title: An Efficient Direct Conversion Strategy to Generate Functional Astrocytes from Human Adult Fibroblasts

status: Public on Jul 19 2024

city: San Antonio

first: Melanie

middle: Anita

last: Carless

state: Texas

address: One UTSA Circle

institute: The University of Texas at San Antonio

summary: Direct reprogramming approaches offer an attractive alternative to stem-cell-derived models, allowing the retention of epigenetic information and age-associated cellular phenotypes, as well as a fast method to reach a target cell type. Several groups have previously generated multiple neuronal subtypes, neural progenitor cells, oligodendrocytes, and other cell types directly from fibroblasts. Other groups have had success at the efficient conversion of embryonic fibroblasts to astrocytes but have not yet achieved similar conversion efficiency for adult human fibroblasts. In order to generate astrocytes for the study of adult-stage disorders, we developed an improved direct conversion strategy employing a combination of small molecules to activate specific pathways that induce trans-differentiation of human adult fibroblasts to astrocytes. We demonstrate that this method produces mature GFAP+/S100β+ cells at high efficiency (40-45%), comparable to previous studies utilizing embryonic fibroblasts. Further, Fibroblast-derived induced Astrocytes (FdiAs) are enriched for markers of astrocyte functionality, including ion-channel buffering, gap-junction communication, and glutamate uptake; and exhibit astrocyte-like calcium signaling and neuroinflammatory phenotypes. RNA-Seq analysis indicates an adult rather than fetal astrocytic gene expression signature, with a greater correlation to temporal lobe astrocytes. Fibroblast-derived induced astrocytes provide a useful tool in studying the adult brain and complement existing in vitro models of induced neurons (iNs), providing an additional platform to study late-stage brain disorders.

overall_design: Fibroblasts (Day 0), and FdiAs at different stages of conversion (Day 10, 30 and 50) were fixed and processed for single-cell RNA-seq using a combinatorial indexing approach to assess gene expression profiles of induced cells.

first: Uchit

last: Bhaskar

middle: A

first: Mark

last: Kos


title: Molecular profiling of pre-and post-5-azacytidine myelodysplastic syndrome samples identifies predictors of response

status: Public on Jul 21 2024

city: NY

first: Christopher

middle: Y.

last: Park

address: 550 First Avenue

country: USA

institute: NYU Grossman School of Medicine

type: Expression profiling by high throughput sequencing

summary: Treatment with the hypomethylating agent 5-azacytidine (AZA) increases survival in high-risk (HR) myelodysplastic syndrome (MDS) patients, but predicting patient response and overall survival remains challenging. To address these issues, we analyzed mutational and transcriptional profiles in CD34+ hematopoietic stem/progenitor cells (HSPCs) before and following AZA therapy in MDS patients. AZA treatment led to a greater reduction in the mutational burden in both blast and hematological responders than non-responders. Blast and hematological responders showed transcriptional evidence of pre-treatment enrichment for pathways such as oxidative phosphorylation, MYC targets, and mTORC1 signaling. While blast non-response was associated with TNFa signaling and leukemia stem cell signature, hematological non-response was associated with cell-cycle related pathways. AZA induced similar transcriptional responses in MDS patients regardless of response type. Comparison of blast responders and non-responders to normal controls, allowed us to generate a transcriptional classifier that could predict AZA response and survival. This classifier outperformed a previously developed gene signature in a second MDS patient cohort, but signatures of hematological responses were unable to predict survival. Overall, these studies characterize the molecular consequences of AZA treatment in MDS HSPCs and identify a potential tool for predicting AZA therapy responses and overall survival prior to initiation of therapy.

overall_design: To investigate whether transcriptional profiles from paired pre-/post AZA MDS samples predict treatment responses to AZA, we generated RNA-seq data from 14 MDS patients before and after treatment with AZA. We also generated RNA-seq data from 6 age-matched healthy donors as control. Bone marrow mononuclear cells (BMMCs) were isolated from BM samples using Ficoll-Paque Plus (GE Healthcare, Massachusetts, US) density gradients per manufacturer’s instructions. CD34+ cells were isolated by immunomagnetic separation using an autoMACs Pro Separator (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) following the manufacturer´s instructions, which were used for library preparation and sequencing.

first: Mónica

last: del Rey González

first: Sohini

last: Chakraborty

middle: Y

first: Jesús

middle: M

last: Hernández Rivas


title: CTCF-dependent insulation of Hoxb13 and the heterochronic control of tail length [nCATS]

status: Public on Jul 18 2024

city: Lausanne

first: Lucille

last: Lopez-Delisle

address: Station 19

country: Switzerland

institute: EPFL

summary: In mammals, tail length is controlled by several genetic determinants, amongst which Hox13 genes located at the posterior extremities of Hox clusters, whose main function are to terminate the extension of the body axis. In this view, the precise timing in the transcriptional activation of these genes may impact upon body length. Unlike other Hox clusters, HoxB lacks all posterior genes between Hoxb9 and Hoxb13, two genes separated by a ca. 70 kb large DNA segment containing an unusually high number of CTCF sites, suggesting it isolates Hoxb13 from the rest of the cluster, thereby delaying its negative impact on trunk extension. We deleted the spacer DNA to induce a potential heterochronic gain of function of Hoxb13 at physiological concentration and observed a shortening of the tail as well as other abnormal phenotypes, which were all rescued by inactivating Hoxb13 in-cis with the deletion. A comparable gain of function was observed in mutant ES cells grown as pseudo-embryos in vitro, which allowed us to examine in details the importance of both the number and the orientation of CTCF sites in the insulating activity of the DNA spacer. A short cassette containing all the CTCF sites was sufficient to insulate Hoxb13 from the rest of HoxB and additional modifications of this CTCF cassette showed that two CTCF sites in convergent orientations are already capable of importantly delaying Hoxb13 activation in these conditions. We discuss the relative importance of genomic distance versus number and orientation of CTCF sites in preventing Hoxb13 to be activated too early during trunk extension and hence to modulate tail length.

overall_design: nCATS of the HoxB Del/Del(i9-13)Ins(CBS5-10) ES cell line

first: Célia

last: Bochaton

first: Jozsef

last: Zakany

first: Hocine

last: Rekaik

first: Denis

last: Duboule


title: CTCF-dependent insulation of Hoxb13 and the heterochronic control of tail length [RNA-seq embryo]

overall_design: RNA-seq of HoxB mutated and wild-type littermates headless embryos at E9.5 and E10.5


title: scCRISPR-screen in human monocyte derived macropahges

city: South San Francisco

first: Sandra

last: Melo

address: 1 DNA Way

institute: Genentech, Inc.

summary: To determine whether the hits derived from a genome-wide murine BMDM screen regulated human macrophage phenorypes, we established a framework that coupled genetic perturbation with phenotypic analysis at single-cell resolution in primary human monocyte-derived macrophages (MDM scCRISPR-seq).

overall_design:
MDM scCRISPR-seq combines highly efficient electroporation-based CRISPR gene editing with a perturbation-specific barcoding strategy using cell hashing antibodies, allowing for a pooled sequencing format of 84 conditions per donor. Our sgRNA library consisted of 4 sgRNAs per gene, targeting previsously identified hits in murine BMDMs along with negative controls including non-targeting controls (NTC) as well as positive controls of the interferon gamma signaling pathway (IFNGR1, SOCS1). CRISPR perturbed cells derived from two healthy donors were subjected to IFNγ stimulation or left untreated followed by cell hashing, pooling and scRNAseq analysis
***Raw data will be deposited at EGA***

first: Haag

last: Simone

first: Xie

last: Shiqi

first: Melo

middle: C

last: Sandra


title: CTCF-dependent insulation of Hoxb13 and the heterochronic control of tail length [CUT&RUN]

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: CUT&RUN of CTCF in HoxB mutated mESC

first: Pierre

last: Osteil


title: Pre-existing skin-resident CD8 and γδ T cell circuits mediate immune response in Merkel cell carcinoma and Predict immunotherapy efficacy [scRNA_blood]

status: Public on Jul 24 2024

city: Chicago

first: Jaehyuk

last: Choi

state: Illinois

address: 303 E. Superior St. 5-220

department: Dermatology

institute: Northwestern University

summary: Merkel Cell Carcinoma (MCC) is an aggressie skin cancer with limiterd treatment and low response rates to Immune Checkpoint Blockade (ICB) therapy. We compiled the largest sc database of MCC tumor samples and matched blood to identify tumor gener modules that Predict ICB response.

overall_design:
MCC tumor and matched blood samples were used for bulk and single-cell RNA-seq.

**Raw data is being deposited in dbGaP**

first: Zachary

middle: Z

last: Reinstein

first: Yue

last: Zhang

first: Oscar

last: Ospina

first: Matt

last: Nichols

first: Victoria

last: Chu

first: Alvaro

last: de Mingo Pulido

first: Karol

last: Prieto

first: Johnathan

last: Ngyuen

first: Rui

last: Yin

first: Carlos

last: de Moran Segura

first: Ahmed

last: Usman

first: Brittney

last: Sell

first: Spencer

last: Ng

first: Janis

last: de la Iglesia

first: Sunandana

last: Chandra

first: Jeffery

last: Sosman

first: Raymond

middle: J

last: Cho

middle: B

last: Cheng

first: Ellie

middle: N

last: Ivanova

first: Sergei

last: Koralov

first: Robbert

last: Slebos

first: Christine

middle: H

last: Chung

first: Nikhill

middle: I

last: Khushalani

first: Jane

middle: L

last: Messina

first: Amod

last: Sarnaik

middle: S

last: Zager

first: Vernon

middle: K

last: Sondak

first: Charles

last: Vaske

first: Sungjune

last: Kim

first: Andrew

last: Brohl

first: Xinlei

last: Min

first: Brian

middle: G

last: Pierce

first: Brooke

last: Fridley

first: Kenneth

last: Tsai


title: CTCF-dependent insulation of Hoxb13 and the heterochronic control of tail length

first: Lucille

last: Lopez-Delisle

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

overall_design: Refer to individual Series


title: A brain organoid/ALL co-culture model reveals the AP-1 pathway as critically associated with CNS-involvement of BCP-ALL

status: Public on Jul 16 2024

city: Kiel

first: Lennart

middle: TN

last: Lenk

state: SH

address: Rosalind-Franklin-Str. 9

department: Pediatric Hematology/Oncology

country: Germany

institute: UKSH Campus Kiel

summary: Central nervous system (CNS) involvement remains a clinical hurdle in treating childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The disease mechanisms of CNS leukemia are primarily investigated using 2D cell culture and mouse models. Given the variations in cellular identity and architecture between the human and murine CNS, it becomes imperative to seek complementary models to study CNS leukemia. Here, we present a first-of-its-kind 3D co-culture model combining human brain organoids and BCP-ALL cells. We noticed signicantly higher engraftment of TCF3::PBX1+ leukemia cell lines and patient-derived xenograft (PDX) cells in human induced pluripotent stem cell (iPSC)-derived cerebral organoids when compared to cell lines derived from chronic myeloid leukemia (CML), a subtype typically not associated with CNS involvement. Next, to validate translatability between organoid co-culture model and in vivo murine models, we confirmed that targeting CNS leukemia relevant pathways like CD79a/Igα or CXCR4-SDF1 reduced the invasion of BCP-ALL cells into organoids. RNA sequencing and further validations of organoid-invading leukemia cells compared to the non-invaded fraction revealed significant upregulation of the AP-1 transcription factor-complex members in organoid-invading cells. Moreover, we detected a significant enrichment of AP-1 pathway genes in ALL-PDX cells recovered from the CNS compared to spleen blasts of mice transplanted with TCF3::PBX1+ PDX cells, substantiating the role of AP-1 signaling in CNS disease. Accordingly, we found significantly higher levels of the AP-1-gene JUN in patients initially diagnosed as CNS-positive compared to CNS-negative cases as well as CNS-relapse cases in a cohort of 100 BCP-ALL patients. Our results suggest iPSC-derived brain organoids as a novel model to investigate CNS-involvement and identify the AP-1 pathway as a critical driver of CNS-disease in BCP-ALL.

overall_design: Patrient-derived xenograft cells from five TCF3::PBX1+ BCP-ALL patients were transplanted into NOD-SCID-GAMMA (NSG)-mice. Comparative RNA sequencing of leukemic cells recovered from the CNS versus the spleen and bone marrow was performed

first: Denis

last: Schewe

first: Ameera

last: Alsadeq


title: A molecular pathway for cancer cachexia-induced muscle atrophy revealed at single nucleus resolution [scRNA]

status: Public on Jul 12 2024

city: Dallas

first: kenian

last: chen

state: TX

address: 5323 Harry Hines Boulevard

department: Population and Data Science

institute: UTsouthwestern

summary: Cancer cachexia is a prevalent and often fatal wasting condition that cannot be fully reversed with nutritional interventions. Muscle atrophy is a central component of the syndrome, but the mechanisms whereby cancer leads to skeletal muscle atrophy are not well understood. We performed single nucleus multi-omics on skeletal muscles from a mouse model of cancer cachexia and profiled the molecular changes in cachexic muscle. Our results revealed the activation of a denervation-induced gene program that upregulates the transcription factor myogenin. Further studies showed that a myogenin-myostatin pathway promotes muscle atrophy in response to cancer cachexia. shRNA inhibition of myogenin or inhibition of myostatin through overexpression of its endogenous inhibitor follistatin prevented cancer cachexia-induced muscle atrophy in mice. Our findings uncover a molecular basis of cancer cachexia-induced muscle atrophy and highlight potential therapeutic targets for this disorder.

overall_design: Comparative gene expression profiling analysis of single nucleus RNA-seq data for three biological replicates of Gastrocnemius-plantaris (GP) muscles from control and a mouse model with cancer cachexia (KIC) at 66 days months of age.

first: Yichi

last: Zhang

first: Matthieu

last: Dos Santos

first: Kenian

last: Chen

first: Lin

last: Xu


title: A molecular pathway for cancer cachexia-induced muscle atrophy revealed at single nucleus resolution [snATAC]

overall_design: Comparative gene expression profiling analysis of single nucleus RNA-seq data for three biological replicates of Gastrocnemius-plantaris (GP) muscles from control and a mouse model with cancer cachexia (KIC) at 66 days of age.


title: A molecular pathway for cancer cachexia-induced muscle atrophy revealed at single nucleus resolution [Bulk_RNAseq]


title: CTCF-dependent insulation of Hoxb13 and the heterochronic control of tail length [scRNA-seq]

first: Célia

last: Bochaton

first: Alexandre

last: Mayran

first: Jozsef

last: Zakany

first: Hocine

last: Rekaik

last: Duboule


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

city: Houston

first: Srikanth

last: Kodali

address: 1 Baylor Plaza

institute: Baylor College of Medicine

summary: We performed RNA-seq to profile transcripts sequestered in P-bodies of AML cells

overall_design: RNA-seq

first: Yingzhi

last: Cui

first: Bruno

last: Di Stefano


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

summary: We performed RNA-seq to profile transcriptional changes in AML cells following P-body dissolution induced by DDX6 knockdown.


title: Effect of  CLK2 expression in the regulation of intron retention during heat shock [RNA-seq]

status: Public on Jul 15 2024

city: Wuhan

first: Wei

last: Wang

state: Hubei

address: 13 Hangkong Road

country: China

institute: Huazhong University of Science and Technology Tongji Medical College

summary: Intron retention (IR) constitutes a less explored form of alternative splicing, wherein introns are retained within mature mRNA transcripts. Our investigation demonstrates that the CDC-like kinase 2 (CLK2) undergoes liquid-liquid phase separation (LLPS) within nuclear speckles in response to heat shock (HS). The formation of CLK2 condensates depends on the intrinsically disordered region (IDR) located within the N-terminal amino acids 1-148. Phosphorylation at residue T343 sustains CLK2 kinase activity and facilitates autophosphorylation, thus inhibiting the LLPS activity of the IDR. These CLK2 condensates initiate the reorganization of nuclear speckles, transforming them into larger, rounded structures. Moreover, these condensates facilitate the recruitment of splicing factors into these compartments, potentially restricting their access to mRNA for intron splicing. Consequently, the formation of CLK2 condensates promotes the IR.

overall_design: We established the HeLa cells expressing the GFP (Control) , CLK2 (WT) or CLK2 knockdown (KD) and subjected them to heat shock (HS). These cells were subjected to RNA-seq for the alternative splicing.  analysis. The nuclear and cytoplasmic RNAs were exrracted from CLK2 (T343A) expressing HeLa cells (T343A-C, T343-N) and subjected to RNA-seq.


title: RNA-seq of worker antennae of honeybees (Apis mellifera) with a dsx stop/stop mutation

status: Public on Jul 10 2024

city: Düsseldorf

first: Anna

last: Wagner

address: Universitätsstraße 1

department: Evolutionary Genetics

institute: Heinrich-Heine University

summary: Transcriptional profiling of the antennae of adult honeybee workers with a dsx stop/stop mutation and wild-type workers was performed by RNA-Seq. Gene expression of the dsx stop/stop and wild type female workers was compared.

overall_design: Study of antenna mRNA profiles of adult honey bee workers with a dsx stop/stop mutation and wild-type workers.


title: Determinants of selectivity in the dicing mechanism III

status: Public on Jul 13 2024

city: Hong Kong SAR

first: Truc

middle: Cong

last: LE

address: Clearwater Bay, Kowloon

department: Life Science

country: Hong Kong

institute: The Hong Kong University of Science and Technology

type: Non-coding RNA profiling by high throughput sequencing

summary: The RNase III enzyme, DICER, is instrumental in the production of small RNAs, including miRNAs and siRNAs, by cleaving their precursors, such as pre-miRNAs, shRNAs, and long duplex RNAs. Utilizing High-throughput (HT) cleavage assays, our study delves into the cleavage activity of DICER. We challenge the widely accepted 2-nt loop counting rule in the previous study, revealing a divergent mechanism, the bipartite base pairing rule. This rule directs DICER's cleavage sites via the RNase III domain. Moreover, we demystify the recognition mechanism of the previously identified YCR motif. Building on this understanding, a secondary YCR motif that also influences DICER's cleavage sites has been discovered. We also address a long-debated issue concerning DICER's cleavage sites on long stem RNAs, such as pre-siRNAs or long shRNAs/pre-miRNAs. Our study shows that the dsRBD plays a crucial role in determining the cleavage sites of DICER in long-stem RNAs. In sum, our research provides a comprehensive understanding of several fundamental DICER mechanisms, challenging the long-standing model of the loop counting rule. This newfound knowledge reshapes our understanding of DICER's mechanisms, providing a robust foundation for future studies investigating the vast number of DICER mutations linked to various diseases.

overall_design: small RNA sequencing

first: Thi

last: Le

first: Cong-Truc

first: Tuan-Anh

last: Nguyen


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

summary: To investigate small non-coding RNA changes in AML cells following loss of P-bodies, we knocked down DDX6 in MOLM-13 cells.

overall_design: Transcriptomic profiling of for small non-coding RNAs

first: Junchao

last: Shi

first: Qi


title: The evolving genetic landscape of dyskeratosis congenita (DC) and DC-like (DCL) disorders

city: London

first: Hemanth

last: Tummala

state: London

address: Blizard Institute

department: Genomics and Child Health

country: United Kingdom

institute: QMUL

summary: Dyskeratosis congenita (DC) is a complex inherited bone marrow failure syndrome which is principally a disorder of telomere maintenance. To date approximately 35% of cases remain uncharacterised at the genetic level. Whole exome sequencing on a large collection of uncharacterized DC and DC-like (DCL) families (n=167) has revealed several novel pathogenic variants within known susceptibility loci, POT1 and ZCCHC8, as well as the novel locus POLA1.  Functional characterisation of identified POLA1 and POT1 pathogenic variants, uncovered their effect on protein-protein interactions that have critical implications for telomere maintenance. ZCCHC8 variants disrupt protein interactions that affect nuclear exosome targeting (NEXT) complex stoichiometry and its binding with the human silencing hub (HUSH) complex chromatin modifier MPP8. Global transcriptomic analysis revealed signatures of pervasive transcription that include several short (snRNA and snoRD) and long non-coding RNA (transposable elements; LINE-1) driving inflammation in ZCCHC8 patient blood cells. In summary, our studies inform the current genetic architecture of DC and DCL disorders, by revealing novel gene loci such as POLA1 and extend our current knowledge on disease mechanisms beyond the regulation of long non-coding RNA TERC.

overall_design: RNAseq  of whole blood extracted RNA from control and ZCCHC8 patients For each group, samples were processed from three different individuals.

first: Inderjeet

last: Dokal


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

summary: We performed ATAC-seq to profilechromatin changes in AML cells following P-body dissolution induced by DDX6 knockdown.

overall_design: ATAC-seq


title: RNA sequestration in P-bodies sustains myeloid leukemia

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: CTCF-dependent insulation of Hoxb13 and the heterochronic control of tail length [RNA-seq gastruloid]

first: Lucille

last: Lopez-Delisle

first: Célia

first: Denis


title: Targeted disruption of BCL11A-XL specific zinc finger motif for de-repression of fetal globin expression

status: Public on Aug 01 2024

city: Vellore

first: Mohankumar

last: Murugesan

state: Tamilnadu

address: Christian Medical College

department: Center for Stemcell Research

country: India

institute: CHRISTIAN MEDICAL COLLEGE AND HOSPITAL

summary: BCL11A is a major gamma globin repressor widely used target for the clinical amelioration of beta-hemoglobinopathies. The longer BCL11A-XL isoform has three distinct ZnF domains at the C-terminal which has been implicated in DNA interactions with the HBG region. These unique ZnF domains harbor missense mutations in neurodevelopmental disorder patients and have been shown to elevate HbF levels with normal hematological parameters. However, the therapeutic potential of targeting the BCL11A-XL-specific ZnF domains without affecting other isoforms remains unexplored. Hence, wedisrupted the BCL11A-XL specific ZnF domains usingCRISPR/Cas9 and observed high HbF induction albeit with defects in Hematopoietic Stem and Progenitor Cells(HSPC) engraftment and erythroid maturation. Subsequently, we observed that base substitutions at the key residues of ZnF-domainsinvolved in DNA recognition effectively upregulated HbF with minor defects. A ZnF4 mutation resulted in minimal changes to erythroid gene expression, no impact on erythroid maturation in vitro, but a reduction in HSPC engraftment in vivo. A modification of ZnF6showeda potential elevation of HbF levels, which was unexpected as this region does not directly interact with DNA at the HBG promoter.Our strategy of specifically altering ZnF domains of BCL11A-XL will potentially identify residues critical for fetal globin repression but less important in the other biological functions of BCL11A-XL for the treatment of beta-hemoglobinopathies.

overall_design: Erythroid cells (Phase 1 end) obtained from ZnF4-1 base-edited HSPC is the study sample. The AAVS1 edited (negative control), the Binding site (-115 cluster) disruption in HBG promoter and the BCL11A total knockout are the other controls used in the experiment to compare the Whole transcriptome analysis. Each sample is performed in duplicates.

first: Vignesh

last: Rajendiran

first: Nivedhitha

last: Devaraju


title: MDA-MB-468 cells treated with docetaxel or EZH2i+AKTi

status: Public on Jul 08 2024

city: Boston

first: Amy

middle: E

last: Schade

state: MA

address: 77 Avenue Louis Pasteur

department: Medicine

institute: Brigham and Women's Hospital


title: MDA-MB-468 cells treated with docetaxel or EZH2i+AKTi II

summary: Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype and has the highest rate of recurrence1. The predominant standard of care for advanced TNBC is systemic chemotherapy with or without immunotherapy, however responses are typically short-lived1,2. Thus, there is an urgent need to develop more effective treatments. PI3K pathway components represent plausible therapeutic targets, as approximately 70% of TNBCs have PIK3CA/AKT1/PTEN alterations3–6. However, unlike hormone receptor-positive tumors, it is still unclear if or how PI3K pathway inhibitors will be effective in triple-negative disease7. Here we identify a promising AKT inhibitor-based therapeutic combination for TNBC. Specifically, we show that AKT inhibitors potently synergize with agents that suppress the histone methyltransferase, EZH2, and promote robust tumor regression in multiple TNBC models in vivo. AKT and EZH2 inhibitors exert these effects by first cooperatively driving basal-like TNBC cells into a more differentiated, luminal-like state, which cannot be effectively induced by either agent alone. Once differentiated, these agents kill TNBCs by hijacking signals that normally drive mammary gland involution. Finally, using machine learning approach, we developed a classifier that can be used for patient selection. Together these findings identify a promising therapeutic strategy for this highly aggressive tumor type and illustrate how deregulated epigenetic enzymes can insulate tumors from oncogenic vulnerabilities. These studies also reveal how developmental tissue-specific cell death pathways may be co-opted for therapeutic benefit.

overall_design: HCC38, HCC1395, and HCC1937 cells were treated EZH2i (5 uM tazemetostat), AKTi (5 uM ipatasertib) or EZH2i+AKTi.  On day -5, MDA-MB-468 cells were seeded at 40% confluency and treated with 5uM tazemetostat or DMSO. On day -3, cells were passaged again at 40% confluency and maintained in DMSO or tazemetostat. On day -1, cells were seeded into 6cm plates at 250,000 cells per plate and maintained in DMSO or tazemetostat. On day 0, cells were treated in media containing 2% FBS with AKTi (ipatasertib) and/or EZH2i (tazemetostat). Cells were collected at 24 hours, cells were isolated and then prepared for ATACseq.

first: Karen

last: Cichowski


title: MDA-MB-468 cells treated with docetaxel or EZH2i+AKTi I

overall_design: MDA-MB-468 cells were treated with either docetaxel or EZH2i+AKTi. For cells treated with docetaxel, cells were seeded into 10cm plates after 24 hr they were dosed with 10 nM docetaxel. RNA was collected at 8 and 24 hours of docetaxel treatment. For cells treated with EZH2i+AKTi, On day -5, MDA-MB-468 cells were seeded at 40% confluency and treated with 5uM tazemetostat or DMSO. On day -3, cells were passaged again at 40% confluency and maintained in DMSO or tazemetostat. On day -1, cells were seeded into 6cm plates at 250,000 cells per plate and maintained in DMSO or tazemetostat. On day 0, cells were treated in media containing 2% FBS with AKTi (ipatasertib) and/or EZH2i (tazemetostat). Cells were collected at 24 hours, RNA was isolated and then sequenced using NGS.


title: Soybean cell atlas gene expression profile

city: Tucson

first: Sergio Alan

last: Cervantes-Perez

state: Arizona

address: 1140 E South Campus Dr

department: School of Plant Sciences

institute: University of Arizona

summary: The nuclei of Glycine max from different tissues were collected. The samples were: soybean seed mid-maturation stage (10mm), seed late cotyledon stage (5mm), seed early cotyledon stage (3mm), seed heart stage (1mm), soybean green pods without seeds (stage), soybean flower bud (early flowering stage), soybean shoot apical meristem (stage), soybean trifoliate leaf (R5 stage), and soybean true leave (stage). The library construction was performed applying 10 Genomics technology.

overall_design: Glycine max seedlings were grown on B&D-N media and growth chambers in the dark and then collected the nuclei.TBD. For the nuclei isolation was used mechanical and chemical steps and the libraries were made following 10X genomics recommendations.  The pre-processing and processing of the datasets were performed with Cell Ranger and Seurat.

first: Sergio

middle: A

first: Sandra

last: Thibivilliers

first: Sutton

last: Tennant

first: Marc

last: Libault


title: Neoadjuvant PARPi or Chemotherapy in Ovarian Cancer Informs Targeting Effector Treg Cells for Homologous-Recombination-Deficient Tumors [Singleron scTCR-seq]

first: Qinglei

last: Gao

address: Jiefang Avenue

department: Department of Gynecological Oncology

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

summary: Homologous recombination deficiency (HRD) is prevalent in cancer, sensitizing tumor cells to PARP inhibition. However, the impact of HRD and related therapies on the tumor microenvironment (TME) remains elusive. Our study generates single-cell gene expression and T-cell receptor profiles, along with validatory multimodal datasets from > 100 high-grade serous ovarian cancer (HGSOC) samples, primarily from a phase II clinical trial (NCT04507841). Neoadjuvant monotherapy with the PARP inhibitor niraparib achieves impressive 62.5% and 73.6% response rates per RECIST v1.1 and GCIG CA125, respectively. We identify effector regulatory T cells (eTreg) as key responders to HRD and neoadjuvant therapies, co-occurring with other tumor-reactive T cells, particularly terminally exhausted CD8+ T cells (Tex). TME-wide interferon signaling correlates with cancer cells upregulating MHC-II and co-inhibitory ligands, potentially driving Treg and Tex fates. Depleting eTregs in HRD mouse models, with or without PARP inhibition, significantly suppresses tumor growth without observable toxicities, underscoring the potential of eTreg-focused therapeutics for HGSOC and other HRD-related tumors.

overall_design:
scTCR-seq of sorted T cells from treatment-naïve tumor samples of ovarian cancer patients using the Singleron Biotechnologies platform

Submitter states: Raw sequencing data have been deposited in the Genome Sequence Archive for Human at the National Genomics Data Center (https://ngdc.cncb.ac.cn/gsa-human) with accession number HRA007181, which will be provided for scientific research upon request complying with the law due to human patient privacy concerns.

first: Yikai

last: Luo

first: Yu

last: Xia

first: Dan

last: Liu

first: Xiong

last: Li

first: Huayi

first: Jiahao

first: Dongchen

last: Zhou

last: Dong

first: Ding

last: Ma

first: Yong

last: Fang

first: Han

last: Liang


title: Neoadjuvant PARPi or Chemotherapy in Ovarian Cancer Informs Targeting Effector Treg Cells for Homologous-Recombination-Deficient Tumors [bulk TCR-seq]

overall_design:
Batch 1: Bulk TCR-seq of sorted CD4+ T cells from PBMC samples of ovarian cancer patients with paired scTCR-seq data of tumor samples.

Batch 2: Bulk TCR-seq of sorted CD3+ T cells from patient-matched PBMC and tumor samples of ovarian cancer patients.

Submitter states: Raw sequencing data have been deposited in the Genome Sequence Archive for Human at the National Genomics Data Center (https://ngdc.cncb.ac.cn/gsa-human) with accession number HRA007230, which will be provided for scientific research upon request complying with the law due to human patient privacy concerns.


title: Transcriptomic profiling of medulla and cervical spinal cord to study respiratory neuropathology in Spinocerebellar ataxia type 7

status: Public on Jul 04 2024

city: Durham

first: Debolina

middle: D

last: Biswas

state: North Carolina

address: 203, Research Drive, Room 141

institute: Duke Univeristy

summary: Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurological disorder caused by a deleterious CAG repeat expansion in the coding region of the ataxin-7 gene. The number of CAG repeats determines the severity and age of onset of the disease. Infantile onset SCA7 leads to severe clinical manifestations including dysphagia, aspiration pneumonia and respiratory distress, but the exact cause of respiratory impairment remains unclear. Using the infantile SCA7 mouse model – the SCA7266Q/5Q mouse, we examined the impact of mutant ataxin-7 on the respiratory control centers. Specifically, we examined the effect of pathological poly-Q-ataxin-7 expression on hypoglossal (XII) and phrenic motor units. Further, we identified the transcript profile of the medulla and cervical spinal cord and, investigated the XII and phrenic nerve structure as well as the neuromuscular junctions in the diaphragm and tongue. SCA-7 astrocytes showed significant intranuclear and cytoplasmic inclusions of ataxin-7 in the XII and putative phrenic motor nuclei. Transcriptomic analysis revealed dysregulation of genes involved in amino acid and neurotransmitter transportation, and myelination. Additionally, SCA7 mice demonstrated blunted efferent output of the XII nerve and demyelination in both XII and phrenic nerves. Finally, there was an increased number of NMJ clusters with higher expression of synaptic markers in SCA7 mice compared to WT controls. Thus, pathological ataxin-7 expression disrupts myelination and neurotransmitter transportation, and impairs glial cell function in the respiratory control centers. These pre-clinical findings elucidate the underlying pathophysiology responsible for dysphagia, aspiration and respiratory failure in infantile SCA7.

overall_design: The medulla and cervical spinal cord tissues are harvested and flash frozen from SCA7 and wild-type C57Bl/6J mice at different age groups- p8, 5 weeks, and 9-weeks of age. RNA was extracted using Qiagen miRNeasy kit.

first: Mai

last: ElMallah

first: Yihan

last: Shi


title: Postnatal Allergic Inhalation Induces Glial Inflammation in the Olfactory Bulb and Leads to Autism-Like Traits in Mice

status: Public on Jul 06 2024

city: Fukuoka

first: Eizo

last: Tanaka

state: Fukuoka

address: 3-1-1, Maidashi, Higashi-ku

department: Neurology

country: Japan

institute: Kyushu University Graduate School of Medical Sciences

summary: Autism spectrum disorder (ASD) is one of the most prevalent neurodevelopmental disorders. To clarify its detailed pathophysiology, we studied the association between neonatal allergic exposure and behavioral changes. Adult female C57BL/6J mice were immunized with adjuvant (alum) or ovalbumin emulsified by adjuvant. After immunization, mice were mated, and offspring were born in full-term. Postnatal mother and infant were simultaneously inhaled with allergen. After weaning, behavioral testing and histopathological analysis were conducted for male offspring. As a result, ovalbumin-inhaled allergic offspring showed decreased sociability and increased repetitive behavior, so-called ASD-like phenotype in mice. Histopathological analysis revealed that allergic mice showed increase of astroglia, microglia, and eosinophilic infiltration in the olfactory bulb, as well as increase of eosinophils in the nasal mucosa. Allergic mice also showed decrease of dendritic spine density and proportion of mature spines, suggesting the impairment of stimuli-induced synaptogenesis. In conclusion, postnatal allergic exposure induces autistic phenotype as well as allergic rhinitis, followed by glial inflammation in the olfactory bulb parenchyma.

overall_design: Whole olfactory bulb were isolated from PBS- and OVA- inhaled mice at P28. Extracted RNA were subjected to RNA array assay. Gene expression of PBS (control group) and OVA (allergy group) treated mice were compared respectively.

first: Ryo

last: Yamasaki

first: Ban-yu

last: Saitoh

first: Amina

last: Abdelhadi

first: Satoshi

last: Nagata

first: Sato

last: Yoshidomi

first: Yuka

last: Inoue

first: Koichiro

last: Matsumoto

first: Jun-ichi

last: Kira

first: Noriko

last: Isobe


title: Neoadjuvant PARPi or Chemotherapy in Ovarian Cancer Informs Targeting Effector Treg Cells for Homologous-Recombination-Deficient Tumors [10x scRNA-seq]

first: Qinglei

last: Gao

overall_design:
scRNA-seq of unsorted cells from tumor samples of ovarian cancer patients using 10x genomics 3' v3 chemistry

Submitter states: Raw sequencing data have been deposited in the Genome Sequence Archive for Human at the National Genomics Data Center (https://ngdc.cncb.ac.cn/gsa-human) with accession number HRA007180, which will be provided for scientific research upon request complying with the law due to human patient privacy concerns.

first: Yikai

last: Luo

first: Yu

last: Xia

first: Dan

last: Liu

first: Xiong

last: Li

first: Huayi

first: Jiahao

first: Dongchen

last: Zhou

last: Dong


title: Oral squamous cell carcinomas drive monocytes into immunosuppressive CD25+CD163+CD206+ macrophages

status: Public on Jul 03 2024

city: Madrid

first: Pedro

middle: Antonio

last: Reche

state: Comunidad de Madrid

address: Pza Ramon y Cajal, s/n

department: Immunology & O2, Faculty of Medicine

country: Spain

institute: Complutense University of Madrid

summary: Tumor-associated macrophages (TAMs) are major cellular components in the tumor microenvironment of oral squamous cell carcinomas (OSCCs). Most of these TAMs derive from circulating monocytes that differentiate in situ. In this work, we show that cell culture media (CM) derived from two OSCC cell lines, H413 and TR146, promote monocyte differentiation into M2 macrophages, characterized by a high expression of CD163, CD206 and a low expression of CD11c, CD86 and HLA-DR. These monocyte-derived macrophages (moMΦ) differentiated by CM from H413 cells were unable to activate allogeneic T cells, and inhibited T cell activation and proliferation induced by CD3/CD28 stimulation. By culturing monocytes with fractionated H413-CM, we found that soluble proteins mediated CD163+CD206+ moMΦ differentiation, discarding a role for small metabolites and extracellular vesicles. Differential proteomic analyses on H413-CM fractions revealed the presence of several proteins, including the complement factor H or plasminogen activator inhibitor, as potential candidates to induce CD163+CD206+ moMΦ differentiation. Finally, RNAseq transcriptomic analyses of H413 CM conditioned moMΦ, identified a signature expression profile involving cytokines and cytokine receptors, which surprisingly included IL2RA (CD25). CD25 enhanced expression was confirmed on H143-CM moMΦ. Collectively, these data indicate that the CM from OSCC cell lines promotes the differentiation of monocytes into functionally immunosuppressive macrophages and overall, our work contributes to the understanding of how OSCCs create an immunosuppressive cellular environment that favors tumor growth.

overall_design: The effect of conditioned media (CM) from H413 cell cultures (H413-CM) in macrophage differentiation was investigated. Monocytes from 3 different healthy donors were cultured for 5 days in complete RPMI (non-treated, NT) or H413-CM. Next, gene expression profiling analyses of these cells were carried out using data obtained from RNA-seq experiments.

first: Hector

middle: F

last: Pelaez-Prestel

first: Fernando

last: Gonzalez-Martin

first: Alvaro

last: Ras-Carmona

first: Almudena

last: Rocha

first: Carlos

last: Cabañas

first: Esther

middle: M

last: Lafuente


title: Tissue niche influences immune and metabolic profiles to Staphylococcus aureus biofilm

city: White/Caucasian

first: Tammy

last: Kielian

state: NE

address: 985900 Nebraska Medical Center

institute: UNMC

summary: Infection is a devastating post-surgical complication, often necessitating additional procedures and prolonged antibiotic therapy. This is especially relevant for craniotomy and prosthetic joint infections, both of which are characterized by biofilm formation on the bone or implant surface, respectively, with S. aureus representing a primary cause. The effectiveness of immune responses to these infections is predicated on both host- and pathogen-derived signals in the infection microenvironment. However, the extent to which these signals differ across distinct tissue niches and influence immune function remains relatively unknown.  Using mouse models of S. aureus craniotomy and prosthetic joint infection complemented with patient samples from both infectious modalities, we show profound metabolomic, transcriptomic, and functional differences that are dependent on tissue niche. These signatures were both spatially and temporally distinct, differing not only between surgical site infections but evolving over time within a single model. These findings highlight the unique immune attributes of biofilms that are heavily influenced by the local tissue microenvironment, which will likely have important implications when designing therapeutic approaches to target specific infections.

overall_design: To investigate differential leukocyte responses to tissue niche, bulk RNA-seq was performed on FACS-isolated granulocytes from two surgical site infection mouse models, namely, the galea of craniotomy infection and the tissue of prosthetic joint infection. Craniotomy samples were also collected from a TNF KO genetic mouse background to assess the role of this cytokine in granulocyte responses during infection.

first: Zachary

last: Van Roy

first: Prabakar

last: Arumugam

first: Blake

middle: P

last: Bertrand

first: Dhananjay

last: Shinde

first: Vinai

middle: C

last: Thomas


title: Alternative activation of mast cells by CD4+ T helper cells

city: Toulouse

first: Eric

last: Espinosa

state: France

address: IRSD  INSERM U1220 105 Avenue de Casselardit

department: IMMUNOLOGY

country: France

institute: INSERM

summary: Effector CD4+ T lymphocytes (Teff) infiltrate sites of inflammation and orchestrate the immune response by instructing local leukocytes. Mast cells (MCs) are tissue sentinel cells strategically located near blood vessels and T cell rich areas. MC/Teff cells interactions shape Teff cell responses but in turn, Teff cell action on MC is still poorly understood.  Here, we analyzed the human MC/Teff cells interplay through the application of RNAseq and functional assays and showed that activated Teff cells induced a specific transcriptomic program in MCs driving them toward an inflammatory phenotype. Teff cells affected key MC immune functions as they induced prostaglandin, inflammatory cytokines and chemokines production and fostered FceRI-dependent degranulation. Moreover, Teff cell induced in MCs the capacity to regulate T cell responses through a wide-range of dedicated soluble and membrane ligands. Cell-Cell communication inference based on transcriptomic data indicated that IFN-g, IL-21, IL-27 and IL-1b were the main driver of MC differentiation program.

overall_design: To analyze the effects of activated CD4+ Teff cells on MCs, freshly isolated human effector/memory CD4+ T cells were cocultured with primary human MCs for 24 hours with or without anti-CD3/CD28 coated beads. In parallel experiments, MCs were stimulated with two typical well-known MC stimuli (IL-33 and IgE/Ag) as points of comparison. MCs were then FACS-sorted and processed to mRNA deep sequencing (RNAseq). We then performed gene expression profiling analysis of RNA-seq data for MCs (from 4 different donors : MC186, MC187, MC188, MC189) stimulated with 5 different conditions (unstimulated, cocultured with unstimulated Teff cells, cocultured with anti-CD3/28 bead-stimulated Teff cells, stimulated with IgE/Ag, stimulated with IL-33)

first: Edouard

last: Leveque

first: Louise

last: Battut

first: Camille

last: Petitfils

first: Salvatore

last: Valitutti

first: Gilles

last: Dietrich

first: Nicolas

last: Cenac


title: m6A-eCLIP analysis of mouse BMDM

status: Public on Jul 01 2024

city: South San Francisco

first: Sandra

last: Melo

address: 1 DNA Way

institute: Genentech, Inc.

summary: N6-methyladenosine (m6A) is the most abundant mRNA modification in mammalian cells, mediated co-transcriptionally by a methyltransferase ‘writer’ complex containing METTL3 as the S-adenosyl methionine (SAM)-binding subunit as well as adaptors such WTAP and ZC3H13.

overall_design:
To profile m6A mRNA modification sites transcriptome-wide with single nucleotide resolution, we performed enhanced crosslinking and immunoprecipitation (m6A-eCLIP) in mouse BMDMs. All samples were treated with LPS for 3h. This dataset is part of our larger project which also contains human samples.
contributor: Eclipse BioInnovations Inc, San Diego CA

first: Simone

last: Haag


title: Splice-switching antisense oligonucleotide controlling tumor suppressor REST is a novel therapeutic medicine for neuroendocrine cancer

status: Public on Jul 02 2024

city: Suita

first: Masahito

last: Shimojo

address: 1-6 Yamadaoka

institute: Osaka University

type:
Expression profiling by array
Non-coding RNA profiling by array

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

overall_design: Refer to individual Series


title: m6A-eCLIP analysis of human macrophages

summary: N6-methyladenosine (m6A) is the most abundant mRNA modification in mammalian cells, mediated co-transcriptionally by a methyltransferase ‘writer’ complex containing METTL3 as the S-adenosyl methionine (SAM)-binding subunit as well as adaptors such WTAP and ZC3H13

overall_design:
To profile m6A mRNA modification sites transcriptome-wide with single nucleotide resolution, we performed enhanced crosslinking and immunoprecipitation (m6A-eCLIP) in WTAP-, ZC3H13-, METTL3-deficient and NTC control cells derived from two healthy donors. Additionally we profiles BMDMs from 2 independent mice. All samples were treated with LPS for 3h.
**Raw data will be submitted to EGA**

first: Haag

last: Simone

last: Melo Carlos


title: Mitochondria-localized MBD2c facilitates mtDNA transcription and drug resistance [Bisulfite-Seq]

status: Public on Jun 30 2024

city: Hefei

first: Yijie

last: Hao

address: Huangshan Road 443

institute: USTC

type: Methylation profiling by high throughput sequencing

summary: Mitochondria contain a 16kb-dsDNA genome encoding 13 proteins essential for respiration, whereas its regulatory mechanism and potential role in cancer development remain elusive. Although Methyl-CpG-binding protein (MBD) proteins are essential for nuclear transcription, their role in mitochondrial DNA (mtDNA) transcription is unknown. Here, we report that the MBD2c splicing variant translocates into mitochondria to mediate mtDNA transcription and increase mitochondrial respiration in triple negative breast cancer (TNBC) cells. Specifically, MBD2c binds D-loop regions in mtDNA to recruit SIRT3, which in turn deacetylates TFAM, a primary mitochondrial transcription factor, and activates its function. TFAM activation subsequently enhances transcription of the whole mitochondrial genome. Furthermore, MBD2c overexpression recovered the decreased mtDNA-encoded RNA and protein levels induced by the DNA synthesis inhibitor, cisplatin (CDDP), in vitro and in vivo, preserving mitochondrial gene expression and respiration, consequently enhancing TNBC cells drug resistance and proliferation. These data collectively demonstrate that MBD2c positively regulates mtDNA transcription, thus connecting epigenetic regulation by deacetylation with cancer cell metabolism, suggesting druggable targets to overcome resistance.

overall_design: Bisulfite-Seq (BS-seq) in purfied mtDNA from MDA-MB-468 cells, to facilitate site-specific identification of 5mC modifications in mitochondrial DNA.

first: Shengqi

last: Shen

first: Linchong

last: Sun

first: Ping

last: Gao

first: Huafeng

last: Zhang


title: Whole exome sequencing of NT2.5 and NT2.5-LM cell lines

city: Los Angeles

first: Aaron

last: Baugh

address: 1441 Eastlake Ave

institute: University of Southern California

summary: NT2.5-LM is a cell line derived from the parental NT2.5 cell line that provides a more proliferative and metastatic murine model of breast cancer. We used whole exome sequencing to examine mutational differences.

overall_design: One microgram or more of mouse genomic DNA from each sample was analyzed by whole exome sequencing using the SureSelectXT Mouse All Exon kit (Agilent), followed by next generation sequencing using the NovaSeq 6000 S4 flow cell (Illumina) with a 2x150bp paired-end read configuration, per the manufacturer’s instructions. bcl2fastq v2.15.0 (Illumina) was used to convert BCL files to FASTQ files using default parameters. Running alignments against the mm10 genome was done by bwa v0.7.7 (mem) along with Piccard-tools1.119 to add read groups and remove duplicate reads. GATK v3.6.0 base call recalibration steps were used to create a final alignment file. MuTect2 v3.6.0 was used to call somatic variants against a panel of normal using default parameters. snpEFF (v4.1) was used to annotate the variant calls and to create a clean tab separated table of variants. IGV v2.13.2 was used to identify breast cancer specific mutations from MuTect2 files. SnapGene Viewer v.6.2 was used to visually align and determine the mutations between the two cell lines against the mRNA sequences of selected genes. Annotations were created to visualize mutational differences.


title: Unveiling the molecular effects of replacement and legacy PFASs: Transcriptomic analysis of zebrafish embryos reveals surprising similarities and potencies

city: Saskatoon

first: Hannah

last: Mahoney

state: Saskatchewan

address: 120 Veterinary Rd

department: Vaccine and Infectious Disease Organization

country: Canada

institute: University of Saskatchewan

summary: The prevalence of per- and poly fluoroalkyl substances (PFASs) in the environment has prompted restrictions on legacy PFASs due to their recognized toxic effects. Consequently, alternative “replacement” PFASs have been introduced and are prevalent in environmental matrices. Few studies have investigated the molecular effects of both legacy and replacement PFASs under short-term exposures. This study aimed to address this by utilizing transcriptomic sequencing to compare the molecular impacts of exposure to concentrations 0.001–5 mg/L of the legacy PFOS and two of its replacements, PFECHS and FBSA. Using zebrafish embryos, the research assessed apical effects (mortality, morphology, and growth), identified differentially expressed genes (DEGs) and enriched pathways, and determined transcriptomic points of departure (tPoDs) for each compound. Results indicated that PFOS exhibited the highest relative potency, followed by PFECHS and then FBSA. While similarities were observed among the ranked DEGs across all compounds, over-representation analysis revealed slight differences. Notably, PFOS demonstrated the lowest tPoD identified to date. These findings raise concerns regarding the safety of emerging replacement PFASs and challenge assumptions about PFAS toxicity solely resulting from their accumulative potential. As replacement PFASs proliferate in the environment, this study underscores the need for heightened scrutiny of their effects and questions current regulatory thresholds.

overall_design: To investigate the effect of sub-lethal and environmentally relevant PFAS concentrations, zebrafish embryos were exposed for 72 hours to 4 concentrations of PFOS, PFECHS, and FBSA. Three replicates of each treatment including a solvent control, were sent for transcriptomic sequencing.

first: Phillip

last: Ankley

first: Catherine

last: Roberts

first: Alicia

last: Lamb

first: Matthew

last: Schultz

first: Yutong

last: Zhou

first: John

last: Giesy

first: Markus

last: Brinkmann


title: scRNAseq of NT2.5 and NT2.5-LM breast tumors

summary: Syngeneic NT2.5 and NT2.5-LM breast tumors established in female NeuN mice were collected and sent for scRNAseq to examine various cell populations

overall_design: For library preparation, 10x Genomics Chromium Single Cell 3′ RNA-seq kits v3 were used. Gene expression libraries were prepared per the manufacturer’s instructions. 4 biological replicates totaling 8 processed tumors were sequenced in 2 batches: Run A - 2 NT2.5 tumors, 2 NT2.5-LM tumors; Run B - 2 NT2.5 tumors, 2 NT2.5-LM tumors. These tumors were taken as a subset from a larger batch of tumors that include various mouse treatments, with each batch having an equal assortment of samples from multiple treatment groups to reduce technical biases. Here, we restrict our analysis to replicates under the vehicle treatment condition. Illumina HiSeqX Ten or NovaSeq were used to generate total reads. Paired-end reads were processed using CellRanger v3.0.2 and mapped to the mm10 transcriptome with default settings. ScanPy v1.8.1 and Python v3 was used for quality control and basic filtering.


title: Bulk RNA-sequencing of mouse prostate tumors initiated by transplantation of engineered organoids.

status: Public on Aug 02 2024

city: New York

first: Charles

last: Sawyers

state: NY

address: 417 E 68th Street, ZRC-661

department: Human Oncology and Pathogenesis

institute: Memorial Sloan Kettering Cancer Center

summary: Purpose: Profile global expression of prostatic tumors to compare with published datasets. Methods: Prostate tumors were flash frozen followed by processed for total RNA using the RNeasy kit (Qiagen). Samples were submitted for polyA library preparation and bulk RNA-sequencing. Conclusions: Global gene expression profiling and comparison of prostate tumor genotypes suggests differential enrichment of neuroendocrine signatures that match established human neuroendocrine prostate cancer expression signatures.

overall_design: To investigate the molecular landscape of transplanted PtPM and RPM orthotopic prostate tumors, n = 10 PtPM and n = 8 RPM tumors isolated at early or late time points were subjected bulk RNA-sequencing.

first: Rodrigo

last: Romero

first: Tania

middle: J

last: Gonzalez-Robles

last: Sawers

first: Kelly

last: Ruggles

first: Dana

last: Pe'er


title: Interferon Regulatory Factor 4 Modulates Epigenetic Silencing and Cancer-Critical Pathways in Melanoma Cells

city: Istanbul

first: Tolga

last: Emre

address: Bogazici University, Kuzey Kampus, Kuzey Park Binasi, No 203

country: Turkey

institute: Bogazici University

summary: Interferon Regulatory Factor 4 (IRF4) is a transcription factor initially identified as a key controller in lymphocyte differentiation and function, and subsequently as a dependency factor and therapy target in lymphocyte-derived cancers. In melanocytes, IRF4 takes part in the regulation of pigmentation. While numerous genetic studies have implicated IRF4 also in melanoma skin cancers, how IRF4 contributes to melanoma has remained largely elusive. In this study, we first confirmed that not only IRF4 expression is common in melanoma, but also IRF4 can act as a dependency factor for melanoma cells. Furthermore, to discover the functions of IRF4 in melanoma cells, we analyzed IRF4 target genes, which pointed to IRF4 as an upstream regulator of epigenetic silencing. Accordingly, we show that IRF4 regulates the expression of essential factors for DNA methylation (DNMT1, DNMT3B, UHFR1) and for repressive histone H3 lysine 27 methylation (EZH2), and consequently, the levels of the corresponding methylation marks. Moreover, expression of several tumor suppressor genes known to be silenced by these epigenetic modifications are regulated by IRF4. These include cyclin-dependent kinase inhibitors, PI3K pathway regulator PTEN, and primary cilium component WDR19. As a result, IRF4-dependent epigenetic regulation in melanoma cells modulates the activity of key oncogenic pathways, such as WNT/b-Catenin and PI3K-AKT, impacting on cell proliferation and survival. Moreover, IRF4 modulates the activity of pertinent epigenetic drugs in melanoma cells, encouraging further studies on the therapeutic targeting of IRF4 in melanoma.

overall_design: To investigate the effects of IRF4 in melanoma cell lines, SKMEL28 and SKMEL5 cells were transduced with shLuc( non-targeting control) or shIRF4. RNA-seq was performed for gene expression profiling. For RNA-seq, samples were from 3 biological replicates and 2 different cell lines 72 hours after transduction with shIRF4 or shLuc lentiviruses. For comparative analysis for each cell line, differentially expressed genes were identified in IRF4 knockdown cells compared to control.

first: Erdem

last: YILMAZ

first: N.C. Tolga


title: Transcriptome Profile of Mouse Trophoblast Giant Cells:Effects of Oxycodone on these and other Placental Lineages

city: Columbia

first: Trupti

last: Joshi

state: Missouri

address: 103 Math Science Building

department: Department of Biomedical Informatics, Biostatistics and Medical Epidemiology (BBME)

institute: University of Missouri-Columbia

summary: Pregnant women may be prescribed opioid drugs for pain or may abuse these drugs. The placenta may be key to understanding how opioids cause adverse pregnancy outcomes. Maternal oxycodone (OXY) exposure of pregnant mice leads to disturbances in the layer of invasive parietal trophoblast giant cells (pTGC) that forms the interface between the placenta and uterus and is analogous to extravillous trophoblasts of the human placenta. These cells are probably crucial to coordinating the metabolic needs of the conceptus with those of the mother and are also primary participants in the placenta-brain axis. Their large nuclear size, however, has precluded both single cell (sc) and single nucleus (sn) RNA-seq analyses beyond embryonic day (E) 8.5. Here we compared the transcriptomes of placentas from pregnant mice exposed to OXY with unexposed controls at E12.5, with particular emphasis on the pTGC. The non-fluidic Parse snRNA-seq approach permitted characterization of the nuclear transcriptomes of the major placental cell lineages and their presumed progenitors at E12.5. OXY exposure had negligible effect on components of the placental labyrinth, including the two syncytial cell layers, but caused transcriptomic changes consistent with metabolic stress throughout the spongiotrophoblast. Most notably, there was loss of the majority of pTGCs, whose normal gene expression is consistent with elevated energy demand relating to biosynthesis of multiple secretory products, especially hormones, and endoduplication of DNA. This unusual sensitivity of pTGC may put the pregnancy and future health of the offspring at particular risk to OXY exposure.

overall_design: To characterize the transcriptome profile of parietal trophoblast giant cells and determine how oxycodone exposure affected placenta and decidual cells at embryonic age 9.5 and 12.5, three replicates were tested per group with Parse Biosciences single nuclei RNAseq approach. Thus, a total of 12 samples were tested at embryonic age 9.5 and 12 at embryonic age 12.5. Single nucleotide RNAseq thus represents two embryonic ages (9.5 and 12.5), two types of tissues (placenta and decidual samples), and two treatments (oxycodone vs. control).

first: Zhen

last: Lyu

first: Jessica

last: Kinkae

first: Nathan

last: Bivens

first: R

first: Cheryl

last: Rosenfeld


title: Combined Inhibition of MNK Signaling and BET Proteins Reveals TGM2 as a Novel Vulnerability in Melanoma

city: MONTREAL

first: Antoine

last: MEANT

state: QC

address: 3755 Chemin de la Côte-Sainte-Catherine

institute: Lady Davis Institute - McGill University

summary: Melanoma is the deadliest form of skin cancer. MAPK-targeted therapies (MAPKi) and immune checkpoint inhibitors (ICI) have shown clinical benefit but are limited by resistance mechanisms that remain poorly defined. MNK1/2 inhibitors (MNKi) have shown promising effects in pre-clinical tumor models, particularly in melanoma. Herein, we find that bromodomain and extra-terminal domain protein inhibitors (BETi) in combination with MNKi reduce the proliferation of melanoma cells, including cells with acquired MAPKi resistance. Transcriptomic and proteomic analyses reveal that tissue transglutaminase TGM2 and inhibition of FAK activation are downstream effectors of this combination. We further demonstrate that TGM2 is overexpressed in MAPKi-resistant melanoma cells and that silencing TGM2 inhibits the proliferation of therapy-resistant melanoma cells. Our results introduce TGM2 as a new vulnerability in therapy-resistant melanoma development and suggest that a combination of MNKi and BETi may address the clinical need for novel therapies targeting unresponsive and drug-resistant melanoma.

overall_design: To characterize the molecular mechanisms underlying MNKi and BETi combination in BRAF-mutated melanoma cells, we used a transcriptomic approach. We determined total mRNA expression among the conditions using RNA-sequencing (RNA-seq).

last: Méant

first: Omar

last: Moussa

first: Benjamin

last: Lebeau

first: Feiyang

last: Cai

first: Christophe

last: Gonçalves

first: Vincent

middle: R

last: Richard

first: Sathyen

last: Prabhu

first: Marios

last: Langke

first: Elizabeth

last: Guettler

first: Jie

last: Su

first: Natascha

last: Gagnon

first: Rene

last: Zahedi

first: Christoph

middle: H

last: Borchers

first: Wilson

last: Miller Jr.

first: Sonia

middle: V

last: del Rincón

first: Michael

last: Witcher


title: Neoadjuvant PARPi or Chemotherapy in Ovarian Cancer Informs Targeting Effector Treg Cells for Homologous-Recombination-Deficient Tumors [Singleron scRNA-seq]

city: Wuhan

first: Qinglei

last: Gao

summary: Homologous recombination deficiency (HRD) is prevalent in cancer, sensitizing tumor cells to PARP inhibition. However, the impact of HRD and related therapies on the tumor microenvironment (TME) remains elusive. Our study generates single-cell gene expression and T-cell receptor profiles, along with validatory multimodal datasets from > 100 high-grade serous ovarian cancer (HGSOC) samples, primarily from a phase II clinical trial (NCT04507841). Neoadjuvant monotherapy with the PARP inhibitor niraparib achieves impressive 62.5% and 73.6% response rates per RECIST v1.1 and GCIG CA125, respectively. We identify effector regulatory T cells (eTreg) as key responders to HRD and neoadjuvant therapies, co-occurring with other tumor-reactive T cells, particularly terminally exhausted CD8+ T cells (Tex). TME-wide interferon signaling correlates with cancer cells upregulating MHC-II and co-inhibitory ligands, potentially driving Treg and Tex fates. Depleting eTregs in HRD mouse models, with or without PARP inhibition, significantly suppresses tumor growth without observable toxicities, underscoring the potential of eTreg-focused therapeutics for HGSOC and other HRD-related tumors.

overall_design:
scRNA-seq of sorted T cells from treatment-naïve tumor samples of ovarian cancer patients using the Singleron Biotechnologies platform

Submitter states: Raw sequencing data have been deposited in the Genome Sequence Archive for Human at the National Genomics Data Center (https://ngdc.cncb.ac.cn/gsa-human) with accession number HRA007216, which will be provided for scientific research upon request complying with the law due to human patient privacy concerns.

first: Yikai

last: Luo

first: Yu

last: Xia

first: Dan

last: Liu

first: Xiong

last: Li

first: Huayi

first: Jiahao

first: Dongchen

last: Zhou

last: Dong

first: Ding

last: Ma

first: Yong

last: Fang

first: Han

last: Liang


title: CTCF-dependent insulation of Hoxb13 and the heterochronic control of tail length [ChIPM]

city: Lausanne

first: Lucille

last: Lopez-Delisle

address: Station 19

institute: EPFL

summary: In mammals, tail length is controlled by several genetic determinants, amongst which Hox13 genes located at the posterior extremities of Hox clusters, whose main function are to terminate the extension of the body axis. In this view, the precise timing in the transcriptional activation of these genes may impact upon body length. Unlike other Hox clusters, HoxB lacks all posterior genes between Hoxb9 and Hoxb13, two genes separated by a ca. 70 kb large DNA segment containing an unusually high number of CTCF sites, suggesting it isolates Hoxb13 from the rest of the cluster, thereby delaying its negative impact on trunk extension. We deleted the spacer DNA to induce a potential heterochronic gain of function of Hoxb13 at physiological concentration and observed a shortening of the tail as well as other abnormal phenotypes, which were all rescued by inactivating Hoxb13 in-cis with the deletion. A comparable gain of function was observed in mutant ES cells grown as pseudo-embryos in vitro, which allowed us to examine in details the importance of both the number and the orientation of CTCF sites in the insulating activity of the DNA spacer. A short cassette containing all the CTCF sites was sufficient to insulate Hoxb13 from the rest of HoxB and additional modifications of this CTCF cassette showed that two CTCF sites in convergent orientations are already capable of importantly delaying Hoxb13 activation in these conditions. We discuss the relative importance of genomic distance versus number and orientation of CTCF sites in preventing Hoxb13 to be activated too early during trunk extension and hence to modulate tail length.

first: Célia

last: Bochaton

first: Hocine

last: Rekaik

first: Jozsef

last: Zakany

first: Denis

last: Duboule


title: Genome-wide CRISPR KO screen in BMDMs [Bulk CIRSPR Screen]

first: Sandra

last: Melo

summary: Macrophages adopt dynamic functional states to maintain tissue homeostasis or respond to environmental challenges. Here we performed a genome-wide CRISPR screen in primary BMDMs identifiying negative regulators of iNOS expression in response to IFNg stimulation.

overall_design: We performed multiple pooled genome-wide CRISPR knockout (KO) screens using an optimized Streptococcus pyogenes Cas9 single guide RNA (sgRNA) library paired with delivery of recombinant Cas9 protein via electroporation. Our library consisted of 8 combined lentivirus sgRNA library pools expressing 152 339 sgRNAs targeting 18776 genes and non-targeting control sgRNAs. BMDMs were sorted and sgRNAs isolated and sequenced from 3 sources: (1) iNOS high cells post-IFNγ stimulation (iNOS_high); (2) cells from the last day of differentiation prior to IFNγ stimulation (reference); and (3) cells collected three days after lentiviral transduction as a library control (input)

first: Marinella

last: Callos

first: Mike

last: Costa

first: Aaron

last: Lun

first: Jaclyn

last: Lock

first: Julia

last: Kuhn

first: Christopher

last: Bohlen

first: Simone

last: Haag

first: Aditya

last: Murthy


title: A non-canonical repressor function of JUN restrains YAP activity and suppresses YAP-dependent liver cancer growth [JUN_WT_M14_in_JUN_KO]

city: Jena

first: Bjoern

last: von Eyss

address: Beutenbergstr. 11

institute: Leibniz Institute on Aging

summary: The transcriptional coactivators YAP/TAZ are the critical downstream regulators of the Hippo pathway that regulate gene expression in response to changes in pathway activity, mainly by binding to TEAD transcription factors. Uncontrolled transcriptional output of YAP/TAZ can lead to rapid induction of aggressive tumor growth. AP-1 is a dimeric basic leucine zipper (bZIP) transcription factor complex with JUN and FOS proteins being the most abundant members of this family. Unlike FOS, JUN can also form homodimers, but in the cell, JUN presumably preferentially forms heterodimers with members of the FOS family, which act as potent transcriptional activators. Previous studies identified substantial co-occupancy of YAP/TAZ and AP-1 at genomic sites, and they demonstrated that JUN/FOS heterodimers cooperate with the YAP/TAZ and TEAD transcription factors to drive YAP/TAZ target gene expression. Here, we now elucidate a negative feedback mechanism in which high YAP activity is restrained by the recruitment of homodimeric JUN::JUN/NCOR1 repressor complexes and show that this noncanonical JUN function is part of a tumor suppressor mechanism in the liver.

overall_design: To analyze the genomice binding sites c-JUN WT or c-JUN M14, MCF10A JUN KO cells were infected with a JUN WT or JUN M14 expression vector, and CUT&RUN experiments for JUN were performed

first: Yuliya

last: Kurlishchuk

first: Anita

last: Cindric-Vranesic

first: Marco

last: Gründl

first: Björn


title: A non-canonical repressor function of JUN restrains YAP activity and suppresses YAP-dependent liver cancer growth [iYAP5SA_10A_JUN_oe_antibodies_for_JUN_YAP_TEAD]

overall_design: To analyze the genomice binding sites of YAP, TEAD1 and c-JUN, CUT&RUN experimentes were performed in MCF10A cells which carry a doxycycline-inducible YAP 5SA, and were superinfected with and empty vector control or a JUN overexpression vector.


title: A non-canonical repressor function of JUN restrains YAP activity and suppresses YAP-dependent liver cancer growth [CutRun_in_constitutive_5SA_MCF10A]

overall_design: To analyze the genomice binding sites of YAP, TEAD1, c-JUN,c-FOS, CUT&RUN experimentes were performed in MCF10A cels stably overexpressing YAP 5SA.


title: A non-canonical repressor function of JUN restrains YAP activity and suppresses YAP-dependent liver cancer growth [TruSeq]

overall_design: To analyze the impact of JUN overexpression on YAP target gene expression, MCF10A cells expressing a doxycycline-inducible YAP5SA allele were infected with a JUN overexpression vector or an empty vector control. YAP induction was induced by doxycycline addition (100 ng/ml) and control cells were treated with solvent control (EtOH).


title: A non-canonical repressor function of JUN restrains YAP activity and suppresses YAP-dependent liver cancer growth


title: A non-canonical repressor function of JUN restrains YAP activity and suppresses YAP-dependent liver cancer growth [CUT&RUN]

overall_design: To analyze the genomice binding sites of YAP, c-FOS, NCOR1, ARID1A, and c-JUN, CUT&RUN experimentes were performed in MCF10A cells which carry a doxycycline-inducible YAP 5SA, and were superinfected with and empty vector control or a JUN overexpression vector.


title: A non-canonical repressor function of JUN restrains YAP activity and suppresses YAP-dependent liver cancer growth [3primeSeq_siY]

overall_design: To analyze the impact of JUN overexpression on gene expression, 10A cells expressing the necessary SAM expression constructs (dCas9-VP64 and MCP-p65-HSF1) were infected with a non-targeting control or a sgJUN#1 targeting the activating dCas9 complex to the JUN promoter.


title: A non-canonical repressor function of JUN restrains YAP activity and suppresses YAP-dependent liver cancer growth [SLAM_Seq]


title: O-GlcNAcylation Mediates Wnt-stimulated Bone Formation via Rewiring Aerobic Glycolysis in Osteoblast-lineage Cells

status: Public on Jun 28 2024

city: Chendu

first: Fangyuan

last: Shen

state: --- Select a state ---

address: No.29 Jiuyanqiao Wangjiang Road, Chengdu, 610064

institute: Sichuan University

summary: As the efficient therapeutic treatment for osteoporosis, Wnt signaling is considered to induce bone formation through aerobic glycolysis. However, the mechanism underlying the regulatory role of Wnt in orchestrating glucose metabolism during osteogenesis remains unclear. O-GlcNAcylation, a dynamic posttranslational modification (PTM) on proteins, controls multiple critical biological processes including gene transcription, translation, and cell fate determination. Here, we report Wnt3a either induces O-GlcNAcylation rapidly via the Ca2+-PKA-Gfat1 axis, or increases it in a Wnt-β-catenin dependent manner during prolonged stimulation. Importantly, O-GlcNAcylation is found indispensable for osteoblastogenesis both in vivo and in vitro. Genetic ablation of O-GlcNAcylation in osteoblast-lineage cells diminishes bone formation and delays bone fracture healing in response to Wnt stimulation. Notably, Wnt3a-induced O-GlcNAcylation enhances aerobic glycolysis by stabilizing PDK1 partially through Serine 174 (S174) site, which consequently facilitates osteogenesis. These findings highlight that O-GlcNAcylation is indispensable in Wnt-induced glucose metabolism during osteogenesis, indicating its potential as a therapeutic target for osteoporosis.

overall_design: For RNA -Seq experiments, ST2 cells were exposed to vehicle, OSMI (20 μM), or rhWnt3a (50ng/ml) for 72 hours prior to obtaining total RNA.

first: Chengjia

last: You

first: Ling

last: Ye

first: Yu

last: Shi


title: A non-canonical repressor function of JUN restrains YAP activity and suppresses YAP-dependent liver cancer growth [3prime_Seq_JUNoe]


title: Transcriptomic analysis of Drosophila leg disc associated myoblasts

city: clermont ferrand

first: yoan

last: renaud

address: place henri dunand

institute: igred

summary: Adult leg muscle precursors are associated to leg imaginal disc, here we provide bulk RNA sequecing of FACS sorted myoblasts from dissected imaginal leg discs at beginning of pupation.

overall_design: Drosophila leg discs were dissected from 0h after pupae formation. Leg disc cells were dissociated in collagenase and papain solution and cell-sorted using. RNA from GFP positive myoblasts and from control cells (whole leg disc cells) were extracted for analysis by deep sequencing, in triplicate, using Illumina NextSeq sequencing platform.

first: Blandine

last: Moucaud

first: Yoan

last: Renaud

first: Cedric

last: Soler


title: The effects on the deficient of GDH1 on gene expression in LN229 cells.

city: Liaocheng

first: Rui

last: Yang

state: Shandong

address: Hunan Road

department: Biomedical Laboratory, Medical School

institute: Liaocheng University

summary: Glutamine is an important nutriment for cancer cell growth that provides biological sources for nucleic acid and fatty acid synthesis, but the role of glutaminolysis in signal transduction and glioblastoma (GBM) progression remains little known. Here, we investigated whether GDH1, a key glutamate dehydrogenase, affects the expression levels of signal transduction factors by mRNA expression profiling of wild-type and GDH1 knockdown cells of the glioblastoma cell line LN229 cells

overall_design: Divide Ln229 cells into two groups: one group is wild-type cells, and the other group is subjected to GDH1 gene knockdown. Three biological replicates are set for each group for cultivation treatment. Collect cell precipitates and extract RNA for transcriptome sequencing analysis.


title: Comprehensive alpha, beta, and delta cell transcriptomics reveal an association of cellular aging with MHC class I upregulation

status: Public on Jun 27 2024

city: Paris

first: Vincent

last: Laville

address: 25-28 rue du Dr Roux

institute: Institut Pasteur

summary:
This study aimed to evaluate the efficacy of a purification method developed for isolating alpha, beta, and delta cells from pancreatic islets of adult mice, extending its application to islets from newborn and aged mice. Furthermore, it sought to examine transcriptome dynamics in mouse pancreatic endocrine islet cells throughout postnatal development and to evaluate age-related alterations in intercellular communication within these cell populations.
We leveraged the high surface expression of CD71 on beta cells and CD24 on delta cells to FACS-purify alpha, beta, and delta cells from newborn (1-week-old), adult (12-week-old), and old (18-month-old) mice. Bulk RNA sequencing was conducted on these purified cell populations, and subsequent bioinformatic analyses included differential gene expression, overrepresentation, intersection, and intercellular communication analysis.
Alpha, beta, and delta cells from newborn and aged mice were successfully FACS-purified using the same method employed for adult mice. Our analysis of the age-related transcriptional changes in alpha, beta, and delta cell populations revealed a decrease in cell cycling and an increase in so-called “neurogenesis” processes during the transition from newborn to adult mice. Progressing from adult to old mice, we observed an increase in β-2 microglobulin and major histocompatibility complex (MHC) Class I expression. Computational modeling of cell interactions suggested shifts in TGFβ and BMP signaling underpinning these age-related changes.
Our study demonstrates the effectiveness of our cell sorting technique in purifying endocrine subsets from mouse islets at different ages. We provide a valuable resource for better understanding endocrine pancreas aging and identified increased β-2 microglobulin and MHC Class I expression as a common hallmark of old alpha, beta, and delta cells, with potential implications for immune response regulation.

overall_design: To investigate transcriptomic changes in mouse pancreatic islets during aging, we leveraged data from RNA-seq experiments performed in three replicates of alpha, beta and delta cells at three different timepoints (1 week, 12 months and 18 months old).

first: Willem

last: Staels

first: Claire

last: Berthault

first: Raphael

last: Scharfmann


title: Plakophilin 4 controls the spatio-temporal activity of RhoA at adherens junctions to promote cortical actin ring formation and tissue tension

status: Public on Jun 24 2024

city: Halle

first: Markus

last: Glaß

address: Kurt-Mothes-Str. 3a

department: Institute of Molecular Medicine

institute: Martin Luther University Halle-Wittenberg

summary: Plakophilin 4 (PKP4) is a component of cell-cell junctions that regulates intercellular adhesion and Rho-signaling during cytokinesis with an unknown function during epidermal differentiation. Here we show that keratinocytes lacking PKP4 fail to develop a cortical actin ring, preventing adherens junction maturation and generation of tissue tension. Instead, PKP4-depleted cells display increased stress fibers. PKP4-dependent RhoA localization at AJs is required to activate a RhoA-ROCK2-MLCK-MLC2 axis and organize actin into a cortical ring. AJ-associated PKP4 provided a scaffold for the Rho activator ARHGEF2 and the RhoA effectors MLCK and MLC2, facilitating the spatio-temporal activation of RhoA signaling at cell junctions to allow cortical ring formation and actomyosin contraction. In contrast, association of PKP4 with the Rho suppressor ARHGAP23 reduced ARHGAP23 binding to RhoA which prevented RhoA activation in the cytoplasm and stress fiber formation. These data identify PKP4 as an AJ component that transduces mechanical signals into cytoskeletal organization.

overall_design: PolyA RNA-seq of murine keratinocytes, expressing Pkp4 wildtype cells, Pkp4-knockout cells, or Pkp4-knockout keratinocytes overexpressing GFP-PKP4 (Rescue cells). Three biological replicates per condition.

first: Lisa

last: Müller

first: Mechthild

last: Hatzfeld


title: Computational identification of surface markers for isolating distinct subpopulations from heterogeneous cancer cell populations [scRNA-seq]

status: Public on Jun 26 2024

city: Austin

first: Amy

last: Brock

state: Texas

address: 2515 Speedway

institute: The University of Texas at Austin

summary: Intratumoral heterogeneity reduces treatment efficacy and complicates our understanding of tumor progression. There is a pressing need to understand how heterogeneous tumor cell subpopulations coexist within a tumor, yet biological systems to study these processes in vitro are limited.  With the advent of single-cell RNA sequencing (scRNA-seq), it has been found that some cancer cell lines contain distinct subpopulations. These heterogeneous cell lines provide a unique opportunity to study coexistence and evolution between genetically similar cancer cell subpopulations in controlled experimental settings. Here, we present scEMD, a computational package that returns candidate surface makers maximally unique to transcriptomic subpopulations in scRNA-seq which may be used for FACS isolation. scEMD was experimentally validated using the MDA-MB-231 and MDA-MB-436 cell lines. ESAM and BST2/Tetherin were experimentally confirmed to identify and separate scRNA-seq cluster-matched subpopulations of MDA-MB-231 and MDA-MB-436 cells, respectively. Identification and enrichment of distinct subpopulations within cell line models using this computationally efficient and experimentally validated workflow paves the way for studies on the generation and maintenance of cellular heterogeneity in low-complexity in vitro systems.

overall_design: Single-cell RNA sequencing of MDA-MB-231, MDA-MB-436, BT474, Hs5788t, HCC38, and MDA-MB-453 cell ines

first: Tyler

last: Jost

first: Andrea

last: Gardner


title: Computational identification of surface markers for isolating distinct subpopulations from heterogeneous cancer cell populations [3’-Tag RNAseq]

overall_design: tagSeq on 2 isolated populations from the MDA-MB-231 breast cancer cell line and 2 isolated populations from the MDA-MB-436 breast cancer cell line.


title: Mitochondrial signatures shape phenotype switching and apoptosis in response to PLK1 and RSK inhibitiors in melanoma

status: Public on Jun 14 2024

city: Montreal

first: Simon-Pierre

last: Gravel

address: 2940 ch. de la polytechnique

department: Faculty of pharmacy

institute: University of Montreal

summary: PLK1 inhibitors are emerging anti-cancer agents being tested in monotherapy and combination therapies for various cancers. Although PLK1 inhibition in experimental models shows potent antitumor effects, translation to the clinic has been hampered by low antitumor activity and tumor relapse. Here, we report the identification of mitochondrial protein signatures that determine sensitivity to approaches targeting PLK1 in human melanoma cell lines. In response to PLK1 inhibition or gene silencing, resistant cells adopt a pro-inflammatory and dedifferentiated phenotype, while sensitive cells engage apoptosis. Mitochondrial DNA depletion and silencing of the ABCD1 transporter sensitize cells to PLK1 inhibition and attenuate the associated pro-inflammatory response. We also found that non-selective inhibitors of the p90 ribosomal S6 kinase (RSK) exert their anti-proliferative and pro-inflammatory effects via PLK1 inhibition. This work reveals overlooked impacts of PLK1 on phenotype switching and suggests that mitochondrial precision medicine can help improve response to targeted therapies.

overall_design: Human melanoma cell lines were treated with p90 ribosomal S6 kinase (RSK) inhibitors for 72 h. The MeWo cell line was treated with DMSO 0.1% or the RSK inhibitor BI-D1870 3 µM. The A-375 cell line was treated with DMSO 0.1% or the RSK inhibitor LJH685 at 3 µM. The 3 samples per condition are from 3 independent experiments in which a pair of plates is treated with DMSO or a RSK inhibitor. A-375 and MeWo samples cannot be compared as they were analysed by RNA-seq separately.

first: Émilie

last: Lavallée

first: Maëline

last: Roulet-Matton

first: Viviane

last: Giang

first: Roxana

last: Cardona

first: Dominic

last: Chaput


title: Single neuron analysis of aging associated changes in learning reveals escalating impairments in transcriptional plasticity

city: Baltimore

first: Carrie

last: McCracken

state: MD

address: 670 W. Baltimore St

department: Institute for Genome Sciences

institute: Institute of Genome

summary: Molecular mechanisms underlying aging associated impairments in learning and long-term memory storage are poorly understood. Here we leveraged an identified motor neuron L7,  mediating long-term sensitization of siphon-withdrawal reflex, a form of non-associative learning in sea slug Aplysia, to assess the impact of aging on transcriptional changes during learning. RNAseq analysis of single L7 motor neuron isolated following short-term or long-term sensitization training from 8,10 and 12 months old Aplysia corresponding to mature, late mature and senescent stages have identified progressive impairments in transcriptional plasticity during aging. Specifically, we uncover modulation of the expression of multiple lncRNAs, and mRNAs encoding transcription factors, regulators of translation, RNA methylation, and cytoskeletal rearrangements during learning and their deficits during aging. Our comparative gene expression analysis also revealed the recruitment of specific transcriptional changes in two other neurons, a motor neuron L11 and giant cholinergic neuron R2 whose roles in long-term sensitization were previously not known. Taken together, our analyses establish cell type specific escalating impairments in the expression of learning and LTM relevant components of transcriptomes during aging.

overall_design: Single neuron (L7MN) isolated from long-term or short-term sensitized  or untrained Aplysia that are of 8,10 and 12 months old were used for total RNAseq Analysis.

first: Kerriann

last: Badal

first: Robert

last: Hawkins

first: Sathyanarayanan

last: Puthanveettil


title: A single cell transcriptomic map of the developing Atoh1-lineage uncovers neural fate decisions and neuronal diversity in the hindbrain.[RNA-Seq]

status: Public on Jun 23 2024

city: Houston

first: Huda

last: Zoghbi

state: Tx

address: 1250 Moursund St

institute: Baylor College of Medicine

summary: Proneural transcription factors set up the molecular cascade to orchestrate neuronal diversity. One such transcription factor, Atoh1, gives rise to cerebellar excitatory neurons and over 30 distinct nuclei in the brainstem critical for hearing, breathing, and balance. Although the neurons that arise from the Atoh1-lineage have been qualitatively described, the transcriptional programs that drive their fate decisions and the full extent of their diversity remain unknown. Here, we analyzed single-cell RNA-sequencing and ATOH1 DNA binding in Atoh1-lineage neurons of the developing mouse hindbrain. This high-resolution dataset revealed new markers for specific brainstem nuclei and demonstrated transcriptionally heterogeneous progenitors require ATOH1 for proper migration. Moreover, we identified a sizable proliferating unipolar brush cell progenitor in the mouse Atoh1-lineage that was previously described as the origin of one medulloblastoma subtype. Collectively, our data reveal unprecedented insight into the developing mouse hindbrain and provide markers for functional assessment of less studied neuronal populations.

overall_design: Atoh1-lineage neurons labeled by TdTomato and/or GFP  in mouse hindbrain were enriched by FACS, followed by scRNA-seq with 10X chromium 3' v3.1 kit and NovaSeq 6000. Single-cell transcriptomic profiles across embryonic stages (E9.5, E.10.5, E11.5, E12.5, E13.5, E14.5, and E16.5) were compared

first: Jessica

last: Butts

first: Sih-Rong

last: Wu

middle: Y


title: Expression Patterns of Mal genes and Association with Differential Maltose and Maltotriose Transport rate of Two Saccharomyces pastorianus Yeasts

status: Public on Jun 17 2024

city: San Nicolás de los Garza

first: Benito

last: Pereyra Alférez

state: Nuevo León

address: Av. Pedro de Alba S/N

department: Instituto de Biotecnología

country: Mexico

institute: Universidad Autónoma de Nuevo León

summary: Beer brewing is a well-known process that still faces great challenges, such as the total consumption of sugars present in the fermentation media. Lager-style beer, major worldwide beer type, is elaborated by Saccharomyces pastorianus (Sp) yeast which must ferment high maltotriose content worts, but its consumption represents a notable problem, especially among Sp strains belonging to group I. Factors like fermentation conditions, presence of maltotriose transporters, transporter copy number variation, and genetic regulation variations contribute to this issue. We assess the factors affecting fermentation in two Sp yeast strains: SpIB1, with limited maltotriose uptake, and SpIB2, known for efficient maltotriose transport. Here, SpIB2 transported significantly more maltose (28%) and maltotriose (32%) compared to SpIB1. Furthermore, SpIB2 expressed all MAL transporters (ScMALx1, SeMALx1, ScAGT1, SeAGT1, MTT1, and MPHx) on the first day of fermentation, while SpIB1 only exhibited ScMalx1, ScAGT1, and MPH2/3 genes. Some SpIB2 transporters had polymorphic transmembrane domains (TMD) resembling MTT1, accompanied by higher expression of these transporters and its positive regulator genes like MAL63. These findings suggest that, in addition of the factors mentioned above, positive regulators of Mal transporters contribute significatively to phenotypic diversity in maltose and maltotriose consumption among the studied lager yeast strains.

overall_design:
The goal is to assess the underling genes involved in the phenotypic diversity of maltose and maltotriose consumption of two lager yeast strains (SpIB1 and SpIB2). Both yeasts were subjected to lager fermentation in a standard wort for 5 days and samples were collected during day 2 and 5.
We then performed a gene expression analysis using the RNA-seq data from both yeasts on days 2 and 5 of fermentation.

first: Jorge

last: García

first: César

middle: I

last: Hernández Vásquez

first: Esmeralda

last: del Rocio Pérez Ortega

first: Luis

last: Damas Buenrostro

first: Adriana

last: Martzinez Segundo


title: A single cell transcriptomic map of the developing Atoh1-lineage uncovers neural fate decisions and neuronal diversity in the hindbrain.

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

overall_design: Refer to individual Series


title: The role of SMAD7 on epigenetic regulation of TGF-β targets in the metastasis of ovarian cancer

status: Public on Jun 12 2024

city: Taipei

first: Hung-Cheng

last: Lai

address: No.250, Wuxing St.

department: Department of Obstetrics and Gynecology, School of Medicine, College of Medicine,

country: Taiwan

institute: Taipei Medical University

summary: Targeting TGF-β signaling and its downstream effectors, such as SMAD7, may offer promising therapeutic strategies for reversing epigenetic alterations and inhibiting metastatic progression in ovarian cancer.

overall_design: TGF-β signaling, a key regulator of EMT, may directly influence DNA methylation in the E-cadherin promoter. SMAD7 was overexpressed to investigate if sustained activation of TGF-β signaling contributes to DNA methylation of the E-cadherin promoter in ovarian cancer in a mesenchymal ovarian cancer cell

first: Michael

last: Chan

first: Lin-Yu

last: Chen


title: Redistribution of SOD3 expression due to R213G polymorphism affects pulmonary interstitial macrophage reprogramming in response to hypoxia

city: Aurora

first: Caitlin

middle: Vanessa

last: Lewis

state: Colorado

address: University of Colorado Denver AMC, 12700 East 19th Avenue, Mail Stop B131

department: Pediatrics

institute: University of Colorado

summary: The extracellular isoform of superoxide dismutase (SOD3) is decreased in patients and animals with pulmonary hypertension (PH). The human R213G single nucleotide polymorphism (SNP) in SOD3 causes its release from tissue extracellular matrix (ECM) into extracellular fluids, without modulating enzyme activity, and increases cardiovascular disease risk in humans and chronic hypoxic PH in mice. Given the importance of interstitial macrophages (IM) to PH pathogenesis, this study aimed to determine whether R213G SOD3 worsens PH via increased IM accumulation and altered reprogramming. R213G mice and wild-type (WT) controls were exposed to hypobaric hypoxia for 4 or 14 days compared to normoxia. Flow cytometry demonstrated a transient increase in IMs at day 4 in both strains. Contrary to our hypothesis, the R213G SNP did not augment IM accumulation. To determine strain differences in the IM reprogramming response to hypoxia, we performed RNAsequencing on IMs isolated at each time point. We found that IMs from R213G mice exposed to hypoxia activated ECM-related pathways and a combination of alternative macrophage and proinflammatory signaling. Furthermore, when compared to WT responses, IMs from R213G mice lacked metabolic remodeling and demonstrated a blunted anti-inflammatory response between the early (day 4) and later (day 14) time points. We confirmed metabolic responses using Agilent Seahorse assays whereby WT, but not R213G, IMs upregulated glycolysis at day 4 that returned to baseline at day 14. Finally, we identify differential regulation of several redox-sensitive upstream regulators that could be investigated in future studies.

overall_design:
To investigate the effect of the R213G SOD3 polymorphism on pulmonary interstitial macrophage (IM) transcriptional responses to hypoxia, we exposed R213G and C57BL/6J (WT) mice to 4 days (peak inflammation) or 14 (resolution) days of hypobaric hypoxia.
IMs were isolated from mouse lungs by FACS and RNAseq performed on each population. 4 mice (2 male and 2 female) were pooled for each sample and n=3-4 samples analyzed per group.


title: Tapeworm infection affects sleep behavior in three-spined stickleback

status: Public on Jun 13 2024

city: Münster

last: Peuß

address: Hüfferstrasse 1

department: Institute for Evolution and Biodiversiity

institute: University of Münster

summary: Sleep is a complex and conserved biological process that affects several body functions and behaviors. Evidence suggests that there is a reciprocal interaction between sleep and immunity. For instance, fragmented sleep can increase the probability of parasitic infections and reduce the ability to fight infections. Moreover, viral and bacterial infections alter the sleep patterns of infected individuals. However, the effects of macro-parasitic infections on sleep remain largely unknown. In this study, we investigated whether macro-parasite infections could alter the sleep of their hosts. We experimentally infected three-spined sticklebacks (Gasterosteus aculeatus) with the tapeworm Schistocephalus solidus and used a hidden Markov model to characterize sleep behavior in sticklebacks. One to four days after parasite exposure, infected fish showed no difference in sleep compared with non-exposed fish, whereas fish that were exposed-but-not-infected slept less during daytime. 29-32 days after exposure, infected fish slept more than uninfected fish, while exposed-but-not-infected fish slept less than non-exposed fish. Using brain transcriptomics, we identified immune- and sleep-associated genes that potentially underlie the observed behavioral changes. These results provide insights into the complex association between macro-parasite infection, immunity, and sleep in fish and may thus contribute to a better understanding of reciprocal interactions between sleep and immunity.

overall_design: Stickleback, Gasterosteus aculeatus, were either exposed to the parasite Schistocephalus solidus or not exposed to the parasite ("control"[Con]). Exposed fish were catogorized as "infected"[Inf] (parasite present) or "exposed" [Exp] (parasite not present) based on the presence of the parasite Schistocephalus solidus upon dissection of the fish. Fish were monitored for their activity at different time points and dissection of the brain for RNAseq analysis was performed subsequently of the activity measures.

first: Marc

last: Bauhus

first: Sina

last: Mews

first: Joachim

last: Kurtz

first: Alexander

last: Brinker

first: Jaime

last: Anaya-Rojas


title: Deconvoluted methylation profiles discriminate between closely related melanocytic nevi

city: Marseille

first: Daniel

last: Aldea

address: 27 Bd Jean Moulin

institute: Aix-Marseille University

type: Methylation profiling by genome tiling array

summary: Congenital melanocytic nevi (CMN) and common acquired melanocytic nevi (AMN) are skin conditions that result from an excess of melanocytes arising during fetal development or detected postnatally. Appropriately weighted whole-genome methylation analysis can be used as a basis for further research in dermatopathology and, in this application, provides new insights into nevus biology.

overall_design:
To investigate molecular differences between congenital melanocytic nevi (CMN) and acquired melanocytic nevi (AMN) through the study of their DNA methylation profiles

Please note that the processed data files (containing beta values) have been updated on Oct 2, 2024.

first: Nicolas

last: Macagno

first: Elise

last: Marechal

first: Mathias

last: Moreno

first: Pauline

last: Romanet

first: Morgane

last: Pertuit

first: Jérémy

last: Garcia

first: Nathalie

last: Degardin

first: Stéphanie

last: Mallet

first: Isabelle

last: James

first: Guillaume

last: Captier

first: Anne

last: Barlier

first: Heather

last: Etchevers