title: Yellow fever vaccine propagation in primary human hepatocytes triggers antiviral and cytolytic responses

city: Bochum

first: André

last: Gömer

address: University Street 150

department: MMV

institute: Ruhr University Bochum

summary: Despite the long-standing success of the yellow fever virus (YFV) 17D vaccine, the precise virus and host determinants which confer its virulence attenuation remain incompletely defined. To provide insights, we performed YFV genome-level evolutionary analyses combined with investigations of vaccine hepatotropism. Evolutionary analyses confirmed vaccine-specific mutations are selected against in virulent YFV isolates, with genomic signatures of purifying selection most pronounced in the envelope (E) protein, where vaccine-specific mutations are concentrated. Structural prediction and mapping using Alphafold3 confirmed vaccine-specific mutations form a surface-exposed patch on domain III of the E protein, likely overlapping the flavivirus receptor binding domain. In tissue culture, 17D infections exhibited enhanced cell-to-cell spread and cytolysis compared to virulent YFV strains. In humans, YFV infections cause severe liver damage while vaccination has an excellent safety profile. Consequently, we next investigated whether 17D displays impaired tropism for primary human hepatocytes (PHH) or induces distinct cell-intrinsic responses to virulent strains. Surprisingly, ex vivo infections confirmed robust propagation of both 17D and virulent YFV strains in PHH. RNA-sequencing revealed consistent and shared induction of IFNB and IFNL1-4, modulating gene programs associated with antiviral responses, cellular immunity, chemotaxis and inflammation, cell-death, metabolism and protein translation. Consistent with differences in spreading efficiency, subtle differences in virion production kinetics and the magnitude and tempo of PHH transcriptional responses were observed. Antiviral responses to 17D were activated earlier while responses to virulent YFV were delayed but enhanced. In summary, we confirm the YFV vaccine exhibits comparable hepatotropism to virulent strains and induces virtually identical host responses. Mechanistically, these data suggest that impaired hepatotropism and associated cytopathology are not correlates of 17D’s virulence attenuation, implying unknown barriers to systemic dissemination mitigate viscerotropic spread and associated liver pathology upon vaccination.

overall_design: PHH were infected with the YFV vaccine strain (17D) or virulent YFV strains (Asibi and Uganda) at MOI 0.5 or left uninfected (Mock). Total RNA was harvested from infected and timepoint matched uninfected PHH at 24, 48 and 72 hours post infection

first: Leyla

last: Sirkinti

first: Csaba

last: Miskey

first: Daniel

last: Todt

first: Brown

last: Richard


title: Investigating the Mechanisms of Lactulose on Gut Health and Uric Acid Metabolism in Geese via Transcriptomics

status: Public on Aug 26 2025

city: Chuzhou

first: xiaorong

last: he

address: No. 9 Donghua Road, Fengyang County

country: China

institute: Anhui Science and Technology University

type: Expression profiling by high throughput sequencing

summary: In recent years, the increasing incidence of goose gout has caused serious economic losses. In this study, we explored the effects of lactulose on intestinal health and uric acid metabolism in geese, and used RNA-seq analysis to reveal its potential mechanism. 180 one-day-old Yangzhou geese were divided into three groups: control group (CG, basal diet), high protein model group (MG, containing 21 % crude protein diet), lactulose group (LS, containing 21 % crude protein diet + 0.3 % lactulose). Results showed that compared with the CG group and MG group, the LS group had significantly lower serum uric acid levels and intact ileal mucosal structure. Through bioinformatics analysis of transcriptome (GO and KEGG enrichment analysis, GSEA and trend analysis), we found key pathways and genes closely related to intestinal health and uric acid metabolism. These key findings include: zinc ion import across plasma membrane biological process, ABC Transporters pathway, Wnt signaling pathway, as well as ABCG2, a key gene for uric acid transport, and genes closely related to intestinal structural integrity (KLF2, LRP6, TJP1, TJP3, PROM1). The results showed that lactulose could effectively reduce the serum uric acid level of geese by improving intestinal morphology.

overall_design: Yangzhou geese were divided into three groups : control group ( CG, basal diet ), high protein model group ( MG, containing 21 % crude protein diet ) and lactulose group ( LS, containing 21 % crude protein diet + 0.3 % lactulose ) for RNA-seq analysis.

first: Xiongrong

last: He

first: Pengfei

last: Ye


title: GPRC5B preserves a mature β-cell state in obesity by controlling MafA expression [dof105_scRNA-seq]

city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

country: Germany

institute: MPI for heart and lung research

summary: In vitro studies have implicated orphan receptor GPRC5B in β-cell survival, proliferation and insulin secretion, but its relevance for glucose homeostasis in vivo is largely unknown. Using tamoxifen-inducible, β-cell-specific GPRC5B knockout mice (Ins-G5b-KOs) we show here that loss of GPRC5B does not affect β-cell function in the lean state, but results in strongly reduced insulin secretion and disturbed glucose tolerance in mice subjected to high fat diet for 16 weeks. Flow cytometry and single-cell expression analyses in islets from obese mice show a reduced β-cell abundance and a less mature β-cell phenotype in Ins-G5b-KOs. Expression of β-cell-specific transcription factor MafA is reduced both on the RNA and protein level, as are transcripts of MafA target genes. Mechanistically, we show that phosphorylation of cAMP response element-binding protein (CREB), a major regulator of MafA expression, is reduced in islets of obese Ins-G5b-KOs, and that this phenotype precedes the downregulation of MafA and MafA target genes. Taken together, GPRC5B helps to maintain mature β-cell function in obesity through cAMP/CREB-dependent regulation of MafA expression.

overall_design: Single cell mRNA sequencing in pancreatic islet cells from high fat diet-fed (16 weeks) control mice and beta cell-specific GPRC5B knockout mice

first: Tianpeng

last: Wang

first: Nina

last: Wettschureck

last: Guenther


title: A Single-Cell Transcriptomic Atlas of the Pigtail Macaque Placenta in Late Gestation

city: Seattle

first: Kristina

last: Adams Waldorf

state: Washington

address: 750 Republican Street, E Building, Room E360

department: OBGYN

country: USA

institute: University of Washington

summary: The placenta is a complex organ with multiple immune and non-immune cell types that promotes fetal tolerance and facilitates nutrient and oxygen transfer. Diverse placental structures have evolved to accomplish the singular goal of protecting and nourishing the fetus. The nonhuman primate placenta is the most similar to the human placenta, but the extent to which single cell populations recapitulate those in the human is unknown. As the nonhuman primate is a key experimental model of pregnancy complications, it is essential to understand the degree of similarity in single-cell populations across the maternal-fetal interface. We constructed a single-cell atlas of the placenta from the pigtail macaque in the third trimester, comprised of three different tissues at the maternal-fetal interface: the chorionic villi, chorioamniotic membranes, and the maternal decidua. Each tissue was separately dissociated into single cells and processed through the 10X Genomics single-cell RNA-Seq (scRNA-Seq) pipeline. Samples were aggregated, filtered, and analyzed using Seurat. We performed unsupervised clustering followed by cluster annotation. Additional analyses included determining the maternal-fetal origins of cell populations, scRNA velocity, gene ontology enrichment, and cell-cell communication. The single-cell populations in the pigtail macaque were strikingly similar in their identity and relative frequency to that observed in humans. The third trimester pigtail macaque single-cell atlas enables the identification of cellular sub-clusters that are analogous to the human and provides a powerful resource for understanding the impact of infectious disease and other experimental perturbations on the nonhuman primate placenta.

overall_design: Ten pregnant pigtail macaques either received (1) choriodecidual and intra-amniotic saline infusions after surgical implantation of choriodecidual and amniotic catheters (n=5) or (2) five subcutaneous inoculations of media along the forearm (n=5). The gestational age at tissue collection spanned the late second and third trimester (118-150 days gestation; term gestation = 172 days).

first: Amanda

last: Li

first: Richard

first: Hazel

last: Huang

first: Hong

last: Zhao

first: Briana

last: Del Rosario

first: Miranda

first: Edmunda

first: Andrew

last: Vo

first: Gygeria

last: Manuel

first: Orlando

last: Cervantes

first: Raj

middle: P

last: Kapur

first: Jeff

last: Munson

first: Austyn

last: Orvis

first: Michelle

last: Coleman

first: Melissa

last: Berg

first: Britni

last: Curtis

first: Brenna

last: Menz

first: Jin

last: Dai

first: Inah

last: Golez

first: Solomon

last: Wangari

first: Chris

last: English

first: Audrey

last: Baldessari

first: Lakshmi

last: Rajagopal

first: John

last: Cornelius

middle: M


title: RNA Sequencing Analysis of vascular smooth muscle cell from Wild Type and TIGAR-/- mice

city: nanjing

first: li

middle: chen

last: yu

address: Longmian Avenue

institute: nanjing medical university

summary: We identified that global TIGAR deficiency inhibited VSMC dedifferentiation. The purpose of this experiment was to identify how TIGAR inhibited VSMC dedifferentiation.We then performed gene expression profiling analysis using data obtained from RNA-seq of VSMC from WT and TIGAR KO mice stimulated with LPS.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for vascular smooth muscle cells from WT and TIGAR KO mice stimulated with PDGF-BB.

first: Chenyu

last: Li


title: Transcriptome Reprogramming Associated with Autoimmunity Reveals Key Points of the Growth-Defence Trade-off in Arabidopsis thaliana

city: Singapore

first: Donghui

last: HU

state: Singapore

address: 16 Science Drive 4 Block S1A #06-12

department: department of biological sciences

country: Singapore

institute: National University of Singapore

summary: Growth and defence are inversely correlated processes requiring balance for optimal fitness. Using DANGEROUS MIX (DM) autoimmune plants with stunted growth in Arabidopsis thaliana, we investigated the balancing act through transcriptome analysis of three DM cases and identified two major modules representing defence and growth. Removing all three ADR1s (helper NLRs) reversed the expression patterns and shifted the trade-off balance, revealing ADR1s' dual role in enhancing immune responses and actively suppressing growth-related genes.

overall_design: Comparative transcriptome and chromatin accessibility analysis of autoimmune DM lines versus wild-type controls, with and without ADR1 triple knockout

last: Hu

first: Jinge

last: Wang


title: RNA-seq of Arabidopsis thaliana seedlings infected with Pseudomonas syringae pv. tomato DC3000 carrying effector AvrRpt2 or AvrRps4

summary: Time-course RNA-seq analysis of Arabidopsis thaliana Col-0 seedlings infected with Pseudomonas syringae pv. tomato DC3000 carrying effector genes AvrRpt2 or AvrRps4. Samples were collected at 6, 12, and 20 hours post-inoculation using a transient pathogen flood assay following Ishiga et al., 2011. This study aims to characterise the temporal dynamics of plant immune responses and identify transcriptional changes associated with effector-triggered immunity.

overall_design: Time-course study with pathogen inoculation at three time points (6h, 12h, 20h) plus mock controls, with three biological replicates per condition


title: A fully iPSC-derived 3D model of the human blood-brain barrier for exploring neurovascular disease mechanisms and therapeutic interventions

city: Munich

first: Rainer

last: Malik

address: Feodor-Lynen-Str. 17

department: Institute for Stroke and Dementia Research

institute: Klinikum der Universität München

summary: Integrity of the blood-brain barrier (BBB) is critical for brain homeostasis, and its malfunction contributes to neurovascular and neurodegenerative disorders. So far, mechanistic studies on BBB function have been mostly conducted in rodent and non-physiological in vitro models, which recapitulate some disease features, but have limited translatability to humans and pose challenges for drug discovery. Here we report on a fully human iPSC-derived, microfluidic 3D BBB model consisting of endothelial cells (EC), mural cells, and astrocytes. Our model expresses typical cell fate markers, forms a barrier in vessel-like tubes, and enables perfusion, including with human blood. We optimized iPSC differentiations and validated cellular fates by comparison to published datasets and extensive benchmarking vs. primary cells with proteomic profiles provided in an online database. To demonstrate suitability for translational research, we applied the model to investigate deficiency of FOXF2, a major risk gene for cerebral small vessel disease. Deletion of FOXF2 in EC induced key features of BBB dysfunction, including compromised cell junction integrity and enhanced caveolae formation. Proteomic analysis further revealed dysregulation of endocytosis and cell junction pathways. Disease features phenocopied those seen in mice with endothelial cell-specific Foxf2 deficiency, validating the relevance of our in vitro model to investigate in vivo phenotypes of neurovascular disease. Moreover, lipid-nanoparticle-based treatment with Foxf2 mRNA rescued BBB deficits in our FOXF2 KO model, demonstrating its potential for drug development.

overall_design: bulk RNA-seq of human endothelial and mural cells derived from iPSCs, which were then compared to published datasets as described in the publication

first: Judit

last: González-Gallego

first: Katalin

last: Todorov-Völgyi

first: Martin

last: Dichgans

first: Dominik

last: Paquet


title: Transcriptome of the hindgut of Aedes aegypti wild type females fed on sucrose ad libitum

city: New York

first: Laura

middle: B.

last: Duvall

state: NY

address: 1212 Amsterdam Ave

institute: Columbia University

summary: Aedes aegypti mosquitoes are efficient vectors of human disease including Zika, Dengue and Chikungunya. After taking a blood meal, proteins from the blood are broken down into amino acids that are essential for egg development and growth. We have discovered a novel role for a neuropeptide receptor in the hindgut that coordinates blood utilization. The hindgut is not a tissue normally implicated in blood digestion, therefore we conducted RNA-seq profiling of the hindgut in wild type females to obtain a transcriptomic profile of these tissues in non-blood fed conditions.

overall_design: RNA-seq profiling of the hindgut of wild type 5-15 day old Aedes aegypti female mosquitoes fed on sucrose ad libitum

first: Chloe

last: Greppi

first: Kyle

last: Frank

first: Victoria

last: Saltz

middle: B


title: Comprehensive single cell RNA-seq datasets of mice tracheas in 4 time points from E11.5 to E14.5

city: Kobe

first: Shogo

last: Nakayama

state: Hyogo

address: Chuo-ku, Minatojima-minamimachi2-2-3, Lung Development and Regeneration

country: Japan

institute: RIKEN BDR

summary: To explore the molecular signatures of tracheal cells during tracheal development, we performed single cell RNA-seq of developing whole tracheas. We identified 18 cell clusters that constitue  trachea tissue. Using sub-clustering analysis of chondrocyte progenitors at E12.5, we found Wnt-responsive chondrocytes that higly express wnt signaling target genes such as Lef1, Notum, Axin2.

overall_design: Transcriptomic analyses at single cell resolution of mouse tracheal cells in 4 time points (E11.5, E12.5, E13.5 and E14.5)

first: Mitsuru

last: Morimoto


title: A Single-Short Partial Reprogramming of the Endothelial Cells in vivo decreases Blood Pressure and Vascular Contractility Through the Attenuation of Endothelial-to-Mesenchymal Transition in Male Hypertensive Mice

city: Cincinnati

first: Yunguan

state: oh

address: 3333 Burnet Avenue

institute: Cincinnati Children's Hospital Medical Center

summary: Endothelial cell (EC) reprogramming represents an innovative strategy in regenerative medicine to prevent deleterious effects induced by cardiovascular diseases. Here, we showed for the first time, that a single-short partial reprogramming of ECs, via the overexpression of three master transcription factors Oct-3/4, Sox-2, and Klf-4 (OSK), was able to bring ECs back to a youthful phenotype in hypertensive mice. Accordingly, we effectively generated in vitro ECs with endothelial progenitor cell (EPC)-like features that expressed lower migratory capability and prevented cellular senescence. Furthermore, in vivo OSK treatment of hypertensive mice normalized blood pressure and resistance arteries hypercontractility, via the attenuation of the process of endothelial-to-mesenchymal transition (EndMT) and alleviation of elastin breaks in arteries from hypertensive mice. Human hypertensive ECs treated with OSK recovered their ability to activate endothelial nitric oxide synthase (eNOS) and enhanced nitric oxide (NO) production, with a decrease in reactive oxygen species (ROS) generation. In addition, single-cell RNA analysis showed that OSK could alleviate EC senescence and mitigate the EndMT process, facilitating the restoration of EC phenotypes in human hypertensive ECs.

overall_design: Human male and female normal and hypertensive aortic endothelial cells were treated with OSK or control EGFP sendai virus for 3 or 7 days. The attentuation of EndMT in HAoECs by OSK were determined using scRNA-seq.

first: Pernomian

last: Laena

first: Nguyen

last: Vi

first: Wang

last: Yunguan

first: Tan

last: Wenbin

first: Wenceslau

last: Caimilla


title: Spatial regulation of CD8+ T cells at the HLA-E-NKG2A axis drives HIV persistence in Lymph Node B Cell Follicles

city: Durban

first: Veron

last: Ramsuran

state: KZN

address: Mazisi Kunene Road

country: South Africa

institute: UKZN

type: Genome binding/occupancy profiling by high throughput sequencing

summary: B cell follicles (BCF) in the lymph node are a major sanctuary for HIV reservoirs. Immune regulatory mechanisms hindering cytolytic CD8+ responses at these sites, are poorly characterized, likely enabling HIV persistence. Spatial transcriptomics and high- dimensional histocytometry were used to define CD8+ T cell function and immune regulation in LN follicles of people Living with HIV (PLWH), at various stages of ART- treatment. Histocytometry demonstrated that CD8+ T cells infiltrating BCF mostly lacked granzyme B expression, coinciding with reduced chromatin access at cytolytic gene loci in dissociated lymph node cells. Spatial transcriptomics confirmed the immune regulatory microenvironment of HIV-infected BCF, particularly exhibiting upregulation of HLA-E. Additional FACS analysis identified a subset of LN CD8+ T cells expressing the NKG2A-interacting partner of HLA-E, with reduced granzyme B expression. These findings reveal regulation of follicular CD8+ T cells at the HLA-E- NKG2A axis as a key mechanism for HIV immune evasion.

overall_design: Samples were extracted from participant lymph nodes and peripheral blood. ATAC-Seq data was generated from 6 Lymph Node residing CD8+ T cells and 5 Peripheral blood CD8+ T cells, with two samples paired between peripheral Lymph Node and Blood samples.  RNA-Seq data were generated from 3 Lymph Node CD8+ T cells and 4 Peripheral Blood CD8+ T cells, with three paired samples between the Lymph Node and Blood samples.

first: Zaza

last: Ndlovu


title: SPOP is a key trigger of pathological cardiac hypertrophy and heart failure

city: Monrovia

first: Hao

last: Wu

state: CA

address: 702 Oakdale Ave

institute: harbin medicine univercity

summary: Cardiac hypertrophy is a pathological feature of heart failure, and dysregulation of protein turnover and autophagy has been implicated in its progression. Speckle-type POZ protein (SPOP), a substrate adaptor for the Cullin3-RING E3 ubiquitin ligase complex, plays an emerging role in protein homeostasis and transcriptional regulation. This study investigates the function of SPOP in cardiac hypertrophy and heart failure. RNA sequencing was performed to examine transcriptional changes in mouse hearts with cardiac-specific overexpression  of SPOP, revealing differential expression of genes involved in autophagy, mitochondrial metabolism, and cardiac remodeling. These data provide new insights into the transcriptional landscape regulated by SPOP in the hypertrophic heart.

overall_design: Cardiac-specific SPOP overexpression  was generated using the αMHC-Cre system. Male mice aged 8–10 weeks were harvested  for RNA extraction and sequencing. Total RNA was isolated using TRIzol, and libraries were prepared using poly(A) selection and sequenced on an Illumina platform. Differential gene expression analysis was conducted using DESeq2, and pathway enrichment was performed using GO and KEGG annotations. The dataset includes control (α-MHC-Cre) and SPOP overexpression(cKI) mice under  sham conditions.

first: Yuting

last: Zhuang

first: Ying

last: Yue

first: Jiangqi

last: Liu

first: Yanjiu

last: Lv


title: A Single-Short Partial Reprogramming of the Endothelial Cells decreases Blood Pressure via attenuation of EndMT in Hypertensive Mice

city: Columbia

first: Wenbin

last: Tan

state: SC

address: 6311 Garners Ferry Road

department: Cell Biology and Anatomy

summary: Small artery remodeling and endothelial dysfunction are hallmarks of hypertension. Evidence supports a likely causal association between cardiovascular diseases and endothelial-to-mesenchymal transition (EndMT), a cellular transdifferentiation process in which endothelial cells (ECs) partially lose their identity and acquire mesenchymal phenotypes. EC reprogramming represents an innovative strategy in regenerative medicine to prevent deleterious effects induced by cardiovascular diseases. We hypothesized that arteries from hypertensive mice present high levels of EndMT and that specific EC reprogramming can decrease blood pressure values and restore vascular function in resistance arteries in hypertensive mice.  Here, we demonstrated OSK overexpression induced partial EC reprogramming in vitro, and these cells had lower migratory capability. Using spatial whole transcriptome atlas, we showed that OSK treatment of hypertensive BPH/2J mice attenuated EndMT and elastin breaks,  and by other assays, we showed that OSK treatment reduced blood pressure and resistance arteries hypercontractility. OSK-treated hypertensive HAoECs showed high eNOS activation and NO production, with low ROS formation. Single-cell RNA analysis showed that OSK alleviated EC senescence and EndMT, restoring their phenotypes in HAoECs from hypertensive patients. Overall, these data indicate that OSK treatment and EC reprogramming can decrease blood pressure and reverse hypertension-induced vascular damage.

overall_design: All animal procedures and protocols used were approved by the Animal Care and Use Committee at the University of South Carolina School of Medicine Columbia. Lentiviral vector production and utilization in cells and mice had the School of Medicine Columbia University of South Carolina ethics committee approval (IACUC #2596-101690-041122, and IBC protocol #300322). Experiments were conducted following the National Institutes of Health Guide for the Care and Use of Laboratory Animals and Animal Research Reporting of in Vivo Experiments (ARRIVE) guidelines. Male and female BPN/3J (RRID:IMSR_JAX:003004) and BPH/2J (RRID:IMSR_JAX:003005) mouse strains were obtained from The Jackson Laboratory and maintained as an inbred colony at the Animal Facility (School of Medicine Columbia, University of South Carolina). In addition, all male and female mice were used at 40-44 weeks of age for the intravenous (i.v.) treatment with the LVCO (control) or LV-OSK. One intravenous (i.v.) injection of lentiviral vectors (100 µL) carrying control plasmid (LVCO) or containing the transcription factors Oct-3/4, Sox-2 and Klf-4 (LV-OSK) was performed in male and female BPN/3J and BPH/2J mice through the tail vein. The i.v. injection consisted of 5 µL LVCO or LV-OSK (stock solution at 2.81x108 TU/mL) combined with 95 µL sterile saline. All mice were maintained on a 12-hour light/dark cycle with water and standard chow diet ad libitum. Digital spatial profiling (DSP) was performed on formalin-fixed paraffin-embedded (FFPE) thoracic aortas isolated from the i.v. LV-OSK-treated (40-44-week-old) mice, and the Mouse Whole Transcriptome Atlas (RNA v1.0) was applied to the samples (Nanostring, Seattle, WA, USA) in this study.

first: Laena

last: Pernomian

first: Emily

middle: W

last: Waigi

first: Vi

last: Nguyen

first: Camilla

middle: F

last: Wenceslau


title: Impact of RORgt in regulating CD4 T cells primed under Th17 conditions

city: Orlando

first: Kunal

last: Dhume

state: FLorida

address: 6900 lake Nona Blvd

department: Burnett School of Biomedical Sciences

institute: University Of Central Florida

summary: CD4 T cells are an essential component of antiviral adaptive immune cell responses. Central to the functional capabilities of CD4 T Cells is their ability to differentiate into distinct subsets. Subset differentiation and maintenance is dependent on the coordinated and sequential activation of certain master regulator transcription factors. While antiviral responses are predominantly Th1, prior studies have shown that both T-bet and Eomes, two critical regulators of Th1 responses are dispensable for CD4 T cell mediated protection. CD4 T Cells lacking both T-bet and Eomes produce strong and highly protective Th17 response characterized by high expression of the transcription factor RORγt. Here we determine the impact of RORγt regulation in  CD4 T Cells primed under Th17 conditions and also deficient in T-bet and Eomes. Our studies reveal RORγt regulates several hundred genes that in Th17 CD4 T Cells, thus providing a potential molecular framework for targeting protective anti-viral immunity.

overall_design: RNA-seq profiling and comparison of day 4 CD4 T cells primed under Th17 condiitions and lacking either T-bet and Eomes or T-bet, Eomes and RORγt

first: Caroline

last: Finn

first: Eugene

last: Baffoe

first: Lauren

middle: A

last: Kimball

first: Siva

middle: N

last: Annamalai

first: Verónica

last: Urdaneta-Páez

first: Jash

last: Trivedi

first: Taj

last: Azarian

first: Tara

last: Strutt

first: Karl

middle: K

last: McKinstry


title: Multi-omics analysis identifies progenitors of CD4-CTLs [scATAC-seq invitrodiff]

city: New Delhi

first: Veena

middle: S.

last: Patil

state: Delhi

address: Aruna Asaf Ali Marg

country: India

institute: National Institute of Immunology

summary: The CD4+ T cells expressing cytolytic molecules such as granzymes and perforins have been detected in many diseases and are shown to be enriched in the effector memory expressing CD45RA (TEMRA) in humans, but their origin remains elusive. However, their gene expression profile in comparison to classically defined cytotoxic T lymphocytes (CTLs), the CD8-CTLs has not been well demonstrated, hence their relevance remains debatable.  Thus in this study by parallelly analysing the CD4-CTLs and CD8-CTLs, we demonstrate that they are indistinguishable for the cytolytic program with both showing similar gene expression profile and T cell antigen-receptor (TCR) clonal expansion. Further using an integrative multi-omics approach combining the transcriptome, TCR repertoire, and open chromatin profile of CD4+ naïve (CD4-TN) and memory subsets, we discovered a stem-cell memory subset that is pre-committed to CTL program within the CD4+ T cell lineage. Through an in vitro differentiation model we developed CD4+ T cells with cytolytic potential from CD4-TN cells. The in vitro differentiated cells followed the trajectory of different developmental memory subsets from ex vivo CD4+ T cells for transcriptomic patterns as well as open-chromatin landscape. Thus, through this model, we deciphered the molecular signatures of early commitment of CD4-TN cells to cytotoxicity program. Of particular interest was the expression of both longevity as well as cytotoxicity associated gene sets by the in vitro differentiated CD4-CTLs, hence generating long-lived CD4-CTL effectors. This specific property can be further explored for vaccine development as well as testing the efficacy and cell based therapies for precision medicine.

overall_design: CD4-Naive T cells (CD3+CD4+CD45RA+CCR7+CD95-) were isolated from PBMCs of healthy human donors and activated for 48 hrs using aCD3/aCD28 Dynabeads in a Th1 polarizing milieu of cytokines and antibodies. The cells were then cultured in the Th1 polarizing condition for 8 days (2+8 day cycle) before doing scRNA-Seq (Day 10). The same cycle was repeated for a second round before subjecting the cells to another scATAC-Seq on Day 20.

first: Raunak

last: Kar

first: Shreya

last: Sinha

first: Zainab

last: Khatun

first: Anjali

last: Sharma

middle: S


title: Multi-omics analysis identifies progenitors of CD4-CTLs [scRNA-seq in vitro]

overall_design: CD4-Naive T cells (CD3+CD4+CD45RA+CCR7+CD95-) were isolated from PBMCs of healthy human donors and activated for 48 hrs using aCD3/aCD28 Dynabeads in a Th1 polarizing milieu of cytokines and antibodies. The cells were then cultured in the Th1 polarizing condition for 8 days (2+8 day cycle) before doing scRNA-Seq (Day 10). The same cycle was repeated for a second round before subjecting the cells to another scRNA-Seq on Day 20.


title: Multi-omics analysis identifies progenitors of CD4-CTLs [scRNA-seq ex vivo]

overall_design: PBMCs were isolated from healthy human blood bank donors (n=8 for ex vivo TSCM and TEMRA, n=10 for ex vivo whole CD4), and pure population of interest was sorted using FACS. scRNA-Seq was performed on these sorted cells after pooling cells from all donors for individual compartments for equal representation across donors.


title: Multi-omics analysis identifies progenitors of CD4-CTLs [bulk TCR]

overall_design: TCR analysis of purified populations of human CD4 memory cell subsets including CD4-Naïve and CD8-TEMRA by bulk TCR-sequencing of both TCRa and TCRb from 10 healthy donors.


title: Multi-omics analysis identifies progenitors of CD4-CTLs [scATAC-seq ex vivo]

overall_design: PBMCs were isolated from healthy human blood bank donors (n=8), and pure population of interest was sorted using FACS. scATAC-Seq was performed on these sorted cells after pooling cells from all donors for equal representation across donors.


title: A distinct subset of stem-cell memory is poised for the cytotoxicity program in CD4+ T cells in humans

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

overall_design: Refer to individual Series


title: Multi-omics analysis identifies progenitors of CD4-CTLs [bulk RNA-seq]

overall_design: Transcriptome analysis of purified populations of human CD4 memory cell subsets including CD4-Naïve and CD8-TEMRA by bulk RNA-sequencing from 10 healthy donors.


title: Melatonin enhances function of porcine immature Sertoli cells via modulating gene expression and metabolism

city: Hubei

first: Cai

middle: Xia

last: Yang

address: Jingzhou

institute: Yangtze University

summary: Melatonin (MT), a neurohormone synthesized and secreted primarily by the pineal gland, is of vital function to animal reproduction. However, the effect of MT treatment on porcine immature Sertoli cells (iSCs) remains unclear. Here, MT treatment (10nM, 36h) could elevate mitochondrial function and reduce oxidative stress, therefore inhibit apoptosis of porcine iSCs. Transcriptome profiling identified 39 differentially expressed genes (DEGs) (33 known and 9 novel) (10nM MT vs. Control), mainly involved in GO terms of steroid metabolic process, glutamine metabolic process, oxidoreductase activity and G protein coupled receptor binding, and KEGG pathways of steroid biogenesis, pyruvate metabolism, NF-kappa B and AMPK signal pathways. RT-qPCR validated 6 DEGs (Phgdh, Scd, Hmgcs1, Cytb, Pck2 and Sqle) induced by MT to be with the similar change trend to RNA-seq results. The protein level of HMGCS1 and the estradiol level were confirmed to be significantly decreased by MT (10nM, 36h) treatment of porcine iSCs. Inhibition of HMGCS1 in porcine iSCs could significantly reduce the level of estradiol. The levels of cholesterol content within cells and lactate in culture medium were unchanged by MT (10nM, 36h). 14 metabolites were significantly altered by MT (10nM, 36h) treatment of porcine iSCs. Which were involved in multiple vital metabolic pathways. Collectively, MT treatment of porcine iSCs could promote functions of porcine iSCs via modulating gene expression and metabolism.

overall_design: In this study, porcine immature testicular Sertoli cells (iSCs) were treated with 10 nM melatonin (MT) for 36 h, and the total RNA of 3 control samples (Control-1, Control-2, Control-3) and 3 MT-treated samples (MT-1, MT-2, MT-3) were extracted using Trizol reagent for constructing library and performing RNA-seq.

first: Caixia

first: Zhiqiang

last: Du


title: Regulation of undruggable c-MYC with controlled photothermal therapy for efficient cancer immunotherapy

city: Kuopio

first: Tiit

last: Örd

address: Neulaniementie 2

country: Finland

institute: A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland

summary: Cancer immunotherapies have revolutionized the treatment of metastatic cancers however most patients cannot benefit from current immunotherapies due to a suppressive tumor immune microenvironment (TIME). Photothermal therapy (PTT) with nanoparticles (NPs) as photothermal conversion agents has shown potential to induce immunogenic cell death (ICD) by remodeling TIME. PTT with different temperatures have been applied and mild temperature-PTT was recently claimed as the most promising approach. However, the optimal PTT temperature and related mechanisms in activation of immunotherapy have remained unclear. In the present study, black porous silicon (BPSi) NPs coated with polyethylene glycol and cancer cell membranes were utilized as photothermal agents. These NPs have good biocompatibility, high photothermal conversion efficacy, and homotypic tumor targeting ability. It was found that the PTT with the BPSi NPs could efficiently induce ICD, which was largely affected by the applied temperature in PTT. As compared to the mild temperature (46°C) and high temperature (54 °C) PTT, medium temperature PTT at 50 °C was the optimal treatment to induce effective ICD in vitro and inhibit tumor growth in vivo. Furthermore, it was unveiled that PTT could efficiently regulate undruggable c-MYC by tuning temperatures. c-MYC was upregulated by PTT at 46°C while downregulated by PTT at 50 °C. The activities of MYC-induced pathways such as p53 were also significantly enhanced. These findings not only discover the mechanisms in activating immunotherapy with NPs-based PTT but also suggest new directions for developing more potent therapy regimens to treat metastatic tumors.

overall_design: RNA-seq of MDA-MB-231 breast cancer cells incubated with photothermal therapy nanoparticles at different treatment temperatures.

first: Emilia

last: Happonen

first: Yijing

last: Dang

first: Jiajia

last: Wang

first: Silja

last: Saarela

first: Zakir

last: Mohammed

first: Milla-Iida

last: Laari

first: Yiannis

last: Constantinou

first: Konstantin

last: Tamarov

first: Henri

last: Hakkarainen

first: Ahmed

last: Montaser

last: Leinonen

first: Jenni

last: Küblbeck

first: Mari

last: Taipale

first: Minna

middle: U

last: Kaikkonen

first: Ruixian

last: Liu

first: Juha

last: Klefström

first: Haijun

last: Yu

first: Zhiai

last: Xu

first: Vesa-Pekka

last: Lehto

first: Wujun


title: Effect of Toosendanin on mRNA m6A Modification and Expression in Human hepatoma cell line HepG2

city: Fujian

first: Dongcheng

last: Zhang

address: Overseas Education College of Fujian Medical University, No. 1 Xuefu North Road, University Town, Fuzhou, Fujian Province, P.R.China

institute: Fujian Medical University

summary: mRNA-seq and high throughput MeRIP-seq methods with bioinformatics analysis demonstrated a possible mechanism for the inhibition of cell cycle of hepatoma HepG2 cells induced by toosendanin.

overall_design: The aim of the present study was to elucidate the toxic effects of toosendanin on human hepatoma cells.

first: Yifan

last: Lin

first: Jian

first: Xinyue

last: Gao

first: Jiaqi

last: Li


title: A CRISPR-Activation CROP-seq Screen Identifies HMGN1 as a Dosage Sensitive Regulator of Heart Defects in Down Syndrome (scATAC-Seq)

city: San Francisco

first: Angelo

last: Pelonero

address: 1650 Owens Street

department: Cardiovascular Disease Institute

institute: Gladstone Institutes (UCSF)

summary: Congenital heart defects (CHD) are the most common form of developmental abnormalities, occurring in ~1% of live births, and can arise due to altered dosage of genes essential for cardiogenesis. Aneuploidy accounts for nearly 15% of CHD and the most frequent form involves trisomy of chromosome 21 (Ch21), resulting in Down Syndrome (DS). Here we used single cell RNA-seq, CRISPR-activation with single cell RNA-seq, single cell ATAC-seq and Cut&Tag epigenomic profiling to define the molecular disruptions occuring with Trisomy 21 and upregulation of the Ch21 epigenetic factor HMGN1. These experiments were performed in human pluripotent stem cells (hiPSCs) or in directed differentiation to cardiomyocyte lineages at days 10 and 20.

overall_design: We used human pluripotent stem cells (hIPSCs) from an individual who was mosaic for Down Syndrome. Thus we compare cells that are trisomic for Ch21 with cells that are isogenically similar and disomic for Ch21. As well, we generated a CRISPR-activation knock-in to disomic cells and used sgRNAs directed to chromosome 21 genes. Sample replicates are defined and all single cell samples were processed with 10X Genomics pipelines. Cut&Tag samples were processed with Epicypher reagents. All samples were isolated with Accutase or Trypsin reagents.

first: Sanjeev

last: Ranade

first: Deepak

last: Srivastava


title: Targeting DDX3X suppresses progression of KRAS-driven lung cancer by disrupting antioxidative homeostasis and inducing ferroptosis

city: Guangzhou

first: Dian

last: Meijuan

address: NANFANG HOSPITAL SOUTHERN MEDICAL UNIVERSITY

institute: the sourthern medical university

summary: Approximately 30% of human cancers carry various RAS mutations, including KRAS, NRAS, and HRAS. Among these mutations, KRAS is the most prevalent isoform detected in lung cancer. While several small molecular inhibitors targeting specifically KRASG12C have been developed and tested clinically, alternative approaches are still necessary due to expected drug resistance. In this study, we present evidence that the loss of DDX3X significantly delays tumor progression in various KRAS-driven lung cancer models. Inhibition of DDX3X disrupts cysteine and glutathione metabolism, thereby inducing ferroptosis in lung cancer cells. This effect is primarily mediated by the downregulation of Cystathionine-β-synthase (CBS), the rate-limiting enzyme in cysteine generation. Mechanistically, DDX3X directly binds to the transcription factor JUND, which mediates the transcriptional regulation of METTL16, a key N6-methyladenosine methyltransferase, and subsequently regulates m6A modification and translation of CBS transcripts. This cascade induces hypermethylation and high expression of CBS, consequently triggering cysteine production and maintaining antioxidative homeostasis, which is essential for the survival of KRAS-driven lung cancer cells. Finally, we demonstrate that a newly developed DDX3X PROTAC degrader J10 efficiently delays lung cancer progression with multiple advantages compared to DDX3X small molecular inhibitor RK-33 and limited side effects. These findings unveil the potential of DDX3X as a valuable target for adjuvant therapies in managing KRAS-driven lung cancer.

overall_design: To study the role of DDX3X in lung cancer progression, we undertook a label-free proteomic analysis using DDX3X knockdown (shDDX3X) A549 cells.

first: Meijuan

last: Dian


title: MED26-mediated phase separation regulates erythropoiesis by tuning transcription pausing

city: Beijing,haidian

first: Shicong

last: Zhu

state: Beijing

address: Yi heyuan street, 5th.

department: School of life science and technology

institute: Peking university

summary: The Mediator complex plays a pivotal role in various aspects of transcription and development. However, it remains unclear whether individual Mediator subunits play differential roles during the progression of the developmental process. In this study, we found that the protein level of MED26, a subunit in the core Mediator complex, remained relatively abundant in terminal erythropoiesis, while that of most Mediator subunits drastically diminished. MED26 exhibits pronounced phase separation capability, which is essential for erythroid differentiation of primary human erythroblasts. Interestingly, we found that ~60% of chromatin sites with MED26 or MED1 occupancy did not co-localize. Moreover, MED26-enriched loci were associated with RNA polymerase II pausing, and MED26 preferentially interacted with transcription pausing factors, including PAF1, via its TFIIS domain. During erythropoiesis, both the relative abundance of MED26 and the Pausing Index (PI) increased. Erythroid differentiation was promoted by small molecule-induced PI elevation or overexpression of MED26—full-length or the IDR alone—indicating that MED26-associated pausing is necessary for erythropoiesis. In conclusion, this study revealed that MED26 tunes transcription pausing to facilitate erythroid development via a phase separation mechanism.

overall_design: To investigate the genes regulated by MED26, we compared the different expression genes in wild type K562 and MED26-KO K562 cell lines. Furthermore, to investigate the direct different binding sites of MED26 and MED1 at different erythroid differentiation stages, we conducted MED26,MED1 Cut and Tag assay.

first: Xiaoting


title: Genome-wide DNA replication profiling and full-length total RNA sequencing from the same single cell

city: Tsu-shi

first: Rawin

last: Poonperm

state: Mie

address: 1577 Kurimamachiya

department: Graduate School of Bioresources

institute: Mie University

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Multi-omics analysis identifies progenitors of CD4-CTLs [bulk ATAC-seq]

first: Veena

last: Patil

overall_design: Open chromatin analysis of purified populations of human CD4-Naïve (TN), CD4-TSCM, CD4-TCM, CD4-TEM, CD4-TEMRA-P and CD4-TEMRA-E by bulk ATAC-sequencing from 10 healthy donors.

first: Raunak

last: Kar

first: Shreya

last: Sinha

first: Zainab

last: Khatun

first: Anjali

last: Sharma


title: mRNA microarray analysis in colorectal cancer tissues

city: Nanjing

first: yueming

last: sun

state: jiangsu

address: Nanjing medical university

institute: The First Affiliated Hospital of Nanjing Medical University

type:
Expression profiling by array
Non-coding RNA profiling by array

summary: Colorectal cancer (CRC) is one of the most common malignant tumors of digestive tract, and its incidence rate and mortality rate are all on the rise. Despite the substantial  development in early diagnosis and therapeutic approaches, CRC continues to have high incidence and mortality rates. Most advanced patients have a poor prognosis because of advanced recurrence and metastasis.One of the main reasons is that the mechanisms underlying CRC remain unclear.  To further investage the role of circRNA in CRC, a mRNA micoarray was performed.

overall_design: Fresh tumor and matched normal tissues were collected from CRC patients in the First Affiliated Hospital   of NanjingMedical University after surgery . None of the patients received chemotherapy nor   radiotherapy preoperatively before surgery. All samples were frozen and stored at -80◦C conservation.

first: Junwei

last: Tang

first: Yueming

last: Sun


title: Hepatotoxicity evaluation of co-exposure of microplastics and TBBPA by 3D biofabricated multicellular organoids

city: Beijing

first: Shaojun

last: Liang

address: 18 Shuangqing Road

institute: Chinese academy of sciences

summary: We combined human induced pluripotent stem cells (hiPSCs) and 3D printing to mass-produced liver Disse space-like organoids in microspheres hydrogels (DOs). After 5 days of co-culture, the cells within the microspheres were aggregated and developed into liver organoids, composed of iPSC-derived hepatocytes, endothelial cells, and hepatic stellate cells (LX2). These organoids mirrored the hepatotoxicity of polystyrene microplastics (PS-MPs), TBBPA, and complexes MPTA with healthy and patient-derived organoids.

overall_design: Healthy person hiPSCs (N_iPSCs) and alcoholic liver disease patient hiPSCs (AC19-iPSCs) were differentiated into hepatic endoderm and endothelial cells. With hepatic endoderm, endothelial cells, LX-2 and alginate / laminin511, we fabricated healthy and patient-derived liver Disse space-like organoids: N_DOs and AC19_DOs. N_DOs were incubated with 60 ng/mL of polystyrene microplastics (MPs)(named N_MPs), 100 nM of TBBPA (named N_TBBPA), MPTA at 60 ng/mL/100 nM (N_MPTA), and 0.01% DMSO (N_Ctrl) for 3 days. The AC19_DOs were exposed to MPTA at 60 ng/mL/100 nM (AC19_MPTA) and 0.01% DMSO (AC19_Ctrl). The media were refreshed every day. To reveal the features of N_DOs and AC19_DOs, we compared them with the corresponding pre-printed cell mixtures (N_BPs and AC19_BPs) and primary human hepatocytes (PHH).

first: Rui

last: Yao

first: Yixue

last: Luo


title: Time-resolved small-RNA sequencing identifies microRNAs critical for formation of embryonic stem cells from the inner cell mass of mouse embryos

city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

institute: MPI for heart and lung research

summary: Cells of the inner cell mass (ICM) acquire a unique ability for unlimited self-renewal during transition into embryonic stem cells (ESCs) in vitro, while preserving their natural multi-lineage differentiation potential. Several different pathways have been identified to play critical roles in the formation of ESCs but the function of non-coding RNAs in this process is poorly understood. Here, we describe several microRNAs (miRNAs) that are crucial for efficient generation of mouse ESCs from ICMs. Using small-RNA sequencing, we characterize dynamic changes in miRNA expression profiles during outgrowth of ICMs in a high-resolution, time-course dependent manner. We detected several waves of miRNA transcription during ESC formation, to which miRNAs from the imprinted Dlk1-Dio3 locus contribute extensively. Stringent in silico and functional analyses reveal that Dlk1-Dio3 locus-embedded miRNAs (miR-541-5p, miR-410-3p, and miR-381-3p), miR-183-5p and miR-302b-3p promote, while miR-212-5p and let-7d-3p inhibit efficient ICM transition to ESCs. Collectively, these findings offer new mechanistic insights into the role of miRNAs during ESC derivation.

overall_design: Genome-wide expression profiles of microRNAs during embryonic stem cell (ESC) derivation at different time-points (days 0, 0.5, 1, 2, 3, and 5, and ESCs at passages 2, 4, and 15) were generated at by small-RNA deep sequencing, in duplicate, using Illumina GAIIx.

first: Sharif

last: Moradi

last: Guenther

first: Samira

last: Soori

first: Ali

last: Sharifi-Zarchi

first: Carsten

last: Kuenne

first: Vahid

last: Khoddami

first: Pouya

last: Tavakol

first: Susanne

last: Kreutzer

first: Thomas

last: Braun

first: Hossein

last: Baharvand


title: The Role of NF-κB Signaling Pathway in Lacrimal Gland Damage in Primary Sjögren’s Syndrome: an in vivo study

status: Public on Aug 25 2025

city: Hangzhou

first: Jiajun

last: Xie

state: Zhejiang

address: No. 88 Jiefang Rd

institute: Zhejiang University, Eye Center of Second Affiliated Hospital, School of Medicine, China

summary: Primary Sjögren’s syndrome (pSS) is an autoimmune disease characterized by inflammation and damage to exocrine glands, leading to symptoms such as dry eye and reduced tear production. The NF-κB signaling pathway plays a critical role in the inflammatory processes underlying pSS. The aims of the present in vivo study were to investigate the role of NF-κB signaling in lacrimal gland damage and tear secretion in the NOD/Ltj mouse, and to explore the therapeutic potential of NF-κB inhibition. The animal model was established using NOD/Ltj mouse and lacrimal gland pathological change was showed by H&E. Bioinformatics analysis was conducted through RNA sequencing (RNA-seq) and confirmed by RT-qPCR and Western blot. The NOD/Ltj mouse were treated with NF-κB inhibitor JSH-23. Tear secretion assays, ELISA, TUNEL staining, immunofluorescence, immunohistochemistry, and flow cytometry were employed to assess the role of NF-κB signaling in tear production, inflammatory cytokine expression, cell apoptosis, differentially expressed genes (DEGs), and cell differentiation in lacrimal gland damage in pSS mouse. NF-κB signaling was significantly activated in NOD/Ltj mouse lacrimal gland and pSS-related dry eye. NF-κB blockage decreased cell apoptosis, reduced inflammatory cytokine expression, and inhibited Th17 cell differentiation in NOD/Ltj mouse lacrimal glands, as well as restored tear production and normalized the expression of DEGs CARD14 and CCL19. NF-κB signaling played a pivotal role in lacrimal gland damage in pSS model NOD/Ltj mouse by involvement of cell apoptosis, inflammatory cytokine expression and Th17 cell differentiation. Targeting NF-κB signaling may offer a promising therapeutic approach for managing pSS-related dry eye.

overall_design: RNA-seq profiling of lacrimal glands of NOD/Ltj mouse and ICR mouse.

first: Huina

last: Zhang

first: Wenyue

last: Shen

first: Juan

last: Ye


title: Extracellular vesicles from chylomicron-treated endothelial cells drive macrophage inflammation

city: New York

first: Genome Technology Center

last: (GTC)

address: 550 First Avenue

institute: NYU Langone Health

type: Non-coding RNA profiling by high throughput sequencing

summary: Movement of circulating lipids into tissues and arteries requires transfer across the endothelial cell barrier. This process allows the heart to obtain fatty acids (FAs), its chief source of energy, and apolipoprotein B (apoB)-containing lipoproteins to cross the arterial endothelial barrier leading to cholesterol accumulation in the subendothelial space. Multiple studies have established elevated postprandial triglyceride-rich lipoproteins (TRLs) as an independent risk factor for cardiovascular disease (CVD). We explored how chylomicrons affect ECs and transfer their FAs across the EC barrier. We had reported that media from chylomicron-treated ECs leads to lipid droplet (LD) formation in macrophages. To determine the responsible component of this media, we assessed whether removing the extracellular vesicles (EVs) would obviate this effect. EVs from control and treated cells were then characterized by protein, lipid and microRNA (miR) content. We also studied the EV-induced transcription changes in macrophages and ECs and whether knockdown of scavenger receptor-BI (SR-BI) altered these responses. In addition, using chylomicrons labeled with [13C]oleate, we studied the uptake and release of this labed by ECs.Chylomicron treatment of ECs led to an inflammatory response that included production of EVs that drove macrophage LD accumulation. The EVs contained little free fatty acids and triglyceride, but abundant phospholipids and diacylglycerols. In concert with this, [13]C labeled chylomicron triglycerides exited ECs primarily in phospholipids. EVs from chylomicron treated versus untreated ECs were larger, more abundant, and contained specific miRs. Treatment of macrophages and naïve ECs with media from chylomicron-treated ECs increased expression of inflammatory genes. EC chylomicron metabolism produces EVs that increase macrophage inflammation and create LDs. Media containing these EVs also increases EC inflammation, illustrating an autocrine inflammatory process. FAs within chylomicron triglycerides are converted to phospholipids within EVs. Thus, EC uptake of chylomicrons constitutes an important pathway for vascular inflammation and tissue lipid acquisition.

overall_design: Bulk RNA seq of endothelial cells (Ecs) and peritoneal macrophages (PMACs).Also, bulk sequencing of circulating microRNAs in the media of Ecs.

first: Dimitris

last: Nasias

first: Ira

last: Goldberg

first: Ainara

middle: G

last: Cabodevilla


title: Nanoparticle-boosted myeloid-derived suppressor cell therapy for immune reprogramming in multiple sclerosis

city: Chicago

first: Zongmin

last: Zhao

state: Illinois

address: 833 S Wood St

department: Department of Pharmaceutical Sciences

institute: University of Illinois Chicago

summary: Massive immune cell infiltration and persistent inflammation in the central nervous system (CNS) are key hallmarks of multiple sclerosis, an incurable autoimmune disease. Here, we report a myeloid-derived suppressor cell (MDSC)-based therapeutic strategy, named CNS Immune Targeting Enabled by MDSCs (CITED), which employs surface-decorated MDSCs carrying rapamycin nanoparticles (NPs) for targeted multimodal immune reprograming in the CNS. We show that NP decoration enhances MDSC immunomodulatory function, facilitates their trafficking to inflamed CNS regions, and increases NP accumulation within the CNS. In an experimental autoimmune encephalomyelitis (EAE) model, CITED exhibited robust therapeutic efficacy, resulting in reduced disease progression, improved motor function, and diminished myelin damage. Mechanistic studies reveal that CITED exerts its therapeutic effects by targeted reprogramming both innate and adaptive immune responses in CNS. Specifically, CITED inhibits immune cell infiltration, rebalances CD4 T cell phenotypes, and promotes the polarization of myeloid cells toward anti-inflammatory phenotypes. Collectively, CITED could provide a broadly effective approach for targeted immune restoration in multiple sclerosis and potentially other autoimmune diseases.

overall_design: RNA seq profilling of myeloid derived suppressor cells (MDSC), MDSCs conjugated with blank NP(MDSC_NP_Blk) and MDSCs carrying rapamycin nanoparticles (MDSC_NP_CITED).

first: Endong

first: Hanan

last: Algarni

first: Chih-Jia

last: Chao

first: Shan

last: He

first: Luyu

first: Aditi

last: Upadhye

first: Qing

last: Bao

first: Dahee

last: Jung

first: Shubhi

last: Srivastava

first: Edidiong

last: Udofa

first: Philana

last: Phan

first: Steve Seung-Young

last: Lee

first: Jalees

last: Rehman


title: Single-cell RNA and DNA methylome profiling reveal liquid helium vitrification enhances porcine parthenogenetically activated blastocyst viability [Bisulfite-Seq]

first: Baigao

last: Yang

address: Yuaningyuan 2

institute: Institute of Animal Sciences of CAAS

type: Methylation profiling by high throughput sequencing

summary: Transcriptome and methylation sequencing technologies were used to reveal the effects of vitrification on gene expression and genomic methylation levels in pig blastocysts.

overall_design: A fresh control group A, a vitrified freezing group B, a vitrified freezing group C, and a vitrified freezing group H were set up.

first: Xueming

first: Hongmei

last: Pan


title: Single-cell RNA and DNA methylome profiling reveal liquid helium vitrification enhances porcine parthenogenetically activated blastocyst viability [RNA-Seq]


title: Junction-binding–defective Cdc13 promotes adaptive mutation and transcriptomic rewiring under telomere stress

city: Bloomington

first: Hengyao

last: Niu

state: IN

address: Simon Hall MSB1, Room 415 212 S. Hawthorne Drive

department: Molecular and Cellular Biochemistry

institute: Indiana University

summary: Telomeres protect chromosome ends from degradation and inappropriate DNA repair. In budding yeast, the telomeric protein Cdc13 binds G-rich overhangs to maintain telomere integrity. Here, we show that Cdc13 also weakly interacts with adjacent duplex DNA at the ss/dsDNA junction, a property essential for inhibiting Exo1- and Sgs1–Dna2–mediated resection. Mutation of a conserved residue (K504E) disrupts this junction recognition, impairing resection inhibition, RPA displacement, and cooperation with Ku. cdc13-K504E cells are hypersensitive to Exo1 overexpression and accumulate non-pigmented revertant colonies during chronic growth. These white colonies exhibit faster growth, altered telomere-proximal gene expression, upregulation of metabolic pathways, and increased 4N DNA content. Our findings identify a dual DNA-binding mode of Cdc13 that protects telomeres by blocking resection and regulating Ku positioning, and reveal a stress-induced adaptive state associated with telomere dysfunction.

overall_design: 3 biological replicates per condition; RNA-seq of WT-red, K504E-red, K504E-white colonies.

first: Zhitong

last: Feng


title: A Hedgehog-Induced Homeodomain Cascade Couples Progenitor Identity to Neuronal Diversification

city: Eugene

first: Chris

last: Doe

state: Oregon

address: 1440 Franklin Blvd

institute: University of Oregon

summary: How progenitor identity instructs neuronal diversification remains unclear. We uncover a homeodomain transcription factor (HDTF) cascade that links the identity of a single lamina progenitor type to the diversification of all five lamina neuron subtypes in the Drosophila visual system. Hedgehog signaling induces a pan-class HDTF—expressed across all subtypes of a neuronal class, here all lamina neurons—in progenitors, promoting cell-cycle exit. This progenitor-derived pan-class HDTF is inherited by newborn lamina neurons, where it activates subtype-specific, differentially expressed (DE) HDTFs and acts combinatorially with these DE-HDTFs to specify distinct neuronal subtypes. Loss of DE-HDTFs induces predictable, subtype-specific fate conversions—morphological and functional—reducing neuronal diversity. Strikingly, we find mouse retinal neurons express analogous pan-class and DE-HDTFs, indicating an evolutionarily conserved strategy. With HDTFs broadly expressed, our findings define a progenitor-to-newborn HDTF cascade that provides a generalizable mechanism by which progenitor identity drives neuronal diversification from a limited progenitor pool.

overall_design: 4508 lamina neurons of adult female Drosophila flies were sequenced using 10x Genomics pipeline followed by scanpy analysis.

first: Chundi

last: Xu

first: Peter

last: Newstein


title: Immune and Transcriptional Landscape of Endometrial Receptivity at P+5 Phase: A Triad Analysis of EIN, RIF, and Control Endometrial Profiles

city: beijing

first: Tao

last: Tao

state: Beijing city

address: No. 1 Shuaifuyuan, Dongcheng District, Beijing

department: Department of Obstetrics and Gynecology

institute: Peking Union Medical College Hospital

summary: This study delineates the immune and transcriptional landscape of endometrial receptivity at the P5 phase via triad analysis of Endometrial Intraepithelial Neoplasia/Early Endometrial Cancer (EIN/ECa, N = 64), Repeated Implantation Failure (RIF, N = 25), and control (Ctrl., N = 3)) endometrial profiles. RNA sequencing and deconvolution algorithms identified differential gene expression, immune cell composition, and pathway enrichments. EIN/ECa exhibited dysregulated complement/coagulation cascades and proliferation-related genes, suggesting a pro-proliferative/inflammatory microenvironment. RIF showed immune pathway perturbations, with abnormal TLR4 and CXCL10 expression disrupting maternal-fetal tolerance. Integrative analysis revealed distinct molecular signatures: EIN/ECa prioritized oncogenic proliferation, while RIF focused on immune tolerance defects.These findings illuminate phase-specific endometrial receptivity mechanisms, providing targets for improving fertility outcomes in high-risk groups.

overall_design: These findings illuminate phase-specific endometrial receptivity mechanisms, providing targets for improving fertility outcomes in high-risk groups.RNA Sequencing and Data Analysis RNA was extracted from the endometrial tissue using the RaPure Total RNA Micro Kit & DNase Set (Magen). RNA quantification was performed using the Qubit RNA HS kit (Thermo Fisher Scientific). The extracted RNA samples were reverse transcribed into cDNA using the MALBAC Platinum Micro RNA Amplification Kit (Yikon Genomics). The cDNA products were purified using DNA Clean & Concentrator (Zymo Research). Library preparation involved DNA fragmentation, end repair, adaptor ligation, and PCR enrichment and purification. The resulting libraries were quantified using the Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific) and then sequenced on the Illumina MiSeq DX platform. Sequencing reads were analyzed using a computational model developed by Yikon Genomics in collaboration with Xiangya Hospital of Central South University, designed to predict endometrial receptivity status (rsERT). This model utilizes a panel of over 12,000 differentially expressed biomarker genes spanning the pre-receptive, receptive, and post-receptive phases of the endometrial window of implantation (WOI). The expression profile of each sample was mapped to the three WOI phases, and the predicted deviation from the optimal WOI was calculated. Software and Bioinformatics Analysis Processed data were uploaded to ChromGo (Yikon Genomics，Suzhou, China) for data partitioning, bioinformatics analysis, and determination of the recommended time for optimal endometrial receptivity. Experimental Design and Quality Control The experimental design encompassed RNA sequencing (RNA-seq) and established quality control procedures, including the aforementioned RNA extraction and library preparation methods. Differentially Expressed Gene (DEG) Identification Differentially expressed genes (DEGs) were identified using the following criteria: |log2FC| > 1 and adjusted p-value (padj) < 0.05. Functional Enrichment Analysis Functional enrichment analysis was performed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA). Immune Cell Deconvolution Analysis Immune cell deconvolution analysis was performed on the bulk RNA-seq data using established methods21,22.

first: Yuanzheng

last: Zhou

first: Chengyan

last: Deng

first: Duoduo

last: Zhang

first: Zhengyi

last: Sun

first: Dongyan

last: Cao

first: Qian

last: Liu


title: RoCK and ROI: a single-cell RNA-sequencing method with enhanced transcriptome information via targeted enrichment of transcripts and preselected, region-specific sequencing V

city: Zürich

first: Giulia

middle: Arianna

last: Moro

address: Winterthurerstrasse 190

department: DMLS

country: Switzerland

institute: University of Zurich

summary: Many scRNAseq-based methods suffer from a high number of non-detects and a bias towards the 3' or 5' end of transcripts. We developed a method termed RoCK and ROI to address these two biases. RoCK and ROI leads to the enrichment of the transcriptome profile of cells with information on transcripts of interest

overall_design: RoCK and ROI is based on barcoded beads of the BD Rhapsody platform. For RoCKseq specific capture, TSO oligos on BD Rhapsody beads are modified with a/ multiple capture sequence(s) specific to transcripts of interest. ROIseq, on the other hand, is based on specific primers which are spiked in during library generation, directing reads to regions of interest (ROIs). For this set of experiments, RoCK and ROI was performed to enhance the detection of seven transcripts of interest in HeLa cells.

first: Izaskun

last: Mallona

first: Mark

middle: D

last: Robinson

first: Erich

last: Brunner

first: Konrad

last: Basler


title: Chemoresistance of TP53 mutant AML requires the mevalonate byproduct, GGPP, for induction of an adaptive stress response

city: Philadelphia

first: Robert

middle: L

last: Bowman

state: PA

address: 421 Curie Blvd

department: Cancer Biology

institute: University of Pennsylvania School of Medicine

summary: Acute myeloid leukemia with mutations in TP53 (TP53mut AML) is fatal with a median survival of 6 months. RNA sequencing on purified AML patient samples showed that TP53mut AML had higher expression of mevalonate pathway genes. Using novel, isogenic TP53mut AML cell lines and primary samples, we determined that TP53mut AML resistance to AML chemotherapy cytarabine (AraC) correlated with increased mevalonate pathway activity, a lower induction of reactive oxygen species (ROS), and a mitochondrial response with increased mitochondrial mass and oxidative phosphorylation. Pretreatment with the statin class of mevalonate pathway inhibitors reversed these effects and chemosensitized TP53mut AML. The geranylgeranyl pyrophosphate (GGPP) branch of the mevalonate pathway was required for TP53mut AML chemoresistance. In addition to its role in mitochondria biogenesis, we identified a novel function of GGPP in regulating one-carbon metabolism and subsequent glutathione and polyamine synthesis for management of AraC-induced ROS. However, statins alone were inadequate to fully reverse chemoresistance in vivo and in a retrospective study of 364 TP53mut AML patients who received chemotherapy concurrently with a statin. Finally, we identified clinical settings and strategies to successfully target the mevalonate pathway, particularly to address the unmet need of TP53mut AML

overall_design: RNA-seq profiling of purified leukemic blasts from acute myeloid leukemia patients at diagnosis. Patients were stratified based on outcome and TP53 mutation status.

first: Sarah

last: Skuli

first: Martin

last: Carroll


title: ACTL6A regulates the Warburg effect through coordinated activation of AP-1 signaling in head and neck squamous cell carcinoma

city: Palo Alto

first: Andrey

last: Finegersh

address: 900 Blake Wilbur Dr, 3rd Floor

institute: Stanford University

summary: ACTL6a is an essential component of SWI/SNF and expressed on the chromosome 3q26 cytoband, which is amplified in head and neck squamous cell carcinomas (HNSCC). While ACTL6A is emerging as an oncogene, its role as a treatment target and mechanisms of transcription factor induction remain unknown. Here, we show that ACTL6A expression is a mediator of the Warburg effect, with ACTL6A knockdown inducing mitochondrial dependency and significantly decreasing levels of aerobic glycolysis. Using ATAC-seq, we identify ACTL6A as a mediator of chromatin accessibility of AP-1 transcription factor sites and find that it regulates upstream MAPK signaling through induction Ras and Galectin-1. These effects sensitize ACTL6A over-expressing cells to inhibition of glycolysis by MEK inhibitors. Our results link SWI/SNF subunit amplification with potentiation of MAPK signaling in HNSCC and provide a novel mechanism by which cancer cells drive aerobic glycolysis and reduce mitochondrial dependency.

overall_design: ACTL6A knockdown of SCC1 cells using siRNA compared to a non-targeting siRNA control. SCC1 cells were incubated for 72 hours and harvested. ATAC-seq and RNA-seq was performed.

first: Mehri

last: Monavarian


title: Interleukin-27-adipose-derived mesenchymal stromal cell-based gene therapy attenuates inflammation in lipopolysaccharide-induced acute respiratory distress syndrome

city: West Lafayette

first: Marxa

middle: Leao

last: Figueiredo

address: 625 Harrison St

department: Basic Medical Sciences

institute: Purdue University

summary: Acute respiratory distress syndrome (ARDS) is a lung inflammatory condition associated with the accumulation of fluid edema and cell infiltrates into the alveolar space along with the dysregulation in the immune response. Current therapeutics are limited to palliative care, i.e., mechanical ventilators, thus highlighting the need of developing targeted therapeutic for ARDS. Interleukin-27 (IL-27) is a multifunctional cytokine with the capability for immune modulation. Our interest lies in exploring the properties of IL-27, particularly as an anti-inflammatory cytokine that functions as an antagonist of IL-6 signaling, as an inducer of anti-viral genes, as a promoter of tissue repair, and as a regulator of both the innate and adaptive immune responses, possessing promising potential as a therapeutic for ARDS. To overcome the challenge of repeated administration due to the short half-life of cytokines, we utilized a cell-based gene therapy approach. IL-27-expressing plasmid was transfected into adipose mesenchymal stromal cells (ASC) that serve as the gene therapy carriers. For in vitro studies, we treated mono- and co-culture lung lipopolysaccharide (LPS)-induced lung epithelial and monocytes/macrophages cell line with IL-27-expressing ASC (IL-27) conditioned media (CM) to determine the effects on pro-inflammatory gene expression. For in vivo studies, male C57BL/6 mice was intratracheally injected with LPS (5mg/kg) and treated either PBS, ASC, or IL-27 ASC (5 x 105 cells/mouse) after 24h of LPS instillation. Measurements of gene expression, histopathological changes, cell infiltrations, and protein content in the bronchoalveolar lavage fluid and RNA-seq analysis were performed for the in vivo studies.  Results indicated that IL-27ASC CM were able to reduce pro-inflammatory gene expression of lung epithelial and macrophages cultured in both mono- and co-culture systems. Additionally, IL-27 ASC were able to reduce pro-inflammatory markers, decrease cell infiltration into the lungs, promote genes and immune cells involved in tissue repair, and rebalance innate and adaptive immunity in an LPS-induced in vivo model. Collectively, our in vitro and in vivo results showed promising potential for IL-27 cell-based gene therapy as a treatment for ARDS.

overall_design: In this study, male C57BL/6 mice (8-10 weeks old) were used to create an in vivo LPS-induced ARDS model. Anesthesia was administered using ketamine and xylazine, and LPS (E. coli O111:B4) was given intratracheally at 5 µg/g body weight. Mice were randomly assigned to treatment groups, including 1X PBS (control), LPS, LPS + hASC with empty vector, and LPS + hASC with IL-27 vector. At 24 hours post-LPS instillation, treatments were given, followed by air infusion to ensure distribution. Animals were sacrificed at 72 hours post-LPS instillation, and samples (BALF, serum, lung, and organ tissues) were collected for analysis.

first: Grace

middle: E

last: Mulia

last: Figuereido


title: Genome-wide identification of transcriptional targets for H3K18la in adipose stem and progenitor cells

city: Shanghai

first: Hongrui

last: Chen

address: 639 Zhizaoju Road, Shanghai 200011, P.R.China.

department: Plastic and reconstruction surgery

institute: Shanghai ninth people's hospital

summary: In this study, we found that H3K18la level is elevated in adipose stem and progenitor cells of facial infiltrating lipomatosis (FIL-ASPCs). To further explore the potential functional significance of H3K18la in FIL, we performed genome-wide cleavage under targets and tagmentation (CUT&Tag) analysis to identify candidate genes regulated by H3K18la in FIL-ASPCs and CON-ASPCs. Following CUT&Tag, H3K18la-associated DNAs were amplified using non-biased conditions, labeled, and sequenced with Illumina NovaSeq 150PE.

overall_design:
Group 1: FIL-ASPCs. Group 2: CON-ASPCs
CUT&Tag

first: Chen

last: Hua

first: Xiaoxi

last: Lin


title: Colon tissue gene expression associated with vedolizumab treatment effectiveness in ulcerative colitis.

city: Tokyo

first: Mayu

last: Suzuki

address: 2-1-1 Nihonbashi-Honcho, Chuo-ku

department: Japan Medical Office

institute: Takeda Pharmaceutical Co., Ltd.

summary: Transcriptional profiling was performed on colorectal tissues, both inflammatory and non-inflammatory, collected from patients with ulcerative colitis.

overall_design: RNA was extracted from the tissues using RNeasy Mini Kit (Qiagen) and AmpliSeq libraries were prepared with the Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (Thermo Fisher Scientific) using 10 ng of RNA. The resulting libraries were used for sequencing on the Ion 550™ Chip Kit , Ion 550™ Kit-Chef and Ion GeneStudio™ S5 Prime System (Thermo Fisher Scientific), and output data (BAM files) were obtained.

first: Mikihiro

last: Fujiya

first: Kaori

last: Ishiguro


title: Longitudinal multimodal characterization of radiation dermatitis in a C57BL/6J mouse model

city: Bronx

first: Kosaku

last: Shinoda

state: New York

address: 1301 Morris Park Ave

department: Medicine

institute: Albert Einstein College of   Medicine/Montefiore Medical Center

summary: Radiation dermatitis (RD) is a common adverse reaction to radiation therapy (RT), affecting up to 95% of cancer patients. Despite its prevalence, RD’s pathophysiology is complex and remains poorly understood. Without a better molecular understanding of this condition, targeted treatments are difficult to develop and integrate into clinical practice.  Prior animal model studies of RD are scarce, variable in methodology, and limited to outcomes such as clinical grading and histology. To address this gap, we aimed to develop a temporal murine model of RD using clinical grading, objective skin parameters, histopathology, and gene expression analysis of inflammatory markers.

overall_design: RNA-seq profiling of skin biopsies taken from C57BL/6J mice radiated with 25 Gy to the skin of the left flank. Ten-week-old female mice (n=19) were randomly divided into five groups: sham (no RT), and four post-RT groups with weekly data collection for four weeks.

first: Jasmine

middle: H

last: Wong

first: Li

last: Liu

first: Beth

last: McLellan

first: Sharen

last: Rivas


title: Single-dose cathepsin L CRISPR nanotherapy mitigates long COVID–like lung damage in hamsters

city: Milwaukee

first: Victor

last: Jin

state: WI

address: 8701 W Watertown Plank Rd

institute: MCW

summary: We investigate the long-term cellular response in the lungs of golden Syrian hamsters 31 days after SARS-CoV-2 infection.

overall_design: The SARS-CoV-2-infected golden Syrian hamsters received a single-dose CRISPR-CasRx nanotherapy targeting the host enzyme cathepsin L (SCNC), administered 4 hours post-infection, with a non-targeting CRISPR-CasRx nanotherapy serving as the control. At 31 days post-infection, the caudal lung lobe was isolated. Six samples were subjected to single-cell RNA sequencing, with three samples from the SCNC treatment group and the remaining three from the control group.

first: Zhifen

last: Cui

first: Tianxiang

middle: X

first: Qianben

last: Wang


title: An efficient framework to decipher microRNA regulatory programs in functionally divergent naïve T cell subsets [smallRNAseq_mir29cre]

city: Ithaca

first: Andrew

last: Grimson

state: NY

address: 445 Biotechnology Building

department: Molecular Biology & Genetics

institute: Cornell University

summary: Naïve CD8+ T cells are heterogenous, with subsets exhibiting divergent kinetics and functions post-activation. MicroRNAs (miRNAs) are post-transcriptional regulators, and certain miRNAs contribute to specification of different naïve T cell subsets. However, the microRNA regulatory circuits mediating functional specialization of naïve subsets have not been identified. In this work, we profiled microRNA expression in diverse subsets of naïve CD8+ T cells, revealing significant differences in their microRNA expression landscapes. We developed a novel framework, miR-Inf, to decipher microRNA regulatory programs. MiR-Inf has two key components: (i) an efficient method for estimating gene decay rates from the RNA seq profiles to better capture microRNA regulatory effects, and (ii) identification of functional microRNA targets by integrating decay rate data and microRNA expression data. We applied this framework to identify consequential miRNAs in naïve CD8+ T cell subsets and predicted their context-specific targets. Our analyses revealed that miR-29, a microRNA known to be important in CD8+ T cells, likely functions by modulating transcripts encoding epigenetic factors. Collectively, our data and framework defined microRNA regulatory circuits across diverse naïve CD8+ T cell subsets.

overall_design: Small RNAseq of TN/VM CD8+ T cells isolated from Cre- and WT mice , with twp replicates each.

first: Hongya

last: Zhu

first: Ciarán

last: Daly

first: Kristel

last: Yee Mon

first: Cybelle

last: Tabilas

first: Divya

last: Sankaran

first: Erin

last: Wissink

first: Brian

last: Rudd


title: An efficient framework to decipher microRNA regulatory programs in functionally divergent naïve T cell subsets [RNAseq_mir29cre]

overall_design: RNAseq of TN/VM CD8+ T cells isolated from Cre- and WT mice , with twp replicates each.


title: An efficient framework to decipher microRNA regulatory programs in functionally divergent naïve T cell subsets [smallRNAseq_adultNeoTNVM]

overall_design: Small RNAseq of TN/VM CD8+ T cells isolated from neonatal and adult mice


title: An efficient framework to decipher microRNA regulatory programs in functionally divergent naïve T cell subsets [smallRNAseq_cd8dev]

overall_design: Small RNAseq of CD8+ T cells isolated from neonatal (5-7 days post-birth) and adult (12 weeks) mice , with four replicates each.


title: An efficient framework to decipher microRNA regulatory programs in functionally divergent naïve T cell subsets [smallRNAseq_cleandirty]

overall_design: Small RNAseq of TN/VM CD8+ T cells isolated from clean and dirty mice , with twp or three replicates each.


title: RNA sequencing in Saccharomyces cerevisiae with abrogated binding of Nab3 to PIC2

city: Edinburgh

first: Sander

last: Granneman

address: Mayfield Road, Kings Buildings, Waddington building, room 3.06

department: Centre for Synthetic and Systems Biology

country: United Kingdom

institute: University of Edinburgh

summary: Whole-transcriptome sequencing allowed us to compare transcriptomic differences between a Saccharomyces cerevisiae BY4741 parental strain (PIC2-GFP) and two derived mutants lacking Nab3 RNA-binding sites in PIC2. Previous experiments had established that disrupting Nab3 binding to PIC2 while maintaining Nrd1 binding to the same transcript led to severe growth defects, larger cell size, delayed cell cycle and resistance to oxidative stress. Following characterization of a PIC2-overexpressing mutant we established that these phenotypes were not related to Pic2 overabundance. Therefore, the purpose of this experiment was to determine (i) whether the transcriptomic signature of the mutant lacking Nab3 RNA-binding sites in PIC2  mutant could account for the observed phenotypes and (ii) if the transcripts that were differentially expressed in such mutant were targeted by Nab3 and Nrd1.

overall_design: Three biological replicates of RNA-sequencing experiments performed in a parental Saccharomyces cerevisiae strain (PIC2-GFP) and two derived mutants: mut-Nab3-BS and mut-NNS-BS. While mut-Nab3-BS lacks Nab3 RNA-binding sites in PIC2, the other strain has no Nab3 or Nrd1 RNA-binding sites in PIC2 (mut-NNS-BS). Cells were grown in 25 mL-cultures of SC-Ura containing 1% (w/v) raffinose and harvested at by centrifugation (4000 rpm, 5 minutes) at an OD600 of 0.5. Pellets were then snap frozen by submersion in liquid nitrogen and stored at -80ºC. On the day of the experiments, cells were thawed on ice and denatured by the addition of 200 μL of guanidinium thiocyanate (GTC) acid phenol mix. Cell lysis was performed by adding 400 μL of glass beads to each sample and vortexing all tubes at full speed for five 1-minute intervals separated by four 1-minute incubations on ice. An additional 1.5 mL of GTC phenol mix was added to the samples before incubating them at 65ºC for 10 minutes. Samples were then placed on ice for another 10 min. 800 μL of sodium acetate mix (3.3 mL 3 M NaOAc pH5.2, 0.2 mL 0.5M EDTA pH 8, 1mL 1M Tris-HCl pH 8, water to 100 mL) and 1.5 mL of chloroform were pipetted into each extract before vortexing at maximum speed for 5 seconds. Lysates were centrifuged at 4ºC (10000 rpm, 30 minutes) and the resulting aqueous phase was transferred into a new tube containing an equivalent volume of Phenol:Chloroform:Isoamylalcohol mix. The mixtures were vortexed vigorously and centrifuged at 10000 rpm for 5 minutes. Once more, the aqueous phase of each sample was mixed with an equal volume of Chloroform:Isoamylalcohol and vortexed at maximum speed before centrifugation at 10000 rpm for 5 minutes. Finally, the aqueous phase of each sample was relocated to tubes containing 3 volumes of 96% ethanol. The mix was vortexed vigorously and preserved at -80°C for at least 30 minutes or at -20°C overnight. Following ethanol precipitation, samples were spun at 1000 rpm for 30 minutes at 4ºC. After discarding the resulting supernatants, 70% ethanol was pipetted into each tube. After centrifuging samples at 10000 rpm for 5 minutes at 4ºC, ethanol was removed, and pellets were left to air-dry for approximately 5 minutes before resuspension in DEPC water. Finally, RNA concentrations were measured in the Qubit™ 4 fluorometer (Thermo Fisher Scientific) using the Qubit™ RNA HS assay kit (Thermo Fisher Scientific). RNA extracts were treated with RQ1 RNase-Free DNase as per manufacturer’s guidelines. The quality of the RNA samples was then inspected in an Agilent 2100 bioanalyzer (Agilent Technologies) using an RNA 6000 pico assay (Agilent Technologies) according to the manufacturer’s instructions. Samples with RNA integrity number (RIN) scores greater than 7 were submitted to Novogene for paired-end sequencing of 150 bp-reads in a NovaSeq 6000 system (Illumina). Before sequencing, samples underwent a rRNA depletion step and libraries were generated using the TruSeq preparation protocol (Illumina). Raw sequencing outputs were returned as FASTQ files. Hence, after conversion into FASTA format, files were processed using the paired-end (PE) version of the pyCRAC pipeline (Webb et al., 2014). This package demultiplexes barcoded reads and removes adapter sequences applying the previously developed Flexbar tool (Dodt et al., 2012). The resulting reads undergo two quality control steps. Firstly, sequences are filtered based on their length and nucleotide quality values (i.e., PHRED scores). Secondly, since PCR artifacts will display identical random prefixes, they can easily be pinpointed and excluded from downstream analysis (collapsed). The remaining unique reads were subsequently aligned to the corresponding regions of the Saccharomyces_cerevisiae.R64-1-1.75 reference genome with the Novoalign tool (version 2.07). The pyReadCounters function of the pyCRAC package (version 1.5.1; Webb et al., 2014) was used to compute the number of reads that map to genomic features. The resulting tables for all the samples were merged into a single TXT file that was analysed by the DESeq2 R package (Love et al., 2014). The pipeline corrects for size variability by computing a scaling factor for each sample. To obtain such normalisation factor, the software calculates the ratio of the raw count of a given gene in one of the samples to the geometric mean of the counts for that same gene across the compared samples. The median of all the ratios calculated within a sample is defined as the scaling factor for that replicate. Having adjusted the raw reads, the package then performs a logarithmic transformation of the data and calculates a fold change and an FDR for each entry. Statistical significance was assessed for entries which exceeded or were lower than log2 fold change o 1 and -1 respectively and had an FDR value of 0.05 or less.

first: Sofia

last: Serna


title: Mapping of Sen1 RNA-binding sites in Saccharomyces cerevisiae with abrogated binding of Nab3 to PIC2

summary: Using CRAC, we compared the transcriptomic occupancy of Sen1 in a Saccharomyces cerevisiae BY4741 parental strain (PIC2-GFP) and two derived mutants lacking Nab3 RNA-binding sites in PIC2. The purpose of the experiment was two-fold: on the one hand, we aimed to verify that the mutations inserted in the Nab3 binding sequences of PIC2 affected the binding of Sen1 to the PIC2 transcript; on the other hand, we wanted to check whether differential binding of Nab3 to PIC2 would affect how the protein bound other targets in the genome. Sequencing outputs were processed using the pyCRAC pipeline and peak calling was performed with DBPeaks, our newly developed package for identification and comparison of binding sites defined by RNA-binding footprinting techniques (e.g., CRAC, iCLIP, PAR-CLIP, etc.).

overall_design: Two biological replicates of CRAC experiments performed in a parental Saccharomyces cerevisiae BY4741 strain (PIC2-GFP) and two derived mutants: mut-Nab3-BS and mut-NNS-BS. While mut-Nab3-BS lacks Nab3 RNA-binding sites in PIC2, the other strain has no Nab3 or Nrd1 RNA-binding sites in PIC2 (mut-NNS-BS). All strains encoded SEN1-HTF (HIS6-TEV-3xFLAG). When SC-Ura cultures containing 1% (w/v) raffinose reached an OD600 of 1, cells were UV cross-linked (254 nm; 500 mJ/cm2) in the Vari-X-linker (UVO3) and rapidly filtered on 0.8 μm membranes (Merck) using a filtration device connected to a vacuum pump. Membranes were collected in 50 mL tubes, flash frozen by submersion into liquid nitrogen and stored at -80ºC until the day of the experiment.   On the day of the experiment, pellets were resuspended in a volume of lysis buffer (150 mM NaCl, 50 mM Tris pH 7.8, 0.1% (v/v) Nonidet P-40, 1 EDTA-free protease inhibitor cocktail (Roche) per 50 mL and 5 mM β-mercaptoethanol) corresponding to twice their mass. Lysates were then transferred to a fresh 50 mL tubes, where they were mixed with 2 volumes of 0.5 mm zirconia beads (Thistle Scientific), and vortexed at maximum speed for five 1-minute intervals alternated with 1-minute incubations on ice. An additional volume of lysis buffer was pipetted into each sample before partitioning the cell debris by centrifugation at 4ºC (4000 rpm, 15 minutes) and spinning down lighter impurities in an additional centrifugation step (10000 rpm, 20 minutes) at 4ºC. Immunoprecipitation of the labelled protein from the supernatant was achieved during a two-hour incubation of the sample with 75 µL of anti-FLAG® M2 magnetic beads (Sigma-Aldrich, M8823-5ML) which had been previously washed three times with 1 mL TN150 buffer. Immobilised beads underwent three 5-minute washes with TN1000 buffer (50 mM Tris pH 7.8, 1 M NaCl, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol) and three additional 5-minute washes in 2 mL TN150 buffer (150 mM NaCl, 50 mM Tris pH 7.8, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol). Afterwards, beads were resuspended in 550 µL of TN150 and incubated with 1 µL of a 1:100 dilution of RNace-It™ for exactly 5 minutes at 37 °C. To prevent overdigestion of co-immunoprecipitated transcripts and elute the bait proteins from the anti-FLAG beads, the solution was mixed with 0.4 g of guanidium hydrochloride (Gu-HCl) and the necessary volume of NaCl and imidazole (pH 8.0) to reach a final concentration of 300 mM and 10 mM respectively. After removing the beads, samples were pipetted onto 50 µL of Ni-NTA agarose resin (Qiagen) equilibrated with wash buffer I (6 M Gu-HCl, 10 mM imidazole, 300 mM NaCl, 50 mM Tris-HCl pH 7.8, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol) and incubated at 4 °C for two hours while rotating at 12 rpm.   Upon Ni-NTA binding, beads were transferred to Pierce columns (Thermo Fisher Scientific) and washed twice with 500 µL of wash buffer I before undergoing three additional washes of 500 µL with NP-PNK buffer (10 mM MgCl2, 50 mM Tris-HCl pH 7.8, 0.1% (v/v) Nonidet P-40, 5 mM β-mercaptoethanol). Alkaline phosphatase treatment was performed by resuspending immunoprecipitated RNA-protein conjugates in 80 µL of NP-PNK buffer containing 4 units of FastAP thermosensitive alkaline phosphatase (Thermo Fisher Scientific) and 80 units of RNasin® ribonuclease inhibitor (Promega) and incubating the samples at 37 °C for 15 minutes. Dephosphorylation was stopped upon addition of 500 µL of wash buffer. Having equilibrated the Ni-NTA agarose with three washes of 500 µL with NP-PNK, we performed adapter ligation by resuspending the beads 80 µL of reaction mix containing 0.6 µM of the applicable App-PE 3′ adapter (5’App-NAGATCGGAAGAGCACACGTCTG-ddC 3’), 30 units of T4 RNA ligase 2 truncated K227Q (New England Biolabs), 60 units RNasin® (Promega) and 10% (w/v) polyethylene glycol 8000 (PEG 8000). The reaction took place at 25 °C for three hours.  After washing once with 500 µL of wash buffer I and three times with 500 µL of NP-PNK buffer, we 5’end radiolabelled co-immunoprecipitated RNAs by incubating the Ni-NTA resin with 60 µL of NP-PNK buffer containing 30 µCi 32P-γATP (PerkinElmer) and 30 units of T4 polynucleotide kinase (New England Biolabs). After a 40-minute incubation of the reaction at 37ºC, ATP (Roche) was supplemented to a final concentration of 1 mM and the reaction continued for another 20 minutes. The reaction was stopped by washing three times with 500 µL of wash buffer I and three 500 µL washes of NP-PNK buffer followed before the addition of the 5′ linker ligation mix. The reaction took place in a volume of 80 µL of NP-PNK buffer supplemented with 1.25 µM of the fitting adapter, 40 units of T4 RNA ligase 1 (New England Biolabs), 80 units of RNasin® and 10 mM ATP (Roche).  5’ adapter ligation was left to proceed overnight at 16 °C for all bait proteins. The adapter sequences were 5’-ACACGACGCTCTTCCGATCTNNNCGCTTAGCN-3’ for PIC2-GFP_Rep1 and 5’-ACACGACGCTCTTCCGATCTNNNAGAGCN-3’,  5’-ACACGACGCTCTTCCGATCTNNNATTAGCN-3’ or 5’-ACACGACGCTCTTCCGATCTNNNGCGCAGCN-3’ for PIC2-GFP_Rep2; 5’-ACACGACGCTCTTCCGATCTNNNTGGAGCN-3’ or 5’-ACACGACGCTCTTCCGATCTNNNCACTAGCN-3’ for mut-Nab3-BS_Rep1 and  5’-ACACGACGCTCTTCCGATCTNNNGACTTAGCN-3’ or 5’-ACACGACGCTCTTCCGATCTNNNATCACGN-3’ for mut-Nab3-BS_Rep2;  and 5’-ACACGACGCTCTTCCGATCTNNNCACTGTN-3’ or 5’-ACACGACGCTCTTCCGATCTNNNACTCAGCN-3’ for mut-NNS-BS_Rep1 and 5’-ACACGACGCTCTTCCGATCTNNNGTGACAN-3’ for the second mut-NNS-BS library. After pooling suitable libraries together, the samples and its corresponding traces were submitted to Novogene, where paired-end sequencing of 150 bp-reads was performed in a NovaSeq 6000 system (Illumina).


title: Mapping of Nrd1 RNA-binding sites in Saccharomyces cerevisiae with abrogated binding of Nab3 to PIC2

summary: Using CRAC, we compared the transcriptomic occupancy of Nrd1 in a Saccharomyces cerevisiae BY4741 parental strain (PIC2-GFP) and two derived mutants lacking Nab3 RNA-binding sites in PIC2. The purpose of the experiment was two-fold: on the one hand, we aimed to verify that the mutations inserted in the Nab3 binding sequences of PIC2 affected the binding of Nrd1 to the PIC2 transcript; on the other hand, we wanted to check whether differential binding of Nab3 to PIC2 would affect how the protein bound other targets in the genome. Sequencing outputs were processed using the pyCRAC pipeline and peak calling was performed with DBPeaks, our newly developed package for identification and comparison of binding sites defined by RNA-binding footprinting techniques (e.g., CRAC, iCLIP, PAR-CLIP, etc.).

overall_design: Two biological replicates of CRAC experiments performed in a parental Saccharomyces cerevisiae BY4741 strain (PIC2-GFP) and two derived mutants: mut-Nab3-BS and mut-NNS-BS. While mut-Nab3-BS lacks Nab3 RNA-binding sites in PIC2, the other strain has no Nab3 or Nrd1 RNA-binding sites in PIC2 (mut-NNS-BS). All strains encoded NRD1-HTF (HIS6-TEV-3xFLAG). When SC-Ura cultures containing 1% (w/v) raffinose reached an OD600 of 1, cells were UV cross-linked (254 nm; 500 mJ/cm2) in the Vari-X-linker (UVO3) and rapidly filtered on 0.8 μm membranes (Merck) using a filtration device connected to a vacuum pump. Membranes were collected in 50 mL tubes, flash frozen by submersion into liquid nitrogen and stored at -80ºC until the day of the experiment.   On the day of the experiment, pellets were resuspended in a volume of lysis buffer (150 mM NaCl, 50 mM Tris pH 7.8, 0.1% (v/v) Nonidet P-40, 1 EDTA-free protease inhibitor cocktail (Roche) per 50 mL and 5 mM β-mercaptoethanol) corresponding to twice their mass. Lysates were then transferred to a fresh 50 mL tubes, where they were mixed with 2 volumes of 0.5 mm zirconia beads (Thistle Scientific), and vortexed at maximum speed for five 1-minute intervals alternated with 1-minute incubations on ice. An additional volume of lysis buffer was pipetted into each sample before partitioning the cell debris by centrifugation at 4ºC (4000 rpm, 15 minutes) and spinning down lighter impurities in an additional centrifugation step (10000 rpm, 20 minutes) at 4ºC. Immunoprecipitation of the labelled protein from the supernatant was achieved during a two-hour incubation of the sample with 75 µL of anti-FLAG® M2 magnetic beads (Sigma-Aldrich, M8823-5ML) which had been previously washed three times with 1 mL TN150 buffer. Immobilised beads underwent three 5-minute washes with TN1000 buffer (50 mM Tris pH 7.8, 1 M NaCl, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol) and three additional 5-minute washes in 2 mL TN150 buffer (150 mM NaCl, 50 mM Tris pH 7.8, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol). Afterwards, beads were resuspended in 550 µL of TN150 and incubated with 1 µL of a 1:100 dilution of RNace-It™ for exactly 5 minutes at 37 °C. To prevent overdigestion of co-immunoprecipitated transcripts and elute the bait proteins from the anti-FLAG beads, the solution was mixed with 0.4 g of guanidium hydrochloride (Gu-HCl) and the necessary volume of NaCl and imidazole (pH 8.0) to reach a final concentration of 300 mM and 10 mM respectively. After removing the beads, samples were pipetted onto 50 µL of Ni-NTA agarose resin (Qiagen) equilibrated with wash buffer I (6 M Gu-HCl, 10 mM imidazole, 300 mM NaCl, 50 mM Tris-HCl pH 7.8, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol) and incubated at 4 °C overnight while rotating at 12 rpm.   Upon Ni-NTA binding, beads were transferred to Pierce columns (Thermo Fisher Scientific) and washed twice with 500 µL of wash buffer I before undergoing three additional washes of 500 µL with NP-PNK buffer (10 mM MgCl2, 50 mM Tris-HCl pH 7.8, 0.1% (v/v) Nonidet P-40, 5 mM β-mercaptoethanol). Alkaline phosphatase treatment was performed by resuspending immunoprecipitated RNA-protein conjugates in 80 µL of NP-PNK buffer containing 4 units of FastAP thermosensitive alkaline phosphatase (Thermo Fisher Scientific) and 80 units of RNasin® ribonuclease inhibitor (Promega) and incubating the samples at 37 °C for 1 hour. Dephosphorylation was stopped upon addition of 500 µL of wash buffer. Having equilibrated the Ni-NTA agarose with three washes of 500 µL with NP-PNK, we performed adapter ligation by resuspending the beads 80 µL of reaction mix containing 0.6 µM of the applicable App-PE 3′ adapter (5’App-NAGATCGGAAGAGCACACGTCTG-ddC 3’), 30 units of T4 RNA ligase 2 truncated K227Q (New England Biolabs), 60 units RNasin® (Promega) and 10% (w/v) polyethylene glycol 8000 (PEG 8000). The reaction took place at 25 °C for six hours.  After washing once with 500 µL of wash buffer I and three times with 500 µL of NP-PNK buffer, we 5’end radiolabelled co-immunoprecipitated RNAs by incubating the Ni-NTA resin with 60 µL of NP-PNK buffer containing 30 µCi 32P-γATP (PerkinElmer) and 30 units of T4 polynucleotide kinase (New England Biolabs). After a 40-minute incubation of the reaction at 37ºC, ATP (Roche) was supplemented to a final concentration of 1 mM and the reaction continued for another 20 minutes. The reaction was stopped by washing three times with 500 µL of wash buffer I and three 500 µL washes of NP-PNK buffer followed before the addition of the 5′ linker ligation mix. The reaction took place in a volume of 80 µL of NP-PNK buffer supplemented with 1.25 µM of the fitting adapter, 40 units of T4 RNA ligase 1 (New England Biolabs), 80 units of RNasin® and 10 mM ATP (Roche).  5’ adapter ligation was left to proceed overnight at 16 °C for all bait proteins. The adapter sequences were 5’-ACACGACGCTCTTCCGATCTNNNTAAGCN-3’ or 5’-ACACGACGCTCTTCCGATCTNNNCACTAGCN-3’ for PIC2-GFP_Rep1 and 5’-ACACGACGCTCTTCCGATCTNNNTCTCTAGCN-3’ for PIC2-GFP_Rep2; 5’-ACACGACGCTCTTCCGATCTNNNCACTAGCN-3’ and 5’-ACACGACGCTCTTCCGATCTNNNGACTTAGCN-3’ for mut-Nab3-BS libraries 1 and 2 respectively;  and 5’-ACACGACGCTCTTCCGATCTNNNTGGAGCN-3’,  5’-ACACGACGCTCTTCCGATCTNNNACTCAGCN-3’ or 5’-ACACGACGCTCTTCCGATCTNNNACAGTGN-3’ for mut-NNS-BS_Rep1  and 5’-ACACGACGCTCTTCCGATCTNNNGTGACAN-3’ for mut-NNS-BS_Rep2. After pooling suitable libraries together, the samples and its corresponding traces were submitted to Novogene, where paired-end sequencing of 150 bp-reads was performed in a NovaSeq 6000 system (Illumina).


title: Expression Data from Sms-G56S Osteoblasts

city: Hamburg

first: Thorsten

last: Schinke

address: Martinistr. 52

department: Department of Osteology and Biomechanics

institute: University Medical Center Hamburg-Eppendorf

summary:
SMS catalyzes the final step of spermine synthesis. The G56S variant causes the Snyder-Robinson-syndrome. We investigated the effect of the SMS G56S variant on primary murine bone marrow osteoblasts.
We used microarray analysis to detail the global gene expression of primary murine bone marrow osteoblasts isolated from SMS-G56S/0 mice after 10 days of osteogenic differentiation.

overall_design: 4 samples generated from independent experiments were analyzed. The expression of bone-marrow derived osteoblasts at day 10 of osteogenic differentiation derived from Sms-G56S/0 mice was compared to osteoblasts derived from Sms-+/0 wildtype littermates.

first: Yihao

first: Timur

last: Yorgan