title: Autophagy regulates Müller glial cell inflammatory activation

city: St. Louis

first: Philip

last: Ruzycki

state: MO

department: Ophthalmology and Visual Sciences

institute: Washington University in St. Louis

summary: Purpose. Immune privilege in the eye is a compilation of anti-inflammatory mechanisms that protect vision from the damaging sequelae of intense immune responses.  Although the breakdown of privilege can lead to ocular disease, little is known about how these mechanisms are regulated. Since retinal Müller glial cells and autophagy also have anti-inflammatory properties, we tested the idea that Müller cells utilize autophagy to support immune privilege.  Methods. The essential autophagy regulator Atg5 was deleted in retinal Muller cells using a tamoxifen inducible GlastCre ERT X Atg5f/f strain.  Intraocular inflammation was induced by intravitreal injection of LPS and monitored by H&E staining, immunofluorescent confocal microscopy, and flow cytometry.  scRNA-seq was performed on retinal Müller cells isolated from control and Atg5-deficient mice.  Additionally, markers of Müller cell gliosis and function were assessed by western blotting and cytokines were detected by Luminex cytokine/chemokine arrays. In cultured Müller cells siRNA knockdown techniques were used to examine the role of autophagy in regulation of LPS-induced inflammatory pathways.  Results. We observed increased and prolonged intraocular inflammation when Müller cells were autophagy (Atg5) deficient.  Müller cell gliosis (as measured by Gfap expression) was significantly increased and inflammation was sustained over 5 days post-LPS injection compared to control.  The retinae with Atg5-deficient Müller cells also contained increased inflammatory mediators and neuroprotective molecules.  Gene expression analysis revealed a heterogeneous response to LPS in Müller glial cell population that revealed 2 states of activation.  The normal retinae contain Müller cells in both a basal and an activated state, while autophagy deficient retinae (Atg5iΔMüller) contained only activated Müller cells.  Analysis of gliosis markers Gfap and Lcn2 confirmed this heterogeneity showing that in control eyes basal and activated (gliotic) Müller glia were observed; however, with autophagy deficiency nearly all Müller cells were gliotic.  Interestingly, activated cells were largely indistinguishable between autophagy sufficient and deficient Müller cells. In cultured Müller cells, siRNA knockdown of autophagy genes resulted in heightened mTOR activation, increased Gfap expression, and upregulated cytokine/chemokine production.  Conclusions.  Autophagy deficiency in Müller cells leads to enhanced intraocular inflammation though activation of the mTOR pathway. Our data suggests that autophagy defines a novel perspective on Müller glial heterogeneity based on their activation state.  Thus, autophagy restrains cellular activation and inflammation, supporting immune privilege by preventing excessive and potentially destructive immune responses.

overall_design: Single cell suspensions were generated from pooled isolated retinas for each group (n=6) with a Papain Dissociation kit using the protocol provided. The final cell pellet was resuspended in PBS (pH7.4) with 0.1% BSA and cell viability assessed using Trypan blue staining. Only samples with >85% viability were used. Single-cell RNA sequencing (scRNA-seq) was performed using the 10X Genomics platform and reagents (3’v3.1) according to the manufacturer’s instructions. Resulting libraries were sequenced on an Illumina NovaSeq 6000 at the McDonnell Genome Institute at Washington University School of Medicine. Raw fastq files were processed and mapped to the mouse genome (mm10) using CellRanger (v6.0.0). Further analysis was performed using Seurat (v5.2.1) in R. The full code for this analysis is available at https://github.com/p-ruzycki/Atg5KO_LPS. Briefly, raw count matrices from Atg5f/f and Atg5iΔMuller samples were imported and merged for comparative analysis. Quality control was performed by filtering out cells with mitochondrial content ≥25% and cells with fewer than 500 detected genes. Normalization and variance stabilization were conducted using the LogNormalize method in Seurat, and the top 2,000 most variable genes were identified. The dataset was then scaled, and dimensionality reduction was performed using Principal Component Analysis (PCA), retaining the first 11 principal components (PCs) based on an elbow plot. Clustering was performed at multiple resolutions, and a resolution of 1.0 was selected for downstream analysis. Cell types were identified based on FindMarkers results and known marker gene expression, and visualization was conducted using Uniform Manifold Approximation and Projection (UMAP) in Seurat. A subset of non-neuronal cell populations (including Müller glia, myeloid cells, and vascular endothelial cells) was re-clustered and filtered for residual contaminating retinal neurons using Seurat preprocessing steps and reannotated based on FindMarkers results and known marker genes. Further sub setting was performed to isolate Müller glia cells, and these were re-clustered to examine subpopulation structures. Two differential expression analyses (DEA) were performed using Seurat: (1) comparison between Atg5f/f and Atg5iΔMuller in Müller glia (Cluster 1) using the FindMarkers function, where genes with adjusted p-value (p_adj) < 0.05 and log2 fold change (log2FC) ≥ ±1.0 were considered significantly differentially expressed, and (2) comparison between Müller glia subpopulations, where Cluster 2 was compared to Cluster 1 using the same statistical criteria. Volcano plots were generated using ggplot2 (v3.5.1) and ggrepel (v0.9.6) to visualize differentially expressed genes, highlighting selected genes with distinct colors and labeling them using geom_text_repel, with threshold lines indicating statistical significance. Pathway enrichment analysis was conducted using Gene Set Enrichment Analysis (GSEA, v4.3.3), where pathways were considered significantly enriched if they had a normalized enrichment score (NES) ≥2 (upregulated) or ≤-2 (downregulated) with an FDR q-value ≤0.05. Enrichment results were visualized using dot plots, and gene sets were obtained from MSigDB (Molecular Signatures Database).

first: Doggett

last: Teresa

first: Zhou

last: Zhenqing

first: Rebba

last: Sohini

first: Unsinger

last: Jacqueline

first: Ruzycki

last: Philip

first: Ferguson

last: Thomas


title: Androgen deprivation-mediated activation of AKT is enhanced in prostate cancer with TMPRSS2:ERG fusion [RNA-Seq]

status: Public on Sep 10 2025

city: Boston

first: Steven

last: Balk

state: MA

address: 3 Blackfan circle

country: USA

institute: BIDMC

type: Expression profiling by high throughput sequencing

summary: The TMPRSS2:ERG gene fusion (T:E fusion) in prostate adenocarcinoma (PCa) puts ERG under the androgen receptor (AR) regulated expression of TMPRSS2. The T:E fusion is frequently associated with PTEN loss, and is highly correlated with decreased expression of INPP4B, both of which may compensate for ERG-mediated suppression of PI3K/AKT signaling. We confirmed in PCa cells and a mouse PCa model that ERG suppresses AKT activation, and that one potential mechanism is through downregulation of IRS2. In contrast, ERG knockdown did not increase INPP4B, suggesting its decreased expression is indirect and reflects selective pressure to suppress INPP4B function. Notably, INPP4B expression is similarly decreased in PTEN-intact and PTEN-deficient T:E fusion tumors, suggesting selection for a function distinct from regulation of PI3K activity. As ERG expression in T:E fusion tumors is AR regulated, we further assessed the extent to which AR inhibition increased AKT activity in T:E fusion tumors. T:E fusion positive versus negative PDXs had greater increases in AKT activity after castration. Moreover, in a neoadjuvant trial of AR inhibition prior to radical prostatectomy we similarly found greater increases in AKT activation in the T:E fusion tumors. Together these findings indicate that AKT activation may mitigate the efficacy of AR targeted therapy in T:E fusion PCa, and that these patients may most benefit from combination therapy targeting AR and AKT.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: A model with doxycycline (DOX) regulated expression of ERG in mouse prostate, driven by a tetracycline operon regulated ERG (pTET-ERG) combined with a probasin promoter regulated reverse tetracycline transactivator (Pb-rTta), was generated to assess the functions of ERG in driving prostate adenocarcinoma (PRAD) in vivo. When crossed onto a Pten-/+ background and treated with doxycycline for ~1 year, these mice developed PRAD.  To identify ERG-regulated genes that may be contributing to PRAD development in this model, we carried out time course experiments with both short-term ERG induction and long term induction plus short term DOX withdrawal. For the short-term induction, previously untreated mice were treated with DOX or vehicle for 3 or 6 days. Ventral prostate epithelium was then laser capture microdissected and transcriptome profiles were obtained using Affymetrix Mouse Gene 1.0 ST arrays. Another cohort of mice with DOX regulated ERG on the Pten-/+ background were treated with DOX for ~1 year to generate PRAD. DOX was then discontinued in one set of mice and prostates were harvested after 1-2 weeks and analyzed by RNA-seq using the Illumina TruSeq stranded mRNA kit. To identify ERG regulated genes in VCaP cells (expressing the TMPRSS2:ERG fusion), cells were treated with ERG or nontargeted siRNA and assessed by RNA-seq.

first: Sen

last: Chen

first: Carla

last: Calagua

first: Seiji

last: Arai

first: Fang

last: Xie

first: Olga

last: Voznesensky

first: Larysa

last: Poluben

first: Eva

last: Corey

first: Changmeng

last: Cai

first: Yuzhuo

last: Wang

first: David

middle: J

last: Einstein

first: Mary-Ellen

last: Taplin

first: Huihui

last: Ye

first: Adam

middle: G

last: Sowalsky

first: Joshua

middle: W

last: Russo

first: Xin

last: Yua

middle: P


title: Single-cell RNA-sequencing analysis reveals the peripheral immune characteristics of patients with primary membranous nephropathy

city: Nanjing

first: fang

last: lu

address: 300 Guangzhou Road

department: Department of Nephrology

country: China

institute: the First Affiliated Hospital of Nanjing Medical University

summary: Primary Membranous Nephropathy (PMN) is characterized by dysregulated immune responses, with B and T cells playing critical roles in disease pathogenesis. While significant strides have been made in identifying specific autoantigens and leveraging monoclonal antibodies, the precise immunopathogenic mechanisms underlying immune cell involvement in PMN remain elusive. To comprehensively characterize the cellular, molecular, and immunological landscape, we employed single-cell RNA sequencing (scRNA-seq) on peripheral blood mononuclear cell samples (PBMC) obtained from 6 patients with PMN and 3 healthy controls (NC).We examined the heterogeneity and functional diversity of B cells in PMN. These results offer new insight into pathogenesis and identify new therapeutic targets for PMN.

overall_design: Single-cell RNA sequencing (scRNA-seq) was applied to PBMC from 6 MN and 3 control subjects to define the transcriptomic landscape at the single-cell resolution.

last: Lu

first: Suyan

last: Duan

first: Si

first: Ying

last: Pan

first: Changying

last: Xing

first: Huijuan

last: Mao

first: Yanggang

last: Yuan


title: Loss of PTDSS1 in tumor cells improves immunogenicity and response to anti-PD-1 therapy [RNA-seq]

city: Houston

first: Jielin

last: Liu

state: TX

address: 7455 Fannin St

department: Genitourinary Medical Oncology

institute: MD Anderson Cancer Center

summary: PTDSS1 (Phosphatidylserine synthase 1) encodes an enzyme that facilitates production of phosphatidylserine (PS), which mediates a global immunosuppressive signal. Here, based on in vivo CRISPR screen, we identified PTDSS1 as a target to improve anti-PD-1 therapy. Depletion of PTDSS1 in tumor cells increased expression of IFNγ-regulated genes, including B2m, Cxcl9, Cxcl10, and Stat1, even in the absence of IFNγ stimulation in vitro. Loss of PTDSS1 in tumor cells also led to increased expression of MHC-I, which was associated with enhanced cytotoxicity of CD8+ T cells and increased frequency of an iNOS+ myeloid subset. A gene signature derived from the iNOS+ myeloid cell subset correlated with clinical benefit in patients treated with anti-PD-1 therapy. Moreover, genetic and pharmacological inhibition of PTDSS1 in different tumor models improved anti-PD-1 therapy. Together, our results provide insights on a new therapeutic strategy for overcoming immunosuppression by inhibiting PTDSS1 and provide rationale for development of a combination immunotherapy strategy comprised of PTDSS1 inhibition plus PD-1 blockade.

overall_design: RNA-seq to compare the transcriptomes of Ptdss1 WT and KD cells cultured in vitro.


title: Effect of inhibition of fatty acid beta-oxidation on gene expression of double-negative T cells

city: Beijing

first: Zeyu

address: Gongti South Road No.8

institute: Capital medical university

summary: Double negative T cells are a subset of T cells with immunosuppressive functions. Our research found that cellular energy metabolism, especially fatty acid beta oxidation, is closely related to the function of DNT cells. Therefore, we treated DNT cells with Etomoxir, an inhibitor of fatty acid beta oxidation, and performed RNA sequencing. Taken together, our data provides genetic evidence for the regulation of DNT cell survival and function by fatty acid beta oxidation, and allows for further exploration of its inner molecular mechanisms.

overall_design: To investigate the differential gene expression of DNT cells after inhibiting fatty acid beta oxidation, we treated primary DNT cells obtained from the spleen and lymph nodes of C57 mice with/without treatment with the fatty acid oxidation inhibitor etomoxir.

first: Guangyong

last: Sun

first: Dong

last: Zhang


title: AAV9-mediated TNPO3 overexpression in the heart rescues RBM20 cardiomyopathy in mice

city: Heidelberg

first: Julia

last: Kornienko

state: Baden-Württemberg

address: Meyerhofstraße 1

country: Germany

institute: EMBL

summary: The RBM20 gene encodes for an alternative splicing regulator which, when mutated, can cause an aggressive form of DCM. The majority of RBM20 mutations identified in patients are found in the RS-domain, and lead to cytoplasmic mislocalization of the protein. In our previous work (Kornienko et al., Nat Commun, 2023), we identified TNPO3 as a direct nuclear importer of RBM20, and showed that restoring nuclear localization of mislocalizing variants rescues splicing dysfunction in iPSC-CMs. Here, we used our two recently published mouse models with patient-relevant RBM20 variants (Grosch et al., Nat Commun, 2023) to test whether nuclear relocalization could restore the alternative splicing and cardiac dysfunction in vivo. We used AAV9 vectors to overexpress TNPO3 in murine hearts and thereby facilitate nuclear import of RBM20 variants. We show that TNPO3 overexpression could rescue ejection fraction, cardiac volume, and alternative splicing, without having any detectable adverse effects on wild type animals. These results provide the first in vivo evidence that restoring RBM20 localization can significantly improve cardiac function and correct for splicing errors.

overall_design: Mice (males and females, Bl6J) with or without homozygous RBM20 mutation (P635L) were injected with 1e12 v.g. of AAV9 delivering either Tnpo3 cDNA or Ctr contrsuct (pCAG-iCre-T2A-eGFP) in the tail vein at 4 weeks of age. 12 weeks later, when mice were 16 weeks old, the hearts were harvested, and RNA was extracted from LVs for RNA sequencing. N = 4 mire per experimental group were sequenced.

first: Lars

last: Steinmetz


title: Decoding Trp53 regulatory program with knockout cell transcriptomic landscape analyses [scRNA-seq]

city: Hanzhou

first: Guoji

last: Guo

address: Yuhangtang street 866

department: Zhejiang University School of Medicine

institute: Center for Stem Cell and Regenerative Medicine

summary: For decades, researchers have devoted great efforts to study the tumor suppressor gene Trp53, however, our understanding of Trp53 regulation has never reached either organism-scale or single-cell resolution. We constructed single-cell transcriptomic landscape covering 18 organs of Trp53-/- mice models to explore genetic network transformations in the absence of Trp53.

overall_design: Eighteen tissues and organs of Trp53-/- mice at different ages were isolated and dissociated into single-cell suspensions, followed by single-cell cDNA library construction, high-throughput sequencing, and bioinformatics analysis.

first: Xiaoping

last: Han

first: Xinru

last: Wang

first: Yuqing

last: Mei

first: Xueyi

first: Jingjing

first: Peijing

first: Hanyu

last: Wu


title: Decoding Trp53 regulatory program with knockout cell epigenomic landscape analyses [scATAC-seq]

type: Genome binding/occupancy profiling by high throughput sequencing

summary: For decades, researchers have devoted great efforts to study the tumor suppressor gene Trp53, however, our understanding of Trp53 regulation has never reached either organism-scale or single-cell resolution. We constructed single-cell epigenomic landscape covering 17 organs of Trp53-/- mice to explore genetic network transformations in the absence of Trp53.

overall_design: Nineteen tissues and organs of Trp53-/- mice at different ages were isolated and dissociated into single-nucleus suspensions, followed by single-cell DNA library construction, high-throughput sequencing, and bioinformatics analysis.


title: Identification of p53 target genes in various tissues and organs using Cut&tag

summary: For decades, researchers have devoted great efforts to study the tumor suppressor gene Trp53. However, tissue-specific Trp53 regulation still remains elusive.Here, we used Cut&tag to analyze the p53 target gene groups in 18 tissues and organs of adult mice to decipher the tissue heterogeneity of Trp53 regulation.

overall_design: Eighteen tissues and organs of Trp53-/- mice at different ages were isolated, followed by cut&tag DNA library construction, high-throughput sequencing, and bioinformatics analysis.


title: Intratumoral bacteria are immunosuppressive and promote immunotherapy resistance in head and neck squamous cell carcinoma

status: Public on Sep 09 2025

city: Cleveland

first: Daniel

last: McGrail

state: Ohio

address: 9500 Euclid Avenue

department: Center for Immunotherapy and Precision Immuno-Oncology

institute: Cleveland Clinic

summary: Despite the promise of immune checkpoint blockade (ICB) in head and neck squamous cell carcinoma (HNSCC), mediators of response are poorly understood. To address this, we analyzed oropharyngeal HNSCCs treated with neoadjuvant durvalumab (anti-PD-L1) alone or in combination with tremelimumab (anti-CTLA-4) from the CIAO clinical trial (NCT03144778). We found that only the total abundance of intratumoral bacteria predicted ICB response, which was validated in multiple independent patient cohorts. High intratumoral bacteria abundance was associated with an immunosuppressive tumor microenvironment, characterized by an accumulation of neutrophils coupled with depletion of T cells and other adaptive immune cells. Experimental elevation or reduction of intratumoral bacteria abundance in orthotopic models of HNSCC in female mice recapitulated immunological associations observed in patient tumors. Increasing intratumoral bacteria abundance was sufficient to induce resistance to anti-PD-L1 immune checkpoint blockade, irrespective of bacterial species tested. Together, these findings demonstrate that high intratumoral bacteria abundance is a key suppressor of anti-tumor immunity and promotes immunotherapy resistance.

overall_design: For the transcriptome analysis, cells were either mock infected or infected with Fusobacterium or Prevotella at an MOI of 200 for four hours prior to harvesting RNA using a Qiagen RNeasy Mini Kit for RNA sequencing.


title: Black Seed Derivative Thymoquinone (TQ) Suppresses Pancreatic Ductal Adenocarcinoma Associated Oncogenic circRNAs

city: Detroit

first: Yang

last: Shi

state: MI

address: 4100 John R St MM03BI

department: Karmanos Cancer Institute

institute: Wayne State University Schhol of Medicine

type: Non-coding RNA profiling by high throughput sequencing

summary: To reveal the molecular mechanisms underlying TQ tratment on cell proliferation, we performed circRNA-seq analysis on human PDAC cell cultures (MiaPaCa2 and Panc1 cells).

overall_design: Circular RNA expression profiles between TQ treated and untreated (control) PDAC-cells were compared by circRNA-seq.

first: Irfana

last: Muqbil

first: Ramzi

middle: M

last: Mohammad

first: Md. Hafiz

last: Uddin

first: Bin

last: Bao

first: Yin

last: Wan


title: Activation of CISD2 as a Protective Strategy against Doxorubicin-Induced Cardiotoxicity

city: Taipei City

first: Ting-Fen

last: Tsai

address: No. 155, Sec. 2, Linong St. Beitou Dist.,

department: Department of Life Sciences and Institute of Genome Sciences

country: Taiwan

summary: This study investigated transcriptomic changes in mouse cardiac tissues following doxorubicin and hesperitin treatments. Cardiac tissues were collected at 16 and 72 hours post-treatment, and RNA sequencing was performed to identify differentially expressed genes and pathways involved in drug response and cardioprotection.

overall_design: Mouse cardiac tissues were collected at 16 and 72 hours following doxorubicin or hesperitin treatment. Total RNA was isolated from cardiac muscle using TRI Reagent and phenol/chloroform extraction, and RNA integrity was assessed with an Agilent Bioanalyzer (RIN > 8). RNA sequencing was conducted at the Genome Research Center, National Yang Ming Chiao Tung University, generating at least 20 million single-end reads per sample on the Illumina platform. The study design enables transcriptomic comparisons across treatment groups and time points to identify differentially expressed genes and pathways associated with doxorubicin and hesperitin responses.

first: Yi-Ju

last: Chou

first: Chi-Hsiao

last: Yeh

first: Chien-Yi

last: Tung

first: Shu-Ling

last: Fu


title: Study of the transcriptomic changes induced by Aurora A inhibition in the colorectal cancer cell line C10

city: Madrid

first: Anxo

last: Rio-Vilariño

address: Avenida Reyes Católicos, 2

department: Medical Oncology

country: Spain

institute: Instituto de Investigaciones Sanitarias Fundación Jiménez Díaz (IIS-UAM)

summary: Resistance to anti-EGFR therapies poses a significant challenge in the management of metastatic colorectal cancer (mCRC) patients. Our previous research has illustrated that AURKA phosphorylates YAP1, leading to increased stability and the induction of stemness, consequently promoting resistance to cetuximab. The AURKA inhibitor alisertib has demonstrated its ability to reinstate cetuximab efficacy in both cell lines and tumors exhibiting YAP1 activation. Thus, we aimed to investigate the transcriptomic alterations provoked by AURKA inhibition in resistant cell lines to elucidate the underlying changes driving these phenotypic shifts. .

overall_design: The C10 CRC cell line was subjected to treatment with 300nM alisertib for 24 hours. RNA extraction was performed using the NucleoSpin RNA mini kit (Macherey Nagel), followed by the generation of sequencing libraries on the Illumina NovaSeq 6000 platform (Novogene)

first: Arancha

last: Cebrián

first: Jesús

last: García-Foncillas


title: SPANXA2-OT1 promotes macrophage chemotaxis

first: Masanori

last: Aikawa

state: Massachusetts

address: 77 Avenue Louis Pasteur, NRB741

institute: Brigham and Women's Hospital

summary: Coronary artery disease (CAD) remains the leading global cause of death, with macrophages playing a central role in driving inflammation through cytokines, chemokines, and other mediators. Using gene expression meta-analysis and weighted gene co-expression network analysis (WGCNA) of human CAD datasets, we identified 26 lncRNA–mRNA modules and prioritized SPANXA2-OT1 as a key inflammation regulator. Conservation analysis revealed SPANXA2-OT1 to be primate-specific, necessitating human macrophage models derived from PBMCs. IL-1β stimulation induced cytoplasmic SPANXA2-OT1, and antisense oligonucleotide-mediated silencing reduced chemotaxis signatures, validated by RNA-seq and proteomics. Mechanistically, SPANXA2-OT1 directly bound miR-338, as shown by luciferase assays, thereby regulating IL-8 and related chemokines critical for monocyte recruitment. CRISPR/Cas9 deletion of exon 3 further confirmed reduced IL-8 expression and impaired macrophage chemotaxis. Collectively, these findings establish SPANXA2-OT1 as a human-specific regulator of macrophage-driven inflammation in CAD and highlight its promise as a translational biomarker and therapeutic target.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for PBMC derived human primary macrophages stimulated by IL-1beta and treated with negative control antisense oligos (NC_ASO) and SPANXA2-OT1 antisense oligos (SPAN_ASO).

first: Prabhash

last: Kumar Jha

first: Chelvanambi

last: Sarvesh

first: Nakamura

last: Yuto

first: Yuji Umesaki Itto

last: Lucas

first: Vijay

last: Aatira

first: Lupieri

last: Adrien

first: Cantadori Barbeiro

last: Miguel

first: Thanh

last: Dat Le

first: Borges Nascimento

last: Caio

first: Kasai

last: Taku

first: Whelan

last: Mary

first: Hosokawa

last: Daiki

first: Becker-Greene

last: Dakota

first: Sasha

middle: A

last: Singh

first: Elena

first: Uchida

last: Shizuka


title: RNA sequencing reveals the microRNA features of kidney in dengue virus-infected mouse

city: Guangzhou

first: zheng

last: wenjiang

state: Guangdong

department: The First Clinical Medical School

institute: Guangzhou University of Chinese Medicine

summary:
Background: Dengue virus (DENV) spread widely, causing not only dengue fever, dengue haemorrhagic fever, and dengue shock syndrome but also renal damage, which results in a poor prognosis. MicroRNA (miRNA) has attracted attention in recent years due to its regulatory functions in the transcriptomes of viral and host genomes. However, research on the function of miRNAs in the pathogenesis of dengue and dengue-associated nephropathy is still in the early stages.   Methods: We investigated the miRNA libraries from uninfected and DENV-infected mouse kidney tissue with high-throughput miRNA-seq technology, performed genomic analysis, and subsequently, practiced target gene prediction and enrichment analysis of differentially expressed miRNAs (DEmiRNAs).   Result: We identified 32 DEmiRNAs, including 19 with upregulated and 13 with downregulated expression, with miR-206-3p, miR-206, and miR-135a-5p showing significantly upregulated expression, the first two were associated with AKT phosphorylation, and the latter was associated with inflammation, however, those factors have been rarely investigated. MiR-122-5p, a potential biomarker in dengue, was downregulated in the kidney, in contrast to previous studies. Subsequently, we predicted 2209 potential target genes of the differentially expressed miRNAs and explored the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis, showing the pathogenic mechanisms of dengue in which the factors participated, which included viral replication and inflammatory response.   Conclusion: The current study investigates overall miRNA expression in mouse kidneys as a result of DENV-2 infection in vivo. These results may broaden the scope of knowledge about how miRNAs regulate the occurrence and development of DENV infection and in turn enhance the prevention and treatment of severe dengue and associated renal disease.
In this study, RNA-Seq analysis of kidney tissues from a mouse model after DENV infection was performed to identify differential miRNA expression profiles, predicted target genes, and analyzed the biological functions and pathways involved in the regulation of differential miRNAs.

overall_design: Comparative microRNA expression profiling analysis of RNA-seq data for the kidney tissues of dengue virus-infected mice and control mice.

first: Wenjiang

last: Zheng

first: Hui

last: Wu

first: Ting

last: Wang

first: Chengxin

last: Liu

first: Qian

last: Yan

first: Yong

last: Jiang

first: Shaofeng

last: Zhan

first: Geng

last: Li

first: Xiaohong


title: Effect of a 24h corticosterone treatment on differentiated C2C12 myotubes

city: Paris

first: Carine

last: Beaupere

address: 27 rue Chaligny

department: Saint-Antoine Research Center

country: France

institute: INSERM

summary: Long term glucocorticoïds treatment, e.g. corticosterone, induces insulin resistance in vivo and in vitro.  In vivo insulin resistance can trigger pancreatic beta cells adaptation in order to maintain glycemia. In particular, we observed that beta-cell mass adaptation can occur via beta-cell proliferation or via beta-cell neogenesis (de novo beta-cell production). Further, we demonstrated that beta-cell neogenesis was not a direct effect of GC, but rather due to the presence of pro-neogenic factors in the serum of GC-treated mice, suggesting a communication in-between insulin resistant tissues and the pancreas (Courty et al., Diabetes, 2019). Hence, the present work aims to decipher the role of the skeletal muscle in pancreatic adaptation in response to insulin resistance. We used an in vitro modeling in-between C2C12 differentiated myotubes treated for 24h with corticosterone (10-7M) or the same volume of solvant ethanol (VEH). Conditioned medium from corticosterone treated cells was tested on pancreatic buds E11.5 and proven to enhance beta-cell and other pro-endocrine lineage cellular differentiation in comparison to VEH conditioned medium. Subsequently, we analyzed the transcriptome of the C2C12 myotubes (n=3 per condition) using RNAsequencing (Illumina, NOVAseq 6000, 150bp paired-end sequencing). We found an enrichement for genes coding for secreted proteins in the CORT-treated myotubes. We narrowed down to 4 myokines candidates and validated the pro-neogenic potential of the candidate myokines using pancreatic buds E11.5 treated with recombinant proteins.

overall_design:
3 replicates of C2C12 differentiated myotubes (below passage 7) were treated for 24h with either Corticosterone 10-7M or corresponding volumes (1uL/mL) of ethanol 100%.
We then performed RNAseq analysis on 3 independant biological replicates per condition, using Illumina NOVAseq 6000 sequencer, 150bp paired end sequencing.
We compared differentially expressed genes in-between CORT- or VEH- treated cells.

first: Alexandrine

last: Liboz

first: Ghislaine

last: Guillemain

first: Bertrand

last: Blondeau


title: Single-nucleus transcriptome profiling provides insights into the pathophysiology of OSA-related renal injury

city: Zhangzhou

first: Li-Da

last: Chen

address: No 59, Shenglixiroad, Xiangcheng district

institute: Zhangzhou Affiliated Hospital of Fujian Medical University

summary: Obstructive sleep apnea (OSA) has been demonstrated to be associated with renal injury. However, the cellular and molecular mechanisms by which chronic intermittent hypoxia (CIH), a hallmark of OSA, contributes to renal injury remain poorly understood. Twelve male Sprague-Dawley rats were randomized into normoxic control (NC) and CIH groups (n = 6 each), with CIH exposure for 12 weeks. Renal injury was evaluated by Hematoxylin and Eosin staining and a modified Jablonski score. Single-nucleus RNA sequencing (snRNA-seq) was performed. Data analysis included clustering of cells, differential gene expression analysis, and functional enrichment through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Histopathological analysis revealed renal injury with renal tubular epithelial cell edema, necrosis in the CIH group compared to NC. A total of 42,581 cells (19,655 cells from NC group and 22,926 cells from CIH group) were retained after quality control, with 13 distinct renal cell populations identified. CIH exposure resulted in an increased proportion of podocytes, while mesangial cells were reduced compared to the NC group. CIH exposure altered the transcriptomic landscape, with differentially expressed genes (DEGs) observed across multiple cell types. Functional enrichment analysis indicated that CIH-induced DEGs were associated with suppressed metabolic and signaling pathways in proximal tubule and loop of Henle epithelial cells, activation of apoptotic and sodium reabsorption pathways in distal convoluted tubule cells, and enhanced inflammatory and phagocytic signaling in mononuclear phagocytes. Our study provides a high-resolution atlas of CIH-induced transcriptional changes in the rat kidney. The findings reveal cell type-specific responses and pathways potentially contributing to OSA-associated renal injury, offering novel insights into pathogenesis and potential therapeutic targets for preventing renal damage in OSA patients.

overall_design: Twelve male Sprague-Dawley rats were randomized into normoxic control (NC) and CIH groups (n = 6 each), with CIH exposure for 12 weeks.3 renal tissue samples were randomly selected from each group and then subjected for single-nucleus transcriptome  sequencing.


title: MicroRNAs induced by IFNα in HepG2 cells

city: Changchun City

first: Le

state: Jilin Province

address: No. 1 Xinmin Street

institute: the First Hospital of Jilin University

summary: Interferon-α (IFNα) plays a key role in innate immunity by inducing interferon-stimulated genes (ISGs). Beyond protein-coding ISGs, non-coding RNAs such as microRNAs are also modulated by IFNα. Here, we profiled differentially expressed microRNAs in HepG2 cells following IFNα stimulation, providing new insights into interferon signaling and antiviral regulation.

overall_design: RNA sequencing of HepG2 hepatocellular carcinoma cells treated with IFNα (20 ng/mL) for 0, 3, 8, and 24 hours.

first: Guangyun

last: Tan


title: Multi-step genomics on single cells and live cultures in sub-nanoliter capsules

first: Ignas

last: Mazelis

address: 210 Longwood Avenue

department: System Biology

institute: Harvard Medical School

summary: Single-cell genomics encompasses a set of methods whereby hundreds to millions of cells are individually subjected to multiplexed assays including sequencing DNA, chromatin accessibility or modification, RNA, or combinations thereof 1,2. These methods enable unbiased, systematic discovery of cellular phenotypes and their dynamics 1–3. Many functional genomic methods, however, require multiple steps that cannot be easily scaled to high throughput, including assays on living cells. Here we develop capsules with amphiphilic gel envelopes (CAGEs), which selectively retain cells, mRNA, and gDNA, while allowing free diffusion of media, enzymes and reagents. CAGEs enable carrying out high-throughput assays that require multiple steps, including combining genomics with live-cell assays. We establish methods for barcoding CAGE DNA and RNA libraries, and apply them to measure persistence of gene expression programs by capturing the transcriptomes of tens of thousands of expanding clones in CAGEs. The compatibility of CAGEs with diverse enzymatic reactions will facilitate the expansion of the current repertoire of single-cell, high-throughput measurements and extend them to live-cell assays.

sample_organism:
Escherichia coli
Homo sapiens
Mus musculus

overall_design:
RNA-seq data for the establishment of Capsule-based multi-step processing of single-cell in cultures. Data includes inC-RNAseq of fresh, frozen, and fixed cell lines, frozen PBMCs and cell lines cultured in CAGES. Washed cells were encapsulated into CAGEs, lyzed, washed, followed by mRNA capture via oligo-dT RT. cDNA was amplified in CAGEs, barcoded using split-pool implementation. Barcoded cDNA was used for 3’ end RNA-seq library preparation using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina. Data also included single-cell inC-ATACseq data of cell lines, GFP-RFP gDNA amplicon sequencing of individual cells as well as plasmid barcoding test. Raw sequencing data are available via SRA. Fastq were demultiplexed to a sample resolution and contain all 4 reads. Read1 - transcript; read2 - PCR barcode, read3 - index; read - ligation barcode2, ligation barcode1, RT/ATAC/PCR barcode and UMI [for RNAseq].

Plasmid_sequencing: Sequencing data for the establishment of Capsule-based multi-step processing of single-cell and cultures. Data includes inC-seq barcoding of plasmid DNA processed in CAGES. DNA containing CAGEs were washed and plasmid DNA were amplified in CAGEs, barcoded using split-pool barcode addition. Barcoded DNA amplicon were indexed and sequenced using Illumina
Raw sequencing data are available via SRA. Fastq were demultiplexed to a sample resolution and contain all 4 reads. Read1 - gDNA amplicon; read2 - PCR barcode, read3 - index; read - ligation barcode2, ligation barcode1 and CMV gDNA loci.

Fresh_Fixed: RNA-seq data for the establishment of Capsule-based multi-step processing of single-cells and live cultures. Data includes inC-RNAseq of fresh and PFA fixed K562 cells processed in CAGES. Washed cells were encapsulated into CAGEs, lyzed (decrosslinked in PFA case), washed, followed by mRNA capture via oligo-dT RT. cDNA was amplified in CAGEs, barcoded using split-pool implementation. Barcoded cDNA was used for 3? end RNA-seq library preparation using NEBNext® Ultra? II DNA Library Prep Kit for Illumina.
Raw sequencing data are available via SRA. Fastq were demultiplexed to a sample resolution and contain all 4 reads. Read1 - transcript; read2 - PCR barcode, read3 - index; read - ligation barcode2, ligation barcode1, RT/ATAC/PCR barcode and UMI [for RNAseq].

gDNA_Amplicons: Sequencing data for the establishment of Capsule-based multi-step processing of single-cell and cultures. Data includes inC-seq barcoding of GFP and RFP gDNA amplicon derived from a 1:1 mix of K562 and L1210 single-cells processed in CAGES. Washed cells were encapsulated into CAGEs, lyzed, washed, followed by mRNA capture via oligo-dT RT. gDNA targeted with CMV and RFP or GFP loci primers was amplified in CAGEs and barcoded using split-pool CAGE aproach. Barcoded gDNA amplicon were indexed and sequenced using Illumina
Raw sequencing data are available via SRA. Fastq were demultiplexed to a sample resolution and contain all 4 reads. Read1 - gDNA amplicon; read2 - PCR barcode, read3 - index; read - ligation barcode2, ligation barcode1 and CMV gDNA loci.

PBMCs: RNA-seq data for the establishment of Capsule-based multi-step processing of single-cells and live cultures. Data includes inC-RNAseq of cryopreserved PBMCs processed in CAGES. Thawed and washed cells were encapsulated into CAGEs, lyzed, washed, followed by mRNA capture via oligo-dT RT. cDNA was amplified in CAGEs and barcoded using inC-seq split-and-pool implementation. Barcoded cDNA was used for 3? end RNA-seq library preparation using NEBNext® Ultra? II DNA Library Prep Kit for Illumina.
Raw sequencing data are available via SRA. Fastq were demultiplexed to a sample resolution and contain all 4 reads. Read1 - transcript; read2 - PCR barcode, read3 - index; read - ligation barcode2, ligation barcode1, RT and UMI.

Cell_Culturing: RNA-seq data for the establishment of Capsule-based multi-step processing of single-cells and live cultures. Data includes inC-RNAseq of K562, L929 and induced pluripotent stems cells cultured in CAGES. After culturing, cells were lyzed followed by mRNA capture via oligo-dT RT. cDNA was purified, amplified and was used for 3? end RNA-seq library preparation using NEBNext® Ultra? II DNA Library Prep Kit for Illumina.
Raw sequencing data are available via SRA. Fastq were demultiplexed to a sample resolution and contain all 4 reads. Read1 - transcript; read2 - PCR barcode, read3 - index; read4 - UMI [no barcodes were used]

CAGE_Colonies: RNA-seq data for the establishment of Capsule-based multi-step processing of single-cells and live cultures. Data includes inC-RNAseq of mixed K562 and L1210 cells expanded into clones from single cells inside CAGEs in the presence of Decitabine, 5-Azacytidine or Vorinostat.  Cells were pretreated with drugs and encapsulated into CAGEs, followed culturing. Cells were lysed after 0, 2, 4, 6 days followed by oligo dT mRNA capture. cDNA was barcoded using split-pool inC-seq approach and Barcoded DNA was used for scRNA-seq library construction
Raw sequencing data are available via SRA. Fastq were demultiplexed to a sample resolution and contain all 4 reads. Read1 - transcript; read2 - PCR barcode, read3 - index; read - ligation barcode2, ligation barcode1, RT/ATAC/PCR barcode and UMI [for RNAseq].

first: Haoxiang

last: sun

first: Arpita

last: Kulkarni

first: Theresa

last: Torre

first: Allon

last: Klein


title: Aberrant Hippo-YAP/TEAD signaling drives malignant transcriptional reprogramming in external auditory canal squamous cell carcinoma

city: La Jolla

first: Kuniaki

last: Sato

state: CA

address: 3855 Health Sciences Dr Room 2345

department: Moores Cancer Center

institute: University of California San Diego

summary: External auditory canal squamous cell carcinoma (EACSCC) is an extremely rare malignancy. The molecular characteristics and evidence-based therapeutic strategies of EACSCC still remain to be elucidated. To elucidate the transcriptomic aberrations of EACSCC, RNA-seq was performed on non-cancerous ear skin tissues and EACSCC tissues.

overall_design: Library preparation from RNA extracted from cultured cells and RNA sequencing were performed using a DNBSEQ-G400 sequencer. The sequenced reads were aligned to the human reference genome GRCh38 using STAR v2.7.9a with Gencode v38 annotations. Gene count tables were generated with RSEM v1.3.1.

first: Noritaka

last: Komune

first: Mayumi

last: Ono

first: Takashi

last: Nakagawa


title: Synthetic engineering demonstrates that synergy among enhancers involves an increase in transcriptionally productive enhancer-gene contacts [Capture-C]

city: Santander

first: Alvaro

last: Rada-Iglesias

state: Cantabria

address: Albert Einstein 22, PCTCAN

institute: Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), CSIC/University of Cantabria

summary: Enhancers are non-coding cis-regulatory elements that control the expression of distally located genes in a tissue- and time-specific manner. Recent studies indicate that enhancers can differ in their underlying genetic architecture and regulatory properties. However, these different types of enhancers were previously investigated under rather variable conditions (e.g. model organism, cell type, enhancer-promoter distance, type of target promoter, etc.), thus introducing confounding factors that make it difficult to discern the distinct regulatory properties of each enhancer type. To overcome these limitations, here we generated transgenic mouse embryonic stem cells (mESC) lines in which different types of synthetic enhancers (i.e. “typical” enhancer, CTCF-associated enhancer, enhancer cluster/super-enhancer) were built upon the same “core” neural enhancer and inserted at the same distance (i.e. 100 Kb) from a typical developmental gene (i.e. Gata6). Subsequently, the mESC lines were differentiated to systematically compare the regulatory properties of the different enhancer types under identical conditions. Regarding the CTCF-associated enhancer, our data revealed that the addition of a CTCF site to the “core” enhancer increased insulation and led to the formation of a smaller contact domain, while having a rather mild effect on enhancer-promoter contact frequency and target gene expression. On the other hand, in comparison to the “core” enhancer alone, the enhancer cluster synergistically increased target gene expression and burst fraction. Importantly, we found that, in contrast to previous models, the strong regulatory activity of the enhancer cluster can not be explained by changes in enhancer-promoter contact frequency or the formation of transcriptional condensates. Instead, our data suggest that the emergent regulatory properties of enhancer clustering preferentially entail an increase in RNA Polymerase II pause release and, thus, in the fraction of enhancer-promoter contacts that are transcriptionally productive.

overall_design: Capture-C profiles for synthetic engineered mESC differentiated into Neural Progenitors

first: Endika

last: Haro

first: Mariana

last: Iliadou

first: Shyam

last: Ramasasy

first: Thais

last: Ealo

first: Victor

last: Campa

first: Tomas

last: Pachano

first: Marieke

last: Oudelaar


title: Three-dimensional Co-culturing Reveals Human Stem Cell-Derived Somatostatin Interneurons with Subclass Expression

city: Lund

first: Daniella

last: Rylander Ottosson

address: Sölvegatan 19, BMC A11.

department: Exp Med Science Department

country: Sweden

institute: Lund University

summary: Cortical interneuron deficiencies, particularly involving the somatostatin (SST) subtypes, contribute to neurological and neuropsychiatric disorders. These interneurons are difficult to derive in vitro from human embryonic stem cells (hESCs) due to their late embryonic development and dependence on glial interaction. To this end, we developed a three-dimensional co-culture model of hESC-derived neurons, enabling long-term development, functional maturity, and neuron-glial interaction. Under these conditions, hESCs successfully differentiated into functional GABAergic interneurons expressing the SST gene and protein within 50 days. Single-nuclei RNA sequencing revealed transcripts for SST subclasses, including Martinotti, non-Martinotti, and long-projecting neurons, that have not yet been described for hESC cultures. Upon injection into forebrain organoids, the interneuron progenitors spread and functionally matured while retaining their SST subclass identities, suggesting cell-intrinsic fate specification. Our in vitro model provides a robust platform for studying human SST interneurons, offering new avenues for investigating their role in health and disease.

overall_design:
RNA-seq analysis of hESC-derived interneurons in 3D co-cultures with glial cells at 100 DIV and transplanted hESC-derived interneurons in forebrain organoids at 100 DIV

***************************************************************
Raw files for human/patient samples were not submitted to GEO due to concerns about submitting personally identifiable sequence data for open access.
***************************************************************


title: Single-cell RNA sequencing of ICB samples from FUSCC

city: Shanghai

first: Tong

last: Fu

state: Shanghai

address: DongAn Road

institute: Fudan University

summary: Here, we performed single-cell RNA sequencing of pre-treatment tumor samples in 26 patients with early-stage TNBC and 6 patients with advanced TNBC to define anti-PD-1 response-related components in the tumor microenvironment.

overall_design:
We performed single-cell RNA sequencing on pre-treatment tumor samples from 26 early-stage TNBC patients and 6 advanced TNBC patients. The goal was to identify components of the tumor microenvironment related to the response to anti-PD-1 therapy. All samples were collected before any treatment was administered.

***************************************************************
Raw files for human/patient samples were not submitted to GEO due to concerns about submitting personally identifiable sequence data for open access.
***************************************************************

first: Xi

last: Jin

first: Yiyu

last: Chen

first: Yunsong

last: Yang

first: Yizhou

last: Jiang

first: Zhiming

last: Shao


title: Spatial transcriptomics reveals distinct role of monocytes/macrophages with high FCGR3A expression in kidney transplant rejections

city: Loma Linda

first: Yan

last: Chen Wongworawat

address: 11234 Anderson Street, Room 2516

institute: Loma Linda University

summary: Kidney transplant rejections are classified as active antibody mediated rejection (AMR) and cell mediated rejection (TCMR), with AMR primarily driven by antibodies produced by B cells, whereas TCMR is mediated by T lymphocytes that orchestrate cellular immune responses against the graft. Emerging evidence highlights the essential roles of innate immune cells in rejections, especially monocytes/macrophages and natural killer (NK) cells. However, the roles of specific innate immune cell subpopulations in kidney allograft rejection remain incompletely understood. Exploiting the spatial transcriptomics and formalin-fixed paraffin-embedded (FFPE) core needle biopsies from human kidney allografts, we demonstrated that non-rejection, AMR, acute TCMR and chronic active AMR have distinct transcriptomic features. Subclusters of monocytes/macrophages with high Fc gamma receptor IIIA (FCGR3A) expression were identified in C4d-positive active AMR and acute TCMR, and the spatial distribution of these cells corresponded to the characteristic histopathological features. Key markers related to monocyte/macrophage activation and innate alloantigen recognition were upregulated, along with metabolic pathways associated with trained immunity in AMR and TCMR. Taking together, these findings revealed that intragraft monocytes/macrophages with high FCGR3A expression play a critical role in kidney transplant rejections.

overall_design: We selected 8 cases based on histopathological and clinical features (Table 1), representing 4 diagnostic groups: 1) non-rejection conditions; 2) Active AMR; 3) Acute TCMR; 4) Chronic active AMR. The clinical diagnosis is interpreted by our renal pathologists based on the 2018 Banff Criteria. We performed 10x Genomic Visium spatial transcriptomics analysis on H&E - stained sections from archived FFPE core needle biopsies of human kidney allografts following Visium Spatial Gene Expression for FFPE workflow.


title: Effect of depletion of vacuolar protein sorting 13 homolog c (Vps13c) in heart tissue from ventricles of wild-type (WT) and cardiac-specific knockout (Vps13c-cKO) mice.

city: Hefei

first: Yuchen

last: Wang

state: Anhui

address: 81 Meishan Road

department: School of Basic Medical Sciences

institute: Anhui Medical University

summary: Systems genetics strategy across multiple Hybrid Mouse Diversity Panel (HMDP) cohorts pinpointed vacuolar protein sorting 13 homolog c (Vps13c) as a candidate trans-regulator of the mitochondrial transcriptome. Subsequent functional studies revealed that VPS13C influences mitochondrial morphology, bioenergetics, and global gene expression in cardiomyocytes. To assess the in vivo role of Vps13c, we generated Vps13c-flox mice, which were further crossed with Myh6-Cre mice to generate cardiac-specific knockout (Vps13c-cKO) mice. Efficient gene deletion was confirmed by reduced cardiac Vps13c mRNA and protein levels. RNA sequencing of ventricles from WT and Vps13c-cKO mice were performed here to investigate the impact of loss of Vps13c in vivo.

overall_design: RNA seq profiling of ventricles from heart of WT and Vps13c-cKO mice. The experiments were performed at 7 weeks old male mice.

first: Yu-Chen

first: Yue

last: Lu

first: Shichun

last: Shen

first: Yang

last: Cao


title: Pre-occupied ATF4 relies on CEBPγ co-binding for robust transcription activation during integrated stress response [C2C12_Thap_RNA-seq]

first: Peipei

address: No. 115 Jinzun Road, Pudong New Area, Shanghai

institute: Shanghai Jiao Tong University

summary: Upon encountering stress, cells need to rapidly and extensively remodel their transcriptome to restore homeostasis and ensure survival. However, the molecular mechanisms underlying stress-induced transcriptional remodeling are not well understood. In this study, we employed multi-omics approaches to comprehensively profile the changes in transcriptome, epigenetic landscapes, and 3D genome organization during the integrated stress response (ISR), a conserved cellular pathway activated by diverse stress stimuli. Our findings reveal that widespread transcriptional changes occur 4-6 hours after the induction of ISR, coinciding with the genome-wide binding of key transcriptional effector ATF4. Notably, while ATF4 binds to most of its target genes only upon ISR activation, it also occupies the promoters of hundreds of genes under non-stress conditions, priming them for enhanced transcriptional activation in response to stress signals. We further demonstrate that ATF4-mediated gene activation does not rely on traditional transcriptional regulatory mechanisms, such as increased H3K27 acetylation, chromatin accessibility, or remodeling of enhancer-promoter loops. Instead, our data suggest that the nuclear localization of ATF4 induces a pronounced genomic redistribution of transcriptional factor CEBP-γ, from non-ATF4 sites to a subset of ATF4-bound regions, where it collaborates with ATF4 to elicit transcriptional activation. The combined occupancy of ATF4 and CEBP-γ ultimately dictates the transcriptional outcomes during ISR. Interestingly, the preferred binding sites of CEBP-γ in response to stress are not determined by the strength of the CEBP-γ or ATF4 motifs, but rather by the higher-order chromatin architecture surrounding the binding sites. These findings provide novel insights into the molecular mechanisms that govern the rapid and efficient transcriptional remodeling during ISR, with potential implications for other stress responses.

overall_design: RNA-seq was performed on CEBPγ knocked down C2C12 cells and control C2C12 cells treated with DMSO and Thap for 6 hours respectively

first: Qian

last: Bian


title: Pre-occupied ATF4 relies on CEBPγ co-binding for robust transcription activation during integrated stress response [C2C12_Thap_CUT&Tag]

overall_design: CUT&Tag was performed in C2C12 cells treated with DMSO or thapsigargin (Thap) using ATF4 antibody (Santa Cruz Biotechnology # sc-390063).


title: Pre-occupied ATF4 relies on CEBPγ co-binding for robust transcription activation during integrated stress response [HeLa_CCCP_RNA-seq]

overall_design: RNA-seq in HeLa cells treated with DMSO or Carbonyl cyanide 3-chlorophenylhydrazone (CCCP).


title: Pre-occupied ATF4 relies on CEBPγ co-binding for robust transcription activation during integrated stress response [HeLa_CCCP_CUT&Tag]

overall_design: CUT&Tag for the histone modifications H3K27Ac as well as the transcription factors ATF4, CEBPγ in HeLa cells treated with DMSO or Carbonyl cyanide 3-chlorophenylhydrazone (CCCP).


title: Pre-occupied ATF4 relies on CEBPγ co-binding for robust transcription activation during integrated stress response [HeLa_CCCP_ATAC-seq]

overall_design: ATAC-seq in HeLa cells treated with DMSO or Carbonyl cyanide 3-chlorophenylhydrazone (CCCP).


title: Loss-of-function variants in ciliary genes confer high risk for Tetralogy of Fallot

city: Nanjing

first: Jimiao

last: Gao

address: 101Longmian Avenue, Jiangning District, Nanjing 211166, P.R. China

institute: Nanjing Medical University

summary: Tetralogy of Fallot (TOF), the most common severe cyanotic congenital heart disease, has poorly understood genetic causes. Through next-generation sequencing in 131 nonsyndromic TOF patients, we identified an increased burden of rare deleterious variants in ciliary genes and cilium pathway, and observed a multigenic inheritance pattern, with an OR of 1.672 for more than two deleterious variants and a cumulative OR of 3.158 for six variants. Functional validation in single- and double-heterozygous mouse models carrying these variants recapitulated TOF-like phenotypes and impaired normal cilia structure and function, particularly disrupting Hedgehog signaling in cardiomyocytes, downregulating key transcription factors Gata4 and Nkx2-5.

overall_design: scRNA-Seq analysis of mice E10.5 cardiac OFTs from wild-type and ciliary gene knockout/mutant littermates of each strain.

first: Yayun

last: Gu

first: Yuan

last: Lin

first: Zhibin

last: Hu


title: Effect of CDK11 inhibition via OTS964 treatment on gene expression to assess implication on cellular processes including intron retention.

city: Palo Alto

first: Jason

last: Sheltzer

address: 1651 Page Mill Road

department: Radiation Oncology

institute: Stanford University

summary: CDK11 has known to have functions in the cell cycle and RNA processing.  It has been previously shown that, a mutation within CDK11 (G579S) confers resistance to CDK11 inhibition. To further understand the role of CDK11 in cancer, we performed RNA-sequencing in four cancer cell lines (A375, Cal51, DLD1 and MDA-MB-231 cells) in both wild-type and G579S background. Here, we report that treatment with CDK11 inhibitor OTS964 results in a notable increase in intron retention. The expression of CDK11-G579S rescues the abberent splicing in the presence of CDK11 inhibitior, OTS964. GSEA analysis of the transcripts with retained introns have roles in gene expression, splicing and cell cycle progression. In addition to splicing related genes, we also revealed that CDK11 inhibition results in a significant downregulaiton of genes associated with DNA repair. This was an on-target effect of OTS964 treatment as this downregulation was not observed in cells expressing CDK11-G579S. This data shows that CDK11 has improtant roles in RNA splicing and DNA damage repair pathways.

overall_design: RNA-seq profiling of A375, Cal51, DLD1 and MDA-MB-231 cells in a wild-type background (sensitive to CDK11 inhibtion) or with G579S mutation (resistant to CDK11 inhibition)  after 6hr treatment with DMSO contro (0 nM) or OTS964 (100 nM).

first: Lisa

last: Crozier

first: Devon

last: Lukow

first: Sanat

last: Mishra

first: Ryan

last: Hagenson


title: CCL28 contributes to angiogenesis and cardiac repair by CCR10+ endothelial cells after myocardial infarction

city: Hangzhou

first: Rifeng

state: Zhejiang

address: No. 88 Jiefang Road

institute: Zhejiang University

summary: Secretome-based therapies targeting angiogenesis are promising in ischemic heart disease (IHD). The effects of Chemokine C-C motif ligand (CCL) 28 on IHD remain unclear. This study investigated the role of CCL28 in angiogenesis in IHD. The myocardial infarction (MI) and hindlimb ischemia (HI) models were constructed on male wild-type and CCL28-knockout mice. AAV9-TIE1-sh-CCR10 induced endothelial specific CCR10 deficiency in mice by tail vein injection. Recombinant CCL28 protein was used to treat MI and HI in wild-type and db/db mice. Serum levels of CCL28 were evaluated in a cohort of 416 consecutive chronic total occlusions (CTO) patients. The upregulated CCL28/ C-C motif receptor (CCR)10 axis was observed in myocardial and hindlimb ischemia. Additionally, CCL28 was mainly secreted by macrophages and CCR10 was highly expressed in endothelial cells (ECs). Compared to CCR10- ECs, CCR10+ ECs exhibited robust proangiogenic capacity, which are induced by CCL28 through the CCR10/ERK/SOX5 positive feedback signaling. Deletion of CCL28 results in impaired angiogenesis in the ischemic models while the use of recombinant CCL28 protein has therapeutic potential for myocardial and hindlimb ischemia, including in diabetes. Endothelial specific CCR10 deficiency impairs angiogenesis and blocks the therapeutic effect of rCCL28 in the myocardial and hindlimb ischemia. Serum CCL28 levels exhibited the predictive effect on coronary collateral vessels (CCV) in CTO patients. CCR10+ endothelial cells (ECs) are characterized with the enhanced proangiogenic capacity and beneficial for angiogenesis. CCL28 promotes angiogenesis by increasing CCR10+ ECs via the CCR10/ERK/SOX5 positive feedback signaling. This study highlights the angiogenic role of CCL28 and recombinant CCL28 protein might be a potential therapeutic option to attenuate myocardial and hindlimb ischemia, including in diabetes.

overall_design: RNA-seq was performed on CD45-CD31+ ECs magnetic bead sorted from WT and Ccl28-KO mice

first: Kun

last: Yang


title: KOLF2.1J iTF-Microglia: A standardized platform to study microglial transcriptional regulatory networks in CNS disease

city: Huntsville

first: Richard

middle: M.

last: Myers

state: AL

address: 601 Genome Way

institute: HudsonAlpha Institute for Biotechnology

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

overall_design: Refer to individual Series


title: Pre-occupied ATF4 relies on CEBPγ co-binding for robust transcription activation during integrated stress response [HiC]

last: Jiang

overall_design: HiC in C2C12 cells treated with DMSO or Thapsigargin.


title: Pre-occupied ATF4 relies on CEBPγ co-binding for robust transcription activation during integrated stress response [RNA-seq]

overall_design: RNA-seq in C2C12 cells treated with DMSO or Thapsigargin.


title: HNRNPA2B1 enhances immune evasion in colorectal cancer

first: Liu

middle: chu

last: wei

state: Guangdong province

address: 58 ZhongshanSecond Road, Yuexiu District,

institute: The First Affiliated Hospital of Sun Yat-sen University

summary: Immune checkpoint blockade has shown remarkable efficacy in a subset of colorectal cancers (CRCs) with high microsatellite instability (MSI-H), yet the majority of CRCs remain unresponsive, in part due to poor tumor–immune cell crosstalk. Here we identify HNRNPA2B1, an RNA-binding protein prominently upregulated in CRC, as a key modulator of the tumor immune microenvironment. Despite being central within a dysregulated network of RNA-binding proteins, HNRNPA2B1 exhibits minimal cytotoxicity to normal cells. Using mouse models and single-cell RNA-sequencing, we reveal that HNRNPA2B1 depletion enhances Cxcl9/Cxcl10–Cxcr3 signaling, elevates CD8^+ T-cell infiltration and activation, and improves antigen presentation via MHC class I.

overall_design: We performed single-cell RNA-sequencing (scRNA-seq) on subcutaneous tumors derived from Hnrnpa2b1-knockout (KO) and control MC38 cells (n=2 per group).

first: Chuwei

last: Liu

first: Guohui

last: Wan

first: Weiling

last: He

first: Wu

last: Song


title: Genomic analysis to identify correlates of the inflamed tumor phenotype in patients with advanced malignancies receiving immunotherapy

city: Chicago

first: Thomas

last: Gajewski

state: IL

address: 929 E 57th St

institute: University of Chicago

summary: This biomarker study will analyze tissues from patients with cancer who are receiving immunomodulatory treatment (or therapy that could be hypothesized to modulate the immune response), to correlate molecular/genetic features of the tumor cells, the host, and the intestinal microbiome with the immunologic phenotype of the tumor microenvironment. Recent data have suggested that patients with pre-existing T cell infiltration in the tumor microenvironment and/or an “inflamed” gene expression profile in tumor sites have better clinical outcome following treatment with several immunotherapeutic approaches. However, the mechanism by which this spontaneous anti-tumor immune response develops in only a subset of patients is not understood. Potential correlations between mutational patterns in the tumor, germline genetic polymorphisms, peripheral blood immune response parameters, and the composition of intestinal microbiota and the presence or absence of a T cell-infiltrated tumor microenvironment will be sought. Understanding these mechanisms will have important implications for future patient selection for treatment with immunotherapies and highlight new potential pathways for therapeutic development. Mechanisms of secondary resistance to immunotherapies also will be interrogated using tissues obtained at time of recurrence.

overall_design: Adult patients with advanced cancer, willing to provide at least a subset of tissues can be enrolled in the study. Blood, stool, and tumor specimen are collected prior to and during the course of treatment. Genetic and/or immune histochemical analysis are performed with tumor tissue, germline samples to identify patterns of genetic alterations and germline polymorphisms associated with the presence or absence of a T cell-infiltrated tumor microenvironment. In addition, species composition of the intestinal microbiota and circulating  factors in the peripheral blood are analyzed to determine their association with the presence or absence of a T cell-infiltrated tumor microenvironment.

middle: F

first: Daniel

last: Olson


title: Gene expression in tissues of root tips from Pinus pinaster seedlings under nitrate nutrition

city: Malaga

first: Rafael

last: Cañas

address: Facultad de Ciencias, Campus de Teatinos

department: Biología Molecular y Bioquímica

institute: Universidad de Málaga

summary: In the present study, the gene expression changes in four different tissues of root tips from maritime pine were analyzed. The roots were under two nutritional conditions (3 mM KCl or 3 mM NO3K) and harvested after 2 hours of the treatment. This work was funded by Ministerio de Ciencia e Innovación, grant number PID2021-122641NB-C21, MICINN, FEDER, UE.

overall_design: Pine seedlings were subdivided into two groups. The control group was irrigated with 80 mL of 3 mM KCl (C), the second group was supplied with 80 mL of 3 mM NO3K. Root samples were collected 2 hours after the irrigation. Seedling’s root tips were cut and tissue pieces of 5 - 6 mm were imbibed in a specimen holder with Tissue‐Tek optimal cutting temperature embedding medium (Sakura Finetek, Alphen aan den Rijn, The Netherlands) and immediately frozen in liquid nitrogen for cryostat sectioning. The 24 samples were sequenced on an Illumina NovaSeq 6000 as paired-end reads of 150 bp length.

first: David

last: Salas-Vázquez

first: Francisco

middle: R

last: Cantón


title: Pre-occupied ATF4 relies on CEBPγ co-binding for robust transcription activation during integrated stress response [CUT&Tag]

first: Peipei

overall_design: CUT&Tag for the histone modifications H3K27Ac as well as the transcription factors ATF4, CHOP, CEBPβ, CEBPγ in C2C12 cells treated with DMSO or Thapsigargin.

first: Qian

last: Bian


title: Pre-occupied ATF4 relies on CEBPγ co-binding for robust transcription activation during integrated stress response [ATAC-seq]

overall_design: ATAC-seq in C2C12 cells treated with DMSO or Thapsigargin.


title: RNAseq of gingival fibroblasts exposed to cross-linked hyaluronic acid

city: Vienna

first: Reinhard

last: Gruber

address: Sensengasse 2a

country: Austria

institute: Medical University Vienna

summary: Hyaluronic acid was proposed to support soft tissue recession surgery and guided tissue regeneration. Nevertheless, the molecular mechanisms through which hyaluronic acid modulates the response of the connective tissue cells remain elusive. To elucidate the impact of hyaluronic acid on the connective tissue cells, we employed bulk RNA sequencing to unravel the changes in the genetic signature gingival fibroblasts exposed to 1.6% cross-linked hyaluronic acid and 0.2% natural hyaluronic acid hyaluronic acid. Transcriptome-wide changes were modest, even when implementing a minimum of 1.5 log2 fold-change and a significance threshold of 1.0 -log10; only a dozenth gene was differentially expressed and ranked according to the Manhattan distance. Upregulated were PLK3, SLC16A6, IL6, HBEGF, DGKE, DUSP4, PTGS2, FOXC2, ATAD2B, NFATC2, and downregulated were MMP24 and PLXNA2. RT-PCR analysis supported the impact of hyaluronic acid on increasing expression of the selected gene panel. The findings from bulk RNA sequencing suggest that gingival fibroblasts only show a weak response when exposed to hyaluronic acid.

overall_design: Explant cultures of gingiva from three healthy individuals who provided informed consent were used to isolate gingival fibroblasts. Ethical approval from the Ethical Committee of the Medical University of Vienna (EK #631/2007) is available. These cells were isolated and expanded in DMEM supplemented with 10% fetal calf serum (FCS) and 1% antibiotics (Invitrogen Corporation, Carlsbad, CA). A pool of the three donors of gingival fibroblasts, all at low passage, were seeded at 30.000 cells/cm2 into 24-well plates and exposed to hyaDENT BG (Regedent AG; Zurich, Switzerland) a medical device consistent with 16.0 mg/mL of hyaluronic acid cross-linked and 2.0 mg/mL of hyaluronic acid with sodium chloride. The gingival fibroblasts were exposed to a 4.5-fold diluted hyaDENT BG, resulting in a final concentration of a total of 4 mg/mL cross-linked and native HA for 6 hours in serum-free DMEM followed by isolation of total RNA.

first: Layla

last: Panahipour


title: Nuclear Protein Aggregates Disrupt RNA Processing and Alter Biomechanics in a Muscle Cell Model of OPMD

city: Leiden

first: Vered

last: Raz

address: Einthovenweg

department: Human genetics

country: Netherlands

institute: LUMC

summary: RNAsequencing in three subcellular fractions: cytosolic, nuclear and insoluble from vehicle-treated cells and Dox-treated cells that induces the expression of the expanded (A16) PABPN1. The goal of this study was to demonstrate PABPN1 function in A16 cells, and to identify RNA accummulation in each fraction. This allows the identification of molecular signatures.

overall_design: Samples are from cytosolic, nuclear, and insoluble fractions isolated from vehicle and Dox-induced Ala16-PABPN1 in muscle cells. Biological replicates are N=3.

first: Milad

last: Shademan


title: VAV3 antagonizes Skeletal Muscle Disulfidptosis to maintain Glucose Homeostasis in Type 2 Diabetes

city: beijing

first: Guangjian

address: Beijing University of Traditional Chinese Medicine, No. 11 North Third Ring East Road, Beijing City

institute: Beijing University of Chinese Medicine, Beijing

summary: The emergence and progression of type 2 diabetes mellitus (T2DM) are intricately linked to skeletal muscle loss, yet the exact mechanisms remain elusive. Here, our whole-exome and transcriptomic results indicate that VAV3 is mutated and down-regulated in T2DM patients associated with increased sulfur metabolism. Integrated analysis of human transcriptomics, mouse and cellular proteomics, glycol-metabolomics analyses suggest a potential association between VAV3, T2DM and disulfidptosis. Inhibiting disulfidptosis or SLC7A11/3A2 rescues death of Vav3-deleted cells and IR cells. Mechanistically, Vav3 deletion leads to decreased mitochondrial ATP synthesis and reduced GLUT4 expression. Insufficient glucose intake combined with elevated SLC7A11/3A2 expression induces NADPH depletion and cystine accumulation, leading to redox system imbalance and aberrant disulfide bonds formation. This results in F-actin collapse, broken fiber bundles and rapid cell death. Together, our findings reveal unexpected roles of VAV3 in regulating glucose metabolism, disulfidptosis and provide a therapeutic strategy to target skeletal muscle disulfdptosis in T2DM treatment.

overall_design:
Serum transcriptomic analysis indicates a connection between diabetes development, actin cytoskeleton and disulfidptosis.VAV3 mutationT2DM display a distinct gene expression profile associated with disulfidptosis

>>>Submitter states that raw data are not available due to patient privacy concerns.<<<

first: Siyu

last: Tao

first: Jinfang

last: Dou

first: Xianjie

last: Zhen

first: Yuelin

last: Zhang

first: Tao

last: Wu

first: Nannan

first: Yushan

last: Gao

first: Xiuyan

last: Yang


title: The effect of inhibiting the cGAS-STING pathway in a chronic liver disease mouse model

city: Zurich

first: Genome

last: Informatics

address: Winterthurerstrasse 190

department: Functional Genomics Center Zurich (FGCZ)

country: Switzerland

institute: ETH ZURICH / University of Zurich

summary: Hepatocellular carcinoma (HCC) mostly develops on the background of a chronic liver disease (CLD). The hallmarks of CLD includes increased hepatocyte apoptosis, compensatory proliferation and DNA damage. Mice with MCL-1 deficiency in hepatocytes (Mcl-1Dhep mice ) recapitulated these key features of CLD and developed HCC later in life. However, it is unclear how MCL-1 deficiency drives tumorigenesis. We analysed replication stress and the activation the cGAS-STING pathway in Mcl-1Dhep mice. 2-month-old Mcl-1Dhep mice showed increased replication stress, micronucleated hepatocytes and increased cytosolic DNA sensing. We found that an increase in extrachromosomal circular DNA in Mcl-1Dhep liver promoted a crosstalk between hepatocytes and immune cells to drive the immune response which was dependent on the canonical cGAS-STING pathway. We then generated Mcl-1Dhep/STING-/- double knockout mice to study the effects of inhibiting the cGAS-STING pathway in liver homeostasis and tumorigenesis. In fact, deletion of STING in Mcl-1Dhep mice reduced immune cell chemotaxis, as well as tumor incidence. Our findings indicate that the cytosolic DNA sensing pathway may play an important role in inflammation-driven liver carcinogenesis.

overall_design: Knockout mice were generated by crossing transgenic mice with different genetic background.

first: Peter

last: Leary

first: Lap Kwan

last: Chan

first: Achim

last: Weber


title: Loss-of-function variants in ciliary genes confer high risk for Tetralogy of Fallot [RNA-Seq]

first: Jimiao

overall_design: RNA-Seq analysis of mice E10.5 hearts from wild-type and ciliary gene knockout/mutant littermates of each strain.

first: Yayun

last: Gu

first: Yuan

last: Lin

first: Zhibin

last: Hu


title: Effect of in vitro-transcribed U1 snRNA on gene expression

city: Suzhou

first: Fan

address: 100 Chongwen Road

institute: Suzhou Institute of Systems Medicine

summary: To investigate the effect of in vitro-transcribed U1 snRNA on gene expression, we transfected A549 cells with in vitro-transcribed U1 snRNA or control RFP (200 ng/mL). We then performed gene expression profiling analysis using RNA-seq.

overall_design: Gene expression profiling analysis of RNA-seq data for A549 cells transfected with in vitro-transcribed U1 snRNA or RFP

first: Siying

last: Liu


title: Transcriptomic analysis of ipsilateral spinal cord in rats after bone fracture

first: Shaoxun

last: Yuan

address: No. 138, Xianlin Avenue, Qixia District

institute: Nanjing University of Chinese Medicine

summary: To investigate the specific changes in the spinal cord after bone fractures, we obtained L4-L5 spinal cord segments from the same side of SD rats with tibial fractures

overall_design: Established the rat tibial fracture mode,take the L4-L5 spinal cord segment on the same side of the fracture at different time point(0day,3day,7day,14day,28day)

first: Shen

last: Wang

first: Jin

last: Deng

first: Xiaofeng

last: Yin


title: Driver Gene and Potential Biomarker Investigation of Desmoid-type Fibromatosis Through RNA-seq Analysis

city: Seoul

first: Eun-jin

last: Jeong

address: 81 Ilwon-ro, Gangnam-gu

country: South Korea

institute: Samsung Advanced Institute for Health Sciences & Technolohy, Sungkyunkwan University

summary: Desmoid type fibromatosis(DTF), a locally aggressive fibromatosis, lacks clear pathogenesis. RNA-seq analysis of 29 DTF cases identified hub genes like ZNF536, TWIST2, and SHOX2, indicating involvement in fibroblastic proliferation. These genes were validated in other fibrotic diseases. Immunohistochemistry revealed TWIST2 as a promising diagnostic biomarker, emphasizing its potential clinical utility.

overall_design: Desmoid-type fibromatosis (DTF) presents as a locally aggressive yet benign myofibroblastic neoplasm characterized by infiltrative fibroblastic and myofibroblastic proliferation. Although the CTNNB1 mutation suggests involvement of the Wnt/beta-catenin signaling pathway, the precise driver genes and mechanisms driving fibroblastic proliferation remain unclear. To shed light on this, we conducted RNA-seq analysis on 29 DTF cases and 14 reactive fibrosis (RF) samples as controls.

first: Eun jin

first: Ja min

first: Ji min

first: won kyung

last: Kim

first: Chang Ohk

last: Sung

first: Seok hyung


title: Whole transcriptome analysis after TGF-β treatment in FaDu cells

city: HYDERABAD

first: Swati

last: Singh

state: TELANGANA

address: BITS PILANI HYDERABAD CAMPUS, JAWAHAR NAGAR, KAPRA MANDAL, MEDCHAL

department: BIOLOGICAL SCIENCES

country: India

institute: BITS PILANI HYDERABAD CAMPUS

summary: Several long non-coding RNAs (lncRNAs) are differentially expressed in oral cancer, contributing to Oral squamous cell carcinoma (OSCC), an aggressive cancer of the head and neck region. A dysregulated Transforming growth factor-beta (TGF-β) pathway is often observed in OSCC, leading to increased proliferation, migration, invasion, and chemoresistance in OSCC cells. The role of TGF-β regulated lncRNAs in promoting malignancy has already been established in multiple cancers. Such differentially expressed lncRNA involved in cross-talks with the TGF-β pathway is also reported in OSCC. We suspect many such lncRNAs function downstream of the TGF-β pathway in OSCC. We use whole transcriptomic sequencing to identify lncRNAs which are induced upon TGF-β treatment in OSCC via SMAD dependent or SMAD independent pathways to promote OSCC pathogenesis.

overall_design: Cell line: FaDu; Sample names: control (C2, C3, C4) and TGF-β treated (T2, T3, T4); Total RNA was isolated from control (C2, C3, C4), or TGF-β treated (T2, T3, T4) FaDu cells 48 h post-treatment and RNA sequencing was performed.

first: Vivek

last: Sharma


title: BMDMs were transfected with RFP or U1 and then infected with HSV-1 or not

last: Zhang

summary: Test transcripts in mock and HSV-1 infected BMDMs which transfected with RFP or U1 snRNA

overall_design: Gene expression profiling analysis of RNA-seq data for mock and HSV-1 infected BMDMs which transfected with RFP or U1 snRNA


title: α-Hemihydmus musculuse calcium sulfate/ n-hydroxyapatite combined with metformin promotes osteogenesis in vitro and in vivo

city: Zhengzhou

first: yanyong

last: cao

state: henan

address: No. 116, Huayuan Road

institute: Henan Academy of Agricultural Sciences

summary: This study aimed to examine the effects of loading different concentmus musculusions of metformin onto an α-hemihydmus musculuse calcium sulfate/nano-hydroxyapatite (α-CSH/ nHA) composite. The material characteristics, biocompatibility, and bone formation were compared as functions of the metformin concentmus musculusion. X-ray diffraction results indicated that the metformin loading had little influence on the phase composition of the composite. The hemolytic potential of the composite was found to be low, and a CCK-8 assay revealed only weak cytotoxicity. However, the metformin-loaded composite was found to enhance the osteogenic ability of MC3T3- E1 cells, as revealed by alkaline phosphate and alizarin red staining, real-time PCR, and Western blotting, and the optimal amount was 500 μM. RNA sequencing results also showed that the composite material increased the expression of osteogenic-related genes. Cranial bone lacks muscle tissue, and the low blood supply leads to poor bone regenemus musculusion. As most mammalian cranial and maxillofacial bones are membranous and of similar embryonic origin, the Mus musculus cranial defect model has become an ideal animal model for in vivo experiments in bone tissue engineering. Thus, we introduced a Mus musculus cranial defect with a diameter of 5 mm as an experimental defect model. Micro-computed tomography, hematoxylin and eosin staining, Masson staining, and immunohistochemical staining were used to determine the effectiveness of the composite as a scaffold in a Mus musculus skull defect model. The composite material loaded with 500 μM of metformin had the strongest osteoinduction ability under these conditions. These results are promising for the development of new methods for repairing craniofacial bone defects.

overall_design: Six samples were divided into a control group and Met/α-CSH/nHA scaffold group.

first: Sirui

first: Haojie

last: Fu


title: FOSL2 promotes intertumoral infiltration of T Cells and increase pathological complete response rates in locally advanced rectal cancer patients

first: Xu

last: Kailun

state: zhejiang

address: No.88, Jiefang Road

institute: The Second Affiliated Hospital of Medical School of Zhejiang University

summary:
To characterize potential biomarkers and underlying mechanisms that prompt pathological complete response (pCR) rate of neoadjuvant chemoradiotherapy (nCRT) in locally advanced rectal cancer (LARC) patients.LARC patients from two hospitals were enrolled with pre-nCRT biopsy tissues examined by pressure cycling technology (PCT) combined with data-independent acquisition (DIA) mass spectrometry. Tumor regression grade (TRG) evaluation was performed with the surgical tissues after nCRT to estimate the efficacy of nCRT. Proteins up regulated in pCR patients were highlighted and immune infiltration analysis was carried out.   The candidate biomarker FOSL2 (FOS Like 2, AP-1 Transcription Factor Subunit), was selected and verified its role in vitro and vivo.
We then performed gene expression profiling analysis using data obtained from RNA-seq of 2 different cells with or without chemoradiotherapy.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for CT26 cells and its KD cells.

first: Kailun

last: Xu

first: Xiaoyang


title: Bi-steric mTORC1-selective Inhibitors activate 4EBP1 reversing MYC-induced tumorigenesis and synergize with immunotherapy

city: Stanford

first: Dean

last: Felsher

state: California

address: CCSR 1120 269 Campus Drive

department: Medicine/Oncology

summary: We evaluated the therapeutic efficacy and transcriptome changes induced in hepatocellular carcinoma upon treatment of Lap-tTA/Tet-O-MYC transgenic mice with two Bi-steric mTORC1-selective inhibitors, sorafenib, or doxycycline to inactivate MYC via the TET system at different time points post treatment.

overall_design: Lap-tTA/Tet-O-MYC mice were taken off doxycycline to activate the human transgenic MYC oncogene 4 weeks after being born, then after waiting 4-5 weeks, mice were imaged using magnetic resonance imaging (MRI) every week to precisely define tumor volumes before treatment enrollment. Mice bearing HCC tumors with these volumes were assigned to one of the following treatment groups: vehicle control (5/5/90 Transcutol/Solutol HS 15/Water) at 10 mg/kg via I.P., doxycycline  at 100 µg/ml in water (MYC Off), RMC-4627 at 10 mg/kg I.P. 1x/wk, RMC-6272 at 10 mg/kg I.P. 1x/wk, or Sorafenib at 30 mg/kg/day O.G. (oral gavage). Seven days after treatment, mice were given a second dose of their respective treatments and they were euthanized at 4, 12, 24, 48 or 72 hours to collect and freeze in dry ice HCC tumor chunks.

first: Wadie

middle: D

last: Mahauad-Fernandez

first: Yu

middle: C

last: Yang

first: Mallika


title: Patient 6 and patient 10 CAR-NK study

city: Houston

first: Katayoun

last: Rezvani

address: 1515 Holcombe Blvd

department: Department of Stem Cell Transplantation and Cellular Therapy

institute: The University of Texas MD Anderson Cancer Center

summary: To evaluate whether the findings of our pre-clinical study hold true in patients treated with CAR19/IL-15 NK cells, we isolated and profiled CAR-NK cells and B cells from peripheral blood collected at multiple timepoints from two patients diagnosed with CD19+ relapse/refractory hematological malignancies and treated with CAR19/IL-15 NK cells (11). Each patient received one infusion of CAR-NK cells

overall_design: Single cells were sorted into 96 well plates with lysis buffer (10 l of TCL buffer, Qiagen and 1% 2-mercaptoethanol) stored at -80°C immediately until library preparation. cDNA was synthesized as described by the Smart-Seq2 protocol from Picelli et al.1 Briefly, RNA was purified using RNAClean XP beads (Beckman Coulter) and reverse transcribed to cDNA using Maxima RNAse H-minus (ThermoFisher) in the presence of biotinylated oligo-dT30VN, biotinylated template-switching oligonucleotides, and betaine. Next, a PCR preamplification step was done with KAPA HiFi HotStart Ready Mix (Roche Diagnostics) and biotinylated ISPCR primers. DNA products were further purified with Agencourt XP DNA beads, and the concentration of each sample was quantified by Qubit dsDNA HS Assay kits (Invitrogen). Quality of samples was analyzed on a 2100 Bioanalyzer using a High Sensitivity DNA Kit (Agilent Technologies). 0.15 ng of each sample were tagmented and Nextera index adapters were used for barcoding according to the Illumina Nextera XT DNA sample preparation kit (Illumina).   For sequencing, single cell libraries were pooled equally, purified using AMPure XP beads and analyzed on a 2100 Bioanalyzer. Pooled libraries were denatured and diluted to 1.8pM prior to sequencing using 75 cycle paired-end kit on a NextSeq 500 (Illumina) with an average sequencing depth of 1 million reads per cell. The obtained mRNA sequencing data was demultiplexed into individual FASTQ files followed by alignment to the human reference genome hg19.

first: Banerjee

last: Pinaki Prosad


title: Targeting lysozyme 2 in endocardium promotes rapid recovery by modulating remote injury signals

city: Beijing

first: Hui

address: 167 Beilishi Road, Xicheng District

institute: Fuwai hospital

summary: Intrinsic molecular strategy at the organ level to injury stimuli remains elusive. Identifying and modulating such potential molecular strategy for systemic adaptations to injury is theoretically fascinating and therapeutically promising. Here, using integrative single-cell and spatial transcriptomic analysis, we reported that lysozyme 2 (Lyz2), a long-established myeloid marker, surprisingly indicated global injury status (across cell types and anatomic divisions) and influenced cardiac function after injury. We correlated the Lyz2 regulon of the lysosome-matrisome (particularly endocardial lysosomal-degrading Hspgs) with anatomical and functional relevance to cardiac injury and determined that such spatial model is consensus across various injury types, both mouse and human. Modulation of spatial model mediated by Lyz2 via genetic deletion or chemical inhibition in adult mice with myocardial infarction remarkably achieved immediate protective effects, a rare therapeutic benefit among current interventions for heart failure. These results provided the first molecular evidence for the unprecedented molecular strategy adopted by the heart with unparalleled therapeutic promising for organ repair.

overall_design: We leveraged spatial transcriptomics to overview molecular organization with anatomical divisions during cardiac regeneration. We collected a total of 19 specimens from 10 hearts at 8 developmental points, including seven P1 hearts post-AR (P1+1dpr-7dpr) and four hearts as controls (WT: P1, P7; and SH: P1+4,6 dps). Sample sections were collected with the maximum 4-chamber view along the dorsal-ventral axis, and the sectioning depth of individual hearts increased with postnatal days, reflecting an increase in dorsal-ventral dimensions as the heart grew and ensuring comparable histological views across samples. After data trimming for quality control, the joint dataset yielded a pool of 16,654 individual spots (average 5,343 genes per spot) and identified 16 clusters by uniform manifold approximation and projection (UMAP) corresponding to cardiac anatomical divisions in high spatial and molecular resolution, allowing us to study molecular diversity in intact tissue in space without bias.

first: Chenghao

last: Fan

first: Shen

last: Song

last: Han

first: Dongcheng

last: Cai

first: Anteng

last: Shi

first: Fangfang

last: Wan

first: Jie

last: Feng

first: Jiajun

last: Zhong

first: Yifan

last: Xie

first: Fei

first: Jiangping

first: Shengshou

last: Hu

last: Nie


title: Analysis of gene expression in different zones of root nodules formed by Sinorhizobium meliloti and Medicago truncatula

first: Melanie

last: Barnett

address: 371 Jane Stanford Way

department: Biology

institute: Stanford University

summary: The bacterium, Sinorhizobium meliloti, interacts symbiotically with leguminous plants such as Medicago truncatula to form nitrogen-fixing root nodules. During symbiosis, plant and bacterial cells differentiate in a coordinated manner, resulting in specialized plant cells that contain nitrogen-fixing bacteroids. Medicago nodules are organized in structurally distinct tissue zones, representing different stages of bacterial and plant differentiation. We used laser-capture microdissection (LCM) to analyze bacterial and plant gene expression in four root nodule regions. In parallel, we analyzed gene expression in nodules formed by wild type bacteria on six plant mutants with nitrogen fixation deficiencies (dnf). We found that bacteroid metabolism is drastically remodeled during bacteroid differentiation. Many processes required for bacterial growth are down-regulated in the nitrogen fixation zone. The overall transcriptional changes are similar to those occurring during nutrient limitation by the stringent response. We also observed differential expression of bacterial genes involved in nitrogen fixation, cell envelope homeostasis, cell division, stress response and polyamine biosynthesis at distinct stages of nodule development. In M. truncatula we observed the differential regulation of several host processes that may trigger bacteroid differentiation and control bacterial infection. We analyzed plant and bacterial gene expression simultaneously, which allowed us to correlate processes in both organisms.

sample_organism:
Sinorhizobium meliloti
Medicago truncatula

overall_design: Four different tissue regions from 47 day old root nodules formed by Medicago truncatula [Gaertn.] cv Jemalong and Sinorhizobium meliloti CL150 were collected by laser-capture microdissection. For each of the three replicates 9-10 plants were used. In order to identify symbiotically regulated genes we carried out pairwise comparisons between the samples and used hierarchical clustering to group genes with similar expression profiles.

first: Claus

last: Lang

first: Sharon

last: Long


title: RNA-seq data from mice liver fed with MCD

city: HANGZHOU

first: junxia

last: min

state: zhejiang province

address: zhejiang university zijingang campus medcine school research building C155

department: school of medicine

institute: zhejiang university

summary: We fed control mice and Slc7a11-LTG mice methionine and Choline Deficient (MCD) to induce NASH. Whole-genome RNA sequencing were then performed in the livers of control and LTG mice fed with MCD.

overall_design: n=3 male mice each group

last: Shen

first: Zijun

first: Junxia

last: Min

first: Fudi

last: Wang


title: 3D Vasculature-On-A-Chip

city: Kowloon

first: Sin Yen

last: Tan

address: Clear Water Bay

country: Hong Kong

institute: HKUST

summary: We performed transcriptomic analysis to investigate on-chip endothelial cells and fibroblasts.

overall_design: We extracted the RNA samples of on-chip endothelial cells(ECs) and fibroblasts(FBs) respectively from three different time points (Day 1, 3 and 5) and used Smart-seq2 protocol to generate mini bulk RNA-seq data. A total of 94 mini-bulk RNA samples were analyzed. Of these, 46 RNA samples are from ECs and 48 RNA samples are from FBs. 21 out of the 46 ECs samples are ECs cocultured with FB (6 biological replicates for day 1; 8 biological replicates for day 3 and 7 biological replicates for day 5) and the rest are ECs cultured alone (9 biological replicates for day 1; 8 biological replicates for day 3 and 8 biological replicates for day 5). As for FBs, 24 out of 48 FBs samples are FBs cocultured with ECs (9 biological replicates for day 1; 6 biological replicates for day 3 and 8 biological replicates for day 5) while another 26 samples are FBs monocultured (7 biological replicates for day 1; 6 biological replicates for day 3 and 12 biological replicates for day 5).

first: Sin

middle: Y


title: Elevated PRKD2 Promotes Progression and Poor Prognosis of Multiple Myeloma

city: Xuzhou

first: Guihua

last: Zhang

address: Xuzhou

department: Hematology

institute: Xuzhou Cancer Hospital

summary: This study aimed to address the challenges in treating multiple myeloma (MM) and its advanced stage, relapsed and refractory multiple myeloma (RRMM), by identifying new therapeutic targets. Clinical data were collected from 132 MM patients. Through propensity score matching, bone marrow samples from 24 MM patients and 6 control individuals were selected for RNA sequencing analysis. Western blot validation was performed in cell lines to investigate the role of serine/threonine-protein kinase D2 (PRKD2) in MM and RRMM. Additionally, gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, immune infiltration analysis, and drug sensitivity screening were conducted. The study found PRKD2 to be significantly upregulated in MM and RRMM, suggesting its potential as a therapeutic target. Functional assays indicated that disrupting PRKD2 inhibited myeloma cell proliferation, and pathway analysis revealed immune-related pathway alterations associated with PRKD2 expression. Drug sensitivity analysis identified Axitinib as a potential therapeutic agent targeting PRKD2, highlighting its promise for MM treatment.

overall_design: To study the PRKD2 in Nrelapsed and refractory multiple myeloma

first: Hong

last: Liu

first: Jiaqian

last: Qi

first: Zhenyu

last: Li


title: Effect of U1 snRNA knock-in on gene expression during HSV-1 infection

first: Fan

summary: To investigate the effect of U1 snRNA knock-in on gene expression during HSV-1 infection, we obtained BMDMs from WT and U1 snRNA myeloid knock-in (U1mKI) mice, and infected them with HSV-1 (MOI, 1) for 4 hours. Subsequently, we performed gene expression profiling analysis using RNA-seq.

overall_design: Gene expression profiling analysis of RNA-seq data for mock and HSV-1 infected WT or U1mKI BMDMs

first: Shiyou


title: Decoding Trp53 regulatory program in various tissues and organs

city: Hanzhou

first: Guoji

last: Guo

address: Yuhangtang street 866

department: Zhejiang University School of Medicine

institute: Center for Stem Cell and Regenerative Medicine

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

overall_design: Refer to individual Series


title: Deciphering the cis-regulatory code of alternative splicing in human cell lines

status: Public on Sep 08 2025

city: seattle

first: Samantha

last: Koplik

email: skoplik@uw.edu, kopliksam@gmail.com

state: Washington

address: 185 Stevens Way

department: Electrical and Computer Engineering

institute: University of Washington

summary: Alternative splicing (AS) is a key mechanism of gene expression diversity, and dysregulation of AS is implicated in many human diseases. Identifying variants that disrupt splicing and characterizing their impact on isoform abundance across different cell types remains challenging. Here, we perform massively parallel reporter assays (MPRAs) to measure splicing phenotypes of over 87,000 sequence variants in five cell lines of different tissue origin. The MPRA encompasses 2,096 short human exons from 1,733 genes and tens of thousands of single and double variants. Our measurements identify variants that strongly modulate splicing but are currently classified as variants of unknown significance or have not yet been described. We systematically identify putative RNA-binding protein (RBP) binding motifs and quantify effect sizes associated with the disruption of these motifs, suggesting possible mechanisms for variant impact.  A comparison across cell lines enables the identification of variants that differentially modulate splicing.

overall_design: Massively parallel splicing reporter assay (MPRA) testing ~87,000 exon and variant sequences cloned into a Citrine–SMN2 minigene backbone. Constructs were transfected into five human cell lines (HEK293, HeLa, K562, MCF7, HMC3) with biological replicates. Sequencing was used to quantify exon inclusion (PSI) across replicates, and processed PSI values are reported.

middle: E

first: Angela

last: Yu

first: Georg

last: Seelig


title: Integrative bioinformatics analyses identify an essential role of ZBP1 in myocardial I/R injury

city: Shanghai

first: Xiaokai

state: Please Select

address: 180 Fenglin Road

institute: Zhongshan hosipital

summary: Myocardial ischemia-reperfusion (I/R) injury is the primary factor that counteracts the beneficial effects of reperfusion therapy. However, the distinct pathophysiological processes and underlying mechanisms that govern the ischemic and reperfusion phases of myocardial I/R injury remain incompletely understood. Time-series RNA-seq analyses were conducted on myocardial samples from the ischemic regions of wild-type (WT) mice after being subjected to either sham surgery or 30 minutes of ischemia followed by reperfusion (duration: 0, 1, 6, and 24 hours). Principal component analysis (PCA) demonstrated a clear segregation of gene-expression profiles between the ischemic and reperfusion stages of myocardial I/R injury. Zbp1 remained unchanged at 0 hours post-I/R injury but were consistently upregulated at 1, 6, and 24 hours post-I/R injury.

overall_design: To elucidate the dynamic molecular changes following myocardial I/R injury, time-series RNA-seq analyses were conducted on myocardial samples from the ischemic regions of wild-type (WT) mice after being subjected to either sham surgery or 30 minutes of ischemia followed by reperfusion (duration: 0, 1, 6, and 24 hours)