title: TAZ-mediated enhancement of adipogenesis in goat adipose mesenchymal stem cells induced by TNF-α

status: Public on Oct 17 2024

city: Hohhot

first: Dongjun

last: Liu

state: Inner Mongolia

address: 235 Daxue west road  Saihan District

institute: Inner Mongolia University

summary: Obesity is a clinical condition characterised by a chronic, low-grade inflammatory state. Adipose-derived mesenchymal stem cells (ADSCs) have a multidirectional differentiation potential to adipocytes. However, the relationship between adipogenesis and inflammation is not yet fully understood.TAZ (WW domain containing transcription regulator 1) can act as a molecular rheostat to fine-tune the osteoblast and adipocyte differentiation balance. In this study, we investigated the role of TAZ in the adipogenesis of Arbas cashmere goat ADSCs (gADSCs). We observed elevated TAZ expression levels during the adipogenesis of gADSCs. Overexpression and knockdown of TAZ were performed in stably transfected gADSCs cell lines, and RNA-seq analysis revealed that TAZ was associated with adipogenesis. TAZ overexpression promoted adipose differentiation of gADSCs, while TAZ knockdown had the opposite effect. In addition, TAZ overexpression and knockdown altered the expression of YAP1 (Yes1 associated transcriptional regulator) and TAZ in the nucleus. TAZ regulated the mRNA expression level and secretion level of tumor necrosis factor (TNF)-α, promoting adipose differentiation of gADSCs. Notably, TNF-α counteracted the inhibitory effect of sh-TAZ on the adipose differentiation of gADSCs. Collectively, these findings suggest that TAZ regulates adipogenesis in gADSCs by modulating TNF-α, which is of particular significance in several metabolic disorders where the amount or function of adipose tissue is altered.

overall_design: To investigate the effect of TAZ on adipogenesis of gADSCs, we constructed gADSCs cell lines OE-TAZ. RNA-seq was performed with OE-TAZ and Control cells. Based on the RNA-seq data, an expression profile was generated.


title: Identification of Myc-dependent and -independent Notch2 signatures in marginal zone B cells.

status: Public on Oct 18 2024

city: Philadelphia

first: Brian

middle: Thomas

last: Gaudette

state: PA

address: 3620 Hamilton Walk, Pathology and Laboratory Medicine

department: Pathology and Lab Medicine

country: USA

institute: University of Pennsylvania

type: Expression profiling by high throughput sequencing

summary: Marginal zone (MZ) B cells leverage Notch2 signals to attain rapid differentiation responses and positional cues with in the spenic environment. While it is known that a major player in Notch2 signaling in MZ B cells is the proto-oncogene Myc, it is not clear to what extent MZ B cells use Notch2 signals in Myc-independent manner. In this study we conditinally and inducibly deleted Myc in mature B cells and then used in vivo delivery of anti-Notch2 antibodies to separated Myc-dependent and -independent Notch2 signaling in MZ B cells.

overall_design: Myc-floxed mice were mated to hCD20Tam-Cre mice to conditionally and inducibly delete Myc in B cells following tamoxifen treatment. Myc conditional knockout (MycFL/FL.hCD20Tam-Cre mice and Myc+/+.hCD20Tam-Cre  control animals were fed tamoxifen in their diet for 4 weeks and then treated with anti-Notch2 blocking antibodies or isotype control antibodies for 24 hours prior to sacrifice. Marginal zone B cells (CD19+, CD93-, CD1d+, CD23-) and follicular B cells (CD19+, CD93-, CD1d-, CD23+) were purified by FACS sorting and RNA-sequencing library generation.

first: Anneka

last: Allman

middle: T

first: David

first: Ivan

last: Maillard


title: The effects of serotonin by Tph1 on skeletal muscle

city: Buk-gu, Gwangju

first: Suhyeon

last: Park

state: Gwangju

address: 광주광역시 북구 첨단과기로 123 다산빌딩 403호

country: South Korea

institute: GIST

summary: Reducing circulating serotonin by inhibition of Tph1 increases the sensitivity of BAT cells and this drives thermogenesis by fat and glucose oxidation. Here we report Insulin sensitivity changes by regulating Serotonin on skeletal muscle.  Improved glucose tolerance and insulin sensitivity in HFD Tph1 KO mice. Inhibiting Tph1 increases AMPK activity, glucose uptake, myofiber size and decreases lipid droplet accumulation in HFD mice skeletal muscle. Inhibiting Tph1 in muscle showed activation of SIRT1/LKB1/AMPK pathway in skeletal muscle cells and increased p-ACC. Theses results indicates that protected insulin resistance and myosteatosis on skeletal muscle by lack of Serotonin by Tph1 on skeletal muscle.

overall_design: Non-pathogenic mouse strains on a C57BL/6 background; expressing cre recombinase under the Mlc(Myosin Light Chain) promoter in which the exon 4,5 of the Tph1 gene is floxed.

first: Changmyung

last: Oh


title: Genetic changes through spheroid formation in ascites - interaction with ovarian cancer cell line, OV90, and human primary mesothelial cells (HPMC)-

city: Lund

first: Kaname

last: Uno

address: Medicon Village Building 404:B3

department: Clinical Sciences

country: Sweden

institute: Lund University

summary: More than 70% of patients with epithelial ovarian cancer (EOC) are diagnosed with peritoneal metastasis and ascites, which is the accumulation of intraperitoneal fluid containing non-malignant cells. However, the interactions between EOC and non-malignant cells before the development of peritoneal metastasis remain unclear. This study investigated the mechanism by which EOC cells survive through interactions with the surrounding cells in ascites. Whole EOC spheroids were observed, and their invasion into collagen layers and mesothelial monolayers was examined. RNA sequencing revealed the molecular mechanisms associated with aggressiveness of EOC cells. In malignant ascites, almost 100% of EOC cells were spheroids, 60% of which contained mesothelial cells. EOC cells quickly generated aggregated spheroids with mesothelial cells, and these aggregated cancer-mesothelial spheroids (ACMS) rapidly invaded collagen or mesothelial layers. Mesothelial cells forming ACMS initiated the invasion, with EOC cells following the route created by the mesothelial cells. RNA sequence revealed dramatically RNA expression changes in mesothelial cells, whereas the changes in EOC cells were relatively small. TGF-β1-stimulated mesothelial cells showed increased invadopodia formation along with fascin-1 upregulation. These findings suggest that EOC cells alter gene expression in mesothelial cells through ACMS formation, which explains the rapid spread of EOC in the abdominal cavity.

overall_design: We used OV90-GFP cells and three different kinds of primary human mesothelial cell (HPMC). To investigate the genetic changes through spheroid formation, three types of cell components were  incubated for 72 hours to form spheroids in ultra-low attachement 96-well round-bottome plates; (i) OV90-GFP only (2,000 cells/well), (ii) HPMC cells only (4,000 cells/well), (iii) a co-culture of OV90-GFP and HPMC cells (2,000 cells/well each). Before analyzing, these spheroids were disaggregated with trypsin and collected by FACS. Comparative gene expression profile analysis of RNA-seq data for (i) OV90-GFP cells with or without interaction with HPMC, and (ii) HPMC cells with or without interaction with OV90-GFP.

first: Yoshihiro

last: Koya

first: Mai

last: Sugiyama


title: Identification of Notch ligand useage by splenic B cells using Dll1 and Dll4 Notch ligand blockade.

summary: Marginal zone (MZ) B cells leverage Notch2 signals to attain rapid differentiation responses and positional cues with in the spenic environment.To determine the likely molecular source of Notch2 signals we sought to block the Notch ligands delta-like ligand 1 (Dll1) and delta-like ligand 4 (Dll4) in wildtype mice using in-vivo treatment with monoclonal blocking antibodies. Splenic populations of MZ B cells and follicular B cells were sort purified after 12, 24 and 48 hours of blockade and analyzed by RNA-seq.

overall_design: C57BL/6 (10-12 weeks of age) were treated with anti-Dll1, anti-Dll4  blocking antibodies or isotype control antibodies for 12-48 hours prior to sacrifice. Marginal zone B cells (CD19+, CD93-, CD1d+, CD23-) and follicular B cells (CD19+, CD93-, CD1d-, CD23+) were purified by FACS sorting and RNA-sequencing library generation.


title: RNA sequencing of MC38 tumor cells from control-diet- and high-fat-diet-fed C57BL/6J mice

city: San Mateo

first: Sreya

last: Bagchi

state: CA

address: 1202 S Claremont Street

institute: Stanford University

summary: Multiple cancers, including colorectal cancer (CRC), are more frequent and often more aggressive in obese individuals. Here, we show that macrophages accumulate within tumors of obese CRC patients and in obese CRC mice and promote accelerated tumor growth. These changes are initiated by oleic acid accumulation and subsequent tumor cell-derived acid production, and driven by signaling through GPR65, an acid-sensing receptor on CRC-associated macrophages. We demonstrate a similar role for GPR65 in hepatocellular carcinoma (HCC) in obese mice. Tumors in obese patients with CRC or HCC also exhibit increased GPR65 expression, suggesting that the mechanism revealed here likely contributes to tumor growth in a range of obesity-associated cancers and represents a potential therapeutic target.

overall_design: Gene expression profiling of MC38 tumor cells, characterized as CD45-negative cells and dTomato-positive via sorting, from 5 control-diet-fed and 5 high-fat-diet-fed mice 21 days post implantation.

first: Robert

last: Yuan

first: Edgar

last: Engleman


title: RNA sequencing of tumor-associated macrophages from control-diet- and high-fat-diet-fed C57BL/6J mice

overall_design: Gene expression profiling of tumor-associated macrophages, characterized as CD45/CD11b/F4/80-positive cells via sorting, from control-diet- and high-fat-diet-fed mice 21 days post implantation.


title: RNA sequencing of tumor-associated macrophages from Gpr65-wild-type and -knockout C57BL/6J mice fed with high-fat diet

overall_design: Gene expression profiling of tumor-associated macrophages, characterized as CD45/CD11b/F4/80-positive cells via sorting, from Grp65-wild-type and Grp65-knockout high-fat-diet-fed mice 21 days post implantation.


title: GLS2 links glutamine metabolism and atherosclerosis by remodelling artery walls (aorta).

city: Paris

first: Sebastien

last: Dussaud

address: 91 bd de l'hopital

country: France

institute: UPMC

summary: Metabolic dysregulation, including perturbed glutamine-glutamate homeostasis, is common in atherosclerotic cardiovascular disease. Here, we reveal that modulation of glutaminolysis or glutamate availability in culture media is critical for smooth muscle cell line (MOVAS) phenotypic switching. Cells were treated for 24 hours with glutaminase inhibitors (50mM DON, Sigma-SML0601) in presence or absence of 2mM glutamate suplementation (Gibco-35050061). High-throughput transcriptional profiling revealed that modulation of glutamine and glutamate homeostasis impacted genes involved in protein and extracellular matrix organization, cell motility and microtubule. Thus, we uncovered a novel role for glutamine metabolism in the phenotypic switch in SMCs, a hallmark of vascular remodelling.

overall_design: Atherosclerosis-prone Ldlr-/- and Apoe-/- mice were backrossed with or without Gls2-/- mice. Then, hyperlipidemia was induced in these mice by feeding them with an atherogenic diet (Western diet, TD88137, Ssniff) for 12 weeks. Livers were obtained from all four genotypes and aortas were collected from Western died-fed Ldlr-/- and Ldlr-/-Gls2-/- mice.

first: Florent

last: Murcy

first: Coraline

last: Borowczyk

first: Thibault

last: Barouillet

first: Sébastien

first: Laurent

last: Yvan-Charvet


title: Gene expression profile at single nuclei resolution of aortas from Ldlr-/- mice with or without defective hepatic glutaminolysis

city: Valbonne - Sophia-Antipolis

first: Kevin

last: Lebrigand

address: 660 route des lucioles

institute: IPMC/CNRS

summary: Metabolic dysregulation, including perturbed glutamine-glutamate homeostasis, is a hallmark of atherosclerotic diseases. We used single nuclei RNA sequencing (snRNA-seq) to analyze the impact of defective hepatic glutaminolysis on cell diversity in atherosclerotic aorta.

overall_design: single-nuclei transcriptomics (snRNA-seq) from control and Ldlr-/-Gls2shRNA aortas


title: PfSnf2L - an epigenetic regulator of just-in-time expression and gametocyte formation

city: Regensburg

first: Simon

last: Holzinger

address: Universitätsstraße 31

country: Germany

institute: University of Regensburg

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Mapping diverse RNA modifications simulataneously by proximity barcode sequencing

city: San Diego

first: Gudrun

last: Stengel

address: 11535 Sorrento Valley Rd Suite 407

institute: AlidaBio

summary: RNA is subject to a multitude of different chemical modifications that collectively represent the epitranscriptome. Individual RNA modifications including N6-methyladenosine (m6A) on mRNA play essential roles in the posttranscriptional control of gene expression. Recent technological advances have enabled the transcriptome-wide mapping of certain RNA modifications, to reveal their broad relevance and characteristic distribution patterns. However, convenient methods that enable the simultaneous mapping of multiple different RNA marks within the same sample are generally lacking. Here we present EpiPlex RNA modification profiling, a bead-based proximity barcoding assay with sequencing readout that expands the scope of molecular recognition-based RNA modification detection to multiple targets, while providing relative quantification and enabling low RNA input. Measuring signal intensity against spike-in controls provides relative quantification, indicative of the RNA mod abundance at each locus. We report on changes in the modification status of HEK293T cells upon treatment with pharmacological inhibitors separately targeting METTL3, the dominant m6A writer enzyme, and the EIF4A3 component of the exon junction complex (EJC). The treatments resulted in decreased or increased m6A levels, respectively, without effect on inosine levels. Inhibiting the helicase activity of EIF4A3 and EIF4A3 knockdown both cause a significant increase of m6A sites near exon junctions, consistent with the previously reported role of EIF4A3 in shaping the m6A landscape. Thus, EpiPlex offers a reliable and convenient method for simultaneous mapping of multiple RNA modifications to facilitate epitranscriptome studies  .

overall_design: The m6A and Inosine detection in METTL3 knockdown or inhibition, EIF4A3 knockdown, and ADAR knockout HEK293T cells. DMSO and siRNA controls are included in each set of experiments. Each condition and controls contain two biological replicates for the epitranscriptome measurement.

first: Erdem

last: Sendinc

first: Hua

last: Yu

first: Yu-Hsien

last: Hwang-Fu

first: Jerome

last: Santos

first: Zachary

last: Johnson

first: Justin

middle: R

last: Kirstein

first: Jiani

last: Niu

first: Michael

middle: B

last: Chabot

first: Vito

middle: A

last: Cantu

first: Željko

last: Džakula

first: Quan

last: Lam

first: Ananya

last: Anmangandla

first: Timothy

middle: S

last: Burcham

middle: D

last: Miles

first: Andrew

last: Price

first: Byron

middle: W

last: Purse

first: Richard

middle: I

last: Gregory


title: PfSnf2L - an epigenetic regulator of just-in-time expression and gametocyte formation [RNA-seq]

first: Simon

last: Holzinger

summary: The complex life cycle of the malaria parasite Plasmodium falciparum (Pf) and the accompanying transcriptional variation is only explained in part by stage-specific transcription factors like the ApiAP2 family, as these factors are strongly under-represented in Pf. Global and local changes in chromatin structure during life cycle progression suggest the contribution of epigenetic mechanisms as a critical mechanism for fine-tuning gene regulation during malaria parasite development. Pf chromatin is largely unexplored and is distinct from other eukaryotes in many respects. This is likely to accommodate the highly A/T-rich genome which averages over 90% in non-coding regions, the highest of any known eukaryote. We characterized PF3D7_1104200 (PfSnf2L), an ATPase-containing protein that is related to ISWI-type chromatin remodeling enzymes (CREs). Using a conditional knockout (KO) system, we demonstrate that PfSnf2L is essential for parasite development, globally controls just-in-time transcription regulation, and specifically represses gametocyte-specific gene expression. Characterization of the enzymatic activity in vitro, combined with mapping chromatin organization in vivo revealed that PfSnf2L shapes the promotor architecture of stage-specific genes through its nucleosome remodeling activity, thereby influencing gene activation and repression. The unique properties of the Plasmodium Snf2L allowed screening and identification of a specific inhibitor that specifically kills the parasite, phenocopies the loss of correct gene expression timing, and inhibits the formation of gametocytes.

overall_design: Comparative time-resolved gene expression profiling analysis RNA-seq data of 3D7 parasites with PfSnf2L-KO induced vs. non induced, for two different induction timepoints.   Comparative time-resolved gene expression profiling analysis of RNA-seq data of 3D7 parasites treated with a PfSnf2L-inhibitor vs. untreated.

first: Maria

last: Watzlowik

first: Elisabeth

last: Silberhorn

first: Sujaan

last: Das

first: Gernot

last: Laengst


title: Cross-talk between enhancers, structural elements and activating transcription factors maintains the 3D architecture and expression of the CFTR gene.

city: Cleveland

first: Ann

last: Harris

state: Ohio

address: 2109 Adelbert Rd Building

department: Genetics and Genome Sciences

institute: Case Western Reserve University

summary: Here we examine the mechanisms by which multiple cis-regulatory elements (CREs) at the CFTR locus coordinate its expression in intestinal epithelial cells. Using CRISPR/Cas9 to remove CREs, individually and in tandem, followed by assays of gene expression and higher-order chromatin structure (4C-seq), we reveal the cross-talk and dependency of two cell-specific intronic enhancers. The results also suggest an internal monitoring mechanism at the locus whereby alternative enhancers may be recruited when the normal sites are lost. Of note, activating transcription factors such as FOXA2 may contribute to this mechanism.  Also, by deleting the 5' topologically-associating domain (TAD) boundary, we illustrate its relative contribution to normal mechanisms of CFTR gene expression and 3D gene architecture.

overall_design: Analysis of the 3D chromatin architecture at the CFTR locus to determine interaction frequencies between elements which lead to changes in CFTR gene regulatory patterns.

first: Shiyi

last: Yin

first: Monali

last: NandyMazumdar

first: Alekh

last: Paranjapye


title: Multi-organ single-cell RNA-sequencing of early hyperglycaemia responses

city: Oxford

first: Robin

last: Choudhury

address: L6 West Wing, John Radcliffe Hospital, Headley Way

department: Cardiovascular Medicine, Radcliffe Department of Medicine

country: United Kingdom

institute: University of Oxford

summary: Diabetes causes a range of complications that can affect multiple organs. Hyperglycaemia is an important driver of diabetes-associated complications, mediated by biological processes such as dysfunction of endothelial cells, fibrosis and alterations in leukocyte number and function. Here, we dissected the transcriptional response of key cell types to hyperglycaemia across multiple tissues using single-cell RNA-seq.

overall_design: Wild-type C57BL/6J (12-14 weeks old) control or diabetic experimental groups. Diabetes was induced through an intra-peritoneal injection of streptozotocin (STZ) at a low dose range (42 – 45 mg / kg / day) over 5 consecutive days. After 6 weeks post-final STZ injection, diabetic mice with non-fasted blood glucose levels >13.9 mmol/L were sacrificed and tissue collected. Cells were isolated from heart, kidney, liver and spleen and assessed by single-cell RNA-sequencing.

first: Adam

last: Braithwaite

first: Naveed

last: Akbar

middle: P


title: Follicle rupture and luteinization during mammalian ovulation are informed by functionally and molecularly distinct regions of the ovarian follicle wall

city: Somerville

first: Daniela

middle: Dolores

last: Russo

state: Massachusetts

address: 5 Russell Rd

institute: Ragon Institute of MGH, MIT and Harvard

summary: Ovulation refers to the process when the ovarian surface-facing wall of a preovulatory follicle ruptures and releases the cumulus oocyte complex (COC) into the oviduct or fallopian tube in response to hormonal cues. In parallel, the unruptured wall of the follicle within the ovary transitions to becoming a progesterone-producing corpus luteum.  Ovulation is essential for fertilization and eventual pregnancy. Disruption of ovulation, whether purposefully through contraceptive intervention or idiopathically in cases of anovulatory infertility, has translational implications for human health. Importantly, key processes of ovulation, including follicle rupture and luteinization, are recapitulated in models of ex vivo ovulation despite the absence of an intact hypothalamic-pituitary-gonadal axis and intra-ovarian cues. In our study, we used an ex vivo ovulation model to identify functional and molecular differences between distinct regions of the follicle wall, which we refer to as the ruptured and unruptured sides. We observed that the unruptured side of the follicle wall exhibits hallmarks of luteinization after ovulation while the ruptured side exhibits signs of cell death. RNA-sequencing of these specific follicle regions revealed 2,099 differentially expressed genes between follicle sides without hCG exposure and 1,673 between follicle sides 12 hours post-hCG, which were further validated in vivo. We found enriched pathways that recapitulate known ovulation biology, including oxidative stress on the ruptured side and angiogenesis on the unruptured side. We also identified previously unappreciated pathways that may play an important role in ovulation, such as amino acid transport and Jag-Notch signaling on the ruptured side, as well as metal ion processing and IL-11 signaling on the unruptured side. Ultimately our studies demonstrate that our ex vivo model recapitulates known in vivo ovarian biology, identifies pathways that may be novel regulators of ovulation and luteinization, and may have future translational applications for the study of ovulatory disorders and the development of novel non-hormonal contraceptives.

overall_design: Single oocytes and somatic cells from CD-1 mice at various stages of ovarian follicle development were collected. Eight to twelve P6 ovaries were used to obtain all oocytes and somatic cells. In this study we included samples from the the primordial , transitioning from primordial to primary, primary, and secondary stages of follicular development. For the single oocytes, we included 14 primordial stage, 19 transitioning to primary, 20 primary, and 7 secondary replicates. There were a greater number of replicates for somatic cells with 40 primordial, 47 tranisitioning to primary, 56 primary, and 51 secondary being included in our study.Profiling of 54 tissue pieces from cumulus-oocyte complexes. These were extracted from an ex vivo mouse model that involves the isolation and culturing of mouse ovaries in an alginate hydrogel to gain insights into follicle-intrinsic, asymmetric functioning and molecular signaling within distinct regions of the follicle wall.

first: Emily

middle: J

last: Zaniker

first: Jiyang

last: Zhang

first: Kristine

last: Suritis

first: Riley

last: Drake

first: Alex

middle: K

last: Shalek

first: Teresa

last: Woodruff

first: Shuo

last: Xiao

first: Brittany

last: Goods

first: Francesca

middle: E

last: Duncan


title: Glutamine is critical for the maintenance of Type 1 Conventional Dendritic cells in normal tissue and the tumor microenvironment.

status: Public on Oct 16 2024

first: Graham

last: Lobel

address: 421 Curie Blvd

summary: Proliferating tumor cells take up glutamine for anabolic processes, engendering glutamine deficiency in the tumor microenvironment. How this might impact immune cells is not well understood. Using multiple mouse models of soft tissue sarcomas, glutamine antagonists, as well as genetic and pharmacological inhibition of glutamine utilization, we found that the number and frequency of conventional dendritic cells (cDC) is dependent on microenvironmental glutamine levels. cDCs comprise two distinct subsets – cDC1s and cDC2s, with the former subset playing a critical role in antigen cross-presentation and tumor immunity. While both subsets show dependence on glutamine, cDC1s are particularly sensitive. Notably, glutamine antagonism did not reduce the frequency of DC precursors but decreased the proliferation and survival of cDC1s. Further studies suggest a role of the nutrient sensing mTOR signaling pathway in this process. Taken together, these findings uncover glutamine dependence of cDC1s that is coopted by tumors to escape immune responses.

overall_design: In vitro FLT3L-differentiated cDCs generated from wild-type C57BL/6 mice. cDCs were differentiated in either 2 mM or 50 μM Glutamine, or in 2 mM Glutamine with 1 uM DON, and then harvested for single-cell RNAseq.

first: M. Celeste

last: Simon

first: Malay

last: Haldar


title: ZNF207-YWHAZ Enhances Breast Cancer Proliferation and Chemotherapy Resistance by Activating the HIF-1alpha-PPARgamma-PKM2 Signaling Axis.

city: Shenyang

first: Huanyu

last: Wu

state: Liaoning

address: No. 77 Puhe Road

department: Pathology Teaching and Research Office of Basic Medical College

country: China

institute: China Medical University

summary: Hypoxia is closely linked to chemotherapy resistance and accelerates breast cancer progression. However, the underlying mechanism of resistance to hypoxic chemotherapy remains uncertain. ZNF207 was identified as a differentially expressed gene involved in hypoxia and chemotherapy resistance by RNA-sequencing array. ZNF207 expression was elevated in lung, breast, liver, colon, and ovarian cancers, and its positive expression was correlated significantly with advanced TNM stage, lymph node metastasis, and poor prognosis. ZNF207 overexpression promoted the proliferation, invasion capabilities, and stemness of breast cancer cells by activating the HIF-1alpha-PPAR-gamma-glycolysis signaling pathway. Notably, ZNF207 was directly bound to the coiled-coil domain of YWHAZ, thereby accelerating HIF-1alpha deacetylation in an HDAC4-dependent manner. Furthermore, ZNF207 might stabilize YWHAZ by inhibiting its degradation via TRIM67 through a ubiquitin-dependent mechanism. ZNF207 overexpression enhanced resistance to doxorubicin and vinorelbine. Conversely, ZNF207-DeltaGLE overexpression disrupted HIF-1alpha-PPAR-gamma-glycolysis signaling and abolished chemotherapy resistance. Additionally, ZNF207 expression was higher in patients with breast cancer who exhibited poor treatment outcomes (Miller/Payne grades 1-2) than in those with more favorable outcomes (Miller/Payne grades 3-5). Sappanchalcone, a specific ZNF207 inhibitor, impedes breast cancer progression while exerting a synergistic effect with chemotherapy. Our findings revealed that ZNF207 expression was elevated in breast cancer under hypoxic conditions, promoting proliferation and invasion by activating HIF-1alpha through accelerated deacetylation in a positive feedback loop. The interaction between ZNF207 and YWHAZ enhances HIF-1alpha stability, ultimately accelerating therapeutic resistance in breast cancer.

overall_design: we studied the expression and mechanism of ZNF207 in breast cancer through a variety of methods, including its relationship with chemotherapy resistance, interaction with YWHAZ, and the effect of specific inhibitors.


title: A cross-disease human microglial framework identifies disease-enriched subsets and tool compounds for microglial polarization II.

city: New York

first: John

last: Tuddenham

state: NY

institute: Columbia University

summary: Human microglia play a pivotal role in neurological diseases, but few targeted therapies that directly modulate microglial state or function exist due to an incomplete understanding of microglial heterogeneity. We use single-cell RNA sequencing to profile live human microglia from autopsies or surgical resections across diverse neurological diseases and central nervous system regions. We observe a central divide between oxidative and heterocyclic metabolism and identify subsets associated with antigen presentation, motility, and proliferation. Specific subsets are enriched in susceptibility genes for neurodegenerative diseases or the disease-associated microglial signature. We validate subtypes in situ with an RNAscope-immunofluorescence pipeline and leverage our dataset as a classification resource, finding that iPSC model systems recapitulate substantial in vivo heterogeneity. Finally, we identify and validate candidates for chemically inducing subtype-specific states in vitro, showing that Camptothecin downregulates the transcriptional signature of disease-enriched subsets and upregulates a signature previously shown to be depleted in Alzheimer’s.

overall_design: Single-cell RNA-sequencing data on CD45+ cells dissociated from autopsy tissue and surgical resections from a broad collection of diseases and brain regions.

middle: F

first: Marta

last: Olah

first: Vilas

last: Menon

first: Philip

middle: L

last: De Jager


title: Developing enhanced immunotherapy using NKG2A knockout human pluripotent stem cell-derived NK cells

city: Duarte

first: Guihua

last: Sun

address: 1500 East Duarte Rd.

department: Department of Neurodegenerative Diseases

institute: City of hope

summary: Cancer immunotherapy is gaining increasing attention. However, immune checkpoints are exploited by cancer cells to evade anti-tumor immunotherapy. Here, we knocked out NKG2A, an immune checkpoint expressed on natural killer (NK) cells, in human pluripotent stem cells (hPSCs) and differentiated these hPSCs into NK (PSC-NK) cells. We show that NKG2A knockout (KO) enhances the anti-tumor and anti-viral capabilities of PSC-NK cells. NKG2A KO endows PSC-NK cells with higher cytotoxicity against HLA-E-expressing glioblastoma (GBM) cells, leukemia cells, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected cells in vitro. The NKG2A KO PSC-NK cells also exerted potent anti-tumor activity in vivo, leading to substantially suppressed tumor progression and prolonged survival of tumor-bearing mice in a xenograft GBM mouse model. These findings underscore the potential of PSC-NK cells with immune checkpoint KO as a promising cell-based immunotherapy. The unlimited supply and ease of genetic engineering of hPSCs makes genetically engineered PSC-NK an attractive option for easily accessible "off-the-shelf" cancer immunotherapy.

overall_design: Human induced proginotor stem cells from human embronic stem cell (H9), fibroblast (IMR90), and human preferial blood cells (PBMCs) were differentiated into NK cells (iNKs). RNA-seq were perform on parental samples (WTs) and iNKs to investigate their difference in transcriptome profiling.

first: Yue

last: Qin

first: Yanhong

last: Shi


title: Targeting pyruvate metabolism generates distinct CD8+ T cell responses to gammaherpesvirus and B lymphoma

city: Lebanon

first: Edward

last: Usherwood

state: NH

address: Rubin Building, Level 7, 710-53

department: Microbiology and Immunology

institute: Geisel School of Medicine at Dartmouth College

summary: T cells rely on different metabolic pathways to differentiate into effector or memory cells, and metabolic intervention is a promising strategy to optimize T cell function for immunotherapy. Pyruvate dehydrogenase (PDH) is a nexus between glycolytic and mitochondrial metabolism, regulating pyruvate conversion either to lactate or acetyl-CoA. Here, we retrovirally transduced pyruvate dehydrogenase kinase 1 (PDK1) or pyruvate dehydrogenase phosphatase 1 (PDP1), which control PDH activity, into CD8+ T cells to test effects on T cell function. Although PDK1 and PDP1 were expected to influence PDH in opposing directions, by several criteria they induced similar changes relative to control T cells. Seahorse metabolic flux assays showed both groups exhibited increased glycolysis and oxidative phosphorylation. Both groups had improved primary and memory recall responses following infection with murine gammaherpesvirus-68 (MHV-68). However, metabolomics using labeled fuels indicated differential usage of key fuels by metabolic pathways. Importantly, after B cell lymphoma challenge the engineered CD8+ T cells produced smaller effector populations in the tumor, resulting in larger tumors and poorer survival compared with control cells. Overall, this study indicates that PDK1 and PDP1 both enhance metabolic capacity, but the context of the antigenic challenge significantly influences the consequences for T cell function.

overall_design: To investigate the transcriptional changes with the modified PDH phosphorylation status in CD8+ T cells, we retrovirally transduced mouse CD8+ T cells with empty vector, PDK1 or PDP1.

first: Taewook

last: Kang

first: Owen

middle: M

last: Wilkins


title: H3K27ac and H3K27me3 ChIP-seq analysis for MDA-MB-468 cells treated with SAHA and sonidedgib

city: Shanghai

first: Min

last: Huang

address: 555, Zuchongzhi Road

institute: Shanghai Institute of Materia Medica

summary: Epigenetic therapies that cause genome-wide epigenetic alterations, could trigger local interplay between different histone marks, leading to a switch of transcriptional outcome and cellular phenotypes. In human cancers with diverse oncogenic activation, how oncogenic pathways cooperate with epigenetic modifiers to regulate the histone mark interplay is poorly understood. We herein discover that the hedgehog (Hh) pathway reprograms the histone methylation landscape in triple-negative breast cancer (TNBC), which facilitates histone acetylation caused by histone deacetylase (HDAC) inhibitors and gives rise to new therapeutic vulnerability of combination therapies. Specifically, overexpression of zinc finger protein of the cerebellum 1 (ZIC1) in TNBC promotes Hh activation, facilitating the switch of H3K27 methylation (H3K27me) to acetylation (H3K27ac). The mutually exclusive relationship of H3K27me and H3Kac allows their functional interplay at oncogenic gene locus and switches therapeutic outcomes. Using multiple in vivo TNBC models including patient-derived xenograft, we show that Hh signaling-orchestrated H3K27me and H3Kac interplay tailors combination epigenetic drugs in treating TNBC.

first: Xiaomin

last: Wang

first: Jun

last: Xu

first: Ting


title: edu-Ψ-seq Reveals snoRNA-guided mRNA Pseudouridylation that Regulates Protein Synthesis

city: Guangzhou

first: Jian-Hua

last: Yang

address: No. 135, Xingang Xi Road

department: School of Life Sciences

institute: Sun Yat-sen University

summary:
Purpose:Pseudouridine (Ψ) is the most abundant RNA epigenetic modification and plays vital roles in various biological processes. However, its biogenesis and functions in mRNAs remain elusive.
Method:Here, we developed an approach for enriching Ψ sites with deep sequencing (edu-Ψ-seq), which utilizes both exoribonuclease and restriction endonuclease activity to eliminate other nucleosides or reads without Ψ modifications.
Results:Using edu-Ψ-seq approach in the dyskerin pseudouridine synthase 1 (DKC1) knockdown cells, we identified that more than 100 pseudouridines could be catalyzed by DKC1 in human mRNAs and non-coding RNAs (ncRNAs). Surprisingly, we discovered, for the first time, 10 pseudouridines in mRNAs might be guided by 11 small nucleolar ncRNAs (snoRNAs). Interestingly, we found that SNORA10 could guide DKC1 protein to induce the pseudouridylation of RPL15 mRNA. Importantly, loss of SNORA10 impaired the protein synthesis of RPL15 and inhibited cell proliferation.
Conclusion:Overall, our study not only developed a powerful method for detecting Ψ sites but also uncovered another layer of gene expression regulation that involves crosstalk among snoRNAs, mRNA pseudouridylation and protein synthesis.

overall_design: All mRNA profiles of HEK293T, HCT116 and HCT116-shDKC1-1 cells treated with Ψ-seq/edu-Ψ-seq procedures  are generated by deep sequencing. Each replicate consists of two samples (CMC-/CMC+) and were treated with or without N-cyclohexyl-N′-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMC)

first: Jianhua

first: Wujian

last: Zheng


title: Acute myeloid leukemia cells adhere to bone marrow and acquire chemoresistance by downregulating UNC5B expression

city: Jinan

first: bo

middle: lan

last: li

address: Wenhua West Road

institute: Qilu Hospital of Shandong University

summary: Acute myeloid leukemia (AML) is a malignant tumor of the hematological system. Because of its characteristics of recurrence, refractory and chemoresistance, new therapeutic targets need to be identified. Adhesion and proliferation are characteristics of AML cells, and critical steps in inducing chemotherapy resistance. In this study, we reported that UNC5B inhibits AML cell bone marrow adhesion, inhibits AML cell proliferation and increases sensitivity to chemotherapy. Mechanistically, RNA sequencing (RNA-seq) and experimental results revealed that overexpression of UNC5B inhibits adhesion and proliferation signaling pathways and inhibits the expression of MPZL1, CLDN23, IGF2 and WNT7B. In conclusion, our findings suggest that UNC5B serves as a prognostic indicator and a potential therapeutic target for AML.

overall_design: In order to detect the expression of proliferation and adhesion related genes, UNC5B was overexpressed in AML cell lines to detect the expression of adhesion and proliferation related genes

first: Teng

last: Teng

first: Liping

last: Ren

first: Jilong

last: Xiao


title: Generation of human induced pluripotent stem cell-derived 3D human cortical, striatal, mesencephalic and diencephalic organoids

city: STANFORD

first: Yuki

last: Miura

state: California

address: 318 Campus Drive, W200

department: Department of Psychiatry and Behavioral Sciences

summary: Single cell RNA-seq characterization of stem cell derived organoids resembling the parts of human cortico-striatal-thalamo-cortical circuit

overall_design: Single-cell RNA-sequencing of human cortical, striatal, mesencephalic, and diencephalic organoids differentiated from human iPS cells

first: Ji-il

last: Kim

first: Sergiu

last: Pasca


title: Lifespan-regulated CAR-macrophages from Myeloid Progenitors for Enhanced Colorectal Cancer Therapy

city: Nan Jing

first: Chuancheng

last: Gao

address: 163 Xianlin Avenue, Qixia Distric

institute: Nanjing University

summary: To investigate the activation of CAR-macrophages by the antigen CEA, we treated CAR-macrophages with CEA and detected them by RNA-Seq.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for Control and CAR-treated group.

first: Fangling

last: Hong

first: Yao

last: Dong

first: Yong

last: Fu


title: Cystathionine γ-lyase downregulation contributes to hepatocyte injury and dysfunction via multiple signaling pathways by regulation of histone lysine methylation

city: Changsha

first: wei

last: zhang

address: 87 Xiangya Road, Changsha, Hunan, China

institute: Xiangya Hospital Central South University

summary: Chronic liver disease (CLD) is prevalent globally. We sought to elucidate the molecular mechanisms governing the progress of CLD. We confirmed hepatic cystathionine γ-lyase (Cth) expression was significantly down-regulated in some CLDs. Therefore, we subsequently investigated the roles of Cth gene in the regulation of hepatic function and underlying mechanisms by using Cth knockout mice.  Furthermore, we found that Cth deficiency resulted in one-carbon unit reprogramming, and betaine was one of key metabolites in one-carbon reprogramming, which caused changed histone lysine methylation and thereby leading to dysregulation of PPARα, PGC-1α, IRF8 and IRF9, the transcriptional factors governing metabolism, mitochondrial function and apoptosis. Our data immediately highlight a new potential target for therapy of some CLDs.

overall_design: To investigate the role of CTH in chronic liver disease, liver tissues from WT mice, CTH-deficient mice, and CTH-deficient mice supplemented with betaine were analyzed by RNA-Seq.

first: Cichun

last: Wu

first: Wei

last: Zhang

first: Wenhu

last: Liu

first: Zhengshan

last: Tang

first: Shifang

last: Peng

first: Lei

first: Xin

last: Ni


title: A Molecular Switch from Tumor Suppressor to Oncogene in ER-Positive Breast Cancer: Role of Androgen Receptor, JAK-STAT, and Lineage Plasticity [scRNA-seq]

city: Memphis

first: Daniel

middle: Lee

last: Johnson

state: Tennessee

address: 71 S Manassas

department: Molecular Informatics Core

institute: UTHSC

summary: Cancers develop resistance to inhibitors of oncogenes mainly due to target-centric mechanisms such as mutations and splicing. While inhibitors or antagonists force targets to unnatural conformation contributing to protein instability and resistance, activating tumor suppressors may maintain the protein in an agonistic conformation to elicit sustainable tumor growth inhibition. Due to lack of tumor suppressor-agonists, this hypothesis and mechanisms underlying resistance are not understood. In estrogen receptor (ER)-positive breast cancer (BC), androgen receptor (AR) is a druggable tumor suppressor and offers a promising avenue for this investigation. Spatial genomics suggest that the molecular portrait of AR-expressing BC cells in tumor microenvironment corresponds to better overall patient survival, clinically confirming AR’s role as a tumor suppressor. Ligand-activation of AR-positive BC xenografts reprograms cistromes, inhibits oncogenic pathways, and promotes cellular elasticity towards a more differentiated state, while sustained treatment results in cistromes rearrangement towards transcription factor PROP1, transformation of AR into an oncogene, and activation of the JAK/STAT pathway, all culminating in lineage plasticity to an aggressive resistant subtype. While the molecular profile of AR agonist-sensitive tumors corresponds to better patient survival, the profile represented in the resistant phenotype corresponds to shorter survival. Inhibition of activated oncogenes in resistant tumors reduces growth and re-sensitizes them to AR agonists. Together, these findings indicate that persistent activation of a tumor suppressor is not fail-safe and leads to resistance through lineage plasticity-driven tumor metamorphosis. Our work provides a framework to explore the above phenomenon across multiple cancer types and underscores the importance of factoring the sensitization of tumor suppressor targets while developing agonist-like drugs

overall_design:
To determine the effect of AR agonists on the transcriptome at single cell level and also to determine the single cell transcriptome of T47D and WHIM23 xenografts that have developed resistance to AR agonists
G1 = Vehicle
G2 = DHT
G4 = Enobosarm
G4R = Enobosarm Resistant

first: Sarah

last: Asemota

first: Ramesh

last: Narayanan


title: A Molecular Switch from Tumor Suppressor to Oncogene in ER-Positive Breast Cancer: Role of Androgen Receptor, JAK-STAT, and Lineage Plasticity [RNA-seq II]

overall_design:
To investigate the mechanism of resensitization to ruxolitinib, ZR-75-1 parental and enobosarm-resistant cells were treated for 7 days and RNA sequencing was performed.
G1 = Vehicle
G2 = DHT
G4 = Enobosarm
G4R = Enobosarm Resistant


title: NKp46+ ILC3s promote early neutrophil defense against C. difficile infection through GM-CSF secretion

city: Saint Louis

first: Alina

last: Ulezko Antonova

state: MO

address: 660 S. Euclid Ave

department: Path/Immuno

institute: WUSM

summary: Clostridioides difficile infection (CDI) is a common cause of antibiotic-associated colitis. C. difficile proliferates and produces toxins that damage the colonic epithelium, leading to symptoms ranging from mild diarrhea to severe pseudomembranous colitis. The host's innate response to CDI occurs in two phases: an early phase in which neutrophils reduce the bacterial load, and a late phase involving repair mechanisms to restore epithelial integrity. Group 3 innate lymphoid cells (ILC3s) are crucial in protecting the gut from CDI. Previous studies have shown that ILC3 production of IL-22 is essential in the late phase of CDI for epithelial repair and maintaining an intestinal microbiota that competes with C. difficile, preventing its expansion. Our study finds that ILC3s also protect during the early stages of CDI by sustaining neutrophils through GM-CSF. Less neutrophil production, accumulation and activation was evident in ILC3-deficient mice than in wild-type (WT) mice, which led to exacerbated symptoms, impaired pathogen clearance, a compromised epithelial barrier, and increased mortality. The adoptive transfer of ILC3s into ILC3-deficient mice restored neutrophil responses and improved disease outcomes. Both in vitro and in vivo experiments revealed that GM-CSF production by ILC3s is crucial for neutrophil production and effective resistance during CDI. Using mice lacking NKp46+ ILC3s, we found that this subset significantly contributes to GM-CSF production in CDI. These findings highlight the critical role of the ILC3-neutrophil connection in early innate responses to CDI. Enhancing ILC3 production of GM-CSF could be a promising strategy for improving host defense against CDI and other enteric infections.

overall_design: Mice received an oral 4-day antibiotic treatment via drinking water, followed by a single intraperitoneal dose of clindamycin. Mice were either uninfected or infected with C. difficile following antibiotic treatment. Both antibiotic-treated (d0) and infected (d2) mice were euthanized, and colonic lamina propria ILC3s were isolated using FACS sorting. Cells were gated as live, lymphocyte-sized, lineage-negative (CD3, CD19, KLRG1, NK1.1), Thy1.2-high, and CD45-intermediate. High-throughput bulk RNA sequencing was performed on 3 infected and 3 uninfected mouse colonic tissues.

first: José

last: Fachi

last: De Oliveira

first: Marco

last: Colonna


title: GLS2 links glutamine metabolism and atherosclerosis by remodelling artery walls (MOVAS).

city: Paris

first: Sebastien

last: Dussaud

address: 91 bd de l'hopital

institute: UPMC

summary: Metabolic dysregulation, including perturbed glutamine-glutamate homeostasis, is common in atherosclerotic cardiovascular disease. Here, we reveal that modulation of glutaminolysis or glutamate availability in culture media is critical for smooth muscle cell line (MOVAS) phenotypic switching. Cells were treated for 24 hours with glutaminase inhibitors (50mM DON, Sigma-SML0601) in presence or absence of 2mM glutamate suplementation (Gibco-35050061). High-throughput transcriptional profiling revealed that modulation of glutamine and glutamate homeostasis impacted genes involved in protein and extracellular matrix organization, cell motility and microtubule. Thus, we uncovered a novel role for glutamine metabolism in the phenotypic switch in SMCs, a hallmark of vascular remodelling.

overall_design: Immortalized smooth muscle cell line MOVAS (ATCC CRL-2797) were cultured at 37°C and 5% CO2 in standatd growth media consisting of Dulbecco’s Modified Eagle’s Medium (DMEM, Corning), 100 U/ml of penicillin/streptomycin (Corning), 10% Fetal Bovine Serum (Corning) and 0.2mg/mL geneticin (G418, VWR Life Science). Cells used at 60-70% confluency were treated for 24 hours with glutaminase inhibitors (50mM DON, Sigma-SML0601) and supplemented or not with 2mM glutamate (Gibco-35050061)

first: Florent

last: Murcy

first: Coraline

last: Borowczyk

first: Thibault

last: Barouillet

first: Sébastien

first: Laurent

last: Yvan-Charvet


title: Establishment and Characterization of Mouse Metabolic dysfunction-associated steatohepatitis-related Hepatocellular carcinoma organoids

city: Seoul

first: JA-LOK

last: KU

address: 101 Daehak-ro, Jongno-gu

department: Cancer Research Institute

institute: Seoul National University College of Medicine

summary: Metabolic dysfunction-associated steatohepatitis is a form of chronic liver inflammation associated with metabolic syndrome, such as obesity and a major cause of hepatocellular carcinoma. Multi-biotics, a soymilk fermented with lactic acid bacteria, are known to alleviate obesity by lowering lipid profile. In this study, we investigated efficacy of multi-biotics in MASH-related HCC using mouse model fed choline-deficient L-amino acid-defined high-fat diet, and determined that tumor regression was not affected by multi-biotics. We established mouse MASH-related HCC organoids, and performed RNA-sequencing to identify transcriptomic features in MASH-HCC treated multi-biotics. We also examined response of MASH-related HCC to four HCC treatment drugs, and MASH-related HCC treated multi-biotics was good response to Lenvatinib. We established Lenvatinib-resistant MASH-related HCC organoids by administrating repeatedly Lenvatinib, and identified enriched pathways in Lenvatinib-resistant models. This study provides a tool for studying MASH-related HCC treatment and Lenvatinib resistance by establishing the MASH-related HCC and Lenvatinib resistance organoid model.

overall_design: To investigate the effect of multi-biotics on MASH-related HCC, a mouse organoid was established, and changes that occurred when treated with multi-biotics were confirmed through transcriptomic analysis. To investigate the effect of multi-biotics in MASH-related HCC treated with Lenvatinib, we confirmed what transcriptomic changes occurred through RNA-seq.

first: Sumin

last: Kim

first: Nahyun

last: Jeong

first: Jeayeon

last: Park

first: Hyojin

last: Noh

first: Ja

middle: O

last: Lee

first: Su

last: Yu

first: Ja-Lok

last: Ku


title: A Molecular Switch from Tumor Suppressor to Oncogene in ER-Positive Breast Cancer: Role of Androgen Receptor, JAK-STAT, and Lineage Plasticity [RNA-seq I]

first: Daniel

last: Johnson

overall_design:
To determine the effect of androgen receptor (AR) agonists on the transcriptome of T47D cell line xenograft and WHIM23 patient-derived xenograft. The transcriptome of AR agonist resistant tumors were also determined
G1 = Vehicle
G2 = DHT
G4 = Enobosarm
G4R = Enobosarm Resistant

first: Sarah

last: Asemota

first: Ramesh

last: Narayanan


title: Snf2L drives Plasmodium just-in-time gene expression and gametocytogenesis

city: University Park

first: Manuel

last: Llinas

state: Pennsylvania

address: W126 Millennium Science Road

institute: The Pennsylvania State University

summary: The complex life cycle of the malaria parasite Plasmodium falciparum (Pf) involves the differentiation into multiple distinct forms, requiring a tight and dynamic control of gene expression at each stage. However, the full range of variation cannot solely be explained by stage-specific transcription factors, such as ApiAP2-family members, as they are strongly under-represented in Pf. Huge changes in chromatin structure and epigenetic modifications during life cycle progression suggest a central role of these mechanisms in regulating the transcriptional program for parasite development. Pf chromatin is distinct from other eukaryotes with an extraordinarily high AT-content (>80 %) and highly divergent histones lacking typical DNA packaging properties. In addition, the chromatin remodelers, key in shaping chromatin structure, are not conserved and unexplored in Pf. We identified PfSnf2L (PF3D7_1104200) as ISWI-related ATPase actively repositioning Pf nucleosomes in vitro. Knockout studies demonstrate that PfSnf2L is essential, regulating asexual development and sexual differentiation. It globally controls just-in-time transcription by spatiotemporally determining nucleosome positioning at promoters of stage-specific genes. The unique sequence and functional properties of PfSnf2L allows the identification of an inhibitor that specifically kills Plasmodium falciparum, phenocopies the loss of correct gene expression timing, and inhibits the formation of gametocytes.

overall_design: Two biological replicates (with a single paired no-epitope negative control sample) of chromatin immunoprecipitation followed by sequencing (ChIP-seq) across four asexual blood stage timepoints (Early Ring, Late Ring, Trophozoite, and Schizont) was used to identify the genome-wide binding sites of chromatin-assocaited factor, PfSNF2L (gene ID: PF3D7_1104200), Plasmodium falciparum parasite line with a C-terminal 3xHA tag.

first: Maria

middle: T

last: Watzlowik

first: Sujaan

last: Das

first: Simon

last: Holzinger

first: Elisabeth

last: Silberhorn

first: Ritwik

last: Singhal

first: Victoria

last: Bonnell

first: Matthew

last: Gow

first: Andreas

last: Klingl

last: Llinás

first: Markus

last: Meissner

first: Gernot

last: Längst


title: Impaired p53-Mediated DNA Damage Response Contributes to Microcephaly in Nijmegen Breakage Syndrome Patient-Derived Cerebral Organoids

city: Düsseldorf

first: Wasco

last: Wruck

address: Moorenstr. 5

institute: Universitätsklinikum Düsseldorf

summary: Nijmegen Breakage Syndrome (NBS) is a rare autosomal recessive genetic disorder caused by mutations within nibrin (NBN), a DNA damage repair protein. Hallmarks of NBS include chromosomal instability and clinical manifestations such as growth retardation, immunodeficiency, and progressive microcephaly. We employed induced pluripotent stem cell-derived cerebral organoids from two NBS patients to study the etiology of microcephaly. We show that NBS organoids carrying the homozygous 657del5 NBN mutation are significantly smaller with disrupted cyto-architecture. The organoids exhibit premature differentiation, and Neuronatin (NNAT) over-expression. Furthermore, pathways related to DNA damage response and cell cycle are differentially regulated compared to controls. After exposure to bleomycin, NBS organoids undergo delayed p53-mediated DNA damage response and aberrant trans-synaptic signaling, which ultimately leads to neuronal apoptosis. Our data provide insights into how mutations within NBN alters neurogenesis in NBS patients, thus providing a proof of concept that cerebral organoids are a valuable tool for studying DNA damage-related disorders.

overall_design: The transcriptomes of brain organoids  from NBS patients and healthy controls with and without Bleomycin were compared by microarray analysis (Clariom-S Human Gene Expression Array, Affymetrix, Thermo Fisher Scientific).

first: Soraia

last: Martins

first: James

last: Adjaye


title: Deconvolution of the tumor-educated platelet transcriptome reveals activated platelet and inflammatory cell transcript signatures [RNA-seq]

first: Erik

last: Sulman

address: 522 1st Ave, Smilow Bldg, Rm 801C

institute: NYU Grossman School of Medicine

summary: Tumor-educated platelets (TEPs) are a potential method of liquid biopsy for the diagnosis and monitoring of cancer. However, the mechanism underlying tumor education of platelets is not known, and transcripts associated with TEPs are often not tumor-associated transcripts. We demonstrate that direct tumor transfer of transcripts to circulating platelets is an unlikely source of the TEP signal. We use CDSeq, a latent Dirichlet allocation algorithm, to deconvolute the TEP signal in blood samples from patients with glioblastoma. We demonstrate that a significant proportion of transcripts in the platelet transcriptome are derived from non-platelet cells, and the use of this algorithm allows the removal of contaminant transcripts. Furthermore, we used the results of this algorithm to demonstrate that TEPs represent a subset of more activated platelets, which also contain transcripts normally associated with non-platelet inflammatory cells, suggesting that these inflammatory cells, possibly in the tumor microenvironment, transfer transcripts to platelets that are then found in circulation. Our analysis suggests a useful and efficient method of processing TEP transcriptomic data to enable the isolation of a unique TEP signal associated with specific tumors.

overall_design: GS 8-11 glioblastoma sphere cells (1 × 105 per mouse) were implanted into the brains of 6- to 8-week-old Foxn1nu mice, one week after guide screw placement, using a stereotactic apparatus under anesthesia. A minimum of five mice were included in each group. Animals in the treatment group that showed signs of distress or were moribund were euthanized and blood was collected using an 18-gauge needle by cardiac puncture. Five weeks after tumor implantation, approximately 1 ml of blood was collected from each mouse in a BD Vacutainer yellow-top tube containing acid citrate dextrose to prevent platelet activation. Platelet-rich plasma (PRP) was collected by centrifuging the tubes at 100 × g for 15 min. The centrifuge was set to accelerations of 5 and 2 to avoid platelet activation. PRP was separated carefully from the other components of the blood and centrifuged at 1000 × g for 10 min after the addition of 2 µL of 1 mM PGE2 to 2 ml of PRP. RNA was isolated from the resulting platelet pellets using the mirVana™ miRNA Isolation Kit (Invitrogen, USA, Cat #AM1560), followed by standard RNA-seq library preparation and paired-end sequencing using NextSeq 2000 (Illumina). FASTQ files were processed using Trimmomatic version 0.36, STAR (using the hg38 genome as reference), and HTSeq. The DSVD lung adenocarcinoma mouse model was intranasally induced with Ad-CMV-Cre (5 × 107 plaque-forming units) as previously described. Approximately 1 ml of blood was collected from each mouse by cardiac puncture using an 18 g needle, three weeks after tumor implantation. The samples were stained with a panel of 13 barcoded CITE-seq-compatible antibodies. Briefly, a maximum of 1 million cells per sample were resuspended in 100 µL of cell staining buffer (2% BSA / 0.01% Tween, PBS) with 10 µL of Fc receptor block (TrueStain FcX, BioLegend, USA) for 10 min. This was followed by a 30-minute staining with the antibodies at 4°C. A concentration of 1 µg / 100 µl was used for all the antibody markers used in this study. The cells were then washed three times with 1 ml of staining buffer, followed by centrifugation (350 × g for 5 min at 4°C) (50). Stained cells were then used for single-cell reverse transcription using 10X Genomics and libraries were prepared as previously described (32). Briefly, cDNA amplification was performed in the presence of an antibody oligo-specific primer to increase the yield of antibody-derived tags (ADTs). The amplified cDNA was separated by SPRI size selection into cDNA fractions containing mRNA-derived cDNA (>300bp) and ADT-derived cDNAs (<180bp). Sequencing libraries were generated from the mRNA and ADT cDNA fractions, which were quantified, pooled, and sequenced on an Illumina NovaSeq platform (Illumina). CITE-seq data were processed using Seurat v.4 pipeline (51). Platelets were selected from the single-cell dataset by visual inspection of the UMAP plot of the total data set, selecting the cell cluster that contained high levels of platelet markers including Ppbp and Pf4, then removing cells with >2% ribosomal protein transcripts, >2% hemoglobin transcripts, >10% mitochondrial mRNA transcripts, or increased levels of Malat1. Platelet datasets from normal mice and mice with implanted tumors were integrated using the Seurat pipeline. The integrated data were then processed using the standard Seurat pipeline, with markers of Seurat clusters found using the FindConservedMarkers function.

first: Jerome

last: Karp

first: Aram

last: Modrek

first: Ravesanker

last: Ezhilarasan

first: Ze-Yan

last: Zhang

first: Yingwen

last: Ding

first: Melanie

last: Graciani

first: Ali

last: Sahimi

first: Michele

last: Silvestro

first: Ting

last: Chen

first: Shuai

last: Li

first: Kwok-Kin

last: Wong

first: Bhama

last: Ramkhelawon

first: Krishna

last: Bhat


title: PfSnf2L - an epigenetic regulator of just-in-time expression and gametocyte formation [MNase-Seq]

city: Regensburg

first: Simon

last: Holzinger

address: Universitätsstraße 31

institute: University of Regensburg

summary: The complex life cycle of the malaria parasite Plasmodium falciparum (Pf) and the accompanying transcriptional variation is only explained in part by stage-specific transcription factors like the ApiAP2 family, as these factors are strongly under-represented in Pf. Global and local changes in chromatin structure during life cycle progression suggest the contribution of epigenetic mechanisms as a critical mechanism for fine-tuning gene regulation during malaria parasite development. Pf chromatin is largely unexplored and is distinct from other eukaryotes in many respects. This is likely to accommodate the highly A/T-rich genome which averages over 90% in non-coding regions, the highest of any known eukaryote. We characterized PF3D7_1104200 (PfSnf2L), an ATPase-containing protein that is related to ISWI-type chromatin remodeling enzymes (CREs). Using a conditional knockout (KO) system, we demonstrate that PfSnf2L is essential for parasite development, globally controls just-in-time transcription regulation, and specifically represses gametocyte-specific gene expression. Characterization of the enzymatic activity in vitro, combined with mapping chromatin organization in vivo revealed that PfSnf2L shapes the promotor architecture of stage-specific genes through its nucleosome remodeling activity, thereby influencing gene activation and repression. The unique properties of the Plasmodium Snf2L allowed screening and identification of a specific inhibitor that specifically kills the parasite, phenocopies the loss of correct gene expression timing, and inhibits the formation of gametocytes.

overall_design: Comparative nucleosome profiling analysis of MNase-seq data of 3D7 parasites with PfSnf2L-KO induced vs. non induced, for two different induction timepoints.

first: Maria

last: Watzlowik

first: Elisabeth

last: Silberhorn

first: Sujaan

last: Das

first: Markus

last: Meissner

first: Gernot

last: Laengst


title: Deconvolution of the tumor-educated platelet transcriptome reveals activated platelet and inflammatory cell transcript signatures [CITE-seq]

first: Erik

last: Sulman

first: Jerome

last: Karp

first: Aram

last: Modrek

first: Ravesanker

last: Ezhilarasan


title: Gene expression profile of Wild Type (WT) and P2x1 knockout (P2X1-KO) leukemia leukemia-initiating cells Transcriptomes

first: Xiaoxiao

last: He

state: Shanghai

address: 280 South Chongqing Road, Shanghai 200025

department: Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education,Faculty of Basic Medicine

institute: Shanghai Jiao Tong University School of Medicine

summary: To determine the underlying molecular mechanisms that control the homing and self-renewal activities of P2x1-null Mac-1+c-Kit+ LICs, WT and P2x1-null LICs were WT and P2x1-KO  were sorted by flow cytometry, followed by the extraction of total RNA and subjected to the RNA-sequencing.

overall_design: Wild Type (WT) and P2x1 knockout (P2X1-KO) two population AML leukemia leukemia-initiating cells from bone marrow were sorted to measure gene expression.

first: Junke

last: Zheng


title: Hinokiflavone resists HFD-induced obesity by promoting apoptosis in an IGF2BP2-mediated Bim m6A modification dependent manner

city: Yangling

first: Mingkun

last: Chao

address: No. 22 Xinong Road, Yangling, Shaanxi Province 712100, China

institute: NWAFU

summary: Obesity has emerged as a major health risk on a global scale. Natural small molecules, such as hinokiflavone (HF) extracted from plants like cypress, exhibit diverse chemical structures and low synthesis costs. Using HFD-induced obese mice models, we found that HF suppresses obesity by inducing apoptosis in adipose tissue. Adipocyte apoptosis helps maintain tissue health by removing aging, damaged, or excess fat cells, crucial in preventing obesity and metabolic diseases. We found that HF can specifically bind to insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) to promote the stability of N6-methyladenosine (m6A) -modified Bim, inducing mitochondrial outer membrane permeabilization (MOMP). MOMP leads to Caspase9/3-mediated adipocyte mitochondrial pathway apoptosis, alleviating obesity induced by a high-fat diet. HF offers a controlled means of adipocyte apoptosis for weight loss. This study reveals the potential of small molecules like HF in developing new therapeutic approaches in drug development and biomedical research.

overall_design: To investigate the effects and mechanisms of HF on obesity-induced mice with a high-fat diet and 3T3-L1 cells during adipogenesis, RNA-seq and RIP-seq of IGF2BP2 were conducted.

first: Gongshe

last: Yang

first: Weijun

last: Pang

first: Rui

last: Cai


title: Tbet dysregulation compensates for Ets-deficiency in iNKT1 cells

city: Chicago

first: Barbara

last: Kee

state: Illinois

address: 924 E57th st

department: Pathology

institute: The University of Chicago

summary: The goal of this study was to compare the gene expression program of liver iNKT1 cells from Control mice to those with a postselection deletion of the transcription factor Ets1 and heterozybous deletion of Tbx21. Ets1-floxed and Tbx21-floxed alleles were deleted in iNKT1 cells using Tbx21Cre bacterial artifical chromosome transgenic mice that also carry a Rosa26-floxed-stop-YFP reporter. Control iNKT1 cells were Tbx21Cre+ Rosa26-floxed-stop-YFP reporter+ and cKOHet iNKT cells were Tbx21Cre+ Ets1F/F Tbx21F/+ Rosa26-floxed-stop-YFP reporter+.  The differentially expressed genes were compared to those in liver iNKT1 cells with postselection deletion of Ets1 only. We examined gene expression by RNA-sequencing of sorted iNKT1 cells (CD1d-Tet-PBS57+ YFP+) from the liver.  Reads were aligned to the mm10 reference genome using Tophat v 2.1.0.  Reads were assigned to genes using the htseq-count tool from HTSeq v 0.6.1 and gene annotations from Ensembl release 78.   Differential expression was calculated across 3 independent replicates by EdgeR.  We found that iNKT1 cells lacking Ets1 upregulated Tbx21 and its protein Tbet, many Tbet target genes, and CD8 effector T cell associated genes.

overall_design: Expression profiling analysis of RNA from thymic and liver iNKT1 cells isolated from Ctrl (Tbx21Cre; Rosa26-YFP+) and cKO (Tbx21Cre; Rosa26-YFP+; Ets1F/F) mice by RNA-sequencing. 3 independent replicates for each sample.

first: Roxroy

middle: C

last: Morgan

first: Cameron

last: Frank

first: Mikael

last: Sigvardsson

first: Elizabeth

last: Bartom


title: TAK1 operates at the primary cilium in non-canonical TGFB/BMP signaling to control heart development

city: Copenhagen

first: Lars

middle: Allan

last: Larsen

address: Blegdamsvej 3

department: Department of Cellular and Molecular Medicine

country: Denmark

institute: University of Copenhagen

summary: Transforming Growth Factor-Beta-Activated Kinase 1 (TAK1/MAP3K7), along with its upstream regulators TAK1-Binding Protein 2 (TAB2) and the catalytic alpha-subunit of Protein Kinase A (PKA-Cα/PRKACA), has been identified as a pivotal player in regulation of developmental processes. Haploinsufficiency of TAB2 causes Congenital Heart Disease (CHD) and rare variants in PKA-Cα and TAK1 cause cardioacrofacial dysplasia (CAFD), and Frontometaphyseal Dysplasia (FMD) and cardiospondylocarpofacial syndrome (CSCFS), respectively, rare multisystem syndromes, where CHD may appear in the clinical spectrum. We hypothesized that TAK1 plays a significant role in heart development and CHD and addressed this by genetic analysis in CHD patient cohorts and experiments in cell and animal models. Exome sequencing data from 1,471 CHD patients with extracardiac anomalies (syndromic CHD, sCHD), 2,405 patients with nonsyndromic CHD (nsCHD) and 45,082 controls showed increased burden of rare TAB2 and TAK1 variants in sCHD, but not in nsCHD. Detailed characterization of tak1-/- and tab2-/- zebrafish mutants revealed cardiac defects (dilated atrium, trabeculation defects, tachycardia and reduced contractility) as well as  extracardiac developmental anomalies. RNA sequencing of tak1-/- mutant hearts showed downregulation of genes encoding core cardiac transcription factors, sarcomeric proteins and extracellular matrix proteins. Experiments with cell cultures and analysis of zebrafish larvae and gastruloids indicated that TAK1 via TAB2 and PKA-Cα is activated at the primary cilium during cardiomyogenesis and that TAK1 activation at this site is enhanced by cardiomyogenic signaling molecules, including ligands of the TGFB/BMP superfamily. Consistent with these findings, CRISPR/Cas9-mediated editing of TAK1 or administration of small molecule inhibitors targeting TAK1 inhibited ciliary signaling and cardiomyocyte differentiation in vitro, while FMD-causing mutations in TAK1 reduced its ciliary localization. In conclusion, our data establishes a central role for TAK1 and its upstream regulators in cardiac development and syndromic CHD, coordinated via the primary cilium.

overall_design: To investigate the function of tak1 in heart development, we created a zebrafish mutant using CRISPR-cas9. Three groups of hearts from 3 dpf tak1-/- and tak1+/+ zebrafish larvae (15 hearts of each genotype respectively) were extracted as previously described (PMID 25651299) and RNAseq was used to compare the transcription profile between mutant and wild-type.

first: Canan

last: Doganli

middle: A


title: Chitinase 1: A novel therapeutic target in metabolic dysfunction-associated steatohepatitis

first: Jung Hoon

last: Cha

address: 222, Banpo-daero, Seocho-gu

institute: College of Medicine, The Catholic University of Korea

summary: Macrophage profiling was performed using single-nuclei RNA sequencing and flow cytometry. Single-nuclei RNA sequencing demonstrated a notable increase in macrophages numbers, particularly of lipid-associated macrophages, in MASH mice.

overall_design: We established a mouse model of MASH-related liver fibrosis by administering streptozocin (STZ, 0.2 mg) in combination with a high-fat and high-cholesterol (HFHC) diet. Single-nuclei RNA sequencing (sNuc-Seq) was conducted using frozen liver tissue samples from one normal male C57BL/6 J mouse and two mice with MASH-related liver fibrosis.

first: Jung

middle: H

middle: R

last: Park

first: Sung

middle: W

last: Cho

first: Heechul

last: Nam

first: Hyun

first: Eun

middle: S

last: Jung

first: Jeong

last: Jang

first: Jong

middle: Y

last: Choi

first: Seung

last: Yoon

first: Pil

last: Sung

first: Si

last: Bae


title: Gene expression profiles of PD-L1 positive and PD-L1 negative lung neutrophils from Influenza A virus infected mice

city: Tours

first: Guy

last: Ilango

address: 10, boulevard Tonnellé

institute: University of Tours

summary: Neutrophils are often considered as foes during severe lung infections through their ability to secrete inflammatory mediators and participate in lung damages. Here, we used single cell RNA-sequencing (scRNA-seq) to assess the transcriptional heterogeneity of neutrophils in mice with severe lung infection caused by influenza A virus

overall_design: Lung neutrophils from IAV (H3N2/Scotland/74 strain) infected C57BL/6j femelle mice (day 11 post-infection) were isolated using a cell sorter based on PD-L1 expression.

first: Loïc

last: Gonzalez

first: Roxane

last: Lemoine

first: Christophe

last: Paget


title: Hydrogel Microsphere Stem Cell Encapsulation Enhances  Cardiomyocyte Differentiation and Functionality in Scalable Suspension System

city: Auburn University

first: Elizabeth

last: Lipke

state: AL

address: 212 Ross Hall

institute: Auburn University

summary: A reliable suspension-based platform for scaling engineered cardiac tissue (ECT) production from human induced pluripotent stem cells (hiPSCs) is crucial for regenerative therapies. Here, we compared the production and functionality of ECTs formed using our scaffold-based, engineered tissue microsphere differentiation approach with those formed using the prevalent scaffold-free aggregate platform. We utilized a microfluidic system for the rapid (1 million cells/min), high density (30, 40, 60 million cells/ml) encapsulation of hiPSCs within PEG-fibrinogen hydrogel microspheres. HiPSC laden-microspheres and aggregates underwent suspension-based cardiac differentiation in chemically defined media. In comparison to aggregates, microspheres maintained consistent size and shape initially, over time, and within and between batches. Initial size and shape coefficients of variation for microspheres were eight and three times lower, respectively, compared to aggregates. On day 10, microsphere cardiomyocyte (CM) content was 27% higher and the number of CMs per initial hiPSC was 250% higher than for aggregates. Contraction and relaxation velocities of microspheres were four and nine times higher than those of aggregates, respectively. Microsphere contractile functionality also improved with culture time, whereas aggregate functionality remained unchanged. Additionally, microspheres displayed improved β-adrenergic signaling responsiveness and uniform calcium transient propagation. Transcriptomic analysis revealed that while both microspheres and aggregates demonstrated similar gene regulation patterns associated with cardiomyocyte differentiation, heart development, cardiac muscle contraction, and sarcomere organization, the microspheres exhibited more pronounced transcriptional changes over time. Taken together, these results highlight the capability of the microsphere platform for scaling up biomanufacturing of ECTs in a suspension-based culture platform.

overall_design: Bulk RNA-seq analysis was performed on engineered cardiac tissues generated through our scaffold-based microsphere and microrod differentiation approaches, as well as those formed using the widely-used scaffold-free aggregate platform, at days 5 and 15 of differentiation. RNA was isolated from over 30 individual tissues for each condition, all derived from a single batch.

first: Mohammadjafar

last: Hashemi

first: Amit

middle: K

last: Mitra


title: White button mushroom intake modulates PMN-MDSCs, T, and NK cells’ function at the single cell level in prostate cancer patients (validation cohort)

city: Duarte

first: Shiuan

last: Chen

address: 1500 E Duarte Rd

department: Cancer Biology-BRI

institute: City of Hope National Medical Center

summary: In prostate cancer patients following 3 months of white button mushroom consumption in freeze-dried tablet form, we observed the decline of circulating polymorphonuclear MDSCs (PMN-MDSCs), along with an increase of granzyme B positive CD8+ T cells and NK cells. Furthermore, single immune cell profiling of peripheral blood from WBM-treated patients revealed the transcriptional landscape of circulating immune cells. This methods showed a decrease in overall circulating PMN-MDSCs with suppression on the expression of STAT3/IRF1 and TGFβ signaling. Subclusters of PMN-MDSCs showed transcriptional profiles associated with "response to fungus", "neutrophil chemotaxis", "leukocyte aggregation", and "regulation of inflammatory response", which are associated with enhanced immune-modulated anti-tumor activity.

overall_design: Paired whole blood samples of four patients from timepoint of baseline vs. 3 months treatment were collected and processed based single immune cell profiling protocol. The leukocytes from whole blood were enriched according to 10X standard protocol

first: Xiaoqiang

last: Wang

first: Jinhui

first: Xiwei

last: Wu


title: Comparative analysis of gene expression in HepG2-NTCP_HA:HBc upon treatment with CAM-A or CAM-E

city: Strasbourg

first: Frank

last: Jühling

address: 3 Rue Koeberlé

institute: Université de Strasbourg, Inserm, Institut de Recherche sur Les Maladies Virales et Hépatiques UMR_S1110

summary: HepG2-NTCP_HA:HBc cells stably express HBV core antigen. Treatment of these cells with CAM-A compounds (but not CAM-E) induces the abnormal nuclear aggregation of core leading to cell death via apoptosis in a time dependent manner.  The goal of this project is to determine the transcriptomic modifications induced by CAM-A treatment in these cell lines at day 2 and day 3 post-treatment.

overall_design: HepG2-NTCP_HA:HBc cells were treated 24 hours post seeding with DMSO 2% (non treated control), CAM-Ah RG7907, CAM-At ALG5398 and CAM-E ALG26 (negative control) at 1 µM in culture medium in presence of 2% DMSO. Apoptosis activation in all conditions was mesured daily from day 1 to day 6 post-treatment. We observed activation of apoptosis only in CAM-A treated cells  from day 2 post-treatment. Total RNA was extracted from cells at day 2 and 3 post-treatment send for RNAseq analysis.

first: Valerio

last: Taverniti

first: Thomas

last: Baumert

first: Eloi

last: Verrier


title: Recombinant RSV G protein vaccine induces enhanced respiratory disease via IL-13 and mucin overproduction

city: Suita

first: Daisuke

last: Motooka

state: Osaka

address: 3-1, Yamadaoka

country: Japan

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

summary: The G protein expressed on the surface of respiratory syncytial virus (RSV) is important for adhesion to host cells and as a vaccine target antigen. The corresponding vaccines can effectively eliminate RSV, but exacerbate pulmonary immunopathology, such as eosinophil infiltration into the lungs, after the RSV challenge in animal models, raising concerns about enhanced respiratory disease (ERD), creating a need for approaches that mitigate these effects. Herein, we aimed to examine the mechanisms of G protein vaccine-induced ERD in mice, using recombinant G protein as a vaccine antigen. After the RSV challenge, G protein-vaccinated mice exhibited lung weight gain, lung tissue damage, and increased infiltration of eosinophils, neutrophils, and CD4+ T cells into the lungs. We set lung weight gain as the endpoint for ERD and examined the impact of each infiltrating cell on lung weight gain. We observed that CD4+ T cells, but not eosinophils or neutrophils, that infiltrate the lungs are responsible for lung weight gain. In addition, T helper 2 cell-mediated IL-13 induced mucin hypersecretion and lung weight gain. Mucin hypersecretion may contribute to weight gain in the lungs. In conclusion, our results indicate a novel mechanism of G protein vaccine-induced ERD via IL-13 and mucin hypersecretion, which could lead to the development of safe G protein vaccines and the elucidation of the causes of ERD associated with other vaccines.

overall_design:
To thoroughly evaluate the ERD inflammatory response induced by the G protein vaccine, we analyzed the gene expression profile from an RNA-seq data obtained from the lungs of mice subjected to control and experimental treatments (four treatments in total).
Comparative gene expression profiles from lungs of mice vaccinated with PBS or G protein, then treated with an IgG isotype control or anti-CD4 antibody, and subsequently challenged with RSV.
Samples from mice treated with anti-CD4 antibodies served as reference samples.

first: Eigo

last: Kawahara

first: Kota

last: Senpuku

first: Yoshino

last: Kawaguchi

first: Shinya

last: Yamamoto

first: Koubun

last: Yasuda

first: Etsushi

last: Kuroda

first: Noriko

last: Ouji-Sageshima

first: Toshihiro

last: Ito

first: Toshiro

last: Hirai

first: Takehiko

last: Shibata

first: Yasuo

last: Yoshioka


title: A Molecular Switch from Tumor Suppressor to Oncogene in ER-Positive Breast Cancer: Role of Androgen Receptor, JAK-STAT, and Lineage Plasticity [AR low/high]

first: Daniel

last: Johnson

address: 71 S Manassas

institute: UTHSC

summary: Cancers develop resistance to inhibitors of oncogenes mainly due to target-centric mechanisms such as mutations and splicing. While inhibitors or antagonists force targets to unnatural conformation contributing to protein instability and resistance, activating tumor suppressors may maintain the protein in an agonistic conformation to elicit sustainable tumor growth inhibition. Due to lack of tumor suppressor-agonists, this hypothesis and mechanisms underlying resistance are not understood. In estrogen receptor (ER)-positive breast cancer (BC), androgen receptor (AR) is a druggable tumor suppressor and offers a promising avenue for this investigation. Spatial genomics suggest that the molecular portrait of AR-expressing BC cells in tumor microenvironment corresponds to better overall patient survival, clinically confirming AR’s role as a tumor suppressor. Ligand-activation of AR-positive BC xenografts reprograms cistromes, inhibits oncogenic pathways, and promotes cellular elasticity towards a more differentiated state, while sustained treatment results in cistromes rearrangement towards transcription factor PROP1, transformation of AR into an oncogene, and activation of the JAK/STAT pathway, all culminating in lineage plasticity to an aggressive resistant subtype. While the molecular profile of AR agonist-sensitive tumors corresponds to better patient survival, the profile represented in the resistant phenotype corresponds to shorter survival. Inhibition of activated oncogenes in resistant tumors reduces growth and re-sensitizes them to AR agonists. Together, these findings indicate that persistent activation of a tumor suppressor is not fail-safe and leads to resistance through lineage plasticity-driven tumor metamorphosis. Our work provides a framework to explore the above phenomenon across multiple cancer types and underscores the importance of factoring the sensitization of tumor suppressor targets while developing agonist-like drugs

overall_design: To investigate how the transcriptome of androgen receptor (AR)-expressing breast cancer cells are different from AR low/negative cells

first: Sarah

last: Asemota

first: Ramesh

last: Narayanan


title: Novel RNA Biomarkers Improve Discrimination of Children with Tuberculosis Disease from Those with non-TB Pneumonia after In-Vitro Stimulation

city: London

first: Myrsini

last: Kaforou

address: Norfolk Place

institute: Imperial College London

summary: The diagnosis of pediatric tuberculosis (TB) poses a challenge for clinical teams worldwide. TB-mediated changes in the expression of host genes in the peripheral blood can serve as diagnostic biomarkers and can provide better insights into the host immune mechanisms of childhood TB. Peripheral blood mononuclear cells (PBMCs) from children (n=102) with microbiologically confirmed TB disease, ΤΒ infection (ΤΒΙ), pneumonia, and healthy controls (HC) were stimulated with either the Purified Protein Derivative (PPD) or the Early Secretory Antigen 6kDa-Culture Filtrate Protein 10 (ESAT6-CFP10) complex of Mycobacterium tuberculosis (Mtb). RNA was extracted and quantified using gene expression microarrays. Differential expression analysis was performed comparing microbiologically confirmed TB to the other diagnostic groups for the stimulated and unstimulated samples. Using variable selection, we identified sparse diagnostic gene signatures; one gene (PID1) was able to distinguish TB from pneumonia after ESAT6-CFP10 stimulation with an AUC of 100% in the test set, while a combination of two genes (STAT1 and IFI44) achieved an AUC of 91.7% (CI95% 75.0%-100%) in the test set after PPD stimulation.  The number of significantly differentially expressed (SDE) genes was higher when contrasting TB to pneumonia or HC in stimulated samples, compared to unstimulated ones, leading to a larger pool of candidate diagnostic biomarkers. Our approach provides enlightened aspects of peripheral TB-specific responses and can form the basis for a point of care test meeting the World Health Organization (WHO) Target Product Profile (TPP) for pediatric TB.

overall_design: 102 children were enrolled in the study and were classified into 4 different groups, microbiologically confirmed TB disease, TBI, pneumonia and HC. In the patient groups the whole pheripheral blood was collected before treatment for TB disease or TBI was initiated. PBMCs were isolated according to the Histopaque 1077 (Sigma-Aldrich) cell isolation procedure, cultured in 0.2 ml growing medium, and were exposed for 24 hours in triplicate to a) recombinant Mtb antigens, ESAT-6 and CFP-10, b) Tuberculin PPD , or c) remained in the nutrient agent without stimulation. The supernatant was removed from each well and the remaining culture medium was triturated to resuspend the attached cells, transferred to a new vial, and centrifuged. The supernatant was discarded and Trizol reagent was added to the cell pellet. After washing the cell monolayer with PBS, Trizol reagent was added to the culture plate, incubated for 5 minutes at RT, before being added to the cell pellet. The combined Trizol cell lysate was stored at -80oC. Τotal RNA was isolated from pooled replicates for each condition per patient using a modified RNeasy protocol (Qiagen). After QC, RNA was converted to biotinylated cRNA using Illumina TotalPrep RNA Amplification kits (Applied Biosystems) and  the labelled cRNA hybridised to Human HT-12 V4 Expression BeadChip arrays (Illumina) following manufacturer’s instructions. Data were analysed in R.

first: Ortensia

last: Vito

first: Stelios

last: Psarras

first: Angeliki

last: Syggelou

first: Victoria

middle: J

last: Wright

first: Sandra

last: Newton

first: Virginia

last: Amanatidou

first: Katerina

last: Trochoutsou

first: Maria

last: Tsagaraki

first: Michael

last: Levin

last: Tsolia


title: Spatial Multiomics Reveals Metabolic Reprogramming and Calcification Characteristics of Diabetic Macroangiopathy [scRNA-Seq]

city: Zhenjiang

first: Zhongqun

last: Wang

address: Jiefang road 438

institute: Affiliated Hospital of Jiangsu University

summary: BACKGROUND: The diabetic macroangiopathy includes coronary heart disease, cerebrovascular disease and peripheral artery disease. Diabetic macroangiopathy is the main cause of death in diabetic patients. The exact pathogenesis of diabetic macroangiopathy is still not completely clear. METHODS: Single-cell transcriptome, spatial transcriptome, and spatial metabolome sequencing were performed on specimens of the anterior tibial artery from 11 amputated patients for visualization and data analysis through bioinformatics. RESULTS: This study reveals the gene-metabolic network in metabolic reprogramming and calcification characteristics in diabetic macrovasculature. Tissue specificity was found in some metabolic pathways. Alpha-linolenic acid metabolism and linoleic acid metabolism were characteristically enriched in the arteriae externa, and folate biosynthesis was characteristically expressed in calcification region. O-glycan biosynthesis, and primary bile acid biosynthesis are enriched in plaques. The calcification area mainly expresses COL1A2 COL6A2 FN1, MIF, SPP1, TNC as ligands to adjust and control media and plaque. The presence of nuclear enrichment   related lesion changes around calcification resulted in activation of chemokine function, erk1/2 function and phenylalanine pathway. CONCLUSIONS: This study is the first to construct a spatial gene-metabolic map of complete blood vessels, which provides a basis for the subsequent exploration of the mechanism and clinical transformation of diabetic macrovascular disease.

overall_design: The clinical samples and related information obtained in this study were all approved by the Ethics Committee of Jiangsu University, and all patients gave written informed consent. They were diagnosed with diabetes according to the diagnostic criteria of China's Guidelines for the Prevention and Treatment of Type 2 Diabetes (2020), and CT of lower limbs showed significant calcification. In addition, patients who received diabetic foot surgery according to the Guidelines for the Prevention and Treatment of Diabetic Foot in China (2019) were included in the study.The fresh tissues isolated from 4 patients were divided into calcified group (n=3) and noncalcified group (n=3) according to the relative position of CT images and vascular stiffness. After dead cells were removed, the samples were sequenced on the NovaSeq 6000 platform.


title: Spatial Multiomics Reveals Metabolic Reprogramming and Calcification Characteristics of Diabetic Macroangiopathy [Spatial Transcriptomics]


title: Spatial transcriptomic analysis of Gastric adenocarcinoma

first: Daisuke

last: Motooka

summary: This study aims to identify novel cancer stem cell markers by analyzing gastric cancer with spatial transcriptome. This analysis identified a cancer stem cell-associated molecule.

overall_design: The sample used was a gastric cancer case, and the pathological diagnosis was Moderately differentiated tubular adenocarcinoma. The stage of the disease was Stage I. The analysis was performed to include the cancer area. The software used was Spaceranger, R, and compatible packages such as Seurat and monocle3.

first: Akira

last: Ishikawa


title: AAV vector-derived elements integrate into Cas9-generated double strand breaks and disrupt gene transcription

city: Chapel Hill

first: Austin

last: Hepperla

state: NC

address: 7018B Mary Ellen Jones Building

department: Genetics

institute: University of North Carolina at Chapel Hill

summary: We previously found that an adeno-associated virus (AAV) vector containing S. aureus Cas9 and a multi-target guide (g)RNA could integrate into the genome and prematurely terminate transcription of Ube3a-ATS, a long non-coding RNA. Here, we assessed the performance of three additional AAV vectors containing S. aureus Cas9 and twenty-five vectors containing N. meningococcus Cas9, all targeting single sites within Ube3a-ATS. We found that none of these single-target gRNA vectors were as effective as multi-target gRNA vectors at reducing Ube3a-ATS expression in neurons. We also developed a new anchored multiplex PCR sequencing (AMP-seq) method and analysis pipeline to quantify the relative frequency of all possible editing events at target sites, including unresolved double-stranded breaks (DSBs) and capture of foreign DNA. We found that integration of AAV was the most frequent editing event (67-89% of all edits) at three different single target sites, far surpassing insertions and deletions (indels). None of the most frequently observed indels was capable of blocking transcription when incorporated into a Ube3a-ATS minigene reporter, whereas two vector derived elements—the polyA and reverse promoter—reduced downstream transcription by up to 50%. Since not all editing events disrupt gene transcription, our findings suggest that the probability that a gene trapping AAV integration event occurs is influenced by which vector-derived element(s) are integrated and by the number of target sites.

overall_design: AMP-seq on DNA from neurons treated with various NmCas9-gRNA AAVs: NmCas9-AAV gRNA23 (0-7 day treatment), NmCas9-AAV gRNA25 (7 day treatment), NmCas9-AAV gRNA21 (7 day treatment), NmCas9-AAV non-targeting gRNA (7 day treatment), or untreated DNA. AMP-seq on DNA from neurons of P1 (post-natal-day 1) C57BL/6 mice treated with SaCas9-gRNA AAVs (SaCas9-AAV-G3) or SaCas9-AAV non-targeting gRNA.

first: Hannah

last: Bazick

first: Hanqian

last: Mao

first: Jesse

last: Niehaus

first: Justin

last: Wolter

first: Mark

last: Zylka


title: Serum starvation induces quiescence in ovarian cancer

city: Pittsburgh

first: Katherine

last: Aird

state: PA

address: 5051 Centre Ave.

institute: University of Pittsburgh School of Medicine

summary: While prior work in muscle cells indicates that metabolic reprogramming is associated with quiescence, whether metabolic changes occur in cancer to drive quiescence is unclear. We found that the metabolic enzyme ACSS2 is highly upregulated in quiescent ovarian cancer cells and overexpresssion of ACSS2 induces ovarian cancer quiescence.

overall_design: We cultured Ovcar8 high grade serous cancer cells with or without 5% FBS for 48 hours. Controls were untreated. All samples were done in triplicate. Comparative gene expression profiling analysis for control and serum starved Ovcar8 cells.

first: Aird

last: Katherine

first: Buckanovich

last: Ronald


title: Effect of inhibition of SRPK1 and CDK12 on transcription and splicing in castration resistance prostate cancer cells

city: Helsinki

first: Harri

last: Itkonen

address: Haartmaninkatu

country: Finland

institute: University of Helsinki

summary: Inactivation of CDK12 characterizes an aggressive sub-group of castration-resistant prostate cancer (CPRC), and CRPC is currently a lethal disease. Hyper-activation of MYC transcription factor is sufficient to confer the CRPC-phenotype. Here, we show that the loss of CDK12 promotes MYC activity, which renders the cells addicted on the splicing regulatory kinase SRPK1. High MYC expression is associated with increased levels of SRPK1 in patient samples, and over-expression of MYC sensitizes prostate cancer cells to SRPK1 inhibition using pharmacological and genetic strategies. We show that Endovion (SCO-101), a compound currently in clinical trials against pancreatic cancer, phenocopies the effects of the well-characterized SRPK1 inhibitor (SRPIN340) on the nascent transcription, splicing and the overall transcriptional program. Inhibition of SRPK1 with either of the compounds promotes transcription elongation, causes defects in splicing and transcriptionally activates the unfolded protein response. In brief, here we show that CDK12 inactivation promotes MYC-signaling in an SRPK1-dependent manner and identify the clinical grade compound Endovion, which selectively targets the cells with CDK12 inactivation.

overall_design: SLAM-seq after 4 hours treatment of 22RV1 cells with 150nM THZ531, 20micromolar SRPIN340 or 50micromolar Endovion.

first: Jing

last: Liang

first: Aishwarya

last: Gondane


title: Pathogenic mechanism and therapeutic intervention of impaired N7-methylguanosine (m7G) tRNA modification [snRNA-seq]

city: Canton

first: jieyi

last: Ma

state: Guangdong province

address: No.58 Zhong Shan Er Road

department: 1Department of Otolaryngology

institute: the First affiliated hospital of Sun Yat-sen University

summary: The tRNA N7-methylguanosine (m7G) modification is not essential for yeast growth, but in mammals mis-regulations of tRNA m7G modification cause stem cell defect and developmental disorders. Here we found that homozygous mutated WDR4 caused microcephaly, retardation in motor coordination and spatial learning ability in mice, which mimics the symptom in human patients with WDR4 mutation. Further analysis revealed that homozygous mutation of WDR4 impaired tRNA m7G modification and reduced the translation level of genes involved in mTOR signaling pathway in mouse brain, which further caused endoplasmic reticulum (ER) stress and cell death. Our study uncovered a new layer of translation regulation mechanism mediated by tRNA m7G modification, provided strong evidence to support the important physiological function of mis-regulated tRNA modification in neuron disorders.

overall_design: Single-nucleus transcriptomic analysis of mouse brain tissues.

first: Jieyi

first: Shuibin

last: Lin


title: Acetate is a driver of quiescence in ovarian cancer

summary: While prior work in muscle cells indicates that metabolic reprogramming is associated with quiescence, whether metabolic changes occur in cancer to drive quiescence is unclear. Using a multi-omics approach, we found that the metabolic enzyme ACSS2 is both highly upregulated in quiescent cells and required for their survival. ACSS2 converts acetate into acetyl-CoA, and we confirmed increased acetate-derived acetyl-CoA in quiescent cells, demonstrating increased ACSS2 activity. Interestingly, supplementing cells with acetate alone was sufficient to induce a reversible quiescent cell cycle exit.

overall_design: We treated Ovcar8 high grade serous cancer cells with 10mM sodium acetate for 72h. Controls were untreated. All samples were done in triplicate. Comparative gene expression profiling analysis for control and acetate-treated Ovcar8 cells.

first: Ronald

last: Buckanovich


title: DAMPs drive Fibroinflammatory Changes in the glaucomatous ONH

city: Madison

first: Emma

last: Geiduschek

state: WI

address: 1300 University Ave

institute: University of Wisconsin - Madison

summary: Purpose: The optic nerve head (ONH) is well known to be the initial site of glaucomatous damage. However, the molecular mechanisms initiating this pathology are not fully understood. To further understand the initiating factors in glaucomatous damage we utilized a novel mouse model of glaucoma, B6.EDA+/+ mice, that constitutively express FN containing the EDA domain (FN+EDA). FN+EDA is a known damage associated molecular pattern (DAMP) that activates toll-like receptor 4 (TLR4) and elicits a fibro-inflammatory response. Results: B6.EDA+/+ mice exhibit significantly higher IOP, loss of RGCs, thinning of the RNFL, and progressive levels of ON damage at 12 months and 22 months of age compared to C57BL/6J controls. Protein expression of DAMPs FN+EDA and biglycan were significantly increased in B6.EDA+/+ mice compared to C57BL/6J controls.  GSEA analysis identified significantly up- and down-regulated gene groupings at both 12 months and 22 months of age, and IHC staining demonstrated significant increases of IFNα, IFNβ, and pSTAT1 expression in B6.EDA+/+ mice compared to C57BL/6J controls. Conclusions: Our study characterizes glaucomatous changes to the retina, ON, and ONH over the course of two years and identifies novel molecular pathways associated with these pathophysiological changes. These data indicate time specific DAMP production and immune system signaling in a novel mouse model of glaucoma.

overall_design: Methods: Eyes from B6.EDA+/+ and C57BL/6J mice were evaluated for RGC death, RNFL thickness, and ON damage at 12 months and 22 months of age. ONH sections were isolated using laser capture microdissection (LCM) for subsequent RNAsequencing and gene set enrichment analysis (GSEA). GSEA results were confirmed using immunohistochemcial (IHC) staining.

last: Bricco

first: Colleen

last: McDowell


title: Pathogenic mechanism and therapeutic intervention of impaired N7-methylguanosine (m7G) tRNA modification [RNC seq]

overall_design: Ribosome nascent-chain complex-bound mRNA sequencing was used to study the differentially translated genes in the WDR4 mutated and wild type mice brain tissues.