title: Halfpipe: a tool for analyzing metabolic labeling RNA-seq data to quantify RNA half-lives

city: Cologne

first: Achim

last: Tresch

state: North Rhine-Westphalia

address: Paul-Schallueck-Str. 10

department: Institute of Medical Statistics and Computational Biology

institute: University of Cologne

summary: We introduce Halfpipe, a tool for analyzing RNA-seq data from metabolic RNA labeling experiments. Its main features are the absolute quantification of 4sU-labeling-induced T>C conversions in the data, calculating the proportion of newly synthesized transcripts, and estimating (compartment-specific) RNA half-lives. Halfpipe excels at correcting critical biases caused by typically low labeling efficiency. We measure and compare the RNA metabolism in the G1 phase and during the mitosis of synchronized human cells. We find that RNA half-lives of constantly expressed RNAs are similar in mitosis and G1 phase, suggesting that RNA stability of those genes is constant throughout the cell cycle. Our estimates correlate well with literature values and with known RNA sequence features.

overall_design: HeLa-S3 cells were cultured at 37°C in DMEM supplemented with 10% FBS Superior (Sigma, S0615) and 1X penicillin-streptomycin (Gibco, 15070063).  A double thymidine block was used for cell cycle synchronisation at the G1/S boundary. Briefly, Hela-S3 cells were blocked with 2.5 mM thymidine for 18 hours. The cells were released from the block by two washes with pre-warmed medium, followed by incubation for 9 hours in fresh medium supplemented with 24 µM 2-deoxycytidine. The second G1/S block was again performed by adding 2.5 mM thymidine for 18 hours. Finally, cells were released by two washes with pre-warmed media and incubated for 6 hours (late G2) in DMEM containing 24 µM 2-deoxycytidine before labelling with 500 µM 4sU (Glentham Life Sciences, GN6085) for 0, 15, 45, 75, 120 and 180 minutes. At each time point, 3 million cells were fractionated to obtain nuclear and cytosolic fractions based on Mayer et al. (2015) with the following modifications: Nuclear pellets were washed with nuclear wash buffer (PBS with 1X cOmplete Protease Inhibitor (Roche, 11873580001)) and resuspended in 1400 µl TRIzol (Invitrogen, 15596026). Similarly, a time series in the G1 cell cycle phase (12 hours release time after double thymidine blocking) was measured at 0, 15, 45, 75, 145, 180, 240 and 300 minutes. Prior to phenol extraction of RNA, 1.9 µl of 1:100 diluted ERCC ExFold RNA Spike-In Mixes (Invitrogen, 4456739) per 1 million cells was added to each sample. The 4sU RNA was alkylated according to Herzog et al. (2017), and poly-A enriched SLAM-seq libraries were generated using the 3'-QuantSeq-REV protocol (Lexogen) according to the manufacturer's protocol. The final libraries were sequenced on an Illumina NovaSeq6000 sequencer at 150 nucleotide read lengths in paired-end mode.


title: m6A-mediated epi-transcriptomic dysregulation underlies synaptic dysfunction in fragile X syndrome [RNA-seq]

status: Public on Feb 26 2025

city: Cincinnati

first: Ziyuan

last: Guo

address: 3333 Burnet Ave

country: USA

institute: CCHMC

type: Expression profiling by high throughput sequencing

summary: N6-methyladenosine (m6A) is a prevalent mRNA modification essential for post-transcriptional regulation. Fragile X syndrome (FXS), the leading genetic cause of intellectual disability, arises from FMR1 gene silencing and loss of FMRP, which has been shown to antagonize m6A readers. However, the impact of FMRP loss on transcriptome-wide m6A modifications in human FXS remains unknown. Using cortical neurons derived from healthy subjects and FXS patient-induced pluripotent stem cells, we found that FXS neurons exhibited synaptic and neuronal network defects, mirroring clinical EEG data from the same FXS patients. FMRP deficiency led to increased translation of m6A writers, resulting in transcriptome-wide hypermethylation, primarily affecting synapse-associated transcripts and increasing mRNA decay. Treatment with an m6A writer inhibitor rescued synaptic defects in FXS neurons, highlighting its therapeutic potential. Our findings uncover an epi-transcriptomic mechanism by which FMRP deficiency disrupts m6A modifications in FXS, contributing to FXS pathogenesis, and suggest a promising avenue for m6A-targeted therapies.

overall_design: Differentially expressed genes (DEGs) between FXS iPSC-derived cortical neurons and  healthy control cortical neurons were examed to investigate transcriptome-wide gene expression changes.

first: Lu

last: Lu


title: Landscape analysis of m6A modification reveals the dysfunction of bone metabolism in osteoporosis mice

city: Fuzhou

first: 佳泳

last: 吴

address: 20 Cha Zhong Road

country: China

institute: The First Affiliated Hospital of Fujian Medical University

summary: Our research findings indicated that m6A modification is essential in regulating OP, offering potential insights for prevention and treatment.

overall_design: This study aimed to explore the relationship between OP and RNA m6A methylation, investigating its underlying mechanisms through comprehensive bioinformatic analysis and experimental validation.

first: Lifeng

last: Zheng

first: Chao

last: Lan

first: Xinyue

last: Gao


title: Effect of modulation of microRNA-455-3p on gene expression in SW1353 human chondrosarcoma cells

city: Norwich

first: Ian

last: Clark

state: Norfolk

address: Earlham Road

country: United Kingdom

institute: University of East Anglia

summary: MicroRNA-455 is a key microRNA in cartilage, strongly induced by Sox9.  In order to understand its function, we have overexpressed (miR-455-3p mimic) or inhibited (miR-455-3p inhibitor) miR-455 compared to appropriate controls and measured gene expression.

overall_design:
SW1353 cells were plated in 6 well plates and grown for 24 hours.  They were then transfected with miR-455 mimic, mimic control, inhibitor or inhibitor control at 50nM using Lipofectamine 3000 and incubated for 48 hours.
RNA was purified using Trizol and treated with Dnase (TURBO, Ambion).
We then performed RNASeq analysis on the cell-derived RNA from n=3 of each experimental condition

middle: M


title: The influence of microRNA-455-3p on gene expression in the developing chick limb bud

summary: MicroRNA-455 is a key microRNA in cartilage, strongly induced by Sox9.  Overexpression of miR-455-3p in the developing chick limb bud at HH18-19 causes a reduction in limb bud size at HH24-25.

overall_design:
Limb buds were injected at H18-19 and harvested by dissection and snap freezing in liquid nitrogen after 24 hours at H24-25 prior to storage at -80oC
Limb bud tissue was disrupted and homogenized in extractino buffer.   RNA was purified using a combination Qiagen Rneasy Micro.
We then performed RNASeq analysis on tissue from n=4 control mimic vs n=3 miR-455-3p mimic

first: Clark

last: Ian M


title: Gene expression in murine cartilage, 3 months of age, wild type vs miR-455 null

summary: MicroRNA-455 is a key microRNA in cartilage, strongly induced by Sox9.  Using CrispR-Cas9, we produced a global deletion of miR-455 in the mouse and characterised cartilage and bone.  In this study, we compared the cartilage gene expression in 3 month old mice, between wild-type and miR-455 null.

overall_design:
Cartilage was dissected under a microscope from both tibia and femur of the hind limb into RNALater, snap frozen in liquid nitrogen and stored at -80C.
Cartilage from two mouse knees per sample was pooled and ground to a fine powder under liquid nitrogen; RNA was purified using a combination of Trizol and miRVana
We then performed RNASeq analysis on tissue from n=3 wild-type vs miR-455 mice


title: Single-cell RNA sequencing to reveal tissue-specific transcriptomic changes induced by perfluorooctanesulfonic acid (PFOS) in larval zebrafish (Danio rerio)

city: Amherst

first: Kruuttika

last: Satbhai

state: MA

address: 637 N. Pleasant St.

department: Environmental Health Science

institute: University of Massachusetts Amherst

summary: Per- and poly-fluoroalkyl substances (PFAS), a class of ubiquitous, persistent, and toxic anthropogenic chemicals that exert harmful effects on environmental and human health, but the mechanisms that drive these effects are not well understood. Perfluorooctanesulfonic acid (PFOS) is a legacy PFAS that elicits numerous adverse effects on the endocrine, immune, nervous, reproductive, and developmental processes. The aim of this study was to identify transcriptomic changes in zebrafish (Danio rerio) exposed to PFOS that provide insight into sensitive target tissues and molecular mechanisms of developmental toxicity. Zebrafish embryos were exposed to DMSO (0.01%) or PFOS (16 µM) from 3–72 hours post fertilization (hpf). Larvae were collected, cells isolated and assessed for viability, and single-cell gene expression profiling was performed. Data analysis was conducted in three different ways: pseudo-bulk analysis, untargeted cluster analysis, and a targeted cluster analysis where unique clusters were defined for liver (fabp10a, and apoa2). Results from the pseudo-bulk analysis showed that 8.63% (2390/ 27698) genes were significantly differentially expressed between DMSO and PFOS groups. The top five pathways impacted by these significant DEGs were neuron development, visual perception, central nervous system development, eye development, and synaptic signaling. Results from untargeted cluster analysis revealed 22 distinct clusters that were annotated to specific tissues. The top five clusters with the highest number of significant DEGs were digestive organs, muscle, otolith, cardiac muscle, and optic lens. Additionally, we looked at the distribution of pathway-specific genes for epigenetic modifications, Nfe2l2 pathway, and PPAR pathway within clusters to identify sensitive tissues—blood-related tissue, retinal rod, and cardiac muscle were more likely be impacted by epigenetic modifications by DNA methylation; while blood-related tissue and periderm are sensitive to be impacted by both Nfe2l2 and PPAR pathway. Another potential mechanism induced by PFOS is beta-oxidation of fatty acids—which was significantly downregulated in blood-related tissue and upregulated in digestive organs. Results of targeted or curated liver cluster showed 220 DEGs in the liver were significant with PFOS exposure with Notch signaling pathway more likely to be impacted with PFOS exposures. The sensitivity pathways for PFOS-induced toxicity in larval zebrafish are PPAR signaling (overall, and digestive organs) and Notch signaling pathway (liver). Overall, scRNA-seq is a powerful tool to identify tissue-specific changes and novel mechanisms by which toxicants like PFOS disrupt embryonic development.

overall_design: Single cells were obtained from whole body zebrafish larvae exposed to DMSO and PFOS.

first: Satbhai

last: Kruuttika


title: Single-cell RNA sequencing reveals cellular composition of fibrotic fascia associated with gluteal muscle contracture

city: Hengyang

first: Weizhi

last: Zhao

state: Hunan

address: 28 West Changsheng Road

institute: University of South China

summary: Fibrosis is a common and integral pathological feature in various chronic diseases, capable of affecting any tissue or organ. Fibrosis within deep fascia is implicated in many myofascial disorders, including gluteal muscle contracture (GMC), Dupuytren’s disease, plantar fasciitis, iliotibial band syndrome, and chronic muscle pain. In this study, we performed scRNA seq analysis on fibrotic fascia associated with GMC and compared them to nonfibrotic control fascial samples. Our findings show that fibroblast and macrophage cells play a role in pathological tissue remodeling within fibrotic deep fascia. We observed an upregulation of various collagens, proteoglycans, and extracellular matrix (ECM) glycoproteins in contracture deep fascia, attributed to the widespread activation of fibroblast subclusters. Additionally, two pro fibrotic macrophage subpopulations, SPP1+ MP and ECM like MP, appear to facilitate ECM deposition in fibrotic deep fascia by either regulating fibroblast activation or directly contributing to ECM production. SPP1+ MP and ECM like MP cells, as well as the signal interaction between SPP1+ MP and fibroblast cells, present novel and potential therapeutic target for treating GMC and other related myofascial disorders.

overall_design: To investigate the pathogenesis of deep fascial fibrosis, we obtained 10 fibrotic iliotibial band samples from patients with gluteal muscle contracture and 4 non-fibrotic iliotibial band samples from patients with femoral fracture.    Single cell sequencing was performed on 14 samples of iliotibial bands, and the single cell sequencing data of fibrotic and non-fibrotic iliotibial bands were compared and analyzed

first: Zongchao

last: Li

first: Liangjun

first: Danling

last: Wang


title: Characterization of differential gene expression pattern in control and microglia depleted retinas during retinal vascular development in mice

city: Boston

first: Gracia

middle: M.

last: Bonilla

address: 185 Cambridge St

department: Molecular Biology

institute: Massachusetts General Hospital

summary: This study reports the comprehensive gene expression profiling of postnatal day (P) 10 control diet (Control) and Csf1r antagonist (PLX5622, Plexicon Inc. Berkeley, CA) diet fed microglia deplted retinas (referred as PLX5622) during development. Further functional enrichment analysis revealed down-regulation of biological processes linked to phagocytosis and innate immune signaling.

overall_design: Retinal mRNA profiles of P10 control and microglia depleted (PLX5622) mice were generated by deep sequencing, n=4/genotype, using Illumina Nextseq 2000, and the sequencing target was 25 million reads per samples.

first: Gopalan

last: Gnanaguru

first: Ruslan

last: Sadreyev


title: Decreased Th-1 CD4+ T cell function underlies recurrent respiratory infections in 17q12 deletion syndrome

city: NEW HAVEN

first: HYEON JUN

last: SHIN

state: CT

address: 1 Gilbert St

institute: Yale University School of Medicine

summary: The 17q12 deletion syndrome (17q12DS) is a chromosomal aberration with a heterozygous deletion of a 1.4 megabases (Mb)‒spanning DNA sequence on the long arm of chromosome 17. The well-known clinical characteristics of 17q12DS include abnormalities of the kidney and urinary tracts, as well as neurodevelopmental disorders. However, in our cohort of 37 subjects with 17q12DS, we observed increased atopic disorders and recurrent sinopulmonary infections requiring prolonged courses of antibiotic therapies and/ or hospitalizations, raising the possible immune dysregulation in 17q12DS, a point that has not been previously addressed. We thus tested the hypothesis that individuals with 17q12DS[KI1] have altered T-cell function, contributing to recurrent infections and[KI2] atopy. Here we found that individuals with 17q12DS had a substantially decreased frequency of CD4+ T cells producing the T helper (Th) 1 cytokine IFN[KI3]-g but not Th2 and Th17 cytokines compared to age-matched healthy controls (HCs). RNA seq analysis of CD4+ T cells revealed decreased levels of TBX21 encoding the Th1 transcription factor T-bet, IFNG, and other Th1 chemokine encoding genes in subjects with 17q12DS compared to HCs. These findings were validated at the protein level using flow cytometry and multiplex assay. Our study is the first to demonstrated immune alterations in 17q12DS characterized by decreased T-bet and its downstream molecules such as IFN-γ. These findings warrant further investigations into the underlying mechanisms, which would inform precision therapy for individuals with 17q12DS.

overall_design: mRNA profiles of health control (HC) and 17q12 deletion syndrome patient (Pt)

first: Junghee

middle: J

last: Shin

first: Hyeon

first: Alan

last: Gutierrez

first: Noelle

last: Yoo

first: Jennefer

last: Par-Young

first: Lais

last: Osmani

first: Pedro

middle: A

last: Sanchez-Lara

first: Richard

last: Bucala

first: Gary

last: Soffer

first: Insoo

last: Kang


title: Eosinophils respond to extracellular matrix treated muscle injuries but are not required for macrophage polarization

city: Bethesda

first: Kaitlyn

last: Sadtler

state: Maryland

address: 50 S Drive, Room 4341

department: NIBIB

institute: NIH

summary: The immune response to decellularized extracellular matrix muscle injury is characterized by Th2 T cells, Tregs, M2-like macrophages, and an abundance of eosinophils. Eosinophils have previously been described as mediators of muscle regeneration but inhibit skin wound healing. In addition to response to wounding, a large number of eosinophils respond to biomaterial-treated muscle injury, specifically in response to decellularized extracellular matrix. ECM treatment of muscle wounds has been associated with positive outcomes in tissue regeneration, but the detailed mechanisms of action are still being evaluated. Here, we investigate the role of these eosinophils in terms of their immunologic phenotype and subsequent effect on the local tissue microenvironment. These cells have a mixed phenotype showing both type-2 and regulatory gene upregulation and regulate immune infiltration into the scaffold and wound microenvironment. Beyond the local tissue, ECM treatment was seen to induce a transient flux of eosinophils to the lungs but prevented a trauma-associated neutrophilia in the lungs of injured mice. We believe this local and systemic immunomodulation contributes to the regenerative effects of the material and such distal tissue effects should be considered in therapeutic design and implementation.

overall_design: To investigate the reponse of eosinophils to muscle trauma and ECM implantation and subsequent role in macrophage polarization

first: Daphna

last: Fertil


title: Hepatic sphingomyelin phosphodiesterase 3 promotes steatohepatitis by disrupting membrane sphingolipid metabolism

city: Shanghai

first: Cen

last: Xie

address: 501 Haike Road

institute: Shanghai Institute of Materia Medica

summary: Metabolic dysfunction-associated steatohepatitis (MASH) remains a significant health challenge. Herein, we identify sphingomyelin phosphodiesterase 3 (SMPD3) as a key driver of hepatic ceramide accumulation through increasing sphingomyelin hydrolysis at the cell membrane. Hepatocyte-specific Smpd3 gene disruption and pharmacological inhibition of SMPD3 alleviate MASH, whereas reintroducing SMPD3 reverses the resolution of MASH. While healthy livers express low-level SMPD3, lipotoxicity-induced DNA damage suppresses SIRT1, triggering an upregulation of SMPD3 during MASH. This disrupts membrane sphingomyelin-ceramide balance and promotes MASH progression by enhancing caveolae-dependent lipid uptake in hepatocytes and extracellular vesicle secretion from steatotic hepatocytes to worsen inflammation and fibrosis. Thus, SMPD3 acts as a central hub integrating key MASH hallmarks. Notably, we discovered a bifunctional agent that simultaneously activates SIRT1 and inhibits SMPD3, which shows significant therapeutic potential in MASH treatment. These findings suggest that inhibition of hepatic SMPD3 restores membrane sphingolipid balance and holds great promise for developing novel MASH therapies.

overall_design: To investigate the role of SMPD3 in MASH on diet-induced mouse model, we used cre-loxP system to generate mice with Smpd3 specifically knocked out in hepatocytes, which were fed with 9-week HFMCD to induced mouse MASH model.

first: Jie

last: Jiang

first: Yuqing

last: Gao

first: Jiang

first: Yan

last: Huang

first: Rong

last: Yang


overall_design: To investigate the role of SMPD3 in MASH on diet-induced mouse model, we used cre-loxP system to generate mice with Smpd3 specifically knocked out in hepatocytes, which were fed with 24-week GAN diet to induced mouse MASH model.

last: Wang


title: Transcriptome signature of Neuromyelitis spectrum disorder under steroid tapering

city: Suita

first: Daisuke

last: Motooka

state: Osaka

address: 3-1, Yamadaoka

country: Japan

institute: NGS core facility, Research Institute for Microbial Diseases, Osaka University

summary: The advent of biologics is significantly transforming the treatment strategies for NMOSD. However, there are no biomarkers to predict relapses associated with steroid tapering. Therefore, in this study, we analyzed the immune dynamic changes due to steroid reduction. The results of this study identified peripheral blood transcriptome signatures that could lead to the creation of urgently needed NMOSD disease activity biomarkers.

overall_design: We collected peripheral blood mononuclear cells (PBMCs) from NMOSD patients during the steroid tapering period and performed bulk RNA sequencing. PBMCs were collected from 10 NMOSD patients (including one relapse case). The subjects were positive for anti-AQP4 antibodies and had no history of biologics use.

first: Ryohei

last: Yamamura

first: Makoto

last: Kinoshita

first: Yoshiaki

last: Yasumizu

first: Tomohiro

last: Yata

first: Keigo

last: Kihara

first: Naoyuki

last: Shiraishi

first: Yasuko

last: Sugiyama

first: Shohei

last: Beppu

first: Hisashi

last: Murata

first: Naoshi

last: Koizumi

first: Itsuki

last: Sano

first: Toru

last: Koda

first: Tatsusada

last: Okuno

first: Hideki

last: Mochizuki


first: Cen

last: Xie

overall_design: To investigate the role of SMPD3 in MASH on in vitro model, we used primary hepatocytes isolated from cre-loxP system generating mice with Smpd3 specifically knocked out in hepatocytes, which were stimulated with 0.4 mM palmitic acid to induced in vitro MASH model.

first: Jie

last: Jiang

first: Yuqing

last: Gao

first: Jiang

first: Yan

last: Huang

last: Yang


title: Extracellular matrix protein type I collagen and fibronectin are regulated by the endothelin axis in ovarian fibroblasts

city: Rome

first: Valerio

last: Licursi

address: via degli Apuli, 4

department: c/o Dept. Biology and Biotechnologies "C. Darwin", Sapienza University of Rome

country: Italy

institute: Institute of Molecular Biology and Pathology (IBPM), National Research Council (CNR)

summary: The invasive and metastatic spread of ovarian cancer (OC) results from the cooperative interactions between cancer and stroma, which include acellular extracellular matrix (ECM) and cellular components, such as cancer-associated fibroblasts (CAFs). Soluble growth factors secreted by cancer and stromal cells contribute to stroma remodeling through the synthesis and modification of ECM proteins, providing a favorable environment for cancer cell dissemination. Within the OC, the peptide endothelin-1 (ET-1), through two G protein-coupled receptors, ET A R and ET B R, supports tumor growth and progression by acting on both cancer and stromal cells. Many of these effects rely on the functional and molecular link with the protein b-arrestin1 (b-arr1). However, the precise role of ET-1 in regulating the expression of ECM proteins by primary human ovarian fibroblasts (HOFs) remains unclear. In this study, we aimed to elucidate the regulatory role of ET-1 in the expression of type I collagen (Col1) and fibronectin 1 (FN1). We report a significant increase in the expression of both Col1 and FN1 in cells stimulated with ET-1, both at mRNA and protein levels, reaching the amounts of proteins found in ovarian CAFs. The observed effects are associated with the activation of both receptors since they are impaired by treatment with either ET A R or ET B R antagonists in HOFs and CAFs. Furthermore, they are particularly affected by the dual ET A R/ET B R antagonist bosentan, in vitro and in vivo models. At the molecular level, ET-1 activates the Col1 promoter and enhances the expression of this gene through the nuclear function of β-arr1. The change in transcriptome of HOFs silenced for β-arr1 was analyzed to confirm the molecular response triggered by β-arr1, which resulted in the upregulation of ECM remodeling and collagen deposition. In summary, these findings demonstrate that the ET-1 axis may contribute to the remodeling of the ECM and probably to the early stages of OC progression by modulating the expression of Col1 and FN1. The targeting of ET-1 signaling with ET A R/ET B R antagonists may impede HOFs’ ability to produce key ECM proteins in this tumor.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for primary HOFs and its derivative for β-arr1 knock-down through siRNA.

first: Danila

last: Del Rio

first: Laura

last: Rosanò


title: Knockdown of FAF2 Prevents Alcohol-Induced Steatosis in Mouse Liver

city: Indianapolis

first: Zhihong

institute: Indiana University

summary: Alcohol is metabolized in the liver, and chronic consumption can lead to inflammation, scarring, and damage to liver cells. The pathogenesis of alcoholic liver disease (ALD), a complicated condition, is characterized by a succession of histopathological alterations that occur through a multistep and multifactorial process. FAF2/UBXD8/ETEA is a ubiquitin ligase adaptor protein and plays a crucial role in the ubiquitin-mediated degradation of misfolded proteins in the endoplasmic reticulum. Recent GWAS study indicated that FAF2 was associated with ALD, but the exact function of FAF2 in ALD has not been identified yet. The objective of this study was to investigate the role of FAF2 in ALD.Our study revealed a noteworthy rise in hepatic FAF2 protein expression among individuals with ALD and mice subjected to chronic-plus-single binge ethanol feeding. The suppression of FAF2 in mice liver provided protection against alcohol-induced hepatic steatosis.

overall_design: Adeno-associated virus (AAV) subtype 8 carrying either scrambled shRNA (control) or FAF2-specific shRNA #3 was delivered into the livers of wild-type mice through tail vein injection.  After 2 weeks, mice were fed with either control or ethanol-containing diet for 10 days, followed by a single binge of maltose (control) or ethanol .


title: DYRK1A Inhibition via Harmine

city: Seattle,

first: Stephanie

last: Osmond

state: WA

address: 1201 9th Ave

institute: Benaroya Research Institute

summary: Chemical inhibition of DYRK1A (e.g. using harmine) inhibits Th17 and promotes Treg differentiation. To better understand the mechanisms by which DYRK1A regulates Th17 differentiation, we are comparing expression profiles of naïve CD4+ T cells during the course of Th17 differentiation in the presence or absence of harmine.

overall_design: We MACS-isolated naïve CD4+ T cells from 4 mice on C57/Bl6 background. These were plated in pro-Th17 conditions in the presence or absence of 10 uM harmine. Cells were harvested at 0 and 5 hours and RNA isolated by RNeasy Micro & Mini kits. pro-Th17 conditions: 2ug/mL Anti-Il-4 2ug/mL Anti-Il-12 2ug/mL Anti-IFNγ 20ng/mL Il-6 20ng/mL Il1β 0.1ng/mL TGFB

first: Bernard

last: Khor

first: Matt

last: Malueg


title: The Ddx21 mutant peptide is a potent neoantigen in preventive lung cancer vaccines and can activate long-term immune function

city: 郑州市

first: yimeng

last: xia

address: 郑州市经八路

institute: 郑州大学第二附属医院

summary: Preventive vaccine is an important means of tumor prevention. Our previous study showed that the lung cancer cell lines KPL160302S and KPL160424S obtained from the primary culture of lung cancer mice were prepared into preventive vaccines. After inoculation with KPL160302S, the survival period of lung cancer mice was significantly prolonged and the anti-tumor immunity of mice was enhanced. Significantly increased the number of CD8+CD4+T cells, while KPL160424S failed to extend survival and had limited effects on the immune system. In order to explore the cause of this difference, two mutant peptides Ddx21 and Zfp760 were screened as potential neoantigens by bioinformatics method. The results showed that Ddx21MT immunization could significantly up-regulate central memory T cells (TCM) and activate long-term immune function in mice. The magnetic bead sorting of spleen CD4+ and CD8+T cells after enhanced immunization was performed by transcriptome sequencing to explore the differences.

overall_design: The peptides of Ddx21MT and Zfp760MT combined with adjuvant PolyIC were used to immunize 129 mice. After one week of enhanced immunization, spleen cells were selected through magnetic bead sorting to obtain CD4+ and CD8+T cells and sent to transcriptomic sequencing to obtain RNA-seq data of mouse cells in different groups and compare gene expression profile analysis.

first: Zhe

last: Zhang

last: Wang

first: Yimeng

last: Xia


title: Effects of oxidized LDL versus IL-1ß/TNF-ɑ/INFɣ on human gingival mesenchymal stem cells properties

city: Kiel

first: Kristina

last: Schlicht

address: Düsternbrooker Weg 17

country: Germany

institute: UKSH Kiel

summary: Oxidized low-density lipoprotein (oxLDL) is an important player in the course of metabolic inflammatory diseases. oxLDL was identified in the gingival crevicular fluid, denoting possible associations between oxLDL-induced inflammation and periodontal disease. The current investigation compared direct effects of oxLDL to a cytokine cocktail of IL-1ß/TNF-ɑ/INF-γ on gingival mesenchymal stem cells’ (G-MSCs) attributes. G-MSCs exhibited all mesenchymal stem cells’ characteristics. oxLDL- and cytokine-group displayed no disparities in their stemness markers (p>0.05). Next-generation-sequencing revealed altered expression of the TXNIP gene in response to oxLDL treatment compared to controls (p=0.04). Following an initial boosting for up to 5 days by inflammatory stimuli, over 14 day, cellular counts [median count x10-5(Q25/Q75)] were utmost in control- [2.6607(2.0804/4.5357)], followed by cytokine- [0.0433(0.0026/1.4215)] and significantly lowered in the oxLDL-group [0.0274(0.0023/0.7290);p=0.0047). Osteogenic differentiation [median relative Ca2⁺ content(Q25/Q75)] was significantly lower in cytokine- [0.0066(0.0052/0.0105)] compared to oxLDL- [0.0144(0.0108/0.0216)] (p=0.0133), with no differences notable for chondrogenic and adipogenic differentiation (p>0.05).  Within the current investigation’s limitations, in contrast to cytokine mediated inflammation, G-MSCs appear to be minimally responsive to oxLDL-mediated metabolic inflammation, with little effect on their regenerative attributes.

overall_design: Human third passage G-MSCs, isolated from connective tissue biopsies (n=18) and characterized, were stimulated in three groups over 3 days: control-group, cytokine-group (IL-1β[1ng/ml], TNF-α[10ng/ml], IFN-γ[100ng/ml]) or oxLDL-group (oxLDL [50µg/ml]). Next Generation Sequencing and KEGG pathway enrichment analysis, stemness gene expression (NANOG/SOX2/OCT4A), cellular proliferation, colony-formation, multilinear potential and altered intracellular pathways were investigated via histochemistry, next-generation sequencing and RT-qPCR.


title: Transcriptomic analysis of miRNA503 inhibition  in human mesothelioma

city: Napoli

first: Stefania

last: Crispi

address: Via Pietro Castellino,111

department: DISBA

institute: CNR

summary: We analysed the molecular effects on gene modulation exerted by inhibition of miRNA 503 expression in mesothelioma. In vitro results demonstrated that its inhibition determined apoptosis and reduced cancer properties in two different mesothelioma cell lines including one resistant to conventional chemotherapeutic treatment.

overall_design: Human malignant mesothelioma MSTO-211H cells were treated with miRNA 503 inhibitor for 48 hours. Cells treated with the vehicle (DMSO) were used controls. RNA extracted from  treated and control cells were used for transcriptomic analysis using Clariom S Array


title: Colony-stimulating factor 2 (CSF2) as a gut microbiome dependent immune factor that alters molecular and behavioral responses to cocaine

city: Winston-Salem

first: Jonathon

middle: P

last: Sens

state: NC

address: 115 S Chestnut St

department: Physiology/Pharmacology

institute: Wake Forest Baptist Health

summary: Cocaine use disorder is a condition that leads to tremendous morbidity and mortality for which there are currently no FDA-approved pharmacotherapies. Previous research has demonstrated an important role for the resident population of bacteria of the large intestine, collectively dubbed the gut microbiome, in modulating brain and behavior in models of cocaine and other substance use disorders. Importantly, previous work has repeatedly shown that depletion of the gut microbiome leads to increased cocaine taking and seeking behaviors in multiple models. While the precise mechanism of these gut-brain signaling pathways in models of cocaine use is not fully clear, and intriguing possibility is through gut microbiome influences on innate immune system function. In this manuscript we identify the cytokine colony stimulating factor 2 (CSF2) as an immune factor that is increased by cocaine in a gut microbiome dependent manner. Peripherally injected CSF2 crosses the blood-brain barrier into the nucleus accumbens, a brain region central to behavioral responses to cocaine. Treatment with peripheral CSF2 reduces acute and sensitized locomotor responses to cocaine as well as reducing cocaine place preference at high doses. On a molecular level, we find that peripheral injections of CSF2 alter the transcriptional response to both acute and repeated cocaine in the nucleus accumbens. Finally, treatment of microbiome depleted mice with CSF2 reverses the behavioral effects of microbiome depletion on the conditioned place preference assay. Taken together, this work identifies an innate immune factor that represents a novel gut-brain signaling cascade in models of cocaine use and lays the foundations for further translational work targeting this pathway.

overall_design: To define the contribution of CSF2 on the transcriptomic landscape of acute and repeated-cocaine treated mice, we administered exogenous CSF2 (or PBS control) in mice treated with cocaine (or saline control). With a 2x2 design, we transcriptionally  profiled whole nucleus accumbens (NAc) tissue of n=5-6 samples (mice) per group. DIfferential gene expression and gene ontology was performed on the aforementioned RNAseq data.

first: Kelsey

middle: E

last: Lucerne

first: Calista

middle: R

last: Dean

first: Aya

last: Osman

first: Katherine

last: Meckel

first: Yesha

last: Dave

first: Ava

middle: L

last: Shipman

first: Dannis

last: Cazarez

first: Flurin

last: Cathomas

first: Rebecca

middle: S

last: Hofford

first: Drew

middle: D

last: Kiraly


title: Transcriptomic analysis on prostate tissues uncover the diverse gene expression features induced by Lonp1

first: Xingming

address: No. 270, Dong'an Road, Xuhui District, Shanghai

institute: Fudan University Shanghai Cancer Center

summary: Lon protease 1 (LONP1) is an ATP-dependent protease located in the mitochondrial  matrix and plays a crucial role in regulating mitochondrial proteostasis, metabolism and cellular stress responses. Here we show that overexpression of LONP1 is closely related to adverse clinicopathological features and poor prognosis in prostate cancer (PCa) patients. Mechanistically, the findings reveal that LONP1 is implicated in modulating the metabolic switch from oxidative phosphorylation (OXPHOS) to aerobic glycolysis, thereby promoting tumor proliferation, invasion and metastasis both in vitro and in vivo. Meanwhile, we prove that LONP1 as a protease directly targets mitochondrial pyruvate carrier 1 (MPC1), a key metabolic protein in the process of glycolysis, and enhances its degradation, which in turn suppresses tricarboxylic acid (TCA) cycle and ultimately promotes the progression of PCa. Furthermore, using PCa in cancer-prone mice homozygous for a prostate-targeted conditional Pten knockout and Lonp1 knockin, we integrate transcriptomic and proteomic analyses of prostate tumors, upon which reveals that Lonp1 overexpression results in a significant downregulation of NADH: ubiquinone oxidoreductase activity, consequently impeding the electron transfer process and mitochondrial ATP synthesis, associated with metastasis of PCa. Collectively, our results highlight that metabolic reprogramming induced by LONP1 in PCa is closely coupled with disease progression, suggesting that targeting the LONP1-mediated cascade in the mitochondrial may provide therapeutic potential for PCa disease.

overall_design: To investigate whether lonp1 overexpression has a causal role in PCa metastasis, genetically engineered mouse was used to generate a PCa model. Firstly, we crossed Lonp1KI mice withProbasin-Cre mice to induce Lonp1 expression specifically in the prostate epithelium. Subsequently, we generated Pten-/-; Lonp1KI mice by crossing Lonp1KI mice with Ptenfloxed/floxed mice. Transcriptomic analysis was then performed on prostates extracted from 40-week-old wild-type (WT), Lonp1KI, Pten-/- and Pten-/-; Lonp1KI mice.

first: Leilei

last: Du


title: CDK6 kinase inhibition opens up metabolic dependencies in BCR-ABL+ leukemia

city: Vienna

first: Gerwin

last: Heller

address: Waehringer Guertel 18-20

country: Austria

institute: Medical University of Vienna

summary: Metabolic reprogramming and cell cycle deregulation are hallmarks of cancer cells. We here investigate the role of the cell cycle kinase CDK6 in the regulation of cellular energetics in BCR-ABL+ leukemia. Gene expression data and ChIP-Seq analysis from murine BCR-ABL+ cell lines expressing kinase-inactive CDK6 or no CDK6 highlight an activating role for the kinase in regulating the oxidative phosphorylation gene set by interacting with respective promoter regions. Our data imply a competition of CDK6 and Nrf-1, a master regulator of genes required for mitochondrial respiration, at the same sites. In line, cells expressing kinase- inactive CDK6 show signs of a defective electron transport chain and morphologically changed mitochondria. An enhanced cytoplasm/mitochondria ATP ratio together with high levels of pyruvate and lactate point towards a metabolic switch to glycolysis in those cells.  Combinatorial treatment of BCR-ABL+ cell lines with the clinically used CDK4/6 inhibitor palbociclib and the glycolysis inhibitor 2-deoxyglocose (2-DG) reduced proliferation and led to enhanced apoptosis compared to single-agent treatments. Our data, suggest a new therapeutic avenue for hematologic malignancies with high CDK6 expression.

overall_design: RNA-seq of samples from CML patients

first: Karoline

last: Kollmann


title: Epithelial NSD2 maintains FMOs-mediated taurine biosynthesis to prevent intestinal barrier disruption [RNA-Seq]

first: Yue

last: Xu

address: 上海市徐汇区华山路

institute: 上海交通大学

summary: We reported that the deletion of NSD2 in IECs leads to intestinal barrier dysfunction and exacerbates inflammatory infiltration. Further analysis revealed that a deficiency of NSD2 results in decreases of H3K36me2 at protein level and Fmo expression at mRNA level to impede taurine accumulation, both in vitro and in vivo. Supplementation of taurine can effectively alleviate intestinal epithelial damage in NSD2-deficient IBD mice. Thus, these results indicate that NSD2 act as a protector of the intestinal epithelial barrier during intestinal inflammation, which helps maintain intestinal epithelial homeostasis by preventing cell apoptosis.

overall_design: RNA-sequencing of DSS-treated NSD2F/F and NSD2VIL-KO mouse IECs.

first: Chunxiao

last: Ma


title: Transcription-Replication conflicts are linked to histone H3K79 methylation and R-loop dependent nucleosome eviction [BrdUseq]

city: Munich

first: Stephan

last: Hamperl

state: Bayern

address: Feodor-Lynen-Strasse 21

department: Institute of Epigenetics and Stem Cells

institute: Helmholtz-Zentrum Munich

summary: Transcription-replication conflicts (TRCs) can provoke genome instability. Progressing transcription and replication machineries profoundly impact their underlying chromatin template and thus, conflict sites are vulnerable to chromatin and epigenome alterations. Here, we engineered an inducible TRC reporter system using a genome-integrated R-loop-prone sequence. Using this system, we characterized the dynamic changes of the local chromatin structure inflicted by TRCs, leading to reduced nucleosome occupancy and replication fork blockage. Consequently, inducing a handful TRCs on the genome results in a measurable global replication stress response. We also find a TRC-specific increase in H3K79 methylation at the R-loop forming TRC-site. Accordingly, inhibition of the H3K79 methyltransferase DOT1L leads to increased cellular TRCs and exacerbated DNA damage response, suggesting that deposition of this mark is required for effective detection and resolution of TRCs. Thus, our work highlights the molecular dynamics and reveals specific epigenetic modifiers bookmarking TRC sites, relevant to cancer and other diseases.

overall_design: BrdU Immunprecipitation sequencing (BrdU-seq) time course experiment for analysis of nascent DNA replication in synchronized U-2OS Clone#12 cells. G1 cells as well as S-phase released cells for 2h, 4h, 6h, and 8h were analyzed. BrdU was added to the cell culture medium 30 min before harvesting. Cells were treated with  0 or 1 µg/mL Doxycycline. Immunprecipitations were conducted in biological dublicate.

first: Marcel

last: Werner

first: Manuel

last: Trauner

first: Tamas

last: Schauer


title: Epithelial NSD2 maintains FMOs-mediated taurine biosynthesis to prevent intestinal barrier disruption

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: ChIP assays of two-month-old NSD2F/F and NSD2VIL-KO IECs were generated by deep sequencing


title: Eicosatetraynoic Acid Regulates Pro-Fibrotic Pathways in an Induced Pluripotent Stem Cell Derived Macrophage:Human Intestinal Organoid Model of Crohn’s Disease II

city: Ramat Gan

first: Tzipi

last: Braun

address: Derech Sheba 2

country: Israel

institute: Sheba Medical Center at Tel Hashomer

summary: Background: We previously reported Crohn’s Disease (CD) ileal macrophage and fibroblast gene expression modules linked to future strictures, and identified small molecules which may reverse this gene signature. Here we developed a model system to test a lead candidate, eicosatetraynoic acid (ETYA), a Peroxisome Proliferator-Activated Receptor (PPAR) agonist. Methods: CD patient induced pluripotent stem cells (iPSC) were differentiated into macrophages and human intestinal organoids (HIOs).  Macrophages and macrophage:HIO co-cultures were exposed to lipopolysaccharide (LPS) with and without ETYA pre-treatment. Cytospin and flow cytometry characterized macrophage morphology and activation markers, and RNA sequencing defined the global pattern of macrophage gene expression. TaqMan Low Density Array, Luminex multiplex assay, immunohistologic staining, and sirius red polarized light microscopy were performed to measure macrophage cytokine production and HIO pro-fibrotic gene expression and collagen content. Results: iPSC-derived macrophages exhibited morphology similar to primary macrophages and expressed inflammatory macrophage cell surface markers including CD64 and CD68. LPS-stimulated macrophages expressed a global pattern of gene expression enriched in CD ileal inflammatory macrophages and matrisome secreted products, and produced cytokines and chemokines including CCL2, IL1B, and OSM implicated in refractory disease. ETYA suppressed CD64 abundance and pro-fibrotic gene expression pathways in LPS stimulated macrophages. Co-culture of LPS-primed macrophages with HIO led to up-regulation fibroblast activation genes including ACTA2 and COL1A1, and an increase in HIO collagen content.  ETYA pre-treatment prevented pro-fibrotic effects of LPS-primed macrophages. Conclusions: ETYA inhibits pro-fibrotic effects of LPS-primed macrophages upon co-cultured HIO. This model system may be used to screen personalized effects of candidate small molecules upon inflammatory and fibrotic pathways implicated in refractory CD patients.

overall_design: CD patient-induced pluripotent stem cells (iPSC) were differentiated into macrophages. These macrophages were exposed to lipopolysaccharide (LPS) with and without ETYA pre-treatment.

first: Ingrid

last: Jurickova

first: Benjamin

middle: W

last: Dreskin

first: Elizabeth

last: Angerman

first: Erin

last: Bonkowski

first: Kentaro

last: Tominaga

last: Iwasawa

first: Takanori

last: Takebe

first: Yael

last: Haberman

first: James

last: Wells

first: Lee

last: Denson


title: Gene expression profiling of murine BCR-ABL(p185)-positive CDK6-WT, CDK6-K43M and CDK6-KO cells

first: Thorsten

last: Klampfl

address: Veterinaerplatz 1

institute: University of Veterinary Medicine Vienna

summary: The aim of this experiment was to study the effects of a kinase-dead mutant of the cell cycle kinase CDK6 (CDK6-K43M) on gene expression in a murine model of BCR-ABL driven leukemia.

overall_design:
Mouse bone marrow cells from mice homozygously expressing wildtype (CDK6-WT), or mutant (CDK6-K43M) CDK6, or with a homozygous CDK6 knock-out (CDK6-KO) were retrovirally transduced to generate cell lines which express BCR-ABL(p185). Three cell lines were generated per genotype for a total of nine lines. RNA sequencing was performed to study differential gene expression between the genotypes. The CDK6-WT and CDK6-KO samples were submitted to GEO previously as GSM4748865 (CDK6_WT1), GSM4748866 (CDK6_WT2), GSM4748867 (CDK6_WT3), GSM4748868 (CDK6_KO1), GSM4748869 (CDK6_KO2) and GSM4748870 (CDK6_KO3). They were reanalysed in this study together with the CDK6_K43M samples.
SRX9022293 GSM4748866 SAMN15923427
SRX9022294 GSM4748867 SAMN15923426
SRX9022295 GSM4748868 SAMN15923425
SRX9022296 GSM4748869 SAMN15923424
SRX9022297 GSM4748870 SAMN15923423

first: Sophie

last: Nebenführ

first: Karoline

last: Kollmann

first: Markus

last: Zojer

first: Veronika

last: Sexl


title: Influenza Infection Promotes Lung Accrual of Macrophages with an Obese Adipose Tissue Transcriptomic Signature

city: Cincinnati

first: Nathan

last: Salomonis

state: OH

address: 3333 Burnet Avenue

department: Biomedical Informatics

institute: Cincinnati Children's Hospital

summary: Obesity is a risk factor for increased lung damage and disease severity during influenza virus infection. White adipose tissue (WAT) inflammation is a key driver of disease pathogenesis in obesity. While lung tissue immune cell pathogenic mechanisms are dogmatically studied in influenza virus infection, how obesity modifies lung and WAT immune cell character and contribution to amplify disease severity remains unknown. We show that obesity results in lung immune cells having a more inflammatory transcriptome in response to influenza infection than in the lean state. Further, in both lean and obese mice, influenza virus infection induces expression of inflammatory genes in visceral WAT and dominantly modifies the WAT immune cell milieu in obesity. Notably, obese influenza virus-infected mice exhibit a decrease in white adipose tissue macrophage (ATM) populations that inversely correlates with the increase in infiltrating lung macrophage numbers.

overall_design: Comparison of both lung and WAT immune cell transcriptional landscapes uncovered a presence of a macrophage subset in the lungs whose transcriptomic signatures matched those of an inflammatory ATM subset seen in obese mice. Notably, adoptive transfer of ATMs from obese mice into lean influenza-virus infected mice resulted in increased host immune cell lung infiltration. Together, our novel findings provide transcriptomic evidence of obesity-driven changes in immune cell character in the lungs and WAT and identify a previously unrecognized contribution of visceral ATMs to exacerbated influenza disease severity in obesity.

first: Pablo

middle: C

last: Alarcon

first: Cassidy

last: Ulanowicz

first: Maria

last: Moreno-Fernandez

first: William

last: Zacharias

first: Senad

last: Divanovic


title: ATAC seq in murine BCR-ABL(p185)-positive pre/pro B-cells expressing wildtype CDK6 or a kinase-dead mutant (CDK6 K43M)

summary: The aim of this experiment was to study the chromatin accessibility in BCR-ABL(p185)-positive pre/pro B-cells expressing wildtype CDK6 or a kinase-dead mutant (CDK6 K43M)

overall_design:
Mouse bone marrow cells from mice homozygously expressing wildtype (CDK6-WT), or mutant (CDK6-K43M) CDK6 were retrovirally transduced to generate cell lines which express BCR-ABL(p185). ATAC seq was performed in two cell lines per genotype for a total of 4 samples. The sample CDK6_WT_1 was submitted to GEO previously as GSM4748863. This sample was reanalysed in the current study.
SAMN15923430: GSM4748863: SRX9022290


title: Vascular smooth muscle cell phenotype switching during cancer progression.

city: Stanford

first: Caitlin

last: Bell

state: California

address: 241 Pasteur Drive

department: Medicine

institute: Stanford University School of Medicine

summary: Smooth muscle cell plasticy and phenotypic switching play a prominent role in pathogenesis of multiple diseases. We used scRNAseq to look at the diversity of vascular smooth muscle cells in tumorigenesis.

overall_design: The syngeneic colon cancer cell line MC38 was subcutaneously implanted on the flanks of mice with fluorescent SMC lineage tracers. Initial datasets used the wildtype MC38 line implanted in an Myh11-eGFP tracing mouse, while subsequent experiments implanted MC38 transduced with either an shControl lentiviral plasmid or an shBST2 lentiviral plasmid into an Myh11-tdTomato lineage tracing mouse.

first: Bell

last: Caitlin

first: Leeper

last: Nicholas


title: Corticosteroid premedication and the antitumor effect of immune checkpoint blockade in murine hepatocellular carcinoma models

city: Taipei City

first: Nai-Wen

last: Chang

address: 2F., No.7, Zhongshan S. Rd., Zhongzheng Dist.

department: Department of Medical Research

country: Taiwan

institute: National Taiwan University Hospital

summary: This study aims to investigate whether corticosteroid premedication may compromise antitumor efficacy of dual ICB, a regimen that may cause significant irAEs. The results signal a green light for further clinical investigations using corticosteroid premedication to prevent dICB-induced irAEs.

overall_design: Subcutaneous Hepa1-6 and orthotopic BNL 1MEA.7R.1 syngeneic HCC models were used. Low-dose (LD; 10 μg) or high-dose (HD; 200 μg) dexamethasone (Dexa) was intraperitoneally administered before each dose of anti-CTLA-4 and anti-PD-1. Tumor shrinkage, T cell priming, cytokine quantitation, as well as cytotoxicity and single-cell RNA-sequencing (scRNA-seq) of tumor-infiltrating T cells were assessed.


title: CD45+/Col I+ Fibrocytes: Major Source of Collagen in the Fibrotic Lung, but not in Fibroblast Cultures

city: Charleston

first: Stefano

last: Berto

state: SC

address: 68 President Street

department: Neuroscience

institute: Medical University of South Carolina

summary: The role of cells of the hematopoietic lineage in fibrosis is controversial. Here we evaluate the contribution of Col I+/CD45+ cells (fibrocytes) to lung fibrosis. Systemic bleomycin treatment was used to induce fibrosis in a bone marrow transplant and two transgenic mouse models. Lung cells from these mice were analyzed by flow cytometry, both immediately upon release from the tissue or following growth on tissue-culture plastic. Fibrotic and control human lung tissue were also used. Fibroblasts and fibrocytes derived from a transgenic mouse model were compared in terms of their morphology, growth, and adhesion to fibronectin. Single cell RNAseq was performed with the analysis focusing on CD45-/Col I+ “fibroblasts” and CD45+/Col I+ “fibrocytes” in control and fibrotic mouse lung tissue. Finally, we inhibited fibrosis in mice using a novel, water-soluble version of caveolin scaffolding domain (CSD) called WCSD.

overall_design: In both mouse and human lung tissue, we observed a large increase in fibrocyte number and Col I expression associated with fibrosis. In contrast, fibroblast number is not significantly increased. These results on increased fibrocyte number in fibrotic mouse lung tissue were strongly supported by single cell RNAseq. RNAseq also revealed that myfibroblast markers are associated with a fibrocyte cluster, not with a resident fibroblast cluster. Some investigators claim that fibrocytes are not present among primary fibroblasts. However, we find that fibrocytes are the predominant cell type present in these cultures prior to passage.  Fewer fibrocytes are present after one passage, and almost none after two passages. Like the classic description of fibroblasts, passage 0 fibrocytes are spindle-shaped (i.e. not the fried-egg shape of macrophages). Experiments suggest that fibrocytes are crowded out of cultures during passage because fibroblasts have a larger footprint than fibrocytes, even though fibrocytes bind more efficiently to fibronectin. Finally, we observed that in mice treated with bleomycin and WCSD, there was a large decrease in the number of fibrocytes present, but not in the number of fibroblasts, compared to bleomycin alone. In summary, fibrocytes are the major collagen-producing cell type that is increased in number in association with fibrosis as well as the major source of myofibroblasts.  The common observation that collagen-producing spindle-shaped cells associated with fibrosis are CD45- appears to be an artifact of passage in cell culture.

first: Suganya

last: Subramanian

first: Charles

last: Reese

first: Stanley

last: Hoffman


title: Genomic Balancing Act: Deciphering DNA rearrangements in the Complex Chromosomal Aberration involving 5p15.2, 2q31.1 and 18q21.32

city: Houston

first: James

middle: R.

last: Lupski

state: TX

address: One Baylor Plaza

institute: Baylor College of medicine

summary: Despite extensive research into the genetic underpinnings of neurodevelopmental disorders (NDD), many clinical cases remain unresolved. We studied a female proband with a NDD, mildly dysmorphic facial features, and brain stem hypoplasia on neuroimaging. Comprehensive genomic analyses revealed a terminal 5p loss and terminal 18q gain in the proband while a diploid copy number for chromosomes 5 and 18 in both parents. Genomic investigations in the proband identified an unbalanced translocation t(5;18) with additional genetic material from chromosome 2 (2q31.3) inserted at the breakpoint, pointing to a complex chromosomal rearrangement (CCR) involving 5p15.2, 2q31.3, and 18q21.32. Breakpoint junction analyses enabled by long read genome sequencing unveiled the presence of four distinct junctions in the father, who is carrier of a balanced CCR. The proband inherited from the father both the abnormal chromosome 5 resulting in segmental aneusomies of chr5 (loss) and chr18 (gain) and a der(2) homologue. Evidences suggest a chromoplexy mechanism for this CCR derivation, involving double-strand breaks (DSBs) repaired by non-homologous end joining (NHEJ) or alternative end joining (alt-EJ). The complexity of the CCR and the segregation of homologues elucidate the genetic model for this family. This study demonstrates the importance of combining multiple genomic technologies to uncover genetic causes of complex neurodevelopmental syndrome and to better understand genetic disease mechanisms.

overall_design: Trio samples plus three controls

first: Zain

last: Dardas

first: Dana

last: Marafi

first: Ruizhi

last: Duan

first: Jawid

last: Fatih

first: Omnia

middle: F

last: El-Rashidy

first: Christopher

last: Grochowski

first: Claudia

last: Carvalho

first: Shalini

middle: N

last: Jhangiani

first: Weimin

last: Bi

first: Haowei

last: Du

first: Richard

last: Gibbs

first: Jennifer

last: Posey

first: Daniel

last: Calame

first: Maha

last: Zaki


title: Single cell RNA sequencing for CD34 lineage cells from normal and Angiotensin Ⅱ-induced abdominal aneurysmal aorta

city: Hangzhou

first: Hong

last: Wu

address: 79 Qingchun Road

department: Department of Cardiology

institute: the First Affiliated Hospital, Zhejiang University School of Medicine

summary: The development of aneurysm is involved in imbalance between connective tissue repair and degradation. CD34+ cells are a heterogeneous population with stem/progenitor cell properties. However, the role of CD34+ cells in abdominal aortic aneurysms/dissections (AAAs) is unknown. We aimed to uncover the exact role and potential mechanism of CD34+ cells in the development of AAAs.

overall_design: This entry contains 10x Single-cell RNA-seq data of FACS sorted nucleated living tdTomato+ cells from normal or tdTomato+CD45- cells from 4-week Angiotensin Ⅱ-induced mouse  abdominal aorta.

first: Xiaoping

last: Yang

first: Ting

last: Chen

first: Qingzhong

last: Xiao

first: Honghua

last: Ye

first: Qingbo

last: Xu


title: Inhibition of PRC2 enables self-renewal of naïve pluripotent stem cells from chimpanzee

city: Minato-ku

first: Hideki

last: Masaki

state: Tokyo

address: 4-6-1 Shirokanedai

department: Institute of Medical Science

institute: University of Tokyo

summary: Naïve pluripotent stem cells (nPSCs) correspond to nascent epiblast in the pre-implantation embryo. nPSCs from mouse and human differ in self-renewal requirements and potency for trophectoderm generation. Here we investigated chimpanzee (Pan troglodytes) nPSCs. Naïve type colonies emerged after resetting or reprogramming but failed to expand. We found that the block to self-renewal is overcome by inhibition of EZH2, the enzymatic component of Polycomb repressor group 2 (PRC2). Chimpanzee nPSCs are euploid, produce teratomas, and can be capacitated for somatic lineage differentiation in vitro. They show transcriptome relatedness to human nPSCs and early epiblast, with shared expression of a subset of pluripotency transcription factors. Chimpanzee nPSCs differentiate to trophectoderm and form tri-lineage blastoids. We confirmed that PRC2 suppresses self-renewal by genetic deletions. Furthermore, we demonstrate that EZH2 inhibition facilitates feeder-free propagation of human nPSCs. In summary, chimpanzee nPSCs expand the repertoire of systems for studying primate pluripotency and early embryogenesis.

sample_organism:
Pan troglodytes
Homo sapiens

platform_organism:
Pan troglodytes
Homo sapiens

overall_design: To investigate the gene expression profile of putative chimpanzee naïve PSC line, RNAseq was performed with chimpanzee nPSCs and their parental primed iPSC lines. Their gene expression profiles were also compared with human embryo scRNAseq data sets and nPSCs.

first: Tao

last: Huang

first: Ayaka

last: Yanagida

first: Arthur

last: Radley

first: Zhili

last: Ren

first: Francesca

last: Carlisle

first: Paul

last: O'Neill

first: Hiroo

last: Imai

first: Hiromitsu

last: Nakauchi

first: Ge

last: Guo

first: Austin

last: Smith


title: Interleukin-10 expression in the mouse spleen in the LPS-induced model

city: Beijing

first: Shaonan

last: Wen

address: Beijing 100875, China

institute: Beijing Normal University

summary: In the LPS-induced innate immune response, the expression level of IL-10 in the spleen of mice was significantly increased. The expression of this key anti-inflammatory cytokine is regulated by local sympathetic projections; however, the cell type in which it is expressed is unknown. Therefore, in this study, we used the LPS-induced mouse spleen model to analyze the spatial transcriptome, and found that IL10+ cells were mainly B cells. In addition, single-cell sequencing analysis of B cells isolated from the spleen of mice also showed that they expressed IL10. These results collectively suggest that B cells are the predominant cell type expressing IL10 in mouse spleen.

overall_design: Wild-type mice were injected with LPS via tail vein to induce innate immune response. Four hours later, the spleens of mice were collected, and CD45+ CD3-CD19 + B220+ B cells were sorted for single cell sequencing analysis.


title: Human Pluripotent Stem Cell Derived Organoids Reveal a Role for WNT Signaling in Dorsal-Ventral Patterning of the Hindgut

first: Jorge

last: Munera

address: 173 Ashley Avenue

summary: The cloaca is a transient structure that forms in the terminal hindgut giving rise to the rectum dorsally and the urogenital sinus ventrally. Similarly, human hindgut cultures derived from human pluripotent stem cells generate human colonic organoids (HCOs) which also contain co-developing urothelial tissue. In this study, our goal was to identify pathways involved in cloacal patterning and apply this to human hindgut cultures. RNA-seq data comparing dorsal versus ventral cloaca in e10.5 mice revealed that Wnt signaling was elevated in the ventral versus dorsal cloaca. Inhibition of WNT signaling in hindgut cultures biased their differentiation towards a colorectal fate. WNT activation biased differentiation towards a urothelial fate, giving rise to human urothelial organoids (HUOs). HUOs contained cell types present in human urothelial tissues. Based on our results, we propose a mechanism whereby WNT signaling patterns the ventral cloaca, prior to cloacal septation, to give rise to the urogenital sinus.

overall_design: To investigate the function of WNT signaling in the Dorsal-Ventral patterning of the hindgut, D7 spheroid, D10 and D35 organoids were generated from human iPSC 72.3 using different culture conditions. RNA-Seq. data from these organoids samples was used for the PCA and GO analysis.

last: Qu

first: Abdelkader

last: Daoud

first: Daniel

last: Kechele

middle: O

last: Múnera


title: Single Cell sequencing analysis of Mouse Bladder Tumor Samples

first: Ganiraju

last: Manyam

address: 1515 Holcombe Blvd

institute: UT MD Anderson Cancer Center

summary: Single-cell RNA sequencing analysis identifies acute changes in the tumor microenvironment induced by interferon α gene therapy in a murine model of bladder cancer.

overall_design: scRNAseq of Ad-Ctrl and Ad-IFNα treated mice tumors

first: Sharada

last: Mokkapati

first: Alexis

last: Steinmetz


title: BPTF cooperates with MYCN/MYC to link neuroblastoma cell cycle control with epigenetic cell states [Cut&Run]

city: Madrid

first: Jaime

last: Martinez de Villarreal

state: Madrid

address: Melchor Fernández Almagro, 3

department: Molecular Oncology Programme

country: Spain

institute: CNIO

summary: The nucleosome remodeling factor BPTF is required for the deployment of the MYC-driven transcriptional program. Deletion of oneBptfallele delays tumor progression in mouse models of pancreatic cancer and lymphoma. In neuroblastoma, MYCN cooperates with the transcriptional core regulatory circuitry (CRC). HighBPTFlevels are associated with high-risk features and decreased survival. BPTFdepletion results in a dramatic decrease of cell proliferation. Bulk RNA-seq, single-cell sequencing, and tissue microarrays reveal a positive correlation ofBPTFand CRCtranscription factor expression. Immunoprecipitation/mass spectrometry shows that BPTF interacts with MYCN and the CRC. Genome-wide distribution analysis of BPTF and CRC in neuroblastoma reveals a dual role forBPTF: 1) it co-localizes with MYCN/MYC at the promoter of genes involved in cell cycle and 2) it co-localizes withthe CRC at super-enhancers to regulate cell identity. The critical role of BPTF across neuroblastoma subtypes supports its relevance as a therapeutic target.

overall_design: Analysis of the genome wide distribution of BPTF assessed using Cut&Run in neuroblastoma cell lines.  To further address the relevance of BPTF to neuroblastoma cell identity,we used the inducible PRRX1-mediated ADRN-MES transdifferentiation model in KP-N-YN cells.

first: Irene

last: Felipe

last: Martínez-de-Villarreal

first: Francisco

middle: X

last: Real


title: FGF4 stimulation increases variability in mESCs [scRNA-seq]

city: Leiden

first: Max

last: Fernkorn

address: Einsteinweg 55

department: Leiden Academic Centre for Drug Research (LACDR)

institute: University Leiden

summary: FGF4 is essential for Epiblast and Primitive Endoderm formation in the mouse embryo and promotes differentiation of mouse embryonic stem cells. However, different FGF-concentrations regulate gene expression quantitatively remained an open question. We used single-cell RNA sequencing to quantify transcriptional variability and dynamics upon FGF4 stimulation of Fgf4-mutant mESCs.

overall_design: Fgf4-mutant mouse embryonic stem cells in N2B27 + Chiron were stimulated for 24 h with different concentration of FGF4.

first: Christian

last: Schröter


title: The integrated stress response finetunes 18S nonfunctional rRNA decay (selective Ribo-Seq)

city: Frederick

first: Colin

last: Wu

state: MD

department: RNA Biology Laboratory

institute: NCI

summary: 18S nonfunctional rRNA decay (NRD) detects and eliminates translationally nonfunctional 18S rRNA. The underlying mechanisms associated with the detection and turnover of nonfunctional 18S rRNA remain elusive. While NRD has been identified and exclusively studied in Saccharomyces cerevisiae, it is unclear whether this quality control pathway exists in mammals. Here we demonstrate the conservation of 18S NRD in mammalian cells. Using genome-wide CRISPR genetic interaction screens, we identify two molecular events triggered by nonfunctional 18S rRNA— activation of the integrated stress response (ISR) and ubiquitination of ribosomal proteins elicited by GCN2 and RNF10, respectively. Selective ribosome profiling reveals nonfunctional 18S rRNA induces translation arrest at start sites. Biochemical analyses show that activation of the ISR limits translation initiation, attenuating collisions between scanning 43S preinitiation complexes and nonfunctional 80S ribosomes arrested at start sites. Thus, the ISR facilitates the turnover of nonfunctional 18S rRNA and 40S ribosomal proteins by RNF10-mediated ubiquitination. Altogether, these results establish a dynamic feedback mechanism by which cells finetune translation initiation to enable ribosome functionality surveillance through the GCN2-RNF10 axis.

overall_design: To identify ribosome pausing across the transcriptome, we used MS2 affinity pulldowns of 18S-tag constructs followed by ribosome profiling in different genetic backgrounds.

first: Aaztli

last: Coria

first: Akruti

last: Shah


title: The integrated stress response finetunes 18S nonfunctional rRNA decay (SHAP-MaP)

overall_design: To ensure proper helix 9 folding we used in vivo RNA secondary structure probing.


title: The integrated stress response finetunes 18S nonfunctional rRNA decay (Ribo-Seq)


title: The integrated stress response finetunes 18S nonfunctional rRNA decay (DMS-MaPseq)

overall_design: To assess the accessibility of the A-site we transfected cells with 18S:WT and 18S:A1824C, subsequently cells were treated  with and without DMS and underwent MS2-affinity pulldown and libraries of the A-site were prepared as described in the methods.


title: The integrated stress response finetunes 18S nonfunctional rRNA decay

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

overall_design: Refer to individual Series


title: Deriving human intestinal organoids with functional tissue-resident macrophages all from pluripotent stem cells

middle: O.

address: 3333 Burnet Ave Location R3.034

department: Developmental Biology

institute: Cincinnati Children's Hospital Medical Center

summary: Background & Aims: Organs of the gastrointestinal tract contain tissue-resident immune cells that function during tissue development, homeostasis, and disease. However, the majority of published human organoid model systems lack resident immune cells, thus limiting their potential as disease avatars. For example, human intestinal organoids (HIOs) derived from pluripotent stem cells contain epithelial and various mesenchymal cell types but lack immune cells. In this study, we aimed to develop an HIO model with functional tissue-resident macrophages. Methods: HIOs and macrophages were generated separately through the directed differentiation of human pluripotent stem cells and combined in vitro. Following two weeks of co-culture, the organoids were used for transcriptional profiling, functional analysis of macrophages, or transplanted into immunocompromised mice and matured in vivo for an additional 10–12 weeks.  Results: Macrophages were incorporated into developing HIOs and persisted in the 2-week in vitro HIOs and for at least 12 week in HIOs in vivo. These macrophages expressed CD163 and had a transcriptional signature that resembled those of the human fetal intestine, indicating that they were acquiring the features of tissue-resident macrophages. HIO macrophages could phagocytose bacteria and produced inflammatory cytokines in response to proinflammatory signals such as lipopolysaccharide, which could be reversed with IL-10.  Conclusions: We generated an HIO system containing functional tissue-resident macrophages for an extended period. This new organoid system can be used to investigate the molecular mechanisms involved in inflammatory bowel disease.

overall_design: Human intestinal organoids at day 35 with and without an added immune component and iPSC- derived Macrophages at day 28 were dissociated into single cells and were subjected to 10X sequencing.

first: J

middle: G

last: Sanchez

first: James

middle: M

last: Wells


title: CD44 plays a key role in aggravating tubular cell apoptosis in renal ischemia-reperfusion injury through p65/ PGC-1α pathway

city: Guangzhou

first: Jiewu

last: Huang

address: State Key Laboratory of Organ Failure Research

institute: Nanfang Hospital

summary: Acute kidney injury (AKI) is rapidly increasing and becomes a major of public health problem nowadays. However, its underlying mechanism has not been elucidated. Studies have shown that cluster of differentiation-44 (CD44) play a role in the pathological process of AKI. Nevertheless, the molecule mechanism has not been totally clarified. Herein, we found that CD44 is increased in renal tubules in ischemia-reperfusion injury (IRI)-induced AKI mice. Knockout of CD44 improved mitochondrial biogenesis and mitochondrial fatty acid oxidation (FAO), further protecting against renal tubular cell apoptosis and kidney injury. Conversely, ectopic expression of CD44 impaired mitochondrial function and FAO, which exacerbated the pathological process of AKI. Transcriptome sequencing revealed NF-κB p65 is highly responsible for this process. In vitro, we found that CD44 induced activation of NF-κB p65 via mitogen-activated protein kinase (MAPK) ERK1/2 and MAPK p38, further transcriptionally silencing peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) promoters. As a result, downregulation of PGC-1α contributed to impairment of mitochondrial dysfunction and FAO, resulting in the deterioration of AKI. Our study found that inhibiting CD44 is a new potential therapeutic strategy for AKI through promoting mitochondrial biogenesis and FAO. The underlying mechanism is associated with MAPK/p65/ PGC-1α pathway.

overall_design: To verify the potential mechanism by which CD44 aggravates AKI, we established IRI models and performed RNA-seq in WT and CD44KO mice. We compared the gene expression differences between the two groups and performed GSEA enrichment analysis


title: Epigenetic priming promotes acquisition of TKI resistance and oncogene amplification in human lung cancer [RNA-seq]

city: New Haven

first: Eric

last: Sun

address: 10 Amistad St

department: Genetics

institute: Yale University

summary: In mammalian cells, gene copy number is tightly controlled to maintain gene expression and genome stability. However, a near-universal molecular feature across cancer types is oncogene amplification, which promotes cancer progression by dramatically increasing the copy number and expression of tumor-promoting genes. For example, in tyrosine kinase inhibitor (TKI)-resistant lung adenocarcinoma (LUAD), oncogene amplification occurs in over 40% of patients’ tumors. Despite the prevalence of oncogene amplification in TKI-resistant tumors, the mechanisms facilitating oncogene amplification are not fully understood. Here, we find that LUAD tumors exhibit a unique chromatin signature demarcated by strong CTCF and cohesin deposition in drug-naïve tumors, which correlates with the boundaries of oncogene amplicons in TKI-resistant LUAD cells. Throughout the acquisition of TKI resistance, we identified a global chromatin priming effect, marked by a dynamic increase of H3K27Ac, cohesin loading, and inter-TAD interactions, which occurs before the onset of oncogene amplification. Furthermore, we have identified that the protein METTL7A, which was previously reported to localize to the ER and inner nuclear membrane, has a novel chromatin regulatory function in binding to amplified oncogenes and regulating cohesin recruitment and inter-TAD interactions. Surprisingly, we discovered that METTL7A remodels the chromatin landscape prior to any noticeable oncogene copy number gains. Furthermore, while METTL7A depletion has little effect on drug-naïve cells, METTL7A depletion prevents the formation of TKI resistant-clones, highlighting the specific role of METTL7A as cells are acquiring resistance. In summary, we discovered an unexpected mechanism required for the acquisition of TKI resistance regulated by a largely uncharacterized factor, METTL7A. This discovery sheds new light into maintenance of oncogene copy number and paves the way to the development of new therapeutics for preventing TKI resistance in LUAD.

overall_design: RNA-seq: Osimertinib-resistant cells were derived from parental cells over the course of chronic osimertinib treatment. Time-course experiments with Osimertinib treatment specify how many days of treatment occurred before harvesting for RNA.

first: Rebecca

last: Starble

first: Rana

last: Gybli

first: Jonathan

last: Radda

first: Jiuwei

last: Lu

first: Tyler

middle: B

last: Jensen

first: Ning

first: Shuai

last: Zhao

first: Nitin

last: Roper

first: Gang

last: Wang

first: Jikui

last: Song

first: Katerina

last: Politi

first: Siyuan

first: Andrew

middle: Z

last: Xiao


title: Induced expression of ectopic myelin genes by the transcription factor SOX10 in an enteric glial cell line

city: Kiel

first: Francois

last: Cossais

address: Otto-Hahn-Platz 8

department: Institute of Anatomy

institute: Kiel University

summary:
This study investigates the impact of Sox10 overexpression on enteric glial cell (EGC) phenotype using a doxycycline-inducible EGC cell line. Sox10 overexpression upregulated genes were associated with myelin regulation and glial differentiation pathways, resembling Schwann cell or oligodendrocyte-specific SOX10 target genes.
These findings suggest cell-specific regulation of glial phenotype by SOX10 dosage and identify potential target genes, contributing to our understanding of EGC diversity.

overall_design: Rat enteric glial cell line (JUG2) were generated to overexpress SOX10 or GFP after treatment with doxycycline. Genome-wide expression analysis was performed using Clariom S microarray

first: François

first: Meike

last: Kähler

first: Michel

last: Neulist


title: Eicosatetraynoic Acid Regulates Pro-Fibrotic Pathways in an Induced Pluripotent Stem Cell Derived Macrophage:Human Intestinal Organoid Model of Crohn’s Disease

first: Tzipi

last: Braun

address: Derech Sheba 2

institute: Sheba Medical Center at Tel Hashomer

summary: Background: We previously reported Crohn’s Disease (CD) ileal macrophage and fibroblast gene expression modules linked to future strictures, and identified small molecules which may reverse this gene signature. Here we developed a model system to test a lead candidate, eicosatetraynoic acid (ETYA), a Peroxisome Proliferator-Activated Receptor (PPAR) agonist.  Methods: CD patient induced pluripotent stem cells (iPSC) were differentiated into macrophages and human intestinal organoids (HIOs).  Macrophages and macrophage:HIO co-cultures were exposed to lipopolysaccharide (LPS) with and without ETYA pre-treatment. Cytospin and flow cytometry characterized macrophage morphology and activation markers, and RNA sequencing defined the global pattern of macrophage gene expression. TaqMan Low Density Array, Luminex multiplex assay, immunohistologic staining, and sirius red polarized light microscopy were performed to measure macrophage cytokine production and HIO pro-fibrotic gene expression and collagen content. Results: iPSC-derived macrophages exhibited morphology similar to primary macrophages and expressed inflammatory macrophage cell surface markers including CD64 and CD68. LPS-stimulated macrophages expressed a global pattern of gene expression enriched in CD ileal inflammatory macrophages and matrisome secreted products, and produced cytokines and chemokines including CCL2, IL1B, and OSM implicated in refractory disease. ETYA suppressed CD64 abundance and pro-fibrotic gene expression pathways in LPS stimulated macrophages. Co-culture of LPS-primed macrophages with HIO led to up-regulation fibroblast activation genes including ACTA2 and COL1A1, and an increase in HIO collagen content.  ETYA pre-treatment prevented pro-fibrotic effects of LPS-primed macrophages. Conclusions: ETYA inhibits pro-fibrotic effects of LPS-primed macrophages upon co-cultured HIO. This model system may be used to screen personalized effects of candidate small molecules upon inflammatory and fibrotic pathways implicated in refractory CD patients.

overall_design: CD patient-induced pluripotent stem cells (iPSC) were differentiated into macrophages. These macrophages were exposed to lipopolysaccharide (LPS) with and without ETYA pre-treatment. Overall there were 6 macrophages controls with no treatment, 6

first: Ingrid

last: Jurickova

first: Benjamin

last: Dreskin

first: Elizabeth

last: Angerman

first: Erin

last: Bonkowski

first: Kentaro

last: Tominaga

last: Iwasawa

first: Rotem

last: Hadar

first: Takanori

last: Takebe

first: Yael

last: Haberman

first: James

last: Wells

first: Lee

middle: A

last: Denson


title: Photomorphogenesis of Myxococcocus macrosporus: New Insights for Light-Regulation of Cell Development

city: Lexington

first: Kinga

middle: B.

last: Graniczkowska

state: KY

address: 675 Rose St

department: Biology

institute: University of Kentucky

summary: Myxobacteria are social bacteria distinguished among prokaryotes by a multicellular stage in their life cycle known as fruiting bodies formations, which is a response to nutrient deprivation. Additionally, fruiting body formation in Stigmatella aurantiaca is stimulated by light. Genome sequence of Myxococcus macrosporus, revealed the presence of a red-light photoreceptor, bacteriophytochrome (BphP), found in photosynthetic and non-photosynthetic bacteria. BphPs belong to a large phytochrome family of photoreceptors originally discovered in plants, where they are directly linked to the circadian clock genes. BphPs have been implicated in carotenoid synthesis, motility, and photodynamic stress in non-photosynthetic bacteria. The role of the BphPs in M. macrosporus remains unknown although it has been linked to fruiting body formation in related S. aurantiaca. We have observed that M. macrosporus displays differential photomorphogenic behaviors under constant light, and 12:12 hour light/dark (LD) cycle as compared to constant dark (DD) conditions. We hypothesize that BphP plays a role in these behaviors. In this study we demonstrate light and nutrient dependent pattern of fruiting bodies development in Myxococcus macrosporus HW-1. Additionally, we characterized light induced differential gene expression with the special focus on the circadian clock, and carotenoid biosynthesis genes in M. macrosporus and M. xanthus.

sample_organism:
Myxococcus xanthus
Corallococcus macrosporus

overall_design: We incubated vegetative cells of M. xanthus and M. macrosporus in constant darkness (DD) or 12:12 light dark (LD) cycle. Samples of stationary cells were collected at 36 hours post inoculation from DD and LD cultures to investigate impact of light on gene expression of above mentioned myxobacteria.

first: Mitchell

last: Singer

first: Emina

last: Stojković


title: Regulatory T cells in the Tumor Microenvironment Display a Unique Chromatin Accessibility Profile (ATAC-Seq)

city: Pittsburgh

first: Dhivyaa

last: Rajasundaram

state: PA

address: 4401 Penn Avenue

department: Pediatrics

institute: University of Pittsburgh

summary: Regulatory T cells (Tregs) are a suppressive CD4+ T cell population that limit the anti-tumor immune response. In this study, we analyzed the chromatin accessibility of Tregs in the murine tumor microenvironment (TME) to identify tumor-specific accessible peaks and if these are altered over time in the tumor microenvironment, with or without anti-PD1 immunotherapy. We found that Tregs have a more distinct chromatin accessibility signature in the TME compared to Tregs in the periphery than within location over time in the models we accessed. This distinct tumor Treg chromatin accessibility profile highlights reduced accessibility at loci important for an CD4+ conventional T cell (CD4+ Foxp3—) effector phenotype and reinforcement of a suppressive phenotype. Analysis of chromatin accessibility in Tregs in B16 and MC38 tumor models indicated similar regulation independent of tumor type, indicating a tumor-derived Treg effector signature in the models we accessed. We also found that Tregs do not alter their transcriptome nor chromatin accessibility following immunotherapy. We conclude that chromatin accessibility in Tregs is altered in the TME, but is otherwise remarkably stable and appears unaltered by tumor type, over time, or following immunotherapy.

overall_design: Regulatory T cells from the murine B16 and MC38 tumor microenvironment were accessed for chromatin accesibility by bulk ATAC-sequencing.

first: Rebekah

last: Dadey

first: Dario

last: Vignali


title: Regulatory T cells in the Tumor Microenvironment Display a Unique Chromatin Accessibility Profile (RNA-Seq)

overall_design: Regulatory T cells from the murine that were injected with MC38 and treated with isotype or anti-PD1 were accessed for chromatin accesibility by bulk RNA-sequencing.


title: Epigenetic priming promotes acquisition of TKI resistance and oncogene amplification in human lung cancer

first: Eric

last: Sun


title: Effect of deletion of ZHX3 on gene expression in human pancreatic beta cells EndoC-BH1

city: Singapore

first: Adrian

middle: Kee Keong

last: Teo

state: Singapore

address: 61 Biopolis Drive, Proteos

department: Stem Cells and Diabetes Laboratory

country: Singapore

institute: IMCB, A*STAR

summary: ZHX3 is a transcriptional factor whose role in human pancreatic beta cells is not well understood. In attempt to identify the genes that ZHX3 regulates in beta cells, we KO ZHX3 using CRISPR in EndoC-βH1 cells and performed RNA-Seq. We have only identified 36 and 31 genes that were significantly up and downregulated respectively. This is in concordance with our functional data whereby the KO of ZHX3 in EndoC-βH1 did not affect its insulin secretory function. Overall, we conclude that the KO of ZHX3 did not result in major perturbations in EndoC-βH1 cells.

overall_design: ZHX3 was deleted using the CRISPR-Cas9 system with one gRNA targeting the first coding exon of ZHX3 (sgZHX3-3). EndoC-βH1 cells were transduced with either empty lentiCRISPRv2 plasmids or lentiCRISPRv2 plasmids containing sgZHX3-3. The cells were then selected with antibiotics and cultured as stable lines.

first: Wei-Xuan

last: Tan

first: Gokce

last: Oguz

first: Adaikalavan

last: Ramasamy

first: Elhadi

last: Iich

middle: K


title: SNP profiling of blood DNA samples of breast cancer patients and healthy women

city: Central Jakarta

first: Rafika

middle: Indah

last: Paramita

address: Salemba Raya

country: Indonesia

institute: Faculty of Medicine Universitas Indonesia

summary: Illumina Infinium Asian Screening Array (ASA) are used to found any SNPs from Asian population. Therefore, we profiled the blood DNA samples to found SNPs related to breast cancer

overall_design: To investigate the SNPs related to breast cancer, the hybridizations were conducted on Illumina ASA BeadChips. Analysis were performed by using gtc2vcf and PLINK software

first: Sonar

last: Panigoro

first: Noorwati

last: Sutandyo

first: IGN Gunawan

last: Wibisana

first: Rafika Indah


title: Epigenetic priming promotes acquisition of TKI resistance and oncogene amplification in human lung cancer [ChIP-seq]

overall_design: ChIP-seq: ChIP-seq was performed against IgG or MYC-tag in Wild-Type or METTL7A-MYC-tag-FLAG expressing cells.

first: Rebecca

last: Starble

first: Rana

last: Gybli

first: Jonathan

last: Radda

first: Jiuwei

last: Lu

first: Tyler

last: Jensen

first: Ning

first: Shuai

last: Zhao

first: Nitin

last: Roper

first: Gang

last: Wang

first: Jikui

last: Song

first: Katerina

last: Politi

first: Siyuan

first: Andrew

last: Xiao


title: NSUN2 mediated m5C modification regulates myocardial hypertrophy

first: Dongdong

last: Jian

address: 38 Xueyuan Road

department: Biochemistry and Molecular Biology

institute: Peking University Health Science Center

summary: Aims: Mammalian mRNA internal modification m6A has been suggested to be essential in the maintenance of cardiac homeostasis. The role of mRNA cytosine methylation (m5C) in the heart remains unexplored.Methods and results: In contrast with human normal heart, the expression of RNA cytosine methyltransferase NSun2 was found to be significantly elevated in the hypertrophied one. Consistently, NSun2 expression was increased in hypertrophic rat ventricular myocytes induced in vitro, as well as in TAC (transverse aortic constriction) developed hypertrophied murine heart. Cardiomyocyte-specific knockout of NSun2 (Myh6-CreERT2, NSun2 flox+/+) dramatically accelerated heart failure progression in TAC mice. RNA m5C immunoprecipitation followed by sequencing and MeRIP (methylation-specific RNA immunoprecipitation) revealed that NSun2 particularly methylates the mRNA of protein kinase A (PKA) catalytic subunit alpha (PRKACA). Polyribosome and dual luciferase reporter gene analysis further suggested that NSun2 promotes PRKACA translation via RNA methylation. In TAC hypertrophic cardiomyocytes, NSun2 ablation markedly dampened PKA signaling, as evidenced by reduced PKA activity and protein phosphorylation levels of PKA diverse substrates, impaired myocyte contraction and relaxation, and disturbed calcium transients. Cardiac-restricted overexpression of NSun2 via adeno-associated virus 9 (AAV9, NSun2 expression cassette driven by cTnT promoter) sensitized heart hypertrophic response, whereas administration of PKA inhibitor H89 successfully blocked this process, reiterating the importance of NSun2-PRKACA regulation in cardiac hypertrophic program.Conclusions: Our study showed that RNA methyltransferase NSun2 promotes PRKACA translation via RNA methylation, whereby achieving its modulation of PKA signaling within the cardiomyocyte. NSun2-PRKACA regulation is required for cardiac homeostasis maintenance and adaptation to hypertrophic stresses. These findings furnish us with novel understanding of heart function mediated by mRNA cytosine methylation in cardiac homeostasis.

overall_design: NSUN2flox/flox and NSUN2-cKO mice were divided into the following four groups: NSUN2flox/flox group (FF), NSUN2-cKO group (cKO), NSUN2flox/flox+TAC group (FT), and NSUN2-cKO+TAC group (CT). Three weeks after TAC surgery, two mice were selected from each group, and left ventricular myocytes were isolated using the Langendorf method, followed by m5C-RIP-seq.

first: Xiaolei

last: Cheng

first: Liguo

first: Wengong

first: Junyue

last: Xing

first: Hao

last: Tang


title: Regulatory T cells in the Tumor Microenvironment Display a Unique Chromatin Accessibility Profile

first: Dhivyaa

last: Rajasundaram


title: Transcriptomic analysis of ZHX3-depleted HepG2 cells

first: Adrian

last: Teo

summary: The aim of this RNA-Seq analysis was to identify target genes of the transcription repressor ZHX3 in HepG2 cells. To do so, four stable lines were generated in HepG2 using three different ZHX3-targeting CRISPR constructs and an empty vector construct. Bulk RNA-Seq analysis was performed on these four lines. Sequencing was done by NovogeneAIT Genomics using the Illumina® NovaSeq6000-PE150 platform and 63-82 million base pairs were sequenced per sample. A total of 47 and 156 genes were found to be consistently upregulated and downregulated (fold change ≥ 2 and FDR < 0.05), respectively, in all three KO lines as compared to the empty control line. Functional enrichment analysis of the 156 upregulated genes using g:profiler revealed the enrichment of two biological processess, urate transport and urate metabolic process. qPCR analysis validated the upregulation of uric acid transporter gene SLC17A1.

overall_design: Four different HepG2 stable lines were generated by transfecting with four different CRISPR constructs: (1) empty vector control (termed as Empty), (2) sgZHX3-2 (Z2) , (3) sgZHX3-5 (Z5) and, (4) sgZHX3-9 (Z9). Each stable line was seeded in three wells of a 24-well plate (triplicates).

first: Wei

last: Tan

first: Gokce

last: Oguz

first: Adaikalavan

last: Ramasamy


title: Loss of CPT1a Modulates Phosphatidylethanolamine Levels Leading to Microvesicular Steatosis in Female Mice

first: Robert

last: Helsley

address: 741 S. Limestone St.

department: Intern

summary: To investigate the impact of liver-specific deletion of Cpt1a in hepatic lipid metabolism.

overall_design: We performed bulk RNA-sequencing on livers from HFD-fed WT and Cpt1a LKO male and female mice.

first: Helsley

last: Robert

first: Zelows

last: Mikala


title: Identification of genes regulated by the MADS transcription factor (TF), SEPALLATA3 spliced variant, SEP3.3 , in the context of the  triple sep1 sep2 sep3 mutant background, by RNA-Seq analysis

city: Grenoble

first: Veronique

last: Hugouvieux

address: 17 rue des Martyrs

department: IRIG

country: France

institute: CEA Grenoble

summary: These analyses allowed the identification of more than 2000 genes regulated by the MADS TF, SEPALLATA3.3 spliced variant(renamed SEP3Δtet or SEP3deltet).

overall_design: RNA seq analysis were performed on inflorescence meristem and flower buds up to stage 10-11 harvested from Arabidopsis plants grown in parralel in long days conditions. Experiments were performed on 3 independant lines   sep1sep2sep3  expressing SEP3Δtet . These lines are described in Hugouvieux et al, Nucleic Acid Research, 2018:1;46(10):4966-4977. doi: 10.1093/nar/gky205. Libraries and sequencing were performed by Genewiz (Illumina High SEq, 2X150 bp configuration).

first: Véronique

first: Jeremy

last: Lucas

first: Chloé

last: Zubieta


title: RNA Expression Profiles of Vascular Smooth Muscle Cells Isolated from Mice with and without Bmal1 Deletion

status: Public on Feb 25 2025

city: La Jolla

first: Ming

last: He

state: CA

address: 9500 Gilman Dr.

institute: UCSD

summary: To investigate the transcriptional changes in vascular smooth muscle cells (VSMC) induced by diabetic condition medium via Bmal1, we isolated VSMCs from three pairs of Bmal1 flox/flox (wild-type, WT) and Bmal1 Δ/Δ (Bmal1 knockout) littermates. The cells were cultured in diabetic condition medium for seven days. Total RNA was extracted using the RNeasy Mini Plus Kit (Qiagen) and analyzed via RNA sequencing (RNA-seq) to profile transcriptomic changes.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for WT and Bmal1 KO VSMC treated with diabetic conditions medium for 7 days.

first: Zhehao

last: Zhu

first: Fengyuan

last: Huang

first: Zechen

last: Chong

first: Yabing

last: Chen


title: Mapping the initial effects of carcinogen-induced oncogenic transformation in the mouse bladder

city: Bergen

first: Luiza

last: Ghila

address: Haukelandsbakken 15

department: Department of Clinical Science

country: Norway

institute: University of Bergen

summary: BBN is a bladder specific carcinogen that can cause invasive bladder cancer in mice possessing similar genetic alterations as seen in human cancer. We employed a single cell sequencing approach using 10x Genomics scRNAseq to investigate the cellular heterogeneity and molecular modifications induced by long term treatment with a carcinogen (BBN) on the bladder urothelium and sub-urothelium cells. Our data show infiltrations of B- and T-cells in the tumor and decrease of the several other urothelial populations.

overall_design: 3 sample analysed: 2 non-treated controls and 1 BBN-treated. After 22 weeks of BBN treatment, the mucosa layer of mouse bladder was separated from detrusor and digested to prepare a cell suspension and single live cells were sorted using Fluorescence-activated cell sorting (FACS). Then scRNA-seq was performed to analyze the cells. The same was done for two age-matched non-treated mice.

first: Md Kaykobad

last: Hossain

first: Lucas

last: Unger

first: Ulrik

last: Larsen


title: Extracorporeal shock waves effectively suppress colorectal cancer proliferation and growth

first: Xiaoli

last: Zhang

address: 119 Xincun Road, Fengtai District

institute: Beijing Neurosurgical Institute

summary: Shock waves are widely used to treat various diseases and have numerous medical applications. In particular, extracorporeal shock waves (ESV) can substantially inhibit tumour growth. However, the therapeutic efficacy of ESV in colorectal cancer and its underlying mechanisms are not well understood. To address this gap in our knowledge, colorectal cancer cell lines HT29 and SW620 were used to generate xenograft mouse models and examined the therapeutic effects of a stepwise increase in ESV energy on tumour growth. In vivo, 60 mJ ESV significantly delayed xenograft growth compared with 120 and 240 mJ ESV, with no impact on body weight or hepatic and renal function. Transcriptome analysis revealed that 60 mJ ESV suppressed colorectal cancer cell proliferation and induced apoptosis and ferroptosis; these findings were further confirmed by immunohistochemical staining and western blotting. The in vitro study showed that ESV mechanistically suppressed cell proliferation and induced apoptosis and ferroptosis by activating the p53 signaling pathway. In conclusion, 60 mJ ESV substantially inhibited colorectal cancer growth by activating p53 pathway-related proliferation inhibition and cell death. These findings indicate that ESV therapy is a promising therapeutic strategy for colorectal cancer.

overall_design: RNA-Seq profiling of colorectal cancer cell line HT-29-derived xenografts from the control  and 60 mJ extracorporeal shock waves (ESV) groups at day15 of  60 mJ ESV therapy.

first: Chun

last: Ran

first: Qingzhi

last: Song

first: Guoqing

last: Lv


title: Wzi regulates host innate immunity to promote Klebsiella pneumoniae survival in a CPS-dependent and -independent manner

city: Shenzhen

first: Sha

last: Wei

address: Dongmen North Road No. 1017, Shenzhen, China

institute: Shenzhen People’s Hospital

summary: Klebsiella pneumoniae is a major pathogen that causes a variety of human infections, posing a significant public health threat. Understanding its pathogenesis is essential for devising effective treatment strategies. In this study, we aim to identify critical virulence factors in K. pneumoniae through analyzing virulence-associated genes that were identified in three transposon mutagenesis libraries. Two genes, wzi and kvrB, are consistently detected across these libraries, indicating their potential as critical virulence factors. While Wzi has usually been implicated in virulence through CPS, its actual function in K. pneumoniae pathogenicity has rarely been explored. Wzi deficiency reduces CPS production in K. pneumoniae, contrasting with its effect in Escherichia coli. Importantly, Wzi exerts a pivotal role in K. pneumoniae pathogenicity in vitro and in vivo, functioning through both CPS-dependent and -independent pathways. Wzi inhibits the secretion of IFN-γ-related cytokines at early infection stage to promote K. pneumoniae survival in the host. Wzi triggers sustained neutrophil recruitment during infection through the upregulation of CXCL1 expression, resulting in the pulmonary barrier damage and increased K. pneumoniae invasion into the bloodstream. Concurrently, Wzi confers K. pneumoniae to counteract neutrophil-mediated clearance in a CPS-dependent manner. Sequence polymorphisms of wzi significantly affect bacterial resistance to serum killing, with alleles frequently associated with hypervirulent K. pneumoniae exhibiting the highest resistance. Collectively, our findings highlight that the dual role of Wzi as a CPS-dependent and -independent virulence factor that combats host clearance during K. pneumoniae infection, representing a promising target for the development of anti-infective treatment against the bug.

overall_design: RNA-seq profiling of the lungs of mice infected with Klebsiella pneumoniae WT and wzi deletion mutant at 5 and 10 hours post infection.

first: Kai

last: Zhou

first: Tingting

last: Xu

first: Dongliang

last: Li

first: Quanhang

last: Xiang


title: 16S rRNA gene sequencing analysis of the colon contents in mice

city: Chongqing

first: Ruijing

last: Yang

address: yuzhong

institute: Chongqing medical university

summary: We conducted 16S rRNA sequencing analyse on colonic contents to evaluate whether forced loss led to alterations in gut microbiota composition and function.

overall_design: Following forced loss and go-through-tube, the colon contents of the forced loss and go-through-tube mice were isolated and analyzed using 16S rRNA gene sequence.

first: Jing

last: Wu

first: Peng

last: Zheng

first: Xin

last: Wang


title: Molecular mechanisms of lifespan extension in long-lived mitocondrial superoxide dismutase sod-2 mutants

city: Montreal

first: Alain

last: Pacis

state: QC

address: 1010 Sherbrooke #1800

country: Canada

institute: Canadian Centre for Computational Genomics (C3G)

summary: To explore the molecular mechanisms of lifespan extension in sod-2 mutants in an unbiased manner, we used RNA sequencing to identify differentially expressed genes in sod-2 mutants compared to wild-type worms. We found a large number of gene expression changes at day 1 of adulthood and much fewer at day 8. Upregulated genes in sod-2 mutants included genes involved in innate immunity and cuticle development. There were many more differentially expressed genes between day 1 and day 8 of adulthood compared to differences between the genotypes.

overall_design: sod-2 and wild-type N2 worms were collected at day 1 and day 8 of adulthood.

first: Jeremy

last: Van Raamsdonk

first: Ulrich

last: Anglas