title: ICMT deficiency ameliorates weight loss and mortality, but not tumor formation in a mouse model of liver cancer.

city: Krakow

first: Tomasz

last: Gromowski

address: Gronostajowa 7 Str.

country: Poland

institute: Jagiellonian University in Krakow

summary: Liver cancer often arises in the setting of chronic liver disease and remains a major global health challenge with limited effective treatments. Isoprenylcysteine Carboxyl Methyltransferase (ICMT) is an enzyme that catalyzes the methylation of prenylated proteins containing a CAAX motif, such as RAS, a modification thought to be essential for their function. Given its role in oncogenic signaling, ICMT has emerged as a potential therapeutic target in cancer. Notably, ICMT is overexpressed in human hepatocellular carcinoma (HCC), and previous studies have shown that Icmt deletion impairs BrafV600E-driven transformation in murine fibroblasts. In this study, we investigated the role of ICMT in liver cancer using a genetically engineered mouse model with hepatocyte-specific expression of BrafV600E, combined with deletion of Trp53 and Icmt. We assessed the impact of Icmt loss on liver physiology, tumorigenesis, and global transcriptomic and proteomic landscapes. Expression of BrafV600E led to progressive body weight loss, reduced survival, hepatomegaly, vascular congestion, liver fibrosis, and tumor development. These phenotypic changes were associated with widespread alterations in gene and protein expression, particularly affecting pathways related to cell differentiation, coagulation, and metabolism. Strikingly, liver-specific Icmt deletion ameliorated several pathological features, including weight loss, early mortality, fibrosis, and vascular abnormalities. It also partially normalized the transcriptomic and proteomic profiles. However, Icmt deletion did not prevent hepatomegaly or tumor formation. In conclusion, while ICMT inhibition mitigates several deleterious effects of BrafV600E-driven liver pathology, it does not block tumorigenesis. These findings suggest that ICMT is not a suitable therapeutic target for liver cancer prevention or treatment, but may have potential as a target to alleviate liver disease-associated symptoms. This research was supported by a research grant from National Science Centre No. 2020/37/B/NZ4/02533.

overall_design: RNAseq profiling of livers derived from mice with liver-specific (driven by Alb-Cre expression) knockin allele of BrafV600E (BrafCA/+), knockout of two copies of trp53 gene (trp53flx/flx), and one or two copies of Icmt gene (Alb-Cre/ BrafCA/+/ trp53flx/flx/Icmt flx/+ or Alb-Cre/ BrafCA/+/ trp53flx/flx/Icmt flx/flx). Mice with WT Braf alleles served as controls (Alb-Cre/ trp53flx/flx/Icmt flx/+).

first: Jarosław

last: Cisowski

first: Piotr

last: Czarnota

first: Anna

last: Golda

first: Danuta

last: Bryzek

first: Sławomir

last: Lasota

first: Mateusz

last: Wilamowski

first: Joanna

last: Koziel


title: CLIP-seq analysis of FLAG-DHX29 in HEK293T cells

status: Public on Mar 19 2026

city: Kyoto

first: Masanori

last: Yoshinaga

state: Kyoto

address: Yoshida-konoe-cho, Sakyo-ku

department: Graduate School of Medicine

country: Japan

institute: Kyoto University

summary: Synonymous codon usage controls global gene expression in both prokaryotic and eukaryotic species. Non-optimal codons are known to induce mRNA decay; however, the underlying molecular mechanism remains poorly understood in human cells. Through genome-wide CRISPR screening, we identified the RNA-binding protein DHX29 as a critical regulator of codon-dependent gene expression. Cryogenic electron microscopy and selective ribosome profiling demonstrated that DHX29 directly interacts with the A-site entrance of the translating 80S ribosome, the binding site for the eEF1A•GTP•aminoacyl-tRNA ternary complex, suggesting a role in monitoring aminoacyl-tRNA sampling. Proteomic analysis further revealed that DHX29 recruits the GIGYF2•4EHP complex to mediate global suppression of non-optimal mRNAs. These findings establish a mechanistic link between synonymous codon usage and the regulation of gene expression.

overall_design: HEK293T cells stably expressing FLAG-DHX29 were treated with DOX 40 ng/ml for 24 hours. Cell lysate were treated with RNase I digestion at 0.01 unit/ml and supjected to CLIP-seq.

first: Hirotaka

last: Toh

first: Shintaro

last: Iwasaki

first: Osamu

last: Takeuchi


title: In Vivo identification and Engineering of Arabidopsis’s Enhancers Using Agro-STARR-Seq and Deep Learning

city: Shenzhen

first: Yongzhou

last: Bao

address: BuxinRoad NO. 97, Dapeng District

country: China

institute: Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences

summary: In Vivo identification Arabidopsis’s Enhancers Using Agro-STARR-Seq

overall_design: employs Nicotiana benthamiana leaves as a heterologous expression platform, utilizing Agrobacterium-mediated delivery of a custom-designed STARR-Seq T-DNA binary vector optimized specifically for plant systems

first: Zhaohong

last: Li

first: Xiwen

last: Zhang

first: Yanwei

last: Song

first: Hongqiu

last: Cui

first: Yun

last: Chen

first: Weigang

last: Zheng


title: Mapping Endothelial-Macrophage Interactions in Diabetic Vasculature: Role of TREM2 in Vascular Inflammation and Ischemic Respons

city: DUARTE

first: Naseeb

middle: Kaur

last: Malhi

email: naseebmalhi@gmail.com, nmalhi@coh.org

state: CA

address: 1500 E Duarte Road

department: DMRI

country: USA

institute: City of Hope

type: Expression profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: A novel function of “inactive” cytoplasmic ezrin in its closed conformation as an RNA binding protein to mediate osteosarcoma metastasis

city: Washington

first: Aykut

last: Uren

state: DC

address: 3970 Reservoir Road, N.W.

department: Department of Oncology

institute: Georgetown University

summary: Ezrin is a cytoplasmic protein that can exist in multiple conformational forms that are regulated by a post-translational modification. Phosphorylation at the threonine 567 residue confers an open form on ezrin by removing the head-to-tail interaction. Open ezrin was generally accepted as the active form because it can translocate to the plasma membrane and link the actin cytoskeleton to the membrane proteins. Closed ezrin, sequestered in the cytoplasm was considered inactive. Our previous work established that ezrin directly interacts with RNA binding proteins (RBPs), and here we show that ezrin expression correlates with cytoplasmic RBPs in human osteosarcoma samples. Therefore, we hypothesized that closed ezrin is not a dormant protein, but rather biologically active. Purified recombinant ezrin protein in its closed conformation directly bound RNA with 6.1 nM affinity.  Ezrin binding specificity was highest for the guanine-rich sequences and RNAs with G-quadruplex (G4) secondary structures.  Expression of closed ezrin rescued the transcriptome profiles better than open ezrin in an osteosarcoma cell line with its endogenous ezrin knocked out. Closed ezrin also rescued the metastatic ability of osteosarcoma xenographs in zebrafish. Taken together, our data established that the closed conformation of ezrin, previously thought to be inactive, can directly bind RNA and contribute to its metastatic phenotype.

overall_design: We first knocked out the endogenous ezrin expression in K7M2 mouse osteosarcoma cell line by using CRISPR/Cas9 gene editing at the EZRIN gene locus. We then stably rescued the ezrin expression by stably transfecting them with either a wildtype, a T567A mutant, or a T567D mutant EZRIN cDNA carrying vector or left the cells unrestored by transfecting only an empty vector. The resulting cell lines were referred here as COL3WT, COL3A, COL3D, or COL3E. Each cell line has two replicates for the RNAseq analysis.

first: Emre

last: Deniz


title: Expression of CD25 in human lung mast cells and its regulation by IL-33

city: Solna

first: Elin

last: Rönnberg Höckerlind

address: NKs Bioclinicum J7:30, Visionsgatan 4

department: Medicine (Solna)

country: Sweden

institute: Karolinska Institutet

summary: Expression of CD25 in human mast cells is primarily associated with aberrant mast cell in clonal mast cell disorders. The regulation of its expression remains undefined. We investigate the role of IL-33 in regulating CD25 in primary human lung mast cells (HLMCs) and the functional consequence of this regulation.

overall_design: Purified HLMCs were exposed to IL-33 and IL-2 alone or in combination and the effects were assessed by bulk RNA sequencing.


title: Caspase-8 silences cell death-independent constitutive immune activation driven by tonic TNF-α

city: Messina

first: Concetta

last: Beninati

state: ME

address: Via Consolare Valeria 1

department: Human Pathology

country: Italy

institute: University of Messina

summary: Caspase-8 maintains organismal integrity by preventing cell death and secondary inflammation in selected epithelial and endothelial tissues. Here we show that caspase-8 also controls a systemic, cell death-independent inflammatory pathway that is constitutively activated during homeostasis. In vivo, selective caspase-8 inhibition produces, in the absence of other stimuli, marked neutrophilia driven by circulating proinflammatory and chemotactic cytokines. In vitro, caspase-8 inhibition triggers in neutrophils, but not in macrophages, a profound transcriptional response associated with the release of IL-1βand other cytokines. This process requires tonic TNF-α production by neutrophils, which acts autocrinally to sequentially activate RIPK1, RIPK3,MAPKs and NF-κB. The IL-1βrelease induced by caspase-8 inhibition requires gasdermin D and neutrophil serine proteases, but not canonical inflammasome components, such as NLRP3 and ASC, or gasdermin E. Our data unveil the mechanistic features of a neutrophil-centric, pro-inflammatory pathway that can be therapeutically targeted to augment host defenses against pathogenic agents.

overall_design: RNA-seq profiling of murine Neutrophils and Macrophages isolated frombone marrow and treated for 3 h with Caspase-8 inhibitor (z-IETD-fmk 50 µM) or vehicle (DMSO 0,2%).

first: Germana

last: Lentini

first: Francesco

last: Coppolino

first: Agata

last: Famà

first: Giuseppe Valerio

last: De Gaetano

first: Federica

last: Grasso

first: Luigi

last: Fiore

first: Alessia

last: Berbiglia

first: Silvia

last: Carnevale

first: Isabella

last: Venza

first: Egil

last: Lien

first: Terje

last: Espevik

first: Mario

first: Sebastien

last: Jaillon

first: Giuseppe

last: Teti


title: Metabolic Programming Promotes Cellular Uptake of Extracellular Vesicles and Boosts In Vivo Therapeutic Efficacy

city: Chicago

first: Zongjie

last: Wang

state: Illinois

address: 400 N Aberdeen St., STE800

department: Biohub Chicago

institute: Biohub

summary: Extracellular vesicles (EVs) are promising carriers for delivering therapeutic cargos to target cells, yet their in vivo delivery efficacy remains limited despite extensive efforts to optimize their physical and chemical properties. Recognizing that EV uptake is an interactive process between EVs and cells, we hypothesize that modulating cell states can enhance the uptake and therapeutic impact of drug-loaded EVs. In this study, we investigated how the cellular metabolome influences EV uptake in neuroblastoma cells. Our results demonstrate that EV uptake varies significantly depending on the cells' metabolic conditions. Through multi-omic analyses, we identified a metabolite cocktail composed of pyruvate and methionine that enhances EV uptake by 150%. In an animal cancer model, co-administration of these metabolite adjuvants markedly reduced the tumor size. This work highlights the potential of targeting the cellular metabolome to optimize drug delivery and introduces a straightforward adjuvant strategy to enhance nanoparticle delivery efficacy.

overall_design: RNA-seq profiling of BE(2)-C cell cultured in EMEM/F12 and HPLM medium for 7 days.

first: Kangfu

last: Chen


title: Sox8 and Sox9 influence differentiation and nuclear positioning of retinal Muller glia [CUT&RUN]

city: Pittsburgh

first: Clayton

middle: Pio

last: Santiago

state: Pennsylvania

address: 1622 Locust St.

department: Ophthalmology

institute: University of Pittsburgh

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Temporal patterning of retinal progenitor cells governs the sequential generation of retinal cell types, with gliogenesis occurring late in development. Sox8 and Sox9, members of the SoxE transcription factor family, are highly expressed in late-stage retinal progenitor cells and mature Müller glia (MG), yet their functional roles remain incompletely defined. Here we employed gain- and loss-of-function approaches, single-cell multiomic profiling, and injury models to investigate Sox8/9 function. Overexpression of Sox8 and/or Sox9 in early-stage RPCs suppressed early-born cell fates and promoted photoreceptor generation, consistent with a role in late-stage temporal identity. Conversely, conditional deletion of Sox8 and/or Sox9 in late-stage progenitors did not impair MG specification, but caused radial displacement of MG nuclei into the outer retina and modest changes in glial gene expression. Loss of Sox8/9 in mature MG modestly increased proliferation post-injury without inducing neurogenic competence. These findings suggest that Sox8/9 are dispensable for gliogenesis and repression of neurogenic competence, but are essential for proper laminar positioning and maturation of retinal Müller glia.

overall_design: CUT&RUN of Sox8 and Sox9 transcription factors in wildtype retinas at P2 and P17 ages

first: Nicole

last: Pannullo

first: Debarpita

last: Datta

first: Jared

middle: A

last: Tangeman

first: Lizhi

last: Jiang

first: Leighton

middle: H

last: Duncan

middle: P

first: Seth

last: Blackshaw


title: Effect of depletion of PRSS22 on gene expression of HCT116 colorectal cancer cells

city: Guangzhou

first: Zijian

last: Kuang

state: Guangdong

address: No. 382, East Outer Ring Road, Guangzhou Higher Education Mega Center, Panyu District, Guangzhou City

institute: South China University of Technology

summary: Cancer cells exhibit abnormally altered proteome to satisfy the metabolic demands that arise from heightened proliferation. Trypsin-like serine proteases are a class of proteolytic enzymes whose expression is often dysregulated in cancer. Serine protease 22 (PRSS22) has been associated with the tumorigenesis of several types of tumors. In this study, we identified PRSS22 as a key driver of inflammation to cancer transition (ICT) in colorectal cancer (CRC). PRSS22 expression was positively correlated with the pathological progression of colitis-associated ICT. Genetic disabling of PRSS22 inhibited the growth and migration of and caused redox stress in CRC cells. Mechanistically, knocking down PRSS22 promoted the expression of HMOX1, which fine-tuned the inflammatory response and led to ferroptosis. Loss of PRSS22 also prevented the cleavage of osteopontin and the migratory capacity of CRC cells. We also observed a reduction of M0-to-M2macrophage polarization in a co-culture system of CRC cells and THP-1-derived macrophages. Altogether, this study reveals a tumor-promoting function of PRSS22 as positions it as a key driver in CRC progression. Targeting PRSS22 represents a promising therapeutic strategy against CRC.

overall_design: RNA-seq profiling of wildtype HCT116 and their knockdown derivatives (shPRSS22)


title: Mapping the miRNA landscape of primitive macrophage extracellular vesicles highlights their pro-vasculogenic effects in engineered human cardiac tissue

city: Toronto

first: Milica

last: Radisic

state: Ontario

address: 160 College St

country: Canada

institute: University of Toronto

type: Non-coding RNA profiling by high throughput sequencing

summary: Resident cardiac macrophages, derived from primitive yolk sac precursors during embryogenesis, have increasingly been recognized for their distinct phenotype and functions in regulating homeostasis of the human heart. However, the profile of their extracellular vesicles (EVs) in cardiac signalling and regulation remains uncharted. Here, we employ differentiation of human pluripotent stem cell (hPSC)-derived primitive macrophages (Mac) for isolation of their secreted EVs and in-depth characterization of EV-miRNA cargo. Primitive macrophages secreted nanoscale EVs that expressed canonical EV markers, and miRNA sequencing highlighted a diverse and unique profile of miRNAs when compared to EVs sourced from other principal cardiac cell lineages and published data from monocyte-derived cells. In particular, we noted the abundance and enrichment of vascular-modulatory let-7 miRNAs and miR-126-3p. Functional screening of Mac-EVs in a 3D model of in vitro cardiac vasculogenesis confirmed enhanced early endothelial cell organization and branching. Establishing a reference for the human Mac-EV miRNome enables further hypothesis-driven mechanistic tests of Mac-EV miRNAs in mediating cardiac physiology and disease, opening the door to identification of therapeutic targets and modalities for cardiac repair.

overall_design: miRNA-seq profiling of extracellular vesicles (EVs) isolated via PEG-based precipitation from conditioned media of human embryonic stem cell (hESC)-derived primitive macrophages (Mac-EVs), human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM-EVs), primary human cardiac fibroblasts (CF-EVs), and primary human cardiac microvascular endothelial cells (HCMVEC-EVs).

first: Karl

middle: T

last: Wagner

first: Shira

last: Landau

first: Gregory

middle: M

last: Kent

first: David

middle: F

last: Bodenstein


title: The effect of WRI1 and DGAT1 overexpression on lipid metabolism in developing pennycress embryos

city: Upton

first: Jorg

last: Schwender

state: NY

address: 50 Bell Ave.

department: Biology

institute: Brookhaven National Laboratory

summary: Thlaspi arvense (field pennycress) is an emerging oilseed crop closely related to Arabidopsis thaliana. Prior research on oilseed species has demonstrated that seed specific upregulation of WRINKLED1 (WRI1), a key regulator of triacylglycerol (TAG) biosynthesis, can augment seed oil content. This phenomenon extends to the TAG biosynthetic enzyme diglyceride acyltransferase 1 (DGAT1). In this study, we show that stable co-expression of WRI1 and DGAT1 in pennycress during seed development significantly elevates oil content in mature seeds. Utilizing in vitro cultured developing embryos, we thoroughly characterized the alterations in seed composition resulting from WRI1/DGAT1 overexpression, employing a combination of transcriptomic analysis, targeted metabolomics, and metabolic flux analysis.

overall_design: RNAseq profiling of Thlaspi arvense (cultivar Spring32) developing embryos grown in liquid culure for 10 days. WT was compared to line WD3 overexpressing WRI1 and DGAT1.

first: Hai

last: Shi

first: Brice

last: Jarvis

first: Nathaphon

last: Yu King Hing

first: Liza

last: Gautam

first: Cathleen

last: Kuczynski

first: Alexander

last: Hilo

first: Hardy

last: Rolletschek

first: John

last: Sedbrook


title: Gene expression profile at single cell level of mice brain tissues

city: Xi'an

first: Zhe

last: Feng

address: No.620 West Chang'an Street

institute: Shaanxi Normal University

summary: We used single-cell RNA sequencing (scRNA-Seq) to analyze the cells from the NMOSD-like mice brain.

overall_design: Cells from the brain of NMOSD-like mice were analyzed using single-cell RNA sequencing (scRNA-seq).

first: Tingting

last: Cui

first: Zhen

last: Wang

first: Pei

last: Li

first: Jia

last: Chen

first: Luhang

last: Dai

first: Wenyu

last: Huang

first: Shaogang

first: Huanyi

last: Liu

first: Bing

last: Xiao

first: Ye

last: Gong

first: Ke

first: Xiaoli

last: Ding

first: Lei

last: Zhang

first: Xiaochang

last: Xue

first: Luting

last: Yang

first: Rui

first: Ying

last: Fu

first: Yaling

first: Yaping

last: Yan


title: Transfer RNA quantifications in HEK293T cells by tRNA sequencing

first: Masanori

last: Yoshinaga

overall_design: Transfer RNAs in HEK293T cells were quantified by tRNA-seq analysis.

first: Takayuki

last: Ohira

first: Masaki

last: Takegawa

first: Tsutomu

last: Suzuki

first: Osamu

last: Takeuchi


title: SP1 antagonizes H3K27me3 to shape chromatin landscapes for RNA polymerase II recruitment during gastrulation [RNA-seq]

city: chongqing,china

first: xipeng

last: shen

address: yixueyuan road NO.1

department: basic medicine

institute: CQMU

summary: Following implantation, the epiblast gives rise to three germ layers that form the cellular foundation of the entire body plan. However, how transcription is initiated and regulated at developmental genes during this critical period remains unclear. By analyzing RNA polymerase II (Pol II) occupancy in mouse post-implantation embryos, we found that Pol II occupies nearly half of bivalent promoters, in a temporally ordered, stepwise manner. H3K27me3 is deposited first at developmental genes, followed by the sequential acquisition of H3K4me3 and Pol II. Pol II recruitment is quantitatively modulated by the opposing activities of KMT2B-mediated H3K4me3 and EED-dependent H3K27me3: loss of H3K4me3 impairs Pol II binding, while depletion of H3K27me3 enhances its occupancy and leads to premature gene activation. Notably, the transcription factor SP1 promotes Pol II recruitment by antagonizing H3K27me3 deposition. These in vivo findings shed light on chromatin-based mechanisms that regulate transcriptional activation of developmental genes during early embryogenesis.

overall_design: To evaluate the gene expression level in different germ layers in E6.5 and E7.5 embryos, Smart-seq2 was perform in normal development embryos as well as in gene knockout E6.5 epiblast including Jarid2, Kmt2b, Eed and Sp1 with control.

first: Xipeng

last: Shen

first: Yuting

last: Wen

first: Yunlong

last: Xiang


title: Generation of Induced Pluripotent Stem Cells Derived Tolerogenic Dendritic Cells and Their Immunosuppressive Properties.

first: Aiko

last: Hirano

address: 465, Kajii-cho, Kawaramachi-Hirokoji, Kamigyo-ku

department: Inflammation and Immunology, Graduate School of Medical Science

institute: Kyoto Prefectural University of Medicine

summary:
Background:
Dendritic cells (DCs), the primary antigen-presenting cells, regulate immune priming. DCs with a semi-mature phenotype and tolerogenic properties, termed tolerogenic DCs (tolDCs), are promising candidates for cell therapy to induce immune tolerance. Induced pluripotent stem cells (iPSCs) can provide an unlimited source of DCs, and in mice, tolDCs can be generated from bone marrow cells by adding agents such as minocycline and dexamethasone during DC differentiation. On this basis, we aimed to establish a protocol for generating tolDCs from iPSCs.
Methods:
We modified an existing iPSC-to-DC induction protocol by adding minocycline and dexamethasone. Then, we assessed whether the resulting cells exhibited tolDC characteristics through comparison of iPSC-derived DCs (iPS-DCs) and iPSC-derived tolDCs (iPS-tolDCs).
Results:
We established a method for generating iPS-tolDCs from iPSCs. Flow cytometric analysis revealed reduced expression of co-stimulatory molecules and the maturation marker in iPS-tolDCs compared with iPS-DCs. In co-culture experiments, CD4⁺CD45RA⁺ T cells isolated from PBMCs of healthy donors or patients with autoimmune diseases showed reduced proliferation when cultured with iPS-tolDCs compared with iPS-DCs. Gene expression profiles of iPS-tolDCs differed from those of iPS-DCs.
Conclusion:
We developed a protocol for producing tolDCs from iPSCs and demonstrated their tolerogenic phenotype and immunosuppressive effects on T cells. These findings indicate that iPS-tolDCs are a promising candidate for cellular immunotherapy and warrant further investigation.

overall_design: Examination of 3 different cell types: iPSC-derived tolDCs, iPSC-derived DCs, and iPSC-derived monocytes

first: Kazuki

last: Fujioka

first: Takuya

last: Inoue


title: Adeno-associated virus (AAV)-TBX18 does not generate biological pacemaker activity, unlike AAV-Hcn2

city: Amsterdam

first: Vincent

middle: M.

last: Christoffels

state: N/A (NA)

address: Meibergdreef 15  K2-159

department: Medical Biology

country: Netherlands

institute: Amsterdam UMC

summary: Gene therapy-based biological pacemakers have been proposed as an alternative to their hardware-based counterparts. In this context, short-term ectopic expression of the T-box transcription factor 18 (TBX18) in the ventricle reportedly generated potent short-term pacemaker function in various animal models. Here, we investigated the effect of adeno-associated virus (AAV)-mediated long-term expression of TBX18, and compared the outcome to that of the pacemaker ion channel Hcn2. Our findings revealed that CMV-driven ectopic TBX18 expression in mouse hearts led to severe cardiac fibrosis. At lower, non-fibrogenic levels, TBX18 maintained its transcriptional function but failed to induce pacemaker phenotypes. TBX18-expressing cells showed suppressed expression of key working myocardial genes, but the pacemaker gene program was not induced. Electrophysiological studies showed abnormal automaticity in TBX18-expressing cells, combined with prolonged repolarization and various current changes. However, no hyperpolarization-activated funny current was detected. In a complete AV-block rat model, AAV-mediated Hcn2 expression induced robust ectopic pacemaker activity in the presence of isoproterenol, whereas TBX18 expression neither generated such activities, nor augmented Hcn2-mediated pacing. In conclusion, at functional non-fibrogenic levels, TBX18 is neither sufficient nor necessary to induce pacemaker activity. In contrast,  Hcn2 generates reliable pacing, making it  a more viable candidate for biological pacemaker development.

overall_design: RNA-seq profiling of neonatal rat ventricular myocytes transduced with AAV-MCS, AAV-Hcn2 and various AAV-TBX18 vectors.

first: Jianan

last: Wang

first: Karel

last: van Duijvenboden

first: Gerard

last: Boink


title: Effect of DHX29 depletion on gene expression in HEK293T cells

overall_design: Control and DHX29-deficient HEK293T cells were generated by transducing Cas9-expressing cells with vectors expressing control and DHX29-targeting gRNAs, and subjected to RNA-seq analysis.

first: Fabian

last: Hia


title: Effect of DHX29 depletion on gene expression in U937 cells

first: Masanori

last: Yoshinaga

overall_design: Control and DHX29-deficient U937 cells were generated by transducing Cas9-expressing cells with vectors expressing control and DHX29-targeting gRNAs, and subjected to RNA-seq analysis.

first: Ting

last: Cai


title: BRD8 is a therapeutic vulnerability for overcoming resistance to dual ER/HER2 blockade therapy in HR+/HER2+ breast cancer

city: Madison

first: Wei

last: Xu

state: WI

address: 7459 WIMR II

department: McArdle Laboratory for Cancer Research

institute: University of Wisconsin-Madison

summary: Hormone receptor (HR)-positive, HER2-positive breast cancers often develop resistance to endocrine and anti-HER2 therapies due to the heterogeneous expression of estrogen receptor (ER) and HER2 and the crosstalk of these growth-promoting pathways. However, how anti-HER2 agents activate ER and other growth-promoting pathways remains unknown. Single-cell RNA sequencing of BT474 breast cancer cells identified Bromodomain Containing Protein 8 (BRD8), an acetyl-lysine reader protein in the histone acetylase EP400 complex, as a pivotal mediator to activate ER in response to neratinib treatment.  Since EP400 enhances H2AZ deposition and acetylation on chromatin, we performed H2AZ and H2AZac ChIP-sequencing in the presence or absence of BRD8 and neratinib treatment. This will help us address how BRD8 regulates ER as well as other growth factors.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for histone variants H2AZ and H2AZac in BT474 parental or BRD8 knockout cells with or without neratinib treatment


title: Effect of DHX29 depletion on gene expression in K562 cells

overall_design: Control and DHX29-deficient K562 cells were generated by transducing Cas9-expressing cells with vectors expressing control and DHX29-targeting gRNAs, and subjected to RNA-seq analysis.


title: Single-cell RNA sequencing reveals deuterosomal cells during differentiation of hiPSCs into airway multiciliated cells

city: Kyoto city

first: Shimpei

last: Gotoh

state: Kyoto prefecture

address: 53 Kawahara-cho, Shogoin, Sakyo-ku

department: Department of clinical application

institute: Kyoto University CiRA

summary: We performed single-cell RNA sequencing (scRNA-seq) to investigate the differentiation of human induced pluripotent stem cells (hiPSCs) into airway multiciliated cells. The analysis revealed diverse epithelial cell types, including basal, secretory, goblet, and pulmonary neuroendocrine cells, as well as a distinct population of deuterosomal cells. This dataset provides a valuable resource for studying human multiciliogenesis and related diseases.

overall_design: Single-cell libraries were constructed using the Chromium platform (10X Genomics) and the Single Cell 3′ Reagent Kits v3.1 according to the manufacturer’s protocols. Sequencing data were processed using Cell Ranger (v8.0.1) for demultiplexing, alignment to the GRCh38 reference genome (refdata-gex-GRCh38-2020-A, including reporter sequences), and generation of gene count matrices.

first: Haruka

last: Yamaki

first: Koji

last: Tamai


title: In Vivo identification and Engineering of Plant Enhancers Using Agro-STARR-Seq and Deep Learning

first: Yongzhou

last: Bao

summary: In Vivo identification Nicotiana benthamiana Enhancers Using ATAC-Agro-STARR-Seq

overall_design: employs Nicotiana benthamiana leaves as a heterologous expression platform, utilizing Agrobacterium-mediated delivery of a custom-designed STARR-Seq T-DNA binary vector optimized specifically for plant systems

first: Xiwen

last: Zhang

first: Huiming

last: Li

first: Junyan

last: Qi

first: Lingna

first: Yuanyuan


title: Phenotypic Screening of human iPSC-Derived Neurons identifies Thienopyridone as a Neuritogenic Small Molecule

first: Haruhisa

institute: Kyoto University

summary: Human induced pluripotent stem cell (iPSC)-derived neurons provide a platform for modeling a wide range of brain disorders. Among disease-relevant cellular phenotypes, impaired neurite outgrowth has emerged as a robust and quantifiable indicator that reflects core aspects of neurodevelopmental and neurodegenerative disease pathophysiology. In this study, we performed a high-throughput phenotypic chemical screen of over 21,000 small molecules to identify compounds that enhance neurite outgrowth in iPSC-derived cortical neurons.By iterative validation using disease-specific and control iPSC-derived neuronal lines, we identified three bioactive compounds that were sharing a common indazole scaffold. Two hit compounds were further validated in a human neural organoid model, where they proliferated neural stem cells by reproducing the neurite-promoting effect. Transcriptomic profiling revealed activation of signaling pathways associated with neurotrophic stimulation. These findings identified  a novel scaffold for a neurogenic compound, suggesting the potential of this compound as a therapeutic strategy for brain disorders and for promoting neural regeneration.

overall_design: To elucidate the molecular mechanisms underlying the activity of the two  compounds, whice were identified by compound screening, RNA sequencing was performed on compound-treated neural organoids.

first: Keiko

last: Imamura

first: Keisuke

last: Shibata

first: Dang

last: Suong

first: Takeshi

last: Niki


title: The role of CNS1 in shaping the CD4+ T cell and Treg response to influenza

city: Chapel Hill

first: Jason

middle: Robert

last: Mock

state: NC

address: 125 Mason Farm Road

institute: University of North Carolina

summary: Regulatory T cells (Tregs) have critical roles in the recovery from acute lung injury (ALI). Recently, the field has gained critical insights into Tregs in ALI, and the diversity of gene expression and function of Tregs throughout ALI is beginning to be described. However, the origins of these Tregs has remained unknown. Here, we utilized a mouse model lacking the conserved noncoding sequence 1 (CNS1) of the Foxp3 promoter, which results in attenuated peripheral Treg induction, to understand the role(s) of peripheral Tregs in the initiation, pathogenesis, and recovery from acute lung injury. We found that CNS1-deficient mice exhibit more severe ALI and delayed resolution. We find that CNS1 may have roles in shaping the T cell response during influenza and that CNS1-deficient Tregs have several cell-intrinsic maladaptive phenotypes, overall giving us insight into the Treg plasticity that may aid in ALI recovery.

overall_design: To understand the cell-intrinsic effects of CNS1 loss, we generated mixed bone marrow chimeric mice to study the intrinsic differences between wild-type Tregs and Tregs lacking CNS1 in the Foxp3 promoter.  These mixed chimeric mice were then given PR8 induced influenza and two time points bead enrichment of regulatory T cell-enriched lymphocytes were obtained and sent for scMulti-omic analysis and compared to a control mixed chimeric mice not exposed to PR8.

first: Morgan

middle: J

last: McCullough

first: Hong

last: Dang

middle: R


first: Huy

last: Dinh

address: 1111 Highland Avenue

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

overall_design: Refer to individual Series


title: Global transcription changes driven by the second messenger cyclic di-GMP in Acinetobacter baumannii AB5075

city: Southampton

first: Rubén

last: de Dios

state: Hampshire

address: University Road

department: School of Biological Sciences

country: United Kingdom

institute: University of Southampton

summary:
In order to know the effect of the second messenger cyclic di-GMP (cdGMP) on the global transcription of A. baumannii AB5075, we performed a differential RNA sequencing (dRNA-seq) experiment comparing two AB5075 derivatives that allow IPTG-induced overexpression of the phosphodiesterase coding gene rocR (decreases cdGMP levels) or a constitutively active version of the diguanylate cyclase pleD (pleD*, increases cdGMP levels)
A. baumannii AB5075 derivatives carrying either rocR or pleD* inserted in a miniTn7 transposon  were grown in LB supplemented with IPTG 1 mM (3 biological replicates per strain) until reaching mid-exponential phase (approx. OD600 0.6). At this point, cells were harvested, treated with RNAlater for preservation of total RNA and stored at -80 C. After that, total RNA was extracted from each sample.
As a result,  154 genes appeared upregulated in high cdGMP conditions (overexpressing pleD*) whereas 188 were downregulated. In highlights, the gene functional groups showing the strongest transcriptional alterations were those related to biofilm formation (upregulated) and twitching motility (downregulated).

overall_design: Three independent replicates of A. baumannii AB5075 carrying either rocR or pleD* in an IPTG-inducible expression system were grown to mid-exponential phase in LB supplemented with IPTG 1 mM and compared between the two sample groups.

first: Ronan

last: McCarthy


title: Lysosome-Related Organelles Orchestrate Biogenic Crystal Formation in Pigment Cells

city: Rehovot

first: Tsviya

last: Olender

address: 234 Herzl St.

department: Molecular Genetics

country: Israel

institute: The Weizmann Institute

summary: Lysosome-related organelles (LROs) enable cell-type-specific functions by adapting endolysosomal pathways for specialized roles. Here, we show that iridosomes - optically active, guanine crystal-forming organelles in zebrafish iridophores - constitute a previously unrecognized class of LROs. Using transcriptomic profiling of purified iridophores, CRISPR-Cas9 gene perturbation, and cryogenic transmission electron microscopy, we identify a conserved molecular program governing iridosome biogenesis. Key LRO regulators, including rab32a, ap3M2, and hps5, are required for crystal formation, with gene disruptions leading to reduced organelle number and distorted crystal morphology. We also observe hallmark LRO features such as intraluminal vesicles and dynamic pH regulation during maturation. Cross-species transcriptomic analysis reveals that the LRO identity of iridosomes is conserved in vertebrates including fish and reptiles, suggesting ancient evolutionary roots. These findings expand the LRO paradigm to include organelles dedicated to crystalline assembly and establish iridophores as a model for investigating how cells build structurally specialized organelles through coordinated trafficking, acid-base regulation, and biomineralization.

overall_design: Transcriptomic profiling of zebrafish larvae iridophores versus control cell population at 5dpf using bulk-RNAi sequencing

first: Anna

last: Gorelick-Ashkenazi

first: Yuval

last: Barzilay

first: Tali

last: Lerer-Goldshtein

first: Zohar

last: Eyal

first: May

last: Glaser

first: Yonatan

last: Broder

first: Nadav

last: Mishol

first: Rachael

last: Deis

first: Merav

last: Kedmi

first: Dvir

last: Gur


title: Translatome analysis reveals altered spinal astrocyte metabolism in acute and chronic pain

city: Palo Alto

first: Vivianne

middle: L

last: Tawfik

address: 3174 Porter Drive

department: Anesthesiology, Perioperative and Pain Medicine

institute: Stanford University

summary: Chronic pain affects an estimated 20% of people globally, but the mechanisms that differentiate acute pain from chronic pain are not well understood. Astrocytes regulate neuronal signaling in physiological conditions and can drive neuroinflammation in pain states, likely contributing to pain progression. It is therefore important to determine how astrocyte cellular and molecular pathways change in response to pain-inducing peripheral injury, and to identify critical timepoints of astrocyte involvement in pain progression. Here, we utilize translating ribosome affinity purification technology in a mouse model of complex regional pain syndrome to uncover the molecular signature of spinal astrocytes in early (acute) and late (chronic) post-injury phases. We find that astrocytes exhibit a temporally distinct translatomic signature, with the most significant gene expression changes occurring early after injury. We further identify astrocyte lipid metabolism and oxidative phosphorylation as key dysregulated pathways after injury, and discover that orlistat, a clinically approved weight loss drug that reduces systemic lipid load, prevents the development of chronic pain in CRPS mice. Overall, this work highlights the importance of astrocyte lipid metabolism in pain pathophysiology and suggests that targeting this pathway may have therapeutic potential.

overall_design: Using Aldh1l1-CreER x Rpl22-HA mice, spinal cord astrocyte ribosomes were tagged with an HA protein. Tagged ribosomes were then immunoprecipitated using anti-HA antibody and magnetic coated beads. Immunoprecipitated samples were named "Trap" samples, and consisted of astrocytic mRNA only. Respective control samples were named "Input" and consisted of astrocytic mRNA and mRNA from all other cell types in the tissue. Both Trap and Input samples were colleced at 3-, 5-, and 7-weeks post-injury in a mouse model of Complex Regional Pain Syndrome or were collected from uninjured control animals. 3 replicates per group per sex were collected.

first: Janelle

last: Siliezar-Doyle

first: Richard

middle: K

last: Perez

first: Emma

last: Stauffenberg

first: Amy

last: Nippert


title: Inhibition of LXR Signaling in Human Foam Cells Impairs Macrophage-to-Endothelial Cell Crosstalk and Promotes Endothelial Cell Inflammation

city: Dijon

first: Damien

last: LELEU

address: 36 Rue Félix Vionnois

country: France

institute: CHU Dijon Bourgogne

summary: Backgrounds: During atherogenesis, macrophages turn into foam cells by engulfing lipids present within the atheroma plaques. The shift of foam cells towardspro- or anti-inflammatory phenotypes, critical step in disease progression, is still poorly understood. Liver X Receptors (LXRs) play a pivotal role in the macrophage response to lipid, promoting the expression of key genes of cholesterol efflux, mitigating intracellular cholesterol accumulation. LXRs also exert balanced actions on inflammation in human macrophages, displaying both pro- and anti-inflammatory effects. Aims and methods:  Our study explored the role of LXRs in the functional response of human macrophage to lipid-rich plaque environment. We used primary human macrophages treated with atheroma plaque extracts and assessed the impact of pharmacological LXR inhibition by GSK2033 on cholesterol homeostasis and inflammatory response. Ultimately, we evaluated macrophage and endothelial cell crosstalk by assessing the impact of macrophage-conditioned supernatants on human endothelial cell. Results: LXR inhibition by GSK2033 resulted in increased levels of cholesterol and oxysterols in human macrophages, alongside notable changes in the cholesterol ester profile. This was accompanied by heightened secretion of pro-inflammatory cytokines such as IL-6 and TNFα, despite a transcriptional repression of IL-1β. Conditioned media from GSK2033-treated macrophages more effectively activated ICAM-1 and CCL2 expression in endothelial cells. Conclusions/perspectives: Our findings illustrate the intricate relationship between LXR function, cholesterol metabolism, and inflammation in human macrophages. While LXR is required for the proper handling of plaque lipids by macrophages, the differential regulation of IL-1β versus IL-6/TNFα secretion by LXRs could be challenging for potential pharmacological interventions.

overall_design: ATACseq of atheroma plaque conditioned human monocyte derived macrophages (hMDM) treated with or without LXR antagonist (GSK22033). Atheroma plaque sample was obtained from one patient undergoing carotid endarterectomy. hMDM was obtained from buffy coats from one healthy anonymous blood donor.

first: Thomas

last: PILOT

first: Léa

last: MANGIN

first: Kevin

last: VAN DONGEN

first: Lil

last: PROUKHNITZKY

last: DENIMAL

first: Maxime

last: SAMSON

first: Aline

last: LAUBRIET

first: Eric

last: STEINMETZ

first: Mickael

last: RIALLAND

last: PIERRE

last: GROETZ

first: Jean-Paul

last: PAIS DE BARROS

last: GAUTIER

first: Charles

last: THOMAS

first: David

last: MASSON


title: Histone modifications and SP1 balance Pol II recruitment at bivalent promoters during gastrulation

first: xipeng

last: shen

summary: Progressive activation of bivalent genes is crucial for promoting cell differentiation and embryonic development. However, the mechanisms governing RNA polymerase II recruitment at bivalent promoters remain poorly understood. Here, we unveiled the dynamic landscape of Pol II in mouse post-implantation embryos. Our findings indicated that imprinting genes are selectively activated in embryonic and extra-embryonic lineages. Pseudotranscribed genes exhibit significant dynamism across embryonic germ layers, with transcriptional events preceding substantial mRNA production. The recruitment of Pol II to bivalent promoters is synergistically regulated by KMT2B/H3K4me3, EED/H3K27me3, and SP1 in a dose-dependent manner. Specifically, the absence of H3K4me3 and SP1 reduces Pol II recruitment at promoters, while the loss of H3K27me3 increases Pol II occupancy and derepresses developmental gene expression. Our in vivo study not only elucidated the mechanism of Pol II recruitment at bivalent promoters in mammals, but also provided a valuable framework for exploring gene regulation mechanisms in disease contexts.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: We firstly tested the sensitivity of antibodies to capture total RNA Pol II using low-input Hela cell  using STAR chromatin immunoprecipitation–sequencing (STAR ChIP-seq). To investigate the dynamics of RNA Pol II in mouse post-implantation embryos, we separated mouse E6.5 embryos into epiblast (Epi), visceral endoderm (VE), and extra-embryonic ectoderm (ExE), and E7.5 embryos into ectoderm (Ect), mesoderm (Mes), endoderm (End), and ExE and generated ChIP-seq data for Pol II and Ser2p. To investigate the factors involved in regulating Pol II occupancy at bivalent genes, we utilized CRISPR-Cas9 technology combined with electroporation of fertilized eggs to obtain gene knockout embryos including Jarid2, Kmt2b, Eed and Sp1. ChIP-seq of transcription factors Pol II, Ser2p and SP1 as well as histone modifications H3K4me3 and H3K27me3 were performed.

first: Xipeng

last: Shen

first: Yuting

last: Wen

first: Yunlong

last: Xiang


title: Promoter mutagenesis and a massively parallel reporter screen of the MAPT locus identifies cis-regulatory elements and genetic variation effects [Variation and Saturation Mutagenesis MPRA]

status: Public on Mar 18 2026

city: Huntsville

first: Jesse

middle: Nicholas

last: Cochran

state: AL

address: 601 Genome Way

institute: HudsonAlpha Institute for Biotechnology

summary: Tau neurofibrillary tangles are a hallmark of several neurodegenerative diseases called tauopathies, including frontotemporal dementia and Alzheimer's Disease. Ongoing clinical trials for tauopathies seek to reduce Tau in the brain through immunotherapy, antisense oligonucleotides, and siRNA. MAPT codes for Tau, therefore understanding how the MAPT gene is regulated and the effect of genetic variation at its regulatory elements is likely to have high relevance for tauopathies. We screened a ~3 Mb region including the MAPT locus using 2 different massively parallel reporter assay (MPRA) strategies in KOLF2.1J h-NGN2 neurons and HEK293FT cells, identifying previously unannotated cis-regulatory elements (CREs). Using CRISPR interference (CRISPRi) in mixed neuron cultures, we identified a new CRE for MAPT, as well as 2 CREs for another nearby gene of interest, KANSL1. Known genetic variation from the Alzheimer's Disease sequencing project was tested in a separate MPRA at the top CREs near the MAPT gene, identifying variants with altered regulatory effects including those at previously identified CREs for MAPT. Using a saturation mutagenesis screen of a 2,000 bp region encompassing the MAPT promoter, we assessed regulatory effects of each possible single nucleotide variant in this region. We identified several neuron-specific regulatory variant effects at this region, including a high confidence binding site for the transcription factors EGR2, ZBTB14 and TCLF5 at a region of high MPRA activity and genetic conservation.

overall_design: LentiMPRA in KOLF2.1h-NGN2 Neurons (transduced on day 14, collected on day 18) and HEK293FT cells testing known genetic variation at proximal CREs for MAPT as well as a saturation mutagenesis of the MAPT promoter

first: Rebecca

last: Hauser

first: Nicholas


title: Targeting branched-chain amino acids alleviates pulmonary fibrosis

city: wuhan

first: Jie

last: Yao

state: 湖北

address: wuhan hubei province

institute: wuhan university

summary: Purpose: To determine whether BCAAs regulate H3K36me3 modification on collagen genes via KDM4A. Methods:  We transfected two KDM4A siRNAs into MLF cells. After 24 h of transfection, the cells were treated with 20 ng/mL TGF-β1 for 48 h, followed by CUT&Tag assay for H3K36me3. Conclusions: BCAAs promote H3K36me3 modification on collagen genes via KDM4A.

overall_design: Primary mouse lung fibroblasts (MLFs) were isolated from lungs of 6–8-week-old mice using a combination of collagenase I digestion and differential adhesion and cultured in DMEM/F12 medium. CUT&Tag analyses were conducted on indicated MLFs.

first: Miao

last: Lei

last: He

first: Su

last: Fang

first: Wanli

last: Peng

first: Wenxiang

last: Hu


title: Global transcription changes driven by the second messenger cyclic di-GMP in Acinetobacter baumannii AB0057

first: Rubén

last: de Dios

summary: In order to know the effect of the second messenger cyclic di-GMP (cdGMP) on the global transcription of A. baumannii AB0057, we performed a differential RNA sequencing (dRNA-seq) experiment comparing two AB0057 derivatives that allow IPTG-induced overexpression of the phosphodiesterase coding gene rocR (decreases cdGMP levels) or a constitutively active version of the diguanylate cyclase pleD (pleD*, increases cdGMP levels). As a result,  98 genes appeared upregulated >2-fold in high cdGMP conditions (overexpressing pleD*) whereas 143 were downregulated. In highlights, the gene functional groups showing the strongest transcriptional alterations were those related to biofilm formation (upregulated) and twitching motility (downregulated).

overall_design: A. baumannii AB0057 derivatives carrying either rocR or pleD* inserted in a miniTn7 transposon  were grown in LB supplemented with IPTG 1 mM (3 biological replicates per strain) until reaching mid-exponential phase (approx. OD600 0.6). At this point, cells were harvested, treated with RNAlater for preservation of total RNA and stored at -80 C. After that, total RNA was extracted from each sample and sequenced, followed by a differential gene expression analysis.

first: Ronan

last: McCarthy


title: Signaling cascades shape functional subpopulations of cortical astrocytes in male wild-type mice and APP/PS1dE9 Alzheimer’s disease model

city: Evry

first: Eric

last: Bonnet

address: 2 rue Gaston Crémieux

institute: CNRGH

summary: Astrocytes are key partners for neurons and can impact diseases such as Alzheimer's disease (AD), as they exhibit multiple reactive changes. Recent single cell/nucleus genomics analyses evidence astrocyte subpopulations coexisting in normal and AD brains. However, the signaling cascades controlling them, their functional characteristics and roles in AD are still unknown. Here, thanks to astrocyte-specific reporters for STAT3 and NF-kB signaling pathways, two regulators of astrocyte reactivity, we report the presence of three astrocyte subpopulations defined by their signaling activity, in the prefrontal cortex of male APP/PS1dE9 mice. These subpopulations are not triggered by amyloid deposition and are also observed in wild-type mice. They show distinct morphologies, molecular signatures and functional profiles. While NF-kB+ astrocytes have larger territories and higher lysosomal activity, STAT3+ astrocytes display enhanced hemichannel activity. The specific inhibition of these subpopulations reduces amyloid plaque size and impacts anxiety, social preference and social memory in AD but not wild-type mice. Our results show how innate signaling shapes astrocyte subpopulations in the mouse cortex, with distinct functions in health and disease.

overall_design: Experiments were carried out on one-year-old male APP/PS1dE9 (APP) mice (Jankowsky et al., 2004) on the C57BL/6J background and their WT littermates. These mice express a chimeric mouse/human APP695 gene containing the Swedish K670M/N671L mutations (APPswe) and human PSEN1 with a deletion in exon 9 (PS1dE9). Mice were injected with two fluorescent lentivirus based reporters for the STAT3 and NF-KB pathways, which target astrocytes. Astrocytes from the mouse PFC were then sorted based on their expression of fluorescent reporters (GFP=STAT3; CFP=NF-kB), into three subpopulations (STAT3, NF-kB, double). The protocol described in (Ceyzeriat et al., 2018) was used to analyze the transcriptome of the three astrocyte subpopulations, except that the lower number of astrocytes sorted per sample was not compatible with RNA quality assessment, and full length double strand cDNA libraries were amplified with 18 LD-PCR cycles. RNAseq libraries were sequenced on a Novaseq Illumina 6000 platform (2x100 bp). Quality control of sequencing data was performed with FastQC (v0.11.9). Reads were mapped on the GRCm39 (mm39) mouse genome assembly with STAR (v2.7.9a) and quantified with RSEM (v1.3.3).

first: Yiannis

last: Poulot-Becq Giraudon

first: Océane

last: Guillemaud

first: Elisa

last: Degl'Innocenti

first: Vivien

last: Letenneur

first: Karouna

last: Bascarane

first: Tony

last: Barbay

first: Mie

last: Møller Clausen

first: Céline

last: Derbois

first: Martine

last: Guillermier

first: Ludmila

last: Juricek

first: Miriam

last: Riquelme-Perez

first: Tom

last: Lakomy

first: Lucile

last: Benhaim

first: Noëlle

last: Dufour

first: Pauline

last: Gipchtein

first: Fanny

last: Petit

first: Léa

last: Siron

first: Gwennaëlle

last: Aurégan

first: Nathalie

last: Dechamps

first: Marie-Claude

last: Gaillard

first: Alexis

last: Bemelmans

first: Rémi

last: Bos

first: Maria-Angeles

last: Carrillo-de Sauvage

first: Giampaolo

last: Milior

last: Rouach

first: Solène

last: Brohard

first: Kévin

last: Muret

first: Carole

last: Escartin


title: CITEgeist: Cellular Indexing of Transcriptomes and Epitopes for Guided Exploration of Intrinsic Spatial Trends

city: Pittsburgh

first: Alexander

middle: Chih-Chieh

last: Chang

state: PA

address: 5051 Centre Ave

institute: University of Pittsburgh

summary: Spatial transcriptomics provides insights into tissue architecture by linking gene expression with spatial localization. Current deconvolution methods rely heavily on single-cell RNA sequencing (scRNA-seq) references, which are costly and often unavailable, especially if the tissue under evaluation is limited as in a core biopsy specimen. We present a novel tool, CITEgeist, that deconvolutes spatial transcriptomics data using antibody capture from the same slide as the reference, directly leveraging cell surface protein measurements from the same tissue section. This approach circumvents the limitations of scRNA-seq as a reference, offering a cost-effective and biologically grounded alternative. Our method employs mathematical optimization to estimate cell type proportions and gene expression profiles, incorporating sparsity constraints for robustness and interpretability. Benchmarks against state-of-the-art deconvolution methods show improved accuracy in cell type resolution, particularly in dense tumor microenvironments, while maintaining computational efficiency. This antibody-based tool advances spatial transcriptomics by providing a scalable, accurate, and reference-independent solution for deconvolution in complex tissues. We validate this tool by using a combined approach of simulated data and clinical samples by applying CITEgeist to translational pre-treatment and post-treatment ER+ breast tumors from an ongoing clinical trial, emphasizing the applicability and robustness of CITEgeist.

overall_design: 12 breast cancer samples from 6 patients (pre-endocrine therapy biopsy samples and matched post-primary-endocrine therapy surgical samples) were used for analysis

middle: C

first: Brent

last: Schlegel

first: Neil

last: Carleton

first: Priscilla

last: McAuliffe

first: Steffi

last: Oesterreich

first: Russell

last: Schwartz

first: Adrian

middle: V

last: Lee


title: Effect of promoter compatibility and sequence positioning on high-throughput identification of cell type-specific enhancers using STARR-CRAAVT in HepG2 and HaCaT cells

city: Biberach

first: Martin

last: Oti

address: Birkendorfer Str. 65

country: Germany

institute: Boehringer Ingelheim

summary: Tuning transgene expression through novel combinations of enhancers and promoters holds great potential for adeno-associated virus (AAV)-based gene therapies. Here, we present STARR-CRAAVT, a STARR-Seq-based platform to screen for enhancers in the AAV context. Employing two in-vitro models, HepG2 and HaCaT cells, we aimed to implement STARR-CRAAVT for the identification of cell type-specific enhancers. We integrated epigenetic datasets into an in-silico library of putative HepG2 enhancers and captured corresponding fragments from sheared genomic DNA. The fragments were processed into AAV libraries and applied to HepG2 and HaCaT cells. Comparative STARR-CRAAVT analysis revealed a selective activity of the libraries confirming our HepG2-directed in-silico design and the specificity of single enhancer/promoter combinations could be validated using luciferase reporter assays. In addition, we scrutinized the impact of key experimental parameters on enhancer identification and found that enhancer activity was significantly influenced by the used promoter. Furthermore, switching the enhancer location in reporter assays revealed that the level of enhancer activity is highly dependent on the position in the AAV genome. Taken together, this study demonstrates that STARR-CRAAVT can be employed to identify cell type-specific enhancers for AAV vectors and highlights promoter preference and enhancer positioning as key considerations for STARR-CRAAVT campaigns.

overall_design: STARR-Seq (self-transcribing active regulatory region sequencing)-based identification of enhancers in combination with a minimal promoter (minP), an AFP promoter, or a PGK promoter in HepG2 and HaCaT cells.

first: Robert

last: Becker

first: Priyanka

last: Choudhury

first: Benjamin

last: Strobel

first: Stephanie

last: Ketterer

first: Simon

last: Rumpel

first: John

last: Park

first: Sebastian

last: Kreuz

first: Udo

last: Maier

first: Stefan

last: Michelfelder

first: Christian

last: Schön


title: Dendritic cell redundancy enables priming of anti-tumor CD4 T cells in pancreatic cancer

city: Boston

first: Chong

last: Zuo

address: 450 Brookline Ave

institute: Dana Farber Cancer Institute

summary: Pancreatic ductal adenocarcinoma (PDAC) is resistant to current immunotherapies and lacks effective CD8 T cell responses, which is potentially due to a paucity of cross-presenting dendritic cells (cDC1s). Here, we combine an innate immune adjuvant with anti-CTLA-4 and anti-PD-1 to achieve durable remissions and immunologic memory in multiple mouse models of poorly immunogenic PDAC. Tumor regression does not depend on CD8 T cells or tumor cell MHC class I expression but instead requires IFNγ-producing CD4 T cells (Th1s) that were primed by dendritic cells in lymph nodes. Combination immunotherapy induces an influx of activated cDC2s carrying tumor antigen into tumor-draining lymph nodes; cDC2s are required for orthotopic tumor clearance. Intratumoral CD4 T cells and cDC2s remain present in treatment-naïve and chemotherapy-exposed human PDAC. Even in chemotherapy-exposed patients’ blood, cDC2s outnumber cDC1s by ten-fold. Therefore, therapeutic targeting of the cDC2-CD4 T cell axis could be efficacious in pancreatic cancer.

overall_design:
Murine scRNAseq tissue preparation: Mice bearing ZsGreen-expressing 6694c2 tumors were treated as described above. 48 hours after treatment, tumors, spleens, and tumor-draining lymph nodes were harvested from mice. Spleens and lymph nodes were macerated. Tumors were macerated and digested in collagenase IV (Sigma #C5138) and soybean trypsin inhibitor (Life Technologies #17075029). Tumor chunks were removed via filtration with a 40 μM filter, and the resulting single cell suspension was washed with PBS. Part of the tumor, lymph node, and spleen sample was taken for flow cytometry analysis. The rest of the samples were hashed, sorted based on ZsGreen expression on a BD FACS Aria, and analyzed via single-cell sequencing analysis.
Bulk RNA sequencing: 6694c2 and B16F10 tumor cells were treated with IFNγ (100 ng/mL) (Peprotech #315-05), IFNβ (1000 U/mL) (BioLegend #581302), and STING agonist (225 ug/mL) in culture for 24 hrs. Cells were harvested and RNA isolated with a QIAGEN RNAeasy Plus Mini Kit (#74134). RNA was sent to Genewiz for paired-end RNA sequencing. In brief, FASTQ files were put through quality control (fastqc 0.11.9 & multiQC v1.5 for collating fastqc results), adaptor trimming (cutadapt v.1.14), genomic alignment (STAR v2.7.9a), and read count analyses (Rsubread) via the Harvard O2 cluster. Filtering for genes with less than 10 reads combined across samples was conducted, and analysis of differential gene expression was conducted with DESeq293. Plots were generated with ggplot2 in R.

last: Dougan


title: BRD8 is a therapeutic vulnerability for overcoming resistance to dual ER/HER2 blockade therapy in HR+/HER2+ breast cancer

first: Parth

last: Khatri

address: 1111 Highland Ave

department: Biostatistics and Medical Informatics

institute: University of Wisconsin - Madison

summary: Hormone receptor (HR)-positive, HER2-positive breast cancers often develop resistance to endocrine and anti-HER2 therapies due to the heterogeneous expression of estrogen receptor (ER) and HER2 and the crosstalk of these growth-promoting pathways. However, how anti-HER2 agents activate ER and other growth-promoting pathways remains unknown. Single-cell RNA sequencing of BT474 breast cancer cells identified Bromodomain Containing Protein 8 (BRD8), an acetyl-lysine reader protein in the histone acetylase EP400 complex, as a pivotal mediator to activate ER in response to neratinib treatment. BRD8 expression was rapidly induced by various anti-HER2 agents (neratinib, lapatinib, and trastuzumab), and its depletion disrupted the crosstalk between ER and HER2 signaling pathways and rendered HR+/HER2+ cells and tumor organoids more sensitive to anti-HER2 agents. BRD8, ER, and ER target genes are co-induced by neratinib in single-nucleus (sn) RNA sequencing of a patient-derived xenograft (PDX). Concomitantly, SnATAC-seq reveals that the activated genes share open chromatin regions enriched in transcription factors (TF) binding motifs of ER, forkhead box (FOX), and ETS family. Since EP400 enhances H2AZ deposition and acetylation on chromatin, we performed H2AZ and H2AZac ChIP-sequencing in the presence or absence of BRD8 and neratinib treatment. Upon neratinib treatment, BRD8 activates ER, FOX, and ETS target genes through modulating H2AZac deposition and chromatin decompaction. This finding coincides with RNA-sequencing, where BRD8 promotes cell growth in an ER-dependent and independent manner. In line with these findings, patients who responded poorly to the anti-HER2 therapies exhibited higher BRD8 gene signatures. Furthermore, BRD8 knockdown ablates the ER and HER2 signaling crosstalk and re-sensitizes neratinib-resistant HR+/HER2+ cells to neratinib. Together, this work not only explains why ER signaling is activated upon anti-HER2 therapies but also identifies BRD8 as a druggable vulnerability in HR+/HER2+ breast cancer.

overall_design: HCI032 PDX tumors treated with vehicle, fulvestrant, neratinib, or their combination were dissociated into single nuclei. Libraries were constructed using the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle Kit to perform multi-omics snRNA-seq and snATAC-seq.

first: Ang

last: Gao

first: Huy

middle: Q

last: Dinh

first: Wei

last: Xu


title: Deep sequencing data from PFS screen of GeCas12a2 CRISPR-Cas nuclease

city: Salt Lake City

first: Emily

middle: Rose

last: Wilson

state: UT

address: 2000 Circle of Hope

department: Oncological Sciences

institute: Huntsman Cancer Institute

summary: In this work we analyzed possible protospacer flanking sequences recognized by GeCas12a2 Type V CRISPR-Cas nuclease.

overall_design: GeCas12a2 was challenged with a library of targets containing randomized protospacer flanking sequences. Sequences which result in GeCas12a2 activation are consequently depleted from the library. Conditions include a library prepared with GeCas12a2 under non-targeting conditions (NT) and a library with GeCas12a2 under targeting conditions (T). Sequences were extracted from the Illumina read data and their corresponding depletion scores calculated.

first: Jared

last: Thompson

first: Oleg

last: Dmytrenko

first: Friso

last: Schut

first: Xiaoyang

last: Zhang

first: Chase

last: Beisel

first: Yang

last: Liu


title: Ubiquitylation by the GID/CTLH complex regulates the metabolic and innate immune response of macrophages to infection by Mycobacterium tuberculosis

city: Ithaca

first: Nelson

middle: Victor

last: Simwela

address: 930 Campus Rd

department: Microbiology and Immunology

institute: Cornell University

summary: The GID/CTLH E3 ligase complex is implicated in several biological processes, yet its full substrate repertoire remains poorly defined. We recently identified the complex as a broad modulator of macrophage responses to Mycobacterium tuberculosis (Mtb) infection.  Here, we use label-free proteomics and diGly capture analysis of Mtb-infected macrophages to define the GID/CTLH-dependent ubiquitylome. We identify thousands of dynamically altered ubiquitylation sites, with strong enrichment among proteins involved in cellular metabolism and innate immune signaling. Concurrent proteome analysis revealed extensive rewiring in GID/CTLH-deficient macrophages, with >90% of enriched pathways among increased proteins consisting of metabolic targets. Notably, inhibitory phosphatases (PTEN, INPP5D) also emerged as candidate substrates. Functional studies revealed proteasome-dependent stabilization of PTEN and INPP5D in GID/CTLH-deficient macrophages with each phosphatase individually exerting an influence on Mtb intracellular survival. Together, our study defines a GID/CTLH-dependent ubiquitylome in macrophages and identifies the complex as a central regulator of metabolism and antimicrobial immunity.

overall_design: Anlysis of the transcriptional profile of Mtb infected murine macrophages in either wild type state or INPP5D knockout conditions treated or untreated with the INPP5D inhibitor 3AC.

first: Luana

last: Johnston

first: Chris

last: Sassetti

first: David

middle: G

last: Russell


title: Pericytes are organ-specific regulators of tissue morphogenesis

city: Münster

first: Kai

last: Kruse

state: North Rhine-Westphalia

address: Roentgenstr. 20

department: Bioinformatics Core Facility

institute: Max Planck Institute for Molecular Biomedicine

summary: Blood vessels provide a versatile and adaptable transport system, but recent work has established that endothelial cells, which form the innermost lining of the vascular network, are also a source of molecular signals controlling the behavior of other cell types in the surrounding tissue. Pericytes are another essential component of the vessel wall, but comparably little is known about their signaling interactions with other cell populations during organ growth and patterning. Here, we have used tissue-specific and inducible mouse genetics, high-resolution imaging, single-cell RNA sequencing and cell culture experiments to address the function of three pericyte-derived growth factors in the postnatal development of two model organs, namely lung and brain. We found that Pdgfrb-CreERT2-controlled inactivation of the gene for hepatocyte growth factor (HGF) causes no overt alterations in the postnatal brain but impairs alveologenesis in the lung due to defective interaction with AT2 epithelial cells. Likewise, expression of brain-derived neurotrophic factor (BDNF) by pericytes is not required in the postnatal brain but controls lung development through interactions with the receptor tyrosine kinase TrkB in the pulmonary endothelium. Conversely, pericyte expression of the TGFβ family growth factor Nodal is not required for lung morphogenesis but regulates blood vessel growth and barrier function in the postnatal brain, which we attribute to signaling interactions with endothelial cells, astrocytes and microglia. Taken together, our findings establish that pericytes are a critical source of angiocrine signals that control morphogenetic processes in an organ-specific fashion.

overall_design: For the single cell sequencing of postnatal lung, two Hgf lox/lox  control mice and two Hgf iPCKO P21 old pups or two Bdnf lox/lox  control mice and two Bdnf iPCKO P21 old pups were analyzed, respectively. For the single cell sequencing of postnatal brain, the P12 cerebral cortices belonging to mice from the same genotype were pooled together

first: Seyed Javad

last: Rasouli

first: Rodrigo

last: Diéguez-Hurtado

first: Parisa

last: Ghanbari

first: Anusha

last: Aravamudhan

first: Mara-Elena

last: Pitulescu

first: Ralf

middle: H

last: Adams


title: CK2-mediated phosphorylation of SF3B3 enhances its deubiquitination and stability to promote aberrant alternative splicing and ESCC progression

city: Xiamen

first: Guosheng

last: Hu

state: Fujian

address: Xiang'an South Road, Xiang'an District

department: School of Pharmaceutical Sciences

institute: xiamen University

summary: Coactivator-associated arginine methyltransferase 1 (CARM1/PRMT4) is well-known for its essential physiological functions and its pivotal role in the development and progression of various cancers. Targeted inhibition of CARM1 activity has emerged as a promising strategy for cancer treatment. Herein, we report a peptide inhibitor of CARM1, Pi-CARM1. which demonstrates high selectivity for CARM1. The cell-permeable form of Pi-CARM1, Pi-CARM1-TAT, effectively suppresses breast cancer cell proliferation in vitro and significantly reduces tumor growth in mouse models of breast cancer.  Mechanistically, Pi-CARM1-TAT recapitulates the impact of CARM1 on the expression of oncogenic estrogen/ERα-target genes, as well as type I interferon (IFN) and IFN-induced genes (ISGs) in breast cancer cells. Notably, the combination of Pi-CARM1-TAT with endocrine therapy drugs or etoposide shows synergistic effects in inhibiting breast tumorigenesis. In conclusion, we present a novel peptide inhibitor of CARM1, which provides valuable insights and holds substantial therapeutic potential for the development of CARM1-targeted treatments in breast cancer.

overall_design: Nanopore sequencing performed in this study was designed to reveal the alternative splicing  that regulated by SF3B3 knockdown

first: Wen

first: Guo-sheng

first: Du-chuang

last: Wang

first: Jia-yuan

last: Li

first: Xiao-bing


title: Decoding a combinatorial RNA polymerase II CTD grammar that specifies protein-coding gene transcription and mRNA export [ChIP-seq]

city: Austin

first: Qian

last: Zhang

state: TX

address: 2515 Speedway

institute: The Unibersity of Texas at Austin

summary: The C-terminal domain (CTD) of RNA polymerase II orchestrates transcription via specific post-translational modifications (PTMs), notably phosphorylation. The combinatorial phosphorylation of CTD can potentially encode the precise recruitment of transcription machinery, but lacks robust evidence. Addressing the challenge of detecting combinatorial CTD phosphorylation in cells without specific antibodies, we developed a novel genome-wide approach exploiting antibody masking and selective interference removal to identify dual phosphorylation patterns. Using this method, we uncovered pT4pS5 phosphosites occurring exclusively during protein-coding gene transcription but excluded in non-coding genes. Using multiple sequencing methods, the role of T4 phosphorylation in elongation is highlighted. Particularly, pT4pS5 marks recruit the Transcription and Export complex to facilitate mRNA export. These findings provide robust evidence that CTD operates as a combinatorial, gene-specific coding system, encoding precise transcriptional instructions. Our approach offers a versatile framework for mapping all combinatorial PTMs on RNA polymerase II, advancing molecular insights into gene-specific transcriptional regulation.

overall_design: To investigate genomic location of pT4pS5 double phosphorylation, nuclear lysate from HEK293 cells was split into matched aliquots and treated in vitro with either active or catalytically inactive SCP1. Following phosphatase treatment, chromatin immunoprecipitation DNA-sequencing (ChIP-seq), was performed using a pThr4-specific antibody (clone 6D7, Active Motif). By comparing ChIP-seq signals between active and inactive phosphatase treatment conditions, genomic loci harboring pThr4 sites masked by adjacent pSer5 were identified. The experiment was performed with three biological replicates.

first: Kangsan

last: Kim

first: Yan


title: Selective ribosome profiling of the DHX29-bound ribosomes in HEK293T cells

city: Kyoto

first: Masanori

last: Yoshinaga

state: Kyoto

address: Yoshida-konoe-cho, Sakyo-ku

department: Graduate School of Medicine

institute: Kyoto University

summary: Synonymous codon usage controls global gene expression in both prokaryotic and eukaryotic species. Non-optimal codons are known to induce mRNA decay; however, the underlying molecular mechanism remains poorly understood in human cells. Through genome-wide CRISPR screening, we identified the RNA-binding protein DHX29 as a critical regulator of codon-dependent gene expression. Cryogenic electron microscopy and selective ribosome profiling demonstrated that DHX29 directly interacts with the A-site entrance of the translating 80S ribosome, the binding site for the eEF1A•GTP•aminoacyl-tRNA ternary complex, suggesting a role in monitoring aminoacyl-tRNA sampling. Proteomic analysis further revealed that DHX29 recruits the GIGYF2•4EHP complex to mediate global suppression of non-optimal mRNAs. These findings establish a mechanistic link between synonymous codon usage and the regulation of gene expression.

overall_design: HEK293T cells expressing FLAG-DHX29 were lysed, and ribosome footprints were isolated from the input and FLAG-DHX29-bound ribosomes. The ribosome footprints were then subjected to Thor-Ribo-Seq analysis.

first: Mari

last: Mito

first: Yuichi

last: Shichino

first: Shintaro

last: Iwasaki

first: Osamu

last: Takeuchi


title: Transcriptomic profiling of C6/36 cells treated with the METTL3 inhibitor STM2457

city: Hamilton

first: Lucas

last: Tirloni

state: MT

address: 903 S 4th Street

department: LB

institute: NIAID

summary: Mosquitoes transmit viruses such as Zika and chikungunya to millions of people worldwide. Before these viruses can spread to humans, they must successfully replicate inside mosquito cells. Understanding how mosquitoes naturally control viral infections can reveal new ways to limit disease transmission. This study explores how a common chemical modification of RNA, known as m6A, helps regulate virus infection in mosquito cells. By uncovering how RNA modifications affect viral infection in mosquitoes, this work provides new insights into the biology of disease transmission and identifies potential targets for future strategies to reduce the spread of mosquito-borne viruses.

overall_design: C6/36 cells were seeded in 12-well culture plates and grown to approximately 50% confluence prior to treatment. Cells were treated with the METTL3 inhibitor STM2457 at concentrations of 40 µM for 48 h, while control cells were treated with an equivalent volume of DMSO. Total RNA was isolated from cells using TRIzol reagent according to the manufacturer’s instructions and analyzed with an Agilent High Sensitivity RNA ScreenTape System in an TapeStation 4150 (Agilent). The library was constructed using the NEBNextUltraTM II (Directional) RNA with polyA selection library prep kit and sequencing was performed in an Illumina Novaseq 6000 DNA sequencer.


title: RNA-Seq quantitative analysis of the regulatory effects of circRBM6 and circMLLT1 on nasopharyngeal carcinoma cell transcriptomes

city: Guangzhou

first: Fei

state: Guangdong

address: 135 Xingang West Road, Haizhu District

institute: Sun Yat-sen University

summary: Nasopharyngeal carcinoma (NPC) is among the most commonly occurring head and neck malignancies characterized by high metastatic and invasive potential. Recent studies have suggested that dysregulation of circular RNAs (circRNAs) may contribute to tumorigenesis and serve as potential therapeutic targets for NPC. Therefore, further in-depth research is necessary to identify specific circRNAs that could potentially serve as biomarkers for NPC. Here, we identified that two circRNAs, circRBM6 and circMLLT1, were more highly expressed in NPC patients with distant metastasis than in patients without distant metastasis. To shed light on the mechanism underlying circRBM6 and circMLLT1i promote nasopharyngeal carcinoma metastasis, we performed high-throughput RNA-Seq analyses to reveal the differentially expressed genes in NPC cells transfected with control or circRBM6/circMLLT1-expressing vectors. We observed a significant upregulation in the expression of tumour metastasis-related genes in NPC cells with circRBM6 or circMLLT1 overexpression.

overall_design: RNA-seq profiling of 6-10B control cells,  5-8F control cells,  circRBM6-overexpressed 6-10B cells, circRBM6-overexpressed 5-8F cells,circMLLT1-overexpressed 6-10B cells, and the circMLLT1-overexpressed 5-8F cells.

first: Peiling

last: Chen

first: Qing


title: Effect of DHX29 depletion on mRNA stability in HEK293T cells

overall_design: Control and DHX29-deficient HEK293T cells were generated by transducing Cas9-expressing cells with vectors expressing control and DHX29-targeting gRNAs. The cells were treated with 4-thiouridine for 24 hours and then cultured in media containing uridine for 0, 3, and 6 hours. The samples were analyzed using SLAM-seq.

first: Yitong

last: Wu


title: Detection of transcriptomic changes in N9 and N33 GBM cells following treatment with small molecular inhibitors of MK-0752, WP1066 and retinoic acid

city: Beijing

first: Xiaolong

last: Fan

address: No.19, Xinjiekouwai St, Haidian District

institute: BeiJing Normal University

summary: We demonstrated that the cell state of suppressed neurogenesis and precocious astrogenesis in IDH-wildtype glioblastoma (GBM) could be manipulated as a potential therapeutic option. Clinically tested small molecule inhibitors MK-0752 (MK), WP1066 (WP), and retinoic acid (RA) were used to induce de-repression of neuronal differentiation in IDH-wildtype GBM. In order to explore the pathways affected by the two-drug (combination of MK and WP) or three-drug (combination of MK, WP, and RA) treatment, we performed mRNA sequencing on N9 and N33 cells at 48 hours post two-drug or three-drug treatent.

overall_design: Expression profiling of RNA-seq data form N9 and N33 GBM cells treated with small molecular inhibitors of MK-0752, WP1066 and retinoic acid for 48 hours.

first: Jing

last: Lyu


title: Effect of DHX29, GIGYF2, 4EHP, and DHX29/4EHP depletion on gene expression in K562 cells

overall_design: RNA-seq analysis was performed on Control, DHX29-, GIGYF2-, 4EHP-, and DHX29/4EHP-knockdown K562 cells generated by the CRISPRi system.


title: Transcriptomic profiling of CRISPR-Cas9 knockout of RAD51D, LIMCH1, and BRD9 in MC38 colorectal cancer cells reveals regulation of PD-L1 expression

city: Shanghai

first: Chen

last: Qian

address: No. 227 Chongqing South Road, Huangpu District, Shanghai, China

institute: Shanghai Jiao Tong University School of Medicine

summary: Programmed death-ligand 1 (PD-L1) expression in tumor cells plays a critical role in regulating anti-tumor immune responses and determining the efficacy of immune checkpoint blockade therapy. Previous studies have suggested that multiple genes are associated with PD-L1 expression in colorectal cancer, but the underlying regulatory mechanisms remain incompletely understood. In this study, we investigated the transcriptional consequences of knocking out three candidate genes, RAD51D, LIMCH1, and BRD9, which were previously identified to be associated with PD-L1 expression in colorectal cancer. Using the murine colorectal cancer cell line MC38, we generated CRISPR-Cas9–mediated knockout cell lines targeting each gene and performed RNA sequencing to characterize the global transcriptomic alterations. Our results revealed that RAD51D deficiency led to activation of the JAK1–STAT1 signaling pathway, accompanied by increased expression of JAK1, STAT1, and IRF1, which are known transcriptional regulators of PD-L1. Consistently, PD-L1 transcription was upregulated following RAD51D knockout. Similar transcriptomic analyses were performed for LIMCH1 and BRD9 knockout cells to explore their potential regulatory roles in PD-L1 expression and tumor immune signaling. This dataset provides a comprehensive transcriptomic resource for understanding how these genes regulate PD-L1 expression and immune-related signaling pathways in colorectal cancer cells, and may facilitate further studies on the molecular mechanisms underlying tumor immune evasion and responses to immune checkpoint blockade.

overall_design: Murine colorectal cancer MC38 cells were subjected to CRISPR-Cas9–mediated knockout targeting three genes: RAD51D, LIMCH1, and BRD9. For each gene, knockout (KO) cells were generated using specific sgRNAs and compared with wildtype (WT) MC38 cells. Total RNA was extracted from each cell line and subjected to high-throughput RNA sequencing to evaluate transcriptomic changes induced by gene knockout. The experimental groups included: MC38 non-targeting control (WT), MC38 RAD51D KO, MC38 LIMCH1 KO, MC38 BRD9 KO. Each condition was analyzed to identify differentially expressed genes and immune-related pathways, with a particular focus on the JAK1-STAT1-IRF1 signaling axis and PD-L1 transcriptional regulation.

first: Wei

last: Cai


title: MAGED2 mediates AKT pathway regulates the biological function of glioblastoma

city: Dalian

first: Jianxin

state: Liaoning

address: West Lushun South Road

institute: Dalian Medical University

summary: Glioma is the most common primary intracranial malignant tumor in the brain, and according to the fifth edition of the World Health Organization (WHO) classification, glioblastoma multiforme (GBM) is the most malignant grade IV glioma.The median survival period of GBM patients is only 15 months, and the 5-year survival rate is less than 5%. At present, the treatment of glioma is mainly to achieve maximal safe resectionby surgery by surgery, combined with radiotherapy and chemotherapy, to prolong the patient's survival as much as possible.However, due to the high heterogeneity and invasiveness of glioma, it is easy to relapse after surgery.In addition, the clincal efficacy is not ideal because of the blood-brain barrier and drug resistance. Therefore, it is urgent to find novel targets for glioma treatment. MAGE-D2 (MAGE Family Member D2) is a member of the melanoma-associated antigen type II subfamily. MAGE-D2 has been identified as being critical for transcriptional regulation, epigenetic alteration, cell development and differentiation,and is involved in the regulation of cell cycle. Furthermore, numerous studies have shown that MAGE-D2 is a tumor-specific antigen that plays an important role in tumor progression and metastasis, including hepatocellular carcinoma, gastric cancer, and lymphoma. However, the precise function and mechanism of MAGE-D2 in glioma is still unknown.

overall_design: Glioblastoma U87-MG cells with stable knockdown of MAGED2 and shCON as a negative control.  shCON and shMAGED2 cells were extracted for RNA sequencing

first: Songshu

last: Meng


title: DksA enhances hypervirulent Klebsiella pneumoniae intestinal colonization by  remodeling RNA polymerase to promote carbon source utilization

city: Please Select

first: 佳

last: 万

state: Please Select

address: 广东省广州市越秀区中山二路中山医学院科技楼301

institute: 中山医学院

summary: Intestinal colonization by Klebsiella pneumoniae is recognized as a pivotal prerequisite for its systemic dissemination and subsequent invasive infection; however, the metabolic basis and regulatory mechanisms underlying this process remain poorly understood. In this study, we employed transposon insertion sequencing (Tn-seq) to systematically identify metabolic genes associated with intestinal colonization in hypervirulent Klebsiella pneumoniae (hvKp). By integrating high-throughput screening with in vivo phenotypic validation, we identified the global transcriptional regulator DksA as a key factor that markedly enhances hvKp intestinal colonization. Mechanistically, DksA appears to promote colonization by enhancing carbon source metabolism. Combined transcriptomic analyses with homology modeling and molecular dynamics simulations further revealed that DksA induces cascade allosteric remodeling of key RNA polymerase structural domains, thereby enhancing hvKp utilization of common intestinal carbon sources and facilitating intestinal colonization. Collectively, our findings highlight the critical role of transcriptionally regulated metabolic plasticity in hvKp intestinal colonization and provide a theoretical framework and potential targets for developing anti-colonization strategies that exploit pathogen metabolic vulnerabilities.

overall_design: Transcriptional profiling of wild-type and ΔdksA mutant strains of Klebsiella pneumoniae cultured in SPF mouse cecal filtrate.

first: Jia

last: Wan

first: Yixuan

last: Wang


title: Comparative Analysis of Alkyne- and Desthiobiotinylated Photoaffinity Probes for Chemotranscriptomic Profiling

city: Nijmegen

first: Daphne

middle: Ai Lin

last: van den Homberg

address: Heyendaalseweg

department: Physical Organic Chemistry

institute: Radboud University

summary: Understanding small molecule-RNA interactions is a crucial part in drug development and fundamental biology. Chemotranscriptomic profiling is emerging as a powerful platform to interrogate interactions of small molecules with entire transcriptomes. This technique relies on photoaffinity probes that covalently capture small molecule RNA interactions. Most photoaffinity probes bear an alkyne handle that requires additional inefficient functionalization and purification steps after RNA capture. We sought to improve the workflow by directly desthiobiotinylating a photoaffinity probe, omitting these additional alkyne functionalization steps. Here, we compare the suitability of desthiobiotin and alkyne modified Ribocil-derived photoaffinity probes for chemotranscriptomic profiling. Our results demonstrate binding of both photoaffinity probes to their specific target, the FMN riboswitch, using in vitro transcription/translation and RT-qPCR. We also observed high unspecific interactions due to proposed weak and nonspecific binding of the desthiobiotin moiety to RNA analyzed by dot blots and RT-qPCR. Finally, transcriptome-wide sequencing confirmed the unselective interaction of desthiobiotin. These findings suggest that desthiobiotin is an inefficient enrichment handle for the design of photoaffinity probes, resulting in many off-target interactions.

overall_design: Chemotranscriptomic profiling of Ribocil-derived photoaffinity probes of E. coli RNA using next-generation sequencing.

first: Georgia

last: Poulladofonou

first: Aurelia

last: Dekens

first: Willem

middle: A

last: Velema


title: Rice leaf: Control vs. OsSGS3b mutant

city: Matsuyama

first: Masamichi

last: Nishiguchi

address: 3-5-7 Tarumi

institute: Ehime University

summary: Transcriptional profiling of rice leaf comparing control wild type rice with OsSGS3b mutant.

overall_design: Two-condition experiment, control vs. OsSGS3b mutant. Biological replicates: 2 control replicates, 2 SGS3b mutant  replicates.


title: Rice leaf: Control vs. OsRDR1 mutant

summary: Transcriptional profiling of rice leaf comparing control wild type rice with OsRDR1 mutant.

overall_design: Two-condition experiment, control vs. OsRDR1 mutant. Biological replicates: 2 control replicates, 2 OsRDR1 mutant replicates.


title: NK cells promote cardiac cell death and regulate myelopoiesis in myocardial infarction

city: Paris

first: Jean-Sébastien

last: Hulot

address: Rue Leblanc

institute: Paris Centre de Recherche Cardiovasculaire

summary: Ischemic heart failure remains a major clinical challenge, underscoring the need to better understand post-infarction immune mechanisms and identify new therapeutic targets. Both innate and adaptive immunity contribute to adverse cardiac remodeling following myocardial infarction (MI), yet the role of cytotoxic cells such as natural killer (NK) cells remains poorly defined. Here, we show that after acute MI in mice, NK cells are recruited to the ischemic myocardium in a CCR2-dependent manner and become activated. Activated NK cells locally release granzyme B, promoting cardiomyocyte apoptosis, adverse ventricular remodeling, and impaired cardiac function. Genetic deletion or pharmacological depletion of NK cells reduces cardiomyocyte death, attenuates inflammation, limits myocardial injury, and improves cardiac function. In contrast, NK cell activation using an anti-NKG2A monoclonal antibody exacerbates ischemic heart failure. We further demonstrate that NK cells regulate bone marrow myelopoiesis through local GM-CSF production. Finally, we identify a distinct NK cellular and transcriptomic signature in human ischemic heart tissue at early stages. Together, these findings reveal a detrimental role for NK cells following acute MI and highlight NK cells as potential therapeutic targets to limit adverse cardiac remodeling.

overall_design: We performed transcriptomic analysis of human heart biopsies using the nCounter Human Organ Transplant Panel from Nanostring (770 genes) to compare early MI (< 14 days post-MI) and late MI (> 3 months port-MI) against healthy heart tissue.

first: Orianne

last: Domenge

first: Marine

last: Livrozet

first: Alexandre

last: Loupy

first: Marc

last: Diedisheim

first: Hafid

last: Ait-Oufella


title: Extracellular vesicle protein and miRNA signatures as biomarkers for post-infectious ME/CFS patients

city: Berlin

first: Oliver

last: Knight

address: Föhrer Strasse 15

department: Institut für Medizinische Immunologie

institute: Charité – Universitätsmedizin Berlin

summary: Post-infectious Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a chronic disease with a yet unresolved pathophysiology and sparse diagnostic options. Extracellular vesicles (EVs) serve as carriers of disease-specific protein and microRNA (miRNA) signatures. We aim to identify novel miRNA markers as potential biomarker in isolated plasma EVs from female post-COVID-19 ME/CFS patients in comparison to healthy controls (HCs). EVs were isolated from plasma of post-infectious ME/CFS patients and HCs by size exclusion chromatography, and characterized for their number, size, morphology and surface marker expression. Small RNA sequencing identifed four small RNAs, which are significantly differential expressed between ME/CFS patients and healthy donors. Subsequently performed  qPCR studies revealed significant downregulation exclusively for the hsa-let-7b-5p miRNA in EVs from ME/CFS patients versus HCs. Notably, reduced hsa-let-7b-5p expression correlated with impaired physical functioning as well as increased fatigue, pain, and immune activation scores. Our data indicate, that hsa-let-7b-5p could serve as a promising candidate for  development as diagnostic and stratification marker of ME/CFS.

overall_design: Extracellular vesicles were isolated by size exclusion chromatography (SEC) from post-COVID-19 ME/CFS patients and healthy donors (n=3/group). Thereafter, RNA was isolated from ultracentrifugated  EV-containing SEC fractions. miRNAs were isolated via the RNA Clean & Concentrator-5’ Kit (Zymo Research).

first: Martina

last: Seifert

first: Johannes

last: Schäfers

first: Fiona

middle: F

last: Douglas

first: Carl

last: Schwarzburg

first: Diana

last: Boristowski

first: Anne

last: Birke

last: Klein

first: Franziska

last: Sotzny

first: Kerstin

last: Rubarth

first: Lara

last: Windzio

first: Christien

middle: M

last: Beez

first: Claudia

last: Kedor Peters

first: Kirsten

last: Wittke

first: Carmen

last: Scheibenbogen

first: Anna

last: Greco


title: Mitochondrial Abundant Heat Soluble (MAHS) Protein Expression Modulates Metabolic Dynamics in Human Adipose Derived Stem Cells

city: Riverside

first: Joshua

last: Morgan

state: CA

address: 900 University Ave

department: Bioengineering

institute: University of California, Riverside

summary: Effect of expressing MAHS from Ramazzottius varieornatus in human ASC52telo cells.

overall_design: 2nd generation lentiviral expression of RvMAHS tagged with AcGFP in ASC52telo cells compared to AcGFP expression alone

first: Jordan

last: Rolsma

first: Darch

last: William


title: Vagus nerve-mediated neuroimmune modulation accelerates remyelination and functional recovery in a preclinical model of multiple sclerosis

city: Valencia

first: Yaakov

middle: A.

last: Levine

address: 25101 Rye Canyon Loop

institute: SetPoint Medical Corporation

summary: Failure of remyelination contributes to persistent disability in multiple sclerosis (MS), yet no approved therapies directly promote central nervous system repair. Observations of enhanced oligodendrocyte maturation signatures during vagus nerve-mediated neuroimmune modulation (VNIM) in the experimental autoimmune encephalomyelitis model of MS prompted us to test whether VNIM could promote remyelination. Here we show that VNIM, delivered as a single 1-minute treatment using clinically relevant parameters, accelerates remyelination and functional recovery in lysolecithin-induced focal demyelination. VNIM reduced lesion volume, increased remyelinated axons with thicker myelin sheaths, enhanced debris clearance, and promoted blood-brain barrier repair. Single-cell transcriptomic analyses revealed that VNIM reshaped the lesion microenvironment by promoting microglial states associated with debris clearance and pro-repair signaling while advancing oligodendrocyte precursor maturation. Together, these findings identify VNIM as a therapeutic strategy that coordinates glial repair programs to accelerate remyelination, providing preclinical rationale for an ongoing clinical trial in relapsing-remitting MS (ClinicalTrials.gov: NCT06796504). This GEO submission includes single-cell RNA sequencing (scRNA-seq) data from lesion, non-lesion, and naïve spinal cord white matter tissue used in this study.

overall_design: Focal demyelination was induced in the ventral spinal cord white matter of adult mice by lysolecithin (LPC) injection at day 0 (D0). Vagus nerve-mediated neuroimmune modulation (VNIM) or sham treatment was administered at D4. At D8, neutral red-labeled lesion tissue and adjacent non-lesion white matter were separately isolated. In naïve animals, white matter from a comparable spinal cord level was collected. To achieve sufficient cell numbers, lesion and non-lesion tissues were pooled from two animals of the same sex per sample. Single-cell suspensions were processed for 3′ scRNA-seq using the 10x Genomics GEM-X platform. Experimental groups: Naïve, Sham lesion, VNIM lesion, Sham non-lesion, and VNIM non-lesion.

first: Linh

last: Le

first: Chandramohan

last: Natarajan

first: Nicole

last: Hamlin

first: Peder

middle: S

last: Olofsson

first: Kevin

middle: J

last: Tracey

first: Jeffrey

last: Huang

first: David

last: Chernoff


title: CRISPR-Cas12a2

city: Salt Lake City

first: Emily

last: Wilson

address: 2000 Circle of Hope

institute: Huntsman Cancer Institute

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

sample_organism:
Escherichia coli
Homo sapiens

platform_organism:
Escherichia coli
Homo sapiens

overall_design: Refer to individual Series


title: Single-cell RNA sequencing of chondrocytes from osteoarthritis (OA) patients and healthy controls

city: guangzhou

first: cuicui

last: wang

state: guangdong

address: 1 Pan Fu Road

department: rheumatology

institute: Guangzhou First People’s Hospital, The Second Affiliated Hospital of South China University of Technology

summary: Osteoarthritis (OA) is a common degenerative joint disease characterized by progressive cartilage degeneration and osteophyte formation. However, the mechanisms underlying OA progression remain poorly understood. Single-cell RNA sequencing (scRNA-seq) was performed on chondrocytes from three OA patients undergoing total knee arthroplasty and three control donors undergoing arthroscopic surgery for anterior cruciate ligament (ACL) injury to explore cellular heterogeneity and dysregulated pathways. These data provide a resource for investigating the molecular mechanisms underlying OA progression.

overall_design: Human cartilage tissues were obtained from three osteoarthritis (OA) patients undergoing total knee arthroplasty and three control donors undergoing arthroscopic surgery for anterior cruciate ligament (ACL) injury. Chondrocytes were isolated and subjected to single-cell RNA sequencing (scRNA-seq) to characterize cellular heterogeneity and identify dysregulated pathways associated with OA.

first: Cuicui

last: Wang

first: Peiyao

last: Tan


title: High altitude de-acclimatization leads to long-term immune-suppression via upregulating Nrf2 expression in Tregs [RNA-seq]

first: Zhijie

last: Bai

state: 北京Beijing

address: Taiping Road, Haidian District

institute: beijing institute of radiation

summary: Introduction: High-altitude exposure imposes hypoxic, low-pressure, and cold conditions that challenge immune homeostasis. The long-term effects of high-altitude de-acclimatization (HADA) remain poorly understood. Objectives: The purpose of this study is to investigate how HADA affects the immune populations, both quantitatively and functionally, and to dissect the underlying molecular mechanisms involved. Methods: Peripheral blood was collected from both human cohorts and mice models while other immune organs including spleen, thymus and bone marrow were obtained from mice models. Proportions of immune cell populations in peripheral blood and other immune organs were analyzed using flow cytometry. The immune-suppressive functions of Tregs were determined by in vitro co-culture with CD8+ T cells. Transcriptomic and chromatin-accessibility feature induced by HADA were obtained through RNA-seq and ATAC-seq. The functional validation of HADA target gene was performed using specific agonist during in vitro co-culture. Results: HADA perturbed the proportions of immune populations in multiple immune site. Both data from human and mice showed increased regulatory T cells (Tregs) and enhanced immune-suppressive function in the peripheral blood. As a consequence, these Tregs mediated long-term immune suppression, impairing the immunity against tumor cells. Multi-omic analyses predicted Nrf2 as the key mediator of molecular alterations in Tregs caused by HADA, which was further confirmed by the functional assay. Conclusion: High-altitude de-acclimatization mediates the expansion of peripheral Tregs and enhances long-lasting immunosuppressive function of Tregs, increasing the probability of serious health consequences. Nrf2 plays as a key regulator mediating the effects of HADA on Tregs. These findings provide novel insights into altitude-related immune regulation and implications for health management in populations transitioning between high and low altitudes.