title: Discovering Genetic Modulators of the Protein Homeostasis System through Multilevel Analysis

city: Amherst

first: Peter

last: Chien

address: 240 Thatcher Rd, Life Science Laboratories Room N325

institute: University of Massachusetts Amherst

summary: Every protein progresses through a natural lifecycle from birth to maturation to death; this process is coordinated by the protein homeostasis system. Environmental or physiological conditions trigger pathways that maintain the homeostasis of the proteome. An open question is how these pathways are modulated to respond to the many stresses that an organism encounters during its lifetime. To address this question, we tested how the fitness landscape changes in response to environmental and genetic perturbations using directed and massively parallel transposon mutagenesis in Caulobacter crescentus. We developed a general computational pipeline for the analysis of gene-by-environment interactions in transposon mutagenesis experiments. This pipeline uses a combination of general linear models (GLMs), statistical knockoffs, and a nonparametric Bayesian statistical model to identify essential genetic network components that are shared across environmental perturbations. This analysis allows us to quantify the similarity of proteotoxic environmental perturbations from the perspective of the fitness landscape. We find that essential genes vary more by genetic background than by environmental conditions, with limited overlap among mutant strains targeting different facets of the protein homeostasis system. We also identified 146 unique fitness determinants across different strains, with 19 genes common to at least two strains, showing varying resilience to proteotoxic stresses. Experiments exposing cells to a combination of genetic perturbations and dual environmental stressors show that perturbations that are quantitatively dissimilar from the perspective of the fitness landscape are likely to have a synergistic effect on the growth defect.

overall_design:
To investigate the fitness landscapes of Caulobacter crescentus under proteotoxic stresses, we have generated transposon mutagenesis libraries from wild-type, ∆lon, ∆clpA, ∆clpB, and dnaK-NI strains.
We then subjected these libraries to heat, oxidative, and l-canavanine stress at three different levels. (low, medium, high)
Each condition contains 4 replicates. There are 2 control conditions performed with each condition. T0 control obtained from the overnight recovery after thawing. T24 control obtained by growing the libraries for 24 hours in 30C shaker after the overnight recovery.
We then performed transposon sequencing to quantify number of total and unique transposon insertions in each sample/condition.
Sequencing is utilized to quantify the frequency of transposon-induced mutations and identify a set of conditionally essential, beneficial, or detrimental genes for each environment by applying a regularized negative binomial regression combined with local False Discovery Rate (FDR) testing within a general linear model (GLM) framework.

first: Vishal

last: Sarsani

first: Berent

last: Aldikacti

first: Tingting

last: Zhao

first: Shai

last: He

first: Patrick

last: Flaherty


title: Transciptional profiling of polyIC-treated or untreated, polyIC-experience or naive primary murine alveolar macrophages

status: Public on Oct 02 2024

city: New York

first: Alexander

last: Lercher

state: NY

address: 1230 York Avenue

country: USA

institute: The Rockefeller University

type: Expression profiling by high throughput sequencing

summary: Pathogen encounter results in long-lasting epigenetic imprinting that shapes diseases caused by heterologous pathogens. The breadth of this innate immune memory is of particular interest in the context of respiratory pathogens with increased pandemic potential and wide-ranging impact on global health. Here, we investigated epigenetic imprinting across cell lineages in a disease relevant murine model of SARS-CoV-2 recovery. Past SARS-CoV-2 infection resulted in increased chromatin accessibility of type I interferon (IFN-I) related transcription factors and transcriptionally poised antiviral genes in airway-resident macrophages. Mechanistically, viral pattern recognition and canonical IFN-I signaling were required for establishment of this innate immune memory and resulting augmented secondary antiviral responses. SARS-CoV-2-associated innate immune memory in airway-resident macrophages was necessary and sufficient to ameliorate secondary disease caused by the heterologous respiratory pathogen influenza A virus. Insights into how innate immune memory shapes outcome of heterologous secondary diseases could facilitate the development of broadly effective therapeutic strategies.

overall_design: Alveolar macrophages were isolated from naive C57Bl/6J mice and exposed to polyIC for 24h, washed and restimulated with polyIC after 5 days for 6h and compared to unstimulated and/or polyIC-unexperienced controls (n = 3).

first: Charles

middle: M

last: Rice


title: Transciptional profiling of polyIC stimulated alveolar macrophages isolated from naive or SARS-CoV-2 recovered mice

summary: Pathogen encounter results in long-lasting epigenetic imprinting that shapes diseases caused by heterologous pathogens. The breadth of this innate immune memory is of particular interest in the context of respiratory pathogens with increased pandemic potential and wide-ranging impact on global health. Here, we investigated epigenetic imprinting across cell lineages in a disease relevant murine model of SARS-CoV-2 recovery. Past SARS-CoV-2 infection resulted in increased chromatin accessibility of type I interferon (IFN-I) related transcription factors and transcriptionally poised antiviral genes in alveolar macrophages. Mechanistically, viral pattern recognition and canonical IFN-I signaling were required for establishment of this innate immune memory and resulting augmented secondary antiviral responses. SARS-CoV-2-associated innate immune memory in alveolar macrophages was necessary and sufficient to ameliorate secondary disease caused by the heterologous respiratory pathogen influenza A virus. Insights into how innate immune memory shapes outcome of heterologous secondary diseases could facilitate the development of broadly effective therapeutic strategies.

overall_design: Alveolar macrophages were isolated from naive or SARS-CoV-2 (mouse adapted strain MA10) recovered C57B/6J wild type mice via bronchoalveolar lavage and restimulated with polyIC ex vivo for 6h and compared to unstimulated controls (n = 3).


title: Deletion of Gba in neurons, but not microglia, causes neurodegeneration in a Gaucher mouse model

city: Bethesda

first: Hannah

middle: B. D.

last: Duffy

state: MD

address: 10 Center Drive

institute: National Institutes of Health

summary: Gaucher disease, the most prevalent lysosomal storage disease, is caused by homozygous mutations at the GBA gene, which is responsible for encoding the enzyme glucocerebrosidase. Neuronopathic Gaucher disease is associated with microgliosis, astrogliosis, and neurodegeneration. However, the role that microglia, astrocytes, and neurons play in the disease remains to be determined. In the current study, we developed inducible, cell-type specific Gba KO mice to understand the individual impacts of Gba deficiencies on microglia and neurons. Gba was conditionally knocked out either exclusively in microglia or neurons, or throughout the body. These mouse models were developed using a tamoxifen-inducible Cre system, with tamoxifen administration commencing at weaning. Microglia-specific Gba KO mice showed no signs of disease. However, the neuron-specific Gba KO resulted in a shortened lifespan, severe weight loss, and ataxia. These mice also had significant neurodegeneration, microgliosis, and astrogliosis accompanied by the accumulation of glucosylceramide and glucosylsphingosine, recapitulating Gaucher disease-like symptoms. These surprising findings reveal that, unlike the neuron-specific Gba deficiency, microglia-specific Gba deficiency alone does not induce disease. The neuronal Gaucher disease mouse model, with a median survival of 16 weeks, may be useful for future studies of pathogenesis and the evaluation of therapies.

overall_design: In order to explore mechanisms of Gba deficiencies, cell-specific (microglial and neuronal) Gba KO mice were developed. We then performed bulk RNAseq from brain tissue. Each group (neuron-specific Gba KO (Gba nKO), microglia-specific Gba KO (Gba mKO), Gba nKO littermate controls, Gba mKO littermate controls) consisted of 6 samples. CD11b+ cells were also isolated from 4 Gba mKO (Gba mmKO) and 4 control mice for bulk RNAseq.

first: Hannah B D

first: Colleen

last: Byrnes

first: Hongling

last: Zhu

first: Galina

last: Tuymetova

first: Y Terry

last: Lee

first: Frances

last: Platt

first: Richard

middle: L

last: Proia


title: Implication of Acyl Coenzyme A Binding Protein (ACBP) in Cushing Syndrome

city: PARIS

first: Guido

last: Kroemer

address: 15 rue de L'école de médecine

country: France

institute: Centre de Recherche des Cordeliers

summary: Acyl coenzyme A binding protein (ACBP), encoded by the diazepam binding protein (DBI) gene, plays a pivotal role in stimulating appetite and lipo-anabolic reactions, showing correlations with laboratory indications of metabolic syndrome in ostensibly healthy individuals. In a screening effort targeting inhibitors of ACBP/DBI expression among distinct neuroendocrine factors, we discovered that glucocorticoids induce ACBP/DBI secretion in cultured cells.  In mouse models of iatrogenic Cushing syndrome, antibody-mediated neutralization of ACBP/DBI prevented the metabolic consequences of prolonged glucocorticoid administration. Our findings suggest that a surge in extracellular ACBP/DBI may mediate crucial aspects of Cushing syndrome.

overall_design: injection of a neutralizing monoclonal anti-ACBP/DBI antibody to prevent the metabolic consequences of Cushing syndrome.

first: Hui

last: Pan

first: Ai-Ling

last: Tian

first: Vincent

last: Carbonnier

first: Isabelle

last: Martins

first: Oliver

last: Kepp


title: Clearance of p21-highly-expressing senescent cells accelerates cutaneous wound healing

status: Public on Oct 01 2024

city: Farmington

first: Junyu

state: CT

address: 263 Farmington Avenue

institute: UConn Health

summary: Targeting senescent cells for therapeutic purposes is gaining momentum across various organ systems. However, concerns about potential off-target effects have been raised. Previous studies have shown that removing senescent cells expressing high levels of p16 (p16high) can hinder processes like wound healing. Here, we identify a distinct senescent cell population during dermal wound healing characterized by high expression level of p21 (p21high) using the p21-Cre mouse model. Using a standard cutaneous injury model, we find that eliminating p21high cells can expedite wound closure, in contrast to the effects of removing p16high cells. Through Xenium, a single cell spatial imaging platform, we show that p21high cells are distinct from p16high cells, with p21high cells mainly comprising fibroblasts, immune cells, keratinocytes, and endothelial cells with a pro-inflammatory profile. Moreover, inhibition of NF-kB signaling specifically from p21high cells partially contributes to the accelerated wound healing rates. These findings highlight the heterogeneity of senescent cells during wound healing responses within the skin and likely in other conditions.

overall_design: Wound tissue samples were harvested from mice at day 2, 4, and 12 post-injury, as well as from unwounded controls. Total RNA was extracted using TRIzol, purified with chloroform, isopropanol, and ethanol, and dissolved in RNase-free water. 500 ng RNA per sample was prepared for sequencing using the TruSeq Stranded mRNA kit and Illumina NovaSeq 6000. Reads were trimmed with Trimmomatic, mapped to GRCm39 using HISAT2, and gene expression was quantified with htseq-count.

first: Nathan

middle: S

last: Gasek

first: Ming

last: Xu


title: Deep mutational scanning of CYP2C19 reveals a substrate specificity-abundance tradeoff

city: Seattle

first: Douglas

last: Fowler

state: WA

address: 3720 15th Ave NE, Foege Building, S320

department: Genome Sciences

institute: University of Washington

summary: Cytochrome P450s (CYPs) are a family of enzymes responsible for metabolizing nearly 80% of small molecule drugs. Variants in CYPs can substantially alter drug metabolism, which may result in improper dosing and severe adverse drug reactions. CYPs have low sequence conservation, making it difficult to anticipate whether variant effects measured in one CYP may extend to others based on sequence alone. Even closely related CYPs, like CYP2C9 and its closest homolog CYP2C19, have distinct phenotypic properties despite sharing 92% of their sequences. Thus, we used Variant Abundance by Massively Parallel sequencing (VAMP-seq) to measure the protein abundance of 7,660 missense variants in CYP2C19 expressed in cultured human cells. Our results confirmed positions and structural features critical for CYP function and revealed how variants at positions conserved across all eukaryotic CYPs influence abundance. We jointly analyzed 4,670 variants whose abundance was measured in both CYP2C19 and CYP2C9, finding that the homologs have different variant abundances in substrate recognition sites within the hydrophobic core, and that substitutions in some regions reduced abundance in CYP2C19 but not CYP2C9. We also measured the abundance of all single and some multiple WT amino acid exchanges between CYP2C19 and CYP2C9. While most exchanges had no effect, substitutions in substrate recognition site 4 (SRS4) reduced abundance in CYP2C19. When nearby amino acids were exchanged in double and triple mutants, we found distinct interactions between the sites in CYP2C19 and CYP2C9, revealing a region that is partially responsible for the difference in thermodynamic stability between the two homologs. Since these positions are also important for determining substrate specificity, there may be an evolutionary tradeoff between stability and altered enzymatic function. Finally, we used our data to analyze 368 previously unannotated human variants, finding that 43% had decreased abundance. Thus, by comparing variant effects between two closely related, important human genes, we have uncovered regions underlying their functional differences and paved the way for a more complete understanding of one of the most versatile families of enzymes.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: To investigate the effect of thousands of CYP2C19 variants, we measured steady state abundance of GFP-tagged CYP2C19 expressing one variant per HEK293T cell, flow sorted cells according to GFP fluorescence, and sequenced the variant-associated barcode

first: Gabriel

middle: E

last: Boyle


title: Single-cell RNA sequencing of iPSCs-drived human bone marrow organoid

city: Chicago

first: Peng

last: Ji

state: IL

address: 303 E Chicago Ave

department: Pathology

institute: Northwestern University

summary: Human bone marrow organoids were differentiated from the commercially available human iPSC line (SCTi003-A) for single-cell RNA sequencing analysis.

overall_design: Human iPSCs were differentiated into human bone marrow organoids within a 3D hydrogel system for up to 16 days. Ten bone marrow organoids were harvested, digested into a single-cell suspension using collagenase, and then loaded onto the 10X Genomics Chromium platform for single-cell library construction.

first: Ren

last: Kehan

first: Li

last: Ermin

first: Aydemir

last: Inci

first: Han

first: Ji

last: Peng


title: Gene expression profile at single cell level of lung alveolar macrophages (AM) and interstital macrophages (IM) in MMTV-HER2 and wildtype early stage (16-week) or late stage (32-week) mice.

city: The Bronx

first: Michael

last: Papanicolaou

state: New York

address: 1300 Morris Park Avenue

department: Cell Biology

institute: Albert Einstein College of Medicine

summary: Disseminated cancer cells reside in the lung alveolar niche where they are exposed to interactions with alveolar macrophages. Here we report singe-cell RNA-sequencing data of lung macrophages from MMTV-HER2 mice at the early stage (16-weeks) harboring single dormant cancer cells and late stage mice (32-weeks harboring metastatic lesions, as well as wildtype control mice. We discovered alveolar macrophage and interstitial macrophage subsets that change in expression profile and frequency between stages. We find majority of the AMs are homeostatic in phenotype, and emergence of inflammatory AM subsets in late-stage MMTV-HER2 mice.

overall_design: Lungs from MMTV-HER2 mice and wildtype FVB mice from early stage (16-weeks) and late stage (32-week) were dissociated and macrophages were FACs sorted for single cell RNA sequencing.

first: Matthew

last: Park

first: Erica

last: Dalla

first: Julio

last: Aguire-Ghiso

first: Miriam

last: Merad


title: Rapid design of bacteriophage cocktails to suppress the burden and virulence of gut-resident carbapenem-resistant Klebsiella pneumoniae

first: Mark

last: Mimee

address: Cummings Life Science Center 920 E 58th St, Room 1001

department: Microbiology

institute: University of Chicago

summary: Antibiotic use can lead to expansion of multi-drug resistant pathobionts within the gut microbiome that can cause life-threatening infections. Selective alternatives to conventional antibiotics are in dire need. Here, we describe a Klebsiella PhageBank that enables the rapid design of antimicrobial bacteriophage cocktails to treat multi-drug resistant Klebsiella pneumoniae. Using a transposon library in carbapenem-resistant K. pneumoniae, we identified host factors required for phage infection in major Klebsiella phage families. Leveraging the diversity of the PhageBank and experimental evolution strategies, we formulated combinations of phages that minimize the occurrence of phage resistance in vitro. Optimized bacteriophage cocktails selectively suppressed the burden of multi-drug resistant K. pneumoniae in the mouse gut microbiome and drove bacterial populations to lose key virulence factors that act as phage receptors. Further, phage-mediated diversification of bacterial populations in the gut enabled co-evolution of phage variants with higher virulence and a broader host range. Altogether, the Klebsiella PhageBank represents a roadmap for both phage researchers and clinicians to enable phage therapy against a critical multidrug-resistant human pathogen.

overall_design: To further investigate the effect of csrD deletion on cell metabolism, we performed RNA-seq on MH258 and ∆csrD. Cells were grown in vitro to mid-log phase for RNA isolation and RNA sequencing. Differential expression and gene set enrichment analysis was done to analyze how the loss of the carbon storage regulator (csrD) affects phage infection.

first: Ella

last: Rotman

first: Joshua

last: Glazier


title: A measles virus collective infectious unit that caused lethal human brain disease includes many locally restricted and few widespread copy-back defective genomes

city: Rochester

first: Roberto

last: Cattaneo

state: MN

address: 222 3rd Ave SW

department: Molecular Medicine

institute: Mayo Clinic

summary: During virus replication in cultured cells, copy-back defective viral genomes (cbDVG) can arise. CbDVG are powerful inducers of innate immune responses in vitro, but their occurrence and impact on natural infections of human hosts remain poorly defined. We asked whether cbDVG were generated in the brain of a patient who succumbed to subacute sclerosing panencephalitis (SSPE) about 20 years after acute measles virus (MeV) infection. Previous analyses of 13 brain specimens of this patient indicated that a collective infectious unit (CIU) drove lethal MeV spread. In this study, we identified 276 replication-competent cbDVG species, each present in over 100 copies in the brain. Six species were detected in multiple forebrain locations, implying that they travelled long-distance with the CIU. The cbDVG to full-length genomes ratio was often close to 1 (0.6-1.74). Most cbDVG were 324-2000 bases in length, corresponding to 2-12% of the full-length genome; all are predicted to have complementary terminal sequences. If improperly encapsidated, these sequences have the potential to form double-stranded structures that can induce innate immune responses. To assess this, we examined the transcriptome of all brain specimens. Several interferon- and inflammatory response genes were upregulated, but upregulation levels did not correlate with cbDVG levels in the specimens. Thus, the CIU that drove MeV pathogenesis in this brain includes, in addition to two complementary full-length genome populations, many locally restricted and few widespread cbDVG species. The widespread cbDVG species may have been positively selected but how they impacted pathogenesis remains to be determined.

overall_design: RNA was extracted from frozen brain specimens using Trizol extraction protocol. The concentration and integrity of the RNA were assessed on an Agilent Bioanalyzer DNA 100 chip (Agilent). cDNA library prep was conducted using Illumina TruSeq Stranded Total RNA Sample Prep Kit (Illumina) according to the manufacturer’s protocol, which depletes ribosomal RNA. DNA fragmentation of 150 bp and two paired end sequencing (2 x 150) of each library was performed on an Illumina MiSeq or HiSeq4000 platform. Direct RNA library preparation was based on Oxford Nanopore Technologies (ONT) SQK-RNA004 kit and used 1 ug of RNA from brain specimens in 8.5 µL volume. For MeV cbDVG RNA sequencing, we synthesized a cbDVG specific oligo B to form part of the reverse transcriptase adapter (RTA), which has 12 nucleotides complementary to the conserved 3′ end of MeV antigenome and cbDVG. This oligoB-MeV-cbDVG (5′ to 3′) GAGGCGAGCGGTCAATTTTCCTAAGAGCAAGAAGAAGCCaccagacaaagc was combined with oligo A in a 1:1 ratio at 1.4 μM in buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl) by heating to 95°C for 2 minutes and letting them cool down slowly (0.1°C/sec). This mixture was used as a direct replacement to the RTA supplied in the ONT SQK-RNA004 kit. Adapter ligated MeV-cbCVG RNA was directly sequenced on the GridION ONT sequencer using MinION RNA flow cells (FLO-MIN004RA). All libraries were sequenced in separate flow cells using the high-accuracy base-calling model and the default run settings for direct RNA sequencing.

first: Biruhalem

last: Taye

first: Iris

last: Yousaf

first: Chanakha

last: Navaratnarajah

first: Christian K

last: Pfaller

first: Declan C

last: Schroeder


title: Integrating Gene Expression Analysis and Ecophysiological Responses to Water Deficit in Leaves of Tomato Plants

city: Leiden

first: Salma

last: Balazadeh

address: Slviusweg 72

country: Netherlands

institute: Leiden University

summary: Soil water deficit (WD) is one of the most important abiotic stresses affecting plant survival and crop yield. Despite its economic relevance, many gaps remain in our understanding of how crops respond to WD, especially concerning the synergistic coordination of molecular and ecophysiological adaptations delaying plant mortality. In this study, we investigated the gene expression imposed by a progressive WD and combined it with measurements pointing to key ecophysiological thresholds in leaves of tomato plants. We uncovered the transcriptomic changes in mature leaves at four stages defined by physiological markers relating to different WD intensities: partial stomatal closure, complete stomatal closure, after leaf wilting, and beginning of embolism development in the veins. By identifying key transcription factors (TFs) across these progressively worsening WD stages, we investigated the timing and impact of ABA-(in)dependent gene regulatory pathways during WD. In addition, we compared the transcriptome in young developing versus mature leaves and explored the physiological mechanisms that may explain the higher tolerance to dehydration in younger leaves. By correlating the transcriptomic changes to precise ecophysiological measurements, the combined dataset will serve as a framework for future studies comparing leaf molecular and physiological responses to WD at specific intensities.

overall_design: To investigate the temporal transcriptomic response of tomato plants to a progressive water deficit, we subjected 50 day old plants to either well-watered control conditions, or complete water withholding. RNA was harvested from mature leaves from both groups at four timepoints, as counted from the start of water withholding: 4 days, 5 days, 8 days, and 10 days. For the final timepoint, we additionally harvested young developing eaves.

first: Tobias

middle: A

last: de Werk

first: Giovanni

last: Bortolami

first: Maximillan

last: Larter

first: Ajaree

last: Thonglim

first: Bernd

last: Mueller-Roeber

first: Frederic

last: Lens


title: RNA Polymerase II ChIP in mESCs depleted in holo-TFIID [ChIP-Seq]

city: Illkirch

first: Stephane

middle: D

last: VINCENT

address: 1 rue Laurent Fries

department: Development and Stem Cells

institute: IGBMC

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Analysis of RNA Polymerase II genomic distribution in mESCs depleted or not for TAF7 (RT7 line) or TAF10 (RT10).

overall_design: Stéphane D VINCENT

first: Dylane

last: DETILLEUX

first: Matthieu

last: STIERLE

first: Stéphanie

last: LE GRAS


title: Insights into the mechanisms of serplulimab: a distinctive anti-PD-1 monoclonal antibody, in combination with a TIGIT or LAG3 inhibitor in preclinical tumor immunotherapy studies

city: Shanghai

first: Yongqiang

last: Shan

address: 367 Shengrong Rd, Pudong District

country: China

institute: Shanghai Henlius Biologics Co.,Ltd.

summary: Background: Anti-PD-1 therapy has become a cornerstone of tumor immunotherapy. Over a dozen of anti-PD-1/PD-L1 antibodies have currently been marketed. However, these agents exhibit notable disparities in their characteristics and clinical performance. For instance, in the field of small cell lung cancer (SCLC) where the majority of anti-PD-1 antibodies yielded limited success, Serplulimab produced impressive survival improvements and has been approved by NMPA. The marketing authorization application has also been validated by EMA. Nevertheless, the molecular mechanism underpinning Serplulimab’s superiority over its competitors remains elusive.  Methods: We characterized the difference of Serplulimab with approved PD-1/PD-L1 inhibitors such as Pembrolizumab and Nivolumab on their antibody binding features and funcations in vitro and anti-tumor activity in vivo. We also investigated the potential cellular pathways underlying the efficacy of Serplulimab in combination with other immune checkpoint inhibitors. Results: In comparison to competitors, Serplulimab robustly induces PD-1 receptor endocytosis while fostering weaker PD-1-CD28 cis interactions. This phenomenon could mitigate the dephosphorylation of CD28 by SHP2, thereby facilitating sustained and robust T cell activation. Notably, Serplulimab and Pembrolizumab exhibited similar performance in both in vitro Mixed Lymphocyte Reaction assays and in vivo efficacy studies. However, upon co-administration with anti-TIGIT or anti-LAG3, the Serplulimab-containing combination consistently demonstrated superior tumor killing efficacy compared to the Pembrolizumab-based combination. Moreover, our mechanistic investigations have unveiled that the Serplulimab combination effectively reduces tumor microenvironment Treg cell populations, augments effector and memory T cell populations, and more potently modulates genes associated with diverse facets of the immune system, surpassing the effects of the Pembrolizumab combination.  Conclusions: In summary, our data underscore Serplulimab as a highly differentiated PD-1 monoclonal antibody with best-in-class therapeutic potential

overall_design: HuGEMM human PD-1 and TIGIT double knock-in mice (BALB/c background) were used to inoculate CT26 tumor cells subcutaneously inoculated in the right flank with 0.5 × 106 cells in 0.1 mL PBS. Treatment was initiated 8 days later when tumors reached 90–100 mm3. The mice were randomized into groups so that the average tumor sizes of all groups were similar, and treatment by intraperitoneal injections was initiated on day 8. Vehicle group received saline solution (10 μL/g) twice weekly, other group received tested antibodies treatment twice weekly. Tumor tissue RNA was purified and performed with 2x150 paired-end sequencing on MGISEQ-2000RS (MGI).

first: Yizhou

last: Zhang

first: Ruicheng

last: Wei

first: Ge

last: Song

first: Xinyi

last: Yang

first: Mengli

first: Wei

last: Liu

first: Aiying

last: Xiong

first: Xuehan

first: Qianhao

last: Li

first: Wan-Jen

first: Chencheng

last: Han

first: Rui

first: Chen

last: Hu

first: Qingyu

last: Wang

first: Jun

last: Zhu


title: Inter-chromosomal contacts demarcate genome topology along a spatial gradient

city: Toronto

first: Philipp

last: Maass

state: Ontario

address: 686 Bay St

department: Genetics and Genome Biology

country: Canada

institute: The Hospital for Sick Children

summary: Non-homologous chromosomal contacts (NHCCs) between different chromosomes participate considerably in gene and genome regulation. However, due to analytical challenges, NHCCs are currently considered as singular, stochastic events, and their extent and fundamental principles across cell types and sexes remain controversial. To determine the fundamental properties of NHCCs, we developed a supervised and unsupervised learning algorithm, termed Signature. Signature revealed 40,282 NHCCs and their properties across 62 Hi-C datasets of 53 diploid human cell types. Genomic regions of NHCCs are gene-dense, highly expressed, and harbor genes for cell-specific and sex-specific functions. Extensive inter-telomeric and inter-centromeric clustering occurs across cell types and 61 NHCCs are consistently found at the nuclear speckles. These constitutive ‘anchor loci’ facilitate an axis of genome activity whilst cell-type-specific NHCCs act in discrete hubs. Our results suggest that non-random chromosome positioning is supported by constitutive NHCCs that shape genome topology along an off-centered spatial gradient of genome activity.

overall_design: Omni-C datasets of chondrogenic differentiation, CHLA-9, TC-32, and TeloHAEC cells. Each dataset is derived from two biological replicates, each with three technical replicates. Omni-C libraries were sequenced as paired-end runs with 2x150 bp on S4 flowcells of NovaSeq6000 with > 2 billion reads per sample.

first: Milad

last: Mokhtaridoost

first: Jordan

middle: J

last: Chalmers

first: Marzieh

last: Soleimanpoor

first: Brandon

last: McMurray

first: Daniella

middle: F

last: Lato

first: Son

middle: C

last: Nguyen

first: Viktoria

last: Musienko

first: Joshua

middle: O

last: Nash

first: Sergio

last: Espeso-Gil

first: Sameen

last: Ahmed

first: Kate

last: Delfosse

first: Jared

middle: W

last: Browning

first: Rasim

last: Barutcu

first: Michael

last: Wilson

first: Thomas

last: Liehr

first: Adam

last: Shlien

first: Samin

last: Aref

first: Eric

last: Joyce

first: Anja

last: Weise

middle: G


title: Rab35 knock-down during in vitro hepatocyte differentiation

city: Dresden

first: Lenka

last: Belicova

state: Saxony

address: Pfotenhauerstr. 108

country: Germany

institute: MPI-CBG Dresden

summary: We performed siRNA-mediated knockdown of Rab35 GTPase in primary mouse hepatoblasts during their differentiation into hepatocytes and analyzed changes in gene expression profiles in cells treated with Rab35 or control (Luciferase) siRNA at the final day of the culture.

overall_design: Dlk1+ hepatoblasts were isolated from mouse E13.5 embryonic livers. Cells were reverse transfected with control and Rab35-targeting siRNAs (10 nM) and cultured till day 5, then total RNA was extracted. The RNA samples were prepared from 3 independent Dlk1+ hepatoblasts’s isolations for bulk RNA-sequencing.

first: Maarten

last: Bebelman

first: Marino

last: Zerial


title: Hedgehog signal pathway affecting myelin development and repair through cholesterol Deregulation

first: 敏溪

last: 方

address: 杭师大仓前

institute: 浙江大学

summary: we used the active point mutant model RosaM2 to inject tamoxifen at specific postnatal time periods to induce Smo overexpression in brain OPCs. Meanwhile, we used lpc-induced acute demyelination as a pathological model to explore the effect of Smo on OL differentiation under pathological conditions. The cortex and CC of SmoM2-OE (RosaM2; pdgfra-creER) and littermates were then analyzed using rna-sequencing to explore the molecular mechanisms. Our results reveal that activing hedgehog signaling in opcs (1) affects ol proper differentiation and reduces myelination; and (2) alters cholesterol biosynthesis and trafficking in mouse oligodendrocytes. We have refined the regulatory network of oligodendrocyte development and myelination, established the functional link between the Hh signaling pathway, cholesterol homeostasis and myelination, and provided guidance for the application of Hh agonists/antagonists in the treatment of demyelination-related neurodegenerative diseases.

overall_design:
In view of our findings that overexpression of Smoothened(Smo) delays oligodendrocyte differentiation, we tried to identify downstream target genes regulated by Smo through RNA-sequencing. This analysis was collected the bulk RNA of cortex and corpus callosum from P15 SmoM2-OE mice and wild-type controls. We identified 2808 upregulated and 2474 downregulated genes (log2FC≥1, p≤0.05) in SmoM2-OE mice compared to control littermates.

Please note that the data columns (in the Differentially_expressed_genes.csv) belong to the samples as below:
sample title processed data column header
CC+Cortex, P15, Mu, rep1 P15_2
CC+Cortex, P15, Mu, rep2 P15_7
CC+Cortex, P15, WT, rep1 P15_1
CC+Cortex, P15, WT, rep2 P15_3

first: Minxi

last: Fang

first: Lixia

last: Chen

first: Fang

last: Li

first: Sitong

last: Wang

first: Mengsheng

last: Qiu

first: Xiaofeng

last: Xu


title: Liver cancer in ovo models

city: Grenoble

first: Anouk

last: Emadali

address: Allée des Alpes, Site Santé La Tronche, BP170, Cedex 9

department: Institute for Advanced Biosciences

institute: Grenoble University Hospital

summary: Using RNA-seq analysis, we have described in ovo liver cancer models providing the information that could help in preclinical research of new therapies for liver cancer.

overall_design: The white leghorn chicken fertilized eggs were obtained. At EDD9, liver tumor cells were suspended in a graft medium and were deposited onto the CAM.  On EDD18, the tumors were removed. The total RNA was extracted i) from the tumor in ovo samples (n=4 samples per each cell line), and ii) from in vitro tumor cell lines (n=3 samples per each cell line).

first: Zuzana

last: Macek Jilkova

first: Paul

last: Garcia

first: Yan

first: Emilie

last: Montaut

first: Léa

last: Payen-Gay

first: Claire

last: Bardel-Danjean

first: Arnaud

last: Vigneron


title: Spring viraemia of carp virus infection induces hypoxia response in zebrafish by stabilizing hif1α

city: 武汉

first: zixuan

last: wang

state: 湖北省

address: 湖北省武汉市东湖南路7号

institute: 中国科学院水生生物研究所

summary: The hypoxia signaling pathway controls hypoxia adaptation and tolerance of organisms, which is regulated by multiple mechanisms. Viral infection elicits various pathophysiological responses in the host. However, whether viral infection can affect the hypoxia response is still largely unknown. In this study, we found that Spring viraemia of carp virus (SVCV) infection in zebrafish caused symptoms similar to those in zebrafish under hypoxic conditions. Further assays indicated that SVCV infection activated the hypoxia signaling pathway in zebrafish. In addition, SVCV infection caused increased glycolysis and ROS levels in cells. Mechanistically, SVCV-G protein interacted with hif1α-a/b and attenuated their K48-linked polyubiquitination, leading to their stabilization and subsequent enhancement of target gene expression. Moreover, treatment with the HIF1α-specific inhibitor PX478 enhanced the antiviral ability against SVCV infection in zebrafish and zebrafish cells. This study reveals a relationship between SVCV infection and the hypoxia signaling pathway in fish, and provides a strategy for reducing the damage of viral disease in the aquaculture industry.

overall_design:
Zebrafish larvae were placed in disposable cell culture dishes (60 mm in diameter) containing 4 mL of egg water, and 2 mL of SVCV (~5.0×107 TCID50/mL) was added into each dish.
Whole RNA of zebrafish larvae (3 dpf) infected with or without SVCV were purified using RNeasy Mini Kit (QIAGEN NO. 74104). Three biological replicates were used for each sample. The transcriptome library for sequencing was generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA, #E7530L) following manufacturer’s protocol
In order to ensure the quality of information analysis, the software fastp was used to remove the splice sequences, filter the low quality, N bases (indicating that the base information could not be determined), and obtain high quality clean data. clean data, and the number of bases and sequences of the clean data were counted.
At the same time, GC, Q20, Q30 content of the clean data were calculated (GenePlus, Beijing, China). Heatmaps were generated using Multi Experiment Viewer (MeV) software. Gene Ontology (GO) enrichment analyses for the differentially expressed genes (DEGs) were performed using the cluster Profiler version 3.8.

first: Zixuan

first: Xing

last: Liu

first: Wuhan

last: Xiao


title: EWS-WT1 fusion isoforms establish oncogenic programs and therapeutic vulnerabilities in Desmoplastic Small Round Cell Tumors.

city: Charlestown

first: Rui

last: Dong

state: MA

address: 149 13th Street

institute: Massachusetts General Hospital

summary: EWS fusion oncoproteins underlie the pathogenesis of several human malignancies including Desmoplastic Small Round Cell Tumor (DSRCT), an aggressive mesenchymal tumor driven by fusions between the disordered domain of EWS and the developmental transcription factor WT1. Here we combined chromatin occupancy and long-range interaction profiles to identify EWS-WT1-dependent gene regulation networks and directly controlled target genes. We show that EWS-WT1 operates primarily as a powerful activator of distal regulatory elements and controls an oncogenic gene expression program that characterize primary DSRCTs.  Moreover, EWS-WT1 has two isoforms that differ by three amino acids in their DNA binding domain (+/- KTS), as observed for wild type WT1, and we show that each fusion isoform has a specific DNA binding profile that is distinct from its wild type counterparts and requires a functional EWSR1 prion like domain. Remarkably, xenograft experiments using human mesothelial cells, candidate cells of origin of DSRCT, reveal that both isoforms are required to generate viable tumors that resemble DSRCT. Finally, we identify new candidate EWS-WT1 target genes with potential therapeutic implications, including CCND1, whose inhibition by the clinically-approved drug Palbociclib leads to marked tumor burden decrease in DSRCT PDXs in vivo. Taken together, our studies identify gene regulation programs and therapeutic vulnerabilities in DSRCT and provide a mechanistic understanding of the complex isoform-dependent oncogenic activity of EWS-WT1.

overall_design: ChIP-seq,for,histone,marks,in,DSRCT.

first: Gaylor

last: Boulay

first: Miguel

last: Rivera

first: Nicolo

last: Riggi


title: Single-cell RNA-seq characterization of columnar epithelial cells in mouse stomach.

first: Timothy

last: Wang

address: 1130 Saint Nicholas Avenue

institute: Columbia university

summary: We investigated the nature and extent of transcriptional heterogeneity of columnar epithelial cells in mouse stomach at the celluar level using 10x scRNAseq.

overall_design: We performed 10x scRNAseq on columnar epithelial cells in mouse stomach under 3 conditions: Control, Il1b treated, and Il1b treated + p53 KO.

first: Junfei

last: Zhao


title: Transcriptomic Signatures of WNT-Driven Pathways and Granulosa Cell-Oocyte Interaction during Primordial Follicle Activation

city: Fukuoka

first: Tappei

last: Mishina

address: 744 Motooka Nishi-ku

country: Japan

institute: Kyushu University

summary: Primordial follicle activation (PFA) is a pivotal event in female reproductive biology, orchestrating the transition from quiescent to growing follicles. This study employed comprehensive single-cell RNA sequencing to investigate the transcriptomic landscape of ovarian cells, focusing on the process of PFA. The aim of our study was to gain insights into the detailed regulatory mechanisms governing synchronized dormancy and activation between granulosa cells (GCs) and oocytes with decomposition of the PFA process. The Wntless (Wls) conditional knockout (cKO) mouse served as a unique model, suppressing the transition from pre-GC to GC, and disrupting somatic cell-derived WNT signaling in the ovary. Our data revealed immediate transcriptomic changes in GCs post-PFA in Wls cKO mice, leading to a divergent trajectory, while oocytes exhibited modest transcriptomic alterations. Subpopulation analysis identified molecular pathways affected by WNT signaling on GC maturation, along with specific gene signatures linked to dormant and activated oocytes. Despite minimal evidence of continuous up-regulation of dormancy-related genes in oocytes, loss of WNT signaling in (pre-)GCs impacted gene expression on oocytes even before PFA, subsequently influencing them globally. The infertility observed in Wls cKO mice was attributed to compromised GC-oocyte molecular crosstalk and a microenvironment for oocytes. Our study highlights the pivotal role of the WNT-signaling pathway and its molecular signature, emphasizing the importance of intercellular crosstalk between (pre-)GCs and oocytes in orchestrating folliculogenesis.

overall_design: To investigate cell type-specific alteration in gene expression during PFA with and without WNT-signaling, scRNA-seq analysis was performed on the ovaries of control and Wls cKO PD7 mice using the RamDA-seq, a full-length total RNA-sequencing method for single cells

first: Hinako

last: Takase

first: Tetsutaro

last: Hayashi

first: Mika

last: Yoshimura

first: Mariko

last: Kuse

first: Itoshi

last: Nikaido

first: Tomoya

last: Kitajima


title: CLSY docking to Pol IV requires a conserved domain critical for small RNA biogenesis and transposon silencing

city: La Jolla

first: Laura

last: Martins

email: jlaw@salk.edu, lmartins@salk.edu

state: CA

address: 10010 N Torrey Pines Rd

institute: Salk Institute for Biological Studies

type: Non-coding RNA profiling by high throughput sequencing

summary: Eukaryotes must balance the need for gene transcription by RNA polymerase II (Pol II) against the danger of mutations caused by transposable element (TE) proliferation. In plants, these gene expression and TE silencing activities are divided between different RNA polymerases. Specifically, RNA polymerase IV (Pol IV), which evolved from Pol II, transcribes TEs to generate small interfering RNAs (siRNAs) that guide DNA methylation and block TE transcription by Pol II. While the Pol IV complex is recruited to TEs via SNF2-like CLASSY (CLSY) proteins, how Pol IV partners with the CLSYs remains unknown. Here, we identified a conserved CYC-YPMF motif that is specific to Pol IV and is positioned on the complex exterior. Furthermore, we found that this motif is essential for the co-purification of all four CLSYs with Pol IV, but that only one CLSY is present in any given Pol IV complex. These findings support a "one CLSY per Pol IV" model where the CYC-YPMF motif acts as a CLSY-docking site. Indeed, mutations in and around this motif phenocopy pol iv null and clsy quadruple mutants. Together, these findings provide structural and functional insights into a critical protein feature that distinguishes Pol IV from other RNA polymerases, allowing it to promote genome stability by targeting TEs for silencing.

overall_design:
4 smRNAseq libraries were sequenced. We also used 11 published smRNAseq (GSE164601) sequenced along with these 4 smRNAseq samples.
GSM5718042: WT_rep1.fastq_TagDirAll-24nt.ucsc.bedGraph.gz
GSM5718043: WT_rep2.fastq_TagDirAll-24nt.ucsc.bedGraph.gz
GSM5718044: WT_rep3_reseq.fastq_TagDirAll-24nt.ucsc.bedGraph.gz
GSM5718055: clsy3-1.fastq_TagDirAll-24nt.ucsc.bedGraph.gz
GSM5718063: clsy4-1.fastq_TagDirAll-24nt.ucsc.bedGraph.gz
GSM5718049: WT_rep4.fastq_TagDirAll-24nt.ucsc.bedGraph.gz
GSM5718050: WT_rep5.fastq_TagDirAll-24nt.ucsc.bedGraph.gz
GSM5718051: WT_rep6.fastq_TagDirAll-24nt.ucsc.bedGraph.gz
GSM5718060: WT_rep1_reseq.fastq_TagDirAll-24nt.ucsc.bedGraph.gz
GSM5718061: WT_rep2_reseq.fastq_TagDirAll-24nt.ucsc.bedGraph.gz
GSM5718062: WT_rep3_reseq.fastq_TagDirAll-24nt.ucsc.bedGraph.gz

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

first: Ceyda

last: Coruh

first: Julie

last: Law


title: Analysis of nascent transcription in ES cells mutant for specific TFIID subunits [4-sU-Seq]

first: Stephane

last: VINCENT

summary: RNA polymerase II (Pol II) transcription initiation starts with the assembly of the preinitiation complex (PIC) on core promoters. The PIC is composed of six general transcription factors (GTFs). The recognition of the core promoter sequences by the TFIID GTF complex is the first step of the PIC assembly. In metazoans, holo-TFIID is composed of the TATA binding protein (TBP) and of 13 TBP associated factors (TAFs). Genetic depletion of different murine TAFs have shown that TAFs such as TAF7 or TAF10, can be either required, or dispensable for Pol II transcription, depending on different cellular contexts. In this report, we depleted TAF7 and/or TAF10 in the same cellular system; either in mesodermal progenitors during mouse development or in mESCs. In these two models, TAF7 depletion leads to a milder phenotype compared to TAF10 depletion. As TAF10 is also a subunit of the transcriptional co-activator Spt-Ada-Gcn5 acetyl transferase (SAGA), we first showed that the difference in phenotype between the Taf7 and Taf10 mutant is not due to the SAGA effect, at least for mESCs. Immunoprecipitations coupled with mass spectrometry analyses from mESCs lysates assembly of holo-TFIID complex is rapidly affected after induction of the depletion. In line with the model of holo-TFIID sequential assembly, TAF10 depletion leads to an early defect with formation of the core-TFIID, while TAF7 depletion results in the formation of a TAF7-less TFIID. Thus, the difference in phenotype severity correlates with the degree of TFIID disassembly. Surprisingly, no major global changes in Pol II transcription could be observed after either TAF7 or TAF10 depletion. Our data suggest that the inducible loss of fully assembled canonical TFIID does not correlate with the lack of global Pol II transcription activity changes suggesting that partially assembled TFIID complexes can participate in Pol II transcription initiation, with only limited effect on Pol II nascent transcription.

overall_design: We have ES cell lines allowing the conditional deletion of Taf7 or Taf10 upon 4-OHT treatment. Cells were treated 15 minutes with 500µM 4-sU. Total RNA was collected, spiked with 4-sU labelled Drosophila S2 cells . 4-sU labelled RNA molecules were purified, sheared and sequenced.

first: Vincent

last: Hisler

first: Damien

last: Plassard

first: Matthieu

last: Jung

first: Stéphane

last: Vincent


title: Analysis of nascent transcription in ES cells mutant for specific TFIID subunits

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

overall_design: Refer to individual Series


title: Murine gamma delta intraepithelial lymphocyte response to type I IFN and TCR blockade

city: New York

first: Matthew

last: Fischer

address: 1470 Madison Ave

institute: Icahn School of Medicine at Mount Sinai

summary: Interferon alpha (IFNa) is a pro-inflammatory cytokine that is rapidly upregulated as part of an innate immune response. Gamma delta intraepithelial lymphocytes (gd IEL) mount a rapid antimicrobial response to luminal microorganisms and previous report indicate that the T cell receptor (TCR) of these cells is constantly triggered at steady-state. We assessed the contribution of tonic TCR activation in response to acute, systemic IFNa administration among murine gd IELs. Our data demonstrate that inhibiting basal TCR signaling has a minimal effect on the transcriptional profile of gd IELs and acute IFNa exposure induces antiviral gene programs independent of TCR signaling.

overall_design:
Mice were treated mice with 200 ug TCRgd antibody (UC7-13D5) or Armenian hamster IgG i.p. (-48 or -24 h) prior to i.v. administration with 1 ug murine IFNa or phosphate buffered saline (PBS) for 5 h.
RNAseq was performed on sorted gd IELs isolated from murine small intestine from each of the following treatment groups: IgG-PBS, IgG-IFNa, UC713D5-PBS, UC713D5-IFNa

first: Karen

last: Edelblum


title: Analysis of nascent transcription in ES cells mutant for specific TFIID subunits [CUT&Run]

overall_design: We have ES cell lines allowing the conditional deletion of Taf7 or Taf10 upon 4-OHT treatment. In order to analyse the distribution of TBP, TAF7, TAF10 and TAF12 on the chromatin, we carried out CUT&RUN.

first: Dylane

last: Detilleux

first: Stéphanie

last: Le Gras


title: Transcriptome Analysis in Mouse Skin After Exposure to Ultraviolet Radiation from a Canopy Sunbed

city: Ottawa

last: Meier

address: 251 Sir Frederick Banting Driveway

department: Health Canada

institute: Government of Canada

summary: Exposure of skin to ultraviolet radiation (UV-R), in the form of both natural and artificial tanning, strongly increases the risk for developing skin cancer. On a molecular level, exposing cells and tissues to UV-R results in well-documented transcriptional changes. However, many of the previous investigations into these transcriptional responses did not adequately characterize the UV emissions or only assessed a limited number of doses or post-exposure times. As a result, our objectives were to provide a mechanistic study that describes the dose- and time-dependent changes in gene expression that drive adverse short-term (e.g. sunburn) and long-term actinic (e.g. skin cancer) possibly delineating response thresholds. In the current study, we examined transcriptomic expression data from mouse skin following exposure to five erythemally-weighted doses (0, 5, 10, 20, 40 mJ/cm2) of UV-R emitted from a UV-emitting tanning device, with six post-exposure durations of 0, 6, 24, 48, 72 and 96 hours (h).  Surprisingly, the lowest sub-erythemal dose of 5 mJ/cm2, produced 116 significant DEGs at 96 h post-exposure related to genes associated with structural changes associated with UV-R damage. The largest number of significant changes in gene expression were found at the 6 and 48 h post-exposure time points at the doses of 20 and 40 mJ/cm2. At the highest dose of 40 mJ/cm2, 13 differentially expressed genes of interest were commonly perturbed across all post-exposure time points relative to the time-matched control groups.  UV-R exposure induced pathways related to oxidative stress, P53 signaling, inflammation, biotransformation, skin barrier maintenance and innate immunity. The transcriptional data generated in this in vivo study provides mechanistic insight into the short-term and potential long-term health effects of exposure to UV-R tanning that may not be threshold dependent.

overall_design: Mice were exposed to different doses of ultraviolet radiation (UVA96.4% / UVB3.6%) emitted from 8 (100Watt) UV fluorescent lamps from a tanning canopy, including sham-handled control. 5 spectral ultraviolet radiation doses (0 (sham-handled control), 5, 10, 20, or 40 mJ/cm2) were tested in quintuple (5 animals per dose = 25 samples) over 6 timepoints (0, 6, 24, 48, 72, 96 h = 150 samples in total, with a randomized design to avoid batch effects. Five samples were removed due to animal death before the final time point or low RNA yield

first: Sami

last: Qutob

first: Samantha

middle: P

last: Roesch

first: Sandy

last: Smiley

first: Pascale

last: Bellier

first: Andrew

last: Williams

first: Kate

middle: B

last: Cook

first: Andrea

last: Rowan-Carroll

first: Carole

last: Yauk

first: James

last: McNamee


title: TF ChIP-seq Caenorhabditis elegans

status: Public on Oct 06 2024

first: Louis

last: Gevirtzman

address: 3720 15th Ave NE

department: Genome Science

institute: University of Washington

summary: A catalog of transcription factor (TF) binding sites in the genome is critical for  deciphering regulatory relationships.  Here we present the culmination of the efforts of the  modENCODE (Model Organism ENCyclopedia Of DNA Elements) and modERN (model  organism Encyclopedia of Regulatory Networks) consortia to systematically assay TF binding  events in vivo in two major model organisms, Drosophila melanogaster (fly)  and Caenorhabditis elegans (worm).  These datasets comprise 605 TFs identifying 3.6M sites in  the fly and 356 TFs identifying 0.9 M sites in the worm, and represent the majority of the  regulatory space in each genome. We demonstrate that TFs associate with chromatin in clusters  termed “metapeaks”, that larger metapeaks have characteristics of high occupancy target (HOT)  regions, and that the importance of consensus sequence motifs bound by TFs depends on  metapeak size and complexity.  Combining ChIP-seq data with single cell RNA-seq data in a  machine learning model identifies particular TFs with a prominent role in promoting target gene  expression in specific cell types, even differentiating between parent-daughter cells during  embryogenesis.  These data are a rich resource for the community that should fuel and guide  future investigations into TF function.  To facilitate data accessibility and utility, all strains  expressing GFP-tagged TFs are available at the stock centers for each organism.  The chromatin  immunoprecipitation sequencing data are available through the ENCODE Data Coordinating  Center, GEO, and through a direct interface (http://epic.gs.washington.edu/modERN/) that  provides rapid access to processed data sets and summary analyses, as well as widgets to probe  the cell type-specific TF-target relationships.

overall_design: Reanalysis of raw data files in BioProject PRJNA63463  using ENCODE TF/Histone ChIP-Seq pipeline version 1.3.6  https://github.com/ENCODE-DCC/chip-seq-pipeline2

first: Michelle

last: Kudron

first: Alec

last: Victorsen

first: Bridget

last: Lear

first: Jiahao

last: Gao

first: Jinrui

last: Xu

first: Swapna

last: Samanta

first: Emily

last: Frink

first: Adri

last: Tran-Pearson

first: Chau

last: Huynh

first: Dionne

last: Vafeados

first: Ann

last: Hammonds

first: William

last: Fisher

first: Martha

last: Wall

first: Greg

last: Wesseling

first: Vanessa

last: Hernandez

first: Zhichun

last: Lin

first: Mary

last: Kasparian

first: Kevin

last: White

first: Ravi

last: Allada

first: Mark

last: Gerstein

first: LaDeana

last: Hillier

first: Susan

last: Celniker

first: Valerie

last: Reinke

first: Robert

middle: H

last: Waterston


title: SRSF2 transcriptional function maintains genome integrity during cell division

city: Heidelberg

first: Rebecca

middle: Emma

last: Wagner

address: Inf 280 im Neuenheimer Feld

department: Cell Biology and Tumor Biology

institute: DKFZ

summary: To investigate the role of SRSF2 in skin we made use of mice where SRSF2 can be conditionally deleted or a point mutation can be introduced in an undifferentiated population marked by expression of Keratin 14.

overall_design: Mouse back skin was treated topically with 4-hydroxy tamoxifen for up to 28 days. Upon collection mouse back skin was snap frozen. In order to assay the transcriptomic changes caused by SRSF2 loss or mutant SRSF2 total RNA was extracted uing Trizol reagent, according to manufacturer's instructions. RNA-sequencing libraries were then prepared. Comparative gene expression profiling analysis was then performed comparing mice which had SRSF2 deleted or mutated with an appropriate control.

first: Michaela

last: Frye

first: Anke

last: Heit-Mondrzyk


title: Next generation sequencing of white adipose tissues from adipose FoxO1 knockout mice

city: Gainesville

first: Zhiyong

last: Cheng

state: Florida

address: 572 Newell Drive

department: Food Science and Human Nutrition

institute: University of Florida

summary: FoxO1 is a transcription factor known to regulate metabolism and functions of different tissues. To study the role of FoxO1 in adipose biology, this study examined the transcript profile in inguinal and gonadal adipose tissues from conditional adipose FoxO1 knockout mice and floxed FoxO1 (control) mice.

overall_design: RNA samples from inguinal and gonadal adipose tissues from conditional adipose FoxO1 knockout mice and floxed FoxO1 (control) mice were sequenced. RNA-seq experiment is intended to identify genes and pathways that are regulated by FoxO1.

first: L

last: Shi

first: Z

last: Tao

last: Zheng


title: SRSF2 transcriptional function maintains genome integrity during cell division [cell lines]

summary: To investigate the molecular mechanisms through which SRSF2 acts it was depleted in 3 epithelial cell lines; primary human keratinocytes (HK) and two squamous cell carcinoma cell lines SCC25 and FaDu cells. Additionally FaDu cells were used to make clones with stably reduced SRSF2 levels in order to explore how cells can compensate reduced SRSF2 levels. Due to SRSF2's role in transcription Cut&Run using an antibody specific for total Pol II was performed in ctr and SRSF2 depleted FaDu cells.

overall_design: SRSF2 depletion was achieved using an siRNA pool. Total RNA was isolated 48 hours after transfection, using Trizol reagent and RNA-sequencing libraries were prepared. SLAM-sequencing and Cut&Run were also performed 48 hours after transfection and libraries were prepared from Total RNA that had undergone thiol modification or on DNA that was pulled down. RNA was also isolated from stable clones and a control clone and RNA-sequencing libraries were prepared.

first: Etienne

last: Sollier


title: RNA Sequencing FVB.B6 vs. CRYAB-R120G mutant mouse heart apex at 1,3, and 6 months of age

city: Vermillion

last: Bouska

state: SD

address: 414 E Clark St.

institute: Mark Bouska

summary: To investigate the transcriptional response to proteotoxic stress from CRYAB-R120G mutation in the heart with age, RNA sequencing of the heart apex was performed.

overall_design: RNA sequencing of the heart apex for FVB.B6 background and CRYAB-R120G mutant mice from 3 replicates of each genotype at 1,3, and 6 months of age followed by gene expression profiling analysis was performed.

first: Xuejung

last: Wang


title: The effect of co-culture on MMTV-HER2 early lesion or primary tumor cell and alveolar macrophages [bulk RNA-seq]

status: Public on Sep 30 2024

city: The Bronx

first: Michael

last: Papanicolaou

address: 1300 Morris Park Avenue

institute: Albert Einstein College of Medicine

summary: Disseminated cancer cells reside in the lung alveolar niche where they are exposed to interactions with alveolar macrophages. Here we report bulk RNA-sequencing data from co-cultured MMTV-HER2 mammary early lesion cells or mammary primary tumor cells cultured with lung alveolar macrophages. We discovered reciprocal regulation between mammary cells and alveolar macrophages, where early lesion cells instruct alveolar macrophages to maintain an anti-tumor phenotype and alveolar macrophages activate an epithelial to mesenchymal program in early lesion cells. Coveresely, primary tumor cells activate inflammatory prorgams in alveolar macrophages, while alveolar macrophages are not able to activate growth restriction programs in primary tumor cells. Receptor-ligand analysis highlighted two known pathways invovled in quiencesce, namely the TGF-beta pathway and the OSM pathway.

overall_design:
Mammary early lesion cells or mammary primary tumor cells were isolated from the MMTV-HER2 mouse model and lung alveolar macrophages were isolated from congenic wildtype mice. Early lesion or primary tumor cells were co-cultured with alveolar macrophages, and were FACS-sorted and bulk RNA-sequencing was performed. Mono-culture controls of each cell type were also sequenced.
>>>Submitter states that raw data are not available due to file back-up system failure<<<

first: Erica

last: Dalla

first: Julio

last: Aguirre-Ghiso


title: Merkel cells and corpuscles of Stannius as putative targets for polyethylene terephthalate microfibers in sheepshead minnow larvae

city: Jinju

first: June-Woo

last: Park

address: 17, Jegok-gil, Munsan-eup

department: Environmental Risk Research

country: South Korea

institute: Korea Institute of Toxicology

summary:
Purpose: This study aimed to investigate the biological effects of PET microfibers and their underlying mechanisms in early-staged sheepshead minnows (Cyprinodon variegatus).
Method: PET microfibers (about 13 μm diameter × 106 μm length) were prepared by cutting PET threads and treated to sheepshead
minnow larvae at 10 and 100 mg/L for 10 days. mRNA was produced with a TruSeq Standed mRNA library kit, and sequencing using a NovaSeq6000 equipment.
Results and Conclusions: Our study represents a detailed analysis of the transcriptome of sheepshead minnow larvae exposed to PET microfibers by RNA-seq technology. No acute toxicity was found in the minnow, but PET microfibers significantly produced reactive oxygen species and reduced behavioral responses of traveled distance and maximum velocity. The transcriptomic data suggested that Merkel cells (flow sensors) and corpuscles of Stannius (calcium regulator) are putative targets, which were derived from oxidative stress, sensory neuropathy, cognitive impairment, and movement disorders. These findings underscore that although PET microfibers are not directly lethal to sheepshead minnows, they could impact their survival by damaging swimming-related key genes. This study provides new insights into how PET microfibers are toxic to aquatic organisms and disrupt ecosystems
beyond survival and pathological changes.

overall_design: mRNA profiles of sheepshead minnow larvae exposed to PET microfiber for 10 days

first: Jin Soo

last: Choi


title: Next Generation Sequencing Facilitates Quantitative Analysis of BALB/C mice triple-negative breast cancer Transcriptomes

city: Tianjin

first: Jianwen

last: Song

address: Jinnan District

institute: Nankai Unversity

summary: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived tumor transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis

overall_design: Tumor mRNA profiles of 9-week old BALB/C mice were generated by deep sequencing, in triplicate, using Illumina GAIIx.

first: He

first: Xue

last: Meng


title: MBD2 regulates the progression and chemoresistance of cholangiocarcinoma through interaction with WDR5

city: Wuhan

first: Da

state: Hubei

address: 1095 Jiefang Avenue

department: Biliary-Pancreatic Surgery

institute: Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology

summary:
Background: Cholangiocarcinoma (CCA) is a high malignancy, rapid progression tumor of bile duct. Due to its chemoresistance, it always has an extremely poor prognosis. Therefore, it is still need to detailed elucidation the mechanisms of chemoresistance and find the therapeutic targets.
Methods: We analysis the expression of MBD2 in CCA and normal bile duct tissues through public database and immunohistochemical (IHC). The role of MBD2 in CCA cells’ proliferation, migration, chemoresistance ability was validated through CCK-8, plate clone assay, wound healing assay, xenograft mice models. Besides, we construct a primary CCA mice model to further confirm the effect of MBD2. RNA-seq, co-IP-MS were used to find the mechanisms of MBD2 leading to chemoresistance.
Results: MBD2 is up-regulated in CCA. It could promote the proliferation, migration and chemoresistance of CCA cells. Mechanistically, MBD2 could directly interact with WDR5, and binding to the promoter of ABCB1, then promote the tri-methylation of H3K4 in this region through KMT2A, active the expression of ABCB1. Knocking down of WDR5 or KMT2A could block the transcriptional activation of ABCB1 by MBD2. MM-102 is a molecular inhibitor that targets the interaction of WDR5 with KMT2A. MM-102 could inhibit the expression of ABCB1 in CCA cells, and decrease the chemoresistance of CCA to cisplatin.
Conclusion: MBD2 promote the progress and chemoresistance of CCA through interact with WDR5. MM-102 could effectively block this procedure and enhance the sensitivity to cisplatin of CCA.

overall_design: To investigate the factors regulated by MBD2 in CCA cells, RNA-seq analysis of knockdown of MBD2 and negative control TFK1 cells was performed.

first: Junsheng

last: Chen

first: Guanhua

last: Wu

first: Fei

last: Xiong

first: Wenzheng

last: Liu

first: Qi

first: Yiyang

last: Kuai

first: Wenhua

last: Huang

first: Yongqiang

last: Qi

first: Bing

first: Ruizhi

last: He

first: Yongjun


title: The Diagnostic Potential of miRNA Signatures in the Fabry Disease Serum: Automated and Manual Sample Isolations

city: Cambridge

first: Josephine

middle: Yen

last: Fang

address: 40 Landsdowne Street

department: DMPK

institute: Takeda Pharmaceuticals

summary: Fabry disease, an X-linked lysosomal storage disorder caused by galactosidase alpha (GLA) gene mutations, exhibits diverse clinical manifestations, and poses significant diagnostic challenges. Early diagnosis and treatment are crucial for improved patient outcomes, pressing the need for reliable biomarkers. In this study, we aimed to identify miRNA candidates as potential biomarkers for Fabry disease using the KingFisher™ automated isolation method and NanoString nCounter® miRNA detection assay. Clinical serum samples were collected from both healthy subjects and Fabry disease patients. RNA extraction from the samples was performed using the KingFisher™ automated isolation method with the MagMAX mirVana™ kit or manually using the Qiagen miRNeasy kit. The subsequent NanoString nCounter® miRNA detection assay showed consistent performance and no correlation between RNA input concentration and raw count, ensuring reliable and reproducible results. Interestingly, the detection range and highly differential miRNA between the control and disease groups were found to be distinct depending on the isolation method employed. Nevertheless, enrichment analysis of miRNA-targeting genes consistently revealed significant associations with angiogenesis pathways in both isolation methods. Additionally, our investigation into the impact of enzyme replacement therapy on miRNA expression indicated that some differential miRNAs may be sensitive to treatment.    Our study provides valuable insights to identify miRNA biomarkers for Fabry disease. While different isolation methods yielded various detection ranges and highly differential miRNAs, the consistent association with angiogenesis pathways suggests their significance in disease progression. These findings lay the groundwork for further investigations and validation studies, ultimately leading to the development of non-invasive and reliable biomarkers to aid in early diagnosis and treatment monitoring for Fabry disease.

overall_design: A total 39 serum samples were collected, including 20 from healthy controls and 19 from Fabry patients. These were further subdivided between two isolation methods (manual:24 and automated:23) resulting in 47 total data entries. Notably, 9 serum samples were isolated using both automated and manual methods.

middle: Y

first: Saravanan

last: Ayyadurai

first: Alyssa

last: Pybus

first: Hiroshi

last: Sugimoto

first: Mark

last: Qian


title: The glutamate receptor antagonist ifenprodil inhibits hepatitis E virus infection

city: Bochum

first: Eike

last: Steinmann

state: NRW

address: Universitätsstr. 150

department: Molecular Medical Virology

institute: Ruhr-University Bochum

summary: Infection with hepatitis E virus (HEV) represents a global problem, with over 20 million people infected annually. No specific antiviral drugs are available for treating HEV infection, necessitating the development of novel targeted therapeutics. Here, we report that the N-methyl-D-aspartate receptor (NMDAR) antagonist ifenprodil, a clinically approved drug used to treat idiopathic pulmonary fibrosis (IPF), is an HEV inhibitor in liver-derived cells. In vitro investigation demonstrates that ifenprodil suppresses viral protein expression in a dose-dependent manner in human hepatoma cells by inhibiting early stages of viral infection. We also found that ifenprodil modulates host cell intrinsic biological processes distinct from virus-induced innate immunity, inhibiting HEV RNA accumulation in primary human hepatocytes. Finally, the inhibitory effect of ifenprodil in vivo was also tested in rabbits challenged with the HEV-3ra CHN-BJ-R14 strain. Fecal virus shedding was below the limit of detection in two animals for both ribavirin-treated and ifenprodil-treated rabbits compared to vehicle-treated control animals. Our data demonstrate that ifenprodil is an effective anti-HEV compound with potential as a therapeutic candidate for the treatment of HEV infection.

overall_design: We compared host transcriptional responses in vehicle control (DMSO) treated PHH to HEV infected PHH (neHEVCC, Kernow-C1/p6), PHH treated with ifenprodil (20 µM), or PHH where infection and treatment were combined. PHH were derived from three different donors and total RNA was extracted at 48h post infection for subsequent RNA-Sequencing.

first: Mara

last: Klöhn

first: André

last: Gömer

first: Qiyu

first: Richard

last: Brown

first: Daniel

last: Todt

first: Lin

last: Wang


title: Bulk RNA-sequencing of synovial fibroblasts (IC stimulation)

first: Tetsuo

last: Hasegawa

address: Francis Crick Ave

department: Medicine

country: United Kingdom

institute: University of Cambridge

summary: Bulk RNA-sequencing of synovial fibroblasts stimulated with IC

overall_design: Entire synovium was dissected from adult wild-type mice after intravenous IC (or control) injection. Synovium was  diggested with collagenase and fibroblasts were sorted and immediately placed in RNA Later for RNA extraction.

first: Menna

last: Clatworthy

first: Colin

last: Lee


title: Adamts1 Promotes Cyst Expansion in Polycystic Kidney Disease

city: New Haven

first: Vijayakumar

middle: R

last: Kakade

state: Connecticut

address: 300 Cedar St Room S240

department: Internal Medicine/Section of Nephrology

institute: Yale Univeristy

summary: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by mutations in either the PKD1 or PKD2 genes leading to progressive cyst growth and often ESKD. We have previously demonstrated that with loss of Pkd1 the tubules can enlarge without an increase in tubular cell numbers, suggesting that tubular basement membrane remodeling is important for cystic dilation. Here we compared the transcriptomes from inducible tubule-specific knock out of Pkd1 (PkdTKO) and the uninduced littermate mice kidneys as controls. RNA sequencing revealed significant upregulation of 17 metalloproteinases. Of these, A Disintegrin and Metalloproteinase with Thrombospondin Motif 1 (Adamts1), expressed primarily by the proximal tubule, is the most significantly upregulated following loss of Pkd1 expression. Upregulation of Adamts1 in PkdTKO kidneys correlated with a significant increase in the 70 kDa cleavage product of the V1 isoform of versican, which localized to the tubule basement membrane (TBM) and adjacent interstitial mononuclear cells. Simultaneous deletion of both Adamts1 and Pkd1 (P/ATKO) reduced Adamts1 expression levels by >90%, prevented V1 versican cleavage, and reduced interstitial macrophage accumulation and activation. P/ATKO mice demonstrated slower cystic enlargement, improved BUN and creatinine and better survival than did PkdTKO mice. In conclusion, preventing Adamts1 upregulation following loss of tubular Pkd1 expression effectively reduced macrophage accumulation and TBM remodeling, slowing cyst growth and preserving renal function.

overall_design: We used bulk RNA-seq to investigate the role of metalloproteinases in tubule basement membrane remodeling and cystic dilaiton of tubules in 8 weeks old PkdTKO and uninduced mice kidneys.

first: Marcelo

last: Cassini

first: Lloyd

last: Cantley


title: Alteration of gene expression in ubr-5(-) and pabp-2(RNAi)

city: Dallas

first: Benjamin

last: Weaver

address: 6001 Forest Park Rd

department: Pharmacology

institute: University of Texas Southwestern Medical Center

summary: To investigate how UBR-5 and PABP-2 affect gene expression profile, we generated ubr-5(-) c elegans mutant and performed RNA seq in combination with pabp-2(RNAi).

overall_design:
We compared gene expression profile of wild type (A), pabp-2(RNAi) (B), ubr-5(-) (C) and ubr-5(-) pabp-2(RNAi) (D).
glmLRT.txt: Files with Diﬀerential expressed gene list (as indicated in the corresponding sample description field)
igenomes.genes_normalized.counts.CPM.tab, normalized counts

Grant 3:
Welch Foundation
I-2022-20190330
Regulation of Non-Canonical Caspase Functions by Distinct Protein Complexes

first: Jose

last: Calva-Moreno

first: Yi


title: The tumor immune microenvironment in long-term survival resected pancreatic cancer patients

city: Rotterdam

first: Jie

last: Ju

address: Dr. Molewaterplein 40

institute: Erasmus University Medical Center

summary: This study aimed to elucidate immunological and microbial factors contributing to prolonged survival in pancreatic ductal adenocarcinoma (PDAC) patients, providing valuable guidance for treatment strategies that enhance efficacy. To accomplish this, we performed metatranscriptomics analyses and IF staining analyses to map the TIME in PDAC tumors from treatment-naïve short-term survivors (a cancer-specific survival of shorter than six months, STS) and long-term survivors (a cancer-specific survival over five years, LTS). In conclusion, our study unveiled a distinctive, yet intricate tumore immune microenvironment associated with prolonged survival in PDAC. This profile includes a decrease in pro-tumoral M2 and M0 macrophages, along with an increase in plasma B cells, all linked to a better prognosis in PDAC. Future research should be directed towards a more comprehensive understanding of the role of B cells in pancreatic cancer and the modulation of M2 macrophage polarization. Such insights will be invaluable in tailoring personalized therapeutic strategies to enhance patient outcomes.

overall_design: To understand the prerequisites for long-term survival following surgical resection in PDAC patients, we performed the metatranscriptomic analyses and multiplex immunofluorescence, between STS and LTS.  Surgical specimens were formalin-fixed paraffin-embedded (FFPE) and stored. Patients were treatment-naive prior to surgery.

first: Casper

last: van Eijck

first: Freek

last: van ’t Land

first: Maaike

last: Verheij

first: Yunlei

last: Li

first: Michael

last: Doukas

first: Lara

last: Heij

first: Lola

last: Alonso

first: Núria

last: Malats

first: Bas

last: Koerkamp

first: Eveline

last: Vietsch


title: Optimized periphery-core interface increases fitness of the Bacillus subtilis glmS ribozyme

city: Baltimore

first: Sarah

last: Woodson

address: 3400 N. Charles St.

department: Biophysics

institute: Johns Hopkins University

summary: Like other functional RNAs, ribozymes contain a conserved catalytic center supported by peripheral domains that vary among ribozyme sub-families. To understand how core-peripheral interactions contribute to ribozyme fitness, we compared the cleavage kinetics of all single base substitutions at 152 sites across the Bacillus subtilis glmS ribozyme by high-throughput sequencing (ClvSeq). The in vitro activity map mirrored phylogenetic sequence conservation in glmS ribozymes, indicating that biological fitness reports all biochemically important positions. Most deleterious mutations impaired RNA self-assembly. All-atom MD simulations of the complete ribozyme revealed how individual mutations in the core or the IL4 peripheral loop rewire the network of hydrogen bonds around the catalytic site. Remarkably, IL4 mutations introduce a non-native helix interface that corrupts folding of the wild type core, eliminating activity. The results illustrate how competition between native and non-native structures in RNA drives the natural selection of central and peripheral tertiary interaction motifs.

overall_design:
The plasmid variant library was in vitro transcribed, allowed to self-cleave in the presence of Mg2+ and GlcN6P, purified by PAGE and reverse transcribed into 12 barcoded output cDNA sub-libraries that were pooled and amplified for paired-end HiSeq.
Two barcoded versions of the WT ribozyme were added to the purified RNA pools as spike-in controls to estimate absolute RNA concentration.

first: Daniel

last: Yu


title: ITGAV variants are associated with immune dysregulation, brain abnormalities, and very early onset Inflammatory Bowel Disease

city: Toronto

first: Aleixo

last: Muise

state: ON

address: 686 Bay Street, 19-9400 I-M PGCRL

department: Cell Biology

institute: The Hospital for Sick Children

summary: Integrin alpha–V (ITGAV) forms heterodimers with beta subunits to regulate several cellular processes including transforming growth factor β (TGF-β) signaling. We examined three unrelated families with common disease features including two children born with congenital brain anomalies and later development of immune dysregulation, atopy, and colitis, and three fetuses with brain and developmental defects. Using whole exome and RNA sequencing and functional studies, we demonstrate that biallelic ITGAV variants caused abnormal splicing or mislocalized protein that led to dysregulated TGF-β signaling in these families. Furthermore, knockout itgav (-/-) zebrafish embryos developed brain defects with loss of microglia and juvenile itgav (-/-) zebrafish developed colitis. Together, we show the critical role of ITGAV in immune regulation and gut and brain homeostasis and disease pathogenesis.

overall_design:
To investigate the role of itgav in the development of very early onset inflammatory bowel disease,we generated zebrafish itgav knock out line using CRISPR/Cas9 technology.
We then perform gene expression profile analysis using data abtained from RNAseq from wild type zebrafish line, and itgav(-/-) line, at two timepoints (8 dpf and 50 dpf).
Comparative gene expression profiling analysis between wt and itgavKO zebrafish mutant were performed to examine the transcriptomic alterations caused by itgav deficiency.

first: Neil

last: Warner

first: Rei

last: Guan

first: Sina

last: Ghasempour

first: Ryder

last: Whittaker Hawkins

first: Yijing

last: Liang


title: Bulk RNA-sequencing of synovial macrophages (Fcgr2b-KO mice)

first: Tetsuo

last: Hasegawa

summary: Bulk RNA-sequencing of three types of synovial macrophages in Fcgr2b-KO mice stimulated with or without immune complexes

overall_design: Adult Fcgr2b-KO mice were intravenously injected with immune complexes and culled after 8 hours. Synovium was dissected and diggested with collagenase. Three types of macrophages were sorted and immediately placed in RNA Later for RNA extraction.

first: Menna

last: Clatworthy

first: Colin

last: Lee


title: Bulk RNA-sequencing of synovial macrophages (CGRP stimulation)

summary: Bulk RNA-sequencing of synovial macrophages stimulated with CGRP

overall_design: Entire synovium was dissected from adult wild-type mice and stimulated ex vivo with 100 nM CGRP or control for 4 hours. Synovium was  diggested with collagenase and two types of macrophages were sorted and immediately placed in RNA Later for RNA extraction.


title: Bulk RNA-sequencing of synovial macrophages (wild-type)

summary: Bulk RNA-sequencing of three types of synovial macrophages in wild-type C57BL/6 mice stimulated with or without immune complexes

overall_design: Adult C57BL/6 mice were intravenously injected with immune complexes and culled after 8 hours. Synovium was dissected and diggested with collagenase. Three types of macrophages were sorted and immediately placed in RNA Later for RNA extraction.


title: The spatial transcriptomic landscape of fibrosing ILDs

city: London

first: Marco

last: Mura

address: 800 Commissioners Road E

department: Medicine, Respirology

institute: Western University

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

overall_design: Refer to individual Series


title: The elevated YTHDC1 affects trophoblastic function and induces preterm birth in ART conception via augmenting RPL37 translation

city: Shanghai

first: Wei

last: Li

address: 910# Hengshan Road, Shanghai, China.

institute: International Peace Maternity and Child Health Hospital

summary: Here, it is shown that YTHDC1 was significantly increased in the human preterm placenta after ART and the murine preterm placenta after E2 exposure. Overexpression of YTHDC1 significantly promoted the proliferation, migration, and invasion of trophoblast cells, but inhibited their apoptosis. Knockdown or knockout of YTHDC1 played an opposite effect. Mechanistically, E2 promoted YTHDC1 expression through retinoid X receptor alpha (RXRA) upregulation. YTHDC1 could activate WNT and JAK/STAT signaling pathways in the human trophoblast cells. Meanwhile, YTHDC1 could fine-tone the translation of ribosomal protein L37 (RPL37) in a m6A-dependent manner, which then influence the cellular total protein synthesis. Importantly, administration of siRNA targeting YTHDC1 effectively delays preterm birth in vivo. Collectively, these findings uncover the epigenetic mechanism underlying ART preterm birth and provide a potential target for therapeutic intervention.

overall_design: To investigate the biological functions of YTHDC1 in human trophoblast cells, we applied siRNA against YTHDC1 to transiently knockdown YTHDC1 in HTR-8/SVneo cells.    RNA immunoprecipitation followed by sequencing (RIP-seq) was performed using antibody against YTHDC1 to identify the targeted transcripts enriched by YTHDC1 in HTR-8/SVneo cells. m6A-RNA immunoprecipitation followed by sequencing (MeRIP-seq) was performed to identify the m6A sites across mRNA in HTR-8/SVneo cells.  Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed using indicated antibodies to study the H3K27ac status and binding sites of transcription factor RXRA in HTR-8/SVneo cell.

first: Jiuru

last: Zhao

first: Zhiwei

last: Liu


title: Effet of pioglitazone and darglitazone on AML cell lines

city: Boulogne

first: Valerie

last: Bardet

address: 9 avenue Charles de Gaulle

department: CHU A Paré

institute: AP-HP

summary: As PPAR-gamma is a nuclear receptor acting as a transcription factor regulating the expression of multiple genes and to better understand the signaling pathways involved, we performed RNAseq analysis on AML cell lines cultured in the absence or presence of pioglitazone or darglitazone.

overall_design: KG-1a, MV4-11, MOLM-14 and OCI-AML3 were treated either by pioglitazone 10µM, darglitazone 10µM or DMSO as control during 12H.

first: Jaja

last: Zhu

first: Sylvain

last: Clauser


title: Differential neuroprotective and anti-inflammatory effects of dimethyl fumarate, monomethyl fumarate and cannabidiol in neurons and microglia [Mouse]

city: Madrid

first: Alicia

last: Sanchez

address: Joaquín Rodrigo, 2 Majadahonda

country: Spain

institute: Instituto de Investigación Sanitaria Puerta de Hierro-Segovia de Arana

summary: Dimethyl fumarate (DMF) is an immunomodulatory treatment for multiple sclerosis (MS) that can cross the blood-brain barrier, presenting neuroprotective potential. Its mechanism of action is not fully understood and there is a need to characterize if DMF or its bioactive metabolite monomethyl fumarate (MMF) exert neuroprotective properties. The combination of adjuvant agents such as cannabidiol (CBD) could be relevant to enhance neuroprotection. The aim of this study was to compare the effects of DMF, MMF and CBD on neuroprotective and immunomodulatory pathways in neurons and microglia in vitro. We found that DMF and CBD, but not MMF, activated the Nrf2 antioxidant pathway in neurons. Similarly, only DMF and CBD, but not MMF, prevented the LPS-induced activation of the inflammatory pathway NF-kB in microglia. However, the 3 drugs inhibited the production of nitric oxide in microglia and protected neurons against apoptosis. Transcriptomically, DMF, MMF and CBD exhibited differential effects on these pathways, with DMF achieving the most pronounced changes.  Our results show that DMF and MMF, despite being structurally related, present differences in their mechanisms of action that could be relevant for the achievement of neuroprotection in MS patients and the potential of CBD as an adjuvant therapy in neuroprotection.

overall_design: We treated microglial cells activated with LPS with DMF, MMF or CBD  for 4 and 24 hours, to compare the transcriptomic changes induced by each drug. We also included microglia cells treated with the drug vehicles alone (DMSO for DMF and MMF, EtOH for CBD). We carried out 3 independent experiments

last: Sánchez-Sanz

first: Antonio

last: García-Merino

last: Sánchez-López

first: Rafael

last: Muñoz-Viana


title: METTL3/MYCN cooperation drives neural crest differentiation and provides therapeutic vulnerability in neuroblastoma

city: Göteborg

first: Tanmoy

last: Mondal

address: Bruna stråket 16

department: Department of Laboratory Medicine

country: Sweden

institute: University of Gothenburg

summary: Neuroblastoma (NB) is a childhood cancer. Improper differentiation of developing trunk neural crest cells (tNCC) cause NB tumor formation in sympathetic nervous system. N6-methyladenosine (m6A) epitranscriptomic modification control post-transcriptional gene expression but the mechanism which recruit m6A methyltransferase complex to specific locus is less characterized.  We explored if m6A epitranscriptome could fine tune gene regulation in migrating/differentiating tNCC. We uncovered m6A epitranscriptome based regulation of HOX gene expression in tNCC which contributes in their timely differentiation to sympathetic neurons. Posterior HOX genes are m6A modified in MYCN NB cells and tumors. We provided evidence that sustained overexpression of MYCN oncogene could drive METTL3 recruitment in subset of genes to create an undifferentiated state in tNCC. Our study suggests oncogenic transcription factor mediated modulation of m6A epitranscriptome could be a prevalent mechanism in other cancers. METTL3 depletion/inhibition induces DNA damage and differentiation in MYCN overexpressed cells. METTL3 inhibition increases vulnerability to the chemotherapeutic drug in MYCN-amplified Patient-derived xenografts (PDX) cells, suggesting METTL3 inhibition could be a potential therapeutic approach for NB.

overall_design:
RNA-seq was performed in bilogical replicates from total RNA isolated at different stages of hESC differentiation to Sympathtic Neurons (SN) or neural crest stem cells NCSC. RNA from MYCN amplified NB cell line (SKNBE2) and tumor samples were also sequenced. RNA-seq was also performed in cells with METTL3 KD and with induction of MYCN overexpression.
m6A- RNA immunoprecipitaion followed by sequencing  (m6A RIP-seq) was performed with RNA isolated fromf hESC, trunk neural crest cells (tNCC), neural crest stem cells (NCSC),  MYCN amplified NB cell line (SKNBE2) and tumor samples.
Chromatin immunoprecipitaion followed by sequencing for METTL3, MYCN and H3K27ac in hESC and tNCC. In SHEP cells with or without induction of MYCN overexpression ChIP-seq was performed with METTL3 and MYCN antibodies. All the ChIP were performed in duplicates and pooled before library prepation step.

first: Ketan

last: Thombare

first: Roshan

last: Vaid

first: Perla

last: Pucci

first: Kristina

middle: I

last: Lundberg

first: Ritish

last: Ayyalusamy

first: Mohammad

last: Baig

first: Akram

last: Mendez

first: Rebeca

last: Burgos-Panadero

first: Stefanie

last: Höppner

first: Christoph

last: Bartenhagen

first: Daniel

last: Sjövall

first: Aqsa

last: Rehan

first: Sagar

last: Nale

first: Anna

last: Djos

first: Tommy

last: Martinsson

first: Pekka Jaako

last: Jaako

first: Jae-June

last: Dong

first: Per

last: Kogner

first: John

last: Johnsen

first: Matthias

last: Fischer

first: Suzanne

last: Turner


title: Transcriptome analysis of neuroblastoma SH-SY5Y cells upon DDX17 helicase knock-down

city: Lyon

first: Cyril

last: Bourgeois

address: 46 Allée d'Italie

department: LBMC

institute: ENS de Lyon

summary: DDX17 is a DEAD-box RNA helicase protein which is involved in many aspects of RNA metabolism, from gene transcription to RNA processing and decay. As a simple approach to identify DDX17 target genes, we carried out a transcriptome analysis of the human neuroblastoma cell line SH-SY5Y following DDX17 gene knock-down

overall_design: Cells were transfected with a mixture of validated siRNA against DDX17, and cells were harvested after 48h. Total RNA were isolated using TriPure Isolation Reagent (Roche). Directional RNA libraries were prepared from total RNA after removal of ribosomal RNA (lncRNA library, Novogene). High throughput sequencing of 150 bp paired-end reads was carried out on an Illumina Novaseq 6000 platform (Novogene), generating at least 24 Gigabases of raw data per sample, for an average number of 56 millions of matched pairs of reads per sample.

first: Valentine

last: Clerc

first: Xavier

last: Grand


title: Total RNA sequencing of Plasmodium falciparum field parasites 2018-2020 collected from Cambodia and Vietnam

city: Singapore

first: Zbynek

last: Bozdech

address: 60 Nanyang Drive

department: School of Biological Sciences

country: Singapore

institute: Nanyang Technological University

summary: The objective of this study is to characterize the gene expression signatures associated with in vivo artemisinin resistance phenotype and its transcriptional response to Artemisinin Combination Therapy (ACT) or Triple artemisinin combination therapy (TACT).  Using stranded RNA-seq protocol expression profiles of Plasmodb annotated transcripts along with anti-sense, alternatively spliced and non-coding isoforms expressed in clinical isolates were generated for 310 pre-treated, 300 post-treated and 24 timepoints from the intra-erythrocyte development cycle.

overall_design: Plasmodium falciparum isolates were sampled from the whole blood of patients with un-complicated malaria from Cambodia and Vietnam (2 provinces from each country) between the year of 2018 to 2020.

first: Michal

last: Kucharski

first: Sourav

last: Nayak


title: miRNA-mediated gene silencing in Drosophila larval development involves GW182-dependent and independent mechanisms

status: Public on Sep 29 2024

city: Bunkyo-ku

first: Eriko

last: Matsuura

state: Tokyo

address: Yayoi

department: IQB

institute: The University of Tokyo

summary: MicroRNAs (miRNAs) regulate a wide variety of biological processes by silencing their target genes. Argonaute (AGO) proteins load miRNAs to form an RNA-induced silencing complex (RISC), which mediates translational repression and/or mRNA decay of the targets. A scaffold protein called GW182 directly binds AGO and the CCR4-NOT deadenylase complex, initiating the mRNA decay reaction. Although previous studies have demonstrated the critical role of GW182 in cultured cells as well as in cell-free systems, its biological significance in living organisms remains poorly explored, especially in Drosophila melanogaster. Here, we generated gw182-null flies using the CRISPR/Cas9 system and found that, unexpectedly, they can survive until an early second-instar larval stage. Moreover, in vivo miRNA reporters can be effectively repressed in gw182-null first-instar larvae. Nevertheless, gw182-null flies have defects in the expression of chitin-related genes and the formation of the larval trachea system, preventing them from completing larval development. Our results highlight the importance of both GW182-dependent and -independent silencing mechanisms in vivo.

overall_design:
To investigate the function of GW182 in drosophila vivo, gw182 null mutant was generated and RNA-seq was performed.
RNA sequence,of 1st instar larvae of sample1-2(WT +/+), sample2-3(gw null-2 -/+),and sample5-6(gw null-2 -/-): total 6samples

last: Matsuura-Suzuki


title: mRNA profiling in immune mediated necrotizing myopathy

city: chengdu

first: Li

last: Zeng

state: sichuan

address: 32# W. Sec 2, 1st Ring Rd

department: neurology

institute: sichuan provincial people's hospital

summary:
Background: The therapeutic regimen of immune-mediated necrotizing myopathy (IMNM), a category of immune-related myopathies featuring progressive proximal muscle weakness and extremely high level of serum creatine kinase, remains a formidable barrier for clinicians. The proposed study intended to investigate new diagnostic biomarkers based on high-throughput sequencing data of IMNM and generate novel IMNM management strategies by bioinformatics technology.
Methods: Eighteen IMNM patients were synthesized in a local sequencing cohort (high-throughput sequencing data) with Weighted Gene Co-expression Network Analysis (WGCNA) and differential expression analysis to pinpoint robust IMNM-related differentially expressed genes (DEGs). The definitive feature genes were categorized by employing the protein-protein interaction (PPI) network, Least Absolute Shrinkage Selection Operator (LASSO), and support vector machine recursive feature elimination (SVM-RFE), and their sample discriminatory utility was appraised by obtaining the area under the curve (AUC) of the receiver operating curve (ROC) curve. The expression difference of key genes was verified by quantitative real-time polymerase chain reaction (qRT-PCR). Enrichment analysis of predefined gene sets disclosed the hidden functions of diagnostic DEGs. The 28 immune cell abundance patterns in the IMNM cohort were measured using single-sample gene set enrichment analysis (ssGSEA).
Results: We recognized 193 genes that were aberrantly upregulated in the IMNM population and were firmly affiliated with immune-inflammatory responses, regulation of skeletal and cardiac muscle contraction and lipidprotein metabolism. LTK, MYBPH, and MYL4 were authenticated as diagnostic markers for IMNM (all AUC = 1.00), and functional enrichment analysis highlighted that these genes were notably integrated with immune-inflammatory response, autophagy-lysosome pathway and myofiber differentiation and regeneration. Ultimately, ssGSEA revealed a pronounced immune cell infiltrative microenvironment in the IMNM population. The results revealed the most significant relationship among CD4 T cells, CD8 T cells, macrophages, natural killer cells, T helper cells, Th17 cells and dendritic cells.
Conclusion: LTK, MYBPH, and MYL4 might be served as diagnostic molecules for IMNM and possibly yield innovative insights into the mechanisms of progression and therapeutic options for IMNM in the future.

overall_design: Muscle mRNA profiles of IMNM patients compared to healthy controls generated by high throughput sequencing, using illumina Novaseq 6000

first: Song

last: Tan

first: Kai

last: Chen


title: A Stat3-STING-IFN axis controls the metastatic spread of small cell lung cancer

city: Clayton

first: Daniel

middle: James

last: Gough

state: Victoria

address: 27-31 Wright Street

department: Centre for Cancer Research

country: Australia

institute: Hudson Institute of Medical Research

summary:
To define the impact of STAT3-deletion on gene expresssion in primary and metastatic small cell lung cancer
Gene expression was defined for cell lines derived from primary or liver metastases from independent mice by RNA-Seq

overall_design: Comparative gene expression profiling analysis of RNA-seq data for Rb1-/-:Trp53-/-:MYC (RPM) or  Rb1-/-:Trp53-/-:MYC:STAT3-/- (RPMS) cells derived from small cell lung cancer from either lung or liver metastases.


overall_design: RNA sequence, 8samples, sample1-2(yw;control), sample2-3(gw null-1), sample5-6(gw null-2)


title: Differential neuroprotective and anti-inflammatory effects of dimethyl fumarate, monomethyl fumarate and cannabidiol in neurons and microglia

first: Alicia

last: Sanchez

overall_design: We treated primary hippocampal neurons with DMF, MMF or CBD and their respective vehicles (DMSO for DMF and MMF, EtOH for CBD) for 4 and 24 hours, to compare the transcriptomic changes induced by each drug. We carried out 3 independent experiments

last: Sánchez-Sanz

first: Antonio

last: García-Merino

last: Sánchez-López

first: Rafael

last: Muñoz-Viana