title: An essential role for the ultra-conserved poison exon of Tra2b in fertility in mice (RNA-seq)

first: Graham

last: Smith

overall_design: Gene expression profiling by RNA-seq of mouse testes at 12 days post partum. Samples were on a C57/BL6 background, either wild type (n=4) or after conditional deletion of the poison exon within the Tra2b gene.

first: Caroline

last: Dalgliesh

first: Saad

last: Aldalaqan

first: Christian

last: Atallah

last: Best

first: Emma

last: Scott

first: Ingrid

last: Ehrmann

first: Joel

last: Mannion

first: Barbora

last: Badurova

first: Raveen

last: Sandher

first: Yve

last: Mende

first: Brunhilde

last: Wirth

first: Sara

last: Wells

first: Gemma

last: Codner

first: Lydia

last: Teboul

first: Ann

last: Hedley

first: Colin

last: Miles

first: Dirk

last: de Rooij

first: Louise

last: Reynard

first: Mary

last: Herbert

first: David

last: Elliott


title: Cancer mutations rewire the RNA methylation specificity of  METTL3-METTL14

status: Public on Dec 19 2024

city: Dallas

first: Yunsun

last: Nam

state: Texas

address: 5323 Harry Hines Blvd

department: Biochemistry

country: USA

institute: UTSouthwestern Medical Center

summary: Chemical modification of RNAs is important for post-transcriptional gene regulation. The METTL3-METTL14 complex generates most N6-methyladenosine (m6A) modifications in mRNAs, and dysregulated methyltransferase expression has been linked to cancers. Here we show that a changed sequence context for m6A can promote oncogenesis. A gain-of-function missense mutation from cancer patients, METTL14R298P, increases malignant cell growth in culture and transgenic mice, without increasing global m6A levels in mRNAs. The mutant methyltransferase preferentially modifies noncanonical sites containing a GGAU motif, in vitro and in vivo. The m6A in GGAU context is detected by the YTH family of readers similarly to the canonical sites but is demethylated less efficiently by an eraser, ALKBH5. Combining the biochemical and structural data we provide a model for how the cognate RNA sequences are selected for methylation by METTL3-METTL14. Our work highlights that sequence-specific m6A deposition is important and that increased GGAU methylation can promote oncogenesis.

overall_design: We generated four stable HepG2 liver cancer cell lines by integrating lentivirus cassettes expressing EGFP, METTL14 (WT), METTL14 (R298P), or METTL14 (D312A). m6A-seq was performed using three biological replicates from each cell line. The sequencing data included the reads of input sample and RNA immunoprecipitation sample per replicate per cell line. The RNA-seq analysis was performed using the reads of input samples from three replicates of METTL14 (WT), METTL14 (R298P) expressing cell lines.

first: Chi

last: Zhang

first: Rohit

last: Setlem

first: Ashwani

last: Kumar

first: Xunzhi

first: Chao

last: Xing


title: The neuroepithelial origin of ovarian carcinomas explained through anepithelial-mesenchymal-ectodermal transition enhanced by cisplatin

status: Public on Dec 18 2024

city: Herne

last: Diaz-Carballo

state: NRW

address: Düngelstr. 33

department: Medical clinic of hematology & oncology

country: Germany

institute: Marienhospital Herne, Clinic of Ruhr University Bochum

type: Expression profiling by high throughput sequencing

summary: Acquired resistance to platinum-derived cytostatics poses major challenges in ovarian carcinoma therapy. In this work, we show a shift in the epithelial-mesenchymal transition (EMT) process towards an “ectodermal” conversion of ovarian carcinoma cells in response to cisplatin treatment, a progression we have termed epithelial-mesenchymal-ectodermal transition (EMET). EMET appears to occur via the classical EMT as judged by a) the downregulation of several epithelial markers and b) upregulation of Vimentin, accompanied by various embryonal transcription factors and, importantly, a plethora of neuronal markers, consistent with ectodermal differentiation. Moreover, we isolated cells from ovarian carcinoma cultures exhibiting a dual neural/stemness signature and multidrug resistance (MDR) phenotype. We also found that the epithelial cells differentiate from these neural/stem populations, indicating that the cell of origin in this tumor must in fact be a neural cell type with stemness features. Notably, some transcription factors like PAX6 and PAX9 were not localized in the nucleoplasm of these cells, hinting at altered nuclear permeability. In addition, the neuronal morphology was rapidly established when commercially available and primary ovarian carcinoma cells were cultured in the form of organoids. Importantly, we also identified a cell type in regular ovarian tissues, which possess similar neural/stemness features as observed in 2D or 3D cultures. The signature of this cell type is amplified in ovarian carcinoma tumors, suggesting a neuroepithelial origin of this tumor type. In conclusion, we propose that ovarian carcinomas harbor a small population of cells with an intrinsic neuronal/stemness/MDR phenotype, serving as the cradle from which ovarian carcinoma evolves.

overall_design: SKOV3WT cells were incubated in DMEM medium until the cells reached about 70 % of confluence. At this point medium was replaced with DMEM containing 1 µg/ml cisplatin and incubated for 72 h. After this period, cells were renamed as SKOV3 cp. This experiment was repeated several time to get at least 3 biological replicates.  Primary ovarian cancer cell line OC236 revealed a subpopulation of cells with a nested morphology, which are EpCAM negative but positive for a plethora of neuronal markers. In order to isolate such cells, we employed MicroBeads directly conjugated to EpCAM antibody (Cat. # 130–061-101,Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, cells were counted and resuspended in a separation buffer (PBS, pH 7.2, 0.5%BSA, and 2 mM EDTA), and incubated with the appropriate amount of FcR Blocking Reagent (Cat. # 130–059-901 Miltenyi Biotec) andCD326 (EpCAM) MicroBeads for 30 min at 4°C and then separated using MS MACS® Columns and a MiniMACS™ Separator. The eluted fraction was subjected to a second round of separation and positive and negative fractions were collected. The purity of these fractions was evaluated by flow cytometry. This isolation was done three times.

last: Díaz-Carballo

first: Ayesha

last: Safoor

first: Sahitya

last: Saka

first: Adrien

last: Noa-Bolaño

first: Flevy

last: D’Souza

first: Jacqueline

last: Klein

first: Ali

middle: H

last: Acikelli

first: Sascha

last: Malak

first: Udo

last: Rahner

first: Amin

middle: T

last: Turki

first: Anne

last: Höppner

first: Annabelle

last: Kamitz

first: Wanlu

last: Song

first: Ye-guang

last: Chen

first: Lalitha

last: Kamada

first: Andrea

last: Tannapfel

first: Sebastian

last: Brinkmann

first: Crista

last: Ochsenfarth

last: Strumberg


title: Gene expression analysis of nasal mucosa in birch pollen allergic and non-allergic individuals upon nasal provocation with birch pollen

status: Public on Dec 17 2024

city: Krems

first: Christine

last: Hafner

address: Dr.-Karl-Dorrek-Straße 30

country: Austria

institute: Karl Landsteiner University of Health Sciences

summary: Birch pollen is a significant cause of allergic rhinitis, yet the mechanisms of sensitization is to be understood completely. Here, we investigate the changes in gene expression of birch pollen allergic and non-allergic individuals that occur as a result of nasal provocation with birch pollen.

overall_design: This study was conducted specifically outside the birch pollen season to ensure that the results obtained are due to the nasal provocation alone and not to the exposure to birch pollen in the environment. 11 birch pollen allergic and 12 birch pollen non-allergic individuals participated in the study. All participants made two visits where they were subjected to nasal provocation with saline solution at visit 1 (baseline) and birch pollen solution at visit 2. At visit 1, nasal scrapings were collected 15 minutes following nasal provocation. The participants were randomized into four groups according to the time point of sample collection (15, 30, 60 or 120 minutes) following nasal provocation at visit 2. Nasal scrapings were collected at the respective time points. Total RNA was isolated from the nasal scrapings, Sequencing libraries from total RNA were prepared at the Core Facility Genomics, Medical University of Vienna, using the QuantSeq FWD protocol (Lexogen) and the pooled libraries were sequenced on a NextSeq500 instrument (Illumina) in 1x75bp single-end sequencing mode.

first: Srinidhi

last: Sudharson

first: Sophia

last: Derdak

first: Julia

last: Eckl-Dorna

first: José

last: Basílio

first: Heimo

last: Breiteneder


title: IFIT1 Knockdown Mitigates Translational Suppression, Enhancing Myogenic Differentiation and Revitalizing Mesenchymal Stem Cell Therapy for Myocardial Infarction

status: Public on Dec 16 2024

city: Shanghai

first: Lei

last: Hou

state: Shanghai

address: 746 Zhongshan Middle Road

department: Department of Cardiology

country: China

institute: Songjiang Hospital and Songjiang Research Institute, School of Medicine, Shanghai Jiao Tong University

summary: Early transplantation of mesenchymal stem cells (MSCs) showed promise in rescuing cardiac function and alleviating ventricular remodeling post-myocardial infarction (MI) while regulatory T cells (Treg) either. However, the crosstalk between them remains fully elusive. To explore mechanisms optimizing MSC therapy from Treg immunoregulatory and develop potential therapeutic targets, adipose-derived stem cells (ADSCs), an easily accessible MSC, were applied. The Treg depletion diminished the cardioprotective effects of ADSCs, causing both systolic dysfunction and cardiac fibrosis. Tregs promoted ADSC retention by proliferation and migration activation with further myogenic differentiation. RNA-sequencing analysis revealed Treg co-culture induced a myogenic differentiation mRNA profile in ADSCs, significantly downregulating Ifit1. IFIT1 downregulation resulted in cardiomyocyte-like differentiation in ADSCs, evidenced by increased myogenic markers and spontaneous beating. Mechanistically, IFIT1 knockdown provoked upregulation of E2F1 protein without altering E2f1 mRNA levels. IFIT1 specifically bound to E2f1 mRNA to inhibit translation, reducing polysomes while knockdown introduced myogenic translational profile. The rAAV9-sh-Ifit1 injection restored systolic dysfunction, reduced fibrosis, and attenuated cardiac hypertrophy. This study uncovered a novel mechanism whereas Tregs potentiated the therapeutic efficacy of ADSCs in MI by downregulating IFIT1, thereby promoting E2f1 translation and myogenic differentiation, offering a potential target to improve cardiac repair strategies.

overall_design: RNA sequencing analysis was performed on primary ADSCs (C-1,C-2,C-3) and ADSCs after Treg co-culature (T-1,T-2,T-3) 48 hours (n = 3/group).

first: Luyao

last: Wu

first: Yanan

last: Pang

first: Jiachun

last: Xia

first: Sensen

last: Zhang

first: Jumo

last: Zhu


title: Single-cell analysis identifies the CNP/GC-B/cGMP axis as marker and regulator of modulated VSMCs in atherosclerosis

city: Tuebingen

first: Robert

last: Feil

address: Auf der Morgenstelle 34

department: Interfaculty Institute of Biochemistry

institute: University of Tuebingen

summary: A balanced activity of cGMP signaling contributes to the maintenance of cardiovascular homeostasis. Vascular smooth muscle cells (VSMCs) can generate cGMP via three ligand-activated guanylyl cyclases, the NO-sensitive guanylyl cyclase, the atrial natriuretic peptide (ANP)-activated GC-A, and the C-type natriuretic peptide (CNP)-stimulated GC‑B. Here, we studied natriuretic peptide signaling in murine VSMCs and atherosclerotic lesions. Correlative profiling of pathway activity and VSMC phenotype at the single-cell level showed that phenotypic modulation of contractile VSMCs to chondrocyte-like plaque cells during atherogenesis is associated with a switch from ANP/GC‑A to CNP/GC‑B signaling. Silencing of the CNP/GC-B axis in VSMCs resulted in an increase of chondrocyte-like plaque cells. These findings indicate that the CNP/GC‑B/cGMP pathway is a marker and atheroprotective regulator of modulated VSMCs, limiting their transition to chondrocyte-like cells. Overall, this study highlights the plasticity of cGMP signaling in VSMCs and suggests analogies between CNP-dependent remodeling of bone and blood vessels.

overall_design: Primary VSMCs were isolated from murine aortas and cultured for five days as a cell culture model for phenotypic modulation. VSMCs with different phenotypes were identified and compared by scRNA-sequencing.


title: TIRE-seq: an Integrated Sample Extraction and Transcriptomics Workflow for High Throughput Perturbation Studies

city: Melbourne

first: Daniel

middle: Victor

last: Brown

address: 1G Royal Parade

country: Australia

institute: Walter and Eliza Hall Institute

summary: TIRE-seq (Turbocapture Integrated RNA Expression Sequencing) is a novel RNA sequencing method that integrates mRNA purification directly into library preparation, eliminating separate RNA extraction. Compared to traditional protocols like Primeseq, TIRE-seq demonstrates improved sequencing efficiency using crude cell lysates. We validated the method across three biological applications: analyzing transcriptional changes in stimulated human T cells, identifying genes driving murine dendritic cell differentiation, and examining temozolomide's dose-response effects on patient-derived neurospheres. TIRE-seq offers a streamlined, cost-effective approach for gene expression studies, reducing workflow complexity and minimizing sample loss.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design:
These data contain multiple samples. The biological applications include human T cells stimulated with CD3 and CD28 microbead beads and murine dendritic cells stimulated with Flt3L. Multiple cellular subsets and timepoints are sampled.
Samples are bulk RNA-seq but utilise a single-cell style barcoding scheme. See feature_README.txt file at the foot of this record for barcoded sample information.

first: Piper

middle: O

last: O'Keeffe

first: Yasmin

middle: N

last: Nouri

first: Hui

middle: S

last: Saw

first: Zachery

middle: M

last: Moore

first: Tracey

last: Baldwin

first: Sam

middle: W

last: Olechnowicz

first: Jafar

last: Jabbari

first: David

last: Squire

first: Stephen

middle: L

last: Leslie

first: Changqing

last: Wang

first: Yupei

middle: Y

last: You

first: Matthew

middle: E

last: Ritchie

first: Ryan

last: Cross

first: Misty

middle: R

last: Jenkins

first: Cindy

middle: A

last: Audiger

first: Shalin

last: Naik

first: James

last: Whittle

first: Saskia

middle: F

last: Freytag

first: Sarah

last: Best

first: Peter

last: Hickey

first: Daniela

last: Amann-Zalcenstein

first: Rory

middle: B

last: Bowden

middle: V


title: Loss of YTHDC1 m6A reading function promotes invasiveness in urothelial carcinoma of the bladder [RNA-seq]

city: Heidelberg

first: Jinyun

last: Xu

address: Im Neuenheimer Feld 280

institute: German Cancer Research Center

summary: Bladder cancer poses significant clinical challenges due to its high metastatic potential and poor prognosis, especially when it progresses to muscle-invasive stages. Here, we show that the m6A reader YTHDC1 is downregulated in muscle-invasive bladder cancer and is negatively correlated with the expression of epithelial‒mesenchymal transition genes. The functional inhibition or depletion of YTHDC1 increased the migration and invasion of urothelial cells. Integrative analysis of multimodal sequencing datasets provided detailed insights into the molecular mechanisms mediating YTHDC1-dependent phenotypes and identified SMAD6 as a key transcript involved in the invasiveness of urothelial carcinoma of the bladder. Notably, SMAD6 mRNA colocalized less with YTHDC1 in tumoral tissues than in paratumoral tissues, indicating disrupted binding during cancer progression. Our findings establish YTHDC1-dependent m6A reading as a critical epitranscriptomic mechanism regulating bladder cancer invasiveness and provide a paradigm for the epitranscriptomic deregulation of cancer-associated networks.

overall_design: To investigated the functional role of YTHDC1 in urothelial carcinoma of bladder,  we used the healthy urothelial cell model UROtsa to generate cells with reduced YTHDC1 expression via CRISPR-Cas9, and performed an RNA-seq analysis with control and YTHDC1-depleted UROtsa cells.

first: Jonas

last: Koch

first: Claudia

last: Schmidt

first: Malin

last: Nientiedt

first: Manuel

last: Neuberger

first: Philipp

last: Erben

first: Maurice

last: Michel

last: Rodríguez-Paredes

first: Frank

last: Lyko


title: Transcriptome and epigenome dynamic of the clonal heterogeneity of human induced pluripotent stem cells for cardiac differentiation [RNA-seq]

city: Suwon

first: Seunghee

last: Jeong

address: 2066 Seobu-ro, Janna-gu, Suwon, Gyeonggi-do, South Korea

country: South Korea

institute: Sunkyunkwan University

summary: Human induced pluripotent stem cells (hiPSCs) generate multiple clones with inherent heterogeneity, leading to variations in their differentiation capacity. Previous studies have primarily addressed line-to-line variations in differentiation capacity, leaving a gap in the comprehensive understanding of clonal heterogeneity. Here, we aimed to identify predictive biomarkers for cardiomyocyte differentiation capacity by integrating transcriptomic, epigenomic, endogenous retroelement, and protein kinase phosphorylation profiles. We generated multiple clones from a single donor and validated that these clones exhibited comparable levels of pluripotent markers. These clones were then classified into two groups based on their differentiation capacity: productive clone (PC) and non-productive clone (NPC). We performed RNA sequencing and assay for transposase-accessible chromatin with sequencing (ATAC-seq). NPC was enriched in vasculogenesis and cell adhesion, accompanied by elevated ERK1/2 signaling pathway phosphorylation. Conversely, PC exhibited enrichment in embryonic organ development and transcription factor activation, accompanied by increased chromatin accessibility. Integrative analysis of RNA sequencing and ATAC-seq revealed 14 candidate genes correlated with cardiac differentiation propensity. Notably, TEK and SDR42E1 were upregulated in NPC. Our integrative profiles enhance the understanding of clonal heterogeneity and highlight two novel biomarkers associated with cardiomyocytes differentiation. This insight may facilitate the identification of suboptimal hiPSC clones, thereby mitigating adverse outcomes in clinical applications.

overall_design: To assess the differentiation potential of hiPSC clones, we quantified TNNT2-positive cells using flow cytometry between days 12 to 15 of cardiomyocytes (CMs) differentiation. Notably, we observed diverse proportions among the clones, with some consistently exhibiting robust differentiation rates (>60%), while others displayed lower rates (<50%) along with a higher proportion of non-beating cells. This distinct pattern facilitated the categorization of certain clones as productive and others as non-productive based on their respective differentiation rates, indicative of varying capacities for CMs differentiation.These findings highlighted the presence of a heterogeneous population encompassing both CMs and non-CMs among the hiPSC clones, suggesting differing potentials for differentiation. Therefore, we plan to explore the underlying variability by examining genetic and epigenetic influences on differentiation potential using techniques such as RNA-seq and ATAC-seq. Additionally, to characterize the gene expression and chromatin accessibility landscapes comprehensively across all studied hiPSC clones, we will conduct integrative RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin by sequencing (ATAC-seq) analyses.

first: Jihye

last: Yun

first: Jaemin

last: So

first: Jaecheol

last: Lee


title: Influence of trace-elements and glucose on Trichophyton rubrum grown in keratin medium

status: Public on Dec 14 2024

city: Nanchang

first: Oliver

last: Blechert

address: Fusheng Road 666

institute: Affiliated Hospital of Jiangxi University of Chinese Medicine

summary: Trichophyton rubrum is a pathogenic fungus infecting human skin, hairs and nails. These substrates are low in most nutrients required for fungal growth and consequently are colonized only by very few fungal species. Especially, concentration of trace elements is low and a limiting factor for fungal growth. T. rubrum is a highly specialist fungus and adapted to this environment. By in-vitro experiments, we analyzed the influence of trace-elements on mRNA expression. We measured gene expression by RNAseq of two T. rubrum strains, STRB008 and STRB012, in three different cultivation condition, each in 6 replications. Keratin medium, without sugar supply, was used as basic medium. In the second condition, we added trace-elements to the keratin medium. In the third condition, we added glucose. We point to the evolutionary adaption of the fungus to the human skin. T. rubrum has a sophisticated system for the digestion and utilization of human skin protein and a relative low demand for trace-elements.

overall_design: To analyze the influence of trace-elements on mRNA expression of Trichophyton rubrum, the fungus was pre-cultured in liquid keratin medium. Afterwards, the mycel was aliquot to keratin medium, keratin medium with trace-elements and keratin medium with glucose. After 5 days, total RNA for RNAseq has been extracted. Each condition was replicated 6 times for two fungal strains, T. rubrum STRB008 and STRB012.

first: Shuzhen

last: Xiong

first: Ping

last: Zhan


title: Loss of YTHDC1 m6A reading function promotes invasiveness in urothelial carcinoma of the bladder [RIP-seq]

overall_design: To map the binding of YTHDC1 on mRNAs, we performed RIP-seq in wild-type UROtsa cells. As a control, we treated the UROtsa cells with the METTL3 inhibitor STM2457, which should remove a substantial part of the m6A modifications on mRNAs and thus reduce the number of mRNAs bound by YTHDC1.


title: Pol II degradation activates cell death independently from the loss of transcription [Switchover_RNAseq]

status: Public on Dec 13 2024

city: Worcester

first: Michael

state: MA

address: 368 Plantation Street

institute: UMASS Chan Medical School

summary: Pol II-mediated transcription is essential for eukaryotic life. While loss of transcription is thought to be universally lethal, the associated mechanisms promoting cell death are not yet known. Here, we show that death following loss of Pol II is not caused by loss of gene expression. Instead, death occurs in response to the loss of Pol II protein itself. Loss of Pol II protein exclusively activates apoptosis, and using functional genomics, we identified a previously uncharacterized pathway, which we call the Pol II Degradation-dependent Apoptotic Response (PDAR). Using the genetic dependencies of PDAR, we identify clinically used drugs that owe their efficacy to a PDAR-dependent mechanism. Our findings unveil a novel apoptotic pathway that contributes to the efficacy of a wide array of anti-cancer therapies.

overall_design: RNA-seq profiling of U2OS cells expressing a Dox-inducible RPB1 transgene (RPB1-N792D-∆CTD), referred to as "switchover" cells. Cells were incubated with or without 2 µg/mL Dox for 48 hours, followed by exposure to 10 µM ⍺-amanitin for 0, 12, or 24 hours.

first: Nicholas

last: Harper

first: Gavin

last: Birdsall

first: Megan

last: Honeywell

first: Athma

last: Pai

middle: J


title: Pol II degradation activates cell death independently from the loss of transcription [Fractionation_RNAseq]

overall_design: RNA-seq profiling of U2OS cells and U2OS-PTBP1-knockout cells following treatment with or without 1 µM triptolide for 8 hours. Samples were fractionated into cytoplasmic and nuclear compartments.


title: A longitudinal atlas of post-viral lung regeneration reveals persistent injury-associated cell states

city: Philadelphia

middle: Patrick

last: Morley

state: PA

address: 3400 Civic Center Blvd, Bldg 421

department: Penn Cardiovascular Institute

institute: Perelman School of Medicine at the University of Pennsylvania

summary: Functional regeneration of the lung’s gas exchange surface following injury requires the coordination of a complex series of cell behaviors within the alveolar niche. Using a multi-modal approach, we have mapped the temporal sequencing of mouse lung regeneration after acute viral injury, demonstrating that this response is asynchronously phased across different cellular compartments. This longitudinal atlas of regeneration has produced a catalogue of new cell states that reflect transient and persistent transcriptional alterations in daughter cells as they transit across axes of differentiation. These new cell states include an injury-induced capillary endothelial cell (iCAP) that arises after injury, persists indefinitely, and shares transcriptional hallmarks with both developing lung endothelium and the endothelial aberrations found in degenerative human lung diseases. This comprehensive atlas of lung regeneration provides a foundational resource to understand the complexity of the cellular and molecular responses to injury, reveals the critical importance of capillary endothelium in maintaining and rebuilding the alveolar niche after injury, and correlates these responses to those found in development and human lung diseases.

overall_design: Lungs of adult, lineage-traced Ki67-CreERT2; ROSA26-tdTm mice were collected at homeostasis or after PR8 influenza infection (6, 11, 19, 25, 42, 90, or 366 dpi) for single-cell RNA sequencing. In addition, lungs of adult mice with lineage traced CAP1 endothelial cells (Kit-MerCreMer; ROSA26-tdTm) or CAP2 endothelial cells (Car4-CreERT2; ROSA26-tdTm and Ednrb-CreERT2; ROSA26-tdTm) were collected for single-cell RNA sequencing at 19 days post-PR8 influenza infection.

first: Edward

last: Morrisey

first: Terren

middle: K

last: Niethamer

first: Joseph

middle: D

last: Planer

middle: P


title: Pol II degradation activates cell death independently from the loss of transcription [Exosome_knockout_RNAseq]

overall_design: RNA-seq profiling of U2OS cells with CRISPR mediated knockout of either NUP93 or EXOSC5, or cells given a nontargeting sgRNA (NONT). Cells were treated with or without 1 µM triptolide for 8 hours.


title: Pol II degradation activates cell death independently from the loss of transcription [CRISPR_screen]

overall_design: Genome-wide CRISPR KO screen of U2OS-Cas9 cells treated with or without 1 µM triptolide for 32 hours. T0 samples were taken immediately prior to drugging. Drug treated cells were then mechanically separated into live and dead fractions.


title: An essential role for the ultra-conserved poison exon of Tra2b in fertility in mice

city: Newcastle upon Tyne

first: Graham

last: Smith

address: Framlington Place

department: Bioinformatics Support Unit

country: United Kingdom

institute: Newcastle University

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

overall_design: Refer to individual Series


title: Deletion of ddx4 ovary-specific transcript causes downregulation of sycp1 and derepression of DNA transposons in zebrafish ovaries

city: Wuhan

first: Fei

last: Liu

state: Hubei

address: No. 7 Donghu South Road, Wuchang District

institute: Institute of Hydrobiology, Chinese Academy of Sciences

summary: Ddx4 (DEAD-box helicase 4) is a well-established marker gene for early-stage germlines and is required for the development of germ cells. Alternative splicing of ddx4 produces a long transcript that is exclusively expressed in zebrafish ovaries. The function of this ovary-specific transcript (ddx4-L) remains unclear. In this study, using the ddx4-L knockout zebrafish model, we found that elimination of ddx4-L results in a decrease in fertilization rate and in the number of mature eggs laid by the female mutants. Transcriptome analysis was performed to identify the underlying changes of gene expression between WT and ddx4-L knockout ovaries. A total of 1134 differentially expressed genes were identified, of which 524 were upregulated and 610 were downregulated. Functional enrichment analysis showed that the terms of negative regulation of fertilization, sperm-egg recognition process, and oocyte meiosis might be affected by elimination of ddx4-L. Furthermore, we found that Sycp1, a synaptonemal complex protein involved in meiosis and fertility, was dramatically decreased in the ddx4-L knockout ovaries at both mRNA and protein levels. Moreover, the activities of transposable elements were analyzed based on the RNA-seq data. The result showed that elimination of ddx4-L causes the derepression of DNA transposons, a subclass of transposable elements, in zebrafish ovaries. In conclusion, the aforementioned findings indicate that the ovary-specific transcript of ddx4 plays an important role in oocyte development and egg quality, possibly by maintaining sycp1 expression and repressing DNA transposons. Our work provide novel insights into the functions and regulatory mechanisms of ddx4 in zebrafish oogenesis.

overall_design: Ovaries were dissected from three WT and three ddx4-E4Δ zebrafish at 6 mpf, respectively. Six total RNA samples were extracted and subjected to RNA sequencing. Differentially expressed genes (DEGs) and transposable elements (TEs) between WT and ddx4-E4Δ zebrafish ovaries were analyzed.

first: Yuanyuan

last: Chen

first: Daji

last: Luo


title: Differentiation status determines the effects of IFNγ on the expression of PD-L1 and immunomodulatory genes in melanoma

status: Public on Dec 12 2024

city: Reykjavík

first: Berglind

middle: Ósk

last: Einarsdóttir

address: Sturlugata 8

country: Iceland

institute: University of Iceland

summary: Background Melanoma cells frequently dedifferentiate in response to inflammation which can increase responses to certain cytokines. Interferon-γ (IFNγ) is an integral part of the anti-tumour immune response and can directly induce both differentiational changes and expression of immunosuppressive proteins in melanoma cells. How the differentiation status of melanoma cells affects IFNγ responses remains unclear. Methods Dedifferentiation of melanoma cells was induced via either siRNA or shRNA mediated MITF knockdown and the cells were subsequently treated with IFNγ. Effects of MITF knockdown and IFNγ treatment on gene expression were evaluated via qPCR and RNA sequencing. A Luminex assay was used to analyse the effects of dedifferentiation and IFNγ treatment on cytokine secretion. Effects on PD-L1 protein expression were analysed via flow cytometry and western blotting. Inhibition of the JAK kinases, NF-κB and STAT3 with small molecule inhibitors, and siRNA mediated knockdown of STAT1 and IRF1 was applied to investigate the molecular mechanism behind IFNγ induced PD-L1 expression in dedifferentiated melanoma cells. The effects of inhibitor treatments and siRNA mediated knockdowns were evaluated via qPCR and western blotting. Bioinformatic analyses of publicly available RNA sequencing data, consisting of 45 patient derived melanoma cell lines, with or without IFNγ treatment, was conducted to assess the generalisability of the in vitro results.   Results Dedifferentiation renders 624Mel melanoma cells hypersensitive to IFNγ stimulation in a context-dependent manner, resulting in non-additive upregulation of IFNγ-induced genes, increased PD-L1 protein expression and amplified secretion of CCL2, CXCL10 and IL-10. Furthermore, the intensified PD-L1 protein expression occurs through the JAK-STAT1-IRF1 axis. Lastly, dedifferentiated patient derived melanoma cell lines showed enhanced inflammatory signalling in response to IFNγ compared to differentiated cells, and tended to have higher PD-L1 expression, associated with increased IRF1 expression and activity. Conclusions Together, these findings indicate the existence of a molecular context linking dedifferentiation and IFNγ signalling in melanoma which may lead to immune evasion. Furthermore, these results imply that modulating melanoma differentiation may help shape IFNγ responsiveness.

overall_design: Investigate the effects of MITF mediated dedifferentiation on IFNγ responses in melanoma cells. 624Mel melanoam cells were treated with siMITF or siCTRL and subsequently treated with IFNγ. We then performed RNA sequencing and quantified the overall transcriptional changes associated with the combination of dedifferentiation and IFNγ signalling.

first: Teitur

last: Saevarsson

first: Adrian

last: Lopez garcia de Lomana

first: Olafur

last: Sanchez

first: Veerle

last: van Esch

first: Gunnar Bjarni

last: Ragnarsson

first: Siggeir Fannar

last: Brynjolfsson

first: Eirikur

last: Steingrimsson

first: Berglind Osk

last: Einarsdottior


title: Transcriptome and epigenome dynamic of the clonal heterogeneity of human induced pluripotent stem cells for cardiac differentiation [ATAC-Seq]

first: Seunghee

last: Jeong

type: Genome binding/occupancy profiling by high throughput sequencing

first: Jihye

last: Yun

first: Jaemin

last: So

first: Jaecheol

last: Lee


title: Genetic regulation and targeted reversal of lysosomal dysfunction and inflammatory sterol metabolism in pulmonary arterial hypertension

city: Pittsburgh

first: Satoshi

last: Okawa

address: 200 Lothrop Street

institute: University of Pittsburgh

summary: We treated human pulmonary artery endothelial cells (PAECs) with silencing RNA against the nuclear receptor coactivator 7 (NCOA7) or negative control (NC). We further treated the cells to either control or IL-1B conditions (1ng/mL) for 24 hours.

overall_design: We created four groups of cells in triplicate: (1) si-Negative Control (NC) PAECs (2) siNC+IL1B PAECs (3) siNCOA7 PAECs (4) siNCOA7+IL1B PAECs

first: Stephen

last: Chan

first: Lloyd

last: Harvey


title: Formylation occurrence on translation in s. cerevisiae

city: Seoul

first: Tae-Young

last: Roh

state: --- Select One ---

address: 52 Ewhayeodae-gil, Seodaemun-gu

department: Life Sciences

institute: Ewha Womans University

summary: This study elucidates the processes of transcription and translation in Saccharomyces cerevisiae following MTF (Mitochondrial Transcription Factor) expression. We identifed the functional significance of MTF in the translation process through a comprehensive analysis of multi-omics data.

overall_design: RNA-Seq and Ribo-Seq were performed to understand translational mechanism in s. cerevisiae

first: Hyeonji

first: Chang-seok

first: Cheol-Sang

last: Hwang


title: Effect of depletion of FXR on gene expression in islets from FXR knockout mice

city: Shenzhen

first: Lianqi

last: Shao

state: Guangdong

address: 3688 Nanhai Avenue, Nanshan District

department: School of basic medicine

institute: Shenzhen University

summary: FXR (Farnesoid X Receptor), also known as Nr1h4, is a transcription factor who is also one of the bile acid receptor has been reported to be widely involved in regulating blood glucose, bile acid, and the apoptosis of hepatocyte. We have been studing the key role of FXR in Type 2 Diabetes, which can regulate the insulin secretion or even the apoptosis of β-cells. Thus,we measured the gene expression in the islets of FXR KO mice. Our data shows the abnormal expressed gene between the FXR KO mice and the Control group, which may be regulated by FXR. It will be significant promotion for the research in mechanism of FXR regulating the insulin secretion and apoptosis of β-cells.

overall_design: FXR gene knockout mice and corresponding control mice aged three months were selected, and the pancreatic tissue was isolated then digested by collagenase p.The total RNA of each group of islets was extracted separately.After the total RNA extracted from the sample was treated with DNase I, the eukaryotic mRNA was enriched with magnetic beads with Oligo (dT).The interrupting reagent was added to break the mRNA into short fragments, and a strand of cDNA was synthesized with six-base random hexamers using the interrupted mRNA as a template, and the second strand of cDNA was synthesized by adding buffer, dNTPs and DNA polymerase I.After kit purification and recovery, sticky end repair, adding base "A" to the 3 'end and connecting the sequencing connector, the obtained fragment was amplified and enriched by PCR after size selection.The constructed library was qualified by Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System, and then sequenced by Illumina sequencing platform.

first: Xinagchen

last: Kong

first: Dan

last: Yan


title: Glutathione-S-transferase theta 2 (GSTT2) modulates the tumor microenvironment and the response to BCG immunotherapy in a murine orthotopic model of bladder cancer

status: Public on Dec 11 2024

city: Singapore

first: Mugdha

last: Patwardhan

address: 10 Medical Drive,  MD11-B1-0

country: Singapore

institute: National University of Singapore

summary: Whilst M. bovis Bacillus Calmette-Guérin (BCG) therapy remains the gold-standard for treatment of high-risk non-muscle invasive bladder cancer (BC), 30-40% of patients fail therapy, resulting in disease recurrence and progression. Loss of glutathione-S-transferase theta 2 (GSTT2) expression has been associated with improved response to  fewer instillations of BCG. To understand these responses, wild-type (WT) and GSTT2-knockout (KO) mice were implanted orthotopically with MB49-PSA BC cells, and mice were treated with four weekly BCG instillations, after which the  bladders were harvested for single-cell RNA sequencing.

overall_design: Single cells, isolated from the bladder of BCG-treated WT and KO mice, were barcoded using the whole-transcriptome kit from Parse Biosciences and sequenced (HiSeq PE150). The reads were aligned to the mouse genome (GRCm39) with the split-pipe (v0.9.6p) program (from Parse).

first: Toh

middle: Q

last: Kane

first: Edmund

last: Chiong

first: Juwita

middle: N

last: Rahmat

first: Ratha

last: Mahendran


title: Small RNA-seq of small extracellular vesicles derived from adipose-derived stem cells of young and old donors at different in vitro expansion stages

city: Tokyo

first: Toshiya

last: Tsubaki

address: 7-3-1 Hongo, Bunkyo-ku

country: Japan

institute: The University of Tokyo

type: Non-coding RNA profiling by high throughput sequencing

summary: Adipose-derived stem cells (ASCs) and their small extracellular vesicles (sEVs) hold significant potential for regenerative medicine due to their capabilities for tissue repair. However, the effects of ASC heterogeneity on sEV characteristics and microRNA (miRNA) profiles remain unclear. We investigated ASCs heterogeneity and its changes with aging and cell expansion using bulk RNA-seq from young and old donors across various passages. Non-negative matrix factorization (NMF) was used to identify distinct ASC clusters. Small RNA sequencing was performed on sEVs collected from ASC culture supernatants to analyze miRNA profiles. NMF analysis revealed ASC heterogeneity, identifying clusters characterized by ISLR and genes associated with collagen and smooth muscle cells. The miRNA profiles of sEVs matched these ASC clusters, showing variations based on donor age and cell expansion. Functional enrichment analysis suggested that these miRNAs may have distinct functional roles. Our findings show that ASC heterogeneity and sEV miRNA content are significantly influenced by donor age and cell expansion. These variations underscore the necessity of selecting specific ASC-derived sEVs.

overall_design: To explore how donor age and cell expansion influence the miRNA profiles of sEVs derived from ASCs, we isolated sEVs from ASCs of three young and three old donors at different passages. Small RNA sequencing was performed to profile miRNA content in sEVs and assess the correlation with ASC cluster distributions identified from RNA-seq.

first: Taku

last: Saito


title: Targeted Transcriptome Analysis of Beef Cattle Persistently Infected with Bovine Viral Diarrhea Virus

status: Public on Dec 10 2024

city: Knoxville

first: Sonia

middle: Josefina

last: Moisa

state: Knox

address: 2506 River Drive

department: Animal Science

institute: University of Tennessee

summary: Bovine Viral Diarrhea Virus (BVDV) is an endemic virus of North American cattle populations with significant economic and animal health impacts. While BVDV infection has a myriad of clinical manifestations, a unique and problematic outcome is the establishment of a persistently infected (PI) animal following in-utero viral infection. While it is well established that PI animals serve as a constant reservoir of BVDV, the mechanism for the maintained infection remains unknown despite multiple theories. The purpose of this study was to use transcriptome analysis to further define long term immune status of adult PI cattle and offer insight into the potential mechanistic establishment of persistent BVDV infection, in utero. Peripheral blood mononuclear cells were collected from PI beef cattle (N=6) and uninfected controls (N=6) for targeted RNAseq analysis conducted using 54 genes of interest and followed by pathway enrichment analysis. Analysis revealed 29 differentially expressed genes (FDR < 0.05, fold change > 2) representing 14 significant KEGG pathways between PI and control animals (FDR < 0.05). Transcriptome changes indicate chronic upregulation of interferon gamma (IFNG) with unexpected expression of related genes, suggesting a maintained stimulation of the PI immune system resulting in virus-mediated dysregulation of immune function.

overall_design: The goal is to further identify BVDV-host interactions by identifying signaling pathways that are upregulated or suppressed in the PI animals. The hypothesis is that there will be significant gene expression changes and altered signaling pathways of the persistently infected animals.

first: Adkins

last: Morgan

first: Lear

last: Andrea

first: Beever

last: Jon

first: Moisa

last: Sonia


title: RNA-seq of Adipose-derived stem cells from young and old donors during early and late stages of in vitro expansion

overall_design: To investigate the effects of donor age and in vitro cell expansion on the stem cell properties and heterogeneity of ASCs, we collected ASCs from three young and three old donors. We performed RNA-seq at different passages (early and late stages) to analyze gene expression profiles and cluster distributions in each condition.


title: Gene expression dynamics in fibroblasts during early-stage murine pancreatic carcinogenesis

city: Halle

first: Markus

last: Glaß

address: Kurt-Mothes-Str. 3a

department: Institute of Molecular Medicine

institute: Martin Luther University Halle-Wittenberg

summary: Pancreatic ductal adenocarcinoma (PDAC) stands as one of the most lethal malignancies, characterized by aggressive local tumor growth and metastatic spread, in part driven by fibroblast-mediated stromal reactions in the tumor microenvironment. Early detection is critical for improved patient outcomes. In this study, we investigate the molecular cues within fibroblasts that offer promise for early PDAC diagnosis. We employ RNA sequencing of fibroblasts from KPC mouse models at pivotal time points, corresponding to the early preinvasive stages of pancreatic cancer. Our analysis reveals a substantial upregulation of gene sets associated with adipogenesis, fatty acid metabolism, and the ROS pathway in 12-week KPC-fibroblasts, indicative of metabolic reprogramming and redox regulation during early PDAC initiation. We narrow our focus to 26 potential key genes that may facilitate paracrine communication between fibroblasts and preneoplastic cells. Validation of our findings confirms the consistent increase in Angptl4, a gene linked to adipogenesis, and the notable downregulation of Lama2, a crucial extracellular matrix (ECM) gene. This study is the first to observe the elevation of Angptl4 in KPC-fibroblasts, providing essential insights into the role of fibroblasts in early PDAC. Moreover, our results hint at a potential role of fibroblasts in ECM degradation, a process that could support cancer cell proliferation and metastatic invasion. Genes associated with the ECM consistently display downregulation. These discoveries lay the foundation for future investigations into the mechanisms driving fibroblast-mediated elevation of Angptl4 and downregulation of ECM genes and their role in mediating crosstalk between fibroblast and preneoplastic cells. Our investigation into fibroblast transcriptional dynamics during early carcinogenesis offers significant potential for enhancing early diagnostic approaches in combating this deadly malignancy.

overall_design: PolyA RNA-seq of murine pancreatic stellate cells, either from KPC (Ptf1a/p48-Cre; lox-stop-lox-KrasG12D/+; lox-stop-lox-Trp53R172H/+) or control (Ptf1a-Cre) mice, isolated at age 4, 6, 8, 10, and 12 weeks (passage 4)

first: Nupur

last: Ohri

first: Johanna

last: Häußler

first: Bogusz

last: Trojanowicz

first: Monika

last: Hämmerle

first: Yoshiaki

last: Sunami

first: Jörg

last: Kleeff


title: Effects of different temperatures on chondrocyte growth: a transcriptomic analysis

city: Kunming

first: Jingming

last: Xie

state: Yunnan

address: 374# Dianmian Road

department: Orthopedics

institute: The Second Affiliated Hospital of Kunming Medical University

summary: BACKGROUND: We previously demonstrated that temperature-related microwave ablation (MWA) can safely regulate vertebral growth plates of piglets. Therefore, the present study was designed to investigate the effects of different temperatures on chondrocyte proliferation and the molecular mechanisms involved in vitro. METHODS: After treatment for 10 min using different temperatures (37°C, 40°C, 42°C, 44°C, 46°C, 48°C, or 50°C), CCK-8 assay examined the activity of ATDC5 cells at 12 hr. RNA-seq identified differentially expressed genes (DEGs) and the hub genes in ATDC5 cells treated at 37°C, 40°C and 44°C. RT-qPCR verified the differential expression of hub genes in ATDC5 cells. RESULTS: Compared with 37°C, stimulation at 40°C significantly enhanced the activity of ATDC5 cells, and 40°C had no significant effect. In addition, stimulation at 44°C, 46°C, 48°C, or 50°C exhibited the opposite pattern, and ATDC5 cells were particularly less than 50% active after treatment at 46°C, 48°C, or 50°C. Differential expression analysis revealed the presence of 179, 374 and 221 DEGs in 40°C vs. 37°C, 44°C vs. 37°C, and 44°C vs. 40°C, respectively. Enrichment analysis indicated that these DEGs predominantly regulate proliferation, differentiation, necrosis, inflammatory and immune responses, and ECM synthesis/degradation, and that they are associated with the Ras, PI3K/AKT, mTOR, cAMP, and MAPK pathways. Furthermore, Agt, Hspa1a, Hspb1, and Nlrc4 were hub genes in DEGs, and RT-qPCR confirmed that the mRNA expression patterns of these hub genes in ATDC5 cells were essentially consistent with the RNA-seq results. CONCLUSION: Temperature-mediated chondrocyte proliferation was associated with Ras, PI3K/AKT, mTOR, cAMP, and MAPK pathways, and Agt, Hspa1a, Hspb1, and Nlrc4 may be the key regulatory genes.

overall_design: The study groups included 37C, 40C and 44C groups. ATDC5 in the three groups was subjected to temperatures of 37C, 40C and 44C for 10 min, respectively. Each group had four biological replicates, for a total of 12 samples.

first: Wei

last: Zhao

first: Yingsong

last: Wang

first: Jin

last: Zhou

first: Zhi

first: Tao

last: Li

first: Zhiyue

last: Shi

first: Jie

last: Xiao


title: Response of Arabidopsis thaliana to flooding with physical flow

first: Nobuhiro

last: Suzuki

state: 東京都

address: 7-1, Kioicho, Chiyoda

institute: Sophia University

summary: It is the study to investigate the growth and transcriptome of Arabidopsis thaliana exposed to submergence or flooding with physical flow.

overall_design: Four-week-old Arabidopsis plants were grown under controlled conditions, or exposed to submergence or flooding with 8L/s water flow for 30 min. Three replicate were obained from each treatment.

first: Momoko

last: Kaji

first: Kazuma

last: Katano

first: Hiroshi

last: Nitta

first: Ryotaro

last: Yamaji

first: Rio

last: Shimizu

first: Shunsuke

last: Shigaki

first: Hiroyuki


title: Genome-wide CRISPR-Cas9 screen to identify SFV4 receptor

city: Uppsala

first: Miika

last: Martikainen

address: Dag Hammarskjölds Väg 20

department: Immunology, Genetics and Pathology

country: Sweden

institute: Uppsala Universitet

summary: The goal of this study is to identify entry receptor for Semliki Forest virus (SFV, strain SFV4). For this, we used the human genome-wide Brunello CRISPR/Cas9 knockout library in a screen on human osteosarcoma cells (HOS, ATCC, CRL-1543) infected with fully replicative SFV4. While these cells are not a natural target for neurotropic SFV, they offer a highly SFV-permissive and easy to culture model needed for the screen. We performed the screening both with and without the janus kinase (JAK) inhibitor ruxolitinib as we hypothesized that ruxolitinib would limit the host cells capability to resist SFV replication and therefore sensitize the screen more towards factors associated with viral entry

overall_design: Generation of HOS-Cas9/BFP and transduction with lentiviral human genome-wide Brunello CRISPR-library as service by SciLifeLab CRISPR Functional Genomics unit (Karolinska Institutet, Stockholm, Sweden). 40 000 000 cells/sample were used in the screen to maintain full coverage of the library. For the screening cells were given either Ruxolitinib alone (10µM, Selleck Chemicals, #S5243) or Ruxolitinib and SFV4-d1-EGFP (MOI=50). Two days later the surviving cells were harvested sgRNA expression was analyzed with MAGeCK analysis by SciLifeLab CRISPR Functional Genomics unit.

first: Di

last: Yu

first: Magnus

last: Essand


title: Human VCP mutant ALS/FTD microglia display immune and lysosomal phenotypes independently of GPNMB

status: Public on Dec 09 2024

city: London

first: Oliver

last: Ziff

address: Institute of Neurology, Queen Square

institute: University College London

summary: Background: Microglia play crucial roles in mediating neuronal homeostasis but have been implicated in contributing to amyotrophic lateral sclerosis (ALS). However, the role of microglia in ALS remains incompletely understood. Methods: Here, we generated highly enriched cultures of VCP mutant microglia derived from human induced pluripotent stem cells (hiPSCs) to investigate their cell autonomous and non-cell autonomous roles in ALS pathogenesis. We used RNA-sequencing, proteomics and functional assays to study hiPSC derived VCP mutant microglia and their effects on hiPSC derived motor neurons and astrocytes. Results: Transcriptomic, proteomic and functional analyses revealed immune and lysosomal dysfunction in VCP mutant microglia. Stimulating healthy microglia with inflammatory inducer lipopolysaccharide (LPS) showed partial overlap with VCP mutant microglia in their reactive transformation. LPS-stimulated VCP mutant microglia displayed differential activation of inflammatory pathways compared with LPS-stimulated healthy microglia. Conserved gene expression changes were identified between VCP mutant microglia, SOD1 mutant mice microglia, and postmortem ALS spinal cord microglial signatures, including increased expression of the transmembrane glycoprotein GPNMB. While knockdown of GPNMB affected inflammatory and phagocytosis processes in microglia, this was not sufficient to ameliorate cell autonomous phenotypes in VCP mutant microglia. Secreted factors from VCP mutant microglia were sufficient to activate the JAK-STAT pathway in hiPSC derived motor neurons and astrocytes. Conclusions: VCP mutant microglia undergo cell autonomous reactive transformation involving immune and lysosomal dysfunction that partially recapitulate key phenotypes of microglia from ALS models and post mortem tissue and are independent of GPNMB. VCP mutant microglia elicit non cell autonomous responses in motor neurons and astrocytes involving the JAK-STAT pathway.

overall_design: To study differences in ALS or LPS treated microglia and their effects on other CNS cell types we differentiated microglia from human iPSC lines from VCP mutant ALS or healthy control backgrounds and conducted RNA sequencing of microglia monocultures or astrocytes or motor neurons treated with microglia conditioned media. To investigate the role of GPNMB in microglia we conducted RNA sequencing of samples treated with either an siRNA targeting GPNMB or a scrambled control siRNA.

first: Benjamin

last: Clarke

first: Giulia

last: Tyzack

first: Marija

last: Petrić Howe

first: Yiran

first: Pierre

last: Klein

first: Claudia

last: Smith

first: Cameron

last: Hall

first: Adel

last: Helmy

first: Michael

last: Howell

first: Gavin

last: Kelly

first: Rickie

last: Patani


title: Multi-omics dissection of human RAG deficiency reveals distinctive patterns of immune dysregulation but a common inflammatory signature

city: New Haven

first: Andrew

last: Martins

state: Connecticut

address: 100 College Street Rm 1155

department: Immunobiology

institute: Yale School of Medicine

summary: A multi-omics approach has revealed both common and distinctive cellular and molecular signatures of inflammation and immune dysregulation that characterize distinct phenotypes of human RAG deficiency.

overall_design:
PBMC from RAG deficiency patients and controls were processed via the 5' CITEseq technique. Samples are pooled before staining and demlutiplexed based on SNPs inferred from bulk RNAseq.

CodeForUpload.zip contains the R analysis code used in the associated manuscript, which describes the analysis of the submitted data.

CountsH5mat.tar.gz is the cellranger counts output files for the Feature Barcode and mRNA tag libraries. There are 12 counts h5 files within the tar archive, which correspond to the 12 'csp-' and 'rna5p-' libraries in order.

first: Marita

last: Bosticardo

first: Adam

last: Dobbs

first: Ottavia

middle: M

last: Delmonte

first: Can

last: Liu

first: John

middle: S

last: Tsang

first: Luigi

last: Notarangelo


title: An Improved Bacterial Single-cell RNA-seq Reveals Biofilm Heterogeneity

first: Xiaodan

last: Yan

state: Hubei Province

address: Donghu Road, No. 115, Wuchang District,

department: Medical Research Institute

institute: Wuhan University

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


title: RSK1 is an exploitable dependency in myeloid malignancies [CD14_RNAseq]

status: Public on Dec 06 2024

city: St. Louis

first: Tim

last: Kong

state: MO

address: CSRB 8838

institute: Washington University School of Medicine

overall_design: Here, we establish a comprehensive RNA-seq dataset of CD14+ cells encompassing 68 patient samples across the spectrum of myeloproliferative neoplasms with normal donor controls. CD14+ cells were bead-enriched from the CD34- sorted fraction from MPN PBMCs and normal healthy donor (bone marrow) to assess gene expression patterns.

first: Stephen

middle: T

last: Oh


title: RSK1 is an exploitable dependency in myeloid malignancies [CD34RNAseq]

overall_design: Here, we establish a comprehensive RNA-seq dataset of CD34+ cells encompassing 90 patient samples across the spectrum of myeloproliferative neoplasms and secondary acute myeloid leukemia with normal donor controls. CD34+ cells were bead-enriched from MPN/sAML patient PBMCs and normal healthy donor (bone marrow) to assess gene expression patterns.


title: An Improved Bacterial Single-cell RNA-seq Reveals Biofilm Heterogeneity [RNA-seq]

summary: In contrast to mammalian cells, bacterial cells lack mRNA polyadenylated tails, presenting a hurdle in isolating mRNA amidst the prevalent rRNA during single-cell RNA-seq. This study introduces a novel method, Ribosomal RNA-derived cDNA Depletion (RiboD), seamlessly integrated into the PETRI-seq technique, yielding RiboD-PETRI. This innovative approach offers a cost-effective, equipment-free, and high-throughput solution for bacterial single-cell RNA sequencing (scRNA-seq). By efficiently eliminating rRNA reads and substantially enhancing mRNA detection rates (up to 92%), our method enables precise exploration of bacterial population heterogeneity. Applying RiboD-PETRI to investigate biofilm heterogeneity, distinctive subpopulations marked by unique genes within biofilms were successfully identified. Notably, PdeI, a marker for the cell-surface attachment subpopulation, was observed to elevate cyclic diguanylate (c-di-GMP) levels, promoting persister cell formation. Thus, we address a persistent challenge in bacterial single-cell RNA-seq regarding rRNA abundance, exemplifying the utility of this method in exploring biofilm heterogeneity. Our method effectively tackles a long-standing issue in bacterial scRNA-seq: the overwhelming abundance of rRNA. This advancement significantly enhances our ability to investigate the intricate heterogeneity within biofilms at unprecedented resolution.

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

overall_design:
We performed gene expression profiling analysis using data obtained from RNA-seq of exponential growth phase E.coli.
Then we analyzed consistency of RNA-seq data with single-cell sequencing data.


title: An Improved Bacterial Single-cell RNA-seq Reveals Biofilm Heterogeneity [scRNA-seq]

overall_design:
We performed a new single-cell library construction method for three species of bacteria: Escherichia coli MG1655, Caulobacter crescentus strain NA1000, Staphylococcus aureus 25923 and verified the effect of different treatments during library construction on library data.
And then we analyzed the single-cell sequencing datas by Seurat(version 4.3.0; http://satijalab.org/seurat/).


title: Multi-omics analysis of placental metabolomics and transcriptomics datasets reveals comprehensive insights into the pathophysiology of preeclampsia

city: Malatya

first: Berat

last: Dogan

address: Faculty of Eng.

department: Biomedical Eng.

country: Turkey

institute: Inonu University

summary: Preeclampsia, a life-threatening pregnancy complication, remains a major global health concern. Understanding the complex molecular mechanisms underlying this disorder is crucial for improving both diagnostics and therapeutic strategies. In this study, a multi-omics approach based on NMR metabolomics and RNA-seq transcriptomics analyses was conducted to analyze placental tissue samples obtained from patients with preeclampsia and healthy controls. Metabolomics data analysis results indicated alterations in several metabolite levels including lactate, myo-inositol, glutamate, glutamine, valine, leucine, isoleucine, creatinine, alanine, taurine, choline, phosphocholine, glycerophosphocholine, ethanolamine, and dihydroxyacetone. These alterations cause significant disruptions in the Krebs cycle, energy, lipid, and amino acid metabolisms. Concurrently, transcriptomics data analysis identified 10 upregulated and 37 downregulated genes (|log2FC| > 1 and padj < 0.05) in preeclampsia patients. Identified genes were linked to critical roles such as vasoconstriction, angiogenesis, inflammation, hormonal balance, oxidative stress, and collagen integrity. Multi-omics data analysis revealed the association of certain metabolites with several other genes. A gene interaction network formed by these genes resulted in a lower protein-protein interaction enrichment value (p-value < 1e-16) compared to the network formed with the differentially expressed genes (p-value = 0.0183) which suggests the importance of considering multiple omics levels for a comprehensive understanding of the disease.

first: Akın

last: Mumcu

first: Erdinç

last: Sarıdoğan

first: Senem

last: Arda Düz

first: Görkem

last: Tuncay

first: Ali

last: Erdoğan

first: Kadri

last: Karaer

first: Taylan

last: Onat

first: Abdullah

last: Doğan


title: Rejuvenating aged osteoprogenitors for bone repair

city: Lausanne

first: Shukry

middle: James

last: Habib

address: Rue du Bugnon 7

department: Biomedical Sciences

country: Switzerland

institute: University of Lausanne

summary: To investigate cellular population heterogeneity, and  gene expression in osteoprogenitors and other cell types (endothelial, osteogenic, immune) we isolate the calvarial periosteum and sagittal sutures as stem cell compartments that contribute to osteogenesis. We compare cell populations and gene expression between different ages of mouse, and also between different diets (ad libitum vs intermittent fasting)

overall_design: Gene expression analysis of single cell RNA-seq data for cells isolated from the calvarial periosteum and sagittal sutures of young, adult, and aged mice, and those undergoing intermittent fasting

first: Joshua

last: Reeves

first: Pierre

last: Tournier

last: Becquart

first: Robert

last: Carton

first: Yin

last: Tang

first: Alessandra

last: Vigilante

first: Dong

last: Fang


title: PhpNF-Y drives heterochromatic transcription to generate splicing machinery-dependent small RNAs (ChIP-Seq)

city: Bethesda

first: Shiv

last: Grewal

state: MD

address: 9000 Rockville Pike

department: National Cancer Institute

institute: National Institutes of Health

summary: Heterochromatin regions of the genome are transcriptionally repressive in nature. However, constitutive heterochromatin, such as at centromeres and telomeres, coats regions harboring multiple repetitive DNA elements that are preferentially transcribed during S-phase by RNA polymerase II. RNAi machinery processes the transcripts to generate small RNAs (siRNAs) that are crucial for heterochromatin assembly. Despite significant mechanistic advances in the field, the contribution of transcription factor (TF) based mechanisms to repeat element transcription and siRNA production is unclear. Here, we discover that the conserved TF PhpCNF-Y, which contains a histone fold motif, collaborates with a Zn-finger domain-containing factor, Moc3, to access specific sites within the heterochromatin region, regardless of the presence of the repressive histone methylation mark. The cooperative function of the TFs is essential for gaining access to the heterochromatin region, where they bind to specific CCAAT motif sites within the heterochromatic repeat elements cenH and dh. Perturbation of heterochromatin has minimal effect on the binding of the TFs to these specific sites, and they remain bound even outside of the S-phase. We find that PhpCNF-Y and Moc3 mediate the transcription of the repeat elements originating from a bottom strand of cenH, which the spliceosome targets to generate siRNAs in a Swi6HP1-independent manner. This pathway for generating the initial transcripts is crucial for de novo heterochromatin assembly. Collectively, our study uncovers a fundamental transcription factor-based mechanism that gains access to the heterochromatin to enforce transcriptional silencing of repeat elements. Our findings provide significant insights into de novo heterochromatin assembly.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) in fission yeast. Input control and immunoprecipitation samples are provided for each experiment.

first: Manjit

last: Kumar Srivastav

first: H

last: Diego Folco

first: Drisya

last: Vijayakumari

first: Patroula

last: Nathanailidou

first: Shweta

last: Jain

first: Jothy

last: Dhakshnamoorthy

first: David

last: Wheeler


title: PhpNF-Y drives heterochromatic transcription to generate splicing machinery-dependent small RNAs (small RNA-Seq)

overall_design: Small RNA-Seq in fission yeast.


title: An Unbiased Approach to Identifying Cellular Reprogramming-Inducible Enhancers [RNA-seq]

status: Public on Dec 05 2024

city: Athens

first: Dimitris

last: Valakos

address: Soranou Ephessiou 4

department: Center of Basic Research

country: Greece

institute: Biomedical Reasearch Foundation Academy of Athens

summary: Cellular reprogramming of somatic cells towards induced pluripotency is a multistep stochastic process mediated by the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM), which orchestrate global epigenetic and transcriptional changes. We performed a large-scale analysis of integrated ChIP-seq, ATAC-seq and RNA-seq data and revealed the spatiotemporal highly dynamic pattern of OSKM DNA binding during reprogramming. We found that OSKM show distinct temporal patterns of binding to different classes of pluripotency-related enhancers. Genes involved in reprogramming are regulated by the coordinated activity of multiple enhancers, which are sequentially bound by OSKM for strict transcriptional control. Based on these findings, we developed an unbiased approach to identify Reprogramming-Inducible Enhancers (RIEs), constructed enhancer-traps and isolated cells undergoing reprogramming in real time. We used a representative RIE taken from the Upp1 gene fused to GFP and isolated cells at different time-points during reprogramming and found that they have unique developmental capacities as they are reprogrammed with high efficiency due to their distinct molecular signatures. In conclusion, our experiments have led to the development of an unbiased method to identify and isolate reprogrammable cells in real time by exploiting the functional dynamics of OSKM, which can be used as efficient reprogramming biomarkers.

overall_design: We wanted to examine the cells where the Upp1-800 enhancer is activated in cellular reprogramming. Thus, we cloned this element upstream of a minimal promoter and a GFP gene, transduced MEFs with lenti-viral particles bearing the construct and isolated GFP positive (GFP+) and GFP negative (GFP-) cells at day 2 after DOX induction.

first: Eleftheria

last: Klagkou

first: Giannis

last: Vatsellas

last: Thanos


title: Hi-C Calibration by Chemically Induced Chromosomal Interactions

city: State College

first: Yi

last: Li

address: 406 SOUTH FREAR, UNIVERSITY PARK

institute: Penn State University

summary: The genome-wide chromosome conformation capture method, Hi-C, has greatly advanced our understanding of genome organization. However, its quantitative properties, including sensitivity, bias, and linearity, remain challenging to assess. Measuring these properties in vivo is difficult due to the heterogenous and dynamic nature of chromosomal interactions. Here, using Chemically Induced Chromosomal Interaction (CICI) method, we create stable intra- and inter-chromosomal interactions in G1-phase budding yeast across a broad range of contact frequencies. Hi-C analysis of these engineered cell populations demonstrates that static intra-chromosomal loops do not generate Topologically Associated Domains (TADs) and only promote 3D proximity within ~50kb flanking regions. At moderate sequencing depth, Hi-C is sensitive enough to detect interactions occurring in a few percent of cells. It also shows no inherent bias toward intra- versus inter-chromosomal interactions. Furthermore, we observe a linear relationship between Hi-C signal intensity and contact frequency. These findings illuminate the intrinsic properties of the Hi-C assay and provide a robust framework for its calibration.

overall_design: Hi-C experiments are performed on two yeast strains with intra- or inter- chromosomal CICI pairs. For each strain, 5 populations with different absolute contact frequencies between CICI loci are generated and their Hi-C contacts are measured.

first: Fan

last: Zou

first: Lu

last: Bai


title: Transcriptional and metabolic changes following repeated fasting and refeeding of adipose stem cells highlight adipose tissue resilience

city: Oslo

first: Thomas

last: Sæther

state: Oslo

address: Sognsvannsveien 9

department: Dept. of Molecular Medicine

country: Norway

institute: University of Oslo

summary: Background: Obesity and related metabolic disorders have reached epidemic levels, calling for diverse therapeutic strategies. Altering nutrient intake, timing and quantity by intermittent fasting seem to elicit beneficial health effects by modulating endocrine and cell signaling net-works. This study explores the impact of cyclic nutrient availability in the form of every-other-day fasting (EODF) on human adipose stem cells (ASCs). Methods: We subjected ASCs to repeated fasting/refeeding (F/R) cycles, mimicking low glucose/high fatty acid (LGHF) conditions, and assessed phenotypic and transcriptomic changes, lipid storage capacity, insulin sensitivity and differentiation potential. Results: Four consecutive F/R cycles induced significant changes in ad-ipogenic gene expression, with upregulation of FABP4 and PLIN1 during fasting, and increased lipid storage in the ASCs. Upon differentiation, ASCs exposed to LGHF conditions retained a transient increase in lipid droplet size and altered fatty acid metabolism gene expression until day 9. However, these changes dissipated by day 15 of differentiation, suggesting a limited duration of fasting-induced transcriptional and adipogenic memory. Despite initial effects, ASCs showed re-silience, returning to a physiological trajectory during differentiation, with respect to gene ex-pression and lipid metabolism. Conclusions: These findings suggest that the long-term effects of EODF on the ASC niche may be transient; emphasizing the ability of the adipose tissue to adapt and restore homeostasis.

overall_design: Upon confluency, ASCs were either subjected to fasting/refeeding cycles or kept in growth media without FGF2 until such cycles were concluded. Cycled cells were exposed 24h to fasting media, followed by 24h on refeeding media.Fasting was performed in low-glucose media alone or supplemented with 100uM oleic acid. After 4 fasting-refeeding cycles, cells were induced with adipogenic cocktail for maximum 15 days.We then compared gene expression patterns from RNA-seq between treatments at the end of fasting-refeeding (D0), and at days 9 and 15 of differentiation post-cycling (D9 and D15, respectively).

first: Natalia

last: Galigniana

first: Mohamed

last: Abdelhalim

first: Philippe

last: Collas


summary: Cellular reprogramming of somatic cells towards induced Pluripotent Stem Cells (iPSCs) is achieved through the over-expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM). Due to the high complexity of the process, the vast number of the involved molecules and its stochastic and inefficient nature, the molecular background of reprogramming is only partially revealed. The aim of this study is the identification of genomic regulatory elements which are induced during cellular reprogramming, the examination of their action and their association with the expression of important genes. First, we constructed a combined ChIP-seq OSKM dataset using experiments on MEFs (Mouse Embryonic Fibroblasts) from multiple published studies, in order to examine the OSKM binding upon the genome, irrespective of minor discrepancies originating from the different reprogramming systems and protocols used. We also performed detailed RNA-seq and ATAC-seq experiments in order to monitor the functional results of the OSKM genomic binding. Initially, we focused on the ESC-related sites (Embryonic Stem Cells, natural equivalent of iPSCs) and found out that almost 1/3 of them are bound by OSKM as early as at day 1, or since MEFs. Our analysis highlighted the significant role of Myc in reprogramming, which is often overlooked. We saw that, along with Klf4, they “mark” positions where Oct4 and/or Sox2 will bind after reprogramming initiation. Furthermore, we discovered that various genes are regulated by multiple genomic elements, where OSKM bind at different timepoints in reprogramming. For example, many genes involved in the MET pathway (Mesenchymal to Epithelial Transition) are located near both ESC-, MEF- and Transient-OSKM sites, resulting in stably high expression levels which temporarily increase during the intermediate timepoints. Finally, we observed that OSKM often bind on the chromatin in a dynamic fashion. This “on-and-off” association of OSKM with the genome is related to the need of chromatin reorganization to achieve pluripotency. It is logical to assume that part of the inherent stochasticity of cellular reprogramming is based on the low percentage of cells that will achieve to reproduce this dynamic OSKM binding to regulate efficiently their transcriptional programs and to finally transform to iPSCs.

overall_design: We examined the transcriptome of MEFs undergoing reprogramming at different time-points during the process (RNA-seq).

first: Dimitrios

first: Spyros

last: Foutadakis


title: Real-time quantitative PCR analysis of human corneal cell types

city: Boston

first: Pablo

last: Argueso

address: 150 Harrison Avenue

institute: Tufts Medicine

summary: Primary human limbal epithelial cells, corneal fibroblasts and a human neuronal cell line SY-SY5Y were incubated with recombinant human galectin-3 or BSA control and mRNA collected for neurotrophic factor mRNA expression.

overall_design: qPCR gene expression profiling. Cultures from three different donors (primary cells) or cells plated on three separate days (cell line) were used to collect mRNA. Equal amounts of total RNA from each donor were used for PCR arrays.

first: Ashley

last: Woodward

last: Argüeso


title: An Unbiased Approach to Identifying Cellular Reprogramming-Inducible Enhancers[ATAC-seq]

overall_design: We examined the chromatin accessibility of MEFs undergoing reprogramming using ATAC-seq at multiple time-points.


title: PhpNF-Y drives heterochromatic transcription to generate splicing machinery-dependent small RNAs (RNA-Seq)

first: Shiv

last: Grewal

overall_design: RNA-sequencing (RNA-seq)  in fission yeast.

first: Manjit

last: Kumar Srivastav

first: H

last: Diego Folco

first: Drisya

last: Vijayakumari

first: Patroula

last: Nathanailidou

first: Shweta

last: Jain

last: Dhakshnamoorthy


title: Identification of Differentially Expressed Genes in Human Testis Biopsies with Defective Spermatogenesis

city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

institute: MPI for heart and lung research

summary: The study was designed to identify differences in the expressed transcriptome of the two pathological human testis sample groups compared to normal spermatogenesis samples, with the objective of discovering genes related to the pathological condition. Transcriptional differences in human testis biopsies collected from men with normal spermatogenesis (NSP, n=3; intact germ cells, clinical score - 10), spermatid arrest (SDA, n=2; contains somatic cells and germ cells up to round spermatids, clinical score – 4-5) and Sertoli cell-only (SCO, n=2; complete absence of germ cells, clinical score - 0) phenotypes were assessed by RNA sequencing. A total of over 49 million reads were generated in NSP samples, over 24 million reads in SDA samples, and over 13 million reads in SCO samples. Differentially expressed genes (DEG) were identified based on a mean minimum count => 5, FDR <= 0.05 and log2FC fold change of >0.585 or <-0.585. Genes differentially expressed in pathological groups SDA and SCO compared to NSP, and genes differentially expressed in between the two pathological groups SDA and SCO were filtered. The study discovered a number of significantly differentially expressed genes (DEG) between groups as follows: NSP vs SDA n=1,873; SDA vs SCO n= 4,017; and NSP vs SCO n=10,253. A number of genes (n=10) were selected for further analysis by qRT-PCR and the detection of the protein (n=2) localization in testis by immunohistochemistry.

overall_design: Examination of differentially expressed genes and its transcript levels in normal and abnormal spermatogenesis conditions in human

first: Shashika

last: Kothalawala

last: Guenther

first: Hans-Christian

last: Schuppe

first: Adrian

last: Pilatz

first: Florian

last: Wagenlehner

first: Sabine

last: Kliesch

first: Katherine

last: Loveland

first: Liza

last: O’Donnell

first: Daniela

last: Fietz


title: Differential Activity of MAPK signalling Defines Fibroblast Subtypes in Pancreatic Cancer (RNA-Seq)

city: Verona

first: Davide

last: Pasini

state: VR

address: Piazzale Ludovico Antonio Scuro 10

country: Italy

institute: University of Verona

summary: Fibroblast heterogeneity is increasingly recognised across cancer conditions. Given their important contribution to disease progression, mapping fibroblasts’ heterogeneity is critical to devise effective anti-cancer therapies. Cancer-associated fibroblasts (CAFs) represent the most abundant cell population in pancreatic ductal adenocarcinoma (PDAC). Whether CAF phenotypes are differently specified by PDAC cell lineages remains to be elucidated. Here, we reveal an important role for the MAPK signalling pathway in defining PDAC CAF phenotypes. We show that epithelial MAPK activity promotes the myofibroblastic differentiation of CAFs by sustaining the expression and secretion of TGF-β1. We integrate single-cell profiling of post-perturbation transcriptional responses from mouse models with cellular and spatial profiles of human tissues to define a MAPKhigh CAF (mapCAF) phenotype. We show that this phenotype associates with basal-like tumour cells and reduced frequency of CD8+ T cells. In addition to elevated MAPK activity, this mapCAF phenotype is characterized by TGF-b signalling, hypoxia responsive signatures, and immunoregulatory gene programs. Furthermore, the mapCAF signature is enriched in myofibroblastic CAFs from various cancer conditions and correlates with reduced response to immune checkpoint inhibition in melanoma Altogether, our data expand our knowledge on CAF phenotype heterogeneity and reveal a potential strategy for targeting of myofibroblastic CAFs in vivo.

overall_design:
To identify molecular features predictive of cellular dependency on MAPK signalling, we treated 6 human cell lines and 5 patient-derived organoids (PDOs) with increasing doses of the MEK1/2 inhibitor trametinib (MEKi).
To understand whether transcriptional changes from the scRNA-seq experiment could be observed in vitro. we used at first mPSCs exposed to conditioned media from both basal-like and classical cancer cell lines. And subsequently, mPSCs  pre-treated for two days with TGF-β1 to attenuate the polarization towards iCAFs and exposed for 24 hours to either basal-like or classical CM.
We performed gene expression profile with RNA-seq

first: Lisa

last: Veghini

first: Vincenzo

last: Corbo


title: Differential Activity of MAPK signalling Defines Fibroblast Subtypes in Pancreatic Cancer (Spatial Transcriptomics)

overall_design: We performed spatial RNAseq profiling with Visium on 4 PDAC tissue samples (2 Basal-like and 2-classical) to check the presence and the spatial localization of our described mapCAF phenotype in the tissue


title: mRNA juxtacap sequences govern mRNA translation and stability

status: Public on Dec 04 2024

city: New York

first: Ben

middle: R

last: Hawley

state: New York

address: 1300 York Avenue

institute: Weill Cornell Medicine

summary: Here we utilize a highly precise transcription-start site (TSS) mapping method to assess the relative contributions of the first transcribed nucleotides and modifications (the "juxtacap") to mRNA stability and translation. We find that several functionally relevant juxtacaps exist, and the presence of m6Am regulates the effect of adenosine juxtacaps.

overall_design: Development of 5'UTR SLAM-seq for assaying half-lives, and 5'UTR polysome profiling to measure the mean ribosome load (MRL; equivalent to translational efficiency).

first: Samie

last: Jaffrey


title: Differential Activity of MAPK signalling Defines Fibroblast Subtypes in Pancreatic Cancer (scRNA-Seq)

overall_design: Single cell transcriptomic analysis of vehicle treated and Meki treated mouse PDAC


title: Cardiac Applications of CRISPR/AAV-Mediated Precise Genome Editing

status: Public on Dec 02 2024

city: Indianapolis

first: Nathan

last: VanDusen

state: IN

department: Pediatrics

institute: Indiana University School of Medicine

summary: The activity of cardiomyocyte and control enhancers was assessed in mouse cardiomyocytes, using two assay formats: a Tnni1 genomically integrated MPRA and AAV-based episomal MPRAs with various minimal promoters.

sample_organism:
Mus musculus
Adeno-associated virus

platform_organism:
Mus musculus
Adeno-associated virus

overall_design: AAV9 reporter pools were injected at P0 and tissue was harvested for measurement of enhancer activity at P28 by amplicon sequencing. See https://github.com/nvanduse/CASAAV-HDR_Applications for NGS processing scripts.


title: Placental and Immune Cell DNA Methylation Reference Panel for Bulk Tissue Cell Composition Estimation in Epidemiological Studies

status: Public on Dec 03 2024

city: Ann Arbor

first: Kelly

last: Bakulski

state: MI

address: 1415 Washington Heights

department: Epidemiology

institute: University of Michigan

type: Methylation profiling by genome tiling array

summary: To distinguish DNA methylation (DNAm) from cell proportion changes in whole placental villous tissue research, we developed a robust cell type-specific DNAm reference to estimate cell composition. We collated new and existing cell type DNAm profiles quantified via Illumina EPIC or 450k microarrays. To estimate cell composition, we deconvoluted whole placental samples (n=36) with robust partial correlation based on the top 30 hyper- and hypomethylated sites identified per cell type. To test deconvolution performance, we evaluated RMSE in predicting principal components of DNAm variation in 204 external placental samples. We analyzed DNAm profiles (n=368,435 sites) from 12 cell types: cytotrophoblasts (n=18), endothelial cells (n=19), Hofbauer cells (n=26), stromal cells (n=21), syncytiotrophoblasts (n=4), six lymphocyte types (n=36), and nucleated red blood cells (n=11). Median cell composition was consistent with placental biology: 60.9% syncytiotrophoblast, 17.3% stromal, 8.8% endothelial, 3.7% cytotrophoblast, 3.7% Hofbauer, 1.7% nucleated red blood cells, and 1.2% neutrophils. Our expanded reference outperformed an existing reference in predicting DNAm variation (PC1, 15.4% variance explained, IQR=21.61) with cell composition estimates (RMSEP: 8.62 vs. 10.79, p-value<0.001). This cell type reference can robustly estimate cell composition from whole placental DNAm data to detect important cell types, reveal biological mechanisms, and improve causal inference.

overall_design: To identify placental cell type-specific DNA methylation profiles for reference-based deconvolution, we collected human placentas from a convenience sample of complication-free pregnancies. Upon collection, placental villous tissue was excised for whole tissue DNA extraction and for dissociation and magnetic bead-based cell type sorting for cell type-specific DNA extraction. Four placentas were subsampled by quadrant, though this was not a primary variable for analysis in this study.

first: Abraham

last: Campbell

first: A

last: Colacino

first: John

last: Dou

first: C

last: Dolinoy

first: Sung

last: Park

first: Rita

last: Loch-Caruso

first: Vasantha

last: Padmanabhan


title: Transcriptomic analysis the mechanism of detoxification metabolism induced by afidopyropen in Semiaphis heraclei

city: Jinan

first: jianhua

last: zhang

address: gongyebei street No. 23788

institute: Shandong Academy of Agricultural Sciences

summary: In order to clarify the molecular mechanism of metabolic detoxification in Semiaphis heraclei, the transcriptome sequencing analysis was used to screen and identify the relevant genes related to metabolic detoxification after induced by afidopyropen at LD50 dose.

overall_design: Comparative gene expression profiling analysis of RNA-seq data under afidopyropen treatment.

first: Jianhua

last: Zhang

first: Yuanxue

last: Yang

first: Xiaochen

last: Fu

first: Yun

last: Zhou


title: Histone methyltransferase SETDB1 safeguards mouse fetal hematopoiesis by suppressing activation of cryptic enhancers [ChIP-seq]

city: Planegg-Martinsried

first: Gunnar

last: Schotta

address: Grosshaderner Strasse 9

department: Biomedical Center

institute: LMU Munich

summary: The H3K9me3-specific histone methyltransferase SETDB1 is critical for proper regulation of developmental processes, but the underlying mechanisms are only partially understood. Here, we show that deletion of Setdb1 in mouse fetal liver hematopoietic stem and progenitor cells (HSPCs) results in compromised stem cell function, enhanced myeloerythroid differentiation, and impaired lymphoid development. Notably, Setdb1-deficient HSPCs exhibit reduced quiescence and increased proliferation, accompanied by the acquisition of a lineage-biased transcriptional program. In Setdb1-deficient HSPCs, we identify genomic regions that are characterized by loss of H3K9me3 and increased chromatin accessibility, suggesting enhanced transcription factor (TF) activity. Interestingly, hematopoietic TFs like PU.1 bind these cryptic enhancers in wild-type HSPCs, despite the H3K9me3 status. Thus, our data indicate that SETDB1 restricts activation of nonphysiological TF binding sites which helps to ensure proper maintenance and differentiation of fetal liver HSPCs.

overall_design: H3K9me3 analysis of Setdb1 mutant vs Setdb1 con fetal liver LSK cells at E13.5

first: Maryam

last: Kazerani

first: Filippo

last: Cernilogar


title: Histone methyltransferase SETDB1 safeguards mouse fetal hematopoiesis by suppressing activation of cryptic enhancers [RNA-seq]

overall_design: RNAseq of FACS sorted E13.5 fetal liver LT-HSCs and MPPs from Setdb1 mutant vs control


title: Histone methyltransferase SETDB1 safeguards mouse fetal hematopoiesis by suppressing activation of cryptic enhancers [ATAC-seq]

overall_design: ATAC-seq of Setdb1 mutant vs Setdb1 control fetal liver LSK cells at E13.5


title: Discovery, Validation and Mechanistic study of XPO1 inhibition in the Treatment of Triple Negative Breast Cancer

city: Minneapolis

first: R. Stephanie

last: Huang

state: MN

address: 308 Harvard St. SE

institute: University of Minnesota

summary: Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with limited treatment options. This study investigates the potential of repurposing selinexor, an XPO1 inhibitor, as a novel therapeutic option for TNBC. By utilizing a computational drug repurposing approach, XPO1 inhibitors were identified to be preferentially sensitive in TNBC compared to other breast cancer subtypes and its efficacy validated in an independent patient dataset and across various TNBC cell lines. Using RNA-sequencing after longitudinal XPO1 inhibition in a panel of TNBC cell lines and western blotting, we reveal that XPO1 inhibitor induces TNBC cell death by inhibiting the NF-kB pathway through nuclear retention of NFKBIA, a key regulator of this pathway. These findings suggest that XPO1 inhibitors could be an effective targeted therapy for TNBC and paves the way for more personalized treatment strategies offering hope for better outcomes in TNBC patients.

overall_design: 4 cell lines treated with either drug or DMSO control; 2 time points; conducted in triplicate

first: Amy

middle: L

last: Paulson

first: Robert

middle: F

last: Gruener

first: Adam

last: Lee


title: Integrative Analysis of Genome-Wide Epigenetic and Transcriptomic Alterations Reveals Molecular Markers for Diagnosing Pediatric Obstructive Sleep Apnea in Black Females

city: Cincinnati

first: Matthew

last: Weirauch

state: Ohio

address: 3333 Burnet Avenue

department: Center for Autoimmune Genomics and Etiology (CAGE)

institute: Cincinnati Children's Hospital Medical Center

type: Methylation profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Integrative Analysis of Genome-Wide Epigenetic and Transcriptomic Alterations Reveals Molecular Markers for Diagnosing Pediatric Obstructive Sleep Apnea in Black Females [WGBS]

summary:
This study explores the epigentic and transcriptomic changes associated with pediatric obstructive sleep apnea in Black female patients. By analyzing saliva samples, the study identifies dysregulated pathways and specific molecular markers, emphasizing the need for accessible diagnostic tools and addressing healthcare disparities in underrepresented population.
The non-invasive approach using saliva samples offers potential for future research and improved diagnostics for pediatric OSA.

overall_design: Saliva samples were obtained from both healthy individuals (controls) and children diagnosed with obstructive sleep apnea (OSA). The DNA was extracted from the samples and subsequently fragmented into base pairs ranging from 100 to 300. Bi-sulfite conversion was performed specifically for non-methylated DNA. Paired-end libraries were created from the converted DNA and then subjected to sequencing using an Illumina HiSeq 2000 instrument.

first: Bala

last: Koritala

first: Sreeja

last: Parameswaran

first: Leah

last: Kottyan

first: David

last: Smith


title: Integrative Analysis of Genome-Wide Epigenetic and Transcriptomic Alterations Reveals Molecular Markers for Diagnosing Pediatric Obstructive Sleep Apnea in Black Females [RNA-Seq]

overall_design: Saliva samples were collected from both healthy controls and children with OSA. RNA was extracted and sequenced pair-end 150 base reads using combinatorial Probe-Anchor Synthesis (cPAS) technology.


title: Dysregulated regulatory T cell responses expand fibroblasts and lead to chronic rejection after heart transplantation

city: Pittsburgh

first: Khodor

middle: I

last: Abou-Daya

state: PA

address: 200 Lothrop Street

department: Surgery

institute: Starzl Transplantation Institute

summary: Chronic rejection after organ transplantation manifests as immunosuppressant-resistant graft vascular remodeling and fibrosis, which remains the dominant driver of mortality after the first year of heart transplantation. Single-cell RNAseq analysis of MHCII-mismatched heart transplants developing chronic rejection identified graft IL-33 as a stimulator of tissue repair pathways in infiltrating myeloid cells and regulatory T cells (Tregs). Using IL-33-deficient donor mice, it was revealed that graft fibroblast-derived IL-33 potently induced Amphiregulin (Areg) expression by recipient Treg. Areg is an epidermal growth factor secreted by multiple immune cells to shape immunomodulation and tissue repair. In particular, Areg is proposed to play a major role in Treg-mediated muscle, epithelium, and nerve repair. Assessing recipient mice with Treg-specific deletion of Areg demonstrated that this pathway surprisingly contributes to chronic rejection. Specifically, heart transplants from recipients with Areg-deficient Tregs exhibited less vasculopathy and vessel-associated fibrotic niches infiltrated by recipient T cells. Mechanistically, we show that Areg does not impact Treg suppressive function, but that IL-33 mediated Treg-secretion of Areg increases fibroblast proliferation and migration. In total, these studies identify how a dysregulated repair response involving interactions between IL-33+ fibroblasts in the allograft and recipient Treg contributes to the progression of chronic rejection.

overall_design:
The overall purpose of this study was to elucidate how local IL-33 supports Treg-mediated tissue repair responses and the ways that these processes become dysregulated following transplant. We hypothesized that the recipient immune responses to persistent MHC differences will instigate immune-mediated injuries that disrupts effective repair of the damages sustained during transplantation. On day 14 post-transplantation we applied scRNA-seq with cell hashing to immune cells (lympholyte-M seperation) from BM12 chronic murine heart rejection transplant model that were compare to  BM12 heart grafts deficient in IL-33 (n=3/group), enabling us to understand the full scope of IL-33 in regulating immune remodeling of graft tissue. IL-33 modulated over 700 genes in heart-infiltrating Tregs, including the growth factor amphiregulin (Areg), yet when B6 Foxp3YFP-CrexAregfl/fl and Foxp3YFP-Cre mice receiving Bm12 hearts were assessed at day 100 (n=6-10), the deletion of Areg in Tregs protected against chronic rejection. Our analyzed data reflects bioinformatic computational tools, in vitro assays, and immunofluorescence. Age and sex matched mice were randomly assigned to experimental groups and the studies were not blinded.
HTO Barcode Hashtagged Sample
TotalSeq-A0301 ACCCACCAGTAAGAC BM12 WT 1
TotalSeq-A0302 GGTCGAGAGCATTCA BM12 WT 2
TotalSeq-A0303 CTTGCCGCATGTCAT BM12 WT 3
TotalSeq-A0304 AAAGCATTCTTCACG BM12 IL33 KO 4
TotalSeq-A0305 CTTTGTCTTTGTGAG BM12 IL33 KO 5
TotalSeq-A0306 TATGCTGCCACGGTA BM12 IL33 KO 6

first: Heth

last: Turnquist


title: Osmotic disruption of chromatin induces Topoisomerase 2 activity at sites of transcriptional stress [RNA-Seq]

status: Public on Dec 01 2024

city: Brighton

first: William

last: Gittens

state: I am not in the U.S. or Canada

address: Science Park Road

department: Genome Damage and Stability Centre

institute: University of Sussex

summary: Transcription generates superhelical stress in DNA that poses problems for genome stability, but determining when and where such stress arises within chromosomes is challenging. Here, using G1-arrested S. cerevisiae cells, and employing rapid fixation and ultra-sensitive enrichment, we utilise the physiological activity of endogenous topoisomerase 2 (Top2) as a probe of transcription-induced superhelicity. We demonstrate that Top2 activity is surprisingly uncorrelated with transcriptional activity, suggesting that superhelical stress is obscured from Top2 within chromatin in vivo. We test this idea using osmotic perturbation—a treatment that transiently destabilises chromatin in vivo—revealing that Top2 activity redistributes within sub-minute timescales into broad zones patterned by long genes, convergent gene arrays, and transposon elements—and also by acute transcriptional induction. We propose that latent superhelical stress is normally absorbed by the intrinsic topological buffering capacity of chromatin, helping to avoid spurious topoisomerase activity arising within the essential coding regions of the genome.

overall_design: Stranded total RNA-seq of G1-arrested S. cerevisisae, grown in media containing glucose, raffinose, or galactose.

middle: H

middle: J

last: Neale


title: Gpnmb and Spp1 mark a conserved macrophage injury response masking fibrosis-specific programming in the lung

city: Denver

last: Janssen

state: CO

address: 1400 Jackson St

institute: National Jewish Health

summary: Macrophages are required for healthy repair of the lungs following injury, but they are also implicated in driving dysregulated repair with fibrosis. How these two distinct outcomes of lung injury are mediated by different macrophage subsets is unknown. To assess this, single-cell RNA sequencing was performed on lung macrophages isolated from mice treated with lipopolysaccharide or bleomycin. Macrophages were categorized based on anatomic location (airspace versus interstitium), developmental origin (embryonic versus recruited monocyte-derived), time after inflammatory challenge, and injury model. Analysis of the integrated dataset revealed that macrophage subset clustering was driven by macrophage origin and tissue compartment rather than injury model. Gpnmb-expressing recruited macrophages that were enriched for genes typically associated with fibrosis were present in both injury models. Analogous GPNMB-expressing macrophages were identified in datasets from both fibrotic and non-fibrotic lung disease in humans. We conclude that this subset represents a conserved response to tissue injury and is not sufficient to drive fibrosis. Beyond this conserved response, we identified that recruited macrophages failed to gain resident-like programming during fibrotic repair. Overall, fibrotic versus non-fibrotic tissue repair is dictated by dynamic shifts in macrophage subset programming and persistence of recruited macrophages.

overall_design: Male resident interstitial macrophage(IM)  lineage tracing mice (were treated with intratracheal lipopolysaccharide or bleomycin.  Lungs were harvested 3, 6, and 15 days after lipopolysaccharide treatment and 3, 7, and 14 days after bleomycin treatment.  Lungs were also harvested from untreated mice (day 0).  For each timepoint and group, leukocytes were isolated from lungs by FACS sorting to separate: 1)all CD45+ cells, 2) all airspace macrophages, 3) resident interstitial macrophages, or 4)recruited (monocyte-derived) interstitial macrophages. Sorted cells were analyzed by scRNAseq

first: Emily M.

last: King

first: Yifan

last: Zhao

first: Camille M.

last: Moore

first: Benjamin

last: Steinhart

first: Kelsey C

last: Anderson

first: Brian

last: Vestal

first: Peter K.

first: Shannon A.

last: McManus

first: Christopher M.

last: Evans

first: Kara

last: Mould

first: Elizabeth

last: Redente

first: Alexandra L.

last: McCubbrey

first: William J.