title: Gasdermin C promotes stemness and immune evasion in pancreatic cancer via pyroptosis-independent mechanism

first: Christopher

last: Heeschen

summary: Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic and lethal disease. Gasdermins are primarily associated with necrosis via membrane permeabilization and pyroptosis, a lytic pro-inflammatory type of cell death. In this study, we report GSDMC upregulation during PDAC progression. GSDMC directly induces genes related to stemness, EMT, and immune evasion. Targeting Gsdmc in murine PDAC models reprograms the immunosupressive tumor microenvironment, resulting in diminished tumor initiation, growth, metastasis, and enhanced response to PD-1 checkpoint inhibition. Mechanistically, we discover that ADAM17 cleaves GSDMC, releasing a C-terminal fragment that translocates to the nucleus and binds to promoter regions of stemness, metastasis, and immune evasion-related genes. Pharmacological inhibition of GSDMC cleavage or hindrance of its nuclear translocation was equally effective in suppressing downstream targets and inhibiting PDAC progression. Our findings position GSDMC as a potential therapeutic target for enhancing treatment response in this deadly disease.

overall_design: Chromatin Immunoprecipitation DNA sequencing (ChIP-seq) for GSDMC antibodies targeting either N-terminal fragment or C-terminal fragment

first: Renfei

last: Šabanović


title: Senescence-specific translation dysregulation desensitizes cells to stress by inhibiting activation of the integrated stress response

status: Public on Sep 18 2024

city: Baltimore

first: Manolis

last: Maragkakis

address: 251 Bayview Boulevard

country: USA

institute: National Institute on Aging

type: Expression profiling by high throughput sequencing

summary: Senescence is a state of indefinite cell cycle arrest associated with aging, cancer, and age-related diseases. Here we find that translational deregulation and a corresponding maladaptive integrated stress response (ISR) is a hallmark of senescence that desensitizes senescent cells to stress. We present evidence that senescent cells maintain high levels of eIF2? phosphorylation, typical of ISR activation, but translationally repress production of the stress response transcription factor 4 (ATF4) by ineffective bypass of the inhibitory upstream open reading frames (uORFs). Surprisingly, ATF4 translation remains inhibited even after acute proteotoxic and amino acid starvation stressors, resulting in a highly diminished stress response. We also find that stress augments the senescence associated secretory phenotype with sustained remodeling of inflammatory factors expression that is suppressed by non-uORF carrying ATF4 mRNA expression. Our results thus show that senescent cells possess a unique response to stress that entails an increase in their inflammatory profile.

overall_design: Ribosome sequencing analysis of cycling, senescent, quiescent, and Tg-stressed IMR90 fibroblasts. RNA and Ribo-seq material harvested in parallel. Ribo-seq footprints generated by incubating RNA with RNAse I at 4C for 20 minutes.

first: Matthew

middle: J

last: Payea

first: Showkat

middle: A

last: Dar

first: Carlos

last: Anerillas

first: Jennifer

middle: L

last: Martindale

first: Cedric

last: Belair

first: Rachel

last: Munk

first: Sulochan

last: Malla

first: Jinshui

last: Fan

first: Yulan

last: Piao

first: Xiaoling

last: Yang

first: Abid

last: Rehman

first: Nirad

last: Banskota

first: Kotb

last: Abdelmohsen

first: Myriam

last: Gorospe


title: Hepatic transcriptome of pulmonary TB infection.

status: Public on Sep 17 2024

city: Leicester

first: Mrinal

last: Das

state: Leicestershire

address: University Road, Leicester

country: United Kingdom

institute: University of Leicester

summary: Pulmonary tuberculosis (TB) generates chronic systemic inflammation and metabolic dysregulation. The liver is the master regulator of metabolism and to determine the impact of pulmonary TB on this organ we undertook unbiased mRNA  analyses of the liver in mice with TB. Pulmonary TB led to upregulation of genes in the liver related to interferon signalling and glycolysis, and downregulation of genes encoding gluconeogenesis rate-limiting enzyme

overall_design: Uninfected, and infected fro 21 day and 42 day

middle: K

first: Andrea

middle: M

last: Cooper


title: Transcriptomic and metabolomic reveal OsCOI2 as the jasmonate-receptor master switch in rice root

city: Montpellier

first: Anne-Sophie

last: PETITOT

address: 911 avenue Agropolis

department: UMR DIADE

country: France

institute: IRD

summary: Jasmonate is an essential phytohormone involved in plant development and stress responses. Jasmonate perception occurs through the COI nuclear receptor allowing to target the JAZs repressors for degradation by the 26S proteasome. Consequently, repressed transcription factors are released and expression of JA-responsive genes is induced. In rice, three OsCOI genes have been identified, OsCOI1a and the closely related OsCOI1b homolog, and OsCOI2. While a role for OsCOI-1a and -1b genes was previously established in plant defense or leaf senescence, evidence for an important role of OsCOI2 in plant development, jasmonate-signaling and defence emerged only recently. To decipher the role of OsCOI2 in regulating jasmonate responses, we used rice mutant lines, edited both in the OsCOI1a and OsCOI1b genes, or in the OsCOI2 gene. Rice roots from WT and oscoi lines were treated by jasmonate to establish the molecular responses of each line, at the transcriptomic and metabolomic levels. RNA-seq data indicate that OsCOI2 is the main actor of the large transcriptional reprogramming observed after a JA challenge in rice roots. A series of transcription factors with an OsCOI2-dependent expression was identified, including transcription factors known to have a role in root development or stress response. An OsCOI2-dependent expression was detected for genes involved in specific processes or pathways such as photosynthesis, cell-growth and secondary metabolite biosynthesis (phenylpropanoids and diterpene phytoalexins). Functional redundancy between OsCOI1a/b and OsCOI2 can be shown for some genes, even if a lower response usually occurred in oscoi2 plants compared to oscoi1ab plants. Metabolic data revealed a shift from the primary metabolism to the secondary metabolism that is mainly OsCOI2-dependent. Differential accumulation of oryzalexins was also observed in oscoi lines.These data validate the major role of OsCOI2 in JA responses in rice roots. OsCOI2 is the major regulator of specialized metabolic pathways related to development and production of secondary metabolites. This study suggests that OsCOI2 controls the growth-defense trade-off in response to JA.

overall_design: To decipher the molecular responses controlled by the OsCOI receptors in rice roots, we used rice edited lines, either edited in both the oscoi1a and oscoi1b genes, or edited in the oscoi2 gene. Rice roots were submitted to JA treatment to compare the molecular responses of the oscoi edited plants to the WT plants.

first: Mohamad

last: Cheaib

first: HieuTran

last: N'Guyen

first: Chris

last: Richter

first: Manish

last: Raorane

first: Björn

middle: H

last: Junker

first: Alexandre

last: Soriano

first: Pierre

last: Larmande

first: Marie

last: Coudrec

first: Julien

last: Serret

last: Petitot

first: Antony

last: Champion


title: Innate cells and STAT1-dependent signals orchestrate vaccine-induced protection against invasive Cryptococcus infection

city: Newark

first: Adriana

last: Messyasz

address: 185 South Orange Avenue

institute: Rutgers-New Jersey Medical School

summary: Fungal pathogens are underappreciated causes of significant morbidity and mortality worldwide. In previous studies we determined that a heat-killed, Cryptococcus neoformans fbp1-deficient strain (HK-fbp1) is a potent vaccine candidate. We determined that vaccination with HK-fbp1 confers protective immunity against lethal Cryptococcosis in an interferon γ (IFNγ)-dependent manner. In this study, we set out to uncover cellular sources and relevant targets of the protective effects of IFNγ in response to the HK-fbp1 vaccine. We found that early IFNγ production peaks at day 3 and that monocytes and neutrophils are important sources of this cytokine after vaccination. Neutralization of IFNγ at day 3 results in impaired CCR2+ monocyte recruitment and reduced differentiation into monocyte-derived dendritic cells (Mo-DC). In turn, depletion of CCR2+ cells prior to immunization results in impaired activation of IFNγ-producing CD4 and CD8 T cells. Thus, monocytes are important targets of innate IFNγ and help promote further IFNγ production by lymphocytes. We employed monocyte-fate mapper and conditional STAT1 knockout mice to uncover that STAT1 activation in CD11c+ cells, including alveolar macrophages, Mo-DCs, and monocyte-derived macrophages (Mo-Mac) is essential for HK-fbp1 vaccine-induced protection. Altogether, our aggregate findings suggest critical roles for innate cells as orchestrators of vaccine-induced protection against Cryptococcus infection.

overall_design: We hypothesize that the important roles observed for both CCR2+ and CD11c+ cells might be connected to the interrelationship of these populations where CCR2+ monocytes give rise to important CD11c+ effectors that no longer express CCR2. Thus, we set out to examine whether monocytes give rise to alveolar macrophages after HK-fbp1 immunization. To this end, we employed CX3CR1CreER tamoxifen- inducible cre mice crossed to Rosa26floxedTdTomato mice. This model allows us examine murine macrophage and monocyte fate mapping analysis by administration of tamoxifen.  We injected mice with 2.5mg tamoxifen on day -1 on CX3CR1CreER x RosaR26TdTomato mice (monocyte fate mapper), and immunized mice with HK-fbp1 on day 0. On day 30 post vaccination, we sacrificed mice, and sorted tissue-derived alveolar macrophage (TD-AMs, CD45+DAPI-CD11c+SiglecFhi TdTomato-) and monocyte-derived AMs (Mo-AMs, CD45+DAPI-CD11c+SiglecFhi TdTomato+) the lung. Naive control is from Dr. Dane Park's group previous sorted naive AMs. We then performed gene expression profiling analysis on these sorted cell population.

first: Keyi

last: Wang

first: Vanessa

last: Espinosa

first: Alexander

last: Lememze

first: Chaoyang

last: Xue

first: Amariliz

last: Rivera


title: Quantification of subcellular mRNA kinetics in mouse embryonic stem cells (flavopiridol)

city: Berlin

first: Markus

last: Landthaler

address: Hannoversche Str. 28

department: Berlin Institute for Medical Systems Biology

country: Germany

institute: Max Delbrueck Center

summary: In this work, we quantified mRNA flow rates between subcellular compartments in mouse embryonic stem cells. Combining metabolic RNA labeling, biochemical cell fractionation and RNA sequencing with mathematical modeling we were able to determine the kinetic rates of nuclear pre-, nuclear mature, cytosolic and membrane mRNA derived from more than 9000 protein-coding genes. For more than 5000 genes, we additionally estimated the transcript elongation rate. For most of the transcripts, mature nuclear half-lives are the longest, suggesting nuclear retention to be the rate-limiting step in the mRNA life cycle. Genes encoding transcription factors and immediate early genes possess fast kinetic rates, specifically a short nuclear half-life. Differentially localized mRNAs exhibit distinct combinations of rate constants, suggesting modular control within subcellular compartments. We show that membrane stability is high for membrane-localized mRNA and that cytosolic stability is high for cytosol-localized mRNA. Genes encoding target signals, such as signal peptides or transmembrane domains, have low cytosolic and high membrane half-lives with only slight differences between target signals. Nuclear-encoded mitochondrial proteins show long nuclear mature half-lives and otherwise similar features of cytoplasmic kinetics that do not resemble co-translational targeting to the mitochondria.

overall_design:
To obtain global insights into the nucleocytoplasmic kinetics of mRNA we conducted a time-resolved SLAM-seq experiment with subcellular fractionation in mouse embryonic stem cells (mESCs).
To experimentally validate our metabolic labeling-derived subcellular flow rates with an orthogonal method, we inhibited transcription using flavopiridol followed by subcellular fractionation and RNA sequencing, resulting in a second set of subcellular rates (this repository). For the SLAM-seq dataset, please go to GSE252199.
Three independently passaged biological replicates of mESCs (~3.5 × 107 cells per replicate) were cultured in “2i + LIF” ES medium supplemented with 1µM flavopiridol to block the transcription. Cells were harvested at 0, 30, 60, 120 and 180 min after addition of flavopiridol followed by cell fractionation as described above. Obtained fractions were mixed with Trizol LS and RNA was isolated following the manufacturer instructions. One microgram of total RNA was used as an input for TruSeq Stranded mRNA Library Prep Kit according to the instructions of the manufacturer. The multiplexed libraries were sequenced using HiSeq 4000 for pair-end 75 cycles.
Reads were aligned with STAR (v2.7.6) and counted with RSEM (v1.3.3). Library sizes were normalized using genes with known half-lives greater than 14 hours (Herzog et al, 2017). Samples of each time series were further normalized to the corresponding t=0 time point. We fitted a simple exponential decay model per compartment for roughly 10,000 genes, giving the aggregated subcellular turnover rates, in contrast to the rates of transition from one compartment to the next.

first: David

last: Steinbrecht

first: Igor

last: Minia

first: Miha

last: Milek

first: Johannes

last: Meisig

first: Nils

last: Bluethgen


title: Quantification of subcellular mRNA kinetics in mouse embryonic stem cells

overall_design:
To obtain global insights into the nucleocytoplasmic kinetics of mRNA we conducted a time-resolved SLAM-seq experiment with subcellular fractionation in mouse embryonic stem cells (mESCs).
mESCs were exposed to 4-thiouridine (4sU) for various time points to label newly synthesized RNA. Subsequently, we performed subcellular biochemical fractionation to generate cytosolic, membrane and nuclear fractions (Fig. 1A). 4sU concentrations were carefully chosen such that they allowed for sufficient labeling rates while minimizing toxicity elicited by 4sU. Consequently, we used higher concentrations of 4sU (500 µM) for labeling periods less than one hour, and lower concentrations (100 µM) thereafter.
To quantify the kinetics of mRNA globally, we next extracted RNA from each fraction and from the unfractionated cells, followed by iodoacetamide (IAA) alkylation and poly(A)-selected strand-specific RNA library preparation
Based on a binomial mixture model by Juerges et al. (2018) we determined the conversion rate per sample. With the conversion rates, T and T2C conversion counts the share of new to total RNA was calculated on intron/exon level, which was used later on as input for the kinetic model.To integrate the subcellular transcriptome and metabolic labeling data and estimate rates of subcellular mRNA kinetics, we developed transcript-wise mathematical models.


title: The effects of a low isoleucine diet on the liver expression of aged C57BL/6J.Nia mice of both sexes

city: Madison

first: Chung-Yang

last: Yeh

state: WI

address: 2500 Overlook Ter

department: Department of Medicine

institute: University of Wisconsin-Madison

summary: Dietary isoleucine is a significant determinant of metabolic health and longevity. In this study, we found that even at an advanced age, both male and female mice received significant metabolic benefits from a low isoleucine diet.  We collected liver tissues for transcriptomic analysis and the results found that the majority of age-driven changes is ameliorated with the administration of low isoleucine diet.

overall_design: In this transcriptomic study, we treated 20-month-old C57BL/6J.Nia mice of both sexes with either a control or a low isoleucine diet, and 6-month-old C57BL/6J.Nia mice with a control diet. They ate their respective diets for 4 months before being sacrificed after a overnight fast (16 hr) and refeeding (3 hr). The liver was flash frozen in L2N and the RNA extracted.

first: Dudley

last: Lamming


title: Retinoic acid and TGF-β orchestrate organ-specific programs of tissue-residency [dnRA_Antag]

status: Public on Sep 16 2024

city: Melbourne

first: Laura

last: Mackay

state: Victoria

address: 792 Elizabeth street

department: Microbiology and Immunology

country: Australia

institute: The Peter Doherty Institute for Infection and Immunity

summary: Tissue-resident memory T (TRM) cells are integral to tissue immunity, persisting in diverse anatomical sites where they adhere to a common transcriptional framework. How these cells integrate distinct local cues to adopt the common TRM cell fate remains poorly understood. Here, we show that while skin TRM cells strictly require TGF-β for tissue residency, those in other locations utilize the metabolite retinoic acid (RA) to drive an alternative differentiation pathway, directing a TGF-β-independent tissue residency program in the liver and synergizing with TGF-β to drive the TRM cells in the small intestine.

overall_design: Congenically marked naïve P14 T cells were transferred into LCMV-infected mice. At >30 d p.i., mice were treated with DMSO or AGN194310 (RA antag.) every other day for 6 d and CD69+ P14 TRM cells were sorted from the liver and SI the following day

first: Luke

middle: C

last: Gandolfo

first: Lachlan

last: Dryburgh

first: Andreas

last: Obers

first: Maximilien

last: Evrard

first: Thomas

middle: N

last: Burn

first: Jan

last: Schroeder


title: OLFM4 is a direct target of the cGAS-STING pathway and acts as an extracellular inhibitor for the Hippo signaling in EBV-associated cancer

city: Shanghai

first: Fuping

last: Wen

address: Yanchang Middle Road

country: China

institute: Shanghai Tenth People's Hospital

summary: Gene expression profile regulated by EBV infection

overall_design: To investigate the effect of EBV on gastric cancer cells, we treat HGC-27 cells with EBV for 24h.We then collect the control and EBV treated cells and performed gene expression profiling analysis. Each treatment have 3 biological replicates.

first: Zhaocai

last: Zhou


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

overall_design: Refer to individual Series


title: Profiling the functionality of designed transcription activation domain sequences to identify their grammar rules and exceptions

city: Indianapolis

first: Caleb

last: Class

state: IN

address: 4600 Sunset Ave

department: Pharmaceutical Sciences

institute: Butler University

summary: To investigate the functionality of sequences as transcription activation domains (TADs), a library was screened for growth on aureobasidin-containing growth media. Libraries of designed sequences were investigated to identify certain features associated with TAD functionality, such as balance between acidic and aromatic amino acids, and preferential location of certain types of amino acids.

overall_design: DNA-Seq of yeast cell line Y2H Gold containing designed TAD sequences, at baseline and after growth on growth medium containing antibiotic aureobasidin for up to four days. Five biological replicates were taken at each time point, indicated by biorep barcodes appended to the sequences. Ability to grow on antibiotic-containing media leads to increase in sequence read counts after a certain growth period, demonstrating the functionality of certain TAD sequences

middle: G

last: Cooper

first: Tamara

middle: Y

last: Erkina

first: Alexandre

last: Erkine


summary: Transcriptome analysis of the function of OLFM4 MV on gastric cancer HGC-27 cells

overall_design: To investigate the effect of OLFM4 over expression MV on gastric cancer cells, we treated HGC-27 cells with control MV or OLFM4 over expressing MV for 24h.Then collect cells and performed gene expression profiling analysis. Each treatment have 3 biological replicates.


title: Relocalizing transcriptional kinases to activate apoptosis

city: Stanford

first: Sai

last: Gourisankar

state: CA

address: 240 Pasteur Dr, Rm 4700

department: Chemical Systems Biology

institute: Stanford

type: Genome binding/occupancy profiling by high throughput sequencing


title: RNA-seq and ATAC-seq of V-SVZ derived aNS cells

city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

institute: MPI for heart and lung research


title: Joint single-nucleus RNA sequencing and single-nucleus ATAC sequencing of neuroblastoma cell lines

city: Boston

first: Allan

last: Goldstein

state: MA

address: 185 Cambridge St

department: Pediatric Surgery

institute: Massachusetts General Hospital

summary: This study was undertaken to assess transcriptional and epigenetic heterogeneity a the  level of individual cells within neuroblastoma cell lines, and to compare cell lines with MYCN amplificaion to cell lines without MYCN amplification. Methods: We used 10X Genomics multiome sequencing technology to perform joint gene expression and ATAC profiling on thousands of nuclei isolated from the following human neuoblastoma cell lines: SHSY5Y, SK-N-AS, SK-N-SH, SK-N-DZ, Be-2c, and CHP134. Results: We found considerable gene expression and epigeneic heterogeneity both within and between neuroblastoma cell lines. Conclusion: Joint single-nucleus RNA sequencing and single-nucleus ATAC sequencing has demonsrated that neuroblastoma cell lines are heterogeneous, which may have implications for therapeutic strategies.

overall_design: Use of 10X Genomics muliome sequencing technology (GEX + ATAC) to analyze single-cell transcriptomics and single-cell epigenetics of neuroblastoma cell lines.

first: Richard

last: Guyer


title: Retinoic acid and TGF-β orchestrate organ-specific programs of tissue-residency

city: New York

last: Lareau

state: New York

address: 417 E 68th St, Zuckerman - ZRC 1132

institute: Memorial Sloan Kettering

overall_design: OT-I WT and OT-I Tbx21-/- cells were co-transferred into LCMV-OVA infected mice and isolated from the SI for ATAC sequencing 8 days post LCMV-OVA infection.

first: Frank

last: Buquicchio

first: Ansuman

middle: T

last: Satpathy

last: Mackay


title: NEAT1 promotes genome stability via m6A methylation-dependent regulation of CHD4

city: Würzburg

first: Redmond

last: Smyth

state: Bayern

address: Josef-Schneider-Straße 2, D15

institute: Helmholtz Institute for RNA-based Infection Research

summary: Long non-coding (lnc)RNA emerge as regulators of genome stability. The nuclear enriched abundant transcript 1 (NEAT1) locus encodes two lncRNA isoforms that modulate gene expression, growth and proliferation in mammals. Interestingly, NEAT1 transcripts are overexpressed in many tumours and induced by DNA damage, suggesting a genome-protective function. However, the precise role of NEAT1 in the DNA damage response (DDR) is unclear. Here, we investigate the expression, modification levels, localization and structure of NEAT1 in response to DNA double-strand breaks (DSBs) induced by the topoisomerase-II inhibitor etoposide or the locus-specific endonuclease AsiSI. We find that induction of DSBs increases both the levels and N6-methyladenosine (m6A) marks on NEAT1, which promotes alterations in NEAT1 secondary structure and accumulation of hyper-methylated NEAT1 at a subset of promoter-associated DSBs to facilitate efficient DSB signalling. The depletion of NEAT1, in turn, delays the response to DSBs and triggers elevated DNA damage. The genome-protective role of NEAT1 is mediated by the RNA methyltransferase 3 (METTL3) and involves spreading of the chromodomain helicase DNA binding protein 4 (CHD4) upon release from NEAT1. Together, we describe a novel RNA-dependent DDR pathway that couples NEAT1 to the recognition and repair of DSBs.

overall_design: Nano-DMS-MaP analysis of Neat1 structure under different cellular treatments

first: Victoria

last: Mamontova

first: Barbara

last: Trifault

first: Anne-Sophie

last: Gribling-Burrer

first: Patrick

last: Bohn

first: Pit

last: Preckwinkel

first: Werner

last: Schmitz

first: Peter

last: Gallant

first: Dimitrios

last: Papadopoulos

first: Martin

last: Eilers

first: Tony

last: Gutschne

middle: P

first: Kaspar

last: Burger


title: Retinoic acid and TGF-β orchestrate organ-specific programs of tissue-residency [dnRARa]

first: Laura

overall_design: Effector P14 T cells were transduced with either empty (Ctrl-RV) or dnRARα (dnRARα-RV) retroviruses. Transduced cells were co-transferred into LCMV infected recipient mice and isolated from the SI and liver for RNA sequencing 14 days post LCMV infection.

first: Lachlan

last: Dryburgh

first: Andreas

last: Obers

first: Maximilien

last: Evrard

first: Thomas

last: Burn

first: Jan

last: Schroeder


title: Regulon of SdiA in Salmonella Typhimurium and Typhi

city: Columbus

first: Brian

last: Ahmer

state: OH

address: 776 Biomedical Research Tower, 460 W 12th Ave

department: Microbial Infection and Immunity

institute: The Ohio State University

summary: SdiA is a LuxR-type protein found in some Enterobacteriaceae. SdiA encoding bacteria do not encode a luxI homolog and rely on foreign bacteria for the production of N-acyl homoserine lactones (AHLs), SdiA's ligand. The regulon of Salmonella SdiA is largely unknown. In this study, we measured the sdiA dependent transcriptional changes of two serovars of Salmonella, Typhimurium and Typhi, exposed to synthetic AHLs. This was evaluated in two experiments. First, the wild-type and sdiA mutant were grown in the presence of AHLs. In the second, sdiA mutants harboring an arabinose inducible copy of SdiA on a plasmid and vector control were grown with AHLs and arabinose. From this a putative regulon was established and confirmed with subsequent characterization experiments.

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium

overall_design: two pairs of two experiments are included in this study. The first pair was performed in Salmonella Typhimurium (wild-type 14028, sdiA mutant BA612). The second pair was performed in Salmonella Typhi (wild-type Ty2, sdiA mutant AMS001 and AMS002). In the first experiment, wild-type and sdiA mutant cultures were grown in LB broth in the presence of synthetic AHLs until late expontential phase. In the second experiment, sdiA mutant harboring either a plasmid copy of sdiA or vector control were grown in LB broth in the presence of synthetic AHLs and arabinose until late exponential phase. The plasmid copy of sdiA was placed in front of the araBAD promoter, which is induced by  arabinose.  Three biological replicates were collected per strain/condition

first: Schwieters

last: Andrew

first: Ahmer

last: Brian


title: Neuronal lineage tracing from progenitors in human cortical organoids reveals mechanisms of neuronal production, diversity, and disease

city: Cleveland

first: Luke

last: Bury

address: Adelbert Road 2109, BRB 743

department: Genetics and Genome Sciences

institute: Case Western Reserve University

summary: The contribution of progenitor subtypes to generate the billions of neurons produced during human cortical neurogenesis is not well understood. We developed the Cortical ORganoid Lineage Tracing (COR-LT) system for human cortical organoids. Differential fluorescent reporter activation in distinct progenitor cells leads to permanent reporter expression, enabling the progenitor cell lineage of neurons to be determined. Surprisingly, nearly all excitatory neurons produced in cortical organoids were generated indirectly from intermediate progenitor cells, while astrocytes and inhibitory neurons were produced both directly and indirectly. Additionally, neurons of different progenitor lineages were transcriptionally distinct. Isogenic lines made from an autistic individual with and without a likely pathogenic variant in the CTNNB1 gene demonstrated that the variant substantially altered the proportion of neurons derived from specific progenitor cell lineages, as well as the lineage-specific transcriptional profiles of these neurons, suggesting a pathogenic mechanism for this mutation. These results suggest individual progenitor subtypes play unique roles in generating the diverse neurons of the human cerebral cortex.

overall_design: Cortical organoids derived from human induced pluripotent stem cells possessing the the COR-LT system were assessed via scRNA-seq

first: Shuai

last: Fu

first: Anthony

last: Wynshaw-Boris


city: Wuerzburg

address: Am Hubland

department: Chair for Biochemistry and Molecular Biology

institute: University of Wuerzburg

summary: Long non-coding (lnc)RNA emerge as regulators of genome stability. The nuclear enriched abundant transcript 1 (NEAT1) is overexpressed in many tumours and responsive to genotoxic stress. However, the mechanism that links NEAT1 to DNA damage response (DDR) is unclear. Here, we investigate the expression, modification levels, localization and structure of NEAT1 in response to DNA double-strand breaks (DSBs). DNA damage increases the levels and N6-methyladenosine (m6A) marks on NEAT1, which promotes alterations in NEAT1 structure, accumulation of hyper-methylated NEAT1 at promoter-associated DSBs and DSB foci formation. The depletion of NEAT1 delays DSB signalling and elevates DNA damage. The genome-protective role of NEAT1 is mediated by the RNA methyltransferase 3 (METTL3) and involves the release of the chromodomain helicase DNA binding protein 4 (CHD4) from NEAT1 to fine-tune histone deacetylation, which links NEAT1 to DDR.

overall_design: Profiling of double-strand breaks (DSBs) was done in U2OS cells using BLISS sequencing. Samples were analyzed following transfection with a control siRNA (siCtrl) or an siRNA targeting NEAT1 (siNEAT1), and with or without etoposide treatment.

first: Lea

last: Boten

first: Daniel

last: Solvie

first: Carsten

last: Ade


title: Tenascin C Deletion Impairs Tendon Healing and Functional Recovery After Rotator Cuff Repair

city: Saltlake city

first: Michael

last: Jurynec

state: UT

institute: University of Utah

summary: The biological factors that affect healing after rotator cuff repair (RCR) are not well 63 understood. Genetic variants in the extracellular matrix protein Tenascin C (TNC) are associated 64 with impaired tendon healing and it is expressed in rotator cuff tendon tissue after injury, 65 suggesting it may have a role in the repair process. The purpose of the current study was to 66 determine the role of TNC on tendon healing after RCR in a murine model. The supraspinatus 67 tendon was transected and repaired on the left shoulder of Wild-Type (WT-RCR) and Tenascin 68 C null (Tnc - -RCR) mice. Controls included the unoperated, contralateral shoulder of WT-RCR 69 and Tnc - -RCR mice and unoperated shoulders from WT and Tnc - mice. We performed 70 histologic, activity testing, RNA-seq, and biomechanical analyses. At 8-weeks post-RCR, Tnc71 mice had severe bone and tendon defects following rotator cuff repair. Tnc- -RCR mice had 72 reduced activity after rotator cuff repair including reduced wheel rotations, wheel duration, and 73 wheel episode average velocity compared with WT-RCR. Loss of Tnc following RCR altered 74 gene expression in the shoulder, including upregulation of sex hormone and WNT pathways and 75 a downregulation of inflammation and cell cycle pathways. Tnc - mice had similar biomechanical 76 properties after repair as WT. Further research is required to evaluate tissue specific alterations 77 of Tnc, the interactions of Tnc and sex hormone and inflammation pathways as well as possible 78 adjuvants to improve enthesis healing in the setting of reduced TNC function.

overall_design: RNA sequencing and enrichment analysis 148 WT-RCR (n=3) and Tnc - -RCR (n=4) were sacrificed at 2 weeks postoperative and used 149 for RNA-seq. RCR was performed on seven mice (WT-RCR (n=3) and Tnc - -RCR (n=4)) and Page 7 of 24 John Wiley & Sons, Inc. Journal of Orthopaedic Research For Peer Review 150 mice were euthanized 2 weeks post-RCR. Whole shoulder joints were dissected and total RNA 151 was isolated as previously described.11 RNA sequencing, quality control, and alignment was 152 performed as previously described.16 Fold‐changes were calculated by comparing counts in Tnc153 -RCR samples relative to WT-RCR. Genes with an adjusted p < 0.05 were considered 154 differentially expressed. We used Ingenuity Pathway Analysis (Qiagen) software for functional 155 pathway analysis of the differentially expressed genes identified in this study. The log2 fold 156 change and adjusted p values using Bonferroni method from Deseq2 analysis were used to 157 calculate directionality (Z score) and set canonical pathway p-values (

first: Robert

middle: Z

last: Tashjian

first: Jared

last: Zitnay

first: Nikolas

last: Kazmers

first: Shivakumar

middle: R

last: Veerabhadraiah

first: Antonio

last: Zelada

first: Matthew

last: Honeggar

first: Peter

last: Chalmers

first: Heath

middle: B

last: Henninger


title: Differential blood transcriptional signatures induced by SARS-CoV-2 Variants Alpha and Delta show reduced increased inflammatory with reduced interferon-stimulated responses and heme synthesis gene expression as compared with wild type strains

city: Karachi

first: Asghar

last: Nasir

state: Sindh

address: Stadium Road

department: Pathology and Laboratory Medicine

country: Pakistan

institute: Aga Khan University

summary: Blood transcriptomes were determined in COVID-19 symtomatic cases and asymtomatic cases using the Clariom S RNA microarray, Affymetrix assay.

overall_design: COVID-19 cases with a positive respiratory samples SARS-CoV-2 and healthy Controls cases were recruited. Blood transcriptomes were analysed using Clariom S RNA Microarray, Affymetrix Inc.

first: Mariam

last: Merchant

first: Javaria

last: Ashraf

first: Kiran

last: Iqbal

first: Maliha

last: Yameen

first: Rabia

last: Hussain

first: Zahra

last: Hasan


title: Chromatin remodeller Chd7 is developmentally regulated in the neural crest by tissue-specific transcription factors

city: Oxford

first: Tatjana

last: Sauka-Spengler

state: Oxfordshire

address: Radcliffe Department of Medicine

department: University of Oxford

institute: MRC Weatherall Institute of Molecular Medicine

summary: Neurocristopathies such as CHARGE syndrome result from aberrant neural crest development. A large proportion of CHARGE cases are attributed to pathogenic variants in the gene encoding CHD7, chromodomain helicase DNA binding protein 7, which remodels chromatin. While the role for CHD7 in neural crest development is well documented, how this factor is specifically upregulated in neural crest cells is not understood. Here, we use epigenomic profiling of chick and human neural crest to identify a cohort of enhancers regulating Chd7 expression in neural crest cells and other tissues. We functionally validate upstream transcription factor binding at candidate enhancers, revealing novel epistatic relationships between neural crest master regulators and Chd7, showing tissue-specific regulation of a globally-acting chromatin remodeller for the first time. Furthermore, we find conserved enhancer features in human embryonic epigenomic data and validate the activity of the human equivalent CHD7 enhancers in the chick embryo. Our findings embed Chd7 in the neural crest gene regulatory network and offer potentially clinically relevant elements for interpreting CHARGE syndrome cases without causative allocation.

overall_design: 10X single-cell RNA-seq of chicken embryos

first: Ruth

last: Williams

first: Andrew

last: Price


title: PD1 and CD73 on naïve CD4+ T cells synergistically limit responses to self

city: Augusta

first: Klaus

last: Ley

state: GA

address: 1410 Laney Walker Blvd

institute: Immunology Center of Georgia

summary: Vaccination with self and foreign peptides induces weak and strong expansion of antigen-specific CD4 T cells, respectively, but the mechanism is not known. We tested how much the naïve CD4 T cell repertoires specific for self antigens are shaped by negative selection in the thymus by computational analysis of the entire mouse MHCII peptidome and found that negative selection only partially explains the difference between responses to self and foreign. In naïve (uninfected, unimmunized) mice, RNA-Seq identified higher PD1 and CD73 expression on self-specific compared to foreign-specific CD4 T cells. Pharmacological or genetic blockade of PD1 and CD73 significantly increased the vaccine-induced expansion of self-specific CD4 T cells and moved their transcriptomes toward those of foreign-specific CD4 T cells. We conclude that PD1 and CD73 synergistically limit CD4 T cell responses to self. This discovery has implications for the development of tolerogenic vaccines and cancer immunotherapy.

overall_design: Experiment 1: m25+ CD4 T cells from naive C57BL/6J mice were tetramer sorted and subjected to 10x genomics single cell sequencing. A pseudobulk analysis was performed. Experiment 2: m25+ and p6+ CD4 T cells were sorted by tetramers from draining lymph nodes (inguinal and para-aortic) of C57BL/6J mice that were immunized with m25 or p6 (i.m. into both thighs, 1x in CFA) and treated with anti-PD1 and CD73 inhibitor or isotype control/vehicle.

first: Felix

middle: S

last: Nettersheim

first: Simon

last: Brunel

first: Robert

last: Sinkovits

first: Sujit

last: Silas Armstrong

first: Payel

last: Roy

first: Monica

last: Billitti

first: Kouji

last: Kobiyama

first: Ahmad

last: Alimadadi

first: Sergei

last: Bombin

first: Lihui

last: Lu

first: Chris

last: Benedict

first: Alessandro

last: Sette


title: Toggling of BRD4 functions is triggered through its phosphorylation by JNK [RNAseq-JQ1]

city: Bethesda

first: Dinah

middle: S.

last: Singer

state: MD

address: Building 10, Room 4B-17

department: Division of Cancer Biology

institute: National Cancer Institute

summary: BRD4 is a key regulatory factor in multiple cancers and cellular stress responses with pleotropic functions. It regulates chromatin remodeling and transcription through its histone acetyltransferase (HAT) and kinase activities, respectively. The mechanism responsible for switching BRD4 from a chromatin to transcriptional regulator is currently unknown. Here, we report that in response to a broad range of stimuli, this switch is mediated by the JNK kinase which directly interacts with BRD4. JNK specifically phosphorylates human BRD4 at Thr1186 and Thr1212, triggering transient BRD4 release from chromatin. JNK phosphorylation of BRD4 halts its HAT-mediated chromatin regulation and activates its transcription-enhancing kinase function. BRD4 release from chromatin is necessary to toggle between its enzymatic activities: chromatin-bound BRD4 is kinase inactive and RNA Pol II-bound BRD4 does not acetylate chromatin. BRD4 release from chromatin augments its interaction with and phosphorylation of key transcriptional regulators RNA Pol II, PTEFb and c-MYC. The PP4 phosphatase dephosphorylates JNK phosphorylated BRD4 in the nucleoplasm, which promotes its interaction with RNA Pol II at transcriptionally active sites. Accordingly, JNK-mediated release of BRD4 from chromatin leads to significantly elevated transcription of BRD4-regulated immune and inflammatory response genes through enhanced BRD4-Pol II interaction at the promoters of these genes.  JNK phosphorylation of BRD4 occurs during T-cell activation and is required for epithelial to mesenchymal transition (EMT) in prostate cancer cells. These findings thus characterize a novel mechanism that triggers the transition of BRD4 from a chromatin regulator to transcriptional activator during stress/immune/inflammatory responses and EMT.

overall_design: This RNA-seq analysis is aimed at identifying genome-wide differential gene expression when HCT116 cells are treated JQ1, a small molecule BET inhibitor that blocks BRD4 from binding chromatin. The analysis includes samples where untransfected (E) and wild type BRD4 transfected (B) HCT116 cells are treated with or without 500nM JQ1 (J)  for 18 hrs. The analysis was done to compare the DEGs from JQ1 treated cells with the DEGs identified upon the release of BRD4 from chromatin through JNK activiation.

first: Devaiah

last: Ballachanda

first: Amit

last: Singh

first: Jie

last: Mu

first: Qingrong

last: Chen

first: Daoud

last: Meerzaman


title: TASOR expression in naive embryonic stem cells safeguards their developmental potential [CUT&TAG]

city: Dallas

first: Laura

last: Banaszynski

state: TX

address: 5323 Harry Hines Blvd

department: Green Center for Reproductive Biology Sciences

institute: UT Southwestern Medical Center

summary: The seamless transition through stages of pluripotency relies on a delicate balance between transcription factor networks and epigenetic silencing mechanisms that ensure proper regulation of the developmental program, critical for normal development. Here, we uncover the pivotal role of the transgene activation suppressor (TASOR), a component of the human silencing hub (HUSH) complex, in sustaining cell viability during the transition from naive to primed pluripotency, despite its rapid downregulation during this transition. Loss of TASOR in naive cells triggers replication stress, disrupts H3K9me3 heterochromatin formation, and compromise the transcriptional and post-transcriptional silencing of LINE-1 (L1) transposable elements (TEs), with these effects become more pronounced in primed cells. Remarkably, the survival of Tasor-knockout cells during naive to primed transition can be restored through the inhibition of cysteine-aspartic acid protease (Caspase) or deletion of mitochondrial antiviral signaling protein (MAVS). This suggests that unscheduled L1 expression activates an innate immune response, leading to programmed cell death, specifically in cells exiting naïve pluripotency. Additionally, we propose that HUSH-promoted H3K9me3 in naïve PSCs sets the stage for ensuing DNA methylation in primed cells, establishing long-term silencing during differentiation. Our findings shed insights on the crucial impact of epigenetic programs established in early developmental stages on subsequent phases, underscoring their significance in the developmental process.

overall_design: CUT&TAG analysis in mouse embryonic stem cell lines and epiblast stem cell lines (WT, TASORKO, TASORKO+addback, TASORKO+MET)).

first: Carlos

last: Pinzon-Arteaga

first: Ryan

last: O'Hara

first: Jun

last: Wu


title: Chromatin remodeller Chd7 is developmentally regulated in the neural crest by tissue - specific transcription factors (ATAC-Seq)

first: Tatjana

last: Sauka-Spengler

summary: Neurocristopathies such as CHARGE syndrome result from aberrant neural crest development. A large proportion of CHARGE cases are attributed topathogenic variantsin the gene encoding CHD7, chromodomain helicase DNA binding protein 7, which remodels chromatin. While the role for CHD743in neural crest development is well documented,how this factor is specifically upregulated in neural crest cells is not understood. Here, we use epigenomic profiling of chick and human neural crest to identify a cohort of enhancers regulating Chd7 expression in neural crest cells and other tissues. We functionally validate upstream transcription factor binding at candidate enhancers, revealing novel epistatic relationships between neural crest master regulators and Chd7, showing tissue-specific regulation of a globally-acting chromatin remodeller for the first time. Furthermore, we find conserved enhancer features in human embryonic epigenomic data and validate the activity of the human equivalent CHD7 enhancers in the chick embryo. Our findings embed Chd7 in the neural crest gene regulatory network and offer potentially clinically relevant elements for interpreting CHARGE syndrome cases without causative allocation.

overall_design: Single cell multiome of human foetal heads

first: Daniel

last: Fountain

first: Ruth

last: Williams

first: Ivan

last: Candido-Ferreira


title: NEAT1 promotes genome stability via m6A methylation-dependent regulation of CHD4 [Cut&Run]

first: Martin

last: Eilers

summary: Long non-coding (lnc)RNA emerge as regulators of genome stability. The abundant nuclear enriched transcript 1 (NEAT1) locus encodes two lncRNA isoforms that modulate gene expression, growth and proliferation in mammals. Interestingly, NEAT1 transcripts are overexpressed in many tumours and induced by DNA damage, suggesting a genome-protective function. However, the precise role of NEAT1 in the DNA damage response (DDR) is unclear. Here, we investigate the expression, modification levels, localization and structure of NEAT1 in response to DNA double-strand breaks (DSBs) induced by the topoisomerase-II inhibitor etoposide or the locus-specific endonuclease AsiSI. We find that induction of DSBs increases both the levels and N6-methyladenosine (m6A) marks on NEAT1, which promotes alterations in NEAT1 secondary structure and accumulation of hyper-methylated NEAT1 at a subset of promoter-associated DSBs to facilitate efficient DSB signalling. The depletion of NEAT1, in turn, delays the response to DSBs and triggers elevated DNA damage. The genome-protective role of NEAT1 is mediated by the RNA methyltransferase 3 (METTL3) and involves spreading of the chromodomain helicase DNA binding protein 4 (CHD4) upon release from NEAT1. Together, we describe a novel RNA-dependent DDR pathway that couples NEAT1 to the recognition and repair of DSBs.

overall_design: Mapping of DNA-protein interaction in U2OS DIvA cells upon DSB induction via AsiSI with or without STM2457 treatment

first: Victoria

last: Mamontova

first: Peter

last: Gallant

first: Kaspar

last: Burger


title: TASOR expression in naive embryonic stem cells safeguards their developmental potential [RNA-seq]

overall_design: RNA-seq analysis in mouse embryonic stem cell lines and epiblast-like cells (WT, TASORKO, WT+addback, TASORKO+addback).


title: NEAT1 promotes genome stability via m6A methylation-dependent regulation of CHD4 [seCLIP-seq]

overall_design: Mapping of RNA-protein interaction in U2OS wt cells with or without Etoposide treatment

first: Dimitrios

last: Papadopoulos


title: NEAT1 promotes genome stability via m6A methylation-dependent regulation of CHD4 [CHART-seq]

overall_design: Accessing lncRNA NEAT1 accumulation on the chromatin in U2OS wt and DIvA cells upon DSB induction via 4-OHT with or without pretreatment with STM2457


title: TASOR expression in naive embryonic stem cells safeguards their developmental potential [ATAC-seq]

overall_design: ATAC-seq analysis in mouse embryonic stem cell lines and epiblast like stem cells (WT, TASORKO, TASORKO+addback, TASORKO+MET).


title: ATAC-seq of V-SVZ derived aNS cells

first: Stefan

last: Günther

summary: Developmental transcription factors act in networks, but how these networks achieve cell- and tissue specificity is still poorly understood.  Here we explored pre-B cell leukemia homeobox 1 (PBX1) in adult neurogenesis combining genomic, transcriptomic, and proteomic approaches. ChIP-Seq analysis uncovered PBX1 binding to a wide range of different sites. Integration of PBX1 ChIP-seq with ATAC-seq data predicted interaction partners, which were subsequently validated by mass spectrometry. Spatial transcriptomics revealed distinct temporal expression dynamics of Pbx1 and interacting factors. Among these were class I bHLH proteins TCF3, TCF4 and TCF12. RNA-seq upon Pbx1, Tcf3 and Tcf4 knockdown identified proliferation- and differentiation associated genes as shared targets. Neuronal differentiation was reduced upon depletion of either factor, suggesting functional cooperation between PBX1 and TCF3/4. Notably, while physiological PBX1-TCF interactions have not yet been described, chromosomal translocation resulting in genomic TCF3::PBX1 fusion characterizes a subtype of acute lymphoblastic leukemia. Introducing Pbx1 into Nalm6 cells, a pre-B cell line expressing TCF3 but lacking PBX1, upregulated leukemogenic genes including BLK and NOTCH3, arguing that functional PBX1-TCF cooperation likely extends to hematopoietic contexts. Our study hence uncovers a PBX1-TCF module orchestrating the balance between progenitor cell proliferation and differentiation in adult neurogenesis with implications for leukemia etiology.

overall_design: ATAC-seq of wild-type (WT) V-SVZ derived cells, cultured for 48 h as free-floating adult neurospheres (aNS), was performed in two biological replicates.

first: Dorothea

last: Schulte

first: Vera

last: Laub


title: RNA-seq of V-SZV derived aNS cells with siRNA mediated depletion of Pbx1, Tcf3 and Tcf4

overall_design: RNA-seq of wild-type (WT) V-SVZ derived aNS, or of aNS 48 h post transfection with siRNA directed against Pbx1, Tcf3, Tcf4 or control siRNAs, was performed in triplicates each.


title: Uncovering functional lncRNAs by unconventional scRNA-seq analysis

city: Pamplona

first: Maite

last: Huarte

state: Navarra

address: c/ Avd Pio XII, 55

country: Spain

institute: Centro de Investigación Médica Aplicada

summary: Long non-coding RNAs (lncRNAs) play fundamental roles in cellular processes and pathologies, regulating gene expression at multiple levels. Despite being highly cell-type specific, their study at single-cell level has been challenging due to their less accurate annotation and low expression. Here, we show that single-cell RNA-seq (scRNA-seq) preprocessing workflows using the pseudoaligner Kallisto enhance the detection and quantification of lncRNAs. Further, using single-cell multiome data, we demonstrate that the ATAC-seq profiles exhibit higher concordance when the scRNA-seq is processed by Kallisto. We then experimentally confirmed the expression patterns of cell-type specific lncRNAs exclusively detected by Kallisto and unveiled biologically relevant lncRNAs, such as AL121895.1, a previously undocumented cis-repressor lncRNA, whose role in proliferation of breast cancer cells was detected by Kallisto and overlooked by other pipelines. Our results emphasize the necessity for an alternative scRNA-seq preprocessing workflow tailored to lncRNAs that sheds light on the multifaceted roles of lncRNAs.

overall_design:
5 scRNA-Seq samples

**Raw data were removed Jun 30, 2024 due to privacy concerns, and added back on Oct 10, 2024 with revised ethical requirements**

first: Enrique

last: Goñi

first: Aina

last: Mas

first: Amaya

last: Abad

first: Marta

last: Santisteban

first: Puri

last: Fortes

first: Mikel

last: Hernaez


title: Inceptor binds to and directs insulin towards lysosomal degradation in beta-cells

city: Neuherberg

first: Heiko

last: Lickert

address: Ingolstaedter Landstraße 1

department: Institute of Diabetes and Regeneration Research

institute: Helmholtz Zentrum München German Research Center for Environmental Health

summary: Blunted first-phase insulin secretion and insulin deficiency are indicators of β-cell dysfunction and diabetes manifestation. Thus, insights into molecular mechanisms that regulate insulin homeostasis might provide entry sites  to replenish insulin content and restore β-cell function. Here, we identify the insulin inhibitory receptor (short: inceptor; encoded by the gene IIR) as an insulin-binding receptor that regulates insulin stores by lysosomal degradation. Using human induced pluripotent stem cell (iPSC)-derived islets, we show that IIR knockout (KO) results in enhanced stem cell (SC)-β-cell differentiation and survival. Strikingly, extended in vitro culture of IIR KO SC-β-cells leads to greatly increased insulin content and glucose-stimulated insulin secretion (GSIS). We find that inceptor localises to clathrin-coated vesicles (CCVs) close to the plasma membrane (PM) and in the trans-Golgi network (TGN), as well as in secretory granules (SGs), where it acts as a sorting receptor to direct proinsulin and insulin towards lysosomal degradation. Targeting inceptor using a monoclonal antibody (mAB) increases proinsulin and insulin content and improves SC-β-cell GSIS.

overall_design: Stage 6 iPSC derived islet-like clusters (SC-islets) from IIR KO and WT cell lines were analyzed by scRNA-seq

first: Johanna

last: Siehler

first: Sara

last: Bilekova

first: Prisca

last: Chapouton

first: Alessandro

last: Dema

first: Pascal

last: Albanese

first: Chirag

last: Jain

first: Michael

last: Sterr

first: Stephen

middle: J

last: Enos

first: Chunguang

last: Chen

first: Adrian

last: Villalba

first: Leopold

last: Schomann

first: Sreya

last: Bhattacharya

first: Jin

last: Feng

first: Melis

last: Akgün Canan

first: Federico

last: Ribaudo

first: Ingo

last: Burtscher

first: Christin

last: Ahlbrecht

first: Oliver

last: Plettenburg

first: Thomas

last: Kurth

first: Raphael

last: Scharfmann

first: Stephan

last: Speier

first: Richard

last: Scheltema

first: Sem

last: Tamara

first: Chetna

last: Malhotra


title: Beyond Glucose: The Crucial Role of Redox Signaling in β-Cell Metabolic Adaptation

status: Public on Sep 15 2024

city: Praha

first: Blanka

last: Holendova

address: Videnska 1083

country: Czech Republic

institute: Institute of Physiology, CAS

summary: Objective: Redox signaling mediated by reversible oxidative cysteine thiol modifications is crucial for driving cellular adaptation to dynamic environmental changes, maintaining homeostasis, and ensuring proper function. This is particularly critical in pancreatic β-cells, which are highly metabolically active and play a specialized role in whole organism glucose homeostasis. Glucose stimulation in β-cells triggers signals leading to insulin secretion, including changes in ATP/ADP ratio and intracellular calcium levels. Additionally, lipid metabolism and reactive oxygen species (ROS) signaling are essential for β-cell function and health. Methods: We employed IodoTMT isobaric labeling combined with tandem mass spectrometry to elucidate redox signaling pathways in pancreatic β-cells. Results: Glucose stimulation significantly increases ROS levels in β-cells, leading to targeted reversible oxidation of proteins involved in key metabolic pathways such as glycolysis, the tricarboxylic acid (TCA) cycle, pyruvate metabolism, oxidative phosphorylation, protein processing in the endoplasmic reticulum (ER), and insulin secretion. Furthermore, the glucose-induced increase in reversible cysteine oxidation correlates with the presence of other post-translational modifications, including acetylation and phosphorylation. Conclusions: Proper functioning of pancreatic β-cell metabolism relies on fine-tuned regulation, achieved through a sophisticated system of diverse post-translational modifications that modulate protein functions. Our findings demonstrate that glucose induces the production of ROS in pancreatic β-cells, leading to targeted reversible oxidative modifications of proteins. Furthermore, protein activity is modulated by acetylation and phosphorylation, highlighting the complexity of the regulatory mechanisms in β-cell function.

overall_design: Isolated islets were cultivated in CMRL medium at non/stimulating glucose concentration for 24 h. The islets were washed with PBS and used immediately for RNA extraction.  Subsequent comparative gene expression profiling analysis of RNA-seq data was performed for WT islets treated with non/stimulating glucose.

first: Barbora

last: Salovska

first: Stepanka

last: Benakova

first: Lydie

last: Plecita


title: Regulation of histone deacetylase activity by serine phosphorylation conserved in Arabidopsis and human

city: Wuhan

first: Xiangsong

last: Chen

state: Hubei

address: Bayi Road

institute: Wuhan university

summary: In this study, we uncovered a novel phosphorylation site that plays pivotal roles in regulating the activity of Arabidopsis HDA9 and human HDAC3

overall_design: Profiling genomewide H3K9ac by Cut&Tag in wild type Col-0, hda9 mutant, and the two complementation lines  of HDA9-S193A and HDA9-S193D

first: Xiaojing

last: Liu


title: Single-cell and bulk transcriptional profiling of mouse ovaries reveals novel genes and pathways associated with DNA damage response in oocytes

city: Bar Harbor

first: Ewelina

last: Bolcun-Filas

state: ME

address: 600 Main St

institute: The Jackson Laboratory

summary: Immature oocytes enclosed in primordial follicles stored in female ovaries are under constant threat of DNA damage induced by endogenous and exogenous factors. Checkpoint kinase 2 (CHEK2) is a key mediator of the DNA damage response in all cells. Genetic studies have shown that CHEK2 and its downstream targets, p53 and TAp63, regulate primordial follicle elimination in response to DNA damage, however the mechanism leading to their demise is still poorly characterized. Single-cell and bulk RNA sequencing were used to determine the DNA damage response in wildtype and Chek2-deficient ovaries. A low but oocyte-lethal dose of ionizing radiation induces a DNA damage response in ovarian cells that is solely dependent on CHEK2. DNA damage activates multiple ovarian response pathways related to apoptosis, p53, interferon signaling, inflammation, cell adhesion, and intercellular communication. These pathways are differentially employed by different ovarian cell types, with oocytes disproportionately affected by radiation. Novel genes and pathways are induced by radiation specifically in oocytes, shedding light on their sensitivity to DNA damage, and implicating a coordinated response between oocytes and pre-granulosa cells within the follicle. These findings provide a foundation for future studies on the specific mechanisms regulating oocyte survival in the context of aging, as well as therapeutic and environmental genotoxic exposures.

overall_design: To investigate DNA damage response in ovaries female mice were irradiated with 0.5Gy (IR) or 0Gy (nonIR). Wildtype (WT) and Chek2-/- (CHEK2KO) mice were used in this study (N=6 per group). Ovaries were collected at 6hrs after treatment for bulk RNA-sequencing (100bp-paired ends)

first: Monique

last: Mills

first: Chihiro

last: Emori

first: Parveen

last: Kumar

first: Zachary

last: Boucher

first: Joshy

last: George


title: Toggling of BRD4 functions is triggered through its phosphorylation by JNK [RNA-seq]

first: Dinah

last: Singer

summary: BRD4 is a key regulatory factor in multiple cancers and cellular stress responses with pleotropic functions. It regulates chromatin remodeling and transcription through its histone acetyltransferase (HAT) and kinase activities, respectively. The mechanism responsible for switching BRD4 from a chromatin to transcriptional regulator is currently unknown. Here, we report that in response to a broad range of stimuli, this switch is mediated by the JNK kinase which directly interacts with BRD4. JNK specifically phosphorylates human BRD4 at Ser1117, Thr1186 and Thr1212, triggering transient BRD4 release from chromatin. JNK phosphorylation of BRD4 halts its HAT-mediated chromatin regulation and activates its transcription-enhancing kinase function. BRD4 release from chromatin is necessary to toggle between its enzymatic activities: chromatin-bound BRD4 is kinase inactive and RNA Pol II-bound BRD4 does not acetylate chromatin. BRD4 release from chromatin augments its interaction with and phosphorylation of key transcriptional regulators RNA Pol II, PTEFb and c-MYC. The PP4 phosphatase dephosphorylates JNK phosphorylated BRD4 in the nucleoplasm, which promotes its interaction with RNA Pol II at transcriptionally active sites. Accordingly, JNK-mediated release of BRD4 from chromatin leads to significantly elevated transcription of BRD4-regulated immune and inflammatory response genes through enhanced BRD4-Pol II interaction at the promoters of these genes.  JNK phosphorylation of BRD4 occurs during T-cell activation and is required for epithelial to mesenchymal transition (EMT) in prostate cancer cells. These findings thus characterize a novel mechanism that triggers the transition of BRD4 from a chromatin regulator to transcriptional activator during stress/immune/inflammatory responses and EMT.

overall_design: This RNA-seq analysis is aimed at identifying genome-wide differential gene expression when BRD4 is stimulated to be released from the chromatin. The analysis includes samples where wild type (W) and mutant (T) BRD4 is exogenously expressed and treated with or without Anisomycin (A), a chemical which leads to the release of BRD4 from chromatin. An empty vector expressing control (N) is similarly treated.

first: Devaiah

last: Ballachanda

first: Amit

last: Singh

first: Jie

last: Mu

first: Qingrong

first: Daoud

last: Meerzaman


title: Toggling of BRD4 functions is triggered through its phosphorylation by JNK [ChIP-seq]

overall_design: This ChIP-seq project is aimed at identifying novel binding sites of BRD4 across the genome when it is stimulated to be released from the chromatin. The project includes samples where wild type (WT) and mutant (TM) BRD4 is exogenously expressed and treated with or without Anisomycin (A), a chemical which leads to the release of BRD4 from chromatin. An empty vector expressing control (EV) is similarly treated.


title: Toggling of BRD4 functions is triggered through its phosphorylation by JNK.

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

overall_design: Refer to individual Series


title: RNC-seq of S1K247Q and S1WT

city: 上海

first: 利

last: 赵

state: State/Province

address: 梅陇路130号

institute: 华东理工大学

summary: RNC-seq experiment of the simulated acetylation mutant S1K247Q of S1 protein

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

overall_design: Escherichia coli K-12 MG1655 S1WT and S1K247Q overexpression strains grown in LB medium are used for RNC-seq

first: Li

last: Zhao

first: Bin-Cheng

last: Yin

first: Bang-Ce

last: Ye

first: Di

last: You


title: RNA-seq of S1K247Q and S1WT

summary: RNA-seq experiment of the simulated acetylation mutant S1K247Q of S1 protein

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


title: Alternative splicing and its regulation in the malaria vector Anopheles gambiae

city: Armilla

first: Elena

last: Gómez-Díaz

state: Granada

address: Avda. del Conocimiento, 17

department: Institute of Parasitology and Biomedicine Lopez-Neyra (IPBLN)

institute: Spanish National Research Council (CSIC)

summary:
Introduction: Alternative splicing (AS) is a highly conserved mechanism that allows for the expansion of the coding capacity of the genome, through modifications of the way that multiple isoforms are expressed or used to generate different phenotypes. Despite its importance in physiology and disease, genome-wide studies of AS are lacking in most insects, including mosquitoes. Even for model organisms, chromatin associated processes involved in the regulation AS are poorly known.
Methods: In this study, we investigated AS in the mosquito Anopheles gambiae in the context of tissue-specific gene expression and mosquito responses to a Plasmodium falciparum infection, as well as the relationship between patterns of differential isoform expression and usage with chromatin accessibility changes. For this, we combined RNA-seq and ATAC-seq data from A. gambiae midguts and salivary glands, infected and non-infected.
Results: We report differences between tissues in the expression of 392 isoforms and in the use of 247 isoforms. Secondly, we find a clear and significant association between chromatin accessibility states and tissue-specific patterns of AS. The analysis of differential accessible regions located at splicing sites led to the identification of several motifs resembling the binding sites of Drosophila transcription factors. Finally, the genome-wide analysis of tissue-dependent enhancer activity revealed that approximately 20% of A. gambiae transcriptional enhancers annotate to a differentially expressed or used isoform, and that their activation status is linked to AS differences between tissues.
Conclusion: This research elucidates the role of AS in mosquito vector gene expression and identifies regulatory regions potentially involved in AS regulation, which could be important in the development of novel strategies for vector control.

overall_design: RNA-seq of salivary glands from A. gambiae infected and non-infected.

first: Bárbara

last: Díaz-Terenti

first: José Luis

last: Ruiz


title: Single cell RNA-sequencing reveals molecular signatures that distinguish allergic from irritant contact dermatitis

city: Worcester

first: Michael

last: Frisoli

state: Massachusetts

address: 364 Plantation St.

department: Dermatology

institute: University of Massachusetts Chan Medical School

summary: Allergic and irritant contact dermatitis can be challenging to distinguish. We used single cell RNA sequencing (scRNA-seq) and insterstitial fluid proteomic analysis to compare suction blister biopsy skin samples from human volunteers with induced allergic and irritant contact dermatitis reactions.

overall_design:
Volunteers were first sensitized to the allergen, squaric acid dibutyl ester (SADBE).  Skin reactions were induced by 2-day application of individual patches containing SADBE, an acetone vehicle control, and the irritant sodium lauryl sulfate (SLS).  Skin reactions as well as a nonlesional control site were sampled by suction blister biopsy. A 4-day allergic reaction was also sampled by 2-day allergen patch application followed by 2 days of rest before the suction blister biopsy. Suction blister samples were collected by aspirating fluid from the blister. Samples were centrifuged to pellet cells, which were analyzed by scRNA-seq. The suction blister fluid supernatant was analyzed by Olink proteomic extension assay.

>>>Submitter states that raw data will be submitted to dbGaP due to patient privacy concerns.<<<

first: Wei-Che

last: Ko

first: Nuria

last: Martinez

first: Khashayar

last: Afshari

first: Yuqing

last: Wang

first: Manuel

last: Garber

first: John

middle: E

last: Harris


title: microRNAs binding with 3'UTR of tapasin

city: Halle (Saale)

first: Yuan

state: Deutschland

address: Heinrich-Lammasch Platz, 4

type: Non-coding RNA profiling by high throughput sequencing

summary:
Background Dysregulation of major histocompatibility complex (MHC) class I antigen processing and presentation machinery (APM) components in the tumor as one main molecular mechanism of immune escape leading to deactivation of T cell immune surveillance could be due to post-transcriptional regulation via immune-modulatory microRNAs (miRNA). It is now well established from a variety of studies that several miRNAs could effectively modulate the expression of some MHC class I APM components in tumors. Tapasin is an important APM molecule involved in the association of MHC class I with transporter associated with antigen processing (TAP) and peptide loading. Since so far no detailed investigation of the posttranscriptional regulation of tapasin exists, the aim of this study is to identify and functionally characterize miRNAs targeting tapasin in melanoma.
Methods Using miRNA trapping by RNA in vitro affinity purification (miTRAP) and in silico as well as small RNA sequencing, miRNAs will be identified, which bind to the 3'untranslated region (3' UTR) of tapasin. Dual luciferase assays will be performed to determine binding of the miRNA. In silico analysis was performed to predict the effect of miRNAs on the survival of melanoma patients in correlation to tapasin. RT-qPCR, Western blot, flow cytometry and other functional assays were performed after transfecting miRNA mimics in three melanoma cell lines.
Results Using the combination strategy of miTRAP and RNA seq we identified miR-155-5p to bind to the 3’UTR of tapasin, which was further confirmed by in silico analysis and dual luciferase reporter assay.  Transfection of miR-155-5p mimics demonstrated that miR-155-5p upregulate tapasin protein level, which was accompanied by an upregulation of the MHC class I (HLA-ABC) surface expression. Simultaneously, in several different types of cancer, including melanoma, the expression of miR-155-5p is significantly positively correlated with the patient's survival and HLA-A protein.
Conclusion Our data revealed for the first time a positive role of miR-155-5p in the posttranscriptional regulation of tapasin in melanoma and provide further insights into the miR-155-5p-mediated induction of HLA-ABC surface expression. This might lead to a better T cell response, avoidance tumor cell escape, improvement of patients' survival and thus might be a potential therapeutic target.

first: Maria-Filothei

last: Lazaridou


title: Macropinocytosis mediates resistance to loss of glutamine transport in triple-negative breast cancer

city: Kensington

first: Jeff

last: Holst

state: NSW

address: Cnr of High and Botany

institute: University of New South Wales

summary: Triple-negative breast cancer (TNBC) relies on glutamine uptake by the transporter ASCT2 to sustain their unique glutamine metabolism and growth. Despite previous data showing cell growth inhibition after ASCT2 knockdown, ASCT2 CRISPR knockout was well-tolerated by breast cancer cell lines. Despite the loss of a glutamine transporter and low rate of glutamine uptake, intracellular glutamine steady state levels were higher in ASCT2 knockout compared to control TNBC cells. Proteomics data revealed upregulation of macropinocytosis, reduction in glutamine efflux and glutamine synthesis in ASCT2 knockout cells. Loss of ASCT2 in TNBC cell line HCC1806 induced a 5-10-fold increase in macropinocytosis across 5 separate ASCT2 knockout clones, compared to a modest 2-fold increase in the shRNA ASCT2 knockdown. By comparison, ASCT2 knockout impaired cell proliferation in a non-macropinocytic breast cancer cell line, HCC1569. These data suggest that macropinocytosis provides a novel resistance mechanism to strategies targeting glutamine uptake alone. Despite this adaptation, TNBC cells continue to rely on glutamine metabolism for their growth, which suggests therapeutic targeting may need to focus on downstream glutamine metabolism pathways.

overall_design:
To investigate the impact of glutamine transporter, ASCT2, on breast cancer cells we generated CRISPR/Cas9 breast cancer knockout lines as well as dox-inducible ASCT2 knockdown cells.
We performed mRNA sequencing analysis on the triple negative breast cancer line, HCC1806 for the wildtype line and six ASCT2 knockout clones as well as shCtrl and shASCT2 cells.

first: Kanu

last: Wahi

first: Natasha

last: Freidman

first: Fabio

last: Zanini


title: Analysis of gene expression of mice's thyroid glands according to the state of the Pink1 Knock-out

city: Seoul

first: HAN SAI

last: LEE

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

country: South Korea

institute: Seoul National University Seoul Metropolitan Government Boramae Medical Center

summary: To investigate the differential gene expression in thyroid glands between C57BL/6 mice of Pink1 Knock-out and Pink1 Wild-type, RNA-sequencing was conducted.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for thyroid glands in C57BL/6 mice of Pink1 Knock-out or Wild-type.

first: Han

last: Lee

first: Jinju

first: Young

last: Song


title: scRNA-seq of TNF receptor-deficient NK cells during Salmonella Typhimurium infection

city: Woolloongabba

first: Timothy

middle: Ryan

last: McCulloch

state: QLD

address: 37 Kent St

department: Frazer Institute

institute: University of Queensland

summary: Natural killer (NK) cell function is regulated by a balance of activating and inhibitory signals, however the role of tumor necrosis factor (TNF) on NK cells remains poorly understood. The goal of this study was to use single-cell RNA sequencing to delineate the transcriptional changes induced in NK cells by TNF signalling through either TNFR1 or TNFR2. Our findings highlight the different regulatory pathways induced by each receptor which play dichotomous roles on the outcome of NK cell function and anti-bacterial immunity.

overall_design: We generated novel mouse models of NK specific TNF signalling deficiency by crossing Ncr1cre mice to either TNFR1 or TNFR2 flox mice. These mice, along with Ncr1cre used as WT controls, were infected with 5 x 10^6 Salmonella Typhimurium SL3261 by i.p. infection to induce systemic infection. At day four post infection, NK cells were isolated from mouse spleens. Each biological replicate (n = 3-4) were stained with TotalSeq HTO antibodies (Biolegend), pooled, and processed for 10x scRNA-seq.

first: Fernando

last: Souza-Fonseca-Guimaraes


title: VTA dopamine neurons are hyperexcitable in 3xTg-AD mice due to casein kinase 2-dependent SK channel dysfunction

status: Public on Sep 14 2024

city: Oklahoma City

first: Willard

last: Freeman

address: 825 NE 13th St

institute: Oklahoma Medical Research Foundation

summary: Alzheimer's disease (AD) patients exhibit neuropsychiatric symptoms that extend beyond classical cognitive deficits, suggesting involvement of subcortical areas. Here, we investigated the role of midbrain dopamine (DA) neurons in AD using the amyloid + tau-driven 3xTg-AD mouse model. We found deficits in reward-based operant learning in AD mice, suggesting possible VTA DA neuron dysregulation. Physiological assessment revealed hyperexcitability and disrupted firing in DA neurons caused by reduced activity of small-conductance calcium-activated potassium (SK) channels. RNA sequencing from contents of single patch-clamped DA neurons (Patch-seq) identified up-regulation of the SK channel modulator casein kinase 2 (CK2). Pharmacological inhibition of CK2 restored SK channel activity and normal firing patterns in 3xTg-AD mice. These findings shed light on a complex interplay between neuropsychiatric symptoms and subcortical circuits in AD, paving the way for novel treatment strategies.

overall_design: Single cells extracted from 3xTg-AD and control (WT) mice via Patch-seq.

first: Harris

last: Blankenship

first: Kelsey

middle: A

last: Carter

first: Kevin

middle: D

last: Pham

first: Nina

last: Cassidy

first: Andrea

last: Markiewicz

first: Michael

middle: I

last: Thellmann

first: Amanda

middle: L

last: Sharpe

last: Beckstead


title: Specific ablation of BFAR on CD8+ T cells promotes CD8+T cell antitumor immunity

city: Shanghai

first: Siyu

last: Pei

address: 320 Yueyang Road

institute: Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences

summary: To investigate the role of aging in the regulation of CD8+ T cell anti-tumor function, we sorted CD8+T cells from Rag1-/- mice that were received intravenously injection of young/old mice-derived CD8+ T cells on the same day with tumor cell inoculation and carried out single-cell RNA sequencing, and the young group showed increased highly cytotoxic TRM and reduced exhausted CD8+ T cells in tumor microenvironment. Our study also found that BFAR deficiency reinvigorates tumor infiltrating CD8+ T cells into a more young state, as reflected by increased TRM.

overall_design: Young/aged tumor infiltrating CD8+T cells were isolated from Rag1-/- mice that were received intravenously injection of young (8-weeks)/old mice (18-months-old)-derived CD8+ T cells on the same day with MB49 tumor cell inoculation and and analyzed by single-cell RNA sequencing technology, and WT/BFAR-deficient CD8+ T cells isolated from subcutaneous MB49 tumors in WT or BFAR-CD8-KO mice. Each sample contains CD45+ CD8+ T cells combined from 3 mice.

first: Yichuan

last: Xiao


title: Expression data of young and aged mice derived CD8+ T cells stimulated in TCR or TRM condition for 3 days

summary: Ageing can compromise antitumor immunity, but the underlying mechanism by which ageing causes CD8+ T cell dysfunction and thus limits their antitumor activity remains poorly understood. We found that ageing greatly impaired the generation of CD8+ tissue-resident memory T (TRM) cell subset in non-lymphoid tissues (NLTs). And here we presented that in vitro differentiation of TRM cells was inhibited in the aged CD8+ T cell group compared to the young CD8+ T cell group.

overall_design: mRNA profiles of primary CD8+T cells from young (6-weeks-old) and aged (18-months-old) mice that were cultured under TCR condition (α-CD3/28: 1μg/ml) or TRM condition (α-CD3/28: 1μg/ml, IL-15: 20ng/ml, TGFβ: 5ng/ml) for 3 days in vitro.

first: Pei

last: Siyu


title: 3D structure of the genome of F. graminearum strain PH-1

city: VILLENAVE D ORNON

first: Nadia

last: PONTS

state: Select State

address: 71 avenue E. Bourlaux

institute: INRAE

summary: Fusarium graminearum is an efficient plant pathogen found in crops worldwide. Major causal agent of Fusarium head blight on wheat in Europe, this filamentous fungus can also produce toxic mycotoxins and a variety of secondary metabolites in developing kernels. These secondary metabolites are produced as the result of a cascade of coordinated regulations of an arsenal of genes dispersed accross the genome. Previous study highlighted the roles of the histone marks H3K27me3 and H3K4me3 in regulating the metabolism of F. graminearum. Here, we applied HiC in combination to transcriptomics to explore the functional components of 3D organization of the four chromosomes in the nucleus. We show that the orchestration of genome regulation is mediated by histone-dependant highly topologically structured chromatin that organizes chromosomes in territories to potentiate coordinated genome regulation.

overall_design: In vitro 3 day-old mycelia (liquid modified MS) were subjected to in situ HiC-seq; two biological replicates were run

last: Ponts

first: Aurélie

last: Etier

first: Fabien

last: Dumetz


title: Transcriptomic analysis of gene expression pathways impacted by metaxin-2 (MTX-2) deficiency in a C. elegans mutant (mtx-2 KO, (gk444)) vs Wild-type worms (N2 Bristol)

status: Public on Sep 13 2024

city: Paris

first: Chloé

last: Talarmin-Gas

state: Ile de France

address: 2, rue ambroise Paré

department: Biochemistry

institute: APHP

summary: MandibuloAcral Dysplasia associated to MTX2 gene (MADaM) is a recently described progeroid syndrome (accelerated aging disease) whose clinical manifestations include skin abnormalities, growth retardation, and cardiovascular diseases. We previously proposed that mtx-2-deficient C. elegans could be used as a model for MADaM and to support this, we present here our comprehensive phenotypic characterization of these worms using atomic force microscopy (AFM), transcriptomic, and oxygen consumption rate analyses. AFM analysis showed that young mtx-2-less worms had a significantly rougher, less elastic cuticle which becomes significantly rougher and less elastic as they age, and abnormal mitochondrial morphology. mtx-2 C. elegans displayed delayed development, decreased pharyngeal pumping, significantly reduced mitochondrial respiratory capacities, and transcriptomics analysis identified perturbations in the aging, TOR, and WNT-signaling pathways. The phenotypic characteristics of mtx-2 worms shown here are analogous to many of the human clinical presentations of MADaM and we believe this validates their use as a model which will allow us to uncover the molecular details of the disease and develop new therapeutics and treatments.

overall_design: To investigate the different genes expressed in the nematode C. elegans due to the absence of the mtx-2 gene (an outer mitochondrial membrane gene). We performed gene expression profiling analysis using data obtained from triplicate samples of RNA-Seq of mutant worms (mtx-2 KO, gk444), at L4 stage compared to Wild-type worms (N2 Bristol).

first: Benjamin

last: Saintpierre

first: Franck

last: Letourneur

first: Chloe

last: Talarmin


title: Nonfunctional coq10 mutants maintain the ERMES complex and reveal true phenotypes associated with the loss of the coenzyme Q chaperone protein Coq10

city: Los Angeles

first: Guillaume

last: Chanfreau

state: California

address: 607 Charles E Young Dr E

institute: University of California, Los Angeles

summary: Coenzyme Q (CoQ) is a redox-active lipid molecule that acts as an electron carrier in the mitochondrial electron transport chain. In Saccharomyces cerevisiae, CoQ is synthesized in the mitochondrial matrix by a multi-subunit protein-lipid complex termed the CoQ synthome, the spatial positioning of which is coordinated by the Endoplasmic Reticulum-Mitochondria Encounter Structure (ERMES). The MDM12 gene encoding the cytosolic subunit of ERMES, MDM12, is co-expressed with COQ10, which encodes the putative CoQ chaperone Coq10, via a shared bidirectional promoter. Deletion of COQ10 results in respiratory deficiency, impaired CoQ biosynthesis, and reduced spatial coordination between ERMES and the CoQ Synthome. While Coq10 protein content is maintained upon deletion of MDM12, we show that deletion of COQ10 by replacement with a HIS3 marker results in diminished Mdm12 protein content. Since deletion of individual ERMES subunits prevents ERMES formation, we asked whether some or all of the phenotypes associated with COQ10 deletion result from ERMES dysfunction. To identify the phenotypes resulting solely due to the loss of Coq10, we constructed strains expressing a functionally impaired (coq10-L96S) or truncated (coq10-R147*) Coq10 isoform using CRISPR-Cas9. We show that both coq10 mutants preserve Mdm12 protein content and exhibit impaired respiratory capacity like the coq10Δ mutant, indicating that Coq10’s function is vital for respiration regardless of ERMES integrity. Moreover, the maintenance of CoQ synthome stability and efficient CoQ biosynthesis observed for the coq10-R147* mutant suggests these deleterious phenotypes in the coq10Δ mutant result from ERMES disruption. Overall, this study clarifies the role of Coq10 in modulating CoQ biosynthesis.

overall_design: The coq10 point mutations,  coq10-R147* and coq10-L96S were introduced chromosomally using CRISPR-Cas9. For the Coq10 knockout the COQ10 ORF was replaced with the HIS3 gene. Cells were grown in YPGal in biological triplicates. Differential gene expression analysis was performed on on these strains and their triplicates.

first: Noelle

last: Novales

last: Feustel

last: He

middle: F

first: Catherine

last: Clarke


title: Ulp1 Regulates Cell Proliferation through INO1 in Pichia pastoris

city: hangzhou

first: bo

last: zhong

state: zhejiang

address: No. 928, 2nd Street, Xiasha Higher Education Park, Qiantang District, Hangzhou City, Zhejiang Province

institute: Zhejiang Sci-Tech University

summary: Pichia pastoris is extensively employed as a host for expressing foreign proteins, and its secretion system is especially advantageous for efficient protein purification. Ulp1 is essential for the cell cycle. In Saccharomyces cerevisiae, the absence of Ulp1 leads to G2/M cell cycle arrest. Similarly, in Pichia pastoris, we also observed G2 phase stalling in Ulp1 knockout cells. RNA-seq data revealed significant changes in seven genes, including INO1. Interestingly, overexpressing INO1 in Ulp1 knockout cells restored cell proliferation, suggesting that Ulp1 regulates cell proliferation through INO1. However, adding extra inositol to Ulp1 knockout cells was not able to restore cell proliferation, indicating that the enzyme activity of INO1 is not essential for cell proliferation. Our data provide new insights into Ulp1 functions in the cell cycle and raise new questions about yeast cell cycle regulation.

overall_design: Transcriptome sequencing and analysis results of wild-type Pichia pastoris X33 strain, Pichia pastoris x33 Ulp1 knockout strain, Pichia pastoris X33 Ulp1 knockout strain overexpressing INO1 strain, Pichia pastoris X33 Ulp1 knockout strain supplemented with inositol culture in culture medium.

first: Junjie

last: Yang

first: Bo

last: Zhong

first: Lan

first: Guodong

last: Chen


title: RUNX2 as a novel Biomarker for early identification of Patients Progressing to Advanced-Stage Mycosis Fungoides

city: Aarhus

first: Morten

middle: Muhlig

last: Nielsen

address: Palle Juul-Jensens Bouleward 99

department: Department of Molecular Medicine

country: Denmark

institute: Aarhus University

summary: Here, we used spatial transcriptomics on skin samples at time-of-diagnosis to enable prediction of patients who later progressed to advanced stages of MF. Formalin-fixed, paraffin-embedded skin biopsies at time of diagnosis from six patients with MF who progressed to advanced stages of disease within 4 months to 12 years after diagnosis, and nine patients who remained in early-stage disease over 9 to 27 years were analyzed using the GeoMx Digital Spatial Profiler to capture spatially resolved high-plex RNA gene expression data. Five different regions of interest (the epidermis, the basal layer of epidermis, CD4+ T-cells and neighboring cells, and Pautrier’s microabscesses) were profiled for further assessment.

overall_design: Open mechanistic study

first: Maria

last: Danielsen

first: Thomas

last: Emmanuel

middle: M

first: Lise

last: Lindhl

last: Gluud

first: Niels

last: Odum

first: Line

last: Raaby

first: Torben

last: Steiniche

first: Lars

last: Iversen

first: Rikke

last: Bech

first: Terkild

last: Buus

first: Claus

last: Johansen


title: Genomic analysis of mouse pancreatic KPC tumor grafts upon stroma targeting

city: Houston

first: Yulin

last: Dai

address: 7000 fannin, suite 600

department: School of Biomedical Informatics

institute: University of Texas health science center at Houston

summary: During carcinoma progression, mesenchymal stromal cells (MSCs) become recruited to tumors and contribute to the pool of cancer-associated fibroblasts (CAFs). Sub-populations of CAFs, have diverse and incompletely understood effects on disease progression and resistance to therapy. Adipose stromal cells (ASCs), the MSCs from fat tissue increasingly recruited by carcinomas in the context of obesity, have been shown to support cancer progression. Here, the roles of perivascular CAFs expressing platelet-derived growth factor receptor beta (Pdgfrb) and of CAFs expressing non-glycanated decorin (ngDCN), a marker of ASCs, were analyzed. We used mice orthotopically grafted with pancreatic ductal adenocarcinoma (KPC) cells. Ablation of cells expressing thymidine kinase under the control of Pdgfrb promoter with ganciclovir resulted in suppression of primary pancreatic tumor growth. However, ablation Pdgfrb+ cells induced spontaneous metastases to the liver. For depletion of ngDCN+ cells, we used a hunter-killer peptide D-CAN, which has been previously reported to induce apoptosis of ASCs and CAFs in mouse models. Here, D-CAN also had a negative effect on pancreatic tumor growth, however, there was no significant effect on metastatic dissemination. Single cell RNA sequencing of tumors demonstrated that targeting of Pdgfrb+ and ngDCN+ stromal cells had distinct effects on sub-populations of CAFs. Endothelial cell and cancer cell survival was decreased by depletion of either Pdgfrb+ or ngDCN+ stroma. However, pathway analysis revealed that depletion of Pdgfrb+ and ngDCN+ stromal cells has different effects on the markers of cancer cell aggressiveness. D-CAN treatment, and to a less extent Pdgfrb+ cell ablation, suppressed macrophage M2 polarization and increased infiltration of cytotoxic T-lymphocytes. This observation, confirmed in the MyCap graft model of prostate cancer, suggested that ablation of ngDCN+ stroma should synergize with immune checkpoint inhibitors. We tested the effect of D-CAN on the efficacy of anti-PD-L1 antibody in the orthotopic KPC model. Compared to D-CAN and anti-PD-L1 antibody alone, combination treatment had a synergistic effect on tumor growth. Importantly,  liver metastases were suppressed by the D-CAN / anti-PD-L1 antibody combination, but not by individual agents. We conclude that improved approaches to target mesenchymal stroma in tumors may be effective in combination with immunotherapy.

overall_design: In mice orthotopically grafted with KPC cells, stromal cells were depleted with TK/GCV or peptide D-CAN.

first: Kolonin

last: Mikhail

first: Rupert

last: Joseph


title: Environmental fluoxetine promotes skin cell proliferation and wound healing

city: Kingston-Upon-Hull

first: Quentin

last: Rodriguez-Barucg

address: University of Hull, Cottingham Road

department: HYMS Centre for Biomedicine

institute: University of Hull

summary: This study investigates the effects of environmentally-relevant concentrations of fluoxetine (FLX, commercial name: Prozac) on wound healing. Pollution of water systems with pharmaceutical and care products, including antidepressants such as FLX and other selective serotonin reuptake inhibitors, is a growing environmental concern. Environmentally-relevant FLX concentrations are known to impact physiological functions and behaviour of aquatic animals, however, the effects of exposure on humans are currently unknown. Using a combination of human skin biopsies and keratinocyte cell lines, we show that exposure to environmental FLX enhances wound closure. We show dose-dependent increases in wound closure with FLX concentrations from 125 ng/l. We demonstrate that the mechanisms underlying enhanced wound closure are increased cell proliferation and serotonin signalling. Transcriptomic analysis revealed 350 differentially expressed genes after exposure. Downregulated genes were enriched in pathways related to mitochondrial function and metabolism, while upregulated genes were associated with cell proliferation and tissue morphogenesis. Kinase profiling showed altered phosphorylation of kinases related to the MAPK pathway. Consistent with this, phosphoproteomics analyses identified 235 differentially phosphorylated proteins after exposure, with enriched GO terms related to cell cycle, division, and protein biosynthesis. These findings collectively show that exposure to environmental FLX promotes wound healing through modulation of serotonin signalling, gene expression and protein phosphorylation, leading to enhanced cell proliferation. Our results open the door for further research to investigate the effects of environmental FLX on cell metabolism and proliferation, and clinical applications of FLX in the setting of wound healing. We argue that there is an urgent need to develop models that can inform on the risks, and potential benefits, of environmental pollution with selective serotonin reuptake inhibitors.

overall_design: We sought to understand the molecular mechanisms underlying the enhancement of cell proliferation and scratch closure when HaCaT cells were exposed to ER-FLX concentrations

first: Pedro

last: Beltran-Alvarez


title: Effect of mitochondrial transfer from bone marrow stroma cells to anti-tumor T cells on T cell differentiation and function

city: Regensburg

first: LIT

last: NGS-Core

address: Franz-Josef-Strauß-Allee 11

department: NGS-Core

institute: Leibniz Institute for Immunotherapy

summary: Mitochondrialloss and dysfunctiondrive T cell exhaustion, representing major barriers to successful T cell-based immunotherapies. We found that bone marrow stromal cells (BMSCs) transfer stromal cell mitochondria into CD8+ T cells. CD8+ T cells with donated mitochondria displayed enhanced mitochondrial respiration and spare respiratory capacity. When transferred into tumor-bearing hosts, these supercharged T cells expanded more robustly, infiltrated the tumor more efficiently, and exhibited fewer signs of exhaustion compared to T cells that did not take up mitochondria. As a result, mitochondria-boosted CD8+ T cells mediated superior antitumor responses, prolonging animal survival.

overall_design:
We co-cultured activated CD8+ T cells from 3 donor mice with BMSCs in which mitochondria are stably labeled with dsRed.
After co-culture, we sorted dsRed+ (MitoPositive) T cells that had taken up mitochondria from BMSCs and dsRed-negative (MitoNegative) T cells.
Sorted T cells were either processed for immediate scRNA-Seq ("d0") or transferred into B16KVP tumor-bearing B6 mice and re-isolated by flow cytometry on d7.
For multiplexing of d0 samples, biological replicates were marked using hashtags 1,3,5 and 2,4,6 for MitoPositive and MitoNegative cells, respectively.
Transferred T cells were isolated from spleen (#1 and #2) and tumor (#3 and #4 for MitoPositive and MitoNegative T cells, respectively) and pooled by donor after mulitplexing.

first: Jeremy

last: Baldwin

first: Nicholas

last: Strieder

first: Inmaculada

last: Hernandez-Lopez

first: Luca

last: Gattinoni


title: Intercellular nanotube-mediated mitochondrial transfer enhances T-cell metabolic fitness and antitumor efficacy

summary: Mitochondrial loss and dysfunction drive T cell exhaustion and represent major barriers to successful T cell immunotherapies. We found that mesenchymal stromal cells (MSC) establish nanotubular connections with T cells in a TLN2-dependent manner and leveraged these intercellular highways to supply new mitochondria to CD8+ T cells. Acquisition of MSC mitochondria increased T cell basal mitochondrial respiration and spare respiratory capacity. When transferred into tumor-bearing hosts, tumor-specific CD8+ T cells with donated mitochondria expanded more robustly, infiltrated the tumor more efficiently, and exhibited fewer signs of exhaustion compared to CD8+ T cells that did not take up mitochondria. As a result, mitochondria-boosted CD8+ T cells mediated superior antitumor responses, prolonging animal survival. To unravel the mechanisms behind mitochondrial transfer we performed RNA sequencing on CD8+ T cells that acquired donor mitochondria (MitoPositive), or did not acquire donor mitochondira (MitoNegative) directly after after co-culture (0hr timepoint).

overall_design: conditions: control, Mito_positive, Mito_negative, three replicates each

middle: G

first: Christoph

last: Heuser


title: Microarray analysis of mouse bone marrow hematopoietic progenitors in steady state conditions

city: Barcelona

first: Cristina

last: Lopez-Rodriguez

address: Carrer Dr. Aiguader, 88

department: Medicine and Life Sciences

institute: Universitat Pompeu Fabra

summary: Microarray analysis of differentially expressed genes in wild-type and NFAT5-deficient hematopoietic stem cells (HSC) and multipotent progenitors (MPP)

overall_design: 12 samples of bone marrow hematopoietic stem cells in steady-state conditions

last: López-Rodríguez

first: Jose

last: Aramburu

first: Laia

last: Traveset


title: Microarray analysis of mouse bone marrow hematopoietic progenitors

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

overall_design: Refer to individual Series


title: Microarray analysis of mouse bone marrow hematopoietic progenitors after bone marrow transplant

overall_design: 12 samples of bone marrow hematopoietic stem cells after bone marrow transplantation


title: Testosterone acts through membrane protein GPRC6A to cause cardiac edema in zebrafish emrbyos

status: Public on Sep 12 2024

first: Daniel

last: Gorelick

state: texas

address: 1 Baylor Plaza BCM229

institute: Baylor College of Medicine

summary: Classically, intracellular nuclear androgen receptors (ar) are considered to be the key mediators of androgen actions. However, there have been several reports that androgens can also exert their effects by binding to integral membrane proteins. Despite in vitro cloning and characterization of several putative membrane androgen receptors, their functions in vivo remain poorly understood. We used a chemical-genetic screen in zebrafish and discovered that the G-protein coupled receptor, GPRC6A, mediates androgen action during embryonic development. We exposed zebrafish embryos to testosterone (30 μM) from 2-4 hours post-fertilization (hpf) until 72 hpf. We found that testosterone exposure caused cardiac edema in wild-type and ar mutant zebrafish embryos, but there was a significant reduction in this phenotype in the grprc6a mutant embryos, suggesting that gprc6a regulates androgen action independently of nuclear androgen receptors. Additionally, we exposed wild-type embryos to testosterone together with GPRC6A antagonists and observed a significant suppression of the cardiac edema phenotype. These results suggest that testosterone causes cardiac edema in zebrafish embryos by acting via the integral membrane protein GPRC6A, independently of nuclear androgen receptors. Overall, our study provides insights into non-genomic androgen signaling during embryonic development and identifies GPRC6A as a key receptor mediating androgen action.

overall_design: To identify gene targets of Testosterone-GPRC6A, we performed RNAseq on 3 day post fertilization zebrafish embryos following 3 day exposure to either: testosterone, testosterone + strong GPRC6A antagonist EGCG, testosterone + weak GPRC6A antagonist calindol, 0.1% DMSO (vehicle control). We then performed gene expression analysis comparing each chemical exposure to vehicle with 3 biological replicates for each condition.

first: Shayan

last: Shahriar

first: Vahid

last: Zadmajid