title: A comprehensive evaluation of metabolically engineered Cynara cardunculus calli as platform for valuable fatty acid derivatives production

city: Milano

first: Raul

last: Pirona

address: Via Alfonso Corti 12

institute: IBBA-CNR

summary: Increasing global demand for energy along with climate change claims for sustainable and renewable energy sources. In this study, we investigated transcriptomic changes in two cardoon (Cynara cardunculus L. var. altilis) calli cell culture lines engineered for production of valuable fatty acids, therefore suitable for large-scale production of biofuel precursors, as alternative to triacylglycerols (TAGs) engineered plants. Namely, to gain insight in the molecular effects of genetic transformation, we analyzed the transcriptome of SAD-OE line (overexpressing stearic acid desaturase), and FAD-KD line (a fatty acid desaturase RNA interference line, FAD2.2-RNAi) accumulating oleic and linoleic acids, respectively. Differentially expressed genes (DEGs) involved in fatty acid metabolism and streamed pathways such as glycolysis, pyruvate, glycerolipid, and glycerophospholipid metabolism, were enriched in the KEGG analysis in both lines. In addition to analyzing DEGs, we used qRT-PCR to monitor key regulatory and biosynthetic genes involved in TAGs formation and assembly, aiming to understand their transcriptional dynamics over ten days of cell growth in liquid culture. In transgenic scaled up lines, upon observing increased lipid accumulation, we investigated how genetic transformation influenced sugar metabolism. Fructan depletion analysis further supported their role as carbon sources for TAG biosynthesis. Confocal microscopy confirmed a significantly higher accumulation of oil bodies in transgenic lines compared to wild-type (WT) calli. Overall, this study presents a comprehensive characterization of a cardoon cell-based platform for large-scale vegetable oil production. Unlike whole plants, these engineered cell cultures do not suffer from severe oxidative stress or growth and metabolic impairments, making them a promising alternative for sustainable fatty acid biosynthesis.

overall_design: RNA-seq experiments on different transformed calli for stearic acid desaturase overexpression (SAD-OE), and fatty acid desaturase RNAi silencing (FAD-KD). four samples with three biological replicates were sequenced: Empty vector transformed calli, SAD-OE transformed calli, FAD-KD transformed calli, WT non trasformed calli.

first: Elisa

last: Cappetta

first: Monica

last: De Palma

first: Rosa

last: D'alessandro

first: Antonio Luca

last: Langellotti

first: Alfredo

last: Ambrosone

first: Teresa

last: Docimo


title: Glutamine: A novel player in maintaining skeletal strength and body fitness in obese mice

status: Public on Oct 14 2025

city: Prague

first: Yusuf

middle: Caglar

last: Odabasi

state: Vestec

address: Prumyslova 595

department: BIOCEV

country: Czech Republic

institute: IBT

type: Expression profiling by high throughput sequencing

summary:
Background and aims:
Glutamine plays a key role in cellular metabolism and tissue homeostasis. In obesity, circulating glutamine levels decline, accompanied by impaired bone homeostasis and increased fracture risk. While dietary glutamine supplementation shows metabolic benefits, its effects on bone and fat metabolism remain unclear. This study investigates whether glutamine supplementation mitigates obesity-induced alterations in bone and fat metabolism.

Methods:

C57BL/6J male mice were subjected to a 2-month dietary intervention with either high-fat diet (HFD) or HFD supplemented with glutamine (HFD + G) and low-fat diet (LFD) as a control group. Body weight, fat mass, glucose tolerance, white adipose tissue (WAT) morphology, and bone parameters were analyzed. Functional assays of adipose-derived mesenchymal stem cells (AT-MSCs) and bone marrow stromal cells (BMSCs) assessed metabolic phenotype and differentiation potential. Glutamine turnover was evaluated, and findings were extended to human BMSCs to assess sex-specific patterns of glutaminolysis.
Results:

Glutamine supplementation attenuated body weight gain, fat mass, and WAT weight, along with improved glucose tolerance compared to HFD-fed mice. In WAT, glutamine reduced adipocyte hypertrophy and inflammation, while in AT-MSCs it suppressed obesity-driven hyper-metabolic phenotype by shifting cells toward quiescence. In bone, glutamine improved bone quality, along with reduced bone marrow adiposity and decreased bone resorption. BMSCs from glutamine-treated mice showed decreased adipogenic and increased osteogenic potential, supported by enhanced glutamine turnover, which maintained the stemness of the cells and reduced the inflammation induced by obesity. In human BMSCs, glutamine metabolism displayed sex-specific differences, underscoring its physiological relevance.

overall_design: High throughput sequencing expression profiling by high throughput sequencing (Bulk RNA-seq data analysis)

first: Martina

last: Dzubanova

first: Michaela

last: Ferencakova

first: Dung

middle: K

last: Nguyen

first: Kristina

last: Bardova

first: Elena

last: Golovina

first: Heleen

last: Fehervary

first: Andrea

last: Benova

middle: C

first: Radek

last: Sindelka

first: Frantisek

last: Spoutil

first: Jan

last: Prochazka

first: Tomas

last: Cajka

first: Harry

last: van Lenthe

first: Martin

last: Rossmeisl

last: Kopecky

last: Tencerova


title: Notch interaction with RUNX factors regulates initiation of the T-lineage program [RNA-seq]

city: Isehara

first: Hiroyuki

last: Hosokawa

address: 143 Shimokasuya

department: Immunology

country: Japan

institute: Tokai Univ

summary: RUNX factors play an essential role in the development of T cell. Dynamic interacting partner switching of RUNX1 induces the redeployment of RUNX1 at the T-lineage commitment checkpoint. Here, we attempted to reveal functional differences in RUNX factors between lymphoid progenitor (LP) and Noth-stimulated the earliest T progenitor stage, phase1. We identified CTCF as a LP-specific RUNX1-interacting partner. Indeed, LP-specific RUNX1 binding genomic sites had significantly enriched CTCF consensus motif and co-occupied with CTCF. After Notch stimulation, Notch1-IC directly interacted with RUNX1 and recruited it to the Notch-regulated T-signature gene loci with the Mediator/p300 transcriptional activation complex. CRISPR/Cas9-mediated stage-specific deletion of RUNX factors and its binding partners revealed that the RUNX1/CTCF complex in LP and the RUNX1/Mediator/p300 complex in phase1 negatively and positively regulate T-signature genes expression, respectively. Our results indicate that the Notch-mediated functional conversion of RUNX factors; re-organization of protein complexes and redeployment of genomic binding sites, has a crucial role in the initiation of T-lineage program.

overall_design:
Cas9-LPs with or without Notch stimulation were transduced with various sgRNAs.
Total RNA was isolated from samples of 3 × 105 cultured cells using a RNeasy Micro Kit (Qiagen).  500 ng of total RNA was subjected to the 3’mRNA library preparation with QuantSeq 3’ mRNA-Seq Library Prep Kit FWD (LEXOGEN) according to the manufacturer’s instructions.  After the PCR step, size distribution and yield of the library was determined by the D1000 high sensitivity tape station (Agilent) or Agilent High Sensitivity DNA kit on the bioanalyzer (Agilent).  The pooled libraries were loaded on the Illumina Nextseq500 platform and analyzed by 75bp single read.

first: Yuichi

last: Kama

first: Ken-ichi

last: Hirano

first: Kaori

last: Masuhara

first: Yusuke

last: Endo


title: Trimodulin and pneumonia

city: Berlin

first: Peter

last: Pennitz

address: Charitéplatz 1

department: Department of Infectious Diseases, Respiratory Medicine and Critical Care

country: Germany

institute: Charité – Universitätsmedizin Berlin

summary: This RNAseq dataset evaluates gene expression to assess the therapeutic potential of trimodulin in a complex mouse model mimicking critically ill CAP patients infected with S. pneumoniae. In addition, we combined trimodulin with ampicillin to address if trimodulin has further added benefit in addition to reducing bacterial burden.

overall_design: Mice were either infected with S. pneumoniae or sham-infected with PBS as control, 22 h later mice were treated with placebo as control, trimodulin, ampicillin or a combination of both until the 30 hpi analysis time point.

first: Geraldine

last: Nouailles

first: Romina

last: Bischoff

first: Kerstin

last: Linke

first: Alexander

last: Taylor

first: Birgitt

last: Gutbier

first: Cengiz

last: Goekeri

first: Sandra

last: Kunder

first: Anne

last: Voß

first: Theresa

last: Broemel

first: Olivia

last: Kershaw

first: Miha

last: Milek

first: Vadim

last: Farztdinov

first: Michael

last: Mülleder

first: Sabrina

last: Weissmüller

first: Corina

last: Heinz

first: Mayken

last: Visser

first: Fabian

last: Bohlaender

first: Katharina

last: Ahrens

first: Dieter

last: Beule

first: Achim

middle: D

last: Gruber

last: Koenig

last: Witzenrath


title: The Exon Junction Complex coordinates the co-transcriptional inclusion of blocks of neighboring exons

city: New Haven

first: Alexandra

last: Bergfort

state: CT

address: 333 Cedar Street

department: Molecular Biophysics & Biochemistry

country: USA

institute: Yale University

summary: The Exon Junction Complex (EJC) has roles in mRNA export and cytoplasmic quality control. Yet EJC is recruited to pre-mRNA by the spliceosome, prior to the second catalytic step of splicing. Because splicing often occurs co-transcriptionally, EJC can be deposited on nascent RNA almost immediately after introns are synthesized and excised, raising the question of whether EJC regulates downstream RNA processing decisions. Here, we show that degron-mediated depletion of EJC component EIF4A3 leads to substantial skipping of neighboring pairs of two or more exons. These exon skipping events occurred on the same molecule of mRNA, detected by long read sequencing, suggesting that “exon blocks” require EJC for inclusion. Introns flanking EJC-dependent exon blocks were longer and spliced after internal introns. Thus, block exons form a larger EJC-marked exon prior to surrounding splicing events. These findings identify a previously unknown nuclear function of EJC that coordinates co-transcriptional inclusion of exon blocks.

overall_design: To investigate a role of EIF4A3 in co-transcriptional splicing, we genereated HEK293T cells with an EIF4A3-SMASh degron tag and an EIF4A3 overexpressing HEK293 Flp-In T-REx cell line. We extracted mRNA and chromatin bound nascent RNA (nRNA) and conducted Illumina  paired end sequencing and PacBio long-read sequencing.

first: Jackson M.

last: Gordon

first: Chuan-Tien

last: Hung

first: Benhur

last: Lee

first: Karla M.

last: Neugebauer

first: Matthew R.

last: Gazzara

first: Yoseph

last: Barash


title: Notch interaction with RUNX factors regulates initiation of the T-lineage program [ChIP-seq]

first: Hiroyuki

last: Hosokawa

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: Ten million of Cas9-LPs with or without Notch stimulation were fixed with 1 mg/ml disuccinimidyl glutarate (Thermo Scientific) in PBS for 30 min at room temperature followed by an additional 10 min with addition of formaldehyde up to 1 %. The reaction was quenched by addition of 1/10 volume of 0.125 M glycine and the cells were washed with cold PBS. Pelleted nuclei were dissolved in lysis buffer (0.5 % SDS, 10 mM EDTA, 0.5 mM EGTA, 50 mM Tris-HCl [pH 8.0], and PIC) and sonicated on a Bioruptor (Diagenode) for 18 cycles of 30 sec sonication followed by 30 sec rest, with high power. Five micrograms of anti-RUNX1 (abcam; ab23980), anti-CTCF (CST; 3418), anti-Med12 (Bethyl; A300-774A), or anti-p300 Abs (CST; 57625) were prebound to Dynabeads anti-rabbit Ig and then added to the diluted chromatin complexes. The samples were incubated overnight at 4 ℃, then washed and eluted overnight at 65 ℃ in ChIP elution buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 50 mM NaCl, 1 % SDS, and 50 mg/ml proteinase K). Eluted chromatin fragments were cleaned up using ChIP DNA Clean & Concentrator (Zymo). ChIP-seq libraries were constructed using NEBNext Ultra Ⅱ DNA Library Prep with Sample Purification Beads (E7103S, NEB) and NEBNext Multiplex Oligos for Illumina (E7500S, NEB) and sequenced on Illumina NextSeq500 in single read mode with the read length of 75 nt.

first: Yuichi

last: Kama

first: Ken-ichi

last: Hirano

first: Kaori

last: Masuhara

first: Yusuke

last: Endo


title: Cancer suppresses mitochondrial chaperone activity in macrophages to drive immune evasion

city: Columbus

first: Stanley

last: Huang

state: Ohio

address: 2255 Kenny Road

department: Department of Microbial Infection and Immunity

institute: The Ohio State University

summary: Here we uncover that TNF-receptor associated protein-1 (TRAP1) expression in TAMs is markedly reduced in the TME, a process mediated by the TIM4-AMPK signaling pathway. In addition, TRAP1 deficiency leads to increased activity of mitochondrial electron transport chain complexes and an elevated α-ketoglutarate (α-KG) to succinate ratio. The increase in α-KG facilitate JMJD3-mediated histone demethylation, altering genes expression associated with immunosuppressive activation in macrophages.

overall_design: mRNA of naive or YUMM1.7 tumor-conditioned medium (TCM)-treated bone marrow-derived macrophages isolated from TRAP1 WT, TRAP1 KO, JMJD3 WT or JMJD3 conditional KO mice.

first: Haoxin

last: Zhao

first: Yuzhu

last: Wang

first: Wei

last: Cao

first: Yili

last: Ouyang


title: Pseudomonas aeruginosa clinical isolates can encode functional plastic degrading enzymes that allow survival on plastic and augment biofilm formation

city: Southampton

first: Rubén

last: de Dios

state: Hampshire

address: University Road

department: School of Biological Sciences

country: United Kingdom

institute: University of Southampton

summary: Several environmental bacteria encode plastic-degrading enzymes, a potential evolutionary response to the rapid introduction of plastic across global ecosystems. Given the widespread use of plastic in healthcare, we hypothesised that clinical bacterial isolates may also degrade plastic, rendering plastic-containing medical devices susceptible to degradation and failure and potentially offering these pathogens a carbon source that could be used to persist in the hospital-built environment. Here, we mined the genomes of prevalent pathogens and identified several enzymes in different pathogens with homology to known plastic-degrading enzymes. Synthesising and expressing a potential plastic-degrading enzyme derived from a Pseudomonas aeruginosa wound isolate in a heterologous host, we were able to demonstrate  potent plastic degrading activity. We subsequently found that the original P. aeruginosa clinical isolate could reduce the weight of a medically relevant plastic, polycaprolactone (PCL), by 78% in 7 days, and critically could use it as a sole carbon source to grow. We uncovered a direct link to virulence, demonstrating that encoding a plastic degrading enzyme can significantly enhance biofilm formation and pathogenicity in vivo. We also demonstrate that this augmented biofilm phenotype is conserved in another P. aeruginosa PCL-degrading clinical isolate we identified in a screening. We reveal that the mechanism underpinning this enhanced biofilm formation is the incorporation of the plastic breakdown products into the extracellular matrix, leading to enhanced biofilm levels. The level of PCL degradation we show by a clinical isolate and its ability to promote a key virulence and persistence determinant such as biofilm formation indicates that the integrity of any PCL containing medical device, such as sutures or implants, and the condition of patients receiving such devices could be severely compromised by pathogens with this capacity. Given the central role of plastic in healthcare, this should be considered in the future of medical interventions and practice and hospital designs implementing this material

overall_design: PA-W23 cultures (3 biological replicates per condition) were grown to mid‐exponential phase (OD600 0.6–0.7) in 20 ml Starvation media (10% LB + 90% M9 Minimal Media) supplemented with 0.5 g of PCL beads in the 3 treated samples.

first: Sophie

middle: A

last: Howard

first: Evgenia

last: Maslova

first: Ronan

middle: R

last: McCarthy


title: Effect of ATP7A knockdown on gene expression in glioma stem cells

city: PITTSBURGH

first: Fanen

last: Yuan

state: PA

address: 5030 Centra Ave, APT861

institute: University of pittsburgh

summary: Copper-related cell death, cuproptosis, involves accumulation of intracellular copper that triggers mitochondrial lipoylated protein aggregation and destabilization of iron–sulfur cluster proteins. Here, glioblastoma (GBM) stem cells (GSCs) displayed resistance to cuproptosis with circadian variation of intracellular copper levels, unlike differentiated GBM cells (DGCs) or neural stem cells (NSCs). CRISPR screening of copper regulators under concurrent treatment with copper ionophore or clock disruption revealed dependency on ATPase Copper Transporting Alpha (ATP7A). Circadian control of copper homeostasis was mediated by core clock transcription factor, Basic helix-loop-helix ARNT-like protein 1 (BMAL1). In turn, ATP7A promoted tumor cell growth through regulation of fatty acid desaturation. Copper levels negatively fed back onto the circadian clock through SQSTM1 (sequestosome 1/p62)-mediated lysosomal degradation of BMAL1. Targeting cuproptosis and circadian clock or fatty acid desaturation generated synergistic anti-tumor effects. Thus, crosstalk between the circadian rhythm and copper sustains GSCs, reshaping fatty acid metabolism and promoting drug resistance.

overall_design: ATP7A was knockdown by shRNA in GSC387, GSC3565 and GSC738. And then RNA-seq was performed to investigate the effect of ATP7A knockdown on gene expression.

first: Jeremy

middle: N

last: Rich


title: Addressing the specific roles of histone modifications in transcriptional repression [RRBS]

city: London

first: Aina

middle: Maria

last: Mas

address: Chester Beatty Laboratories, 237 Fulham Rd.

institute: Institute of Cancer Research

type: Methylation profiling by high throughput sequencing

summary: Our understanding of epigenetic processes is based on the hypothesis that individual posttranslational modifications of DNA and histones, or combinations thereof, function to direct unique downstream effects on transcription. Still, histone modifications are broadly categorized as repressive or activating, raising the question of potential functional redundancy. Here, we present an approach for addressing this question by substituting the genome-wide H3K27me3 pattern with other histone modifications. By taking advantage of the modular organization of PRC2, we direct de novo recruitment of H3K9me3 and H3K36me3 to PRC2 target genes in H3K27me3 null mouse embryonic stem cells (mESCs). We show that despite accurate genome-wide re-establishment of H3K36me3 at PRC2 target genes, which leads to significant reduction in H3K4me3 levels, the remaining H3K4me3 prevents H3K36me3 from recruiting sufficient DNA methylation to substitute for H3K27me3-mediated repression. In contrast, we demonstrate that H3K9me3 is more efficient in repressing H3K27me3 regulated genes, however this repression is also contingent on H3K4me3 status. Taken together, these results highlight the unique repressive functions of H3K27me3 and suggest that the functional effects of individual posttranslational modifications are highly dependent on the interplay with the existing chromatin environment.

overall_design: Reduced Representation Bisulfite Sequencing on mESC samples, to identify changes in PRC2 target gene CPG island methylation

first: Lin

last: Hedehus

first: Robin

middle: L

last: Armstrong

first: Richard

last: Koche

first: Hua

first: Aktan

last: Alpsoy


title: Gene expression of human iPSC-derived hepatocyte-like cells treated with insulin and dihydrotestosterone

status: Public on Oct 13 2025

city: Suita

first: Yosuke

last: Yoneyama

state: Osaka

address: 2-2 Yamadaoka

department: Graduate School of Medicine

institute: The University of Osaka

summary: We compared the global transcriptome of human iPSC-derived hepatocyte-like cells (HLC), which have homozygousTM6SF2 E167K, cultured in different conditions: control, insulin, dihydrotestosterone, and insulin plus dihydrotestosterone.

overall_design: Comparative gene expression profiling analysis of RNA-seq for liver tissues of rats treated in different protocols.

first: Asei

last: Hirai

first: Takanori

last: Takebe


title: GDF3 promotes adipose tissue macrophage-mediated inflammation via altered chromatin accessibility during aging

city: Minneapolis

first: In Hwa

last: Jang

state: Minnesota

address: 100 Church Street SE

institute: University of Minnesota

summary: SMAD2/3 transcription factors (TFs) have low DNA binding affinity for a specific consensus sequence, allowing them to elicit anti- or pro-inflammatory responses depending on the cellular context. Genes associated with pro-inflammatory processes in immune cells, including myeloid cells, exhibit increased chromatin accessibility with age. We hypothesized that this could affect the transcriptional activity of SMAD2/3 in adipose tissue macrophages (ATMs). To assess chromatin accessibility, ATMs isolated using fluorescence-activated cell sorting (FACS) from young control, old control and old mKO mice were profiled using assay for ATAC-seq.

overall_design: Visceral adipose tissue depots were isolated from mice and digested into stromal vascular fraction and adipocyte fraction (PMID: 33870227).  Then, the stromal vascular fraction was used for fluorescence-activated cell sorting of adipose tissue macrophages: Live CD45+ CD11b+ SiglecF- F4/80+ cells were sorted on a BD FACS Aria II into RPMI with 20% FBS

first: Victor

last: Kruglov

first: Stephanie

middle: H

last: Cholensky

first: Declan

middle: M

last: Smith

first: Suxia

last: Bai

first: Timothy

last: Nottoli

first: David

last: Bernlohr

first: Christina

last: Camell

first: Anna

last: Carey


title: Comparative analysis of isoproterenol and lipopolysaccharide mediated cytoprotective responses in the heart

city: St. Louis

first: Douglas

last: Mann

state: MO

address: 660 S Euclid Ave

institute: Washington University in St. Louis

summary: This study aimed to compare the mechanisms by which isoproterenol and lipopolysaccharide induce cytoprotection in the heart. We performed bulk RNA sequencing to compare the transcriptomic effects of isoproterenol and lipopolysaccharide on the heart, to examine features that are shared and unique to each treatment.

overall_design: Female 10-wk old wild-type (C57BL/6J) mice were treated with lipopolysaccharide (LPS; 2.5 mg/kg) or isoproterenol (ISO; 300 mg/kg). Mice were then euthanized at baseline (no treatment) and at 1 and 7 days later (n=3-4/group) with hearts collected for bulk RNA sequencing.

first: Kenji Rowel

middle: Q

last: Lim


title: Oxidative stress sensing by the translation elongation machinery promotes production of detoxifying selenoproteins

city: Dallas

first: Joshua

last: Mendell

address: 6000 Harry Hines Blvd

department: Molecular Biology

institute: UT Southwestern Medical Center

summary: The special amino acid selenocysteine plays an essential role in metazoan redox biology and is incorporated into nascent protein chains through the recoding of a UGA termination codon. Using GPX1 and GPX4, two selenoenzymes that protect cells from oxidative stress, as fluorescent reporters we carried out the first genome-wide knockout screen to identify regulators of selenocysteine decoding.

overall_design: Two independent clones for each cell line, GPX1, GPX4 and GPX4M, were used for the screen and 1.4x10^8 cells were infected with the Brunello pooled lentiviral CRISPR library (Addgene #73179) in the presence of 5 μg/ml polybrene at a multiplicity of infection between 0.3 to 0.5. Two days after transduction, the cells were selected in medium containing 1 μg/ml puromycin. After 8 days puromycin concentration was lowered to 0.5 μg/ml. After 10 days of selection the top and bottom 0.75% eGFP populations were sorted on a FACS Melody (BD Biosciences). gDNA from sorted pools was isolated by phenol-chloroform extraction. gDNA from 6x10^7 unsorted cells, was isolated with the Masterpure DNA isolation kit (Lucigen). Sequencing libraries were generated from the isolated gDNA through two sequential PCR reactions using the Herculase II DNA polymerase (Agilent). After 18 rounds of initial PCR amplification, 5% of the reaction product was used for a second round of PCR (10cycles) with primers that introduced Illumina sequencing adapters and barcodes. The final PCR products were purified using AMPure XP beads (Beckman Coulter). Libraries were sequenced on a NextSeq2000 (Illumina) with 100bp single-end reads at an average sequencing depth of ~2.2x10^7 reads per sample. sgRNAs sequences were extracted from fastq files through an in-house Galaxy script and normalized reads counts were calculated.

first: Frederick

last: Rehfeld

first: Claire

last: Lundstrom

first: He

last: Zhang

middle: T


title: Addressing the specific roles of histone modifications in transcriptional repression [ChIP-seq and CUT&RUN]

first: Aina

last: Mas

overall_design: Chromatin Immunoprecipitation (ChIPseq) of PRC2 subunit SUZ12 as well as Cleavage Under Targets & Release Using Nuclease (CUT&RUN) for histone modifications H3K4me3, H3K9me3, H3K27me3, H3K36me3 and H2AK119ub1

first: Lin

last: Hedehus

first: Robin

last: Armstrong

first: Richard

last: Koche

first: Hua

last: Wang

first: Aktan

last: Alpsoy


title: Addressing the specific roles of histone modifications in transcriptional repression [RNA-seq]

overall_design: RNAseq on panel of mESC cell lines, to identify changes en gene expression of PRC2 target genes upon changes of histone modification profiles


title: Q373fs variant of RBM20 affects splicing of cardiac genes that are causative for sudden death; Human sudden death case and animal experiment

city: Nishinomiya

first: Takuma

last: Yamamoto

address: 1-1 Mukogawa-Cho

institute: Hyogo Medical University

summary: RBM20 is one of the genes predisposing to dilated cardiomyopathy (DCM). Variants in the RS domain and the RNA-recognition motif have been well studied, but the pathogenicity of variants outside the hotspot remains unknown. A human patient with the Q373fs-RBM20 variant without a typical DCM phenotype was identified in sudden death cohorts. In the mouse experiments, RNA seq analysis revealed that Q374fs-Rbm20 mice showed some different splicing patterns in such as Ttn, Ldb3, Camk2d,Obscn, and Ryr2. The expressions of Casq1, Mybpc2, and Myot were also upregulated in Q374fs-Rbm20 mice. The pathway analysis indicated the involvement of some of the 1770 differentially expressed genes in Cytoplasmic ribosomal proteins Calcium regulation in cardiac cells and Striated muscle contraction. Cardiac dysfunction such as cardiac dilation and extended duration of QRS and QTc were observed by ultrasound echocardiography and Electro-cardiogram. The Q373fs-RBM20 (Q374fs-Rbm20) variant changes gene splicing and affects gene expression of sarcomere structural genes and Ca handling genes and presented cardiac dysfunction, suggesting that Q373fs-RBM20 human patient may cause arrhythmogenic sudden death.

overall_design: Using the CRISPR/Cas system, we generated KI mice harboring the c.1121_1122delAG (p. Q374Rfs) variant in the mouse Rbm20 gene, which corresponded to human c.1118_1119delAG (p. Q373Rfs).


title: Anti-uPAR CAR T cells reverse and prevent aging-associated defects in intestinal regeneration and fitness

city: Cold Spring Harbor

first: Vyom

last: Shah

state: New York

address: 1 bungtown road

department: Beyaz Lab

institute: Cold Spring Harbor Laboratory

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

Intestinal stem cells (ISCs) drive the rapid regeneration of the gut epithelium. However, during aging their regenerative capacity is reduced, possibly through senescence and chronic inflammation, albeit little is known about how aging-associated dysfunction arises in the intestine. We previously identified the urokinase plasminogen activator receptor (uPAR) as a senescence-associated protein in other tissues and developed CAR T cells able to efficiently target it. Harnessing them, here, we identify the accumulation of uPAR-positive cells in the aging gut and uncover their detrimental impact on ISC function in aging. Thus, both therapeutic and prophylactic treatment with anti-uPAR CAR T cells improved barrier function, regenerative capacity, inflammation, and mucosal immune function in aged mice. Overall, these findings reveal the deleterious role of uPAR-positive cells on intestinal aging in vivo and provide proof of concept for the potential of targeted immune-based cell therapies to enhance tissue regeneration in aging organisms.

overall_design: Refer to individual Series


title: Effect of pressure-overload on cardiac endothelial cells in C57BL6/J mice

city: Rouen

first: Ebba

last: Brakenhielm

address: Inserm U1096

country: France

institute: Inserm

summary: We performed single-cell RNA-seq analysis of FACS sorted cardiac blood vascular and lymphatic endothelial cells in the heart of adult C57 female mice in a model of pressure-overload.

overall_design: Cardiac CD31+ CD45neg endothelial cells, and Lyve1/Podoplanin-expressing lymphatic endothelial cells (LEC) were extracted from hearts of mice 8 weeks after pressure-overload or sham surgery in a murine model of transaortic constriction, and analyzed by single-cell RNA-sequencing. Briefly, cardiac single cell suspensions were sorted by FACS (ARIA II, BD Biosciences). BECs were defined as live CD45-/CD31+/Lyve1- cells, whereas LECs were identified as CD45-/CD31+/Lyve1+/Pdpn+ cells. A 1:3 mix of cardiac LECs and BECs were included in each sample prepared using the 10X Genomics Chromium Next Gem Single Cell 3' kit. For each group (Sham or TAC), 1-2 single cell sorting and GEM reactions were performed, using each time cells pooled from 10 mouse hearts. Samples were sequenced using Illumina NextSeq550 at an estimated sequencing depth of 20 000 reads per cell. Reads were aligned with the mouse genome using STARsolo. For low-quality data filtering, genes expressed by <10 cells, and cells expressing <300 genes, or >10% mitochondria-derived UMI counts, were excluded. R package Seurat was used for read normalization, data integration, and principal component-based unsupervised clustering and statistical tests. Among 4178 total sequenced cells, 1577 cells remained post-filtering, including 665 cells from sham-operated controls and 912 cardiac cells from TAC mice.

first: Coraline

last: Heron

first: Theo

last: Lemarcis

first: Oceane

last: Laguerre

first: Benjamin

last: Bourgeois

first: Corentin

last: Thuilliez

first: Chloe

last: Valentin

first: Anais

last: Dumesnil

first: Manon

last: Valet

first: Gaetan

last: Riou

first: Sophie

last: Candon

first: Celine

last: Derambure

first: Caroline

last: Berard

first: Helene

last: Dauchel

first: Virginie

last: Tardif

first: Brakenhielm

last: Ebba

first: Zernecke

last: Alma


title: De novo mutations in the MiDAC HDAC complex cause a neurodevelopmental 2 syndrome resulting from increased deacetylase activity [qPRO-seq]

city: Leicester

first: John

last: Schwabe

state: Leicestershire

address: Lancaster Road

department: Molecular and Cell Biology

institute: University of Leicester

summary: MIDEAS is a scaffold protein that, together with DNTTIP1, mediates assembly of the MiDAC histone deacetylase complex. Mice lacking MiDAC die before birth suggesting a key developmental function. Here, we report two unrelated individuals, with a multisystem disorder characterised by delayed speech development, joint contractures, dysmorphic features and dysmotility of the gut. Both individuals have the same de novo heterozygous missense variant in MIDEAS (p.Tyr654Ser). A cryoEM structure of the MiDAC complex reveals that this amino acid is located in a conserved auto-inhibitory loop that covers the active site of the deacetylase enzyme. We suggest that the variant results in loop displacement leading to elevated deacetylase activity. In support, we observe reciprocal gene expression changes in patient fibroblasts compared with a cell line following rapid MiDAC degradation. Our results establish MIDEAS as a dominant monogenic disease gene and that hyperactivity of the MiDAC complex results in a characteristic multisystem disorder.

overall_design: qPRO nascent transcription profiling of HCT 116 cells CRISPR-engineered to express endogenous DNTTIP1 with C-terminal FKBPF36V/FLAG to allow rapid degradation using dTAGv1 at 2h and 6h.

first: Robert

middle: E

last: Turnbull

first: Kristupas

last: Sirvydis

middle: W


title: H2AK119ub dynamics controls hair follicle stem cell quiescence [RNA-Seq 2]

city: Bronx

first: Deyou

last: Zheng

state: NY

address: 1301 Morris Park Avenue

department: Department of Genetics

institute: Albert Einstein College of Medicine

summary: The transition of stem cells from a quiescent state to an active state is a finely tuned process that requires the dismantling of the quiescence program and the establishment of a cell cycle-promoting transcriptional landscape. Whether epigenetic processes control stem cell states to promote the regeneration of adult tissues remains elusive. In this study, we show that a repressive histone modification, H2AK119ub, is dynamic between quiescent and active hair follicle stem cells (HFSCs) in the adult murine skin. Ablation of H2AK119ub in HFSCs leads to failure in maintaining quiescence leading to premature activation and an eventual exhaustion of HFSC pool. Transcriptional and chromatin studies revealed that H2AK119ub directly represses a proliferation promoting transcriptional program in the HFSCs to preserve quiescence. Lastly, we identify that the inhibitory FGF signaling produced by the hair follicle niche keratinocytes maintains H2AK119ub in quiescent HFSCs. Together, these findings reveal that a repressive histone mark, H2AK119ub, is under the dynamic regulation of inhibitory niche signaling to prevent the untimely establishment of an activated state to preserve SC function and longevity.

overall_design: Control and PRC1 i2KO mice were induced starting at P50. 15 days after completion of induction treatment, the back skin was collected and the adipose layer from the dorsal side was scraped off using a surgical scalpel. The scraped skin sample was washed with PBS prior to incubation with 0.25% Trypsin/EDTA at 37°C for 1 hour on a rotation plate set at 60 r.p.m. After incubation, the epidermal cells, including the HFSCs, was scraped off from the trypsinized skin into the plate. 25 mLs of E-media was added to the cell suspension and was then sequentially strained through 100μm and 40μm filters. The cell pellet was washed twice with DPBS before proceeding with staining with cell surface markers. The cells were stained with 1:200 PerCP-Cy5.5-Sca1 (Thermo Fisher Scientific, 45-5981-82), 1:200 FITC-α6-integrin (eBioscience, 11-0493-81), 1:100 APC-EpCAM (Biolegend, 324207) and 1:20 Alexa700-CD34 (eBioscience, 56-0341-82) in staining buffer (HBSS + 2% Fetal Bovine Serum) for 30 minutes on ice and then washed twice with DPBS before cell sorting. HFSCs were sorted by gating on EpCAM (+), Sca1(-), α6-integrin(high) and CD34(+).

first: Pooja

last: Flora

first: Meng Yen

last: Li

first: Yudong

last: Zhou

first: Maria

last: Mercédes

first: Xiang Yu

first: Phillip

last: Galbo Jr

first: Elena

last: Ezhkova


title: HIV-Induced Sialoglycans on Infected Cells Promote Immune Evasion from Myeloid Cell-Mediated Killing

city: Philadelphia

first: Priyankara

middle: J

last: Wickramasinghe

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute

summary: Sialic acid-containing glycans (sialoglycans) on pathological cells interact with Siglecs, glyco-immune checkpoint receptors expressed on myeloid cells such as monocytes and neutrophils. This interaction suppresses the cytotoxic functions of these immune cells. We show that HIV infection reprograms the glycosylation machinery of infected cells to increase the expression of specific sialoglycan ligands for Siglecs-3, -7, and -9. These ligands engage Siglecs on myeloid cells, impairing their ability to target HIV-infected cells. Selective disruption of these interactions using 10-1074-Sia, an HIV-specific antibody conjugated to sialidase—an enzyme that removes sialic acids—significantly enhances monocyte- and neutrophil-mediated killing of HIV-infected cells in autologous assays. Treatment with 10-1074-Sia in humanized mice infected with HIV reduces viral load and decreases inflammation. These findings reveal a novel immune evasion mechanism exploited by HIV to evade myeloid cell immune surveillance and highlight the potential of targeting sialoglycan-Siglec interactions to improve immune clearance of HIV-infected cells.

overall_design: RNAseq profilling of non-activated primary CD4 T cells infected with HIV-DH12 virus

first: Shalini

last: Singh

first: Mohamed

last: Abdel-Mohsen


title: H2AK119ub dynamics controls hair follicle stem cell quiescence [CUT&Seq]

overall_design: C57BL6/J (wild type) mice were acquired from either The Jackson Laboratory or Charles River Laboratories and aged till P56., The back skin was collected and the adipose layer from the dorsal side was scraped off using a surgical scalpel. The scraped skin sample was washed with PBS prior to incubation with 0.25% Trypsin/EDTA at 37°C for 1 hour on a rotation plate set at 60 r.p.m. After incubation, the epidermal cells, including the HFSCs, was scraped off from the trypsinized skin into the plate. 25 mLs of E-media was added to the cell suspension and was then sequentially strained through 100μm and 40μm filters. The cell pellet was washed twice with DPBS before proceeding with staining with cell surface markers. The cells were stained with 1:200 PerCP-Cy5.5-Sca1 (Thermo Fisher Scientific, 45-5981-82), 1:200 FITC-α6-integrin (eBioscience, 11-0493-81), 1:100 APC-EpCAM (Biolegend, 324207) and 1:20 Alexa700-CD34 (eBioscience, 56-0341-82) in staining buffer (HBSS + 2% Fetal Bovine Serum) for 30 minutes on ice and then washed twice with DPBS before cell sorting. HFSCs were sorted by gating on EpCAM (+), Sca1(-), α6-integrin(high) and CD34(+).


title: Single-cell RNA sequencing on cord blood-derived HPSCs cultured with or without liproxstatin-1 supplementation in chemically-defined conditions

city: Boston

first: Vijay

middle: G

last: Sankaran

state: MA

address: 1 Blackfan Circle

institute: Boston Children's Hospital / Broad Institute

summary: The aim for this dataset is to demonstrate that blockade of ferroptosis with the small molecule liproxstatin-1 can significantly augment the expansion of human HSCs derived from either cord blood or adult sources using diverse methods, including widely applied serum-free cultures and recently reported chemically-defined conditions. We find that human HSCs treated with liproxstatin-1 display increased ribosome biogenesis and cholesterol biosynthesis, accompanied by elevated 7-dehydrocholesterol levels - a potent endogenous inhibitor of ferroptosis. This study demonstrates the effectiveness of mitigating ferroptosis to enhance human HSC expansion in the setting of therapeutic genome modifications, paving the path to clinical applications.

overall_design: Single-cell transcriptomic profiling by high throughput sequencing

first: Lucrezia

last: della Volpe


title: Profiling of histone modifications in prostate cancer cells in response to  androgen deprivation and the onset of the neuroendocrine phenotype

city: Paris

first: Antonin

last: Morillon

state: Paris

address: 26 rue d'Ulm

institute: Institut Curie

summary: Androgen deprivation therapy (ADT), also called chemical castration, is one of the prevailing treatments of local and metastatic prostate cancer enabling inhibition of the Androgen Receptor (AR) pathway and tumor regression. However, the response to therapy varies among patients with some showing symptomatic relief and prolonged survival, while others relapsing in less than 5 years or failing to respond (reviewed in Harris et al, 2009). These clinical trajectories are in part due to emergence of mechanisms allowing maintenance of the AR activity, often observed in CRPC tumors, and progressive cell reprograming enabling androgen-independent growth. In some case CRPC tumors progressively evolve in highly agressive AR-negative neuroendocrine prostate cancer (NEPC). In this project we aimed to profile histone modifications associated with actively transcribed and repressed regions in the dynamic in vitro system recapitulating the CRPC and NEPC onset. Hormone-sensitive LNCaP cells were grown in  DHT-supplemented medium. In this condition cells exhibit an epithelial-like morphology and actively proliferate under the control of AR.  Androgen deprivation results in growth arrest and progressive emergence of the neuroendocrine phenotype characterized by AR signalling inactivation, expression of neuroendocrine genes and change in morphology  (Terry et al, 2013).

overall_design: The experiment was performed in duplicates following the protocol from Henikoff lab (Skene et al, 2018) with modifications described in (Jarroux et al, 2021) in prostate cancer adenocarcinoma cells LNCaP growing in the presence of androgen (DHT) or in hormone-depleted media for 1 month (NE1m). Antibodies used : against H3K27ac (Millipore, MABE647, 1:50), H3K27me3 (Cell signaling, C36B11, 1:50), H3K4me3 (Millipore, 04745, 1:50); IgG (Thermo Fisher, 31235). DNA fragments were purified with NEB Monarch PCR & DNA purification kit following the protocol enrichment for short DNA fragments and was quantified by Qubit HS DNA kit. CUT & RUN libraries were prepared using the TruSeq ChIP-Sample Prep Kit-Set A and PCR Box (Illumina) from 5 ng of DNA with 10 PCR cycles for amplification following the manufacture’s procedures. Concentration and quality were assessed by Qubit with dsDNA HS Assay Kit (Thermo Fisher Scientific) and High Sensitivity DNA Analysis Chip Kit (Agilent Technologies). Libraries were sequenced on a NovaSeq – S1 device in a pair-end 100 bp read length mode.

first: Rocco

last: Cipolla

first: Marina

last: Pinskaya

first: Marc

last: Gabriel


title: H2AK119ub dynamics controls hair follicle stem cell quiescence [RNA-Seq 1]

first: Deyou

overall_design: Control and PRC1 i2KO mice were induced starting at P50. 10 days after completion of induction treatment, the back skin was collected and the adipose layer from the dorsal side was scraped off using a surgical scalpel. The scraped skin sample was washed with PBS prior to incubation with 0.25% Trypsin/EDTA at 37°C for 1 hour on a rotation plate set at 60 r.p.m. After incubation, the epidermal cells, including the HFSCs, was scraped off from the trypsinized skin into the plate. 25 mLs of E-media was added to the cell suspension and was then sequentially strained through 100μm and 40μm filters. The cell pellet was washed twice with DPBS before proceeding with staining with cell surface markers. The cells were stained with 1:200 PerCP-Cy5.5-Sca1 (Thermo Fisher Scientific, 45-5981-82), 1:200 FITC-α6-integrin (eBioscience, 11-0493-81), 1:100 APC-EpCAM (Biolegend, 324207) and 1:20 Alexa700-CD34 (eBioscience, 56-0341-82) in staining buffer (HBSS + 2% Fetal Bovine Serum) for 30 minutes on ice and then washed twice with DPBS before cell sorting. HFSCs were sorted by gating on EpCAM (+), Sca1(-), α6-integrin(high) and CD34(+).


title: De novo mutations in the MiDAC HDAC complex cause a neurodevelopmental 2 syndrome resulting from increased deacetylase activity [RNA-seq]

first: John

last: Schwabe

summary: MIDEAS is a scaffold protein that, together with DNTTIP1, mediates assembly of the MiDAC histone deacetylase complex. Mice lacking MiDAC die before birth suggesting a key developmental function. Here, we report two unrelated individuals, with a multisystem disorder characterized by delayed speech development, joint contractures, dysmorphic features and dysmotility of the gut. Both individuals have the same de novo heterozygous missense variant in MIDEAS (p.Tyr654Ser). A cryoEM structure of the MiDAC complex reveals that this amino acid is located in a conserved auto-inhibitory loop that covers the active site of the deacetylase enzyme. We suggest that the variant results in loop displacement leading to elevated deacetylase activity. In support, we observe reciprocal gene expression changes in patient fibroblasts compared with a cell line following rapid MiDAC degradation. Our results establish MIDEAS as a dominant monogenic disease gene and that hyperactivity of the MiDAC complex results in a characteristic multisystem disorder.

sample_organism:
Homo sapiens
Mus musculus

overall_design:
RNA seq profiling of HCT 116 cells CRISPR-engineered to express endogenous DNTTIP1 with C-terminal FKBPF36V/FLAG to allow rapid degradation using dTAGv1 over 48h. Mouse embryonic fibroblast (MEF) wildtype and MIDEAS knockout RNA seq samples were reanalysed (samples: GSM4295062, GSM4295063, GSM4295064, GSM4295065, GSM4295066, GSM4295067, GSM4295068, GSM4295069, GSM4295070, GSM4295071) from series GSE144748. Samples were aligned using HISAT2 (v12.3.0) to GRCm39 release 112, sorted and indexed using samtools (v1.17) and count files made using LiBiNorm (v2.5; options -z -r pos -I gene_name -s reverse) with GRCm39 release 112 gene transfer format file. Differential gene expression analysis was performed using DESEQ2 (v1.44.0). Normalised counts are supplied in "reanalysis_samples.xlsx".

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

first: Robert

last: Turnbull

first: Kristupas

last: Sirvydis


title: Divergent programming and nonreciprocal plasticity of tissue resident exhaustedand memory CD8+T cell lineages

first: E

middle: JOHN

last: WHERRY

address: 421 Curie Blvd,354 BRB II/III

department: Systems Pharmacology       and Translational Therapeutics,Institute for Immunology

institute: University of Pennsylvania

summary: persistent antigen stimulation during chronicinfectioninduces apopulationof CD39hiCD69+CD103+/–Hobit+/–tissue-resident TEXcells in non-lymphoid tissuesthataredevelopmentally distinctfrombona fideTRMcells generated afteracutelyresolvedinfection.Tissue-resident TEXcells had reduced function and limited developmentalplasticity compared to TRMcells. Moreover, tissue-resident TEXandbona fideTRMcells weregoverned bydistinct transcriptional and epigenetic programs indicating divergent developmentalpaths.Indeed, whereasestablishedTRMcellscould giverise to secondaryantigen-independentmemory CD8+T cellsafter acute infectionas well as multiple subsets of TEXcells including TCF1+progenitorsduring chronic restimulation,establishedTEXcells were unable to form TRMcells andrequired ongoing antigen stimulation to efficientlyacquire residency features.Moreover,bona fideTRMdevelopedindependently of Tox, whereasTox controlled induction of residency-associatedmolecules in developing CD69+CD103+/–tissue-resident TEXcells and was essential for theiraccumulation in nonlymphoid tissues

overall_design: live CD8β+CD44hiCD45.1+Vα2+ P14 cells from each sample (pooled organs from 20 Arm-infected mice or 20 C13 infected mice) d30 post infection were sorted and stained with TotalSeq-A Oligos and custom pool of antibody derived tags.Also TEA sequencing on these samples for GEX+ATAC

first: Simone

last: Park

last: Wherry


title: Interpretable Inflammation Landscape of Circulating Immune cells (ILCIC_RespiratoryDiseases)

city: Barcelona

first: Laura

last: Jiménez-Gracia

address: c/ Baldiri Reixac, 4  Barcelona Science Park - Tower I

country: Spain

institute: Centre Nacional d'Anàlisi Genòmic (CNAG)

summary: Inflammation is a biological phenomenon beneficial for homeostasis, but unfavorable if dysregulated. Although major progress has been made in characterizing inflammation in specific diseases, a global, holistic understanding is still elusive. This is particularly intriguing, considering its function for human health and the potential for modern medicine if fully deciphered. Here, we leveraged advances in single-cell transcriptomics to delineate inflammatory processes of circulating immune cells during infection, immune-mediated inflammatory diseases and cancer. Our single-cell atlas of >6.5 million peripheral blood mononuclear cells from 1047 patients (56% female, 43% male) and 19 diseases allowed us to learn a comprehensive model of inflammation in circulating immune cells. The atlas expanded our current knowledge of the biology of inflammation of immune-mediated diseases, acute and chronic inflammatory diseases, infection and solid tumors, and laid the foundation to develop a disease classification framework using unsupervised as well as explainable machine learning. Beyond a disease-centered analysis, we charted altered activity of inflammatory molecules in peripheral blood cells, depicting discriminative inflammation-related genes to further understand mechanisms of inflammation. We present a rich resource for the community, and  laid the groundwork for learning a classifier for inflammatory diseases, presenting cells in circulation as a potential tool for disease classification.

overall_design: scRNA-seq from 32 human PBMC samples, including 12 Asthma, 12 COPD and 8 HC.

first: Davide

last: Maspero

first: Sergio

last: Aguilar-Fernández

first: Francesco

last: Craighero

first: Juan

last: Nieto

first: Holger

last: Heyn


title: Interpretable Inflammation Landscape of Circulating Immune cells (ILCIC_BRCA)

overall_design: scRNA-seq from 6 human PBMC samples of BRCA patients, at the pre-treatment stage.


title: RNA-mediated mechanisms of regulation of the Androgen Receptor activity in response to androgen deprivation

summary: Androgen deprivation therapy (ADT), also called chemical castration, is one of the prevailing treatments of local and metastatic prostate cancer enabling inhibition of the Androgen Receptor (AR) pathway and tumor regression. However, the response to therapy varies among patients with some showing symptomatic relief and prolonged survival, while others relapsing in less than 5 years or failing to respond (reviewed in Harris et al, 2009). These clinical trajectories are in part due to emergence of mechanisms allowing maintenance of the AR activity, often observed in CRPC tumors, and progressive cell reprograming enabling androgen-independent growth. Activation and repression of gene exprssion by AR require interactions with numerous proteins, and very recently, with RNAs. Notably, Schmidt et al. identified a specific pyrimidine-rich motif within SRA1 and another SRA-like long noncoding RNA, named SLNCR, both binding directly the AR N-terminal domain in melanoma cells  (Schmidt et al, 2016); (Schmidt et al, 2020). In addition to SLNCR, other RNAs, belonging to the long noncoding (lnc)RNA gene family, contain the aforementioned AR-interacting consensus motif, including a paragon lncRNA - HOTAIR that protects AR from proteolysis in the context of castration-resistant prostate cancer (Zhang et al, 2015) but also tethers AR to the GLI2 promoter to activate its transcription in renal cell carcinoma (Bai et al, 2021). The AR NTD is not the only domain reported to bind RNA. The growth arrest specific-5 gene GAS5, another lncRNA with well-known tumor suppressor functions, interacts with the LBD of AR inhibiting its transactivation activity and proliferation of prostate cancer cells (Lv et al, 2021). In this project we aimed to identify RNAs associated with AR in the androgen-dependent and independent  cell using the technique of RNA immunoprecipitation of the transcription factor (RIP).

overall_design: prostate cancer adenocarcinoma cells LNCaP growing in the presence and absence of androgen were treated with low doses of formaldehyde and 10^7 cells per condition were collected for lysis and immunoprecipitation of AR (G Hendrickson et al, 2016) .Associated RNAs were recovered and analyzed by total stranded RNA-sequencing.  THe experiment was performed in duplicates and AR immunoprecipitation was compared to IgG.


title: Dissecting Gene Regulatory Networks Governing Human Cortical Cell Fate

city: San Francisco

first: Alex

last: Pollen

institute: UCSF

summary: Human cortical neurogenesis involves conserved and specialized developmental processes during a restricted window of prenatal development. Radial glia (RG) neural stem cells shape cortical cell diversity by giving rise to excitatory neurons, oligodendrocytes, and astrocytes, as well as olfactory bulb interneurons (INs) and a recently characterized population of cortical INs. Complex genetic programs orchestrated by transcription factor (TF) circuits govern the balance between self-renewal and differentiation, and between different cell fates. Despite progress in measuring gene regulatory network activity during human cortical development, functional studies are required to evaluate the roles of TFs and effector genes in human RG lineage progression. Here we establish a human primary culture system that allows sensitive discrimination of cell fate dynamics and apply single cell clustered regularly interspaced short palindromic repeats interference (CRISPRi) screening to examine the transcriptional and cell fate consequences of 44 TFs active during cortical neurogenesis. We identified multiple TFs, with novel roles in cortical neurogenesis, including ZNF219, previously uncharacterized, that represses neural differentiation and NR2E1 and ARX that have opposing roles in regulating RG lineage plasticity and progression across developmental stages. We also uncovered convergent effector genes downstream of multiple TFs enriched in neurodevelopmental and neuropsychiatric disorders and observed conserved mechanisms of RG lineage plasticity across primates. We further uncovered a postmitotic role for ARX in safeguarding IN subtype specification through repressing LMO1. Our study provides a framework for dissecting regulatory networks driving cell fate consequences during human neurogenesis.

sample_organism:
Macaca mulatta
Homo sapiens

platform_organism:
Macaca mulatta
Homo sapiens

overall_design: (1) 2D Libraries: Perturb-seq targeting 44 TFs on differentiation D0 and D7: Primary human culture derived from cortical RG were transduced with CRISPRi library targeting 44 TFs with capture sequence on each guide expanded for 7 days (D0) and differentiated for 7 days (D7). Cells were sorted based on GFP expression and captured using 10X Chromium v3.1 HT kit for single cell RNA sequencing. (2) HM2D libraries: Perturb-seq targeting 5 TFs with lineage tracing on differentiation D7: Primary human or macaque culture derived from cortical RG were transduced with CRISPRi library targeting 5 TFs with capture sequence on each guide and STICR static barcode library, expanded for 7 days and differentiated for 7 days. Cells were sorted based on GFP and mCherry co-expression and captured using 10X Chromium v3.1 HT kit. (3) Slice culture libraries: Perturb-seq targeting 5 TFs with lineage tracing on differentiation D12-14: The germinal zone of the developing human brain was locally transduced with CRISPRi library targeting 5 TFs and STICR static barcode library, cultured for 12-14 days. Cells were sorted based on GFP and mCherry co-expression and captured using 10X Chromium v3.1 HT kit. (4) Double knockdown libraries: Perturb-seq targeting ARX and LMO1 on differentiation D7: Primary human culture derived from cortical RG were transduced with CRISPRi library targeting ARX and LMO1 expanded for 7 days (D0) and differentiated for 7 days (D7). Cells were sorted based on GFP and mCherry co-expression and captured using Illumina Single Cell CRISPR Library kit.

first: Jingwen

last: Ding


title: Profiling of transcriptomic changes in androgen-dependent prostate cancer cells  in response to anrogen deprivation.

first: Antonin

last: Morillon

summary: Castration-resistant prostate cancer (CRPC) emerges in response to androgen deprivation therapies and exhibits high cellular plasticity eventually progressing into neuroendocrine prostate cancer (NEPC). In this study, we took advantage of the dynamic prostate cancer cell system recapitulating the CRPC onset to identify lncRNAs induced early in response to androgen deprivation. In this system, first, adenocarcinoma LNCaP cells are grown in the presence of dihydrotestosterone (DHT) mimicking primary epithelial androgen-dependent luminal tumors  (NE_0j). Then, they are subjected to androgen deprivation which induces immediate growth arrest, progressive change in cell morphology and properties towards the neuroendocrine-like phenotype (NE-like cells, NE_15j, 1m, 3m) and finally lead to acquisition of androgen independence (NE_6m).  Total RNA-sequencing followed by differential expression analysis of scallop-assembled transcripts and multiple filtering isolated  gencode annotated protein-coding and long noncoding RNA genes but also  15 novel lncRNAs highly upregulated upon androgen deprivation.

overall_design: Cells were cultured and treated in triplicates for each condition to extract RNA. RNA samples were treated with Dnase, quality controlled (RIN above 9)  and used for the library preparation). To perform differential expression analysis a condition of growth in the presence of androgen NE_0j) was considered as a reference to define up- and down-regulated genes in cells grown in the absence of androgen.

first: Rocco

last: Cipolla

first: Virjinie

last: Firlej


title: Revealing heterogeneity and damage response in the adult human utricle [scRNA-Seq]

city: Toronto

first: Alain

last: Dabdoub

state: Ontario

address: 2075 Bayview Ave., A3 42

country: Canada

institute: Sunnybrook Health Sciences Centre

summary: The human utricle is a vestibular organ composed of sensory and non-sensory cells responsible for maintaining balance. Balance sense gradually deteriorates with age. With the aging population expected to double to 2 billion by 2050, vestibular disorders will pose a significant challenge as no pharmaceutical or biological treatments are available, a current unmet medical need. Here we show, for the first time, the cellular and transcriptional profile of the adult human utricle and its response to damage by performing bulk and single-cell RNA-sequencing from patient-derived utricles. We discovered six transcriptionally distinct cell types, including a unique population, demonstrating the heterogeneity and complexity of the adult human utricle. In addition, using an aminoglycoside damage paradigm, we determined the early transcriptional changes of the utricle after damage. Our findings demonstrate that this organ has the capacity to respond to ototoxic damage within 24 h and potentially initiate a regenerative response via an early-responding supporting cell population. This study represents a major step forward in inner ear regenerative medicine. Our results will serve as a foundation for preclinical studies, paving the way to therapeutic strategies for balance recovery.

overall_design: We cultured a total of six organotypic utricles, two with 24h vehicle control, two treated with gentamicin for 24h to induce hair cell damage, and two with no treatment (time 0). Then, we isolated the sensory epithelium and performed scRNA-seq.

first: Luca

last: Emilia

first: Ibeh

last: Neke

first: Yamamoto

last: Ryosuke

first: Bennett

last: Dallas

first: Lin

last: Vincent

first: Chen

last: Joseph

first: Lovett

last: Michael

first: Dabdoub

last: Alain


title: Interpretable Inflammation Landscape of Circulating Immune cells (ILCIC_cirrhosis)

first: Laura

last: Jiménez-Gracia

overall_design: scRNA-seq from 8 human PBMC samples of patients with cirrhosis.

first: Davide

last: Maspero

first: Sergio

last: Aguilar-Fernández

first: Francesco

last: Craighero


title: Interpretable Inflammation Landscape of Circulating Immune cells (ILCIC_HealthyExercise)

overall_design: scRNA-seq from 8 human PBMC samples from healthy donors, undergoing different physical activity (4 active and 4 inactive).

first: Juan

last: Nieto

first: Holger

last: Heyn


title: Revealing heterogeneity and damage response in the adult human utricle

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

overall_design: Refer to individual Series


title: Anti-uPAR CAR T cells reverse and prevent aging-associated defects in intestinal regeneration and fitness

first: Vyom

last: Shah

summary: Intestinal stem cells (ISCs) drive the rapid regeneration of the gut epithelium. However, during aging their regenerative capacity is reduced, possibly through senescence and chronic inflammation, albeit little is known about how aging-associated dysfunction arises in the intestine. We previously identified the urokinase plasminogen activator receptor (uPAR) as a senescence-associated protein in other tissues and developed CAR T cells able to efficiently target it. Harnessing them, here, we identify the accumulation of uPAR-positive cells in the aging gut and uncover their detrimental impact on ISC function in aging. Thus, both therapeutic and prophylactic treatment with anti-uPAR CAR T cells improved barrier function, regenerative capacity, inflammation, and mucosal immune function in aged mice. Overall, these findings reveal the deleterious role of uPAR-positive cells on intestinal aging in vivo and provide proof of concept for the potential of targeted immune-based cell therapies to enhance tissue regeneration in aging organisms.

overall_design: For "control" samples: Young (3 months) and old (20 months) Bl/6J mice had their intestinal crypts isolated and processed for scRNAseq. For "uPAR and UT" experiments: Young (3 months) and old (18 months) mice were treated by tail vein injection with 0.5x10^6 untransduced T cells (UT) or uPAR CAR T cells (m.uPAR-m.28z). The intestinal crypts from these mice were isolated and disociated at 6 weeks after infusion. At this time the young mice were 4.5months old and the old mice were 19.5months old.

first: Corina

last: Amor Vegas


title: Interpretable Inflammation Landscape of Circulating Immune cells

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

Inflammation is a biological phenomenon beneficial for homeostasis, but unfavorable if dysregulated. Although major progress has been made in characterizing inflammation in specific diseases, a global, holistic understanding is still elusive. This is particularly intriguing, considering its function for human health and the potential for modern medicine if fully deciphered. Here, we leveraged advances in single-cell transcriptomics to delineate inflammatory processes of circulating immune cells during infection, immune-mediated inflammatory diseases and cancer. Our single-cell atlas of >6.5 million peripheral blood mononuclear cells from 1047 patients (56% female, 43% male) and 19 diseases allowed us to learn a comprehensive model of inflammation in circulating immune cells. The atlas expanded our current knowledge of the biology of inflammation of immune-mediated diseases, acute and chronic inflammatory diseases, infection and solid tumors, and laid the foundation to develop a disease classification framework using unsupervised as well as explainable machine learning. Beyond a disease-centered analysis, we charted altered activity of inflammatory molecules in peripheral blood cells, depicting discriminative inflammation-related genes to further understand mechanisms of inflammation. We present a rich resource for the community, and  laid the groundwork for learning a classifier for inflammatory diseases, presenting cells in circulation as a potential tool for disease classification.


title: Targeting FABP4 Improves Healthy Aging by Rejuvenating Liver Metabolism

city: Shenzhen

first: Zhihua

last: Wang

address: No. 12, Langshan Road, Xili Street, Nanshan District, Shenzhen

department: Shenzhen Key Laboratory of Cardiovascular Disease

country: China

institute: Fuwai Hospital Chinese Academy of Medical Sciences, Shenzhen

summary:
Introduction: Aging is characterized by progressive metabolic dyshomeostasis that increases morbidity and mortality. Solutions for optimizing healthy aging are challenged by lacking appropriate biomarkers. Moreover, druggable targets to rejuvenate the aging-associated metabolic phenotypes remain unavailable.

Methods: Proteomics analysis was performed in a cohort of young and elderly adults. Circulating levels of insulin-like growth factor 1 (IGF-1) and fatty acid binding protein 4 (FABP4) were evaluated by ELISA. FABP4 was silenced in elderly mice by adeno-associated virus. Metabolic activities were measured by metabolic cages. Cognitive function was evaluated by Morris water maze. Glucose and lipid metabolism were evaluated by biochemistry assays with blood samples. RNA-seq in mouse liver was performed for transcriptome analysis.

Results: Among 9 aging-sensitive proteins shared by both male and female, FABP4 was identified as a reliable aging biomarker in both human and mouse. Silencing FABP4 in elderly mice significantly rejuvenated the aging-associated decline in metabolic activities. FABP4 knockdown reversed the aging-associated metabolic disorders by promoting degradation of cholesterol and fatty acids, while suppressing gluconeogenesis. Transcriptome analysis revealed a restoration of the pro-aging gene reprogramming towards inflammation and metabolic disorders in the liver after FABP4 knockdown. FABP4 overexpression promoted human LO2 cell senescence. Moreover, administration of an FABP4 inhibitor BMS309403 delivered metabolic benefits in elderly mice.

Conclusion: Our findings demonstrate FABP4 as a reliable aging biomarker as well as a practicable target to improve healthy aging in the elderly.

Keywords: Aging; Biomarkers; FABP4; Metabolic disorders; Rejuvenation; Senescence.

overall_design: All animals were housed in a constant environment (room temperature, 24 ± 3 °C; room humidity,55 ± 5%) with a standard 12-h light/12-h dark cycle. Mice were fed with normal chow and with free access to water. A total of thirty 14-month-old male WT mice were randomly divided into two groups as the following: (1) mice injected with AAV9-shRNA-Fabp4 by tail vein and sWAT in situ injection with a dose of 4×10^11 viral genomes, n=15; (2) mice injected with AAV9-shRNA-Neg with the same way and same amount, n=15. The above animals were kept until 20-month-old after AAV injection for sundry experiments. A group of 8-week-old WT mice were regarded as young control group (n=15). The mice were anaesthetized with inhalational anaesthetic and euthanized by cervical dislocation. Liver were immediately dissected and stored at -80°C. Three independent biological replicate samples from Young or Old group were used for RNA extraction. RNA-seq was completed by Novogene.

first: Jian

last: Lv

first: Yimeng

last: Hu

first: Lili

last: Li

first: Jingjing

first: Yuan

last: He

first: Ningning

last: Guo

first: Yu

last: Fang

last: Tong

first: Lixu

last: Tang


first: Alain

last: Dabdoub

overall_design: We cultured six whole organotypic utricles for 24 h, three with vehicle control and three treated with gentamicin to induce hair cell damage. Then, we isolated the sensory epithelium and performed bulk RNA-seq.

first: Luca

last: Emilia

first: Ibeh

last: Neke

first: Yamamoto

last: Ryosuke

first: Bennett

last: Dallas

first: Lin

last: Vincent

first: Chen

last: Joseph

first: Lovett

last: Michael

first: Dabdoub

last: Alain


title: Targeted CRISPR-Cas9 screening identifies core transcription factors controlling murine haemato-endothelial fate commitment [RNA-seq]

status: Public on Oct 12 2025

city: Zürich

first: Nina

last: Schmolka

address: Winterthurerstrasse 190

country: Switzerland

institute: University of Zurich

summary: During development, blood generation begins in the yolk sac with the differentiation of haemato-endothelial mesoderm generating haematopoietic progenitors. This study aimed to identify the crucial molecular regulators of haemato-endothelial mesoderm formation and to extend our knowledge of the process in an unbiased way. We employed a murine embryonic stem cell model that recapitulates embryonic blood development, and performed targeted CRISPR-Cas9 knock out screens focusing on transcription factors and chromatin regulators, which highlighted the transcription factors Smad1, Ldb1, Six4 and Zbtb7b. Embryonic stem cells lacking these regulators gave rise to mesodermal subsets with a defined lineage differentiation bias, while transcriptome analysis of these cells uncovered the precise impact of each factor on gene expression in the developing mesoderm. Our study reveals novel molecular pathways governing mesodermal development crucial to allow endothelial and haematopoietic lineage specification and paves the way for future advances in haematopoietic stem cell applications.

overall_design: Viable single cells were FACS sorted into 384-well cell capture plates (Single Cell Discoveries, Netherlands), in which each well contained 50 nl of barcoded primers and 10 µl of mineral oil (Sigma M8410). After sorting, plates containing cells were centrifuged at 1500x g for 1 minute then placed on dry ice before storage at -80° C. Single-cell RNA sequencing was performed by Single Cell Discoveries according to an adapted version of the SORT-seq protocol57

first: Michael

last: Teske


title: Targeted CRISPR-Cas9 screening identifies core transcription factors controlling murine haemato-endothelial fate commitment [SORT-seq]

overall_design: Viable single cells were FACS sorted into 384-well cell capture plates (Single Cell Discoveries, Netherlands), in which each well contained 50 nl of barcoded primers and 10 µl of mineral oil (Sigma M8410). After sorting, plates containing cells were centrifuged at 1500x g for 1 minute then placed on dry ice before storage at -80° C. Single-cell RNA sequencing was performed by Single Cell Discoveries according to an adapted version of the SORT-seq protocol.


title: Dynamic transcriptional and epigenetic changes define postnatal tendon growth

first: Jenna

last: Galloway

address: 185 Cambridge St.

department: Orthopaedics

institute: Massachusetts General Hospital

summary: Tendons are dynamic structures that efficiently transmit forces and enable movement. From birth, tendons undergo dramatic changes from a principally cellular tissue to a hypocellular one characterized by a dense and highly ordered extracellular matrix. During this time, tendon cells change morphology from rounded to stellate in appearance and their proliferative rates decline. There is also significant expansion and maturation of the extracellular matrix (ECM) as tendons grow in length and diameter and alter their biomechanical properties to sustain increased physical activities. Surprisingly, for such an important stage of tendon maturation, we understand very little about the transcriptional and epigenetic regulators that direct these processes. Here, we present a roadmap of genes that are differentially regulated during the early neonatal and postnatal time period. We find differentially expressed genes fall into specific transcriptional modules, representing expression increases, decreases, or gene sets undergoing dynamic changes over postnatal time. By pairing our transcriptomic data with epigenetic data, we were able to perform an integrative analysis of the datasets and further define modules with highly correlated changes in gene expression and chromatin accessibility.  From this analysis, several new pathways emerge. Among them, we focus on Yap1, a transcriptional co-activator of the Hippo signaling pathway. We observe accessible regions near to differentially expressed genes, containing motifs for TEAD, the transcription factor that binds Yap to regulate transcription. Conditional loss of Yap1 at postnatal stages results in altered expression of Col1a1 and disrupted matrix organization and density, suggesting that Yap is important for refining tendon ECM maturation. Together, our analyses identify a regulator of matrix maturation and provides a rich dataset with which to interrogate transcriptional networks and pathways during this poorly understood time in tendon growth.

overall_design: Tendon samples from weekly timepoints during postnatal development were subjected to both RNAseq and ATACseq to profile transcriptional and chromatin level changes during postnatal tendon growth and development.

first: Heather

last: Dingwall

first: Terence

last: Capellini


title: Distinct Layer 6b transcriptomic subtypes parcellate the cortical mantle

status: Public on Oct 11 2025

city: Vancouver

first: Mark

middle: Steven

last: Cembrowski

state: BC

address: 2350 Health Sciences Mall

department: Cellular and Physiological Sciences

institute: University of British Columbia

summary: Here, we examined the transcriptomic heterogeneity of mouse Layer 6b (L6b) neurons using single-cell spatial transcriptomics.

overall_design: Examination of layer 6b neuron transcriptomes.

first: Margarita

last: Kapustina

first: Brianna

last: Bristow

middle: S


title: 3-D biomechanics and epigenomics reveal atypical fibroblast responses in cardiometabolic disease

status: Public on Oct 10 2025

city: Little Rock

first: Se-Ran

last: Jun

address: 4301 W Markham St

institute: University of Arkansas for Medical Sciences

summary: Cell behavior is influenced by substrate stiffness and cell-cell and cell-environment interactions. The limitations of two-dimensional (2-D) culture, such as its inability to fully capture the complexity of cell interactions and tissue structure, highlight the necessity of three-dimensional (3-D) cell culture. This explicitly applies to "disease modeling in a dish" platforms for translational studies. 3-D bioprinting demonstrates significant potential in recapitulating the intricate physiological environments of human tissues in both healthy and pathological states. With the alarming rise in obesity, addressing systemic pathophysiological dysfunction beyond adipose tissue itself, such as the heart, is inevitable. To capture cellular and tissue-level responses to overnutrition, we used state-of-the-art 3-D bioprinting technology to understand the acute response of 3-D matrix-embedded human cardiac fibroblasts to a "high-fat diet" mimic. Chromatin accessibility profiling revealed that excess fatty acid (FA) exposure in 2-D induces a noncanonical extracellular matrix gene program that is minimally expressed in healthy adult myocardium. In contrast, 3-D cultures exhibited reduced fibroblast proliferation and blunted transcriptional responses to the impact of biomechanical cues under metabolic stress, reflecting a more quiescent and physiologically relevant phenotype. Furthermore, we incorporated human induced pluripotent stem cell-derived cardiac fibroblasts (iPSC-CFs), which mirrored key transcriptional changes, including sex-dependent gene regulation. Notably, male iPSC-CFs showed stronger fibrotic gene induction than females, reinforcing the need to account for biological sex in disease modeling. Together, our results highlight the limitations of 2-D systems and demonstrate that 3-D-bioprinted platforms provide a scalable, physiologically relevant tool for investigating cardiometabolic diseases and therapeutic targets.NEW & NOTEWORTHY Biomechanical cues in conventional 2-D systems can artificially prime fibroblasts toward activation, whereas 3-D-bioprinted hydrogels better preserve physiological phenotypes. The use of Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) to profile chromatin accessibility uncovered noncanonical epigenomic remodeling in response to FA overload, highlighting novel fibrotic gene programs not captured by traditional assays. The identification of sex-specific transcriptional responses in iPSC-derived fibroblasts highlights the importance of incorporating biological variables for personalized cardiometabolic disease modeling and accelerating therapeutic development.

overall_design: ATAC-seq on nuclei isolated from 2D cultured human cardiac fibroblasts (HCFs) treated with fatty acid enriched or vehicle medium

first: Somaya

middle: Y

last: Ibrahim

first: Ryan

last: Holdiness

first: Ashira

last: Thadisena

first: Kristen

last: Boyle

first: Rushita

last: Bagchi


title: Profiling of Androgen Receptor in prostate cancer cells in response to  androgen deprivation and the onset of the neuroendocrine phenotype

first: Antonin

last: Morillon

summary: Androgen deprivation therapy (ADT), also called chemical castration, is one of the prevailing treatments of local and metastatic prostate cancer enabling inhibition of the Androgen Receptor (AR) pathway and tumor regression. However, the response to therapy varies among patients with some showing symptomatic relief and prolonged survival, while others relapsing in less than 5 years or failing to respond (reviewed in Harris et al, 2009). These clinical trajectories are in part due to emergence of mechanisms allowing maintenance of the AR activity, often observed in CRPC tumors, and progressive cell reprograming enabling androgen-independent growth. In some case CRPC tumors progressively evolve in highly agressive AR-negative neuroendocrine prostate cancer (NEPC). In this project we aimed to profile Andrigen Receptor in the dynamic in vitro system recapitulating the CRPC and NEPC onset. Hormone-sensitive LNCaP cells were grown in  DHT-supplemented medium. In this condition cells exhibit an epithelial-like morphology and actively proliferate under the control of AR.  Androgen deprivation results in growth arrest and progressive emergence of the neuroendocrine phenotype characterized by AR signalling inactivation, expression of neuroendocrine genes and change in morphology  (Terry et al, 2013).

overall_design: The experiment was performed in duplicates following the protocol from Henikoff lab (Skene et al, 2018) with modifications described in (Jarroux et al, 2021) in prostate cancer adenocarcinoma cells LNCaP growing in the presence of androgen (DHT) or in hormone-depleted media for 1 month (NE1m). Antibodies: against AR (Abcam, ab108341) and IgG (Thermo Fisher, 31235). DNA fragments were purified with NEB Monarch PCR & DNA purification kit following the protocol enrichment for short DNA fragments and was quantified by Qubit HS DNA kit. CUT & RUN libraries were prepared using the TruSeq ChIP-Sample Prep Kit-Set A and PCR Box (Illumina) from 5 ng of DNA with 10 PCR cycles for amplification following the manufacture’s procedures. Concentration and quality were assessed by Qubit with dsDNA HS Assay Kit (Thermo Fisher Scientific) and High Sensitivity DNA Analysis Chip Kit (Agilent Technologies). Libraries were sequenced on a NovaSeq – S1 device in a pair-end 100 bp read length mode.

first: Rocco

last: Cipolla

first: Marina

last: Pinskaya

first: Marc

last: Gabriel


title: Interpretable Inflammation Landscape of Circulating Immune cells (ILCIC_COVID-HC)

first: Laura

last: Jiménez-Gracia

overall_design: scRNA-seq from 8 human PBMC samples, 4 patients with COVID and 4 healthy controls.

first: Davide

last: Maspero

first: Sergio

last: Aguilar-Fernández

first: Francesco

last: Craighero

first: Juan

middle: C

last: Nieto

first: Holger

last: Heyn


title: Integrative single cell analysis of progressive multiple sclerosis induced neural stem cells reveals epigenetically dysregulated type-I-interferon signaling [scRNA-Seq]

city: Cambridge

first: Irina

last: Mohorianu

address: Puddicombe Way

department: Wellcome-MRC Cambridge Stem Cell Institute

institute: University of Cambridge

summary: Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system, which currently lacks effective therapies that provide regeneration and stop disease progression. A suggested link is anticipated between the development of progressive MS (PMS) and aging, as suggested by recent work identifying hallmarks of cellular senescence in numerous cell types both in vitro with patient cell lines and in vivo in the post-mortem MS brain, including neural stem cells (NSCs). Using an inducible system, directly reprogramming human fibroblasts into induced NSCs (iNSCs), we generated control and PMS iNSC cell lines, which was found to maintain epigenetic age. Here we performed multi-omics, including bulk RNA, single-cell (sc) RNA, single-nucleus (sn) RNA and ATAC, and whole genome bisulfite sequencing. Here, we show that PMS iNSCs have overall increases in senescence and inflammatory associated genes using bulk sequencing. We next identified a unique subcluster of cells within the PMS iNSCs that have a pro-inflammatory and senescent phenotype, associated with interferon gene activation using sc-RNA seq. Within this specific subcluster there is higher expression of genes associated with inflammatory protein secretion, as well as the senescence associated secretory phenotype (SASP). A similar clustering pattern was observed in the ATAC-seq data, a large main cluster and a small inflammatory cluster primarily comprising of PMS iNSCs. Differentially accessible regions within the PMS cluster revealed promoter sites of inflammatory-associated genes to be more accessible correlating with gene expression. Our results highlight a novel cellular mechanism in PMS wherein NSCs are interferon-responsive and senescent-like which may impact other cells within the lesion leading to chronic inflammation.

overall_design: iNSC lines, between passages 25-30, were plated at 500,000 cells per well in a growth factor reduced matrigel coated 6 well plate. After 24 hours, the media was changed to fresh media. Cells were harvested in RLT lysis buffer 72 hours after plating then frozen at -80C until extraction.

first: Bongsoo

last: Park

first: Alexandra

last: Nicaise

first: Dimitrios

last: Tsitsipatis

first: Liviu

last: Pirvan

first: Andi

last: Munteanu

first: Lopez

last: De Novales

first: Pranathi

last: Prasad

first: Julia

last: Whitten

first: Luka

last: Culig

first: Joseph

last: Llewellyn

first: Rosana-Bristena

last: Ionescu

first: Jinshui

last: Fan

first: Supriyo

last: De

first: Marta

last: Suarez Cubero

first: Tommaso

last: Leonardi

first: Valentina

last: Fossati

first: Frank

last: Edenhofer

first: Myriam

last: Gorospe

first: Isabel

last: Beerman

first: Stefano

last: Pluchino


title: Integrative single cell analysis of progressive multiple sclerosis induced neural stem cells reveals epigenetically dysregulated type-I-interferon signaling

first: Irina

last: Mohorianu

overall_design: iNSC lines, between passages 25-30, were plated at 500,000 cells per well in a growth factor reduced matrigel coated 6 well plate. After 24 hours, the media was changed to fresh media. Cells were harvested in RLT lysis buffer 72 hours after plating then frozen at -80°C until extraction. RNA extraction was performed according to the RNeasy kit followed by DNase treatment (Qiagen). This accession refers to the bulk mRNAseq data.

first: Bongsoo

last: Park

first: Alexandra

last: Nicaise

first: Dimitrios

last: Tsitsipatis

first: Liviu

last: Pirvan


title: Targeted CRISPR-Cas9 screening identifies core transcription factors controlling murine haemato-endothelial fate commitment

first: Nina

last: Schmolka

overall_design: Viable FLK1+ single cells from different genotypes ( Ctrl,  Etv2 KO, Ldb1 KO, Six4 KO, Smad1 KO, Zbtb7b KO) were FACS sorted and fixed using the 10x Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit (PN-1000414) and stored at -80°C. The quantity of fixed cell suspension was assessed using an automated cell counter (LUNA-FX7, Logos). Library preparation was conducted using the 10x Chromium Fixed RNA Profiling Reagent Kits for Multiplexed Samples. Briefly, four fixed samples underwent probe hybridization, pooling, and loading onto the chip. Approximately 66,000 cells from the sample pools were loaded into the 10x Chromium X. Library preparation followed the manufacturer's guidelines (Guide CG000527, Rev E).

first: Michael

last: Teske


title: Integrative single cell analysis of progressive multiple sclerosis induced neural stem cells reveals epigenetically dysregulated type-I-interferon signaling [scATAC-seq]

overall_design:
iNSC lines, between passages 25-30, were plated at 500,000 cells per well in a growth factor reduced matrigel coated 6 well plate. After 24 hours, the media was changed to fresh media. Cells were harvested in RLT lysis buffer 72 hours after plating then frozen at -80C until extraction.
Please note that processed data files (generated from both Lane1 and Lane2) are linked to the corresponding *Lane1 sample records.

first: Andi

last: Munteanu

first: Lopez

last: De Novales

first: Pranathi

last: Prasad

first: Julia

last: Whitten

first: Luka

last: Culig

first: Joseph

last: Llewellyn

first: Rosana-Bristena

last: Ionescu


title: Modeling NRF2-Driven Anti-inflammatory, Tumor-promoting Macrophages In Vitro II

city: Zurich

first: Florence

last: Vallelian

address: Rämistrasse 100

institute: University Hospital Zürich

summary: We employed 3D spheroid co-cultures to explore the paracrine and direct cell-contact effects of NRF2-driven macrophages on MC38 tumor growth. We seeded equal numbers of GFP+ MC38 cells and macrophages (WT or Keap1 KO) in ultra–low-adherence plates to form multicellular spheroids. Live-cell imaging showed that spheroids containing NRF2high macrophages reached significantly larger sizes over 120 hours compared to spheroids with WT macrophages. With multiplexed scRNA-seq of spheroids collected at 24 and 120 hours post-formation, we defined the directed effects of NRFhigh stress-TAMs on tumor cells. UMAP density projections visualize that the macrophage genotype did not affect tumor cell gene expression within the first 24 hours despite persistent NRF2-driven macrophage phenotypic diversity. However, by 120 hours, tumor cell transcriptomes diverged markedly, as visualized by an unequal shift in cell densities, which was much more pronounced in the spheroids containing Keap1 KO macrophages. Leiden clustering and subsequent cluster-based GSEA revealed that while most tumor cells co-cultured with wild-type macrophages remained in a proliferative state, those co-cultured with Keap1 KO macrophages transitioned into an epithelial-mesenchymal transition (EMT) state, indicative of high metastatic potential.

overall_design: We seeded equal numbers of GFP+ MC38 cells and macrophages (WT or Keap1 KO) in ultra–low-adherence plates to form multicellular spheroids. We collected these spheroids after 24 and 120 hours post-formation and analyzed them with multiplexed scRNAseq.

first: Dominik

last: Schaer

first: Livio

last: Baselgia

first: Elena

last: Duerst


title: Modeling NRF2-Driven Anti-inflammatory, Tumor-promoting Macrophages In Vitro

summary: To determine whether NRF2-mediated anti-inflammatory differentiation and its downstream consequences on tumor immunology are intrinsic macrophage processes, we conducted a series of in vitro studies. First, we performed scRNA-seq studies of WT, Keap1 KO, and Nrf2 KO BMDMs after short-term culture with GM-CSF for 5 days to capture a broad spectrum of differentiation and polarization states across the macrophage lineage trajectory.

overall_design: scRNA-seq study of WT, Keap1 KO, and Nrf2 KO BMDMs after short-term culture with GM-CSF for 5 days


title: Spatial Zonation of Pro- and Anti-inflammatory Tumor Macrophages After Anti-CD40 II

summary: Because necrosis can be induced by multiple therapeutic modalities, we next analyzed irradiated MC38 tumors to see if they similarly exhibit Spp1+–Cxcl9+ polarization. Therefore, we conducted a spatial RNA-seq analysis on MC38 tumors at 24 hours and 7 days post–irradiation with 12 Gy. As observed under anti–CD40–mediated immunotherapy, we discovered that phenotype heterogeneity among Lyz2+ features increased over time, reproducing the bifurcated polarization trajectory along the Spp1–Cxcl9/Cxcl10 axis. High-resolution macrophage profiling similarly confirmed the regional segregation of Cxcl10+ (better detected in these samples than Cxcl9) and Spp1+ cells.

first: Nadja

last: Schulthess-Lutz


title: Transcriptomic profiling during normothermic machine perfusion of human kidneys reveals a pro-inflammatory cellular landscape and gene expression signature associated with prolonged delayed graft function after transplantation [bulk RNA-seq]

first: Irina

last: Mohorianu

summary: Assessment and treatment of severe ischaemia-reperfusion-injury (IRI) remains an unmet challenge in kidney transplantation. Normothermic machine perfusion (NMP) recapitulates IRI ex situ, but there is limited understanding of the transcriptional pathways, and the associated cellular landscape, driving IRI during NMP and determining its severity. Such knowledge is essential for therapeutic targeting and organ resuscitation during machine perfusion. Using tissue obtained at the time of NMP from kidneys subsequently transplanted as part of a randomised controlled trial, we undertook in-depth transcriptomic analyses comparing kidneys suffering severe IRI, manifesting clinically as the development of delayed graft function (DGF), to kidneys with mild IRI (defined by immediate graft function, IGF) post-transplantation. We validated upregulation of previously described pro-inflammatory and immune transcriptomic pathways, including TNFa via NFkB signaling, Allograft Rejection and Inflammatory Response. Going further, we identified innate immune system driven processes at the core of the transcriptional signature in kidneys suffering severe IRI, such as recruitment and migration of myeloid leucocytes, macrophage activation, phagocytosis and inflammasome activation. Deconvolution using single-cell-RNAseq data showed kidneys with severe IRI and post-transplant DGF were enriched for pro-inflammatory mononuclear phagocytes, myofibroblasts and fibroblasts, but depleted of tubuloepithelial, cell signatures. These transcriptional findings were recapitulated in tissue biopsies obtained during NMP from an external cohort comparing kidneys with high acute tubular injury and severe IRI to kidneys with low acute tubular injury and mild IRI; these kidneys were histologically similar to the DGF/IGF kidneys, respectively. Together, our study characterises the transcriptional signature of severe IRI during NMP, suggesting the role of pro-inflammatory innate/pro-fibrotic cells in this process. We describe a transcriptomic signature that may support future prospective therapeutic trials as a potential efficacy endpoint, and highlight potential cellular targets for therapeutic intervention during NMP in an era of precision medicine.

overall_design: bulkRNA samples of Kidnay biopsies for patients that underwent NMP

first: Harry

middle: V

last: Spiers

first: Sarah

middle: A

last: Hosgood

first: Miguel

last: Larraz

first: Lukas

last: Stadler

first: Ying

last: Zhai

first: Haeun

last: Moon

first: Serena

last: MacMillan

last: Nicholson

first: Vasilis

last: Kosmoliaptsis


title: Androgenic Effects of 11-Oxygenated Androgens in Castration-Resistant Prostate Cancer

city: Québec

first: Chantal

last: Guillemette

state: Québec

address: 2705 blvd Laurier

department: Faculty of Pharmacy

institute: Laval University

summary: Investigations of CRPC cell models demonstrate that both 11oxygenated androgens 11Ketotestesterone (11KT) and 11-hydroxytestosterone (11OHT) drive robust AR-mediated proliferative responses and significantly modulate gene and protein expression profiles, paralleling canonical androgens. They also exhibit unique transcriptional and post-transcriptional effects that include potential AR-independent pathways. Furthermore, the AR antagonist enzalutamide effectively inhibits their androgenic actions, suggesting they are primarily AR-mediated.

overall_design: RNA-seq profiling of the prostate cancer cell lines DU145, LAPC4, VCaP exposed to R1881 or 11ketotestosterone or 11hydroxytestosterone with or without co-exposure to the androgen receptor inhibitor enzalutamide

first: Michèle

last: Rouleau

first: Vincent

last: Gélinas


title: Rebound Polarization of Monocyte-derived Tumor Macrophages Under Repeated Anti-CD40 Immunotherapy

first: Florence

last: Vallelian

summary: To investigate TAM phenotypes and potential stress-induced feedback mechanism upon immunotherapy, we treated MC38 tumor-bearing C57BL/6 mice with a single dose of agonistic anti-CD40 or isotype control on day 11 (1× anti-CD40) or with repeated doses on days  11,  13, and  15 (3× anti-CD40). Twenty-four hours after the last injection, tumors were harvested for flow cytometry and single-cell RNA sequencing (scRNA-seq). A single anti-CD40 injection significantly elevated CD45+F4/80+ macrophage numbers. PCA on CD45+F4/80+ cell RNAseq data , followed by gene set enrichment analysis (GSEA) and transcription factor enrichment, revealed a pronounced transcriptional transformation. After the first round of immunotherapy, most macrophages shifted toward an IFN–STAT1–driven, immune-activated phenotype—reflecting a robust antitumor response. However, repeated anti-CD40 doses induced a rebound in macrophage gene expression, reverting toward the original “tumor-conditioned” state highlighted by strong Spp1 expression. This rebound raised questions about stress signals in the local tumor environment driving macrophage immunosuppression. Across all conditions, we detected a striking polarity along a phenotype gradient from Spp1+ immunocompromised TAMs towards immune-activated Cxcl9+ Cxcl10+ MHC class II macrophages.

overall_design: Mice bearing subcutaneous (s.c.) MC38 tumors were treated intravenously (i.v.) with either isotype control (ctrl)l or agonistic anti-CD40 on day 11 (single injection; 1xCD40) or on days 11, 13, and 15 (multiple injections, 3xCD40). Twenty-four hours after the final injection, tumors were harvested, and CD45+F4/80+ cells were FACS-sorted for multiplexed scRNA-seq.

last: Schaer


title: Rebound Polarization of Monocyte-derived Tumor Macrophages Under Repeated Anti-CD40 Immunotherapy II

summary: To confirm that both pro-inflammatory and immunosuppressive subsets arise from common monocyte precursors, we employed Ms4a3-Cre Rosa26-tdTomato fate-mapping mice. Flow cytometry of dissociated tumors showed that most F4/80+ macrophages were tdTomato+, with subpopulations differing in MHC class II expression. scRNA-seq of sorted CD45+tdTomato+ cells confirmed a bifurcated differentiation trajectory that yields pro-inflammatory, MHC II–high macrophages (Cxcl9+, H2-Eb1), as well as Spp1+ Arg1+ immunocompromised phenotypes.

first: Dominik

last: Baselgia

last: Duerst


title: Spatial Zonation of Pro- and Anti-inflammatory Tumor Macrophages After Anti-CD40

summary: Seeking to understand how local tissue factors reinforce these divergent TAM phenotypes, we next investigated their spatial distribution in MC38 tumors following anti-CD40 treatment (days 6 and 8 post-inoculation). We performed high-definition spatial transcriptomics at a spatial resolution of ~2 µm on a formalin-fixed, paraffin-embedded (FFPE) section. H&E-staining  revealed dense leukocyte infiltrates and a large necrotic core. Spatial mapping showed a clear partition in macrophage phenotypes. The peripheral zone was rich in antigen-presenting and proinflammatory macrophages, while antiinflammatory macrophages were concentrated close to the necrosis-proximal region. This pattern was consistent with the accumulation of Cd74+ and Cxcl9+ macrophages at the tumor edges and Spp1+ macrophages clustering toward the necrotic center. Cells co-expressing pro- and anti-inflammatory markers (Cxcl9+ / Cd74+ and Spp1+) were scattered between the single-positive populations, suggesting a continuous gradient of macrophage states driven by local stress.

overall_design: MC38 cells in culture medium were mixed with Geltrex and injected subcutaneously into the mouse flanks. Agonistic anti-CD40 treatment was administered intravenously according to treatment protocol. Mice were euthanized, and tumors were collected at defined timepoints after antibody administration. The tumor was analyzed using spatial transcriptomics.

first: Livio

last: Schulthess-Lutz


title: NRF2 Engenders Anti-inflammatory Stress Macrophages

summary: We evaluated the function of NRFhigh stress-TAMs and how they respond to immunostimulatory cues. scRNA-seq of CD45+ tumor-infiltrating leukocytes from Keap1flox/flox VavCre and WT mice (n=2 per group) revealed that macrophages were the most abundant leukocyte population in both genotypes. Differential expression, gene set enrichment analysis (GSEA), and cell-by-cell functional scoring indicated a pronounced upregulation of oxidative-stress defense and metabolic adaptation in Keap1 KO macrophages, accompanied by reduced MHC II expression, suppressed interferon-response pathways, and impaired antigen presentation capacity. Despite major shifts in oxidative stress defense and antigen presentation, key macrophage-defining functions such as phagocytic capacity remained comparable between WT and Keap1 KO TAMs. Transcription factor motif analysis further identified NRF2 as a top activated factor and STAT1 as a repressed factor in Keap1 KO TAMs.

first: Elena