title: PDGFC facilitates enzalutamide resistance in prostate cancer through autocrine activation of the RAP1-MAPK pathway

city: chongqing

first: bingqian

last: deng

address: Gaotanyan street 30

institute: Army Medical University

summary: Prostate cancer (PCa) is a highly heterogeneous malignant tumor within the male genitourinary system, characterized by its invasive and metastatic potential. Although the second-generation androgen receptor (AR) antagonist enzalutamide has shown therapeutic efficacy in PCa patients, enzalutamide resistance (EnzaR) will be inevitably develops and the underlying mechanisms are not fully understood. In this study, we confirmed that the expression of Platelet-Derived Growth Factor C (PDGFC) elevated in EnzaR PCa and promoted the proliferation of PCa in vitro and in vivo. Silencing PDGFC in EnzaR cells notably enhances the sensitivity of EnzaR PCa cells to enzalutamide, thereby inhibiting the progressive of PCa. Mechanistically, overexpressed PDGFC activates the PDGFR-RAP1-MAPK signaling cascade in an autocrine fashion within EnzaR cells. PDGFR inhibitors alone or combined with enzalutamide significantly suppressed the progression of EnzaR PCa xenografts. Additionally, we demonstrated that the transcription factor STAT4 binds to a specific DNA sequence in the PDGFC promoter region, which is essential for PDGFC upregulation. Collectively, our results confirmed the pivotal role of PDGFC in PCa cells developing resistant to enzalutamide therapy, suggesting that targeting PDGFC may serve as a promising therapeutic strategy for EnzaR PCa.

overall_design: To investigate the mechanism of PDGFC in enzalutamide resistence cells, we established a stable knockdown of PDGFC in LNCaP-EnzaR cells using shRNA.

first: Bingqian

last: Deng

first: Saipeng

last: Chen

first: Dan

last: Zhong

first: Guojing

last: Song


title: Restoration of endogenous transcription factor activity by nuclease-deficient Cas9VPR prevents cardiac failure upon stress

status: Public on Jan 30 2026

city: Göttingen

first: Laura

last: Zelarayan

address: Robert-Koch-Str 40

country: Germany

institute: University Medical Center Göttingen

type: Expression profiling by high throughput sequencing

summary: Cardiac remodeling is linked to maladaptive transcriptional networks, but identifying and targeting key factors with potential to reverse these processes is still a challenge. We employed network-based analysis of single cell heart transcriptomes to compute the transcription factor activities triggering heart failure. We found that the regulation of KLF15 activity is a major driver of cardiomyocyte failure. Through the use of CRISPR/nuclease-deficient (d)Cas9-based transcriptional regulation, we demonstrated that restoration of endogenous Klf15 expression and activity in cardiomyocytes was sufficient to mitigate pathological transcriptional reprogramming and preserve cardiac function in mice and human myocardial models. We also showed that KLF15 is a downstream targetable factor in the broad TGFβ-mediated stress response in human cardiomyocytes, controlling transcriptional embryonalization. Lastly, we engineered a therapeutically applicable mini-dCas9VPR variant suitable for adeno-associated viral delivery, eliciting endogenous KLF15 transcriptional activation in human cardiomyocytes. This case study presents a therapeutic strategy for restoring the stress-induced downregulation of essential genes to prevent heart failure with broader implications for other disease contexts.

overall_design: Double transgenic Myh6-dCas9VPR/Klf15 knockout (KO) mice were generated by crossing Myh6-dCas9VPR mice with Klf15 KO mice. Wild-type C57BL/6N mice were used as controls. Mice (both male and female, 4-6 months old) were injected via the tail vein with either rAAV2/9-TRISPR-Klf15 or rAAV2/9-TRISPR-NT. Following injection, mice underwent transverse aortic constriction (TAC) or sham surgery. Pressure overload was confirmed by measuring the blood flow velocity ratio between the right and left carotid arteries. Echocardiography was conducted at 4 and 8 weeks post-surgery, and hearts were harvested at 12-16 weeks. Ventricular measurements were taken using a VisualSonics Vevo 2100 Imaging System. All procedures were performed in a blinded manner and in accordance with ethical guidelines from the Lower Saxony State Office for Consumer Protection and Food Safety.

first: Eric

last: Schoger

first: Federico

last: Bleckwedel

middle: C

last: Zelarayán


title: Myocardial Fibroblasts are a Primary Source High Molecular Weight Hyaluronan Production in the Failing Heart [RNASeq_Has2_knockout]

city: Louisville

first: Richa

last: Singhal

state: KY

address: 580 S Preston Street, Baxter II Building, Office 321B

department: Medicine

country: USA

institute: University of Louisville

summary: Background: The extracellular matrix provides organizational context for solid organs. During disease, such as acute myocardial infarction, the composition of the extracellular matrix changes remarkably. One of the most notable changes in the extracellular matrix is in the accumulation of collagen; however, hyaluronan rivals collagen in terms of its abundance. Yet, the extent to which specific cells and enzymes may contribute to such accumulation has been largely unexplored. Here, we hypothesized that activated cardiac fibroblasts produce hyaluronan via hyaluronan synthase 2 (HAS2). Methods and Results: Using immunofluorescence, biochemical extraction, and ELISA, we show that hyaluronan accumulates following myocardial infarction and persists through at least four weeks. Our analyses of failing heart RNA sequencing data suggest fibroblasts are the cells most changed in expression of HAS2. Given these insights, we used HAS2 gain- and loss-of-function approaches to examine the extent to which activated cardiac fibroblasts produced hyaluronan. TGF-induced activation of fibroblasts caused a significant increase in Has2 mRNA and concomitant accumulation of hyaluronan greater than 1 MDa in size. Deletion of Has2 abrogated TGF-induced production of hyaluronan. In addition, overexpression of Has2 was sufficient to cause an increase in hyaluronan accumulation in the absence of TGF-induced activation. Given the magnitude of hyaluronan production, and that hyaluronan has been reported to have biologic activity, we then queried whether the primary functions of fibroblasts (proliferation, migration, and collagen production) were impacted by hyaluronan treatment. Our data indicated negligible impacts of Has2 on proliferation, migration, and collagen production. Exposing fibroblasts to exogenous hyaluronan also had minimal impact on fibroblasts. We also assessed whether fibroblast-borne Hyal2 plays a role in degradation of hyaluronan, and our data indicated little impact of Hyal2 on hyaluronan accumulation (or even any impacts on the transcriptional profile of fibroblasts). Conclusion: Activated fibroblasts produce high molecular weight hyaluronan, and the production of hyaluronan depends on Has2. The robust production of hyaluronan by fibroblasts does not appear to impact fibroblast function.

overall_design: Cardiac fibroblasts were treated with adenovirus to force deletion of Has2.

first: Steven

middle: P

last: Jones

first: Danielle

middle: T

last: Little

middle: A


title: Effect of interference of  PALMD on gene expression in human valvular endothelial cells(VECs)

city: BEIJING

first: YINGCHUN

last: HAN

address: No.2, Anzhen Road, Chaoyang District

country: China

institute: ANZHEN HOSPITAL

summary: To investigate the role of PALMD in VECs, we knockdown PALMD by siRNA.We then performed RNA-seq and gene expression profiling analysis from VECs transfected with siCtrl or siPALMD.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for VECs transfected with siCtrl and siPALMD for 48 hours

first: Yingchun

last: Han

first: Jie

last: Du


title: Single-cell transcriptomic atlas of glial cells in adult mouse dorsal root ganglia identifies multipotent progenitors

status: Public on Jan 29 2026

city: St. Louis

first: Valeria

last: Cavalli

state: MO

address: 1 Bookings Dr.

institute: Washington University in St. Louis

summary: Sensory neuron soma and non-neuronal cells in adult dorsal root ganglia (DRG) derive from multipotent neural crest cells. Satellite glial cells (SGCs), which surround neuronal soma, were suggested to retain developmental potential, but the precise molecular identity of progenitor cells in the adult DRG remains undefined. To address this question, we assembled a comprehensive single-cell transcriptomic atlas by integrating over 200,000 DRG and sciatic nerve transcriptomes across multiple studies and injury paradigms. High-resolution clustering resolved 26 cell types and demonstrated significant transcriptional heterogeneity within SGCs and Schwann cells, including repair and reactive sub-states. Crucially, we identified two distinct populations of progenitor cells that reflect different states in the progenitor trajectory. Functionally, progenitor cell numbers significantly increase after injury, and endothelin signaling regulates glial cell proliferation early in development. This integrated DRG and peripheral nerve cell atlas represents an essential resource for exploring new features of the peripheral nervous system.

overall_design: L4 and L5 DRGs from adult WT (C57BL/6) mice were collected, dissociated into a single cell suspension, and analyzed using 10X Genomics scRNA-seq. Sample treatments include acute intermittent hypoxia (AIH), sciatic nerve crush (SNC), and uninjured control.

first: Pauline

last: Meriau

first: Michael

middle: B

last: Thomsen

first: Oshri

last: Avraham

first: Brent

last: Trauterman


title: Single-cell RNA sequencing combined with multiplex immunofluorescence probes the role of MFAP5+ fibroblasts in the microenvironment of pancreatic ductal adenocarcinoma.

city: Anhui

first: 午洋

last: 张

state: State

address: No.17 Lujiang Road, Hefei City, Anhui Province, China

institute: The First Affiliated Hospital of USTC

summary:
we employed single-cell RNA sequencing to examine the biological characteristics of MFAP5+ fibroblasts in PDAC and their interaction with vascular endothelial cells within tumors. We then utilized a proposed temporal sequencing analysis technique to infer the evolution of cellular subtypes of cancer-associated fibroblasts. To verify our hypothesis, we employed a multiplex immunofluorescence technique to observe the spatial distribution of MFAP5+ fibroblasts and endothelial cells.
This study investigated the tumor heterogeneity of PDAC, elucidating intricate cellular interactions within the tumor microenvironment through a comprehensive analysis of 23,905 cells. The biology of MFAP5+ fibroblasts in pancreatic ductal adenocarcinoma was investigated, and it was found that MFAP5+ fibroblasts engage in active biosignaling exchanges with FABP4+ endothelial cells and other endothelial cells through the activation of key tumor-promoting signaling pathways, such as TGF-β, VEGF, and FGF. These pathways provide support for the tumor's invasive and malignant behavior. Our hypothesis was validated by multiplex immunofluorescence and subsequent semi-quantitative analysis, which demonstrated a notable increase in the prevalence of FABP4+ and VWF+ endothelial cells in the area with elevated MFAP5+ fibroblast expression. Furthermore, our study revealed that vascular endothelial growth factor and FGF2 signaling were highly expressed in the region with high MFAP5+ fibroblast expression.

overall_design: NOT PROVIDED; REQUESTED

first: Wuyang

last: Zhang

first: Min

last: Wang

first: Yuyi

last: Liu

first: Shuai

last: Ming

first: Mengru

last: Li

first: Wei

last: Wei


title: Transcriptome profiling of human gonadal tissue in Differences of Sex Development

city: Campinas

first: Helena

last: Fabbri-Scallet

state: São Paulo

address: Av. Cândido Rondon, 400

department: Center for Molecular Biology and Genetic Engineering

country: Brazil

institute: State University of Campinas

summary: This study investigates the transcriptomic landscape of human gonadal tissues from individuals with Differences of Sex Development (DSD). Bulk RNA sequencing was performed on dysgenetic testes, testes, ovaries, and ovotestes obtained from pediatric and adult patients with 46,XY and 46,XX DSD phenotypes.

overall_design: Bulk RNA-seq of gonadal tissues from 11 DSD patients (14 gonadal samples). Libraries were prepared from FFPE or fresh tissue and sequenced using paired-end Illumina technology.  Gene-level read counts were generated using STAR.


title: Single-cell RNA sequencing of mouse hearts under normal fat diet and high fat diet conditions

city: Newark

first: Alexander

last: Lemenze

state: New Jersey

address: 185 South Orange Ave

institute: Rutgers

summary: To investigate transcriptional changes in the heart induced by diet, we performed single-cell RNA sequencing on pooled mouse hearts from normal fat diet (NFD, 10% kcal fat) and high fat diet (HFD, 60% kcal fat) groups using the Parse Biosciences Evercode WT Mini v3 kit.

overall_design: Whole hearts from C57BL/6J mice fed either NFD or HFD for 4 weeks (n=3 pooled per group) were processed using the Parse Biosciences Evercode Whole Transcriptome Mini v3 kit for single-cell RNA-seq. Libraries were sequenced on Illumina NovaSeq 6000. Two sublibraries were generated.

first: Junichi

last: Sadoshima

first: Shin-ichi

last: Oka

first: Eun-Ah

last: Sung

first: Mainul

last: Hoque


title: Stranded mRNA sequencing of chicken cecal tissue

city: Ames

first: Torey

last: Looft

state: Iowa

address: 1920 Dayton Avenue

department: Food Safety and Enteric Pathogens Research Unit

institute: USDA-ARS

summary: The objective of this study was to determine if Salmonella colonization of chickens could be reduced through competitive exclusion using a defined community of chicken commensal bacteria. One-day old White Leghorn chicks, hatched on-site, were randomly divided into experimental groups and given an oral gavage of either a defined community of 15 bacterial species (DC), cecal contents (CC), or sterile PBS (control; CT). After one week, birds were euthanized for cecal content collection (pre-Salmonella sample) while the remaining birds were orally gavaged 1 X 10^8 colony forming units (CFU) of Salmonella enterica ser. Heidelberg strain 2813 (SH2813). Bacterial counts for three post-Salmonella timepoints (3, 14, and 28 days post inoculation; dpi) were evaluated. Bacteriological enumeration was performed by plating cecal contents onto Salmonella selective agar to determine CFU/g in each group for all collection days. Cecal contents were also used for 16S amplicon sequencing. Cecal tissue was used for stranded mRNA sequencing (RNA-Seq).

overall_design: RNAseq of cecal tissue from birds given 1) consortium of bacteria, 2) unfiltered cecal contents from mature hens, 3) sterile carrier. Samples taken from 10 birds per group prior to Salmonella introduction (dpi) and 12 birds per group each for 3, 14, and 28 dpi.

first: Carmen

middle: L

last: Wickware

first: Dylan

last: Harris

first: Briony

last: Atkinson

first: Melissa

middle: S

last: Monson

first: Shawn MD

last: Bearson

first: Christopher

last: Anderson

first: Adelumola

last: Oladeinde


title: Characterization of non-NF2 related schwannomatosis

city: Boston

first: Sonu

last: Subudhi

state: MA

address: 100 Blossom St, Cox 7

department: Radiation Oncology

institute: Massachusetts General Hospital

summary: The goal of the study is to profile the transcriptome of non-NF2 related schwannomatosis

overall_design: We compared RNASeq data between 3 patient samples vs. 3 normal nerve RNAs.

first: Xu

last: Lei

first: Subudhi

last: Sonu


title: Multiomics identifies unique modulators of calf muscle pathophysiology in peripheral artery disease and chronic kidney disease

city: Gainesville

first: Terence

last: Ryan

address: 1864 Stadium Rd

institute: University of Florida

summary: This project aimed to map transcriptome changes in the skeletal muscle of patients with and without PAD (peripheral artery disease) and CKD (chronic kidney disease)

overall_design: bulk RNA sequencing was performed on gastrocnemius muscle specimens obtained from control, PAD, CKD, and PAD+CKD patients.

middle: E


title: Effects of rocking during sleep on the bulk transcriptome of mouse cerebral cortex.

city: Camerino

first: Michele

last: Bellesi

address: Via Gentile III Da Varano

country: Italy

institute: University of Camerino

summary: Transcriptomic studies have revealed that sleep and wake states are associated with widespread changes in gene expression in the brain across multiple species. While these findings have provided important insights into the molecular correlates of sleep, the transcriptional consequences of experimentally enhancing sleep through non-pharmacological interventions remain poorly characterized. In the present study, we investigated the effects of sleep enhancement induced by vestibular stimulation via rocking on the bulk transcriptome of the mouse cerebral cortex. Mice were exposed to repeated rocking sessions during sleep across multiple days, a protocol previously shown to increase sleep duration and consolidation. Cortical tissue was collected following the stimulation protocol, and bulk RNA sequencing was performed to assess global gene expression changes associated with rocking-induced sleep enhancement. Transcriptomic analysis revealed significant modulation of genes involved in synaptic function, neurotransmission, and plasticity-related pathways, consistent with the observed behavioral and synaptic outcomes of the experimental paradigm. These data provide a molecular framework for understanding how sensory-driven sleep enhancement influences cortical gene expression and support a link between improved sleep and transcriptional programs associated with neuroplasticity.

overall_design: Standard RNA sequencing of motor cortex tissue was conducted to compare transcriptomic changes between sleep (S) and sleep-enhanced (SE) C57BL/6 mice. For each animal, primary motor cortex was immediately extracted and snap frozen.

first: Reyila

last: Simayi

first: Letizia

last: Santoni

first: Sabrina

last: Galizia

first: Riccardo

last: Avvisati

first: Ester

last: Biecher

first: Luisa

last: de Vivo


title: Surprising regulatory plasticity for the conserved HOG pathway in diverse Saccharomyces cerevisiae strains

city: Fayetteville

first: Carson

middle: Len

last: Stacy

state: AR

address: 850 W. Dickson St. Rm 601

department: Biological Sciences

institute: University of Arkansas

summary: Timely signaling pathways activation allows cells to survive diverse environmental stress conditions. Mitogen-activated protein kinases (MAPKs) are a highly conserved class of signaling molecules in eukaryotes with essential functions in cellular responses to stress. In Saccharomyces cerevisiae, the role of MAPK Hog1 as a master regulator of the coordinated response to osmotic stress is well understood. However, recent findings suggest that the role of Hog1 may extend beyond canonical osmoadaptation. This study investigates the role of Hog1 in mediating transcriptional responses to acute oxidative and ethanol stress. By harnessing the natural variation present in wild strains of S. cerevisiae, we use gene knockouts, comparative transcriptomics, and survival assays to determine Hog1’s involvement in stress responses beyond osmoadaptation. Our findings demonstrate that Hog1 mediates transcriptional reprogramming for non-osmotic stress response in a strain-dependent manner. Osmospecificity of Hog1 activity was identified in the DBY8268 laboratory strain, while differential gene expression was observed in HOG1 knockouts of all wild strains tested under both oxidative and ethanol stress. Further, our data indicate that the function of Hog1 in the response to non-osmotic stress is distinct from the canonical response, with effects ranging from altered ribosomal protein expression dynamics to altered environmental stress response (ESR) activity. Differences in expression correlate with fitness defects of hog1∆ mutants. These results suggest a generalized role of the Hog1 MAPK in S. cerevisiae, consistent with an evolutionarily generalized function for this kinase, underscoring the importance of genomic diversity for elucidating stress signalling dynamics in yeast.

overall_design: Bulk RNA-seq was performed on S. cerevisiae cultures comparing HOG1 wild-type and hog1 deletion mutants in three distinct strains: DBY8268-derivative (Laboratory strain), YPS606 (Oak isolate), and M22 (Vineyard isolate). For each strain and genotype, we collected samples under (i) unstressed conditions, (ii) acute exposure to 0.4 M NaCl, 5% (v/v) ethanol, or 0.4 mM hydrogen peroxide. Stressed samples were collected after 30 (ethanol and peroxide) or 45 minutes (NaCl) of exposure. For each condition, 4 biological replicates were sequenced. One strain was resequenced in triplicate once its original samples were discovered to be following sequencing.

first: Amanda

middle: N

last: Scholes

first: Tara

last: Stuecker

first: Lily

last: Pitts

first: Jeffrey

last: Lewis


title: Effects of ACTH and Dexamethasone on the Transcriptomic Profile of the Rat Adrenal Gland: An In Vivo Study

city: Poznan

first: Marcin

last: Rucinski

address: Swiecicki 6

department: Histology and Embriology

country: Poland

institute: Poznan University of Medical Science

summary:
The hypothalamic-pituitary-adrenal (HPA) axis plays a pivotal role in regulating stress responses through ACTH-stimulated glucocorticoid production. Adult male Wistar rats were subjected to acute ACTH stimulation (single injection, 1 h), prolonged ACTH exposure (three injections over 36 h), or Dexamethasone treatment (three injections over 36 h). Plasma corticosterone levels were measured using ELISA. Adrenal transcriptome profiling was performed using Affymetrix Rat Gene 1.1 ST Arrays. Differentially expressed genes (DEGs; |fold change| >= 1.8, adjusted p < 0.05) were analyzed using limma, followed by pathway and network analyses.
Acute ACTH exposure resulted in induction of 569 DEGs (357 upregulated), including immediate-early genes (Nr4a family, AP-1 factors), cAMP-PKA-CREB signalling components, and heat shock proteins. Prolonged ACTH resulted in 98 DEGs (predominantly downregulated), including suppression of mitochondrial genes and upregulation of Polycomb repressive complex 2 components. Dexamethasone treatment yielded 75 DEGs with selective suppression of SREBP-mediated cholesterol biosynthesis and uptake pathways.

overall_design: Adult male Wistar rats (160-200 g) were randomly assigned to four experimental groups (n=3 per group, 12 animals total). Control group (C) received saline injections at 0, 12, and 24 h and were decapitated 12 h after the last injection. Acute ACTH group (ACTH_A) received a single ACTH injection (Synacthen, 5 μg/rat) and were decapitated 1 h post-injection. Prolonged ACTH group (ACTH_P) received ACTH injections at 0, 12, and 24 h and were decapitated 12 h after the last injection. Dexamethasone group (DEX) received Dexamethasone injections (5 μg/rat) at 0, 12, and 24 h and were decapitated 12 h after the last injection. Total RNA was extracted from adrenal glands and analyzed using Affymetrix Rat Gene 1.1 ST Arrays.

first: Emilia

last: Cicha

first: Małgorzata

last: Blatkiewicz

first: Karol

last: Jopek

first: Marta

last: Szyszka

first: Piotr

middle: W

last: Malendowicz

first: Anna

last: Olechnowicz

first: Ludwik

middle: K


title: PPARa-NFkB heterodimer mediates obesity cardiomyopathy through autocrine production of IL-6

first: Jiyeon

last: Park

state: NJ

address: 185 South Orange Ave.

department: Department of Cell Biology and Molecular Medicine

institute: Rutgers New Jersey Medical School

type: Genome binding/occupancy profiling by high throughput sequencing

summary: This study investigates the molecular mechanism of obesity cardiomyopathy. We found that high-fat diet (HFD) consumption stimulates IL-6 production in cardiomyocytes through the formation of a PPARa/NFkB heterodimer. This local autocrine production of IL-6 is a key driver of diastolic dysfunction in the early stages of obesity.

overall_design: ChIP-seq was conducted to identify the genomic binding sites of PPARα, RelA, and p50 in mouse heart tissue. To assess changes in the recruitment of these transcription factors to bona fide PPAR and NF-κB binding elements, we compared mice fed a normal diet (ND) with those fed a high-fat diet (HFD) for one month.

first: Shinichi

last: Oka

first: Yeun-Jun

last: Chung

first: Junichi

last: Sadoshima


title: A Spatiotemporal Cell Atlas of the Developing Rat Tooth Germ Reveals Iron-Deficiency Induced Ameloblast Dysregulation

city: Qingdao

first: Xianwei

last: Yang

address: Hengyushan Road

institute: BGI-Qingdao

summary: Iron deficiency anemia (IDA) during pregnancy is a crucial risk factor for dental caries in offspring, yet the cellular and molecular mechanisms underlying its impact on tooth development remain unclear. To elucidate the pathogenic mechanism in situ spatially, this study established a pregnant rat model of IDA and analyzed the molar tooth germs of 7-day-old offspring using high-resolution spatial transcriptomics (Stereo-seq).

overall_design: Confirmed IDA female rats and Control female rats were co-housed with normal male SD rats at a 2:1 ratio, which is two females per male. Pregnant females were then separated and individually housed until parturition. We randomly selected one offspring from each of the IDA and Control groups at 7 days of age for tissue collection.

first: Rui

last: Han


title: A plant histone H3.3-specific amino acid safeguards the deposition of H3K36 methylation for proper plant development and stress responses [ATXR5 H3K36me3 ChIP-seq]

city: Beijing

first: Xiaoyi

last: Li

address: west beichen

department: Chinese Academy of Sciences

institute: Institute of Genetics and Developmental Biology

summary: Histone variants are critical determinants for chromatin function with diverse regulatory mechanisms. The H3 variants, H3.1 and H3.3, evolved independently in animals and plants. A significant difference between H3.1 and H3.3 is the amino acid variation at position 31, with H3.1 carrying alanine (A), while H3.3 bearing serine (S) in animals or threonine (T) in plants. Both S and T can be phosphorylated, but why plants have selectively adopted T over S remains unclear. Here, we report the specific role of plant H3.3T31 in controlling plant development and stress responses by promoting the deposition of histone H3 lysine 36 trimethylation (H3K36me3) on H3.3. T31 prevents plant-specific H3K27 methyltransferases, ATXR5 and ATXR6, from depositing H3K27 monomethylation (H3K27me1), which inhibits the activity of H3K36 methyltransferase EFS. Substituting H3.3T31 with an S or A residue results in increased ATXR5/6 activity and elevated H3K27me1 levels, leading to a reduction in H3K36me3. Moreover, we show that unlike H3.3 T31S and T31A mutations, cancer-associated G34R and G34W mutations directly disrupt H3K36me3 deposition without affecting H3K27me1. These G34 mutations may also influence H3.3 function through mechanisms beyond the disruption of H3K36me3. Our data suggest a co-evolution of the plant-specific H3.3T31 residue and H3K27 methyltransferases ATXR5/6, which ensures the selective accumulation of H3K27me1 on H3.1 and H3K36me3 on H3.3, thereby enabling proper chromatin function.

overall_design: Examination of H3K36me3 enrichment in seedlings

first: Danhua

last: Jiang

first: Jie

last: Pan


title: Glial UDP-glycosyltransferase Ugt35b regulates longevity by maintaining lipid homeostasis in Drosophila

city: Shanghai

first: Lihong

last: Sheng

address: 131 Dongan Road

department: Institutes of Brain Science

institute: Fudan University

summary: Lipid droplets (LDs) are dynamic organelles essential for lipid storage and organismal survival. Studies have highlighted the importance of glial function in brain LD formation during aging, however, the genes and mechanisms involved remain elusive. Here, we found that Ugt35b, a member of the UDP-glycosyltransferases that catalyze the transfer of glycosyl groups to acceptors, is highly expressed in glia and crucial for Drosophila lifespan. By integrating multi-omics data, we demonstrated that glial Ugt35b plays key roles in regulating glycerolipid and glycerophospholipid metabolism in the brain. Notably, we found that Ugt35b and Lsd-2 are co-expressed in glia and confirmed their protein interaction in vivo. Knockdown of Ugt35b significantly reduces LD formation by downregulating Lsd-2 expression, while overexpression of Lsd-2 partially rescues the shortened lifespan in glial Ugt35b RNAi flies. Our findings reveal the crucial role of glial Ugt35b in regulating LD formation to maintain brain lipid homeostasis and support Drosophila lifespan.

overall_design: To elucidate potential molecular alterations contributing to reduced lifespan, we conducted bulk RNA-seq analysis on the heads of flies pan-glial (Repo-GS) expressing Ugt35b RNAi, subjected to either EtOH or RU486 treatment at day 10.

first: Jianpeng

last: Gao

first: Zhixiang

last: Xu


title: FABP7 Progenitors are a Targetable Metabolic Root in the BRCA1 Breast

city: Pittsburgh

first: Curtis

last: McCloskey

state: PA

address: 5051 Centre Avenue office 2043

institute: University of Pittsburgh

summary: It has been nearly 3 decades since the discovery of the BRCA1/2 genes and their link to breast cancer risk, with prophylactic mastectomy remaining the primary management option for these high-risk mutation carriers. The current paucity of interception strategies is due to undefined, targetable cancer precursor populations in the high-risk breast. Despite known cellular alterations in the BRCA1 breast, epithelial populations at the root of unwarranted cell state transitions remain unresolved. Here, we identify a root progenitor population that is dysregulated in BRCA1 carriers stemming from the metabolic role of BRCA1. This fatty-acid binding protein 7 (FABP7) expressing luminal progenitor population is spatially confined to the mammary ducts, has enhanced clonogenic capacity, and is the predicted origin of mixed basal-luminal differentiation in the BRCA1 but not BRCA2 breast. We show global H3K27 acetylation is reduced within ductal FABP7 cells in BRCA1 carriers in situ, linking to a non-canonical metabolic role of BRCA1 in regulating acetyl-CoA pools and de novo fatty acid synthesis. We demonstrate FABP7 progenitor capacity is preferentially ablated in BRCA1 carriers through inhibition of fatty acid metabolism using an FDA-approved fatty acid synthase (FASN) inhibitor. This study lays the foundation for metabolic control of breast progenitor dynamics to mitigate breast cancer risk in the BRCA1 breast.

overall_design: Single-cell RNA sequencing of primary human breast samples from prophylactic mastectomies in non-carrier, BRCA1 mutation, and BRCA2 mutation carriers, using Multiplexed using lipid-tagged indices (MULTI-seq). Samples were staged for menstrual cycle.

first: Rama

last: Khokha


title: Myeloid Derived Growth Factor is an endogenous, paracrine regulator of neutrophil activation and confined motility

city: Madison

last: Huttenlocher

state: WI

address: 1550 Linden Drive

department: Pediatrics

institute: University of Wisconsin School of Medicine and Public Health

summary: Neutrophils are necessary for host defense but also contribute to tissue damage. Therefore, it is important to understand mechanisms that limit neutrophil-driven inflammation. We previously reported that myeloid derived growth factor (MYDGF) restricts neutrophil inflammation in zebrafish. However, the mechanism of MYDGF’s effects remain unknown. Here, we generated human induced pluripotent stem cells (iPSCs)-derived neutrophils (iNeutrophils) that are deficient in MYDGF to identify neutrophil intrinsic roles for MYDGF. Depletion of MYDGF results in iNeutrophils that have increased ROS production, confined motility and antimicrobial efficacy. Secreted MYDGF was sufficient to rescue motility in a MYDGF-deficient iNeutrophil line and modulate primary human neutrophil behavior, suggesting neutrophil paracrine signaling. MYDGF-KO iNeutrophils exhibit increased calcium flux during confined motility in response to fMLP. Taken together, our data identify MYDGF as an endogenous negative regulator of neutrophil inflammatory function and cell-cell communication.

overall_design: Wildtype and MYDGF-/- bone marrow-derived human iPSCs were differentiated into iNeutrophils and collected at Day 7 for single-cell RNA profiling.

first: Frances

last: Smith

first: Jonathan

middle: H

last: Schrope

first: David

last: Bennin

first: Adam

last: Horn

first: Andrew

last: Wagner

first: Yiran

last: Hou

first: Jack

middle: J

last: Stevens

first: Morgan

last: Giese

first: Huy

middle: Q

last: Dinh


title: Linezolid prevents fibroblast activation and ameliorates tissue fibrosis by inhibition of mitochondrial translation

city: Dusseldorf

first: Alexandru-Emil

last: Matei

address: Merowingerpl. 1A

institute: Heinrich-Heine-University Düsseldorf

summary: Background: Beyond its role as a ribosome-targeting antibiotic, linezolid was recently shown to modulate immune responses by inhibiting mitochondrial translation. Since mitochondrial dysfunction is implicated in various fibrotic diseases, including systemic sclerosis (SSc), this study aimed to evaluate the antifibrotic potential of linezolid and delineate its underlying mechanisms in SSc. Methods: The effects of linezolid on fibrotic tissue remodeling were assessed using multiple experimental systems: human dermal fibroblasts, human macrophages, three-dimensional SSc skin equivalents (SScSE), the murine model of sclerodermatous chronic graft-versus-host disease (sclGvHD) and precision-cut skin slices (PCSS) obtained from SSc patients, by RNA sequencing, immunofluorescence, Western Blot and histology. Mitochondrial function was evaluated using Seahorse assays alongside mitochondrial protein synthesis assessments. Results: Linezolid inhibited TGFβ-induced fibroblast activation in cultured human fibroblasts, SScSE, sclGvHD mice and SSc PCSS, as demonstrated by reversal of profibrotic gene expression programs, downregulation of TGFβ, WNT, and JAK-STAT signaling and by reductions in αSMA expression or stress fiber formation, which led to reduced collagen deposition and ameliorated skin or lung fibrosis in vivo. Mechanistically, linezolid induced metabolic alterations by inhibiting mitochondrial translation in fibroblasts to hamper the oxidative phosphorylation, reduce the NAD⁺/NADH ratio and downregulate glycolysis, an essential metabolic pathway for fibroblast activation. Conclusions: This study provides first evidence that inhibiting mitochondrial translation with linezolid ameliorates fibrotic tissue remodeling. Since linezolid is already clinically approved as a reserve antibiotic, these findings hold translational promise and support the use of linezolid as a novel treatment for fibrotic disorders after further validation.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Mice sample: Allogeneic transplanted mice (cGVHD model) and syngeneic transplanted control mice (Syn) received daily oral gavage of 0.4% methylcellulose (vehicle) starting on day 21 after allogeneic or syngeneic bone marrow transplantation (alloBMT). Treatment groups included linezolid monotherapy (4 mg daily, orally) and vancomycin (4 mg daily, orally). All mice were sacrificed seven weeks post-alloBMT. Fibroblast RNA-seq: Primary dermal fibroblasts were stimulated with TGF-β for 7 days, with or without linezolid, and harvested on day 7 for bulk RNA-seq. Macrophage RNA-seq: iPSC-derived macrophages were exposed to TGF-β for 48 h, with or without linezolid, then collected for bulk RNA-seq. Precision-cut skin slices (PCSS) RNA-seq. PCSS prepared from patient skin were cultured ex vivo and treated for 48 h with either control medium (positive control) or linezolid.

first: Xuezhi

last: Hong

first: Yanhua

last: Xiao

first: Jörg

last: Distler

first: Alexandru


title: PI3 Kinase and IFN- pathways, identified in an unbiased kinase drug screen, are key mediators of cytokine release syndrome

city: Saint Louis

first: Li

last: Ding

address: 660 S. Euclid Avenue

institute: Washington University in St. Louis

summary: Chimeric Antigen Receptor T (CAR-T) cell therapy can induce cytokine release syndrome (CRS), which ranges clinically from mild to life-threatening, and is universally characterized by elevation of multiple cytokines, including IL-6. To clarify mechanisms underlying CRS and identify potential new therapeutic agents, we screened a protein kinase inhibitor library using an in vitro  model system where CART19 cells were co-cultured with CD19+ tumor target cells and cytokine-primed myeloid cells (pMC), and secreted IL-6 levels serving as the CRS readout. We identified duvelisib, a dual PI3Kdg inhibitor, which blocked pMC IL-6 secretion in in vitro and in vivo  without negative impact on CART19 cell function. Immunophenotyping showed increased activation markers on CART19 cells CD137+(4-1BB); CD279+ (PD-1) and pMC cells (CD83+CD86+) when cultured with tumor target cells compared to cultures without target cells; these activation phenotypes were reduced in the presence of duvelisib. Single-cell RNA sequencing analyses identified four transcriptional clusters of pMCs. Among these, the activated pMC1 subset was the primary source of IL-6 expression, which was effectively reduced by duvelisib and by ruxolitinib, a JAK1/2 kinase inhibitor known to block CRS. Transcriptionally, IFNG expression in CART19 cells was significantly downregulated in the presence of duvelisib and ruxolitinib, and cell-cell communication between CART19 cells and pMCs suggests different mechanisms of action for the two inhibitors. Finally, pMC IL-6 production was significantly reduced by CRISPR/Cas9 deletion of IFNG in CART19 cells, the IFN-g receptor in pMC, or treatment with duvelisib which blocks both IFN-g production by CART19 and IFN-g receptor signaling. Our studies suggest that targeting PI3dg and IFN-g signaling pathways reduce CRS without altering CAR-T cell function.

overall_design: 1x106 CART19, RamosCG, and primed myeloid cells (pMC), were co-cultured for 24hrs with DMSO control or 1000nM duvelisib or 1000nM ruxolitinib. ~50,000 cells of each experimental groups were subjected to scRNA sequencing (3’ V3.1 – Dual Index Kit) from 10XGenomics at the Genome Technology Access Center / McDonnell Genome Institute (GTAC@MGI).

first: Amatya

last: Parmeshwar

first: O'Neal

last: Julie

first: Jayasinghe

last: Reyka

first: DiPersio

last: John


title: The antiseizure medication Stiripentol inhibits mitocondrial complex I by blocking early-stage electron transfer

city: Köln

first: Cyrielle

middle: Louise

last: Bouchez

address: Lübecker strasse 5

institute: Uniklinik Köln

summary: The oxidation of NADH is essential for maintaining cellular redox balance and supporting cell metabolism. Mitochondrial complex I (NADH:ubiquinone oxidoreductase) plays a central role in this process by coupling NADH oxidation to electron transfer and protons translocation across the inner mitochondrial membrane. We previously reported that the antiseizure drug stiripentol decreases lactate production and mitochondrial respiration, suggesting an impact on NADH turnover beyond its known inhibition of lactate dehydrogenase. In this study, we identify complex I as a target of stiripentol across multiple species and cell types. Biochemical and spectroscopic analyses demonstrate that stiripentol inhibits NADH oxidation and electron transfer through a mechanism distinct from that of classical ubiquinone pocket inhibitors such as rotenone or piericidin A. Remarkably, stiripentol acts upstream of the ubiquinone reduction site, representing the first example of a complex I inhibitor with a binding site within the N-module. These findings uncover a previously unrecognized mode of complex I inhibition and link stiripentol’s metabolic effects to direct modulation of mitochondrial NADH oxidation. This work broadens the understanding of stiripentol’s mechanism of action and highlights its potential to modulate redox metabolism in cancer cells.

overall_design: We compared control to stiripentol inhibition

first: Thibaut

last: Molinié

first: Guillaume

last: Duranthon

first: Sebastian

last: Lillo

first: Corinne

last: Pellon

first: Nadia

last: El Mammeri

first: Benjamin

last: Dartigues

first: Philippe

last: Pasdois

first: Benoit

last: Pinson

first: Macha

last: Nikolski

first: Anne

last: Devin

first: Arnaud

last: Mourier

first: Roland

last: Ullrich

first: Thomas

last: Daubon


title: Effect of the loss of the sRNA BasA on Borrelia burgdorferi gene expression under two in vitro growth conditions

city: Bryan

first: Jon

last: Skare

state: TX

address: 8447 John Sharp Pkwy

institute: Texas A&M University Health Science Center

summary: Puprose: The goal of this experiment was to determine if the sRNA BasA regulates borrelial gene expression. Methods: We genetically inactivated BasA in B. burgdorferi B31 5A4 NP1 and complemented BasA into the mutant to then examine gene expression differences due to the loss of BasA. We grew our B. burgdoferi in vitro in both BosR-RpoS uninduced (32ºC, pH 7.6, 1% CO2) conditions and induced (37ºC, pH 6.8, 5% CO2) conditions to mimic the tick phase and mammalian phase of the borrelial enzootic cycle, respectively. These strains were prepared for total RNA sequencing. Results: When the BasA mutant was compared to the parent strain 5A4 NP1, 61 transcripts in uninduced conditions and 103 transcripts in induced conditions were signifcantly dysregulated. The majority of the transcripts dysregulated by the loss of BasA in induced conditions are known to be regulated by B. burgdorferi BosR and RpoS. Conclusions: The sRNA BasA regulates important borrelial virulence factors.

overall_design: RNA-seq profiling of B. burgdorferi B31 5A4 NP1, ΔBasA, and the ΔBasA complement derivatives in both BosR-RpoS uninduced in vitro growth conditions (32ºC, pH 7.6, 1% CO2) and BosR-RpoS induced in vitro growth conditions (37ºC, pH 6.8, 5% CO2).

first: Brittany

last: Shapiro


title: ACTB methylation regulates SMARCA4 genomic occupancy to promote translation and reduce adhesion in colorectal cancer cells

city: Be'er Sheva

first: Dan

last: Levy

address: David Ben Gurion Blvd 1

country: Israel

institute: Ben-Gurion University

summary: ACTB is a cytoskeletal protein involved in intracellular trafficking. In recent years, it has become evident that, in addition to its established roles in these compartments, ACTB also participates in the regulation of transcription. However, the molecular mechanisms underlying this function remain poorly understood. The methyltransferase SETD3 has previously been shown to methylate ACTB at H73, thereby regulating ACTB polymerization and smooth muscle contraction. Here, we show that the genomic distribution of ACTB is SETD3-dependent and that this regulation modulates the transcription of genes involved in cell adhesion and mRNA translation in colorectal cancer cells. Proteomic analyses reveal that ACTB and SETD3 interact with multiple large protein complexes, including complexes associated with transcriptional regulation. Specifically, we demonstrate that SETD3-mediated ACTB methylation is required for the co-localization of SMARCA4, a subunit of the SWI/SNF BAF complex, at specific genomic loci. Genomic analyses further show that this co-localization enables the coordinated occupancy of SMARCA4 and H73-methylated ACTB at genes involved in cell adhesion and mRNA translation. Finally, phenotypic assays confirm these regulatory effects. Together, these findings uncover a new mechanistic layer of selective transcriptional regulation mediated by an ACTB–SETD3–SMARCA4 axis in colorectal cancer cells.

overall_design: Cut&Run for Flag_Actin_Wt and BRG1 in Control and SETD3 KOs cells

first: Abeav-Schneiderman

last: Elina


overall_design: RNA-Seq in Actin KO cell stably expressing Empty, Actin WT, Actin H73A mut

first: Elina

last: Abeav-Schneiderman


title: Wnt Activation and Dual SMAD Inhibition For Induction and Maintenance of Hindbrain-like Neural Stem Cell from hiPSCs [RNA-Seq]

city: Kyoto

first: Makoto

last: Ikeya

address: 53 kawahara-cho, shogoin

department: Center for iPS Cell Research and Application(CiRA)

country: Japan

institute: Kyoto University

summary: Neurons exhibit region-specific identities corresponding to functional distinctions across different brain areas. Region-restricted neural stem cells (NSCs) have previously been generated from pluripotent stem cells; however, maintaining their regional identity over extended passages remains challenging. Here, we report the generation of hindbrain-like induced NSCs (Hb-LiNSCs) with upregulated hindbrain-specific markers and downregulated forebrain, midbrain, and spinal cord markers under xeno- and basic fibroblast growth factor-free conditions using three chemicals—CHIR99021 (at a high concentration), a potent activator of the Wnt pathway, A-83-01, a potent inhibitor of the TGF-β/Activin/Nodal pathway, and LDN193189, a potent inhibitor of the bone morphogenetic protein pathway. Hb-LiNSCs maintained their chromosomal integrity, multipotency, and differentiation capacity even after long-term culture for more than 60 weeks. This innovative approach enhances our understanding of neurodevelopmental and neurodegenerative processes in the hindbrain region and paves the way for developing targeted cell-based therapy as well as disease modeling for drug discovery.

overall_design: Human iPSCs were induced into hindbrain like neural stem cells using small molecules then maintained for 60 weeks in culture and sequenced at different timepoints

first: Ziadoon

last: Al-Akashi

first: Denise

last: Zujur

first: Nicholas

last: Boyd-Gibbins

first: Nathalie

last: Wiguna

first: Masato

last: Nakagawa

first: Tetsuhiro

last: Kikuchi

first: Asuka

last: Morizane

first: Jun

last: Takahashi


title: Bridge to transplant using a flow-adaptive extracorporeal total artificial lung system following bilateral pneumonectomy for septic ARDS

city: Chicago

first: Yuanqing

last: Yan

state: IL

address: 303 E Superior Street

institute: Northwestern University

summary: Acute respiratory distress syndrome (ARDS), when complicated by secondary pneumonias caused by nosocomial antibiotic-resistant pathogens and septic shock, can carry mortality rates exceeding 80%. We performed single-cell RNA sequencing and Visium HD spatial transcriptomics on an explanted lung from a 33-year-old patient with influenza-associated ARDS complicated by carbapenem-resistant Pseudomonas aeruginosa pneumonia and refractory septic shock. Our results demonstrated diffuse and uniform tissue destruction in the affected lung, with dense infiltration by neutrophils, monocyte-derived alveolar macrophages, and activated T cells. We also observed marked expansion of aberrant basaloid epithelial cells and CTHRC1-positive myofibroblasts coexisting with widespread inflammation, accompanied by near-complete loss of normal alveolar architecture. The resulting molecular signatures recapitulated those seen in end-stage fibrotic lung disease and were consistent with irreversible injury rather than a recoverable ARDS phenotype.

overall_design: Snap-frozen lung samples were obtained for single-cell RNA sequencing (scRNA-seq) using the 10X Genomics Chromium Single Cell Flex Gene Expression Assay and sequenced on a HiSeq 4000. A total seven samples from different regions were collected for sequencing. Raw scRNA-seq reads were demultiplexed with CellRanger (v7.1.0) and mapped to the human genome (HG38). FFPE samples were used for spatial transcriptomic analysis, and the sequencing reads were mapped to human genome (HG38).

first: Anitha

last: Chandrasekhar

first: Hee Chul

last: Yang

first: Teja

last: Devarakonda

first: Aaron

last: Reynolds

first: Anjana V.

last: Yeldandi

first: Arunachalam

last: Ambalavan

first: Chitaru

last: Kurihara

first: G.R. Scott

last: Budinger

first: Ankit

last: Bharat


title: YAP disrupts bile acid homeostasis to drive cancer-associated cachexia [Crispant_RNAseq]

city: Melbourne

first: Andrew

last: Cox

state: Victoria

address: 305 Grattan St

country: Australia

institute: PeterMacCallum Cancer Centre

summary: Cancer-associated cachexia is a severe metabolic syndrome marked by dramatic loss of adipose and muscle mass. Although preclinical models have advanced our understanding of cachexia, there are still no approved therapies due to the limited insights into the mechanisms underlying tissue wasting. Here, we utilise a YAP-driven model of liver cancer in zebrafish, which rapidly develops cachexia, to uncover an evolutionarily conserved role for bile acid disruption in the onset of cachexia. Spatial transcriptomic analysis revealed that YAP induces a bi-lineage cholangiocarcinoma phenotype, which was associated with bile acid dysregulation. Mechanistically, we establish that both bile acid synthesis (via CYP7A1) and signalling through the bile acid receptor TGR5 are essential for cachexia induction. Notably, we find that the promotion of bile acid excretion with odevixibat ameliorates cachexia. Together, our findings reveal an evolutionarily conserved mechanism by which YAP promotes cachexia and suggest a potential therapeutic strategy to treat the syndrome

overall_design: Bulk RNA sequencing of whole zebrafish larvae at 21 days post-fertilisation (dpf), including wild-type (WT) controls and cachectic tumour-burdened p53 knockout zebrafish with constitutive hepatic YAP overexpression (PY). Samples underwent CRISPR/Cas9-mediated knockouts targeting the melanin transporter SLC45a2 (used as a pigmentation marker), either alone, (SLC45a2-KO), or in combination with the bile acid-responsive metabolic receptor TGR5 (TGR5-KO) or the classical bile acid synthesis enzyme CYP7a1 (CYP7a1-KO).

first: Madeline

last: Webb


title: scRNA-Seq of Retinal microglia from 6 months old Balb/c mice infected with MCMV at birth

city: Philadelphia

first: Jessica

last: McCord

institute: Thomas Jefferson University

summary: We used a single cell sequencing approach using 10x Genomics scRNAseq to understand the transcriptional differences between microglia from MCMV infected animals vs controls. MCMV infection at birth resulted in increased retinal degeneration with age. Microglia from mice infected with MCMV appeared to have an alternative activation state compared to age matched uninfected controls. We utilized a depletion and repopulation strategy to reset the population of microlgia using PLX5622, a CSFR1 inhibitor. This study allowed analysis of the transcriptional changes caused by MCMV infection early in life potentially contributing to neurodegeneration.

overall_design: Balb/c mice were infected with MCMV or injected with PBS (controls) less than 24hrs after birth. PLX5622 (a CSF1R inhibitor) was adminstered 2 months after infection for 7 days to deplete microglia. Microglia repopulated 21 days (3 months of age) and were aged to 6 months. Whole eyes were collected at 6 months from 5 Control (uninfected untreated), Infeted (MCMV infected untreated) and PLX (MCMV infected treated). Retina and RPE/choroid were isolated and Microglia were seperated by FACs. Pooled microglia from 5 Retina and RPE/Choroid from 5 mice per group were analyized by sc- RNA seq.

first: Christopher

last: Snyder


title: FVE and HDA9 form a complex to promote thermomorphogenesis by reducing H2A.Z deposition through histone deacetylation [RNA-Seq]

first: Xiaoyi

last: Li

summary: Many plants, including Arabidopsis thaliana, respond to elevated ambient temperatures by altering their growth through a process known as thermomorphogenesis. This response involves the depletion of the repressive histone variant H2A.Z from the gene bodies of PIF4-regulated auxin-related genes, enabling their transcriptional activation. Interestingly, this activation also requires the histone deacetylase HDA9, raising the question of how histone deacetylation, typically associated with transcriptional repression, can instead promote gene activation. Here, we identify FVE as a co-regulator that partners with HDA9 to activate PIF4 target genes at elevated temperatures. PIF4 directly interacts with and recruits the FVE-HDA9 complex to its target genes to remove acetylation from histone H4 and H2A.Z. We show that H2A.Z acetylation is required for recruiting the SWR1 complex, which deposits H2A.Z. Consequently, FVE-HDA9-mediated deacetylation reduces SWR1 complex binding and limits H2A.Z deposition. Moreover, we demonstrate that in addition to limiting H2A.Z deposition, H2A.Z depletion also results from H2A.Z eviction mediated by the INO80 complex. Together, these findings uncover a dual mechanism contributing to H2A.Z depletion: INO80-mediated active eviction and histone deacetylation-mediated inhibition of H2A.Z deposition, which underlies PIF4 target gene activation and explain the paradoxical role of histone deacetylation in transcriptional activation.

overall_design: For RNA extraction, 7-day-old seedlings grown at 21°C were shifted to 27°C or kept at 21°C with or without mannitol treatment at ZT1, and seedlings were collected at ZT13. The extracted RNA was used for library preparation and sequencing

first: Danhua

last: Jiang

first: Hui


title: ID3 Deficiency Alters Chromatin Accessibility at DSB Sites and Enhances Vulnerability to HDAC Inhibition [ATAC-seq]

city: Heidelberg

first: Ali

last: Bakr

address: Im Neuenheimer Feld 280

department: Cancer Epigenomics

institute: Deutsches Krebsforschungszentrum

summary: The inhibitor of DNA-binding 3 (ID3) plays a crucial role in DNA double-strand break (DSB) repair. We previously reported that ID3 loss reduced chromatin accessibility at DNA repair gene promoters, yet its exact role in DNA repair via chromatin regulation remained elusive. Using the AID-DIvA cell system with inducible DSBs, we show that ID3 directly regulates chromatin accessibility at DSB sites, as demonstrated by reduced chromatin accessibility and lower H3K27ac levels in ID3-knockout (KO) cells. Loss of ID3 renders cells highly sensitive to histone deacetylase (HDAC) inhibition, particularly to the Class I HDAC inhibitor, leading to the accumulation of unrepaired DSBs and delayed cell cycle progression. Transcriptome and proteome analyses revealed that HDAC inhibition in ID3-KO cells results in the downregulation of gene sets involved in the regulation of cell cycle and cell division. The synthetic lethality observed between ID3 loss and Class I HDAC inhibition underscores a novel therapeutic vulnerability in ID3-deficient cancers, driven by compounded defects in chromatin remodeling, cell cycle, and DNA repair. Our study provides new insights into the relationship between chromatin regulation and genome stability, with implications for targeted cancer therapies.

overall_design: ATAC-seq on wild-type and ID3-KO AID-DIvA U2OS cells, either untreated or treated with 4-hydroxytamoxifen (4-OHT). Three replicates for each condition.

first: Giuditta Della

last: Corte


title: Transglutaminase 2 regulates vimentin-dependent proteostasis during macrophage activation in sepsis

city: Yokohama

first: Xian-Yang

last: Qin

address: 1-7-22 Suehiro, Tsurumi

department: RIKEN Center for Integrative Medical Sciences

institute: Institute of Physical and Chemical Research

summary: Sepsis-associated liver dysfunction (SALD) is a life-threatening condition with a high mortality rate and no mechanism-based therapy. In this study, we identified transglutaminase 2 (TG2) as a druggable driver of macrophage-mediated liver inflammation in murine lipopolysaccharide (LPS)-induced sepsis. Pharmacological inhibition of TG2 significantly improved survival and reduced multi-organ inflammation, with the liver as a primary therapeutic target. Mechanistically, TG2 activity was upregulated in macrophages, where it cross-linked vimentin to promote oligomerization and intermediate filament remodeling. Genetic ablation of TG2 or vimentin suppressed macrophage cytokine production and attenuated LPS-induced inflammation. Notably, vimentin-deficient macrophages exhibited enhanced proteasome recruitment to detergent-insoluble protein aggregates, accelerating the degradation of proinflammatory mediators such as TRAF6, thereby dampening NF-κB signaling. Proteomic profiling revealed a previously unrecognized Rab27a-positive vesicle trafficking pathway for inflammatory aggregate clearance. Together, these findings define a TG2–vimentin axis that controls macrophage activation through proteostasis regulation, linking cytoskeletal remodeling to inflammatory signaling. Our study suggests that targeting this axis, either via TG2 inhibition or modulation of vimentin-dependent proteostasis, represents a novel therapeutic strategy for the treatment of SALD.

overall_design: RNA-seq profiling of mouse liver under various inflammatory conditions, including PBS, LPS, and LPS+CTM treatment; with additional groups involving macrophage depletion (PBS and LPS) and TG2 knockout mice (PBS and LPS).

first: Wenkui

last: Yu

first: Harukazu

last: Suzuki

first: Yali

last: Xu

first: Ting

last: Su

first: Mishra

last: Hricha

first: Naoshi

last: Dohmae

first: Takehiro

first: Yuriko

last: Sakamaki

first: Haruyo

last: Aoyagi

first: Hideki

last: Aizaki

first: Hajime

last: Nishimura

first: Erina

last: Furuhata

first: Yuqing

last: Gong

first: Beiyuan

last: Zhang

first: Chenzhe

last: He

first: Yutaka

last: Furutani

last: Tatsukawa


title: PU.1-Activated Genomic Regions Define Low-risk MDS Subsets Characterized by Immune Dysregulation and Disease Progression [MDS-L ChIP-Seq]

city: Milan

first: Serena

last: Ghisletti

address: Via Adamello 16

department: Department of Experimental Oncology

institute: European Institute of Oncology

summary: Myelodysplastic syndromes (MDS) are heterogeneous myeloid neoplasms with an increased risk of progression to secondary acute myeloid leukemia (sAML). This study investigates the genomic correlates of disease progression in MDS by profiling active genomic regulatory regions and their transcriptional impact through H3K27ac ChIP-seq and RNA-seq analysis on CD34+ bone marrow progenitors cells isolated from a prospective cohort of 86 and 357 patients, respectively. Our analysis revealed distinct patterns of genomic region activation and transcriptional regulation across different disease stages (low-risk MDS, high-risk MDS and sAML). Unexpectedly, unsupervised clustering revealed a subset of low-risk MDS patients displaying regulatory and transcriptional profiles similar to those of high-risk MDS and sAML, highlighting early molecular events that may predispose patients to disease progression. This subset is characterized by PU.1 genomic occupancy in regions linked to immune and inflammatory responses, increased T-cell and NK activation, and a higher frequency of SRSF2 mutations. Clinically, patients in this group exhibit greater susceptibility to infections and cardiovascular events, along with an elevated risk of disease progression, resulting in a significantly reduced overall survival. Functional studies demonstrate that PU.1 inhibition suppresses MDS cell proliferation and clonogenicity, as impaired PU.1 binding inhibits the activation of key transcriptional programs involved in disease advancement. Collectively, these findings identify epigenetic factors that predispose low-risk MDS patients to progression into high-risk MDS and, ultimately, sAML. Moreover, they provide proof of concept for targeting PU.1 as a potential strategy to prevent disease progression in low-risk MDS.

overall_design:
To investigate the regulatory mechanisms underlying gene expression in MDS, we performed H3K27ac ChIP-seq on CD34⁺ bone marrow cells from patients at different stage of disease, focusing on transcription factors potentially involved in disease evolution and progression to secondary AML (sAML).
To investigate the functional role of PU.1 in regulating gene expression, we pharmacologically inhibited its activity by treating MDS-L cells with the PU.1 inhibitor DB2115.

first: Veronica

last: Vallelonga

first: Francesco

last: Gandolfi

first: Matteo

last: Zampini

first: Elena

last: Riva

first: Giulia

last: Maggioni

first: Denise

last: Ventura

last: Saba

first: Alberto

last: Termanini

first: Sara

last: Polletti

last: Prosperini

first: Laura

last: Crisafulli

first: Alessia

last: Campagna

first: Ivan

last: Ferrari

first: Nicole

last: Pinocchio

first: Gabriele

last: Todisco

first: Michela

last: Calvi

first: Clara

last: Di Vito

first: Domenico

last: Mavilio

first: Francesca

last: Ficara

last: Giovanni Della Porta


title: FVE and HDA9 form a complex to promote thermomorphogenesis by reducing H2A.Z deposition through histone deacetylation [ChIP-Seq]

first: Xiaoyi

last: Li

overall_design: Examination of H2A.Z enrichment in seedlings

first: Danhua

last: Jiang


title: ID3 Deficiency Alters Chromatin Accessibility at DSB Sites and Enhances Vulnerability to HDAC Inhibition [RNA-seq]

first: Ali

last: Bakr

overall_design:
RNA-seq on wild-type and ID3-KO AID-DIvA U2OS cells, either untreated or treated with mocetinostat and allowed to recover. Three biological replicates for each condition.

RNA-seq on wild-type and ID3-KO U2OS cells, either untreated or treated with mocetinostat. Three biological replicates for each condition.

first: Giuditta Della

last: Corte


title: Cleavage of the RNA polymerase II general transcription factor TFIIB tunes transcription during stress

city: Berkeley

first: Britt

last: Glaunsinger

state: CA

address: 565 Li Ka Shing Center

department: Department of Plant and Microbial Biology

institute: University of California, Berkeley

summary: Cellular stressors often cause widespread repression of RNA polymerase II (RNAP II) activity, which is thought to facilitate a focused transcriptional output towards stress resolution. In many cases, however, the underlying regulatory mechanisms remain unknown. Here, we demonstrate that stress-induced downregulation of the general transcription factor TFIIB tempers expression of specific stimulus response genes. Following a variety of stressors, TFIIB is proteolytically cleaved between its cyclin folds at conserved aspartic acid residue D207 by caspases- 3 and 7. Cleavage in this portion of the protein significantly reduces the ability of TFIIB to form a TBP-TFIIB-DNA promoter complex in vitro. Using both overexpression and endogenous base-editing, we find that B and T cells that are unable to cleave TFIIB upregulate expression of a select gene set during apoptosis. These TFIIB-sensitive genes are primarily short, stimulus-responsive and proto-oncogenic loci, and cleavage of TFIIB temporally restricts their expression. Failure to cleave TFIIB during stress leads to aberrant lymphocyte proliferation during chemical perturbation. Hence, caspase targeting of TFIIB destabilizes transcription to tune gene expression, allowing for proper stress resolution.

overall_design: Jurkat cells stably bearing siRNA-resistant 6xHis-TFIIB-V5 under a doxycycline-inducible promoter were induced to express the transgene at near-endogenous levels. Simultaneously, endogenous TFIIB was depleted via siRNA. ChIP-sequencing of the RNAP II catalytic subunit RPB1 and TFIIB-V5 was subsequently performed after 4h of mock treatment or TRAIL ligand-induced apoptosis.

first: Leah

last: Gulyas

first: Azra

last: Lari

first: Sahil

last: Shah


title: Biopsy-derived colonoid air-liquid interface monolayers reveal persistent mucosal defects in ulcerative colitis patients

city: Vancouver

first: Irvin

last: Ng

state: BC

address: 950 West 28th Avenue Room #211 Room #211

country: Canada

institute: BC Children's Hospital Research Institute

summary:
Background: The Inflammatory Bowel Disease (IBD) ulcerative colitis (UC) is characterized by colonic mucosal inflammation and barrier dysfunction. We hypothesize that UC causes persistent defects in mucosal homeostasis, evident even in the absence of active inflammation, contributing to disease chronicity.

Methods: To test this, we grew patient biopsy-derived sigmoid colonoids into air-liquid interface (ALI) monolayers, characterizing them through microscopy, proteomics, bulk RNAseq, and their susceptibility to UC patient-isolated Escherichia coli pathobiont p19A.

Results: Non-IBD ALI monolayers formed uniform crypt-like structures and a thick mucus layer containing all epithelial-derived proteins previously identified in human colonic mucus. In contrast, ALI monolayers from UC patients displayed a range of impairments, classified as a mild phenotype with distorted architecture and a thinner, more permeable mucus layer to a severe phenotype with defects in cellular differentiation and an inability to produce a mucus layer. Transcriptome analysis identified activated pathways associated with extracellular matrix formation and cell signaling, including numerous cancer-associated genes in UC ALI monolayers, which also proved significantly more susceptible to E. coli p19A.

Conclusions: Taken together, the culturing of patient biopsies into ALI colonoid monolayers provides a powerful model to assess human colonic mucosal development, healing, homeostasis, and mucus barrier function, revealing that UC-derived colonoid monolayers display a range of developmental and functional defects that persist in the absence of inflammation.

overall_design: RNA-seq profiling of non-IBD and ulcerative colitis patient-derived colonoids grown as air-liquid interface (ALI) monolayers collected on Day 14.

first: Ashley

last: Gilliland

first: Yan

last: Chen

first: Xiao

last: Han

first: Ho Pan

last: Sham

first: Itay

last: Kalisky

first: Dominique

last: Lévesque

first: Kevan

last: Jacobson

first: Wei

last: Xiong

first: François-Michel

last: Boisvert

first: Brian

last: Bressler

first: Bruce

last: Vallance


title: In Vitro Fertilization Induces Premature Reproductive Aging and Multigenerational Epigenetic Changes in Female Mouse Offspring

first: Eric

middle: Alejandro

last: Rhon Calderon

state: Pennsylvania

address: 3400 Civic Center Blvd, Bldg 421, Floor 9-175

department: Cell and Developmental Biology

institute: University of Pennsylvania

summary: In vitro fertilization (IVF) is an assisted reproductive technology that has enabled millions of births. Although considered safe, emerging evidence suggests that IVF may have long-term health adverse outcomes in offspring, though its impact on female reproduction, especially on reproductive aging is largely unknown. Here, we use a mouse model to investigate how IVF influences female reproductive and transgenerational health outcomes. We assessed IVF-conceived fetal (E18.5) and adult (12-week and 39-week) female offspring, identifying alterations in ovarian-to-body weight ratios, ovarian morphology, serum sex hormone levels, and transcriptomic and DNA methylation profiles in ovaries, oocytes, and cumulus cells, consistent with biomarkers of accelerated reproductive aging. The offspring of IVF females and wild-type males exhibited altered fetal-to-placental weight ratios, placental morphology, and dysregulated placental gene expression. At 12-weeks-of-age, offspring showed increased body weight, disrupted lipid and glucose metabolism, and transcriptomic and epigenetic changes in liver and reproductive organs. Our results highlight the need for deeper mechanistic understanding of assisted reproduction’s long-term and multigenerational impacts on female reproductive health.

overall_design: Gene expression analysis using Total RNA extracted from F1: E18.5 ovaries (n=10, Natural=4 and IVF=6), 12 weeks ovaries (n=10, Natural=5 and IVF=5), 39 weeks ovaries (n=10, Natural=4 and IVF=6), 12 weeks single oocytes (n=20, Natural=10 and IVF=10), 39 weeks single oocytes (n=20, Natural=10 and IVF=10), 12 weeks cumulus cells (n=4, Natural=2 and IVF=2), 39 weeks cumulus cells (n=4, Natural=2 and IVF=2) and embryonic day 13.5 female gonads (n=9, Natural=4 and IVF=5). F2: 12 weeks testis (n=12, Natural=5 and IVF=7), 12 weeks male livers (n=10, Natural=5 and IVF=5), 12 weeks ovaries (n=9, Natural=4 and IVF=5), 12 weeks female livers (n=10, Natural=4 and IVF=4), using either Illumina NovaSeq6000 or NextSeq1000.

last: Rhon-Calderon

first: Cassidy

last: Hemphill

first: Alexandra

last: Savage

first: Christopher

last: Krapp

first: Zhengfeng

last: Liu

first: Laren

last: Riesche

first: Richard

middle: M

last: Schultz

first: Marisa

last: Bartolomei


title: Dnmt1 mediates epigenetic restriction of invasive traits in clonal crayfish [scRNA-seq]

first: Jaime

last: Diaz-Larrosa

state: Baden Württemberg

address: INF 580

department: Epigenetics

institute: DKFZ

summary: Marbled crayfish (Procambarus virginalis) are a parthenogenetically reproducing, globally invasive freshwater crayfish species. Previous work has shown that the global population is characterized by a monoclonal genome, which raises important questions about the mechanisms that promote their invasiveness. Dnmt1 is an evolutionarily conserved DNA methyltransferase responsible for maintaining 5-methylcytosine (5mC) patterns across the genome. While its relevance in transcriptional regulation and cell fate specification is well established, its role in ecological adaptation remains poorly understood. Here we show that environmental changes lead to significant downregulation of Dnmt1 in marbled crayfish. When mimicking this effect through a dsRNA-based in vivo knockdown of Dnmt1, we observed that invasiveness-related behavioral traits, such as activity and boldness were enhanced. Using image cytometry and single-cell RNA sequencing, we also detected an increase in the most mature immune cell type, the granular cells and depletion of hemocyte-derived neuronal precursors, which support adult neurogenesis. Further analysis by whole-genome bisulfite sequencing showed that these phenotypic changes are underpinned by a global reduction in gene body DNA methylation and a dysregulation of genes involved in nervous and immune system functions. Additionally, we observed nucleosome destabilization as a key mechanism influencing transcriptional changes after methylation loss. Our findings highlight a role for Dnmt1 in the canalization of cellular and organismal phenotypes, and provide a paradigm for how downregulation of Dnmt1 can enhance invasive traits.

overall_design: 28 days post injection of, 3 control animals (injected with dsGFP) and 3 Dnmt1 KD (injected with dsDNM1) were bled and the hemocytes isolated. 10 X genomics were used for single-cell RNA-sequencing.

first: J. Jaime

first: Vitor

last: Carneiro

first: Katharina

last: Hanna

first: Günter

last: Raddatz

first: Frank

last: Lyko


title: Dnmt1 mediates epigenetic restriction of invasive traits in clonal crayfish [RNA-seq]

overall_design: RNA-seq from 2 Control (dsGFP) and 2 Dnmt1-KD (dsDNMT1) animals’ hemocytes 28 days post-injection. These samples were also submitted for WGBS


title: Dnmt1 mediates epigenetic restriction of invasive traits in clonal crayfish [BiSulfite-seq]

type: Methylation profiling by high throughput sequencing

overall_design: 28 days post injection of, 2 control animals (injected with dsGFP) and 2 Dnmt1 KD (injected with dsDNM1) were bled and the hemocytes isolated. After bisulfite-converting the genomic DNA, samples were submitted for sequencing. These samples (before conversion) were split to use for RNA-seq as well


title: A plant histone H3.3-specific amino acid safeguards the deposition of H3K36 methylation for proper plant development and stress responses [Bisulfite-Seq]

city: Beijing

first: Xiaoyi

last: Li

address: west beichen

institute: Institute of Genetics and Developmental Biology

summary: Histone variants are critical determinants for chromatin function with diverse regulatory mechanisms. The H3 variants, H3.1 and H3.3, evolved independently in animals and plants. A significant difference between H3.1 and H3.3 is the amino acid variation at position 31, with H3.1 carrying alanine (A), while H3.3 bearing serine (S) in animals or threonine (T) in plants. Both S and T can be phosphorylated, but why plants have selectively adopted T over S remains unclear. Here, we report the specific role of plant H3.3T31 in controlling plant development and stress responses by promoting the deposition of histone H3 lysine 36 trimethylation (H3K36me3) on H3.3. T31 prevents plant-specific H3K27 methyltransferases, ATXR5 and ATXR6, from depositing H3K27 monomethylation (H3K27me1), which inhibits the activity of H3K36 methyltransferase EFS. Substituting H3.3T31 with an S or A residue results in increased ATXR5/6 activity and elevated H3K27me1 levels, leading to a reduction in H3K36me3. Moreover, we show that unlike H3.3 T31S and T31A mutations, cancer-associated G34R and G34W mutations directly disrupt H3K36me3 deposition without affecting H3K27me1. These G34 mutations may also influence H3.3 function through mechanisms beyond the disruption of H3K36me3. Our data suggest a co-evolution of the plant-specific H3.3T31 residue and H3K27 methyltransferases ATXR5/6, which ensures the selective accumulation of H3K27me1 on H3.1 and H3K36me3 on H3.3, thereby enabling proper chromatin function.

overall_design: Examination of the levels of DNA methylation in seedlings

first: Danhua

last: Jiang


title: A plant histone H3.3-specific amino acid safeguards the deposition of H3K36 methylation for proper plant development and stress responses [H3K36me3_ChIPseq]

overall_design: Examination of H3K36me3 enrichment in seedlings


title: PU.1-Activated Genomic Regions Define Low-risk MDS Subsets Characterized by Immune Dysregulation and Disease Progression [RNA-seq]

first: Serena

last: Ghisletti

summary: Myelodysplastic syndromes (MDS) are heterogeneous myeloid neoplasms with an increased risk of progression to secondary acute myeloid leukemia (sAML). This study investigates the genomic correlates of disease progression in MDS by profiling active genomic regulatory regions and their transcriptional impact through H3K27ac ChIP-seq and RNA-seq analysis on CD34+ bone marrow progenitors cells isolated from a prospective cohort of 86 and 357 patients, respectively. Our analysis revealed distinct patterns of genomic region activation and transcriptional regulation across different disease stages (low-risk MDS, high-risk MDS and sAML). Unexpectedly, unsupervised clustering revealed a subset of low-risk MDS patients displaying regulatory and transcriptional profiles similar to those of high-risk MDS and sAML, highlighting early molecular events that may predispose patients to disease progression. This subset is characterized by PU.1 genomic occupancy in regions linked to immune and inflammatory responses, increased T-cell and NK activation, and a higher frequency of SRSF2 mutations. Clinically, patients in this group exhibit greater susceptibility to infections and cardiovascular events, along with an elevated risk of disease progression, resulting in a significantly reduced overall survival. Functional studies demonstrate that PU.1 inhibition suppresses MDS cell proliferation and clonogenicity, as impaired PU.1 binding inhibits the activation of key transcriptional programs involved in disease advancement. Collectively, these findings identify epigenetic factors that predispose low-risk MDS patients to progression into high-risk MDS and, ultimately, sAML.

overall_design:
To comprehensively characterize the transcriptional alterations occurring during disease progression, we analysed bone marrow progenitors from a prospective, unselected cohort of 357 patients. Our analyses were exclusively performed on isolated CD34+ bone marrow cells, which include the disease-initiating population in MDS from a cohort of 209 MDS patients plus another set of 148 sAML patients.
To investigate the functional role of PU.1 in regulating gene expression, we pharmacologically inhibited its activity by treating MDS-L cells with the PU.1 inhibitor DB2115.
To assess the functional role of PU.1, we employed CRISPR/Cas9-mediated depletion of PU.1 in CD34⁺ MDS-L cells after testing five different sgRNAs targeting PU.1 were tested in bulk cell populations.

***************************************************************
Raw files for human/patient samples were not submitted to GEO due to concerns about submitting personally identifiable sequence data for open access.
***************************************************************

first: Veronica

last: Vallelonga

first: Francesco

last: Gandolfi

first: Matteo

last: Zampini

first: Elena

last: Riva

first: Giulia

last: Maggioni

last: Saba

first: Alberto

last: Termanini

first: Sara

last: Polletti

last: Prosperini

first: Laura

last: Crisafulli

first: Alessia

last: Campagna

first: Ivan

last: Ferrari

first: Nicole

last: Pinocchio

first: Gabriele

last: Todisco

first: Denise

last: Ventura

first: Francesca

last: Ficara

middle: G

last: Porta


title: PU.1-Activated Genomic Regions Define Low-risk MDS Subsets Characterized by Immune Dysregulation and Disease Progression [ChIP-seq]

last: Ghisletti

overall_design:
To investigate the regulatory mechanisms underlying gene expression in MDS, we performed H3K27ac ChIP-seq on CD34⁺ bone marrow cells from patients at different stage of disease, focusing on transcription factors potentially involved in disease evolution and progression to secondary AML (sAML).
To test whether the genomic distribution of PU.1 changes during the disease progression, we performed PU.1 ChIP-seq in CD34+ cells from a subset of LR-MDS and sAML patients.
To investigate the functional role of PU.1 in regulating gene expression, we pharmacologically inhibited its activity by treating MDS-L cells with the PU.1 inhibitor DB2115.

***************************************************************
Raw files for human/patient samples were not submitted to GEO due to concerns about submitting personally identifiable sequence data for open access.
***************************************************************

last: Vallelonga

first: Michela

last: Calvi

first: Clara

last: Di Vito

first: Domenico

last: Mavilio

last: Giovanni Della Porta


title: B cell imprinting in children impairs antibodies to the hemagglutinin stalk

city: New York

first: Patrick

last: Wilson

state: NY

address: 413 E 69th St

institute: Weill Cornell Medicine

summary: Immune imprinting or Original Antigenic Sin (OAS) is a phenomenon where the immune system preferentially recalls its initial response to a related, often evolving pathogen upon subsequent exposure. Despite its important implications for vaccine development, the causes of imprinting remain unclear. To understand the basis and impact of imprinting by influenza A viruses, we characterized the B cell responses of young children following consecutive first infections with divergent H1N1 and H3N2 strains. Children had a primary but otherwise similar B cell response to that of adults. Adult B cells commonly cross-reacted with past strains using more stereotyped and mutated immunoglobulin genes, indicating significant homosubtypic imprinting. In children, following consecutive heterosubtypic primary infections, up to 6% of memory B cells are H1/H3 cross-reactive and bind the highly conserved central stalk epitope, a lead target for broadly protective vaccine candidates. Over 90% of these B cells had higher affinity for the imprinting H3N2 strain, resulting in reduced breadth and neutralization potency against H1N1 strains. Mechanistically, the imprinting H3 and affected H1 strains shared a single residue change in the stalk epitope (D46N) that was central to the nearly universal shift in reactivity, despite differing by only a single atomic group. In conclusion, imprinting by influenza viruses can cause a deleterious shift of nearly the entire memory recall response against key, conserved epitopes.

overall_design: Blood samples were collected from 2- to 6-year-old children following sequential first-time exposures to H3N2 then H1N1 influenza viruses or the reverse order.  Blood from children only infected by H1N1 viruses and H1N1-infected adults were included as controls. Acute (d1) and convalescent (d30) HA-specific B cells were bait-sorted with oligonucleotide-barcoded probes together with bulk-sorted total CD19+ cells for 10X Genomics single-cell RNA sequencing, following a lab-adapted protocol similar to LIBRA-seq. The 5’ transcriptome, B cell receptor immunoglobulin VDJ gene (BCR) repertoire, and surface feature barcode profiles were obtained and analyzed.

first: Jiayi

last: Sun

first: Chloe

last: Troxell

middle: C


title: PU.1-Activated Genomic Regions Define Low-risk MDS Subsets Characterized by Immune Dysregulation and Disease Progression [MDS-L RNA-seq]

first: Serena

overall_design:
To comprehensively characterize the transcriptional alterations occurring during disease progression, we analysed bone marrow progenitors from a prospective, unselected cohort of 357 patients. Our analyses were exclusively performed on isolated CD34+ bone marrow cells, which include the disease-initiating population in MDS from a cohort of 209 MDS patients plus another set of 148 sAML patients.
To investigate the functional role of PU.1 in regulating gene expression, we pharmacologically inhibited its activity by treating MDS-L cells with the PU.1 inhibitor DB2115.
To assess the functional role of PU.1, we employed CRISPR/Cas9-mediated depletion of PU.1 in CD34⁺ MDS-L cells after testing five different sgRNAs targeting PU.1 were tested in bulk cell populations.

first: Veronica

first: Francesco

last: Gandolfi

first: Matteo

last: Zampini

first: Elena

last: Riva

last: Maggioni

last: Saba

last: Termanini

last: Polletti

last: Prosperini

last: Crisafulli

first: Silvia

last: Pedretti


title: PU.1-Activated Genomic Regions Define Low-risk MDS Subsets Characterized by Immune Dysregulation and Disease Progression.

last: Ghisletti

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

overall_design: Refer to individual Series


title: Non-disruptive in vitro monitoring of cellular states with cell-free DNA methylation

city: Berlin

first: Anja

last: Hess

email: ahess@molgen.mpg.de, anja.hess@mail.de

address: Ihnestrasse 63-67

institute: Max-Planck-Institut für molekulare Genetik

summary: Regenerative cell therapies and stem-based in vitro models all aspire the approximation of a given start cell to an alternate (often more mature) cell state. However, ‘monitoring’, hence controlling, such a dynamic process requires disruption of the precious sample, destroying architecture and introducing variation. Here, we establish real time-compatible, fully non-invasive monitoring of cell fates through simple cell-free DNA medium sampling. We demonstrate that cell-free DNA methylation non-disruptively reveals genome-wide DNA methylation transitions during epigenetic drug treatment in stem cells, and reports the gradual maturation of 3D hepatocytes grown in bioreactors. Our cell-free DNA methylation platform enables monitoring a broad variety of in vitro systems, has implications for the real-time evaluation human therapeutic cells, and the potential to be readily integrated into large-scale bioreactor workflows.

overall_design:
Whole genome bisulfite profiling of cell-free and genomic DNA from KH2 mESCs treated with DNMT1-Inhibitor GSK-3484862 for four days, followed by a two day recovery period. DMSO-treated cells served as a negative control.

Whole genome Nanopore-Sequencing of differentiating 3D bioreactor hepatocytes over 20 days.

first: Alexander

last: Kovacsovics

first: Fabian

last: Bachinger

first: Helene

last: Kretzmer

first: Ludovic

last: Vallier

last: Meissner


title: A plant histone H3.3-specific amino acid safeguards the deposition of H3K36 methylation for proper plant development and stress responses [H3K27me1_ChIPseq]

first: Xiaoyi

last: Li

overall_design: Examination of H3K27me1 enrichment in seedlings

first: Danhua

last: Jiang


title: Dnmt1 mediates epigenetic restriction of invasive traits in clonal crayfish [MNase-seq]

first: Jaime

last: Diaz-Larrosa

overall_design: 28 days post injection with dsGFP and dsDNMT1, 4 control and 6 KD animals were bled. Hemocytes were subject to a Micrococcal Nuclease incubation and sonication to release the Nucleosome-bound DNA, which was sequenced.

first: J. Jaime

first: Vitor

last: Carneiro

first: Katharina

last: Hanna

first: Günter

last: Raddatz

first: Frank

last: Lyko


title: A plant histone H3.3-specific amino acid safeguards the deposition of H3K36 methylation for proper plant development and stress responses [G34]

overall_design: To examine transcriptomic changes in h3.3ko;HTR5 G34R, h3.3ko;HTR5 G34W


title: A plant histone H3.3-specific amino acid safeguards the deposition of H3K36 methylation for proper plant development and stress responses [GFP_ChIPseq]

overall_design: Examination of H3.3 enrichment in seedlings


title: A plant histone H3.3-specific amino acid safeguards the deposition of H3K36 methylation for proper plant development and stress responses [T31A_RNAseq]

overall_design: To examine transcriptomic changes in h3.3ko;HTR5 T31A, h3.3ko;HTR5 K36A, and efs mutants


title: Nascent-seq of HeLa WT and SRSF5 KO cells

city: Frankfurt am Main

first: Michaela

last: Müller-McNicoll

address: Max-von-Laue-Str. 13

institute: Goethe University Frankfurt

summary: We used HeLa cells where SRSF5 was knocked out by CRISPR/Cas9 to perform Nascent-seq. WT cells served as control. One hour before harvesting, cells were incubated with 4-sU for exactly 1 hour to label newly transcribed RNAs. 4-sU-labelled RNAs were biotinylated and purified over streptavidin beads and transformed into an RNA-seq library. We compared differential expression and intron retention between WTand SRSF5 KO samples.

overall_design: We sequenced 2 biological replicates for all conditions (WT and KO) with 150-bp paired end reads and 100 Million reads per replicate.


title: A plant histone H3.3-specific amino acid safeguards the deposition of H3K36 methylation for proper plant development and stress responses

overall_design: To examine transcriptomic changes in h3.3ko;HTR5


title: RIPK1 prevents STING-dependent autoinflammation in keratinocytes

city: Ghent

first: Bruno

last: Verstraeten

state: Oost-Vlaanderen

address: Technologiepark 71

country: Belgium

institute: Ghent University

summary: RIPK1 is a key signaling kinase involved in inflammation and regulated cell death. In mouse keratinocytes RIPK1 prevents spontaneous activation of ZBP1-RIPK3-MLKL-mediated necroptosis, resulting in skin inflammation. Interestingly, ex vivo cultured Ripk1-deficient keratinocytes show no spontaneous phenotype, suggesting the need for an additional (exogenous) trigger to initiate ZBP1-mediated necroptosis. We show that inflammatory lesion development in keratinocyte-specific Ripk1 knock-out (Ripk1E-KO) mice is not rescued by antibiotics treatment or in germ-free conditions. Additionally, we excluded the adaptive immune system as a disease driver by crossing Ripk1E-KO animals into a Rag2/Il-2rg-deficient background. Together, these findings demonstrate that Ripk1E-KO mice develop a sterile autoinflammatory skin phenotype.

overall_design: Five groups of samples of skin tissue from keratinocyte-specific knockout (EKO) and wild type mice (four or five replicates/group) : 1) Lesional tissue of RIPK1-EKO mice, 2) Non lesional tissue of RIPK1-EKO mice, 3) Tissue from RIPK1 control mice, 4) Tissue from RIPK1 EKO-STING EKO, 5) Tissue from STING control mice.

first: Simon

last: Verdonck

first: Evelien

last: Dierick

first: Richard

last: De Reuver

first: Katrien

last: Staes

first: Jonathan

last: Maelfait


title: Senolytics rejuvenate aging cardiomyopathy in human cardiac organoids

city: Munich

first: Gianluca

last: Santamaria

state: Germany

address: Ismaninger Strasse 22

department: Department of Medicine I

institute: Klinikum rechts der Isar – Technical University Munich

summary: Human cardiac organoids closely replicate the architecture and function of the human heart, offering a potential accurate platform for studying aging cardiomyopathy. Senolytics have shown potential in addressing age-related pathologies. However, their potential to reverse aging-related human cardiomyopathy remains largely unexplored. We employed human iPSC-derived cardiac organoids (hCardioids) to model doxorubicin-induced cardiomyopathy in an aged context. hCardioids were treated with doxorubicin (DOXO) and subsequently with a combination of two senolytics: dasatinib (D) and quercetin (Q). DOXO-treated hCardioids exhibited significantly increased oxidative stress, DNA damage (pH2AX), cellular senescence (p16) and decreased cell proliferation associated with a senescence-associated secretory phenotype (SASP). DOXO-treated hCardioids were considerably deprived of cardiac progenitors and displayed reduced cardiomyocyte proliferation as well as contractility. These distinctive aging-associated characteristics were confirmed by global RNA-sequencing analysis. Treatment with D+Q reversed these effects, reducing oxidative stress and senescence markers, alleviating SASP, and restoring hCardioids viability and function Additionally, senolytics replenished cardiac progenitors and reversed the cardiomyocyte proliferation deficit. Doxorubicin triggers an age-associated phenotype in hCardioids representing a reliable model of aged cardiomyopathy. Senescence is a key mechanism of the aged phenotype as senolytics rejuvenated aged hCardioids restoring their structure and function while reverting the age-associated regenerative deficit.

overall_design:
To demonstrate that Doxorubicin exposure induces an aging cardiomyopathy phenotype in human cardiac organoids as confirmed by global transcriptome analysis and typical, cellular and molecular age-related features and cell senescence along with a senescence-associated secretory phenotype (SASP) in particular. Senescent cell ablation by senolytics treatment reversed these aging effects, restoring organoid viability and function and replenishing cardiac regeneration potentia
We then performed gene expression profiling analysis using data obtained from RNA-seq of 9  samples at three different experimental conditions
Comparative gene expression profiling analysis of RNA-seq data

first: Mariangela

last: Scalise

first: Eleonora

last: Cianflone

first: Claudia

last: Quercia

first: Loredana

last: Pagano

first: Antonio

last: Chiefalo

last: Stincelli

first: Annalaura

last: Torella

first: Brabara

last: Puccio

first: Hiram

last: P. Guzzi

first: Pierangelo

last: Veltri

first: Antonella

last: De Angelis

first: Konrad

last: Urbanek

first: Georgina

last: Hughes

first: Daniele

first: Fabiola

last: Marino


title: Human BM-MSC and BM-neurosphere transplantation shows limited efficacy as a therapeutic adjunct to surgical decompression in an implantable polymer-induced DCM model.

city: Hong Kong

first: Hyun Woo

last: Kim

address: Pokfulam Road

country: Hong Kong

institute: The University of Hong Kong

summary:
Human bone marrow-derived marrow stromal cell (BM-MSC) and neurosphere (BM-neurosphere) transplantation shows limited efficacy as a therapeutic adjunct to surgical decompression in an implantable polymer-induced degenerative cervical myelopathy (DCM) model
Degenerative cervical myelopathy (DCM) is a prevalent spinal cord disorder in the developed world. The study investigates the therapeutic potential of human bone marrow-derived mesenchymal stem cells (BM-MSC) and BM-neurospheres as adjunct therapies to surgical decompression in a polymer-induced DCM rat model. The study assessed locomotor function, blood-spinal cord barrier (BSCB) integrity, and cell engraftment. Results showed delayed locomotor recovery upon cervical decompression and limited efficacy of BM-MSCs and BM-neurospheres in improving outcomes under the experimental conditions.

overall_design: RNAseq on hBM-MSC (n=3) and hBM-neurospheres (n=3) from cultured from three bone marrow donors


title: Systemic lupus cutaneous photoresponses are characterized by dysregulated epidermal inflammation and inflammatory myeloid cell infiltration

city: Ann Arbor

first: J. Michelle

last: Kahlenberg

state: MI

address: NCRC 20-1841, 2800 Plymouth Rd

department: Department of Internal Medicine – Division of Rheumatology and Department of Dermatology

institute: University of Michigan

summary: For patients with systemic lupus erythematosus (SLE), sun avoidance and sunscreen use are the only preventive therapies for photosensitivity, a process by which exposure to ultraviolet (UV) light triggers cutaneous and systemic flares of disease. Photosensitivity is exceedingly common amongst SLE patients with skin disease, affecting up to 93% of this population. Treatment of skin and systemic disease triggered by the sun can be challenging, and thus there exists a critical need for better therapeutic and preventative strategies. Innovation of targeted therapies for photosensitive skin diseases requires a precise dissection of the cellular players and signaling pathways that contribute to aberrant inflammation following UV exposure. Here, we utilize cutaneous UVB photoexposure of healthy control and SLE patients to provide an atlas of the cutaneous inflammatory response 24 hours after UV exposure in single-cell resolution. We demonstrate that the epidermis is dysregulated in lupus, where epidermal-dermal crosstalk in SLE is dominated by inflammatory keratinocytes following UV exposure in contrast to the healthy control epidermis. In both healthy control and SLE, the downstream consequence of UV exposure is the recruitment of a population of monocyte-derived dendritic cells (moDCs). Though these moDCs are present in relatively equal proportions in SLE and healthy control UV-treated skin, they appear to be derived from distinct circulating monocyte populations. Notably, SLE UV moDCs are characterized by inflammatory and antigen presentation pathways and may represent a precursor to lesional cutaneous lupus CD16+ DCs, identifying a potential link between cutaneous UV responses and cutaneous lupus flare.

overall_design: Following signing of written informed consent, subjects were exposed to varying doses of UVB light via a Kodacel lamp ranging from 10mJ/cm2 to 80mJ/cm2. 24 hours after UV exposure, 6mm punch skin biopsies were taken from UV-unexposed skin as well as the portion of skin corresponding with the minimal erythema dose (MED). Patients who didn’t achieve MED were biopsied at the 80 mJ/cm2 site. All samples from all patients enrolled were included in the analysis. No outliers were excluded. Exclusion criteria for the study were active pregnancy, history of bleeding disorder, use of NSAIDs in the past 72 hours, CLASI>10 or SLEDAI>8 (reflecting high disease activity that could be exacerbated by UV exposure), ongoing use of prednisone >10 mg daily, active Cytoxan treatment or exposure within the past 6 weeks, absence of non-lesional skin on buttocks, or sunburn in the past month.

first: Mitra P.

last: Maz


title: Sympathetic-Epithelial Crosstalk Governs Regionalized CD8+ TRM Cell Immunosurveillance in the Skin

city: Hangzhou

first: Zhang

middle: Zhou

last: melanoma

state: China

address: 西湖大学医学院

institute: Westlake University

summary: Epithelial cells, immune cells, and nerves converge at barrier tissues to collaboratively defend against pathogens and malignancies, but the mechanisms mediating their coordination remain poorly understood. Here we show that in the skin, sympathetic nerves interact with epidermal stem cells (EpiSCs) to regulate CD8+ tissue-resident memory T (TRM) cell density, thereby modulating local immunosurveillance against developing melanoma. Sympathetic nerves anchor on skin epithelium and establish synapse-like connections with EpiSCs, modulating their secretion of chemokine ligand CXCL16 via norepinephrine-ADRB2 signaling. Reduced sympathetic tone elevates CXCL16 secretion, increasing CD8+ TRM cell abundance in the skin epithelium and enhancing local cancer immunosurveillance. Conversely, heightened sympathetic activity during acute stress decreases CD8+ TRM cell number, allowing nascent cancer cells to evade elimination and propagate. Our study unveils a neuro-epithelial-immune axis that governs CD8+ TRM cell dynamics at barrier tissues, and illustrates how regional immunosurveillance can be influenced by systemic neuronal inputs and mental status.

overall_design: To investigate what molecule was responsible for the recruitment of CD8+ T cells in skin epidermis, we treated the Gi-DREADD and control mice with CNO to suppress the activity of sympathetic neuron. EpiSCs were then isolated fluorescence-activated cell sorting (FACS)


title: Ustilago maydis disrupts carbohydrate signaling networks to induce hypertrophy in host cells

city: Köln

first: Gunther

last: Doehlemann

address: Zülpicher Straße 47a

institute: University of Cologne/ Universität zu Köln

summary: Ustilago maydis infection in maize causes hypertrophic leaf tumors; however, the underlying mechanisms driving this excessive cell growth are unknown. In this study, we identify Hap1 (hypertrophy-associated protein 1) as an effector and virulence factor that regulates mesophyll cell hypertrophy. Using CRISPR-Cas9 mutagenesis, we demonstrate that Hap1 contributes to endoreduplication and starch accumulation in infected tissues. Transcriptomics revealed Hap1-dependent upregulation of starch biosynthesis and cell cycle genes, as well as suppression of plant defense. This links Hap1 to metabolic and cell cycle reprogramming, and immune suppression. To identify the target of Hap1 that drives metabolic reprogramming, we investigated its interaction with ZmSnRK1α in maize. We found that Hap1 interferes with the phosphorylation of SnRK1 substrates and that two Hap1-interacting effectors, Hip1 and Hip2, enhance its protein stability. We conclude that Hap1 contributes to the reprogramming of maize metabolism and cell cycle, as well as mesophyll cell hypertrophy, by modulating the SnRK1 signaling pathway to regulate starch biosynthesis and host defense responses.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for maize leaves treated with water (control), SG200, SG200_CR-Hap1. Three biological replicates were generated.

first: Yoon Joo

last: Lee

first: Dong

last: Zhang

first: Sara

last: Stolze

first: Georgios

last: Saridis

first: Malaika

last: Ebert

first: Hirofumi

last: Nakagami