title: SARS-CoV-2 Nsp2 recruits GIGYF2 near viral replication sites and supports viral protein production

city: Seoul

first: Sunghan

last: Lee

state: South Korea

address: Building 203 Room 526, 1, Gwanak-ro, Gwanak-gu

department: School of Biological Sciences

institute: Seoul National University

summary: The SARS-CoV-2 genome encodes 16 non-structural proteins (Nsps), with Nsp2 being the least conserved and least understood. In this study, we found a crucial role of Nsp2 in the SARS-CoV-2 life cycle and demonstrated its interaction with GIGYF2, which causes the relocation of GIGYF2 to near double membrane vesicles (DMVs) and the enhancement of viral protein production. Deletion of the Nsp2-coding region from the viral genome led to a drastic reduction in viral RNA synthesis early in infection (3–4 hours post infection). Through interactome analysis of Nsp2 in virus-infected cells, we identified GIGYF2—a protein involved in translational regulation—as a key partner of Nsp2. We confirm the interaction between GIGYF2 and Nsp2 from both SARS-CoV-1 and SARS-CoV-2. Depleting GIGYF2 or its cofactor ZNF598 phenocopied the replication defects observed with Nsp2 deletion, suggesting their critical roles in viral reproduction. Moreover, GIGYF2 and ZNF598 relocate to areas near DMVs, viral replication sites, upon infection. This relocation does not happen with the Nsp2-deleted virus, indicating Nsp2’s role in directing GIGYF2 to DMVs. Using formaldehyde crosslinking and immunoprecipitation-sequencing experiments (fCLIP-seq), we found that GIGYF2 interacts with viral RNAs, particularly in regions encoding Nsp3, M, and Orf6. Depletion of GIGYF2 resulted in decreased expression of these proteins. Our findings reveal the function of Nsp2 in supporting viral protein production by exploiting GIGYF2 as a host factor.

overall_design: We employed formaldehyde immunoprecipitation and sequencing (fCLIP-seq) using anti-GIGYF2 antibody after SARS-CoV-2 infection. To investigate the function of GIGYF2 and NSP2 in SARS-CoV-2 replication, we performed RNA-sequencing after infection with WT and nsp2-deleted virus. We also performed RNA-sequencing in parental and GIGYF2 shRNA cells after SARS-CoV-2 virus infection.


title: Real-time quantitative PCR analysis of extracellular vesicle miRNA from human U2OS cells culture medium [HRH1]

status: Public on Jul 30 2025

city: Portland

first: Xiao

last: Shi

state: Oregon

address: 3181 SW Sam Jackson PK Rd

department: dept. of psychiatry

country: USA

institute: Oregon Health and Science University

summary: Human U2OS cells were stimulated with selective agonist PEA of Histamine receptor H1 (HRH1) for 24 hours. Extracellular vesicles were isolated from U2OS cell culture media. Total RNAs were purified from extracellular vesicle samples. We used TaqMan Low Density Arrays to quantitate miRNA expression in extracellular vesicles secreted by U2OS cells after GPCR activation.

overall_design: qPCR miRNA expression profiling.  Each GPCR stimulation experiment included its own media control, vehicle control, and treated samples.

first: Aaron

last: Janowsky


title: Real-time quantitative PCR analysis of extracellular vesicle miRNA from human U2OS cells culture medium [AODRA1]

summary: Human U2OS cells were stimulated with selective agonist CCPA of adenosine receptor A1(ADORA1) for 24 hours. Extracellular vesicles were isolated from U2OS cell culture media. Total RNAs were purified from extracellular vesicle samples. We used TaqMan Low Density Arrays to quantitate miRNA expression in extracellular vesicles secreted by U2OS cells after GPCR activation.


title: Cancer-intrinsic Cxcl5 orchestrates global metabolic reprogramming of three dimensional cancers by upregulating Hif1α and Myc expression

city: Daejeon

first: RAMIN

last: SEO

country: South Korea

institute: Korean Advanced Institute of Science and Technology

type: Expression profiling by high throughput sequencing

summary: The tumor microenvironment (TME) of pancreatic cancer is characterized by deposition of extracellular matrix (ECM). In 3D TME comprising cellular and non-cellular components, physical and chemical restriction induce oxygen and nutrients shortage in tumor site. Therefore, it is inevitable for cancer cells to manipulate the biological processes and metabolism for survival. However, it has not been fully explored the metablic phenotype coupling to the components existed in 3D and how the cancer cells exploit the metabolic reprogramming. In this study, we establisehd in vitro 3D tumor spheroid-in-Matrigel culture system recapitulating the 3D TME. We discovered the significant upregulation of Cxcl5 expression in murine pancreatic cancer cell line Panc02 by 3D culture with M1 macrophage conditioned media treatment. The loss of Cxcl5 in 3D Panc02 cells showed dramatical reduction of cancer growth. Importantly, the Cxcl5-deficient 3D cancer cells showed global alteration in metabolism. Additionally, Cxcl5 loss failed to control oxidative stress and in turn, increased ferroptotic cell death. Overexpression of Hif1α and Myc in Cxcl5-deficient cancer cells rescued the impaired metabolic processes, ferroptotic cell death, and cancer growth.

overall_design: 1) To investigate the impact of macrophage soluble factors on Panc02 spheroid growth, we obtained unstimulated or stimulated macrophage conditioned media and treated to Panc02 spheroid. 6.5% FBS containing DMEM was treated to Panc02 spheroid as a control. 2) To investigate the role of Cxcl5 in 3D cancer growth, Cxcl5 was knockout using CRISPR/Cas9 system and cultured in 2D- or 3D-condition 3) To investigate the role of Hif1a and Myc, we overexpressed Hif1a and Myc in Cxcl5-deficient cells respectively.

first: Ramin

last: Seo

first: Suk-jo

last: Kang


title: In vivo efficacy of NRL knockdown with cell-penetrating siRNA in retinal degeneration

city: Ulsan

first: Hyo Kyung

address: UNIST-gil 50

department: Biomedical engineering

institute: UNIST

summary: In retinal degenerative diseases, including retinitis pigmentosa (RP) and age-related macular degeneration (AMD), loss of photoreceptors at the macula leads to blindness. Among photoreceptors, rod cells are more vulnerable to degenerative stress than cone cells. Following disruption of neural retina leucine zipper (NRL) expression induced by knockout or the CRISPR/Cas9 system, rod cells have been shown to gain increased expression of several cone cell markers and demonstrate improved survival under degenerative pressure, consequently preventing secondary cone cell degeneration. Here, we report an mRNA silencing approach in which a cell-penetrating asymmetric small interfering RNA (cp-asiRNA) targeting NRL (cp-asiNRL) in postmitotic photoreceptors was used to suppress the expression of the NRL, which determines the fate of developing photoreceptors to rod cells. In mouse models of RP and neovascular AMD, intravitreal delivery of cp-asiNRL substantially increased the expression of cone-specific genes among the photoreceptor population and improved rod cell survival. Furthermore, in retinal organoids, cp-asiNRL also increased the production of photoreceptors expressing cone-specific markers. Our data suggest that cp-asiNRL-mediated NRL suppression in rod cells may be a promising strategy for treating retinal degenerative diseases.

overall_design: Rho(P23H/+) mice (male and female, one to four months old) were maintained on a C57BL/6J background (Jackson Laboratory). Rho(P23H/+) transgenic mice were purchased from the Jackson Laboratory (stock no. 000664). The P23H Rho mutation in Rho(P23H/+) transgenic mice leads to the misfolding of rhodopsin in rod photoreceptors. This activates the unfolded protein response, ultimately leading to the death of both rod and cone cells approximately 2 weeks after birth. Therefore, the mice were treated by intravitreal injection of cp-asiNRL (0.4 μg/μl) or PBS as a control on day 14 after birth, and the eyes were subsequently analyzed on day 35.

first: Hyungwoo

first: Hyoik

last: Jang

first: Jae-Byoung

last: Chae

first: Chul-Woo

last: Park

first: Taejeong

last: Ha

first: Munjeong

last: Yum

first: Sungmi

first: Sun-Woo

last: Hong

first: Suk-Young

first: Jungmook

last: Lyu

first: Dong-Ki

first: Hyewon

last: Chung

first: Hyo-Kyung

first: Semin


title: Real-time quantitative PCR analysis of extracellular vesicle miRNA from human U2OS cells culture medium [ACKR3]

first: Xiao

summary: Human U2OS cells were stimulated with selective agonist SDF-1 of atypical chemokine receptor 3 (ACKR3) for 24 hours. Extracellular vesicles were isolated from U2OS cell culture media. Total RNAs were purified from extracellular vesicle samples. We used TaqMan Low Density Arrays to quantitate miRNA expression in extracellular vesicles secreted by U2OS cells after GPCR activation.

first: Aaron


title: Histone H4 Lysine 20 Methylation Marks Genes Dynamically Regulated During Erythroid Maturation (CUT&Tag)

city: Rochester

first: Laurie

last: Steiner

state: NY

address: 601 Elmwood Ave

department: Pediatrics

institute: University of Rochester

type: Genome binding/occupancy profiling by high throughput sequencing

summary: We profiled H4K20 mono- and tri- methylation during the well-characterized erythroid differentiation of human hematopoietic stem and progenitor cells.

overall_design: CUT&Tag

first: K

middle: E

last: Murphy

first: Michael

last: Getman

first: Xiurui

last: Lv

first: Z

middle: C

first: Deanna

last: Adib

first: N

last: Raman

first: L

middle: A


title: Histone H4 Lysine 20 Methylation Marks Genes Dynamically Regulated During Erythroid Maturation (CUT&RUN)


title: Transcriptome Analysis of Herbs Pulsatilla chinensis, Bupleurum chinense, and Polyporus umbellatus in the Treatment of Lung and Liver Cancer

city: stockholm

first: mengzhen

last: Li

address: Drottning Kristinas Väg 36A

country: Sweden

institute: KTH Royal Institute of Technology

summary: To explore the treatment mechanisms of herbs Pulsatilla chinensis, Bupleurum chinense, and Polyporus umbellatus on lung and liver cancer. We conducted transcriptomic analysis on A549 and Huh7cells treated with these herbs extracts.

overall_design: We did a comparative gene expression profiling analysis of RNA-seq data for four groups including the Pulsatilla chinensis (BTW) treated group, the Bupleurum chinense (CH) treated group, and the Polyporus umbellatus (ZL) treated group.

first: Adil

last: Mardinoglu


title: Global Chromatin Remodeling and Widespread Differential Methylation Changes Underlie Brown Adipose Dysfunction and Dysregulated Metabolism in Response to Chronic Air Pollution Exposure: A Key Role for Reciprocal Regulation by HDAC9 and KDN2b

city: Baltimore

first: Bongsoo

state: Maryland

address: 251 Bayview Blvd

department: Translational Gerontology Branch

institute: National Institute on Aging

type: Methylation profiling by high throughput sequencing

summary: The Global Burden of Disease implicates air pollution in mediating as much as 20% of Type 2 Diabetes mellitus globally. This profound impact of air pollution is supported by experimental studies suggesting effects on whole body metabolism and insulin resistance. We explored the effect of chronic inhalational air pollution exposure for 12 weeks, using a whole body inhalation device in mice that allows for daily exposure to concentrated ambient particulate matter <2.5 microns (6 hours/day, 5 days a week) on whole body metabolism and brown adipose tissue (BAT) function, with detailed investigation of differentially methylated regions (DMRs), chromatin accessibility (differential accessible regions or DARs), and differential gene expression (DEGs) levels using a combination of whole genome bisulfite sequencing, ATAC-sequencing, and RNA-sequencing respectively. A significant impact of chronic PM2.5 exposure on BAT function and whole-body metabolism through alterations in key BAT transcriptional programs resulting in increased redox stress, lipid deposition, fibrosis and reduced thermogenic function of BAT was observed. Significant differential methylation with evidence for both hypomethylation and hypermethylation involving target regions corresponding to both circadian and Nrf2 target genes including sites enriched for transcription factor binding sites implicated in redox stress, insulin resistance and BAT function in response to PM2.5. ATAC sequencing revealed widespread chromatin remodeling involving intronic regions and enhancer elements and loss of DARs in Ucp1 and Gclc major regulatory genes associated with thermogenesis and glutathione synthesis. DARs-DEGs interaction analysis pointed to functional pathways linked to diabetes development, fatty acid biosynthesis activation and activation of JAK-STAT, and MAPK in PM-exposed mice. Integrated analysis of DARs, DEGs and DMRs uncovered the Histone de-acetylase Hdac9 and Kdm2b, a Histone demethylase that demethylates 'Lys-4' and 'Lys-36' of histone H3. ChiP studies demonstrated that Hdac9 physically interacts with the BAT transcription factor Prdm16 and rorα in BAT decreasing their expression and providing at least one direct mechanism for our observations. Collectively our results results link DNA methylation with control of chromatin accessibility and gene expression changes involved in the alteration of brown fat metabolism linking chronic air pollution exposure with alterations in BAT and whole-body metabolism

overall_design: WGBS and ATAC-seq were performed to compare the epigenome of air pollution exposed mice and control mice

first: Palanivel

last: Rangasamy

first: Jean

last: Dazard

first: Sanjay

last: Rajagopalan


title: A kidney specific fasting-mimicking diet induces podocyte reprogramming and restores renal function in glomerulopathy

city: West Hollywood

first: AGCT

last: Core

state: CA

address: 8687 Melrose Ave, PDC B230

department: BMS

institute: Cedars Sinai Medical Center

summary: Cycles of a fasting-mimicking diet (FMD) promote regeneration and reduce damage in the pancreas, blood, gut, and nervous systems of mice but its effect on kidney disease is unknown, and the FMD has not been tested in rats. Here we show that cycles of a newly developed low-salt FMD (LS-FMD) restore normal proteinuria, nephron structure and function in rats with puromycin-induced nephrosis in the long term. LS-FMD induces expression of nephrogenic markers, resembling renal developmental processes in multiple kidney structures, and activates podocyte-lineage reprogramming pathways, in addition to promoting a quiescent state in mature podocytes.

overall_design: Sprague-Dawley rats were injected with puromycin aminonucleoside. Rats were either fed ad libitum, or a low salts fasting mimicking diet (LS-FMD). A group of healhty rats was used as control. Rats were fed a total of 6 cycles of diet and analyzed 6 weeks post treatment after 5 months from the PAN injection. Another healthy rat was fed one cycle of LS-FMD and analyzed at 72 hours post refeeding.  Kidneys were harvested, embedded in OCT and frozen. 10 um sections were cut and laid on a gene expression slide for Visium spatial transcriptomics.


title: Enzyme-independent functions of HDAC3 in the adult heart

city: Houston

first: Zixiu

last: Cheng

state: Texas

address: TX 77030 one baylor plaza, ABBR R631

institute: BCM

summary: The cardioprotective effects of histone deacetylase (HDAC) inhibitors (HDIs) are at odds with the deleterious effects of HDAC depletion. Here, we use HDAC3 as a prototype HDAC to address this contradiction. We show that adult-onset cardiac-specific depletion of HDAC3 in mice causes cardiac hypertrophy and contractile dysfunction on a high-fat diet (HFD), excluding developmental disruption as a major reason for the contradiction. Genetically abolishing HDAC3 enzymatic activity without affecting its protein level does not cause cardiac dysfunction on HFD. HDAC3 depletion causes robust downregulation of lipid oxidation/bioenergetic genes and upregulation of antioxidant/anti-apoptotic genes. In contrast, HDAC3 enzyme activity abolishment causes much milder changes in far fewer genes. The abnormal gene expression is cardiomyocyte-autonomous and can be rescued by an enzyme-dead HDAC3 mutant but not by an HDAC3 mutant (Δ33-70) that lacks interaction with the nuclear-envelope protein lamina-associated polypeptide 2β (LAP2β). Tethering LAP2β to the HDAC3 Δ33-70 mutant restored its ability to rescue gene expression. Finally, HDAC3 depletion, not loss of HDAC3 enzymatic activity, exacerbates cardiac contractile functions upon aortic constriction. These results suggest that the cardiac function of HDAC3 in adults is not attributable to its enzyme activity but to its nuclear envelope tethering action, which has implications for understanding the cardioprotective effects of HDIs.

overall_design: HDAC3 has enzyme dependent and independent  functions, we compared the effect of  HDAC3 KO and HDAC3 inactive mutant NS-DADm recovery in mouse heart protection. The groups used for RNA seq are WT, HDAC3 KO and NS-DADm(HDAC3 inactive).

first: Sichong

last: Qian

first: Chen

last: Zhang

first: Wenbo

first: Shiyang

last: Song

first: Haiyang

first: Huying

last: Shen

first: Zheng

last: Sun


title: Two-generational impacts of airway exposure to silver nanoparticles in utero on male reproductive function in mice

city: Kawasaki

first: Satoshi

last: Yokota

state: Kanagawa

address: Tonomachi, Kawasaki-ku

department: 1.	Division of Cellular & Molecular Toxicology, Center for Biological Safety & Research

country: Japan

institute: Research, National Institute of Health Sciences

summary: This study investigated the effects of airway exposure to silver nanoparticles (AgNPs) in utero on male reproductive function. Exposure of pregnant ICR mice to AgNPs (low dose: 5 μg/injection; high dose: 50 μg/injection) at embryonic day 7.5 and 14.5, by means of intratracheal administration, resulted in dose-dependent exacerbation of sperm parameters in both F1 and F2 males. Microarray analysis revealed that the high-dose group displayed significant upregulation of four microRNAs (miRNAs) and downregulation of seven miRNAs, as compared to the control group, results that were further verified using quantitative real-time PCR. Interestingly, in the mature sperm as well, the high-dose group displayed significant upregulation of one, and downregulation of three of the miRNAs found to be dysregulated in the testes. Notably, in silico analyses predicted that the target genes of the differentially expressed miRNAs in the sperm were significantly associated with neuronal development. Neuronal migration in the cerebral cortex was dysregulated in the F2 males, accompanied by depression-like behavior. These observations demonstrated for the first time that prenatal exposure to AgNPs can induce germ cell development dysfunction in F1 males, which is involved in developmental toxicity in F2 males born to those F1 males.

overall_design: Effects of prenatal exposure to AgNPs on microRNA (miRNA) expression in the testes.Using total RNA extracted from the testes of 12-week-old mice, the expression of 1,908 mature miRNAs (1,908 probe sets) and 1,176 mouse pre-miRNAs (1,255 probe sets) was profiled using the GeneChip® miRNA Array service, according to the protocol of Filgen (Aichi, Japan). The RNA samples were labeled with biotin using the FlashTag™ Biotin HSR RNA Labeling Kit (Applied Biosystems, Foster City, Union City, USA), according to GeneChip® miRNA Arrays User Manual. Briefly, the labeled RNA was hybridized using GeneChip® Hybridization Oven 645 (Applied Biosystems); the plate used in the microarray was miRNA 4.0 Array (Applied Biosystems). After hybridization, the microarray was washed using GeneChip® Fluidics Station 450 (Applied Biosystems), the slides scanned using GeneChip® Scanner 3000 7G system (Applied Biosystems), and the images digitized using Array-Pro Analyzer Ver. 4.5 (Media Cybernetics, Silver Spring, MD, USA). Finally, the data were normalized and expressed as fold increase in comparison to the control, using the Expression Console™ Software (Applied Biosystems). We identified differentially expressed miRNAs with >1.2-fold upregulation or downregulation, along with a false discovery rate of <0.1.

first: Nozomu

last: Moriya

first: Shigeru

last: Oshio


title: Histone H4 Lysine 20 Methylation Marks Genes Dynamically Regulated During Erythroid Maturation

first: Laurie

last: Steiner

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

overall_design: Refer to individual Series


title: Bacterial type II topoisomerases cleave DNA in a species-specific manner

city: Bethesda

first: Ian

middle: L

last: Morgan

state: MD

address: 50 Center Dr.

institute: National Heart Lung and Blood Institute

summary: The type II topoisomerases, gyrase and topoisomerase IV, are essential enzymes in nearly all bacteria and are the targets of fluoroquinolones, which are some of the most widely prescribed broad-spectrum antibacterials in clinical use. As part of their catalytic cycle, gyrase and topoisomerase IV transiently cleave DNA in a sequence-dependent manner. However, it is unclear whether this sequence-dependence is species-specific. Therefore, using our recently developed SHAN-seq method, we mapped and compared cleavage sites for type II topoisomerases from three different pathogenic bacterial species, Escherichia coli, Bacillus anthracis, and Mycobacterium tuberculosis in the presence of the fluoroquinolone, ciprofloxacin. We found that the enzymes have substantially different DNA cleavage specificities that vary between gyrase and topoisomerase IV, across species, with supercoil chirality, and in response to ciprofloxacin. Our results demonstrate that bacterial species fine-tune the DNA cleavage specificity of their type II topoisomerases. This finding suggests that cleavage specificity may play important physiological roles and, in turn, may affect the susceptibility of bacteria to fluoroquinolone antibacterials.

overall_design: Supercoiled pBR322 DNA was cleaved with DNA gyrase or topoisomerase IV and spiked with three DNA oligonucleotides. The DNA was subject to standard DNA-seq library preparation to label the enzyme-cleaved ends, fragmented, and subject to a second round of standard DNA-seq library preparation to label the fragmented ends. The labelled DNA was pair-end sequenced and mapped to the plasmid and spike-in oligonucleotide reference sequences. The reads from the topo-cleaved plasmid DNA ends were normalized by the reads from the spike-in ends and were used to determine the gyrase or topo IV cleavage levels at each position on the plasmid.

first: Jeffrey

middle: J

last: Jian

first: Neil

last: Osheroff

first: Keir

last: Neuman


first: AGCT

summary: Cycles of a fasting-mimicking diet (FMD) promote regeneration and reduce damage in the pancreas, blood, gut, and nervous systems of mice but its effect on kidney disease is unknown, and the FMD has not been tested in rats. Here we show that cycles of a newly developed low-salt FMD (LS-FMD) restore normal proteinuria, nephron structure and function in rats with puromycin-induced nephrosis in the long term. LS-FMD induces expression of nephrogenic markers, resembling renal developmental processes in multiple kidney structures, and activates podocyte-lineage reprogramming pathways, in addition to promoting a quiescent state in mature podocytes.  In a pilot randomized cross-over study in patients with chronic kidney disease, FMD cycles promote renoprotection by reducing proteinuria and improving endothelial function. These results show that LS-FMD cycles, which promote the reprogramming of multiple renal cell types, leading to glomerular damage reversal, should be tested further in the treatment of progressive kidney diseases.

overall_design: Sprague-Dawley rats were injected with puromycin aminonucleoside. A group of rats (N=3) were analyzed at 14 days (PAN), another group were subjected to one cycle (4 days) of low salts fasting-mimicking diet (LS-FMD) and analyzed at end of diet (EoD, and at 24 hours and 72 hours after re-feeding with regular chow (N=3 each group). A group of N=4 halthy rats were included as baseline controls in the analysis.


title: RNA sequencing of pancreatic progenitor cells differentiated from human iPS cells

city: Nagatsuta-cho, Midori-ku

first: Shoen

last: Kume

state: Kanagawa-pref.

department: Life Science and Technology

institute: Tokyo institute of technology

summary: Our RNA-seq results showed pancreatic differentiation inducer K-3 upregulated expression of genes related to pancreas development.

overall_design: To elucidate the effect of K-3 on the differentiation of human iPS cell-derived pancreatic progenitor (PP) cells, we performed RNA-seq and analyzed the global expression profile at the PP stage.

first: Yukihiro

last: Shimaya


title: Evaluating the bioactivity of the ubiquitous tire preservative 6PPD and degradant, 6PPD-quinone

city: RTP

first: Monique

last: Hazemi

state: NC

address: 109 T.W. Alexander Dr

institute: US EPA

summary: N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6PPD-quinone; 6PPD-Q), a transformation product of an antiozonant chemical widely used in automobile tires (6PPD; N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine) was identified as the causative agent for pre-spawn mortality in coho salmon resulting from exposure to stormwater run-off. This study employed a 24 h, 96-well plate based, exposure to larval fathead minnows (5 days post-fertilization) to examine the effects of 6PPD and 6PPD-Q exposure on global gene expression. Concentration-response modeling of the resulting gene expression changes identified a transcriptomic point of departure of 1.3 uM for 6PPD and 2.5 uM for 6PPD-Q, although they are effectively equivalent considering the range of uncertainty. Examination of the functional roles of differentially expressed genes provided little insight into the mechanism of idiosyncratic toxicity observed in coho salmon and certain other salmonid species. Overall, larval fathead minnows do not appear sensitive to the same mechanisms.

overall_design:
This study was conducted to investigate whether exposure to either the tire rubber antioxidant chemical 6ppd or its quinone-transformation product, 6ppd-q, impacts the transcriptome of fathead minnow and can provide any insight into the mode of toxic action of 6ppd-q or underlying cause of fish species sensitivity differences to 6ppd-q.
Comparative gene expression profiling was performed using data obtained from RNA-seq of larval (five day post hatch) fathead minnow exposed for 24 hr in 1-mL, 96-well plate format to either the tire rubber antioxidant chemical 6ppd (n=30) or its quinone transformation product, 6ppd-quinone (6ppd-q; n=40). Exposures were conducted without feeding (reliant on yolk and not independently feeding at this age) in a total volume of 700 µl with 0.3% DMSO per well. Fish were exposed to either six (6ppd) or eight (6ppd-q) concentrations, with 5 replicate fish exposed per plate and triplicate plates (A, B, C). Fish were pulled from either plate A or C for RNA-seq, including a total of 10 non-exposed control samples. Plates were sealed and placed in a 25°C incubator under a 16:8h light routine for 24 h.

first: Dan

last: Villeneuve

first: Kevin

last: Flynn

first: Mark

last: Jankowski

first: Michelle

last: Le

first: John

last: Hoang


title: Mapping the RNA interactome of DDX5 and its rewiring by the K144N mutation in the RNA helicase domain

status: Public on Jul 31 2025

city: West Lafayette

first: Matt

last: Olson

address: 201 St University St

institute: Purdue University

summary: DDX5 is a multifunctional RNA helicase that regulates RNA metabolism and gene expression programs relevant to cancer and antiviral responses. Despite its importance, the RNA-binding landscape of DDX5 remains poorly defined. Here, we use CLIP-seq to systematically map the endogenous RNA interactome of DDX5 and to determine how it is altered by the K144N point mutation within the conserved RNA helicase domain.

overall_design: We employed the enhanced CrossLinking and ImmunoPrecipitation (eCLIP) protocol (1) to identify RNAs associated with DDX5. Experiments were conducted using HepaRG cell lines engineered to express doxycycline-inducible FLAG-tagged DDX5, including both the wild-type (WT) protein and the catalytically inactive K144N mutant (2). The DDX5-K144N mutant, which is deficient in ATP hydrolysis and RNA binding, served as a negative control in downstream RNA and bioinformatic analyses. The eCLIP assays were performed by Eclipse BioInnovations, a specialized RNA genomics service provider.

first: Ourania

last: Andrisani

first: Majid

last: Kazemian

first: Sagar

last: Uttarkar

first: Zhili

last: Li


title: Transcriptomic analysis of mouse aortas  from NFATc3 flox mice and VSMC-specific NFATc3 knockout mice with BAPN treatment

city: Guangzhou

first: Xiu

last: Liu

state: Guangdong

address: 1838 North Guangzhou Avenue

country: China

institute: Southern Medical University

summary: In this study, we aim to investigate the role of vascular smooth muscle cell (VSMC) NFATc3 in the development of AAD and establish NFATc3 as a novel target to treat AAD. We used VSMC-specific NFATc3 knockout mice in AAD model of BAPN treatment. The molecular mechanisms underlying NFATc3 function were investigated using RNA-seq analysis on aortas with BAPN treatment.

overall_design: RNA-seq of aortas from NFATc3 flox mice and VSMC-specific NFATc3 knockout mice treated with BAPN.

first: Shao-Yi

last: Zheng


title: Prototype LNP-mRNA vaccine candidates [SC_RNAseq]

city: West Haven

first: Sidi

last: Chen

state: CT

address: 850 West Campus Drive

institute: Yale University

summary: We developed prototype LNP-mRNA vaccine candidates against SARS-CoV-2 Delta, SARS-CoV and MERS-CoV, and test how multiplexing LNP-mRNAs can induce effective immune responses in animal models. Triplex and Duplex LNP-mRNA vaccination induced antigen-specific antibody responses against SARS-CoV-2, SARS-CoV and MERS-CoV. Single cell RNA-seq profiled the global systemic immune repertoires and respective transcriptome signatures of vaccinated animals, revealing systemic increase in activated B cells, and differential gene expression across major adaptive immune cells. Sequential vaccination showed potent antibody responses against all three species, significantly stronger than simultaneous vaccination in mixture. These data demonstrated feasibility, antibody responses and single cell immune profiles of multi-species coronavirus vaccination. The direct comparison between simultaneous and sequential vaccination offers insights on optimization of vaccination schedules to provide broad and potent antibody immunity against three major pathogenic coronavirus species.

overall_design: PBMCs were isolated and processed from immunized mice and analyzed by scRNA-seq.

first: Paul

last: Renauer


city: Chicago

first: Gail

middle: B

last: Mahady

state: IL

address: 833 S Wood St

department: Pharmacy Practice

institute: University of Illinois

summary: MCF-7 breast cancer cells were grown and maintained and treated with a combination of Vitamin D2 and D3 and all trans-retinoic acid (ATRA) at 1.5 µg/ml for four hours. The cells were harvested, RNA was isolated and analyzed for integrity using Agilent 4200 TapeStation and RNA Screen Tape. RNA-seq libraries were prepared using a Universal Plus mRNASeq kit, and library fragment size distribution was confirmed by electrophoresis using a 2200 TapeStation system with D1000 ScreenTape. Sequencing was carried out on NovaSeq 6000, SP flowcell, 2x50 nt reads. General quality-control metrics were obtained using FastQC and raw reads were aligned to human reference genome hg38 using STAR and BWA MEM. ENSEMBL genes were quantified using FeatureCounts, and differential expression statistics were computed using edgeR. Specific gene expression was measured and verified using qPCR.

overall_design: mRNA-seq study performed on MCF-7 breast cancer cells treated with a combination of vitamins A and D.  Control samples in triplicate versus treated samples in triplicate.

first: Pinal

last: Kanabar


title: RNA sequencing of sorafenib-sensitive Huh-7 cells versus  sorafenib resistant Huh-7/SR1 and Huh-7/SR2 cells

city: Nedlands

first: Peter

middle: J.

last: Leedman

state: Western Australia

address: 6 Verdun Street

department: Cancer and Cell Biology

country: Australia

institute: Harry Perkins Institute of Medical Research

summary: Sorafenib is the global standard of care for patients with advanced hepatocellular carcinoma. It has marginal efficacy and patients invariably develop therapeutic resistance.  The primary goal of this project was to comprehensively chratcerize the molecular profile of sorafenib resistant Huh-7/SR1 and Huh-7/SR2 cells compared to their parental counterpart Huh-7 cells, and discover potential new druggable targets which can overcome sorafenib resistance.

overall_design: Sorafenib sensitive Huh-7 cells were treated with escalating doses of sorafenib to a maximum concentration of 10 microM for 3 months. Cell titre assay was used to confirm high EC50 of sorafenib in sorafenib-resistant Huh-7 cell lines. The cells were continuously maintained with 10 microM sorafenib. Total RNA was extracted using miRVana miRNA isolation kit. RNA integrity was tested using bioanalyzer. Three replicates of Huh-7 cells, Huh-7/SR1 cells and Huh-7/SR2 cells were send for RNA sequencing to Australian Genome Research Facility.

first: Tasnuva

middle: D

last: Kabir

first: Andrew

last: Woo

first: Samuel

last: Beck


title: A Spatial and Temporal Transcriptomic Atlas of Mouse Intestinal Regeneration

status: Public on Jul 29 2025

city: Piscataway

first: Verzi

last: Michael

state: New Jersey

address: 145 bevier road

department: Genetics

institute: Rutgers University

summary: The highly proliferative intestine is prone to damage, making it an ideal model for studying tissue regeneration. We performed spatial transcriptomics to characterize intestinal healing over a six-day period, revealing shifts in the spatial organization of epithelial and stromal cell populations, as well as changes in gene expression patterns and functional roles across tissue compartments. Furthermore, our analysis recapitulates known features of intestinal regeneration, highlighting the robustness of the dataset and its value as a resource for developing novel therapeutic strategies.

overall_design: 23 C57BL/6J mice were included in the study to generate 4 spatial transcriptomics datasets. Mice were treated with 12Gy whole-body irradiation (IR). Control (non-IR) mice, and mice post-IR days 1 through 6 were analyzed using Visium HD spatial transcriptomics. The platform uses near-genome scale probes to capture transcripts within a 2x2um array. A supplementary file (README_spatial_data_guide.pdf) is provided with instructions for accessing the datasets, and includes a description of the treatment post-IR per mouse, per each dataset.

first: Rachel

last: Ofer

first: Tyler

last: Tran

first: Ping

last: He

first: Xinyu

last: Hu

first: Xia

last: Qiu

first: Emmanual

last: Zachariah

first: Curtis

last: Krier

first: Ankit

last: Saxena

first: Jiekun

last: Yang

first: Michael

last: Verzi


title: RNA sequencing of colonic ILC2s sorted from mice with DSS-induced colitis

city: Jinan

first: Jing

last: Shen

address: Wenhua West Road 44

institute: Shandong University

summary: RNA sequencing was performed for ILC2s sorted from the colon of DSS- or H2O-treated C57BL/6 mice (n = 3 per group).

overall_design: Six- to eight -week-old C57BL/6 mice were fed with 3% DSS or H2O for 6 days, and then colonic ILC2s were sorted and lysed in TRIzol for RNA extraction.

first: Yanyan

last: Cui

first: Qiuheng

last: Tian

first: Yatai

last: Chen

first: Shiyang

last: Li


title: Transcriptome for gene expression in Pardosa millvina based on predator exposure

city: Oxford

first: Alexander

middle: Dean

last: Berry

state: OH

address: 501 E HIGH ST

department: Biology

institute: Miami Unviersity

summary: We compare levels of gene expression in predator (Tigrosa helluo) exposed female Pardosa milvina compared to unexposed spiders and the offspring of predator exposed spiders to the offspring of control spiders.

overall_design: Whole body mRNA analysis of spiders exposed to predator cues and their offspring compared to unexposed spiders.

first: Ann

last: Rypstra


title: Nox1 and Npy1r mark regional colon stem cell populations that serve as cancer origins in vivo [scRNA_seq]

first: Bernett

last: Lee

address: 8A Biomedical Grove #03-06 Immunos

country: Singapore

institute: Singapore Immunology Network

summary: Current colorectal cancer (CRC) mouse models lack either colon specificity, limiting progression towards more advanced disease, or preclude evaluation of resident stem cells as cancer origins. Here, we report identification of Nox1 and Npy1r as cell-surface markers enriched in Lgr5+ stem cells predominantly within the caecum and exclusively within the mid-distal colorectum respectively. Selective dysregulation of Wnt signalling in Nox1+ or Npy1r+ stem cells using new CreERT2 mouse lines drives colon cancer initiation, predominantly within the caecum and the rectum respectively, establishing these stem cell populations as important sources of colon cancer. Selective conditional activation of Wnt signalling and oncogenic K-RAS in combination with loss of Trp53 in these stem cell compartments resulted in development of advanced, invasive cancers. This study establishes the new CreERT2 drivers as valuable tools for studying stem cell contributions to colon cancer.

overall_design: Crypts from individual regions of the colon and small intestine were isolated and dissociated into single cells. The single cell transcriptomics were analyzed using scRNA-Seq.

first: Maxime

last: Gasnier

first: Tanysha

first: Swathi

last: Yada

first: Sowmya

last: Sagiraju

first: Yusuke

last: Yoshikawa

first: Stefano

last: Perna

first: Grace

middle: H

last: Lim

first: Nick

last: Barker


title: Nox1 and Npy1r mark regional colon stem cell populations that serve as cancer origins in vivo [RNA_seq]

overall_design: RNA-seq profiling of GFP-sorted epithelial cell from small intestine and colon Lgr5-2A-eGFP mice.


title: Nox1 and Npy1r mark regional colon stem cell populations that serve as cancer origins in vivo [RNA-seq2]

overall_design: RNA-seq profiling of GFP-sorted epithelial cell from small intestine and colon Lgr5-2A-eGFP mice. Small intestine is separated in 3 equal parts (proximal, middle and distal). Colon is separated in 2 parts (proximal and distal).


title: Exploration potential regulatory mechanisms in ovarian injury of mice exposed to low-pressure hypoxia through whole transcriptome sequencing analysis [miRNA-seq]

city: Lanzhou

first: mengwen

last: Jiao

state: Gansu

address: No. 333, Nanbinhe Road, Qilihe District

department: Department of Reproductive Center

type: Non-coding RNA profiling by high throughput sequencing

summary: Chronic high-altitude hypoxia causes significant ovarian tissue damage. This study investigates the underlying mechanisms of ovarian injury in mice exposed to hypoxia, aiming to guide targeted therapy development. Mice were divided into either normal pressure (MP, n=15) or hypobaric hypoxia (PU, n=15) and samples from both groups were analyzed using whole-transcriptome sequencing data. Hub genes were identified through differential expression analysis and protein-protein interaction (PPI) network construction before their expression patterns were examined. Subsequently, functional enrichment was conducted. Key miRNAs, lncRNAs, circRNAs, and transcription factors (TFs) were ascertained. Finally, RT-qPCR and Western blotting (WB) were performed. Hmgcr, Ptgs2, and Mmp3 were identified as hub genes, all showing pronounced lower expression in PU samples. RT-qPCR analysis confirmed the downregulation of these genes, with WB analysis further demonstrating a significant reduction in their protein levels in PU samples. These hub genes were predominantly enriched in "oxidative phosphorylation" pathway. Furthermore, lncRNA-miRNA-mRNA, circRNA-miRNA-mRNA, and TF-mRNA regulatory networks were constructed. These networks highlighted key regulatory molecules, including the miRNA mmu-miR-144-3p, 2 lncRNAs (Miat and Neat1), 8 circRNAs (eg. novel_circ_041272-mu-miR-144-3p-Ptgs2), and 1 TF (Etv4). These results provide key insights for targeted ovarian injury therapies under low-pressure hypoxia.

overall_design: Female 8-week-old C57BL/6 mice were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Animal License No.: SCXK (Beijing) 2024-0001). Notably, the mice were maintained under standard laboratory environment with a 12-hour light/dark cycle (07:00–19:00), ambient temperature maintained at 22±2°C, and a relative humidity of 45%–55%. The mice were provided with ad libitum access to standard laboratory chow and drinking water. The experimental animals were randomly divided into two groups: the MP group (Normal Pressure, n=15) and the PU group (Pressure Under Vacuum, n=15). The PU group was placed in a hypobaric hypoxia animal chamber (Guizhou Fenglei Aviation Mechanical Manufacturing Company, Guizhou, China) simulating the conditions of an altitude of 6500 meters, with a pressure of 43.12 kPa. The control group was housed in a normal environment at an altitude of 1588 meters  and a pressure of 83.56 kPa, in Lanzhou, China. All mice were maintained for 15 consecutive days. All animal experiments were strictly conducted in compliance with the international ARRIVE 2.0 guidelines (Animal Research: Reporting of In Vivo Experiments) and were approved by the Institutional Ethics Committee of The 940th Hospital of Joint Logistic Support Force of Chinese People's Liberation Army (Approval No. 2023KYLL299).Animal welfare measures were implemented according to the 2020 American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals.The detailed procedures were as follows: mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (40 mg/kg), followed by cervical dislocation performed by trained personnel, with subsequent collection of blood and ovarian tissue samples for analysis.These samples were used for evaluating the mouse model conditions and performing whole-transcriptome sequencing.

first: Mengwen

first: Yuewen

last: Zhang

first: Xuan

last: Hou

first: Min

last: He

first: Ling

last: Wang

first: Shaokang

last: Mo


title: Exploration potential regulatory mechanisms in ovarian injury of mice exposed to low-pressure hypoxia through whole transcriptome sequencing analysis [RNA-seq]

institute: The 940th Hospital of Joint Logistic Support Force of Chinese People's Liberation Army


title: A pan-immunotherapy signature to predict intratumoral CD8+ T cell expansions [bulkTCRseq_FTY]

city: Noda-shi

first: Mikiya

last: Tsunoda

state: Chiba

department: Molecular Preventive Medicine

institute: The University of Tokyo

summary: Effective cancer immunotherapy relies on the clonal proliferation and expansion of CD8+ T cells in the tumor.  However, our insights into clonal expansions are limited, owing to an inability to track the same clones in tumors over time. Here, we developed a multi-site tumor mouse model system to track hundreds of expanding and contracting CD8+ T cell clones over multiple timepoints in tumors of the same individual. Through coupling of clonal expansion dynamics and single-cell RNA/TCR-seq data, we identified a transcriptomic signature in PD-1+Ly108+ precursor exhausted cells that strongly predicts rates of intratumoral clone expansion. The signature correlates with expansion in mice, both with and without immunotherapies, and in patients undergoing PD-1 blockade therapy. Expression of the signature during treatment corresponds with positive clinical outcomes. Downregulation of the signature precedes clone contraction – a phase in which clones contract but maintain revivable precursor exhausted cells in the tumor. LAG-3 blockade re-activates the expansion signature, re-expanding pre-existing clones, including previously contracted clones. These findings reveal how the study of clonal expansion dynamics provide a powerful ‘pan-immunotherapy’ signature for monitoring immunotherapies with implications for their future development.

overall_design:
Lewis lung carcinoma (LLC) cells (5 × 10^5 cells /mouse) were inoculated subcutaneously (s.c.) into the left and right flanks of C57BL/6 mice (n=6). After 14 days, the left tumor was surgically removed, and a week later, the right tumor was harvested with the mice. CD8+ T cells obtained from the left and right tumor were analyzed by bulk TCR-sequencing.
For the FTY720 experiment, FTY720 (Selleck) was injected intraperitoneally at a dose of 20μg per mice on days 14, 16, 18 and 20.

first: Munetomo

last: Takahashi

first: Hiroyasu

last: Aoki

first: Masaki

last: Kurosu

first: Haru

last: Ogiwara

first: Shigeyuki

last: Shichino

first: David

last: Bending

first: Shumpei

last: Ishikawa

first: James

last: Thaventhiran

first: Kouji

last: Matsushima

first: Satoshi

last: Ueha


title: A pan-immunotherapy signature to predict intratumoral CD8+ T cell expansions [bulkRNAseq]

overall_design:
Lewis lung carcinoma (LLC) cells (5 × 10^5 cells /mouse) were inoculated subcutaneously (s.c.) into the left flank and right flank of C57BL/6 mice (untreated : n=5, aLAG3 : n=6). Tumors were excised on day 14 and day 21. T cells obtained from each tumor were analyzed by bulk RNA-sequencing.
For treatment, anti-LAG-3 (clone C9B7W, BioLegend or Selleck) was injected intraperitoneally at a dose of 200μg per mouse on days 14 and 16 after tumor inoculation.


title: A pan-immunotherapy signature to predict intratumoral CD8+ T cell expansions [bulkTCR_control]

overall_design: Lewis lung carcinoma (LLC) cells (5 × 10^5 cells /mouse) were inoculated subcutaneously (s.c.) into the left and right flanks of C57BL/6 mice (n=7). After 14 days, the left tumor was surgically removed, and a week later, the right tumor was harvested with the mice. CD8+ T cells obtained from the left and right tumor were analyzed by bulk TCR-sequencing.


title: Single-Cell Transcriptional Analysis of Murine Mesenteric Lymph Nodes Following Oral Lyso-phosphatidylserine Nanoparticle Administration Reveals Cellular Heterogeneity in Tolerance Features

city: Buffalo

first: Jonathan

last: Bard

state: New York

address: 701 Ellicott Street

department: Genomics and Bioinformatics Core

institute: University at Buffalo

summary: Unwanted immune responses against self-proteins or exogenous therapeutic proteins can trigger autoimmune diseases or compromise the safety and efficacy of life-saving therapeutic biologics. To address this issue, we utilized the ability of phosphatidylserine (PS) to convert an immunogen into a tolerogen and rationally developed a LysoPS-containing tolerogenic nanoparticle platform to prevent and mitigate unwanted immune responses. We demonstrated that prophylactic oral treatment with LysoPS-containing proteins prevents unwanted immune responses by inducing tolerance. However, the biological processes and cellular communication involved in LysoPS-mediated oral tolerance remain unclear. Therefore, this study aimed to characterize immune cell interactions and the potential tolerogenic mechanism in mice treated with LysoPS using Single-cell RNA sequencing (scRNA-seq). Our data showed that LysoPS nanoparticles increased the expression of RNAs associated with tolerogenic features in B cells, T cells, and NK cells, primarily through TGF-β responses.

overall_design: Animals were continuously fed Buffer, Free OVA, and LysoPS-OVA for four weeks via oral administration. Mesenteric lymph nodes (MLNs) were collected, enzymatically digested, and purified into a single live cell suspension before scRNA-seq. Six animal per group were used in this study and animal from the same group were pooled together for scRNA

first: Vincent

last: Chak

first: Edwin

last: Ovalle

first: Elizabeth

last: Wohlfert

first: Jason

middle: G

last: Kay

first: Sathy

last: Balu-Iyer


title: Targeting nucleoporin 214 selectively eliminates leukemia stem cells by inducing ferroptosis [H3K27ac CUT-Tag-seq]

city: Chongqing

first: Zhe

last: Chen

address: 1 Yixueyuan Road, Yuzhong District

institute: Chongqing Medical University

summary: Leukemia stem cells (LSCs) give rise to mature leukemia blasts and are the major cause of therapy resistance and relapse in acute myeloid leukemia (AML). We propose a novel strategy to eliminate heterogeneous LSCs but spare hematopoietic stem cells (HSCs) by targeting essential genes that are highly expressed in LSCs than that in HSCs. Nucleoporin 214 (NUP214) is determined as a dose-dependently essential gene (DDEG) in LSCs. Homozygous deletion of Nup214 eliminates LSCs and HSCs; however, Nup214 haploinsufficiency impairs LSC maintenance but spares HSCs by selectively inducing ferroptosis of LSCs. Mechanistically, NUP214 locates to chromatin and interacted with Sub1 to inhibit the transcription of heme oxygenase-1 (Hmox1) and arachidonate 15-lipoxygenase (Alox15), thus protecting LSCs against lipid peroxidation. We screened and identified a small molecule 0449-b as a molecular glue that links NUP214 and an NEDD8 E3 ligase, Rbx2, to induce neddylation and degradation of NUP214. 0449-b shows efficacy in selective eradication of LSCs but has a negligible effect on normal hematopoietic stem and progenitor cells. These biological and chemical findings provide a valuable strategy to eliminate LSCs by targeting DDEGs.

overall_design: LSCs (Lin-Sca-1-c-Kit+CD16/32+CD34+) from MLL-AF9-driven Nup214fl/+ and Nup214fl/+,Tie2-Cre (Nup214-HET) AML mice were sorted, followed by H3K27ac-CUT&Tag-seq.


title: Targeting nucleoporin 214 selectively eliminates leukemia stem cells by inducing ferroptosis [ATAC-seq]

overall_design: HSCs (CD48-CD150+LSK) from Nup214fl/+(FL) and Nup214fl/+,Tie2-Cre (HET) mice were sorted, followed by ATAC-seq. LSCs (Lin-Sca-1-c-Kit+CD16/32+CD34+) from Nup214fl/+(FL) and Nup214fl/+,Tie2-Cre (HET) AML mice (MLL-AF9-driven) were sorted, followed by ATAC-seq.


title: mTORC1-Driven DDA1 Phosphorylation Promotes DNA Repair and Glioblastoma Progression

city: Beijing

first: Xing

address: Capital Medical University

institute: Capital Medical University

summary: DDA1 is a protein involved in protein degradation, cell cycle regulation, and DNA damage repair. Interestingly, recent studies have revealed its differential expression across various tumor types. However, how DDA1 functions as a tumorogenic factor remains to be elucidated. Through studies in multiple glioblastoma cell models, we identified a physical association between cytoplasmic DDA1 and Raptor, a key component of lysosome-associated mTORC1. We report that amino acid stimulation triggers phosphorylation of DDA1 at serine 33, promoting its nuclear translocation and involvement in DNA damage repair. Integrated genomic and transcriptomic analyses revealed that the mTORC1-DDA1S33-DNA repair axis regulates the expression of a subset of metabolic genes, including ENO2, a glycolytic enzyme; CA12, which contributes to intracellular and extracellular pH homeostasis; and NMRK1, a key enzyme in nicotinamide riboside metabolism. Notably, DDA1 deficiency markedly impaired glioblastoma growth and triggered a compensatory upregulation of metabolic activity to sustain tumor cell survival. These metabolic genes likely supply essential nutrients required for effective DNA repair. Our findings establish DDA1 as a previously unrecognized phosphorylation target downstream of mTORC1, serving as a critical mediator of mTORC1-driven DNA damage response and a key regulator of glioblastoma progression, thereby expanding our understanding of gliomagenesis.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for LN229, T98G, and U118-MG cells treated with sgCon and sgDDA1, and total RNAs were extracted for cDNA synthesis, library construction and sequencing.

first: Xiaozhong

last: Peng


title: Targeting nucleoporin 214 selectively eliminates leukemia stem cells by inducing ferroptosis [RNA Pol II CUT-Tag-seq]

overall_design: LSCs (Lin-Sca-1-c-Kit+CD16/32+CD34+) from MLL-AF9-driven Nup214fl/+ and Nup214fl/+,Tie2-Cre (Nup214-HET) AML mice were sorted, followed by RNA Pol II-CUT&Tag-seq.


title: TRAP-seq in 3D Angiogenesis Assays Reveals Distinct Biological Processes Associated with Early and Late Morphogenesis that are Disrupted with PIK3CA-H1047R

city: Albany

first: Ramon

last: Bossardi Ramos

state: Ny

address: 43 New Scotland Avenue

department: Department of Molecular and Cellular Physiology

institute: Albany Medical College

summary: Endothelial cells (ECs) are often a minority cell type in a tissue, limiting the utility of bulk sequencing approaches.  Single cell sequencing lacks sensitivity and requires disruptive tissue digestion techniques.  TRAP seq (Translating Ribosome Affinity Purification) or “RiboTag” has been used to overcome these limitations. Co-culture systems allow primary ECs to differentiate and undergo tubular morphogenesis in cell culture, however similar limitations exist with these in vitro assays, as ECs are under-represented by as much as a factor of 10 in many assays.  We sought to use TRAP seq to better understand the gene expression landscapes that drive these morphogenic events. We found TRAP seq selectively enriches for endothelial RNA in both the planar and fibrin bead co-culture assays. Intriguingly, there are distinct changes in blood vessel development and in the mitotic cell cycle, unique to early and late phases of morphogenesis. To determine how these biological processes are altered by a known disruptor of vascular morphogenesis, we expressed PIK3CAH1047R in ECs, implicated as a driver in several types of vascular malformations. We found PIK3CAH1047R expression results in profound changes in endothelial cell expression with enrichment in genes associated with blood vessel morphogenesis gene ontology terms. There is a cohort of genes which showed dysregulation in both morphogenesis assays and these include numerous genes known to be important for tip/stalk specification during developmental angiogenesis, with a profound penetrance of NOTCH pathway dysregulation.

overall_design: HUVECs are infected with the pHAGE_Rpl22-3X-HA plasmid and seeded for either 2D culture or 3D planar co-culture. As biological variability is a factor when using primary cells, experiments were conducted using 4 independent pools of primary HUVECs from two different commercial vendors, each pool representing at least three donors of mixed races and sexes.  All cells from one lot are seeded simultaneously for all time points (day 1, day 4 or day 8). To ensure representative numbers of endothelial and fibroblast mRNAs in the 2D endothelial samples, identical numbers of endothelial and fibroblasts as used in 3D culture, were plated separately at the same time and media as the co-culture assays and were lysed in a common fraction of lysis buffer. Lysates were made from 2D and 3D cultures at day 1, and additional 3D cultures at day 4 and day 8 time points.

first: Samantha

last: King

first: Qing-fen

last: Li

first: Ian

last: Tschang

first: Ekta

last: Singh

first: Kevin

last: Pumiglia


title: Primary adventitial fibroblast response to TGFb is dependent on vascular site and implicates functional developmental epigenomic memory

city: Stanford

first: Chad

middle: S

last: Weldy

address: 870 Quarry Road, CVRC Falk

department: Medicine, Cardiovascular Medicine

institute: Stanford University

summary: We have shown that vascular SMCs, fibroblasts, and endothelial cells have transcriptional and epigenomic features that are distinct to vascular site. Further, TF motif accessibility analysis in fibroblasts reveals an increased accessibility of AP1, and SMAD2:3:4, and TCF21 motifs in the ascending fibroblast population compared to carotid and descending fibroblasts, with further gene regulatory network analysis suggesting distinct regulatory TF module activity in ascending fibroblasts highlighting master regulator of fibroblast activation Meox1. This differential chromatin accessibility would suggest the potential for heightened biological response to TGFb in ascending fibroblasts. To evaluate the functional effect of this differential chromatin accessibility and to identify if differential gene expression is retained following removal from vascular site flow conditions, we isolated and cultured primary adventitial fibroblasts from healthy 14 week old C57BL/6 mice from the ascending and descending aorta and stimulated them with control or TGFb (10ng/mL, 48hrs) and performed bulk RNA sequencing (n = 3 per condition).

overall_design: We isolated and cultured primary adventitial fibroblasts from healthy 14 week old C57BL/6 mice from the ascending and descending aorta and stimulated them with control or TGFb (10ng/mL, 48hrs) and performed bulk RNA sequencing (n = 3 per condition)


title: The histone-methyltransferase DOT1L cooperates with LSD1 to control cell division in blast-phase MPN [ChIP-seq]

city: Hannover

first: Florian

last: Perner

address: Carl-Neuberg-Str. 1

department: Hematology, Hemostasis, Oncology and Stem Cell Transplantation

country: Germany

institute: Hannover Medical School

summary: Persistence of JAK2-mutated clones that may undergo clonal evolution and malignant transformation remains a challenge in myeloproliferative neoplasms (MPN), Novel therapeutic approaches to attenuate clonal evolution and progression to blast-phase are therefore urgently needed. LSD1 (KDM1A) inhibitors reduce symptoms and clonal burden in MPN, but whether these compounds may be effective in advanced disease stages remained so far elusive. Using a chromatin-focused CRISPR-Cas9 screen, we identified the histone methyltransferase DOT1L as a synthetic lethal target under pharmacologic LSD1 inhibition. DOT1L knockout impaired cellular fitness, reduced proliferation, and prolonged survival in xenografts. Furthermore, genetic inactivation of DOT1L increased LSD1 inhibitor sensitivity up to 100-fold resulting in cell cycle arrest and apoptosis induction in TP53 mutant blast-phase MPN. Mechanistically, we have identified a novel, non-canonical function of DOT1L which co-occupied LSD1-bound enhancers and contributed to the repression of transcriptional programs independent of its enzymatic activity. DOT1L loss cooperated with LSD1 inhibitors to activate tumor suppressive programs, while pharmacologic inhibition of DOT1Ls catalytic activity failed to elicit comparable effects. These findings indicate that leveraging DOT1L targeting via protein degradation or RNA interference, rather than conventional enzymatic inhibition, could enhance the therapeutic efficacy of LSD1 inhibitors in blast-phase MPN.

overall_design: ChIP sequencing of DOT1L, LSD1, H3K79me2 and H3K4me1 in different cell lines (HEL, SET2, MOLM13) and primary MPN-BP patient samples.


title: Evolution of SARS-CoV-2 T cell responses as a function of multiple COVID-19 boosters

city: La Jolla

first: Alessandro

last: Sette

address: 9420 Athena Cir

department: Center for Infectious Disease and Vaccine Research

institute: La Jolla Institute for Immunology

summary: We investigated the long-term impact of repeated COVID-19 vaccinations on adaptive immunity through a three-year study of 78 individuals without reported symptomatic infections. We observed distinct dynamics in Spike-specific responses across multiple vaccine doses. While antibody levels increased and stabilized with each booster, T cell responses quickly plateaued and remained stable. Notably, approximately 30% of participants showed evidence of asymptomatic infections. Single-cell RNA sequencing revealed a diverse landscape of Spike-specific T cell phenotypes, without signs of exhaustion or functional impairment. Individuals with evidence of asymptomatic infection displayed unique immunological features, including increased frequencies of Th17-like CD4+ T cells and GZMKhi/IFNR CD8+ T cell subsets. In this group, repeated vaccinations correlated with an increase in regulatory T cells, potentially indicating a balanced immune response that may mitigate immunopathology. By regularly stimulating T cell memory, boosters contribute to a stable and enhanced immune response, which may provide better protection against symptomatic infections.

overall_design: Single-cell RNA-seq profiling of CD4+ and CD8+ T cells reactive to SARS-CoV-2 Spike (S) as captured by applying the activation-induced marker (AIM) assay to PBMC samples from multiple visits (time-points) of 54 donors after receiving COVID-19 vaccine boosters and reporting no disease symptoms.

first: Ricardo

last: da Silva Antunes

first: Vicente

last: Fajardo-Rosas

first: Esther

last: Dawen Yu

first: Rosa Isela

last: Gálvez

first: Adam

last: Abawi

first: E. Alexander

last: Escarrega

first: Amparo

last: Martínez-Pérez

first: Emil

last: Johansson

first: Benjamin

last: Goodwin

first: April

last: Frazier

first: Jennifer

middle: M

last: Dan

first: Shane

last: Crotty

first: Grégory

last: Seumois

first: Daniela

last: Weiskopf

first: Pandurangan

last: Vijayanand


title: The histone-methyltransferase DOT1L cooperates with LSD1 to control cell division in blast-phase MPN [RNA-seq]

first: Florian

last: Perner

overall_design: 1. RNAseq of Human Erythroleukemia cells (HEL cell line) with a CRISPR-Cas9-mediated knockout of DOT1L (or non-targeting control) treated with the LSD1-inhibitor Bomedemstat for 24h and 5d. 2. RNAseq of MPN-BP primary patient sample with a shRNA-mediated knockdown of DOT1L (or scrambled control) cultured in methylcellulose for 7 days under puromycine selection for shRNA-expressing cells.


title: TCR sequences of peripheral blood CD8+ T cells and regulatory T cells before and after chemoimmunotherapy in KORNELIA phase II trial

city: Daejeon

first: Seung Hyuck

last: Jeon

address: 291 Daehak-ro, Yuseong-gu

institute: Korea Advanced Institute of Science and Technology

summary: We aimed to analyze the effects of chemoimmunotherapy on peripheral blood T cell clonotypes in HER2-negative breast cancer patients.

overall_design: CD8+ T cells and regulatory T cells were sorted from PBMCs using a FACS Aria II cell sorter. Sequencing libraries were prepared using the SMARTer Human TCR a/b Profiling Kit.

first: Koung Jin

last: Suh

first: Sungmin

last: Jung

first: Minwoo

first: Ho Cheol

last: Jang

first: Eui-Soon

last: Kim

first: Se Hyun

first: So Yeon

last: Park

first: Milim

first: Jee Hyun

first: Eui-Cheol

last: Shin


title: Single cell gene expression analysis of X-linked Alport syndrome kidney organoid models and isogenic controls

city: Paris

first: Hassan

last: Saei

address: 24 boulevard de Montparnasse

department: Imagine Institute of Genetic Diseases

country: France

institute: Universite Paris Cite

summary: In study we investigated podocyte maturation, with a particular focus on type IV collagen maturation in the glomerular basement membrane (GBM) of kidney organoids over extended culture periods (comparing Control Organoid d22 R2 with Control Organoid d38). The secondary aim was to compare the gene expression profiles of podocytes in X-linked Alport syndrome (XLAS) organoid models with isogenic controls at an early culture time point (day 22). Two distinct batches of sample collection were conducted to capture early and later stages of GBM development. In the first batch, three libraries were generated by collecting 16 organoids from one isogenic control, one severe XLAS model, and one moderate XLAS model on day 22 of differentiation. In the second batch, two libraries were created to evaluate the impact of culture duration on GBM maturation by harvesting an additional isogenic control organoid at both day 22 and day 38 of culture. Organoids were dissociated using a two-step enzymatic and mechanical protocol to obtain single-cell suspensions, with cell viability ranging between 70% and 85%. This experimental design enabled a comparative analysis of GBM maturation dynamics between early and late time points and across different severities of XLAS. Findings from this study provided insights into the cellular mechanisms underlying GBM abnormalities in Alport syndrome and supported the potential for GBM maturation in kidney organoids over time.

overall_design: Two separate batches of sample collection were performed for this project. In the first batch, 16 organoids were collected on day 22 of differentiation, including one isogenic control organoids, one severe XLAS organoid, and one moderate XLAS organoid, resulting in three libraries. This project aimed to compare XLAS severe and moderate organoid models at early time point (day 22 of differentiation). An additional isogenic control was harvested on day 22 (Control_d22_2) and kept in culture for 38 days (Control_d38) and harvested at this later time point (two library, 5 libraries in total).  This batch was used to compare isogenic controls at day 22 and day 38 of culture to study the gene expression profile in podocytes to confirm the glomerular basement membrane maturation in prolonged culture. Libraries obtained from these two batches were sequenced together.

first: Saei

last: Hassan

first: Antignac

last: Corinne

first: Dorval

last: Guillaume

first: Mollet

last: Geraldine


title: Pulsatile stretch stimulates endothelial PDGFB secretion to maintain arterial vascular smooth muscle

city: Dallas

first: Thomas

last: Carroll

address: 6000 Harry Hines Blvd.

institute: UT Southwestern

summary: The endothelial cells (ECs) that line blood vessels are remarkably sensitive to mechanical force. Hemodynamic force impacts ECs in three dimensions: laminar shear stress, intraluminal pressure, and circumferential stretch. The magnitude of these forces in arteries is pulsatile in nature—a quality that is lost in veins. The role of pulsatility in shaping these vessels is not well understood. Here we examine loss of pulmonary arterial pulsatility following the Glenn surgery (surgical palliation of single ventricle congenital heart disease). Using cultured pulmonary arterial ECs, we define the transcriptional changes driven by pulsatility within each dimension. We identify pulsatile stretch as a critical stimulus for endothelial secretion of PDGFB—a known driver of vascular smooth muscle cell (VSMC) proliferation—and show that loss of arterial pulsatility in vivo leads to thinning of VSMCs. This work provides a mechanistic understanding of how arterial pulsatility maintains the structure that supports arterial hemodynamic force.

overall_design: Human pulmonary artery endothelial cells (HPAECs) exposed to either static, pulsatile, or non-pulsatile conditions of flow, stretch, or pressure

first: Stephen

last: Spurgin

first: Chris

last: Cheney

first: Ondine

last: Cleaver


title: Understanding molecular response of Arabidopsis to salt, heat and highlight stress combinations.

city: Columbia

first: Ron

last: Mittler

state: Missouri

address: University of Missouri,1201 Rollins St

department: Division of Plant Science and Technology

institute: University of Missouri - Columbia

summary: Plants in nature are exposed to combinations of abiotic and biotic stresses. Climate change driven increase in frequency of abiotic stress combinations is being detrimental to the plant survival and growth. Previously we found that Arabidopsis plants when exposed to combination of abiotic stresses termed as multifactorial stress combination (MFSC), the accumulation of multiple mild abiotic stresses becomes damaging to plants causing severe reduction in plant survival which otherwise not detrimental when exposed as single stresses. Here in this study, we investigated the role of a transcription factors Basic helix loop helix (bHLH35) in MFSC tolerance in Arabidopsis seedlings.

overall_design: In this study, Arabidopsis thaliana wild-type (Col0), and mutants of bHLH35 gene plants were subjected to salt stress (S; 50mM), high light stress (HL; 700 µE), heat stress (HS; 33 °C) and different combinations of these three stresses (S+HL, S+HS, HL+HS, S+HL+HS). Sterilized seeds of above-mentioned genotypes were germinated on ½ MS (murashige and Skoog media) with or without salt, at 21°C temperature and 150 µE light intensity. 7 days old seedling were subjected to HL and HS for 3 days (in combinations mentioned above) to determine the impact of stresses on survival. Seedlings for control were grown on ½ MS media without salt at 21°C temperature and 150 µE light intensity. To understand the transcriptomic response of Arabidopsis genotypes, 7 days old seedling growing on ½ MS with or without salt were exposed to HS and HL stress (in combinations mentioned above) for 1.5 hrs and seedlings from all the stress treatments and control were snap frozen in liquid nitrogen. Total RNA from the seedlings were isolated using Qiagen RNeasy® Plant Mini Kit. The RNAseq analysis was performed by Novogene Co. RNA libraries were prepared using standard Illumina protocols and RNA-Seq was performed using a NovaSeq 6000 PE150 platform by Novogene Co. Ltd. (https://en.novogene.com/; Sacramento, CA, USA). Read quality control was performed using Trim Galore v0.6.4 and FastQC v0.11.9.

first: Ranjita

last: Sinha

first: Abdul

last: Ghani

first: Sai Preethi

last: Induri

first: Trupti

last: Joshi

first: Felix

last: Fritschi

first: Sara

middle: I

last: Zandalinas


title: The study investigates which genes change within cells when using a coronavirus spike protein for 48 h.

city: Tokyo

first: Yoichi

last: Teratake

institute: National Center for Global Health and Medicine

summary:
Several genes showed significant changes after 48 h of spike protein addition
These results revealed that various gene expressions change due to infection with the coronavirus pseudotyped virus.

overall_design: Comparing expression profiles among comparable samples using Affymetrix Human Clariom-S data.


title: The study investigates which genes change within cells when using a coronavirus spike pseudotyped virus.

summary:
Several genes showed significant changes, with the gene expression of SMAD5 being the most elevated among them
These results revealed that various gene expressions change due to infection with the coronavirus pseudotyped virus.


title: Steroid Receptor Coactivator-2 Controls Human Extravillous Trophoblast Cell Proliferation, Migration, and Invasion

city: HOUSTON

first: Vineet

middle: Kumar

last: Maurya

state: TX

address: ONE BAYLOR PLAZA

department: Molecular & Cell Biology

institute: BAYLOR COLLEGE OF MEDICINE

summary: Abnormal placentation is a fundamental cause of pregnancy complications including preeclampsia, miscarriage, and intrauterine growth restriction. Proliferation, differentiation, migration, and invasion are key properties of trophoblast cells that regulate trophoblast functions. Imbalance of these cellular processes is linked to placental pathologies. Using HTR-8/SVneo immortalized human trophoblast cells, we demonstrate that steroid receptor coactivator-2 (SRC-2), a transcriptional regulator of nuclear receptors, is essential for extravillous trophoblast cell proliferation, migration, and invasion. Genome-wide transcriptomics identified an SRC-2–dependent transcriptome in HTR-8/SVneo cells that includes a diverse spectrum of proteins involved in placental tissue development and function. To emphasize the utility of this transcriptomics data set, we demonstrate that WNT family member 9A (WNT9A) is not only a molecular target of SRC-2 but also crucial for maintaining many of the cellular properties of human extravillous trophoblast cells.

overall_design: To investigate the SRC-2 gene signature in regulating human extravillous trophoblast cells, HTR8/Svneo cells were used. Total RNA was isolated from NT siRNA and SRC-2 siRNA-treated HTR8 cells and subjected to the RNAseq assay.

first: John

last: Lydon


title: Targeting FGFR4 Abrogates HNF1A-driven Metastasis in Pancreatic Ductal Adenocarcinoma

city: BUFFALO

first: Eduardo

last: Cortes

state: NY

address: ELM AND CARLTON STREETS

department: Biostatistics and Bioinformatics

institute: ROSWELL PARK CANCER INSTITUTE

summary: Background: We previously identified an oncogenic role for the transcription factor HNF1A in pancreatic ductal adenocarcinoma (PDAC). However, the role of HNF1A in the metastatic progression of PDAC remains unknown and targeting modalities for HNF1A-dependent phenotypes have yet to be identified. Methods: Transwell chambers were used to assess the effects of HNF1A and FGFR4 modulation on the migration and invasion of ATCC and patient-derived PDAC cells in vitro. An intrasplenic injection xenograft model was used to evaluate the impact of HNF1A knockdown and overexpression on metastatic tumor burden. Single-cell RNA sequencing, tissue microarray (TMA) data, and UMAP spatial profiling were used to identify FGFR4 as an HNF1A target gene upregulated in metastatic cells. RNAi and two FGFR4 inhibiting modalities (H3B-6527 and U3-1784) were utilized to demonstrate the efficacy of FGFR4 inhibiting agents at reducing HNF1A38 driven metastasis. Results: Knockdown of HNF1A significantly decreases and HNF1A overexpression significantly increases PDAC cell migration and invasion. In vivo studies show that HNF1A knockdown significantly abrogates metastasis, while overexpression significantly promotes metastasis. Single-cell RNAseq shows that FGFR4 is upregulated in metastatic PDAC cells and staining for HNF1A and FGFR4 in a PDAC TMA reveals significant correlation between HNF1A and FGFR4 in PDAC patients. Further, knockdown and inhibition of FGFR4 significantly decreases HNF1A45 mediated cell migration and invasion, and blocks HNF1A-driven metastasis in vivo. Conclusions: These findings demonstrate that HNF1A drives PDAC metastasis via upregulation of FGFR4, and FGFR4 inhibition is a potential mechanism to target metastasis in PDAC patients.

overall_design: RNA-seq profiling of 3 Controls and 3 HNFA Kos.

first: Katherine

last: Crawford

first: Kennedy

last: Humphrey

first: Jianxin

last: Wang

first: Vishnu

last: Kumarasamy

first: Michael

last: Feigin

first: Agnieszka

middle: K

last: Witkiewicz

first: Eric

last: Knudsen

first: Ethan

middle: V

last: Abel


title: Synovial joint cavitation during mouse embryo limb skeletogenesis entails Na/K-ATPase pump expression and osmoregulatory activity

city: Shenyang

first: Lutian

last: Yao

address: 155 Nanjing st

department: Department of Orthopedic

institute: the First Affiliated Hospital of China Medical University

summary: The developmental biology of limb synovial joints and their water/lubricant-filled cavity remains unclear. Joint formation is known to start with emergence at each prescribed anatomical site of a compacted Gdf5-expressing mesenchymal interzone flanked by cartilaginous skeletal primordia. We and others previously showed that the interzone cells serve as progenitors and generate most joint tissues over time, including articular cartilage and ligaments. But how is the cavity formed within the compacted interzone? Because water accrual would be needed to create and inflate the cavity and in turn distance the flanking skeletal elements, cavity formation may require energy-consuming osmoregulatory mechanisms able to draw water and exert mechanical force. Our data now reveal that interzone cells in cavitating knee and digit mouse embryo joints strongly express the Na/K-ATPase pumps Atp1a1, Atp1b1 and Atp1b3, members of a key cell surface osmoregulatory protein family that regulates fluid tonicity and drawing of water. There was also local and specific expression of the water channel Aquaporin 1 (Aqp1). Single cell RNA seq (scRNAseq) showed that Gdf5+ interzone cells expressing those genes culminated in number at cavitation and were characterized by other osmoregulatory and mechano-sensing genes also. Indeed, when pregnant mice were administered ouabain -a physiological glycoside that normally limits Na/K-ATPase pump activity and osmoregulatory processes-, limb joint cavitation in the embryos was inhibited as was lubricant gene expression. Joint development is known to depend on signals from Indian hedgehog-expressing growth plate chondrocytes in flanking skeletal primordia. Interference with hedgehog signaling did coordinately inhibit expression of pump, mechano-sensing and lubricant genes as well as cavitation. Together, our data suggest a novel understanding of joint cavitation as an active energy-requiring osmoregulatory process able to accrue a water-based fluid, distance the flanking articulating surfaces and establish a synovial cavity in close coordination with long bone development and growth.

overall_design: cell atlas of mouse knee joint cells at E16.5 timepoint

first: Maurizio

last: Pacifici


title: Effect of conditional SMC specific deletion of ADAR1 with constitutive deletion of RNA sensor MDA5 on mouse aortic single cell transcriptomic profile and effect on atherosclerosis

first: Chad

last: Weldy

state: CA

summary: Mapping the genomic architecture of complex disease has been predicated on the understanding that genetic variants influence disease risk through modifying gene expression. However, recent discoveries have revealed that a significant burden of disease heritability in common autoinflammatory disorders and coronary artery disease is mediated through genetic variation modifying post-transcriptional modification of RNA through adenosine-to-inosine (A-to-I) RNA editing. This common RNA modification is catalyzed by ADAR enzymes, where ADAR1 edits specific immunogenic double stranded RNA (dsRNA) to prevent activation of the double strand RNA (dsRNA) sensor MDA5 (IFIH1) and stimulation of an interferon stimulated gene (ISG) response. Multiple lines of human genetic data indicate impaired RNA editing and increased dsRNA sensing by MDA5 to be an important mechanism of coronary artery disease (CAD) risk. Here, we provide a crucial link between observations in human genetics and mechanistic cell biology leading to progression of CAD. Through analysis of human atherosclerotic plaque, we implicate the vascular smooth muscle cell (SMC) to have a unique requirement for RNA editing, and that ISG induction occurs in SMC phenotypic modulation, implicating MDA5 activation. Through culture of human coronary artery SMCs, generation of a conditional SMC specific Adar1 deletion mouse model on a pro-atherosclerosis background with additional constitutive deletion of MDA5 (Ifih1), and with incorporation of single cell RNA sequencing cellular profiling, we further show that Adar1 controls SMC phenotypic state by regulating Mda5 activation, is required to maintain vascular integrity, and controls progression of atherosclerosis and vascular calcification. Through this work, we describe a fundamental mechanism of CAD, where cell type and context specific RNA editing and sensing of dsRNA mediates disease progression, bridging our understanding of human genetics and disease causality.

overall_design: To investigate the role of SMC specific ADAR1 and RNA sensor MDA5 on vascular function and progression of atherosclerosis, we used the Adar1 flox mouse crossed onto an Myh11 CreERT2 background, with additional constitutive either homozygous or heterozygous deletion of Mda5 (Ifih1), that has additional hyperlipidemia with ApoE-/- and SMC lineage tracing with ROSA tdTomato alleles. In our prior experiments, we performed homozygous deletion of Adar1 by treating 8 week old male mice with tamoxifen (Adar1 fl/fl, Myh11CreERT2, ApoE-/-,  tdTomato+/+). These mice develop severe disease and single cell RNAseq was performed of the ascending aorta and aortic root at 2 weeks post tamoxifen. In the submitted data, SMC Adar1 deletion was performed with additional constitutive homozygous deletion of MDA5 (Adar1 fl/fl, Ifih1-/-, Myh11CreERT2, ApoE-/-,  tdTomato+/+). Control mice were wild type for the Adar1 fl/fl allele and Ifih1 allele ( Myh11CreERT2, ApoE-/-,  tdTomato+/+) and were similarly treated with tamoxifen at 8 weeks of age. To then evaluate the effect of SMC Adar1 haploinsufficiency in atherosclerosis, in our prior experiments we treated mice that were heterozygous for the Adar1 flox allele (Adar1 fl/WT, Myh11CreERT2, ApoE-/-,  tdTomato+/+) with tamoxifen at 8 weeks of age, and then put on a high fat diet for 16 weeks to induce atherosclerosis. In the submitted dataset, these SMC Adar1 het mice where then crossed onto the Mda5 het background where scRNAseq was performed at the same 16 week timepoint.


title: The epigenomic landscape of single vascular cells reflects developmental origin and identifies disease risk loci

summary: Vascular sites have distinct susceptibility to atherosclerosis and aneurysm, yet the biological underpinning of vascular site-specific disease risk is largely unknown. Vascular tissues have different developmental origins that may influence global chromatin accessibility, and understanding differential chromatin accessibility, gene expression profiles, and gene regulatory networks (GRN) on single cell resolution may give key insight into vascular site-specific disease risk. Here, we performed single cell chromatin accessibility (scATACseq) and gene expression profiling (scRNAseq) of healthy adult mouse vascular tissue from three vascular sites, 1) aortic root and ascending aorta, 2) brachiocephalic and carotid artery, and 3) descending thoracic aorta. Through a comprehensive analysis at single cell resolution, we discovered key regulatory enhancers to not only be cell type, but vascular site specific in vascular smooth muscle (SMC), fibroblasts, and endothelial cells. We revealed an epigenetic ‘memory’ of embryonic origin with differential chromatin accessibility of key developmental transcription factors such as Tbx20, Hand2, Gata4, and Hoxb family members and discovered transcription factor motif accessibility to be cell type and vascular site specific. Notably, we identify ascending fibroblasts to have distinct epigenomic patterns, highlighting SMAD2/3 function to suggest a differential susceptibility to TGF, a finding we confirmed through in vitro culture of primary adventitial fibroblasts. Finally, to understand how vascular site-specific enhancers may regulate human genetic risk to disease, we integrated genome wide association study (GWAS) data for ascending and descending aortic dimension, and through using a novel technique, we employed ChromBPNet, a distinct base resolution deep learning model to predict variant effect on chromatin accessibility We trained nine ChromBPNet models to predict variant effect in SMC, Fibroblasts, and Endothelial cells within ascending aorta, carotid, and descending aorta sites of origin. We reveal that although cell type remains a primary influence on variant effect, vascular site modifies effect within cell type and highlights genomic regions that are enriched for specific TF motif footprints — including MEF2A, SMAD3, and HAND2. This work supports a paradigm that the epigenomic and transcriptional landscapes of vascular cells are cell type and vascular site-specific and that these vascular site-specific enhancers govern complex genetic drivers of vascular site-specific disease risk.

overall_design: Tissues were collected and dissociated for single cell capture as previously described. Briefly, vascular tissue was washed three times in PBS, tissues were then placed into an enzymatic dissociation cocktail (2 U ml−1 Liberase (5401127001; Sigma–Aldrich) and 2 U ml−1 elastase (LS002279; Worthington) in Hank’s Balanced Salt Solution (HBSS)), and minced. After incubation at 37 °C for 1 h, the cell suspension was strained and then pelleted by centrifugation at 500g for 5 min. The enzyme solution was then discarded, and cells were resuspended in fresh HBSS. To increase biological replication, 16 mice were used to obtain single-cell suspensions for each vascular tissue (aortic root/ascending aorta, brachiocephalic/carotid, and descending thoracic aorta) with two captures per vascular site. Cells were FACS sorted and live cells were identified as previously described. Briefly, cells were sorted on a BD Aria II instrument, where cells were gated on forward/side scatter parameters to exclude small debris and then gated on forward scatter height versus forward scatter area to exclude obvious doublet events. Approximately 100-150,000 live cells were sorted for each vascular site, where a portion of cells were taken directly to scRNAseq capture. For single cell ATAC, cells were collected in BSA-coated tubes, and nuclei isolated per 10X recommended protocol, and captured on the 10X scATAC platform.  All single-cell capture and library preparation was performed in the Quertermous lab and sequencing was performed at MedGenome (Foster City, CA). Cells were loaded into a 10x Genomics microfluidics chip and encapsulated with barcoded oligo-dT-containing gel beads using the 10x Genomics Chromium controller according to the manufacturer’s instructions. Single-cell libraries were then constructed according to the manufacturer’s instructions (Illumina). Libraries from individual samples were multiplexed into one lane before sequencing on an Illumina platform with targeted depth of 50,000 reads per cell for RNA and 75,000 reads/cell for ATAC. Post filtering for non-cells, mean number of reads within peaks per cell in scATAC data was 18,000-20,000 as was seen in our prior report.


title: Targeting nucleoporin 214 selectively eliminates leukemia stem cells by inducing ferroptosis [CUT&Tag 7]

first: Zhe

last: Chen

summary: Leukemia stem cells (LSCs) give rise to mature leukemia blasts and are the major cause of therapy resistance and relapse in acute myeloid leukemia (AML). We propose a novel strategy to eliminate heterogeneous LSCs but spare hematopoietic stem cells (HSCs) by targeting essential genes that are highly expressed in LSCs than that in HSCs. Nucleoporin 214 (NUP214) is determined as a dose-dependently essential gene (DDEG) in LSCs. Homozygous deletion of Nup214 eliminates LSCs and HSCs; however, Nup214 haploinsufficiency impairs LSC maintenance but spares HSCs by selectively inducing ferroptosis of LSCs. Mechanistically, NUP214 locates to chromatin and interacted with Sub1 to inhibit the transcription of heme oxygenase-1 (Hmox1) and arachidonate 15-lipoxygenase (Alox15), thus protecting LSCs against lipid peroxidation. We screened and identified a small molecule 0449-b as a molecular glue that links NUP214 and an NEDD8 E3 ligase, Rbx2, to induce neddylation and degradation of NUP214. 0449-b shows efficacy in selective eradication of LSCs but has a negligible effect on normal hematopoietic stem and progenitor cells. These biological and chemical findings provide a valuable strategy to eliminate LSCs by targeting DDEGs.

overall_design: HSCs (CD48-CD150+LSK) from Nup214fl/+ mice and LSCs (Lin-Sca-1-c-Kit+CD16/32+CD34+) from MLL-AF9-driven Nup214fl/+ mice were sorted, followed by Ruvbl2-CUT&Tag-seq


title: Evolution of a transplantable cancer by adaptation [CTVT_RNAseq_cells]

city: Edinburgh

first: Jose

middle: Ignacio

last: de las Heras

address: Max Born Crescent

department: Wellcome Trust Centre

country: United Kingdom

institute: University of Edinburgh

summary: The histocompatibility barrier prevents transmission of both normal and tumour cells between individuals 1, however clonally transmissible cancers in dogs, Tasmania devils and soft-shell clams can naturally transmit as allografts 2. To understand how tumours can escape the histocompatibility barrier, we have serially passaged a mouse melanoma first into syngeneic mice and then into progressively more mismatched mouse strains until a fully allo-transplantable tumour emerged. Here we show that tumour passaging selected for cells with an anti-viral inflammatory signature, characterised by expression of MHC-I, PD-L1, Qa-1 non-classical MHC molecules and the upregulation of intergenic endogenous retroviral elements (ERVs). Knock-out of RNA sensor RIG-I 3 in the transplantable tumour cells resulted in reduced expression of MHC-I, PD-L1 and Qa-1. Antibody-mediated blockade of both PD-L1 and Qa-1 induced tumour regression after transplantation. A similar inflammatory signature was found in human melanomas that respond to anti-PD1 antibody treatment. Thus, an inflammatory anti-viral signature likely induced by RIG-I sensing of ERVs facilitates escape of the mouse melanoma from allogeneic rejection. The results underscore the immunological plasticity of melanoma and reveal an unanticipated role for inflammation in allograft immune evasion.

overall_design: RNAseq profiling of YUMM1.7 tumour cells after serial passaging into mice. RNAseq was performed on polyA RNA extracted from tumour cells in vitro. Cells were obtained from dissociation of tumours and grown in vitro for 1-3 passages. Different tumours of the same passage are indicated with the letters A to H. The first batch was sequenced in January 2021 and the second batch in April 2022. The F2 tumour cells were resequenced in April 2022 for reference.

first: Ahmed

last: Rokan

first: Ariberto

last: Fassati


title: Targeting nucleoporin 214 selectively eliminates leukemia stem cells by inducing ferroptosis [CUT&Tag 6]

overall_design: HSCs (CD48-CD150+LSK) from Nup214fl/+ mice and LSCs (Lin-Sca-1-c-Kit+CD16/32+CD34+) from MLL-AF9-driven Nup214fl/+ mice were sorted, followed by Sub1-CUT&Tag-seq


title: Targeting nucleoporin 214 selectively eliminates leukemia stem cells by inducing ferroptosis [Cut&Tag 5]

overall_design: HSCs (CD48-CD150+LSK) from Nup214fl/+ mice and LSCs (Lin-Sca-1-c-Kit+CD16/32+CD34+) from MLL-AF9-driven Nup214fl/+ and Nup214fl/+,Tie2-Cre (Nup214-HET) AML mice were sorted, followed by NUP214-CUT&Tag-seq


title: Targeting nucleoporin 214 selectively eliminates leukemia stem cells by inducing ferroptosis [RNA-seq 3]

overall_design: HSCs (CD48-CD150+LSK) from Nup214fl/+ and Nup214fl/+,Tie2-cre (Nup214-HET) mice were sorted, followed by RNA-seq


title: Dimethyl itaconate alleviates Escherichia coli-induced endometritis by activating the guanosine-CXCL14 axis via increasing the abundance of Muribaculaceae

city: Jilin Province

first: He

middle: Yu

last: hang

address: 5451 Xi'an Road

department: College of Veterinary Medicine

institute: Jilin University

summary: Endometritis, a prevalent reproductive system disease with high incidence, leads to reproductive dysfunction and potential miscarriage in both humans and animals, impacting human health and the dairy industry, resulting in significant economic loss. Gut microbiota plays an important role in the pathogenesis of endometritis. However, the specific microbial and metabolic mediators participating in the pathogenesis remain to be identified. In this study, we found that the concentration of itaconate in the feces of mice increased after endometritis induced by Escherichia coli (E. coli) infection, suggesting a potential association between itaconate and inflammatory processes. Here, we investigated the specific role of dimethyl itaconate (DI), a derivative of itaconate with potent anti-inflammatory and immunomodulatory properties on endometritis. Oral administration of DI ameliorated the symptoms of E. coli-induced endometritis in mice. The protective effect was abolished by antibiotic-induced depletion of the gut microbiota, and fecal microbiota transplantation (FMT) from DI-treated mice to recipient mice ameliorated E. coli-induced endometritis. Integrative multiomics revealed that the addition of DI led to an enrichment of Muribaculaceae and the microbial purine metabolite guanosine in the feces. Muribaculum intestinale (DSM 28989), a bacterium of Muribaculaceae or guanosine supplementation alleviated E. coli-induced endometritis in mice. Mechanistically, DI promoted the multiplication of Muribaculaceae, and supplementation of M. intestinale upregulated the level of guanosine in the uterus. Transcriptome data and antibody-neutralizing experiment demonstrated the protective effect of guanosine in E. coli-induced endometritis was mediated by activating the expression of CXCL14 in uterine epithelial cells. In conclusion, our study elucidates the significant role of the gut microbiota and its metabolites in safeguarding the uterus against pathogen infections, providing substantiation for the regulation of distal organ by the gut microbiota, and establishing a basis for preventive measures against related diseases through the gut-X axis.

overall_design:
DE:Administer 400mg/kg DI (Dimethyl itaconate) orally daily for seven consecutive days.
E:Administer PBS orally daily for seven consecutive days.

first: Yuhang

last: He

first: Xufeng

last: Xie

first: Jiapei

last: Cai

first: Yongguo

last: Cao


title: Antifungal drug responses in two Candida glabrata strains

first: Edward

middle: William Joseph

last: Wallace

address: Waddington Building, Max Born Crescent

department: Institute for Cell Biology, School of Biological Sciences

summary: Transcriptomic responses of Candida glabrata strains, BG2 and CBS138, to two azole antifungal drugs, Fluconazole and Voriconazole.

overall_design: Measurements of the transcriptomic responses of two Candida glabrata strains, BG2 and CBS138, to two azole antifungal drugs, Fluconazole and Voriconazole and a mock treatment, after a 4 hour growth, in 3 biological replicates.

first: Weronika

middle: A

last: Danecka

first: Delma

last: Childers

middle: W


title: Targeting nucleoporin 214 selectively eliminates leukemia stem cells by inducing ferroptosis [RNA-seq 4]

overall_design: LSCs (Lin-Sca-1-c-Kit+CD16/32+CD34+) from MLL-AF9-driven Nup214fl/+ and Nup214fl/+,Tie2-cre (Nup214-HET) AML mice were sorted, followed by RNA-seq


title: Targeting nucleoporin 214 selectively eliminates leukemia stem cells by inducing ferroptosis [RNA-seq 2]

overall_design: At 3 days after pIpC administration, HSCs (CD48-CD150+LSK) from Nup214fl/fl and Nup214fl/fl,Mx1-cre mice were sorted, followed by RNA-seq


title: Targeting nucleoporin 214 selectively eliminates leukemia stem cells by inducing ferroptosis [RNA-seq 1}

overall_design: HSCs (CD48-CD150+LSK) from wild-type C57BL/6J mice and LSCs (Lin-Sca-1-c-Kit+CD16/32+CD34+) from MLL-AF9-driven AML mice were sorted, followed by RNA-seq


title: Long non-coding RNA TUG1 regulates multiple glycolytic enzymes in hepatocellular carcinoma cells via sponging microRNA-122-5p

city: Bangkok

first: Thammachanok

last: Boonto

address: 254 Phaya Thai Rd, Khwaeng Wang Mai, Pathum Wan

department: Biochemistry

country: Thailand

institute: Faculty of Medicine, Chulalongkorn University

summary: Hepatocellular carcinoma (HCC) remains the third leading cause of cancer deaths; however, its therapeutic options are limited. Understanding the molecular mechanisms of HCC could shed light on new therapies. Emerging studies indicate the important role of long-noncoding RNAs (lncRNAs) in the pathogenesis of HCC. One of the early discovered lncRNAs, taurine-upregulated gene 1 (TUG1), has been up-regulated in HCC tissues, but its impact on HCC cells’ transcriptomics remains unexplored. We established TUG1-knockdown HCC cells and performed RNA-seq analysis. KEGG analysis revealed the significant enrichment of downregulated genes involved in glycolysis. Accordingly, TUG1-depleted HCC cells showed impairments in glucose uptake, ATP synthesis, and lactate production. Clinical HCC tissue data revealed positive gene expression correlations between TUG1 and glycolysis genes identified by RNA-seq. To identify a molecular function of TUG1 in glycolysis, we explored the competing endogenous (ceRNA) model and identified five microRNAs (miRNAs) with the highest number of TUG1 binding sites using bioinformatic tools. Amongst these miRNAs, microRNA-122-5p (miR-122-5p) displayed an inverse relationship for gene expression with most TUG1-regulated glycolysis genes, including PKM, ALDOA, ENO2, and PFKM. Dual luciferase assays confirmed the direct interaction between TUG1 and miR-122-5p and between miR-122-5p and the 3’ untranslated regions (3’UTR) of PKM and ALDOA. We further showed that inhibition of miR-122-5p alleviated the suppression of glycolysis induced by TUG1 depletion. Together, our RNA-seq analysis of TUG1-depleted HCC cells in combination with clinical data reveals the crucial function of TUG1 in promoting glycolysis via sponging miR-122-5p, which is a negative regulator of multiple glycolytic enzymes.

overall_design: To investigate the influence and the molecular function of  lncRNA TUG1 on biological pathway in HCC cell lines, we established TUG1-knockdown HepG2 lines with shRNA.  Three technical replicated were performed for each group (shCon,  sh1-TUG1, and sh2-TUG1) to obtaine the expression profile followed by trascriptomic analysis.

first: Chaiyaboot

last: Ariyachet


title: Gene expression profile at single cell level of  purified "Tumor"  and "Stroma" cells in ASD+/+ and ASD+/- murine RMS tumors [scRNA-seq]

city: Memphis

first: Hongjian

last: Jin

state: TN

address: 262 Danny Thomas Place

department: Center for Applied Bioinformatics

institute: St Jude Children's Research Hospital

summary: We used scRNA-seq to compare intratumoral heterogeniety between ASD+/+ and ASD+/- tumors. We found that RMS populations were similar between genotypes and that neutrophils were strongly enriched in ASD+/- tumor stroma

overall_design: Tumors were digested and then isolated by Fluorescence-activated cell sorting (FACS) into "Tumor" (PECAM1-;tdTomato+) and "Stroma" (PECAM1-;tdTomato-) cells prior to scRNA-seq analysis

first: Randolph

last: Larsen

first: Patrick

last: Schreiner

first: Mark

middle: E

last: Hatley