title: In situ targeted gene expression analysis of Marjolin ulcers

summary: Marjolin's ulcer is a a rare and aggressive cutaneous malignancy that can arise on previously injured skin, established scars, and chronic non-healing wounds. It is most often found in burn scars, but it can also occur in other types of wounds, including venous stasis ulcers, pressure sores, and vaccination sites. The most common histological type of Marjolin’s ulcers is squamous cell carcinoma, however basal cell carcinomas, malignant melanomas, and sarcomas have also been reported. All parts of the body could potentially be affected yet the lower extremities are the anatomic sites most commonly involved. While SCCs commonly have a metastasis rate of 0.5 to 3.0 percent, those arising from burn scars metastasize at a rate in excess of 30 percent. The 5-year survival after a diagnosis of Marjolin’s ulcer was found to be 50 percent. The transformation to a malignancy can occur either chronically, over a period of more than 35 years, or occasionally within a year of the original injury. The exact mechanism of this transformation is not fully understood, but it is thought to be related to chronic keratinocyte dysfunction during the healing process of severe burn wounds. Surgical excision is the main treatment for Marjolin's ulcer and provides the best chance of survival. In a previous study, we investigated the bulk transcriptional changes that lead to Marjolin's ulcer by comparing global gene expression changes between squamous cells present in a squamous cell carcinoma versus those present within Marjolin's ulcer (MU) (Sinha et. al. JBCR 2017). This imparted novel insights into mechanisms underlying divergent clinical features of these cutaneous cancers. The goal of our current study is to characterize a new case of Marjolin's ulcer in a patient under our care by analyzing the cell types, their frequencies, and their individual transcriptional responses within a burn scar versus within Marjolin's ulcer. To achieve this, we conducted single-cell RNA sequencing on two excised tissues: 1. a sample from the center of the tumor (tumor core), and 2. another sample from the margin of tumor-free scar tissue.

overall_design: The Xenium In Situ platform employs a microscopy-based method for analysis. A formalin-fixed and paraffin-embedded (FFPE) MU tissue block was obtained. Adhering to the manufacturer's protocols, the tissue underwent processing, including sectioning, organizing the sections, and performing Hematoxylin and Eosin (H&E) staining to ensure quality. The tissue sections were precisely trimmed to match the dimensions of the Xenium slide, with careful attention to include areas of both the burn scar and the tumor core. Once positioned on Xenium slide, the section was stained with Hematoxylin and Eosin (H&E) for tissue morphology analysis. Subsequent steps involved the hybridization and ligation of specific DNA probes to target mRNA, followed by a process of rolling circle amplification. The slide underwent several rounds of fluorescent probe hybridization and imaging in the Xenium Analyzer. This process, leveraging each gene's unique optical signature, allowed for the decoding of target genes and the creation of a spatial transcriptomic map of the entire tissue section.

first: Sarthak

last: Sinha

first: Nicole

last: Rosin

first: Keerthana

last: Chockalingam

first: Rohit

last: Arora

first: Lindsay

last: Burnett

first: Temple-Oberle

last: Claire

first: Vincent

last: Gabriel


title: Single-cell transcriptomics of Marjolin ulcers

status: Public on Jul 21 2024

city: Calgary

first: Jeff

last: Biernaskie

state: Alberta

address: 3330 Hospital Drive NW

department: Comparative Biology and Experimental Medicine

country: Canada

institute: University of Calgary

type: Expression profiling by high throughput sequencing

overall_design: During surgery, we collected two full-thickness punch biopsies from the resected tissue: one from the center of the tumor (tumor core) and one from the margin of tumor-free scar tissue. To examine molecular changes within specific cell compartments, we isolated single-cells from both biopsies and processed according to 10X Genomics Chromium Single Cell 3’ Reagent Guidelines v3 Chemistry as per the manufacturer’s protocol. In brief, single cells were sorted based on forward versus side scatter gating into 0.1% BSA–PBS and partitioned into Gel Bead-In-EMulsions (GEMs) using 10x GemCodeTM Technology. Next-Generation Sequencing was performed using the Illumina NovaSeq S2 Flow cells. All raw FASTQs were aligned to the human reference genome generated using cellranger mkref pipeline. The resulting gene barcode matrix was processed using Seurat R toolkit.


title: A small intestinal bile acid modulates the gut microbiome to improve host metabolic phenotypes following bariatric surgery

city: Boston

first: Yingjia

last: Chen

address: 75 Franis St

country: USA

institute: Brigham and Women's Hospital

summary: Bariatric surgical procedures such as sleeve gastrectomy (SG) provide effective type 2 diabetes remission in human patients. Previous work demonstrated that gastrointestinal levels of the bacterial metabolite lithocholic acid (LCA) are decreased after SG in mice and humans. Here, we show that LCA worsens glucose tolerance and impairs whole body metabolism. We also show that taurodeoxycholic acid (TDCA), which was the only bile acid whose concentration was increased in the murine small intestine post-SG, suppresses the bacterial bile acid-inducible (bai) operon and production of LCA both in vitro and in vivo. Treatment of diet-induced obese (DIO) mice with TDCA reduces LCA levels and leads to microbiome-dependent improvements in host glucose handling. Moreover, TDCA abundance is decreased in small intestinal tissue from T2D patients. This work has revealed that TDCA is an endogenous inhibitor of LCA production and suggests that TDCA may contribute to the glucoregulatory effects of bariatric surgery.

overall_design: 5 biological replicates of mouse distal ileum samples from TDCA- or vehicle-treated DIO mice were used for RNA-sequencing.

first: A

middle: S

last: Devlin

first: Eric

middle: G

last: Sheu


title: Gene expression profile of inflammatory monocytes in cachectic blood cancer mice (scRNA-Seq)

city: Hachioji

first: Yoshihiro

last: Hayashi

state: Tokyo

address: 1432-1 Horinouchi

country: Japan

institute: Tokyo University of Pharmacy and Life Sciences

summary: Many patients with advanced cancers develop cachexia. Despite the high prevalence and catastrophic impact on quality of life and survival, the critical pathobiological mediators responsible for the cancer cachexia development remain poorly defined. Here, we show that a distinct subset of neutrophil-like monocytes, which we term cachexia-inducible monocytes (CiMs), emerges in advanced cancer pathogenesis and promotes skeletal muscle loss. Unbiased transcriptome analysis revealed that IL36G-producing CD38+ CiMs are induced during cachexia progression in the pathogenesis of chronic monocytic blood cancer characterized by persistent monocytosis and cachexia. The emergence of CiMs and activation of CiM-related gene signature in inflammatory monocytes were confirmed in multiple mouse models and patients with advanced solid cancers. Notably, genetic inhibition of IL36G/IL1RL2 signaling attenuated skeletal muscle loss and rescued cachexia phenotypes derived by both blood and solid cancers in mice. These data highlight the critical role for a subset of neutrophil-like monocyte induced in cancer pathogenesis.

overall_design: Single cell RNA-Seq of inflammatory monocytes from control mice and CMML mice at the time of weight loss onset.


title: Gene expression profile of inflammatory monocytes in cachectic blood cancer mice [bulk RNA-seq]

overall_design: Expression profiles of mRNA in inflammatory monocytes from control mice and CMML mice with or without cachexia.


title: Integrative genomic, virulence, and transcriptomic analysis of emergent Streptococcus dysgalactiae subspecies equisimilis (SDSE) emm type stG62647 isolates causing human infections

status: Public on Jul 20 2024

city: Houston

first: James

middle: M.

last: Musser

state: Texas

address: 6670 Bertner Ave.

department: Pathology and Genomic Medicine

institute: Houston Methodist Hospital and Research Institute

summary: Streptococcus dysgalactiae subspecies equisimilis (SDSE) is a Gram-positive bacterial pathogen that infects humans and is closely related to group A streptococcus (GAS). Compared to GAS, far less is known about SDSE pathobiology. Increased rates of invasive SDSE infections have recently been reported in many countries. One SDSE emm type, stG62647, is known to cause severe diseases, including necrotizing soft-tissue infections, endocarditis, and osteoarticular infections. To increase our understanding of the molecular pathogenesis of stG62647 SDSE isolates causing human infections, we sequenced to closure the genomes of 120 stG62647 SDSE isolates. The genomes varied in size from 2.1 to 2.24 megabase pairs. Consistent with previous data, the great majority of stG62647 isolates had IS1548 integrated into the silB gene, thereby inactivating it. Regions of difference in gene content, including putative mobile genetic elements, were the largest source of genomic diversity. All 120 stG62647 isolates were assayed for virulence using a well-established mouse model of necrotizing myositis. An unexpectedly wide range of virulence was identified (20% to 95%), as assessed by near-mortality data. To explore the molecular mechanisms underlying virulence differences, we analyzed RNAseq transcriptome profiles for 38 stG62647 isolates (comprising the 19 least and most virulent) grown in vitro. Genetic polymorphisms were identified from whole-genome sequence data. Collectively, the results suggest that these SDSE isolates use multiple genetic pathways to achieve an altered virulence phenotype. Our study integrates genomic, mouse virulence, and RNAseq data to advance our understanding of SDSE pathobiology and its molecular pathogenesis.

sample_organism: Streptococcus dysgalactiae subsp. equisimilis

overall_design: 120 stG62647 SDSE isolates identified in a comprehensive sample of 499 isolates cultured from humans and recovered between 2010 and 2018 in French Brittany were tested for virulence using a mouse model of necrotizing myositis. Of these 120, 19 isolates with near_mortality rates ranging between 20% to 30% (classified as low-virulence isolates), and 19 isolates with near_mortality rates ranging between 70% and 95% (high-virulence isolates), were grown in triplicate, in THY broth in vitro, and RNA-seq cDNA libraries were generated at the mid-exponential phase of growth (ME, OD600=1).

first: J

middle: M

last: Eraso

first: S

middle: B

last: Beres


title: Hydrogen Peroxide Regulation of Root Growth and Stem Cell Differentiation by XAANTAL1 in Arabidopsis thaliana

city: Mexico City

first: Adriana

last: Garay Arroyo

state: Mexico City

address: C.U., Coyoacán

department: Ecología Funcional

country: Mexico

institute: Instituto de Ecología, Universidad Nacional Autónoma de México, UNAM

summary: The balance between cell proliferation, differentiation and elongation rates emerges from gene regulatory networks coupled to various signal transduction pathways, including reactive oxygen species (ROS) and transcription factors, to respond to environmental cues. The Arabidopsis thaliana primary root has become a valuable system to unravel such networks and the role of transcription factors mediating the inhibition of primary root growth by ROS is just beginning to be studied. In this study, we demonstrate that the MADS-box transcription factor XAANTAL1 (XAL1) mediates the role of hydrogen peroxide (H2O2) in primary root growth and root stem cell niche identity. Interestingly, our findings suggest that XAL1 acts as a positive regulator of H2O2 concentration in the root meristem by directly regulating genes involved in oxidative stress response, such as PEROXIDASE 28 (PER28). Moreover, we found that XAL1 is necessary for the H2O2-induced inhibition of primary root growth through the negative regulation of peroxidase and catalase activities. Furthermore, we found that XAL1 together with RETINOBLASTOMA-RELATED (RBR), is also necessary to positively regulate the differentiation of columella stem cells triggered by a moderate oxidative stress induced by H2O2 treatment.

overall_design: The XAANTAL1 loss-of-function mutant xal1-2 exhibits a short root phenotype due to a slower cell division rate, a shorter meristem and a longer cell cycle duration in comparison to the WT (Col-0) (Tapia-López et al., 2008). However, little is known about the genes involved in the mutant phenotype. Thus, we conducted an RNA-Seq analysis to identify differrentially expressed genes (DEGs) (fold change>1.5; p<0.05) in 8 days-old primary roots of xal1-2 mutant in comparison to WT roots.

first: Garay-Arroyo

last: Adriana

first: Zluhan-Martínez

last: Estephania

first: Castañón-Suárez

last: Claudio

first: Gutiérrez-Rodríguez

last: Mario


title: Structurally Diverse PFAS Produce Unique Transcriptomic Changes Linked to Developmental Toxicity in Zebrafish

city: Corvallis

first: Robyn

middle: Leigh

last: Tanguay

state: Oregon

department: Environmental Molecular toxicology

institute: Oregon State University

summary: Transcriptomic data for 48 hours post-fertilization (hpf) wildtype zebrafish developmentally exposed to 15 PFAS, and for 48, 72, and 96 hpf wildtype zebrafish developmentally exposed to nafion byproduct 2.

overall_design: Three separate experiments: A (Control-A compared to PFAS 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13), B (Control-B compared to PFAS 2, 4, 14, 15), and C (Control-C compared to nafion byproduct 2 across developmental stages; s071-100). In all experiments, wildtype zebrafish embryos were statically exposed to PFAS beginning at 6 hours post-fertilization (hpf) until the sampling timepoint, when samples were extracted (Direct-zol RNA MiniPrep kit; Zymo) and processed through library prep (Lexogen QuantSeq 3' mRNA-seq FWD) and sequencing on an Illumina NextSeq 2000 (100 bp single end). For experiments A and B, all samples were collected at 48 hpf and each sample was split between two sequencing runs. Experimental Design for A consisted of controls and treated samples being collected across multiple days with intentional temporal variation, whereas B consisted of controls and treated samples all collected on a single day. For experiment C, samples were collected at 48, 72, and 96 hpf.

first: Yvonne

last: Rericha

first: Lindsey

last: St. Mary

first: Lisa

last: Truong

first: Ryan

last: McClure

first: Jared

last: Martin

first: Scott

last: Leonard

first: Preethi

last: Thunga

first: Michael

middle: T

last: Simonich

first: Katrina

last: Waters

first: Jennifer

middle: A

last: Field

middle: L


title: MARK2/MARK3 kinases are catalytic co-dependencies of YAP/TAZ in human cancer [CUT&RUN]

city: Cold Spring Harbor

first: Olaf

last: Klingbeil

state: New York

address: 1 Bungtown Road

institute: Cold Spring Harbor Laboratory

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The effect of gene perturbation on the histone mark H3K27ac was evaluated in human Cas9 expressing MDA-MB231 cancers

overall_design: Human pancreatic cancer cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). CUT&RUN was performed according to manufacturer’s instructions (EpiCypher-CUTANA Kit) and Sequencing libraries were generated using  Ultra II DNA Library Prep Kit (NEBNext)

first: Chris

last: Vakoc


title: Differentiating visceral sensory ganglion organoids from induced pluripotent stem cells[chip_scRNAseq]

status: Public on Jul 19 2024

city: Seoul

first: Hwee-Seon

last: Park

address: 103, Daehak-ro, Jongno-gu, Seoul

department: Department of Biomedical Sciences

country: South Korea

institute: Seoul National University

summary: The ability to generate visceral sensory neurons (VSN) from induced pluripotent stem (iPS) cells may help to gain insights into how the gut-nerve-brain axis is involved in neurological disorders. We established a protocol to differentiate human iPS-cell-derived visceral sensory ganglion organoids (VSGOs). VSGOs exhibit canonical VSN markers, and single-cell RNA sequencing revealed heterogenous molecular signatures and developmental trajectories of VSGOs aligned with native VSN. We integrated VSGOs with human colon organoids on a microfluidic device and applied this axis-on-a-chip model to Alzheimer's disease. Our results suggest that VSN could be a potential mediator for propagating gut-derived amyloid and tau to the brain in an APOE4- and LRP1-dependent manner. Furthermore, our approach was extended to include patient-derived iPS cells, which demonstrated a strong correlation with clinical data.

overall_design: Five chips were collected to create each library. For each chamber of the axis-on-a-chip, single-nucleus sequencing of VSGO and single-cell RNA sequencing of iPSC-derived colon organoids were performed. Additional information can be found in the supplementary files attached to the article.

first: Kyusik

last: Ahn

first: Sieun

last: Choi

first: Hojeong

last: Lee

first: Hyunjung

first: Seok Beom

last: Hong

first: Jihui

last: Han

first: Jong Won

first: Jinchul

first: Jaehoon

last: Song

first: Kyunghyuk

first: Bukyung

last: Cha

first: Minseop

last: Kim

first: Hui-Wen

last: Liu

first: Hyeonggyu

first: Sang Jeong

first: Seok

last: Chung

first: Jong-Il

first: Inhee

last: Mook-Jung


title: RNA-sequencing of siNT and siRAD52 transfected HeLa cells

city: New York

first: Simon

last: Powell

state: NY

address: 1250 1st Ave

institute: Memorial Sloan Kettering Cancer Center

summary: Gene expression profiling by RNA sequencing of control (siNT) and RAD52-depleted (siRAD52) HeLa cells.

overall_design: To test how RAD52 knockout alters transcriptional profiles, we transfected HeLa cells with the siRAD52 and siNT and collected their RNA for high-throughput sequencing.


title: MARK2/MARK3 kinases are catalytic co-dependencies of YAP/TAZ in human cancer [RNA-seq]

first: Olaf

summary: The effect of gene perturbation on the transcriptome was evaluated in human Cas9 expressing cancer cell lines

overall_design: Human pancreatic cancer cell lines were cultured in DMEM,RPMI or HITES  supplemented with 5 or 10% fetal bovine serum (FBS). RNA-extraction (Triazol) and Poly-A followed by library preparation was performed in accordance to manufacturer’s instructions (Illumina Truseq Kit).

first: Christopher

middle: R


title: Differentiating visceral sensory ganglion organoids from induced pluripotent stem cells

first: Hwee-Seon

overall_design:
We established a novel protocol to differentiate visceral sensory ganglion organoid (VSGOs) and induced-visceral sensory neurons (i-VSNs) from induced pluripotent stem cells (iPSCs).

VSGOs and i-VSNs among two isogenic iPSC lines (E3, E4), progenitors of VSGOs (Otic-epibranchial progenitor domain(OEPD), Epibranchial placode(EpP) were analyzed using single cell and single nucleus RNA sequencing. Additional information can be found in the supplementary files attached to the article.

first: Kyusik

first: Sieun

first: Hojeong Lee

first: Hyunjung

first: Seok Beom


title: Neoadjuvant immune checkpoint blockade in women with mismatch repair deficient endometrial cancer: a phase I study

city: Groningen

first: Marco

last: de Bruyn

address: Hanzeplein 1

department: Obstetrics&Gynecology

country: Netherlands

institute: University Medical Center Groningen

summary: Neoadjuvant immune checkpoint blockade (ICB) has shown unprecedented activity in mismatch repair deficient (MMRd) colorectal cancers, but its effectiveness in MMRd endometrial cancer (EC) remains unknown. In this investigator-driven, phase I, feasibility study (NCT04262089), 10 women with MMRd EC of any grade, planned for primary surgery, received two cycles of neoadjuvant pembrolizumab (200 mg IV) every three weeks. All patients completed treatment without severe toxicity. A partial radiologic response was observed in 3/10 patients, 5/10 patients had stable disease and 2/10 patients were non-evaluable on Magnetic Resonance Imaging (MRI). Pathologic response was observed in 5/10 patients, with 2 patients showing a major pathologic response. No patient achieved a complete pathologic response. At median duration of follow-up of 22.5 months, two non-responders experienced disease recurrence. Monoclonal T cell expansion significantly correlated with treatment response. Tumour-draining lymph nodes displayed clonal overlap with intra-tumoural T cell expansion. All pre-specified endpoints were reached. Neoadjuvant ICB with pembrolizumab proved safe and induced pathologic, radiologic, and immunologic responses in MMRd EC, warranting further exploration of extended neoadjuvant treatment.

overall_design: Intratumoral T cells of 3 patients following neoadjuvant treatment were isolated by Fluorescence-activated cell sorting (FACS) according to the presence or abscence of CD45, CD3, CD4, CD8, CD19, CD14, CD56, EpCAM, CXCR5 and PD1 and analyzed using scRNAseq and scTCRseq.

first: Anneke

last: Eerkens

first: Koen

last: Brummel

first: Annegé

last: Vledder

first: Sterre

last: Paijens

first: Marta

last: Requesens

first: Domenik

last: Loiero

first: Nienke

last: van Rooij

first: Annechien

last: Plat

first: Floris-Jan

last: Haan

first: Patty

last: Klok

first: Refika

last: Yigit

first: Thijs

last: Roelofsen

first: Natacha

last: de Lange

first: Rie

last: Klomp

first: David

last: Church

first: Arja

last: ter Elst

first: René

last: Wardenaar

first: Diana

last: Spierings

first: Floris

last: Foijer

first: Viktor

middle: H

last: Koelzer

first: Tjalling

last: Bosse

first: Joost

last: Bart

first: Mathilde

last: Jalving

first: Anna

middle: K

last: Reyners

first: Hans

middle: W

last: Nijman


title: Tissue-resident memory T cells- iPSC-cardiomyocyte co-cultures

city: london

first: Paula

last: Longhi

address: charterhouse sq

country: United Kingdom

institute: Queen Mary University of London

summary: The main aim of this study was to investigate a direct alteration of the electrical properties of coupled cardiomyocytes by tissue-memory T cells from epicardial adipose tissue.

overall_design: Epicardial adipose tissue was obtained from patients undergoing heart surgery. Immune cells were siolated and CD4 (CD3+ CD4+CD69+PD1+CCR7-CD45RO+) and CD8 (CD3+ CD4+CD69+PD1+CCR7-CDR5RO+) tissue-resident memory T cells were purified by FACS. As control, momory, non-TRM cells were also purified (CD3+ CD69-PD1-CCR7-CDR5RO+). Purified T cells were then co-cultures with iPSC-derived Atrial cardiomyocytes for 18 hours. Cells were washed and iPSC colected for RNA extraction.

first: Vishal

last: Vyas


title: Comparing transcriptional profiles of CD14+  monocytes from melanoma patients and healthy donors by scRNA-seq.

city: Mannheim

first: Volker

last: Ast

address: Theodor-Kutzer-Ufer 1-3

department: NGS Core Facility

country: Germany

institute: University Hospital Mannheim

summary: Myeloid-derived suppressor cells (MDSC), a heterogeneous population of myeloid cells, accumulate in the melanoma microenvironment. With the ability to inhibit anti-tumor T cell responses, MDSC have been shown to promote immunosuppression, enhancing tumor progression and tumor cell resistance to the immunotherapy. Novel markers are still needed to define MDSC. In this study,we wanted to find possible markers for M-MDSC by comparing cd14+ monocytes from melanoma patients and healthy donors by scRNA-seq.

overall_design: Peripheral blood mononuclear cells (PBMC) were isolated from whole blood of 2 treatment-naive stage IV melanoma patients and 2 healthy individuals via density gradient centrifugation.CD14+ monocytes were isolated from PBMC by MACS (Miltenyi Biotec) according to the manufacturer’s instructions. 10x Genomics Chromium single-cell 3′ library kit was used to prepare sequencing library. 10000 cells were loaded on the chips.

first: Feyza

last: Gul Ozbay Kurt

last: Umansky


title: The human mitochondrial mRNA structurome reveals mechanisms of gene expression

first: Silvi

last: Rouskin

state: MA

address: 77 Ave Louis Pasteur

department: Microbiology

institute: Harvard

summary: The mammalian mitochondrial genome encodes thirteen proteins of the oxidative phosphorylation system, crucial in aerobic energy transduction. These proteins are translated from 9 monocistronic and 2 bicistronic transcripts, whose native structures remain unexplored, leaving fundamental molecular determinants of mitochondrial gene expression unknown. To address this knowledge gap, we developed a mitoDMS-MaPseq approach and used DREEM clustering to resolve the native mt-mRNA structurome in human mitochondria. In this way, we gained insights into mt-mRNA biology and translation regulatory mechanisms, including a unique translational frameshifting for the ATP8/ATP6 transcript. Furthermore, absence of the mt-mRNA maintenance factor LRPPRC led to a mitochondrial transcriptome structured differently, with specific mRNA regions exhibiting increased or decreased structuredness. This highlights the role of LRPPRC in maintaining mRNA folding to promote mt-mRNA stabilization and efficient translation. In conclusion, our mt-mRNA folding maps reveal novel mitochondrial gene expression mechanisms, serving as a detailed reference and tool for studying them in different physiological and pathological contexts.

overall_design: To investigate the structure of human mitochondrial mRNAs. Briefly, mitochondria isolated from HEK-293T cells were incubated with DMS and RNA was extracted from isolated mitochondria. An off the shelf IDT RNAseq library preparation kit was used in conjunction with TGIRT to mediated mutagenesis where DMS modifications occur. This sequencing data was then aligned to the mitochondrial genome and structural models were developed using Detection-of-RNA-folding-Ensembles-using-Expectation-Maximization (DREEM, Rouskin Lab) and RNAstructure (Mathews lab).

first: J

middle: C

last: Moran

first: Amir

last: Brivanlou

first: Michele

last: Brischigliaro

first: Flavia

last: Fontanesi

first: Antoni

last: Barrientos


title: Epitranscriptomic Reader YTHDF2 Regulates SEK1(MAP2K4)-JNK-cJUN Inflammatory Signaling in Astrocytes during Neurotoxic Stress

city: Athens

first: Anumantha

last: Kanthasamy

state: GA

address: B122 Life Sciences Bldg University of Georgia

institute: University of Georgia

summary: As the most abundant glial cells in the CNS, astrocytes dynamically respond to neurotoxic stress, however, the key molecular regulators controlling the inflammatory status of these sentinels during neurotoxic stress have remained elusive. Herein, we demonstrate that the m6A epitranscriptomic mRNA modification tightly regulates the pro-inflammatory functions of astrocytes.  Specifically, the astrocytic neurotoxic stresser, manganese (Mn),  downregulated the m6A reader YTHDF2 in human and mouse astrocyte cultures and in the mouse brain. Functionally, YTHDF2 knockdown augmented, while its overexpression dampened, neurotoxic stress induced proinflammatory response, suggesting YTHDF2 serves as a key upstream regulator of inflammatory responses in astrocytes. Mechnistically, YTHDF2 RIP-sequencing identified MAP2K4 (MKK4; SEK1) mRNA as a YTHDF2 target influencing inflammatory signaling. Our target validation revealed Mn-exposed astrocytes mediates  proinflammatory response by activating the phosphorylation of SEK1, JNK, and cJUN signaling.  Collectively, YTHDF2 serves a key upstream ‘molecular switch’ controlling SEK1(MAP2K4)-JNK-cJUN proinflammatory signaling in astrocytes.

overall_design: Human U251-MG astrocytoma cells were treated with manganese chloride (100 µM) and YTHDF2-RIP sequencing was performed to identify YTHDF2 target mRNAs that may control astrocytic inflammation. We expected a loss of enrichment in manganese-treated samples, concurrent with an upregulation of its cognate protein. 3 samples per group (control vs manganese), both inputs and immunoprecipitated fractions.

first: Emir

last: Malovic

first: Alyssa

last: Ealy

first: Phillip

middle: J

last: Hsu

first: Souvarish

last: Sarkar

first: Cameron

last: Miller

first: Dharmin

last: Rokad

first: Cody

last: Goeser

first: Aleah

last: Hartman

first: Allen

last: Zhu

first: Bharati

last: Palanisamy

first: Gary

last: Zenitsky

first: Huajun

last: Jin

first: Vellareddy

last: Anantharam

first: Arthi

first: Chuan

last: He

first: Ahyoung

last: Jan


title: The single-cell transcriptomic atlas iPain identifies senescence of nociceptors as a therapeutical target for chronic pain treatment

city: Solna

first: Saida

last: Hadjab

address: Solnavägen 9

department: Neuroscience

country: Sweden

institute: Karolinska Institutet

summary: Chronic pain remains a significant medical challenge with complex underlying mechanisms, and an urgent need for new treatments. Our research built and utilized the iPain single-cell atlas to study chronic pain progression in dorsal root and trigeminal ganglia. We discovered that senescence of a small subset of pain-sensing neurons may be a key driver of chronic pain. This mechanism was observed in animal models after nerve injury and in human patients with chronic pain or diabetic neuropathy. Notably, treatment with senolytics, drugs that remove senescent cells, reversed pain symptoms in mice post-injury. These findings highlight the crucial role of cellular senescence in chronic pain development, demonstrate the therapeutic potential of senolytic treatments, and underscore the value of the iPain atlas for future pain research.

overall_design: DRG was isolated from mice with or without CCI procedure. Nuclei were extracted from frozen DRG. Pooled nuclei were used for snMultiomic. For smartseq3, NeuN positive nuclei were sorted and analyzed.


title: Comparative Analysis Highlights Uniconazole’s Efficacy in Enhancing the Cold Stress Tolerance of Mung Beans by Targeting Photosynthetic Pathways

city: Zhanjiang

first: Mingming

last: Chen

address: Haida Street No.1

country: China

institute: Guangdong Ocean University

summary: Soybean (Glycine max) and mung bean (Vigna radiata) are key legumes with global importance, but their mechanisms for coping with cold stress—a major challenge in agriculture—have not been thoroughly investigated, especially in a comparative study. This research aimed to fill this gap by examining how these two major legumes respond differently to cold stress and exploring the role of uniconazole, a potential stress mitigator. Our comprehensive approach involved transcriptomic and metabolomic analyses, revealing distinct responses between soybean and mung bean under cold stress conditions. Notably, uniconazole was found to significantly enhance cold tolerance in mung bean by upregulating genes associated with photosynthesis, while its impact on soybean was either negligible or adverse. To further understand the molecular interactions, we utilized advanced machine learning algorithms for protein structure prediction, focusing on photosynthetic pathways. This enabled us to identify LOC106780309 as a direct binding target for uniconazole, confirmed through isothermal titration calorimetry. This research establishes a new comparative approach to explore how soybean and mung bean adapt to cold stress, offers key insights to improve the hardiness of legumes against environmental challenges, and contributes to sustainable agricultural practices and food security.

overall_design: RNA sequencing


title: Crnic Institute Human Trisome Project - Trisomy 21 Model Atlas: PolyA RNA-seq from adult mouse brain hippocampus

status: Public on Jul 18 2024

city: Aurora

first: Matthew

middle: D

last: Galbraith

state: CO

department: Department of Pharmacology; Linda Crnic Institute for Down Syndrome

institute: University of Colorado Anschutz Medical Campus

summary: Analysis of steady-state mRNA levels in adult mouse brain hippocampus at week 17-18 from wild-type C57BL/6J, Dp16, and interferon receptor dosage-normalized Dp16^2xIFNRs mice.

overall_design: Comparison of wild-type (n=6), Dp16 (n=6), and Dp16^2xIFNRs (n=5).

first: Katherine

last: Waugh

first: Kelly

last: Sullivan

first: Joaquin

last: Espinosa


title: Crnic Institute Human Trisome Project - Trisomy 21 Model Atlas: PolyA RNA-seq from adult mouse kidney tissue

summary: Analysis of steady-state mRNA levels in adult mouse kidney tissue at 17-18 weeks from wild-type C57BL/6J, Dp16, and interferon receptor dosage-normalized Dp16^2xIFNRs mice.

overall_design: Comparison of wild-type (n=6), Dp16 (n=6), and Dp16^2xIFNRs (n=6).


title: MARK2/MARK3 kinases are catalytic co-dependencies of YAP/TAZ in human cancer

first: Olaf

last: Klingbeil

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

overall_design: Refer to individual Series

first: Christopher

last: Vakoc


title: Lymphatic-localized Treg-mregDC crosstalk limits antigen trafficking and restrains anti-tumor immunity [TCR-Seq]

city: Xiamen

first: Shuqin

last: Li

state: Fujian Province

address: Xiang'an south rd, Xiang'an district

department: Life Science

institute: Xiamen University

summary: To investigate the clonality of tumor Treg cells, we performed TCRseq on tumor Treg cells from Apc Min/+ mice versus normal colonic Treg cells from WT mice.

overall_design: Using TCRseq to compare the TCR repertoire of tumor Treg cells from Apc Min/+ mice versus normal colonic Treg cells from WT mice.

first: Kairui

last: Mao

first: Siyuan

last: You


title: Crnic Institute Human Trisome Project - Trisomy 21 Model Atlas: PolyA RNA-seq from adult mouse brain cortex

summary: Analysis of steady-state mRNA levels in adult mouse brain cortex at week 17-18 from wild-type C57BL/6J, Dp16, and interferon receptor dosage-normalized Dp16^2xIFNRs mice.


title: Crnic Institute Human Trisome Project - Trisomy 21 Model Atlas: PolyA RNA-seq from adult mouse spleen tissue

summary: Analysis of steady-state mRNA levels in adult mouse spleen tissue at 17-18 weeks  from wild-type C57BL/6J, Dp16, and interferon receptor dosage-normalized Dp16^2xIFNRs mice.


title: LX1 Dual Targets AR Variants and AKR1C3 in Advanced Prostate Cancer Therapy

city: Sacramento

first: Allen

last: Gao

state: California

address: 4645 2nd Ave, Research III Bldg

department: Urologic Research

institute: University of California Davis

summary: The development of resistance to current standard-of-care treatments, such as androgen receptor (AR) targeting therapies, remains a major challenge in the management of advanced prostate cancer. There is an urgent need for new therapeutic strategies targeting key resistant drivers such as AR variants like AR-V7 and steroidogenic enzymes such as AKR1C3 to overcome drug resistance and improve outcomes for patients with advanced prostate cancer. Here we have designed, synthesized, and characterized a novel class of LX compounds targeting both the AR/AR-Variants and AKR1C3 pathways. Molecular docking and in vitro studies demonstrated that LX compounds bind to the AKR1C3 active sites and inhibit AKR1C3 enzymatic activity. LX compounds were also shown to reduce AR/AR-V7 expression and inhibit their target gene signaling. LX1 inhibited the conversion of androstenedione into testosterone in tumor-based ex vivo enzyme assays. In addition, LX1 inhibited the growth of cells resistant to antiandrogens including enzalutamide, abiraterone, apalutamide and darolutamide in vitro. A synergistic effect was observed when LX1 was combined with antiandrogens and taxanes, indicating the potential for this combination in treating resistant prostate cancer. Treatment with LX1 significantly decreased tumor volume, serum PSA levels, as well as reduced intratumoral testosterone levels, without affecting mouse body weight. Furthermore, LX1 was found to overcome resistance to enzalutamide treatment, and its combination with enzalutamide further suppressed tumor growth in both the CWR22Rv1 xenograft and LuCaP35CR PDX models. Collectively, the dual effect of LX1 in reducing AR signaling and intratumoral testosterone, along with its synergy with standard therapies in resistant models, underscores its potential as a valuable treatment option for advanced prostate cancer.

overall_design: To investigate the dual targeting effects of LXs compounds in advanced prostate cancer, we employed castration-resistant prostate cancer cells with enzalutamide resistance C4-2B MDVR cells treated with DMSO, LX1 and LX5 at 5μM for 48h incubation, and LuCaP35CR patient-derived xenograft tumor models treated with vehicle control and LX1 at 90mg/kg, p.o.. We then performed gene expression profiling analysis using data obtained from RNA-seq.

first: Pui-Kai

first: Shu

last: Ning

first: Cameron

last: Armstrong

first: Enming

last: Xing


title: Regulated and adaptive in vivo insulin secretion from islets only containing β-cells

city: Geneva

first: Marta

last: Perez Frances

state: Geneva

address: 1, rue Michel-Servet

country: Switzerland

institute: University of Geneva

summary: The activity of pancreatic islets’ insulin-producing β-cells is closely regulated by systemic cues and, locally, by adjacent islet hormone-producing “non-β-cells” (namely α-, δ- and γ-cells). Still, it is unclear whether the presence of the non-β-cells is a requirement for accurate insulin secretion. Here, we generated and studied a mouse model in which adult islets are exclusively composed of β-cells, and human pseudoislets containing only primary β-cells. Mice lacking non-β-cells had optimal blood glucose regulation. They exhibited enhanced glucose tolerance, insulin sensitivity and restricted body weight gain under high-fat diet. The insulin secretion dynamics in islets composed of only β-cells was like in intact islets, both in homeostatic conditions and upon extreme insulin demand. Similarly, human β-cell pseudoislets retained the glucose-regulated mitochondrial respiration, insulin secretion and exendin-4 responses of human islets comprising all four cell types. Together, the findings indicate that non-β-cells are dispensable for blood glucose homeostasis and β-cell function. This is particularly relevant in diabetes, where non-β-cells become dysfunctional and worsen the disease’s pathophysiology. These results support efforts aimed at developing diabetes treatments by generating β-like cell clusters devoid of non-β-cells, as for example from human embryonic stem cells and/or by in situ conversion of non-β-cells into insulin producers.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for FAC-sorted pancreatic beta-cells of control mice with intact islets and DT-treated beta-only mice, both under 2-hour refed or 8-hour fasting conditions.

last: Perez-Frances

first: Eva

last: Bru-Tari

first: Christian

last: Cohrs

first: Maria

middle: V

last: Abate

first: Léon

last: van Gurp

first: Kenichiro

last: Furuyama

first: Stephan

last: Speier

first: Fabrizio

last: Thorel

first: Pedro

last: Herrera


title: Longitudinal Immune Profiling and Corticosteroid Modulation of the Immediate Innate Immune Response to ChAdOx1 nCoV-19

city: Ulju-gun

first: Wonhyo

last: Lee

state: Ulsan

address: Unist-gil 50

department: Biological Sciences

institute: UNIST

summary:
ChAdOx1 nCoV-19, a chimpanzee adenovirus (ChAd)-vector vaccine expressing the SARS-CoV-2 spike protein, has elicited robust immunological responses in large populations. Despite its proven efficacy and safety, some recipients have reported immediate inflammatory reactions post-vaccination. We comprehensively examined the immune landscape following ChAdOx1 nCoV-19 vaccination based on the epigenomic profiles of monocytes.
Glucocorticoids are wildly used as steroid anti-inflammatory drugs to modify acute and chronic inflammation. Recently, dexamethasone is used to modify inflammation in COVID19 and side-effects after COVID19 vaccination. ChAdox1 nCoV-19 induces interferon responses and inflammation in human. In addition to IFNa, TNF and IL1b treatment to mimic inflammatory environment gives general effects of glucocorticoids in complex inflammatory context.

overall_design:
Peripheral blood samples were obtained from five healthy volunteers at four post-ChAdOx1 nCoV-19 vaccination time points: days 0 (baseline), 1, 3, and 14.
To mimic COVID19 and inflammatory environment, we pre-treated combination of IFNa(1000U), TNF(20ng), and IL1b(20ug) to 1 million of PBMC derived CD14+ cells with 100mg/ml of MCSF. In order to investigate glucocorticoid effects in chromatin-remodeling level, we treated 100nM of Dexamethasone to each condition at 3h after cytokine treatment. Cells are havested 12h after dexamethasone treatment.

first: Sung

last: Park


title: Effect of fasting on transcription in intestinal cells dissected from adult C. elegans

city: Mainz

first: Jan

last: Padeken

address: Ackermannweg 4

institute: Institute of Molecular Biology

summary: Chromatin architecture is a fundamental mediator of genome function. Fasting is a major environmental cue across the animal kingdom. Yet, how it impacts on 3D genome organization is unknown. Here, we show that fasting induces an intestine-specific, reversible, and large-scale spatial reorganization of chromatin in C. elegans. This fasting-induced 3D genome reorganization requires inhibition of the nutrient-sensing mTOR pathway, through the regulation of RNA Pol I, but not Pol II nor Pol III, and is accompanied by remodeling of the nucleolus. By uncoupling the 3D genome configuration from the animal´s nutritional status we find that the spatial reorganization of chromatin correlates with the expression of metabolic and stress-related genes, potentially supporting the transcriptional response in fasted animals. Our work documents the first large-scale chromatin reorganization triggered by fasting and reveals that mTOR and RNA Pol I shape genome architecture in response to nutrients.

overall_design: RNA was extracted from intestines dissected from day 1 N2 wild type adults. Briefly, synchronized L1s by hypochlorite treatment were grown on OP50-seeded plates until day 1 of adulthood. Next, a fraction of adults was immediately dissected for the fed state, the rest was washed 3 times with M9 buffer for 10 minutes and fasted in M9 buffer for 12 hours on a roller at 20°C prior to intestine dissection.

first: Nada

last: Al-Refaie

first: Francesco

last: Padovani

first: Johanna

last: Hornung

first: Lorenz

last: Pudelko

first: Francesca

last: Binando

first: Andrea

last: del Carmen Fabregat

first: Qiuxia

last: Zhao

first: Benjamin

last: Towbin

first: Elif

middle: S

last: Cenik

first: Nicholas

last: Stroustrup

first: Kurt

last: Schmoller

first: Daphne

last: Cabianca


title: Spatial transcriptomic of EAT and Atrial appendage from patienst with Atrial Fibrillation

first: Paula

last: Longhi

summary: To examine biological changes and regionality dictating structural remodelling in Atrial Fibrillation, we performed NanoString GeoMx Digital Spatial Profiling (DSP) of tissue biopsies from AF and SR patients. Regions were selected from adipose tissue and atrail appendage tissue close or far apart from tissue interphase.

overall_design: AA tissue with surrounding EAT samples were selected from 4 patients of which 2 were from AF patients and 2 from SR patients. Freshly cut paraffin-embedded sample slides were sent to NanoString Technologies. Sample slides were deparaffinised and stained with fluorescent antibody markers to distinguish between the adipose tissue (FABP4) and cardiac tissue (troponin). Specific regions of interest (ROIs) were then pre-selected either side of the border zones of the AA/EAT and deep in the AA and deep in the EAT. Thus 4 ROIs were selected per slide in triplicates.

first: Vishal

last: Vyas


title: Lymphatic-localized Treg-mregDC crosstalk limits antigen trafficking and restrains anti-tumor immunity

first: Shuqin

last: Li


title: Tumor RNA sequencing identifies a group of Mycosis Fungoides patients with failure of skin-directed therapies

city: Tampere

first: Henry

middle: Juho

last: Barton

state: Pirkanmaa

address: Hämeenkatu 14 C 33

country: Finland

institute: Genevia Technologies Oy

summary: Mycosis fungoides (MF), the most common type of primary cutaneous T-cell lymphoma, has a heterogeneous clinical course, and the factors determining disease progression from the indolent to the aggressive disease stages are not well known. Our previous study described prognostic groups of MF cases according to the transcription factors Twist and Zeb1. Using Twist expression, we differentiated distinct groups based on their global RNA expression. Using the same dataset with 40 formalin-fixed paraffin-embedded MF samples, we performed RNA sequencing using bioinformatics tools. Based on the consensus clustering, we defined four clusters with different disease pathologies and prognostic groups. Cluster 3 showed the shortest time to skin-directed therapy failure (p=0.031), and with Cluster 1 they included all the MF-related deaths (2/40). Cluster 3 presented a higher proportion of lymphocytic infiltrates per sample (p<0.001), with the highest percentages (>60 %) and a higher rate of CD30+ tumor cells (over 10 %, p=0.048). Cluster 3 samples also showed upregulated genes related to keratinocyte, basal cell, and melanocyte activities. This is the first study to describe the association between gene expression, tumor cell density, reactive cell infiltration, and failure of skin-directed therapies

first: Marjaana

last: Häyrinen

first: Hanna-Riikka

last: Teppo

first: Liisa

last: Väkevä

first: Annamari

last: Ranki

first: Katja

last: Porvari

first: Jenni

last: Kiiskilä

first: Milla

middle: E

last: Kuusisto

first: Hanne

last: Kuitunen

first: Siria

last: Lemma

first: Helka

last: Sahi

first: Kirsi-Maria

last: Haapasaari

first: Outi

last: Kuittinen


title: Expression data from S100A9 and HMGB1 treated human CD14+ monocytes

first: Carolina

last: Delatorre

institute: University Heidelberg

summary:
TLR4 signaling was reported to be involved in the upregulation of PD-L1 expression and induction of immunosuppressive properties of human CD14+ monocytes. Damage associated molecular patterns (DAMPs) S100A8, A9, and HMGB1, which act as endogenous TLR4 ligands, drive MDSC activation and were shown to be markedly expressed in the tumor microenvironment (TME) of solid tumors.
The role of S100A8/9 and HMGB1 in the acquisition of MDSC immunosuppressive activity remains to be better defined.

overall_design: CD14+ monocytes were treated with recombinant (r) S100A9 and HMGB1 in the presence of GM-CSF with or without FPS-ZM1 or TAK-242 for 72 h. Monocytes were also stimulated with GM-CSF only for 72h (Control). After 72h, total RNA was isolated from monocytes and gene expression profiling was performed.

first: Feyza

middle: G

last: Özbay Kurt


title: Double strand break repair protein TIRR regulates RNA metabolism in response to DNA damage and cell stress [RNA-Seq]

city: Oxford

first: Irina

last: Chelysheva

address: Roosevelt Dr

department: NDM

institute: University of Oxford

summary: Tudor interacting repair regulator (TIRR) is a well-studied p53 binding protein 1 (53BP1) interactor in response to DNA double strand breaks (DSBs). TIRR is also an RNA binding protein, however its role in RNA regulation in response to cellular insults is not understood.  Here we show that TIRR binds to specific group of mRNAs in response to DNA damage. TIRR bound transcripts encode for transcription factors and RNA polymerase II (RNAPII) transcription regulators. Furthermore, TIRR modulates the formation of RNA processing bodies (P bodies/PBs), in response to DSBs. TIRR also regulates the formation of the closely related but distinct compartments known as stress granules (SGs) in response to cell stress. Furthermore, TIRR dissociates from 53BP1 in response to sodium arsenite treatment independently of Ataxia Telangiectasia Mutated Protein (ATM) signalling. Finally, TIRR interacts with the nuclear export protein Exportin-1 (XPO1) upon DNA damage. TIRR bound RNA co-localises with PB and SGs and TIRR depletion leads to nuclear RNA retention. This work reveals that TIRR orchestrates mRNA nuclear export, metabolism and storage in PBs/SGs. Hence TIRR regulates RNA mediated DNA damage and cell stress responses independently of its canonical 53BP1 binding role.

overall_design: RNA immunoprecipitation and sequencing (RIP-Seq) experiments were perfomed to identify specific RNAs bound to TIRR after DNA damage using TIRR-GFP and GFP cells.  Hela cells with integrated inducible TIRR-GFP or GFP only (as a control for background) were used to induce expression of TIRR-GFP or GFP with doxycycline. DNA double strand breaks were induced with Etoposide (Eto). Total RNA-sequencing was performed in stably integrated inducible shRNA Hela cells, expressing shRNA for TIRR or GFP as a control. TIRR knockdown was induced using doxycycline for 48 hours. RNA was collected before andafter DNA damage,inducedby IR and followed by 1 hour incubation,or by ETO incubation for 2 hours. It was thensubjected to paired-end total RNA sequencing on the Illumina NovaSeq6000.

first: Ruth

middle: F

last: Ketley

first: Monika

last: Gullerova

first: Michelle

last: Glossop


title: Lymphatic-localized Treg-mregDC crosstalk limits antigen trafficking and restrains anti-tumor immunity [RNA-seq]

first: Shuqin

last: Li

summary: To investigate the mechanism by which Treg cell positioning is regulated in our manuscript,we performed RNAseq on tumor Treg cells from  Apc Min/+ Foxp3 DTR mice versus normal colonic Treg cells from Foxp3 DTR mice.

overall_design: Comparative gene expression profiling analysis of RNAseq data for Treg cells from Apc Min/+ Foxp3 DTR colon tumor or Foxp3 DTR mice.

first: Kairui

last: Mao

first: Siyuan

last: You


title: Double strand break repair protein TIRR regulates RNA metabolism in response to DNA damage and cell stress


title: Lymphatic-localized Treg-mregDC crosstalk limits antigen trafficking and restrains anti-tumor immunity [scRNA-seq]

summary: Tumor microenvironment (TME) plays critical roles in both tumor progression and immunotherapeutic efficacy. Here using single-cell RNA sequencing (scRNA-seq), we described the TME heterogeneity and found a specific state of CCL22+ dendritic cell (mregDC) cross talk close to regulatory T cells (Treg). We analyzed cell–cell communication mediated by ligand–receptor interactions and finally found that mregDCs probably attract Treg cells via chemotaxis and physically interact with them in tumor. Within this cross talk, Treg cells received activation signals from mregDCs and upregulated suppressive and adhesion molecules, including PD-1, ICOS, CTLA-4 and OX-40, further enhancing their interaction with mregDCs. In turn, the association of Treg cells with mregDCs restrained the trafficking of tumor antigen-bearing dendritic cells to draining mesenteric lymph nodes and thus dampened the presentation of tumor antigens to initiate anti-tumor immune responses in a cell contact dependent manner.

overall_design: We analyzed the single cell transcriptomes of wild-type colon and Apc Min/+ colon tumor, a spontaneous mouse colorectal tumor model. Tumor single cell suspensions were enriched for immune cells and rapidly processed for analysis on the inDrops scRNA-seq platform. After filtering to exclude putative cell doublets and stressed or dead cells, a total of 23,922 cells passed the quality control, of which 14,445 were from tumor and the remaining 9,477 cells were from normal colon tissue.


title: Crnic Institute Human Trisome Project - Trisomy 21 Model Atlas: PolyA RNA-seq from adult mouse liver tissue

first: Matthew

last: Galbraith

summary: Analysis of steady-state mRNA levels in adult mouse liver tissue at 17-18 weeks from wild-type C57BL/6J, Dp16, and interferon receptor dosage-normalized Dp16^2xIFNRs mice.

first: Katherine

last: Waugh

first: Kelly

last: Sullivan

first: Joaquin

middle: M

last: Espinosa


title: Transcriptomic Evaluation of a Stress Vulnerability Network Using Single-Cell RNA Sequencing in Mouse Prefrontal Cortex

city: Iowa City

first: Benjamin

last: Hing

state: IA

address: 51 Newton Road, BSB 6-509

institute: University of Iowa

summary: We used single cell RNA sequencing (scRNA-Seq) to study transcriptomic changes within individual cell types underlying stress vulnerability network activity.

overall_design: The prefrontal cortex was extracted from mice two days after forced interaction test when local field potential recordings were taken from  the nucleus accumbens, basolaterl and central amygdala, ventral hippocampus and ventral tegmental area.

first: Sara

last: Mitchell

first: Maureen

last: Eberle

first: Yassine

last: Filali

first: Molly

last: Matkovich

first: Mukundan

last: Kasturirangan

first: Ian

last: Hultman

first: Whitney

last: Wyche

first: Alli

last: Jimenez

first: Radha

last: Velamuri

first: Micah

last: Johnson

first: Sanvesh

last: Srivastava

first: Rainbo


title: Double strand break repair protein TIRR regulates RNA metabolism in response to DNA damage and cell stress [RIP-seq]

first: Irina

last: Chelysheva

summary:
Tudor interacting repair regulator (TIRR) is a well-studied p53 binding protein 1 (53BP1) interactor in response to DNA double strand breaks (DSBs). TIRR is also an RNA binding protein, however its role in RNA regulation in response to cellular insults is not understood.
Here we show that TIRR binds to specific group of mRNAs in response to DNA damage. TIRR bound transcripts encode for transcription factors and RNA polymerase II (RNAPII) transcription regulators. Furthermore, TIRR modulates the formation of RNA processing bodies (P bodies/PBs), in response to DSBs. TIRR also regulates the formation of the closely related but distinct compartments known as stress granules (SGs) in response to cell stress. Furthermore, TIRR dissociates from 53BP1 in response to sodium arsenite treatment independently of Ataxia Telangiectasia Mutated Protein (ATM) signalling.
Finally, TIRR interacts with the nuclear export protein Exportin-1 (XPO1) upon DNA damage. TIRR bound RNA co-localises with PB and SGs and TIRR depletion leads to nuclear RNA retention.
This work reveals that TIRR orchestrates mRNA nuclear export, metabolism and storage in PBs/SGs. Hence TIRR regulates RNA mediated DNA damage and cell stress responses independently of its canonical 53BP1 binding role.

overall_design: RNA immunoprecipitation and sequencing (RIP-Seq) experiments were perfomed to identify specific RNAs bound to TIRR after DNA damage using TIRR-GFP and GFP cells.  Hela cells with integrated inducible TIRR-GFP or GFP only (as a control for background) were used to induce expression of TIRR-GFP or GFP with doxycycline. DNA double strand breaks were induced with Etoposide (Eto). Total RNA-sequencing was performed in stably integrated inducible shRNA Hela cells, expressing shRNA for TIRR or GFP as a control. TIRR knockdown was induced using doxycycline for 48 hours, before inducing damage using IR (10Gy). RNA was collected before and 6 hours after IR and subjected to paired-end total RNA sequencing.

first: Ruth

last: Ketley

first: Monika

last: Gullerova


title: ABCA7-dependent induction of neuropeptide Y is required for synaptic resilience in Alzheimer's disease through BDNF/NGFR signaling

first: Caghan

last: Kizil

department: Neurology & TAUB Institute

institute: Columbia University

summary: Genetic variants in ABCA7, an Alzheimer's disease (AD)-associated gene, elevate AD risk, yet its functional relevance to the etiology is unclear. We generated a CRISPR-Cas9-mediated abca7 knockout zebrafish to explore ABCA7's role in AD. Single-cell transcriptomics in heterozygous abca7+/- knockout combined with Aβ42 toxicity revealed that ABCA7 is crucial for neuropeptide Y (NPY), brain-derived neurotrophic factor (BDNF), and nerve growth factor receptor (NGFR) expressions, which are crucial for synaptic integrity, astroglial proliferation, and microglial prevalence. Impaired NPY induction decreased BDNF and synaptic density, which are rescuable with ectopic NPY. In induced pluripotent stem cell-derived human neurons exposed to Aβ42, ABCA7-/- suppresses NPY. Clinical data showed reduced NPY in AD correlated with elevated Braak stages, genetic variants in NPY associated with AD, and epigenetic changes in NPY, NGFR, and BDNF promoters linked to ABCA7 variants. Therefore, ABCA7-dependent NPY signaling via BDNF-NGFR maintains synaptic integrity, implicating its impairment in increased AD risk through reduced brain resilience.

overall_design: WT and abca7+/- zebrafish injected with Amyloid-beta 42 peptide as described (Bhatatrai et al. 2016) and cells were dissociated, sorted by FACS as described (Cosacak et al. 2019). The single-cell encapsulation and cDNA synthesis done by following 10X Genomics' workflows. The reads were aligned to zebrafish genome (GRChZ 11, v 105) was used for gene annotation and assigning reads to genes. Cellranger software version 6.1.2 was used to process the data

first: Elanur

last: Yilmaz

first: Huseyin

last: Tayran

first: Mehmet

middle: I

last: Cosacak

first: Prabesh

last: Bhattarai


title: Analysis of deregulated genes upon Septin9 depletion in C2C12 and primary isolated myoblasts

status: Public on Jul 17 2024

city: Berlin

first: Paul-Lennard

last: Mendez

address: Thielallee 63

department: Institute for Chemistry and Biochemistry

institute: Freie Universität Berlin

summary: Controlled myogenic differentiation is integral to the development, maintenance and repair of skeletal muscle, necessitating precise regulation of myogenic progenitors and resident stem cells. The transformation of proliferative muscle progenitors into multinuclear syncytia involves intricate cellular processes driven by cytoskeletal reorganization. While actin and microtubles have been extensively studied, we illuminate the role of septins, an essential yet still often overlooked cytoskeletal component, in myoblast architecture. Notably, Septin9 emerges as a critical regulator of myoblast differentiation during the initial commitment phase. Knock-down of Septin9 in C2C12 cells and primary mouse myoblasts accelerates the transition from proliferation to committed progenitor transcriptional programs. Furthermore, we unveil significant reorganization and downregulation of Septin9 during myogenic differentiation. Collectively, we propose that filmamentous septin structures and their orchestrated reorganization in myoblasts are part of a temporal regulatory mechanism governing the differentiation of myogenic progenitors. This study sheds light on the dynamic interplay between cytoskeletal components underlying controlled myogenic differentiation.

overall_design: Bulk RNA-seq of C2C12 cells and primary myoblasts was performed in duplicates for a total of 2 conditions (4 C2C12 samples and 4 primary myoblast samples). C2C12 and primary myoblasts were subjected to Septin9 siRNA treatment and non-targeting siRNA was used as a control. C2C12 cells were myogenically differentiatied for 12 hours thorugh serum deprivation (sample collection 60 hours after Septin9 depletion) and primary myoblasts were cultured under proliferatve conditions (sample collection 48 hours after Septin9 depletion).

first: Ugorets

last: Vladimir

first: Mendez

last: Paul-Lennard

first: Knaus

last: Petra


title: Paternal under and over nutrition modify semen composition and preimplantation embryo developmental kinetics in mice

city: Manchester

first: Hannah

last: Morgan

address: Oxford Road

institute: University of Manchester

summary: The importance of parental diet in relation to eventual offspring health is increasing in prominence due to the increased frequency of reproductive age adults consuming poor diets. Whilst maternal health and offspring outcome have been studied in some detail, the paternal impacts are not as well understood. A father’s poor nutritional status has been shown to have negative consequences on fetal growth and development and ultimately impact the long-term adult health of the offspring.  In this study we examined sperm and seminal plasma mediated mechanisms of preimplantation embryo development alterations in response to sub-optimal paternal diets. Male mice were fed a diet to model either under (low protein diet (LPD)) or over (high fat/sugar (Western) diet (WD)) nutrition, LPD or WD supplemented with methyl-donors or a control diet before mating with age-matched control fed females. Male metabolic health was influenced by WD and MD-WD; with significant changes in serum lipids and hepatic 1-carbon metabolites. Sperm RNA sequencing revealed significant changes to mRNA profiles in all groups when compared to CD (LPD: 32, MD-LPD: 17, WD: 53, MD-WD: 35). In vitro embryo development was revealed all sub-optimal diets with and without methyl-donors increased the rate of embryo development. As embryo development can be directed by sperm and seminal plasma mediated mechanism, we also examined the proteomic profile of the seminal plasma, revealing a significant number of changes in all groups compared to control (LPD: 13, MD-LPD: 27, WD: 24, MD-WD: 19). Male diet also altered uterine gene expression examined via qPCR, specifically alterations in Cd14 and Ptgs1 were observed in response to male WD matings. Our current study shows that paternal nutritional status has the potential to influence male metabolic and reproductive factors, which can ultimately alter embryonic development and the post-mating maternal environment. This study highlights potential direct (sperm mediated) and indirect (semen mediated) pathways in which a father's poor diet could shape the long-term health of his offspring.

overall_design: To investigate murine sperm RNA profile changes in response to sub-optimal diets and dietary supplementations. We fed male mice either control diet (NPD; 18% casein, 21% sugar, 0% milk fat, 0% cholesterol), isocaloric low protein diet (LPD; 9% casein, 24% sugar, 0% milk fat, 0% cholesterol), ‘Western’ diet (WD; 19% casein, 34% sugar, 20% milk fat, 0.15% cholesterol) or LPD or WD supplemented with methyl-donors [an addition of 5 g/kg diet choline chloride, 15 g/kg diet betaine, 7.5 g/kg diet methionine, 15 mg/kg diet folic acid, 1.5 mg/kg diet vitamin B12 to either LPD (MDL) or WD (MDWD)] for a minimum of 8 weeks before live caudal sperm was retrieved for total RNA extraction.

first: Nadine

last: Holmes

first: Sonal

last: Henson

first: Fei

last: Sang

first: Victoria

last: Wright

first: Adam

last: Watkins


title: TMK4-mediated Phosphorylation of FIP37 Regulates Auxin-triggered N6-methyladenosine mRNA Modification of Auxin Biosynthetic Genes in Arabidopsis

city: Changsha

first: Chongsheng

last: He

state: Hunan

address: 27 Tianma road

institute: Hunan university

type: Methylation profiling by high throughput sequencing

summary: Using m6A-seq, RNA-seq, we show that auxin can induce transcriptome-wide m6A reprogramming.

overall_design: This series includes samples for two experiments: m6A-seq (two biological replicates for mock and NAA treated 15-day-old wild-type seedlings plus input controls); RNA-seq (three biological replicates for mock and NAA treated 15-day-old wild-type.).

first: Bin

last: Li

first: Chong

last: Xie

first: Donghao

first: Tongda

last: Xu

first: Xiaoying

last: Zhao

first: Fan

last: Zhu

first: Qiting

last: Zhou

first: Xiushan

first: Xuanming

last: Liu


title: Different effects of bariatric surgery on epigenetic plasticity in skeletal muscle of individuals with and without type 2 diabetes

city: Nuthetal

first: Heike

last: Vogel

state: BRB

address: Arthur-Scheunert-Allee 114-116

department: Department of Experimental Diabetology

institute: German Institute of Human Nutrition Potsdam-Rehbruecke

summary: Bariatric surgery is highly effective for the treatment of obesity in individuals without (OB ) and in those with type 2 diabetes (T2D ). However, whether bariatric surgery triggers similar or distinct molecular changes in OB and T2D remains unknown. Given that individuals with type 2 diabetes often exhibit more severe metabolic deterioration, we hypothesized that bariatric surgery induces distinct molecular adaptations in skeletal muscle, the major site of glucose uptake, of  OB and T2D after surgery-induced weight loss.  All participants (OB, n=13; T2D, n=13) underwent detailed anthropometry before and one year after the surgery. Skeletal muscle biopsies were isolated at both time points and subjected to transcriptome and methylome analyses using a comprehensive bioinformatic pipeline.

overall_design: Skeletal muscle biospsies (m. vastus lateralis) were collected from 26 individuals (13 with obesity; OB and 13 with obesity and type 2 diabetes; T2D ) in two time points. Before bariatric surgery (w0); One year or 52 weeks after bariatric surgery (w52). Next, total RNA was isolated from  all samples followed by library preparation and RNA sequencing.

first: Meriem

last: Ouni

first: Leona

last: Kovac

first: Sofiya

last: Gancheva

first: Sabine

last: Kahl

first: Markus

last: Jähnert

first: Ratika

last: Sehgal

first: Lucia

last: Mastrototaro

first: Matthias

last: Schlensak

first: Frank

last: Granderath

first: Killian

last: Rittig

first: Michael

last: Roden

first: Annette

last: Schürmann


title: ATAC-seq analysis of mutant histone variant H2BE in neuronal chromatin

city: Philadelphia

first: Emily

last: Feierman

address: 3400 Civic Center Blvd

institute: University of Pennsylvania

overall_design: H2BE-KO primary cortical neurons were isolated from E16.5 mice, infected with WT H2BE, H2BE-L3P, or GFP control, and cultured for 12 days for ATAC-sequencing (n=2 biological replicates per group).


title: ChIP-seq analysis of histone variant H2BE in neuronal chromatin

overall_design: WT primary cortical neurons were isolated from E16.5 mice and cultured for 12 days for H2BE ChIP-seq. Rabbit IgG serves as negative control.


title: Knockdown of microglial iron import gene, DMT1, worsens cognitive function and alters microglial transcriptional landscape in a sex-specific manner in the APP/PS1 model of Alzheimer's disease

city: Nashville

first: Alyssa

last: Hasty

state: TN

address: 702 LIght Hall

department: Molecular Physiology and Biophysics

institute: Vanderbilt University

summary: Background Microglial cell iron load and inflammatory activation are significant hallmarks of late-stage Alzheimer’s disease (AD). In vitro, microglia preferentially upregulate iron importer, divalent metal transporter 1 (DMT1, gene name Slc11a2) in response to inflammatory stimuli, and excess iron can augment cellular inflammation, suggesting a feed-forward loop between iron import mechanisms and inflammatory signaling. However, it is not understood whether microglial iron import mechanisms directly contribute to inflammatory signaling and chronic disease in vivo. These studies determined the effects of knocking down microglial iron import gene, Slc11a2, on AD-related cognitive decline and microglial transcriptional phenotype.  Methods In vitro experiments and RT-qPCR were used to assess a role for DMT1 in amyloid-β-associated inflammation. To determine the effects of microglial Slc11a2 knockdown on AD-related phenotypes in vivo, triple-transgenic Cx3cr1Cre-ERT2;Slc11a2flfl;APP/PS1+ or – mice were generated and administered corn oil or tamoxifen to induce knockdown at 5-6 months of age. Both sexes underwent behavioral analyses to assess cognition and memory (12-15 months of age). Hippocampal CD11b+ microglia were magnetically isolated from female mice (15-17 months) and bulk RNA-sequencing analysis was conducted.  Results DMT1 inhibition in vitro robustly decreased Aβ-induced inflammatory gene expression and cellular iron levels in conditions of excess iron. In vivo, Slc11a2KD APP/PS1 female, but not male, mice displayed a significant worsening of memory function in Morris water maze and a fear conditioning assay, along with significant hyperactivity compared to control WT and APP/PS1 mice. Hippocampal microglia from Slc11a2KD APP/PS1 females displayed significant increases in genes Enpp2, Ttr, and iron-export gene, Slc40a1, compared to control APP/PS1 cells. Slc11a2KD cells from APP/PS1 females also exhibited decreased expression of markers related to disease-associated microglia (DAMs), such as Apoe, Ctsb, Csf1, and Hif1α. Conclusions This work suggests a sex-specific role for microglial iron import gene Slc11a2 in propagating behavioral and cognitive phenotypes in the APP/PS1 model of AD. These data also highlight an association between loss of a DAM-like phenotype in female microglia and cognitive deficits in Slc11a2KD APP/PS1 mice. Overall, this work illuminates an iron-related pathway in microglia that may serve a protective role during disease and offers insight into mechanisms behind disease-related sex differences.

overall_design: To investigate the effect of DMT1 (gene, Slc11a2) knockdown on microglial gene expression in an Alzheimer's disease model, CD11b+ microglial cells were magnetically sorted and collected from bilateral hippocampi isolated from female mice. Experimental groups were: Control WT, Control APP/PS1, Slc11a2-KD WT, Slc11a2-KD APP/PS1.

first: Katrina

last: Volk Robertson

first: Alec

last: Rodriguez

first: J

middle: P

last: Cartailler

first: Shristi

last: Shrestha

first: Kyle

last: Schroeder

first: Arianna

last: Valenti

first: Fiona

last: Harrison


title: Cryopreservation of stem cell-derived beta-like cells enriches for beta cells with improved function

city: Gainesville

first: Holger

middle: Andreas

last: Russ

state: FL

address: 1275 Center Drive, Rm. #J545

department: Pharmacology and Therapeutics

institute: University of Florida

summary: The generation of stem cell-derived beta-like cells (sBC) holds extreme promise as not only an abundant insulin-producing cell source for replacement therapy of Type 1 diabetes (T1D), but also as an invaluable model system for investigating human beta cell development, immunogenicity, and function. Several groups have developed methodology to direct differentiate human pluripotent stem cells into pancreatic cell populations that include glucose responsive sBC. Nevertheless, the process of generating sBC poses significant experimental challenges. It involves lengthy differentiation periods, experiences substantial variability in efficiency, and inconsistencies in obtaining functional sBC. Here, we described a simple and effective cryopreservation approach for sBC cultures that yields homogeneous sBC clusters that are enriched for insulin expressing cells while simultaneous depleting proliferative progenitors. Thawed sBC display enhanced glucose-stimulated insulin release compared to controls in vitro and can effectively engraft and function in vivo. Collectively, this approach alleviates current challenges with inefficient and variable sBC generation while improving their functional state. We anticipate that these findings can inform ongoing clinical application of sBC for the treatment of patients with T1D and serve as an important resource for the wider diabetes field that will allow for accelerated research discoveries.

overall_design: Singe cell RNA-seq libraries were created using the Chromium Next GEM Single Cell 5’ Kit v2 from 10X Genomics (PN-1000265). Libraries were derived from 2 matched day 30 controls and F/T samples for a total of 4 samples. scRNA-seq libraries were sequenced via paired-end sequencing through the Illumina ® Novaseq platform by Novogene Corporation Inc. Sequencing reads were uploaded to 10X Genomic’s cloud analysis platform and processed with the Cell Ranger Count v7.1.0 pipeline using the Human GRCh38 v3.0.0 reference genome to create unique molecular identifier (UMI) gene count matrices per sample. IThese individual sample matrices were then aggregated together and processed using the 10X Genomics Loupe Browser v7.0.1 platform to perform filtering, normalization, clustering and to create t-SNE projections. Cells that contained a UMI count lower than 260 or higher than 200,000, had fewer than 20 or greater than 12,000 genes expressed, or a proportion of mitochondrial genes greater than 20% were removed from the analysis.

first: Jessie

last: Barra

first: Austin

middle: T

last: Kratz

first: Roberto

last: Castro-Gutierrez

first: James

last: Proia

first: Gurprit

last: Bhardwaj

first: Ed

last: Phelps

middle: A


title: miRNAs regulated systemically by nitrogen availability in shoots and roots of Medicago truncatula

city: Gif-sur-Yvette

first: Thomas

last: Blein

address: Avenue des Sciences

country: France

institute: CNRS

type: Non-coding RNA profiling by high throughput sequencing

summary: Using a dedicated split-root approach, we identified miRNAs regulated systemically by nitrogen availability in both shoots and roots of the Medicago truncatula model legume, depending on the CRA2 pathway, highlighting the phosphate-related miR399.

overall_design: small RNA-seq of distant split-root or shoots of medicago truncatulla control (A17) or cra2 mutant where local split-roots were subjected to nitrogen satiety or deficitconducted in triplicate.

first: Pervent

last: Marjorie

first: Lepetit

last: Marc

first: Parrinello

last: Hugues

first: Blein

last: Thomas

first: Frugier

last: Florian


title: Glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA) with leptomeningeal dissemination and ZMIZ1::RET fusion

city: Rome

first: Evelina

last: Miele

address: Piazza di Sant'Onofrio 4

department: Oncoematologia

country: Italy

institute: Ospedale Pediatrico Bambino Gesù

type: Methylation profiling by genome tiling array

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

overall_design: Refer to individual Series


title: SMARCB1 loss defines a novel PTCL-NOS subtype displaying an immunosuppressive TME susceptible to HDAC inhibitors

city: Münster

first: Carolin

last: Walter

address: Domagkstraße 9

department: Medical Faculty of the WWU Münster


title: SMARCB1 loss defines a novel PTCL-NOS subtype displaying an immunosuppressive TME susceptible to HDAC inhibitors [single-cell RNA-seq]

summary: Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) are aggressive and heterogeneous tumors with poor outcome and scarce genetic characterization. We analyzed by immunohistochemistry tumor tissue of adult and pediatric PTCL-NOS patients  and discovered frequent loss of SMARCB1 positivity, mostly associated with pediatric cases (45%). Using a genetically engineered mouse model (PTCL-NOSSmarcb1-) and single-cell RNA sequencing, we investigated the transcriptional landscape of this Smarcb1-negative PTCL-NOS tumor and the functional interactions between tumor and tumor microenvironment (TME). We unrevealed an immunosuppressive, exhausted and proinflammatory TME, characterized by high myeloid cell infiltration (predominantly myeloid derived suppressor cells, MDSC) and reduced lymphoid infiltration.   In addition, using a multidrug epigenetic screen in vitro, we identified histone deacetylase inhibitors (HDACi) as promising agents against PTCL-NOSSmarcb1-. Treatment of PTCL-NOSSmarcb1- mice with SAHA, a pan-HDACi, triggered TME remodelling, promoting the replenishment of T- and B-cell compartments and the limitation/reversion of the exhaustion phenotype.   In conclusion, we have identified a novel PTCL-NOS subtype characterized by the loss of SMARCB1 at pediatric ages, presenting an exhausted and immunosuppressive TME. Administration of SAHA reshaped the TME increasing lymphoid cells recruitment  into the tumor bed, turning the tumor from cold to hot. These results provide the rationale for further investigations based on combination therapies.

overall_design: We used a mouse model, Cd4-cre::Smarcb1fl/fl, to profile the transcriptome of  PTCL-NOS, by single cell RNA-sequencing (scRNA-seq). We crossed the Smarcb1 fl/fl strain with a mouse line harboring the Cre coding region under the control of the Cd4 cell specific promoter. All mice were intensively monitored until signs indicating tumor growth were observed. Tumors were dispersed into single cells and further processed for scRNA-seq (10X Genomics Platform). Two of the mice presenting this tumor were treated with the pan HDACi ihnibitor SAHA and equally processed for scRNA-seq analyses.

first: Kornelius

last: Kerl

first: Natalia

last: Moreno

first: Marta

last: Interlandi


title: Cell-type-specific ageing effects in the human OFC and implication for psychiatric disease

city: Munich

first: Natan

last: Yusupov

address: Kraepelinstr. 2-10

department: Dept. Genes and Environment

institute: Max Planck Institute of Psychiatry

summary: We evaluated biological aging using two epigenetic clocks (Horvath’s multi-tissue clock and CorticalClock (Shireby et al.)Brain 2020) calculated from DNA methylation measured in human post-mortem brain (orbitofrontal cortex; BA11) in a trans-diagnostic psychiatric sample including healthy controls. Epigenome-wide DNA methylation profiling was performed with the Illumina Infinium MethylationEPIC BeadChip (version 1.0; Illumina, San Diego, CA, USA) according to manufacturer’s guidelines. The total cohort consisted of 91 individuals. Five individuals were run in duplicate (as internal control). Individuals were either diagnosed a psychiatric disease (1) or psychiatrically healthy (0). Independent of the epigenetic clock used, we did not find evidence for accelerated epigenetic aging in individuals diagnosed with a psychiatric disorder.

overall_design: The study sample (N=91, 5 additional technical replicates) included participants from NSW Brain Tissue Resource Centre in Sydney, Australia. DNA from fresh-frozen postmortem tissue of the orbitofrontal cortex (BA11) was extracted and underwent bisulfite conversion. The bisulfite-converted DNA was used to assess epigenome-wide DNAm via Illumina Infinium MethylationEPIC BeadChip v1.0 (Illumina, San Diego, CA, USA).

first: Anna

last: Fröhlich

first: Nathalie

last: Gerstner

first: Miriam

last: Gagliardi

first: Maik

last: Ködel

first: Natalie

last: Matosin

first: Darina

last: Czamara

first: Susann

last: Sauer

first: Simone

last: Roeh

first: Vanessa

last: Murek

first: Janine

last: Knauer-Arloth

first: Michael

last: Ziller

first: Elisabeth

middle: B

last: Binder


title: RNA-seq analysis of WT and H2BE-KO primary cortical neurons

first: Emily

last: Feierman

summary: Effect of H2BE loss on neuronal transcription

overall_design: WT and H2BE-KO primary cortical neurons were isolated from E16.5 mice and culture for 12 days for RNA-sequencing (n=4 biological replicates per genotype).


title: SMARCB1 loss defines a novel PTCL-NOS subtype displaying an immunosuppressive TME susceptible to HDAC inhibitors [bulk RNA-seq]

first: Carolin

last: Walter

summary: Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) are aggressive and heterogeneous tumors with poor outcome and scarce genetic characterization. We analyzed by immunohistochemistry tumor tissue of adult and pediatric PTCL-NOS patients and discovered frequent loss of SMARCB1 positivity, mostly associated with pediatric cases (45%). Using a genetically engineered mouse model (PTCL-NOSSmarcb1-) and single-cell RNA sequencing, we investigated the transcriptional landscape of this Smarcb1-negative PTCL-NOS tumor and the functional interactions between tumor and tumor microenvironment (TME). We unrevealed an immunosuppressive, exhausted and proinflammatory TME, characterized by high myeloid cell infiltration (predominantly myeloid derived suppressor cells, MDSC) and reduced lymphoid infiltration. In addition, using a multidrug epigenetic screen in vitro, we identified histone deacetylase inhibitors (HDACi) as promising agents against PTCL-NOSSmarcb1-. Treatment of PTCL-NOSSmarcb1- mice with SAHA, a pan-HDACi, triggered TME remodelling, promoting the replenishment of T- and B-cell compartments and the limitation/reversion of the exhaustion phenotype. In conclusion, we have identified a novel PTCL-NOS subtype characterized by the loss of SMARCB1 at pediatric ages, presenting an exhausted and immunosuppressive TME. Administration of SAHA reshaped the TME increasing lymphoid cells recruitment into the tumor bed, turning the tumor from cold to hot. These results provide the rationale for further investigations based on combination therapies.

overall_design: We analyzed gene expression profiling (bulk RNA-seq) of the T15 cell line,  derived from a murine Smarcb1-negative PTCL model (doi.org/10.1172/JCI37210). Additionally, bulk RNA of T15 cells treated with SAHA and T15-Smarcb1 cells, in which a Doxycycline-inducible Smarcb1 gene was transduced, was analyzed and compared to the T15 control. Total RNA from the cells was extracted and submitted to RNA-Sequencing.

first: Kornelius

last: Kerl


title: Analysis of histone variant H2BE in neuronal chromatin

summary: WT and H2BE-KO primary cortical neurons were isolated from E16.5 mice and culture for 12 days for ATAC-sequencing (n=3 WT biological replicates, 4 KO biological replicates).

overall_design: Emily Feierman


title: EGFR-MEK1/2 cascade negatively regulates bactericidal function of bone marrow macrophages in mice with Staphylococcus aureus osteomyelitis

city: Guangzhou

first: mingchao

last: Jin

state: Guangdong Province

address: No.1838 North of Guangzhou Avenue

department: Division of Orthopedics and Traumatology, Department of Orthopedics

institute: Nanfang Hospital, Southern Medical University

summary: Ojectives: To further discover the mechanism of EGFR-MEK1/2 activation might suppress the production of mitochondrial reactive oxygen species (mtROS) upon Staphylococcus aureus (S. aureus) challenge. Methods: Bone marrow derived macrophages (BMDMs) were isolated from C57BL/6 mice (8-10 weeks old) and cultured for 7days. On day 8, cells in S.aureus-infected group were infected with S. aureus at multiplicity of infection (MOI) of 10 for 1 h, and then treated with 20 μg/mL lysostaphin and 50 μg/mL gentamicin to remove extracellular bacteria. After being washed with PBS for three times, Cells were then cultured for additional 12 h. Afterwards, cells were washed with PBS, lysed in Trizol, and sent for next-generation sequencing. RNA was quality-assessed with Agilent 2100 using RNA 6000 Nano kit, with RNA integrity number above 9 for library construction. The total RNA samples from three independent experiments were used for library construction according to the protocol of Illumina NovaSeq 6000 platform and then sequenced on a Thermal Cycler using HighSensivity DNA assay Kit. The FASTq sequencing files were aligned to the mouse reference genome (GRCm38, mm10) using STAR aligner for further analysis. The resulting gene profiles were analyzed using limma package (3.54.0) to determine differentially expressed genes (DEGs) between S. aureus infected BMDMs and control ones. Gene oncology (GO) analysis of DEGs (p value < 0.05, and │Log2(fold change)│>1) was performed by clusterProfiler package (Version 4.7.1) in R platform (Version 4.2.3). Results: GO analysis of DEGs associated with cellular response to environmental stimulus and regulation of DNA damage checkpoint showed that Map3k20, Chek2 and Fbxo4 were significantly down-regulated. Chek2 is a central effector of DNA damage response, mediating DNA checkpoints activation under stress conditions.

overall_design: BMDMs in the Control group and the S.aureus-infected group were respectively performed total RNA sequencing after treatment.

first: Mingchao

first: Xiaohu

last: Wu

first: Jin

last: Hu

first: Yijie

last: Chen

first: Bingsheng

last: Yang

first: Chubin

last: Cheng

first: Mankai

first: Xianrong

last: Zhang


title: CUT&Tag seq analysis of histone variant H2BE in neurons [H2BE_CT_antibodyValidation]

summary: Analysis of histone variant H2BE in neuronal chromatin

overall_design: WT and H2BE-KO primary cortical neurons were isolated from E16.5 mice and cultured for 12 days for validation of anti-H2BE antibody for CUT&Tag sequencing (n=1 biological replicates per genotype). KO serves as negative control to validate antibody.


title: CUT&Tag seq analysis of histone variant H2BE in neurons

summary: Histone variant H2BE Analysis of histone variant H2BE in neuronal chromatin

overall_design: WT and H2BE-KO primary cortical neurons were isolated from E16.5 mice and culture for 12 days for H2BE CUT&Tag sequencing (n=3 biological replicates per genotype). KO serves as negative control to validate antibody.


title: Single-nucleus transcriptomic analysis of cortical tissue from WT and H2BE-KO young adult mice

summary: Single-nucleus Drop-seq was used to profile transcriptional changes upon loss of H2BE in nuclei isolated from the cortex of young adult mice

overall_design: Nuclei were isolated from cortices of 2-4 month old WT and H2BE-KO mice. Nuclei capture and library preparation was performed using single-nucleus Drop-seq (n=3 biological replicates per genotype).


title: Scribble loss promotes pancreatic cancer development through intrinsic and extrinsic mechanisms

city: CAMBRIDGE

first: Giulia

last: Biffi

state: Cambridgeshire

address: Cancer Research UK, Robinson Way

institute: University of Cambridge


title: Different effects of bariatric surgery on epigenetic plasticity in skeletal muscle of individuals with and without type 2 diabetes [methylation array]

first: Heike

last: Vogel

summary: Bariatric surgery is highly effective for the treatment of obesity in individuals without (OB ) and in those with type 2 diabetes (T2D ). However, whether bariatric surgery triggers similar or distinct molecular changes in OB and T2D remains unknown. Given that individuals with type 2 diabetes often exhibit more severe metabolic deterioration, we hypothesized that bariatric surgery induces distinct molecular adaptations in skeletal muscle, the major site of glucose uptake, of  OB and T2D after surgery-induced weight loss.   All participants (OB, n=13; T2D, n=13) underwent detailed anthropometry before and one year after the surgery. Skeletal muscle biopsies were isolated at both time points and subjected to transcriptome and methylome analyses using a comprehensive bioinformatic pipeline.

overall_design: Skeletal muscle biospsies (m. vastus lateralis) were collected from 26 individuals (13 with obesity; OB and 13 with obesity and type 2 diabetes; T2D ) in two time points. Before bariatric surgery (w0); One year or 52 weeks after bariatric surgery (w52). Next, genomic DNA was isolated from  all samples followed bisulfite treatment and hybridization on EPIC v1 arrays.

first: Meriem

last: Ouni

first: Leona

last: Kovac

first: Sofiya

last: Gancheva

first: Sabine

last: Kahl

first: Markus

last: Jähnert

first: Ratika

last: Sehgal

first: Lucia

last: Mastrototaro

first: Matthias

last: Schlensak

first: Frank

last: Granderath

first: Killian

last: Rittig

last: Roden

first: Annette

last: Schürmann


title: RNA-Seq comparison of SCRIB WT and KO KPC tumors

summary: We investigated the role of SCRIB in pancreatic cancer looking at SCRIB WT and SCRIB KO KPC tumors

overall_design: 9 samples total (4 WT and 5 KO tumors) were analyzed

first: Joaquín

last: Henriquez

first: Pedro

last: Perez-Mancera

first: Camino

last: Bermejo-Rodriguez

first: David

last: Tuveson


title: Endogenously produced itaconate negatively regulates innate-driven cytokine production and drives global ubiquitination in human macrophages

city: Indianapolis

middle: Charles

last: Dorsey

state: IN

address: Eli Lilly and Company

department: Department of Immunology and Biotechnology

institute: Eli Lilly and Company

summary: A wide variety of electrophilic derivatives of the Kreb’s cycle-derived metabolite, itaconate, are immunomodulatory, yet these derivatives have overlapping and sometimes contradictory activities. Therefore, we generated a genetic system to interrogate the immunomodulatory functions of endogenously produced itaconate in human macrophages. Endogenous itaconate is driven by multiple innate signals restraining inflammatory cytokine production. Endogenous itaconate directly targets cysteine 13 in IRAK4 disrupting IRAK4 autophosphorylation and activation, drives the degradation of NFκB, and modulates global ubiquitination patterns. As a result, cells unable to make itaconate overproduce inflammatory cytokines such as TNFα, IL6, and IL-1β in response to these innate activators. In contrast, the production of IFNβ, downstream of LPS, requires the production of itaconate. These data demonstrate that itaconate is a critical arbiter of inflammatory cytokine production downstream of multiple innate signaling pathways laying the groundwork for the development of itaconate mimetics for the treatment of autoimmunity.

overall_design: WT and IRG1 KO THP-s cells were differentiated with PMA for 72 hr. Cells were subsequently stimulated with LPS and IFNg for 6, 12, and 24 hr before RNA was collected and analyzed by RNA sequencing. Each condition has 3 replicates.

first: Luke

last: Bourner

first: Linda

last: Chung

first: Ying

last: Wang


title: Chemoproteomic profiling unveils binding and functional diversity of endogenous proteins that interact with endogenous triplex DNA

city: Nanjing

first: Hongzhan

last: Xu

address: 163 Xianlin Road

institute: Nanjing university

summary: Triplex DNA structures, formed by a third DNA strand wrapped around the major groove of double helix, are key molecular regulators and genomic threats that are pharmacologically exploitable. However, the regulatory network governing triplex DNA dynamics are poorly understood. Here we performed chemoproteomic profiling of triplex DNA interactome in live cells to address this knowledge gap. We developed and validated a chemical probe that exhibits exceptional specificity for recognizing triplex DNA structures. By employing a co-binding-mediated proximity capture strategy, we enriched triplex DNA interactome for quantitative proteomics analysis. This enabled the identification of a comprehensive list of triplex DNA interacting proteins, characterized by diverse binding properties and regulatory mechanisms in native chromatin context. As a demonstration, we further validated DDX3X as the first ATP-independent helicase capable of resolving triplex DNA structures with 5' overhangs on the third DNA strand to prevent triplex-DNA-induced DNA damages. Overall, our triplex DNA interactome offers a valuable resource for investigating the biology of triplex DNA in both health and disease.

overall_design: HeLa cells were cultured in MEM media supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C with 5% CO2. For the CUT&Tag of DNMT1 protein, HeLa cells were treated with 10 uM BQQ or DMSO for 4 hours before experiment. For the CUT&Tag of DDX3X protein, HeLa cells were transfected with DDX3X-Flag plasmid for 48 hours before experiment. For the CUT&Tag of gammaH2AX protein, HeLa cells were transfected with DDX3X siRNA or scrambled siRNA for 48 hours before experiment. The antibodies used in this experiment included: Anti-DNMT1 (Active Motif, 39204, 1:100 dilution), Anti-Flag (Abclonal, AE092, 1:100 dilution), Anti-gammaH2AX (Abcam, ab303656, 1:500 dilution), Goat anti-rabbit-IgG antibody (Beyotime, A0468, 1:400 dilution) and Goat anti-mouse-IgG antibody (Beyotime, A0428, 1:400 dilution).

first: Jing

last: Ye

first: Kuixing

last: Zhang

first: Jiayu

last: Chen


title: Expression analysis of RB1 region in bladder cancer cases

city: Houston

first: Bogdan

last: Czerniak

state: TX

address: 1515 Holcombe

department: Pathology

institute: UT MD Anderson Cancer Center

type: Expression profiling by genome tiling array

summary: We investigated a sequence of genetic hits in the region of RB1 and identified alternative target genes referred to as forerunner (FR) genes whose silencing was associated with the development of the clonal plaque-like mucosal field effects initiating bladder carcinogenesis.

overall_design: A study using total RNA from 10 bladder cancer cell lines, 22 fresh frozen samples of bladder tumors, 2 samples adjacent to tumors, 5 control samples from normal ureters, and 1 control normal urothelial cell line

first: Tadeusz

last: Majewski

first: Keith

last: Baggerly


title: RNA-Seq comparison of SCRIB KD and Control Renilla KPC organoids

first: Giulia

last: Biffi

summary: We investigated the role of SCRIB in pancreatic cancer looking at SCRIB KD and Control Renilla KPC organoids

overall_design: 6 samples total of were analyzed, from 3 KPC organoid lines each transfected with expressing plasmids for shRNA targetting the Renilla locus or for shRNA targeting Scrib (#5523)


title: Broad-spectrum antibiotics disrupt homeostatic efferocytosis [bulk RNA-seq]

status: Public on Jul 16 2024

city: New York

first: Justin

middle: Shaun Arnold

last: Perry

state: New York

address: 408 East 69th Street, 408-15

department: Immunology Program

institute: Sloan Kettering Institute for Cancer Research

summary: The clearance of apoptotic cells, termed efferocytosis, is essential for tissue homeostasis and prevention of autoimmunity. Although past studies have elucidated local molecular signals that regulate homeostatic efferocytosis1 in a tissue, whether signals arising distally also regulate homeostatic efferocytosis remains elusive. Here, we find that large peritoneal macrophages (LPMs) display impaired efferocytosis in broad-spectrum antibiotics (ABX)-treated, vancomycin-treated, and germ-free mice in vivo. Mechanistically, the microbiota-derived short-chain fatty acid butyrate directly boosted efferocytosis efficiency/capacity in mouse and human macrophages, and rescued ABX-induced LPM efferocytosis defects in vivo. Bulk mRNA sequencing of butyrate-treated macrophages in vitro and single cell mRNA sequencing of LPMs isolated from ABX-treated and butyrate-rescued mice revealed regulation of efferocytosis-supportive transcriptional programs. Specifically, we found that the efferocytosis receptor T-cell immunoglobulin and mucin domain containing 4 (TIM-4, Timd4) was downregulated in LPMs of ABX-treated mice but rescued by oral butyrate and TIM-4 was required for the butyrate-induced enhancement of LPM efferocytosis capacity. LPM efferocytosis was impaired beyond withdrawal of ABX and ABX-treated mice exhibited significantly worse disease in a mouse model of lupus. Our results demonstrate that homeostatic efferocytosis relies on distal metabolic signals and suggest that defective homeostatic efferocytosis may explain link between ABX use and inflammatory disease.

overall_design: Bulk mRNAseq of bone marrow-derived macrophages treated with vehicle or butyrate.

first: PedroH

last: Saavedra

first: AlissaJ

last: Trzeciak

first: Allie

last: Lipshutz

first: AndrewW

last: Daman

first: Anya

last: O’Neal

first: Zong-Lin

last: Liu

first: Zhaoquan

first: Jesús

last: Romero-Pichardo

first: Waleska

last: Rojas

last: Zago

first: Marcel

middle: R

last: van den Brink

first: Steven

last: Z Josefowicz

first: Christopher

last: Lucas

last: Anderson

first: Alexander

middle: Y

last: Rudensky


title: Broad-spectrum antibiotics disrupt homeostatic efferocytosis [scRNA-seq]

overall_design: Single cell RNA sequencing (scRNAseq) of total peritoneal cells isolated from wildtype untreated mice, mice treated with broad-spectrum antibiotics, and mice treated with both broad-spectrum antibiotics and oral butyrate. Each mouse (n = 2 each condition) was labeled separately by staining with MHC Class I- barcoded antibodies bearing hashtags 1-7 (BioLegend, A0301-A0307). Library prep and sequencing were performed by the Single Cell Research Initiative (SCRI) at MSKCC as follows: scRNAseq was performed on a Chromium instrument (10X Genomics) following the user guide manual for 3′ v3.1. Cells were captured in droplets, then subjected to reverse transcription and cell barcoding. Following this step, emulsions were broken, and cDNA purified using Dynabead MyOne SILANE, followed by PCR amplification per manual instruction. Approximately 30,000 cells were targeted for each sample. Samples were multiplexed together on one lane of a 10X Chromium following a published cell hashing protocol69. Final libraries were sequenced on the Illumina NovaSeq S4 platform (R1 – 28 cycles, i7 – 8 cycles, R2 – 90 cycles).

first: Pedro

middle: H

first: Alissa

middle: J

first: Andrew

middle: W

last: Wang

middle: D