303 E Superior Street, Lurie 8-220

institute: Northwestern University

summary: Background: Pathological accumulation of aggregated α-synuclein (aSYN) is a common feature of Parkinson’s disease (PD). However, the mechanisms by which intracellular aSYN pathology contributes to dysfunction and degeneration of neurons in the brain are still unclear. A potentially relevant target of aSYN is the mitochondrion. To test this hypothesis, genetic and physiological methods were used to monitor mitochondrial function in substantia nigra pars compacta (SNc) dopaminergic and pedunculopontine nucleus (PPN) cholinergic neurons after stereotaxic injection of aSYN pre-formed fibrils (PFFs) into the mouse brain.  Methods: aSYN PPFs were stereotaxically injected into the SNc or PPN of mice. Twelve weeks later, mice were studied using a combination of approaches, including immunocytochemical analysis, cell-type specific transcriptomic profiling, electron microscopy, electrophysiology and two-photon-laser-scanning microscopy of genetically encoded sensors for bioenergetic and redox status.  Results: In addition to inducing a significant neuronal loss, SNc injection of PFFs induced the formation of intracellular, phosphorylated aSYN aggregates selectively in dopaminergic neurons. In these neurons, PFF-exposure decreased mitochondrial gene expression, reduced the number of mitochondria, increased oxidant stress, and profoundly disrupted mitochondrial adenosine triphosphate production. Consistent with an aSYN-induced bioenergetic deficit, the autonomous spiking of dopaminergic neurons slowed or stopped. PFFs also up-regulated lysosomal gene expression and increased lysosomal abundance, leading to the formation of Lewy-like inclusions. Similar changes were observed in PPN cholinergic neurons following aSYN PFF exposure.   Conclusions: Taken together, our findings suggest that disruption of mitochondrial function is a proximal step in the cascade of events induced by aSYN pathology leading to dysfunction and degeneration of neurons at-risk in PD.

overall_design: To investigate the effects of aSYN pathology on the gene expression profile of SNc dopaminergic neorons, we isolated mRNAs from SNc dopaminergic neorons in aSYN monomer-injected (10 mice, as control) or PFF-injected mice (10 mice), and performed comparative gene expression profiling analysis using data obtained from RNA-seq.

first: Tatiana

last: Tkatch

first: Zhong

last: Xie

first: Fanni

last: Geibl

first: Martin

last: Henrich

first: D


title: Gut Adaptation After Gastric Bypass in Humans Reveals Metabolically Significant Shift in Fuel Metabolism

city: Boston

first: Margaret

last: Stefater-Richards

state: MA

address: 300 Longwood Ave

institute: Boston Children's Hospital

summary:
Objective: Roux-en-Y gastric bypass surgery (RYGB) is among the most effective therapies for obesity and type 2 diabetes (T2D), and intestinal adaptation is a proposed mechanism for these effects. We hypothesized that intestinal adaptation precedes and relates to metabolic improvement in humans after RYGB.
Methods: This was a prospective, longitudinal first-in-human study of gene expression (GE) in Roux limb (RL) collected surgically/endoscopically from 19 patients with and without diabetes. GE was determined by microarray across 6 postoperative months, including at an early postoperative (1 month  15 days) timepoint.
Results: RL GE demonstrated tissue remodeling and metabolic reprogramming, including increased glucose and amino acid utilization. RL GE signatures were established early, before maximal clinical response, and persisted. Distinct GE fingerprints predicted concurrent and future improvements in HbA1c and in weight. Human RL exhibits GE changes characterized by anabolic growth and shift in metabolic substrate utilization. Paradoxically, anabolic growth in RL appears to contribute to the catabolic state elicited by RYGB.
Conclusions: These data support a role for a direct effect of intestinal energy metabolism to contribute to the beneficial clinical effects of RYGB, suggesting that related pathways might be potential targets of therapeutic interest for patients with obesity with or without T2D.


title: Loss of SMARCB1 evokes targetable epigenetic vulnerabilities in Epithelioid Sarcoma [ChIP-Seq]

city: Heidelberg

first: Thomas

middle: Georg Philipp

last: Grunewald

address: Im Neuenheimer Feld 280

department: Division of Translational Pediatric Sarcoma Research

institute: German Cancer Research Center (DKFZ)

summary: Dysfunction of epigenetic modulators such as the SWI/SNF complex is involved in a wide spectrum of cancer entities, yet their precise role in carcinogenesis is not clear to date Among SWI/SNF-mutant entities, SMARCB1-deficient cancers such as Epithelioid Sarcoma (EpS) are characterized by this genetic event in an otherwise rather silent mutational landscape. However, its oncogenic role remains unclear. Here, we generate a panel of SMARCB1 re-expressing Epithelioid Sarcoma (EpS) cell lines and employ a functional multi-omics approach to characterize and compare the function of the residual SMARCB1-deficient and the physiological SWI/SNF complex in EpS. We show that SWI/SNF holds canonical characteristics of both tumor-suppressors and proto-oncogenes due to its multi-faceted role in the regulation of the epigenome. Our data indicates that the loss of SMARCB1 causes an overall loss of SWI/SNF chromatin affinity at cis-regulatory enhancer elements, inducing a preference for uncontrolled proliferation and cell cycle progression as opposed to development and differentiation. As epigenetic regulation is a dynamic complex, we further demonstrate that EpS cell lines depend on continuous residual SWI/SNF action to maintain clonogenicity and proliferation. Consequently, our models exhibit markedly increased sensitivity to pharmacological inhibition of the residual SWI/SNF when compared with SWI/SNF-proficient cancer entities. Collectively, our results shed new light on the pleiotropic, deregulated pathways upon SWI/SNF dysfunction in EpS and provide inroads for further therapeutic approaches.

overall_design: ChIP-Seq for H3K4me3, H3K27me3, H3K27ac, BRG1, SMARCB1 (aliases: BAF47/hSNF5/INI-1) in stable isogenic VA-ES-BJ EpS cell lines in 2 biological replicates with/without (tetON) re-expression of SMARCB1.

first: Jia xiang

last: Jin

first: Fabia

last: Fuchslocher

middle: G

last: Grünewald


title: Antigen experience history directs distinct functional states of CD8+ CAR T cells during the anti-leukemia response [ATAC-seq]


title: Loss of SMARCB1 evokes targetable epigenetic vulnerabilities in Epithelioid Sarcoma [ATAC-Seq]

overall_design: ATAC-Seq for 4 stable isogenic EpS cell lines (FU-EPS-1, HS-ES-2M, NEPS, VA-ES-BJ) with 2 biological replicates each for 3 conditions: after 4d BRG1/BRM-inhibition with BRM014, after 4d SMARCB1 re-expression, control with no treatment.


title: Staggered immunization with mRNA vaccines encoding SARS-CoV-2 polymerase or spike antigens broadens the T cell epitope repertoire

status: Public on Nov 08 2024

city: Los Angeles

first: Owen

last: Witte

address: 675 Charles E Young Dr S, 5720 MRL Bldg; MC 729022

department: Microbiology, Immunology, and Molecular Genetics

institute: University of California, Los Angeles

summary: The infrequently altered SARS-CoV-2 protein RNA dependent RNA polymerase (RdRp) is a promising target for T cell-targeting mRNA vaccines. We used single cell RNA sequencing (scRNA-seq) to define the transcriptional profile and TCR repertoire of CD8+ cells in mice immunized with a mRNA vaccine encoding SARS-CoV-2.

overall_design: CD8+ splenocytes were isolated from mice immun ized with a RdRp-encoding mRNA vaccine and analyzed using scRNAseq and scVDJ-seq.

first: Evan

last: Abt

first: Ting-Ting

last: Wu

first: Wendy

last: Tran


title: RNA-seq of control and PEBP1 knockout human RPE1 cells treated with or without oligomycin

city: Haining

first: Mikael

last: Bjorklund

address: International Campus

institute: Zhejiang University-University of Edinburgh Institute

summary: This experiment compares the transcriptional response of control and PEBP1 KO cells to ATP synthase inhibitor oligomycin.

overall_design: Three control KO replicates treated with and without oligomycin, three PEBP1 KO replicates treated with and without oligomycin

first: Ling

last: Cheng

first: Yidi

last: Kong


title: scEGOT: single-cell trajectory inference framework by entropic Gaussian mixture optimal transport

city: Kyoto

first: Yukihiro

last: Yabuta

state: Kyoto

address: Yoshida-Konoe-cho, Sakyo-ku

department: Anatomy and Cell Biology

institute: Kyoto University, Graduate school of medicine

summary: Time-series single-cell RNA sequencing (scRNA-seq) data in biology has opened the door to elucidate cell differentiation processes. In this context, the optimal transport theory has attracted attention to interpolate scRNA-seq data in adjacent times and infer the trajec- tories of cell differentiation. This paper presents scEGOT, a novel comprehensive single-cell trajectory inference framework based on entropic Gaussian mixture optimal transport (EGOT). The method- ology facilitates the inference of cell differentiation pathways and dynamics from time-series scRNA-seq data. The scEGOT frame- work provides comprehensive outputs, including cell state graphs, velocity fields of cell differentiation, time interpolations of scRNA- seq data, space-time continuous videos of cell differentiation with gene expressions, gene regulatory networks, and reconstructions of Waddington’s epigenetic landscape. These outputs allow us to un- derstand the dynamics of the cell differentiation process from mul- tiple perspectives. To demonstrate that scEGOT is a powerful and versatile tool for single-cell biology, it was applied to time-series scRNA-seq data of the human primordial germ cell-like cell (human PGCLC) induction system. This application provided new insights into the mechanism of PGCLC/somatic cell segregation. In particu- lar, we identified the PGCLC progenitor population and the time point of the segregation. In addition, we found novel key genes that may be critical for human PGCLC differentiation.

overall_design: Time-series single-cell RNA sequencing (scRNA-seq) data. For the aggregates of PGCLC induction at day 0.5, 1, 1.5 and 2, the aggregates were collected.

first: Toshiaki

last: Yachimura

first: Hanbo

last: Wang

first: Yusuke

last: Imoto

first: Momoko

last: Yoshida

first: Sohei

last: Tasaki

first: Yoji

last: Kojima

first: Mitinori

last: Saitou

first: Yasuaki

last: Hiraoka


title: The DNA demethylase TET1 modifies the impact of maternal folic acid status on embryonic brain development

city: Leuven

first: Lehua

last: Chen

address: Herestraat 49

country: Belgium

institute: KU Leuven

type: Methylation profiling by high throughput sequencing

summary: Folic acid (FA) is well known to prevent neural tube defects (NTDs), but we do not know why many human NTD cases still remain refractory to FA supplementation. Here, we investigate how the DNA demethylase TET1 interacts with maternal FA status to regulate mouse embryonic brain development. We determined that cranial NTDs display higher penetrance in non-inbred than in inbred Tet1-/- embryos and are resistant to FA supplementation across strains. Maternal diets that are either too rich or deficient in FA are linked to an increased incidence of cranial deformities in wild type and Tet1+/- offspring and to altered DNA hypermethylation in Tet1-/- embryos, primarily at neurodevelopmental loci. Excess FA in Tet1-/- embryos results in phospholipid metabolite loss and reduced expression of multiple membrane solute carriers, including a FA transporter gene that exhibits increased promoter DNA methylation and thereby mimics FA deficiency. Moreover, FA deficiency reveals that Tet1 haploinsufficiency can contribute to DNA hypermethylation and susceptibility to NTDs. Overall, our study suggests that epigenetic dysregulation may underlie NTD development despite FA supplementation.

overall_design: 129S6.cg Tet1 HET dams were adapted to different FA diets: 30 ppm, 3 ppm, 0.1 ppm minimally for 4 weeks. After a successful timed-mating, dams were euthanized at E11.5 for brain tissue collection. WT and Tet1 KO brains from 30 ppm and 3 ppm FA groups were selected for WGBS.

first: Bernard

last: van der Veer


title: Integrative analysis of patient-derived tumoroids and ex vivo organoid modeling of ARID1A loss in bladder cancer reveals therapeutic molecular targets

status: Public on Nov 07 2024

city: Rotterdam

first: Tokameh

last: Mahmoudi

address: Dr. Molewaterplein 40

department: Department of Urology, Department of Biochemistry, Department of Pathology

country: Netherlands

institute: Erasmus MC Cancer Institute

summary: Somatic mutations in ARID1A (AT-rich interactive domain-containing protein 1A) are present in approximately 25% of bladder cancers (BC) and are associated with poor prognosis. With a view to discover effective treatment options for ARID1A-deficient BC patients, we set out to identify targetable effectors dysregulated consequent to ARID1A deficiency. Integrative analyses of ARID1A depletion in normal organoids and data mining in publicly available datasets revealed upregulation of DNA repair and cell cycle-associated genes consequent to loss of ARID1A and identified CHEK1 (Checkpoint kinase 1) and chromosomal passenger complex member BIRC5 (Baculoviral IAP Repeat Containing 5) as therapeutically drug-able candidate molecular effectors. Ex vivo treatment of patient-derived BC tumoroids with clinically advanced small molecule inhibitors targeting CHEK1 or BIRC5 was associated with increased DNA damage signalling and apoptosis, and selectively induced cell death in tumoroids lacking ARID1A protein expression. Thus, integrating public datasets with patient-derived organoid modelling and ex-vivo drug testing can uncover key molecular effectors and mechanisms of oncogenic transformation, potentially leading to novel therapeutic strategies. Our data point to ARID1A protein expression as a suitable candidate biomarker for the selection of BC patients responsive to therapies targeting BIRC5 and CHEK1.

overall_design: Transcriptomic profiles of patient-derived bladder cancer tumoroid (PDT) and normal bladder organoid (NO) lines were characterised by quant-seq on total RNA. Additionally, the effect of ARID1A-depletion was investigated by comparing ARID1A-depleted normal organoids vs. parental or shRNA controls. Chromatin accessibility profiling by ATAC-seq was performed on a patient-derived tumoroid line depleted for ARID1A or E2F8 vs. shRNA control.

first: Tahlita

last: Zuiverloon

first: Pantelis

last: Hatzis


title: NOTCH1 Drives Sexually Dimorphic Immune Responses in Hepatocellular Carcinoma

city: New York

first: Amaia

last: Lujambio

state: New York

address: 1470 Madison Avenue

institute: Icahn School of Medicine at Mount Sinai

summary: We study the role of NOTCH1 in immune escape in HCC.

overall_design: Examination of transcriptional profiles in NOTCH1 murine tumors

first: Katherine

last: Lindblad

first: Alexander

last: Tsankov


title: Aging modifies endometrial dendritic cell function and unconventional double negative T cells in the human genital mucosa.

city: Detroit

first: Marta

last: Rodriguez-Garcia

state: MI

address: 275 E Hancock St

institute: Wayne State University

summary: Immune function in the genital mucosa balances reproduction with protection against pathogens. As women age, genital infections, and gynecological cancer risk increase, however, the mechanisms that regulate cell-mediated immune protection in the female genital tract and how they change with aging remain poorly understood. Unconventional double negative (DN) T cells (TCRαβ + CD4-CD8-) are thought to play important roles in reproduction in mice but have yet to be characterized in the human female genital tract. Using genital tissues from women (27–77 years old), here we investigated the impact of aging on the induction, distribution, and function of DN T cells throughout the female genital tract.

overall_design: Hysterectomy samples of endometrium and endocervix of female donor, determined to be healthy according to study criteria, (no cancer or active infections) were enzymatically digested to generate single-cell suspensions. To enrich for immune cells, the single cell suspensions were filtered to remove cells larger than 30mm, followed by depletion of red blood cells and fibroblasts through magnetic bead selection. The cells were subsequently stained with CITE-seq antibodies (AbSeq) and sample multiplexing antibodies (hs_SampleTag). Single-cell suspensions were processed on the BD Rhapsody system according to manufacturer’s protocols. cDNA libraires were constructed using BD Rhapsody Whole Transcriptome (WTA) Amplification KIT (BD Biosciences, Catalog# 633801).

first: Ragav Siddharth

last: Parthasarathy


title: Enhanced RNAi does not provide efficient innate antiviral immunity in mice in vivo. [smallRNA-Seq spleen]

city: Prague

first: Filip

last: Horvat

address: Videnska 1083

country: Czech Republic

institute: Institute of Molecular Genetics of the ASCR

type: Non-coding RNA profiling by high throughput sequencing

summary: In RNA interference (RNAi), long double-stranded RNA (dsRNA) is cleaved by Dicer endonuclease into small RNA interfering RNAs (siRNAs), which guide degradation of complementary RNAs. While RNAi mediates antiviral innate immunity in plants and many invertebrates, vertebrates adopted sequence-independent response and their Dicer produces siRNAs inefficiently because it is adapted to process small hairpin microRNA precursors in the gene-regulating microRNA pathway. Mammalian RNAi is thus a rudimentary pathway of unclear significance. To investigate its antiviral potential, we modified mouse Dicer locus to express a truncated variant (DicerΔHEL1) known to stimulate RNAi. Next, we analyzed how DicerΔHEL1/wt mice respond to four RNA viruses: Coxsackievirus B3 (CVB3) and encephalomyocarditis virus (ECMV) from Picornaviridae; tick-borne encephalitis virus (TBEV) from Flaviviridae; and lymphocytic choriomeningitis virus (LCMV) from Arenaviridae. Increased Dicer activity in DicerΔHEL1/wt mice had no antiviral effect. This result supports insignificant antiviral function of endogenous mammalian RNAi in vivo. However, we also report that sufficiently high expression of DicerΔHEL1 suppressed LCMV in embryonic stem cells and in a transgenic mouse model. Altogether, mice with increased Dicer activity offer a new benchmark for identifying and studying viruses susceptible to mammalian RNAi in vivo.

overall_design: Bulk small RNA-seq of mouse samples extracted with two different protocols, two Dicer genotypes, spleen infected with LCMV virus: - Qiazol extraction, spleen, LCMV infection = 2x Dicer wt/wt; 2x Dicer tg(O-HA)/tg(O-HA) - TrapR extraction, spleen, LCMV infection = 2x Dicer wt/wt; 2x Dicer tg(O-HA)/tg(O-HA)

first: Petr

last: Svoboda

first: Radek

last: Malik

first: Marcos Iuri

last: Roos Kulmann

first: Eliska

last: Taborska


title: Mapping the glial transcriptome in Huntington’s disease using snRNAseq: Selective disruption of glial signatures across brain regions

city: Edinburgh

first: Anna

last: Williams

state: Edinburgh

address: 5 Little France Drive, Centre for Regenerative Medicine

institute: University of Edinburgh

summary: Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease with a fatal outcome. There is accumulating evidence of a prominent role of glia  in the pathology of HD, and we investigated this by conducting single nuclear RNA sequencing (snRNAseq) of human post mortem brain in four differentially affected regions; caudate nucleus, frontal cortex, hippocampus and cerebellum. Across 127,205 nuclei from donors with HD and age/sex matched controls, wefound heterogeneity of glia which is altered in HD. We describe prominent changes in the abundance of certain subtypes of astrocytes, microglia, oligodendrocyte precursor cells and oligodendrocytes between HD and control samples, and these differences are widespread across brain regions. Furthermore, we highlight possible mechanisms that characterise the glial contribution to HD pathology including depletion of myelinating oligodendrocytes, an oligodendrocyte-specific upregulation of the calmodulin-dependent 3’,5’-cyclic nucleotide phosphodiesterase 1A (PDE1A) and an upregulation of molecular chaperones as a cross-glial signature and a potential adaptive response to the accumulation of mutant huntingtin (mHTT). Our results support the hypothesis that glia have an important role in the pathology of HD, and show that all types of glia are affected in the disease.

overall_design: Fresh frozen post mortem brain tissue from caudate nucleus, cerebellum, hippocampus and frontal cortex of patients with Huntington's disease, and age and sex-matched control donors.

first: Sunniva M. K.

last: Bøstrand

first: Luise A.

last: Seeker

first: Nadine

last: Bestard-Cuche

first: Nina-Lydia

last: Kazakou

first: Sarah

last: Jäkel

first: Boyd

last: Kenkhuis

first: Neil C.

last: Henderson

first: Susanne T.

last: de Bot

first: Willeke M. C.

last: van Roon-Mom

first: Josef

last: Priller


title: Ezrin drives adaptation of monocytes to the inflamed lung microenvironment

city: New Haven

first: Giulia

last: Biancon

state: Connecticut

address: 300 George Street

department: Internal Medicine - Yale Cancer Center

institute: Yale University

summary: Ezrin, an actin-binding protein, plays a crucial role in organizing the cellular cortical cytoskeleton and plasma membrane during cell migration, adhesion, and proliferation. While there is a good understanding of ezrin's function in cell types such as epithelial cells and lymphocytes, its role in monocytes/macrophages (MΦs) is less understood. Here, we used a monocyte/MΦ-specific ezrin knock-out mouse model to investigate the contribution of ezrin to monocyte recruitment and adaptation to the lung extracellular matrix (ECM) in response to lipopolysaccharide (LPS). Our study revealed that LPS induces ezrin expression in monocytes/MΦs, and that ezrin is essential for monocytes to adhere to lung ECM, proliferate, and differentiate into tissue-resident interstitial MΦs.  Notably, ezrin is not required for monocyte extravasation into the lung parenchyma. Mechanistically, the loss of ezrin in monocytes disrupts activation of FAK and AKT signaling, which are necessary for lung-recruited monocytes and monocyte-derived MΦs to adhere to the ECM, proliferate, and survive. In summary, our data suggest that ezrin plays a role beyond structural cellular support, influencing diverse monocytes/MΦ processes and signaling pathways in response to infections, driving their adaptation to the lung microenvironment.

overall_design: In this study, we performed high-throughput sequencing of RNA isolated from lung monocyte and macrophage populations (monocyte derived macrophages -moMs, transitional interstitial macrophages (tr/tnsIMs), mature interstitial macrophages (matIMs) of (WT) and B6.Ezrinfl/fl.Cx3cr1tm1.1(cre)Jung/J (Ez-KO) mice that were exposed to LPS for 24h. Sequencing was performed performed in 3 biological replicates per genotype, celltype and timepoint.

first: Ravindra

last: Gudneppanavar

first: Caterina

last: Di Pietro

first: Hasan

last: Öz

first: Ping-Xia

last: Zhang

first: Ee-Chun

last: Cheng

first: Pamela

last: Huang

first: Toma

last: Tebaldi

first: Stephanie

last: Halene

first: Adam

middle: D

last: Hoppe

first: Catherine

last: Kim

first: Anjelica

last: Gonzalez

first: Diane

middle: S

last: Krause

first: Marie

last: Egan

first: Neetu

last: Gupta

first: Thomas

last: Murray

first: Emanuela

last: Bruscia


title: Kinase activity of nuclear TYRO3 promotes colon cancer malignancy

city: Tainan

first: Shaw-Jenq

last: Tsai

address: 1 , University Road

department: Department of Physiology, College of Medicine

country: Taiwan

institute: National Cheng Kung University

summary: The goal of this study is to compare the different biological functions between nuclear (ICD)-TYRO3- and kinase dead (KD)-TYRO3-overexpressed cells by BRD3 occupancy profiling. We revel that ICD-TYRO3-overexpressed cell were particularly enriched in negatively regulate cell death, regulation of cell cycle, regulation of vascular permeability, positively regulation of EMT, cell-cell adhesion, and regulation of phosphorylation.

overall_design: ChIP-seq profiles of nuclear TYRO3 or kinase-dead TYRO3 overexpressing HCT116 cells

first: Pei-Ling

last: Hsu


title: HSP and CD279 gene expression as candidate biomarkers in symptomatic LGLL patients

city: Milano

first: Andrea

last: Franceschini

state: Milano

address: via Ripamonti 435

institute: European Institute Of Oncology

summary: Stemming from preliminary results obtained by single cell-RNAseq we investigated by qPCR HSP and IFIT gene families in 27 LGLL patients (23 T-LGLL and 4 NK-LGLL), including 11 with neutropenia and/or thrombocytopenia and 16 asymptomatic for the disease. HSP90AA1 and HSPA1B, among HSP family and CD279 exhibit a significantly higher expression in CD3+ CD57+ sorted cells of symptomatic LGLL patients compared to asymptomatic patients and healthy controls. Also, monocytes derived from symptomatic LGLL patients express high levels of CCL3, CCL4 and CCL5 mRNA and of IL-1β, IL-6, TNF, and PD-L1 mRNA, thus confirming a pro-inflammatory cytokine profile reminiscent of a non-classical phenotype. HSP90AA1 and HSPA1B, among HSP family and CD279 exhibit a significantly higher expression in CD3+ CD57+ sorted cells of symptomatic LGLL patients compared to asymptomatic patients and healthy controls. Also, monocytes derived from symptomatic LGLL patients express high levels of CCL3, CCL4 and CCL5 mRNA and of IL-1β, IL-6, TNF, and PD-L1 mRNA, thus confirming a pro-inflammatory cytokine profile reminiscent of a non-classical phenotype. HSP90AA1 and HSPA1B, among HSP family and CD279 exhibit a significantly higher expression in CD3+ CD57+ sorted cells of symptomatic LGLL patients compared to asymptomatic patients and healthy controls. Also, monocytes derived from symptomatic LGLL patients express high levels of CCL3, CCL4 and CCL5 mRNA and of IL-1β, IL-6, TNF, and PD-L1 mRNA, thus confirming a pro-inflammatory cytokine profile reminiscent of a non-classical phenotype.

overall_design: Single cell samples of TLGLL patients peripheral blood: 2 Symptomatic T-LGLL patients with neutropenia, 2 asymptomatic T-LGLL gamma delta patients, 2 secondary patients that developed T-LGLL after being treated for B-NHL, 2 healthy controls.

first: Giovanna

last: Talarico

first: Patrizia

last: Mancuso

first: Francesco

last: Bertolini


title: Effect of Mac1 deletion on copper homoestasis and virulence in Fusarium oxysporum f. sp. lycopersici [RNA-seq]

city: Córdoba

first: Antonio

last: Di Pietro

state: Córdoba

address: Campus Rabanales, Edificio Gregor Mendel, 1

department: Genetics

institute: Universidad de Cordoba

summary: The transcription factor Mac1 is a key regulator of copper homeostasis, controlling the transcriptional response to copper-limiting conditions in fungi. It has been described as a virulence factor in human pathogens such as Candida albicans and Aspergillus fumigatus, but surprisingly its role in plant pathogenesis is poorly understood. Targeted deletion of Mac1 in the soilborne pathogen Fusarium oxisporum results in an inability to grow under copper-limited conditions and kills tomato plants. The aim of this RNA-seq analysis is to elucidate the role of Mac1 in gene regulation under copper starvation and during infection of tomato plants by F. oxysporum.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for Fusarium oxysporum f. sp. lycopersici 4287 WT and mac1 knock-out mutant  grown in liquid culture with and witout added copper (Cu) and tomato root tissue after 2 and 6 days post infection (dpi)

first: Rafael

last: Palos-Fernández

first: María

middle: V

last: Aguilar-Pontes

first: Gema

last: Puebla-Planas

first: Harald

last: Berger

first: Lena

last: Studt-Reinhold

first: Joseph

last: Strauss

first: Manuel

last: López-Berges