title: Genome-wide DNA replication profiling and full-length total RNA sequencing from the same single cell [03_CBMS1_mESC]

city: Tsu-shi

first: Rawin

last: Poonperm

state: Mie

address: 1577 Kurimamachiya

department: Graduate School of Bioresources

institute: Mie University

summary: Single-cell sequencing has advanced our understanding of cell-type diversity and heterogeneity. However, existing single-cell multi-omics methods lack DNA data resolution and full-length total RNA detection. Here, we introduce single-cell (sc)Repli-RamDA-seq (scRR-seq), a novel multi-omics method that enables high-resolution DNA replication profiling and full-length total RNA sequencing from the same single cell, made possible by the clean separation of DNA and RNA. scRR-seq generates DNA replication and RNA sequencing data comparable to individually obtained scRepli-seq and scRamDA-seq data, respectively. scRR-seq also allows one to tell the cell-cycle stage of a given S-phase cell and detects copy-number variation (CNV) in non-S-phase cells. scRR-seq outperforms other scRNA-seq methods in the number of transcripts identified and full-length total RNA detection. Furthermore, scRR-seq allowed haplotype-specific analysis and the identification of novel S-phase markers. Taken together, scRR-seq is a robust single-cell multi-omics method with promising potential for comprehensive genome and transcriptome analysis.

overall_design: We performed simultaneous DNA-seq and RNA-seq on the same cells using the scRR-seq protocols (scRR1 and scRR3). The following cell types were used for the experiments: (1) hTERT-RPE1 cells collected from mid-S phase, (2) mouse 8-cell embryos, (3) CBMS1 mouse embryonic stem cells collected from the entire S phase, (4) hTERT-RPE1 cells collected from the entire S phase, (5) HAP1 cells collected from mid-S phase, and (6) IMR-90 cells treated with or without 0.4 µM aphidicoline, collected from the G1 phase.  Single cells were collected into 0.2 ml PCR tubes by cell sorter or manually picked and lysed in RamDA-seq lysis buffer (TOYOBO, #RMD-101) containing 2 mg/ml Dynabeads™ MyOne™ Carboxylic Acid (Invitrogen, #65011). Genomic DNA was trapped on the beads, while RNA was released into the lysis buffer. The isolated DNA on magnetic beads was subjected to whole-genome amplification (WGA) and Next-Generation Sequencing (NGS) library preparation, as described in scRepli-seq (Takahashi et al., Nat Genet 2019). The DNA-seq (scRepli-seq) data was used to assess genome-wide DNA copy numbers, karyotype G1-phase cells, and analyze the replication state of S-phase cells. In parallel, the RNA isolated in the supernatant was subjected to scRamDA-seq (Hayashi et al., Nat Commun 2018) using the GenNext®RamDA-seq™ Single Cell Kit (TOYOBO, #RMD-101) according to the manufacturer’s instructions with modifications.

first: Shin-ichiro

last: Takebayashi


title: Effect of LINC01871 silencing on CD4+ T cell activation.

status: Public on Aug 27 2025

city: Turku

first: Riitta

last: Lahesmaa

address: Tykistökatu 6A

department: Turku Bioscience

country: Finland

institute: University of Turku

type: Expression profiling by high throughput sequencing

summary: Long intergenic noncoding RNAs (lincRNAs) play crucial roles in regulating biological processes in health and disease. However, little is known about the contribution of lincRNAs in T-cell activation. Here, we identified a lincRNA, LINC01871, which is highly induced upon T-cell activation and is predominantly located in the cytoplasm. The anti-inflammatory cytokine TGF-β was found to suppress its expression. Silencing LINC01871 led to a modest decrease in IL-2 secretion. RNA-seq and proteomic analyses of LINC01871-deficient CD4+ T cells revealed several targets, including genes associated with autophagy and membrane organization such as ATG2B; TRIM5; SNX30; TIMM8B; and ATP10A. Notably, LINC01871 expression was highly specific to T cells in several cross tissue single cell RNA-seq atlases. Furthermore, ex-vivo CD4+ T cells from children progressing to beta-cell autoimmunity showed higher LINC01871 expression as compared to their age, sex and HLA-risk matched controls. These data suggest that LINC01871 has an in-vivo function in T-cell-mediated immunity.

overall_design: CD4 T cells were isolated using magnetic bead-based positive selection kit from Dynal. Cells were nucleofected with the three antisense locked nuelceic acid (LNAs) targeting the lincRNA LINC01871 or non targeting control LNAs (NT). After 24 hours of rest, cells were activated with CD3/CD28 for 48 hours. The cells were then reactivated for 30 minutes and RNA samples were extracted. There were three biological replicates* three LNAs  =9 samples.

first: Kedar

last: Batkulwar

first: Ubaid Ullah

last: Kalim

first: Tommi

last: Välikangas

first: Omid

last: Rasool


title: Genome-wide DNA replication profiling and full-length total RNA sequencing from the same single cell [IMR-90 G1]

country: Japan


title: GPRC5B preserves a mature β-cell state in obesity by controlling MafA expression

status: Public on Aug 26 2025

city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

country: Germany

institute: MPI for heart and lung research

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

overall_design: Refer to individual Series


title: Sclerotic GvHD and Scleroderma Share Dysregulated Gene Expression that is Ameliorated by EREG Therapeutic Antibody

status: Public on Aug 29 2025

city: New Haven

first: Ian

last: Odell

state: CT

address: 330 Cedar St. LMP-5040

department: Dermatology

country: USA

institute: Yale University

summary: Immune driven fibrotic skin diseases including scleroderma/systemic sclerosis (SSc) and chronic graft-versus-host-disease (cGvHD) cause skin stiffening that has major impact on patient quality of life and associated patient mortality. Therapies to improve sclerotic skin resulting from these diseases are largely ineffective. We previously showed that EREG, a DC3 dendritic cell-derived EGFR ligand, is elevated in the skin and lung of patients with SSc and required for maintenance of skin fibrosis. Here, we developed a fully human anti-EREG neutralizing antibody that has both high affinity and specificity. We found this therapeutic antibody to be functional and safe in vivo using human EREG knock-in mice. To understand the antifibrotic mechanism of targeting EREG, we aligned skin single-cell transcriptomic profiles of SSc, morphea (localized scleroderma) and SclGvHD with disease biomarkers. EREG expression in the skin was elevated in all three fibrotic diseases and a driver of TNC production by myofibroblasts in all three fibrotic diseases. TNC is a pro-inflammatory extracellular glycoprotein that functions as an endogenous TLR4 ligand which induces expression of TLR4 target genes CCL2 and IL6. Examination of skin explants from patients with active SclGvHD treated with anti-EREG therapeutic antibody by spatial transcriptomics demonstrated upregulation of matrix degradation by increased MMP and decreased TIMP1 expression. Protein measurements showed reduced secretion of EREG targets TNC, CCL2, and TIMP1 in all patients, and type I collagen and FN1 in 3/4 patients. Thus, sclerotic skin treated with the anti-EREG therapeutic antibody reduced inflammatory and fibrosis biomarkers associated with EGFR and TLR4 signaling.

overall_design: Skin biopsies from 3 patients with sclerotic graft-vs-host disease analyzed using 10x genomics platform

middle: D


title: Genome-wide DNA replication profiling and full-length total RNA sequencing from the same single cell [02_Mouse8cell_embryos]


title: Multi-omics analysis identifies progenitors of CD4-CTLs [invitrodiff_n_scm_scRNA]

city: New Delhi

first: Veena

middle: S.

last: Patil

state: Delhi

address: Aruna Asaf Ali Marg

country: India

institute: National Institute of Immunology

summary: The CD4+ T cells expressing cytolytic molecules such as granzymes and perforins have been detected in many diseases and are shown to be enriched in the effector memory expressing CD45RA (TEMRA) in humans, but their origin remains elusive. However, their gene expression profile in comparison to classically defined cytotoxic T lymphocytes (CTLs), the CD8-CTLs has not been well demonstrated, hence their relevance remains debatable.  Thus in this study by parallelly analysing the CD4-CTLs and CD8-CTLs, we demonstrate that they are indistinguishable for the cytolytic program with both showing similar gene expression profile and T cell antigen-receptor (TCR) clonal expansion. Further using an integrative multi-omics approach combining the transcriptome, TCR repertoire, and open chromatin profile of CD4+ naïve (CD4-TN) and memory subsets, we discovered a stem-cell memory subset that is pre-committed to CTL program within the CD4+ T cell lineage. Through an in vitro differentiation model we developed CD4+ T cells with cytolytic potential from CD4-TN cells. The in vitro differentiated cells followed the trajectory of different developmental memory subsets from ex vivo CD4+ T cells for transcriptomic patterns as well as open-chromatin landscape. Thus, through this model, we deciphered the molecular signatures of early commitment of CD4-TN cells to cytotoxicity program. Of particular interest was the expression of both longevity as well as cytotoxicity associated gene sets by the in vitro differentiated CD4-CTLs, hence generating long-lived CD4-CTL effectors. This specific property can be further explored for vaccine development as well as testing the efficacy and cell based therapies for precision medicine.

overall_design: CD4-Naive (CD3+CD4+CD45RA+CCR7+CD95-) and CD4-TSCM (CD3+CD4+CD45RA+CCR7+CD95+) were isolated from PBMCs of healthy human donors and activated for 48 hrs using aCD3/aCD28 Dynabeads in a Th1 polarizing milieu of cytokines and antibodies. The cells were then cultured in the Th1 polarizing condition for 8 days (2+8 day cycle) before doing scRNA-Seq (Day 10).

first: Raunak

last: Kar

first: Shreya

last: Sinha

first: Zainab

last: Khatun

first: Anjali

last: Sharma

middle: S


title: GPRC5B preserves a mature β-cell state in obesity by controlling MafA expression [dof92_RNA-seq]

summary: In vitro studies have implicated orphan receptor GPRC5B in β-cell survival, proliferation and insulin secretion, but its relevance for glucose homeostasis in vivo is largely unknown. Using tamoxifen-inducible, β-cell-specific GPRC5B knockout mice (Ins-G5b-KOs) we show here that loss of GPRC5B does not affect β-cell function in the lean state, but results in strongly reduced insulin secretion and disturbed glucose tolerance in mice subjected to high fat diet for 16 weeks. Flow cytometry and single-cell expression analyses in islets from obese mice show a reduced β-cell abundance and a less mature β-cell phenotype in Ins-G5b-KOs. Expression of β-cell-specific transcription factor MafA is reduced both on the RNA and protein level, as are transcripts of MafA target genes. Mechanistically, we show that phosphorylation of cAMP response element-binding protein (CREB), a major regulator of MafA expression, is reduced in islets of obese Ins-G5b-KOs, and that this phenotype precedes the downregulation of MafA and MafA target genes. Taken together, GPRC5B helps to maintain mature β-cell function in obesity through cAMP/CREB-dependent regulation of MafA expression.

overall_design: mRNA sequencing in isolated pancreatic beta cells from control mice and mice with beta cell-specific GPRC5B deficiency

first: Tianpeng

last: Wang

first: Nina

last: Wettschureck

last: Guenther


title: Genome-wide DNA replication profiling and full-length total RNA sequencing from the same single cell [hTERT-RPE1 Whole-S]

last: Poonperm


title: GPRC5B preserves a mature β-cell state in obesity by controlling MafA expression [dof105_scRNA-seq]

overall_design: Single cell mRNA sequencing in pancreatic islet cells from high fat diet-fed (16 weeks) control mice and beta cell-specific GPRC5B knockout mice


title: Multi-omics analysis identifies progenitors of CD4-CTLs [bulk TCR]

overall_design: TCR analysis of purified populations of human CD4 memory cell subsets including CD4-Naïve and CD8-TEMRA by bulk TCR-sequencing of both TCRa and TCRb from 10 healthy donors.


title: Multi-omics analysis identifies progenitors of CD4-CTLs [scRNA-seq ex vivo]

overall_design: PBMCs were isolated from healthy human blood bank donors (n=8 for ex vivo TSCM and TEMRA, n=10 for ex vivo whole CD4), and pure population of interest was sorted using FACS. scRNA-Seq was performed on these sorted cells after pooling cells from all donors for individual compartments for equal representation across donors.


title: Multi-omics analysis identifies progenitors of CD4-CTLs [scRNA-seq in vitro]

overall_design: CD4-Naive T cells (CD3+CD4+CD45RA+CCR7+CD95-) were isolated from PBMCs of healthy human donors and activated for 48 hrs using aCD3/aCD28 Dynabeads in a Th1 polarizing milieu of cytokines and antibodies. The cells were then cultured in the Th1 polarizing condition for 8 days (2+8 day cycle) before doing scRNA-Seq (Day 10). The same cycle was repeated for a second round before subjecting the cells to another scRNA-Seq on Day 20.


title: Multi-omics analysis identifies progenitors of CD4-CTLs [bulk ATAC-seq]

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: Open chromatin analysis of purified populations of human CD4-Naïve (TN), CD4-TSCM, CD4-TCM, CD4-TEM, CD4-TEMRA-P and CD4-TEMRA-E by bulk ATAC-sequencing from 10 healthy donors.


title: Genome-wide DNA replication profiling and full-length total RNA sequencing from the same single cell [01_RPE1_midS]

overall_design:
We performed simultaneous DNA-seq and RNA-seq on the same cells using the scRR-seq protocols (scRR1 and scRR3). The following cell types were used for the experiments: (1) hTERT-RPE1 cells collected from mid-S phase, (2) mouse 8-cell embryos, (3) CBMS1 mouse embryonic stem cells collected from the entire S phase, (4) hTERT-RPE1 cells collected from the entire S phase, (5) HAP1 cells collected from mid-S phase, and (6) IMR-90 cells treated with or without 0.4 µM aphidicoline, collected from the G1 phase.  Single cells were collected into 0.2 ml PCR tubes by cell sorter or manually picked and lysed in RamDA-seq lysis buffer (TOYOBO, #RMD-101) containing 2 mg/ml Dynabeads™ MyOne™ Carboxylic Acid (Invitrogen, #65011). Genomic DNA was trapped on the beads, while RNA was released into the lysis buffer. The isolated DNA on magnetic beads was subjected to whole-genome amplification (WGA) and Next-Generation Sequencing (NGS) library preparation, as described in scRepli-seq (Takahashi et al., Nat Genet 2019). The DNA-seq (scRepli-seq) data was used to assess genome-wide DNA copy numbers, karyotype G1-phase cells, and analyze the replication state of S-phase cells. In parallel, the RNA isolated in the supernatant was subjected to scRamDA-seq (Hayashi et al., Nat Commun 2018) using the GenNext®RamDA-seq™ Single Cell Kit (TOYOBO, #RMD-101) according to the manufacturer’s instructions with modifications.

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

last: Takebayashi


title: Targeting DDX3X suppresses progression of KRAS-driven lung cancer by disrupting antioxidative homeostasis and inducing ferroptosis

city: Guangzhou

first: Dian

last: Meijuan

address: NANFANG HOSPITAL SOUTHERN MEDICAL UNIVERSITY

country: China

institute: the sourthern medical university

summary: Approximately 30% of human cancers carry various RAS mutations, including KRAS, NRAS, and HRAS. Among these mutations, KRAS is the most prevalent isoform detected in lung cancer. While several small molecular inhibitors targeting specifically KRASG12C have been developed and tested clinically, alternative approaches are still necessary due to expected drug resistance. In this study, we present evidence that the loss of DDX3X significantly delays tumor progression in various KRAS-driven lung cancer models. Inhibition of DDX3X disrupts cysteine and glutathione metabolism, thereby inducing ferroptosis in lung cancer cells. This effect is primarily mediated by the downregulation of Cystathionine-β-synthase (CBS), the rate-limiting enzyme in cysteine generation. Mechanistically, DDX3X directly binds to the transcription factor JUND, which mediates the transcriptional regulation of METTL16, a key N6-methyladenosine methyltransferase, and subsequently regulates m6A modification and translation of CBS transcripts. This cascade induces hypermethylation and high expression of CBS, consequently triggering cysteine production and maintaining antioxidative homeostasis, which is essential for the survival of KRAS-driven lung cancer cells. Finally, we demonstrate that a newly developed DDX3X PROTAC degrader J10 efficiently delays lung cancer progression with multiple advantages compared to DDX3X small molecular inhibitor RK-33 and limited side effects. These findings unveil the potential of DDX3X as a valuable target for adjuvant therapies in managing KRAS-driven lung cancer.

overall_design: To study the role of DDX3X in lung cancer progression, we undertook a label-free proteomic analysis using DDX3X knockdown (shDDX3X) A549 cells.

first: Meijuan

last: Dian


title: Nanoparticle-boosted myeloid-derived suppressor cell therapy for immune reprogramming in multiple sclerosis

status: Public on Aug 25 2025

city: Chicago

first: Zongmin

last: Zhao

state: Illinois

address: 833 S Wood St

department: Department of Pharmaceutical Sciences

institute: University of Illinois Chicago

summary: Massive immune cell infiltration and persistent inflammation in the central nervous system (CNS) are key hallmarks of multiple sclerosis, an incurable autoimmune disease. Here, we report a myeloid-derived suppressor cell (MDSC)-based therapeutic strategy, named CNS Immune Targeting Enabled by MDSCs (CITED), which employs surface-decorated MDSCs carrying rapamycin nanoparticles (NPs) for targeted multimodal immune reprograming in the CNS. We show that NP decoration enhances MDSC immunomodulatory function, facilitates their trafficking to inflamed CNS regions, and increases NP accumulation within the CNS. In an experimental autoimmune encephalomyelitis (EAE) model, CITED exhibited robust therapeutic efficacy, resulting in reduced disease progression, improved motor function, and diminished myelin damage. Mechanistic studies reveal that CITED exerts its therapeutic effects by targeted reprogramming both innate and adaptive immune responses in CNS. Specifically, CITED inhibits immune cell infiltration, rebalances CD4 T cell phenotypes, and promotes the polarization of myeloid cells toward anti-inflammatory phenotypes. Collectively, CITED could provide a broadly effective approach for targeted immune restoration in multiple sclerosis and potentially other autoimmune diseases.

overall_design: RNA seq profilling of myeloid derived suppressor cells (MDSC), MDSCs conjugated with blank NP(MDSC_NP_Blk) and MDSCs carrying rapamycin nanoparticles (MDSC_NP_CITED).

first: Endong

last: Zhang

first: Hanan

last: Algarni

first: Chih-Jia

last: Chao

first: Shan

last: He

first: Luyu

first: Aditi

last: Upadhye

first: Qing

last: Bao

first: Dahee

last: Jung

first: Shubhi

last: Srivastava

first: Edidiong

last: Udofa

first: Philana

last: Phan

first: Steve Seung-Young

last: Lee

first: Jalees

last: Rehman


title: Extracellular vesicles from chylomicron-treated endothelial cells drive macrophage inflammation

city: New York

first: Genome Technology Center

last: (GTC)

address: 550 First Avenue

institute: NYU Langone Health

type: Non-coding RNA profiling by high throughput sequencing

summary: Movement of circulating lipids into tissues and arteries requires transfer across the endothelial cell barrier. This process allows the heart to obtain fatty acids (FAs), its chief source of energy, and apolipoprotein B (apoB)-containing lipoproteins to cross the arterial endothelial barrier leading to cholesterol accumulation in the subendothelial space. Multiple studies have established elevated postprandial triglyceride-rich lipoproteins (TRLs) as an independent risk factor for cardiovascular disease (CVD). We explored how chylomicrons affect ECs and transfer their FAs across the EC barrier. We had reported that media from chylomicron-treated ECs leads to lipid droplet (LD) formation in macrophages. To determine the responsible component of this media, we assessed whether removing the extracellular vesicles (EVs) would obviate this effect. EVs from control and treated cells were then characterized by protein, lipid and microRNA (miR) content. We also studied the EV-induced transcription changes in macrophages and ECs and whether knockdown of scavenger receptor-BI (SR-BI) altered these responses. In addition, using chylomicrons labeled with [13C]oleate, we studied the uptake and release of this labed by ECs.Chylomicron treatment of ECs led to an inflammatory response that included production of EVs that drove macrophage LD accumulation. The EVs contained little free fatty acids and triglyceride, but abundant phospholipids and diacylglycerols. In concert with this, [13]C labeled chylomicron triglycerides exited ECs primarily in phospholipids. EVs from chylomicron treated versus untreated ECs were larger, more abundant, and contained specific miRs. Treatment of macrophages and naïve ECs with media from chylomicron-treated ECs increased expression of inflammatory genes. EC chylomicron metabolism produces EVs that increase macrophage inflammation and create LDs. Media containing these EVs also increases EC inflammation, illustrating an autocrine inflammatory process. FAs within chylomicron triglycerides are converted to phospholipids within EVs. Thus, EC uptake of chylomicrons constitutes an important pathway for vascular inflammation and tissue lipid acquisition.

overall_design: Bulk RNA seq of endothelial cells (Ecs) and peritoneal macrophages (PMACs).Also, bulk sequencing of circulating microRNAs in the media of Ecs.

first: Dimitris

last: Nasias

first: Ira

last: Goldberg

first: Ainara

middle: G

last: Cabodevilla


first: Dian

last: Meijuan

first: Meijuan

last: Dian


title: Genome-wide DNA replication profiling and full-length total RNA sequencing from the same single cell [HAP1 mid-S]

first: Rawin

last: Poonperm

first: Shin-ichiro

last: Takebayashi


title: A distinct subset of stem-cell memory is poised for the cytotoxicity program in CD4+ T cells in humans

first: Veena

last: Patil


title: Multi-omics analysis identifies progenitors of CD4-CTLs [scATAC-seq ex vivo]

overall_design: PBMCs were isolated from healthy human blood bank donors (n=8), and pure population of interest was sorted using FACS. scATAC-Seq was performed on these sorted cells after pooling cells from all donors for equal representation across donors.

first: Raunak

last: Kar

first: Shreya

last: Sinha

first: Zainab

last: Khatun

first: Anjali

last: Sharma


title: Genome-wide DNA replication profiling and full-length total RNA sequencing from the same single cell

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: A CRISPR-Activation CROP-seq Screen Identifies HMGN1 as a Dosage Sensitive Regulator of Heart Defects in Down Syndrome (scRNA-Seq)

city: San Francisco

first: Angelo

last: Pelonero

state: CA

address: 1650 Owens Street

department: Cardiovascular Disease Institute

institute: Gladstone Institutes (UCSF)

summary: Congenital heart defects (CHD) are the most common form of developmental abnormalities, occurring in ~1% of live births, and can arise due to altered dosage of genes essential for cardiogenesis. Aneuploidy accounts for nearly 15% of CHD and the most frequent form involves trisomy of chromosome 21 (Ch21), resulting in Down Syndrome (DS). Here we used single cell RNA-seq, CRISPR-activation with single cell RNA-seq, single cell ATAC-seq and Cut&Tag epigenomic profiling to define the molecular disruptions occuring with Trisomy 21 and upregulation of the Ch21 epigenetic factor HMGN1. These experiments were performed in human pluripotent stem cells (hiPSCs) or in directed differentiation to cardiomyocyte lineages at days 10 and 20.

overall_design: We used human pluripotent stem cells (hIPSCs) from an individual who was mosaic for Down Syndrome. Thus we compare cells that are trisomic for Ch21 with cells that are isogenically similar and disomic for Ch21. As well, we generated a CRISPR-activation knock-in to disomic cells and used sgRNAs directed to chromosome 21 genes. Sample replicates are defined and all single cell samples were processed with 10X Genomics pipelines. Cut&Tag samples were processed with Epicypher reagents. All samples were isolated with Accutase or Trypsin reagents.

first: Sanjeev

last: Ranade

first: Deepak

last: Srivastava


title: Multi-omics analysis identifies progenitors of CD4-CTLs [scATAC-seq invitrodiff]

overall_design: CD4-Naive T cells (CD3+CD4+CD45RA+CCR7+CD95-) were isolated from PBMCs of healthy human donors and activated for 48 hrs using aCD3/aCD28 Dynabeads in a Th1 polarizing milieu of cytokines and antibodies. The cells were then cultured in the Th1 polarizing condition for 8 days (2+8 day cycle) before doing scRNA-Seq (Day 10). The same cycle was repeated for a second round before subjecting the cells to another scATAC-Seq on Day 20.


title: RoCK and ROI: a single-cell RNA-sequencing method with enhanced transcriptome information via targeted enrichment of transcripts and preselected, region-specific sequencing V

city: Zürich

first: Giulia

middle: Arianna

last: Moro

address: Winterthurerstrasse 190

department: DMLS

country: Switzerland

institute: University of Zurich

summary: Many scRNAseq-based methods suffer from a high number of non-detects and a bias towards the 3' or 5' end of transcripts. We developed a method termed RoCK and ROI to address these two biases. RoCK and ROI leads to the enrichment of the transcriptome profile of cells with information on transcripts of interest

overall_design: RoCK and ROI is based on barcoded beads of the BD Rhapsody platform. For RoCKseq specific capture, TSO oligos on BD Rhapsody beads are modified with a/ multiple capture sequence(s) specific to transcripts of interest. ROIseq, on the other hand, is based on specific primers which are spiked in during library generation, directing reads to regions of interest (ROIs). For this set of experiments, RoCK and ROI was performed to enhance the detection of seven transcripts of interest in HeLa cells.

first: Izaskun

last: Mallona

first: Mark

last: Robinson

first: Erich

last: Brunner

first: Konrad

last: Basler


title: Impact of RORgt in regulating CD4 T cells primed under Th17 conditions

city: Orlando

first: Kunal

last: Dhume

state: FLorida

address: 6900 lake Nona Blvd

department: Burnett School of Biomedical Sciences

institute: University Of Central Florida

summary: CD4 T cells are an essential component of antiviral adaptive immune cell responses. Central to the functional capabilities of CD4 T Cells is their ability to differentiate into distinct subsets. Subset differentiation and maintenance is dependent on the coordinated and sequential activation of certain master regulator transcription factors. While antiviral responses are predominantly Th1, prior studies have shown that both T-bet and Eomes, two critical regulators of Th1 responses are dispensable for CD4 T cell mediated protection. CD4 T Cells lacking both T-bet and Eomes produce strong and highly protective Th17 response characterized by high expression of the transcription factor RORγt. Here we determine the impact of RORγt regulation in  CD4 T Cells primed under Th17 conditions and also deficient in T-bet and Eomes. Our studies reveal RORγt regulates several hundred genes that in Th17 CD4 T Cells, thus providing a potential molecular framework for targeting protective anti-viral immunity.

overall_design: RNA-seq profiling and comparison of day 4 CD4 T cells primed under Th17 condiitions and lacking either T-bet and Eomes or T-bet, Eomes and RORγt

first: Caroline

middle: M

last: Finn

first: Eugene

last: Baffoe

first: Lauren

middle: A

last: Kimball

first: Siva

middle: N

last: Annamalai

first: Verónica

last: Urdaneta-Páez

first: Jash

last: Trivedi

first: Taj

last: Azarian

first: Tara

last: Strutt

first: Karl

middle: K

last: McKinstry


title: Longitudinal multimodal characterization of radiation dermatitis in a C57BL/6J mouse model

city: Bronx

first: Kosaku

last: Shinoda

state: New York

address: 1301 Morris Park Ave

department: Medicine

institute: Albert Einstein College of   Medicine/Montefiore Medical Center

summary: Radiation dermatitis (RD) is a common adverse reaction to radiation therapy (RT), affecting up to 95% of cancer patients. Despite its prevalence, RD’s pathophysiology is complex and remains poorly understood. Without a better molecular understanding of this condition, targeted treatments are difficult to develop and integrate into clinical practice.  Prior animal model studies of RD are scarce, variable in methodology, and limited to outcomes such as clinical grading and histology. To address this gap, we aimed to develop a temporal murine model of RD using clinical grading, objective skin parameters, histopathology, and gene expression analysis of inflammatory markers.

overall_design: RNA-seq profiling of skin biopsies taken from C57BL/6J mice radiated with 25 Gy to the skin of the left flank. Ten-week-old female mice (n=19) were randomly divided into five groups: sham (no RT), and four post-RT groups with weekly data collection for four weeks.

first: Jasmine

middle: H

last: Wong

first: Li

last: Liu

first: Beth

last: McLellan

first: Sharen

last: Rivas


title: Interleukin-27-adipose-derived mesenchymal stromal cell-based gene therapy attenuates inflammation in lipopolysaccharide-induced acute respiratory distress syndrome

city: West Lafayette

first: Marxa

middle: Leao

last: Figueiredo

state: IN

address: 625 Harrison St

department: Basic Medical Sciences

institute: Purdue University

summary: Acute respiratory distress syndrome (ARDS) is a lung inflammatory condition associated with the accumulation of fluid edema and cell infiltrates into the alveolar space along with the dysregulation in the immune response. Current therapeutics are limited to palliative care, i.e., mechanical ventilators, thus highlighting the need of developing targeted therapeutic for ARDS. Interleukin-27 (IL-27) is a multifunctional cytokine with the capability for immune modulation. Our interest lies in exploring the properties of IL-27, particularly as an anti-inflammatory cytokine that functions as an antagonist of IL-6 signaling, as an inducer of anti-viral genes, as a promoter of tissue repair, and as a regulator of both the innate and adaptive immune responses, possessing promising potential as a therapeutic for ARDS. To overcome the challenge of repeated administration due to the short half-life of cytokines, we utilized a cell-based gene therapy approach. IL-27-expressing plasmid was transfected into adipose mesenchymal stromal cells (ASC) that serve as the gene therapy carriers. For in vitro studies, we treated mono- and co-culture lung lipopolysaccharide (LPS)-induced lung epithelial and monocytes/macrophages cell line with IL-27-expressing ASC (IL-27) conditioned media (CM) to determine the effects on pro-inflammatory gene expression. For in vivo studies, male C57BL/6 mice was intratracheally injected with LPS (5mg/kg) and treated either PBS, ASC, or IL-27 ASC (5 x 105 cells/mouse) after 24h of LPS instillation. Measurements of gene expression, histopathological changes, cell infiltrations, and protein content in the bronchoalveolar lavage fluid and RNA-seq analysis were performed for the in vivo studies.  Results indicated that IL-27ASC CM were able to reduce pro-inflammatory gene expression of lung epithelial and macrophages cultured in both mono- and co-culture systems. Additionally, IL-27 ASC were able to reduce pro-inflammatory markers, decrease cell infiltration into the lungs, promote genes and immune cells involved in tissue repair, and rebalance innate and adaptive immunity in an LPS-induced in vivo model. Collectively, our in vitro and in vivo results showed promising potential for IL-27 cell-based gene therapy as a treatment for ARDS.

overall_design: In this study, male C57BL/6 mice (8-10 weeks old) were used to create an in vivo LPS-induced ARDS model. Anesthesia was administered using ketamine and xylazine, and LPS (E. coli O111:B4) was given intratracheally at 5 µg/g body weight. Mice were randomly assigned to treatment groups, including 1X PBS (control), LPS, LPS + hASC with empty vector, and LPS + hASC with IL-27 vector. At 24 hours post-LPS instillation, treatments were given, followed by air infusion to ensure distribution. Animals were sacrificed at 72 hours post-LPS instillation, and samples (BALF, serum, lung, and organ tissues) were collected for analysis.

first: Grace

middle: E

last: Mulia

middle: L

last: Figuereido


title: Mapping of Sen1 RNA-binding sites in Saccharomyces cerevisiae with abrogated binding of Nab3 to PIC2

city: Edinburgh

first: Sander

last: Granneman

address: Mayfield Road, Kings Buildings, Waddington building, room 3.06

department: Centre for Synthetic and Systems Biology

country: United Kingdom

institute: University of Edinburgh

summary: Using CRAC, we compared the transcriptomic occupancy of Sen1 in a Saccharomyces cerevisiae BY4741 parental strain (PIC2-GFP) and two derived mutants lacking Nab3 RNA-binding sites in PIC2. The purpose of the experiment was two-fold: on the one hand, we aimed to verify that the mutations inserted in the Nab3 binding sequences of PIC2 affected the binding of Sen1 to the PIC2 transcript; on the other hand, we wanted to check whether differential binding of Nab3 to PIC2 would affect how the protein bound other targets in the genome. Sequencing outputs were processed using the pyCRAC pipeline and peak calling was performed with DBPeaks, our newly developed package for identification and comparison of binding sites defined by RNA-binding footprinting techniques (e.g., CRAC, iCLIP, PAR-CLIP, etc.).

overall_design: Two biological replicates of CRAC experiments performed in a parental Saccharomyces cerevisiae BY4741 strain (PIC2-GFP) and two derived mutants: mut-Nab3-BS and mut-NNS-BS. While mut-Nab3-BS lacks Nab3 RNA-binding sites in PIC2, the other strain has no Nab3 or Nrd1 RNA-binding sites in PIC2 (mut-NNS-BS). All strains encoded SEN1-HTF (HIS6-TEV-3xFLAG). When SC-Ura cultures containing 1% (w/v) raffinose reached an OD600 of 1, cells were UV cross-linked (254 nm; 500 mJ/cm2) in the Vari-X-linker (UVO3) and rapidly filtered on 0.8 μm membranes (Merck) using a filtration device connected to a vacuum pump. Membranes were collected in 50 mL tubes, flash frozen by submersion into liquid nitrogen and stored at -80ºC until the day of the experiment.   On the day of the experiment, pellets were resuspended in a volume of lysis buffer (150 mM NaCl, 50 mM Tris pH 7.8, 0.1% (v/v) Nonidet P-40, 1 EDTA-free protease inhibitor cocktail (Roche) per 50 mL and 5 mM β-mercaptoethanol) corresponding to twice their mass. Lysates were then transferred to a fresh 50 mL tubes, where they were mixed with 2 volumes of 0.5 mm zirconia beads (Thistle Scientific), and vortexed at maximum speed for five 1-minute intervals alternated with 1-minute incubations on ice. An additional volume of lysis buffer was pipetted into each sample before partitioning the cell debris by centrifugation at 4ºC (4000 rpm, 15 minutes) and spinning down lighter impurities in an additional centrifugation step (10000 rpm, 20 minutes) at 4ºC. Immunoprecipitation of the labelled protein from the supernatant was achieved during a two-hour incubation of the sample with 75 µL of anti-FLAG® M2 magnetic beads (Sigma-Aldrich, M8823-5ML) which had been previously washed three times with 1 mL TN150 buffer. Immobilised beads underwent three 5-minute washes with TN1000 buffer (50 mM Tris pH 7.8, 1 M NaCl, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol) and three additional 5-minute washes in 2 mL TN150 buffer (150 mM NaCl, 50 mM Tris pH 7.8, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol). Afterwards, beads were resuspended in 550 µL of TN150 and incubated with 1 µL of a 1:100 dilution of RNace-It™ for exactly 5 minutes at 37 °C. To prevent overdigestion of co-immunoprecipitated transcripts and elute the bait proteins from the anti-FLAG beads, the solution was mixed with 0.4 g of guanidium hydrochloride (Gu-HCl) and the necessary volume of NaCl and imidazole (pH 8.0) to reach a final concentration of 300 mM and 10 mM respectively. After removing the beads, samples were pipetted onto 50 µL of Ni-NTA agarose resin (Qiagen) equilibrated with wash buffer I (6 M Gu-HCl, 10 mM imidazole, 300 mM NaCl, 50 mM Tris-HCl pH 7.8, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol) and incubated at 4 °C for two hours while rotating at 12 rpm.   Upon Ni-NTA binding, beads were transferred to Pierce columns (Thermo Fisher Scientific) and washed twice with 500 µL of wash buffer I before undergoing three additional washes of 500 µL with NP-PNK buffer (10 mM MgCl2, 50 mM Tris-HCl pH 7.8, 0.1% (v/v) Nonidet P-40, 5 mM β-mercaptoethanol). Alkaline phosphatase treatment was performed by resuspending immunoprecipitated RNA-protein conjugates in 80 µL of NP-PNK buffer containing 4 units of FastAP thermosensitive alkaline phosphatase (Thermo Fisher Scientific) and 80 units of RNasin® ribonuclease inhibitor (Promega) and incubating the samples at 37 °C for 15 minutes. Dephosphorylation was stopped upon addition of 500 µL of wash buffer. Having equilibrated the Ni-NTA agarose with three washes of 500 µL with NP-PNK, we performed adapter ligation by resuspending the beads 80 µL of reaction mix containing 0.6 µM of the applicable App-PE 3′ adapter (5’App-NAGATCGGAAGAGCACACGTCTG-ddC 3’), 30 units of T4 RNA ligase 2 truncated K227Q (New England Biolabs), 60 units RNasin® (Promega) and 10% (w/v) polyethylene glycol 8000 (PEG 8000). The reaction took place at 25 °C for three hours.  After washing once with 500 µL of wash buffer I and three times with 500 µL of NP-PNK buffer, we 5’end radiolabelled co-immunoprecipitated RNAs by incubating the Ni-NTA resin with 60 µL of NP-PNK buffer containing 30 µCi 32P-γATP (PerkinElmer) and 30 units of T4 polynucleotide kinase (New England Biolabs). After a 40-minute incubation of the reaction at 37ºC, ATP (Roche) was supplemented to a final concentration of 1 mM and the reaction continued for another 20 minutes. The reaction was stopped by washing three times with 500 µL of wash buffer I and three 500 µL washes of NP-PNK buffer followed before the addition of the 5′ linker ligation mix. The reaction took place in a volume of 80 µL of NP-PNK buffer supplemented with 1.25 µM of the fitting adapter, 40 units of T4 RNA ligase 1 (New England Biolabs), 80 units of RNasin® and 10 mM ATP (Roche).  5’ adapter ligation was left to proceed overnight at 16 °C for all bait proteins. The adapter sequences were 5’-ACACGACGCTCTTCCGATCTNNNCGCTTAGCN-3’ for PIC2-GFP_Rep1 and 5’-ACACGACGCTCTTCCGATCTNNNAGAGCN-3’,  5’-ACACGACGCTCTTCCGATCTNNNATTAGCN-3’ or 5’-ACACGACGCTCTTCCGATCTNNNGCGCAGCN-3’ for PIC2-GFP_Rep2; 5’-ACACGACGCTCTTCCGATCTNNNTGGAGCN-3’ or 5’-ACACGACGCTCTTCCGATCTNNNCACTAGCN-3’ for mut-Nab3-BS_Rep1 and  5’-ACACGACGCTCTTCCGATCTNNNGACTTAGCN-3’ or 5’-ACACGACGCTCTTCCGATCTNNNATCACGN-3’ for mut-Nab3-BS_Rep2;  and 5’-ACACGACGCTCTTCCGATCTNNNCACTGTN-3’ or 5’-ACACGACGCTCTTCCGATCTNNNACTCAGCN-3’ for mut-NNS-BS_Rep1 and 5’-ACACGACGCTCTTCCGATCTNNNGTGACAN-3’ for the second mut-NNS-BS library. After pooling suitable libraries together, the samples and its corresponding traces were submitted to Novogene, where paired-end sequencing of 150 bp-reads was performed in a NovaSeq 6000 system (Illumina).

first: Sofia

last: Serna


title: Mapping of Nrd1 RNA-binding sites in Saccharomyces cerevisiae with abrogated binding of Nab3 to PIC2

summary: Using CRAC, we compared the transcriptomic occupancy of Nrd1 in a Saccharomyces cerevisiae BY4741 parental strain (PIC2-GFP) and two derived mutants lacking Nab3 RNA-binding sites in PIC2. The purpose of the experiment was two-fold: on the one hand, we aimed to verify that the mutations inserted in the Nab3 binding sequences of PIC2 affected the binding of Nrd1 to the PIC2 transcript; on the other hand, we wanted to check whether differential binding of Nab3 to PIC2 would affect how the protein bound other targets in the genome. Sequencing outputs were processed using the pyCRAC pipeline and peak calling was performed with DBPeaks, our newly developed package for identification and comparison of binding sites defined by RNA-binding footprinting techniques (e.g., CRAC, iCLIP, PAR-CLIP, etc.).

overall_design: Two biological replicates of CRAC experiments performed in a parental Saccharomyces cerevisiae BY4741 strain (PIC2-GFP) and two derived mutants: mut-Nab3-BS and mut-NNS-BS. While mut-Nab3-BS lacks Nab3 RNA-binding sites in PIC2, the other strain has no Nab3 or Nrd1 RNA-binding sites in PIC2 (mut-NNS-BS). All strains encoded NRD1-HTF (HIS6-TEV-3xFLAG). When SC-Ura cultures containing 1% (w/v) raffinose reached an OD600 of 1, cells were UV cross-linked (254 nm; 500 mJ/cm2) in the Vari-X-linker (UVO3) and rapidly filtered on 0.8 μm membranes (Merck) using a filtration device connected to a vacuum pump. Membranes were collected in 50 mL tubes, flash frozen by submersion into liquid nitrogen and stored at -80ºC until the day of the experiment.   On the day of the experiment, pellets were resuspended in a volume of lysis buffer (150 mM NaCl, 50 mM Tris pH 7.8, 0.1% (v/v) Nonidet P-40, 1 EDTA-free protease inhibitor cocktail (Roche) per 50 mL and 5 mM β-mercaptoethanol) corresponding to twice their mass. Lysates were then transferred to a fresh 50 mL tubes, where they were mixed with 2 volumes of 0.5 mm zirconia beads (Thistle Scientific), and vortexed at maximum speed for five 1-minute intervals alternated with 1-minute incubations on ice. An additional volume of lysis buffer was pipetted into each sample before partitioning the cell debris by centrifugation at 4ºC (4000 rpm, 15 minutes) and spinning down lighter impurities in an additional centrifugation step (10000 rpm, 20 minutes) at 4ºC. Immunoprecipitation of the labelled protein from the supernatant was achieved during a two-hour incubation of the sample with 75 µL of anti-FLAG® M2 magnetic beads (Sigma-Aldrich, M8823-5ML) which had been previously washed three times with 1 mL TN150 buffer. Immobilised beads underwent three 5-minute washes with TN1000 buffer (50 mM Tris pH 7.8, 1 M NaCl, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol) and three additional 5-minute washes in 2 mL TN150 buffer (150 mM NaCl, 50 mM Tris pH 7.8, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol). Afterwards, beads were resuspended in 550 µL of TN150 and incubated with 1 µL of a 1:100 dilution of RNace-It™ for exactly 5 minutes at 37 °C. To prevent overdigestion of co-immunoprecipitated transcripts and elute the bait proteins from the anti-FLAG beads, the solution was mixed with 0.4 g of guanidium hydrochloride (Gu-HCl) and the necessary volume of NaCl and imidazole (pH 8.0) to reach a final concentration of 300 mM and 10 mM respectively. After removing the beads, samples were pipetted onto 50 µL of Ni-NTA agarose resin (Qiagen) equilibrated with wash buffer I (6 M Gu-HCl, 10 mM imidazole, 300 mM NaCl, 50 mM Tris-HCl pH 7.8, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol) and incubated at 4 °C overnight while rotating at 12 rpm.   Upon Ni-NTA binding, beads were transferred to Pierce columns (Thermo Fisher Scientific) and washed twice with 500 µL of wash buffer I before undergoing three additional washes of 500 µL with NP-PNK buffer (10 mM MgCl2, 50 mM Tris-HCl pH 7.8, 0.1% (v/v) Nonidet P-40, 5 mM β-mercaptoethanol). Alkaline phosphatase treatment was performed by resuspending immunoprecipitated RNA-protein conjugates in 80 µL of NP-PNK buffer containing 4 units of FastAP thermosensitive alkaline phosphatase (Thermo Fisher Scientific) and 80 units of RNasin® ribonuclease inhibitor (Promega) and incubating the samples at 37 °C for 1 hour. Dephosphorylation was stopped upon addition of 500 µL of wash buffer. Having equilibrated the Ni-NTA agarose with three washes of 500 µL with NP-PNK, we performed adapter ligation by resuspending the beads 80 µL of reaction mix containing 0.6 µM of the applicable App-PE 3′ adapter (5’App-NAGATCGGAAGAGCACACGTCTG-ddC 3’), 30 units of T4 RNA ligase 2 truncated K227Q (New England Biolabs), 60 units RNasin® (Promega) and 10% (w/v) polyethylene glycol 8000 (PEG 8000). The reaction took place at 25 °C for six hours.  After washing once with 500 µL of wash buffer I and three times with 500 µL of NP-PNK buffer, we 5’end radiolabelled co-immunoprecipitated RNAs by incubating the Ni-NTA resin with 60 µL of NP-PNK buffer containing 30 µCi 32P-γATP (PerkinElmer) and 30 units of T4 polynucleotide kinase (New England Biolabs). After a 40-minute incubation of the reaction at 37ºC, ATP (Roche) was supplemented to a final concentration of 1 mM and the reaction continued for another 20 minutes. The reaction was stopped by washing three times with 500 µL of wash buffer I and three 500 µL washes of NP-PNK buffer followed before the addition of the 5′ linker ligation mix. The reaction took place in a volume of 80 µL of NP-PNK buffer supplemented with 1.25 µM of the fitting adapter, 40 units of T4 RNA ligase 1 (New England Biolabs), 80 units of RNasin® and 10 mM ATP (Roche).  5’ adapter ligation was left to proceed overnight at 16 °C for all bait proteins. The adapter sequences were 5’-ACACGACGCTCTTCCGATCTNNNTAAGCN-3’ or 5’-ACACGACGCTCTTCCGATCTNNNCACTAGCN-3’ for PIC2-GFP_Rep1 and 5’-ACACGACGCTCTTCCGATCTNNNTCTCTAGCN-3’ for PIC2-GFP_Rep2; 5’-ACACGACGCTCTTCCGATCTNNNCACTAGCN-3’ and 5’-ACACGACGCTCTTCCGATCTNNNGACTTAGCN-3’ for mut-Nab3-BS libraries 1 and 2 respectively;  and 5’-ACACGACGCTCTTCCGATCTNNNTGGAGCN-3’,  5’-ACACGACGCTCTTCCGATCTNNNACTCAGCN-3’ or 5’-ACACGACGCTCTTCCGATCTNNNACAGTGN-3’ for mut-NNS-BS_Rep1  and 5’-ACACGACGCTCTTCCGATCTNNNGTGACAN-3’ for mut-NNS-BS_Rep2. After pooling suitable libraries together, the samples and its corresponding traces were submitted to Novogene, where paired-end sequencing of 150 bp-reads was performed in a NovaSeq 6000 system (Illumina).


title: Mapping of Nab3 RNA-binding sites in Saccharomyces cerevisiae with abrogated binding of Nab3 to PIC2

summary: Using CRAC, we compared the transcriptomic occupancy of Nab3 in a Saccharomyces cerevisiae BY4741 parental strain (PIC2-GFP) and two derived mutants lacking Nab3 RNA-binding sites in PIC2. The purpose of the experiment was two-fold: on the one hand, we aimed to verify that the mutations inserted in the Nab3 binding sequences of PIC2 had indeed abrogated binding of Nab3 to the PIC2 transcript; on the other hand, we wanted to check whether differential binding of Nab3 to PIC2 would affect how the protein bound other targets in the genome. Sequencing outputs were processed using the pyCRAC pipeline and peak calling was performed with DBPeaks, our newly developed package for identification and comparison of binding sites defined by RNA-binding footprinting techniques (e.g., CRAC, iCLIP, PAR-CLIP, etc.).

overall_design: Two biological replicates of CRAC experiments performed in a parental Saccharomyces cerevisiae BY4741 strain (PIC2-GFP) and two derived mutants: mut-Nab3-BS and mut-NNS-BS. While mut-Nab3-BS lacks Nab3 RNA-binding sites in PIC2, the other strain has no Nab3 or Nrd1 RNA-binding sites in PIC2 (mut-NNS-BS). All strains encoded NAB3-HTF (HIS6-TEV-3xFLAG). When SC-Ura cultures containing 1% (w/v) raffinose reached an OD600 of 1, cells were UV cross-linked (254 nm; 500 mJ/cm2) in the Vari-X-linker (UVO3) and rapidly filtered on 0.8 μm membranes (Merck) using a filtration device connected to a vacuum pump. Membranes were collected in 50 mL tubes, flash frozen by submersion into liquid nitrogen and stored at -80ºC until the day of the experiment.   On the day of the experiment, pellets were resuspended in a volume of lysis buffer (150 mM NaCl, 50 mM Tris pH 7.8, 0.1% (v/v) Nonidet P-40, 1 EDTA-free protease inhibitor cocktail (Roche) per 50 mL and 5 mM β-mercaptoethanol) corresponding to twice their mass. Lysates were then transferred to a fresh 50 mL tubes, where they were mixed with 2 volumes of 0.5 mm zirconia beads (Thistle Scientific), and vortexed at maximum speed for five 1-minute intervals alternated with 1-minute incubations on ice. An additional volume of lysis buffer was pipetted into each sample before partitioning the cell debris by centrifugation at 4ºC (4000 rpm, 15 minutes) and spinning down lighter impurities in an additional centrifugation step (10000 rpm, 20 minutes) at 4ºC. Immunoprecipitation of the labelled protein from the supernatant was achieved during a two-hour incubation of the sample with 75 µL of anti-FLAG® M2 magnetic beads (Sigma-Aldrich, M8823-5ML) which had been previously washed three times with 1 mL TN150 buffer. Immobilised beads underwent three 5-minute washes with TN1000 buffer (50 mM Tris pH 7.8, 1 M NaCl, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol) and three additional 5-minute washes in 2 mL TN150 buffer (150 mM NaCl, 50 mM Tris pH 7.8, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol). Afterwards, beads were resuspended in 550 µL of TN150 and incubated with 1 µL of a 1:100 dilution of RNace-It™ for exactly 5 minutes at 37 °C. To prevent overdigestion of co-immunoprecipitated transcripts and elute the bait proteins from the anti-FLAG beads, the solution was mixed with 0.4 g of guanidium hydrochloride (Gu-HCl) and the necessary volume of NaCl and imidazole (pH 8.0) to reach a final concentration of 300 mM and 10 mM respectively. After removing the beads, samples were pipetted onto 50 µL of Ni-NTA agarose resin (Qiagen) equilibrated with wash buffer I (6 M Gu-HCl, 10 mM imidazole, 300 mM NaCl, 50 mM Tris-HCl pH 7.8, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol) and incubated at 4 °C overnight while rotating at 12 rpm.   Upon Ni-NTA binding, beads were transferred to Pierce columns (Thermo Fisher Scientific) and washed twice with 500 µL of wash buffer I before undergoing three additional washes of 500 µL with NP-PNK buffer (10 mM MgCl2, 50 mM Tris-HCl pH 7.8, 0.1% (v/v) Nonidet P-40, 5 mM β-mercaptoethanol). Alkaline phosphatase treatment was performed by resuspending immunoprecipitated RNA-protein conjugates in 80 µL of NP-PNK buffer containing 4 units of FastAP thermosensitive alkaline phosphatase (Thermo Fisher Scientific) and 80 units of RNasin® ribonuclease inhibitor (Promega) and incubating the samples at 37 °C for 1 hour. Dephosphorylation was stopped upon addition of 500 µL of wash buffer. Having equilibrated the Ni-NTA agarose with three washes of 500 µL with NP-PNK, we performed adapter ligation by resuspending the beads 80 µL of reaction mix containing 0.6 µM of the applicable App-PE 3′ adapter (5’App-NAGATCGGAAGAGCACACGTCTG-ddC 3’), 30 units of T4 RNA ligase 2 truncated K227Q (New England Biolabs), 60 units RNasin® (Promega) and 10% (w/v) polyethylene glycol 8000 (PEG 8000). The reaction took place at 25 °C for six hours.  After washing once with 500 µL of wash buffer I and three times with 500 µL of NP-PNK buffer, we 5’end radiolabelled co-immunoprecipitated RNAs by incubating the Ni-NTA resin with 60 µL of NP-PNK buffer containing 30 µCi 32P-γATP (PerkinElmer) and 30 units of T4 polynucleotide kinase (New England Biolabs). After a 40-minute incubation of the reaction at 37ºC, ATP (Roche) was supplemented to a final concentration of 1 mM and the reaction continued for another 20 minutes. The reaction was stopped by washing three times with 500 µL of wash buffer I and three 500 µL washes of NP-PNK buffer followed before the addition of the 5′ linker ligation mix. The reaction took place in a volume of 80 µL of NP-PNK buffer supplemented with 1.25 µM of the fitting adapter, 40 units of T4 RNA ligase 1 (New England Biolabs), 80 units of RNasin® and 10 mM ATP (Roche).  5’ adapter ligation was left to proceed overnight at 16 °C for all bait proteins. The adapter sequences were 5’-ACACGACGCTCTTCCGATCTNNNCGCTTAGCN-3’ and 5’-ACACGACGCTCTTCCGATCTNNNGACTTAGCN-3’ or 5’-ACACGACGCTCTTCCGATCTNNNTAAGCN-3’ for PIC2-GFP repeats 1 and 2 respectively; 5’-ACACGACGCTCTTCCGATCTNNNCACTAGCN-3’ and 5’-ACACGACGCTCTTCCGATCTNNNACTCAGCN-3’ for mut-Nab3-BS replicates 1 and 2 respectively;  and 5’-ACACGACGCTCTTCCGATCTNNNGTGACAN-3’ for both mut-NNS-BS libraries. After pooling suitable libraries together, the samples and its corresponding traces were submitted to Novogene, where paired-end sequencing of 150 bp-reads was performed in a NovaSeq 6000 system (Illumina).


title: RNA sequencing in Saccharomyces cerevisiae with abrogated binding of Nab3 to PIC2

summary: Whole-transcriptome sequencing allowed us to compare transcriptomic differences between a Saccharomyces cerevisiae BY4741 parental strain (PIC2-GFP) and two derived mutants lacking Nab3 RNA-binding sites in PIC2. Previous experiments had established that disrupting Nab3 binding to PIC2 while maintaining Nrd1 binding to the same transcript led to severe growth defects, larger cell size, delayed cell cycle and resistance to oxidative stress. Following characterization of a PIC2-overexpressing mutant we established that these phenotypes were not related to Pic2 overabundance. Therefore, the purpose of this experiment was to determine (i) whether the transcriptomic signature of the mutant lacking Nab3 RNA-binding sites in PIC2  mutant could account for the observed phenotypes and (ii) if the transcripts that were differentially expressed in such mutant were targeted by Nab3 and Nrd1.

overall_design: Three biological replicates of RNA-sequencing experiments performed in a parental Saccharomyces cerevisiae strain (PIC2-GFP) and two derived mutants: mut-Nab3-BS and mut-NNS-BS. While mut-Nab3-BS lacks Nab3 RNA-binding sites in PIC2, the other strain has no Nab3 or Nrd1 RNA-binding sites in PIC2 (mut-NNS-BS). Cells were grown in 25 mL-cultures of SC-Ura containing 1% (w/v) raffinose and harvested at by centrifugation (4000 rpm, 5 minutes) at an OD600 of 0.5. Pellets were then snap frozen by submersion in liquid nitrogen and stored at -80ºC. On the day of the experiments, cells were thawed on ice and denatured by the addition of 200 μL of guanidinium thiocyanate (GTC) acid phenol mix. Cell lysis was performed by adding 400 μL of glass beads to each sample and vortexing all tubes at full speed for five 1-minute intervals separated by four 1-minute incubations on ice. An additional 1.5 mL of GTC phenol mix was added to the samples before incubating them at 65ºC for 10 minutes. Samples were then placed on ice for another 10 min. 800 μL of sodium acetate mix (3.3 mL 3 M NaOAc pH5.2, 0.2 mL 0.5M EDTA pH 8, 1mL 1M Tris-HCl pH 8, water to 100 mL) and 1.5 mL of chloroform were pipetted into each extract before vortexing at maximum speed for 5 seconds. Lysates were centrifuged at 4ºC (10000 rpm, 30 minutes) and the resulting aqueous phase was transferred into a new tube containing an equivalent volume of Phenol:Chloroform:Isoamylalcohol mix. The mixtures were vortexed vigorously and centrifuged at 10000 rpm for 5 minutes. Once more, the aqueous phase of each sample was mixed with an equal volume of Chloroform:Isoamylalcohol and vortexed at maximum speed before centrifugation at 10000 rpm for 5 minutes. Finally, the aqueous phase of each sample was relocated to tubes containing 3 volumes of 96% ethanol. The mix was vortexed vigorously and preserved at -80°C for at least 30 minutes or at -20°C overnight. Following ethanol precipitation, samples were spun at 1000 rpm for 30 minutes at 4ºC. After discarding the resulting supernatants, 70% ethanol was pipetted into each tube. After centrifuging samples at 10000 rpm for 5 minutes at 4ºC, ethanol was removed, and pellets were left to air-dry for approximately 5 minutes before resuspension in DEPC water. Finally, RNA concentrations were measured in the Qubit™ 4 fluorometer (Thermo Fisher Scientific) using the Qubit™ RNA HS assay kit (Thermo Fisher Scientific). RNA extracts were treated with RQ1 RNase-Free DNase as per manufacturer’s guidelines. The quality of the RNA samples was then inspected in an Agilent 2100 bioanalyzer (Agilent Technologies) using an RNA 6000 pico assay (Agilent Technologies) according to the manufacturer’s instructions. Samples with RNA integrity number (RIN) scores greater than 7 were submitted to Novogene for paired-end sequencing of 150 bp-reads in a NovaSeq 6000 system (Illumina). Before sequencing, samples underwent a rRNA depletion step and libraries were generated using the TruSeq preparation protocol (Illumina). Raw sequencing outputs were returned as FASTQ files. Hence, after conversion into FASTA format, files were processed using the paired-end (PE) version of the pyCRAC pipeline (Webb et al., 2014). This package demultiplexes barcoded reads and removes adapter sequences applying the previously developed Flexbar tool (Dodt et al., 2012). The resulting reads undergo two quality control steps. Firstly, sequences are filtered based on their length and nucleotide quality values (i.e., PHRED scores). Secondly, since PCR artifacts will display identical random prefixes, they can easily be pinpointed and excluded from downstream analysis (collapsed). The remaining unique reads were subsequently aligned to the corresponding regions of the Saccharomyces_cerevisiae.R64-1-1.75 reference genome with the Novoalign tool (version 2.07). The pyReadCounters function of the pyCRAC package (version 1.5.1; Webb et al., 2014) was used to compute the number of reads that map to genomic features. The resulting tables for all the samples were merged into a single TXT file that was analysed by the DESeq2 R package (Love et al., 2014). The pipeline corrects for size variability by computing a scaling factor for each sample. To obtain such normalisation factor, the software calculates the ratio of the raw count of a given gene in one of the samples to the geometric mean of the counts for that same gene across the compared samples. The median of all the ratios calculated within a sample is defined as the scaling factor for that replicate. Having adjusted the raw reads, the package then performs a logarithmic transformation of the data and calculates a fold change and an FDR for each entry. Statistical significance was assessed for entries which exceeded or were lower than log2 fold change o 1 and -1 respectively and had an FDR value of 0.05 or less.


title: ACTL6A regulates the Warburg effect through coordinated activation of AP-1 signaling in head and neck squamous cell carcinoma

city: Palo Alto

first: Andrey

last: Finegersh

address: 900 Blake Wilbur Dr, 3rd Floor

institute: Stanford University

summary: ACTL6a is an essential component of SWI/SNF and expressed on the chromosome 3q26 cytoband, which is amplified in head and neck squamous cell carcinomas (HNSCC). While ACTL6A is emerging as an oncogene, its role as a treatment target and mechanisms of transcription factor induction remain unknown. Here, we show that ACTL6A expression is a mediator of the Warburg effect, with ACTL6A knockdown inducing mitochondrial dependency and significantly decreasing levels of aerobic glycolysis. Using ATAC-seq, we identify ACTL6A as a mediator of chromatin accessibility of AP-1 transcription factor sites and find that it regulates upstream MAPK signaling through induction Ras and Galectin-1. These effects sensitize ACTL6A over-expressing cells to inhibition of glycolysis by MEK inhibitors. Our results link SWI/SNF subunit amplification with potentiation of MAPK signaling in HNSCC and provide a novel mechanism by which cancer cells drive aerobic glycolysis and reduce mitochondrial dependency.

overall_design: ACTL6A knockdown of SCC1 cells using siRNA compared to a non-targeting siRNA control. SCC1 cells were incubated for 72 hours and harvested. ATAC-seq and RNA-seq was performed.

first: Mehri

last: Monavarian


title: ALDH1a3 inhibitor effects on Breast cancer Cells Transcriptome

city: Plainsboro

first: Yong

last: Wei

state: NJ

address: LTL227 Wathington Road

department: Molecular Biology

institute: Princeton University

summary: Aldehyde dehydrogenase (ALDH1) activity has long been established as a pro-tumorigenic feature of many cancers, yet the identification of specific isoforms that are enriched in cancer, the mechanism of action of this isoform(s), and viable therapeutic strategies to target this pathway have long remained absent. Whereas one of the well-established functions of the ALDH1a family is the conversion of retinaldehyde into retinoic acid to activate nuclear retinoid signaling, the retinoid pathway is paradoxically hypothesized as a cell-intrinsic tumor suppressor pathway. Here we resolve this long-standing conflict by showing that while ALDH1a3 is broadly overexpressed across diverse cancer types, ALDH1a3 expressing tumor cells often lose the sensitivity to retinoid signaling. Instead, all-trans retinoic acid produced by ALDH1a3 acts in a paracrine fashion to suppress anti-tumor immunity and promote tumor growth. We further used structure-based high throughput screening to develop a series of first-in-class, therapeutically viable antagonists of ALDH1a3 with potent anti-tumor immunotherapeutic activity, an excellent pharmacological profile and no evidence of toxicity. Findings of this study resolve prior contradictions in the retinoid pathway through the development of highly specific and potent ALDH1a3 inhibitors.

overall_design: To determine the effects of ALDH1a3 inhibitor MBE1.5 on gene expression regulation in breast cancer cells, 4 breast cancer cell lines including 3 ALDH1a3 positive cells and 1 ALDH1a3 negative cells are treated with MBE1.5 vs DMSO control for 9 hours. Total RNA were harvested from the cells and whole transcriptome expression were determined by RNAseq.

first: Mark

last: Espisito

first: Cao

last: Fang

first: Yibin

last: Kang


title: A Cell Type Enrichment Analysis Tool for Brain DNA  Methylation Data (CEAM) [UKBBN2]

status: Public on Aug 23 2025

city: Maastricht

first: Ehsan

last: Pishva

state: Limburg

address: Universiteitssingel 40

department: Psychiatry and Neuropsychology

country: Netherlands

institute: Maastricht University

summary: DNA methylation signatures are highly cell type-specific, yet most epigenome-wide association studies (EWAS) are performed on bulk tissue, potentially obscuring critical cell type-specific patterns. Existing computational tools for detecting cell type-specific DNAm changes are often limited by the accuracy of cell type deconvolution algorithms. Here, we introduce CEAM (Cell-type Enrichment Analysis for Methylation), a robust and interpretable framework for cell type enrichment analysis in DNA methylation data. CEAM applies over-representation analysis with cell type-specific CpG panels from Illumina EPIC arrays derived from nuclei-sorted cortical post-mortem brains from neurologically healthy aged individuals. The constructed CpG panels were systematically evaluated using both simulated datasets and published EWAS results from Alzheimer’s disease, Lewy body disease, and multiple sclerosis. CEAM demonstrated resilience to shifts in cell type composition, a common confounder in EWAS, and remained accurate across a wide range of differentially methylated positions, underscoring its flexibility. Application to existing EWAS findings generated in neurodegenerative diseases revealed enrichment patterns concordant with established disease biology, confirming CEAM’s biological relevance. The workflow is publicly available as an interactive Shiny app (https://um-dementia-systems-biology.shinyapps.io/CEAM/) enabling rapid, interpretable analysis of cell type-specific DNAm changes from bulk EWAS.

overall_design: We aimed to profile DNA methylation of four major cell types in the healthy human brain to define cell type-specific CpGs for our enrichment tool. Purified cell type fractions were obtained through performing FANS on post-mortem cortical brain samples of neurologically healthy individuals. Subsequently the purified cell nuclei were analyzed using the Infinium MethylationEPIC array in a cohort of 9 female and 10 male individuals resulting in 4(cell types)*19(individuals) = 76  samples. (one sample was deemed an outlier by the "outlyx" function leaving 75 samples for the analysis)

first: Joshua

last: Müller

first: Valentin

middle: T

last: Laroche

first: Jennifer

last: Imm

first: Luke

last: Weymouth

last: Harvey

first: Adam

middle: R

last: Smith

first: Daniel

last: van den Hove

first: Katie

last: Lunnon

first: Rachel

last: Cavill


title: Multi-omics analysis identifies progenitors of CD4-CTLs [bulk RNA-seq]

first: Veena

last: Patil

summary: The CD4+ T cells expressing cytolytic molecules such as granzymes and perforins have been detected in many diseases and are shown to be enriched in the effector memory expressing CD45RA (TEMRA) in humans, but their origin remains elusive. However, their gene expression profile in comparison to classically defined cytotoxic T lymphocytes (CTLs), the CD8-CTLs has not been well demonstrated, hence their relevance remains debatable.  Thus in this study by parallelly analysing the CD4-CTLs and CD8-CTLs, we demonstrate that they are indistinguishable for the cytolytic program with both showing similar gene expression profile and T cell antigen-receptor (TCR) clonal expansion. Further using an integrative multi-omics approach combining the transcriptome, TCR repertoire, and open chromatin profile of CD4+ naïve (CD4-TN) and memory subsets, we discovered a stem-cell memory subset that is pre-committed to CTL program within the CD4+ T cell lineage. Through an in vitro differentiation model we developed CD4+ T cells with cytolytic potential from CD4-TN cells. The in vitro differentiated cells followed the trajectory of different developmental memory subsets from ex vivo CD4+ T cells for transcriptomic patterns as well as open-chromatin landscape. Thus, through this model, we deciphered the molecular signatures of early commitment of CD4-TN cells to cytotoxicity program. Of particular interest was the expression of both longevity as well as cytotoxicity associated gene sets by the in vitro differentiated CD4-CTLs, hence generating long-lived CD4-CTL effectors. This specific property can be further explored for vaccine development as well as testing the efficacy and cell based therapies for precision medicine.

overall_design: Transcriptome analysis of purified populations of human CD4 memory cell subsets including CD4-Naïve and CD8-TEMRA by bulk RNA-sequencing from 10 healthy donors.

first: Raunak

last: Kar

first: Shreya

last: Sinha

first: Zainab

last: Khatun

first: Anjali

last: Sharma


title: A Cell Type Enrichment Analysis Tool for Brain DNA  Methylation Data (CEAM) [BDR]

overall_design: We aimed to profile DNA methylation of the four major cell types in the healthy human brain to define cell type-specific CpGs for our enrichment tool. Purified cell type fractions were obtained through performing FANS on post-mortem cortical brain samples of neurologically healthy individuals. Subsequently the purified cell nuclei were analyzed using the Infinium MethylationEPIC array in a cohort of 9 female and 10 male individuals resulting in 4(cell types)*20(individuals) = 80 samples.


title: Fibroblast growth factors induce preadipocyte UCP1 expression and imprint on post-differentiation white and brown adipocyte function

status: Public on Aug 22 2025

city: Freising

first: Katharina

last: Küllmer

state: Bavaria

address: Gregor-Mendel Straße 2

institute: Technical University of Munich

summary: Objective: The presence of uncoupling protein 1 (Ucp1) is a hallmark of thermogenic adipocytes and enables heat production by dissipating energy from mitochondrial proton motive force as heat. The purpose of its recently discovered presence in preadipocytes in response to certain fibroblast growth factors (FGFs) remains elusive. Methods: In this study, we systematically investigated the potential of all paracrine FGFs to invoke Ucp1 expression in murine preadipocytes derived from interscapular brown and inguinal white adipose tissue. Results: The factors FGF2, FGF4, FGF8, and FGF9 induced Ucp1 expression in undifferentiated preadipocytes, with FGF2 acting most potently and rapidly. This premature Ucp1 induction did not translate into increased Ucp1 protein abundance or thermogenic activity after full adipogenic differentiation. Notably, preadipocyte treatment with FGFs and parallel Ucp1 expression led to a sustained suppression of interferon-stimulated genes after differentiation. Preadipocyte Ucp1 was required and sufficient for this lasting imprint. Conclusion: Thus, FGF-induced Ucp1 expression in preadipocytes programs a lasting post-differentiation anti-inflammatory status.

overall_design: Following primary preadipocyte isolation (stromal vascular fraction) from iBAT and iWAT (C57BL/6N, wild-type), cells were treated upon confluency with 5.5 nM FGF2 or let untreated for 48 hours.

last: Kuellmer

first: Tobias

last: Fromme


title: Preadipocyte uncoupling protein 1 expression invoked by fibroblast growth factors imprints on post-differentiation white and brown adipocyte function

summary: The presence of uncoupling protein 1 (UCP1) is a hallmark of thermogenic adipocytes and enables heat production by dissipating energy from mitochondrial proton motive force as heat. The purpose of its recently discovered presence in preadipocytes in response to certain fibroblast growth factors (FGFs) remains elusive. In this study, we systematically investigated the potential of all paracrine FGFs to invoke Ucp1 expression in murine preadipocytes derived from interscapular brown and inguinal white adipose tissue. The factors FGF2, FGF4, FGF8, and FGF9 induced Ucp1 expression in undifferentiated preadipocytes, with FGF2 acting most potently and rapidly. This premature Ucp1 induction did not translate into increased Ucp1 thermogenic activity after full adipogenic differentiation. Notably, preadipocyte treatment with FGFs and parallel Ucp1 expression led to a sustained suppression of interferon-stimulated genes after differentiation. Preadipocyte Ucp1 was required and sufficient for this lasting imprint. Thus, FGF-induced Ucp1 expression in preadipocytes programs a lasting post-differentiation anti-inflammatory status.

overall_design: Primary white preadipocytes were isolated from murine Ucp1_KI (luciferase reporter, heterozygous) mice. Cells were treated with 5.5 nM FGF1, FGF2, FGF4, FGF8, or FGF9, or left untreated as control. Treatments were performed in triplicate for 48 hours in DMEM supplemented with 10% FBS, starting at ~80% confluency. RNA was directly isolated using the Promega kit and submitted for library preparation and sequencing (Novogene). Samples: Replicate 1: S1 (control), S2 (FGF1), S3 (FGF2), S4 (FGF4), S5 (FGF8), S6 (FGF9); Replicate 2: S7 (control), S8 (FGF1), S9 (FGF2), S10 (FGF4), S11 (FGF8), S12 (FGF9); Replicate 3: S13 (control), S14 (FGF1), S15 (FGF2), S16 (FGF4), S17 (FGF8), S18 (FGF9)


title: CDK1 regulation of P53 phosphorylation mediates cell cycle arrest and apoptosis of macrophages infected with clinical isolates of Mycobacterium tuberculosis from the Xinjiang region, China

city: Urumqi

first: Banghao

last: Sun

address: No. 8, Xinyi Road, Xinshi District

institute: Xinjiang Medical University School of Basic Medicine

summary: THP-1 Macrophages were infected with Mycobacterium tuberculosis clinical isolates from Xinjiang, China, and H37Rv for 24 hours, respectively, and their transcriptomes were sequenced to investigate the specific biological processes that occur after infection of macrophages with Mycobacterium tuberculosis clinical isolates from Xinjiang, China.

overall_design: L3-L6 are samples of macrophages infected with clinical isolates of Mycobacterium tuberculosis from Xinjiang, China;V1-V3 are samples of macrophages infected with H37Rv；C1-C3 are samples of macrophages not infected with any tuberculosis


title: A CRISPR-Activation CROP-seq Screen Identifies HMGN1 as a Dosage Sensitive Regulator of Heart Defects in Down Syndrome (scATAC-Seq)

first: Angelo

last: Pelonero

overall_design: We used human pluripotent stem cells (hIPSCs) from an individual who was mosaic for Down Syndrome. Thus we compare cells that are trisomic for Ch21 with cells that are isogenically similar and disomic for Ch21. As well, we generated a CRISPR-activation knock-in to disomic cells and used sgRNAs directed to chromosome 21 genes. Sample replicates are defined and all single cell samples were processed with 10X Genomics pipelines. Cut&Tag samples were processed with Epicypher reagents. All samples were isolated with Accutase or Trypsin reagents.

first: Sanjeev

last: Ranade

first: Deepak

last: Srivastava


title: A dual-mechanism antimicrobial peptide with antimutagenic activity targets the replisome and induces cell envelope stress

city: Trondheim

first: Amanda

middle: Holstad

last: Singleton

address: Erling Skjalgsons gate 1

department: Department of Clinical and Molecular Medicine

country: Norway

institute: Norwegian University of Science and Technology

summary: To combat the growing threat of multidrug-resistant bacteria, we need to develop novel antibiotics with unique modes of action. This study investigates the antibacterial properties of BTP-001 towards Escherichia coli. BTP-001 is a β-clamp targeting antimicrobial peptide containing an AlkB homologu 2 PCNA-interacting motif (APIM) linked to a cell penetrating peptide composed of 11 arginine residues (R11). Our data indicates that R11 mediates energy-dependent transport of BTP-001 across the cell membrane, possibly via iron transport systems such as the TonB-system. The full-length BTP-001 peptide rapidly affects the bacterial membrane, inducing increased expression and activation of the Cpx, E and Rcs cell envelope stress responses, which trigger the production of reaction oxygen species (ROS). This contributes to a rapid bactericidal effect as evidenced by increased short-term survival by addition of a ROS scavenger. Furthermore, we show that BTP-001 reduces the development of resistance to ciprofloxacin likely by binding to the β-clamp via APIM, thereby inhibiting translesion synthesis. In addition, our multi-omics data suggests that the -clamp is part of ribosomal complexes, and that regulation of translation is targeted by BTP-001.  In conclusion, BTP-001 exhibits a multifaceted mode of action which strengthens its potential as a novel therapeutic drug against antibiotic resistant bacteria

overall_design: To elucidate BTP-001’s mode of action, we conducted RNA-sequencing (RNA-seq) of E. coli MG1655 treated with 2 doses of the novel antibiotic peptide BTP-001 and 1 dose of the cell penetrating peptide R11. We chose a low dose of BTP-001 (1 µM) with no impact on growth and a high dose (3 µM; equivalent to the minimum inhibitory concentration [MIC]) that affected the growth but did not yield extensive lethality during batch cultivation in bioreactors. The R11 dose (1 µM) reflects the BTP-001 low dose. The high dose (3 µM) stalled growth for around 10 min before the culture recovered and eventually reached the same growth rate as the control. All experiments were conducted with continuous dissolved oxygen and off-gas analysis. All treatments were given at OD600 = 0.2 to ensure exponential growth during the entire sampling period. To investigate the temporal effects of BTP-001 on E. coli, the low dose of BTP-001 (1 µM) culture was sampled for transcriptomics  over a time course between 1 to 120 min after treatment.  The high dose of BTP-001 (3 µM) and R11 (1 µM) were sampled for transcriptomics 10 min after treatment.

last: Holstad Singleton

first: Olaug

last: Torheim Bergum

first: Jana

last: Scheffold

first: Synnøve

last: Brandt Ræder

first: Lilja

last: Brekke Thorfinnsdottir

first: Lisa

last: Røst

first: Caroline

last: Krogh Søgaard

first: Per

last: Bruheim

first: Marit

last: Otterlei


title: Zika virus exploits autophagy for replication in human trabecular meshwork and autophagic inhibition at autolysomal maturation ameliorates Zika-induced ocular complications.

status: Public on Aug 21 2025

city: Columbia

first: Pallav

last: Singh

state: Missouri

address: CE720 Clinical Support and Education

department: Institute for Data Science and Informatics

institute: University of Missouri

summary: Zika virus (ZIKV) infection causes severe ocular and neurological pathologies in infants following in-utero exposure. ZIKV is also the lone member of Flavivirus, which is known to cause congenital glaucoma. However, the cellular and molecular basis of congenital ZIKV ocular infection and glaucoma are not well understood. Autophagy has been shown to play dual roles in viral infection and glaucoma pathogenesis. However, how ZIKV interferes with autophagic pathways in the trabecular meshwork (TM) and whether autophagy contributes to ZIKV-induced ocular complications remains elusive. In this study, we investigated the role of autophagic pathways in ZIKV pathogenesis in the anterior segment (AS) of the eye utilizing a primary human trabecular meshwork (HTMC) and a IFNAR1-/- mouse model of ocular ZIKV infection. Our results from this study show that ZIKV permissively infects HTMC, induces a dysregulated immune response, and triggers autophagic activity in TM and mouse AS tissue. Our data suggest that although ZIKV initially activates autophagy in TM, it impairs the overall autophagic flux during the course of infection by modulating the VSP39-HOPS complex and STX17-SNARE complex to avoid autolysosomal maturation. We discovered that ZIKV utilizes late-endosomes/lysosomes for their replication and spread in TM. We further demonstrated that modulation of autophagy at the autolysosomal maturation stage with an FDA-approved drug, hydroxychloroquine, or Bafilomycin A1, inhibits ZIKV replication in TM and ameliorates ZIKV-induced ocular pathology in the mouse eyes.Our study is the first to demonstrate the mechanistic insight by which ZIKV manipulates autophagic activity in TM and ocular milieu, and we suggest that the precise modulation of autophagy could be a potential therapeutic avenue for developing new strategies to treat ZIKV-induced ocular complications.

overall_design: HTMC cells were either mock treted or Infected with Zika Virus in presence and absence of Hydroxychloroquine (HCQ) and Rapamycin (Rapa) and RNAseq was performed.

first: Pawan

first: Faraz

last: Ahmad

first: Trupti

last: Joshi


title: Myocardial Reprogramming by HMGN1 Underlies Heart Defects in Trisomy 21

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

overall_design: Refer to individual Series


title: Multiomic assessments of LNCaP and derived cell strains reveal determinants of prostate cancer pathobiology [scRNA-seq]

city: Seattle

first: Brian

last: Hanratty

state: WA

address: 1100 Fairview Avenue

department: Division of Human Biology

institute: Fred Hutchinson Cancer Cancer

summary: Comparative Molecular Analyses of LNCaP and Substrains Identify Drivers of Cancer Be-havior and Therapy Resistance

overall_design: To collect single cell suspension, cells were trypsinized and washed once in PBS and re-suspended in cell culture media followed by hemocytometer cell count to ensure cell viability above 70%.  The cell suspension were loaded into Chromium Next GEM Chip G (10x Genomics , PN#1000120) with 3′ v.3.1 beads (10x Genomics , PN#2000164) and partitioning oil.  Individual cells were barcoded using a 10× Chromium Controller (10x Genomics). RNA from the barcoded cells were subsequently reverse-transcribed and amplified with a Chromium Single Cell 3′ v3.1 Reagent Kit (10x Genomics, PN#1000123).  Libraries were subsequently constructed from 25% of amplified cDNA traces from pre-vious step with reagents from a Chromium Single Cell 3′ v3.1 Library Kit (10x Genomics, PN#1000190). The cDNA and library traces were quantitated and identified by Tapestation.  Sequencing were performed with an Illumina NextSeq 2000 P3.  All cells grown in RPMI1640 supplemented with 10%FBS and 1%Pen/Strep antibiotic.

first: Armand

last: Bankhead

first: Tony Lok Heng

last: Chu

first: Pete

last: Nelson


title: Multiomic assessments of LNCaP and derived cell strains reveal determinants of prostate cancer pathobiology [ChIP-Seq]

summary: Overall, the LNCaP line has served as a remarkably versatile PC model, though the molecular characteristics of several important substrains have not been established nor compared in a systematic manner.  In this study, we used genome-scale approaches to characterize the genomic and transcriptomic features of the parental LNCaP_FGC line, and a spectrum of 12 derivative models that have been deployed for studies of PC.  These studies provide insights into the biochemical features that endow PC with the potential to resist AR-directed therapy and identify features that may underlie the metastatic and treatment resistant phenotypes observed in patients. An important but under-appreciated feature of LNCaP concerns the remarkable heterogeneity of the original LNCaP isolate and the current LNCaP_FGC line which was never subjected to clonal selection. This feature, coupled with the inherent DNA mismatch repair deficiency and genomic instability, has produced a remarkable spectrum of sublines that have provided important insights into PC pathobiology, but also challenge reproducibility and the accurate interpretation of experimental findings.

overall_design: ChIP-Seq of LNCaP Sublines using AR antibody ab108341

first: Arnab

last: Bose

last: Bankhead III

first: Michael

last: Yang

first: Ilsa

last: Coleman

first: Rabeya

last: Bilkis

first: Wanting

last: Han

first: Jared

last: Jucas

first: Dapei

last: Li

first: Thomas

last: Persse

first: Patty

last: Galipeau

middle: C

last: Haffner

first: Gavin

last: Ha

first: Peter


title: Multiomic assessments of LNCaP and derived cell strains reveal determinants of prostate cancer pathobiology [CRISPR Screen]

overall_design: CRISPR Screen of LNCaP_FGC Using the Brunello Library


title: Effect of BRAF and MEK inhibition in KRAS mutant NSCLC cell line NCI-H358

city: Berkeley

first: Paul

last: Severson

address: 91 Bolivar Dr.

institute: Plexxikon Inc

summary: Paradoxical RAF activation by chemical RAF inhibitors (RAFi) is a well-understood ‘on-target’ biological and clinical phenomenon.  In this experiment, we profile the time-dependent gene expression changes that are specific to dabrafenib, selumetinib and the combination of dabrafenib plus selumetinib.

overall_design: NCI-H358 cells were treated with DMSO, 1uM dabrafenib, 30 nM selumetinib, or 1uM dabrafenib plus 30 nM selumetinib for 4 hours or 24 hours. Four independent biological replicates were prepared making a total of 32 samples.

first: Andrew

last: Kidger

first: Simon

last: Cook


title: E7386 enhances lenvatinib’s antitumor activity in preclinical models and human hepatocellular carcinoma.

city: Barcelona

first: Ana

last: Hernández de Sande

address: C/ del Rosselló, 153

country: Spain

institute: IDIBAPS

summary: To study the antitumor and antiangiogenic activity of combining E7386 (an oral protein-protein interaction inhibitor targeting CREB-binding protein (CBP)/β-catenin) with lenvatinib and explore a novel mechanism of action of E7386 that boosts antitumor response.

overall_design:
We transcriptomically profiled four HCC cell lines treated with DMSO or E7386
We transcriptomically profiled HCCs obtained from genetically engineered mouse models overexpressing MYC-lucOS;CTNNB1 and treated with Vehicle, E7386, Lenvatinib or E7386+Lenvatinib

first: Agavni

last: Mesropian

first: Albert

last: Gris-Oliver

first: Ugne

last: Balaseviciute

first: Amaia

last: Lujambio

first: Josep M

last: Llovet


title: Cyclic di-AMP drives secondary differentiation in Chlamydia trachomatis

city: Omaha

first: Jeonghoon

last: Lee

state: Nebraska

address: 985900 Nebraska Medical Center

department: Pathology, Microbiology, and Immunology

institute: University of Nebraska Medical Center

summary: The obligate intracellular bacterium Chlamydia alternates between two functional forms during its developmental cycle: elementary body (EB) and reticulate body (RB). However, the molecular mechanisms governing the transitions between these forms are unknown. Here, we present evidence cyclic di-AMP (c-di-AMP) is a key factor in triggering the transition from RB to EB (i.e., secondary differentiation) in the chlamydial developmental cycle. We made strains producing different levels of c-di-AMP, which we linked to changes in secondary differentiation status. Increases in c-di-AMP resulted in an earlier increase in transcription of EB-associated genes, and this was further manifested in earlier production of EBs. In contrast, when c-di-AMP levels were decreased, secondary differentiation was delayed. Based on these data, we conclude there is a threshold level of c-di-AMP needed to trigger secondary differentiation in Chlamydia. This is the first study to define a mechanism of secondary differentiation in Chlamydia.

overall_design: The transformants of dacAop and dacA-KD were infected into HeLa cells. At 10 hpi, the constructs were induced or not with 5 nM aTc.  RNA samples were prepared from dacAop-infected cells and dacA-KD-infected cells at 16 hpi and 24 hpi, respectively. 20 μg RNA samples were treated with DNase to remove DNA contamination by using DNA-free Turbo kit (Thermo) according to the manusfacturer’s instruction. Ribosomal RNA was depleted from samples using the MICROBEnrich (Thermo) and MICROBExpress kits (Thermo) following the manufacturer’s instructions. RNA samples were processed for sequencing by the UNMC Genomics Core Facility.

first: Scot

middle: P

last: Ouellette

first: Junghoon


title: Base barrier cells provide compartmentalization of choroid plexus, brain and CSF

city: Zwijnaarde

first: Roosmarijn

middle: E.

last: Vandenbroucke

state: Gent

address: Technologiepark-Zwijnaarde 71

department: VIB Center for Inflammation Research

country: Belgium

institute: VIB-UGent

summary: The choroid plexus (ChP), located in the brain ventricles, is largely composed of ChP epithelial cells that produce the cerebrospinal fluid (CSF) and form the blood–CSF barrier. At the ChP–brain attachment sites, we have discovered unique fibroblasts referred to as ChP base barrier cells (BBCs). We show that ChP BBCs originate from meningeal mesenchymal precursors, arrive early during development, remain throughout life and are conserved across species. ChP BBCs are transcriptionally similar to meningeal arachnoid barrier cells and are interconnected by both adherens and tight junctions. Notably, we provide evidence that the BBCs function as a barrier, controlling communication between the periphery and central nervous system. Moreover, during inflammatory insult, we observed a loss of barrier integrity and immune cell crossing. Altogether, our research revealed a barrier at the ChP base, crucial in protecting the central nervous system by compartmentalizing the ChP stroma, brain parenchyma and CSF.

overall_design: We performed scRNA-Seq to investigate the cellular heterogeneity within the Choroid Plexus of mice of various ages. After an initial analysis, we focused in on the various Fibroblast subsets present in our dataset and compared them to various cell types from other public datasets.

first: Daan

last: Verhaege

first: Clint

last: De Nolf


title: Antisense transposon insertions into host genes trigger piRNA mediated immunity

city: Vienna

first: Dominik

last: Handler

address: Dr. Bohr-Gasse 3

country: Austria

institute: IMBA-Institute of Molecular Biotechnology of Austrian Academy of Sciences

summary: Transposable elements pose a persistent threat to genome integrity, yet how host defense systems adapt to newly invading elements remains poorly understood. Here, we reveal how Drosophila melanogaster acquired PIWI-interacting RNA (piRNA)-mediated immunity against the recently invading endogenous retrovirus tirant. By integrating genetics, small RNA profiling, and population genomics, we identify two distinct modes of de novo piRNA biogenesis. The primary mechanism involves antisense insertions into the flamenco cluster, a well-established master locus for transposon control. Strikingly, we also find that antisense tirant insertions into 3′ UTRs of host genes robustly trigger piRNA production, a process driven by host gene transcription but independent of gene identity. These findings challenge prevailing models that link piRNA precursor specification to genomic origin or nuclear processing context. Instead, they uncover a flexible, general mechanism in which transposition into host gene exons represents a critical vulnerability for transposons: by generating chimeric antisense transcripts that are exported to the cytoplasm, transposons inadvertently initiate their own silencing, enabling rapid and adaptive genome defense against new invaders.

overall_design: small RNA-seq profiling from the Drosophila Synthetic Population Resources (DSPR) founder strains

first: Rafanel

last: Baptiste

first: Protsenko

last: Liudmila

first: Handler

last: Dominik

first: Brennecke

last: Julius

first: Senti

last: Kirsten-André


title: Multiomic assessments of LNCaP and derived cell strains reveal determinants of prostate cancer pathobiology (RNA-Seq)

first: Brian

last: Hanratty

overall_design: RNA sequencing of LNCaP-FGC and 12 LNCaP sublines using Illumina stranded mRNA TruSeq Library prep and sequenced on Illumina NovaSeq 6000.

first: Arnab

last: Bose

first: Armand

last: Bankhead III

first: Michael

last: Yang

first: Ilsa

last: Coleman

first: Rabeya

last: Bilkis

first: Wanting

last: Han

first: Jared

last: Jucas

first: Dapei

last: Li

first: Thomas

last: Persse

first: Patty

last: Galipeau

last: Haffner

first: Gavin

last: Ha

first: Peter

middle: S

last: Nelson


title: Multiomic assessments of LNCaP and derived cell strains reveal determinants of prostate cancer pathobiology

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

WGS data for this study is available at: PRJNA1219540


title: Stress-Induced Iron-Sulfur Cluster Damage as a Conserved Trigger of the Stringent Response

status: Public on Aug 20 2025

city: RENNES

first: Charlotte

last: Michaux

address: 2 avenue du Pr Leon Bernard

department: Inserm

country: France

institute: University of Rennes

summary: Pathogenic bacteria rely on the stringent response to adapt to hostile environments encountered within the host. However, the mechanisms by which host-induced stress activates this response remain poorly understood. Here, we identify iron-sulfur (Fe-S) cluster damage as a conserved trigger of the stringent response in major Gram-negative pathogens, including Salmonella enterica, Enterobacter cloacae, and Klebsiella pneumoniae. We demonstrate that Fe-S cluster disruption—triggered by oxidative stress or metal imbalance—limits intracellular pools of sulfur-containing and branched-chain amino acids, thereby activating the (p)ppGpp synthetase RelA. We further show that during Fe-S cluster stress, (p)ppGpp plays a dual role: enhancing bacterial fitness and promoting virulence by upregulating the Salmonella SPI-2 type III secretion system. These findings reveal a conserved mechanism by which pathogenic bacteria integrate host-associated stresses into an adaptive transcriptional response that promotes fitness and virulence, highlighting Fe-S cluster integrity as a central hub for environmental sensing during infection.

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium

overall_design: RNA-seq profiling of wild-type salmonella and their relA knockdown derivative with or without the presence of manganese

first: Eva

last: Michaud

first: Lorena

last: Ricci

first: Claire

last: Lallement

first: Lars

last: Barquist

first: Vincent

last: Cattoir

first: Régis

last: Hallez

first: Severin

last: Ronneau


title: Loss of UXS1 Selectively Depletes Pyrimidines and Induces Replication Stress in KEAP1-Mutant Lung Cancer [RNA-seq]

city: South San Francisco

first: Melat

middle: T.

last: Gebru

state: California

address: 1170 Veterans Blvd

department: Oncology

institute: Calico Life Sciences LLC.

summary: Kelch-like ECH associated protein 1 (KEAP1) is the third most commonly mutated gene in non-small cell lung cancer (NSCLC) and is associated with poor prognosis. Here, we investigated synthetic lethal interaction genes in KEAP1-mutated cancer cells and identified a dependency on UDP xylose synthase 1 (UXS1), which converts UDP-glucuronic acid (UDP-GlcA) to UDP- xylose in the proteoglycan synthetic pathway. UDP glucose dehydrogenase (UGDH), a transcriptional target of NRF2 that converts UDP-glucose to UDP-GlcA, was highly expressed in KEAP1-mutant tumors. Upon UXS1 knockdown, depletion of UDP-xylose occurred in both KEAP1-mutant and wildtype (WT) cells, whereas UDP-GlcA accumulated to a greater extent in the KEAP1-mutant setting. The resulting shortage of available UDP and other pyrimidines slowed S-phase progression and stalled DNA replication fork marks, causing cells to undergo prolonged cell-cycle exit or apoptosis. Dependency on UXS1 was rescued by knocking out UGDH to prevent UDP-GlcA accumulation and UDP depletion. DNA replication stress in UXS1-depleted cells sensitized them to clinical cell-cycle checkpoint inhibitors. Further, CRISPR screening experiments identified genes that modulate UXS1 dependency. While liver had the highest normal tissue expression of UGDH, UXS1 knockout in the liver did not result in hepatotoxicity. Taken together, these data demonstrate that UXS1 is a selective dependency in KEAP1-mutant tumors and loss of UXS1 creates additional therapeutically exploitable vulnerabilities in KEAP1-mutant tumors.

overall_design: Transcriptome profiling of NCI-H2122 and A549 control NTC and UXS1 knockdown cells 5 and 6 days after induction of knockdown by doxycycline treatment respectively.

first: Timothy

last: Hoffman

first: David

last: Stokoe


title: Loss of UXS1 Selectively Depletes Pyrimidines and Induces Replication Stress in KEAP1-Mutant Lung Cancer

overall_design: Human whole genome CRISPR screen using Avana library. NTC or shUXS1 expressing NCI-H460 or NCI-H2122 cells were transduced with CRISPR library and treated with dox to induce shUXS1 for 11 days.


title: Targeting RAC1 as a novel strategy to reduce inflammation and liver damage in acute liver failure.

city: Derqui

first: Juan

last: Bayo

state: Buenos Aires

address: Av Peron 1500

department: IIMT

country: Argentina

institute: Universidad Austral-Conciet

summary:
Background & Aims:
The Rho GTPase RAC1 regulates key drivers of acute liver failure (ALF) such as oxidative stress and inflammation. The goal of this study was to determine the therapeutic potential of RAC1 pharmacological inhibition in ALF.
Methods:
The involvement of RAC1 in human ALF was explored by analyzing public transcriptome datasets. Murine models of ALF were induced by administration of either Concanavalin A, acetaminophen or D-Galactosamine/Lipopolysaccharide. In vitro assays were conducted on hepatocytes and macrophages primary cultures and cell lines. 1D-142 was used to explore the effect of RAC1 pharmacological inhibition. RNA-Seq analysis was performed and correlated with ALF human datasets to explore the therapeutic potential of RAC1 inhibition. Liver explants from patients with liver failure were treated in vitro with 1D-142.
Results:
RAC1-related pathways are upregulated in human ALF samples and correlate with immune activation and oxidative stress. Administration of 1D-142 ameliorated liver damage across all ALF murine models. 1D-142 treatment diminishes reactive oxygen species formation and reduces inflammatory cells migration and suppresses cytokine production both in vivo and in vitro. Transcriptome analysis of treated livers indicates that RAC1 pharmacological inhibition modulates dysregulated biological processes observed in ALF patients. Liver explants from ALF patients treated with 1D-142 exhibited reduced necrosis and reduced expression of pro-inflammatory genes.
Conclusions:
RAC1 plays a critical role in driving inflammation and oxidative stress in ALF. Targeted inhibition of RAC1 with 1D-142 protects against ALF in murine models; thus, RAC1 inhibition could be a potential therapeutic option for ALF.

overall_design: Transcriptomic Profiling of C57BL/6J Mouse Livers During Acute Liver Failure Induced by acetaminophen or concanavalin A and Treated with the RAC1 Inhibitor 1D-142

first: Barbara

last: Bueloni

first: Esteban

last: Fiore

first: Guillermo

last: Mazzolini


title: Multiomic assessments of LNCaP and derived cell strains reveal determinants of prostate cancer pathobiology (ATAC-Seq)

first: Brian

last: Hanratty

overall_design: ATAC-Seq of LNCaP Sublines using OMNI-ATACseq protocol

first: Arnab

last: Bose

first: Armand

last: Bankhead III

first: Michael

last: Yang

first: Ilsa

last: Coleman

first: Rabeya

last: Bilkis

first: Wanting

last: Han

first: Jared

last: Jucas

first: Dapei

last: Li

first: Thomas

last: Persse

first: Patty

last: Galipeau

last: Haffner

first: Gavin

last: Ha