title: Androgen receptor determines skeletal muscle sexual dimorphism [ChIL-seq]

first: Hiroshi

last: Sakai

overall_design: ChIL-seq of androgen receptor (AR) and H3K27ac in mouse skeletal muscle with or without dihydrotestosterone (DHT) treatment. ChIL-seq against AR and H3K27ac in human skeletal muscles.

first: Yuuki

last: Imai


title: Salivary miRNA in ovarian cancer

status: Public on Aug 03 2026

city: Pisa

first: Francesca

last: Scebba

address: Piazza martiri della libertà 33

country: Italy

institute: Scuola Superiore Sant'Anna

type: Non-coding RNA profiling by high throughput sequencing

summary: This study was aimed at identifing a salivary microRNA signature able to distinguish ovarian cancer from breast cancer patients and healthy subjects.Ovarian cancer is one of the most lethal cancers among female patients and the fifth cause of cancer-related deaths in women. The significant mortality is largely caused by late diagnosis, due to confusing, unspecific symptoms and the absence of effective tools for early diagnosis. MicroRNAs are small non-coding RNA molecules that regulate gene expression. They have been extensively studied as promising circulating biomarkers for the diagnosis and monitoring of various diseases. Among body fluids, saliva accurately reflects systemic pathophysiological conditions including cancer, making it a promising diagnostic resource for screening purposes. Finding a subset of differentially expressed miRNA in saliva of ovarian cancer's patients compared both to healthy subjects and to women affected by a different gynaecological cancer could help in the early screening purpose.

overall_design: miRNA-seq profiling was performed on saliva samples belonging to female individuals of three different experimental groups. The control group was made of healthy subjects while pathologic individuals groups were made of women affected by ovarian cancer and women affected by breast cancer. Unstimulated saliva was collected, cellular debry was removed and total RNA was isolated.

first: Angeloni

last: Debora

first: Marzia

last: Robotti

first: Alberto

last: Mercatanti

first: Milena

last: Rizzo

first: Angelo

last: Lasala

first: Silvia

last: Cateni


title: Lamins gate nuclear and chromatin structures for cardiomyocyte maturation genes.

status: Public on Aug 05 2026

city: Baltimore

first: Yixian

last: Zheng

state: MD

address: 3520 San Martin Dr

country: USA

institute: Carnegie Institution of Washington

type: Expression profiling by high throughput sequencing

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: mtDNA transfer dynamics in vivo can induce dose-dependent tumor cell dedifferentiation and heterogeneity

city: Los Angeles

first: Michael

last: Teitell

state: CA

address: 675 Charles E Young Drive South

department: Pathology and Laboratory Medicine

institute: University of California Los Angeles

summary: Tumor heterogeneity limits treatment efficacy and drives therapy resistance. Although mitochondrial genome (mtDNA) mutations occur in ~60% of solid tumors, their contribution to heterogeneity is unexplored. We deployed established rho0 B16 melanoma and 4T1 breast cancer mouse models and engineered isogenic tumor cells carrying unique homoplasmic single-nucleotide mtDNA variants used as tracking barcodes. Using single-cell ATAC-seq with mitochondrial genotyping (mtscATAC-seq / mgatk) and single-cell multiome (ATAC + gene expression) profiling, we mapped and quantified horizontal mtDNA exchange within tumors in vivo. Respiration-deficient mutant tumor cells selectively and stably acquired host mtDNA, and mtDNA transfer induced dose-dependent tumor cell dedifferentiation and heterogeneity.

overall_design: Single-cell ATAC-seq with mitochondrial genotyping of B16 melanoma (C57BL/6) and 4T1 mammary carcinoma (BALB/c) tumors composed of isogenic rho0-derived cells carrying defined homoplasmic mtDNA variants (WTP, E9, FMI.12, MB77); and 10x single-cell multiome (ATAC + gene expression) of GFP-labeled B16 tumors (FMI.12, and co-injected E9 + FMI.12) grown in NOD scid hosts.

first: Berney

last: Peng

first: Vahan

last: Martirosian

first: Eaint Honey Aung

last: Win-Evirgen

first: Christy

last: Lee

first: Thang

middle: L

last: Nguyen

first: Amy

middle: K

last: Yu

first: Kristin

middle: J

last: Pischel

first: Natalia

last: Garcia Dutton

first: Weihong

last: Yan

first: Paul

middle: T

last: Spellman

first: Caleb

middle: A

last: Lareau

first: Jingyi Jessica

last: Li


title: Single-cell transcriptomic analysis of GFP-positive and GFP-negative CD45-positive and CD45-negative cardiac cells isolated from TAC-operated REFLEX mice

city: Shimotsuke

first: Takayuki

last: Isagawa

state: Tochigi

address: 3311-1 Yakushiji

department: Center for Molecular Medicine

country: Japan

institute: Jichi Medical University

summary: Cardiac remodeling induced by pressure overload is accompanied by extensive changes in intercellular communication among cardiac cell populations. To identify cells that physically interact with vascular endothelial cells in vivo, we utilized the REFLEX system combined with HUNTERuni-seq, a single-cell transcriptomic approach for profiling interacting cell populations. Cdh5-CreERT2;REFLEX mice were subjected to transverse aortic constriction (TAC), and GFP-positive cells representing endothelial-interacting cells were isolated from the heart. Single-cell RNA sequencing was performed to characterize the cellular composition and transcriptional states of interacting and non-interacting cell populations in the pressure-overloaded myocardium. These data provide a resource for investigating endothelial-centered cellular interaction networks during cardiac remodeling.

overall_design: Cdh5-CreERT2;REFLEX mice were treated with tamoxifen and subjected to transverse aortic constriction (TAC) to induce pressure-overload cardiac remodeling. Hearts were harvested after TAC, dissociated into single-cell suspensions, and separated into CD45-positive and CD45-negative fractions. Within each fraction, GFP-positive cells labeled by the REFLEX system and corresponding GFP-negative cells were isolated by fluorescence-activated cell sorting (FACS). Single-cell RNA sequencing libraries were generated using the HUNTERuni-seq workflow and sequenced on an Illumina platform. The resulting datasets were used to compare transcriptional profiles of interacting and non-interacting cell populations in the pressure-overloaded heart.

first: Tatsuyuki

last: Sato


title: CD97/ADGRE5 attenuates the induction of adaptive type 2 immune responses in allergic asthma

city: Leipzig

first: Knut

last: Krohn

address: Philipp-Rosenthal-Straße 55

department: Medizinische Fakultät

country: Germany

institute: University Leipzig

summary: Allergic asthma results from an uncontrolled type 2 immune response to inhaled allergens. Here, we investigate the role of CD97/ADGRE5, expressed in mouse and human immune and lung epithelial cells, in this disease. Female Cd97-/- mice exhibit an exacerbated asthmatic phenotype across multiple models, primarily due to CD97 loss on immune cells. A single CD97 antibody treatment before allergen sensitization worsens allergic responses, highlighting a role for CD97 in early immune regulation. Post-sensitization, Cd97-/- mice display higher frequencies of lung conventional type 2 and monocyte-derived dendritic cells (DCs). Allergen-pulsed Cd97-/- bone marrow-derived DCs are more activated, promote enhanced proliferation and type 2 cytokine secretion by CD4⁺ OT-II cells, and induce stronger airway inflammation. Consistently, ADGRE5 expression is reduced in airway mucosa-derived mononuclear phagocyte subsets in human asthmatics after allergen-induced exacerbation. These results identify CD97 as a key regulator of DC-driven type 2 allergic responses and a potential target in asthma.

overall_design: RNA-seq profiling of CD11c+ OVA-pulsed BMDCs from wildtype and Cd97-/- DCs.

first: Gabriela

last: Aust

first: Marianne

last: Quaas

first: Tobias

last: Polte


title: Atorvastatin modulates oncogenic and protective transcriptomic signatures in hexachlorobenzene-induced preneoplastic liver lesions in rats

city: Mannheim

first: Lucia

last: Coli

address: Theodor-Kutzer-Ufer 1-3

institute: Universitätsklinikum Mannheim

summary:
Background & Aims: Hepatocellular carcinoma (HCC) is the most prevalent type of primary liver cancer.Globally, liver cancers represent the fourth most common cause of cancer-related deaths. Although statins have been shown to be effective in preventing HCC, their underlying molecular mechanisms are not yet fully elucidated. The aim of this  study was to evaluate the hepatic gene expression profile and its relationship to early hepatocarcinogenic  changes in a Hexachlorobenzene (HCB)-induced preneoplastic liver lesion model in rats treated with atorvastatin (AT).
Materials   & Methods: Male Wistar rats weighing 160 g b.w. were randomly assigned to four experimental groups ( n= 5 per group) : control, HCB (100 mg/kg b.w.), AT (5 mg/kg b.w.), and AT + HCB, treated for 30 days. The analysed parameters were liver weight, body weight, hepatosomatic index, liver histology, and expression levels of target proteins, c-Myc, TGF-β1, and p53 by immunofluorescence (n = 3 per group). Transcriptomic analysis  was performed by RNA sequencing on liver tissue (n = 2 per group). Differentially expressed genes (DEGs) were identified using DESeq2 (|log2FC| >  1, adjusted p < 0.05).. Gene set enrichment analysis (GSEA) and KEGG pathway overrepresentation  analysis were conducted  using the WebGestalt platform .
Results: HCB treatment significantly increased the hepatosomatic index (28%, p<0.01) and induced preneoplastic hepatic  lesions, both effects   were prevented by AT co-administration. Transcriptomic analysis of HCB-treated livers identified 6,396 DEGs, of which 1,352 overlapped with the 1,612-gene KEGG “Pathway in cancer” set (83.9% concordance; p = 5.19×10⁻⁵⁴, FDR = 1.82×10⁻⁵¹). HCB activated multiple oncogenic pathways, including RHO GTPase cycle, TGF-β signalling, and receptor tyrosine kinase signalling, AT co-treatment downregulated these HCB-induced pathways and upregulated protective PPAR signalling, autophagy, and cellular stress response pathways. Consistent with these transcriptomic findings, AT co-administration reduced TGF-β1 and c-Myc protein levels and attenuated the HCB-induced accumulation of total p53 protein levels observed in liver tissue.
Conclusions: In this experimental model, Atorvastatin exerted  a protective  effect against  HCB-induced preneoplastic hepatic changes  through the modulation of oncogenic and cytoprotective transcriptomic programmes. These findings provide mechanistic insights into statin-mediated hepatoprotection and support further investigation of AT as a potential chemopreventive agent in high-risk populations. However, additional validation in models that better reflect the chronic liver injury context of human HCC is needed.

overall_design: Male Wistar rats were used from the vivarium of the Faculty of Pharmacy and Biochemistry of the University of Buenos Aires. At the beginning of the treatment, they had a weight of 160 g, were housed with free access to standard chow (Purina 3; Cabañas S.C.A., Argentina) and water. They were kept under a 12-hour light-dark cycle, at a temperature of 19 ± 2 °C and a humidity of 45-75%. Twenty male Wistar rats were randomly assigned to four experimental groups (n = 5 per group): (i) Control (vehicle only), (ii) Atorvastatin (AT, 5 mg/kg b.w./day, administered in drinking water), (iii) Hexachlorobenzene (HCB, 100 mg/kg b.w., administered by oral gavage three times per week for 30 days), and (iv) AT + HCB.  Animals were sacrificed on day 37 by intraperitoneal injection of ketamine/xylazine.  AT was administered in the drinking water at a dose of 5 mg per 20 mL, with each rat receiving 20 mL of water per day. HCB was dissolved in water containing 0.5% Tween-20 (0.5 ml/100 ml) at a concentration of 40 mg/ml; HCB doses were selected based on previously published studies from this group. For transcriptomic analysis, liver samples from two independent animals per experimental group were selected for RNA sequencing (n = 2 biological replicates per group; no technical replicates). Libraries were sequenced by Azenta Life Sciences on an Illumina HiSeq platform using 2 x 150-bp paired-end reads. The sequencing run generated 249,181,156 reads in total, corresponding to 27,998,827-33,891,151 reads per library.

first: Ezequiel

last: Ridruejo

first: Jimmy

last: Daza

first: Giselle

last: Romero Caimi

first: Timo

last: Itzel

first: Andreas

last: Teufel

first: Laura

last: Alvarez


title: An Integrated Spatial Multi-Omics Workflow for Sequential RNA and Protein Profiling in FFPE Tumor Tissue

city: Darlinghurst

first: Merrin Mary

last: Eapen

state: NSW

address: 384 Victoria St

country: Australia

institute: Garvan Institute of Medical Research

summary: Understanding complex cellular niches, such as tertiary lymphoid structures (TLS), requires spatially resolved, multi-omic approaches that link transcriptional states and protein expression levels within individual cells. Here, we present an integrated spatial multi-omics workflow that enables sequential mapping of hundreds of genes via the In Situ platform (Xenium) and over 40 protein markers via Imaging Mass Cytometry™ (IMC™) technology on a single formalin-fixed, paraffin-embedded (FFPE) tissue section. We applied this multi-modal approach to colorectal liver metastases (CRLM) and matched adjacent normal liver tissues. Our results demonstrate that the sequential application of these technologies maintains tissue morphology and the assay’s technical sensitivity. Further, high-dimensional data integration was performed through optimised computational co-registration at a single-cell level. Through this approach, we observed a correlation between β-catenin proteomic levels and malignant cell states, characterised immune cell phenotypes in TLS, and identified biological discrepancies between RNA and protein levels for key checkpoint molecules (PD-L1, TIM-3, and IDO). Overall, this technical framework enables robust profiling of functional cellular states, mapping chemokines, and more sensitive detection of clinically relevant targets missing in single-modality methods.

overall_design: Sequential Xenium In Situ spatial transcriptomics and Imaging Mass Cytometry was performed on formalin-fixed paraffin embedded (FFPE) tissue sections from patients with colorectal liver metastasis (CRLM). Xenium spatial transcriptomics was performed on three CRLM tumor samples and one matched adjacent normal liver sample. Xenium gene expression profiling was performed using a custom standalone panel of 462 genes (Tumor 1, Tumor 2 and Adjacent Normal Liver Sample) or using a 319 genes (Tumor 3).

first: Qanber

last: Raza

first: Lucy

last: Chhuo

first: Annabel

last: Faulkner

first: Abhishek

last: Singh

first: Hao

last: Xu

first: Atefeh

last: Khakpoor

first: Erin

last: Coll

first: Liang

last: Lim

first: Nick

last: Zabinyakov

last: Qiao

first: Anna

last: Di Bartolomeo

first: Jacob

last: George

first: Christina

last: Loh

first: Helen

middle: M

last: McGuire

first: Ankur

last: Sharma


title: Meteorin-like regulates cardiac repair via macrophage modulation following myocardial infarction

city: Barcelona

first: Ana

last: Planavila Porta

state: Catalunya

address: Avinguda Diagonal, 643

department: Bioquímica i Biomedicina Molecular

country: Spain

institute: Universitat de Barcelona

summary:
After myocardial infarction (MI), the heart undergoes a reparative process that includes an initial acute inflammatory phase followed by a subsequent reparative phase. The transition between these phases is crucial for cardiac recovery, but the key factors remain
unclear. Meteorin-like (Metrnl) promotes anti-inflammatory macrophage polarization in the myocardium, yet its role in the acute phase post-MI is unknown. We observed that macrophages infiltrating the ischemic myocardium produced elevated levels of Metrnl in
both the heart and circulation 4 days post-MI. The absence of Metrnl in Metrnl⁻/⁻ mice altered myocardial healing and remodeling, with an increased presence of macrophages with a more pro-inflammatory phenotype. Conversely, cardiac Metrnl overexpression
restored myocardial repair and promoted a shift toward a more anti-inflammatory macrophage phenotype. Mechanistically, Metrnl regulated macrophage-dependent production of Oncostatin M (Osm), a key cytokine in the early inflammatory phase post-
MI that induces cardiomyocyte production of Reg3β. Reg3β, in turn, limits classical macrophage activation and polarization while modulating their trafficking, ultimately influencing the duration and intensity of the pro-inflammatory phase post-MI. Thus,
Metrnl plays a crucial role in cardiac repair by modulating the acute phase following myocardial infarction through its regulation of macrophage populations.

overall_design: Single nuclei RNA sequencing (snRNA-Seq) profiling of the heart (left ventricle, septum and right ventricle) from wild-type (Wt) or Meteorin-like null (Metrnl⁻/⁻)mice treated with AAV9-Null or AAV9-Metrnl to overexpress Metrnl gene product in the heart. All mice underwent a myocardial infarction (MI). The euthanasia and tissue collection was performed 4 days after the MI.

first: Gemma

last: Ferrer-Curriu

first: Albert

last: Blasco-Roset

first: Artur

last: Navarro-Gascon

first: Carolina

last: Soler-Botija

first: Francisco Javier

last: Godoy-Nieto

first: Marta

last: Monguió-Tortajada

first: Rubén

last: Cereijo

first: Tania

last: Quesada-López

first: F

last: Rueda

first: Santiago

last: Roura

last: Gálvez-Montón

first: Francesc

last: Villarroya

first: Antoni

last: Bayés-Genís

last: Planavila


title: RNA‑seq analysis of microglia from Fpr2 knockout and wild‑type mice following LPS‑induced acute inflammation

city: Shanghai

first: Wang

last: Hanqi

address: 800 Dongchuan Road, Minhang District

country: China

institute: Shanghai Jiao Tong University

summary: To investigate the molecular mechanisms by which formyl peptide receptor 2 (FPR2) regulates microglial activation and neuroinflammation in an LPS‑induced acute depression model, we performed RNA‑sequencing of microglia isolated from three groups of mice: wild‑type saline‑treated (WT‑Saline), wild‑type LPS‑treated (WT‑LPS), and Fpr2 knockout LPS‑treated (Fpr2‑/‑‑LPS). Transcriptomic profiling revealed that LPS challenge induced widespread changes in immune‑ and inflammation‑related pathways, including the NLR signaling pathway, cytokine‑cytokine receptor interaction, and chemokine signaling. FPR2 deficiency partially reversed these LPS‑induced alterations, particularly suppressing the upregulation of genes involved in the NLRP3 inflammasome pathway (e.g., Nlrp3, Il1b, Csf1). These RNA‑seq data provide a global view of how FPR2 deletion reshapes the microglial transcriptome under inflammatory conditions and support the conclusion that FPR2 acts upstream of the CSF1/NLRP3 axis to mediate depressive‑like behaviors.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for WT-Saline, WT-LPS, and Fpr2-/--LPS mouse brain microglia to elucidate the impact of Fpr2 knockout on the LPS-induced microglial transcriptome.

first: Hanqi

last: Wang

first: Yang

last: Yu


title: Deletion of a Distal IRF4 Element Prevents Inflammation-induced Reprogramming of Human Regulatory T-cell Fate

city: San Francisco

first: Qizhi

last: Tang

address: 513 Parnassus Ave HSW 1102

department: Diabetes Center

institute: University of California, San Francisco

summary: Adoptive regulatory T cell (Treg) therapy is an emergent treatment paradigm for restoring immune tolerance in patients with autoimmune and inflammatory diseases. While preclinical models have demonstrated the remarkable efficacy of Treg cell therapy, lineage-tracing studies have also revealed that murine Tregs can lose lineage identity and adopt a proinflammatory state. Whether human Tregs exhibit a similar susceptibility to inflammatory cytokine-mediated lineage decommitment remains inconclusive. Here, we present an in vitro model of human Treg destabilization characterized by silencing of the Treg lineage-defining transcription factor, FOXP3, loss of suppressive activity, and gain of proinflammatory functions. Analysis of single-cell ATAC and transcriptomic profiles of Tregs during destabilization revealed a switch in chromatin accessibility between two elements at the IRF4 locus. Excision of the distal IRF4 element enabled Tregs to resist inflammatory cytokine-induced reprogramming, presenting a cell-engineering strategy to design more effective Treg therapies.

overall_design: Primary human Tregs (CD4+ CD25+ CD127-/low) were isolated and stimulated with anti-CD3/CD2/CD28 ImmunoCult on days 0, 9, and 18. Cells were treated with IL-2 only or IL2 + either IL-6, IL-1B, IL-23, or IL-6 + IL-1B + IL-23. Primary human Tconv treated with IL-2 grown under the same conditions are included as a reference control. On day 25, cells were harvested and bulk ATACseq was performed.

first: Patrick

last: Ho

first: Alexander

last: Vu

first: Joey

last: Leung

first: Connor

last: Tomaka

first: Wendy

last: Rosenthal

first: Vasilis

last: Ntranos

first: Jeffery

last: Bluestone


title: Gut microbial interaction networks control autoimmunity to neuroretina

city: Bethesda

first: Guangpu

last: Shi

state: Maryland

address: 10 Center Drive, 10/10N248

department: National Eye Institute

institute: National Institutes Of Health

summary: The gut microbiome influences the development of immune-mediated inflammatory diseases. One such condition is autoimmune uveitis, a sight-threatening ocular inflammation driven by retina-specific T cells.  Using a model of spontaneous experimental autoimmune uveitis (sEAU) we showed that gut commensals provide immune stimuli that trigger the disease. Here we report that uveitis-promoting microbes are present in human gut flora and that colonization of germ-free (GF) mice with commensal flora from healthy human donors was sufficient to provoke disease. Severity of sEAU correlated with expansion of Akkermansia and contraction of short-chain fatty acid (SCFA)–producing Firmicutes, followed by decreased SCFA levels and a dominant gut Th1 effector response. Mechanistic gain-of-function experiments, enriching GF sEAU mice with Akkermansia, reproduced these microbiome, metabolite and immune phenotype shifts, and exacerbated disease. We propose that Akkermansia promotes autoimmunity by outcompeting SCFA-producers and enhancing Th1-type responses. Notably, an inverse correlation between Akkermansia (Verrucomicrobia) and Firmicutes was also present in fecal microbiome of patients with uveitis, multiple sclerosis and Crohn’s disease. These findings reveal a stereotypic gut microbial interaction network that regulates systemic immune balance, and may represent an ecologically conserved mechanism through which the gut microbiome modulates autoimmune and inflammatory diseases.

overall_design: Cells from colon lamina propria (CLP) were labeled with TotalSeq C anti-mouse HashTags 1-10 (C0301-C0310, clones M1/42; 30-F11) and 14 cell surface antibodies at the dilution of 1:100. Cell surface anti-mouse antibodies: CD4 (DNA_ID C0001, clone RM4-5), CD8a (C0002, 53-6.7), Ly-6C (C0013, HK1.4), CD11b (C0014, M1/70), Ly-6G (C0015, 1A8), CD90.2 (C0075, 30-H12), CD19 (C0093, 6D5), CD11c (C0106, N418), F4/80 (C0114, BM8), I-A/I-E (C0117, M5/114.15.2), NK-1.1 (C0118, PK136), TCRβ chain (C0120, H57-597), TCR g/d (C0121, GL3), and CD3 (C0182, 17A2). Labeled cells were sorted for live CD45+ and CD4+ populations with PE anti-mouse CD45.2 (clone 104), eFluorTM 450 anti-mouse CD4 (clone RM4-5) and 7-AAD using FACSAria III/Fusion sorters. Post-sort viability exceeded 80%. Hash-tagged samples were pooled at equal ratios within each experimental group, combining CD45+ and CD4+ fractions at 1:1 ratio to enrich for CD4+ T cells. Pooled cells were resuspended in PBS with 0.04% BSA and loaded onto a 10x Genomics Chromium X instrument at 700-1200 cells/µL for single cell 5’ gene expression profiling. Two sequencing libraries (PBS_CLP and AKK_CLP) were prepared using the Chromium Next GEM Single Cell 5’ Reagent Kits v2 (Dual Index) following the manufacturer’s protocol, sequenced on Illumina NovaSeq 6000 platform, and submitted as separate GSM records. Three biological replicates were demultiplexed from each library during downstream analysis. Six filtered feature-barcode matrices were provided as processed data, each corresponding to an individual biological replicate derived from the pooled libraries. Hashtag ids were included in meta data.

first: Amy

last: Zhang

first: Reiko

last: Horai

first: Yingyos

last: Jittayasothorn

first: Jonathan

last: Badger

first: Zhichao

last: Wu

first: Akriti

last: Gupta

first: Vijayaraj

last: Nagarajan

first: John

last: McCulloch

first: Shilpa

last: Kodati

first: H Nida

last: Sen

first: Jung Wha

last: Lee

last: Jacobs

first: Xiaoyan

last: Xu

first: Mary

last: Mattapallil

first: Zixuan

last: Peng

first: Biying

first: Robert

last: Palmer

first: Nadim

last: Majdalani

first: Kenya

last: Honda

first: Colm

last: O'hUigin

first: Rachel

last: Caspi


title: Methylation-mediated regulation of tumor-suppressor function of the miR-379/656 (C14MC) cluster and its clinical utility in hepatocellular carcinoma

city: Mysuru

first: Prasanna

last: Kumar S

address: Department of Biochemistry, JSS Medical College, Mysuru

country: India

institute: JSS Medical College

summary: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality. Several microRNAs (miRNAs) play key roles in HCC development and progression. The role of epigenetic processes like DNA methylation in the regulation of miRNAs is critical to HCC pathogenesis. In this study, we show that the miR-379/656 cluster (C14MC) acts as a tumor suppressor cluster and is epigenetically regulated by DNA methylation.  We demonstrated that C14MC is downregulated in HCC cell lines through nCounter assay and in clinical samples from The Cancer Genome Atlas (TCGA) liver hepatocellular carcinoma tissues. The C14MC promoter was identified and characterized through cloning and dual luciferase assay. Furthermore, we demonstrated that the loss of C14MC tumor suppressor function is directly regulated by the hypermethylation of promoter-bound CpGs, as shown in artificial methylation experiments. The reactivation of specific C14MC miRNAs, like miR-299-5p and miR-376c-3p via mimics, abrogated the expression of several target oncogenes, including PARP1, SPP1, RAD21, and CENPA that regulate critical molecular pathways such as the p53 signaling and NF-kappa B signaling pathways in HCC. Additionally, overexpression of miR-299-5p and miR-376c-3p inhibited HCC cell migration and invasion, suggesting that overexpression of candidate C14MC miRNAs can mitigate cancer hallmarks in HCC cells. Additionally, we have performed whole transcriptomic sequencing for RNA isolated from HCC spheroids generated from HepG2 cells to identify the DEGs regulated by C14MC. Furthermore, we checked for the clinical correlation of C14MC targets and their target genes in terms of survival outcomes and identified the key genes associated with prognostic potential in HCC.  We conclude from our experimental findings that C14MC is a tumor-suppressor miRNA cluster and is regulated epigenetically by methylation in HCC. Several of these miRNAs and their targets can be used for early HCC diagnosis and prognosis. Thus, targeting C14MC can be useful in HCC management.

overall_design: We performed whole transcriptomic analysis to identify the differentially expressed mRNA in HCC 3D-spheroid models. HepG2 monolayer culture was used to generate a 3D spheroid using hanging drop and forced suspension methods. Whole RNA was isolated from trizol method. Control liver RNA was used from commercial liver RNA (TakaraBio, Japan). RNA-seq was performed to identify the expression of the C14MC target genes in HCC tumouroids. We performed RNA-seq on an Illumina NovaSeq V1.5 instrument according to the manufacturer’s protocol. Briefly, the ribosomal RNA depletion was carried out using Qiagen FastSelect rRNAHMR (Qiagen, Germany), and the mRNA libraries were prepared using the NEB Ultra II directional RNA-Seq Library Kit (NEB, USA). The first-strand and second-strand cDNA synthesis were carried out using reverse transcriptase and DNA Polymerase I enzymes, respectively, followed by adapter ligation. Prior to sequencing, the fragment sizes were analysed using HS NGS Fragment kit (1-6000bp) (Agilent, USA) on a Fragment analyser. Subsequently, the mapping of the aligned paired reads was performed against the reference genome (GRCh38) using STAR (v.2.7.11b). The DETGs were identified using DESeq2. The fold change was calculated using the thresholds (−2≤log2(FC)≥+2, p≤ 0.05).

first: Shreyas

middle: H

last: Karunakara

first: Manju

last: Moorthy

first: Gopalakrishna

last: Ramaswamy

last: Santhekadur


title: DNA methylation profiling of Arabidopsis BR-regulated ces-D and ces-qM mutants II

city: Freising

first: Brigitte

last: Poppenberger

state: Freising

address: Liesel-Beckmann-Str. 1

institute: Technical University of Munich

type: Methylation profiling by high throughput sequencing

summary: When organisms encounter pathogens, they rapidly activate complex defense programs to ensure survival. While these immune responses are vital, they often also incur trade-offs, such as reduced growth and development and must therefore be tightly controlled 1,2. In this study, we reveal that the steroid hormones brassinosteroids (BRs) contribute to this control in Arabidopsis thaliana by epigenetically repressing immunity-related genes. We provide evidence that the BR-regulated basic helix-loop-helix (bHLH) transcription factor CESTA (CES), along with its homologs BR ENHANCED EXPRESSION (BEE)1–3, mediate DNA methylation changes at transposable element (TE)-rich loci containing nucleotide-binding leucine-rich-repeat (NLR)-type receptor genes, including SUPPRESSOR OF NPR1-1 CONSTITUTIVE 1 (SNC1). These CES-induced methylation changes are correlated with altered splicing of SNC1 pre-mRNA, a process that requires the BR receptor BR INSENSITIVE 1 (BRI1). In support, we show that CES associates with components of the chromatin remodelling and splicing machinery. Together, our findings reveal a previously unrecognized BR-induced mechanism that modulates the epigenetic and post transcriptional regulation of immune genes, enabling plants to prioritize growth over defense.

overall_design: Twelve Arabidopsis thaliana samples were analyzed using whole-genome bisulfite sequencing (WGBS). The experiment included ces-D (gain-of-function) and ces-qM (loss-of-function) mutants and their wild-type Col-0 controls, each either treated with 10µM 2,4-Epibrassinolide for 3h, or mock DMSO (n = 2 replicates each) for a total of 12 samples. DNA was extracted from 2-week-old aerial tissue. Bisulfite conversion and library preparation were performed by BGI Genomics using the DNBseq platform. Sequencing was conducted on the Illumina HiSeq X Ten. Reads were quality-filtered, aligned to the TAIR10 reference genome using Bismark, and methylation probabilities were calculated using METHimpute. DMRs were called using jDMR-grid with genotype-specific controls to mitigate batch effects. Processed data include bigWig files with methylation tracks and CX-format methylation reports from METHimpute.

middle: S

last: Piecyk

first: Veronica

middle: E

last: Ramirez


title: scRNAseq of Kaede red+ CD45+ cells from draining-lymph nodes of YUMMER1.7 tumors day 21 post implantation

city: New York City

first: Ines

last: Delclaux

state: New York

address: 550 1st avenue

institute: NYU Langone Health

summary: Successful neoadjuvant immune checkpoint blockade (ICB) therapy depends on the maintenance of a pool of stem-like CD8+ T cells that are enriched in tumor-draining lymph nodes (LN). Upon ICB, stem-like CD8+ T cells activate and proliferate to generate effector T cells that seed the intratumoral response. Yet, how the stem-like niche is maintained over time in cancer remains poorly understood. Here, we identify a continuous lymphatic circuit that sustains the stem-like CD8+ T cell pool. Using photoconvertible fate-mapping, we demonstrate that effector CD8+ T cells egress from the tumor microenvironment via tumor-associated lymphatic vessels, and migrate back to the draining LN. These tumor-specific, migratory effector T cells avoid chronic antigen stimulation in the tumor, re-express the transcription factor associated with self-renewal, TCF1, and subsequently enter a stem-like state in the LN. We find that chronic antigen presentation in LNs by dendritic cells drives the local proliferation and transient residence of tumor-egressing T cells thereby inflating the LN stem-like population. Consequently, maintenance of stem-like T cells and ICB response depends on constitutive lymphatic transport, which sustains both effector egress and antigen presentation in the LN. Finally, we find that metastasis disrupts the stem-like LN niche in patients and impairs response to ICB in mice. We, therefore, define a continuous, peripheral lymphatic circuit that recycles tumor-experienced effector T cells to fuel durable systemic anti-tumor immune responses, providing mechanistic insight into the enhanced efficacy of neoadjuvant immunotherapy and highlighting the importance of preserving lymphatic transport for optimal cancer immunotherapy outcomes.

overall_design: On day 21 after implantation of 500K Yummer1.7 in Kaede-Tg mice (n=3) we photoconverted the tumors and 24 hours later single cell suspensions were enriched for CD45+ using a QuadroMACs magnetic separator (CD45-APC, APC beads; Miltenyi Biotec). We stained with DAPI and sorted Kaede red+CD45+ cells from the LN (LN). Sorted cells were pooled and loaded onto a 10X Genomic Chromium Instrument to generate single-cell gel beads in emulsion. Libraries were prepared using Single Cell 3’ Reagent kits (10x Genomics) and sequenced using Illumina Novaseq 6000.

first: Katherine

last: Ventre

first: Naomi

middle: R

last: Besson

first: Tara

last: Muijlwijk

first: Milad

last: Ibrahim

first: Guanning

last: Wang

first: Taylor

last: Heim

first: Maria

last: Steele

first: Alexander

middle: C

last: Huang

first: Markus

last: Schober

first: Iman

last: Osman

first: Amanda

middle: W

last: Lund


title: Mechanical Isolation Establishes and Maintains the Cardiac Pacemaker Cell Phenotype.

city: Chapel Hill/ 27599

first: Michael

last: Bressan

state: NC

address: 100 Mason Farm Rd

department: Cell Biology and Physiology

institute: University of North Carolina Chapel Hill

summary: Electrical impulses generated in the sinoatrial node (SAN) are responsible for the rhythmic beating of the heart. These impulses are produced by specialized heart cells known as cardiac pacemaker cells (CPCs). While the ionic mechanisms controlling CPC function have been studied for over a century, the upstream cellular events that shape and maintain the SAN's unique electrical properties are still not well understood. We have discovered that abnormal activation of the core mechanotransduction pathways within CPCs leads to significant electrical dysfunction in the SAN. Mechanistically, our data shows that entire systems of ion channels essential for electrical oscillation, along with major transcription factor networks associated with CPC lineage commitment, are rapidly downregulated in response to cellular strain. These findings suggest that mechanical uncoupling and/or the suppression of mechanotransductive signaling pathways serve as a previously unrecognized critical regulatory mechanism necessary for supporting cardiac pacemaking.

overall_design: RNAseq profiling of the embryonic sinoatrial node and atrial myocardium from HH stage 30 chick embryos.  Samples were either freshly isolated, treated for 14hrs with vehicle, or treated for 14 hrs with CN03 (0.25ug/ml).

first: Ashlyn

last: Laidmen

first: Trevor

last: Henley

first: Kathryn

last: Scherrer


title: Microglial sTREM2 limits dyskinesia and acts on TrkB to support circuit plasticity.

city: New York

first: Li

last: Gan

state: NY

address: 413 East 69th Street, 9th Floor BB904

department: Helen and Robert Appel Alzheimer’s Disease Research Institute

institute: Weill Cornell Medicine

summary: Microglia continuously survey the brain and shape neuronal activity, but their contribution to experience-dependent synaptic plasticity is unclear. Levodopa-induced dyskinesia (LID) is a disabling complication of late-stage Parkinson’s disease (PD) that is linked to maladaptive striatal remodeling and is often assumed to reflect detrimental neuroinflammation. Here we identify a dyskinesia-associated microglial gene program in the striatum of PD patients and show that microglia instead act as a protective brake on LID. In a mouse model, microglial depletion exacerbated dyskinesia, whereas microglial repopulation mitigated it. Delivery of AAV expressing soluble TREM2 (sTREM2) similarly reduced LID without impairing the therapeutic benefit of levodopa. Single-nucleus transcriptomics revealed that microglial loss drives extensive remodeling of both direct and indirect spiny projection neurons (SPNs), while repopulation or sTREM2 reverses a large fraction of LID-associated transcriptional changes. Mechanistically, sTREM2 directly engages TrkB and potentiates BDNF-dependent TrkB–ERK signaling, consistent with positive allosteric modulation. Functionally, sTREM2 enhances BDNF-TrkB-dependent hsciippocampal synaptic plasticity and acutely rebalances striatal dendritic excitability in a compartment- and cell type-specific manner. These findings reveal an unexpected neuroimmune pathway in which microglia restrain maladaptive plasticity via sTREM2–TrkB signaling, with therapeutic implications.

overall_design: Droplet-based single nuclear RNA sequencing of striatum from mice with lesioned saline-treated (n=5), lesioned L-DOPA-treated LID (n=5), non-lesioned saline-treated (n=5), non-lesioned L-DOPA-treated LID (n=5), lesioned saline-treated control diet (n=3), lesioned L-DOPA-treated LID control diet (n=3), lesioned L-DOPA-treated LID PLX diet (n=3), lesioned L-DOPA-treated LID PLX reversal diet (n=3), lesioned acute L-DOPA treatment (n=3), lesioned L-DOPA-treated GFP-injected LID (n=4), lesioned saline-treated GFP-injected (n=4), lesioned L-DOPA-treated sTREM2-injected LID (n=4), lesioned saline-treated sTREM2-injected (n=4), non-lesioned saline-treated GFP-injected (n=4), non-lesioned saline-treated sTREM2-injected (n=4).

first: Caitlin

last: Castagnola

first: Roberta

last: Marongiu

first: Yuansong

last: Wan

first: Eileen Ruth

last: Torres

last: Fan

first: Minwoo Wendy

last: Jang

first: Qiaoling

last: Cui

first: Jihye

last: Kim

first: Pearly

last: Ye

first: Jingjie

last: Zhu

first: Alisa

last: Panichkina

first: Alexandra

middle: B

last: Fall

first: Kendra

last: Norman

first: Beichen

last: Gao

first: Nessa

last: Foxe

first: Man Ying

last: Wong

first: Daphne

first: Maitreyee

last: Bhagwat

first: Shiaoching

last: Gong

first: Anna

middle: G

last: Orr

first: Francis

last: Lee

first: D James

last: Surmeier

last: Kaplitt


title: Transcriptome of Fetal Cortex of Tree Shrew Underlying the Emergence and Evolution of Outer Subventricular Zone [TSlayer_RNAseq]

city: Kunming

first: lei

last: shi

state: Yunnan

address: No.17 Longxin Road

institute: Kunming Institute of Zoology

summary: A defining feature of primates is the expansion and increased complexity of the cerebral cortex, processes that are largely associated with evolutionary adaptations of neural progenitor-enriched outer subventricular zone (OSVZ). Yet, the genetic mechanisms underlying the emergence of OSVZ remain unknown. The tree shrew is an extremely well-suited species to investigate this question. In addition to being a closer outgroup of primates than rodents are, its brain exhibits an intermediate phenotype; while being lissencephalic (smooth) similar to the mouse brain, it nonetheless possesses the OSVZ. Here, we applied laser micro-dissection technology to conduct comprehensive gene expression analysis in different cortical layers of tree shrew brain across three key developmental stages. We discovered  distinctive gene expression profiles in different cortical layers, including those enriched in synapse-related-pathways in CP, VZ and ISVZ, while OSVZ exhibited unique enrichment in neurogenesis processes related genes. Utilizing a comparative transcriptome approach, we show that cell-cycle related genes are already involved in tree shrew OSVZ. On the other hand, extra-cellular-matrix (ECM) associated pathways appear unique to primate OSVZ. These novel results begin to reveal the shared genetic toolkit underlying the evolution of OSVZ. Furthermore, we discovered distinctive expression patterns of several zinc finger proteins (ZNFs) that are shared between tree shrew and primate OSVZ, indicating their significance in the evolution of OSVZ. As a proof-of-concept, overexpression ZNF664 significantly increased the proliferation of progenitors in the mouse sub-ventricular zone. Together, these results reveal the shared molecular basis of OSVZ between primates and tree shrew, providing an innovative insight into the brain complexification in primates.

overall_design: We performed RNAseq on micro-dissected cortical layers from  fetal cortex of Chinese tree shrew (Tupaia belangeri chinensis) at three different embryonic days including embryonic 28 days (E28),embryonic 37 days (E37) and embryonic 42 days (E42).

first: Lei

last: Shi


title: c-Rel is a thermoregulatory knob that fine-tunes NF-κB signaling

first: Myong-Hee

last: Sung

address: 251 Bayview Boulevard

department: National Institute on Aging

institute: National Institutes of Health

summary: Thermal regulation is a mechanism for warm-blooded animals to maintain homeostasis against ambient temperature stress. Also, core body temperature rises with fever, an integral part of host responses to infection. Although responses to heat or cold shock have been described, little is understood about how physiological temperature variations affect inflammatory responses. We tracked real-time responses of NF-κB RelA and c-Rel transcription factors to inflammatory and pyrogenic cytokines TNF-α or IL-1β using endogenous knock-in reporter cells in temperatures ranging from 24°C to 40°C. Quantitative live-cell imaging showed that c-Rel signaling, but not RelA, increases with rising temperature. Computational modeling and transcriptomic analysis indicate that the divergent signaling behaviors of these canonical NF-κB subunits cannot be explained solely by accelerated biochemical reactions in higher temperatures. Rather, cytokine receptor-proximal adaptors are likely to amplify c-Rel signaling for precise analog behaviors. The temperature-sensitivity of c-Rel has broad implications for inflammatory cascades in elevated temperatures.

overall_design: Immortalized mouse ear fibroblasts were seeded at medium confluence in 6-cm tissue culture dishes and cultured overnight at 37 °C in humidified incubator with 5% CO2 to ensure recovery and stable basal transcriptional activity. The following day, cultures were transferred to incubators pre-equilibrated to 24 °C, 34 °C, 37 °C, or 40 °C and were allowed to adapt for 2 hours to establish temperature-specific steady states prior to stimulation. Cells were then either left untreated (0h) or stimulated with recombinant murine TNF-α (10ng/mL) or IL-1β (10ng/mL) for 1 hour or 3 hours. At each time point, cells were lysed directly on the plate using RLT buffer (Qiagen). Lysates were stored at -80°C until processing. Total RNA quantity and purity were assessed using a NanoDrop spectrophotometer (ThermoFisher). Three independent biological replicates were collected for each experimental condition. In total, 60 RNA-seq libraries were generated: 48 stimulated samples (4 temperatures x 2 cytokines x 2 time-points x 3 biological replicates) and 12 unstimulated baseline controls collected at 0h across the four temperatures (3 replicates each).

first: Yeuran

last: Oh

first: Shah Md Toufiqur

last: Rahman

first: Kyu-Seon

first: Daisy

last: Dahiya


title: Next Generation Sequencing to evaluate effects of AhR in intestinal epithelial cells on intestine

city: Maastricht

first: Han

last: Jin

address: P. Debyelaan 25

country: Netherlands

institute: Maastricht University

summary: In this study, Ahr fl/fl control mice and AhR fl/fl Villin Cre+ mice were injected with PCSK9 overexpressing AAV8 particles and  placed on a HFD for 12 weeks. Intestines from these mice were used to perform NGS analysis.

overall_design: Intestines from Ahr fl/fl control mice and AhR fl/fl Villin Cre+ mice fed a HFD for 12 weeks were evaluated.

first: Emiel

middle: P

last: Van der Vorst


title: Exercise-associated epigenetic remodeling and TCR repertoire dynamicsin Lynch Syndrome carriers (MEDIP-seq)

city: Houston

first: Eduardo

last: Vilar-Sanchez

state: TX

address: 1515 Holcombe Blvd

institute: The University of Texas MD Anderson Cancer Center

summary: Lynch Syndrome (LS) carriers are at elevated cancer risk. Recent data suggest exercise as a non-pharmacologic preventive strategy, yet the epigenetic and immunological mechanisms underlying its protective effects in this population remain unclear. Here, we perform integrative multi-omics profiling of DNA methylation, gene expression, and the T cell receptor (TCR) repertoire in LS carriers participating in a 52-week aerobic cycling intervention. We identify compartment-specific DNA methylation changes, including innate immune activation in cfDNA and oncogenic pathway repression in tissue. Transcriptomic integration highlights ISL1 as a key exercise-repressed gene and FLCN as a CRC-associated methylation target. TCR analysis reveals increased systemic repertoire diversity and tissue-specific clonal convergence, thus suggesting antigen-driven recruitment. These findings uncover epigenetic and immune remodeling as potential mechanisms of exercise-mediated protection in LS.

overall_design: We included the same cohort of participants previously enrolled in the CYCLE-P trial (ClinicalTrials.gov identifier: NCT03495674)9, which is a prospective, non-randomized, single-site controlled study conducted at the University of Texas MD Anderson Cancer Center from April 2018 to January 2019. Written informed consent was obtained from all individuals and was approved by the University of Texas MD Anderson Cancer Center (MDACC) institutional review board. Briefly, 21 participants diagnosed with LS were enrolled in a 52-week aerobic exercise intervention study involving cycling classes (n =11), followed by enrollment into a control group (n =10). Of the original 21 participants, only 16 had complete paired blood samples collected at both baseline and 52-week follow-up timepoints, including 9 from the exercise group and 7 from the control group. Among these, 15 participants (9 from the exercise group and 6 from the control group) had paired colonic tissue DNA from both time points, enabling comprehensive epigenetic analysis across cfDNA and tissue compartments. Colonic tissue and peripheral blood samples were collected at baseline (Visit 1) and at 52 weeks (Visit 3) during routine cancer surveillance visits.


title: Cell-type-specific transcriptional responses to clozapine and risperidone in mouse frontal cortex

first: Lucía

last: Prohens Coll

state: Barcelona

address: Casanova 143, Facultat de Medicina, Dept de Fonamens Clínics, Unitat de Farmacologia, Planta 1, ala Nord

department: Basic Clinical Practice

summary: Treatment-resistant schizophrenia (TRS) represents a major clinical challenge, with clozapine being the only antipsychotic with proven efficacy. To investigate the cellular mechanisms underlying clozapine’s unique therapeutic profile, we analyzed transcriptional responses to clozapine and risperidone across major brain cell types. Microglia, astrocytes and neurons were acutely enriched from mouse frontal cortex using an immunopanning approach following 14 days of antipsychotic treatment. Bulk RNA sequencing was performed on each enriched cell population, and cell-type-specific transcriptional programs were characterized using differential expression analysis, gene set enrichment analysis and functional network integration. Deconvolution approaches were applied to validate cell-type enrichment. This dataset enables the exploration of shared and drug-specific molecular pathways across brain cell types and provides a resource to study cellular mechanisms relevant to treatment-resistant schizophrenia.

overall_design: Male and female mice were treated daily with vehicle, clozapine (10 mg/kg) or risperidone (1 mg/kg) for 14 days. Frontal cortices from five animals per group were pooled to generate one biological replicate, yielding two biological replicates per treatment. Microglia, astrocytes and neurons were sequentially enriched from each pooled sample using an immunopanning protocol. RNA was extracted from each cell type and processed for bulk RNA sequencing. In total, 18 RNA-seq samples were generated (3 treatments × 3 cell types × 2 biological replicates).

first: L

last: Prohens

first: N

last: Rodríguez

first: S

last: Mas


title: Physiomimetic 3D skeletal muscle reveals senescence-driven matreotype remodeling driving paracrine senescence and regenerative failure

city: Seoul

first: Tae-Hwan

last: Gil

address: 46, Gaeunsa 2-gil, Seongbuk-gu, Seoul, Republic of Korea

country: South Korea

institute: Korea university, College of Medicine

summary: We developed a physiomimetic, 3D engineered skeletal muscle platform by embedding C2C12 myoblasts within muscle tissue–derived extracellular matrix (mdECM) to model how cellular senescence remodels the muscle niche and propagates dysfunction. Senescence was induced in 3D constructs by a single 20 Gy X-ray irradiation, resulting in impaired myogenic differentiation and structural maturation. To define senescence-associated transcriptional programs and paracrine senescence, we performed bulk RNA-sequencing on (i) irradiated senescent (SnC) versus (ii) non-irradiated normal 3D muscle constructs, and (iii) normal constructs exposed in a co-culture to senescent construct–derived secretome (SASP). These datasets enable quantitative characterization of senescence-driven gene expression changes linked to ECM remodeling, inflammation, and loss of myogenic/contractile programs in a physiologically relevant 3D muscle model.

overall_design: Bulk RNA-seq was performed using a physiomimetic 3D engineered skeletal muscle platform generated by embedding C2C12 myoblasts within muscle tissue–derived extracellular matrix (mdECM). Senescence was induced in 3D constructs by a single 20 Gy X-ray irradiation (SnC). To standardize culture conditions, two 3D constructs of the same condition were cultured together in the same dish, and each construct was harvested as an individual sample. RNA-seq was conducted on irradiated senescent 3D muscle constructs (SnC, n = 2) and non-irradiated normal 3D muscle constructs (Normal, n = 2). In addition, to model paracrine effects, a co-culture condition (SnC-Co) was established by culturing a normal 3D muscle construct in the same dish with a senescent 3D muscle construct to expose it to senescent construct–derived secreted factors (SASP); in this condition, only the normal 3D muscle construct was collected for downstream analysis.

first: Myungji

last: Kim

first: Jinah

last: Jang

first: Ok Hee

last: Jeon


title: Exercise-associated epigenetic remodeling and TCR repertoire dynamicsin Lynch Syndrome carriers (RRBS)

overall_design: Genomic DNA was extracted from paired normal and tumor colonic tissues of 3 patients with LS–associated CRC.


title: Plac1 Ablation Disrupts Gene Expression and Signaling Pathways Essential for Prenatal Development: Emergence of a Preeclampsia Transcriptomic Profile

city: Baltimore

first: Supriyo

last: De

address: 251 Bayview Blvd

department: Laboratory of Genetics and Genomics

institute: NIA-IRP, NIH

summary: Plac1 is an X-linked gene essential for placental and embryonic development. A knockout (KO) mouse model was used to identify Plac1-regulated gene expression at E16.5 and E18.5 using differential gene microarray. Genes showing at least 1.5-fold change in expression and FDR < .05 were considered significant. At E16.5, 717 genes were downregulated and 798 were upregulated in KO placentas versus wild type (WT), whereas at E18.5, 1122 genes were downregulated and 1149 were upregulated. GO, KEGG, and IPA analyses revealed that downregulated genes were enriched for Rho GTPase-mediated and actin-cytoskeleton based processes that transmit extracellular cues through canonical signaling pathways, including Integrin, GPCR, Wnt, Notch, VEGF, BMP and TGF-beta, documented to impact trophoblast development, vasculogenesis, vascular tone, branching morphogenesis, and immunomodulation. Furthermore, a preeclampsia transcriptomic-like signature was induced that strengthened over time. By contrast, upregulated genes reflected immune activation and adaptations to oxidative stress resulting from impaired placental function. These findings indicate that Plac1 supports mechanosensitive signaling required to maintain placental structure and regulatory function. Its absence disrupts essential regulatory processes and triggers cellular stress and immune activation, contributing to fetal growth restriction, increased risk for embryopathy and preeclampsia, consistent with the Developmental Origins of Health and Disease (DOHaD) framework.

overall_design: Placental RNA was extracted from Embryonic Days 16.5 and 18.5 from C57/Bl6 (Wild-type, N=5) and Plac1 null (knock-out, N=5) mice.

first: Suzanne

last: Jackman

first: Xiaoyuan

last: Kong

first: Yulan

last: Piao

first: Alexei

last: Sharov

first: Elin

last: Lehrmann

first: Andrew

last: Varshine

first: Ramaiah

last: Nagaraja

first: David

last: Schlessinger

first: Michael

last: Fant


title: Comparative Immunotoxicity and Transcriptomic Profiling of Seven PFAS in THP-1 Macrophages

city: Tampere

first: Dario

last: Greco

address: Arvo ylpön Katu 34

department: Faculty of Medicine and Health Technology

country: Finland

institute: Tampere University

summary: Per- and polyfluoroalkyl substances (PFAS) are a large class of persistent synthetic chemicals widely used across industrial and consumer products. While legacy PFAS, such as perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), have been extensively studied and linked to immunotoxicity, far less is known about the immune-related hazards of newer-generation alternatives. In this study, we systematically evaluated the immunotoxic potential of seven PFAS, including three perfluoroalkyl acids (PFOA, PFOS, PFHpS) and four (poly-)ether PFAS (PFESA BP2, PFESA BP1, DOPFLCA, 8:2 Cl-PFESA) using a THP-1 macrophage model with dose-range exposures for up to 72 hours. Despite an equipotent dosing scheme, the molecular responses to different PFAS congeners were distinct. RNA sequencing revealed that PFOA and PFHpS, but not PFOS, elicited a larger number of differentially expressed genes than ether PFAS. We observed that the absolute PFAS concentration correlated more strongly with the number of DEGs than viability-based potency, suggesting differences in the underlying mechanisms of toxicity. The shared transcriptional signature highlighted PPAR signalling at the core of molecular responses to both legacy and newer-generation alternatives, while an adverse outcome pathway analysis revealed functional differences between the congeners. Together, these findings reveal compound-specific and structure-related differences in immunomodulatory potential among PFAS, with clear distinction by PFAS type. This study provides comprehensive comparative insights to inform hazard evaluation and support the development of safer PFAS alternatives.

overall_design: Transcriptomic profiles were generated from PMA-differentiated THP-1 cells (macrophage phenotype) exposed to seven PFAS compounds including PFOA, PFOS, PFESA-BP2, PFESA-BP1, PFHpS, branched ADONA, and 8:2Cl-PFESA. For each PFAS, cells were treated with three equipotent and subtoxic concentrations, alongside matched vehicle controls. Exposures were performed for 24 h, 48 h, and 72 h to capture time-dependent responses. Each condition, including controls, was generated in three independent biological replicates. All experimental procedures,including cell differentiation, PFAS preparation, dosing, exposure duration, RNA extraction, and sequencing, were performed using standardized protocols to ensure reproducibility and interoperability. The resulting dataset provides a structured, multiconcentration, multitimepoint transcriptomic resource suitable for mechanistic and comparative toxicogenomic analyses

first: Nicoletta

last: D'Alessandro

first: Luca

last: Mannino


title: OCIAD2 promotes epithelial to mesenchymal transition during mesendoderm specification of human pluripotent stem cells

city: Bengaluru

first: Kajal

last: Kamat

state: Karnataka

address: Srirampura cross, Near Rachenahalli lake, jakkur P.O.

department: Moleculr Biology and Genetics Unit

institute: Jawaharlal Nehru Centre for Advanced Scientific Research

summary: The mitochondrial protein OCIAD2 is misexpressed in several cancers and neurodegenerative diseases. However, insights into its physiological role, particularly in the context of stem cell differentiation, remain unexplored. Our analyses report that OCIAD2 is expressed in undifferentiated human embryonic stem cells (hESCs) and is upregulated upon mesendoderm specification. Using bulk transcriptome analysis of CRISPR-Cas9 based OCIAD2 knockout and constitutive OCIAD2 expressing hESC lines, we identify that OCIAD2 modulation does not affect stem cell identity but alters their mesendoderm differentiation potential. Our results highlight that OCIAD2 regulates TGFβ, AKT, BMP signalling pathways to promote epithelial to mesenchymal transition during early mesendoderm specification of pluripotent stem cells.

overall_design: Bulk RNA-seq of human pluripotent stem cell lines: BJNhem20, BJNhem20 OCIAD2 CRISPR 33 and BJNhem20 OCIAD2 OV at day 0 and at day 2 of directed mesendoderm differentiation

first: Maneesha

last: Inamdar


title: Laser capture microdissection-based transcriptomic analysis of neoplastic mast cells reveals novel molecular alterations across canine mast cell tumor grades

city: Ghent

first: Elise

last: Callens

address: Ottergemsesteenweg 460

country: Belgium

institute: Ghent University

summary: Mast cell tumors (MCTs) are one of the most common skin neoplasms in dogs, yet their biological behaviour and the underlying molecular mechanisms remain poorly understood. In this study, neoplastic mast cells (MCs) were isolated from formalin-fixed paraffin-embedded (FFPE) canine MCT biopsies using laser capture microdissection (LCM), followed by RNA sequencing of these cells. In this way, MC-specific mechanisms were investigated in high- versus low-grade canine MCTs. To this end, 32 canine cutaneous MCTs were analysed, of which 18 were graded as low- and 14 as high-grade tumors based on histopathology. A total of 74 differentially expressed genes were identified including MOB3B, EXOC6B, BAIAP2, and TRMO, which have established associations with several human cancers. Gene Set Enrichment Analysis revealed significant enrichment of tumorigenic pathways, including KIT signalling, Ras signalling, ubiquitin-mediated proteolysis, and neurotrophin signalling in high-grade MCTs. Additionally, immune-related pathways, including IL-2 signalling, natural killer cell-mediated cytotoxicity, and leukocyte transendothelial migration, underscored the role of immune interactions in high-grade MCTs. This is the first study to focus on gene expression patterns derived from LCM-isolated neoplastic MCs and their role in the molecular mechanisms driving MC malignancies.

overall_design: RNA-sequencing of laser capture microdissected mast cell-rich regions within low- or high-grade canine mast cell tumor FFPE biopsies

first: Shana

last: De Vos

first: Guillaume

last: Cattebeke

first: Koen

last: Deserranno

first: Jeroen

last: Carlier

first: Ann-Sophie

last: Vander Plaetsen

first: Dieter

last: Deforce

first: Hilde

last: de Rooster

first: Ward

last: De Spiegelaere

first: Filip

last: Van Nieuwerburgh


title: Genome-wide chromatin recording resolves dynamic cell state changes

city: Pasadena

first: Yodai

last: Takei

state: California

address: 1200 E California Blvd

institute: California Institute of Technology

summary: Understanding how the chromatin state of a cell influences its future behavior is a major challenge throughout biology. However, most chromatin profiling methods are limited to endpoint assays. Here, we present LagTag, a method for recovery of earlier and endpoint chromatin states in the same mammalian cells. In this approach, transient expression of bacterial adenine methyltransferase fusions records the DNA binding profiles of chromatin-associated proteins of interest at earlier timepoints. Subsequent tagmentation and sequencing recovers the earlier chromatin profile from adenine methylation profiles, alongside endpoint profiles of endogenous chromatin-associated proteins. We verified that LagTag profiles aligned with those from established methods in mouse and human cells. More importantly, LagTag was able to record and recover dynamic chromatin state transitions during mouse embryonic stem cell differentiation, capturing transcriptional signatures from pre- and post-differentiation timepoints in the same cells. LagTag thus provides a foundation for temporally resolved chromatin profiling.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: CUT&Tag and LagTag profiling in E14, mESC-Gata4, and HCT116 cells. CUT&Tag was performed for RNAPIISer2p, Lamin B1, or histone modifications (H3K4me3, H3K27me3) in E14, mESC-Gata4 (before and after differentiation), and HCT116 cells. LagTag was performed using m6A antibody in engineered cell lines (E14, mESC-Gata4, HCT116 cells, expressing M.EcoGII-mCherry, M.EcoGII-RNAPIISer2p_mintbody, M.EcoGII-LMNB1, M.EcoGII-TAF3_domains, M.EcoGII-CBX7_domains, or EspM-TAF3_domains) along with RNAPIISer2p antibody when noted.

first: Jordan

last: Lay

first: James

last: Linton

first: Duncan

last: Chadly

first: Yoshiki

last: Ochiai

first: Ron

last: Hadas

first: Andrew

last: Perez

first: Mario

last: Blanco

first: Paola

last: Laurino

first: Mitchell

last: Guttman

last: Elowitz


title: RNA-sequencing analysis of gene expression evolution in mole rats

city: London

first: Diego

last: Villar

address: 4 Newark Street, Blizard Institute

country: United Kingdom

institute: Queen Mary University of London

summary: Understanding the genetic basis of phenotypic adaptation poses a significant challenge in evolutionary genomics. Mutations in regulatory sequences that influence gene expression, such as tissue-specific enhancers, are widely recognised as key drivers of phenotypic evolution. However, because cis-regulatory elements evolve rapidly, linking changes in their activity to corresponding changes in gene expression across species is challenging. This study aims to identify and connect accelerated evolution (shifts) in gene expression and cis-regulatory elements, and assess their potential impact on phenotypic adaptations. Using African mole-rats as a model, we profiled gene expression in heart and liver tissues across two mole-rat species and two rodent outgroups, and applied a phylogenetic comparative approach to identify genes that have undergone expression shifts within the mole-rat clade and specific genera. The identified genes are associated with known adaptative functions, including metabolic rewiring such as increased glycolysis in the heart. We then integrated evolution of gene expression and the activity of promoters and enhancers into a unified phylogenetic framework by: (1) characterising the regulatory landscape around transcription start sites of genes with shifted expression across species, and (2) directly integrating regulatory and gene expression shifts. With both approaches, we identified a coordinated shift in regulatory activity and expression levels. Genes showing expression shifts enriched for shifted cis-regulatory elements, and the number of shifted promoters and enhancers correlated with the magnitude of gene expression changes. Our findings represent one of the first systematic demonstrations of parallel evolution acting on gene regulation and expression. Together, these results provide new insights into the molecular mechanisms underlying phenotypic evolution in mole-rats, and highlight the value of combining regulatory and expression-level data in comparative genomics.

sample_organism:
Mus musculus
Cavia porcellus
Heterocephalus glaber
Fukomys damarensis

overall_design: RNA-sequencing of gene expression levels in heart and liver tissue from two mole-rat species and two outgroups (rRNA-depleted total RNA)

first: Maelle

last: Daunesse

first: Elise

last: Parey

first: Camille

last: Berthelot


title: Blood gene expression under immunotherapy as potential biomarker for immune checkpoint blockade in non-small-cell lung cancer

city: Heidelberg

first: Fabienne Dorothea

last: Lusky

address: Röntgenstr. 1

institute: Thoraxklinik at Heidelberg University Hospital

summary:
Abstract
Introduction: PD-(L)1 immunotherapy (IO) is a mainstay first-line (1L) treatment for advanced non-small-cell lung cancer without actionable alterations (aNSCLC), but reliable biomarkers are lacking. This study explored the potential utility of longitudinal blood gene expression profiling in this setting.
Methods: 165 patients with aNSCLC and 1LIO as monotherapy (n=60), or combined with chemotherapy (CHTIO, n=71) were prospectively analyzed along with patients receiving 2LIO (n=34) and age-matched controls (n=21). PAXgene blood RNA was analyzed by quantitative PCR (qPCR) and Affymetrix U133 Plus 2.0 microarrays, including median fold changes [FC] and odds ratio (OR) between groups, with control of the false discovery rate (FDR). Progression-free survival (PFS) was verified through review of radiological images based on RECIST v1.1. Routine blood cell counts and PD L1 tumor proportion score (TPS) were available from the patients’ records.
Results: Significantly higher neutrophil (FC 1.66, p<0.001) and lower lymphocyte counts (FC 0.65, p<0.001) in the blood of untreated patients vs. age-matched controls were significantly associated with shorter PFS (hazard ratio [HR] 1.94 for neutrophil/lymphocyte ratio [NLR] ≥5, p<0.001) and reduced expression of GATA3, FOXP3 and other T-cell specific genes (p<0.01). After 4 cycles of 1L treatment, blood neutrophil counts normalized (FC 0.59 vs. baseline, p<0.001), but lymphopenia persisted. Furthermore, IO monotherapy, but not CHTIO, was accompanied by an upregulation of T cell specific genes, like PD1, GATA3 and FOXP3, whose higher expression after 4 cycles showed stronger associations with longer PFS (HR 0.28-0.31, p<0.05) and long-term responses (LTR) >1year (OR 12-19x, p<0.01) than NLR and PD L1 TPS. Affymetrix profiling revealed profound deregulation of many immunologic pathways in the patients’ blood before and during treatment, whose prognostic value was linked to IO monotherapy, but not CHTIO, and more pronounced after 4 treatment cycles (n=19285 genes associated with LTR, p<0.05, FDR <0.1) compared to baseline (n=610 genes). Gene expression changes, NLR and PD-L1 TPS were independent IO biomarkers in multivariable analyses.
Conclusions: Transcriptomic perturbations are pronounced and readily detectable by qPCR in the blood of NSCLC patients. While the prognostic utility of immunologic gene expression alterations at baseline in the blood was limited by the patients’ initial leucocytosis, an upregulation of PD1, GATA3, FOXP3 and other T-cell related genes specifically under IO monotherapy was strongly associated with clinical benefit and could serve as a novel IO biomarker independent of blood cell counts and tumor PD L1 expression.

overall_design:
165 patients with aNSCLC and 1LIO as monotherapy (n=60), or combined with chemotherapy (CHTIO, n=71) were prospectively analyzed along with patients receiving 2LIO (n=34) and age-matched controls (n=21). PAXgene blood RNA was analyzed by quantitative PCR (qPCR) and Affymetrix U133 Plus 2.0 microarrays, including median fold changes [FC] and odds ratio (OR) between groups, with control of the false discovery rate (FDR). Progression-free survival (PFS) was verified through review of radiological images based on RECIST v1.1. Routine blood cell counts and PD L1 tumor proportion score (TPS) were available from the patients’ records.

We took samples from patients at baseline and after 4 treatment cycles (follow-up, 111/165), additionally there are samples from a control group with smokers (11) and non-smokers (10).
All 165 patients underwent qPCR-based gene expression analysis, while global gene expression profiling using Affymetrix RNA microarrays was possible for 76 patients both at baseline (76/165 or 46%) and after 4 cycles (76/111 or 67%). All Data generated from Affymetrix gene expression profiling is accessible via GEO.

first: Fabienne

first: Petros

last: Christopoulos


title: Few-shot Learning-driven discovery of Lutein suppresses Th1-mediated inflammation via glucose metabolism

city: Hangzhou

first: Jiajia

last: Wang

address: 866 Yuhangtang Road

institute: Zhejiang University

summary: Artificial intelligence (AI)-driven drug discovery is often hindered by the “few-shot” data bottleneck and the limited representational power of traditional two-dimensional models, challenging the accurate identification of functional molecules. In this study, we addressed these challenges by establishing a high-precision screening platform, which leverages transfer learning to recognize the key 3D molecular features of T cell inhibitors. Using this approach, we identified Lutein as a novel, specific immunomodulatory agent from a natural product library. Integrated multi-omics analyses revealed that Lutein activates peroxisome proliferator-activated receptor gamma (PPARγ), suppressing glucose uptake and glycolysis, thereby selectively inhibiting Th1 cell differentiation. In a dextran sulfate sodium (DSS)-induced mouse model of ulcerative colitis, Lutein treatment significantly restored Th1-mediated immune balance and alleviated pathological tissue damage. Our findings not only highlight the great potential of “few-shot” AI strategies that leverage transfer learning and 3D molecular features for the discovery of bioactive natural compounds, but also identify Lutein as a promising therapeutic candidate for ulcerative colitis.

overall_design: CD4⁺ T cells were differentiated under Th1 conditions, with or without 10 µM lutein for 5 days. Total RNA was extracted and quality-checked before library preparation using the NEBNext® Ultra™ RNA Library Prep Kit (Novogene).

first: Zonghui

last: Wei


title: Tissue-specific transcriptomic insights into peel thickness in muscadine (Muscadinia rotundifolia)

city: GAINESVILLE

first: FAHONG

last: YU

state: FL

address: 2033 Morway RD

institute: UNIVERSITY OF FLORIDA

summary: Muscadine grape (Muscadinia rotundifolia) is a native fruit crop valued for its natural stress tolerance and disease resistance, yet its characteristically thick peel  and slippery taste poses challenges for fresh-market appeal. The genetic and developmental basis of muscadine peel development remains unknown. To address this, we conducted the first tissue-specific, cross-cultivar transcriptomic analysis of the muscadine berry, profiling gene expression across four fruit layers (epidermis, hypodermis, mesocarp, and endocarp) in three cultivars with contrasting peel characteristics. Transcriptome comparisons revealed conserved enrichment of cuticle- and cell wall- related pathways in outer fruit tissues, along with cultivar-specific differences in gene expression. We identified several genes and pathways associated peel thickness, as well as candidate genes that may contribute to slippery texture characteristic of muscadine berry. Loss-of-function mutants for PAL1 and XTH7 exhibited a significant reduction in silique valve thickness relative to wild-type controls, suggesting a role in regulating cuticle deposition. This study provides foundational insight into the molecular basis of muscadine berry surface development and identifies new candidate genes for further investigation in fruit-bearing systems

overall_design: we conducted a tissue-specific transcriptome analysis of four major muscadine tissues (epidermis, hypodermis, mesocarp, and endocarp) in three muscadine cultivars with contrasting skin thickness and fruit firmness phenotypes: ‘Alachua’ (thick), ‘Granny Val’(intermit), and ‘Supreme’ (thin). By combining phenotypic data associated with the different muscadine tissue layers with RNA-seq analysis, we aimed to identify candidate genes associated with the peel thickness, leathery skin phenotype, fruit firmness, and slip skin trait, all of which are characteristic of muscadine.

first: Alana

last: Brinley

first: Shirin

last: Shahkoomahally

first: Fahong

last: Yu

first: Islam

last: El-Sharkawy

first: Ali

last: Sarkhosh

first: Tie

last: Liu


title: Transcriptome profiling of Col-0, ces-D, 35S:CES_YFP, ces-tM, ces-qM mutants in A. thaliana

city: Freising

first: Brigitte

last: Poppenberger

state: Bavaria

address: Liesel-Beckmann-Str. 1

department: TUM School of Life Sciences

institute: Technical University of Munich

summary: When organisms encounter pathogens, they rapidly activate complex defense programs to ensure survival. While these immune responses are vital, they often also incur trade-offs, such as reduced growth and development and must therefore be tightly controlled 1,2. In this study, we reveal that the steroid hormones brassinosteroids (BRs) contribute to this control in Arabidopsis thaliana by epigenetically repressing immunity-related genes. We provide evidence that the BR-regulated basic helix-loop-helix (bHLH) transcription factor CESTA (CES), along with its homologs BR ENHANCED EXPRESSION (BEE)1–3, mediate DNA methylation changes at transposable element (TE)-rich loci containing nucleotide-binding leucine-rich-repeat (NLR)-type receptor genes, including SUPPRESSOR OF NPR1-1 CONSTITUTIVE 1 (SNC1). These CES-induced methylation changes are correlated with altered splicing of SNC1 pre-mRNA, a process that requires the BR receptor BR INSENSITIVE 1 (BRI1). In support, we show that CES associates with components of the chromatin remodelling and splicing machinery. Together, our findings reveal a previously unrecognized BR-induced mechanism that modulates the epigenetic and post transcriptional regulation of immune genes, enabling plants to prioritize growth over defense.

overall_design: RNA-seq was performed on 3-week-old Arabidopsis thaliana wild-type (Col-0) and mutant lines (ces-D, CESoe (35S), ces-qM, ces-tM), either mock-treated or inoculated with Hyaloperonospora arabidopsidis (Hpa) isolate Noco2. Infections were carried out by spraying 2-week-old plants with a spore suspension (5 × 10⁴ conidiospores/mL), followed by incubation at 18°C under high humidity. RNA was extracted from aerial tissues using the E.Z.N.A. Plant RNA Kit, and library preparation followed a modified SCRB-seq protocol. Sequencing was performed on an Illumina HiSeq1500. Five biological replicates were included per genotype and condition.

first: Veronica

last: Ramirez

first: Rashmi

last: Hazarika

first: Frank

last: Johannes


title: OCT4 Negatively Regulates the Transcriptional Programming of the Human Adenovirus Early Region 3 Immune Evasion Genes

city: San Rafael

first: Mohamed Ismail

last: Ashrafali

address: 770 Lindaro St

department: Biology Research

institute: BioMarin Pharmaceutical

summary: Genomes of viruses are constrained by the virions’ nanoscale, and viral nucleotide sequences without function are a luxury. Yet the double-stranded DNA genome of human adenovirus (HAdV) contains large regions without known purpose. Using TRANSFAC and ChIP-Seq analysis, we identified binding of OCT4 (octomer-binding transcription factor 4) to a noncoding region of HAdV-D37 DNA. Manipulation of OCT4 expression impacted viral E3 gene transcription and gp19k protein expression, altering subsequent MHC class 1 expression. These effects were specific to OCT4 binding to the adenovirus 5ʹ inverted terminal repeat (ITR) within nucleotides 101-159. Using targeted mutations to OCT4, we found one of two OCT4 binding motifs in the ITR to be crucial for repression of E3 gene expression. In OCT4-siRNA treated cells, E3 RID-α gene expression was also upregulated to inhibit pro-apoptotic signals, suggesting that OCT4 binding also indirectly represses viral replication. Consistent with a role for transcription factors in epigenetic modification during infections, OCT4 knockdown also reduced histone H3 acetylation and DNA methylation. In stem cells, OCT4 sustains pluripotency, whereas in somatic cells, OCT4 plays a dispensable role in self-renewal and maintenance. Herein, we show that OCT4 binding also confers a previously unidentified function to non-coding adenovirus DNA.

sample_organism:
Homo sapiens
Human adenovirus sp.

platform_organism:
Homo sapiens
Human adenovirus sp.

overall_design: A549 cells were infected with HAdV-D37 at 5 moi for 4 hrs and ChIP-Seq was carried out to identify the binding of OCT4.

first: Ashrafali

last: Ismail

first: Meeta

last: Mistry

first: James

last: Chodosh

first: Jaya

last: Rajaiya


title: Macrophage and Triple-Negative Breast Cancer Cell Phenotype Changes in a 3D Co-Culture Model

city: Grand Forks

first: Colin

last: Combs

state: North Dakota

address: 1301 N Columbia Rd

department: Biomedical Sciences

institute: University of North Dakota

summary: Tumor-associated macrophage (TAM) infiltration is a characteristic of triple-negative breast cancer (TNBC) related to drug resistance and poor prognosis. Unraveling intricate cell-cell interactions in the tumor microenvironment (TME) remains challenging, especially when using a standardized 3D culture system. This study used the TNBC cell line, MDA-MB-231, and polarized M1-like or M2-like macrophages derived from THP-1 monocytes to establish 3D co-culture spheroids for mimicking the TME environment. Drug efficacy, epithelial-mesenchymal transition (EMT) in cancer cells, macrophage phenotypes, and RNA sequencing of spheroids were performed. We observed that M2 macrophages increased the viability and proliferation rate of MDA-MB-231 cells in the 3D spheroids, while both M1 and M2 macrophages increased the chemosensitivity of MDA-MB-231 cells to doxorubicin and paclitaxel. Interestingly, instead of maintaining their phenotypes, M1 and M2 macrophages lost some polarization when 3D co-cultured with MDA-MB-231 cells. Compared with 2D cultures, an expected mesenchymal transition was observed in 3D spheroid MDA-MB-231 cells. However, both M1 and M2 macrophages induced a partial epithelial reversion in co-cultured spheroids. Deconvolution of our bulk RNA sequencing results verified the existence of phenotypic transitions between M1 and M2 macrophages when co-cultured with MDA-MB-231 cells in 3D spheroids. In conclusion, our findings suggest that a 3D co-culture system of polarized macrophages and breast cancer cells can serve as an effective platform for studying the dynamic cellular phenotype changes that occur in a heterogeneous environment contributing to chemoresistance. This 3D co-culture system may provide a valuable tool for drug screening to identify targeted therapies for cancer.

overall_design: Twenty four bulk RNA-seq libraries were prepared from MDA-MB-231 cells in either a 2D or 3D conformation, M1 and M2-macrophage polarized THP-1 cells, or a co-culture of MDA-MB-231 cells and polarized macrophages. Each group includes four biological replicate samples.

first: Chen

last: Cheng

first: Brett

last: McGregor

first: Junguk

last: Hur


title: Comparative transcriptional study after treatment with the HDAC inhibitor Trichostatin A

city: Elche

first: Miguel

last: Saceda

state: Valencian Community

address: Unidad de Investigación del Hospital geneCami de la Almazara 11, edificio Anexo II

department: Unidad de Investigación

institute: Fundacion para el Fomento de la Investigacion Sanitaria y Biomédica de la Comunidad Valenciana

summary:
Based on the hypothesis that a universal mechanism of cell death induction by HDAC inhibitors exists, a comparative study of differential gene expression after treatment with TSA has been conducted in both hematopoietic and solid tumor cell lines sensitive to TSA-induced cell death, in certain resistant cell lines, and in a sensitive cell line in the presence of the serine protease inhibitor AEBSF, which blocks TSA-induced cell death in all tested human and mouse models sensitive to the drug. The aim was to identify deregulated genes common to all TSA-sensitive models but not present in resistant lines, and whose deregulation is also prevented in the presence of AEBSF.
We used Affymetrix arrays to analyze samples from cell lines derived from hematopoietic neoplasms and solid tumors, both murine and human, which were either sensitive or resistant to Trichostatin A-induced cell death, as well as some sensitive cell lines treated with the serine protease inhibitor AEBSF, which is capable of blocking this cell death in all tested systems. The aim was to identify a set of genes differentially regulated in all TSA-sensitive models, not deregulated in the resistant ones, and whose regulation was abolished in sensitive cells treated with AEBSF.

overall_design: L1210, L1210R, RWP-1, PANC-1, IMIM-PC-1, IMIM-PC-2, Hs766T, and BxPC3 cells were treated with TSA (1 µM) in the presence or absence of AEBSF (100 µM) for 6 hours. RNA was isolated using the NZY Total RNA Isolation Kit (NZYtech, Lisbon, Portugal) and analyzed using Affymetrix GeneChip® microarrays (PROGENIKA Biopharma S.A., Bizkaia, Spain).  cRNA synthesis followed the Affymetrix IVT Labeling Kit protocol, and purification was performed using the Affymetrix GeneChip Simple Cleanup Module. Hybridization and chip processing were performed according to the manufacturer’s instructions. Software used included AGCC 1.1 and Expression Console™ (EC 1.1, Affymetrix®), with comparative analysis conducted using GeneChip® Operating Software (GCOS 1.4, Affymetrix®).  The microarrays employed were Mouse430_2.na21 for L1210 and L1210R cells, and HG-U133A_2.na22 and HG-U133_Plus_2.na26 for human pancreatic cell lines.

first: María

last: Fuentes-Baile


title: DNA methylation profiling of Arabidopsis BR-regulated ces-D and ces-qM mutants

overall_design: Twelve Arabidopsis thaliana samples were analyzed using whole-genome bisulfite sequencing (WGBS). The experiment included ces-D (gain-of-function) and ces-qM (loss-of-function) mutants and their wild-type Col-0 controls (n = 2 replicates each), as well as bri1-1 and brl-tM (BR receptor mutants) and their own Col-0 controls (n = 2 replicates each). DNA was extracted from 3-week-old aerial tissue. Bisulfite conversion and library preparation were performed by BGI Genomics using the DNBseq platform. Sequencing was conducted on the Illumina HiSeq X Ten. Reads were quality-filtered, aligned to the TAIR10 reference genome using Bismark, and methylation probabilities were calculated using METHimpute. DMRs were called using jDMR-grid with genotype-specific controls to mitigate batch effects. Processed data include bigWig files with methylation tracks and CX-format methylation reports from METHimpute.

first: Robert

last: Piecyk


title: Efficacy,safety and biodistribution of intranasally delivered adenoviral COVID-19 vaccines in hamsters

city: Kuopio

first: Nihay

last: Laham Karam

address: Neulaniementie 2

department: A.I. Virtanen Institute

institute: University of Eastern Finland

summary: Coronavirus disease 2019 (COVID-19) continues to cause outbreaks around the world despite the vast COVID-19 vaccination programs. Thus, it is important to continue developing better vaccines to protect vulnerable populations. Here, we studied the efficacy, safety, persistence and biodistribution of an adenovirus-based COVID-19 vaccine delivered intranasally. Our results confirmed that mucosal delivery leads to effective protection against infection in hamsters. A comparative study of different adenoviral vectors demonstrated that specifically Ad5-S-3M, expressing the SARS-CoV-2 spike glycoprotein ectodomain with inclusion of three common receptor binding domain mutations, induced a robust humoral immune response and effectively protected hamsters from SARS-CoV-2 infection. In addition, longitudinal analysis of vaccinated hamsters’ tissues demonstrated durable vaccine transgene expression and persistent vector DNA in the nasal turbinates. This persistence may be beneficial in increasing the duration of immunity. Expression of the S-3M did not cause any aberrant changes in the hamster tissues and resulted in very few changes in cellular mRNA expression in target tissues. Safety and biodistribution assessment demonstrated restricted y tissue distribution of intranasally delivered vector and no significant changes in clinical blood chemistry, underlining the safety of the vaccine. These results further support that intranasal delivery is a promising approach for mucosal vaccine administration against respiratory infection pathogens and extend our knowledge of Adeno-vector persistence.

overall_design: Hamsters were vaccinated intra-nasally with Adeno-coS or Adeno-LacZ . Vaccine was  delivered 3 times with all vaccinations given 14 days after the previous one.  Nasal turbinates were collected after 43, 57 or 113 days from first vaccination. Both female and male hamsters were used and n=5 for each time point of Adeno-coS vaccinated hamster, n=3 for each time point of Adeno-LacZ and n=4 for unvaccinated control hamsters.

last: Laham-Karam

first: Emma

last: Koivunlehto

first: Petri

last: Mäkinen

first: Erika

last: Gurzeler

first: Einar

last: Niskanen

first: Rikku

last: Fagerlund

first: Tobias

last: Freitag

first: Erkko

last: Ylösmäki

first: Joaquim

last: Segales

first: Kari

last: Alitalo

first: Kalle

last: Saksela

first: Seppo

last: Ylä-Herttuala

first: Aino

last: Sormunen

first: Ahmed

last: Tawfik

first: Tiia

last: Suomalainen

first: Hassan

last: Ugurulu

first: Jimi

last: Virkkala

first: Jordi

last: Rodon

first: Guillermo

last: Cantero

first: Nuria

last: Roca

first: Tuija

last: Kekarainen


title: HNRNPH1-mediated splicing events regulate EIF4G1 transcript variant composition and the organization of the AURKA 5’UTR

city: Bethesda

first: Natasha

last: Caplen

address: 37 Convent Dr, Bldg 37, Rm 6128

department: National Cancer Institute

institute: National Institutes of Health

summary: HNRNPH1 is a regulator of alternative splicing, but few studies have defined the splicing events it mediates. Here, we used short- and long-read RNA sequencing to interrogate the transcriptome-wide effects of HNRNPH1 depletion and its regulation of specific splicing events. Differential alternative splicing analysis revealed effects on the transcriptome that involved all splice event categories. We confirmed HNRNPH1’s regulation of a splicing event involving TCF3-exons 18a and 18b that encode distinct TCF3 transcription factor isoforms. Extending this finding, we present evidence that in neuroblastoma, HNRNPH1 is a MYCN target, potentially explaining the higher levels of HNRNPH1 and TCF3-exon 18a transcript variants in this tumor type. Analysis of two skipped exon events determined that HNRNPH1 regulates the splicing of exons encoding part of the EIF4G1 translation initiation factor’s N-terminus and an exon included in the 5’UTR of specific transcript variants encoding the mitotic kinase AURKA. Using reporter constructs, we show this AURKA 5’UTR exon enhances expression, suggesting HNRNPH1 could contribute to regulating AURKA protein levels. Our findings highlight HNRNPH1’s roles in regulating the expression of proteins with diverse cellular functions.

overall_design: RNA-seq profiling of HNRNPH1 depletion by siRNA in cell lines HEK-293T and HT-1080.

first: Tayvia

last: Brownmiller

first: Erica

last: Pehrsson


title: Massively parallel characterization of RNA G-quadruplex stability and molecular recognition

city: Chapel Hill

first: Daniel

last: Dominguez

address: 120 Mason Farm

department: Pharmacology

institute: UNC Chapel Hill

summary: RNA G-quadruplexes (rG4s) have been implicated as important regulators of RNA metabolism and are promising targets for RNA-targeted therapeutics. rG4s typically require a canonical (G≥2N1-7)4 motif, but the sequence features that affect rG4 stability and recognition by RNA-binding proteins (RBPs) and rG4-binding ligands are not fully understood. To interrogate sequence-level drivers of rG4 folding, we applied a reverse-transcriptase stop sequencing strategy to a library of ~3,000 synthetic rG4s with varied G-tract lengths, loop lengths, and loop compositions, permitting massively parallel quantification of rG4 stability. Our data confirm known sequence-level features and characterize novel combinatorial impacts of these features. We also assessed systematically mutagenized natural rG4s, revealing unexpected mutations that significantly affect rG4 stability, including contributions from flanking sequences outside of the rG4. We further used our strategy to assess rG4 recognition preferences of the model rG4-ligand pyridostatin, revealing a preferential stabilization of rG4s containing mixed-length G-tracts. We further demonstrated the potential for large-scale protein binding assays with our library to reveal rG4 features recognized by RBPs, specifically G3BP1 and FMRP. Our approach and data provide a generalizable framework to study sequence-level drivers of rG4 stability, binding by RBPs, and ligand interactions, defining basic principles of rG4 formation and downstream biology.

overall_design: Sequencing of a synthetic RNA G-quadruplex pool (stG4 pool) prepared with either guanine or 7-deaza-guanine (7dG). This dataset includes two main approaches: i) RT stop profiling in different cation (KCl, LiCl), temperature, and/or pyridostatin treatments. Comparison of sequence frequencies between guanine and 7dG RNA pools afford parallel quantification of rG4 stability across these RT conditions. ii) RNA bind-n-seq (RBNS) experiments with the stG4 pool with G3BP1 (full length and domains) and the low complexity domain of FMRP. This includes sequenced protein bound sequences with guanine and 7dG, as well as guanine and 7dG RNA inputs.

first: Justin

last: Martyr

first: Bryan

last: Guzmán

first: Alli

last: Jimenez

first: Rebekah

last: Rothacher

first: Francisco

middle: F

last: Cavazos Jr.

first: Yue

last: Hu

first: Rhese

middle: D

last: Thompson

first: Anthony

last: Mustoe

first: Maria

last: Aleman


title: Autonomous circadian bladder capacity rhythm independent of the central clock

city: Tsukuba

first: Shuhei

last: Suzuki

address: 1-1-1 Tennodai, Tsukuba, Ibaraki

institute: University of Tsukuba

summary: This study investigates the autonomous regulatory function of the bladder for its capacity circadian rhythm using a central nervous Bmal1 conditional knockout (cKO) mouse. Under constant darkness (DD), these mice lose behavioral rhythmicity but preserve rhythmicity of the bladder capacity and bladder clock gene expression for short term. However, prolonged exposure to DD results in loss of bladder capacity rhythm and decreased clock gene amplitude. RNA-seq analysis in the bladder of Bmal1 cKO mice revealed that clock genes and extracellular matrix-related genes were disrupted in rhythmicity under long DD. These findings highlight that the bladder has an autonomous regulatory function for the diurnal rhythm of bladder capacity independent of the central clock. However, the vulnerability of peripheral bladder clocks is revealed in the long-term absence of central clock input. This study provides molecular insights into how circadian dysregulation may contribute to bladder dysfunction.

overall_design: Investigation of changes in whole bladder RNA by modulation of light stimulation in central nervous system Bmal1 cKO mice.

first: Hiromitsu

last: Negoro

first: Haruna

last: Nonaka

first: Ichiro

last: Chihara

first: Yoshiyuki

last: Nagumo

first: Shigeki

last: Shimba

first: Kenichi

last: Kimura

first: Hiromi

last: Yanagisawa

first: Hiroyuki

last: Nishiyama


title: Perturbation of epithelial and limbal stem cell identity in pathologic corneal neovascularization

city: Nijmegen

first: Jo Huiqing

last: Zhou

address: Geert Grooteplein 26/28

institute: Radboud University

summary: We have employed a single cell sequencing approach using 10x Genomics scRNAseq to study a corneal suture model that damages the epithelium and induces sustained inflammation and neovascularization, focusing on the impact to limbal epithelial stem cells.  Additionally, we evaluated the potential of duloxetine, an FDA-approved medicine, in promoting wound healing and corneal homeostasis at the single-cell level.

overall_design: Following 10 days of no administration (no suture group), topically administered PBS (suture group) and topically administered 10 µM duloxetine (treatment group), in vivo mouse corneas were dissected from harvested eyes. Cells were then retrieved from these corneas and analyzed using scRNA-seq.

first: Mojdeh

last: Abassi

first: Julian

last: Arts

first: Dina

last: Javidjam

first: Petros

last: Moustardas

first: Ava

last: Dashti

first: Huiqing

first: Neil

last: Lagali


title: Development of an antisense oligonucleotide targeting EFEMP1 in models relevant to macular degeneration

city: Boston

first: Anil

last: Upreti

state: MA

address: 20 Staniford St

department: Ophthalmology, Harvard Medical School

institute: Schepens Eye Research Institute, Mass Eye and Ear

summary: Antisense oligonucleotides (ASOs) are RNA targeting therapies with substantial potential for the medical treatment of genetically defined inherited ocular conditions and beyond. The eye is, in principle an ideal model for ASO therapeutic development due to the ease of administration and potential limited dosing frequency due to ASO durability. Autosomal dominant drusen (ADD) is an inherited retinal dystrophy characterized by early onset formation of numerous drusen with subsequent choroidal neovascularization (CNV). ADD is caused by a mutation in the epidermal growth factor-containing fibrillin-like extracellular matrix protein 1 (EFEMP1). EFEMP1R345W is expressed at high levels in the retinal pigmented epithelium (RPE) and accumulates both intracellularly and in the extracellular matrix (ECM) of the RPE during ADD. Furthermore, EFEMP1 has been reported to be significantly upregulated in serum and RPE/choroid during age-related macular degeneration (AMD) yet the consequence of EFEMP1 overexpression has not been fully elucidated. Here, we report the development of a biallelic EFEMP1 targeted ASO that has shown robust EFEMP1 knockdown and modulation of disease relevant factors in induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE), human microvascular retinal endothelial cells (HMREC), ex vivo human choroidal explants and in vivo mouse models. We further demonstrate that EFEMP1 is upregulated at the protein level during AMD. Herein, we report that EFEMP1 knockdown reduces complement factor 3 (C3) levels, a current target for AMD, and significantly reduces proliferation and migration in HMREC and a human choroidal explant sprouting. In summary, we propose a EFEMP1 targeted ASO as a therapeutic modality for DHRD and AMD.

overall_design: RNA-seq profiling of ARPE19 vs ARPE19 R345W cells after 30d days of culture in transwells. We also show data from HMRECs after 48 hours comparing EFEMP1 ASO (50nM) vs ASO mismatch control (50nM).

first: William

last: P. Miller

first: Said

last: Arevalo-Alquichire

first: Paula

last: Perez-Corredor

first: Michael

last: O'Hare


title: Integrating Machine Learning and Transcriptomics to Enhance β-Carotene Production in Yeast

city: Pathumthani

first: Peerapat

last: Khamwachirapithak

address: Phahonyothin Road, Khlong Luang

country: Thailand

institute: Thammasat University

summary: Optimization of microbial production is essential for the application of synthetic biology in industrial and sustainable biosynthesis. β-carotene is a high-value compound that can be heterologously produced in budding yeast Saccharomyces cerevisiae, providing an alternative to natural extraction. However, intelligent design of strain and process to reduce the production cost remains a great challenge. In this study, we aimed to enhance β-carotene production by integrating systems biology and machine learning-guided strain design of the β-carotene-producing yeast. We identified transcriptional alterations in the β-carotene-producing yeast including genes associated iron deficiency, while no significant changes in the mevalonate (MVA) pathway. We then fine-tuned gene expression of rate-limiting enzymes in the mevalonate pathway through combinatorial construction of promoters and terminators. XGBoost training was applied in the DBTL cycle to facilitate rapid optimization. In the second DBTL cycle, fine-tuning MVA gene expression resulted in a 139% improvement in β-carotene titer. We then supplemented β-carotene production with iron, guided by transcriptional insights in alternations of genes related to iron uptake in β-carotene-producing yeast, resulting in an 70.54% improvement in β-carotene titer at 48 hours. Moreover, integrating the fine-tuned MVA cassette with iron supplementation yielded up to 72.07 mg/L of β-carotene at 72 hours, representing a 67.79% increase compared to that of the parent strain without MVA gene adjustments and iron supplementation. Our study highlights the potential of combining machine learning and omics approaches with synthetic biology to enhance non-native biochemical production in yeast.

overall_design: RNA-seq comparing gene expressions of beta-carotene-producing yeast and its parental strain.

first: Kittapong

last: Sae-Tang

first: Suriyaporn

last: Bubphasawan

first: Jongrak

last: Choompuper

first: Xin-Qing

last: Zhao

first: Verawat

last: Champreda

first: Weerawat

last: Runguphan