title: Identification of Resistance Mechanisms to KRAS Inhibition in Pancreatic Cancer

city: Cincinnati

first: Andrew

last: Waters

state: Ohio

address: Vontz Center for Molecular Studies, Rm 1234, 3125 Eden Ave

institute: University of Cincinnati

summary: KRAS inhibitors (KRASi) targeting various KRAS mutations have entered clinical trials for pancreatic cancer. Despite promising preliminary clinical responses, most patients relapse due to intrinsic and acquired resistance. Thus, combination treatments are essential to extend the efficacy of KRAS-targeted therapies. To further determine genetic mechanisms of KRASi resistance, we performed KRASi-anchored CRISPR-Cas9 loss-of-function screens in KRAS G12D-, KRAS G12R-, and KRAS Q61H-mutant PDAC cell lines, using six KRASi, to identify genes, that when lost, modulate sensitivity to KRAS inhibition. We pharmacologically validated several hits from our screens, including EGFR, CK2 p110 alpha and p110 gamma, and YAP, by combining targeted inhibitors with KRASi. We determined that KRAS Q61H-mutant PDAC cell lines are intrinsically less dependent on KRAS for survival than other KRAS mutational subtypes. Further, we found that EGFR inhibitor erlotinib synergized with the RAS(ON) multi-selective inhibitor RMC-7977 in KRAS Q61H-mutant PDAC cell lines, and in cell lines with highly active EGFR, by mitigating ERK rebound activity. We also developed KRASi-resistant cell lines and observed sustained ERK MAPK dependence in the KRASi-resistant cell lines despite decreased ERK activity. Our findings enhance the understanding of KRASi intrinsic and acquired resistance and identify therapeutic vulnerabilities that can potentially be exploited for KRASi combination therapies in patients with PDAC.

overall_design: RNA-Seq of 12 PDAC cell lines with the goal of identifying differences between KRAS Q61H-mutant and non-KRAS Q61H-mutant cell lines.


title: Efficient Chemical Reprogramming of Human T Cells into Functional Megakaryocytes and Platelets

status: Public on Nov 27 2025

city: Beijing

first: Jinhua

last: Qin

address: Taiping Road

country: China

institute: Beijing Institute of Transfusion

type: Expression profiling by high throughput sequencing

summary: The generation of megakaryocytes (MKs) from human somatic cells through chemical reprogramming represents a promising strategy for developing alternative platelet sources. Building on our prior chemical reprogramming protocol for converting erythroblasts to MKs, we established a robust method that successfully generated induced MKs (iMKs) from human cord blood-derived CD3⁺ T cells, which is a more abundant source. This method utilized a five- small-molecule (5M) cocktail containing a reprogramming booster, AZD4205, to promote erasure of T cell identity and facilitate fate transition towards MKs. T cell-derived iMKs exhibited characteristic MK cellular and molecular signatures, demonstrating the capacity to produce proplatelets and release functional platelets in vitro and in vivo. ScRNA-sequencing further revealed that iMKs were heterogeneous with distinct functional profiles, including cycling, immune, and thrombopoiesis-biased MKs. Our findings highlight an optimized chemical reprogramming pathway that enables efficient conversion of T cells to MKs, providing a practical and convenient approach to generate clinically relevant MKs and platelets.

overall_design: We performed Bulk RNA-seq and single cell RNA-Seq to analyse the molecular mechanism of  chemical reprogramming system.Bulk RNA-seq:CD3⁺ T cells cultured under medium containing 4M (Bix01294, RG108, PD0325901, and VPA) and/or AZD4205 conditions for 7 days were collected for bulk RNA-seq. CD3⁺ T cells induced with 5M (Bix01294, RG108, PD0325901, VPA, and AZD4205)were collected for bulk RNA-seq. Single-cell RNA-seq: Isolated CD3+ T cells (day 0) and viable cells from the iMK induction culture on day 7 and day 14 were collected and analyzed using the Chromium system (10X Genomics) according to the manufacturer’s instructions for an expected capture rate of approximately 10,000 single cells per sample.

first: Xuetao

last: Pei

first: Yanhua

last: Li


title: COPG1 Is a Selectively Essential Regulator  of Cancer Progression and Chemoresistance: A Comprehensive Pan-Cancer Study

city: 양산시

first: Susmita

last: Sen

address: 경남 양산시 물금읍 범어리 2762-1

department: Department of Anatomy

country: South Korea

institute: Pusan National University

summary: The coatomer complex, a key regulator of intracellular vesicular transport, has recently been implicated in cancer progression, yet a comprehensive pan-cancer analysis of its subunits has been lacking. Here, we systematically profiled genetic alterations, expression patterns, prognostic relevance, and functional dependencies of all COPI and COPII coatomer subunits across 33 cancer types using more than 10,000 tumor samples from The Cancer Genome Atlas (TCGA), complemented by functional perturbation data from CRISPR (n = 1,178) and RNAi (n = 707) screens in DepMap. Gene amplification—most notably of COPB2—was the most frequent alteration and was associated with poor survival in bladder and esophageal cancers. Mutations in COPA and SEC31A also demonstrated prognostic significance in endometrial carcinoma. Expression analyses revealed broad upregulation of coatomer genes across cancer types, with COPG1 and COPB1 emerging as strong risk-associated genes (HR > 2). Integrative functional dependency analyses further identified COPG1 and COPE as selectively essential in multiple cancer contexts, with COPG1 loss notably linked to increased drug sensitivity. Functional validation in hepatocellular carcinoma (HCC) showed that COPG1 knockdown impaired malignant phenotypes and reduced tumorigenicity in vivo. Mechanistically, COPG1 depletion induced Golgi disruption and ER stress, increased ROS production, and suppressed PI3K–AKT signaling. Moreover, COPG1 knockdown sensitized HCC cells to sorafenib and doxorubicin, whereas COPG1 overexpression conferred resistance. Collectively, our pan-cancer analysis reveals context-dependent roles of coatomer subunits and identifies COPG1 as a novel oncogenic driver and potential therapeutic target in HCC, mediating chemoresistance through redox modulation and PI3K–AKT pathway inhibition.

overall_design: Total RNA was extracted using the RNeasy Mini Kit (Qiagen). RNA quality was assessed by running 1 μl of RNA on a Bioanalyzer system (Agilent, CA, USA) to ensure the RIN and rRNA ratio. We used 100 ng of total RNA to prepare the sequencing libraries using the MGIEasy RNA Directional Library prep kit (MGI). They were quantified using the Agilent 2100 BioAnalyzer (Agilent) according to the manufacturer’s library quantification protocol. Following the cluster amplification of the denatured templates, sequencing was performed as paired-end (2 × 150 bp) using the MGI DNBSEQ-T7 platform. Data processing was performed as previously reported(15). Briefly, raw sequencing reads were processed by FastQC (RRID:SCR_014583) and aligned by STAR (RRID:SCR_004463) to the human reference genome hg38 with Ensembl annotation. Transcript abundance was quantified using RSEM5 in the units of TPM. Differential gene expression analysis was performed using DEseq2 (RRID:SCR_015687).

first: Van-Thanh

last: Duong

first: Youngin

last: Hwang

first: Seungmi

last: Kim

first: Euijin

last: Lee

first: Myoung-Eun

last: Han

first: Dongjun

first: Yun Hak

first: Sik

last: Yoon

first: Sae-Ock

last: Oh


title: CDCA7 facilitates MET1 mediated CG methylation maintenance at pericentromeric and centromeric chromatin [RNA-seq]

city: Los Angeles

first: Shuya

last: Wang

state: California

address: 610 Charles E Young Dr S

department: Molecular, Cell and Developmental Biology

country: USA

institute: University of California, Los Angeles

summary: DNA methylation is a conserved epigenetic modification critical for transposable element (TE) silencing and genome stability. However, how methyltransferases maintain CG methylation within compact heterochromatin remains unclear. In humans, CDCA7 is required for DNA methylation inheritance at juxta-centromeres, and mutations impairing its binding to heterochromatic nucleosomes lead to satellite DNA hypomethylation and ICF syndrome. In Arabidopsis thaliana, we identified two CDCA7 orthologs, CDCA7A and CDCA7B, as key regulators of DNA methylation. Loss of both causes CG hypomethylation specifically at pericentromeric and centromeric regions. Machine learning analysis revealed that nucleosome density, H1 enrichment, the H2A.W variant, and H3K9me2 levels predict methylation loss in cdca7a cdca7b mutants. Notably, H1 depletion restores methylation in these mutants, indicating that CDCA7A and CDCA7B enable MET1 access to H1-containing nucleosomes and are particularly required for methylation maintenance in compact chromatin. Additionally, in h1.1 h1.2 mutants, CG methylation increases modestly at pericentromeric regions but significantly at centromeric sites, revealing an inhibitory role for H1 in centromeric methylation. This hypermethylation is abolished in h1.1 h1.2 cdca7a cdca7b quadruple mutants, demonstrating that CDCA7A and CDCA7B promote MET1 activity at heterochromatic nucleosomes independently of H1. Together, these results establish CDCA7A and CDCA7B as conserved and versatile chromatin remodelers that facilitate MET1 activity within heterochromatin.

overall_design: Employ WGBS and RNA-seq to study the role of CDCA7 in DNA methylation maintenance


title: CDCA7 facilitates MET1 mediated CG methylation maintenance at pericentromeric and centromeric chromatin [BiSulfite-seq]

type: Methylation profiling by high throughput sequencing


title: Detached osteoclasts in the granulation tissue surrounding sequestra in medication-related osteonecrosis of the jaw

city: Kobe

first: Takuo

last: Emoto

state: Hyogo

address: 7-5-1 Kusunoki-cho, Chuo-ku

department: Division of Cardiovascular Medicine, Department of Internal Medicine

country: Japan

institute: Kobe University Graduate School of Medicine

summary: Medication-related osteonecrosis of the jaw (MRONJ) is a rare but serious complication of anti-resorptive agents used for osteoporosis and bone metastasis. As the global population ages, the number of patients with MRONJ continues to rise. Resorptive osteoclasts firmly attached to bone surfaces cannot be detected using single-cell RNA-Seq (scRNA-Seq); however, we hypothesized that multinucleated giant and detached osteoclasts (DetOCs) can be detected by this method. scRNA-Seq was performed on granulation tissues surrounding sequestra from four patients with MRONJ and radicular cysts, a common odontogenic infectious disease with normal bone metabolism. Our results show that DetOCs were detected exclusively in the granulation tissues of MRONJ. These DetOCs expressed well-known osteoclast markers as well as COL27A1. Subcluster analysis revealed that SPP1+ TREM2+ macrophages differentiated into DetOCs and expressed several types of collagens. Immunostaining confirmed the expression of COL27A1 in DetOCs in MRONJ, as well as in osteoclasts in mandibular cancer. Overall, we successfully identified DetOCs in the granulation tissues surrounding sequestra in MRONJ, and our scRNA-Seq analysis suggests the potential to classify osteoclast subtypes based on the expression of collagen types.

overall_design: Single-cell gene expression profiling was performed using the Chromium Fixed RNA Profiling workflow (CG000527; 10x Genomics). Library preparation involved the Chromium Fixed RNA Kit (Human Transcriptome) and the Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit. Following hybridization of detection probes to RNA within fixed cells, adjacent probes were joined through a ligation reaction. Samples were then pooled to normalize cell counts across groups and processed on the Chromium X instrument, where cells were encapsulated with barcoded primer beads. Annealing and extension reactions added cell-identifying adapter sequences, and indexed primers were used for PCR amplification and library purification, generating sequencing-ready libraries.

first: Chika

last: Nakanishi

first: Takeshi

last: Yoshida

first: Masaya

last: Akashi


title: The transcriptomic data of HaCaT cells with and without DNA methyltransferase inhibitors (DNMTi) treatment

city: Shiyan

first: Qin

last: Shanshan

address: Renmin Road 30, Maojian District

institute: Institute of Basic Medical Sciences, School of Basic Medical Sciences, Hubei University of Medicine

summary: This study aimed to explore the genes repressed by DNA methylation, since their expression will be activated by DNMTi treatment.

overall_design: DNA methylation negatively regulates gene expression at pre-transcription level. Thus, we aimed to identify potent genes upregulated by 5-Aza-CdR (25, 50 μM) in HaCaT cells.

first: Shanshan

first: Dandan


title: The transcriptomic data of TCN1 overexpression in HaCaT cells

summary: This study aimed to explore the underlying molecular mechanism of TCN1 in driving psoriasis progression. TCN1 has been identified as an autoantigen and an endogenous activator of the IL-17 signaling pathway.

overall_design: The estabilished TCN1 overexpression  and negative control  cell lines (HaCaT cells) were seeded into 6-well plates and grown overnight for total RNA harvest. The differentally expressed genes by TCN1 overexpression were screened by RNA sequencing. The transcriptome sequencing studies were performed by deep sequencing, using Illumina NovaSeq X Plus.

last: Qin


title: Rapid clearance of bacteria from maternal bloodstream after delivery in pregnancies complicated by pre-labor premature rupture of the membranes.

city: Chicago

first: William

last: Ackerman

state: Illinois

address: 820 South Wood Street, MC 808, Dept. of Obstetrics & Gynecology

department: Obstetrics and Gynecology

institute: University of Illinois Chicago College of Medicine

summary: Objective: We investigated whether birth in patients whose pregnancies are complicated by pre-labor premature rupture of the membranes (PPROM) is associated with translocation of microbes in maternal circulation and a systemic inflammatory process. Research Design and Methods: 66 pregnant patients admitted for PPROM [gestational age (GA) median [IQR]: 32±1 weeks] had maternal blood (MB) retrieved prior to birth and within 1-hour post-partum. Immediately after birth, fetal membranes (FM) and placental tissue were frozen in sterile fashion. Bacterial load was assessed by 16S rDNA gene amplification by qPCR. Multiplexed 16S amplicon libraries were sequenced on the Illumina platform and enrichment analysis performed using LfSe and EdgeR. Pro- and anti-inflammatory cytokines were measured in MB using multiplex immunoassay. Histological chorioamnionitis (HCA) and cord blood haptoglobin and IL-6 indicated fetal exposure to antenatal inflammation (Triple I). Results: Bacterial DNA was identified in MB both before and after birth. A significant decrease in MB bacterial DNA was observed within 1-hour post-delivery (p=0.004). Conversely, MB cytokines IL-6 and Il-10 increased significantly (p<0.05 for all) especially in pregnancies exposed to Triple I. Compared to placenta, FM carried a higher bacterial load (p<0.001) regardless of presence or absence of antenatal inflammation. FM had higher bacterial biodiversity with top representation from Mycoplasma spp although Mycoplasma was not found in MB either pre- or post-delivery. While no taxa were systematically enriched in MB post-delivery, sample-by-sample analysis identified three cases where a MB OTU had a match in FM and four cases with a match in placenta. Yet, the most abundant MB OTUs had no match in either tissues. Conclusion: In patients with PPROM, bacterial DNA is present in MB prior to birth but is rapidly removed after delivery in association with a robust immediate postpartum cytokine surge.

sample_organism:
human metagenome
human blood metagenome

platform_organism:
human metagenome
human blood metagenome

overall_design: Using a prospective observational study design, we enrolled 66 consecutive pregnant patients who were admitted on the antepartum service of our academic university hospital for management of pre-labor premature rupture of the membranes (PPROM) at ≤34 weeks gestational age (GA). Subjects were eligible for enrollment if older than 18 years of age, pregnant between 230/7 to 336/7 weeks gestational age and at high risk of preterm birth secondary to PPROM. Exclusion criteria were anticipated delivery within 2 hours, need for close medical supervision (e.g. cardiac and renal disease, congestive heart failure, history of asthma), maternal viral infection (HIV, hepatitis B or C), known active infections or antibiotic use 7 days prior to admission, need for emergent delivery (e.g. cord prolapse, abruption) and known fetal malformations. Maternal blood (MB) was retrieved via sterile venous puncture prior to birth and within 1-hour post-partum (PP). An alcohol swab was used to prepare the skin area before venipuncture. The blood was collected in serum and plasma citrate tubes (BD Vacutainer, Franklin Lakes, NJ) and transported to the laboratory. An aliquot of whole blood from the plasma tube was first saved for bacterial DNA isolation. The remainder blood volume was spun at 1,500 g for 15 min and plasma and serum aliquoted for cytokine measurements. Cord blood was collected at delivery from all newborns of mothers enrolled in this study. After delivery of the placenta, a loop of the umbilical cord was placed on a sterile field and cord blood was collected by sterile puncture of the umbilical vein. The cord blood was processed as described above for MB. In a subset of deliveries (n=31 newborns: 23 singletons and 4 pairs of twins), a biopsy from FM (full thickness amniochorion) and from the villous tissue in center of the placental disk were retrieved in strict sterile fashion within minutes from delivery. Collection of delivery tissues was contingent on availability of research staff at delivery. Following dissection of the decidua basalis, the villous trophoblast tissue was frozen immediately in liquid nitrogen. The fetal membrane biopsy was rinsed briefly in sterile saline to remove any AF and adherent blood prior to freezing in sterile DNA-free tubes.

first: Irina

middle: A

last: Buhimschi

middle: E

first: Catalin

middle: S


title: Comparison of expression profiles of Control and CDDO-Im treatment microglia under IFN-gamma or Amyloid beta stimulation

city: Sendai

first: Masayuki

last: Yamamoto

state: Miyagi

address: 2-1 Seiryo-cho, Aoba-ku

institute: Tohoku University Graduate School of Medicine

summary: Alzheimer’s disease (AD) is characterized by amyloid-beta (Amyloid beta) plaques and neurofibrillary tangles, accompanied by elevated oxidative stress and inflammation. Microglia, the resident macrophages in the brain, play a key protective role by clearing plaques and damaged neurons. NRF2 (Nuclear factor erythroid 2-related factor 2) is a master regulator of cytoprotection against oxidative stress, whose activation alleviates oxidative damage, neuroinflammation, and cognitive deficits in AD models. However, direct targets of NRF2 in microglia remain unclear. In this study, we demonstrate that NRF2 activation by CDDO-Im significantly suppresses inflammation in human microglial cells (HMC3) stimulated by IFN-gamma or Amyloid beta. Through integrative RNA-sequencing and ChIP-sequencing analysis of NRF2, we identified five representative NRF2 directly target genes involved in inflammation (e.g., IL6, CDK6) and another five in autophagy (e.g., TFE3, SQSTM1). Public single-cell transcriptomic data further underscored the critical role of microglia in NRF2-mediated autophagy regulation within AD brains. Our findings revealed new direct NRF2 target genes, highlighting dual role of NRF2 in suppressing inflammation and enhancing autophagy, thus providing novel insights for therapeutic interventions in AD.

overall_design: mRNA profiles of control and CDDO-Im treatment human microglia cell line (HMC3) under IFN-gamma or Amyloid beta- stimulated condition were generated by deep sequencing, in triplicate.

first: ChingTung

last: Chu

first: Akria

last: Uruno

first: Fumiki

last: Katsuoka


title: NRF2 binding in HMC3 human microglia

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Alzheimer’s disease (AD) is characterized by amyloid-beta (Aβ) plaques and neurofibrillary tangles, accompanied by elevated oxidative stress and inflammation. Microglia, the resident macrophages in the brain, play a key protective role by clearing plaques and damaged neurons. NRF2 (Nuclear factor erythroid 2-related factor 2) is a master regulator of cytoprotection against oxidative stress, whose activation alleviates oxidative damage, neuroinflammation, and cognitive deficits in AD models. However, direct targets of NRF2 in microglia remain unclear. In this study, we demonstrate that NRF2 activation by CDDO-Im significantly suppresses inflammation in human microglial cells (HMC3) stimulated by IFN-γ or Aβ. Through integrative RNA-sequencing and ChIP-sequencing analysis of NRF2, we identified five representative NRF2 directly target genes involved in inflammation (e.g., IL6, CDK6) and another five in autophagy (e.g., TFE3, SQSTM1). Public single-cell transcriptomic data further underscored the critical role of microglia in NRF2-mediated autophagy regulation within AD brains. Our findings revealed new direct NRF2 target genes, highlighting dual role of NRF2 in suppressing inflammation and enhancing autophagy, thus providing novel insights for therapeutic interventions in AD.

overall_design: Chromatin occupancy of NRF2 under CDDO-Im-treated condition were generated by deep sequencing, in dupliplicate


title: Identification of differentially expressed genes in psoriasis by RNA sequencing

summary: This study aimed to explore the novel differentially expressed genes in psoriasis.

overall_design: 6 normal skin tissues and 8 psoriasis tissues were collected for RNA sequencing. The transcriptome sequencing studies were performed by deep sequencing, using Illumina NovaSeq X Plus.

last: Li


title: The sleep–wake history contributes to rhythmic BMAL1 chromatin binding in the cerebral cortex but not in the liver

city: Lausanne

first: Carlos

middle: Eduardo Sousa

last: Neves

state: Vaud

address: Genopode, UNIL-Sorge

department: Faculté de biologie et de médecine

country: Switzerland

institute: University of Lausanne

summary: The timing and quality of sleep is regulated by circadian- and sleep–wake-driven processes. The core clock gene Bmal1 not only affects the circadian timing of sleep, but also the response to sleep deprivation (SD), which, in turn, causes long-term changes in cortical Bmal1 expression. We aimed at separating the circadian- and sleep–wake-driven contributions to BMAL1 binding to its target genes in the cerebral cortex by scheduling 6 SDs at 4 h intervals across the daily 12 h light/12 h dark cycle. We show that BMAL1 rhythmically bound its tissue-specific targets with tissue-specific dynamics, reaching peak binding 2 to 4 h later in the cortex than in the liver, while trough times did not differ. The SDs affected BMAL1 binding most significantly in the cortex, causing 80% of rhythmically bound regions to lose rhythmicity, suggesting BMAL1 binding has a prominent sleep–wake-driven component in this tissue. Analyses of the promoters of other core clock-genes indicate that BMAL1 binding to Bhlhe41 and Nr2d1 have a strong sleep–wake-driven component, while for Per2 two binding regions were identified one with circadian- and the other with sleep–wake-driven binding dynamics. Our results attest to a nonadditive interaction of time-of-day and time-spent-awake affecting the core molecular circadian circuitry. It further highlights that rhythms in gene expression in peripheral tissues are an emergent property of molecular interactions beyond that of the core molecular clock circuitry.

overall_design: 6h sleep deprivation prior to the sampling of cortex and liver tissue ‘around the clock’ (ZT-2,-6,-10,-14,-18,-22). ChIP-seq with BMAL1 antibody was performed in triplicates. Input triplicates were sequenced as one.

first: Charlotte

middle: N

last: Hor

first: Sonia

last: Jimenez

first: Paul

last: Franken


title: Exploiting the polypharmacology of alectinib for synergistic RNA splicing disruption with RBM39 degraders

city: London

first: Yurui

last: Ma

state: london

address: du cane road

country: United Kingdom

institute: imperial college london

summary: Precise control of pre-mRNA splicing is critical for transcriptome integrity, and its disruption is increasingly recognised as a vulnerability in cancer. Here, we identify a functional interplay between two key splicing regulators, RBM39 and serine/arginine protein kinase 1 (SRPK1), and show that dual targeting of these factors severely compromises splicing fidelity in high-risk neuroblastoma. We use the molecular glue indisulam to degrade RBM39 and repurpose the clinical ALK inhibitor alectinib which potently inhibits SRPK1. Co-treatment with indisulam and alectinib inhibited cell proliferation, induced apoptosis, and caused G2–M arrest in multiple cancer cell lines, including MYCN-amplified neuroblastoma. RNA sequencing revealed enhanced splicing defects preferentially in DNA repair and genome maintenance related genes following combination treatment, leading to R-loop accumulation and increased DNA damage. In the Th-MYCN/ALKF1174L neuroblastoma mouse model, combination therapy induced complete tumour regression and significantly improved survival rates compared with monotherapies. These findings demonstrate that combining indisulam and alectinib is a promising approach to treat aggressive malignancies such as high-risk neuroblastoma, exploiting the previously untapped polypharmacology of alectinib as a clinical RNA splicing inhibitor and supporting the therapeutic value of co-targeting interdependent splicing factors for synergistic benefit.

overall_design: RNA-seq profiling of IMR32 cells treated with DMSO, 4uM Alectinib, 1.25uM Indisulam or the combination for 24h

first: Evon

last: Poon

first: Chenchen

last: Jin

first: Barbara

last: Martins da Costa

first: Yuewei

last: Xu

first: Sadiya

last: Quazi

first: Nikolaos

last: Zourdoumis

first: Chiharu

last: Wickremesinghe

first: Louis

last: Chesler

first: Hector

middle: C

last: Keun

first: Anke

last: Nijhuis


title: YKT6 promotes bladder cancer progression by stabilizing β-Catenin via USP7-mediated deubiquitination

city: Wuhan

first: Yu

last: Xiao

address: Donghu Road 169

department: Department of Urology

institute: Zhongnan Hospital of Wuhan University

summary: Bladder cancer (BLCA) remains a highly lethal genitourinary malignancy with complex tumor biology and limited therapeutic strategies. This study investigates the oncogenic role of YKT6, a SNARE protein, in BLCA progression and molecular mechanisms. We demonstrate that YKT6 is significantly upregulated in BLCA tissues and cell lines, correlating with advanced tumor grade, aggressive histology, and poor patient prognosis. Comprehensive transcriptomic and functional analyses reveal that YKT6 promotes BLCA cell proliferation, migration, and metastasis both in vitro and in vivo. Mechanistically, YKT6 activates the Wnt/β-catenin signaling pathway through a novel mechanism involving USP7-mediated deubiquitination of β-catenin. By recruiting USP7, YKT6 inhibits β-catenin’s proteasomal degradation, thereby stabilizing the protein and driving nuclear accumulation. This stabilization leads to increased expression of oncogenic target genes and induces epithelial-mesenchymal transition (EMT). Pharmacological interventions demonstrated that Wnt signaling inhibition reverses YKT6-driven malignant effects, while pathway activation restores tumor progression in YKT6-silenced cells. USP7 knockdown abrogates YKT6-mediated β-catenin stabilization, confirming their functional interdependence. Clinically, the YKT6/USP7/β-catenin axis strongly correlates with poor prognosis, highlighting its potential as a diagnostic and therapeutic target. Our findings unveil YKT6 as a novel regulator of Wnt signaling through USP7-dependent deubiquitination, offering promising insights for precision BLCA therapy.

overall_design: To uncover the underlying mechanism by which YKT6 promotes BLCA tumor growth and metastasis, RNA-seq analysis was performed on 5637 cells transfected with siNC or siYKT6.

first: Sheng

last: Tu

first: Xinghuan

last: Wang

first: Kaiyu

last: Qian


title: Quantitative analysis of small RNA pseudouridylation reveals interplay of PUS enzymes in tRNA anticodon stem-loop

first: Wenqing

last: Liu

address: No. 5, Yiheyuan Road, Haidian District, Beijing

institute: Peking University

summary: Pseudouridine (Ψ) is an abundant modification in small RNA and is catalyzed by multiple pseudouridine synthases (PUSs). However, the substrate specificity of human PUSs are not fully understood. In this study, we adopted PRAISE, a quantitative Ψ detection method, to profile pseudouridylation in small RNA, including cytosolic and mitochondrial tRNAs, snRNA, and snoRNA. We found that pseudouridylation of snoRNAs can be mediated not only by RNA-guided DKC1, but also stand-alone enzyme PUS7. Interestingly, we found that several PUS enzymes, which install nearby Ψ sites within tRNA anticodon stem-loop, can influence pseudouridylation level catalyzed by other PUSs. Typical examples include PUS1, RPUSD1, and PUS7, revealing a novel interplay among human PUSs during Ψ formation. For the three RluA family enzymes, we found that in addition to the canonical substrate Ψ30, RPUSD1 also catalyzes Ψ72 of tRNA-Arg isoacceptors. RPUSD2 pseudouridylates Ψ31 of mt-tRNALeu(CUN), and Ψ32 of mt-tRNAPro and mt-tRNACys, and hence is functionally similar to yeast Pus9. RPUSD3 does not modify tRNA at all, consistent with its lack of a catalytic residue. Together, using quantitative Ψ profiling, our study characterized the tRNA substrates of PUS enzymes and revealed unexpected interplay among human PUSs in tRNA pseudouridylation.

overall_design: We detected the Ψ sites in small RNAs of HEK293T cells to profile the quantitative landscape. We then identified the DKC1-dependent sites of snRNA and snoRNA in DKC1 knockdown cell lines. After that, we identified substrates of 6 PUS enzymes in PUS1, PUS7, TRUB1, RPUSD1, RPUSD2, and RPUSD3 cell lines.


title: Genetic deficiency of C–C chemokine receptor 4 protects against angiotensin II-induced abdominal aortic aneurysm formation in mice

first: Takuo

last: Emoto

summary: Background: The dysregulated immune system, which drives chronic vascular inflammation and remodeling, plays a critical role in the pathogenesis of abdominal aortic aneurysm (AAA). C–C chemokine receptor 4 (CCR4), which is predominantly expressed on T cells and mediates their responses, has been shown to protect against inflammatory diseases including atherosclerosis. However, its role in AAA remains unknown. Methods: By analyzing hypercholesterolemic CCR4-deficient (Ccr4-/-) mice on an apolipoprotein E-deficient (Apoe-/-) background, we investigated the role of CCR4 in the development of angiotensin II-induced AAA, with a particular focus on T cell-mediated immune responses, by performing histological analysis, flow cytometry, biochemical assays, and single-cell RNA sequencing analysis. Results: Genetic deletion of CCR4 on an Apoe-/- background dramatically reduced the incidence and severity of angiotensin II-induced AAA without affecting mortality due to rupture, along with attenuated inflammatory cell recruitment and preserved elastic lamellae. This protective effect was associated with a shift in the T helper cell balance toward T helper type 1 cell predominance, characterized by promoted IFN-γ production and suppressed B cell-mediated immunity including lower plasma IgE levels. Single-cell RNA sequencing analysis of abdominal aortic tissues revealed lower proportions of myofibroblasts and modulated smooth muscle cells highly expressing genes associated with extracellular matrix remodeling in angiotensin II-infused Ccr4-/-/Apoe-/- mice, potentially driven by downregulated TGF-β signaling due to T helper type 1 cell-derived IFN-γ predominance. Conclusions: Our data suggest that CCR4 blockade may serve as a promising immunotherapeutic approach for AAA.

overall_design: Abdominal aortas were isolated from angiotensin II-infused Apoe-/- or Ccr4-/-Apoe-/- mice and frozen in liquid nitrogen within 6 hours after sample collection. For single cell RNA-seq, the libraries were prepared as according to the 10x Genomics Chromium Fixed RNA Profiling for Multiplexed Samples v1 guidelines (CG000527).

first: Aga

last: Krisnanda

first: Naoto

last: Sasaki


title: Autophagy Impairment in Retinal Ganglion Cells Following Hypoglycemia in Mice.

first: Raphaël

last: Roduit

address: Avenue de France 15

department: Lausanne University Ophtalmology

institute: Jules-Gonin Eye Hospital

summary: Diabetic retinopathy (DR), caused by hypo- and hyperglycemia, is a leading cause of blindness. Hypoglycemia induces endoplasmic reticulum stress and retinal cell death in mice, and low glucose conditions induce macroautophagy/autophagy defects in 661W photoreceptor cells and retinal explants. To further understand the effect of hypoglycemia on the mouse retina, we studied autophagy in vivo. Retinal explants cultured under low glucose conditions exhibited autophagy activation; a similar pattern was observed in the presence of autophagy modulators. Intraocular injection of rapamycin and 5 h hypoglycemia had similar effects, specifically in the ganglion cell layer, as shown by green fluorescence protein (GFP) colocalization with different ganglion cell markers. We then isolated retinal ganglion cells (RGCs) from two mouse lines and cultured them at different glucose concentrations. We also assessed autophagosome/lysosome fusion in C57BL/6 RGCs infected with the RFP-GFP-LC3 lentivirus and cultured under different conditions. p62 protein expression and GFP fluorescence increased when RGCs were cultured with 1 or 25 mM glucose in the presence of rapamycin, whereas no sign of autophagy was detected with 5 or 25 mM glucose alone. The fusion process was defective at 1 and 25 mM glucose in the presence of chloroquine, but normal with 5 and 25 mM glucose alone. This study showed that 5 h hypoglycemia induces autophagosome formation in mouse RGCs; however, a defect in the fusion process inhibits the protective effect of autophagy. Therefore, the modulation of both autophagic and apoptotic pathways might be important to avoid complications associated with DR.

overall_design: RNA-Seq analyses on six samples after laser capture microdissection of ganglion cell layer from 3 GFP-LC3 mice underwent a 5 h hypoglycemia and 3 controls mice that were maintain euglycemic during 5 h.


title: Metabolic regulation of Th2 cells by L-phenylalanine

city: Davos

first: Abhijeet

last: Kulkarni

address: Herman-Burchard-Strasse 9

institute: SIAF

summary: RNA-Seq of in vitro differentiated Th2 cells subjected to L-phenylalanine treatment

overall_design: In vitro long differentiated Th2 cells from 5 healthy donors were incubated in RPMI+10%Fetal Calf Serum +100IU recombinant human Interleukin 2 with Vehicle or 1mM additional L-Phenylalanine with or without simultaneous activation with anti-CD2, anti-CD3 and anti-CD28 antibody beads (1:1 beads:cells).

first: Nino

last: Stocker

first: Urszula

last: Radzikowska

first: Ines

last: Jardon Parages

first: Milena

last: Sokolowska


title: Integrated analysis of differential m6A methylation and transcriptome profiles in ovaries of young and aged Cele black sheep

city: Alar City, Xinjiang Uygur Autonomous Region

first: linlin

last: pei

state: Aral

address: Alar City Happy Road Street

institute: Tarim university

summary: Ovarian aging results in follicular failure and atresia, leading to a decline in both the quantity and quality of oocytes. N6-methyladenosine (m6A) has been demonstrated to play a regulatory role in the reproductive functions of female animals; however, the regulatory mechanisms of m6A in the ovaries of sheep remain unclear. In the present study, ovarian tissues from 1~2 year-old and 5~6 year-old Cele black sheep were collected and analyzed using MeRIP-seq and RNA-seq sequencing. A total of 109 differentially methylated genes were identified, including 7 genes that were up-regulated in both methylation and mRNA expression, 55 genes that were up-regulated in methylation while down-regulated in mRNA expression, and 33 genes that were down-regulated in both methylation and mRNA expression. Additionally, 14 genes exhibited down-regulation in methylation and up-regulation in mRNA expression. Differential methylation peaks (DMPs) were found to be significantly enriched in the BMP signaling pathway, uterine embryonic development, histone acetyltransferase activity, and VEGF signaling pathway by GO and KEGG enrichment analysis. Differentially expressed genes (DEGs) were significantly enriched in negative regulation of the I-kappaB kinase/NF-kappaB signalling pathway, positive regulation of leukocyte adhesion to vascular endothelial cells, histone methyltransferase binding, transcriptional co-repressor activity, and cellular senescence. The results suggest that differentially expressed genes may influence various biological functions, including transcription, translation, aging, development, regeneration, and immune responses in the ovary of sheep. In this study, we constructed the first comprehensive whole transcriptome analysis map of the sheep ovary, which provides a theoretical foundation for investigating the transcriptional regulation mechanisms of m6A modification in the ovary.

first: Linlin

last: Pei

first: Chunjie


title: Genomic localization and functions of MBD-2 with and without NuRD in DNA methylation-free C. elegans [ChIP-Seq]

city: Hong Kong

first: Wing Yee Karen

last: Yuen

address: 4S15, Kadoorie Biological Science building, The University of Hong Kong, Pok Fu Lam Road

department: School of Biological Sciences

country: Hong Kong

institute: The University of Hong Kong

summary: While mammalian MBD2 is a well-established 5-Methylcytosine reader and component of the essential Nucleosome Remodeling and Deacetylase (NuRD) complex, the function and binding pattern of C. elegans MBD2 (Cel-MBD-2) in its 5mC-free genome remained uncharacterized. In our study, Cel-mbd-2 null mutation resulted in a wide range of severe morphological defects and sterility. We have also confirmed that Cel-MBD-2 is a key transcriptional modulator that can regulate genes in both directions. The MBD-2 binding patterns in the gene promoter or gene body are linked with the gene regulatory directions and display distinct correlations with histone modifications within and outside the context of NuRD complex.

overall_design: ChIP-seq of Cel-MBD-2 using anti-FLAG magnetic beads targeting GFP::3xFLAG::MBD-2

first: Hok Ning

last: Tsui


title: Genomic localization and functions of MBD-2 with and without NuRD in DNA methylation-free C. elegans [RNA-Seq]

summary: While mammalian MBD2 is a well-established 5-Methylcytosine reader and component of the essential Nucleosome Remodeling and Deacetylase (NuRD) complex, the function and binding pattern of C. elegans MBD2 (Cel-MBD-2) in its 5mC-free genome remained uncharacterized. In our study, Cel-mbd-2 null mutation resulted in a wide range of severe morphological defects and sterility. Coiled-coild domain deletion of Cel-mbd-2 phenocopied the null mutant and led to similar transcriptomic changes. We have also confirmed that Cel-MBD-2 is a key transcriptional modulator that can regulate genes in both directions. The MBD-2 binding patterns in the gene promoter or gene body are linked with the gene regulatory directions and display distinct correlations with histone modifications within and outside the context of NuRD complex.

overall_design:
RNA-seq of N2 wildtype and mbd-2 null mutant synchronized young adult worms. The mutant is maintained as a heterozygous stock balanced with hT2 balancer, we performed RNA-seq for both homozygous null mutants sired by heterozygous null mutant (P0) and homozygous null mutants sired by homozygous null mutant (F1) adult worms.
RNA-seq of gfp::3xflag::mbd-2 (control) and gfp::3xflag::mbd-2?CC (coiled-coil domain deletion) mutant synchronized young adult worms. The mutant is maintained as a heterozygous stock balanced with hT2 balancer, we performed RNA-seq for both homozygous coiled-coil domain deletion mutants sired by heterozygous coiled-coil domain deletion mutant (P0) and homozygous coiled-coil domain deletion mutants sired by homozygous coiled-coil domain deletion mutant (F1) adult worms.


title: Genome-wide screen identifies Runx2 as a novel regulator of hematopoietic stem cell expansion and T-cell commitment

city: Oxford

first: Catherine

last: Chahrour

address: MRC Weatherall Institute of Molecular Medicine

institute: University of Oxford

summary: Self-renewing multipotent hematopoietic stem cells (HSCs) are a rare but important cell population which can reconstitute the entire blood and immune system following transplantation. Due to their rarity, it has been difficult to comprehensively study the mechanisms regulating HSC activity. However, recent improvements in hematopoietic stem and progenitor cell (HSPC) culture methods using polyvinyl alcohol (PVA)-based media now facilitate large-scale ex vivo HSC expansion. Here we performed a genome-wide CRISPR knockout (KO) screen in primary mouse HSPCs to discover novel regulators of ex vivo expansion. The screen identified Runx2 as a strong negative regulator of HSC expansion, which we validated using ex vivo and in vivo assays. Loss of Runx2 increased the frequency of immunophenotypic HSCs in HSPC cultures by ~3-fold. Following expansion, Runx2-KO HSCs engrafted at ~5-fold higher levels in competitive transplantation assays. Non-cultured Runx2-KO HSCs also displayed enhanced reconstitution potential, but loss of Runx2 did not alter blood parameters. Notably however, T-cell reconstitution was diminished from Runx2-KO HSCs, and we further validated an additional role for Runx2 in T-cell commitment using ex vivo and in vivo assays. In summary, we have identified a multifaceted role for Runx2 in HSCs, as a negative regulator of HSC self-renewal and as a facilitator of T-cell commitment. These results will contribute understanding transcriptional regulation of hematopoiesis and improve HSC therapies.

overall_design: RNA-seq profiling of CD201+CD150+cKit+Sca1+Lineage- sorted HSCs, either from Runx2-flox mice or with CRISPR-mediated Runx2 knockout. CUT&Tag profiling for RUNX2 and H3K4me3 in in CD201+CD150+cKit+Sca1+Lineage- sorted HSCs

first: Grace

last: Meaker

first: Matthew

last: Nicholls

first: Ian

last: Hsu

first: Alastair

last: Smith

first: Yavor

last: Bozhilov

first: Maurice

last: Leung

first: Hugo

last: Vassort

first: Leonid

last: Olender

first: Oliver

last: Beaven

first: Xinran

last: Huang

first: Elizabeth

last: Brown

first: Marlies

last: Vanden Bempt

first: Hwei Minn

last: Khoo

first: Joydeep

last: Bhadury

first: Thomas

last: Milne

first: Adam

last: Wilkinson


title: Mesenchymal IL-33 licenses reparative macrophages to facilitate endovascular injury-induced neointimal hyperplasia [scRNA-Seq]

city: Hangzhou

first: Zhewen

last: Qin

state: ZHE JIANG

address: kaixuan street

institute: Zhejiang University School of Medicine

summary: Immune remodeling is crucial for tissue repair, but the interaction between arterial macrophages and mesenchymal stromal cells (MSCs) in neointimal hyperplasia is not well understood. Herein, we identified Il33-expressing MSCs serve as the key regulators of arterial immune responses following endovascular injury via facilitating the recruitment of reparative macrophages. Using temporal-resolving scRNA-seq and multi-color flow cytometry, we revealed a distinct subset of LY6C-CD63+CD72+ reparative macrophages essential for vascular smooth muscle cell (VSMC) proliferation and neointimal formation. These ST2-expressing macrophages are recruited and activated by MSC-derived IL-33, which is upregulated via NFκB signaling following injury, and in turn amplify the osteopontin (OPN) production that drives VSMC proliferation. Furthermore, hydrogel-mediated local delivery of siRNAs targeting Il33 or Spp1 reduced neointimal hyperplasia, confirming the importance of the IL-33-ST2-OPN axis in pathological vascular remodeling. Our findings uncover a critical role of mesenchymal IL-33 in orchestrating immune cell recruitment and promoting VSMC proliferation, suggesting that targeting this pathway may offer therapeutic potential for preventing restenosis and other vascular diseases.

overall_design: Arterial cells were isolated from murine femoral arteries at 2 (D2) and 7 (D7) days post wire-induced femoral artery injury (FAI) or Sham operations. The alive singlets were isolated using fluorescence-activated cell sorting (FACS) and analyzed by single cell RNA sequencing (scRNA-seq). One sample with cells which were pooled from 6 mice was prepared for each time point.


title: Mesenchymal IL-33 licenses reparative macrophages to facilitate endovascular injury-induced neointimal hyperplasia [RNA-Seq]

overall_design: PDGFRb+ MSCs were isolated from the femoral arteries of 8-week-old male mice at 0 (Sham) and 7 (femoral artery injury, FAI) days post wire injury surgery for bulk RNA sequencing (Bulk RNA-seq). Sham group and FAI group had 4 and 3 samples respectively, and each sample was pooled from 5 mice.


title: Exosomal CLOCK-stabilizing lncRNA confers innate memory in tuberculosis resisters [RNAseq]

city: beijing

first: lu

last: z

state: - 选择 -

address: beijing

institute: institution of microbology

summary: Innate immune cells develop enhanced responsiveness to secondary challenges through trained immunity, a process reliant on memory of prior encounters. However, the intercellular regulatory mechanisms and mediators dictating host-protective trained immunity against infections remain incompletely understood. By profiling plasma exosomal long non-coding RNA (lncRNA) repertoires in a tuberculosis (TB) resister cohort resistant to Mycobacterium tuberculosis (Mtb) infection, we identify lncRNA TB Resister-derived CLOCK Regulator 1 (TRCR1) as an immune trainer for monocytes during their differentiation into macrophages. Mechanistically, internalized exosomal TRCR1 collaborates with RNA-binding protein FXR2 to stabilize CLOCK mRNA by forming condensed lncRNA-protein-mRNA complexes in the cytosol, thereby facilitating the expression of CLOCK, a circadian regulator with histone acetyltransferase activity. This upregulated CLOCK amplifies histone H3 acetylation (K9/K14) at promoters of cytokine-encoding and autophagy-related genes, establishing epigenetic memory in monocytes. We further demonstrate that Mtb-secreted antigens induce human lung epithelial cells to release TRCR1-enriched exosomes, which educate monocytes to develop augmented antimicrobial capacity. In mice, TRCR1 training strengthens host anti-Mtb immunity and improves bacille Calmette-Guérin (BCG) vaccine efficacy. Collectively, our findings unveil an intercellular immune training axis wherein exosomal TRCR1 orchestrates CLOCK-mediated epigenetic programming to potentiate innate memory, providing a strategy for refining BCG vaccination and preventing infectious diseases.

overall_design: CD14+ monocytes enriched from PBMCs of healthy donors were seeded in 6-well plates at a density of 5 × 105 cell per well, cultured with RPMI 1640 (Gibco, Cat# C11875500BT) containing 10% exosome-deleted fetal bovine serum (FBS; VivaCell, Cat# C3801), 4 mM L-glutamine, 1% Penicillin-Streptomycin Solution (Hyclone, Cat# SV30010), and endogenous homeostatic level (10 ng/mL) of recombinant human macrophage colony stimulating factor (M-CSF, Cat# PHC9501; Gibco) at 37˚C with 5% CO2. Monocytes were treated with 30 μg/mL pooled exosomes derived from HC, LTBI, TB, or RSTR group (n = 3 biological pools prepared for each group as described above), or an equal volume of PBS as a control (n = 3 replicates) for 48 hours. Total RNA was then isolated from monocytes using the FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme, Cat# RC112-01) following the manufacturer’s instructions. RNA sequencing was conducted using the Illumina Novaseq 6000 platform with paired-end 150-bp reads. The adaptors and low-quality bases, which were evaluated using FastQC (v0.11.3) software (https://github.com/s-andrews/FastQC), were trimmed by Trimmomatic (v0.39) software (https://github.com/timflutre/trimmomatic) with the following parameters: TRAILING:20, MINLEN:235, and CROP:235. The trimmed reads were then aligned to the Human Reference Genome (GRCh38) using HISAT2. Subsequently, the HTseq (v0.11.1) software was used to convert the aligned short reads into read counts for each sample. Genes with fewer than ten counts in two or more samples were removed. The data were then analyzed using R (v3.4.4) and the DEseq2 (v1.18.1) package. DEGs in each exosome-treated group were identified using the Wald statistics test. Genes with a fold-change > 2 and a BH-adjusted P < 0.05 compared to the PBS control group were considered as DEGs. Cluster analysis and visualization were performed using the pheatmap Bioconductor package (v1.0.12.4).

first: Shanshan

last: Yu

first: Qiyao

last: Chai

first: Zhe

last: Lu

first: Cui

middle: H

last: Liu

first: Yu

last: Pang


title: Dysbiosis and Metabolic Dysregulation of Salivary Microbiota in Schizophrenia

city: hangzhou

first: zhu

last: rui

address: jinghuilu 300

institute: zhejiang university

summary: Background: Schizophrenia (SZ) is a chronic, severe mental disorder that presents significant challenges to diagnosis and effective treatment. Emerging evidence suggests that gut microbiota may play a role in the disease's pathogenesis. However, fewer studies have directly investigated the potential links between oral microbiota and SZ. Purpose: This study aimed to explore the relationship between salivary microbiota dysbiosis and SZ, examining microbial and metabolic alterations that may contribute to SZ pathophysiology. Methods: Salivary samples from 30 hospitalized patients diagosed with SZ and 10 healthy controls were collected. The microbial and metabolic profiles were analyzed using 16S rRNA gene sequencing and metabolomic profiling. Clinical parameters, including oral health status, were also evaluated to minimize variability in sampling. Results: Patients with SZ exhibited significantly poorer oral health compared to healthy controls, with more missing teeth and worse periodontal status. Microbiota sequencing revealed notable alterations in the overall structure and composition of the salivary microbiome in SZ patients, characterized by increased abundance of specific genera such as Neisseria and Porphyromonas. Metabolomic analysis indicated significant differences between the SZ and control groups, with upregulation of key metabolic pathways, including “β-alanine metabolism” and “vitamin digestion and absorption”. Correlations between microbial dysbiosis and elevated levels of certain metabolites, such as L-methionine sulfoxide (L-MetO) and tyramine, were observed, suggesting links to oxidative stress. Conclusion: The study highlights the presence of significant dysbiosis and metabolic dysfunction in the salivary microbiota of SZ patients, suggesting that alterations in the oral microbiome may contribute to SZ pathogenesis. These results provide new insights into potential diagnostic biomarkers and therapeutic targets for SZ. Further studies with larger sample sizes are required to validate these findings.

first: Jie

last: wang

first: Yongming

last: Li


title: MHC II-restricted neoantigen vaccine reverses immune microenvironment and overcomes resistance to immune checkpoint inhibitors in cold tumors.

city: Nanjing

first: Chun

address: No. 8 Xuefu Road, Pukou

institute: Nanjing University

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

overall_design: Refer to individual Series


title: MicroRNAs transcriptome analysis at the post-mortem interval estimation

city: Busan

first: Jungho

last: Kim

address: 57, Oryundae-ro, Geumjeong-gu, Busan, Republic of Korea

department: Department of Clinical Laboratory Science, College of Health Sciences

institute: Catholic University of Pusan

type: Non-coding RNA profiling by high throughput sequencing

summary: To identify potential microRNA candidates for the post mortem interval estimation using a transcriptome-based approach, we extracted total RNAs from myocardial tissue samples of BALB/c mice

overall_design: Through miRNA sequencing, we identified potential differentially expressed miRNAs

first: Sunyoung

last: Park

first: Haneul

last: Lee


title: Identification of novel genetic interactions with Recql4 in a genome-wide CRISPR/Cas9 synthetic lethal screen.

city: Meblourne

first: Alistair

middle: Morgan

last: Chalk

state: VIC

address: 1G Royal Parade

department: Bioinformatics

country: Australia

institute: WEHI

summary: This study was performed to provide a detailed understanding of the functions and pathways that intersect with the essential DNA replication factor RECQL4. Mutations in RECQL4 cause Rothmund-Thomson Syndrome (RTS), a familial cancer syndrome associated with early onset osteosarcoma in a high proportion of patients. Using murine RECQL4 mutations that closely map to recurrent human RTS mutations, we performed an unbiased genome-wide screen to identify genes that, when deleted, rescued the phenotypes associated with RECQL4 mutation.

overall_design: Hoxb8 immortalised R26-CreERT2 Recql4 KO sgKlhdc3 Double Knock-Out (DKO) cells and Recql4 Re-Expressing (RRE) Recql4R26-CreERT2 Recql4 KO sgKlhdc3 cells, stably expressing Cas9 were infected with the BRIE lentiviral Mouse CRISPR Knockout Pooled Library, selected with puromycin for 3 days and grown for 14 days with samples collected at day 0 (is day 3 puromycin) , day 2, day 5 and day 8 to identify guides specifically depleted in the R26-CreERT2 Recql4 KO sgKlhdc3 rescued cells.

first: M

middle: F

last: Smeets

first: A

middle: M

first: S

last: Taylor

last: Gupte

first: C

middle: R

last: Walkley


title: DGP1 family members mediate large-scale metabolic remodeling by integrating two environmental signals in rice

city: Yangzhou

first: Mingnan

last: Qu

state: Jiangsu

address: Pazhou Dadao Rd, 17-19

institute: Yangzhou Univerisity

summary: Low-phosphate (LP) and red-light (RL) remould cellular metabolism in rice (Oryza sativa). Herein, DEEP GREEN PANICLE1 (DGP1) family includes DGP1, DGP1B and DGP1C. LP treatment (10 µM KH2PO4) robustly upregulates DGP1 expression (>20-fold), RL treatment (50 µmol m-2 s-1) induces DGP1 (>80-fold) and DGP1B (>90-fold). The TIGR01589 domain of DGP1s targets cytosolic enzymes to mediate a process of nuclear translocation, including in glycolysis (PK1/4 and GAPC1/2/3), TCA cycle (PDC1, CS, SCSb, FH and MDH) and biosynthesis of non-aromatic amino acids (LeuC2, OCTase and P5CS). Meanwhile, it intervenes photorespiratory pathway by blocking the recruitment of enzyme component P subunit in the glycine decarboxylase complex. Intriguingly, cytosol-to-nucleus relocation of PK1-PK3 and PK3-PK4 complexes facilitates the shikimate pathway toward multiple groups of anti-microbial metabolites. Phenotypically, LP or RL treatment largely modifies cellular metabolome in wild-type plants displaying chemical disease resistance with reduced photosynthesis/photorespiration traits, whereas LP-treated dgp1 mutant or RL-treated dgp1 dgp1b mutant impairs these responses. Overall, rice DGP1s switch primary-secondary metabolism responding ambient changes of light quality and Pi status. The TIGR01589 domain is highly conserved in dicots and monocots with diverse activation-expression modes, suggesting the DGP1 family may extensively mediates environment-triggered metabolic reprogramming in higher plants.

overall_design: To better delineate the regulatory role of DGP1 in cellular metabolism, we carried out transcriptome deep sequencing of rice leaves from the DGP1-OE1 overexpressor and WT ZH11 seedlings.

first: Zhan

last: Xu

first: Huanfang

first: Mengmeng

last: Hou


title: Replication timing analysis of Recql4 -/- Klhdc3 -/- vs control using TIGER on whole genome sequencing in triplicate

summary: Abstract TBC

overall_design: Whole genome sequencing of immortalized myeloid cell lines from WT and Recql4-/- Klhdc3-/- genotypes; cell division time 15-18 hours; >25% S-phase in the culture

first: P

last: Buco

last: Koren


title: m6A RNA modification cooperates with alternative polyadenylation to control T cell homeostasis via mTOR signaling

city: shanghai

first: Xingli

last: Zhang

address: 280 Chongqing South Rd

institute: Shanghai Jiao Tong University School of Medicine

summary: To identify all of the APA targets of NUDT21 or METTL3 and obtain  gene expression profile, RNA from WT, Nudt21-deficient and Mettl3-deficient naïve CD4+ T cells stimulated with anti-CD3/CD28 for 0hr or 48hr were subject to RNA-Seq. By using a custom-designed algorithm and Gene ontology (GO) analysis, we found mRNA 3'UTR shortening phenomenon after TCR stimulation and both METTL3 and NUDT21 regulate 3'UTR length. In addition, we found most of up-regulated genes after Nudt21 deficiency are associated with immune activation pathway.

overall_design: mRNA profiles of WT, Mettl3-deficient and Nudt21-deficient naïve CD4+ T cells stimulated with anti-CD3/CD28 for 0hr or 48hr were generated by RNA-sequencing analysis.

first: Gaoyang

last: Wang

first: Haixin

first: Youqiong

last: Ye

first: Hua-Bing


title: C-Kit is Essential for Vascular Smooth Muscle Cell Phenotypic Switch In Vitro And In Vivo After Injury

status: Public on Nov 26 2025

city: Munich

first: Gianluca

last: Santamaria

state: Germany

address: Ismaninger Strasse 22

department: Department of Medicine I

country: Germany

institute: Klinikum rechts der Isar – Technical University Munich

summary: Pathological vascular remodeling—central to restenosis, atherosclerosis and vascu-lo-proliferative diseases—depends on phenotypic switching of vascular smooth muscle cells (VSMCs) from a quiescent, contractile state to a synthetic, proliferative program. Although the receptor tyrosine kinase c-KIT is implicated in proliferation, migration, and tissue repair, its role in VSMC plasticity has yet to be fully understood. Using c-Kit haploinsufficient mice subjected to right carotid artery ligation (CAL) and primary aortic VSMC cultures, we show that c-Kit is required for the contractile-to-synthetic transition. In vitro, c-Kit haploinsufficiency halved c-Kit expression, reduced BrdU incorporation, and blunted PDGF-BB–induced repression of contractile genes. c-Kit–deficient VSMCs ex-hibited a senescence program with increased p16INK4a/p21 expression and up-regulated senescence-associated secretory phenotype (SASP) mediators. RNA-seq of carotid arteries 7 days post-ligation revealed that wild-type arteries activated cell-cycle pathways and suppressed contractile signatures, whereas c-Kit–deficient carotid arteries failed to fully engage proliferative programs and instead maintained contractile gene expression. At 28 days post CAL in vivo, c-Kit haploinsufficiency produced markedly reduced neointima, fewer Ki67+ VSMCs, more p16INK4a+ cells, and impaired re-endothelialization. Because progenitor-to-VSMC differentiation contributes to remodeling, we tested adult cardiac stem/progenitor cells (CSCs) as a model system of adult progenitor differentiation. Wild-type CSCs efficiently generated induced VSMCs (iVSMCs) with appropriate smooth-muscle gene up-regulation; c-Kit–deficient rarely did so. Restoring c-Kit with a BAC transgene rescued both smooth-muscle differentiation and proliferative competence of c-Kit–deficient iVSMCs. Collectively, our data identify c-Kit as a gatekeeper of repar-ative VSMC plasticity. Adequate c-Kit enables progenitor-to-VSMC commitment and expansion of newly formed VSMCs while permitting injury-induced proliferation and matrix synthesis; reduced c-Kit locks cells in a hypercontractile, senescence-prone state and limits neointima formation. Modulating the c-Kit axis may therefore offer a strategy to fine-tune vascular repair while mitigating pathological remodeling.

overall_design: We combine (i) primary mouse aortic VSMCs challenged with platelet-derived growth factor BB (PDGF-BB), (ii) a right carotid artery ligation model with RNA-seq at day 7 and morpho-functional analyses at day 28, and (iii) progenitor-to-VSMC differentiation assays with genetic rescue to test whether c-Kit dosage controls the contractile-to-synthetic switch and reparative proliferation. We identify c-Kit as a dosage-dependent regulator that constrains senescence and enables progenitor commitment and VSMC expansion fol-lowing injury.

first: Siracusa

last: Chiara

first: Canino

last: Giovanni

first: Scalise

last: Mariangela

first: Marino

last: Fabiola

first: Pagano

last: Loredana

first: Santamaria

last: Gianluca

first: Torella

last: Annalaura

first: De Rosa

last: Salvatore

last: Daniele

first: Cianflone

last: Eleonora


title: RNAseq profiling of eSCC human cell line (KYSE-410) treated with pharmacological inhibitors of SHIP2

city: Anderlecht

first: Louison

last: Descampe

address: Route de Lennik 808

department: IRIBHM

country: Belgium

institute: Université Libre de Bruxelles

summary: In our study, we used mRNA sequencing to investigate the effect of SHIP2 inhibition on the transcriptome of KYSE-410 cells.

overall_design: RNA-seq profiling was performed in human esophageal squamous cell carcinoma (eSCC) cell line (KYSE-410). Cells were treated with two different SHIP2 inhibitors : K149 2uM (Echelon, B-0345) or AS1949490 20 uM (Tocris, 3718). DMSO 0,1% was used as control. Cells were collected 48h after the treatment and RNA was extracted using the MicroElute Total RNA kit (Omega Biotek, R6831-02) according to the manufacturer’s recommendations with DNase I digestion protocol on column.

first: Raquel

last: Ramos

first: Giacomo

last: Bregni

first: Nadia

last: Gillet

first: Alizée

last: Vercauteren Drubbel

first: Benjamin

last: Beck


title: Identification of novel genetic interactions with Recql4 in a genome-wide CRISPR/Cas9 rescue screen.

first: Alistair

last: Chalk

overall_design: Hoxb8 immortalised R26-CreERT2 Recql4fl/+ (control) and R26-CreERT2 Recql4fl/R347X (mutant) myeloid cells stably expressing Cas9 were infected with the BRIE lentiviral Mouse CRISPR Knockout Pooled Library, selected with puromycin for 4 days, then tamoxifen treated to delete the Recql4 floxed allele and grown for 14 days with samples collected at day 0 (when tamoxifen is added), day 4 and day 9 to identify guides specifically enriched in the Recql4 R347X mutant cells.

first: M

last: Smeets

first: A

first: S

last: Taylor

last: Gupte

first: C

last: Walkley


title: SHIP2-PLK1 crosstalk promotes sensitivity to dual inhibition in esophageal squamous cell carcinoma


title: Synergy between regulatory elements can render cohesin dispensable for distal enhancer function

city: San Francisco

first: Karissa

middle: Lynn

last: Hansen

state: CA

address: 555 Mission Bay Blvd S, 312

department: CVRI

institute: UCSF

summary: Enhancers are critical genetic elements controlling transcription from promoters, yet how they convey regulatory information across large genomic distances remains unclear. Here, we engineer pluripotent stem cells in which cohesin loop extrusion can be inducibly disrupted without confounding cell cycle defects. Transcriptional dysregulation is cell type-specific, and not all loci with distal enhancers depend equally on cohesin extrusion. Using comparative genome editing, we demonstrate that enhancer-promoter communication over just 20 kilobases can require cohesin. However, promoter-proximal elements can support long-range, cohesin-independent enhancer action – even across strong CTCF insulators. Finally, transcriptional dynamics and the emergence of embryonic cell types remain largely robust despite disrupted extrusion. Beyond establishing novel strategies to study cohesin in enhancer biology, our work provides mechanistic insight into cell type- and genomic context-specificity.

overall_design:
Stranded mRNA-seq and CUT&Tag for H3K27ac and H3K27me3 in mouse ES cells, ES cells exiting pluripotency, neural progenitors, and neural progenitors differentiating into astrocytes in control (DMSO/untreated) and NIPBL-depleted (dTAG-13/IAA) cells.
Bulk ATAC-seq from control (NIPBL) and NIPBL-depleted (dTAG-13) gastruloids. NIPBL depletion was initiated either 48, 72, or 96 hours after aggregation of mouse embryonic stem cells.
ChIP-seq for CTCF in ES cells with a mutation of the CTCF-binding site in the Car2 promoter.
4C-seq in ES cells (WT SRR2 and homozygous SRR2 deletion) from control (DMSO/untreated) and NIPBL- or RAD21-depleted (dTAG or IAA) cells. Viewpoints include the Sox2 promoter, Sox2 SCR, Car2 promoter, Car2 enhancer 1, and Car2 enhancer 2.
Cells harboring an dTAG-inducible (FKBP) or auxin-inducible degron (AID) on endogenous Nipbl or Rad21 alleles and were treated with either DMSO (control) or dTAG-13/IAA (NIPBL or RAD21 depletion). WT parental cells are E14Tg2a (129 X,Y background).

middle: L

first: Elphège

middle: P

last: Nora


title: Advancing Personalized Medicine in Alzheimer’s Disease: Liquid Biopsy Epigenomics Unveil APOE ε4-Linked Methylation Signatures

city: Pamplona

first: Maite

last: Mendioroz

address: Calle Irunlarrea, 3

department: Neuroepigenetics

country: Spain

institute: Miguel Servet Foundation - Navarrabiomed

type: Methylation profiling by genome tiling array

summary: Recent studies show that patients with Alzheimer’s disease (AD) harbor specific methylation marks in the brain that, if accessible, could be used as epigenetic biomarkers.  Liquid biopsy enables the study of circulating cell-free DNA (cfDNA) fragments originated from dead cells, including neurons affected by neurodegenerative processes. Here, we isolated and epigenetically characterized plasma cfDNA from 35 patients with AD and 35 cognitively healthy controls by using the Infinium® MethylationEPIC BeadChip array. Bioinformatics analysis was performed to identify differential methylation positions (DMPs) and regions (DMRs), including APOE ε4 genotype stratified analysis. Plasma pTau181 (Simoa) and cerebrospinal fluid (CSF) core biomarkers (Fujirebio) were also measured and correlated with differential methylation marks. Validation was performed with  bisulfite pyrosequencing and bisulfite cloning sequencing. Epigenome-wide cfDNA analysis identified 102 DMPs associated with AD status. Most DMPs correlated with clinical cognitive and functional tests including 60% for Mini-Mental State Examination (MMSE) and 80% for Global Deterioration Scale (GDS), and with AD blood and CSF biomarkers.  In silico functional analysis connected 30 DMPs to neurological processes, identifying key regulators such as SPTBN4 and APOE genes. Several DMRs were annotated to genes previously reported to harbor epigenetic brain changes in AD (HKR1, ZNF154, HOXA5, TRIM40, ATG16L2, ADAMST2) and were linked to APOE ε4 genotypes. Notably, a DMR in the HKR1 gene, previously shown to be hypermethylated in the AD hippocampus, was validated in cfDNA from an orthogonal perspective. These results support the feasibility of studying cfDNA to identify potential epigenetic biomarkers in AD. Thus, liquid biopsy could improve non-invasive AD diagnosis and aid personalized medicine by detecting epigenetic brain markers in blood.

overall_design: Here, we isolated and epigenetically characterized plasma cfDNA from 35 patients with AD and 35 cognitively healthy controls by using the Infinium® MethylationEPIC BeadChip array. Bioinformatics analysis was performed to identify differential methylation positions (DMPs) and regions (DMRs), including APOE ε4 genotype stratified analysis. After quality control and sample tracking, seven samples were discarded from downstream analysis. Finally, cfDNA from 30 controls and 33 patients with AD was used for differential methylation analysis.


title: Single-cell Multiomics Analysis Reveals CTCF as a Key Regulator of Lung Morphogenesis and Progenitor Maintenance

city: shenzhen

first: Jiang

last: Yamei

address: longfei

institute: The Chinese University of Hong Kong-Shenzhen

summary: Lung development generates a complex tree-like architecture through proximal-distal patterning and branching morphogenesis. However, the gene regulatory programs governing embryonic lung development remain poorly understood. Here, we present a comprehensive single-cell multi-omics atlas of mouse embryonic lungs, integrating gene expression and chromatin accessibility profiles. Through systematic analysis, we identify 13 distinct cell types and map cis-regulatory elements, peak-to-gene linkages, and transcription factors underlying lung development. Leveraging this multi-modal dataset, we uncover lineage-determining transcription factors  driving cell differentiation, including the Activated Protein-1 complex. We further delineate gene regulatory networks involving diverse transcription regulators, including CCCTC-binding factor. Using the Ctcf conditional knockout mouse, coupled with histological and multi-omics analyses, we demonstrate that CCCTC-binding factor  orchestrates lung progenitor maintenance and branching morphogenesis by modulating both gene expression and chromatin accessibility. Thus, our study provides a multi-omics resource and mechanistic insights for transcriptional regulation of lung morphogenesis.

overall_design: To comprehensively study the heterogeneity and regulatory mechanisms of lung morphogenesis, we performed multi-omics profiling of nuclei extracted from embryonic day 12.5 (E12.5) lungs using 10x Genomics paired snRNA-seq and snATAC-seq techniques.

first: Shenfei

last: Sun

first: Yamei

last: Jiang

first: Fujing

last: Huang

first: Wei

last: Wei

first: Mathias

last: Hochgerner

first: Tianmin

last: Xu

first: Xiaoting

last: Wang

first: Kaijun

last: Lin

first: Xinna

last: Zhang

first: Yanli

first: Hua

last: He

first: Miao

last: Yu

first: Xiaofang

last: Tang

first: Xinhua


title: Blood cells genome-wide methylation profiles of BRCA1 mutation and epimutation carries, women with neither BRCA1 mutation nor epimutation prior BC diagnosis and healthy women with neither BRCA1 mutation nor epimutation (Controls)

city: Szczecin

first: Tomasz

middle: K

last: Wojdacz

state: Zachodniopomorskie

address: Unii Lubelskiej 1

department: Independent Clinical Epigenetics Laboratory

country: Poland

institute: Pomeranian Medical University

summary: Genome-wide DNA methylation patterns were quantified in peripheral blood samples collected from 112 individuals (with determined BRCA1 mutation and epimutation status) using the Illumina Infinium HumanMethylationEPIC BeadChip

overall_design: In this study, we recruited 43 healthy women with BRCA1 germline mutations, 29 heathy women with BRCA1 epimutation, 19 women with neither BRCA1 mutation nor epimutation prior breast cancer diagnosis and 21 healthy women with neither BRCA1 mutation nor epimutation (controls). From each participant we collected peripheral blood sample for DNA extraction and performed DNA methylation analyses using Illumina Infinium HumanMethylationEPIC BeadChip.

first: Katarzyna

last: Sokolowska

first: Jacek

last: Antoniewski

first: Marta

last: Sobalska-Kwapis

first: Dominik

last: Strapagiel

first: Jan

last: Lubiński

last: Huzarski


title: RNAseq profiling of eSCC human cell lines (KYSE-410 and KYSE-30) transfected with siRNA targeting INPPL1 (siSHIP2) ; and a non-targeting siRNA (siCT)

first: Louison

last: Descampe

summary: In our study, we used mRNA sequencing to investigate the effect of SHIP2 knockdown on the transcriptome of KYSE-410 cells.

overall_design: RNA-seq profiling was performed in human esophageal squamous cell carcinoma (eSCC) cell lines KYSE-410 and KYSE-30. KYSE-410 cells were transfected with two independent siRNAs targeting INPPL1 (siSHIP2 [HSS_105471] and siSHIP2#2 [HSS_179945], Invitrogen), whereas KYSE-30 cells were transfected with siSHIP2 (HSS_105471). A non-targeting siRNA (siCT, ON-TARGETplus D-001810-01-05, Dharmacon) was used as control. Cells were collected 48h after transfection and RNA was extracted using the MicroElute Total RNA kit (Omega Biotek, R6831-02) according to the manufacturer’s recommendations with DNase I digestion protocol on column.

first: Raquel

last: Ramos

first: Giacomo

last: Bregni

first: Nadia

last: Gillet

first: Alizée

last: Vercauteren Drubbel

first: Benjamin

last: Beck


title: RNAseq profiling of eSCC human cell line (KYSE-410) knocked-out for SHIP2 using CRISPR/Cas9 technology

summary: In our study, we used mRNA sequencing to investigate the effect of SHIP2 ablation on the transcriptome of KYSE-410 cells.

overall_design: CRISPR/Cas9 technology was used for SHIP2 ablation in KYSE-410 cells using the CRISPR/Cas9n plasmids. Plasmids were co-transfected with a GFP-based CRISPR reporter (Addgene, #48138) using the Lipofectamine CRISPRMAX according to the manufacturer’s instructions. GFP-positive cells were subjected to FACS sorting Fluorescence-activated cell sorting using a FACSAria III (BD Biosciences, San Jose, CA, USA) and collected in a 96-well plate. Cells were expanded and positive clones were screened by Western blot for SHIP2. Once expanded RNA from validated clones (D4, G4, G10 and H3) as well as control colony (transfected with the same vector devoid of gRNA) was extracted using the MicroElute Total RNA kit (Omega Biotek, R6831-02) according to the manufacturer’s recommendations with DNase I digestion protocol on column.


title: Mitofusin agonists enhance long-term engraftment and potency of HSC cultures in vivo.

city: Rye

first: Larry

last: Luchsinger

state: NY

address: 601 Midland Ave

department: Stem Cell Regenerative Research

institute: New York Blood Center

summary: Umbilical cord blood (CBU) is a valuable source of hematopoietic stem cells (HSCs) due to its superior donor compatibility and lower incidence of graft-versus-host disease. However, its limited HSC content restricts its use in adult transplantation, necessitating new targets for ex vivo expansion and improved HSC potency. Mitofusin 2 (MFN2), a mitochondrial membrane fusion protein, is necessary for preserving HSC function and agonists of mitofusin activity have been characterized. We report that ex vivo culture of CBU HSCs with mitofusin agonists (MAs) enhances long-term repopulating activity by over five-fold in both primary and secondary transplantation assays without changes of total nucleated cells or phenotypic HSCs. Mechanistically, MA-treated HSCs show suppressed protein synthesis, increased autophagic flux, and elevated lysosomal acidification. Transcriptomic analysis implicates downregulation of MTOR signaling, and immunoprecipitation studies confirm a direct interaction between MFN2 and MTOR. These data support a model in which fusion-competent MFN2 sequesters MTOR, promoting a catabolic state that preserves HSC potency. Our findings suggest a novel MFN2–MTOR regulatory axis that enhances the functional expansion of human HSCs for potential therapeutic application.

overall_design: 10K cord blood CD90+ HSCs were cultured in the presence of 0.01% DMSO or 5nM Compund B mitofusin agonist (MA). Phenotypic CD90+ HSC resorted following 7 days of culture were isolated for total RNA using Trizol and subjected to NGS library preparation.

first: Daniel

last: Jin

first: Alyssa

last: Biondo


title: An Intergenic Silencer Region Regulates HBG Expression

city: Augusta

first: Sergei

last: Bombin

institute: Augusta University

summary: The most common genetic disorders worldwide are the β-hemoglobinopathies, including sickle cell disease and β-thalassemia caused by HBB gene mutations. Five functional β-like globin genes on chromosome 11 are expressed during development in a tissue and stage-specific manner. The HBG genes encoding g-globin polypeptide chains to form fetal hemoglobin (HbF), undergo gene silencing during the first year of life. Our study aimed to characterize an upstream DNA regulatory region that can be targeted for HBG reactivation during adult development. We investigated the intergenic region between HBE1 and HBG2 which we termed the fetal chromatin domain (FCD) to define its role in globin gene regulation. Using normal human CD34+ stem cells to establish erythroid progenitors, we characterized the binding of different transcription factors to the FCD, which facilitated long-range chromatin looping between the FCD and locus control region and HBG. Using CRISPR/Cas9 genome editing technology, the FCD was knocked out in KU812 and CD34+ stem cells. We observed enhanced HBG transcription and  HbF expression in both lines. For FCD knockout CD34+ stem cells, flow cytometry showed increased HbF positive cells and HbF expression and enhanced long-range interactions between the locus control and HBG region.  Single cell RNA-sequencing revealed normal erythropoiesis in the FCD-KO line and partial reversal of HBG silencing. These results support a putative silencer role of the FCD in regulating HBG transcription and HbF expression.

overall_design: The FCD knockout or AAVS1 edited controls were generated in CD34+ stem cells by CRISPR/Cas9 in the Hematopoietic Progenitor/Stem Cell Gene Editing core of the Fred Hutchinson Cancer Center. CD34+ stem cells were then grown in primary culture system.  and harvested on Day 10 for scRNA-seq. Cells were resuspended in ice-cold cryopreservation solution comprised of 50% fetal bovine serum, 40% growth media, and 10% DMSO and store at -80°C until shipping to Active Motif (Carlsbad, CA). Clustering algorithms are applied to identify distinct cell populations and further characterization of gene expression patterns within these clusters was performed. The resulting data were used to compare gene expression patterns across erythroid cell populations and to identify differentially expressed genes.

first: Betty

last: Pace

first: Xingguo

last: Zhu


title: Protocol for Quantifying Silent Ribosome Induction using Polysome Profiling

city: Basel

first: Attila

last: Becskei

state: Basel-Stadt

address: Spitalstrasse 41

department: Biozentrum

institute: University of Basel

summary: Translationally silent ribosomes have become an important focus in RNA biology, yet their quantification remains challenging. Lacking mRNAs, they represent a hibernating ribosome state. This protocol outlines how to measure silent ribosome induction under diverse conditions by analyzing RNA from polysome-profiling fractions in yeast and mammalian cells, using RNA-seq with removal of anomalously amplified rRNAs and qPCR for validation.

overall_design: For an example of the execution of this protocol, please refer to Rahaman, et. al.1

first: Sayanur

last: Rahaman

first: Samuel

last: Mondal

first: Nicole

last: Schiffelholz


title: Transcriptional Response and Adaptive Evolution to Oxidative Stress in Synechococcus elongatus PCC 7942

city: Richland

first: Predictive Phenomics

last: Initiative

state: WA

address: 902 Battelle Boulevard

institute: Pacific Northwest National Laboratory

summary: Cyanobacteria inhabit dynamic redox environments influenced by diurnal cycles, environmental fluctuations, and nutrient availability, necessitating tight redox regulation. This RNA-seq study examines transcriptional regulation of redox control in the model cyanobacterium Synechococcus elongatus PCC 7942 under controlled turbidostatic conditions. We manipulated oxygen concentrations to generate oxidative stress across distinct phases, measuring impacts on growth, physiology, and genome-wide gene expression. Transcriptional profiling reveals independently regulated gene modules that define graded transcriptional responses aligned with redox-regulated patterns activated under fluctuating light conditions. Our findings demonstrate a coordinated, multi-phase response involving ROS formation, detoxification, and damage repair. Oxidative stress induces transcriptional repression of essential cellular processes. This dataset includes wild-type strains under acute oxidative stress and experimentally evolved populations that underwent multiple rounds of elevated oxygen exposure. The evolved strains demonstrated improved tolerance to elevated oxygen levels, enhanced sucrose productivity under high-stress conditions, and accumulated DNA mutations in genes related to molybdopterin biosynthesis and glycogen metabolism.

sample_organism: Synechococcus elongatus PCC 7942 = FACHB-805

overall_design: Time-series transcriptional analysis of S. elongatus PCC 7942 CscB/SPS strain examining both acute oxidative stress response and adaptive evolution under controlled turbidostatic conditions. Oxygen concentrations were systematically manipulated to increase oxidative stress and define distinct phases of inhibition impacting growth, physiology, and gene expression. The starting wild-type (WT) population was grown between 0-78.4% oxygen before being transferred to 0% oxygen, establishing baseline stress response patterns. This oxygen stress cycle was repeated in adapted population one (0-93% oxygen cycling to 0%) and adapted population two (0-99.8% oxygen cycling to 0%). A final sample was collected at 0% oxygen for adapted population 3. Each adapted population was derived from the previous population, representing progressive adaptation to increasingly severe oxidative stress. Through transcriptional analysis at multiple time points across these populations, we identified independently regulated gene set modules revealing coordinated multi-phase responses to ROS formation, detoxifying ROS, and repairing damage. Multiple rounds of induced oxidative stress resulted in adaptive mutations and evolved phenotypes. Two technical replicates were analyzed for each condition sample.

first: Pavlo

last: Bohutskyi

first: Zachary

last: Johnson

first: Marci

last: Garcia


title: Effect of depletion of ANAPC1P2  on OVCAR3 cells.

city: Pune

first: Dr. Sharmila

last: Bapat

state: Maharashtra

address: NCCS campus, SPPU, Ganeshkhind road.

country: India

institute: National Centre for Cell Science

summary: ANAPC1P2 is a chimeric pseudogene that gives rise to a novel chimeric long non-coding transcript, vA-202 (RMND5A-ANAPC1). Notably, vA-202 functions as a γ-irradiation–responsive lncRNA, becoming induced under genotoxic stress conditions. In this study, we generated ANAPC1P2-knockdown OVCAR3 cell lines (OVCAR3_KD) and performed RNA-sequencing alongside their corresponding control lines. The resulting transcriptomic datasets reveal the global changes in gene expression that occur upon loss of this lncRNA, including both upregulated and downregulated targets.

overall_design: RNA-seq profiling of OVCAR3 cells and its derivative having shRNA knockdown of ANAPC1P2 (OVCAR3_KD).

first: Sharmila

first: Jadhav

last: Amruta


title: Chromatin folding principles underlying the generation of antibody diversity

city: Baltimore

first: Noah

last: Ollikainen

address: 251 Bayview Blvd.

institute: National Institutes of Health

summary: Achieving a diverse repertoire of antigen receptors for effective adaptive immunity requires rearrangement of gene segments into unique combinations via V(D)J recombination.  To illuminate precise mechanisms underlying this process, we combined biophysical simulations with experimental data to model chromatin folding and dynamics of the mouse immunoglobulin heavy chain gene (Igh) locus.  Simulations that best recapitulated experimental data on locus structure and recombination of wild-type and mutated Igh alleles identified three novel chromatin folding principles.  First, we found that prominent structural features of the Igh locus, such as the 3’-anchored stripe, required cohesin loading throughout the locus rather than at the stripe anchor.  Second, the Eµ enhancer was best modeled as a bi-directional loop extrusion blocker though it does not bind the known extrusion blocker CTCF.  Third, we found that utilization of VH genes to obtain maximum diversity required both widespread cohesin loading as well as long-range associations between H3K27ac-marked regions.  Our findings provide a conceptual framework to understand chromatin folding principles that enable antibody diversity and reveal mechanisms of long-range genome communication.

overall_design: Capture Hi-C of the Igh locus in an IGCR1-deleted pro-B cell line

first: Fei

last: Ma

first: Fatima

middle: Z

last: Braikia

first: Ranjan

last: Sen


title: The effect of phosphorus use efficiency affected by humic acid application on root gene expression in Rosa rugosa

first: Yong

last: Xu

address: 88 South Daxue Road

institute: Yangzhou University

summary: In the comparisons of CK_vs_HA1 and CK_vs_HA2,3877 and 374 differentially expressed genes were identified, respectively.the expression levels of phosphate transporter genes such as RrPHT1.2, RrPHT1.3, and RrPHT3.6 in rose roots were significantly upregulated compared to CK, with more pronounced effects in the HA2 treatment.

overall_design: one-year-old R. rugosa plants with uniform growth were selected and cultivated in pots within a greenhouse. Rose plants were watered once a week with Hoagland's nutrient solution, 200 ml per pot, for 37 weeks. The rose plants were divided into three groups and underwent a sand culture experiment with combined applications of HA and P fertilizer. Rose plants were watered once a week with Hoagland's nutrient solution, 200 ml per pot, for 37 weeks. The composition of Hoagland's nutrient solution is shown in Table S1. The three experimental groups were categorized based on the P content in Hoagland's nutrient solution and the proportion of P replaced by HA. The control group (CK) received 100% P (0.092 g NH4H2PO4). The HA1 group received 60% P + HA (0.0552 g NH4H2PO4 + 0.0368 g HA, equivalent to 40% P). The HA2 group received 40% P + HA (0.0368 g NH4H2PO4 + 0.0552 g HA, equivalent to 60% P).Approximately 1 g of tender roots from each plant (as per section 2.3) was immediately placed on ice, rinsed with deionized water, and stored at –80°C for RNA-seq analysis.

first: Yang

last: Yang

first: Meng

last: Li

first: Liguo

last: Feng


title: Variability in intrinsic promoter strength underlies the temporal hierarchy of the Caulobacter SOS response induction

city: Bengaluru

first: Anjana

last: Badrinarayanan

state: Karnataka

address: Bellary Road

institute: National Centre for Biological Sciences

summary: Bacteria encode for gene regulatory networks crucial for sensing and repairing DNA damage. Upon exposure to genotoxic stress, these transcriptional networks are induced in a temporally structured manner. A case in point is of the highly conserved SOS response that is regulated by the LexA repressor. Studies have proposed that affinity of LexA towards promoters of SOS response genes is the primary determinant of its expression dynamics. Here, we describe an additional level of regulation beyond LexA properties that modulates the SOS response gene expression pattern. Using transcriptomic analyses, we reveal a distinct temporal hierarchy in the induction of SOS-regulated genes in Caulobacter crescentus. We observe that LexA properties are insufficient in predicting the temporal hierarchy of these genes. Instead, we find that intrinsic promoter strength underlies the order of gene activation, with differential sigma factor association as a key factor in modulating gene expression timing. Our findings highlight a novel regulatory layer in SOS dynamics and underscore the importance of promoter properties in shaping bacterial stress responses.

overall_design: RNA-seq was performed on Caulobacter crescentus strains with or without exposure to mitomycin C DNA damage.

first: Aditya

last: Kamat

first: Asha

last: Joseph

first: Deeksha

last: Rathour


title: Single-Cell Integration of Chromatin Accessibility and Transcriptomics Reveals Regulatory Networks in Ovarian Tumor-Infiltrating Adaptive NK Cells

city: Huddinge

first: Dhifaf

last: Sarhan

state: Stockholm

address: Alfred Nobels allé 8, floor 8, room 83112

department: Dept. of Laboratory Medicine, Division of Pathology

country: Sweden

institute: Karolinska Institutet

summary: Natural killer (NK) cells are traditionally recognized for their rapid, non-specific responses against virus-infected or malignantly transformed cells, functioning as key effectors of innate immunity. However, a distinct subset known as adaptive NK (aNK) cells has been shown to acquire memory-like properties following viral infections, indicating their capacity for antigen-specific immune recall. Intriguingly, aNK cells have also been identified within the tumor microenvironment, where they can mediate tumor-specific recall responses. Yet, the regulatory mechanisms managing their function in tumor-infiltrating aNK cells remain largely unknown. In this study, we integrated publicly available multiomics datasets from ovarian cancer, including single-cell chromatin accessibility (scATAC-seq) and single-cell RNA sequencing (scRNA-seq), to identify chromatin-accessible regions and construct transcription factors (TF)-gene regulatory networks. To validate and extend these findings, we performed Smart-seq3 on NK cells isolated from ovarian tumors and applied SCENIC analysis to identify TF-driven gene regulation. By integrating results from both analyses, we identified PRDM1 and STAT2 as key TFs, along with their downstream targets CRCP and MTFP1, specifically enriched in tumor-infiltrating aNK cells. The expression levels of CRCP and MTFP1 positively correlated with NK cell infiltration in ovarian cancer tissues, suggesting their potential functions in supporting tumor-specific NK cell memory responses. In addition, external validation using data from the PROMIX clinical trial demonstrated that PRDM1 and STAT2 expression levels are positively associated with both overall survival and aNK cell-associated transcriptional features.

overall_design:
Two high-grade serous ovarian cancer (HGSOC) patient samples were obtained from the IPLA clinical trial and subjected to Smart-seq3 sequencing, combined with ovarian tumor-infiltrated NK cells from 5 patitents from Smart-seq2 sequencing.

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

first: Yizhe

last: Sun

last: Wan

first: Solrun

last: Kolbeinsdottir

first: Kang

last: Wang

first: Shruti

last: Khare

first: Okan

last: Gultekin

first: Sahar

last: Salehi

first: Kaisa

last: Lehti

first: Ramanuj

last: Dasgupta

first: Theodoros

last: Foukakis

first: Martin

last: Enge


title: Exosomal CLOCK-stabilizing lncRNA confers innate memory in tuberculosis resisters [lncRNAseq]

city: beijing

first: lu

last: z

address: beijing

institute: institution of microbology

summary: Innate immune cells develop enhanced responsiveness to secondary challenges through trained immunity, a process reliant on memory of prior encounters. However, the intercellular regulatory mechanisms and mediators dictating host-protective trained immunity against infections remain incompletely understood. By profiling plasma exosomal long non-coding RNA (lncRNA) repertoires in a tuberculosis (TB) resister cohort resistant to Mycobacterium tuberculosis (Mtb) infection, we identify lncRNA TB Resister-derived CLOCK Regulator 1 (TRCR1) as an immune trainer for monocytes during their differentiation into macrophages. Mechanistically, internalized exosomal TRCR1 collaborates with RNA-binding protein FXR2 to stabilize CLOCK mRNA by forming condensed lncRNA-protein-mRNA complexes in the cytosol, thereby facilitating the expression of CLOCK, a circadian regulator with histone acetyltransferase activity. This upregulated CLOCK amplifies histone H3 acetylation (K9/K14) at promoters of cytokine-encoding and autophagy-related genes, establishing epigenetic memory in monocytes. We further demonstrate that Mtb-secreted antigens induce human lung epithelial cells to release TRCR1-enriched exosomes, which educate monocytes to develop augmented antimicrobial capacity. In mice, TRCR1 training strengthens host anti-Mtb immunity and improves bacille Calmette-Guérin (BCG) vaccine efficacy. Collectively, our findings unveil an intercellular immune training axis wherein exosomal TRCR1 orchestrates CLOCK-mediated epigenetic programming to potentiate innate memory, providing a strategy for refining BCG vaccination and preventing infectious diseases.

overall_design: Serum exosomes were isolated and purified from HC, LTBI, TB, and RSTR group following the aforementioned protocol. For exosomal lncRNA sequencing, each group contained 3 biological pools, with each pool prepared by pooling exosome samples from 5 donors in HC, LTBI, and TB groups, and 4 donors in the RSTR group. Total exosomal RNA from each pool was extracted and purified using the RNeasy Mini Kit (Qiagen, Cat# 74104) following the manufacturer's instructions. Subsequently, the purified RNA was randomly fragmented in a fragmentation buffer. Then, a series of steps were carried out, including first- and second-strand cDNA synthesis, end repair of the cDNA, ligation of the adapter to the cDNA, purification, and PCR enrichment. These steps were performed using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Cat# E7760S) according to the manufacturer's instructions. After purification with magnetic beads, the final RNA library was obtained and sequenced on an Illumina Novaseq 6000 platform. The Trim Galore (v0.6.10) software (https://github.com/FelixKrueger/TrimGalore/) was utilized to remove sequencing adaptors and filter out low - quality reads. The trimmed reads were then aligned to the Human Reference Genome (GRCh38) using the HISAT2 (v2.2.1) software (https://daehwankimlab.github.io/hisat2/). Subsequently, the HTseq (v0.11.1) software (https://github.com/htseq/htseq/) was employed to convert the aligned short reads into read counts for each sample. To identify novel long lncRNAs, transcriptomes were assembled separately for each sample using the StringTie (v1.3.3) software (https://github.com/gpertea/stringtie). Default parameters were used, and the Ensembl reference annotation (GRCh38.p14) was provided to guide the assembly process. All individual transcriptomes were then merged with the reference Ensembl transcriptome using the StringTie software's merge function. A cutoff of 1 transcript per million (TPM) and a minimum transcript length of 200 nucleotides were applied, with all other parameters set to their default values. Then, computational methods, namely the Coding Potential Calculator (CPC), Coding-Non-Coding Index (CNCI), Coding Potential Assessment Tool (CPAT), and Pfam, were used to distinguish non-coding genes from protein-coding genes. The different types of lncRNAs, including long intergenic non-coding RNA (lincRNA), intronic lncRNA, anti-sense lncRNA, and sense lncRNA, were selected using the CuffCompare (v2.2.1) software (https://cole-trapnell-lab.github.io/cufflinks/cuffcompare/). To identify differentially expressed genes (DEGs), genes with fewer than ten counts in two or more samples were removed. The data were then analyzed using R (v3.4.4) (https://www.r-project.org/) and the DEseq2 (v1.18.1) package. DEGs in the LTBI, RSTR, and ATB groups were identified using the Wald statistics test. Genes with a fold - change > 2 and a Benjamini–Hochberg (BH)-adjusted P < 0.05 compared to the HC group were considered as DEGs.

first: Shanshan

last: Yu

first: Qiyao

last: Chai

first: Zhe

last: Lu

first: Cui

last: Liu

first: Yu

last: Pang


title: SREBP signaling regulates lung resident memory B cell metabolism to sustain allergic memory

city: Indianapolis

first: Wei

last: Luo

state: Indiana

address: 950 W Walnut Street - R2 E302c

department: Microbiology and Immunology

institute: Indiana University

summary: IgE drives allergic airway disease, yet the mechanisms sustaining IgE memory remain incompletely understood. Using a murine model of allergy, we identified CCR6⁺ CD69⁺ lung-resident memory B cells (BRM) that persist after sensitization and expand rapidly upon allergen re-exposure, generating allergen-specific IgG1 and IgE. Transcriptomic analysis revealed that BRM adopt a distinct lipid metabolic program regulated by sterol regulatory element-binding protein (SREBP) signaling. Conditional deletion of this pathway revealed stage-specific roles: it is essential for GC-derived BRM formation, dispensable for maintenance, yet critical during memory recall to support BRM expansion, plasma cell differentiation, and local antibody production. Loss of SREBP signaling during memory recall impaired fatty acid metabolism, protein synthesis and effector differentiation in BRM, while also reducing plasma cell metabolic fitness, resulting in diminished IgG1 and IgE production. These findings identify a lung BRM subset as a key driver of pathogenic antibody responses and establish SREBP signaling as a stage-specific regulator of BRM, highlighting sterol pathways as potential therapeutic targets for allergic disease.

overall_design: Wild-type and SCAP cKO mice were intranallly treated with house dust mite  (HDM) extract to establish airway allergy, and rechallenged with the same allergen six weeks later to induce memory recall response. For SCAP cKO, SCAP was deleted before memory recall. CCR6⁺ CD69⁺, CCR6⁻ CD69⁻ lung resident csMBCs and circulating csMBCs from were sorted for bulk RNA-seq.

first: Alex

last: Maldeney