title: Study the role of different concentrations of VFAs on chromatin accessibility in ovine ruminal epithelial cells at am and pm using ATAC-seq

status: Public on Jan 08 2025

city: Yangzhou

first: Yongkang

last: Zhen

state: — Please Select —

address: Yangzhou University

institute: Yangzhou University

summary: This experiment employed ATAC-seq (Assay for Transposase Accessible Chromatin with sequencing) to explore the mechanism of how different concentrations of VFAs regulate ruminal epithelial chromatin accessibility under the Grain-diet and Hay-diet patterns in both am and pm. Cells from Grain-am, Grain-pm, Hay-am, and Hay-pm treatment groups were havest for ATAC-seq experiments, n=3 pooled biological replicates per library.

overall_design: In Grain-diet group, 71.1 mM acetate (AA, prepared with sodium acetate, 241245, Sigma-Aldrich, St. Louis, MO), 20.1 mM propionate (PA, prepared with sodium propionate, P1880, Sigma-Aldrich), 16.0 mM butyrate (BA, prepared with sodium butyrate, 303410, Sigma-Aldrich), 1.7 mM valerate (VA, 75054, Sigma-Aldrich), 1.1 mM isobutyrate (IBA, I1754, Sigma-Aldrich), and 5.3 mM isovalerate (IVA, 129542, Sigma-Aldrich) were added into the culture medium (Grain-VFAs culture medium, GVCM); in Hay-diet group, 60.8 mM AA, 14.6 mM PA, 9.2 mM BA, 1.2 mM VA, 1.0 mM IBA, and 3.0 mM IVA were added into the culture medium (Hay-VFAs culture medium, HVCM) . The total VFAs concentration in the GVCM was 115.78 mM, while the total VFAs concentration in the HVCM was 89.79 mM. After preparation, the pH value of the culture medium was adjusted to 7.2 using 7.5% NaHCO₃ solution (C0220, Beyotime). Cells were cultured in 6-well plates, when the cells were reached at 70% density, the culture medium was changed to DMEM/F12 containing 100 nM dexamethasone (DEX) and 1% penicillin-streptomycin solution without FBS (Synchronous culture medium, SCM). ORECs were cultured with GVCM and HVCM for 24 hours after synchronized to the initial circadian rhythm using SCM contained 100 nM DEX. After 24 hours (T0), cells in the Grain-diet and Hay-diet groups were harvest at 32h (T8) and 44h (T20), which were defined as am and pm, respectively. Finally, cells from Grain-am, Grain-pm, Hay-am, and Hay-pm treatment groups were rapidly frozen under -80°C conditions for subsequent ATAC-seq experiments, n=3 pooled biological replicates per library.


title: Effect of neutrophil depletion on gene expression im macrophages and fibroblasts from mouse lung and skin

status: Public on Jan 09 2025

city: New Haven

first: Andrés

last: Hidalgo

address: 10 Amistad Street

country: USA

institute: Yale University

type: Expression profiling by high throughput sequencing

summary: To investigate the effect of the absence of neutrophils in the transcriptome of fibroblast and macrophage from lung and skin we injected DT to Mrp8 CRE+ iDTR and  Mrp8 CRE- iDTR mice every two days for a week and isolated lung and skin fibroblasts and macrophages by cell sorting . We then performed gene expression profiling analysis using data obtained from RNA-seq of macrophages and fibroblast 7 days after DT treatment and compared the transcriptomes of macrophages and fibroblasts in control or neutrophil depletion animals. We focused on the role of neutrophils on the regulation of extracelular matrix genes in fibroblasts and macrophages and found that neutrophil depletion does not significantly affect the RNA level of matrix-related transcripts in those cells.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for lung and skin macrophages and fibroblast from control and neutrophil depleted animals

first: Ivan

last: Ballesteros

first: Andrea

last: Rubio-Ponce


title: 18S rRNA sequencing data of ruminal protozoal profiles under grain-diet and hay-diet feeding patterns at six different timepoints in sheep

country: China

summary: In order to investigate the diurnal oscillations of ruminal protozoa, and their responses to the changes in different feeding patterns, we conducted an animal experiment by feeding the sheep ad libitum with a hay-based diet (50% of alfalfa hay and 46% of oats hay) and a grain-based diet (45% of corn meal and 11% of soybean meal) for 30 days, and ruminal fluid samples were collected at six different timepoints from T2 to T22 in one day, and the composition and diversity of the protozoal communities in rumen microbiomes of the sheep in the Grain-diet and Hay-diet groups at different timepoints were analyzed through 18S rRNA sequencing.

overall_design: A total of 16 healthy sheep with similar body weight (nearly 65 kg) and aged around 3.5 years old were selected in this study. All sheep were placed with permanent rumen fistula at 2 years old. The inner diameter of the fistula was 4 cm. Prior to the animal trial, all sheep were maintained under the same farming conditions in individual tie-stalls and fed a hay-based diet (Hay-diet group) ad libitum for 14 days to ensure stable feeding. Then, 8 sheep were randomly divided into the Hay-diet group and continued to feed hay-based diet. The other 8 sheep were shifted to feed on a grain-based diet (Grain-diet group) with a forage/concentrate of 40:60 (dry matter basis). The feeding trial lasted for 30 days, adequate feed was provided daily at 8 am, and all sheep had free access to feed and water throughout the study. All sheep were vaccinated and had no history of antimicrobial agents (antibiotics, antifungals, or antivirals) administration or clinical signs of diseases. Fasting was not required for the sheep before sampling, and the ruminal fluid samples of 16 sheep were collected from 2:00 to 22:00 at a 4-hour interval (a total of 6 timepoints were selected at T2, T6, T10, T14, T18, and T22) on sampling day. A 50 mL rumen fluid sample was collected from the rumen fistula, samples were then filtered through four layers of sterile cheesecloth and stored at -80°C for 18S rRNA gene sequencing.


title: Study the role of different concentrations of VFAs on histone modifications in ovine ruminal epithelial cells at am and pm using CUT&Tag-seq

type: Genome binding/occupancy profiling by high throughput sequencing

summary: This experiment employed CUT&Tag-seq (Cleavage Under Targets and Tagmentation with sequencing) to explore the mechanism of how different concentrations of VFAs regulate ruminal epithelial histone modifications under the Grain-diet and Hay-diet patterns in both am and pm. Cells from Grain-am, Grain-pm, Hay-am, and Hay-pm treatment groups were havest for CUT&Tag-seq experiments, n=3 pooled biological replicates per library. The primary histones used for CUT&Tag were Acetyl-Histone H3 (Lys27) Rabbit mAb (H3K27ac, 8173S, CST), Acetyl-Histone H3 (Lys9) (C5B11) Rabbit mAb (H3K9ac, 9649S, CST), and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb (H3K4me3, 9751S, CST).

overall_design: In Grain-diet group, 71.1 mM acetate (AA, prepared with sodium acetate, 241245, Sigma-Aldrich, St. Louis, MO), 20.1 mM propionate (PA, prepared with sodium propionate, P1880, Sigma-Aldrich), 16.0 mM butyrate (BA, prepared with sodium butyrate, 303410, Sigma-Aldrich), 1.7 mM valerate (VA, 75054, Sigma-Aldrich), 1.1 mM isobutyrate (IBA, I1754, Sigma-Aldrich), and 5.3 mM isovalerate (IVA, 129542, Sigma-Aldrich) were added into the culture medium (Grain-VFAs culture medium, GVCM); in Hay-diet group, 60.8 mM AA, 14.6 mM PA, 9.2 mM BA, 1.2 mM VA, 1.0 mM IBA, and 3.0 mM IVA were added into the culture medium (Hay-VFAs culture medium, HVCM) . The total VFAs concentration in the GVCM was 115.78 mM, while the total VFAs concentration in the HVCM was 89.79 mM. After preparation, the pH value of the culture medium was adjusted to 7.2 using 7.5% NaHCO₃ solution (C0220, Beyotime). Cells were cultured in 6-well plates, when the cells were reached at 70% density, the culture medium was changed to DMEM/F12 containing 100 nM dexamethasone (DEX) and 1% penicillin-streptomycin solution without FBS (Synchronous culture medium, SCM). ORECs were cultured with GVCM and HVCM for 24 hours after synchronized to the initial circadian rhythm using SCM contained 100 nM DEX. After 24 hours (T0), cells in the Grain-diet and Hay-diet groups were harvest at 32h (T8) and 44h (T20), which were defined as am and pm, respectively. Finally, cells from Grain-am, Grain-pm, Hay-am, and Hay-pm treatment groups were rapidly frozen under -80°C conditions for subsequent CUT&Tag-seq experiments (H3K27ac, H3K9ac, and H3K4me3), n=3 pooled biological replicates per library.


title: CCL7 promotes macrophage polarization and synovitis to exacerbate rheumatoid arthritis II

city: Guangzhou

first: Jun

last: Chen

address: No. 1023-1063, Satai South Road, Baiyun District

institute: Southern Medical University

summary: Macrophage polarization and synovitis play significant roles in rheumatoid arthritis (RA). Chemokines are involved almost throughout the entire pathology of synovitis. However, the interaction between chemokines and macrophage polarization in RA has been seldom reported. This study found that chemokine C-C motif ligand 7 (CCL7) secreted by polarized M1 macrophages enhances M1 polarization, creating a positive feedback loop and revealing the role of CCL7 in the progression of rheumatoid arthritis. In this research, RNA sequencing indicated that M1 polarized macrophages secrete a large amount of CCL7, which further enhances M1 polarization.  Through immunohistochemistry and enzyme-linked immunosorbent assay, it was found that the expression of CCL7 in the synovial tissue and serum of RA patients and mice was upregulated. Intra-articular injection of CCL7 recombinant protein exacerbated M1 polarization of macrophages, inflammation, fibrosis in the synovial tissue, and worsened arthritis pain in mice, which was improved after the injection of CCL7 neutralizing antibody. In addition, CCL7 promoted M1 polarization of macrophages and reversed the M2 polarization induced by IL-4, and it also promoted macrophage proliferation and migration. Although it had an inhibitory effect on chondrocyte activity, it did not show a significant impact on chondrocyte metabolism. Mechanistically, CCL7 targets CCR1 to promote M1 polarization of macrophages, a process partly mediated through the activation of the JAK2/STAT1 pathway. When stimulated by CCL7, macrophages secreted a vast amount of pro-inflammatory factors, exacerbating synovitis and cartilage damage, leading to the aggravation of RA disease. Our research established a positive feedback loop between M1 polarization of macrophages and CCL7, indicating that blocking CCL7 could improve the progression of RA, suggesting CCL7 as a potential target for RA treatment.

overall_design: To study the relationship between CCL7 and macrophage polarization, and to explore the effect and mechanism of CCL7 on rheumatoid arthritis. To explore potential therapeutic targets for rheumatoid arthritis.

first: Wenquan

last: Liang

first: Tianyu


title: 16S rRNA sequencing data of ruminal bacterial profiles under grain-diet and hay-diet feeding patterns at six different timepoints in sheep

summary: In order to investigate the diurnal oscillations of ruminal bacteria, and their responses to the changes in different feeding patterns, we conducted an animal experiment by feeding the sheep ad libitum with a hay-based diet (50% of alfalfa hay and 46% of oats hay) and a grain-based diet (45% of corn meal and 11% of soybean meal) for 30 days, and ruminal fluid samples were collected at six different timepoints from T2 to T22 in one day, and the composition and diversity of the bacterial communities in rumen microbiomes of the sheep in the Grain-diet and Hay-diet groups at different timepoints were analyzed through 16S rRNA sequencing.

overall_design: A total of 16 healthy sheep with similar body weight (nearly 65 kg) and aged around 3.5 years old were selected in this study. All sheep were placed with permanent rumen fistula at 2 years old. The inner diameter of the fistula was 4 cm. Prior to the animal trial, all sheep were maintained under the same farming conditions in individual tie-stalls and fed a hay-based diet (Hay-diet group) ad libitum for 14 days to ensure stable feeding. Then, 8 sheep were randomly divided into the Hay-diet group and continued to feed hay-based diet. The other 8 sheep were shifted to feed on a grain-based diet (Grain-diet group) with a forage/concentrate of 40:60 (dry matter basis). The feeding trial lasted for 30 days, adequate feed was provided daily at 8 am, and all sheep had free access to feed and water throughout the study. All sheep were vaccinated and had no history of antimicrobial agents (antibiotics, antifungals, or antivirals) administration or clinical signs of diseases. Fasting was not required for the sheep before sampling, and the ruminal fluid samples of 16 sheep were collected from 2:00 to 22:00 at a 4-hour interval (a total of 6 timepoints were selected at T2, T6, T10, T14, T18, and T22) on sampling day. A 50 mL rumen fluid sample was collected from the rumen fistula, samples were then filtered through four layers of sterile cheesecloth and stored at -80°C for 16S rRNA sequencing.


title: Combinatorial tagging generates a multi-purpose knock-in mouse model revealing phase separation-dependent germ granules in RNA homeostasis and germline development

city: Hefei

first: Caoling

last: Xu

state: Anhui

address: 96 Jinzhai Road

department: Department of Life Sciences and Medicine

institute: University of Science and Technology of China

summary: A large resource of epitope-tagged or Cre/CreERT2-expressing mouse models are available for study of germ granules or germline development. The germ granules are proteinaceous, membraneless organelles implicated in germ cell differentiation and maturation; however, their protein and RNA transcript constituents, and their mechanistic insight remain not fully understood. Herein, we generated a versatile germline mouse model through combinatorial tagging of DDX4 for simultaneous  expression of three cistronic coding products (C-terminal tagged DDX4 - DDX45HA, EGFP, and CreERT2) under the driving control of endogenous Ddx4 promoter. By leveraging the high-affinity HA tag, we optimized an efficient workflow for purification of germ granules (Chromatoid body, CB) from spermatids, and characterized the landscape of protein constituents and RNA transcripts in CB. Moreover, we explored and ascertained that DDX4-mediated, phase-separation dependent CB integrity is functionally important for recruiting distinctive long RNA transcripts and biogenesis of pachytene- and TE-derived piRNAs. Together, our study generated a versatile germline mouse model with a multiplicity of applications for germline study, and provided mechanistic insight into germline development as dictated by germ granules.

overall_design: We have designed a combinatorial tagging knock-in mouse model：Ddx45XHA-EGFP-CreERT2.We found that homozygous knock-in male mice are sterile. Abnormal sperm acrosome and chromatoid body were found in homozygous mice. We want to isolation RNA from round spermatids and chromatoid body for RNA-seq to explore the mechanism of sterility of homozygous knock-in male mice.

first: Jianqiang

last: Bao

first: Lan

last: Meng


title: The role of USF3 in γδ T cells

city: zhuhai

first: xianghong

last: wang

state: Please Select

address: 79 Kangning Road, Xiangzhou District

institute: Zhuhai People’s Hospital

summary: Explore the role of USF3 in γδ T cells.

overall_design: Isolate splenic cells from 6-week-old USF3-f/f and USF3-f/f; CD2-cre mice, sort γδ T cells using flow cytometry, then split the sorted cells with Trizol and perform RNA-seq.


title: Effect of HERV-R env Knockout on Tumorigenic Characteristics of Colorectal Cancer Cells

city: Busan

first: Min-Hye

last: Kim

address: 262, Gamcheon-ro, Seo-gu

country: South Korea

institute: Kosin University College of Medicine

summary: To investigate the cancer properties of HERV-R knockout in the colon cancer cell line DLD1, we knocked out HERV-R from Crispr-cas9 in DLD1. We performed RNA-seq to profile gene expression in DLD1, DLD1R (HERV-R KO) cells, respectively.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for DLD1 cells and its KO derivatives (DLD1R).

first: Eun-Ji

last: Ko

first: HyoJin

last: An

first: Heui-Soo

first: Sun-Hee

last: Leem

first: Hee-Jae

last: Cha


title: CYP3A5 promotes glioblastoma stemness and chemoresistance through fine-tuning NAD+/NADH ratio

city: Tianjin

first: Wentao

last: Hu

state: Tianjin

address: No. 94, Weijin Road, Nankai District, Tianjin

institute: School of medicine, NANKAI university

summary:
Background.
Glioblastoma multiforme (GBM) exhibits a cellular hierarchy with a subpopulation of stem-like cells known as glioblastoma stem cells (GSCs), that drive tumor growth and contribute to treatment resistance. NAD(H) emerged as a crucial factor influencing GSCs.
Methods.
A multistep process of machine learning algorithms was implemented to construct the glioma stemness-related score (GScore). Further in silico and patient tissue analyses validated the predictive ability of the GScore and identified a potential target, CYP3A5. Loss-of-function or gain-of-function genetic experiments were performed to assess the impact of CYP3A5 on the self-renewal and chemoresistance of GSCs both in vitro and in vivo. Mechanistic studies were conducted using nontargeted metabolomics, RNA-seq, seahorse, transmission electron microscopy, immunofluorescence, flow cytometry, ChIP‒qPCR, RT‒qPCR, western blotting, etc. The efficacy of pharmacological inhibitors of CYP3A5 was assessed in vivo.
Results.
Based on the proposed GScore, we identify a GSC target CYP3A5, which is highly expressed in GSCs and temozolomide (TMZ)-resistant GBM patients. This elevated expression of CYP3A5 is attributed to transcription factor STAT3 activated by EGFR signaling or TMZ treatment. Depletion of CYP3A5 impairs self-renewal and TMZ resistance in GSCs. Mechanistically, CYP3A5 maintains mitochondrial fitness to promote GSC metabolic adaption through the NAD⁺/NADH-SIRT1-PGC1α axis. Additionally, CYP3A5 enhances the activity of NAD-dependent enzyme PARP to augment DNA damage repair. Treatment with CYP3A5 inhibitor alone or together with TMZ effectively suppresses tumor growth in vivo.
Conclusion.
Together, this study suggests that GSCs activate STAT3 to upregulate CYP3A5 to fine-tune NAD⁺/NADH for the enhancement of mitochondrial functions and DNA damage repair, thereby fueling tumor proliferation and conferring TMZ resistance, respectively. Thus, CYP3A5 represents a promising target for GBM treatment.

overall_design: To invertigate the role of CYP3A5 in stemness maintenance and chemoresistance of GSCs, we established U87 spheres in which target gene has been knocked out by sgRNA.

first: Jialin

last: Liu

first: Chunsheng

last: Kang

first: Ling


title: CCN2 regulates smooth muscle cell trans-differentiation and lipid accumulation in atherosclerosis

city: Atlanta

first: Qian

state: GA

address: 101 Woodruff Circle

institute: Emory University

summary: Accruing evidence illustrates an emerging paradigm of dynamic vascular smooth muscle cell (SMC) trans-differentiation during atherosclerosis progression. However, the molecular regulators that govern SMC phenotype diversification remain poorly defined. This study aims to elucidate the functional role and underlying mechanisms of cellular communication network factor 2 (CCN2), a matricellular protein, in regulating SMC plasticity in the context of atherosclerosis.

overall_design: CCN2-floxed mice were crossed with Myh11-Cre-ERT2 transgenic mice to generate male CCN2-floxed mice harboring SMC-specific Cre (CCN2fl/fl-Myh11-Cre-ERT2). Smooth muscle cell (SMC)-specific deletion of CCN2 was achieved by intraperitoneal (IP) injection of five consecutive doses of tamoxifen to 6-8 weeks old male CCN2fl/fl-Myh11-Cre-ERT2 mice. Age-matched male CCN2-floxed mice without Cre expression subjected to the same tamoxifen regimen were used as controls. After one-week washout period, SMC-specific CCN2-knockout mice, henceforth CCN2SMC∆ mice and CCN2-floxed control mice, henceforth CCN2fl/fl mice were used for the modeling of atherosclerosis (injected once via tail vein with AAV-PCSK9 (rAAV8/D377Y-mPCSK9,1 × 1011 VG) before being fed for 5 months with high fat diet). Then the aorta of mice were harvested for single cell isolation and sent for Sc-RNA-seq.


title: Transcriptome responses to Salmonella enterica serovar Reading in vaccinated and unvaccinated turkeys

city: Ames

first: Melissa

last: Monson

state: Iowa

address: 1920 Dayton Ave

institute: USDA-ARS National Animal Disease Center

summary: The 2017-2019 foodborne outbreak of Salmonella enterica serovar Reading (S. Reading) in North America revealed the need for effective control of this serovar in turkey production. This study evaluated two live-attenuated Salmonella vaccines against an outbreak-associated strain of S. Reading in turkeys. At 1 day and 3 weeks of age, male turkey poults were either mock-vaccinated or given either an internally developed cross-protective vaccine and a commercially available vaccine. At 7 weeks of age, poults were challenged with S. Reading; one mock-vaccinated group was mock-challenged. Along with assessment of Salmonella colonization and dissemination, acute transcriptomic responses in the cecal tonsil were characterized at 2 days post inoculation and revealed decreased expression of genes encoding intestinal transporters and tight junction proteins. Vaccination with either vaccine mitigated most of the transcriptional changes in intestinal health-related genes induced by S. Reading in turkey cecal tonsil.

overall_design: RNA-seq profiling of 12 samples of turkey cecal tonsil from 4 experimental groups: mock-vaccinated/mock-challenged (M/M), mock-vaccinated/S. Reading-challenged (M/R), BBS 866-vaccinated/S. Reading-challenged (BBSoo/R), and AviPro Megan Egg-vaccinated/S. Reading-challenged (AME oo/R).

middle: S

first: Manoj

last: Gurung

first: Bradley

middle: L

last: Bearson

first: Samuel

middle: J

last: Whelan

first: Julian

middle: M

last: Trachsel

first: Torey

last: Looft

first: Matthew

last: Sylte

first: Shawn


title: Disruption of Immune Responses By Type I Diabetes Exacerbates SARS-CoV-2 Mediated Lung Injury [bulk RNA-seq]

city: Philadelphia

first: Sara

last: Kass-Gergi

state: PA

address: 3900 Spruce St

department: Medicine

institute: University of Pennsylvania

summary: COVID-19 commonly presents as pneumonia, with those most severely affected progressing to respiratory failure. Patient responses to SARS-CoV-2 infection are varied, with comorbidities acting as major contributors to varied outcomes. Focusing on one such major comorbidity, we assessed whether pharmacological induction of Type I Diabetes Mellitus (T1DM) would increase the severity of lung injury in a murine model of COVID-19 pneumonia utilizing wild type mice infected with mouse-adapted SARS-CoV-2. Hyperglycemic mice exhibited increased weight loss and reduced blood oxygen saturation in comparison to their euglycemic counterparts, suggesting that these animals indeed experienced more severe lung injury. Transcriptomic analysis revealed a significant impairment of the adaptive immune response in the lungs of diabetic mice compared to those of control. In order to expand the limited options available for tissue analysis due to biosafety restrictions, we also employed a new technique to digest highly fixed tissue into a single cell suspension, originally designed for scRNA-Seq, which we then adapted for flow cytometric analysis.  Flow immunophenotyping and scRNA-Seq confirmed impaired recruitment of T cells into the lungs of T1DM animals. Additionally, scRNA-Seq revealed a distinct, highly inflammatory macrophage profile in the diabetic cohort that correlates with the more severe infection these mice experienced clinically, allowing insight into a possible mechanism for this phenomenon. Recognizing the near certainty that respiratory viruses will continue to present significant public health concerns for the foreseeable future, our study provides key insights into how T1DM results in a much more severe infection and identifies possible targets to ameliorate comorbidity-associated severe disease.

overall_design: We assessed whether pharmacological induction of T1DM would increase the severity of lung injury in a murine model of COVID-19 pneumonia by infecting STZ treated mice with MA30-SARS-CoV-2 and harvesting lungs from WT and T1DM mice at day 7 post infection for bulk RNA seq

first: Andrew

middle: E

last: Vaughan


title: Epigenetic features support the diagnosis of B-cell prolymphocytic leukemia and identify two clinico-biological subtypes

city: Barcelona

first: Jose Ignacio

last: Martin-Subero

state: Barcelona

address: c/Rosselló, 149-153

country: Spain

institute: IDIBAPS

summary: The recognition of B-cell prolymphocytic leukemia (B-PLL) as a separate entity is controversial based on the current classification systems. Here, we analyze the DNA methylome of a cohort of 20 B-PLL cases diagnosed according to the ICC/WHO-HAEM4R guidelines, and compare them with chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), splenic marginal zone lymphoma (SMZL), and normal B cell subpopulations. Unsupervised principal component analyses suggest that B-PLL is epigenetically distinct from CLL, MCL and SMZL, which is further supported by robust differential methylation signatures in B-PLL. We also observe that B-PLL can be segregated into two epitypes with differential clinico-biological characteristics. B-PLL epitype 1 carries lower IGHV somatic hypermutation and a less profound germinal center-related DNA methylation imprint than epitype 2. Furthermore, epitype 1 is significantly enriched in mutations affecting MYC and SF3B1 as well as gain 1q, and displays DNA hypomethylation and gene upregulation signatures enriched in MYC targets. Despite the low sample size, patients from epitype 1 have an inferior overall survival than those of epitype 2. This study provides relevant insights into the biology and differential diagnosis of B-PLL, and potentially identifies two subgroups with distinct biological and clinical features.

overall_design: Illumina Infinium MethylationEPIC v1.0 commercial kit

first: Eleni

last: Charalampopoulou


title: Conditioned Extracellular Vesicles Derived from Dedifferentiated Fat Cells Promote Bone Regeneration by Altering MicroRNAs

city: Shanghai

first: Yingyi

last: Shen

address: Shanghai Songjiang

institute: cloudseq

type: Non-coding RNA profiling by high throughput sequencing

summary: The identification of suitable seed cells represents a critical scientific challenge in the field of oral and maxillofacial bone tissue regeneration. Dedifferentiated fat (DFAT) cells have biological characteristics similar to adipose-derived stem cells (ASCs), but possess the advantages of minimally invasive acquisition, strong proliferation and high homogeneity. Compared to cell therapy, extracellular vesicles (EVs) have lower immunogenicity, better stability and more convenient transportation and storage, which makes EV-based cell-free therapy to be a promising alternative strategy. It is not clear whether EVs derived from DFAT cells can be used as an effective therapy for bone regeneration. The purpose of this study was to verify the effect of EVs derived from DFAT cells on promoting osteogenic differentiation of bone marrow derived mesenchymal stem cells (BMSCs) to provide a reference for the treatment of alveolar bone loss.

overall_design: DFAT cells from Sprague-Dawley (SD) rats were isolated and cultured by ceiling culture. DFAT cells were conditioned for 14 days using an osteogenic induction medium. EVs derived from conditioned DFAT cells were extracted by ultracentrifugation and then identified and characterized by Western blot, transmission electron micro-scope (TEM) and nanoparticle tracking analysis (NTA). After labeling the conditioned EVs with the green fluorescent dye PKH-67, the EVs were cocultured with BMSCs for 12 hours and then the cells were observed using an laser scanning confocal microscopy (LSCM). CCK-8 kit assay was used to explore the effect of conditioned EVs on the proliferation of BMSCs. Alkaline phosphatase (ALP) staining, Alizarin Red S (ARS) staining and real-time quantitative polymerase chain reaction (qRT-PCR) were used to verify the effect of conditioned EVs on osteogenic differentiation of BMSCs.

first: YingYi

first: Fang

last: Qu

first: Yaqin

last: Wu

first: Chun

last: Xu


title: Molecular correlates for HPV-negative head and neck cancer engraftment prognosticate patient outcomes

city: Toronto

first: Laurie

last: Ailles

state: ON

address: 101 College St.

department: Princess Margaret Cancer Centre

country: Canada

institute: University Health Network

summary: There is a pressing need to improve risk stratification and treatment selection for HPV-negative head and neck squamous cell carcinoma (HNSCC) due to the adverse side effects of treatment. One of the most important prognostic features is metastases in lymph nodes. Previously, we demonstrated that tumor formation in patient-derived xenografts (i.e. engraftment) was associated with poor clinical outcomes in patients with HPV-negative HNSCC. However, assessing engraftment is challenging to implement in clinical settings. Here, we performed transcriptomic and proteomic profiling of 88 HNSCC patients and found the relationship between engraftment and clinical outcomes was recapitulated by molecular phenotype. We identified LAMC2 and TGM3 as candidate prognostic biomarkers and validated their utility in an independent cohort containing 404 HPV-negative HNSCC patients. Strikingly, these markers significantly improve prediction of outcomes beyond nodal status alone and can significantly stratify patients without any nodal involvement. Overall, our study demonstrates how the molecular characteristics of engraftment can inform patient prognostication.

overall_design: RNA-seq of Cal33 and Cal27 tongue squamous cell carcinoma cells and 61137 and 61162 carcinoma-associated fibroblast cells in mono-culture or co-culture.

first: Stephanie

last: Poon

first: Kevin

last: Nixon

first: Joseph

last: Walton


title: Identification of WOX5 targets in the root of Arabidopsis thaliana

city: Freiburg im Breisgau

first: Thomas

last: Laux

address: Schänzlestr. 1

department: Institut für Biologie III

country: Germany

institute: Albert-Ludwigs-Universität Freiburg

type: Genome binding/occupancy profiling by genome tiling array

summary: A chromatin immunoprecipitation-based microarray experiment (ChIP-chip) was performed to identify WOX5 direct target. genes

overall_design: WOX5 ChIP was performed with a WOX5 antibody for two independent biological replicates for each genotype of 35S:WOX5-GR and wox5-1. On each array precipitated DNA and input samples were hybridized in different colors.

first: Ernst

last: Aichinger

first: Edwin

last: Groot

first: Ning

last: Zhang


title: Tet2-mediated clonal hematopoiesis improves neurological deficits and is associated with inflammation resolution in the subacute phase of stroke

city: Charlottesville

first: Nicholas

middle: W

last: Chavkin

address: 415 Lane Road

institute: University of Virginia

summary: Recent work has revealed that clonal hematopoiesis (CH) is associated with a higher risk of numerous age-related diseases, including ischemic stroke, however little is known about whether it influences stroke outcome. Studies suggest that leukocytes carrying CH driver mutations have an enhanced inflammatory profile, which could conceivably exacerbate brain injury after a stroke. Using a mouse model of Tet2-mediated CH, we tested the hypothesis that CH would lead to a poorer outcome after ischemic stroke by augmenting brain inflammation. In contrast to our hypothesis, Tet2-mediated CH had no effect on acute stroke outcome but led to reduced neurological deficits during the subacute phase. This improved neurological outcome was associated with lower levels of brain inflammation during this time, suggesting that Tet2-mediated CH may promote inflammation resolution in the brain post-stroke. While additional mechanistic studies are required, these findings may suggest that Tet2-mediated CH has beneficial actions on the post-stroke brain.

overall_design: RNA was extracted from brains using the methods described above and total RNA was sent to Azenta Life Sciences (formally Genewiz) for library construction, sequencing and analysis. For this, RNA samples were firstly quantified using Qubit 2.0 Fluorometer (Life Technologies, CA, USA) and RNA integrity was measured using the RNA Screen Tape on Agilent 2200 TapeStation (Agilent Technologies, CA, USA). rRNA depletion was performed using QIAseq® FastSelect™−rRNA HMR kit (Qiagen, MD, USA) according to the manufacturer’s protocol. RNA sequencing libraries were constructed with the NEBNext Ultra II RNA Library Preparation Kit for Illumina by following the manufacturer’s recommendations. Briefly, enriched RNAs are fragmented for 15 min at 94 °C. First strand and second strand cDNA are subsequently synthesized. cDNA fragments are end repaired and adenylated at 3’ends, and universal adapters are ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, CA, USA), and quantified using Qubit 2.0 Fluorometer (ThermoFisher Scientific, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were multiplexed and clustered on 1 lane of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq 4000 instrument according to manufacturer’s instructions. The samples were sequenced using a 2x150 Pair-End (PE) configuration.

first: Megan

last: Evans

first: Kenneth

last: Walsh


title: FN1 Shapes the Behavior of Papillary Thyroid Carcinoma through Alternative Splicing of EDB Region

city: Changsha

first: Mian

last: Liu

address: Road Xiangya 87

institute: Xiangya hospital, Central South University

summary: Papillary thyroid cancer (PTC) is often characterized by the indolent behavior with slow cell proliferation, however, small tumors still have a tendency to metastasize to the cervical lymph node, and the molecular mechanisms underlying that remain poorly understood. FN1 is an extracellular matrix (ECM) protein with the controversial role in tumors, containing three alternative splicing domains. In this study, FN1 was as the hottest gene of PTC and distinctive expression in PTC cells. The alternatively splicing EDB region of FN1 was exclusively expressed in tumors, which impacted on integrin β1 (ITGB1) bonding FN1 and it secretion process, resulting in completely distinct roles of two isoforms of FN1 including and skipping EDB domain. EDB(-)FN1 intracellularly inhibited tumor proliferation, whereas extracellular EDB(+)FN1 activated migration, invasion and lymphoangiogenesis in vitro and in vivo. Mechanistically, EDB(-)FN1 upregulated p21 in p53 signaling pathway, however, EDB(+)FN1 secretes into ECM promoted VEGFC expression. Collectively, FN1 is a potential determinant behind the characteristic behavior of PTC with its two isoforms playing distinct roles owing to the alternative splicing of EDB domain.

overall_design: To investigate the function of FN1 different isoforms related to EDB domain in PTC, we established K1 cell lines in which FN1 has been knocked down by shRNA, and selected knockdown monoclonal cell, then, cultured EDB(+)FN1 and EDB(-)FN1 overexpression cells by lentivirus infection.

first: Shi

last: Chang


title: A mRNA vaccine induces antimycobacterial immunity by activating DNA damage repair and autophagy

first: Dan

last: Chen

address: 2005, Songhu Road

department: Department of Microbiology

institute: Fudan University

summary: Effective vaccines are urgently needed for the control of tuberculosis (TB). Here, we report that a mRNA TB vaccine is highly effective and exhibits both prophylactic and therapeutic activities in the zebrafish model of TB.  Adult zebrafish immunized with the mRNA vaccine survived significantly longer than the DNA vaccine after Mycobacterium marinum challenge, and post-infection treatment with the mRNA vaccine drastically reduced the bacterial burden. The mRNA vaccine activated multiple DNA break repair systems that are essential for the normal development and function of adaptive immunity, but not the canonical DNA damage responses that promote cell death, demonstrating a profound connection between DNA damage repair and the activation of immune responses under physiological processes of immunization. Remarkably, the mRNA vaccine induced autophagy in granulomas, coinciding with bacterial killing and cell survival.  Collectively, these findings demonstrate that the mRNA vaccine elicits potent innate and adaptive immunity, conferring effective host protection against mycobacterial challenge.

overall_design: To understand the immune therapeutic mechanisms mediated by the L3T vaccine, we performed RNA-seq analysis of kidneys isolated from Mycobacterium marinum infected zebrafish treatment with theL3T mRNA vaccine and the LPP control, PBS control,  and untreatment control (infection 14 days), as well as healthy control.  Adult zebrafish (AB strain, 3 to 4 months old) were infected with indicated M. marinum M strain for two weeks and were then injected twice with 3-day intervals (day 14 and 17) of the L3T mRNA (0.5μg) at the dorsal muscle using PV830 Pneumatic PicoPump microinjector, and negative control zebrafish were immunized with LPP-GFP mRNA or sterile PBS. After 7 days of  the final immunization, kidneys from 5 zebrafish were pooled as one sample and three samples (total 15 kidneys) collected for each group were subjected to RNA-seq analysis. We also collected the zebrafish kidneys infected with M. marinum M strain 14 days before treatment  to RNA-seq as mock (named as M.m infection group) and healthy kidneys as blank control (named as Blank group), Comparative gene expression profiling analysis of RNA-seq data for L3T mRNA immunized group and its vector control immunized group (LPP-GFP mRNA immunized group) or PBS immunized group.

first: Weili

last: Huang

first: Bo

last: Yan

first: Jun

first: Lu


title: An mRNA vaccine induces antimycobacterial immunity by activating DNA damage repair and autophagy

overall_design: To understand the immune protective mechanisms mediated by the L3T vaccine, we performed RNA-seq analysis of kidneys isolated from zebrafish immunized with the mRNA vaccine and the LPP control.  For mRNA vaccination, mRNA candidate vaccines were injected with 1μg LPP-mRNA in the dorsal muscle using PV830 Pneumatic PicoPump microinjector (World Precision Instruments, Sarasota, FL), negative control zebrafish were immunized with LPP-GFP mRNA, immunized twice with two-week intervals (week 0 and 2).After 12 days of  the final immunization, kidneys from 5 zebrafish were pooled as one sample and three samples (total 15 kidneys) collected for each group were subjected to RNA-seq analysis. Comparative gene expression profiling analysis of RNA-seq data for L3T mRNA immunized group and its vector control immunized group (LPP-GFP mRNA immunized group).


title: KDM6A facilitates Xist upregulation at the onset of X inactivation [RNA-Seq]

city: Riverside

first: Wenxiu

last: Ma

address: 900 University Ave

institute: University of California Riverside

summary: X chromosome inactivation (XCI) is a female-specific process in which one X chromosome is silenced to balance X-linked gene expression between the sexes. XCI is initiated in early development by upregulation of the lncRNA Xist on the future inactive X (Xi). A subset of X-linked genes escape silencing and thus have higher expression in females, suggesting female-specific functions. One of these genes is the highly conserved gene Kdm6a, which encodes a histone demethylase that removes methyl groups at H3K27 to facilitate gene expression. Here, we investigate the role of KDM6A in the regulation of Xist. We observed impaired upregulation of Xist during early stages of differentiation in hybrid mouse ES cells following CRISPR/Cas9 knockout of Kdm6a. This is associated with reduced Xist RNA coating of the Xi, suggesting diminished XCI potency. Indeed, Kdm6a knockout results in aberrant overexpression of genes from the Xi after differentiation. KDM6A binds to the Xist promoter and knockout cells show an increase in H3K27me3 at Xist. These results indicate that KDM6A plays a role in the initiation of XCI through histone demethylase-dependent activation of Xist during early differentiation.

overall_design:
Here, we investigated the role of the histone demethlyase KDM6A in the onset of X chromosome inactivation by establishing female mouse stem cells derived from inter-species crosses with stable knockout of KDM6A via CRISPR/Cas9.
We then perfromed gene expression profiling followed by allelic analysis using RNA-seq at different timepoints during differentiation. Further, we characterized changes in H3K27me3 and KDM6A enrichment across the genome using CUT&RUN by comparing wild-type and knockout cells.

first: Josephine

last: Lin

first: Jinli

first: Li

first: He

last: Fang

first: Rui

first: Camille

last: Groneck

first: Galina N

last: Filippova

first: Xinxian

last: Deng

first: Christine M

last: Disteche

first: Joel B

last: Berletch


title: CCL7 promotes macrophage polarization and synovitis to exacerbate rheumatoid arthritis

summary: Macrophage polarization and synovitis play significant roles in rheumatoid arthritis (RA). Chemokines are involved almost throughout the entire pathology of synovitis. However, the interaction between chemokines and macrophage polarization in RA has been seldom reported. This study found that chemokine C-C motif ligand 7 (CCL7) secreted by polarized M1 macrophages enhances M1 polarization, creating a positive feedback loop and revealing the role of CCL7 in the progression of rheumatoid arthritis.  In this research, RNA sequencing indicated that M1 polarized macrophages secrete a large amount of CCL7, which further enhances M1 polarization.  Through immunohistochemistry and enzyme-linked immunosorbent assay, it was found that the expression of CCL7 in the synovial tissue and serum of RA patients and mice was upregulated. Intra-articular injection of CCL7 recombinant protein exacerbated M1 polarization of macrophages, inflammation, fibrosis in the synovial tissue, and worsened arthritis pain in mice, which was improved after the injection of CCL7 neutralizing antibody. In addition, CCL7 promoted M1 polarization of macrophages and reversed the M2 polarization induced by IL-4, and it also promoted macrophage proliferation and migration. Although it had an inhibitory effect on chondrocyte activity, it did not show a significant impact on chondrocyte metabolism. Mechanistically, CCL7 targets CCR1 to promote M1 polarization of macrophages, a process partly mediated through the activation of the JAK2/STAT1 pathway. When stimulated by CCL7, macrophages secreted a vast amount of pro-inflammatory factors, exacerbating synovitis and cartilage damage, leading to the aggravation of RA disease. Our research established a positive feedback loop between M1 polarization of macrophages and CCL7, indicating that blocking CCL7 could improve the progression of RA, suggesting CCL7 as a potential target for RA treatment.

overall_design: To study the relationship between CCL7 and macrophage polarization, and to explore the effect and mechanism of CCL7 on rheumatoid arthritis. To explore potential therapeutic targets for rheumatoid arthritis.

first: Xiaojia

last: Li

first: Wenquan

last: Liang

first: Tianyu


title: Integrated Multi-Omics Analysis Reveals Epigenetic and Sex-Specific Alterations in Colorectal Cancer Cachexia

city: Fayetteville

first: Nicholas

middle: P

last: Greene

state: Arkansas

address: 155 Stadium Dr. HPER Building 321

institute: University of Arkansas

summary: Cancer cachexia (CC) is experienced by 80% of cancer patients, representing up to 40% of cancer-related deaths. Evidence suggests biological sex dimorphism associated with CC. The initial triggering factors of muscle wasting, biological sex differences, and the epigenetic regulation during the onset and progression of cancer cachexia are not fully understood. The purpose of this study was to define the time course of C26-induced CC in males and females using transcriptomics, compare the transcriptomic changes with a more sever APC+/Min genetic model and identify the impact of epigenetic regulations associated to CC.

overall_design: At week-old Balb/c male and female mice (n=6-8/group) receivd 1x10^6 C26 cells injected bilateral, and tumors were developed for 10-, 20-, and 25-days, at endpoints, Tibialis Anterior (TA) muscle were collected.  The control mice received PBS injections  and TAs were colllected 25-days after PBS injections (controls). TA was also collected from 20-weeks-old APC+/Min mice (C57 black mice background) and their wild types (controls). RNA was extracted for RNA sequencing.

first: Kevin

middle: A

last: Murach

first: Ana

middle: R

last: Cabrera


title: Topologically associating domains (TADs) are insufficient for the transcription of β-globin gene and sub-TADs are required for this process

first: Jin

last: Kang

address: 2, Busandaehak-ro 63beon-gil, Geumjeong-gu

department: Department of Molecular Biology

institute: Pusan National University

summary: We found that TAD and sub-TAD contribute to enhancer-promoter interaction for gene transcription.

overall_design: H3K27me3 and H3K27ac ChIP-seq was performed in control and CTCF motifs-deleted (C3,7; dTAD or C4,5; dSub-TAD) MEL/ch11 cells to disrupt TAD or sub-TAD around the human β-globin locus.

first: AeRi

last: Kim


title: KDM6A facilitates Xist upregulation at the onset of X inactivation [CUT&Run]

first: Wenxiu

overall_design: Here, we investigated the role of the histone demethlyase KDM6A in the onset of X chromosome inactivation by establishing female mouse stem cells derived from inter-species crosses with stable knockout of KDM6A via CRISPR/Cas9.

first: Josephine

first: Jinli

last: Zhang

first: Chizuru

last: Kinoshita

first: Jessica E

last: Young


title: Fexuprazan safeguards the esophagus from hydrochloric acid-induced damage by suppressing NLRP1/Caspase-1/GSDMD pyroptotic pathway

city: Seoul

first: Seo Yeon

address: 50, Yonsei-ro, Seodaemun-gu, Seoul, Republic of Korea

institute: Yonsei universtiy

summary:
In this study, we aimed to investigate whether all available P-CABs exhibit anti-inflammatory effects in gastroesophageal reflux-induced esophagitis and to elucidate the underlying mechanisms.
Het-1A cells, normal esophageal epithelial cells, were treated with HCl (pH 4) for 30 min. Esomeprazole, a representative PPI, and three currently marketed P-CABs (vonoprazan, tegoprazan, and fexuprazan) were used for pretreatment. Total RNA sequencing was performed using Het-1A cells pretreated with 1% DMSO or fexuprazan, followed by exposure to HCl.

overall_design: Comprehensive RNA sequencing (RNA-seq) of cells pretreated with either 1% DMSO or fexuprazan followed by HCl treatment.

first: Seo-Yeon

first: Sang-Kil

last: Lee

first: Jung-Ho

last: Yoon

first: Da-Hyun

last: Jung

first: Ga-Hee

first: Chul-Hoon


title: CyroSCAPE: Scalable Immune Profiling Using Cryopreserved Whole Blood for Multi-omic Single Cell and Functional Assays

city: Seattle

first: Allen Institute

last: For Immunology

state: Washington

address: 615 Westlake Ave N, Seattle, WA

institute: Allen Institute

summary: The field of single cell technologies has rapidly advanced our comprehension of the human immune system, offering unprecedented insights into cellular heterogeneity and immune function. While cryopreserved peripheral blood mononuclear cell (PBMC) samples enable deep characterization of immune cells, challenges in clinical isolation and preservation limit their application in underserved communities. We present CryoSCAPE (Cryopreservation for Scalable Cellular And Proteomic Exploration), a scalable method for immune studies of human PBMC with multi-omic single cell assays using direct cryopreservation of whole blood. This method, optimized for scalability and cost-effectiveness, allows for high-throughput single cell sequencing and functional assays while addressing sample handling challenges in the clinic. Comparative analyses and functional assays of human PBMC from cryopreserved whole blood demonstrate the efficacy of this methodology in capturing cell proportions and molecular features, showcasing its potential to democratize access to single-cell assays and enhance our understanding of immune function across underserved populations.

overall_design: PBMCs collected from CyroSCAPE preservation method as described in publication, processed using 10x Genomics' FLEX kit.

first: Alexander

middle: T

last: Heubeck

first: Cole

last: Phalen

first: Neel

last: Kaul

first: Peter

last: Wittig

first: Palak

last: Genge

first: Jessica

last: Garber

first: Morgan

last: Weiss

first: Zach

last: Thompson

first: Claire

last: Gustafson

first: Julian

last: Reading

last: Skene


title: AMPK related of circRNA expression in C57 male mice myocardium

city: zunyi

first: Yang

last: Song

address: ddd

institute: dd

summary: To identify adenosine monophosphate-activated protein kinase (AMPK)-related circular RNA (circRNA) that was differentially expressed in the mouse myocardial ischaemia-reperfusion injury (MIRI) model, and map the AMPK-related circRNA network to provide novel insights for the prevention and treatment of MIRI.

overall_design: 32 male C57BL/6 mice were randomly divided into four groups: blank control (Sham) group (n=8), model (MIRI) group (n=8), (MIRI+AICAR) group (n=8), and (MIRI+Compound C) group (n=8). In addition to the Sham group, the other three groups constructed myocardial infarction models by ligating the left anterior descending branch of the mice. After successful modeling, the mice were returned to the thermostat box for 30 min and then the ligature wire was removed to achieve the purpose of myocardial reperfusion, and the mice were continued to be kept for 1 week for the ultrasound examination of the mouse heart, and the mouse heart tissues were obtained for pathological staining, ultramicrotome, serum myocardial markers, and RNA sequencing(RNA-seq), capture differentially expressed circRNA molecules, analyze the main biological processes involved in the differential genes by gene ontology (GO) function, Kyoto Encyclopedia of Genes and Genomes (KEGG) function, qRT-PCR validation of gene RNA-seq sequencing results, and construction of AMPK-related circRNA relationship network.

first: Yi

last: Zhao

first: XiaoDi

last: Zhang

first: DengShen


title: Systematic identification and characterization of high efficiency Cas9 guide RNAs for therapeutic targeting of ADAR

city: Dublin

first: Kory

last: Melton

state: CA

address: 6253 Dougherty Road

institute: Self

summary: Therapeutic targeting of the adenosine deaminase ADAR has great potential in cancer and other indications; however, it remains unclear what approach can enable effective and selective therapeutic inhibition. Herein, we conduct multi-staged guide RNA screening and identify high efficiency Cas9 guide RNAs to enable a CRISPR/Cas-based approach for ADAR knockout.  Through characterization in human primary immune cell systems we observe similar activity with two-part guide RNA and single guide RNA, dose responsive activity, similar guide activity rank order across different cell types, and favorable computational off-target profiles of candidate guide RNAs. We determine that knockout of ADAR using these guide RNAs induces pharmacodynamic responses primarily consisting of immunological responses such as a type I interferon response, consistent with the known function of ADAR as a key regulator of dsRNA sensing. We observe similar biological effects with targeting only the p150 isoform or both p110 and p150 isoforms of ADAR, indicating that at least in the contexts evaluated, loss of p150 ADAR mediates the primary response. These findings provide a resource of well-characterized, high efficiency ADAR-targeting Cas9 guide RNAs suitable for genomic medicines utilizing different delivery modalities and addressing different therapeutic areas.

overall_design:
To characterize the mechanism of action for gRNAs which knockout ADAR we editted primary human macrophages and evaluated the transcriptional responses via RNA-Seq.
Primary human CD14+ cells were treated with RNP with various guides for 24 hours. Then gene expression analysis was performed using data obtained from RNA-Seq of the cells after 48 hours of culture.

first: Benjamin

last: Gowen

first: Spencer

last: Wei

first: Weng In

last: Leong

first: Prachi

last: Khekare

first: Shannon

last: McCawley

first: Pierre

last: Boivin

first: Vihasi

last: Jani

first: Aaron J.

last: Cantor

first: Jean

last: Chan

first: Mary

last: Haak-Frendscho

last: Janatpour

first: Akshay

last: Tambe


title: PDHX Acetylation Facilitates Tumor Progression by Disrupting PDC  Assembly and Activating Lactylation Mediated Gene Expression

city: Hefei

first: Zetan

last: Jiang

address: huangshan Road

institute: UCTC

summary: Deactivation of the mitochondrial pyruvate dehydrogenase complex (PDC) is important for the metabolic switching of cancer cell from oxidative phosphorylation to aerobic glycolysis. Studies examining PDC activity regulation have mainly focused on the phosphorylation of pyruvate dehydrogenase (PDH, E1), leaving other post-translational modifications (PTMs) largely unexplored. Here, we demonstrate that the acetylation of Lys 488 of pyruvate dehydrogenase complex component X (PDHX) commonly occurs in hepatocellular carcinoma (HCC), disrupting PDC assembly and contributing to lactate-driven epigenetic control of gene expression. PDHX, an E3-binding protein (E3BP) in the PDC, is acetylated by the p300 at Lys 488, impeding the interaction between PDHX and dihydrolipoyl transacetylase (DLAT, E2), thereby disrupting PDC assembly to inhibit its activation. PDC disruption results in the conversion of most glucose to lactate, contributing to the aerobic glycolysis and H3K56 lactylation-mediated gene expression, facilitating tumor progression. These findings highlight a previously unrecognized role of PDHX acetylation in regulating PDC assembly and activity, linking PDHX Lys 488 acetylation and histone lactylation during HCC progression and providing a potential biomarker and therapeutic target for further development.

overall_design: RNA-seq analysis of HepG2 cells with endogenous PDHX knockdown further expressing PDHX-WT or PDHX-K488R.

first: Nanchi

last: Xiong

first: Weidong

last: Jia

first: Huafeng


title: The chromatin accessibility landscape of mouse oocytes during configuration transition [BS-seq]

city: Nanjing

first: Jiashuo

last: Li

address: 101Longmian Avenue, Jiangning District, Nanjing 211166, P.R. China

institute: Nanjing Medical University

type: Methylation profiling by high throughput sequencing

summary: The transition of chromatin configuration in mammalian oocytes from a non-surrounded nucleolus (NSN) to a surrounded nucleolus (SN) is critical for acquiring the developmental competence. However, the genomic and epigenomic features underlying this process remain poorly understood. In the present study, we first establish the chromatin accessibility landscape of mouse oocyte from NSN to SN stage. Through the integrative analysis of multi-omics, we find that establishment of DNA methylation in oocytes is independent on the dynamics of chromatin accessibility. In contrast, histone H3K4me3 status is closely associated with the dynamics of accessible-regions during configuration transition. Furthermore, by focusing on the actively transcribed genes in NSN and SN oocytes, we discover that chromatin accessibility coupled with histone methylation (H3K4me3 and H3K27me3) participates in the transcriptional control during phase transition. In sum, our data serve as a broad resource for probing configuration transition in oocytes, and provide the potential evidence to elucidate the mechanism determining chromatin dynamics and oocyte quality.

first: Shuai

last: Zhu

first: Qiang

last: Wang


title: The chromatin accessibility landscape of mouse oocytes during configuration transition (RNA-Seq)

overall_design: Transcriptome profiling by high throughput sequencing


title: The effect of piR_004153 on mesenchymal stromal cells

city: Warsaw

first: Agnieszka

last: Piekiełko-Witkowska

address: Marymoncka 99/103

department: Department of Biochemistry and Molecular Biology

country: Poland

institute: FCentre of Postgraduate Medical Education

summary:
Mesenchymal stromal cells (MSCs) are multipotent cells that reside in various tissues, including bone marrow, adipose tissue or dental pulp. They can differentiate into different cell lineages, including osteoblasts, chondrocytes, and adipocytes. MSCs are recruited and stimulate the progression of renal cancer. However, the exact mechanisms that govern MSCs homing to renal tumors are largely unknown. Here, we found that renal cancer cells secrete piR_004153 encapsulated in exosomes that are taken-up by MSCs to induce transcriptional reprograming, and stimulate their migration towards renal cancer cells.
To explore the impact of piRNA on MSCs, we transfected them with piR_004153 or non-targeting scrambled control oligonucleotide and analyzed their transcriptome using microarrays.

overall_design: MSCs derived from bone marrow were cultured with the following transfection of piR_004153 (n=5 samples) or negative control (n=5 samples). 48h after transfections cells were used for RNA isolation. Microarray expression analysis was done on RNA samples using the Affymetrix Gene GeneChip® Instrument System

first: Joanna

last: Bogusławska

first: Piotr

last: Popławski

first: Helena

last: Kossowska

first: Roksana

last: Iwanicka-Rokicka

first: Alex

last: Białas

first: Marta

last: Koblowska


title: The chromatin accessibility landscape of mouse oocytes during configuration transition (CUT&Tag)

overall_design: histone modification profile by high throughput sequencing


title: The chromatin accessibility landscape of mouse oocytes during configuration transition

overall_design: chromatin accessibility by high throughput sequencing


title: ARID1A IDR Targets EWS-FLI1 Condensates and Finetunes Productive Chromatin Remodeling

first: Bing

address: South Chongqing Road 280

department: Department of Biochemistry and Molecular Cell Biology

institute: Shanghai Jiao Tong University

summary: The chimeric transcription factor EWS-FLI1 undergoes liquid-liquid phase separation (LLPS) and binds to neomorphic microsatellite DNA regions, which in turn recruits cBAF to activate an oncogenic transcriptional program in Ewing sarcoma. To identify the specific subunit of cBAF involved in this interaction, we employed unbiased TurboID analysis, revealing ARID1A as a potential mediator. We discovered that an intrinsic disordered region of ARID1A, known as IDR1, undergoes homotypic phase separation and incorporates into EWS-FLI1 droplets. This finding suggests the direct involvement of IDR1 in recruiting cBAF to phase-separated EWS-FLI1. Additionally, we uncovered a surprising function of IDR1 in regulating the productive chromatin remodeling activity of cBAF. This novel discovery of ARID1A IDR1's dual role opens avenues for developing effective treatment strategies for relevant tumors.

overall_design: To investigate the role of ARID1A IDR1b in EWS-FLI1 mediated gene transcriptional regulation, we overexpressed EWS-FLI1 and ARID1A WT or ΔIDR1b in ARID1A-knockout 293T (named 12KO) cell line.

first: Jingdong

last: Xue

first: Siang

last: Lv

first: Jun

last: Wu


title: Gene expression in Pan02 tumors from mice that were either untreated, treated with a TGFb vaccine, with an anti-IL-6R blocking antibody or the combination of the TGFb vaccine and an anti-IL-6R blocking antibody

city: Odense

first: Lars

last: Grøntved

address: Campusvej 55

department: Biochemistry and Molecular Biology

country: Denmark

institute: University of Southern Denmark

summary: To investigate changes in the tumor microenvironment at a gene expression level induced by IL-6R blockade and compare it to treatment with the TGFb vaccine in a murine model of PDAC (Pan02)

overall_design: Pan02 tumor-bearing mice were either left untreated or vaccinated with TGFβ vaccine on days 8 and 15 post-inoculation. Anti-IL-6R antibody was administered on day 8 post-inoculation and every 3rd-4th day for a total of six injections. Tumors were harvested (n=3-4 per group) on day 24 post-inoculation, RNA was extracted, and bulk RNAseq was performed.

first: Maria

last: Perez-Penco

first: Mads

last: Hald Andersen


title: A microRNA cluster downstream of Fezf2 coordinates projection identity and dendritic morphology in cortical projection neurons

city: Stanford

first: Suzanne

last: Tharin

state: California

address: 450 Jane Stanford Way

institute: Stanford

summary: Cortical projection neurons comprise classes of neurons connecting the cerebral cortex to distant targets in the nervous system. Each projection neuron class acquires a distinct molecular identity and develops characteristic patterns of axonal projection and dendritic arborization that determine their inputs, outputs, and distinct functions. How different aspects of neuron identity, including axonal and dendritic morphology, are coordinated appropriately in each specific neuron type is not known. A network of transcription factors, including the selector gene Fezf2, is central to specifying cortical projection neuron fates. However, regulation down-stream of these fate-determinant transcription factors to control different aspects of neuron identity is not understood, particularly as it relates to the development of distinct dendritic arbors that determine the inputs to each projection neuron class. Here we show that the miR-193b~365 microRNA cluster downstream of Fezf2 cooperatively represses the signaling molecule Mapk8 to regulate dendritic development in a neuron subtype-specific manner.

overall_design: Corticospinal motor neuron (CSMN) and callosal projection neurons (CPN) samples were isolated by FACS, then suspended in a transposition mix to generate DNA fragments. The fragments generated in the CSMN and CPN samples were dependent on the chromatin state of the nuclei. The DNA fragments were amplified using indexing primers, and purified to give the final ATAC-seq library.

first: Tharin

last: Suzanne

first: Asha

last: Iyer

first: Lee

last: Vaasjo

first: Verl

last: Siththanandan

first: Rajan

last: KC

first: Abbigail

last: Thurmon

first: Mauren

last: Akumuo

first: Victoria

last: Lu

first: Ramesh

last: Nair

last: Galazo


title: RNA-seq profiling identifies lncRNAs in serum as potential biomarker for muscle-invasive bladder cancer

city: Jinan

first: Xiumei

last: Jiang

state: Shandong

address: Wenhua Xi Road

institute: Department of Clinical Laboratory, Qilu Hospital, Shandong University

summary: To investigate the expression profiles of lncRNAs in muscle-invasive bladder cancer .

overall_design: We performed gene expression profiling analysis using data obtained from RNA-seq of muscle-invasive bladder cancer patients and nonmuscle-invasive bladder cancer patients.


title: Activated PRDM1-CREBBP Axis Contributes to Apoptosis and Impaired Invasion of Trophoblasts: A Novel Mechanism Underlying Preeclampsia

city: Shenyang

first: XUAN

last: ZHANG

state: Liaoning

address: Shenyang 110004, People's Republic of China.

institute: Shengjing Hospital of China Medical University

summary: To analyze the gene expression patterns in placentas collected from four PE patients and six healthy individuals.


title: RNA-sequencing analysis gene expression changes of astrocytes treated with TGF- β1 in response to oxygen-glucose deprivation/reperfusion

city: shenzhen

first: Qiang

last: Zhang

state: Guangdong

address: 628 Zhenyuaan Rd.

department: Emergency medicine

institute: The seventh Affiliated Hospital of Sun Yat sen University

summary: Astrocytes are abundant glial cells in the central nervous system (CNS) that play important roles in cerebral ischemia-reperfusion injury. Following brain ischemia, astrocytes can trigger endogenous neuroprotective mechanisms such as neurogenesis, regulation of inflammation, transfer of mitochondria, and defense against oxidative stress. Transforming growth factor beta 1 (TGF-β1) is known as an injury-related cytokine, particularly associated with neurogenesis, neuronal migration, inflammatory reactions, and astrocyte scar formation in response to brain injury. TGF-β1 is closely related to ischemia-reperfusion brain injury and plays a significant role as both effectors and targets of I/R brain injury. Upregulation of endogenous TGF-β1 in neurons may contribute to preventing apoptosis after ischemic insult. TGF-β1 exerts dynamic effects in tissues through autocrine and paracrine signaling pathways as a secretory factor. The current study suggests that the multiple functions exerted by astrocytes might potentially be mediated by TGF-β1 signaling, raising the idea that astrocytes could be a potential therapeutic target for neuroprotection as sources or targets of TGF-β1. However, few studies are currently available on the effects of TGF-β1 on astrocytes after ischemia-reperfusion brain injury. Although current research shows that transforming growth factor-beta acts as a neuroprotective agent in cerebral ischemia, its specific mechanism is still not completely clear. In this study, RNA sequencing analysis was performed to investigate the potential mechanism of astrocytes pretreated with TGF-β1. Our study found that TGF-β1 mediates the upregulation of DUSP4 in astrocytes, which plays a neuroprotective role after ischemia-reperfusion injury. Overexpression of TGF-β1 inhibits the activation of astrocytes, accompanied by decreased levels of inflammatory factors and reactive oxygen species (ROS), while promoting the transfer of mitochondria between astrocytes and neurons. This enhanced neuron survival and axonal regeneration after injury. Hence, this study provides further insights into strategies for inhibiting neurological impairment and suggests a potential therapeutic target after ischemia-reperfusion injury.

overall_design: Astrocytes were used as the research subjects. Comparative gene expression profiling analysis of RNA-seq data was performed on astrocytes after oxygen-glucose deprivation/reperfusion (OGD/R) treatment and astrocytes treated with TGF-β1 after OGD/R. Each group consisted of three duplicate samples. The divided experimental groups was as follows: OGD/R rep1 ,OGD/R rep2,OGD/R rep3, OGD/R + TGF-β1  rep1, OGD/R + TGF-β1 rep2,OGD/R + TGF-β1  rep3.


title: CPL mitigates myeloproliferative neoplasms through dual targeting of cell senescence and NF-κB signaling

first: Liu

middle: Ming

last: Jie

address: 44 wenhua west road

institute: Shandong University

summary: To investigate the therapeutic effect of CPL on myeloproliferative neoplasms. We then performed gene expression profiling using data obtained by RNA-seq in SET-2 cells treated with or without CPL.

overall_design: Comparative gene expression profiling of RNA-seq data from SET-2 cells treated and untreated with CPL

first: Mingjie

last: Liu

first: Baobing

last: Zhao


title: Characterization of the gene regulatory cascade downstream of master-regulator STZ

first: Souvik

last: Dhar

state: Gwanak gu

address: 1, Gwanak ro

institute: Seoul National University

summary: STZ acts as a brige between PEP1-induced transcription factor and the PEP1-suppressed cell-cycle genes. In the conditional STZ overexpression seedlings (p35S::XVE>>pLexA::STZ), we observed an early differentiation of tissues. To identify the genetic cascade regulated by STZ, we prepare samples at 6h and 24h-post estradiol induction. The total RNA ws extracted and bulk RNA sequiencing was performed.

overall_design: We took 4-5 days old Arabidopsis seedling and treated them with 20 µM estradiol for 6h and 24h. Following STZ-induction, we dissected the apical 2/3 portion of the root and the RNA was extrcted using RNeasy Plant Mini kit (Qiagen). The purification of the samples and sequencing was done by Macrogen (Korea).

first: Ji-Young

last: Lee


title: Characterization of PEP1-influenced transcriptional reprogramming at the Arabidopsis root meristem

summary: The apical root growth is immensely affected by pathogen-associated molecular patterns (PAMPs) or plant-derived damage-associated molecular pattern (DAMPs). Upon perceiving the danger signals, elevated defense machinery often compromises root apical growth, resulting in the meristem's aberrant proliferative and formative divisions. In our previous study, we reported that one of the DAMP signals, PEP1, reprograms developmental pathways in the root apical meristem resulting in the reduction of stele cell numbers, alteration of the xylem vessel organizations, and disruption of cell-to-cell symplastic connections. However, a comprehensive atlas of the gene regulatory networks associated with the root growth-defense tradeoffs is yet to be elucidated. To gain an insight into the dynamic molecular framework that modulates root development under elevated pathogenic signals, we performed a time course RNA-sequencing analysis in the root meristem after PEP1 treatment. Based on expression dynamics of the DEGs (fold change ≥ 1.5; p < 0.05), 12 clusters were obtained following K-means algorithm. Intriguingly, among the PEP1-induced gene clusters, we observed that a group of zinc finger proteins, comprising one of the most prominent transcription factor families, are highly enriched. On the contrary, the PEP1-repressed gene clusters included various developmental regulators expressed in the vascular tissues. Indeed, an in vitro network analysis using selected PEP1-induced and repressed clusters indicated STZ as a potential key mediator of the defense-induced developmental reprogramming pathway in the root stele. We also revealed the involvement of two related homologs of STZ, ZAT6, and AZF3, in this trade-off pathway. Our study provides a comprehensive overview of transcription factors that respond to the early hours of PEP1 induction and hijack cellular machinery, thereby contributing to cellular reorganization and growth in the root apical meristem.

overall_design: We took 4-5 days old Arabidopsis seedling and treated them with 1µM PEP1 for 3h, 6h, 12h, and 24h. Following PEP1 treatment we dissected the root meristem from each mock or PEP1 treated samples. The RNA was isolated using RNeasy Micro kit (Qiagen). The purification of the samples and sequencing was done by Macrogen (Korea).


title: Transcriptomic effects of alginate hydrogel applied to the production of bovine embryos: Different uses during in vitro culture

city: Québec

first: Ricardo

middle: Perecin

last: Nociti

state: Québec

address: 2425, Rue de l'Agriculture

department: Animal Science

institute: Université Laval

summary: In vitro produced blastocysts are exposed to different stimuli when compared with in vivo ones. This includes culture of in vitro embryos in a sturdy petri-dish, while in vivo embyos develop in a soft and dynamic structure. Here we hypothesized that a softer environment could differently modulate the in vitro produced early embryo. To that aim, prezumptive zygotes were produced by in vitro fertilization and divided into three groups: 1) Cultured in a regular petri dish - Control (CON); 2) Cultured on top of an alginate hydrogel surface (TOP); 3) Encapsulated inside an alginate hydrogel sphere (ENC) and cultured. We observed a decrease in blastocyst rate in TOP and ENC compared with the CON. Profiling of 383 bovine miRNAs, we found 3 miRNAs involved in cell proliferation being differently modulated by the TOP and ENC groups (miR-1246; miR-1260b, and miR-541). Analyzing global levels of DNA methylation and hydroxymethylation, we observed increased levels of the two marks in the TOP group when compared with the CON and ENC systems. RNA sequencing (RNA-seq) analysis carried out using blastocysts showed alterations in several developmentally important genes among the three groups. In summary, these results indicate that alginate hydrogels can support embryo culture, even though they negatively impact development into blastocyst, which is paralleled by epigenetic and transcriptomic changes.

overall_design: 1) Cultured in a regular petri dish - Control (CON-2D); 2) Cultured on top of an alginate hydrogel surface (TOP-2.5D); 3) Encapsulated inside an alginate hydrogel sphere (ENC-3D) and cultured.

first: Giuliana

last: de A Ferronato

first: Juliano

middle: C

last: da Silveira


title: Cancer-associated Fibroblast Induces Acinar-to-ductal Cell Transdifferentiation and Pancreatic Cancer Initiation via LAMA5/ITGA4 axis

city: Omaha

first: Moorthy Palanimuthu

last: Ponnusamy

state: Nebraska

address: 42nd and Emile

institute: University of Nebraska Medical Center

summary: Pancreatic ductal adenocarcinoma (PDAC) is characterized by desmoplastic stroma surrounding most tumors. Activated stromal fibroblasts, namely Cancer-associated fibroblasts (CAFs), play a major role in PDAC progression. Here we analyzed if CAFs influence acinar cells and impact PDAC initiation, namely “Acinar to Ductal Metaplasia” (ADM). ADM connection with PDAC pathophysiology is indicated but not yet established. Hence, we hypothesized that CAF secretome might play a significant role in ADM in PDAC initiation. Mouse and human acinar cell organoids, acinar cells cocultured with CAFs and exposed to CAF-conditioned media (CAF-CM), acinar cell explants, and CAF cocultures, etc., were examined by qRT-PCR, RNA-seq, immunoblotting and confocal microscopy. Data from LC-MS/MS analysis of CAF CM and RNAseq data of acinar cells post-CM exposure were integrated using bioinformatics tools to identify molecular mechanism for CAF-induced ADM. Using confocal microscopy, immunoblotting, and qRT-PCR analysis, we validated the depletion of key signaling axis in cell-line, acinar explant coculture, and mCAFs. Close association of acino-ductal (UEA1, Amylase, Ck19) markers and mCAFs (α-SMA) in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1Cre (KPC) and LSL-KrasG12D/+; Pdx1Cre (KC) autochthonous progression tumor tissue was observed. Caerulein treatment-induced mCAFs increased Ck19 and decreased Amylase in wild-type (WT) and KC pancreas. Likewise, acinar-mCAF cocultures revealed induction of ductal transdifferentiation in cell-line, acinar-organoid, and explant coculture formats in WT and KC mice pancreas. Proteomic and transcriptomic data integration revealed a novel Laminin5/Integrinα4/Stat3 axis responsible for CAF-mediated acinar to ductal cell transdifferentiation. Results collectively suggest the first evidence for CAF-influenced acino-ductal phenotypic switchover, thus highlighting the role of the tumor micro-environment in the inception of pancreatic carcinogenesis.

first: Seema

last: Parte

first: Annant

middle: B

last: Kaur

first: Rama

middle: K

last: Nimmakayala

first: Ayoola

middle: O

last: Ogunleye

first: Ramakanth

last: Chirravuri

first: Raghupathy

last: Vengoji

first: Frank

last: Leon

first: Palanisamy

last: Nallasamy

first: Sanchita

last: Rauth

first: Zahraa

last: Alsafwani

first: Subodh

last: Lele

first: Jesse

last: Cox

first: Ishfaq

last: Bhat

first: Shailender

last: Singh

first: Surinder

last: Batra

first: Moorthy


title: Unsupervised Clustering Reveals Noncanonical Myeloid Cell Subsets in the Brain Tumor Microenvironment

city: Tampere

first: Reetta

last: Nätkin

address: Arvo Ylpön katu 34

department: Faculty of Medicine and Health Technology, Prostate Cancer Research Center

country: Finland

institute: Tampere University and Tays Cancer Center

summary: The tumor immune microenvironment (TiME) of human central nervous system (CNS) tumors remains to be comprehensively deciphered. Here, we employed flow cytometry and RNA sequencing analysis via data-driven approaches to dissect a diverse TiME and uncover noncanonical immune cell types in human CNS tumors. Myeloid subsets comprised classical microglia, monocyte-derived macrophages, neutrophils, and two noncanonical myeloid subsets: CD3+ myeloids and CD19+ myeloids. T lymphocyte subsets included double-negative (CD4- CD8-) T cells (DNTs). Noncanonical myeloids and DNTs were validated with an independent dataset, suggesting that our DNT phenotype represents γδ T cells. While the proportions of classical myeloids agreed with reported malignancy type-associated TiMEs, unexpectedly high lymphocyte frequencies were detected in gliosarcoma, which also showed a unique expression pattern of immune-related genes. Our findings highlight the potential of data-driven approaches in resolving CNS TiME and reveal the mosaic of immune cell types constituting TiME.

overall_design: To compare the TiME phenotypes characterised by flow cytometry (FCM) and to computationally asses the TiME at transcriptome level, bulk RNA sequencing was performed from the FCM cohort samples for which tumor material was available.

first: Ismaïl

last: Hermelo

first: Tuomo

last: Virtanen

first: Iida

last: Salonen

first: Sofia

last: Randelin

first: Aliisa

last: Tiihonen

first: Suvi

last: Lehtipuro

first: Laura

last: Kummola

first: Kristiina

last: Nordfors

first: Hannu

last: Haapasalo

first: Miina

last: Rauhala

first: Juha

last: Kesseli

first: Matti

last: Nykter

first: Joonas

first: Kirsi

last: Rautajoki


title: Maternal Malnutrition in Mice Impairs Nephrogenesis by Disrupting DNA Methylation of Regulatory Regions

city: Jerusalem

first: Yaniv

last: Makayes

address: Kalman Ya'Akov Man Street

country: Israel

institute: Hadassah Hebrew University Medical Center

summary: Maternal caloric restriction during pregnancy significantly impacts kidney development, influencing susceptibility to chronic kidney disease in adulthood. This study explores DNA methylation changes in nephron progenitor cells resulting from caloric restriction and their implications for kidney health. Global DNA hypomethylation is observed in nephron progenitors from caloric-restricted embryos, with specific genomic regions displaying distinct methylation patterns, including hypomethylation and hypermethylation. Differentially methylated regions exhibit enhanced chromatin accessibility, indicating biological relevance. Hypomethylated regions are enriched for genes associated with developmental processes, reflecting changes in gene expression and highlighting their functional relevance in kidney development. The study also reveals that supplementing methionine, an essential amino acid, restores disrupted DNA methylation patterns, particularly in enhancer regions, emphasizing methionine's critical role in regulating nephron progenitor cell epigenetics and ensuring proper kidney development. The intricate relationship between maternal nutrition, dynamic DNA methylation, and kidney development is highlighted, emphasizing the enduring impact of early-life nutritional challenges on kidney function. This research elucidates epigenetic mechanisms as mediators for the lasting effects of maternal caloric restriction on kidney health. The study contributes valuable insights into the origins of chronic kidney diseases during early developmental stages, offering potential interventions to mitigate adverse outcomes.

overall_design: Methylation profiling by high throughput DNA sequencing from nephron progenitor cells was acquired from 2 experimental groups each containing 4 samples. Pregnant females were randomly assigned to either a caloric restriction diet (CRD) group or a control, unrestricted diet (URD) group for the duration of the experiment. On embryonic day seventeen (E17.5), the embryonic kidneys were taken, the NPCs were sorted, and the DNA was extracted. Bisulfite conversion of genomic DNA and reduced representation bisulfite sequencing were performed.

first: Makayes

last: Yaniv

first: Abergel

last: Eden

first: Amleh

last: Athar

first: Fok

last: Rimma

first: Azria

last: Batia

first: Ansari

last: Ihab

first: Bergman

last: Yehudit

first: Nechama

last: Morris

first: Volovelsky

last: Oded


title: Renal cancer cells secrete exosomal ncRNAs that affect tumor microenvironment

first: Agnieszka

last: Piekiełko-Witkowska

institute: Centre of Postgraduate Medical Education

summary: Cancer cells secrete multiple molecules that affect tumor microenviroment. In our previous study we analyzed the secretory proteome and metabolome of renal cancer cells (PMID: 36604669). Small non-coding RNAs (sncRNAs) encompass a variable group of oligonucleotides, including miRNAs, piRNAs, and tRNA-derived fragment that can affect the expression of target genes. Here, we aimed to analyzed sncRNAs that are secreted by renal cancer cells and can affect tumor microenvironment. To this end, we: i) collected the conditioned media (CM) from two renal cancer-derived cell lines, Caki-1 and KIJ265T, as well as RPTEC, the control non-tumorous kidney cell line derived from proximal tubules; ii) used the CM for isolation of exosomal total RNA; iii) performed RNAseq to target and analyze the profiles of sncRNAs. We identified multiple miRNAs, piRNAs and tRNAs that were differently secreted by renal cancer cells when compared with non-tumorous proximal tubules. The analysis was followed by qPCR validation and functional analysis to reveal sncRNAs that affect tumor microenvironment.

overall_design:
Caki-1 and RPTEC/TERT1 cells were cultured in accordance with manufacturer’s protocol (ATCC). KIJ265T were cultured as previously described (Boguslawska J, Piekielko-Witkowska A, Wojcicka A, Kedzierska H, Poplawski P, Nauman A. Regulatory feedback loop between T3 and microRNAs in renal cancer. Mol Cell Endocrinol. 2014;384(1–2):61–70). For the collection of conditioned media (CM), 10^6 cells were seeded at 75cm2 flasks, cultured for 24h, rinsed once with PBS and four times with DMEM (low glucose, no glutamine, no phenol red) (Gibco/Thermo Fisher Scientific, Paisley, UK) with GLUTAMAX (Gibco/Thermo Fisher Scientific, Paisley, UK). Following the addition of 15ml of DMEM (no phenol red) supplemented with GLUTAMAX, the cells were cultured for another 24h. CM were collected (three independently cultured batches per each cell line), centrifuged, aliquoted, and stored at -80oC.
Exosomes were purified from CM, with the following isolation of total exosomal RNA.
RNAseq was performed using Illumina NextSeq 500 sequencing platform.

first: Piotr

last: Popławski

first: Joanna

last: Bogusławska

first: Małgorzata

last: Grzanka

first: Karolina

last: Hanusek

first: Alex

last: Białas

first: Beata

last: Rybicka

first: Anna

last: Adamiok-Ostrowska

last: Życka-Krzesińska

first: Helena

last: Kossowska

first: Marta

last: Koblowska


title: Hypoxia drives the formation of lung micropapillary adenocarcinoma through hypoxia-inducible factor-1a (RNA-seq)

city: Suita

first: Daisuke

last: Motooka

state: Osaka

address: 3-1, Yamadaoka

country: Japan

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

summary: Micropapillary adenocarcinoma (MPC) is an aggressive histologic subtype of lung adenocarcinoma (LUAD). MPC is composed of small clusters of cancer cells exhibiting inverted polarity. However, the mechanism underlying its formation is poorly understood. Here we show that hypoxia is involved in MPC formation. Spatial transcriptome analysis with human LUAD tissue suggested that hypoxia-inducible factor (HIF)-1 signaling is activated in MPC. Hypoxia induced the formation of MPC-like structures (MLSs) with inverted polarity in a three-dimensional culture system using A549 human LUAD cells, and HIF-1α was indispensable for MLS formation. RNA sequencing analysis demonstrated that A549 cells forming MLSs exhibited a gene expression signature similar to that of lung MPC. Moreover, MLS formation enhanced the resistance of A549 cells to natural killer cell cytotoxicity. Our findings suggest that hypoxia drives lung MPC formation through HIF-1α and that immune escape from natural killer cells might underlie the aggressiveness of MPC.

overall_design: To characterize the hypoxia-induced MLSs in detail, we performed RNA-seq analysis in A549 cells cultured in the usual 2D culture system or the 3D culture system under normoxia or hypoxia.

last: Umeda

first: Akikazu

last: Harada

first: Satoshi

last: Nojima

first: Eiichi

last: Morii


title: Histone H3 acetylation (pan-acetyl) of WT, Mpc2-deficient basal cells and acetate treated Mpc2-deficient basal cells

city: Shanghai

first: Yalin

last: He

address: No. 320, Yueyang Road, Xuhui District

institute: Center for Excellence in Molecular Cell Science

summary: To further access whether changes in histone acetylation are directly associated with cell differentiation, we performed CUT&Tag experiment on WT cells, Mpc2-deficient cells, and acetate treated Mpc2-deficient cells  by using antibody against H3 pan-acetylation. Mpc2 mutant cells displayed a decrease in the peaks of histone H3 acetylation near transcription start sites compared to control cells, whereas this reduction could be partially reversed by acetate supplementation. Pathway analysis using differentially acetylated genes between control and Mpc2 mutant cells revealed alterations in differentiation-related signaling pathways, such as TGF-β and Notch. Indeed, Mpc2 mutant BCs showed insufficient H3 acetylation at the promoters of key differentiation related genes, such as Hes1 and HeyL, downstream transcriptional factor of Notch signaling, and the decrease could be partially reversed by acetate treatment.

overall_design: Primary airway basal cells were isolated from WT and p63CreER; Mpc2f/f mice, followed by ALI culture. WT, Mpc2-deficient and acetate treated Mpc2-deficient cells were collected on day 3 of ALI culture for analysis.

first: Yawen

last: Li


title: Gene expression analysis during mouse airway basal cell differentiation

summary: To investigate the dynamic gene expression change of tracheal epithelial cells during differentiation, we performed RNA-seq analysis on cells from day 1, day 3, and day 5 of ALI culture, corresponding to the stages of basal cell proliferation, epithelial cell stratification, and early luminal cell specification in vivo. Gene expression and Go analysis revealed that metabolic pathways, such as ATP biosynthesis, cellular respiration, and TCA cycle activity, were highly activated in cells from day 3 of culture compared to those from day 1 of culture.Consistently, the expression of TCA cycle related genes was significantly upregulated during epithelium regeneration, such as Sdhd and Idh2, which encode key enzymes of tricarboxylic acid cycle (TCA cycle); Mpc2 and Pdgb, which function in the mitochondrial pyruvate oxidation process. The activity of these metabolic pathways was further elevated  in cells from day 5 of culture.

overall_design: For investigating the dynamic changes in gene expression during tracheal epithelial cell differentiation, we isolated primary airway basal stem cells and cultured them using ALI (air-liquid interface) culture. We collected cells at three time points during the ALI culture: day 1, day 3, and day 5, corresponding to the stages of basal cell proliferation, epithelial cell stratification, and luminal cell specification, respectively, mirroring the in vivo differentiation process.


title: IFN-III primes pDCs for TLR7 activation and antagonizes TGF-β mediated-immune suppression in tumors.

city: LYON cedex

first: Jenny

last: VALLADEAU-GUILEMOND

address: 28 rue Laennec

department: TERI

country: France

institute: CRCL

summary: Conventional dendritic cell (cDC1, cDC2) and plasmacytoid dendritic cell (pDCs) subsets have specialized functions that can be modulated by the tumor microenvironment (TME). These cells produce different interferons that are central to antitumor immune responses. While the role of Type I Interferons (IFN-I) in tumor immunity is well characterized, the role of Type III interferons (IFN-III) produced by cDC1 in the TME and associated with good outcome remains completely unknown. Here, we demonstrate that IFN-III orchestrates pDCs survival, activation and TLR7 expression in the blood and in tumors, enhancing their capacity to respond to TLR7-ligand. Importantly, IFN-III prevents the inhibition of pDCs induced by the TME or by TGF-β, a dominant TME inhibitory cue, and restores their production of IFN-⍺. Because of the essential role of TGF-β in immune regulation, this mechanism is expected to impact other pathological situations.

overall_design: Expression profiling by high throughput sequencing

first: Candice

last: Sakref

first: Alexis

last: Saby

first: Céline

last: Rodriguez

first: Lyvia

last: Moudombi

first: Elisa

last: Gobbini

first: Aurélien

last: Voissiere

first: Laurie

last: Besson

first: Maude

last: Ardin

first: Léo

last: Laoubi

first: Stéphane

last: Depil

first: Margaux

last: Hubert

first: Nathalie

last: Bendriss-Vermare

first: Christophe

last: Caux

last: Valladeau-Guilemond


title: miR-146b-5p downregulates IRAK1 and ADAM19 to suppress trophoblast proliferation, invasion and migration in miscarriage.

city: Nanning

first: lihong

last: pang

state: Guangxi

address: NO.6 Shuangyong Road

department: Prenatal Diagnosis and Genetic Diseases

institute: Guangxi Medical University

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

overall_design: Refer to individual Series


title: Gene expression analysis of basal cells expanded with UK-5099 or dual SMAD inhibitors

first: Yalin

last: He

summary: Dual SMAD inhibitors (TGF-beta inhibitor A-8301 and BMP inhibitor DMH-1) were previously reported to promote long-term expansion of basal cells. Since UK-5099 showed a comparable effect to dual SMAD inhibitors in promoting basal cells expansion, we performed RNA-seq  to determine if cells expanded by these two methods exhibit a similar transcriptional profile. The Pierson correlation test indicated that cells expanded by UK5099 displayed a transcriptional profile similar to those expanded with dual-SMAD inhibitors. The expression levels of canonical basal cell marker genes, such as Krt5 and p63, were comparable in cells expanded using both methods, as well as genes related to the TCA cycle and glycolysis. Interestingly, despite the similarity in the expression of canonical basal cell markers, cells expanded via UK-5099 exhibited higher activity in differentiation-related signaling pathways, such as the Hippo and TGF-β signaling pathways.This observation suggests that mitochondrial pyruvate transport is a critical metabolic checkpoint for basal cell differentiation.

overall_design: Primary mouse airway basal cells were isolated and then cultured with UK-5099 and dual SMAD inhibitors for 4 days

last: Li


title: Transcription Factor Condensates Mediate Clustering of MET Regulon and Enhancement in Gene Expression [MeDIP]

city: University Park

first: James

last: Lee

address: 406 South Frear Building

institute: Pennsylvania State University

summary: Some transcription factors (TFs) can form liquid-liquid phase separated (LLPS) condensates. However, the function of these TF condensates in 3D genome organization and gene regulation remains elusive. In response to methionine (met) starvation in budding yeast, Met4 and a few sequence-specific co-activators, including Met32, induce a set of genes involved in met biosynthesis. Here, we show that the endogenous Met4 and Met32 form puncta-like structures that significantly overlap in yeast nuclei upon met depletion. Recombinant Met4 and Met32 form mixed droplets with LLPS properties in vitro. In relation to chromatin, Met4 puncta co-localize with target genes, and at least a subset of these target genes are clustered in 3D in a Met4-dependent manner. A MET3pr-GFP reporter inserted near several native Met4 binding sites becomes co-localized with Met4 puncta and displays enhanced transcriptional activity. A Met4 variant with a partial truncation of an intrinsically disordered region (IDR) shows less puncta formation, and this mutant selectively reduces the reporter activity near Met4 binding sites to the basal level. Overall, these results support a model where Met4 and co-activators form condensates to bring multiple target genes into a vicinity with higher local TF concentrations, which facilitates a strong response to met depletion (-met).

overall_design: MeDip of MTAC strains with MET13, MET6 as VP in MET active (-met) and inactive (+met) conditions with +/- IAA for Met4 depletion.

first: Lu

last: Bai

first: Leman

last: Simpson

first: Yi


title: miR-146b-5p downregulates IRAK1 and ADAM19 to suppress trophoblast proliferation, invasion and migration in miscarriage [circRNA, lincRNA, and mRNA profiles]

summary: A large proportion of miscarriages are classified as unexplained miscarriages (UM) since no cause is identified. No reliable biomarkers or treatments are available for these pregnancy losses. While our transcriptomic sequencing has revealed substantial upregulation of miR-146b-5p in UM villous tissues, its role and associated molecular processes have yet to be fully characterized. Our work revealed that relative to samples from normal pregnancy (NP), miR-146b-5p was significantly elevated in villous tissues from UM patients and displayed promising diagnostic potential. Moreover, miR-146b-5p agomir contributed to higher rates of embryonic resorption in ICR mice. When overexpressed in HTR-8/SVneo cells, miR-146b-5p attenuated the proliferative, invasive, and migratory activity of these cells while suppressing the expression of MMP9 and immune inflammation-associated cytokines, including IL1B, IL11, CXCL1, CXCL8, and CXCL12. Conversely, inhibition of its expression enhanced proliferation, migration, and invasion abilities. Mechanistically, IRAK1 (IL-1 receptor-associated kinase-1) and ADAM19 (a disintegrin and metalloproteinase 19) were identified as miR-146b-5p targets regulating trophoblast function, and silencing IRAK1 had similar effects as miR-146b-5p overexpression, while IRAK1 overexpression could partially reverse the inhibitory impact of this miRNA on trophoblasts. miR-146b-5p may inhibit trophoblast proliferation, migration, invasion, and implantation-associated inflammation by downregulating IRAK1 and ADAM19, participating in the pathogenesis of miscarriage and providing a critical biomarker and a promising therapeutic target for UM.

overall_design: Maternal and gestational age-matched placental villus tissues from normal pregnancy (NP, n=5) and recurrent miscarriage (RM, n=5) were collected for RNA-seq of the whole transcriptome (including circRNA, lincRNA, miRNA and mRNA profiles) . To minimize the effect of maternal age and gestational age on miscarriage incidence or gene expression, specific criteria were used to match villus tissues from UM with those from NP. These criteria included a difference in maternal age ≤ 2 years and a difference in gestational age, as determined by ultrasound of ≤ 1 week.


title: Dysregulated alveolar epithelial maintenance in Hermansky-Pudlak syndrome pulmonary fibrosis

city: Philadelphia

first: Sylvia

middle: N

last: Michki

address: 3615 Civic Center Blvd

department: Pulmonary & Sleep Medicine

institute: Children's Hospital of Philadelphia

summary: Hermansky-Pudlak syndrome (HPS) is a genetic disorder of intracellular endosomal trafficking defects associated with pulmonary fibrosis. Double mutant HPS1/2 mice exhibit spontaneous fibrosis and single mutant HPS1 and HPS2 mice display increased fibrotic sensitivity. To identify mechanisms of AT2 cell dysfunction contributing to fibrosis in HPS, we examined lungs from aged HPS1/2 mice and observed regions of AT2 cell loss adjacent to areas of marked AT2 cell hyperplasia as compared to wild type (WT). Overall, there was accelerated loss of AT2 cells in HPS1/2, HPS1, and HPS2 mice with aging based on immunohistochemistry and lineage tracing studies. Lung organoids generated with HPS2 AT2 cells with WT fibroblasts were smaller in size and displayed significantly decreased colony forming efficiency compared to organoids with WT AT2 cells. Utilizing an H1N1 PR8 influenza model to examine AT2 cell regeneration after injury, there was a significant decrease in the percentage of proliferating AT2 cells in HPS1 and HPS2 mice compared to WT mice. RNA sequencing of AT2 cells isolated from unchallenged mice demonstrated upregulation of genes associated with proliferation and cellular senescence in HPS AT2 cells. Collectively, AT2 cells in HPS mice exhibit dysregulated proliferation with transcriptomic features suggesting subpopulations of senescent and hyperproliferative cells. Dysregulated maintenance of the alveolar epithelium with accelerated senescence and aberrant proliferation of AT2 cells appears to be a shared pathway underlying HPS and other sporadic and genetic forms of pulmonary fibrosis.

overall_design: To investigate age and genotype-dependent mRNA expression differences in AT2 cells captured from the lungs of WT and HPS-affected mouse lungs

first: Joanna

middle: Y

last: Wang

first: Brandon

last: Banaschewski

first: Sneha

last: Sitaraman

first: Jarod

last: Zepp

first: David

last: Frank

first: Lisa

last: Young