title: Acutely denervated muscle EVs reshape neuronal mitochondrial metabolism via retrograde signaling to rescue peripheral nerve injury

city: Xi'an

first: Qi

last: Liu

address: No.127, Changle West Road

institute: Air Force Medical University

summary: Peripheral nerve injury causes muscle atrophy due to slow axonal regeneration, highlighting an unmet therapeutic need. Although neuromuscular interactions are classically viewed as unidirectionally nerve-dominated, we show that acutely denervated muscle (adMu) regulates nerve regeneration via a retrograde signaling pathway. adMu initiates a trans-tissue regulatory mechanism through extracellular vesicles (EVsadMu) that orchestrate neural energy homeostasis to accelerate regeneration. Functional profiling identifies IDH2 and CS as key metabolic enzymes within EVsadMu. Neurons treated with EVsadMu exhibit a 1.39-fold increase in NADPH/NADP+ ratio via IDH2, along with a 1.18- and 1.27-fold increase in NADH/NAD+ and FADH2/FAD ratios via CS, fueling the TCA cycle to enhance mitochondrial bioenergetics. This restores redox balance and energy supply, driving axonal regeneration. In sciatic nerve injury models, EVadMu-microneedle conduits significantly promote energy metabolism and functional recovery. Together, our findings position adMu as a metabolic signaling center enabling retrograde regulation for nerve regeneration, offering potential for clinical translation.

overall_design: RNA sequencing of gastrocnemius muscle at different time points after denervation


title: Abnormal shear stress induces ferroptosis in endothelial cells via KLF6 downregulation

status: Public on Dec 09 2025

city: 北京市

first: ZHIYU

last: FAN

state: 北京

address: 雁栖湖校区：北京市怀柔区怀北镇怀北庄380号

country: China

institute: University of Chinese Academy of Sciences

type: Expression profiling by high throughput sequencing

summary: Stable laminar flow maintains vascular tone regulation, while abnormal blood flow, such as disturbed flow or extreme shear stress, causes endothelial dysfunction, but the underlying mechanism is yet to be explored. We used a microfluidic device to deform flat microchannel into tunnel-like macrochannel. The cross-sectional area of this macrochannel varies with the flow rate and the thickness of the deformable layer, creating three levels of shear stress: low (0.99 dynes/cm², LSS), medium (4.78 dynes/cm², MSS), and high (24 dynes/cm², HSS). Comparing different shear stress exposure to endothelial cells for 24 h, prominent ferroptosis features emerged under either LSS or HSS compared to MSS. These features included increased C11 BODIPY-labeled lipid peroxidation and 4-hydroxynonenal accumulation, CoQ10 depletion, reduced SLC7A11 protein expression, and diminished cell death with Ferrostatin-1 treatment. RNA-seq analysis (LSS/MSS) showed that LSS significantly downregulated transcription of cholesterol homeostasis and unfolded protein response (UPR). Compared to MSS, Western blot results showed that both LSS and HSS reduced the expression of two key enzymes (MVD and IDI1) in the mevalonate pathway, as well as the expression of two main UPR signaling regulators (PERK and BiP). Based on the binding prediction between transcription factors and gene promoters from differentially expressed genes identified through RNA-seq, we found KLF6 to be a key transcription factor. It regulates the PERK-mediated UPR and the mevalonate pathway, which are associated with SLC7A11 expression and CoQ10 synthesis, respectively. The overexpression of KLF6 restored SLC7A11 and CoQ10 levels under both LSS and HSS, significantly reducing foam cell formation, monocyte adhesion, and lipid peroxidation. Our findings reveal KLF6 as a crucial regulator of atherosclerosis induced by abnormal shear stress.

overall_design: Total RNA was extracted from the shear stress treated endothelial cells using RNAsimple Total RNA Kit (DP419,TIANGEN BIOTECH) according to the manufacturer's protocol. RNA quantification and purity were determined using Nanodrop One (Thermo Fisher Scientific). Qualified RNA samples were sent to BGI-Shenzhen (Shenzhen, China) for strand-specific library construction using the NEBNext Ultra II RNA Library Prep Kit (NEB #E7770), followed by sequencing on an Illumina NovaSeq 6000 platform with 150-bp paired-end reads. Raw data processing and downstream analyses were conducted on BGI's Dr. TOM platform (https://biosys.bgi.com).

first: Jingang

last: Cui

first: Zhiyu

last: Fan

first: Suoqi

last: Ding

first: Jiazhen

last: Zhang

first: Huihong

last: Shen

first: Syeda Armana

last: Zaidi

first: Yongsheng


title: RNA-sequencing analysis of wild-type, Lao1 knockout and Lao1/Il4i1 double knockout mice under normal and DSS-treated conditions

city: Tokyo

first: Itsarapan

last: Fhaikrue

country: Japan

institute: Tokyo University of Agriculture and Technology

summary: RNA-sequencing was performed on intestinal tissues from wild-type, Lao1 knockout, and Lao1/Il4i1 double knockout germ-free mice under normal and 24-hour DSS treatment conditions to identify regulators of intestinal stress response. Lao1 controls 80.1% of DSS-induced transcriptional differences, with Il4i1 adding 14.7%. Lao1 mainly regulates oxidative phosphorylation and cell migration pathways, while Il4i1 modulates lipid metabolism and ER stress responses. These findings establish Lao1 and Il4i1 as hierarchical regulators of intestinal stress responses.

overall_design: RNA-seq was performed on colon tissues from three genotypes (WT, LAO1KO, and DKO) under two circumstances (Normal and DSS treatment), each with three biological replicates, for a total of 18 samples. To prevent microbiome influencing effects, germ-free male C57BL/6J mice (8 weeks old) were employed.

first: Kentaro

last: Nagaoka

first: Yuki

last: Yamamoto


title: Hyperosmotic stress induces a large-scale rewiring of 3D chromatin interactions [mAID2 Hi-C]

city: Chapel Hill

first: John Patrick

last: Flores

state: NC

address: 104 Manning Dr

country: USA

institute: UNC Chapel Hill

summary: Cells rapidly adapt to hyperosmotic stress through coordinated molecular responses. To determine how three-dimensional (3D) chromatin structure contributes to this process, we profiled chromatin interactions, architectural protein occupancy, and transcriptional dynamics in human cells exposed to sorbitol-induced hyperosmotic stress. We performed time-resolved Hi-C to measure stress-induced remodeling of chromatin loops and domains, CUT&Tag to quantify CTCF, RAD21, YAP1, and H3K27ac occupancy at loop anchors, and RNA-seq to capture stress-responsive transcriptional programs. These data reveal global loss of pre-existing chromatin contacts and concurrent formation of de novo, transient loops enriched for retained CTCF and cohesin, as well as transcriptional responses that are temporally layered and largely decoupled from loop remodeling. This dataset provides a resource for investigating how nuclear architecture and transcriptional regulation respond to hyperosmotic stress.

overall_design: eGFP-YAP HEK293 cells were serum-starved for 1 hour and then exposed to 200 mM sorbitol to induce hyperosmotic stress. Chromatin organization was profiled by in situ Hi-C at eight time points (0 min, 10 min, 30 min, 1 h, 3 h, 6 h, 12 h, and 24 h) to capture the formation, loss, and recovery of chromatin interactions. To assess the generality of chromatin remodeling, additional Hi-C experiments were performed in HEK293 wild-type and HCT116 cells with and without 1 hour of sorbitol treatment. To probe the requirement for architectural proteins, we also performed Hi-C in HCT116 mAID2-CTCF and mAID2-RAD21 degron cell lines treated with 1 μM 5-Ph-IAA to rapidly and reversibly degrade CTCF or RAD21 prior to sorbitol exposure. Transcriptional responses were measured by stranded RNA-seq across a time course (0 h, 1 h, 3 h, 6 h, 9 h, 12 h, and 24 h). To quantify changes in architectural protein occupancy, CUT&Tag for CTCF and RAD21 was performed in untreated and 1-hour sorbitol-treated HEK293 cells.

first: JP

first: Andrea A.

last: Perreault

first: Zack

last: Drum

first: Chenxi

last: Xu

first: Doris

last: Cruz Alonso

first: Gelila

last: Petros

first: Yijia

last: Wu

first: Ivana Y.

last: Quiroga-Barber

first: Isha

last: Sahasrabudhe

first: Justin

last: Demmerle

first: Gang Greg

last: Wang

first: Danfeng

last: Cai

first: Douglas

last: Phanstiel


title: Mechanism through which MTFP1 is involved in bovine oocyte maturation [RNA-seq]

city: Hefei

first: TC

last: H

address: 130 Changjiang West Road

institute: AAU

summary: Oocyte maturation is a complex  process that is regulated by a variety of factors. Mitochondria are a key factor affecting oocyte maturation in vitro. Mitochondrial fission process 1 (MTFP1) is located on the inner mitochondrial membrane and mediates the fission of this membrane. However, the role and mechanism of action of MTFP1 in bovine oocyte maturation are still unclear. We found that MTFP1 is expressed at all stages of bovine oocyte maturation. Moreover, MTFP1 knockdown decreased oocyte maturation efficiency. These observations, combined with our transcriptome sequencing results, showed that MTFP1 knockdown caused mitochondrial dysfunction, impaired nuclear cytoplasmic maturation, promoted mitochondrial fusion, induced mitophagy and decreased oocyte apoptosis. In summary, the inner mitochondrial membrane protein MTFP1 plays a crucial role in bovine oocyte maturation. The results provide a reference and theoretical basis for improving the quality of in vitro oocyte maturation and breeding efficiency in beef cattle

overall_design: Transcriptome differences between negative control group and MTFP1 knockdown group in MII stage of bovine oocytes matured in vitro

first: Tiancang

last: Han

first: Yuhan

last: Zhao

first: Zhaoyang

last: Sun

first: Anhui

last: Jiao

first: Bingbing

first: Qingshan

last: Gao


title: CDK4-driven ketogenesis rewires histone modification to promote leukemia

city: Wuhan

first: Jin

address: 115 East Lake Road, Wuhan, Hubei, 430071, P. R. China

institute: Wuhan university

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Ketogenic nutrition has shown promise as an adjunct to conventional cancer therapies, its efficacy varies across cancer types depending on their metabolic characteristics.1,2 How ketogenic metabolites affect tumorigenesis, and whether ketone metabolism becomes deregulated in cancer cells, remain incompletely understood. Using T-cell acute lymphoblastic leukemia (T-ALL) as a model, we demonstrate that the ketone body β-hydroxybutyrate (BHB) is elevated in cancer cells and exerts a strong pro-leukemogenic effect. Remarkably, the key ketogenic enzyme β-hydroxybutyrate dehydrogenase 1 (BDH1) is significantly upregulated, enabling carbohydrate-driven BHB synthesis in leukemia cells. Mechanistically, aberrant CDK4 activation enables direct phosphorylation of BDH1 and enhances its protein stability. BDH1-mediated production of BHB augments histone β-hydroxybutyrylation (Kbhb) and activates transcription of pro-leukemogenic genes. Either BDH1 deficiency or CDK4 inactivation significantly suppresses T-ALL progression in vivo. Notably, ketogenic diet markedly reduces the anti-leukemic efficacy of the clinically approved CDK4/6 inhibitor palbociclib. Together, these findings highlight how oncogenic CDK4 drives cancer cell ketogenesis through BDH1 phosphorylation and promotes tumor progression via an epigenetic mechanism. Moreover, the implementation of ketogenic nutrition in cancer therapy should be approached with caution and guided by the molecular pathogenesis of individual cancer types.

overall_design: Fragmentation of the genome of Jurkat was achieved using ultrasonication, followed by immunoprecipitation with H3K9bhb and H3K9ac antibodies

first: Hudan

last: Liu


title: ATP6V0D2 drives triple-negative breast cancer progression and inhibits cisplatin sensitivity by promoting PHLPP2 lysosomal degradation

city: Shijiazhuang

first: Zhenchun

last: Song

state: Hebei Province

address: 169 Tianshan Street, Yuhua District, Shijiazhuang City

department: breast center

institute: The Fourth Affiliated Hospital of Hebei Medical University

summary: Triple-negative breast cancer (TNBC) faces therapeutic challenges due to limited treatment options and frequent chemoresistance. In this study, we identify ATP6V0D2 as a key factor upregulated in TNBC, where its expression correlates with poor prognosis. Transcriptomic and functional analyses linked ATP6V0D2 to PI3K/AKT signaling and platinum drug resistance. Mechanistically, ATP6V0D2 enhanced lysosomal function to promote degradation of the tumor suppressor PHLPP2, thereby sustaining AKT phosphorylation. Functional assays showed that ATP6V0D2 knockdown increased cisplatin sensitivity both in vitro and in vivo. Furthermore, pharmacological inhibition of V-ATPase with bafilomycinA1 sensitized TNBC patient-derived organoids to cisplatin. Clinically, our analysis of 91 TNBC patients who received cisplatin-based neoadjuvant chemotherapy found that high ATP6V0D2 expression was inversely correlated with pathological complete response (pCR), whereas PHLPP2 levels showed positive association, supporting the proposed mechanistic link. Together, these findings highlight the impact of ATP6V0D2 on TNBC progression and cisplatin sensitivity, nominating ATP6V0D2 as a promising therapeutic target.

overall_design: Total RNA was extracted and purified from MDA-MB-231 cells transfected with control siRNA or ATP6V0D2-targeting siRNA, and gene expression analysis was conducted with three replicates.

first: Jingyu

last: Zhang

first: Congbo

last: Cao

first: Hongyu

last: Yang

first: Liu

first: Meiqi

first: Xiaohan

first: Xin

last: Hao

first: Yuanyuan

first: Zhenchuan


title: Molecular and phenotypic blueprint of human hematopoiesis links proliferation stress to stem cell aging.

city: Milan

first: Raffaella

last: Di Micco

address: Via Olgettina, 60

country: Italy

institute: San Raffaele Telethon Institute for Gene Therapy

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

overall_design: Refer to individual Series


title: The effect of ALKBH5 overexpression on gene expression in the Huh-7 cell line

city: Changchun

first: Wei

last: Huang

state: Jilin

address: Xinmin Street No. 1163

department: Modern biology

institute: nstitute of Frontier Medical Science, Jilin University

summary: Huh-7 is a sorafenib-sensitive hepatocellular carcinoma cell line. Short-term exposure (48 hours) of Huh-7 cells to sorafenib induces transcriptome-wide m6A hypomethylation and specifically upregulates the expression of the m6A demethylase ALKBH5. Functional analyses demonstrate that ALKBH5 overexpression promotes cellular resistance to sorafenib. To elucidate the molecular mechanisms by which ALKBH5 triggers sorafenib resistance in Huh-7 cells, we performed RNA sequencing (RNA-seq) on ALKBH5-overexpressing Huh-7 cells. RNA-seq data analysis revealed that ALKBH5 overexpression enhances the expression of epithelial-mesenchymal transition (EMT)-related genes and upregulates genes associated with the Wnt/β-catenin signaling pathway.

overall_design: RNA-seq profiling of parental Huh7 cells and ALKBH5-overexpressing counterparts

first: Meng

last: Li

first: Fei


title: Novel SLCO2B1 mRNA isoform acts as a noncoding RNA and drives cancer progression through FMR1-mediated protein biosynthesis

city: Shanghai

first: Wenying

last: Qiu

address: Dongan road 270

institute: Fudan University Shanghai Cancer Center and Institutes of Biomedical Sciences

summary: The eukaryotic 5’ untranslated region (5’ UTR) canonically influences mRNA translation efficiency. However, the role of the 5’ UTR structure in blocking mRNA translation and turning a coding RNA into a noncoding RNA remains undocumented. Herein, we discovered a novel mRNA isoform of the human solute carrier organic anion transporter family member 2B1 (SLCO2B1) gene, named SLCO2B1-isoformNovel (SLCO2B1-isoN), whose 5’ end stem‒loop abrogates its translational capacity and turns it into a noncoding RNA. The SLCO2B1-isoN noncoding isoform stabilizes fragile X messenger ribonucleoprotein 1 (FMR1) to trigger hepatocellular carcinoma (HCC) progression by facilitating de novo protein biosynthesis. Targeting SLCO2B1-isoN effectively inhibited orthotopic tumor xenograft growth in vivo. In conclusion, this study revealed a previously unrecognized noncoding isoform of SLCO2B1 mRNA with a 5’ end stem‒loop structure and highlighted the dual characteristics of mRNAs harboring protein-coding and noncoding isoforms, providing new insights into the richness of genetic information in protein-coding genes.

overall_design: CUT&Tag was performed using antibodies against ZEB1, SETD1B, H3K9ac, H3K27me3, H3K4me3 to identify the binding sites across DNA in MHCC97H or HepG2 cells.

first: Xianghuo

last: He


title: Persistent myeloid cell reprogramming despite miltefosine treatment in Leishmania-infected macaques

city: Quebec

first: Jerome

last: Estaquier

state: Quebec

address: 2705 Boul Laurier

department: Maladies infectieuses et immunitaires

country: Canada

institute: Centre de recherche du CHU - Université Laval

summary: Visceral leishmaniasis (VL) is a neglected tropical disease caused by protozoan parasites. An inflammatory immune response, associated with tissue injury, is occurring shortly after infection. In this study, using a rhesus macaque (RMs) model of VL, we evaluated the impact of miltefosine (HePC) therapy administrated during the acute phase of infection. Despite drug therapy, parasites persist in multiple tissues, including the spleen, bone marrow, peripheral (PLN) and mesenteric lymph nodes (MLNs). Parasite burden inversely correlate with cellular HePC levels. Notably, L. infantum remains detectable three months post-treatment. Single-cell transcriptomic analysis reveals significant cellular heterogeneity and reprogramming of splenic myeloid cell populations that persist post-treatment. This included the emergence of inflammatory macrophages, immature plasmacytoid dendritic cells (pDCs), and type 2 dendritic cells (DCs). Flow cytometric sorting of splenic neutrophils, macrophages, and DCs confirms the presence of L. infantum post-treatment, highlighting the challenge of parasite clearance. Collectively, our findings reveal a disrupted innate immune landscape following L. infantum infection that persists after treatment, enlightening myeloid cell reprogramming that may contribute in sustaining chronic infection and parasite persistence.

overall_design: Ten female rhesus macaques (Macaca mulatta, Chinese origin, 40 months old, ~4.5 kg) were used in this study. Animals were inoculated intravenously with Leishmania infantum. One day post-infection, animals received a 21-day treatment with miltefosine (HePC, 5 mg/kg by nasogastric gavage). Spleen mononuclear cells were isolated by Ficoll-Paque density gradient centrifugation (Sigma). Cell suspensions were stained with a CD45 antibody. Four samples (Naive, Infected, and two treated RMs) were pooled and loaded onto a single BD Rhapsody cartridge (~40,000 pooled cells loaded). Libraries were generated following the manufacturer’s protocol, and sequencing was performed on an Illumina NovaSeq 6000 targeting ~50,000 reads per cell.

first: Morgane

last: Picard

first: Steven

last: Boutrais

first: Vasco

last: Rodrigues

first: Yasmina

last: Fortier

first: Chloé

last: Borde

first: Calaiselvy

last: Soundaramourty

first: Julien

last: Clain

first: Charles

last: Joly-Beauparlant

first: Gina

last: Racine

first: Ouafa

last: Zghidi-Abouzid

first: Arnaud

last: Droit

first: Alain

last: Pruvost

first: Maria

last: Paola Costi

first: Ricardo

last: Silvestre

first: Anabela

last: Cordeiro da Silva

first: Jane

last: MacDougall

first: Sónia

last: André

first: Jérôme


title: Decoding muscle-resident Schwann cell dynamics during neuromuscular junction remodeling [scRNA-seq]

city: Ann Arbor

first: Susan

middle: V

last: Brooks

state: Michigan

address: 109 Zina Pitcher Pl

department: Molecular and Integrative Physiology

institute: University of Michigan

summary: Understanding neuromuscular junction (NMJ) repair mechanisms is essential for addressing degenerative neuromuscular conditions. Here, we focus on the role of muscle-resident Schwann cells in NMJ reinnervation. Using an accepted model of progressive NMJ degeneration, Sod1-/- mice, we identified a clear NMJ regenerative window that allowed us to define cellular and molecular regulators of synapse remodeling and muscle fiber reinnervation. High-resolution imaging and single-cell RNA sequencing provide a detailed analysis of Schwann cell number, morphology, and transcriptome revealing multiple subtypes, including a previously unrecognized terminal Schwann cell (tSC) population expressing a synapse promoting signature. We also discovered a novel SPP1-driven cellular interaction between myelin Schwann cells and tSCs and show that it promotes tSC proliferation and reinnervation following nerve injury in wild type mice. Our findings offer important insights into molecular regulators critical in NMJ reinnervation that are mediated through tSCs to maintain NMJ function.

overall_design: Male C57BL/6J mice (10–16 weeks old) were subjected to bitlateral peroneal nerve injuries, a 10 μL intramuscular injection of SPP1 neutralizing antibody (2 μg; AF808, R&D Systems) was administered into the tibialis anterior (TA) muscle using a Hamilton syringe and repeated every two days following the initial injury. Control mice received goat anti-mouse IgG (2 μg; 115-005-003, Jackson Immuno) on the same schedule. Muscles were harvested for scRNA sequnecing 7 days post injury (7dpi).

first: Steve

middle: D

last: Guzman


title: IDH mutations are rare events in SHH medulloblastoma

city: Hamburg

first: Alicia

last: Fürst

address: Martinistraße 52

country: Germany

institute: University Medical Center Hamburg-Eppendorf

type: Methylation profiling by genome tiling array

summary: Hotspot mutations in IDH1 or IDH2 have been discovered as a frequent oncogenic event in gliomas in 2009. According to the most recent version of the WHO classification of brain tumors from 2021, such mutations define what is now called astrocytoma, IDH-mutant and oligodendroglioma, IDH-mutant and 1p/19q-codeleted. Approximately 85% of these mutations are IDH1:p.R132H mutations with other amino acid exchanges at codon 132 of IDH1 and codon 172 of IDH2 being rare. Of note, no other brain tumor entity has been reported so far to carry any recur-rent IDH mutations. Here, we describe a series of 12 medulloblastomas with four novel and eight previously published cases. Eight of these cases carry IDH1:p.R132C mutations, one of them harbors an IDH1 R132L and one of them an IDH2:p.R172M mutation. Patients have a mean age of 27 years (range: 6-50) and suffer all from medulloblastoma of the SHH type.

overall_design: We analysed DNA methylation of medulloblastoma with IDH mutation.

first: Ulrich

last: Schüller


title: Extracellular vesicles derived from antral follicles significantly change the transcriptional profile of cumulus cells and oocytes during pre-in vitro maturation in cattle

city: Québec

middle: Perecin

last: Nociti

state: Québec

address: 2425, Rue de l'Agriculture

department: Animal Science

institute: Université Laval

summary: Cumulus-oocyte complexes (COCs) used for in vitro production (IVP) of bovine embryos originate from antral follicles of different sizes, leading to variations in developmental competence. To address this, pre-in vitro maturation (pre-IVM) allows oocytes with additional time to acquire competence. Given the role of follicular fluid-derived extracellular vesicles (EVs) in ovarian follicle communication, which has been shown to vary in content and function across folliculogenesis, we investigated whether EVs from early versus late antral follicles influence COCs during pre-IVM. EV supplementation significantly altered gene expression in cumulus cells and oocytes. In cumulus cells, affected pathways included MAPK signaling, Gap junctions, Cytokine-cytokine receptor interaction, Axon guidance, cAMP, and Cushing syndrome. In oocytes, fewer genes were altered, with effects on p53 signaling and Cholesterol metabolism. Despite these changes, no significant effects of the EV treatment were noted on

overall_design: In the present experiment, COCs were subjected to pre-IVM in the absence (control) or presence of EVs from the follicular fluid of either early (7.0-8.5 mm) or late (≥ 12.0 mm) follicles. Afterwards, COCs were used for i) analysis of chromatin remodeling and meiotic progression; ii) IVP encompassing IVM, IVF and IVC; iii) isolation of cumulus cells and oocytes at the end of pre-IVM for analysis of the transcriptional profile by RNA sequencing (RNA-Seq). F

middle: P

last: Nocit

first: Marcos

middle: R

last: Chiaratti

first: Gisele

last: Mingoti


title: CUT&Tag analysis of the inhibitory effects of Olig2 on astrocyte-to-neuron conversion (2)

city: Guangzhou

first: Zheng

last: Wu

state: Guangdong

address: 601 West Huangpu Road

institute: Jinan University

summary: Collectively, our study reveals the role of Olig2 in hindering the in vivo AtN conversion and idendifies an inducible barrier to neuronal reprogramming.

overall_design: WT mouse cortex was injected with AAV9 hGFAP-Ngn2-tdTomato. Virus-infected brain tissue was collected 7 days post injection (n = 12 mice), and dissociated into single-cell suspension, followed by tdTomato+ cells collecting by fluorescent-assisted cell sorting. The tdTomato+ cells were then subjected to CUT&Tag sequencing.

first: Chuying

last: Lai

first: Kaiyu

last: Hou

first: Gong

last: Chen


title: Niclosamide synergizes with sunitinib against renal cell carcinoma via regulation of DNA repair and cell cycle pathways

city: Seoul

first: Aeryang

last: Jung

address: 222, Banpo-Daero

department: Urology

country: South Korea

institute: The Catholic University, Seoul

summary: Tyrosine kinase inhibitors (TKIs), such as sunitinib and sorafenib, are standard treatments for renal cell carcinoma (RCC). However, most patients treated with these drugs eventually develop drug resistance and relapse; therefore, new treatment options for RCC are urgently required. Recent studies have focused on combination therapies targeting distinct molecular pathways that may produce synergistic effects and help overcome drug resistance in RCC.  Niclosamide, an anthelmintic agent, is effectice against various cancers; however, its potential in combination with sunitinib for treating RCC has not been evaluated. In this study, we assessed the therapeutic efficacy of this combination in RCC. Niclosamide alone inhibited RCC cell proliferation, whereas its combination with sunitinib produced a synergistic anticancer effect, both in vitro and in vivo. RNA sequencing (RNA-seq) and bioinformatic analyses showed that niclosamide modulated critical pathways, including BRIP1- and FANCA-mediated DNA repair and E2F2-regulated cell cycle progression. These findings suggest that niclosamide may be repurposed as an anticancer agent to enhance the efficacy of targeted therapies for RCC, supporting its potential for clinical application as part of a combination treatment strategy.

overall_design: ACHN renal cell carcinoma cells were treated with vehicle control, niclosamide (1 µM), sunitinib (2.5 µM), or the combination of niclosamide and sunitinib. Total RNA was extracted from two biological replicates for each condition (n=2 per group). Libraries were prepared using the NEBNext Ultra Directional RNA-Seq Kit (New England BioLabs, Rowley, MA, USA) and subjected to high-throughput sequencing on the Illumina HiSeq 2500 platform with paired-end 100 bp reads.The study aimed to identify transcriptomic alterations induced by single-agent or combination treatments.

first: Yong Hyun

last: Park

first: Ae Ryang

first: Ga Eun

last: Kim

first: Mee Young

first: Dongho

last: Shin


title: CFTR mutation abrogates Prevotella regulation of epithelial cell defense against Staphylococcus aureus

city: Aurora

first: Sarah

middle: E

last: Clark

state: CO

address: 12700 E 19th Ave, Mail Stop 8333

institute: University of Colorado School of Medicine

summary: Background: The oral anaerobe Prevotella melaninogenica is enriched in the lungs of people with cystic fibrosis (pwCF), yet its functional impact on respiratory tract homeostasis remains incompletely understood. Prior studies identified immune modulatory effects following lung exposure to Prevotella, but the relevance of these findings for CF infections is unknown.  Methods: The impact of P. melaninogenica on infection with the CF pathogen Staphylococcus aureus was evaluated using a mouse lung infection model and human respiratory tract cystic fibrosis transmembrane conductance regulator (CFTR) mutant and isogenic wild-type (WT)-corrected CFBE41o- epithelial cells. RNA-sequencing was performed to compare Prevotella-induced signaling programs in WT-corrected versus CFTR mutant cells.  Results: P. melaninogenica significantly reduced S. aureus lung infection, which was associated with elevated S. aureus killing by lung neutrophils and impaired S. aureus adherence to epithelial cells. Live or heat-killed Prevotella were sufficient to mediate these effects, which were dependent on the toll-like receptor TLR2. Prevotella impairment of S. aureus adherence also required CFTR function, as this effect was lost in CFTR mutant cells but restored by CFTR modulator therapy. RNA-sequencing identified several antibacterial defense pathways selectively upregulated by Prevotella in WT corrected epithelial cells, correlating with higher IL-8 and IL-6 cytokine production.  Conclusions: P. melaninogenica enhanced neutrophil and epithelial defense against S. aureus, but these benefits were lost with CFTR dysfunction. CFTR modulator therapy rescued Prevotella responsiveness in respiratory epithelial cells, highlighting the potential for synergistic effects of host-microbiome interactions and CFTR targeted therapies.

overall_design: CFBE41o- CFTR mutant and isogenic WT corrected CFBE41o- epithelial cells in submerged culture were either untreated or treated with heat-killed Prevotella melaninogenica for 24 hours. The experiment is designed to capture the CFTR-dependent gene expression changes in unstimulated and Prevotella stimulated cells.

first: Maksym

last: Goryachok

first: Ana

last: Fairbanks-Mahnke

first: Samantha

last: Fulte

first: Emily

last: Tamkin

first: Arianna

last: McCarty

first: Eric

last: Larson

first: Paul

middle: J

last: Planet


title: Effect of BHB on transcriptional changes of antigen-stimulated CART19 cells

city: Philadelphia

first: Marco

last: Ruella

state: PA

address: 3400 Civic Center Blvd

institute: University of Pennsylvania

summary: This study examines the impact of β-hydroxybutyrate (BHB) on T cell function using RNA-seq, ATAC-seq, and H3K27ac CUT&RUN profiling to characterize BHB-induced transcriptional and epigenetic remodeling.

overall_design: Plates were coated with CD19 protein and incubated overnight at 4 °C. After blocking with 2% BSA the following day, human CAR T19 cells were added and cultured for 48 h in the presence of vehicle or 5 mM β-hydroxybutyrate (BHB).

first: Shan

last: Liu

first: Puneeth

last: Guruprasad

first: Maayan

last: Levy


title: Genome-wide tongue epithelia transcriptomic profiles from K14-rtTA;TRE-FLBmi-1 + 4-nitroquinoline 1-oxide (4-NQO), 25 weeks (KrTB-N (25w)), KrTB+Doxycycline+4-NQO, 4 weeks (KrTB-DN (4w)), and KrTB+Doxycycline+4-NQO, 10 weeks (KrTB-DN (10w)).

city: New York City

first: Jorge

last: Baquero

state: NY

address: 1300 York Avenue

department: Pharmacology

institute: Weill Cornell Medical College

summary: We developed a transgenic mouse model (truncated K14-rtTA; TRE/Bmi1, KrTB) containing a doxycycline- (dox) controlled, Tet-responsive element system to selectively overexpress BMI1 only in the basal epithelial stem cells (SCs) of the tongue. Here we used this transgenic mouse line to delineate BMI1 actions in early-stage oral squamous cell carcinoma (OSCC). We observed more oncogenic changes in mice with ectopic BMI1 expression after 4 weeks of 4-nitroquinoline 1-oxide (4-NQO) treatment. We detected increased proliferation and oxidative stress, as well as increased expression of multiple transcripts and proteins linked to human OSCCs, in murine tongue epithelia with high BMI1 expression during early carcinogen-induced tumorigenesis. Furthermore, increases in mRNAs encoding multiple metabolic targets, such as SLC16A3, PKM, and GPI1, were accelerated upon BMI1 overexpression in our 4-NQO model.

overall_design:
Examination of genome-wide transcript levels in mouse tongue epithelia samples. 12 samples were analyzed, 4 biological replicates per condition (KrTB-DN (4w) vs. KrTB-N (25w) and KrTB-DN (10w) vs. KrTB-N (25w)).

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The table below lists GEO accessions reused/reanalyzed for this study.
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first: Xiao-Han

last: Tang

first: Daniel

last: Galke

first: Theresa

last: Scognamiglio

first: Tuo

last: Zhang

first: Lorraine

last: Gudas


title: Bipolar and schizophrenia risk gene AKAP11 encodes an autophagy receptor coupling the regulation of PKA kinase network homeostasis to synaptic transmission

city: New York

first: Cong

last: Xiao

state: MA

address: Hess 9th floor,1470 Madison AVE

institute: Icahn School of Medicine-Mt Sinai

summary: Human genomic studies have identified protein-truncating variants in AKAP11 associated with both bipolar disorder (BD) and schizophrenia (SCZ), implicating a shared disease mechanism driven by loss-of-function. AKAP11, a protein kinase A (PKA) adaptor, plays a key role in degrading the PKA-RI complex through selective autophagy. However, the neuronal functions of AKAP11 and the impact of its loss-of-function remains largely uncharacterized. Through multi-omics approaches, cell biology, and electrophysiology analysis in mouse models and human induced neurons, we delineated a central role of AKAP11 in coupling PKA kinase network regulation to synaptic transmission. Loss of AKAP11 distorted compartment-specific PKA and GSK3 activities and impaired cellular functions that significantly overlap with pathways associated with BD and SCZ. Moreover, we identified interactions between AKAP11, the PKA-RI adaptor SPHKAP, and the ER-resident autophagy-related proteins VAPA/B, which co-adapt and mediate PKA-RI complex degradation in neurons. Notably, AKAP11 deficiency impaired neurotransmission, providing key insights into the mechanism underlying AKAP11-associated psychiatric diseases.

overall_design: RNA extracted from the AKAP11 conditional knockout and control mice, each group has 4 replicates.

first: You-Kyung

last: Lee

first: Xiaoting

last: Zhou

first: Le

last: Wang

first: Meghan G.

last: McReynolds

first: Zhiping

last: Wu

last: Purisic

first: Henry

last: Kim

first: Xianting

last: Li

last: Pang

first: Jinye

last: Dai

first: Junmin

last: Peng

last: Yang

first: Zhenyu

last: Yue


title: Activated protein C promotes human lung cancer progression through the release of tumor extracellular vesicles and transfer of microRNA-200a

city: Kalyani

first: Kaushik

last: Das

state: West Bengal

address: N.S.S. Kalyani Nadia

country: India

institute: National Institute of Biomedical Genomics

type: Non-coding RNA profiling by high throughput sequencing

summary: Blood coagulation and cancer are intricately related. Hypercoagulation associated with cancer leads to aberrant thrombin generation, which contributes to thrombosis. Thrombin also activates anticoagulant protein C and the activated protein C (aPC), in addition to regulating the coagulation pathway, it also elicits cell signaling by binding to endothelial cell protein C receptor (EPCR) and activating protease-activated receptor 1 (PAR1)-mediated cell signaling. Earlier studies showed that aPC promotes lung adenocarcinoma survival and metastasis. However, the underlying mechanism remains largely unknown. Our present study provides mechanistic insight into how aPC promotes lung adenocarcinoma survival, metastasis, and drug resistance. Our study shows that aPC, through EPCR-PAR1-driven activation of RhoA-ROCKII-JNK1/2-MLC2 signaling, triggers extracellular vesicle (EV) release from lung adenocarcinoma cells. aPC-EVs, via the transfer of microRNA (miR)-200a, promote proliferation, migration, and invasion of normal lung epithelial cells. They also confer resistance to lung cancer against chemotherapeutic agents. Inhibition of miR-200a functions through the incorporation of anti-miR-200a abrogates aPC-EVs-mediated tumorigenic effects. Furthermore, loading miR-200a mimic into control EVs showed similar phenotypic responses to that of aPC-EVs. miR-200a is shown to target SOX17 in the recipient cells, leading to tumorigenesis. miR-200a upregulation and SOX17 downregulation are consistently observed in lung cancer tissues in the UALCAN portal database of clinical specimens. Consistent with these findings, our in vivo studies in BALB/c nude mice showed that aPC-EVs from lung cancer cells promote tumor growth, metastasis, and drug resistance through miR-200a transfer. Targeting EV biogenesis, EV’s miR-200a, and/or EV uptake mechanisms may offer novel therapeutic strategies in limiting lung tumorigenesis, thereby increasing patients’ survival.

overall_design: Deep sequencing of microRNAs isolated from the EVs of A549 lung cancer cells after treatment with a control vehicle (Con) or activated protein C (aPC)

first: Das

last: Kaushik


title: RRAD-reduction reveals efficacy of targeting L-type calcium channel regulation for treatment of heart failure

city: Lexington

first: Jonathan

last: Satin

state: KY

address: 741 S. Limestone

department: Physiology

institute: University of Kentucky

summary: Heart failure with reduced ejection fraction (HFrEF) is a major health problem. Increasing L-type calcium channel (LTCC) activity deteriorates heart function; however, myocardial RRAD knockout (cRADΔ/Δ) instills tonic modulated LTCC current (ICa,L) that preserves healthy myocardium. Thus, we chose to challenge the dogma that enhanced trigger Ca2+ is maladaptive. The study objective was to test the hypothesis that modulated ICa,L in cRADΔ/Δ mice rescues dilated cardiomyopathy by providing tonic modulated trigger Ca2+. Methods and Results Mouse and human models were tested. The muscle lim protein knockout mouse (MLPKO) is a murine model of dilated cardiomyopathy (DCM) and HFrEF. The experimental timeline was to induce cRADΔ/Δ after onset of DCM (2.5 months of age) and follow subjects for up to 1-year. Longitudinal echocardiography and cardiac magnetic resonance imaging (CMR) showed that cRADΔ/Δ intervention rescued systolic function. Patch clamp recordings of isolated cardiomyocytes of MLPKO with cRADΔ/Δ demonstrated augmented LTCC activity, along with rescue of dysfunctional Ca2+ handling and sarcomere function. Bulk RNAseq of hearts demonstrated downregulated pathological signaling cascades and pro-hypertrophic gene expression which comported with the reduction in eccentric hypertrophy observed with gravimetrics, CMR, and echocardiography. RRAD knockdown effects translate from mouse to human heart. Ventricle slices from HFrEF patients were treated with lentiviral shRNA targeting RRAD and recapitulated the inotropic and lusitropic effects observed in the mouse model of DCM. Conclusions Induction of cardiomyocyte-restricted RAD knockout in MLPKO mice after onset of DCM rescued cardiac dysfunction and attenuated pathological remodeling. cRADΔ/Δ intervention provided positive inotropy and lusitropy and reverted transcriptional signatures towards healthy myocardium. This study introduces targeting myocardial RAD regulation of the LTCC as a novel therapeutic strategy for systolic heart failure.

overall_design: Comparative gene expression profile analysis of bulk RNA-seq data of left ventricle heart tissue from control group: MLPKO-RADfloxed/floxed and MLPKO-RADfloxed/floxed-MYH6_MerCreMer. Tamoxifen was adminstered to both groups at 10 weeks of age, inducing cRADKO in the latter group (an inducible double-knockout of MLP and cardiomyocyte-restricted RAD). Hearts were collected 1 month post-tamoxifen: a control group MLPKO and a double knockout MLPKO-cRADΔ/Δ(n=5 hearts each group).

first: Garrett

last: Elmore

first: Andrea

last: Sebastian

first: Solomon

last: Harrar


title: P-bodies act as sites for dynamic RNA processing

city: Manchester

first: Christopher

last: Grant

state: Greater Manchester

address: Oxford Rd

department: Faculty of Biology, Medicine and Health

country: United Kingdom

institute: University of Manchester

summary: Processing bodies (PBs) are cytoplasmic granules that function in the cellular response to stress conditions by regulating mRNA metabolism. They are thought to act as sites where non-translating mRNAs are localised to undergo degradation or sequestration, but their exact intracellular roles remain unclear. We used SH-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) to study PB-localisation and global mRNA fate during glucose depletion conditions that induce PB formation. This enabled us to differentiate newly synthesized and pre-existing RNAs and to separately track mRNA synthesis and degradation. We show that pre-existing mRNAs localise to PBs with differing kinetics with some transcripts localising over the time-course of glucose starvation and some transcripts localising in a more dynamic manner. We identified a small number of transcripts that are transiently enriched in PBs and appear to depend on this localisation for decay. However, most transiently localised transcripts are not destabilized following glucose starvation and PBs appear to act as temporary storage sites for transcripts that later undergo alternative fates. For other transcripts, both their pre-existing and newly made transcripts accumulate in PBs over the time-course of glucose depletion and we suggest that these transcripts are important for adaptation once the nutrient stress is relieved. Together, our data indicate that different classes of mRNAs behave differently following nutrient depletion with P-bodies predominantly acting as triage sites for mRNAs with a more limited role in mRNA-degradation.

overall_design: SLAM-seq profiling of total and P-body–associated mRNAs in Saccharomyces cerevisiae W303 cells, with total RNA sampled under glucose-replete and glucose-depletion conditions and P-body–associated RNA sampled under glucose-depletion at 0, 2.5, 5, 10, 20, and 40 minutes of starvation.

first: Matthew

middle: W

last: Feng

first: Simon

last: Hubbard

first: Mark

last: Ashe

first: Chris

middle: M


title: Molecular and phenotypic blueprint of human hematopoiesis links proliferation stress to stem cell aging

first: Raffaella

last: Di Micco

summary: Hematopoietic stem/progenitor cell (HSPC) aging has been associated with myeloid skewing, reduced clonal output, and impaired regenerative capacity, but quantitative immunophenotypic and functional analysis across human aging is lacking. Here, we provide a comprehensive phenotypic, transcriptional, and functional dissection of human hematopoiesis across the lifespan. Although primitive hematopoietic stem cell (HSC) numbers were stable during aging, overall cellularity was reduced, especially for erythroid and lymphoid lineages. Notably, HSPC from aged individuals had similar repopulating frequency of younger counterparts in primary and secondary xenografts; yet aged HSC displayed impaired differentiation capacity, changes in chromatin accessibility, dysregulation of cell cycle, and a reduced capacity to counteract activation-induced proliferative stress with concomitant accumulation of DNA damage and senescence-like features upon xenotransplantation. This age-associated phenotypic and functional decline was recapitulated by enforcing proliferative stress in vivo on human HSPC. Overall, our work sheds light on dysregulated responses to activation-induced proliferation underlying HSPC aging and establishes xenotransplantation-based models as suitable for studying age-associated hematopoietic defects.

overall_design: Human BM HSPC cells were pre-stimultation in cell culture over night and then transplanted in NSG mice, and purified after 20 weeks from mouse BM.

first: Giacomo

last: Farina

first: Edoardo

last: Carsana

first: Raffaellla


title: Stress resilience is associated with transcriptional remodeling in the VTA

city: Princeton

first: Adelaide

last: Minerva

state: New Jersey

address: Washington Road

department: Princeton Neuroscience Institute

institute: Princeton University

summary: Individual responses to chronic stress vary, with some individuals remaining resilient while others exhibit susceptibility. The ventral tegmental area (VTA), a region involved in reward learning, and the lateral habenula (LHb), a region involved in aversive learning, have been implicated in the pathophysiology of stress-related mood disorders. Here, we seek to understand the molecular adaptations in these regions at the level of single cells that mediate susceptibility versus resilience. In particular, it remains unclear whether, at the level of gene expression, different cell-types within different brain regions mediate stress susceptibility versus resilience, or if these phenotypes are mediated by distinct trajectories within the same cell-types. To address this gap, we performed single-nucleus RNA-sequencing of LHb and VTA of mice subjected to chronic social defeat stress. While we found minimal gene expression changes in the LHb after stress, the VTA exhibited widespread, cell-type-specific transcriptional remodeling in resilient individuals and few gene expression changes in susceptible individuals. Across VTA cell-types, resilience was associated with the coordinated upregulation of genes involved in intercellular signaling and neural communication, with maintenance of receptor-ligand interaction strength in resilience that was not present in susceptibility. Within VTA neurons, gene expression changes were most prominent in glutamatergic and dopaminergic clusters. Multivariate analyses of dopamine and glutamate subclusters showed that resilient neurons diverged more from control than susceptible neurons, but along a similar trajectory, supporting a model in which resilience reflects greater stress-related adaptations in these cell-types. Together, these findings highlight the VTA as a key site of molecular plasticity in stress resilience and therefore a potential therapeutic target.

overall_design: VTA and habenula nuclei from C57BL/6 mice were isolated following chronic social defeat stress (CSDS) and analyzed using snRNA-seq. For VTA, we analyzed nuclei from control (3 male, 3 female), susceptible (2 male, 2 female), and resilient (3 male, 3 female) samples. Each VTA sample contained pooled nuclei from two animals of the same sex and experimental condition. For habenula, we analyzed nuclei from control (3 male, 3 female), susceptible (3 male, 3 female), and resilient (3 male, 3 female) samples. Each habenula sample contained nuclei from one animal.

first: Brenna

last: McMannon

first: Rixing

last: Lin

first: Anna

last: Zhukovskaya

first: Ilana

middle: B

last: Witten

first: Catherine

last: Peña


title: Increased Elmo1 Expression worsens the AKI-CKD transition via ROS

city: Chapel Hill

first: Jiayi

last: Zhou

address: 703, 160 Medical Dr.

department: Pathology and Lab Medicine

institute: University of North Carolina Chapel Hill

summary: Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) and a key contributor to the progression toward chronic kidney disease (CKD), with oxidative stress playing a central role in this transition. Engulfment and Cell Motility 1 (ELMO1) regulates cytoskeletal remodeling and reactive oxygen species (ROS) production through RAC1 activation, yet its involvement in redox imbalance and kidney injury remains unclear. To investigate this, a unilateral renal IRI model was established in wild-type (WT) and ELMO1-overexpressing (Elmo1 H/H) mice. Renal function was evaluated using plasma cystatin C, estimated glomerular filtration rate (eGFR), and urinary albumin-to-creatinine ratio (UACR), while structural and ultrastructural kidney changes were assessed by Masson’s trichrome staining and electron microscopy. Redox-related gene expression was analyzed via RT-qPCR, and global transcriptional alterations were examined using RNA sequencing.

first: Yuye

last: Wang

first: John

last: Hagaman

first: Qing

last: Ma

first: J. Charles

last: Jennette

first: Meitong

last: Chen

first: Xianwen

last: Yi

first: Yukako

last: Kayashima

first: Nobuyo

last: Maeda-Smithies

first: Feng

last: Li


title: CUEDC1 promotes glycolytic metabolism reprogramming through the CUEDC1/CACNG4/PI3K axis to promote ER-positive breast cancer growth

city: Chongqing

first: Jian

address: 重庆市渝中区医学院路1号

institute: 重庆医科大学

summary:
Background The reprogramming of energy metabolism, particularly glycolysis, upholds the malignancy of tumors. The relationship between CUE Domain Containing protein-1 (CUEDC1) and glycolysis, along with its influence on the development of ER-positive breast cancer (ER+ BRCA), is not well defined. This investigation explores the functional involvement of CUEDC1 in glycolysis regulation and uncovers a previously unidentified pathway contributing to the progression of ER+ BRCA.
Methods Immunohistochemistry, western blotting and qRT-PCR were used to detect the expression of CUEDC1 in ER+ BRCA tissues and cell lines. A series of molecular analyses, including dual-luciferase reporter assays, RNA-seq, and ChIP, were performed to elucidate the potential mechanisms underlying CUEDC1’s involvement in ER+ BRCA progression. Metabolic assays focusing on glycolysis were employed to investigate the functional roles of CUEDC1 and Calcium Voltage-Gated Channel Auxiliary Subunit Gamma 4 (CACNG4). The Connectivity Map (CMap) database was utilized to screen CUEDC1 inhibitors.
Results CUEDC1 is aberrantly upregulated in ER+ BRCA tissues and cells. Increased CUEDC1 can promote enhanced tumor growth and lead to poor clinical outcomes in ER+ BRCA patients. The MYC associated zinc finger protein (MAZ) can up-regulate CUEDC1 gene transcription in ER+ BRCA cells by directly binding to its promoter. We determined that CUEDC1 directly modulated CACNG4 to enhance PI3K/AKT pathway activation, thereby facilitating tumor growth of ER+ BRCA. Clinical observations also revealed a positive correlation between CACNG4 expression and CUEDC1, with both factors being strongly associated with poor prognosis in ER+ BRCA patients. Mechanistically, The CUEDC1/CACNG4/PI3K signal axis enhanced glycolysis through upregulating GLUT1, a crucial protein in glucose metabolism, thereby supporting tumor growth of ER+ BRCA. Furthermore, methotrexate was identified as a potential inhibitor of CUEDC1. Importantly, the combination of Ipatasertib (a PI3K/AKT pathway inhibitor), with methotrexate effectively suppressed growth of ER+ BRCA in a mouse model.
Conclusions Our research reveals that the enhanced CUEDC1 is intricately linked to the proliferation of ER+ BRCA. CUEDC1-driven CACNG4 expression stimulates PI3K/AKT pathway activity and its downstream signaling, which facilitates tumor progression and positions CUEDC1-driven glycolysis as a key therapeutic target in addressing this malignancy.

overall_design:
I. Sample Information for Transcriptome Sequencing (RNA-seq)
This study performed transcriptome sequencing on the estrogen receptor-positive breast cancer (ER+ BRCA) cell line T47D. The specific sample grouping and design are as follows:
Sample Type: In vitro cultured ER+ BRCA cell line (T47D). The core reason for selecting this cell line is its high endogenous CUEDC1 expression level (verified by Western blotting and qRT-PCR), which makes it suitable for investigating the downstream regulatory mechanism of CUEDC1.
Grouping Setup: A total of 2 groups, with 3 biological replicates in each group (to ensure data reproducibility and reliability):
Control Group (shNC Group): T47D cells stably transfected with non-targeting short hairpin RNA (shRNA) lentivirus, serving as the negative control.
CUEDC1 Knockdown Group (shCUEDC1 Group): T47D cells stably transfected with CUEDC1-specific shRNA lentivirus to reduce CUEDC1 expression. The knockdown efficiency was verified by qRT-PCR and Western blotting, showing that the levels of CUEDC1 mRNA and protein were reduced by > 70%.
II. Analysis Content Based on Transcriptome Sequencing Data
Focusing on the transcriptomic differences between CUEDC1-knockdown and control T47D cells, the study conducted the following analyses:
1. Screening of Differentially Expressed Genes (DEGs)
Differential analysis of the sequencing data was performed using the DESeq2 tool. With the screening criteria of |log₂ fold change (FC)| > 1 and adjusted P-value < 0.05, a total of 154 differentially expressed genes were identified: upregulated genes accounted for 50, while downregulated genes accounted for 104.
2. Identification of Key Downstream Target Genes of CUEDC1
Core target genes were identified through "intersection analysis of differentially expressed genes and CUEDC1-related genes": first, a gene set related to CUEDC1 expression in breast cancer was extracted from the UALCAN database; then, overlap analysis was conducted between this gene set and the aforementioned 154 DEGs. Finally, CACNG4 (Calcium Voltage-Gated Channel Auxiliary Subunit γ4) was determined as the key downstream target gene of CUEDC1. After CUEDC1 knockdown, the mRNA and protein levels of CACNG4 were significantly downregulated; conversely, CUEDC1 overexpression significantly upregulated CACNG4.
3. Functional Pathway Enrichment Analysis (KEGG Analysis)
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed on the differentially expressed genes using the clusterProfiler tool. Key findings are as follows: the PI3K/AKT signaling pathway was significantly enriched (P < 0.05), suggesting that CUEDC1 may affect the biological functions of ER+ BRCA by regulating this pathway; combined with subsequent verification experiments (detection of PI3K, AKT, and phosphorylated AKT (p-AKT) levels by Western blotting), it was further confirmed that CUEDC1 activates the PI3K/AKT pathway through its target gene CACNG4.
4. Correlation Analysis of Glycolysis-Related Genes
Based on the research background that "tumor metabolic reprogramming (especially enhanced glycolysis) promotes the malignant progression of ER+ BRCA", genes related to glucose metabolism among the differentially expressed genes were analyzed: first, key genes involved in glucose metabolism (such as GLUT1, LDHA, HK2, etc.) were screened out; correlation analysis revealed that GLUT1 (Glucose Transporter 1) had the strongest positive correlation with CACNG4 expression (verified by the TCGA database and confirmed by qRT-PCR and Western blotting experiments); combined with functional experiments (detection of glucose uptake, lactate production, and extracellular acidification rate (ECAR)), it was confirmed that the CUEDC1/CACNG4/PI3K axis promotes the glycolysis process of ER+ BRCA cells by upregulating GLUT1.

first: Zexiu

last: Lu

first: Ming

last: Lei

first: Ao

last: Deng

first: Gang

last: Tu

first: Manran

last: Liu

first: Lingfeng

last: Tang


title: Effect of msbB knockout on gene expression of Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S

city: College Station

first: Ling

last: Yang

state: TX

address: 206 Olsen Blvd, Reynolds HSC  Rm159

institute: Texas A&M university

summary: The Salmonella enterica serovar Typhimurium (ST) mutant lacking the msbB gene (ΔmsbB) has been widely studied as a candidate for attenuated bacterial vectors in therapeutic applications. Deletion of msbB results in LPS with under-acylated lipid A, which lowers endotoxicity while maintaining structural integrity. This attenuation has traditionally been attributed to reduced TLR4 activation due to weaker interaction between the modified lipid A and TLR4. In our study, we confirmed that ΔmsbB ST was less lethal than wild-type (WT) ST in a mouse sepsis model. However, this difference persisted even in TLR4- and caspase-11-deficient mice, suggesting that LPS signaling is not the primary determinant of virulence. In vitro, bone marrow–derived macrophages (BMDMs) from TLR4- or caspase-11-deficient mice showed only modest reductions in ST-induced cell death and cytokine production. Importantly, ΔmsbB ST behaved similarly to WT ST in these assays, further indicating that LPS-mediated signaling is not central to the observed attenuation. Additionally, the mutant exhibited increased outer membrane permeability, likely contributing to its heightened antibiotic sensitivity—and reduced motility due to lower flagellin protein levels.

overall_design: RNAseq profiling of wildtype ST and ST-ΔmsbB, two samples cultured from two different colony of each strain were sent for sequencing

first: Zhuodong

last: Chai

first: Jianqian

last: Qi

first: Yan

last: Zhang

first: Yuqi

first: Zhenyu

first: Yinan

last: Wei


title: scRNA of Naive endothelial cells (EC) from ears of wild-type mice.

first: Vanessa

last: Cristaldi

address: 522 First Avenue

department: Dermatology

institute: NYU Langone Health

summary: In order to determine transcriptional profile of naïve endothelial cell in the ear

overall_design: Ears were collected, were pooled and stained (DAPI-, CD31+, CD45-) and sorted separately.

first: Amanda

last: Lund


title: Effects of TCDD on gene expression in hepatocytes isolated from WT and PkmΔDRE mice.

city: East Lansing

first: Rance

last: Nault

state: United States

address: 1355 Bogue St. B403

department: Department of Pharmacology and Toxicology

institute: Michigan State University

summary: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) reprograms central carbon metabolism by switching pyruvate kinase expression from isoform M1 (Pkm1) to M2 (Pkm2) mediated by aryl hydrocarbon receptor (AhR) binding to a dioxin response element (DRE) located between exon 3 and 4 within the Pkm locus. To further investigate the consequences of Pkm isoform switching in TCDD elicited hepatotoxicity, we examined gene expression in primary hepatocytes isolated from mice with the Pkm locus DRE excised (PkmΔDRE). Wild-type and PkmΔDRE hepatocytes were treated with 10 nM TCDD for 2, 4, 8, 12, 24, 48, 72, 96 and 120 hours. Central carbon metabolite changes were also assessed in WT and PkmΔDRE mice treated with 30 µg/kg TCDD every 4 day for 28 days. While AHR target genes were comparably induced, some genes exhibited divergent expression patterns in PkmΔDREmice compared to wild-types following treatment with TCDD. Notably, antioxidant gene expression was delayed in PkmΔDRE hepatocytes. Metabolomic analysis also revealed differences in glycolytic, TCA cycle and pentose phosphate pathway metabolite levels in TCDD treated WT and PkmΔDRE liver extracts. In addition, amino acid metabolism and serine/glycine synthesis were also elevated, especially in PkmΔDRE. These findings indicate PKM2 induction affects the transcriptional and metabolic coordination of hepatic responses to TCDD.

overall_design: RNA-Seq profiling of WT and PkmΔDRE hepatocytes treated with DMSO vehicle or TCDD for 2, 4, 8, 12, 24, 48, 72, 96 and 120 hours.

first: Karina

last: Orlowska

first: Tim

last: Zacharewski


title: A spatial atlas of colorectal cancer reveals the influence of stromal niches on tumour differentiation

city: Clayton

first: Andrew

last: Pattison

state: Vic

address: Wellington Rd

department: Anatomy and developmental biology

country: Australia

institute: Monash University

summary: Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer-related mortality. CRC can be classified into DNA mismatch repair proficient (MMRp) and deficient (MMRd) subtypes, with only ~50% of MMRd tumours responding to immunotherapy. Tumour architecture is spatially heterogeneous, ranging from the necrotic core to the invasive front, accompanied by diverse stromal and immune responses that influence tumour progression and treatment outcomes. To explore the spatial organisation of the tumour-immune microenvironment, we profiled the expression of ~1,000 genes in 846,469 cells from 23 tumour and normal tissue samples using the CosMx Spatial Molecular Imager (SMI).   Using a custom bioinformatic pipeline, we performed quality control, cell type annotation, identified 9 spatial niches and provide an interactive web application to explore this data. Spatial niches included normal colonic crypts, tumour masses, neutrophil-rich regions, and lymphoid aggregates. Differential gene expression analysis revealed niche-specific changes to chemokine signalling, T cell infiltration and myeloid cell polarisation could either support or inhibit tumours depending on the niche. To expand the analysis, we integrated public single-cell RNA-Seq datasets, identifying increased tumour stemness in samples with higher neutrophil content that appeared to be driven by inflammation-mediated stromal reprogramming. Additionally, samples enriched in lymphoid aggregates, as defined by a spatial lymphoid aggregate signature, displayed heightened interferon signalling and an increased abundance of proliferating B cells. Finally, we identify gene expression patterns that distinguish tumour cells from normal epithelial cells within tumours. Our analysis reveals that many of the most pronounced changes in tumour gene expression originate from alterations in the tumour microenvironment, while also revealing tumour-intrinsic gene expression. By studying the spatial niches of CRC, we reveal niche-specific dynamics that drive tumours through innate stromal signalling and suggest both tumour-specific and stromally-mediated potential therapeutic targets.

overall_design: Slides were run using two revisions (referred to as V1 and V2) of the CosMx 1,000 gene panel23. V1 was run on slides 1-5 as part of the nanostrong technology access program (TAP) and V2 was run at the the Centre of Excellence in spatial biology at Griffith Univeristy. All cells underwent immunofluorescence (IF) imaging for 5 protein markers including DAPI (nuclear) and a membrane stain to allow the AtoMx software to perform cell segmentation and identify the nuclear and cytoplasmic regions of cells. Additional stains included PanCK and CD45 (all slides), CD3 (slides 1-3) and CD68 (slides 5-9). Transcript-specific probes are hybridized to the RNA molecules on FFPE slides, detected by the CosMx SMI, and cleaved to allow subsequent rounds of cyclical hybridizations and readouts. This allows for gene identification, detection of the spatial location of RNA molecules and assignment cells.

first: Rebekah

last: Engel

first: Lochlan

last: Fennell

first: Thierry

last: Jardé

first: Helen

last: Abud


title: Exploring tumor dynamics and responses of prostate cancer to IL-27 based treatment combinations through biodynamic imaging and RNA sequencing analyses

city: West Lafayette

first: Harish

last: Kothandaraman

state: Indiana

address: 201S University St

department: Purdue Institute for Cancer Research

institute: Purdue University

summary: We use Biodynamic imaging and transcriptomics to study the response of cells to various drugs applied ex-vivo to measure and correlate the intracellular motion as a biomarker of tumor samples therapies. Prostate Cancer has been used as a model in this study. The control and IL-27 treated tumors were exposed to chemo/immunotherapy drugs ex vivo and BDI was used to profile changes in the TME. The integration of these techniques will enable informed decision-making regarding combination treatment strategies for PCa.

overall_design: This study used a subcutaneous PCa in vivo model. The control and IL-27 treated tumors were exposed to chemo/immunotherapy drugs ex vivo and BDI was used to profile changes in the TME. Additionally, RNA-sequencing was used to elucidate the molecular changes occurring in the cells in response to various treatment combinations. The mouse adenocarcinoma cell line TRAMP-C2-Ras was used to create a vector for delivering various drugs to the Male C57/BL6 mice (8-10 weeks of age). Stranded RNASeq libraries were created and paired-end sequencing was performed to study the effects of the drugs in a tumor microenvironment. Biodynamic analysis produces Doppler spectra as functions of time for each tested well. The average spectra of all cold (empty vector treated tumors) and hot (IL-27 treated) tumors are identified, and we see the migration of infiltrated immune cells, notably T-Cells that were promoted by the IL-27 treatment of the mouse in vivo. We use RNASeq to look for enhanced T-Cell components in Hot tumors relative to the Cold tumors.

first: Shreya

last: Kumar

first: Dawith

last: Lim

first: Nadia

middle: A

last: Lanman

first: David

last: Nolte

first: John

last: Turek

first: Marxa

middle: L

last: Figueiredo


title: CUT&Tag analysis of the Olig2-targeted genes in adult oligodendrocyte lineage cells

first: Zheng

last: Wu

institute: Jinan University

summary: In summary, our study identifies and characterizes the direct targets of Olig2 in adult oligodendrocyte lineage cells.

overall_design: Sox10-CreERT2 mice were crossed with Ai14 reporter mice to generate double-transgenic offspring. Tamoxifen induction labeled oligodendrocyte lineage cells with tdTomato. Ten days post-induction, whole brains were dissociated into single-cell suspensions, and tdTomato+ cells were isolated and collected by FACS for CUT&Tag sequencing.

first: Chuying

last: Lai

first: Kaiyu

last: Hou

first: Gong

last: Chen


title: Utilization of interstitial fluid extract (IFE) to model cancer in the context of old age in vitro.

city: Tampa

first: Ana

last: Da Silva Gomes

state: Florida

address: 12902 Magnolia Drive

department: Molecular Oncology

institute: H Lee Moffitt Cancer Center

summary: Interstitial fluid is the biological fluid that occupies the spaces between cells within tissues, acting as a vital medium for the exchange of nutrients and waste products. It reflects not only the composition of specific microenvironment of an organ but is also reflective of the tumor milieu, including secreted factors and small molecules from both tumor and normal cells. As such, interstitial fluid captures a biologically relevant snapshot of the organ’s microenvironment, whether cancer is present or not. Here we introduce interstitial fluid extract (IFE) and present a protocol for its isolation from the lungs of metastasis-bearing mice, followed by ex vivo treatment of cultured cells and high-throughput transcriptomic analysis. This approach is highly flexible, supporting a variety of downstream applications and adaptable to different experimental formats such as mouse strain, cancer cell type, and organ of interest. By comparing the molecular profiles of IFE-treated cells with those of cells sorted directly from metastasis-bearing lungs, the protocol helps validate the biological relevance of the IFE model. This approach is particularly valuable for studying in vivo-like conditions where direct in vivo experiments are challenging or not feasible, such as in the context of old age, offering a practical alternative for investigating complex tumor microenvironments and cellular responses.

overall_design: 1)Global analysis of RNA levels to determine transcriptional reprogramming in human lung adenocarcinoma cell line A549 upon treatment with 10 % human serum derived from 6 pools of 30 young (<30) and 6 pools of 30 old (>60) healthy donors for 3 days. 2) Global analysis of RNA levels to determine transcriptional reprogramming in human lung adenocarcinoma cell line A549 upon treatment cortisol (0.5 µM) or treatment with vehicle (ethanol) for a total of 3 days. 3) Global analysis of RNA levels to determine transcriptional reprogramming in human lung adenocarcinoma cell line A549 harboring a) non-targeting shRNA (shNT) treated with 10 % human serum derived from 6 pools of 30 young (<30) healthy donors b) A549 shNT and A549 bearing shRNAs targeting the glucocorticoid receptor (shGR#1 and shGR#2) with treated with 10 % human serum derived from 6 pools of 30 old (>60) healthy donors. The treatments were performed for a total of 3 days.

last: Gomes

first: Stanislav

last: Drapela

first: Didem

last: Ilter


title: Ecological history shapes transcriptome variation in quiescent Saccharomyces cerevisiae

city: Kraków

first: Dominika

last: Wloch-Salamon

state: Małopolskie

address: Gronostajowa 7,, 14

country: Poland

institute: Jagiellonian University

summary: Quiescence is a pivotal state for all living organisms and cells. Recent research indicates lack of one type of qui-escent cells. That is, even if the primary feature of quiescence, restarting divisions is maintained, quiescent cells within populations exhibit variation in their cellular architecture and characteristics. While it is known that the process of quiescent enter is influenced by a combination of nutrient starvation and temporal factors; the under-lying mechanisms remain to be fully elucidated. In this study we compare the transcriptomes of known to be homogenous fraction of dense quiescent yeast isolated from populations of different ecological history. These populations have undergone experimental enrichment of certain types of quiescent cells during cycles of growth and starvation for 300 generations. Transcriptome analysis revealed discrepancies in terms of characteristics as-sociated mainly with energy turnover processes, biosynthesis and the cell wall maintenance. The results of this study suggest that quiescent cells possess the capacity to adapt their transcriptome activity in accordance with their evolutionary history.

overall_design: RNA-seq profiling of  “homogenous” fraction of quiescent cells within the lines that differ about 300 generations of different selection-enrichment experimental procedure, derived from our previ-ous study (Wloch et all 2017) .

first: Hanna

last: Tutaj

first: Agnieszka

last: Marek

first: Katarzyna

last: Tomala


title: ZAD mediates chromatin association and insulator activity of Drosophila Pita and is functionally interchangeable with the human ZFP276 ZAD-like domain

city: Moscow

first: Natalia

last: Klimenko

address: 34/5 Vavilova Street

country: Russia

institute: Institute of Gene Biology (IGB) of the Russian Academy of Sciences

summary: Drosophila Pita belongs to large group of architectural proteins that have C-terminal cluster of C2H2-type zinc-finger domains and N-terminal zinc finger-associated domain (ZAD) that can homodimerize. Despite widespread distribution of ZADs in insects, only one human protein, Zinc-finger protein 276 (ZFP276), has a ZAD-like domain. The functional role of Pita has been studied so far only in the formation of the boundaries of regulatory domains in Bithorax complex. Here we tested the role of ZAD in the functional activity of the Pita protein. Using CRISPR/Cas9, we replaced the promoter region of the pita gene with attP, allowing expression of modified protein variants. Pita null mutants die at a late embryonic stage, whereas flies expressing a Pita mutant lacking ZAD, PitaΔZ, show reduced viability. ChIP-seq studies showed that PitaΔZ effectively binds to promoters of housekeeping genes and insulators in cooperation with other known architectural C2H2 proteins and CP190. However, ZAD is required for the binding of Pita to a subset of chromatin regions and its function as an insulator protein. The ZAD-like domain from human ZFP276 can almost completely compensate for the ZAD functions in the Pita protein. The results directly demonstrate that ZAD is required for insulator activity of Pita and its ability to efficiently bind to specific genomic regions. The ZAD-like domain of human ZFP276 may function similarly to the ZAD of Pita, raising the question of why ZADs have not spread to mammalian C2H2 proteins as they have to most insects.

overall_design: ChIP-seq signal of the Pita with FLAG and Pita antibodies in whole bodies of adult flies in Pitawt, PitaΔZ, and PitaZ276 lines

first: Oksana

last: Maksimenko


title: Cut&tag of H3K4me3 for KDM5A-knockout 293T cell and control 293T.

city: Taiyuan

first: Yuan

last: Fu

state: Shanxi

address: No. 56, Xinjian South Road, Yingze District

department: Department of Biochemistry and Molecular Biology, School of Basic Medicine

institute: Shanxi Medical University

summary: KDM5A-KO HEK293T cells were purchased from Beyotime (Shanghai, China). These KDM5A-KO HEK293T cells are polyclonal cells infected with lentivirus expressing the single guide RNA (sgRNA) and Cas9 and containing a puromycin resistance cassette and were characterized via a T7EI assay. The sgRNA sequence used to generate the KDM5A-KO cells was TCTCCGCCAAAGGCCGGATG. The sequences of the PCR primers specific for KDM5A were as follows: forwards, 5’CTTCAGCCAGCTTTTCTCCA3’; and reverse, 5’TCAGTTCCCAAGTTCTAGGC3’. H3k4me3(CST)  cut&tag was performed for further analysis.

overall_design: We compared the h3K4me3 of control and KDM5A-KO cells. Two biological replicates are set for each group, C1 and C2 were control groups from control. And K1, K2 were from KDM5A-knockout groups.

first: Jianting

last: li


title: Space radiation induces distinct senescent phenotypes: Implications for space travel [bulk RNA-Seq]

city: Minneapolis

first: Louise

middle: Eileen

last: Pitcher

state: MN

address: 312 Church Street SE RM 4-210 NILS HASSELMO HALL

department: Biochemistry, Molecular Biology and Biophysics

institute: Masonic Institute on the Biology of Aging and Metabolism

summary: As Earth’s magnetic field and ozone continue to weaken, space radiation begins pose a significant threat to the health of not only space travelers, but the world’s population. Space radiation and its high-energy and high-charge ions create distinct clusters of DNA and concentrated macromolecular damage that results in the accumulation of senescent cells (SnCs) known to play a critical role in promoting multimorbidity. Here we demonstrate that human fibroblasts exposed to different forms of space radiation acquire senescence-associated phenotypes including morphological alterations and the accumulation of SA-ßgal+ cells more efficiently than ꝩ-irradiation. Bulk and single cell RNA (scRNAseq) sequencing analysis revealed that space irradiated human fibroblasts up-regulated senescent-like phenotypes to a greater extent than γ-irradiation and enriched pathways associated with chronic activation and adaptation of the integrated stress response and NADPH-coupled redox metabolism. Healthy cells treated with conditioned media from irradiated SnCs manifested pro-inflammatory transcriptional profiles dependent on both radiation and cell type. Finally, treatment with known senotherapeutics demonstrated radiation-specific effects in primary dermal fibroblasts. Our data demonstrate that space radiation differentially induces senescent phenotypes in human cells compared to γ-irradiation that may play a key role in the pathogenic effects of space travel.

overall_design: Primary Human dermal cells (HDFs) were cultured in alpha-MEM with nucleosides and glutamine (Gibco), supplemented with 10% FBS, 1% penicillin/streptomycin (Gibco), and non-essential amino acids. The cells were maintained at 37°C, 5% CO2 and 20% O2.  All cells were seeded at 50% confluence and replicate cultures were shipped overnight to the National Aeronautics and Space Administration (NASA) Laboratory at Brookhaven National Laboratory. Upon arrival cells were allowed to recover from shipment overnight prior to radiation exposure and irradiation occurred as described previously. Briefly, irradiations occurred using particle beams for Hydrogen (H), Silicon (Si), Iron (Fe) using particle energies of 150, 238 and 600 MeV/n with LET values of 0.5, 79 and 174 ke V/µm respectively. For γ-irradiation, a 137Cs source (JL Shepard and Associates, CA) was used, all cells regardless of types of radiation exposure received final doses of 4.0Gy. Following exposure to space radiation, a media change was preformed, and cells were allowed to rest overnight prior to overnight shipment back to the University of Minnesota where they were allowed to rest another 13 days to allow senescence induction. During senescence induction medium was replaced and collected every 2-3 days and routine passaging was conducted.

first: Bipasha

last: Mukherjee

first: Ashley

last: Saathoff

first: Zachary

last: Eduvas

first: Nino

last: Giorgadze

first: Sandeep

last: Burma

first: Paul

last: Robbins

first: Xu

last: Zhang

first: Josh

middle: O

last: Bartz

first: Tamar

last: Pirtskhalava

first: Amyn

last: Habib

first: Tamara

last: Tchkonia

first: James

last: Kirkland

first: Xiao

last: Dong

first: Nathan

middle: K

last: LeBrasseur


title: Space radiation induces distinct senescent phenotypes: Implications for space travel [scRNA-Seq]

overall_design: IMR-90 human fibroblasts were obtained from ATCC and cultures in MEM (Gibco), supplemented with 10% FBS, 1% penicillin/streptomycin (Gibco), and non-essential amino acids (Gibco) and sodium pyruvate (Gibco). The cells were maintained at 37°C, 5% CO2 and 20% O2.  All cells were seeded at 50% confluence and replicate cultures were shipped overnight to the National Aeronautics and Space Administration (NASA) Laboratory at Brookhaven National Laboratory. Upon arrival cells were allowed to recover from shipment overnight prior to radiation exposure and irradiation occurred as described previously. Briefly, irradiations occurred using particle beams for Hydrogen (H), Silicon (Si), Iron (Fe) using particle energies of 150, 238 and 600 MeV/n with LET values of 0.5, 79 and 174 ke V/µm respectively. For γ-irradiation, a 137Cs source (JL Shepard and Associates, CA) was used, all cells regardless of types of radiation exposure received final doses of 4.0Gy. Following exposure to space radiation, a media change was preformed, and cells were allowed to rest overnight prior to overnight shipment back to the University of Minnesota where they were allowed to rest another 13 days to allow senescence induction. During senescence induction medium was replaced and collected every 2-3 days and routine passaging was conducted.


title: Bovine intramuscular and subcutaneous preadipocytes have different origins

city: BLACKSBURG

first: Zhendong

last: Tan

state: VA

address: 175 West Campus Drive

institute: Virginia Tech

summary: Abstract Background: Intramuscular fat (IMF) and subcutaneous fat (SF) are two major adipose depots in cattle, each playing distinct roles in meat quality and systemic metabolism. The development of adipose tissue requires proliferation and differentiation of preadipocytes into adipocytes. However, the identity and characteristics of preadipocytes forming IMF and SF, especially the former, remain poorly defined. Results: In this study, we derived four single-preadipocyte clones from stromal vascular fraction (SVF) of IMF and SF tissues of four adult steers and characterized their gene expression signatures. All single-cell clones of IMF and SF preadipocytes were capable of forming adipocytes, as evidenced by lipid droplets accumulation and upregulation of adipocyte marker genes such as PPARG, CEBPA, FABP4, and ADIPOQ upon adipogenic induction. Transcriptomic analysis revealed that although both IMF and SF preadipocyte clones expressed classical preadipocyte markers such as PDGFRA, DLK1, PPARG, CD34, and ZNF423, they had significantly different gene expression profiles. Over 2,000 genes were differentially expressed between IMF and SF preadipocyte clones. Surprisingly, many muscle-specific genes such as MYOG, CKM, MYH2, and MYH3 were expressed at high levels in IMF preadipocyte clones while barely detectable in SF preadipocyte clones. These muscle-specific genes remained elevated in adipocytes differentiated from IMF preadipocyte clones. Functional enrichment analysis revealed that genes upregulated in IMF preadipocyte clones compared to SF preadipocyte clones were significantly enriched in pathways such as PI3K-Akt signaling, MAPK signaling, calcium signaling, cholesterol biosynthesis, muscle structure development, and neuromuscular junction formation, further suggesting that IMF preadipocytes bear characteristics of muscle cells. Differentiation assays demonstrated that IMF SVF cells had strong adipogenic potential, as they readily formed adipocytes upon adipogenic induction, but showed little myogenic potential, as they did not form myotubes upon myogenic induction. In contrast, bovine satellite cells, the widely considered myogenic progenitor cells in skeletal muscle, exhibited both myogenic and adipogenic potential, forming both myotubes and adipocytes under respective induction conditions. Conclusions: These results demonstrate that IMF and SF preadipocytes differ significantly in gene expression profiles with the former expressing many muscle-specific genes. Our findings suggest that IMF preadipocytes may originate from muscle satellite cells or share a common progenitor, whereas SF preadipocytes arise from non-myogenic adipose progenitor cells.

overall_design: Single-cell-derived preadipocyte clones were established from stromal vascular fraction (SVF) cells isolated from intramuscular fat (IMF) and subcutaneous fat (SF) tissues of adult cattle. Each clone was induced to undergo adipogenic differentiation, followed by RNA sequencing to compare gene expression profiles between IMF and SF-derived preadipocytes.

first: Binod

last: Pokhrel

first: Honglin

last: Jiang


title: NAD⁺ Reduction in Glutamatergic Neurons Induces Lipid Catabolism and Neuroinflammation in the Brain Via SARM1

city: BLOOMINGTON

first: Hui-Chen

last: Lu

state: IN

address: 400 EAST SEVENTH STREET, MULTI-DISCIPLINARY SCIENCE BUILDING 2

department: Gill Institute for Neuroscience

institute: Indiana University Bloomington

summary: NAD⁺ homeostasis is vital for neuronal health, as demonstrated by the opposing roles of nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2), a NAD⁺-synthesizing enzyme, and sterile alpha and TIR motif-containing protein 1 (SARM1), a NAD⁺ hydrolase. Neurodegenerative insults that decrease NMNAT2 activate SARM1, leading to axon loss. To understand how the NMNAT2–SARM1 axis influences brain energy metabolism, we employed multi-omics approaches to investigate the metabolic changes resulting from neuronal NMNAT2 loss. Loss of NMNAT2 in glutamatergic neurons leads to a significant metabolic shift in the cerebral cortex from glucose to lipid catabolism, reduced lipid abundance, and pronounced neurodegenerative phenotypes and motor behavioral deficits. These metabolic disturbances are accompanied by altered glial expression of enzymes regulating glucose and lipid metabolism, enhanced inflammatory signaling, and disrupted astrocytic transcriptomic profiles related to cholesterol synthesis and immune activation. Notably, SARM1 deletion in NMNAT2-deficient mice restored lipid metabolism, astrocyte transcriptomic profiles, and mitigated neurodegeneration and motor behaviors. These findings suggest that neuronal NAD⁺ depletion triggers maladaptive, SARM1-dependent metabolic reprogramming, shifting energy use from glucose to lipids, which in turn promotes inflammation and neurodegeneration.

overall_design: Astrocytes were acutely isolated from the cerebral cortices of postnatal day 16 (P16) mice from three genotypes: Control (Ctrl-2): Nmnat2f/+; NexCre+/-; Sarm1+/-, cKOA: Nmnat2f/f; NexCre+/-; Sarm1+/-, cKOB: Nmnat2f/f; NexCre+/-; Sarm1-/-. Brains were dissected and the cortex region was used for astrocytes isolation using magnetic cell sorting (MACS) with anti-ACSA2 microbeads (Miltenyi Biotec). Total RNA was extracted after the sorting and then sent out for RNAseq analysis.

first: Zhen-Xian

last: Niou

first: Andrea

last: Enriquez

last: Espinas


title: miRNA sequencing analysis of kidney tissues in MRL/lpr lupus-prone mice and C57BL/6 controls

city: Beijing

first: Zhiying

last: Gao

state: Beijing

address: Peking Union Medical College Hospital (Dongdan campus), No.1 Shuaifuyuan Wangfujing Dongcheng District, Beijing,China 100730

department: Department of Nephrology

institute: Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences

summary: To investigate the molecular mechanisms of LN, we performed miRNA sequencing on the kidneys of 17-week-old female MRL/lpr and C57BL/6 mice.

overall_design: miRNA sequencing was performed to assess differential gene expression in the kidneys of 17-week-old female MRL/lpr lupus mice versus C57BL/6 wild-type mice

first: Yan

last: Qin

first: Xuemei

last: Li


title: Metabolites produced by agat+ cells support regeneration in the planarian Schmidtea mediterranea

city: Kansas City

first: Alejandro

last: Sánchez Alvarado

institute: Stowers Institute for Medical Research

summary: Planarians exhibit extraordinary regenerative abilities driven by pluripotent neoblasts, yet the roles of post-mitotic progenitor and differentiated cells in this process remain incompletely understood. Here, we investigate the function of agat+ cells, epidermal progenitors expressing members of the arginine:glycine amidinotransferase (agat) gene family, in the regeneration of Schmidtea mediterranea. Comprehensive analysis of all five planarian agat paralogs revealed that agat-1, -2, -3, and -4 are co-expressed in subepidermal populations enriched at wound sites, while agat-5 is minimally expressed. RNAi-mediated knockdown of agat-1 and agat-2 resulted in severe defects: agat-1 primarily disrupted tissue homeostasis, whereas agat-2 specifically impaired blastema formation and stem cell maintenance during regeneration. Transcriptional profiling of isolated agat+ cells demonstrated enrichment for metabolic and transport-related genes, including those involved in creatine and ornithine metabolism, as well as secretory pathway components. Functional assays showed that creatine supplementation partially rescued regeneration defects following agat-2 knockdown, implicating creatine and related metabolites as critical factors for regenerative success. These findings redefine agat+ cells as a heterogeneous, metabolically active, and potentially secretory population that supports regeneration beyond their canonical role as transitional progenitors. Our results highlight the importance of differentiated support cells and their metabolic outputs in tissue regeneration, providing new insight into the coordination between progenitor populations and stem cells during whole-body regeneration in planarians.

overall_design: Homeostatic whole worms were ACME dissociated and then HCR labeled with either piwi-1 (SMED30007406) or pooled agat-1/2/3 probes. Cells were FACS sorted into positive and negative groups and RNA-seq was performed on the sorted cells.

first: Aubrey

last: Kent

first: Carlos

last: Guerrero-Hernández

first: Carolyn

last: Brewster

first: Sean

last: McKinney

first: Jason

last: Morrison

first: Mary

middle: C

first: Eric

last: Ross

first: Frederick

middle: G

last: Mann Jr

first: Blair

last: Benham-Pyle


title: PROTACs therapeutically target the polyglutamine androgen receptor inspinal and bulbar muscular atrophy models

city: Ann Arbor

first: Andrew

last: Lieberman

state: Michigan

address: 1150 W Medical Center Drive

institute: University of Michigan

summary: Spinal and bulbar muscular atrophy (SBMA) is a CAG/polyglutamine (polyQ) repeat expansion disorder in which the mutant androgen receptor (AR) protein triggers progressive degeneration of the neuromuscular system in men. As the misfolded polyQ AR is the proximal mediator of toxicity, therapeutic efforts have focused on targeting the mutant protein, but these prior efforts have met with limited success in SBMA patients. Here, we examine the efficacy of proteolysis targeting chimeras (PROTACs), small molecule AR degraders that rapidly and potently promote AR ubiquitination and degradation by the proteasome. We identify ARD-1676 as a PROTAC that clears polyQ AR in an over-expression system, in patient iPSC-derived induced motor neurons and skeletal muscle cells, and in a gene targeted mouse model of disease. Furthermore, we demonstrate that 24-hour treatment with ARD-1676 rescues transcriptional dysregulation in SBMA induced skeletal muscle cells. These data provide evidence of therapeutic efficacy and in vivo target engagement, establishing PROTACs as potential therapeutic agents for the treatment of SBMA.

overall_design: Control (H179 and H180) and SBMA (SB3 and SB17) iSKMs treated with vehicle (DMSO) or ARD-1676 were lysed in TRIzol (Invitrogen 15596018) according to manufacturer’s instructions for RNA extraction. The pool was subjected to 151bp paired-end sequencing according to the manufacturer’s protocol (Illumina NovaSeq 6000). BCL Convert Conversion Software v4.3.13 (Illumina) was used to generate de-multiplexed fastq files. The reads were trimmed using Cutadapt v4.8. The reads were evaluated with FastQC (v0.11.8) [21] to determine quality of the data. Reads were mapped to the reference genome GRCh38 (ENSEMBL 113) using STAR v2.7.8a and assigned count estimates to genes with RSEM v1.3.3. Alignment options followed ENCODE standards for RNA-seq. QC metrics from several different steps in the pipeline were aggregated by multiQC v1.20. Library prep and next-generation sequencing was carried out in the Advanced Genomics Core at the University of Michigan  The DESeq2 library was used to run differential expression (DE) analysis with default settings in R [26]. EnhancedVolcano was used to make the volcano plot. Genes were filtered with p adjusted value < 0.05 as determined by xx  and |log2(fold-change)| > 1. Gene Ontology (GO) enrichment analysis was performed on this subset of DE genes using ToppGene.

first: Agamjot

last: Sangotra

first: Curtis

last: Kuo


title: Investigation of the metabolic features of cancer therapy induced senescent cells with TPEF, mass spectrometry and RNA sequencing analysis

city: Milan

first: Italia

last: Bongarzone

state: Milan

address: Via Amedeo, 42

department: Department of Diagnostic Innovation

institute: IRCCS Istituto Nazionale dei Tumori

summary: Background. Despite major advances in cancer therapies, treatment failure remains a significant issue for a subset of patients due to therapy resistance and tumour recurrence. A key contributor to these failures is therapy-induced senescence, wherein damaged cells enter a stable cell cycle arrest while remaining metabolically active, contributing to chronic inflammation and cancer relapse. These senescent cells can promote chronic inflammation and disease relapse. However, the heterogeneous nature of senescence complicates its detection and therapeutic targeting. Methods. We employed a multimodal approach to profile metabolic alterations in senescent cancer cells. Senescent cells were induced either by doxorubicin or γ-irradiation, both widely employed strategies in anti-cancer treatment, across three human cancer cell lines: MCF7, HeLa, and TPC-1. Mitochondrial dysfunction was assessed through MitoTracker and JC-1 staining. Two-photon excitation fluorescence microscopy enabled label-free imaging of mitochondrial coenzymes NAD(P)H and FAD. Lipidomic changes were analyzed using MALDI mass spectrometry imaging following lipid extraction and matrix application. Additionally, transcriptomic profiling via RNA sequencing was performed on control, senescent, and engulfing-senescent MCF7 cells. Bioinformatic analyses included differential gene expression, gene set enrichment analysis, and pathway analysis using GO, KEGG, and Reactome databases Results. Across all models, senescent cells exhibited mitochondrial dysfunction, marked by altered NAD(P)H and FAD distribution and decreased mitochondrial membrane potential. Two-photon excited fluorescence imaging confirmed a broader intracellular redistribution of mitochondrial coenzymes. MALDI analyses revealed consistent lipid remodeling, particularly involving cardiolipin precursors such as PG(18:1/18:1) and PG(18:1/22:6). Transcriptomic profiling of senescent MCF7 cells revealed that a previously observed engulfing subpopulation is marked by enhanced oxidative metabolism and increased lipid catabolism, suggestive of an engulfing phenotype. Conclusion. Our findings delineate both conserved and divergent metabolic features across senescent cell states and highlight specific metabolic vulnerabilities. These insights offer potential avenues for the development of targeted senolytic strategies.

overall_design: To obtain the senescent phenotype, adherent cells were treated with 250 nM doxorubicin (Doxo, Sigma), or irradiated at 10 Gy released by γ-rays from 137Cs sources of IBL147 biological irradiator (0.65 Gy=min). Cells were observed and/or analyzed from 6 to 8 days after treatment. Control (untreated) cells, the proliferation of which was not arrested by treatment, were split when necessary to avoid apoptosis due to overgrowth until observation (typically once after seeding and before the end point), or alternatively collected prior to full confluence, typically 72 or 96 hours after seeding. To obtain a MCF7 population enriched in engulfing cells, MCF7 cells were treated with doxorubicin as described above and, after 6 days of treatment, they were collected through trypsinization and re-seeded. Adherent cells were collected 24 hours after being re-seeded.

first: Silvia

last: Ghislanzoni

first: Federica

last: Padelli

first: Alessia

last: Bertolotti

first: Antonino

last: Belfiore

first: Matteo

last: Niero

first: Arianna

last: Bresci

last: Betti

first: Andrea

last: Masella

first: Dario

last: Polli

first: Luca

last: Agnelli


title: Profiling the Microenvironment in Tuberous Sclerosis Complex-Associated Angiomyolipoma: Insights from Spatial Gene Expression Analysis

city: Suita

first: Daisuke

last: Motooka

state: Osaka

address: 3-1, Yamadaoka

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

summary: Tuberous sclerosis complex (TSC) is a multisystem genetic disorder caused by mutations in TSC1 or TSC2, leading to constitutive activation of the mTORC1 pathway. Among its manifestations, renal angiomyolipomas (AMLs) are a common and distinctive tumor type. However, the molecular differences between TSC-associated AMLs (TSC-AMLs) and sporadic AMLs remain poorly understood. To address this, we performed spatial transcriptomic profiling (CytAssist Visium) on FFPE tissue sections from one case each of TSC-AML, sporadic AML, and renal cell carcinoma (RCC). This approach enabled in situ analysis of gene expression while preserving tissue architecture and cellular context. Our findings provide insight into the unique molecular landscape of TSC-associated tumors and offer a valuable resource for identifying disease-specific biomarkers and therapeutic targets.

overall_design: Formalin-fixed, paraffin-embedded (FFPE) tissue samples from one TSC-associated AML, one sporadic AML, and one RCC case were analyzed using the CytAssist Visium spatial transcriptomics platform. Comparative analysis aimed to identify transcriptional differences among the three tumor types while preserving spatial context.

first: Ryuta

last: Watanabe

first: Tetsuya

last: Fukumoto

first: Noriyoshi

last: Miura

first: Mie

last: Kurata

first: Riko

last: Kitazawa

first: Tadahiko

last: Kikugawa

first: Takashi

last: Saika


title: Gene expression profiling of triple-negative breast cancer cells by Nitidine Chloride(NCD)

city: Chengdu

first: Yumei

last: Wang

address: 1166 LiuTai

institute: Chengdu University of TCM

summary: To obtain the gene expression profiles of triple-negative breast cancer MDA-MB-231 cells perturbed by NCD, we treated MDA-MB-231 with either DMSO or 5 μM NCD for 24 hours. By comparing the expression profiles of DMSO-treated cells with those treated with Nitidine Chloride, we can identify the genes affected by Nitidine Chloride.

overall_design: MDA-MB-231 cells were treated with 0.1% DMSO or 5 μM NCD for 24 hours followed by RNA isolation

first: Li

last: Bai

first: Dong


title: Chronic circadian misalignment accelerates sarcopenia progression in mice

city: Kyoto

first: Kazuhiro

last: Yagita

address: Kawaramachi-Hirokoji, Kamigyo-ku

institute: Kyoto Prefectural University of Medicine

summary: In today's 24/7 society, circadian misalignment caused by environmental and lifestyle factors is associated with various adverse health consequences, underscoring the need to understand tissue-specific pathology. A potential association of environmental circadian misalignment with sarcopenia, or accelerated loss of skeletal muscle strength and mass, is poorly documented. Here, we exposed wild-type C57BL/6J male mice to a chronic (64 weeks) jet lag (CJL) paradigm. CJL caused significant reductions in muscle strength and weight relative to untreated mice. Transcriptomic and histological analysis showed activation of TWEAK/Fn14 signaling and reduced myofiber cross-sectional area, hallmark features of sarcopenia. Furthermore, CJL mice showed increased centrally nucleated fibers, myosin heavy chain co-expressing fibers, and myogenic gene expression, suggesting that insufficient muscle regeneration likely contributes to muscle atrophy. These findings demonstrate that circadian misalignment is an independent risk factor for sarcopenia, underscoring circadian rhythms as a key regulator and actionable target for sarcopenia prevention.

overall_design: Examination of poly(A)+ enriched RNAs in mouse gastrocnemius muscle exposed to chronic circadian misalignment in addition to control. Wild-type male C57BL/6J mice were exposed to chronic jet lag characterized by 8-hour advance every 4 days (referred to as ADV paradigm) for 64 weeks, whereas the control group (LD) were maintained under the normal 12h:12h light-dark condition.

last: Seya

first: Nobuya

last: Koike

first: Naoki

last: Okubo