title: Characterization of Interleukin-27-induced HIV-Resistant Human Monocyte-derived Macrophages: Implication of the Induction of a Novel Subset of Macrophages in vitro

city: Frederick

first: Jun

last: Yang

state: MD

address: P. O. Box B

institute: Leidos Biomedical

summary: M2 macrophages can be polarized into M2a, M2b, M2c, and M2d by various stimuli, and Interleukin (IL)-27-treated M2 macrophages (27-Mac) resists HIV infection. Since IL-27 is a member of IL-6 family of cytokines, and IL-6 polarizes M2 macrophages into M2d, 27-Mac was considered an M2d-like cells. In the current study, we compared 27-Mac and M2d and characterized  27-Mac. Monocytes derived from health donors were differentiated to M2 using M-CSF, and then the resulting M2 were polarized into subtypes using IL-6, IL-27, or BAY60-658 (an adenosine analog which polarizes M2 into M2d). Although IL-6 and IL-27 share gp130 to induce STAT3 signaling pathway, only 27-Mac resisted HIV infection and enhanced the generation of reactive oxygen species (ROS). In contrast, BAY60-6583-polarized M2 macrophages (BAY-M2d) resisted HIV infection without affecting ROS production. The cytokine-producing profile of 27-Mac did not resemble those of the two subtypes. The analysis of the gene expression profiles using single-cell RNA sequencing indicated that 27-Mac clustered differed from other cell types and significantly expressed CD38 and secreted CXCL9 in culture supernatants. These data indicate that 27-Mac is distinct from traditional M2d and provide a new insight into the function of IL-27 and the polarized M2 macrophages.

overall_design: Polarized M2 macrophage using monocytes treated with M-CSF, treated with IL27, IL6 and Bay-60 6583 to differentiated in to 27Mac, 6-M2d and Bay-M2d macrophages. Single cell RNAseq analysis with other assays were performaed to classiterize 27Mac.

first: Qian

last: Chen

first: Hongyan

last: Sui

first: Suranjana

last: Goswami

first: Mayra

last: Marquez

first: Udeshika

last: Kariyawasam

first: Tomozumi

last: Imamichi


title: Ferroptosis Induced by Omega-3 Docosahexaenoic Acid (DHA) and Erastin in Control MDA-MB-231 Cells (sh-LUC) and MDA-MB-231 Cells with knockdown of IGFBP6 gene (sh-IGFBP6)

status: Public on Apr 02 2025

city: Moscow

first: Sergey

last: Nikulin

address: 7, Vavilova st.

department: Faculty of Biology and Biotechnology

country: Russia

institute: HSE University

type: Expression profiling by high throughput sequencing

summary: In this study, we further elucidate the mechanism of action of one of the most effective antitumor PUFAs, ω-3 docosahexaenoic acid, on MDA-MB-231 breast cancer cells, including those with IGFBP6 knockdown. Additionally, we analyzed the effect of reduced IGFBP6 gene expression on the mechanism of ferroptosis in MDA-MB-231 breast cancer cells under the influence of the canonical ferroptosis inducer erastin.

overall_design: Total RNA profiles of control MDA-MB-231 cells (sh-LUC) and MDA-MB-231cells with knockdown of IGFBP6 gene (sh-IGFBP6) treated with erastin and omega-3 docosahexaenoic Acid (DHA).

first: Alexandra

last: Razumovskaya


title: Combination therapies of porcupine inhibition with ruxolitinib, ibrutinib or belumosudil in murine sclerodermatous GvHD

city: Düsseldorf

first: Alexandru-Emil

last: Matei

address: Merowingerpl. 1A

country: Germany

institute: Hiller Research Center, University Hospital Düsseldorf, Medical Faculty of Heinrich Heine University

summary: Chronic graft-versus-host disease (cGvHD) is a frequent complication of allogeneic hematopoietic stem cell transplantation (alloSCT) and is associated with high morbidity and mortality. First drugs have been recently approved for the treatment of cGvHD, but the response rates are suboptimal and treatment discontinuation due to side effects remains frequent. Here, we evaluated the efficacy and safety profile of porcupine inhibition by Wnt-C59 in monotherapy or in combination with ruxolitinib, ibrutinib or belumosudil in experimental sclerodermatous GvHD (sclGvHD). Treatment was well tolerated in all groups with improvements in clinical and histological features. Of these, ruxolitinib in monotherapy, and the combination therapies demonstrated the highest efficacy. The combination therapies of Wnt-C59 with ibrutinib or belumosudil showed additive antifibrotic effects in skin and lungs compared to the respective monotherapies. Our study may have direct translational relevance, since: 1) core signaling pathways targeted by these drugs were enriched in both human and murine sclGvHD, and 2) ruxolitinib, ibrutinib and belumosudil are already approved for the treatment of cGvHD, and porcupine inhibitors are in clinical trials for other fibrotic diseases. Thus, simultaneous targeting of different pathogenetically relevant pathways might be a safe and effective therapeutic approach in sclGvHD.

overall_design: Allogeneic transplanted mice (cGVHD model) and syngeneic transplanted control mice (Syn) received daily oral gavage of 0.4% methylcellulose (vehicle) starting on day 21 after allogeneic or syngeneic bone marrow transplantation (alloBMT). Treatment groups included Wnt-C59 monotherapy (5 mg/kg daily, orally) and a combination of Wnt-C59 (5 mg/kg daily, orally) and belumosudil (150 mg/kg daily, orally). All mice were sacrificed seven weeks post-alloBMT.

first: Xuezhi

last: Hong

first: Yanhua

last: Xiao

first: Jörg

last: Distler

first: Alexandru

middle: E


title: Genome-wide temporal gene expression reveals a post-reproductive shift in the nematode C. briggsae

city: Hamilton

first: Bhagwati

middle: P

last: Gupta

state: ON

address: 1280 main street W, Life Sciences Building LSB322

department: Biology

country: Canada

institute: McMaster University

summary: C. briggsae offers a robust system for comparative investigations of genetic pathways that affect physiological processes. One of these, namely reproduction, directly impacts longevity due to its significant energetic demands. Long-lived mutants often exhibit reproductive impairments, and studies in C. elegans have demonstrated that removal of the germline can promote longevity, underscoring the link between reproduction and aging. Our study focuses on identifying gene expression changes during reproductive and post-reproductive phases in C. briggsae. We conducted whole-genome transcriptome profiling at various adult stages and found that the majority of differentially expressed (DE) genes were downregulated during the reproductive period. Interestingly, this trend reversed post-reproduction, with three-quarters of the genes upregulated—a phenomenon we termed the 'post-reproductive shift'. A similar analysis in C. elegans also showed a downregulation bias during the reproductive period, but the post-reproductive shift was absent. In C. briggsae, DE genes were enriched in processes related to the matrisome, muscle development and function during the reproductive period. Post-reproductive downregulated genes were enriched in DNA damage repair, stress response, and immune response. Additionally, terms related to fatty acid metabolism, catabolism, and transcriptional regulation exhibited complex patterns. Experimental manipulations of the reproductive status of animals predictably altered gene expression, providing in vivo support for the post-reproductive shift. Overall, our study reveals novel gene expression patterns during reproductive and post-reproductive changes in C. briggsae. The data provide a valuable resource for cross-sectional comparative studies in nematodes and other animal models to understand evolution of genetic pathways affecting reproduction and aging.

overall_design: RNA-seq profiling of C. briggsae AF16 hermaphrodites at day 1, 3, 6, and 9 of adulthood.

first: Wouter

last: van den Berg


title: Expression data from wound skin of WT and Spink7 KO mice

city: Chong qing

last: Zhao

address: 30 Main Street, Gao Tanyan

country: China

institute: Army Medical University

summary:
Wound healing is one fundamental physiological process which maintains the integrity of skin through a series of well-orchestrated biological and molecular events,effective resolution of inflammation is crucial for wound healing
We used microarrays to characterize molecular mechanisms of wound healing with Spink7 KO mice of up-regulated genes during this process.

overall_design: Normal and wounds skin between WT and Spink7 KO mice were selected at day 0 of resting stage and day 7 of proliferative phase for RNA extraction and hybridization on Affymetrix microarrays.

first: Tao

last: Wang

first: Guojian


title: A one-base therapeutic insertion in the HBG2 distal promoter reactivates γ-globin expression

city: Guangzhou

first: xiuqin

last: bao

address: 1838 Guangzhou

institute: Southern Medical University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The reactivation of developmental silenced g-globin genes (HBG1/2) has shown promise as a therapeutic strategy for improving symptoms of b-hemoglobinopathies. Currently, the focus of therapeutic targets is primarily on the major fetal hemoglobin suppressors, such as BCL11A and ZBTB7A and of their binding sites on the proximal HBG promoter. However, the role of the distal HBG promoter in regulating gene expression remains to be explored. Here, we discovered an insertion of nucleotide A (insA) between -1368 and -1369 bp upstream of the TSS in HBG2 resulting in remarkable increase in γ-globin expression in HUDEP-2 cells. We also observed elevated γ-globin expression in human CD34+ erythroid progenitor cells from healthy individuals and those with b-thalassemia when introducing insA mutation. Similarly, engrafted NCG-Kit-V831M mice showed increased γ-globin expression. Importantly, neither did insA have any off-target effects nor did it affect the maturation of erythroid cells. Furthermore, we found that the insA mutation created a binding site for the transcription activator FOXO3, which can reactivate g-globin. Additionally, introducing insA specifically and significantly demethylated the -162 CpG site on HBG promoter by reducing the enrichment of DNA methyltransferase 3A (DNMT3A). At the same time, it activated histone modifications and RNA polymerase II (Pol II) in both distal and proximal HBG promoter and inhibited the binding of BCL11A and ZBTB7A on -115 and -200 sites on the HBG promoter respectively. Overall, our study suggests that introducing insA mutation leads to significantly boosted fetal globin levels and uncovers new safe therapeutic target or strategy for β-hemoglobinopathies.

overall_design: Examination of the chromosome accessibility in insA and insA KO HUDEP-2 clones. Wild type DNA was served as conrol.

first: Xiuqin

last: Bao

first: Yuanyi

last: Gao

first: Xiangmin

last: Xu


title: Osteoblast-derived mitochondria formulated with cationic liposome guide mesenchymal stem cells into osteogenic differentiation

city: Seongnam-si

first: Hye-Ryoung

last: Kim

state: Gyeonggi-do

address: 335, Pangyo-ro, Bundang-gu

country: South Korea

institute: CHA University

summary: While mitochondria are known to be essential for intracellular energy production and overall function, emerging evidence highlights their importance in influencing cell behavior intracellularly through mitochondrial transfer. This phenomenon provides a potential basis for the development of treatment strategies for tissue damage and degeneration and inhibition of tumor progression. This study aimed to evaluate whether mitochondria isolated from osteoblasts can promote osteogenic differentiation in mesenchymal stem cells (MSCs). We first compared mitochondria from MSCs, which primarily utilize glycolysis, with those from MG63 cells, which depend on OXPHOS. Mitochondria from both cell types were then encapsulated in cationic liposomes and transferred to MSCs and their impact on differentiation was assessed. Mitochondria delivery from MG63 cells to MSCs grown in both 2D and 3D cultures resulted in increased expression of osteogenic markers including RUNX2, OSX, and OPN and upregulation of genes involved in BMP2 signaling and calcium import. This was accompanied by increased calcium influx and regulated by the Wnt/b-catenin signaling pathway. Transplantation of spheroids containing MSCs with MG63-derived mitochondria in bone defect animal models improved bone regeneration. Our results suggest that delivery of MG63-derived mitochondria effectively guides MSCs toward osteogenesis, paving the way for the development of mitochondria-based therapies.

overall_design: To investigate what changes occur when osteoblast-derived mitochondria are transplanted into mesenchymal stem cells.

first: Seonjeong

last: Woo

first: Sohyun

last: Hwang

first: Hye Jin

first: Keun-Hong

last: Park


title: TFE3 fusion oncoprotein condensates drive epigenetic reprogramming and cancer progression

city: Baltimore

first: Wanlu

last: Chen

address: 615 N. Wolfe Street

country: USA

institute: Johns Hopkins University

summary: Translocation renal cell carcinoma (tRCC) presents a significant clinical challenge due to its aggressiveness and limited treatment options. This cancer is primarily driven by fusion oncoproteins (FOs) arising from chromosomal rearrangements, yet their role in oncogenesis remains incompletely understood. Here we investigate TFE3 fusion in tRCC, focusing on NONO::TFE3 and SFPQ::TFE3, constituting 30-40% of all TFE3 FOs. We find that TFE3 FOs form liquid-like condensates with heightened transcriptional activity, selectively recruiting active transcription markers to TFE3 target genes and promoting cell proliferation, migration, and drug resistance. The coiled-coil domains (CCD) of NONO and SFPQ are essential for condensate formation, prolonging TFE3 FOs' chromatin binding time and enhancing transcription. We comprehensively investigated the genome-wide recruitment of TFE3 FOs and their CCD-altered variants, uncovering widespread changes in chromatin accessibility and genomic binding specifically at TFE3 and AP-1 regulated loci. We also observe altered H3K27ac deposition at enhancers and super-enhancers notably at pro-growth and stemness markers such as BCL2 and CD44, and identify novel oncogenic target genes of TFE3 FOs. Disruption of condensate formation, resulting from CCD domain alterations in FOs, robustly dysregulates their role in chromatin accessibility, chromatin binding, H3K27ac occupancy, and gene expression. Altogether our integrated analyses underscore the critical functions of TFE3 FO condensates in driving RCC progression, offering pivotal insights for future targeted therapeutic strategies.

overall_design: To investigate the function of fusion TFE3 and mutants (NONO::TFE3, NONOΔCCD::TFE3, NONOmut::TFE3, SFPQ::TFE3, SFPQΔCCD::TFE3, TFE3), we rescued these proteins in UOK109 KO cells (knocking out NONO::TFE3). We then performed gene expression profiling analysis using data obtained from RNAseq of these seven cell lines (KO and six rescues). Comparative gene expression profiling analysis of RNA-seq data for KO cells and the rescue cells.

first: Choon Leng

last: So

first: Ye Jin

last: Lee

first: Bujamin

middle: H

last: Vokshi

first: Binglin

last: Huang

first: Yangzhenyu

first: Marie Elena

last: Portuallo

first: Sumaiya

last: Begum

first: Kasturee

last: Jagirdar

first: W. Marston

last: Linehan

first: Vito

middle: W

last: Rebecca

first: Hongkai

last: Ji

first: Eneda

last: Toska

first: Danfeng

last: Cai


title: Expression profiling of purified murine epididymal spermatozoa and murine leukocytes.

city: St. Louis

first: Polina

middle: V

last: Lishko

state: Missouri

address: 425 S. Euclid ave

department: Cell Biology and Physiology

institute: Washington University in St. Louis, School of Medicine

summary: A study utilizing next-generation sequencing provided a comprehensive expression profile of murine spermatozoa, revealing the transcriptional landscape of mature sperm isolated from the corpus and cauda epididymides of C57BL/6 mice. By analyzing RNA content, we identified a distinct set of retained transcripts, including those encoding sperm-specific ion channels, as well as transcripts associated with sperm motility, capacitation, and the acrosome reaction. In addition to spermatozoa, the study also reported the transcriptional profiling of murine leukocytes, providing comparative insights into cell-type-specific gene expression. This study advances our understanding of sperm transcriptomics and its implications for male fertility

overall_design: RNA-seq profiling of C57BL/6 murine spermatozoa and blood leukocytes

first: Elena

last: Gracheva


title: TFE3 fusion oncoprotein condensates drive epigenetic reprogramming and cancer progression [ATAC-Seq]

overall_design:
To investigate the function of fusion TFE3 and mutants (NONO::TFE3, NONOΔCCD::TFE3, NONOmut::TFE3, SFPQ::TFE3, SFPQΔCCD::TFE3, TFE3), we rescued these proteins in UOK109 KO cells (knocking out NONO::TFE3).
We then assess the chromatin accessibility using data obtained from ATAC-seq of these seven cell lines (KO and six rescues). Comparative chromatin accessibility analysis of ATAC-seq data for KO cells and the rescue cells.

last: Gao


title: Modeling SMAD2 mutations in iPSCs provides insights into cardiovascular disease pathogenesis. [ATAC-Seq]

city: Boston

first: Jon

last: Seidman

state: Massachusetts

address: 77 Avenue Louis Pasteur

department: Genetics

institute: Harvard Medical School

summary: Rationale: SMAD2 is a co-regulator that binds a variety of transcription factors and modulates activities during human development. Heterozygous SMAD2 loss-of-function (LoF) variants and missense variants are identified in patients with complex congenital heart disease (CHD), and in patients with arterial aneurysms and dissections. Mechanisms that account for distinct cardiovascular phenotypes caused by SMAD2 variants remain unknown. We aimed to define the transcriptional and epigenetic effects of heterozygous SMAD2 loss-of-function (LoF) and missense variants and identify the involvement of transcription factor networks and genes that contribute to CHD. Compared to LoF variants, we assessed the function of rare SMAD2 missense variants of uncertain significance. Methods: We constructed isogenic induced pluripotent stem cells (iPSCs) with heterozygous or homozygous LoF and rare (MAF ≤ 10-5) missense SMAD2 variants identified in CHD probands. Wildtype and mutant iPSCs were studied by bulk RNA sequencing (RNAseq) and chromatin accessibility (ATACseq). Datasets were integrated with published SMAD2/3 chromatin immunoprecipitation (ChIPseq) studies. Cardiomyocyte differentiation and contractility of mutant iPSCs was assessed. Results: SMAD2 haploinsufficiency reduced open chromatin across promoter regions and altered the expression of 385 direct SMAD regulated genes, including ten previously identified as dominant CHD genes. Motif analyses in regions with differential ATAC peaks predicted that SMAD2 haploinsufficiency disrupts interactions with at least five transcription factors, NANOG, ETS, TEAD3/4, CREB1 and AP1. Compared to SMAD2-haploinsufficient cells, iPSCs with heterozygous R114C or W274C variants exhibited shared and distinct patterns of altered chromatin accessibility, transcription factor binding motifs, and dysregulated genes. Conclusions: SMAD2 haploinsufficiency disrupts chromatin interactions and perturbs transcription factor binding, disrupting normal cardiovascular development. Differences in the molecular consequences of LoF and missense variants likely contribute to clinical phenotypic heterogeneity. Additionally, these data indicate opportunities for molecular functional analyses to improve re-classification of SMAD2 variants of uncertain clinical significance.

overall_design: Examination of genetically engineered iPS cell lines that carry SMAD2 mutations. Two biological replicates for SMAD2 HET LoF, W274C, R114C cell lines and two technical replicates for each

first: Tarsha

last: Ward

first: Lauren

last: Wasson

first: Warren

last: Tai

first: Sarah

last: Morton

first: Christine


title: Bone marrow CD34+/lin- cells of patients with Chronic Phase- Chronic Myeloid Leukemia (CP-CML) after 12 months of nilotinib treatment exhibit a different expression signature compared to the diagnosis and the same cells from healthy subjects

city: Milano

first: Alessandra

last: Trojani

address: Piazza dell'Ospedale Maggiore, 3

country: Italy

institute: ASST Grande Ospedale Metropolitano Niguarda

summary: Chronic Phase-Chronic Myeloid Leukemia (CP-CML) is defined by the presence of BCR-ABL1 fusione gene, which encodes a tyrosine kinase protein that drives the uncontrolled proliferation and survival of leukemic stem cells (LSCs). Nilotinib, a tyrosine kinase inhibitor, targets the activity of BCR-ABL1 by reducing aberrant signalling pathways which drive the regeneration of LSCs. Despite nilotinib’s action, a population of resilient LSCs persist in the bone marrow (BM) and can indeed drives relapse and progression in CML patients.  Our study investigated the gene expression profiling (GEP) of BM CD34+/lin- cells from 79 CP-CML patients at diagnosis compared to the BM CD34+/lin- cells from the same patients after 12 months of nilotinib treatment, and to the normal cell counterpart from 10 donors (CTRLs). GEP analyses identified 3012 significantly differentially expressed genes. Among these, we focused on certain genes involved in crucial KEGG pathways: CML, cell cycle, JAK-STAT, PI3K-Akt, MAPK, Ras, NF-kB, and ABC transporters. We demonstrated the upregulation of several genes at diagnosis compared to both 12 months of nilotinib treatment and the CTRLs. We highlighted that certain transcriptome features characterizing the diagnosis persist after 12 months of nilotinib treatment compared to CTRLs, potentially influencing the self-renewal and survival properties of LSCs.

first: Barbara

last: Di Camillo

first: Roberto

last: Cairoli


title: Hippocampal gene expression in the GFAP-R237H rat model of Alexander disease at early and late stages of disease

city: Madison

first: Tracy

last: Hagemann

state: WI

address: 1500 Highland Ave

department: Waisman Center

institute: University of Wisconsin-Madison

summary: Alexander disease is a rare neurodegenerative disorder caused by mutations in the gene for glial fibrillary acidic protein (GFAP), the major intermediate filament of astrocytes in the central nervous system. GFAP mutation causes a toxic gain-of-function and protein aggregation, ultimately leading to gliosis, astrocyte dysfunction, and neurodegeneration. To better understand the disease process, a rat model has been generated to mimic the common R239H mutation observed in the human disease (R237H in the rat). This study focuses on hippocampus, a brain region with a heavy burden of pathology, to determine the impact of GFAP mutation at presymptomatic (3 weeks of age) and severe stages ( 8 weeks of age) of disease. Transcription profiling shows progressive neuroinflammation and neurodegeneration.

overall_design: Hippocampi were collected from male and female Gfap+/R237H rats and wild type (Gfap+/+) littermates at P21 and P56 (N = 4 per group, with the exception of P21 females, N = 3 wild type, N = 5 R237H rats).

first: Hagemann

last: Tracy


title: Colonic goblet cell-associated antigen passages mediate physiologic and beneficial translocation of live gut bacteria in preweaning mice

first: Rodney

middle: D

last: Newberry

address: 660 S. Euclid Ave

department: Medicine

institute: Washington University School of Medicine

summary: Gut-resident microbes contribute to host health via multiple mechanisms. Some of the most striking gut microbiota induced effects occur in the extraintestinal tissues and are restricted to early life. The mechanisms by which gut residing bacteria induce effects on distant host tissues and why this is restricted to a period in early life are largely unknown. We found that a subset of live gut-resident bacteria spontaneously translocate from gut to extraintestinal tissues in preweaning, but not adult mice. Translocation in preweaning mice appeared physiologic as it did not induce an inflammatory response and was in part controlled by sphingosine-1-phsophate receptor (S1PR) expressing host cells and host goblet cells.  One translocating strain, Lactobacillus animalisWU, contained unique coding sequences for genes in the tyrocidine-gramicidin antibiotic-synthesizing gene cluster as well as five other regions putatively producing secondary metabolites with anti-microbial activity.  Lactobacillus animalisWU exhibited antimicrobial activity against the late-onset sepsis pathogen E. coli ST69 in vitro, and translocation of L. animalisWU protected preweaning mice from systemic E. coli ST69 sepsis in vivo.  These observations demonstrate a previously unappreciated higher-level symbiosis with our gut microbes.

overall_design: To assess the effects induced by translocation of L. animalisWU in preweaning mice, we looked at the MLN transcriptome of preweaning mice fed with L. animalisWU (translocation positive group) or with L. animalisWU and EGF (translocation negative group)

first: Udayan

last: Sreeram

first: Newberry

last: Rodney


title: Decoding Plasma Cell Maturation Dynamics with BCMA

city: Erlangen

first: Hans-Martin

last: Jäck

address: Glückstrasse 6

department: Molecular Immunology

institute: Universitätsklinikum Erlangen

summary: Plasma cells provide protective antibodies following an infection or vaccination. A network of intrinsic and extrinsic factors fine-tunes the generation of a heterogenous plasma cell pool with varying metabolic requirements, transcriptional profiles and lifespans. Among these intrinsic factors, the B cell maturation antigen (BCMA) has been implicated in APRIL-mediated survival of long-lived plasma cells. Therefore, we introduced a tdTomato reporter into the Tnfrsf17 (BCMA) locus, to decode the developmental steps behind the generation of mature plasma cell subsets. We demonstrate that the BCMA:Tom reporter is exclusively detected in plasma cells, with its expression differing between IgH isotypes and increasing with plasma cell maturity. Combined with a Blimp1-GFP reporter, the BCMA:Tom reporter allowed us to track the step-wise maturation from newly formed to late plasma cells in various plasma cell subpopulations. Notably, in vitro-stimulated B cells induce BCMA only after adoptive transfer into recipient mice, emphasizing the impact of the in vivo microenvironment in establishing a mature plasma cell compartment. The BCMA:Tom reporter mouse provides a robust tool for tracking plasma cell development and maturation with flow cytometry or advanced imaging techniques, enabling a deeper understanding of the mechanisms regulating plasma cell heterogeneity and longevity.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for bone marrow plasma cell populations from Blimp1-GFP;BCMA:Tom reporter mice

first: Sebastian

middle: R

last: Schulz

first: Shannon

last: Menzel

last: Jaeck


title: Comparative Analysis of Caco-2 Cells and Human Jejunal and Duodenal Enteroid-derived Cells in Gel- and Membrane-Based  Barrier Models of Intestinal Permeability

city: College Station

first: Ivan

last: Rusyn

state: TX

address: VIDI(Veterinary Medicine 2), 2, 402 Raymond Stotzer Pkwy

department: Interdisciplinary Faculty of Toxicology

institute: Texas A&M University

summary: Studies of gut absorption are necessary for characterization of drug toxicokinetics. While the human colon carcinoma cell line Caco-2 is the most used in vitro model, additional options have been proposed to represent different intestinal segments. Accordingly, we characterized the morphology and tissue specific marker expression of three human intestinal cell types: Caco-2 and primary human enteroid cells from jejunum (J2) and duodenum (D109). Cells were cultured in the OrganoPlate® 3-lane 40 microphysiological system with flow (MPS) or static 24-well Transwells™. In both formats, J2 and D109 cells formed domedlike structures compared to uniform monolayers of Caco-2 cells. In the MPS, only Caco-2 cells formed uniform tubules. Cells grown on Transwells formed a thicker monolayer than in the MPS, with the most notable difference observed using J2 cells. All cells and culture conditions exhibited robust expression of ZO-1 (tight junctions). For cell polarization markers ezrin and villin, cell staining showed that expression was greatest in J2 and D109 cells cultured in the MPS. Regarding the functional marker mucin, J2 cells exhibited the highest expression, regardless of the platform. J2 and D109 cells exhibited poor barrier (70 kDa FITCTRITC-dextran) in MPS, but the tight barriers formed in Transwells. Barrier function and drug transport were also evaluated using caffeine, indomethacin, and propranolol; these compounds showed no appreciable binding to empty MPS or Transwells. Notably, we found that the gel lane in MPS was a sink for these compounds; only a small fraction (~13% over 15 hrs) crossed into the opposite channel, even without cells present. The permeability ratios among cell types in the MPS and Transwells were used to parameterize the probabilistic environmental compartmental absorption and transit model to determine whether in vitro data could reduce uncertainty in predictions. We found that the most accurate prediction of the fraction absorbed was achieved when using Transwell-derived data (Papp) from Caco-2 cells, combined with the experimentally-derived segment-specific (Caco-2, J2, and D109 data) absorption ratios. Overall, we show that marker staining was highest in enteroid-derived cells cultured in the MPS platform, but that studies of drug permeability across cell barriers in this model are challenging.

overall_design: Comparative gene expression profiling analysis of TempO-Seq data for Caco-2, J2, and D109 cells using different platforms.

first: Haley

middle: L

last: Moyer

first: Leoncio

last: Vergara

first: Clifford

last: Stephan

first: Courtney

last: Sakolish

first: Lucie

middle: C

last: Ford

first: Han-Hsuan

last: Tsai

first: Hsing-Chieh

last: Lin

first: Weihsueh

middle: A

last: Chiu

first: Philip

last: Hewitt


title: Ligand-binding pockets in RNA, and where to find them [II]

city: Chapel Hill

first: Kevin

middle: M

last: Weeks

state: NC

address: 250 Bell Tower Drive

department: Chemistry

institute: UNC – Chemistry

summary: RNAs are critical regulators of gene expression, and their functions are often mediated by complex secondary and tertiary structures. Structured regions in RNA can selectively interact with small molecules – via well-defined ligand binding pockets – to modulate the regulatory repertoire of an RNA. The broad potential to modulate biological function via RNA-ligand interactions remains unrealized, however, due to challenges in identifying RNA motifs with the ability to bind ligands with good physiochemical properties (often termed drug-like). Here, we devise fpocketR, a computational strategy that accurately detects pockets capable of binding drug-like ligands in RNA structures. Remarkably few, less than 60, of such pockets hadbeen previously visualized. We experimentally confirmed the ligandability of novel pockets detected with fpocketR using a fragment-based approach introduced here, Frag-MaP, that detects ligand-binding sites in cells. Analysis of pockets detected by fpocketR and validated by Frag-MaP reveals dozens of newly identified sites able to bind drug-like ligands, supports a model for RNA secondary structural motifs able to bind quality ligands, and creates a broad framework for understanding the RNA ligand-ome.

overall_design: We used photo-crosslinking fully functionalize fragment (FFF) probes and mutational profiling reverse transcription (MaP-RT) to determine the nucleotide position of RNA-ligand interactions. The FFF probes tested include a non-selective control (methyl), a positive control (linezolid), and fragments (6-aminoquinoxaline and Enamine Z4225957206).

first: Seth

last: Veenbaas

first: Jordan

middle: T

last: Koehn

first: Patrick

middle: S

last: Irving

first: Nicole

middle: N

last: Lama


title: Sexually dimorphic gene expression responses of bovine embryos to the maternal microenvironment

city: Bonn

first: Dessie Salilew

last: Wondim

address: Endenicher Allee 15

institute: University of Bonn

summary: This study investigated the sexual dimorphic gene expression patterns of day 13 embryos developed in Holsteins Frisian cows and heifers. Accordingly, embryos developed in the cows exhibited sexually dimorphic expression of genes associated with steroid biosynthesis and metabolism as well as genes associated with the formation of the primary germ layer whereas those developed in heifers sexually dimorphic expression of genes involved in developmental processes, gonadal development or female sex differentiation, and transmembrane transports. This result may partly explain the possible causes of a skewed sex ratio during development and at birth.

overall_design: To generate  the day 13 female and male embryos, in vitro-produced presumptive male and female embryos were transferred to multiparous Holsteins Frisian cows and nulliparous heifers on day 2 of the oestrous cycle. The embryos were recovered at day 13 of the gestation period and classified into four groups according to their sex and origin as male or female embryos developed in cows or heifers. The total RNA was isolated from each group of embryos (n =5) was subjected to the next-generation transcriptome sequencing. Adapters and non-informative sequences were removed and clean reads were mapped to the bovine reference genome. Quantification and annotation of expressed genes in each embryo group were performed using the Seqmonk tool and differential expression analysis between the embryo groups was performed using the edgeR Bioconductor package.

first: Dessie

last: Salilew-Wondim

first: Ernst

last: Tholen

first: Christine

last: Größe-Brinkhaus

first: Eva

last: Held-Hoelker

first: Dennis

last: Miskel

first: Franca

last: Rings

first: Karl

last: Shellander

first: Urban

last: Besenfelder

first: Vitezslav

last: Havlicek

first: Dawit

last: Tesfaye

first: Michael

last: Hoelker


title: RNA sequencing analysis of human embryonic stem cell line BG01 treated with anti-MFG-e8 neutralizing antibodies (anti-M8nAb)

city: Seoul

first: Jong-Hoon

last: Kim

address: 145 Anam-ro, Seongbuk-gu

department: Biotechnology

institute: Korea University

summary: To understand the function of MFG-e8, which is secreted from human embryonic stem cells (hESCs), hESC line BG01 cells were treated with anti-M8nAb to deplete the MFG-e8 activity. Depletion of MFG-e8 activity in hESC cultures resulted in decreased cell proliferation, increased cell death, and prominent morphological changes within 2 days. To investigate the transcriptomic changes under depletion of MFG-e8 activity in hESC culture, we performed RNA-seq analysis of BG01 cells treated with anti-M8nAb at two different time point. As a result, we identified differentially expressed genes in anti-M8nAb treated BG01 cells compared to isotype IgG treated control cells. Transcriptome of anti-M8nAb tretaed BG01 cells were enriched with the genesets related with cell death, cell cycle arrest, and epithelial-to-mesenchymal transition compared to isotype IgG treated control cells. In conclusion, these RNA-seq analysis results can provide insights into how MFG-e8 affect signaling mechanisms in hESCs.

overall_design:
To investigate the function of MFG-e8 in maintaining hESC culture, we treated hESC line BG01 with 5 μg/ml anti-M8nAb to deplete MFG-e8 activity. Normal mouse IgG (5 μg/ml) was treated as isotype control of anti-M8nAb. (2 biological replications for each samples)
We performed gene expression profilling analysis using RNA-seq data of the BG01 cells treated with anti-M8nAb or isotype IgG for 12 or 24 hours.

first: Hyojin

first: Youngseok

last: Lee


title: WDR36 regulates trophectoderm differentiation during human preimplantation embryonic development through glycolytic metabolism

city: Nanjing

first: Xi

last: Wang

address: Longmian Avenue 101, Jiangning District

institute: Nanjing Medical University

summary: Mammalian pre-implantation development is a complex process involving sophisticated regulatory dynamics. WD repeat domain 36 (WDR36) is known to play a critical role in mouse early embryonic development, but its regulatory functions in human embryogenesis is still elusive due to limited access to human embryos. The human pluripotent stem cell-derived blastocyst-like structure, termed a blastoid, offer an alternative means to study human development in a dish. In this study, after verifying that WDR36 inhibition disrupted polarization in mouse early embryos, we further demonstrated that WDR36 interference could block human blastoid formation, dominantly hindering the trophectoderm lineage commitment. Both transcriptomics and targeted metabolomics analyses revealed that WDR36 interference downregulated glucose metabolism. WDR36 can interact with glycolytic metabolic protein LDHA, thereby positively regulating glycolysis during the late stage of human blastoid development. Taken together, our study has established a mechanistic connection between WDR36, glucose metabolism, and cell fate determination during early embryonic lineage commitment, which may provide potential insights into novel therapeutic targets for early adverse pregnancy interventions.

overall_design: We treated iWDR36 iPS cell-derived blastoids with Dox to knockdown WDR36 gene, starting on Day 3. Then, we collected samples at Day 4 and Day 6. We compared the gene expression profiles between these samples as well as transcriptome data set of natural blastocysts from the literature.

first: Shiyu

last: An

first: Shuyue

last: Hou

first: Yang

last: Yang


title: Effects of dihydrotestosterone on glioblastoma cells U87

city: shanghai

first: Zhen

address: No. 6600, Nanfeng Road, Fengxian District

department: Department of central laboratory

institute: Shanghai University of Medicine & Health Science Affiliated Sixth People’s Hospital South Campus

summary: We analyzed the whole transcriptome of glioblastoma cell U87 samples cultured with different dihydrotestosterone levels. Differentially expressed mRNAs were selected for deep analysis. Especially the Differentially expressed genes that associated with tumor progression and androgen synthesis

overall_design: The experiment was then divided into three groups: control group(3samples), charcoal-stripped fetal bovine serum group (CFBS,3samples), and high-level androgen group (Double Hydrogen Testosterone (DHT,3samples), 20μmol/L). U87 cells were cultured for 72h.

first: Yuanchi

last: Cheng


title: Chip-seq analysis of linker histone-like protein, HP1BP3

city: Minato-Ku

first: Mitsuru

last: Okuwaki

state: Tokyo

address: 5-9-1 Shirokane, Minato-ku

country: Japan

institute: Kitasato University

summary: HP1BP3 has been subbested to be a linker histone, because it exhibits linker histone-like sequence. To investigate the genome-wide distribution pattern of HP1BP3, Chip-Seq analyses have been conducted. In addition, we found that the function of HP1BP3 as a linker histone was regulated by linker histone chaperon NPM1 and TAF-I. Therefore, we also investigated the effects of histone chaperon knockdwon on the distribution pattern of HP1BP3. As a control, the analyses included the experiments with H1.2, a linker histone variant.

overall_design: Growing HeLa cells were transfected witn either control siRNA, NPM1 siRNA, SET or siRNA. The cells were fixed with 0.5% formardehyde 72 h post siRNA transfection and chromatin was digested by extensive sonication. The extracts were subjected to immunoprecipitation with anti-HP1BP3 and anti-H1.2 antibodies. The preciptiated DNA was extracted and used for libruary preparation for Chip-Seq analysis.


title: Mitochondrial Metabolism Shifts Characterized by Quantitative Mass Spectrometry in Insulin Signaling-regulated Longevity of C.elegans

city: Hangzhou

first: Xuanxuan

last: Guo

state: Zhejiang

address: No 600 Dunyu Road

institute: westlake university

summary: Aging is a time-related multifaceted physical functional decline process affected by combination of genetic and environmental factors. As unique bioenergetics factories in eukaryotic cells, mitochondria are proven to keep a close link with aging by increasing evidence. This study systematically investigated the mitochondrial functions in daf-2 induced aging C.elegans. We find that daf-2 worms are able to maintain a youthful mitochondrial morphology and have higher levels of ATP production, basal and maximal respiration at an old age. Quantitative proteomic analyses of isolated mitochondria identified 113 up-regulated and 98 down-regulated proteins in the daf-2 mutants revealed a significantly changes in metabolic activity and translation level in daf-2 mitochondria. These results revealed a shift of metabolic activity in the daf-2 mitochondria, especially in the metabolism of branched-chain amino acid (BCAA), ROS, β-alanine, propionate and fatty acid (FA), which are important for the daf-2 longevity. daf-2 worms with downregulated metabolic enzymes exhibited enhanced protein refolding activity, correlating with lifespan alterations. The amount of mitochondrial ribosomes is approximately 10-20% less in the daf-2 mutants compared to the WT. Slightly decreasing translation elongation factor in normal C.elegans does extend its lifespan. Alteration at mRNA level of mitochondrial translation repressed worms also point to the fatty acid metabolism change in cytoplasm. Our study indicates that mitochondria contribute to daf-2 induced longevity of C.elegans through metabolic disorder and further highlights the crucial role of mitochondria in aging.

overall_design: To gain a deeper understanding of the reasons for lifespan changes in daf-2-induced metabolic abnormalities worms, we choose several genes  to perform mRNA-Seq analyses after RNAi treatmentatment.


title: A stable Netrin-1 fluorescent reporter chicken reveals cell-specific molecular signatures during optic fissure closure.

city: Midlothian

first: Joe

last: Rainger

state: Scotland

address: Easter Bush Campus

department: The Division of Functional Genetics and Development

country: United Kingdom

institute: The Roslin Institute

summary: Coloboma is a developmental eye disorder characterised by failure of epithelial fusion during optic fissure closure (OFC). The incomplete identification of genes and molecular pathways that directly regulate fusion restricts current understanding of OFC mechanisms and coloboma causation. The recent identification of pioneer cells at the edges of the fissure margins provides an opportunity to molecularly characterise OFC. Here we describe the generation of a pioneer cell reporter chicken line (NTN1-T2A-eGFP) by targeting green fluorescent protein into the Netrin-1 locus using CRISPR/Cas9 methodology. Our strategy gave 100% transmission of heterozygous (NTN1T2A-eGFP/+) embryos in which GFP localisation faithfully replicated endogenous NTN1 expression. Furthermore, all NTN1T2A-eGFP/+ embryos and hatched birds were phenotypically normal. We then optimised the isolation of GFP expressing cells from embryonic eyes using spectral fluorescence cell-sorting and performed transcriptomic profiling that revealed multiple new pioneer cell markers. Our approach implicated several novel pathways, including HGF/MET signalling, that plausibly mediate fusion in OFC. This work is the first to characterise pioneer cells directly at the molecular level and provides a new chicken reporter line with broad experimental utility.

overall_design:
Optic fissure tissue from 10x NTN1-T2A-GFP gene edited Hy-Line chicken embryos at Hamburger-Hamilton stage 28 were pooled for each biological replicate.
For each replicate, NTN1-expressing GFP-positive cells and the control GFP-negative cells were sorted. Subsequently, mRNAseq was performed on each replicate. A total of 5 biological replicates of each GFP-positive and GFP-negative cells were included in the study. Differential gene expression analysis was conducted by comparing the gene expression profile of GFP-positive cells to the control GFP-negative cells.

first: Brian Ho Ching

last: Chan

first: Hollly

last: Hardy

first: Teresa

last: Requena Navarro


title: Localization of H2AS40Gc in MDA-MB-231 and expression profile  in H2AS40-knockdown cells

city: Imabari-shi

first: Koji

last: Hayakawa

state: Ehime

address: 1-3 Ikoino-oka, Bldg. A2, Room 630

institute: Okayama University of Science

summary: We report the genome-wide distribution of H2AS40Gc, H3K4me3, H3K27me3, and H3K36me3 in the breast cancer cell line MDA-MB-231 and the expression profile of MDA-MB-231 depleted S40-type H2A. We found that H2AS40Gc positively correlated with H3K27me3 and regulated the expression of genes related to proliferation and migration.

overall_design: ChIP-seq of H2AS40Gc, H3K4me3, H3K27me3, H3K36me3 in MDA-MB-231 and RNA-seq in H2AS40-knockdown cells


title: Pharmacologic degradation of WDR5 suppresses oncogenic activities of SS18-SSX and provides a therapeutic of synovial sarcoma [RNA-seq]

city: Durham

first: Yao

last: Yu

state: North Carolina

address: Medical Science Res Bldg III, 3 Genome Court

institute: Duke University

summary: Disease-causing aberrations of childhood cancers recurrently target the chromatin pathway genes leading to epigenetic dysregulation. For example, almost 100% of patients with synovial sarcoma (SS) carry an abnormal gene fusion termed SS18-SSX, which produces a disease-specific oncofusion protein that is incorporated into the SWI/SNF chromatin-remodeling complex and profoundly alters its functionalities. Thus, targeting epigenetic dependencies in childhood cancer holds great promise for improving current treatment of the affected patients. Leveraging on the cancer cell dependency dataset, medicinal chemistry tool compounds and genomic profiling approaches, we here show WDR5, a chromatin factor involved in the deposition of histone H3 lysine 4 (H3K4) methylation, to be an unexplored vulnerability of SS. Mechanistically, WDR5 and SS18-SSX associate with one another and colocalize significantly at the target oncogenes where WDR5 is essential for optimal chromatin binding of the SS18-SSX-containing chromatin modelers. Using the WDR5-targeted Proteolysis Targeting Chimera (PROTAC) compound, we further show that pharmacologic degradation of WDR5 in SS cells not only suppressed the SS18-SSX-related oncogenic gene-expression programs but additionally led to p53 activation and tumor cell senescence through downregulation of ribosomal proteins (RPs) and activation of nucleolar stress response. Importantly, the WDR5-targeted PROTAC significantly suppressed malignant growth of SS in vitro and in vivo. Taken together, we report WDR5 as a SS dependency and demonstrate the WDR5 PROTAC degrader to be a promising strategy for the treatment of SS.

overall_design: To investigate the gene-regulatory role for WDR5 in synovial sarcoma (SS) oncogenesis, we employed a WDR5-targeting PROTAC degrader (2.5 uM of MS67) and matched non-PROTAC analogs (2.5 uM of MS67N1 or MS67N2) for a two-day or four-day treatment in comparison to DMSO. Also, parental HSSY-II SS cells were stably transduced with a previously reported shRNA targeting WDR5 (shWDR5) or a scramble control (shScr). Cells were collected and subject to total RNA preparation and RNA-seq.

first: Greg

first: Bo

last: Pan


title: Pharmacologic degradation of WDR5 suppresses oncogenic activities of SS18-SSX and provides a therapeutic of synovial sarcoma [CUT&Tag]

overall_design: We used CUT&Tag to assess the genome-wide binding of WDR5, SS18-SSX, BAF155, H3K4me2, H3K4me3 and/or H2AK119ub1 in cells (HSSY II, SYO1 and/or U2OS), either parental, or mock- or compound-treated, or stably transduced with a previously reported shRNA targeting SSX (shSSX) or a scramble control (shScr).


title: Dopamine-driven Increase in IL-1b in Myeloid Cells is Mediated by Differential Dopamine Receptor Expression and Exacerbated by HIV

city: Omaha

first: Howard

last: Fox

state: Nebraska

address: 42nd and Emile

institute: University of Nebraska Medical Center

summary: The catecholamine neurotransmitter dopamine is classically known for regulation of central nervous system (CNS) functions such as reward, movement, and cognition. Increasing evidence also indicates that dopamine regulates critical functions in peripheral organs and is an important immunoregulatory factor. We have previously shown that dopamine increases NF-κB activity, inflammasome activation, and the production of inflammatory cytokines such as IL-1b in human macrophages. As myeloid lineage cells are central to the initiation and resolution of acute inflammatory responses, dopamine-mediated dysregulation of these functions could both impair the innate immune response and exacerbate chronic inflammation. However, the exact pathways by which dopamine drives myeloid inflammation are not well defined, and studies in both rodent and human systems indicate that dopamine can impact the production of inflammatory mediators through both D1-like dopamine receptors (DRD1, DRD5) and D2-like dopamine receptors (DRD2, DRD3, and DRD4). Therefore, we hypothesized that dopamine-mediated production of IL-1b in myeloid cells is regulated by the ratio of different dopamine receptors that are activated. Our data in primary human monocyte-derived macrophages (hMDM) indicate that DRD1 expression is necessary for dopamine-mediated increases in IL-1b, and that changes in the expression of DRD2 and other dopamine receptors can alter the magnitude of the dopamine-mediated increase in IL-1b. We confirm our previous studies that mature hMDM have a high D1-like to D2-like receptor ratio and expand upon this finding to show that this ratio is different in monocytes versus peripheral blood mononuclear cells (PBMCs). We further confirm in human microglia cell lines that a high ratio of D1-like to D2-like receptors promotes dopamine-induced increases in IL-1b gene and protein expression using pharmacological inhibition or overexpression of dopamine receptors to diminish the dopamine-mediated increase of IL-1b. RNA-sequencing of dopamine-treated microglia shows that genes encoding functions in IL-1b signaling pathways, microglia activation, and neurotransmission increased with dopamine treatment. Finally, using HIV as an example of a chronic inflammatory disease that is substantively worsened by comorbid substance use disorders (SUDs) that impact dopaminergic signaling, we show increased effects of dopamine on inflammasome activation and IL-1b in the presence of HIV in both human macrophages and microglia. These data suggest that use of addictive substances and dopamine-modulating therapeutics could dysregulate the innate inflammatory response and exacerbate chronic neuroimmunological conditions like HIV. Thus, a detailed understanding of dopamine-mediated changes in inflammation, in particular pathways regulating IL-1b, will be critical to effectively tailor medication regimens.

overall_design: To expand on our examination of the impact of the D1-like to D2-like receptor ratio on dopaminergic inflammation in microglia, we isolated and analyzed RNA from vehicle and dopamine treated C20 microglia-like cells (cell line) by RNA sequencing to identify pathways that could be regulating dopamine-mediated inflammation.

first: Stephanie

last: Matt

first: Peter

middle: J

last: Gaskill

first: Meng

last: Niu

first: Xiaoke

last: Xu


title: Transcriptome profiling of the liver and white adipose tissue of DIO mice treated with halofuginone.

city: HeFei

first: Zhihua

last: Wang

state: Anhui

address: No. 443, Mount Huangshan Road, Shushan District

institute: University of Science and Technology of China

summary: This study is focused on evaluating the effects of halofuginone, administered at a dosage of 100 μg/kg, on gene expression changes in the liver and white adipose tissue of diet-induced obese (DIO) mice. Halofuginone is a bioactive monomer derivative obtained from the traditional Chinese herb Dichroa febrifuga. Previous studies have predominantly highlighted halofuginone's pharmacological actions by inhibiting EPRS, which subsequently activates the amino acid starvation and integrated stress response pathway. By examining differentailly expressed genes in both metabolic tissues, we aim to uncover the comprehensive effects of halofuginone on liver and adipose tissue metabolism. This investigation is intended to provide a foundational understanding of the intricate relationship between the amino acid starvation pathway and overall metabolic processes. The outcomes of this study are expected to shed light on how halofuginone influences metabolic health, thereby laying the groundwork for future therapeutic strategies targeting metabolic disorders through the modulation of amino acid starvation responses.

overall_design: Mice were intraperitoneally injected with 100 μg/kg halofuginone or vehicle every two day. Samples were collected after 8 weeks.

first: Zhenghong

last: Liu

first: Suowen

first: Jianping

last: Weng


title: Single-cell transcriptomics analysis reveals distinct pathophysiology of the primary coenzyme Q10 nephropathy in pediatric SRNS patient

first: Sowon

last: Choi

state: Seoul

address: 329, Daehak-ro 103, Jongno-gu

institute: Seoul National University

summary:
Background:
Coenzyme Q10 (COQ10) nephropathy is a well-known cause of hereditary steroid-resistant nephrotic syndrome (SRNS), primarily impacting podocytes. This study aimed to elucidate variations in individual cell-level gene expression in COQ10 nephropathy using single-cell transcriptomics.
Methods:
We conducted single-cell sequencing of kidney biopsy specimen on a 5-year-old boy diagnosed with a COQ10 nephropathy caused by compound heterozygous COQ2 mutation. The analysis focused on the proportion of cell types, differentially expressed genes in each cell type, changes in gene expression related to mitochondrial function and oxidative phosphorylation (OXPHOS), and pathway analysis.
Results:
Our findings revealed a uniform downregulation of mitochondrial gene expression across various cell types in the context of these mutations. Notably, there was a specific increase in glycolysis-related gene expression in podocytes. The study also highlighted an altered immune cell population proportion attributed to the COQ2 gene mutation. Pathway analysis indicated a downregulation in OXPHOS and an upregulation of various synthesis pathways, particularly in podocytes.
Conclusion:
This study enhances our understanding of the pathogenesis of COQ10 nephropathy and underscore potential therapeutic targets for primary COQ10 nephropathy.

overall_design: We conducted single-cell sequencing of kidney biopsy specimen on a 5-year-old boy diagnosed with a COQ10 nephropathy.

first: PeongGang

last: Park

first: YoHan

last: Ahn

first: SeongHeon

last: Kim

first: Chaeyoon

first: HyunJe

first: HeeGyung

last: Kang


title: Comparative Analysis of Proximal Tubule Cell Sources for in vitro Studies of Renal Toxicity

first: Ivan

last: Rusyn

summary: The kidneys are essential for eliminating drugs and chemicals from the human body, with renal epithelial cells being particularly vulnerable to damage caused by xenobiotics and their metabolites. Predicting renal toxicity before clinical trials remains a pressing challenge, necessitating more predictive in vitro models. However, the abundance of commercially available renal proximal tubule epithelial cell (RPTEC) sources complicates the selection of the most predictive cell types. This study compared RPTEC sources, including primary cells (Lonza) and various RPTEC lines, such as ciPTECs (Cell4Pharma), TERT1/RPTECs (ATCC), and HEK293 (GenoMembrane), alongside HepG2 cells for specificity comparison. We tested these cells' responses to 12 drugs to evaluate their predictive capabilities. Cells were cultured in 96-well or 384-well plates and exposed to drugs for 72 hours at concentrations ranging from 0.3 to 300 µM. The CellTiterGlo® assay measured cell viability, and transcriptome data from unexposed cells analyzed gene expression profiles using the TempO-seq® S1500+ panel. Cell viability data determined points of departure (PODs), compared to human serum Cmax,free values to assess safety margins. Gene expression showed primary kidney cells closely matched the human kidney medulla, followed by TERT1 and ciPTEC lines, with HEK lines showing the least correlation. This study highlights differences among RPTEC sources and their utility in drug safety studies for the renal proximal tubule, emphasizing unique gene expression patterns related to metabolism, ion transport, and cell development, which are vital for improving predictive models in renal toxicity assessment.

overall_design: Comparative gene expression profiling analysis of TempO-Seq data for different cells, and cells that over-expressed certain transporter proteins.

first: Courtney

last: Sakolish

first: Han-Hsuan

last: Tsai

first: Hsing-Chieh

last: Lin

first: Piyush

last: Bajaj

first: Weihsueh

last: Chiu


title: Pharmaceutical inhibition of the Chk2 kinase mitigates cone photoreceptor degeneration in an iPSC model of Bardet-Biedl syndrome

city: Montreal

first: Gilbert

last: Bernier

state: Quebec

address: 5690 rosemont boulevard

institute: HMR

summary: Bardet-Biedl syndrome (BBS) is a syndromic ciliopathy leading to progressive blindness starting in childhood, but the mechanism leading to photoreceptor degeneration in BBS is unknown. The basal body of the photoreceptor primary cilium originates from the centrosome’s mother centriole, and the BBS-related proteins form a complex present at basal body. Centrosomes organize microtubules of the mitotic spindle and are required for proper cell division. We show here that immature cones, but not rods, from bbs10-/- mouse pups present an early-onset DNA damage response (DDR) that becomes persistent and localizes to the basal body. Using patient-derived induced pluripotent stem cells (iPSCs), we found that BBS10 retinal progenitor cells (RPCs) also present a DDR that correlates with activation of the mitotic spindle checkpoint. Pharmaceutical inhibition of the cell cycle checkpoint kinase 2 (Chk2) in BBS10 RPCs mitigates cell death and genomic instability and largely restores the perturbed phospho-proteome. Drug treatment of BBS10 retinal organoids improves tissue organization, cone photoreceptor survival, and outer segment maturation. These findings reveal an important function for BBS10 in the maintenance of RPCs and cone photoreceptors genomic stability during development and may open new therapeutic avenues to delay photoreceptor degeneration in BBS.

overall_design: DIV 15 RPCs from 2 biological replicates for each genotype (CTL1, CTL2, BBS1 and BBS2 iPSC lines, N =  8 samples) and each treatment (BBS1+C2i, BBS2+C2i, BBS1+ATMi, BBS2+ATMi, N= 8 samples).

first: Andrea

last: Barabino

first: Alisar

last: Katbe

first: Roy

last: Hanna


title: mRNA expression profiles of oxygen-induced retinopathy (OIR) mouse retinas after knockdown treatment of lncRNA-MM2P

city: Changsha

first: Zicong

last: Wang

state: Hunan

address: No.139，Renmin Road Central Changsha-Hunan-China

institute: The Second Xiangya Hospital of Central South University

summary: The purpose of this study is to investigate the mRNA expression profiles of OIR retinas after knockdown treatment of lncRNA-MM2P. We established an OIR model in C57BL/6J mice. The expression of lncRNA-MM2P in the retinas of OIR mice was knocked down by intravitreal injection of self-complementary adeno-associated virus (scAAV). Retinas of four eyes from two OIR mice were collected as one sample. The control group was treated with control scAAV and named as AAV_NC, the knockdown group was treated with knockdown scAAV and named as AAV_sh. The total RNA was extracted from total of eight samples (four samples each group) for RNA sequencing.

overall_design: RNA sequencing of retinas from 17-day old oxygen-induced retinopathy (OIR) C57BL/6J mice which were knocked down of lncRNA-MM2P by scAAV or treated with control scAAV.

first: Yedi

last: Zhou


title: Bulk profiling of genome-wide DNA methylation distributions in mouse embryonic stem cells (mESCs) cultured in different media pulses

city: Utrecht

first: Peter

last: Zeller

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

type: Methylation profiling by high throughput sequencing

summary: We used bulk bisulfite sequencing to analyze genome-wide distribution of DNA methylation in 6 different mESC pre-culture conditions.

overall_design: mESCs were dissociated using TrypLE, DNA was extracted and 100 ng DNA was used as input for bisulfite conversion and sequencing.

first: M

last: Blotenburg

first: V

last: Bhardwaj

first: P


title: Bulk profiling of genome-wide H3K27me3 distributions in mouse embryonic stem cells (mESCs) cultured in different media pulses

summary: We used bulk ChIC-seq to analyze genome-wide distribution of H3K27me3 in 6 different mESC pre-culture conditions.

overall_design: mESCs were dissociated using TrypLE, ethanol-fixed and incubated with anti-H3K27me3 antibody. 1000 cells were Fluorescence-activated cell (FACS)-sorted into tubes and used as input for ChIC-seq processing and sequencing.


title: Gene expression profile at single cell level of mouse embryonic stem cells (mESCs) cultured in different media pulses

summary: We used single cell RNA sequencing (scRNA-seq) to analyze gene expression in 6 different mESC pre-culture conditions.

overall_design: mESCs and gastruloids were dissociated using TrypLE, dapi-stained and Fluorescence-activated cell (FACS)-sorted into 2 384-well plates per condition, processed according to the CELseq2 protocol and analyzed using scRNAseq.


title: BDNF secreted by mesenchymal stem cells improves the quality and development potential of aged oocytes by activating the ERK1/2 signaling pathway

city: Jinan

first: Jing

state: (optional)

address: #44 Wenhua Xi Road

institute: Shandong University

summary: Decreased oocyte quality is a major determinant of age-associated fertility decline which lacks an effective treatment strategy. The secretome of mesenchymal stem cells (MSC-sec) contains various bioactive factors and has the potential to improve oocyte quality. In this study, MSC-sec treatment significantly increased first polar body emission, improved spindle assembly, reduced aneuploidy rate, and promoted maternal mRNA degradation in aged mouse oocytes, whereas the addition of BDNF antibody blocked the effects of MSC-sec. Furthermore, BDNF treatment alone also improved the oocyte quality from aged mice. Mechanistically, both MSC-sec and BDNF activated the ERK1/2 signaling pathway to increase the expression of DAZL and BTG4 in aged oocytes. Furthermore, injection of MSC-sec or BDNF into aged mouse ovaries significantly improved oocyte quality and early embryonic development. Finally, we demonstrated that BDNF treatment increased both the fertilization rate and blastocyst formation of aged human oocytes. Our study identified BDNF as the functional component of MSC-sec to improve the quality and development potential of aged oocytes by activating the ERK1/2 signaling pathway, suggesting that BDNF has the potential to mitigate age-related decline in oocyte quality.

overall_design: To detect the maternal mRNA degradation, we subjected young, aged and aged-sec(aged+MSC-sec) oocytes in MⅡ stage to global RNA-seq analyses. Following the administration of IVM with or without hUC-MSC-sec, RNA sequencing was conducted on 20 MII stage oocytes obtained from four mice in each group, which were classified as young, aged, or aged with hUC-MSC-sec(aged-sec).  The high throughput sequencing and subsequent data analysis was performed by BGI Genomics Co., Ltd (Shenzhen, China) using the DNBSEQ platform.

first: Cheng

last: Li


title: Bisulfite-Free Whole-Genome Mapping of 5-Methylcytosine at Single-Base Resolution by NTD-seq

city: wuhan

first: neng

middle: bin

last: xie

address: 115 donghu road

institute: wuhan university

summary: 5-Methylcytosine (5mC) is a crucial epigenetic modification plays a significant role in the regulation of gene expression. Accurate and quantitative detection of 5mC at single-base resolution is essential for understanding its epigenetic functions within genomes. In this study, we develop a novel NaegleriaTET-assisted deaminase sequencing (NTD-seq) method for the base-resolution and quantitative detection of 5mC in genomic DNA. The TAD-seq method utilizes a Naegleria TET-like dioxygenase (nTET) to oxidize 5mC, generating 5-methylcytosine oxidation products (5moC). We also engineered a variant of the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A), creating an A3A mutant (A3Am). Treatment with A3Am results in the conversion of cytosine to uracil, while 5moC remains unchanged. Consequently, TAD-seq enables the direct deamination of cytosine to uracil by A3Am, which is sequenced as thymine, whereas 5mC, once oxidized to 5moC by nTET, resists deamination and is sequenced as cytosine. Therefore, the cytosines that persist in the sequencing data represent the original 5mC sites. We applied NTD-seq to HEK293T cells, generating a base-resolution map of 5mC that exhibits strong concordance with maps generated by conventional BS-seq. NTD-seq emerges as a powerful, bisulfite-free approach for the single-base resolution mapping of 5mC stoichiometry in genomic DNA.

overall_design: Single-base resolution mapping of 5-methylcytosine in cell line HEK293T by NTD-seq and BS-seq.

first: Nengbin

last: Xie

first: Min

first: Bifeng

last: Yuan

first: Tongtong

last: Ji


title: Mapping cell diversity and position in temporomandibular joint osteoarthritis with pain at single cell resolution

city: Los Angeles

first: Ziying

last: Lin

state: CA

address: 2250 Alcazar St. CSA Clinical Sciences Annex

institute: University of Southern California

summary: Temporomandibular joint (TMJ) osteoarthritis (OA) is a highly prevalent disorder affecting patient’s quality of life due to joint pain and dysfunction. A comprehensive understanding of cell type diversity and their dynamics of TMJ along joint degeneration and pain is lacking. Here we established an inflammatory TMJOA mouse model via intra-articular injection of CFA (Complete Freund’s Adjuvant). TMJOA mice exhibited OA and orofacial pain, recapitulating hallmark symptoms in patients. We performed single-cell transcriptomic profiling of TMJ followed by the validation. We revealed cellular diversity, anatomic position, and cell dynamics of TMJ at single cell resolution along the joint degeneration and pain.

overall_design: Here we established an inflammatory TMJOA mouse model via intra-articular injection of CFA (Complete Freund’s Adjuvant).  We performed single-cell transcriptomic profiling of TMJ followed by the validation using mouse genetic labeling, tissue clearing, light sheet and confocal 3D imaging, multiplex RNAscope and immunodetection.


title: Nucleolus and centromere Tyramide Signal Amplification-Seq reveals variable localization of heterochromatin in different cell types.

city: Boston

last: DCIC

state: MA

address: 10 Shattuck St

institute: 4D Nucleome - Data Coordination and Integration Center

summary: Genome differential positioning within interphase nuclei remains poorly explored. We extended and validated TSA-seq to map genomic regions near nucleoli and pericentric heterochromatin in four human cell lines. Our study confirmed that smaller chromosomes localize closer to nucleoli but further deconvolved this by revealing a preference for chromosome arms below 36-46 Mbp in length. We identified two lamina associated domain subsets through their differential nuclear lamina versus nucleolar positioning in different cell lines which showed distinctive patterns of DNA replication timing and gene expression across all cell lines. Unexpectedly, active, nuclear speckle-associated genomic regions were found near typically repressive nuclear compartments, which is attributable to the close proximity of nuclear speckles and nucleoli in some cell types, and association of centromeres with nuclear speckles in hESCs. Our study points to a more complex and variable nuclear genome organization than suggested by current models, as revealed by our TSA-seq methodology.

overall_design: Nucleolar and PCH TSA-Seq to map the mean cytological proximity of chromosomes genome-wide relative to nucleoli and pericentric heterochromatin (PCH) in four human cell lines - K562, HFFc6, HCT116, H1-hESC. Different condition were used: 1) Condition C: labeling with 1:3000 tyramide biotin, 50% sucrose and 0.0015% hydrogen peroxide, 2) Condition E: labeling with 1:300 tyramide biotin, 50% sucrose and 0.0015% hydrogen peroxide (minor modification: 150ul of Dynabeads M-270 streptavidin (Invitrogen, catalog no. 65306) was used to purify the biotinylated DNA) 3) Condition AI for HFFc6, H1, and K562 and 4) Condition A for HCT116 cells.

first: Pradeep

last: Kumar

first: Omid

last: Gholamalamdari

first: Yang

last: Zhang

first: Liguo

first: Anastassiia

last: Vertii

first: Tom

last: Schaik

first: Daan

last: Peric-Hupkes

first: Takayo

last: Sasaki

first: David

last: Gilbert

first: Bas

last: Steensel

first: Jian

last: Ma

first: Paul

last: Kaufman

first: Andrew

last: Belmont


title: Assessment of Immunological Responses to Water Electrospray: A Novel Non-Chemical Biocide for Safe Disinfection

city: Daejeon

first: Keun Bon

last: Ku

address: 291 Daehak-ro

department: Biological Sciences

institute: Korea Advanced Institute of Science and Technology

summary: Water electrospray is small charged droplets generated by a laboratory-designed prototype module. Extensive investigations have explored its physical properties and identified a potential non-chemical biocide against air-borne pathogens. However, since humans can inhale water electrospray particles through breath, humans' safety and potential toxicity warrant investigation. Hence, to offer insights into the effect of water electrospray on humans, we analyzed the immunopathological response to water electrospray using a nasal challenge mouse model. For this, we accessed the fundamental immune responses following the water electrospray intranasal challenge by comparing its effect with sodium hypochlorite, a biocidal agent with known toxicity. Our results indicate that water electrospray did not induce pathological immune reactions: challenged mice did not exhibit body weight loss and increased inflammatory cytokine production. Furthermore, histopathological analysis revealed that water electrospray did not damage the lung tissue, whereas sodium hypochlorite–treated mice showed significant lung tissue damage with signs of neutrophils and eosinophils infiltration. Finally, transcriptomic analysis on lung tissue confirmed the absence of pathological immune response in water electrospray-treated mice compared with sodium hypochlorite–treated mice. Together, we provide evidence that water electrospray is a safe technology for its use in disinfecting air-borne pathogens with little or no effect on immune system activation at the preclinical level.

overall_design: The treatment groups were categorized into distilled water, water electrospray, and sodium hypochlorite, with three mice assigned to each group. A total of five intra-nasal treatments were administered to each group. Subsequently, the lung tissues of the respective mice were obtained, and RNA was isolated for analysis.

first: Keun

middle: B

first: Jihwan

last: Chae

first: Won

last: Park

first: Jeongwoo

last: La

first: Seung

last: Lee

first: Heung

middle: K


title: The roles of Ptpn22 Ser452 phosphorylation  in tumor-infiltrating CD8+ T cells from mouse

first: Richard

last: Ainsworth

address: 8700 Beverly Blvd

institute: Cedars-Sinai Medical Center

summary: Protein Tyrosine Phosphatase Non-receptor type 22 (PTPN22) is encoded by a major autoimmunity gene and is a known inhibitor of T cell receptor (TCR) signaling for cancer immunotherapy. However, little is known about PTPN22 phosphorylation in tumor infiltrating T cells. Here we characterize a phosphorylation site at Ser452 situated in the interdomain of PTPN22, and its roles in altering T cell function and tumor growth. Tumor infiltrating CD8+ T cells from PepWT or PepS452A mice were collected and subjected to next-generation sequencing (NGS). The results revealed it differentially regulates the genes expression of metabolic process, cytokine prodcution and adaptive immunity etc. This study explores the function of a novel phosphorylation site of PTPN22 that is involved in complex regulation of CD8+ T cell activity, fuctions and tumor growth.

overall_design:
MC38 tumor cells were injected into PepWT or PepS425A mice and tumors were collected after 18days and digested by collagenase to get the single cell suspensions.
CD8+ T cells were sorted out by cell sortor and used for bulk RNA sequencing,

first: Nunzio

last: Bottini

first: Shen

last: Yang

middle: I


title: A one-base therapeutic insertion in the HBG2 distal promoter reactivates γ-globin expression [ChIP-Seq]

city: Guangzhou

first: Xiuqin

last: Bao

address: No. 1838, Guangzhou avenue north, baiyun district, Guangzhou

institute: Southern Medical University

summary: The reactivation of developmental silenced g-globin genes (HBG1/2) has shown promise as a therapeutic strategy for improving symptoms of b-hemoglobinopathies. Currently, the focus of therapeutic targets is primarily on the major fetal hemoglobin suppressors, such as BCL11A and ZBTB7A and of their binding sites on the proximal HBG promoter. However, the role of the distal HBG promoter in regulating gene expression remains to be explored. Here, we discovered an insertion of nucleotide A (insA) between -1368 and -1369 bp upstream of the TSS in HBG2 resulting in remarkable increase in γ-globin expression in HUDEP-2 cells. We also observed elevated γ-globin expression in human CD34+ erythroid progenitor cells from healthy individuals and those with b-thalassemia when introducing insA mutation. Similarly, engrafted NCG-Kit-V831M mice showed increased γ-globin expression. Importantly, neither did insA have any off-target effects nor did it affect the maturation of erythroid cells. Furthermore, we found that the insA mutation created a binding site for the transcription activator FOXO3, which can reactivate g-globin. Additionally, introducing insA specifically and significantly demethylated the -162 CpG site on HBG promoter by reducing the enrichment of DNA methyltransferase 3A (DNMT3A). At the same time, it activated histone modifications and RNA polymerase II (Pol II) in both distal and proximal HBG promoter and inhibited the binding of BCL11A and ZBTB7A on -115 and -200 sites on the HBG promoter respectively. Overall, our study suggests that introducing insA mutation leads to significantly boosted fetal globin levels and uncovers new safe therapeutic target or strategy for β-hemoglobinopathies.

overall_design: Examination of the histone modifications and RNA polymerase II in HUDEP-2 cells. Input DNA was served as conrol.

first: Yuanyi

last: Gao

first: Xiangmin

last: Xu


title: A one-base therapeutic insertion in the HBG2 distal promoter reactivates γ-globin expression [RNA-Seq]

overall_design: Total RNA were extracted from insA (n=3) or wild type (WT, n=3) HUDEP-2 cells after differentiation and  performed by deep sequencing, using DNBSEQ-7.


title: Generation of transient totipotent blastomere-like stem cells by short-term high-dose Pladienolide B treatment

city: Nanjing

first: Xi

last: Wang

address: Longmian Avenue 101, Jiangning District

institute: Nanjing Medical University

summary: As an alternative model for study of the dynamic process of early mammalian embryonic development, multiple progresses have been made in using mouse embryonic stem cells (mESCs) to generate embryo-like structure, especially by modifying the starting cells. A previous study has shown that as long as continuously treating mESCs with a low-dose splicing inhibitor, pladienolide B (PlaB), a totipotent state comparable to 2- and 4-cell embryos at molecular levels can be reached. However, these totipotent mESCs mare less proliferative, with the culture time being limited to about 10-15 passages. Here we reported that transiently PlaB-treated mESCs were sufficient for generating a novel type of totipotent-like stem cells from classical mESCs, and these cells were endowed with the ability to differentiate into both embryonic and extra-embryonic cell lineages, we termed them as transient totipotent-like stem cells (TTLCs). Similarly, TTLCs could self-organize to blastoids and recapitulated key preimplantation developmental processes. Transcriptome analysis also showed that TTLC-blastoids bear similarities with mouse E3.5, E4.5 blastocyst and TBLC-blastoids. Moreover, we found that TTLC-blastoids could develop beyond implantation stage, forming egg-cylinder structures both in vivo and in vitro. In summary, our findings provided a rapidly alternative cell type to generate blastoids for the study of early mouse embryogenesis.

overall_design: We chose TT2-mESCs treated with 0 nM or 20nM PlaB for 6 h in 2D culture, and TTLC-blastoids from day 1 to day 5 in 3D microwell culture. We compared the gene expression profiles between these samples as well as transcriptome data sets from the literature.


title: MYC recruits tumor-associated macrophage to sustain metastatic malignancy of lung adenocarcinoma micropapillary subtype through epigenetic reprogramming

first: Feng

last: Jiang

state: Jiangsu

address: Baiziting 42

institute: Jiangsu Institute of Cancer Research

summary: Current comprehension of micropapillary lung adenocarcinoma (MP-LUAD) remains circumscribed to the realms of biological behaviors and genomic landscapes. Previous studies from our institute have shown that there exist subtle and unactionable genomic alterations between MP and non-MP early-stage lung adenocarcinoma (LUAD). Therefore, deciphering the unknown non-genomic regulatory network of MP-pattern malignancy may offer opportunities to uncover more tractable therapeutic targets for MP-LUAD patients.Bulk RNA-seq after microdissection showed aberrant activation of the MYC pathway in MP. The CDX mouse model showed that MYC overexpression induced a MP-pattern malignancy in tumor tissues, but in vitro experiments lacked consistency. Through screening via single-cell RNA-se, retrospective cohort and TCGA dataset, we found aberrant accumulation of tumor-associated macrophages (TAMs) around MP tissues. in vitro co-culture and in vivo dependency experiments demonstrated that MYC overexpression in tumor cells exhibited a MP-pattern malignancy in collaboration with TAMs. Follow-up experiments revealed that, MYC interacts with the pioneer transcription factor FOSL2 induced by TGFβ secreted from TAMs, transcriptionally regulating the expression of MP-pattern genes.Our study uncovered copy number amplification-induced activation of MYC in MP-LUAD, which by recruiting TAMs, cooperatively transcriptionally regulates MP pattern genes and promotes a metastatic malignancy.

overall_design:
Bulk RNA-seq was performed on microdissected RNA samples from 3 lung adenocarcinoma tissues containing micropapillary and acinar components. RNA-seq was also performed on A549 cell line samples subjected to the following manipulations: 1) transfection with pcDNA3 control plasmid, 2) transfection with MYC overexpression plasmid, 3) transfection with pcDNA3 control plasmid and co-culture with M2-like macrophages for 72 hours, 4) transfection with MYC overexpression plasmid and co-culture with M2-like macrophages for 72 hours.
Anti-H3K27ac CUT&Tag-seq was performed on microdissected samples from 1 lung adenocarcinoma tissues containing micropapillary and acinar components. A549 cell line samples were subjected to the following manipulations: 1) transfection with pcDNA3 control plasmid, 2) transfection with MYC overexpression plasmid, 3) transfection with pcDNA3 control plasmid and co-culture with M2-like macrophages for 72 hours, 4) transfection with MYC overexpression plasmid and co-culture with M2-like macrophages for 72 hours.
Anti-cMyc ChIP-seq was performed on microdissected DNA samples from 1 lung adenocarcinoma tissue containing a micropapillary component as well as on DNA samples from A549 cell line transfected with MYC overexpression plasmid and co-cultured with M2-like macrophages for 72 hours.

first: Gaochao

last: Dong

first: Lin

first: Xuming

last: Song


title: scRNA-seq of lymph node stromal cells from naïve mice and heart allograft transplanted mice with/without anti-CD40L

city: Baltimore

first: yang

last: song

state: MD

address: HSF-3, 3rd Floor; 670 W Baltimore St.

department: Department: Institute for Genome Sciences

institute: University of Maryland in Baltimore

summary: Lymph node stromal cells (LNSCs) are the master regulator of the LN microenvironment. Single-cell genomics has revealed a multitude of LNSCs subsets, each playing unique and critical roles in immune responses and tissue homeostasis. However, the role of specific LNSC subsets controlling tolerance within draining LNs (DLNs) in heart allograft transplanted recipients remains to be explored. In order to assess the phenotype of LNSCs and effect of LNSCs on transplant tolerance or allograft rejection after heart transplantation, we performed single cell RNA sequencing (scRNA-seq) of LNSCs from naive mice and heart allograft transplanted mice with/without anti-CD40L (day8 after heart transplantation, anti-CD40L (250 μg, day 0)).

overall_design: In order to investigate the effect of heart transplantation (HTx) on LNSC phenotypes, three groups were prepared (naïve C57BL/6 mice, C57BL/6 mice after HTx, and anti-CD40L-treated C57BL/6 mice after HTx). C57BL/6 mice were transplanted with heart allograft from BALB/c mice. Recipients of CD40L-treated group were treated with anti-CD40L (250 μg, day 0). On day8, drainage LNs were hervested and enzymatically digested. CD45- cells were purified with the CD45 negative selection beads and were stained with anti-mouse CD45 before sorting for viable CD45- cells. For scRNA-seq, about 1.0 x 104 CD45- LNSCs were run on the 10X Chromium Controller (10X Genomics) to partition single cells into nanoliter-scale droplets containing uniquely barcoded beads and processed for sequencing library preparation using the Chromium Single Cell 3’ Reagent Kit (v3 chemistry) (10x Genomics, Pleasanton, CA). Generated cDNA libraries were sequenced on a NovaSeq 6000 sequencing system. Reads were demultiplexed, aligned to the GRCm38 mm10 assembly reference genome, and filtered; and cell barcodes and UMIs were quantified using the Cell Ranger 6.1 with default parameters (https://support.10xgenomics.com/single-cell-gene-expression/software/overview/welcome).

first: Jonathan

last: Bromberg

first: Yang

first: Reza

last: Abdi

first: Yuta

last: Yamamura


title: Redundant MYC orchestrates M2-like macrophages induced chromatin remodeling to sustain micropapillary-pattern malignancy in Lung Adenocarcinoma

summary: Current comprehension of micropapillary (MP)-subtype lung adenocarcinoma (LUAD) remains circumscribed to biological behaviors and genomic landscapes. Unraveling the core regulatory programs underlying MP-pattern malignancy offers opportunities to identify more feasible therapeutic targets for patients with MP-LUAD. We show aberrant activation of the MYC pathway in MP-subtype LUAD compared with other subtypes. MP-pattern malignancy cannot be solely induced by aberrant MYC expression in vitro or xenograft mouse models but requires the assistance of M2-like macrophages to accomplish. Redundantly expressed MYC aggregates M2-like macrophages derived from the bone marrow to secrete TGFβ, inducing the expression of FOSL2 in tumor cells, thereby remodeling chromatin accessibility in MP-pattern gene promoters to assist MYC in occupying de novo transcriptional regulation. Moreover, effective alleviation of MP-pattern malignancy can be achieved by disrupting the TGFβ-FOSL2 axis. These findings elucidate functions of M2-like macrophage-TGFβ-FOSL2 axis in MYC-redundant MP-subtype LUAD, defining targetable vulnerabilities.


title: A TBX2-driven signaling switch from Androgen Receptor to Glucocorticoid Receptor confers therapeutic resistance in Prostate Cancer

city: Lubbock

first: Srinivas

last: Nandana

state: TX

address: CBB Lbk Genl 3601 4th St Stop 6540

department: Cell Biology Biochemistry

institute: Texas Tech University Health Science Center

summary: Recent studies suggest that glucocorticoid receptor (GR) activation can cause enzalutamide resistance in advanced prostate cancer (PCa) via functional bypass of androgen receptor (AR) signaling. However, the specific molecular mechanism(s) driving this process remain unknown. We previously reported that the transcription factor TBX2 is over-expressed in castrate resistant prostate cancer (CRPC). Our current study demonstrates that TBX2, which has known repressor and activator functions, is a molecular switch that represses AR levels while inducing GR expression to bypass AR-signaling. Mechanistically, our studies revealed that TBX2 binds to the promoters of both AR and GATA2, an AR coregulator, thereby resulting in a two-tiered repression of AR expression.  Concurrently, TBX2 upregulated the GR via direct GR promoter binding and TBX2-GR protein-protein interaction. Together, TBX2-driven repression of the AR and activation of GR resulted in enzalutamide resistance. Significantly, we report that SP2509, an allosteric inhibitor of the demethylase-independent function of LSD1, a TBX2-interacting protein in the COREST complex, disrupted both TBX2-LSD1 and TBX2-GR protein-protein interactions thereby uncovering a unique mode of SP2509 action in CRPC. Taken together, our study provides key insights into a potential therapeutic modality for targeting the AR- to GR- signaling switch via disruption of TBX2-GR and TBX2-LSD1 protein-protein interactions.

overall_design:
To investigate the effects of genetic modulation of TBX2 in human prostate cancer cells,  we blocked endogenous TBX2 in PC3 human prostate cancer cell lines using a dominant negative construct (TBX2DN) resulting in PC3TBX2DN cells and compared gene expression with the control PC3Neo cells. These cell lines were created and maintained in DMEM with 10%FBS , 1% PS and 5µg/ml puromycin.
This was followed by RNA extraction in triplicates for each of the two cell lines i.e. PC3TBX2DN and the PC3Neo control followed by comparative gene expression from the data obtained from RNA-Seq analysis.

first: Sayanika

last: Dutta

first: Girijesh

last: Patel

first: Hamed

last: Khedmatgozar

first: Manisha

last: Tripathi


title: Mapping microRNA-target interactions in rare cell types with Spy3-AGO2 Pull-down

city: San Diego

first: Norjin

last: Zolboot

address: 10550 North Torrey Pines Rd, DNC-222

department: Neuroscience

institute: The Scripps Research Institute

summary: To illuminate the molecular mechanisms driving neuronal differentiation we generated a mouse line amenable to mapping miRNA-target interactions in rare cell types. Biochemical approaches to purify AGO2-miRNA-target complexes have successfully mapped MTIs in abundant populations of neurons. However, due to their technical complexity and high background, these approaches are not suitable for mapping interactions in rare cell populations such the many neuronal subtypes that compose the mammalian brain. We therefore generated a mouse line with a conditional SpyTag3, which is small and offers near-infinite affinity for pull-downs, in the endogenous Ago2 gene. We then developed a method Spy3-AGO2 pull-down and sequencing (SAPseq), which we have used to accurately map miRNA-target interactions in developing Purkinje cells, a rare population of cells in the cerebellum.

overall_design: To identify the miRNA-target interactions occuring in developing cerebellar Purkinje cells (PCs) we delivered PC-specific AAV expressing Cre to conditional Spy3-AGO2 mouse pups at P0 and then isolated their cerebella at P7 and P15. We then performed AGO2 pull-down using a SpyCatcher3 resin followed by RNA sequencing to generate miRNA libraries and target libraries

first: Yao

last: Xiao

first: Ian

last: MacRae

first: Giordano

last: Lippi


title: Cytokine, cfDNA levels and tumoral DNA profiling in plasma as biomarkers of response to immunotherapy in hepatocellular carcinoma

city: Derio

first: Ana

middle: Maria

last: Aransay

state: BIZKAIA

address: Parque tecnologico de Bizkaia, Building 801-A

department: Genome Analysis Platform

country: Spain

institute: CIC bioGUNE

type: Genome variation profiling by high throughput sequencing

summary: Background: Immunotherapy has increased the expected survival of patients with advanced HCC. However, objective radiological response to these therapies has been reported to occur in around 20% of patients. Our aim was to identify potential serological markers of response to ICIs. Methods: 25 patients with advanced HCC treated with immunotherapy were prospectively in-cluded. Cytokine and cfDNA/ctDNA levels were measured prior to first treatment administration (basal) and after 3 months of treatment. Basal ctDNA profiling was also analyzed. Results: Basal levels of CTLA-4, cfDNA and ctDNA were significantly different in patients presenting progres-sive disease as best radiological response. The percentage of patients with basal mutations in CDKN2A was significantly higher in patients presenting progressive disease and they have a significantly lower number of CNV. Levels at 3 months of starting the treatment of MCP-1, TNF-alpha, cfDNA and ctDNA were also significantly different between patients with and without progressive disease. Higher cfDNA and ctDNA levels were associated with a poorer overall survival. Conclusion: Analysis of cfDNA and cytokines could help to stratify patients according to expected response to immunotherapies.

overall_design: Onco-500 TruSight was performed in basal samples (plasma and whole blood) of 21 patients with HCC treated with immunotherapy.

first: Elena

last: Vargas-Accarino

first: Mónica

last: Higuera

first: María

last: Bermúdez

first: Mònica

last: Pons

last: Torrens

first: Jose

middle: E

last: Martín

first: Xavier

last: Merino

first: Beatriz

last: Mínguez


title: Senescence prostate cancer cell promotes resistance to immune checkpoint blockade through ATR activation

city: Tianjin

first: Yu

last: Zhao

state: Tianjin

address: 238 Baidi Road

department: Institute of Radiation Medicine

institute: Chinese Academy of Medical Sciences and Peking Union Medical College

summary: Background: Checkpoint blockade immunotherapy represented by PD-1/PD-L1 or CTLA4 antibody treatment, has been of tremendous success for multiple cancers. Most patients with prostate cancer (PCa) either do not respond to CTLA4 immune checkpoint blockade or develop resistance to it, often because of low CTLA4 antigen presentation in cancer cells.

overall_design: Conclusions: We demonstrate that senescence-related ATR activation plays a vital role in regulating CTLA4 expression and immune response of PCa, which may provide a general strategy to overcome immunotherapy resistance.

first: Chenyi

last: Shao


title: ROCK1 orchestrates B cell differentiation in response to PAMPs and heme by controlling the heme-regulated proteins Bach2 and HRI

city: Brooklyn

first: Juan

last: Rivera-Correa

state: NY

address: 285 Jay st, Room A-302-E

institute: NYC College of Technology

summary: Protective humoral responses require that B cells successfully complete their differentiation programs even when exposed to hostile environments generated during severe infections, like the massive hemolysis triggered by malaria. The mechanisms utilized by differentiating B cells to withstand damaging conditions replete in PAMPs and DAMPs are poorly understood. Here we demonstrate that the serine-threonine kinase ROCK1 enables B cells to execute their differentiation programs upon exposure to PAMPs and high levels of heme, a critical DAMP, by controlling two key heme-regulated molecules, Bach2 and HRI. ROCK1 restrains plasma cell differentiation by phosphorylating Bach2. As B cells differentiate in the presence of PAMPs and heme, furthermore, ROCK1 limits the proinflammatory potential of B cells and restrains mTORC1 activity by controlling the assembly of multimolecular complexes that contain the adaptor p62, raptor, ripoptosome components, and molecules involved in RNA metabolism and proteostasis. ROCK1 regulates formation of these complexes by controlling the interplay between HSPs and the stress kinase HRI. Thus, ROCK1 helps B cells cope with intense pathogen-driven destruction by coordinating the activity and localization of key molecules that mediate cell-fate decisions, effector functions, and RNA and protein homeostasis. These ROCK1-dependent mechanisms may be widely employed by cells to handle severe environmental stresses and these findings may be broadly relevant for infections, vaccine development, and immune-mediated diseases marked by chronic tissue damage like autoimmune disorders.

overall_design: Mice with B cell-specific deletion of ROCK1 (CD23-Cre.Rock1flox/flox) were subjected to Plasmodium yoelii 17XNL (P. yoelii), a non-lethal malaria infection model characterized by severe transient anemia. Rock1flox/flox mice were employed as controls. Immune responses were monitored by FACS at various timepoints and histopathological analyses. Antibody responses and cytokine production were assessed by ELISA. The molecular mechanisms employed by ROCK1 to regulate B cell differentiation were examined by FACS-sorting B cell populations from spleens followed by RNA-sequencing analyses and in vitro experiments coupled with transcriptomic, phospho-proteomic, and biochemical strategies.

first: Alessandra

last: Pernis


title: Gene expression in small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) cell line COV434

city: Madison

first: Jessica

middle: Diane

last: Lang

state: Wisconsin

address: 1111 HIGHLAND AVE WIMR 2766

department: Pathology and Laboratory Medicine

institute: University of Wisconsin-Madison

summary: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare and aggressive form of ovarian cancer. SCCOHT tumors have inactivating mutations in SMARCA4 (BRG1), one of the two mutually exclusive ATPases of the SWI/SNF chromatin remodeling complex. To address the role that SMARCA4 loss plays in SCCOHT tumorigenesis, we performed RNA-seq in a SCCOHT cell line +/- SMARCA4 re-expression. SMARCA4 re-expression was acheived by integration of pIND20-BRG1 and inducing with doxycycline. The goal of this study was to generate a high sequencing depth RNA-seq dataset to determine how restoration of SMARCA4 results in changes in gene expression and RNA splicing.

overall_design: COV434 pIND20 BRG1-2.1 and parental COV434 cell lines were induced as previously described (PMID:29440177) with and without doxycycline to re-express SMARCA4. Experiment was repeated in independent triplicate.

first: Lang

last: Jessica

first: Raupach

last: Elizabeth

first: Pirrotte

last: Patrick

first: Trent

last: Jeffrey


title: ATAC-seq to investigate the effect of DGCR8 knockout and heterozygosity DNA accessibility in PA-1 cells

city: Edinburgh

first: Silvia

last: Peluso

state: Midlothian

address: Western General Hospital, Crewe Road

institute: MRC HGU at the University of Edinburgh

summary: High-throughput profiling to study the effect of DGCR8 heterozygosity on genome-wide chromatin accessibility.

overall_design: Comparison between ATAC-seq samples from wildtype and DGCR8 heterozygous PA-1 cells.

first: Sara

last: Ribela


title: Transcriptional profile of brain region involved in habitual learning following methamphetamine self-administration and dopamine receptor antagonist

first: Michael

last: McCoy

state: Maryland

address: Molecular Neuropsychiatry Branch, 251 Bayview Blvd. Suite 200, 251 Bayview Blvd. Suite 200, 251 Bayview Blvd. Suite 200

institute: NIDA

summary: Methamphetamine (METH) is a powerful stimulant that has caused addiction (compulsive drug seeking and taking behavior) in millions of people world-wide. METH abuse is also associated with negative impact on the brain. One feature of addiction is uncontrollable drug seeking despite adverse consequences and becomes habitual. To mimic this in a rat model, rats with a history of METH use are given the opportunity to earn METH accompanied by aversive shocks on their feet. Rats that continue to take METH are shock-resistant (SR) and rats that reduce their METH intake are shock-sensitive (SS ).Rats that self-administered saline are controls (CT). Thereafter, rats were injected intraperitoneally with the dopamine D1 receptor antagonist, SCH23390. SCH23390 caused substantial reduction of METH taking in a dose-dependent fashion. Stopping SCH23390 administration led to re-emergence of compulsive METH taking in the shock-resistant rats.

overall_design: RNA-seq

first: Subramaniam

last: Jayanthi

first: Jean

last: Cadet


title: Whole-genome transcriptome profiling of dark-grown cryptochrome 2 over-expressing indica rice seedlings

city: Agra

first: Sushma

last: Mishra

state: Uttar Pradesh

address: Dayalbagh

department: Botany

country: India

institute: Dayalbagh Educational Institute, Agra, India

summary: We found that indica rice cryptochrome 2 overexpression (OsiCRY2) seedlings exhibited partial photomorphogenesis in dark. Therefore, we decided to use microarray technique to determine the genes whose expression would have been altered in dark.

overall_design: Whole-genome transcriptome profiling of 7-d-old  dark-grown transgenic seedlings vis-a-vis wild-type  was performed in duplicates using the GeneChip® Rice Affymetrix microarray.  Genome Array

first: Pooja

last: Sharma

first: Jitendra

last: P. Khurana


title: Neutrophil Extracellular Traps Mediate Acute Liver Failure in Regulation of miR-223/neutrophil Elastase Signaling in Mice

first: DEWEI

last: YE

state: Guangdong

address: 280 Waihuan East Road, Guangzhou Higher Education Mega

institute: Guangdong Pharmaceutical University

summary: Marked enhancement of neutrophil infiltration of the liver is a hallmark of acute liver failure (ALF), a severe life-threatening disease with varying etiologies. However, the mechanisms underlying the regulation of neutrophil entry into the liver and their pathophysiological role during ALF development remain poorly characterized.

overall_design: Comparative gene expression profiling analysis of RNA-seq data using bone marrow cells from miR-223 KO and WT control mice

first: Dewei

last: Ye

first: Jianyu

last: Yao

first: Wenfa

last: Du

first: Cuishan

last: Chen