title: Mapping protein selectivity landscapes using multi-target selective screening and next-generation sequencing of combinatorial libraries

city: Beer-Sheva

first: Michal

last: Levi

address: David Ben-Gurion Blvd. 1

department: Biotechnology engineering

country: Israel

institute: Ben Gurion university

summary: Characterizing the binding selectivity landscape of interacting proteins is crucial both for elucidating the underlying mechanisms of their interaction and for developing selective inhibitors. However, current mapping methods are laborious and cannot provide a sufficiently comprehensive description of the landscape. Here, we introduce a novel and efficient strategy for comprehensively mapping the binding landscape of proteins using a combination of experimental multi-target selective library screening and in silico next-generation sequencing analysis. We map the binding landscape of a non-selective trypsin inhibitor, the amyloid protein precursor inhibitor (APPI), to human serine proteases including kallikrein-6 and mesotrypsin. We then use this map to dissect and improve the affinity and selectivity of APPI variants toward each of the proteases. Our strategy can be used as a platform for the development of a new generation of target-selective probes and therapeutic agents based on selective protein–protein interactions.

overall_design: This study investigates the binding selectivity of human amyloid protein precursor inhibitor (APPI) variants toward the human serine proteases: kallikrein-6 (KLK6 ) and mesotrypsin. A yeast surface display (YSD) library containing 3.5 million APPI-3M variants, each with 0–2 amino acid mutations, was generated. These variants were subjected to pairwise selective screening using fluorescence-activated cell sorting (FACS) and labeled protease pairs to isolate variants with enhanced or diminished selectivity for specific proteases. The sorted variants were analyzed using next-generation sequencing (NGS) on the Illumina Miseq platform to identify mutations associated with differential binding selectivity.

first: Si

last: Naftaly

first: Itay

last: Cohen

first: Anat

last: Shahar

first: Alexandra

last: Hockla

first: Evette

last: Radisky

first: Niv

last: Papo


title: SPOCK2 controls the proliferation and function of immature pancreatic β-cells through MMP2.

status: Public on Feb 12 2025

city: Poznan

first: Katarzyna

last: Blaszczyk

state: Wielkopolska

address: ul. Uniwersytetu Poznańskiego 6/1.87

department: Faculty of Biology, Institute of Molecular Biology and Biotechnology, Department of Gene Expression

country: Poland

institute: Adam Mickiewicz University

type: Expression profiling by high throughput sequencing

summary: Human pluripotent stem cell-derived β-cells (SC-β-cells) represent an alternative cell source for transplantation in diabetic patients. While mitogens could in theory be employed to expand β-cells, adult β-cells very rarely replicate. Conversely, newly formed β-cells, including SC-β-cells, display higher proliferative capacity and distinct transcriptional and functional profiles. Through bidirectional expression modulation and single-cell RNA-seq, we identified SPOCK2, an ECM protein, as inhibitor of immature β-cell proliferation. Human β-cells lacking SPOCK2 demonstrated elevated MMP2 expression and activity, leading to β-integrin-FAK-c-JUN pathway activation. Treatment with MMP2 protein resulted in pronounced short- and long-term SC-β-cell expansion, significantly, increasing glucose-stimulated insulin secretion in vitro and in vivo. This suggests that SPOCK2 mediates fetal β-cell proliferation and maturation. Together, we identified molecular mechanism that specifically regulates SC-β-cell proliferation and function, highlighting a unique signaling milieu of SC-β-cells with promising potential for robust derivation of fully functional cells for transplantation.

overall_design: bulk RNA-sequencing of two independent samples of EndoC-BH1 with SPOCK2 KD, GIPZ control, SPOCK2 OE and WT cells and scRNA-sequencing of stem cells derived WT and SPOCK2 KO β-cell spheroids.

first: Malgorzata

last: Borowiak


title: FBXW7 Mutations Reprograms Glucose Metabolism by Activating the ETV6-GLUT Axis in Endometrial Cancer

status: Public on Feb 11 2025

city: Shanghai

first: Siqi

last: Fei

address: No. 2005, Songhu Road

country: China

institute: Fudan University

summary: Endometrial cancer (EC) is associated with metabolic reprogramming, driven in part by mutations in FBXW7, a key tumor suppressor. In this study, we reveal that FBXW7 regulates glucose metabolism by targeting ETV6 for ubiquitination and proteasomal degradation. Loss of FBXW7 leads to ETV6 stabilization, which in turn upregulates GLUT1 expression and promotes its localization to the plasma membrane. This enhances glucose uptake and reprograms cellular metabolism toward increased aerobic glycolysis and oxidative phosphorylation. Functional studies demonstrate that ETV6 overexpression accelerates EC cell proliferation, colony formation, and tumor growth in vitro and in vivo, effects that are significantly attenuated by pharmacological inhibition or genetic knockdown of GLUT1. Mechanistically, ETV6 directly binds to the GLUT1 promoter via its ETS and PNT domains, driving its transcriptional activation. Furthermore, metabolomics analyses reveal alterations in glycolytic and TCA cycle intermediates, highlighting the broad impact of the FBXW7-ETV6-GLUT1 axis on metabolic pathways. These findings establish ETV6 as a central mediator of FBXW7-deficiency-driven metabolic reprogramming and tumor progression in EC. Targeting GLUT1 provides a potential therapeutic strategy to mitigate ETV6-induced tumor growth and metabolic remodeling in endometrial cancer.

overall_design: RNA seq of  Human Ishikawa Cells in control and experiment ETV6 overexpress group


title: V. cholerae transcriptome of WT and dusB deletion mutants in MH with no treatment or after 10min with 2mM H2O2

city: Paris

first: Louna

last: Fruchard

address: 28 Rue Du Docteur Roux

country: France

institute: Institut Pasteur

summary: We study gene expression levels of V. cholerae N16961 hapR+ corrected strain in exponential phase before and after lethal hydrogen peroxide treatment

sample_organism: Vibrio cholerae O1 biovar El Tor str. N16961

overall_design: Bacterial cells were grown to OD 0,25, treated or not 10min with 2mM hydrogen peroxide and RNAseq was performed

first: Zeynep

last: Baharoglu


title: Cellular and molecular characterisation of cone photoreceptor migration in normal and degenerate murine retinae

city: North Coogee

first: Denise

last: Anderson

state: WA

address: 71 Caledonia Loop

country: Australia

institute: INSiGENe Pty Ltd

summary: Spatiotemporal cues guiding neuronal migration are critical for the proper development of laminated neural structures, including the retina. Cone photoreceptors, a type of retinal neuron responsible for bright light photopic and colour vision, undergo a unique postmitotic, postnatal migration, yet the cellular and molecular mechanisms that initiate this migration process are still poorly understood. In this study, we explored the role of key phototransduction genes, Cnga3 and Pde6c, in postnatal cone migration by examining two mouse models of complete colour vision blindness, Cnga3-/- and Pde6ccpfl1, which have previously been shown to exhibit aberrant cone localization in adult animals. Retinal development was assessed at postnatal days 8, 12, and 16 using immunohistochemistry and confocal imaging. We found that cone migration and cone soma length were significantly affected in the disease models, indicating that these genes are essential for appropriate postnatal cone migration and final positioning. Furthermore, single-cell RNA sequencing from P12 wildtype and diseased cones identified significant changes to genes involved with the cytoskeleton and its regulation. Our findings strongly indicate a connection between the role of phototransduction genes with cytoskeletal function and cone migration in healthy and degenerating cones.

overall_design: Fresh retinas were dissected out of the mouse eyes and dissociated with a papain digestion. Fluorescence-activated cell sorting (FACS) was performed to sort GFP+ cones into a 96-well plate for each mouse line. Sample preparation and sequencing was performed using an adapted approach of SMARTseq2 and MARS-seq.

first: Alicia

middle: A

last: Brunet

first: Livia

middle: S

last: Carvalho


title: Identifying genetic expression patterns as marker for sensitivity to oncolytic viruses using patient-derived pancreatic ductal adenocarcinoma organoids

city: Rotterdam

first: Marco

last: Huberts

state: Zuid-Holland

address: Doctor Molewaterplein 40

department: Viroscience

country: Netherlands

institute: Erasmus Medical Centre

summary: Purpose: Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers, with a poor prognosis. Viro-immunotherapy using oncolytic viruses (OVs) represents a promising treatment for PDAC. However, PDACs from different patients respond differently to OV therapies, highlighting the need for predictive biomarkers to optimize outcomes. This study aimed to identify gene expression profiles (GEPs) that could serve as predictors for OV sensitivity. Experimental design: Patient-derived organoids (PDOs) from ten PDAC patients were assessed for sensitivity to various OVs: Newcastle Disease Virus, a Reovirus strain entering cells independently of JAM-A expression, the Edmonston vaccine strain of Measles Virus, and a gorilla-derived adenovirus. To identify GEPs with predictive value, OV sensitivity was correlated with the transcriptome of uninfected PDOs. These GEPs were analyzed in the Gene Ontology database to reveal biological processes associated with them. Results: PDOs showed heterogenic responses to OVs, with nine of ten being sensitive to at least one. Analysis of the generated GEPs using the Gene Ontology database identified intracellular aberrations as the primary determinant of OV sensitivity. Sensitivity to all OVs was associated with GEPs related to embryonic development, while varied sensitivity of the ten PDOs to four OVs was linked to cell cycle regulation, metabolism, and proliferation. Conclusions: This study revealed that OV sensitivity was associated with GEPs comprising genes involved in multiple biological processes. Further investigation into screening tumors for the identified GEPs may yield biomarkers for selecting the most effective OV for PDAC. This approach is a stepping stone toward personalized OV treatments.

overall_design: RNA-seq profiling of ten uninfected PDAC PDOs

first: Elham A

last: Farshadi

first: Farzana

last: Mohammad

first: Jie

last: Ju

first: Andrew

last: Stubbs

first: Ron

last: AM Fouchier

first: Bernadette G

last: van den Hoogen


title: Next Generation Sequencing Facilitates Quantitative Analysis of KMT2C Knockdown/Control VCaP Cell Line Transcriptomes

first: Jianpo

last: Lian

address: Huangpu

institute: Tongji hospital

summary: Prostate cancer (PCa) is a leading cause of male morbidity and mortality. Epigenetic modifier abnormalities are becoming a driving event in prostate cancer (PCa). The specific role of KMT2C, a histone methyltransferase that is frequently aberrant in various tumors, is poorly understood in PCa. This study aimed to reveal the potential carcinogenic role of KMT2C in PCa. Herein we confirmed the KMT2C overexpression in PCa, at transcript and protein level. Knockdown KMT2C in VCaP and LNCaP cells attenuated malignant phenotype, suppressing cell viability, clony, and migration. Consistently, stable KMT2C depletion effectively decreases tumor growth by about 70% in vivo. Mechanically, the results suggest that CLDN8 and ITGAV are two key downstream genes of KMT2C.

overall_design: Retinal mRNA profiles of KMT2C knockdown and control VCaP cells were generated by deep sequencing, using Illumina NovaSeq 6000.

first: Denglong

last: Wu


title: mRNA export factors store nascent transcripts within nuclear speckles as an adaptive response to transient global inhibition of transcription.

city: Melbourne

first: Mark

last: Dawson

state: VIC

address: 305 Grattan Street

institute: Peter MacCallum Cancer Centre

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Infertility and treatments utilized have minimal effects on first trimester human placenta DNA methylation and gene expression.

city: Los Angeles

first: Margareta

middle: D

last: Pisarska

state: CA

address: 8635 West Third Street Suite 160

department: Obstetrics and Gynecology

country: USA

institute: Cedars-Sinai Medical Center

type: Methylation profiling by genome tiling array

summary: OBJECTIVE: To determine whether deoxyribonucleic acid (DNA) methylation alterations exist in the first trimester human placenta between conceptions using fertility treatments and those that do not and, if so, whether differences are the result of underlying infertility or fertility treatments. We also assessed whether significant alterations led to changes in gene expression using bulk total RNA-seq from the same cohort (see Lee et al 2019, PMID: 30611556, NCBI GEO GSE215421). DESIGN: We compared DNA methylation of the first trimester placenta from singleton pregnancies that resulted in live births from unassisted, in vitro fertilization (IVF), and non-IVF fertility treatment (NIFT) conceptions using the Infinium MethylationEPIC BeadChip array. Differentially methylated probes (DMPs) were identified using a generalized linear model adjusted for fetal sex. Associated genes for significant CpG sites were cross-references in RNA-seq results to determine whether methylation alterations lead to differences in gene expression. SETTING: Academic medical center. PATIENTS:  A total of 138 singleton pregnancies undergoing chorionic villus sampling resulting in a live birth were recruited for methylation analysis (56 unassisted, 38 NIFT, and 44 IVF conceptions). INTERVENTIONS AND TREATMENTS: In vitro fertilization-conceived pregnancy or pregnancy conceived via NIFT, such as ovulation induction and intrauterine insemination. RESULTS: Of the 741,145 CpG probes analyzed in the placenta, few were significant at Bonferroni <0.05: 185 CpG sites (0.025%) significant in pregnancies conceived with the fertility treatments (NIFT + IVF) versus unassisted conceptions; 28 in NIFT versus unassisted; 195 in IVF versus unassisted; and only 13 (0.0018%) in IVF versus NIFT conceptions. CONCLUSION: Underlying infertility is the most significant contributor to the minimal differences in first trimester placental methylation, and not the specific fertility treatment used, such as IVF.

overall_design: Patients were recruited with informed consent on the day of their appointment for chorionic villus sampling, a diagnostic prenatal test performed at late first trimester. Leftover chorionic villi (placental tissue) discarded by the clinical lab was collected for research and stored at -80C until normal karyotype was confirmed and birth outcomes were known. Only singleton pregnancies with normal karyotypes that resulted in live births were used. We measured DNA methylation of human first trimester placenta using the human Infinium MethylationEPIC BeadChip array. We compared pregnancies conceived unassisted (n=56 without fertility treatment or "spontaneous"), with in vitro fertilization (n=44 IVF), or with non-IVF fertility treatment (n=38 NIFT), see original publication (PMID: 36379261). The unassisted group, lacking an infertility diagnosis, was a control for infertility phenotype. The NIFT group was another control to help distinguish DNA methylation profiles due to infertility phenotype (affecting both NIFT and IVF groups) versus IVF technology (IVF group only). DNA methylation sex differences in the unassisted group were also analyzed (PMID: 39152463).

first: Tania

middle: L

last: Gonzalez

first: Amelia

middle: M

last: Schaub

first: Bora

last: Lee

first: Jinrui

last: Cui

first: Kent

last: Taylor

first: Anna

middle: E

last: Dorfman

middle: O

last: Goodarzi

first: Erica

middle: T

last: Wang

first: Yii-Der Ida

last: Chen

first: Jerome

middle: I

last: Rotter

first: Rimsha

last: Hussaini

first: Paige

last: Harakuni

first: Mayaal

middle: H

last: Khan

first: Stephen

last: Rich

first: Charles

middle: R

last: Farber

first: John

last: Williams 3rd


title: The liver clock drives circadian rhythms in white adipose tissue [WTKOLRE]

city: HUDDINGE

first: Ivan

middle: Martinov

last: Vlassakev

address: HÄLSOVÄGEN 7C

department: Department of Medicine

country: Sweden

institute: Karolinska Institutet

summary: Circadian rhythms are integral to maintaining metabolic health by temporally coordinating metabolic functions across tissues. However, the mechanisms underlying circadian cross-tissue coordination remain poorly understood. In this study, we uncover a central role for the liver clock in regulating circadian rhythms in white adipose tissue (WAT). Using a liver-specific Bmal1 knockout mouse model, we show that hepatic circadian control is crucial for lipid metabolism in WAT. In addition, by utilizing a model where functional clocks are restricted to the liver, we demonstrate that the liver clock alone drives circadian gene expression in WAT, including Cebpa, a key regulator of adipogenesis. Mechanistically, we show that circadian liver-to-WAT communication is mediated through secreted proteins, including the adaptor protein 14-3-3η (Ywhah). The clinical relevance of these findings is supported by human cohort data, which correlate liver YWHAH expression with lipid metabolic pathways in WAT and cardiometabolic risk factors. These findings provide a mechanistic framework for how the liver clock coordinates WAT physiology and highlights its central role in cardiometabolic health, offering potential insights for targeted circadian-based therapies for metabolic disorders.

overall_design: Bulk SmartSeq3 from AML12 cells WT and Bmal1-KO.

first: Christina

last: Savva

first: Gianluca

last: Renzi

first: Jacob

middle: G

last: Smith

first: Carolina

last: Greco

first: Christopher

last: Litwin

first: Qiang

last: Zhang

first: Leandro

last: Velez

first: Angela

last: Ma

first: Martin

last: O. Bergo

first: Oscar

last: Wiklander

first: Pura

last: Muñoz-Cánovez

first: Niklas

last: Mejhert

first: Marcus

last: Seldin

first: Johan

last: Björkegren

first: Paolo

last: Sassone-Corsi

first: Kevin

middle: B

last: Koronowski

first: Salvador

last: Aznar Benitah

first: Paul

last: Petrus


title: GDF10 promotes rodent cardiomyocyte maturation during the postnatal period

city: Cincinnati

first: Matthew

last: Weirauch

state: Ohio

address: 3333 Burnet Avenue

department: Center for Autoimmune Genomics and Etiology (CAGE)

institute: Cincinnati Children's Hospital Medical Center

summary: Cardiomyocytes and cardiac fibroblasts undergo coordinated maturation after birth, and cardiac fibroblasts are required for postnatal cardiomyocyte maturation in mice. Here, we investigate the role of cardiac fibroblast-expressed Growth Differentiating Factor 10 (GDF10) in postnatal heart development. In neonatal mice, Gdf10 is expressed specifically in cardiac fibroblasts, with its highest expression coincident with onset of cardiomyocytes cell cycle arrest and transition to hypertrophic growth. In neonatal rat ventricular myocyte cultures, GDF10 treatment promotes cardiomyocyte maturation indicated by increased binucleation, downregulation of cell cycle progression genes and upregulation of cell cycle inhibitor genes. GDF10 treatment leads to an increase in cardiomyocyte cell size together with increased expression of mature sarcomeric protein isoforms and decreased expression of fetal cardiac genes. RNAsequencing of GDF10-treated NRVM shows an increase in gene expression related to myocardial maturation, including upregulation of sodium and potassium channel genes. In vivo, loss of Gdf10 leads to a delay in myocardial maturation indicated by a decrease in cardiomyocyte cell size and binucleation as well as increased mitotic activity at postnatal (P) day 7. Further, induction of mature sarcomeric protein isoform gene expression is delayed, and expression of cell cycle progression genes is prolonged. However, by P10 indicators of cardiomyocyte maturation and mitotic activity are normalized in Gdf10-null hearts relative to controls. Together, these results implicate Gdf10 as a novel crosstalk mediator between cardiomyocytes and cardiac fibroblasts, required for appropriate timing of cardiomyocyte maturation steps including binucleation, hypertrophy, mature sarcomeric isoform switch and cell cycle arrest in the postnatal period.

overall_design: We performed RNA-seq in primary rat cardiomyocytes with or without GDF10 treatment. Experiments were performed in four replicates per condition

first: Katherine

last: Yutzey


title: Vhl and Rassf1a mutations cause DNA damage, chromosomal instability and induce gene expression changes characteristic of ccRCC [scRNA-seq]

city: Freiburg

first: Patrick

last: Metzger

address: Breisacher Str. 153

department: Institute for Medical Bioinformatics and Systems Medicine

country: Germany

institute: University Hospital Freiburg

summary: RASSF1A is frequently biallelically inactivated in clear cell renal cell carcinoma (ccRCC) due to loss of chromosome 3p and promoter hypermethylation. Here we investigated the cellular and molecular consequences of single and combined deletion of the Rassf1a and Vhl tumor suppressor genes to model the common ccRCC genotype of combined loss of function of RASSF1A and VHL. In mouse embryonic fibroblasts and in primary kidney epithelial cells, double deletion of Rassf1a and Vhl caused accumulation of chromosomal segregation defects and increased formation of micronuclei, demonstrating that pVHL and RASSF1A function to maintain genomic integrity. Combined Rassf1a and Vhl deletion in renal epithelial cells in vivo increased proliferation and caused mild tubular disorganization, but did not lead to the development of kidney tumors. Single cell RNA-sequencing unexpectedly revealed that Rassf1a deletion or Vhl deletion both induce the expression of an overlapping set of genes in a sub-population of proximal tubule cells. Many of these genes are also upregulated in the Vhl/Trp53/Rb1 deficient mouse model of ccRCC. In other subsets of proximal tubule cells, combined Vhl/Rassf1a deletion induced the expression of additional genes that were not upregulated in each of the single knockouts. The expression of the human homologues of Rassf1a-regulated genes correlate negatively with RASSF1 expression levels in human ccRCC, suggesting that the loss of RASSF1A function establishes a ccRCC-characteristic gene expression pattern.

overall_design: single cell RNA-Seq of murine renal proximal tubule epithelial cells with Vhl, Rassf1a, or Vhl/Rassf1a KO

first: Antonella

last: Catalano

first: Laura

last: Haas

first: Kyra

last: Zodel

first: Mojca

last: Adlesic

first: Francesca

last: Cuomo

first: Asin

last: Peighambari

first: Hsin

last: Huang

first: Stefan

last: Haug

first: Anna

last: Köttgen

first: Natalie

last: Köhler

first: Melanie

last: Boerries

first: Ian

middle: J

last: Frew


title: The NAE1-mediated neddylation operates as an essential posttranslational modification checkpoint for effector CD8+ T cells

city: Columbus

first: Gang

last: Xin

state: OH

address: 460 W 12th Ave

department: MI&I

institute: The Ohio State University

summary: Optimal activation of CD8+ T cells is crucial for immunity-mediated destruction of cancer, requiring a substantial amount of proteins involved in metabolism, proliferation, and effector function. Despite extensive studies emphasizing the role of transcriptional regulation in this process, paired transcriptomic and proteomic analyses reveal that the RNA profile is poorly correlated with protein levels. This discrepancy underscores the importance of posttranslational modifications (PTMs) in controlling protein abundance during activation. However, the impact of PTMs on the CD8+ T cell protein dynamic remains underexplored. We identify that neddylation, a recently discovered PTM, is activated in response to T cell receptor (TCR) stimulation and enriched in effector CD8+ T cells from colon cancer patients. Mechanistically, we found the rate-limiting enzyme of neddylation, neural precursor cell expressed developmentally down-regulated protein 8 activating enzyme E1 (NAE1), is induced by the NFATc1, a critical transcription factor downstream of TCR signaling. Our observation revealed that genetic ablation of NAE1 significantly disturbed the proteomic landscape related to activation and mitochondrial function. As a result, CD8+ T cells lacking NAE1 exhibited severely compromised activation, proliferation, and survival, which was accompanied by impaired mitochondrial function. Consistently, deletion of NAE1 in CD8+ T cells abolished their antitumor function and promoted tumor progression. By contrast, the overexpression of NAE1 significantly improved the function of tumor-infiltrating CD8+ T cells. Overall, we uncovered neddylation, a previously underappreciated PTM, as a proteomic checkpoint for CD8+ T cell activation. Enforced expression of NAE1 offers promising therapeutic potential for boosting the antitumor CD8+ T cell responses.

overall_design: RNASeq profile of in vitro activated CD8+ T cells from Nae1WT and Nae1T-KO mice at day 2

first: Jin

last: Jiacheng

first: Zhang

last: Ruohan

first: Li

last: Jianying

first: Gao

last: Fengxia

first: Liao

last: Zhiwei

first: Yu

last: Yanbao

first: Wang

last: Yi

first: Buvvi

last: Donna

first: Ma

last: Ruilin

last: Qin

last: Shuaixin

first: Lio

last: Jerry

first: Novais

last: Fernanda

first: Huang

last: Stanley

first: Zhu

last: Jiangjiang

first: Ghoneim

last: Hazem

first: Wen

last: Haitao

last: Zihai

first: Sun

last: Nuo

first: Xin

last: Gang


title: The liver clock drives circadian rhythms in white adipose tissue [AML12]

first: Ivan

last: Vlassakev

first: Christina

last: Savva

first: Gianluca

last: Renzi

first: Jacob

last: Smith

first: Carolina

last: Greco

first: Christopher

last: Litwin

first: Qiang

last: Zhang

first: Leandro

last: Velez

first: Angela

last: Ma

first: Martin

last: O. Bergo

first: Oscar

last: Wiklander

first: Pura

last: Muñoz-Cánovez

first: Niklas

last: Mejhert

first: Marcus

last: Seldin

first: Johan

last: Björkegren

first: Paolo

last: Sassone-Corsi

first: Kevin

last: Koronowski

first: Salvador

last: Aznar Benitah

first: Paul

last: Petrus


title: Mitochondrial dysfunction drives a neuronal exhaustion phenotype in methylmalonic aciduria

city: Bethesda

first: Matthew

middle: CS

last: Denley

state: MD

address: 35 Convent Dr

institute: NIH

summary: Methylmalonic aciduria (MMA) is an inborn error of metabolism resulting in loss of function of the enzyme methylmalonyl-CoA mutase (MMUT). Despite acute and persistent neurological symptoms, the pathogenesis of MMA in the central nervous system is poorly understood, which has contributed to a dearth of effective brain specific treatments. Here we utilised patient-derived induced pluripotent stem cells and in vitro differentiation to generate a human neuronal model of MMA. We reveal strong evidence of mitochondrial dysfunction caused by deficiency of MMUT in patient neurons. By employing patch-clamp electrophysiology, targeted metabolomics, and bulk transcriptomics, we further expose an altered state of excitability, which we suggest may be connected to metabolic rewiring which is exacerbated by application of 2-dimethyloxoglutarate. Our work provides first evidence of mitochondrial driven neuronal dysfunction in MMA, which through our comprehensive characterisation of this paradigmatic model, enables first steps to identifying effective therapies.

overall_design: 2x control iPSC-derived glutamatergic neurons and 2x patient iPSC-derived glutamatergic neurons. The sample number of samples were treated with 0.1 mM dimethyl-2-oxoglutarate


title: Sex differences in the neuroinflammatory signaling pathway: Effect of miRNAs on fatty acid synthesis in microglia

city: Fukuoka

first: Akiko

last: Mizokami

address: 3-1-1 Maidashi, Higashi-ku

department: Faculty of Dental Science

country: Japan

institute: Kyushu University

summary: Studies have shown that Alzheimer’s disease is more prevalent and severe in women than men. This could be due to the different gene and protein expression in the microglia, the brain’s primary immune cells. In this study, we analyzed microglial microRNAs from the hippocampi of adult male and female mice and identified 30 miRNAs that were expressed differently in male and female mice. The target genes of the male-enriched miRNAs belong to the fatty acid biosynthesis and metabolism pathways. As sex hormones contribute to sex differences, we investigated whether testosterone regulates the fatty acid biosynthesis pathway in the mouse microglial cell line MG6. Testosterone reduced the expression levels of fatty acid synthase, which in turn suppresses the NF-κB inflammatory signaling pathway. This suggests that testosterone, through the upregulation of specific miRNAs, plays a crucial role in suppressing inflammatory responses in male microglia, which could explain the lower susceptibility of men towards Alzheimer’s disease.

overall_design: Hippocampi were isolated from 8-month-old male and female mice (five mice per group) and pooled to generate one sample per sex.


title: Patient-Derived Meningioma Organoids: A Reliable Model for Studying Human Tumor Pathophysiology

city: Atlanta

first: Youssef

last: Zohdy

state: GA

address: 1365 Clifton Road NE

department: Neurosurgery

institute: Emory University

summary:
Introduction: Meningiomas are the most common primary central nervous system tumors, constituting 39.7% of intracranial tumors. Although generally benign, some exhibit aggressive be-havior and risk of recurrence, necessitating adjuvant therapy and repeat surgical in-terventions. Molecular studies have identified tumor-driving mutations, leading to tar-geted therapies and clinical trials. However, translating preclinical findings into clinical success is often hindered by limitations in current meningioma tumor models. This study aims to develop and validate a standardized protocol for establishing patient-derived meningioma organoids (MEN-O) that faithfully replicate human disease.
Methods: We obtained meningioma samples from neurosurgical resections and MEN-O were developed using an optimized culture protocol. Histological and immunohistochemical analyses were used to assess the resemblance of MEN-O to original tumor tissues. RNA sequencing compared transcriptional signatures between MEN-O and corresponding patient-resected tissues.
Results: MEN-O were successfully established from patient-resected samples and maintained in culture for up to four weeks, showing stable growth and structural integrity. Histopathological analysis revealed that MEN-O preserved key architectural features, including cellular organization, nuclear mor-phology, and proliferation rates. Immunohistochemical staining for meningioma-specific markers, such as progesterone receptor, confirmed similar expression patterns to parental tumors. Transcriptomic profiling demonstrated that MEN-O retained the transcriptional signatures of original tissues, including genes associated with meningioma pathology (NF2, CDKN2A, TP53). Differential expression and deconvolution analyses showed that MEN-O contained diverse cell populations, including tumor and stromal cells, while preserving the immune microenvironment, as validated by histopathological and tran-scriptomic profiling.
Conclusion: We established a robust, reproducible protocol for generating MEN-O, which faithfully replicates the histopathological, molecular, and cellular characteristics of original tumors. MEN-O provide a valuable model for studying meningioma biology and evaluating therapeutic strategies.

overall_design: RNA-seq profiling of human resected meningiomas and correspondingly created tumor organoid cultures

first: Arman

last: Jahangiri

first: Fadi

last: Jacob

first: Aliaksandr

last: Aksionau

first: Ali

last: Alawieh

first: Amelia

last: Tong

first: Bethany

last: Chern

first: Justin

last: Maldonado

first: Kimberly

last: Hoang

first: Edjah

last: Nduom

first: Brian

last: Howard

first: Daniel

last: Barrow

first: Stewart

last: Neill

first: Zhexing

last: Wen

first: Gustavo

last: Pradilla

first: Tomas

last: Garzon-Muvdi


title: The liver clock drives circadian rhythms in white adipose tissue [WT_LKO]

first: Ivan

last: Vlassakev

overall_design: Bulk SmartSeq3 from epidydemal white adipose tissue from male wild-type, and Liver-specific Bmal1-KO mice.

first: Christina

last: Savva

first: Gianluca

last: Renzi

first: Jacob

last: Smith

first: Carolina

last: Greco

first: Christopher

last: Litwin

first: Qiang

last: Zhang

first: Leandro

last: Velez

first: Angela

last: Ma

first: Martin

last: O. Bergo

first: Oscar

last: Wiklander

first: Pura

last: Muñoz-Cánovez

first: Niklas

last: Mejhert

first: Marcus

last: Seldin

first: Johan

last: Björkegren

first: Paolo

last: Sassone-Corsi

first: Kevin

last: Koronowski

first: Salvador

last: Aznar Benitah

first: Paul

last: Petrus


title: Microenvironmental arginine restriction sensitizes pancreatic cancers to polyunsaturated fatty acids by suppression of lipid synthesis

city: Chicago

first: Patrick

middle: Bryan

last: Jonker

state: IL

address: 929 E 57th St

institute: University of Chicago

summary: Nutrient limitation in the microenvironment of poorly perfused tumors constrains the metabolism of cancer cells. Identifying these microenvironmental constraints can provide new insight into the nutritional biochemistry of tumors and reveal metabolic liabilities of cancer cells. We have found that limitation of arginine in pancreatic cancers inhibits fatty acid synthesis by suppressing the lipogenic transcription factor SREBP1. SREBP1-driven fatty acid synthesis produces saturated and monounsaturated fatty acids. Producing these fatty acids enables cells to maintain a balance of differently saturated fatty acids needed for lipid homeostasis, even upon exposure to environments enriched in one specific class of fatty acids. Given the constraints on lipid synthesis in the microenvironment, we asked if pancreatic cancers are sensitive to exposure to fats with imbalanced levels of saturated and unsaturated fats. We found microenvironmental constraints on lipid synthesis sensitize pancreatic cancer cells and tumors to exposure to fat sources that are enriched in polyunsaturated fatty acids. Thus, amino acid restriction in the tumor microenvironment constrains lipid metabolism in pancreatic cancer, which renders pancreatic tumors incapable of maintaining lipid homeostasis upon exposure to polyunsaturated-enriched fats.

overall_design: Mouse pancreatic cancer cells (mPDAC1) were grown in RPMI-1640 or in tumor interstitial fluid medium (TIFM), or in TIFM 1.19 mM arginine. Cells in each medium were grown with or without exogenous lipids using lipid stripped fetal bovine serium (FBS) for 24 hours in triplicate. After 24 hours, RNA was harvested and sent for library prep and sequencing.

first: Patrick B.

first: Alex

last: Muir


title: Gene expression profile at single cell level of mouse liver in whole and fibrotic niche of the liver

city: Daejeon

first: Kwon Yong

last: Tak

address: Gu Sung Street

country: South Korea

institute: KAIST

first: Myungsun

last: Park


title: Vhl and Rassf1a mutations cause DNA damage, chromosomal instability and induce gene expression changes characteristic of ccRCC [bulk RNA-seq]

last: Metzger

first: Antonella

last: Catalano

first: Laura

last: Haas

first: Kyra

last: Zodel

first: Mojca

last: Adlesic

first: Francesca

last: Cuomo

first: Asin

last: Peighambari

first: Hsin

last: Huang

first: Stefan

last: Haug

first: Anna

last: Köttgen

first: Natalie

last: Köhler

first: Melanie

last: Boerries

first: Ian

last: Frew


title: Gene expression profile at single cell level of mouse liver in whole and fibrotic niche of the liver (ATAC-Seq)

summary: Age-dependent fibrosis accumulated in the liver includes driver cell types of aging. We used single cell RNA sequencing (scRNA-seq) from specially digested liver to analyze the age-related fibrosis.

overall_design: The liver was digested to enriched the fibrotic niche. Experiment was done in young (6 months) and old (24 months) mouse to see age-relation in fibrosis

first: Kwon

middle: Y

first: Juyeon

last: Kim

first: Chuna

first: Jong-eun


title: IFNγ stimulation of monoallelic PSMB10 deficiency primary skin fibroblasts

city: Borstel

first: Yuqin

last: Wang

address: Parkallee 26

institute: FZB

summary:
Immunoproteasomes are specialized multiprotein proteases, that degrade intracellular proteins thereby generating antigenic peptides for HLA class I antigen presentation. Mutations in immunoproteasomal genes are associated with systemic autoinflammatory diseases.   We identified a newborn with severe T lymphopenia and found a de novo heterozygous PSMB10 p.Gly209Arg variant. Molecular dynamics analysis predicted and biochemical studies confirmed the variant to impact assembly and function of the immunoproteasome resulting in impaired cytokine responses to interferons.   Skin fibroblasts obtained from the patient (PSMB10G209R) and a healthy individual of comparative age (PSMB10WT) were treated with or without 75U/ml recombinant Human IFN-γ. 5 different cell passages for independent treatments to assess significance of alterations. RNA was isolated from cells to investigate the alteration in IFN response between PSMB10WT and PSMB10G209R.
Skin fibroblasts obtained from the patient (PSMB10G209R) and a healthy individual of comparative age (PSMB10WT) were treated with or without 75U/ml recombinant Human IFN-γ. 5 different cell passages for independent treatments to assess significance of alterations. RNA was isolated from cells to investigate the difference of IFN response between PSMB10WT and PSMB10G209R.

overall_design: 5 passages(7, 8, 9, 10, 12) of PSMB10G209R skin fibroblasts and 5 passages(7, 8, 9, 10, 12) of PSMB10WT skin fibroblasts were used as biological replicates for hIFN-γ treatment. Cells were planted in 6 well plates 16 hours before treatment. Medium was replaced with or without 75U/ml recombinant human IFN-γ for 24 hours. Total RNA isolated from cells were isolated and subjected to Agilent microarray analysis.


title: Mild and ultrafast GLORI enables absolute quantification of m6A methylome from low-input samples

city: Beijing

first: Bo

last: Lu

address: Yiheyuan Road No.5

department: School of Life Sciences

institute: Peking University

type: Methylation profiling by high throughput sequencing

summary: Methods that enable absolute quantification of N6-methyladenosine (m6A) RNA modification have emerged as powerful tools in the field of epitranscriptomics. We previously reported GLORI, a chemical-assisted approach firstly achieved quantitatively transcriptome-wide m6A measurement at single-base resolution. Despite its advantages, GLORI suffers from lengthy reaction time and severe RNA degradation. Here, we present two updated GLORI approaches: GLORI 2.0 is an ultra-fast and mild version that preserves RNA integrity and enhances sensitivity for both transcriptome-wide and locus-specific m6A detection; GLORI 3.0 further utilizes a novel reverse transcription-silent carrier RNA to achieve high-quality m6A quantification from ~ 1,000 cells. Using limited RNA input extracted from single mouse dorsal hippocampus, we measure m6A methylome in the synaptic and cytoplasmic fractions and reveal a high modification level in synapse-related gene sets. We envision that the updated GLORI methods will greatly expand the applicability of absolute quantification of m6A in biology.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: m6A profiling analysis of human cells and mice hippocampal using updated GLORI


title: Optimizing murine sample sizes for RNA-seq studies revealed from large-scale comparative analysis

city: Tarrytown

first: Gabor

last: Halasz

state: New York

address: 777 old saw mill river road

institute: Regeneron Pharmaceuticals

summary: In order to determine the N, the usual approach is to perform a power calculation, which involves understanding the variability between samples and the expected effect size. Here, we focused on bulk RNA-seq experiments, which have become ubiquitous in biology, but which have many unknown or difficult to estimate parameters, and so the required analyses to determine the minimum N is typically lacking. We therefore performed two N=30 profiling studies between wild-type mice and mice in which one copy of a gene had been deleted, to determine how many mice would be required to minimize false positives and to maximize true discoveries found in the N of 30 experiment

overall_design:
Dchs1+/- and Fat+/- heterozygous mice were generated using Regeneron’s (Tarrytown, NY) VelociGene® technology (ID #20561), and  Wild-type littermates were used as controls. All experiments were performed on 100% C57BL/6NTac background.
RNA-Seq was performed on RNA purified  from heterozygous and homozygous Dchs1 or Fat4 heterozygous mice tissues (heart, kidney, liver, and lung).

first: Gurinder

last: Atwal

first: David

last: Glass


title: Transcriptomic analysis of Myotis yumanensis innate immune response

city: Lyon

first: Lucie

last: Etienne

address: 46 Allée d'Italie

institute: Centre International de Recherche en Infectiologie

summary: Bats are natural hosts for a wide diversity of viruses. While many of these viruses are highly pathogenic in humans, most do not appear to cause major symptoms in bats. These modern bat-specific characteristics are the result of past virus-host (co)evolution and virus-driven host adaptations. Innate immunity is the first line of defense against viruses in mammals, we aim at characterizing bat innate immunity in response to viruses. Using genome-wide and gene candidate evolutionary analyses, we found that many bat antiviral genes have undergone multiple duplication events in a lineage-specific manner, specifically in the Myotis bat lineage. We focus on Myotis yumanensis as a  model in the Myotis lineage. We performed transcriptomic analyses and observed the upregulation of most mammalian genes implicated in the different steps of the innate immune response from sensing to interferon-stimulated genes (ISGs), showing the conservation of the core innate immunity. Our study will contribute to identifying adaptations that shaped bat innate immunity. These adaptations may contribute to the bat-virus specificity and influence viral emergence to another mammalian host

overall_design: To investigate potential gene candidate implicate in innate immune responce of Myotis yumanensis, we use M. yumanensis primary cell lines (fibroblast-like) treated or not  for 6h with universal type I interferon or poly I:C to stimulate an innate immune response. Cell were then harvested for RNA extraction and sequencing.

first: Sarah

last: MAESEN

first: Amandine

last: LE CORF

first: Clara

last: LOYER

first: Juan

last: VAZQUEZ

first: Elise

last: LAUTERBUR

first: Gena

last: SANDOVAL

first: Andrea

last: CIMARELLI

first: Peter

last: SUDMANT

last: ENARD

first: Carine

last: REY

last: ETIENNE


title: BulkRNA Sequencing of human induced pluripotent stem cells derived cardiac progenitors (hiPSC-CPs) from healthy control, tetralogy of Fallot (TOF) patients with DiGeorge Syndrome (DG) and without DiGeorge Syndrome (ND)

city: Hong Kong

first: Chun Ho

last: Chan

address: 5 Sassoon Road Pokfulam

country: Hong Kong

institute: The University of Hong Kong

summary: To assess the transcriptomic changes during in vitro cardiac differentiation

overall_design: hiPSC-CPs from 2 healthy control, 2 TOF-DG patients and 2 TOF-ND patients on Day 5,6,7,8 during in vitro cardiac differentiation

first: Yin

last: Lam

first: Chun

first: Wendy

last: Keung

first: Yiu

middle: F

last: Cheung


title: Expression data from HoxA9wt/Meis1 vs. HoxA9AA/Meis1 mouse models

status: Public on Feb 10 2025

city: Boston

first: Daniel

middle: G.

last: Tenen

state: Massachusetts

address: 3 blackfan st

department: Harvard Medical School Initiative for RNA Medicine

institute: Harvard Medical School

summary:
To verify the impact of eIF4E activation on HoxA9-induced leukemic transformation in vivo, we made a double point mutation (HoxA9AA) to disrupt the physical and functional interaction between eIF4E and HOXA9.
Microarray analysis of transformed leukemic cell lines derived from HoxA9wt/Meis1 and HoxA9AA/Meis1 leukemic cells presented highly similar transcriptome profile, without any significant changes of HoxA9 gene signature.

overall_design: we transplanted CD45.2+ LSK cells transduced with Meis1 and either HoxA9wt-GFP or HoxA9AA-GFP transduced into lethally irradiated CD45.1+ recipient mice. Microarrays were done on RNA from BMCs harvested at disease onset. 3 Hoxa9wt/Meis1 and 3Hoxa9AA/Meis1 individuals (biological replicates) were run.

first: Fang

last: Zhou

first: Biljana

last: Culjkovic-Kraljacic

first: Christian

last: Bach

first: Li

last: Feng

first: Yuta

last: Mishima

first: Katherine

last: Borden


title: Transcriptome Response to Autoantibodies in Human Primary Epidermis Keratinocyte Model for Pemphgus Vulgaris

city: Lübeck

first: Hauke

last: Busch

state: Schleswig-Holstein

address: Ratzeburger Allee 160

department: Lübeck Institute of Experimental Dermatology

institute: University of Lübeck

summary: Pemphigus vulgaris (PV) is an autoimmune disease caused by autoantibodies (AAbs) targeting Desmoglein 1 (DSG1) or Desmoglein 3 (DSG3) on keratinocytes, leading to disrupted cell-cell adhesion and epidermal blistering. To investigate the early signaling events triggered by AAb binding, we examined transcriptomic responses in a human primary epidermis keratinocyte (HPEK) model for PV. After incubating the single-chain variable fragment (scFv) PX43, which targets DSG1 and DSG3, and human IgG as control for 5h, 10h and 24h, differentially expressed genes (DEGs) and regulated pathways was analyzed using DESeq2 and pathway enrichment analysis. Few significantly differentially expressed DEGs (under 10) were identified of PX43 compared to hIgG at all three time points (5h, 10h and 24h) with adjusted pvalue below 0.05 and |log2FC| >1. Upregulated inflammatory related pathways including TNFα signaling were identified only at 24h. Correlation analysis of the log2 transformed transcripts per million (TPM) shows the very high positive correlation of PX43 and hIgG at all time points (spearman correlation above 0.9) These findings indicate that AAbs targeting DSG1 and DSG3 do not drive broad transcriptomic or proteomic changes at the HPEK model.

overall_design: Human skin normal human epidermal keratinocytes from three healthy human donors were grown in 2D until 85% confluenent. Calcium concentration was increased to establish cell-cell adhesion and differentiation. Cells were then incubated with DSG-targeting PX43 (DSG1 and DSG3) antibodies and human IgG as controls, with transcriptome data collected by RNA-seq at 5, 10, and 24, 48 hours post-stimulation.

first: Jennifer

last: Hundt

first: Eliane

last: Mueller

first: Sen

last: Guo

first: Veronika

last: Hartmann

first: Siavash

last: Rahimi

first: William

last: Hariton


title: Phenotypic complexities of rare heterozygous neurexin-1 deletions

city: New York

first: Yu

last: Fan

state: NY

address: 1425 madison Ave

institute: Icahn school of medicine at mount sinai

summary: Given the large number of genes significantly associated with risk for neuropsychiatric disorders, a critical unanswered question is the extent to which diverse mutations --sometimes impacting the same gene-- will require tailored therapeutic strategies. Here we consider this in the context of rare neuropsychiatric disorder-associated copy number variants (2p16.3) resulting in heterozygous deletions in NRXN1, a pre-synaptic cell adhesion protein that serves as a critical synaptic organizer in the brain. Complex patterns of NRXN1 alternative splicing are fundamental to establishing diverse neurocircuitry, vary between the cell types of the brain, and are differentially impacted by unique (non-recurrent) deletions. We contrast the cell-type-specific impact of patient-specific mutations in NRXN1 using human induced pluripotent stem cells, finding that perturbations in NRXN1 splicing result in divergent cell-type-specific synaptic outcomes. Via distinct loss-of-function (LOF) and gain-of-function (GOF) mechanisms, NRXN1+/- deletions cause decreased synaptic activity in glutamatergic neurons, yet increased synaptic activity in GABAergic neurons. Reciprocal isogenic manipulations causally demonstrate that aberrant splicing drives these changes in synaptic activity. For NRXN1 deletions, and perhaps more broadly, precision medicine will require stratifying patients based on whether their gene mutations act through LOF or GOF mechanisms, in order to achieve individualized restoration of NRXN1 isoform repertoires by increasing wildtype, or ablating mutant isoforms. Given the increasing number of mutations predicted to engender both LOF and GOF mechanisms in brain disorders, our findings add nuance to future considerations of precision medicine.

overall_design: scRNA-seq was performed on 4-6 pooled 6-month-old organoids per donor line, per condition (hCS or hSS) for a total of 48 organoids. A minimum of 10,000 dissociated cells were submitted for sequencing.

first: Michael

last: Fernando

first: Yanchun

last: Zhang

first: Alex

last: Tokolyi

first: Aleta

middle: N

last: Murphy

first: Sarah

middle: K

last: Kammourh

first: Peter

last: Deans

first: Sadaf

last: Ghorbani

first: Ryan

last: Onatzevitch

first: Adriana

last: Pero

first: Christopher

last: Padilla

last: Williams

first: Erin

last: Flaherty

first: Iya

middle: A

last: Prytkova

first: Lei

last: Cao

first: David

last: Knowles

first: Gang

last: Fang

first: Paul

last: Slesinger

first: Kristen

last: Brennand


title: Impact of human anti-O-specific polysaccharide (OSP) antibody on Vibrio cholerae O1 in complex media containing mucin

first: Smriti

last: Verma

state: MA

address: 55 Fruit Street

department: Department of Infectious Diseases

institute: Massachusetts General Hospital

summary: Antibodies targeting the O-specific polysaccharide (OSP) of Vibrio cholerae O1 are a main determinant of protection against cholera. These antibodies can agglutinate bacteria, and can also directly inhibit V. cholerae motility, including at sub-agglutinating conditions. In order to evaluate for possible additional impacts of OSP-specific antibody on V. cholerae, we assessed the transcriptional profile of V. cholerae exposed to an anti-OSP human monoclonal antibody (G1), including in the presence of mucin, the principal component of intestinal mucous. We identified a subset of genes whose expression was significantly altered in the presence of anti-OSP antibody and mucin, including those involved in V. cholerae metabolism, transport, stress response, biofilm formation, motility, and secondary messenger signaling. Our results suggest a broad impact of anti-OSP antibodies on V. cholerae in the presence of mucin and identify several possible mechanisms by which anti-OSP antibodies might protect against cholera.

overall_design: Vibrio cholerae C6706 bacteria cultured under toxin-inducing conditions were diluted to a subagglutinating optical density (600 nm) of ≤0.1 and exposed to two concentrations (0.0125 µM and 0.125 µM) of human monoclonal immunoglobulin (Ig) G1 targeting V. cholerae O-specific polysaccharide component of LPS (clone G1 - CF21.2.G01) or anti-flagellin antibody as a control (clone B12 -AT11.1.B12) in either Luria Bertani (LB) broth or LB containing 1% w/v porcine stomach mucin (LBM).

first: Edward

last: Ryan


title: Single cell RNAseq of SOD1 mouse spinal cords

city: Cambridge

first: Timothy

last: Hammond

address: 350 Water Street

institute: Sanofi

summary: Sequencing was performed to better understand the role of neuroinflammation and glial RIPK1 signaling in ALS pathogenesis in the SOD1G93A transgenic mouse model.

overall_design: Starting on postnatal day 70, before noticeable motor deficits, female mice were treated prophylactically with the tool compound GSK’547 (RIPK1i), a highly selective CNS-penetrant RIPK1 kinase inhibitor. The inhibitor was initially dosed in chow and as motor symptoms became more severe, RIPK1i was administered via oral gavage. Spinal cords were removed at day 120 for analysis.


title: Wild-Type vs ORAI 1/2/3 TKO_Pathogenic Th17 cells_ 6h vs 24h

city: Los Angeles

first: Yousang

last: Gwack

address: 10833 Le Conte Ave

institute: Univeristy of California-Los Angeles

summary: Time-Course Transicriptome comparison between murine WT and ORAI TKO pTH17cells at 6 hours and 24 hours following differentiation

overall_design: Naive CD4+ T-cells were isolated and purified from wild-type (control) and ORAI 1/2/3 TKO mice. Naïve CD4+ T cells were enriched by magnetic sorting from single-cell suspensions generated by mechanical disruption of spleens and lymph nodes of adult mice using MagniSort naïve CD4+ T cell enrichment kit (catalog # 8804-6824-74,ThermoFisher Scientific). For effector T cell differentiation, cells were stimulated with anti-CD3 antibody (1452C11, Bio X Cell) and anti-CD28 antibody (Clone 37.51, Bio X Cell) on a plate coated with rabbit anti-hamster antibody (MP Biomedicals). CD4 + CD25 - T cells were cultured with anti-IL-4 antibody (Peprotech) and 10 μg/ml anti-IFN-γ antibody, 30 ng/ml IL-6 (Peprotech), 10 ng/ml IL-23 (R&D Systems) and 10 ng/ml IL-1β (R&D Systems) for pathogenic Th17 differentiation  in T cell medium. Cells were collected at 6h and 24h and prepared for bulk RNA sequencing analysis.

first: Beibei

last: Wu

first: Spyridon

last: Hasiakos

first: Shawn

last: Cokus

first: Matteo

last: Pellegrini

first: Sonal

last: Srikanth


title: TRPC1 links calcium signaling to cellular senescence in the protection against post-traumatic osteoarthritis

city: Münster

first: Joanna

last: Sherwood

address: Domagkstrasse 3

institute: University Hospital Münster

summary: Transient receptor potential channel 1 (TRPC1) is a widely expressed mechanosensitive ion channel located within the endoplasmic reticulum membrane, crucial for refilling depleted internal calcium stores during activation of calcium-dependent signaling pathways. Here, we have demonstrated that TRPC1 activity is protective within cartilage homeostasis in the prevention of cellular senescence associated cartilage breakdown during mechanical and inflammatory challenge. We revealed that TRPC1 loss is associated with early stages of osteoarthritis (OA) and plays a non-redundant role in calcium signaling in chondrocytes. Trpc1-/- mice subjected to destabilization of the medial meniscus induced OA developed a more severe OA phenotype than wild type controls. During early OA development, Trpc1-/- mice displayed an increased chondrocyte survival rate, however remaining cells displayed features of senescence including p16INK4a expression and decreased Sox9. RNA sequencing identified differentially expressed genes related to cell number, apoptosis and extracellular matrix organization. Trpc1-/- chondrocytes exhibited accelerated dedifferentiation, while demonstrating an increased susceptibility to cellular senescence. Targeting the mechanism of TRPC1 activation may be a promising therapeutic strategy in osteoarthritis prevention.

overall_design: RNA sequencing of murine knee joint articular cartilage collected 2 weeks post destabilisation of the medial meniscus (DMM) surgery, comparing wild type and Trpc1-/- male mice operated at 10 weeks of age


title: Single cell RNAseq of iPSC tri-cultures treated with necroptosis inducer

summary: Transcriptomic analysis of iPSC tri-culture (microglia, neurons, astrocytes) stimulated with Tnf, Smac, and Zvad (TSZ) to trigger necroptosis pathway activation, in the presence or absence of a Ripk1 inhibitor

overall_design: Cells were pre-treated for 1 hour with 1 μM Smac mimetic LCL161 (Selleck Chem, S7009), 20 μM zVAD-fmk (Enzo Life Sciences, ALX-260-020), and 1 μM RIPK1 inhibitor GSK’547 prior to a 4 hour incubation with 50 ng/mL recombinant human TNF (R&D Systems, 210-TA). Four hours after treatment, cells were collected and sequenced using the 10x Genomics platform.


title: Gene expression profile at single cell level of mouse liver in whole and fibrotic niche of the liver (scRNA-Seq)

first: Kwon Yong

last: Tak

overall_design: The liver was digested to enriched the fibrotic niche and was sequenced in parallel with control data. Experiment was done in young (6 months) and old (24 months) mouse to see age-relation in fibrosis

first: Kwon

first: Juyeon

last: Kim

first: Myungsun

last: Park

first: Chuna

first: Jong-eun


title: Dynamic investigation of hypoxia-induced L-lactylation

city: Shenzhen

first: Rui

last: Li

address: Lishui street

institute: Peking university

summary: The recently identified histone modification lysine lactylation can be stimulated by L-lactate and glycolysis. Although the chemical group added upon lysine lactylation was originally proposed to be the L-enantiomer of lactate (KL-la), two isomeric modifications, lysine D-lactylation (KD-la), and N-ε-(carboxyethyl) lysine (Kce), also exist in cells, with their precursors being metabolites of glycolysis. The dynamic regulation and differences among these three modifications in response to hypoxia have not been investigated previously. In this study, we demonstrate that intracellular KL-la, but not KD-la or Kce, is upregulated in response to hypoxia. Depletion of glyoxalase enzymes, GLO1 and GLO2, had minimal impact on KD_x001E_la, Kce, or hypoxia-induced KL-la. Conversely, blocking glycolytic flux to L-lactate under hypoxic conditions by knocking out LDHA/B completely abolished the induction of KL-la, but increased KD-la and Kce. We further observed a correlation between the level of KL-la and HIF-1α expression under hypoxic conditions and when small molecules were used to stabilize HIF-1α in the normoxia condition. Our result demonstrated that there is a strong correlation between HIF-1α and KL-la in lung cancer tissues, and that patient samples with higher grade tend to have higher KL-la levels. Using a proteomics approach, we quantified 66 KL-la sites that were upregulated by hypoxia and demonstrated that p300/CBP contributes to hypoxia-induced KL-la. Collectively, our study demonstrates that KL-la, rather than KD-la or Kce, is the prevailing lysine lactylation in response to hypoxia. Our results therefore demonstrate a link between KL-la and the hypoxia-induced adaptation of tumor cells.

overall_design: ChIP-seq of the histone modification H3K18L-la in normoxia and hypoxic MCF-7 cells.

first: Jinjun

last: Gao

first: Ruilong

last: Liu

first: Kevin

last: Huang

first: Ziyuan

first: Xinlei

last: Sheng

first: Kasturi

last: Chakraborty

first: Chang

last: Han

first: Di

last: Zhang

first: Lev

last: Becker

first: Yingming

last: Zhao


title: Keratinocyte response to PX43-induced split formation under different pathway inhibitions

first: Hauke

last: Busch

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

overall_design: Refer to individual Series

first: Jennifer

last: Hundt

first: Veronika

last: Hartmann

first: Uta

last: Radine

first: Sen

last: Guo


title: Host cell gene expression profile in Human respiratory syncytial virus (HRSV) infected HEp2 cells at 4, 8, 12 and 15 hours post infection

city: Singapore

first: Richard

last: Sugrue

address: 60 Nanyang Drive

department: School of Biological Sciences

country: Singapore

institute: Nanyang Technological University

summary: We used the microarray data to analyze host cells response on HEp2 cells infected with HRSV

overall_design: The HRSV infected Hep2 cells were harvested at 4, 8, 12 and 15 hpi and RNA extraction was performed using standard protocol as described by Affymetrix. The aim of this experiment is to analyze host cell response to HRSV infection.

first: Dawn

last: Yeo

first: Hui

last: Chen

first: Boon

last: Tan


title: Experimental and Computational Insights into the Mechanism that CHRONO Regulates the Circadian Clock

city: Hefei,

first: Qin

last: Zhou

state: Anhui

address: 111 Jiulong Road, Hefei, Anhui, China

institute: Anhui university

summary: The pace of the endogenous circadian clock is important for organisms to maintain homeostasis. CHRONO has been shown to play an important role in regulating the period of the mammalian clock and has recently been implicated to function in several important physiological aspects. To function properly, CHRONO needs to enter the nucleus to repress transcription. However, how CHRONO enters the nucleus and regulates the circadian clock remains unknown. Here, we report the nuclear localization signal of CHRONO and the genome-scale cistrome of CHRONO. We show that CHRONO acts as a transcriptional repressor on Jun/Fos complexes in addition to the classical BMAL1/CLOCK complexes. We further developed a mathematical model and the model simulations strongly agreed with our findings that CHRONO acts on both REV-ERBα and BMAL1 to regulate the circadian period. The model also sheds light on the fact that CHRONO cycles in most of the mammalian tissues. Thus, our experimental and computational approaches reveal the molecular mechanisms of an important circadian component, CHRONO.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for Bmal1 as well as Myc-CHRONO in U2OS and U2OS-CHRONO cells. The Bmal1-ChIP sample were time coursed with a circadian cycle.


title: Hypidonehydrochloride (YL-0919) improves MCAO-induced ischemia-reperfusion injury by reducing inflammation

city: Zhangjiakou

first: Xiaojuan

last: Hou

state: Heibei

address: No.11, South Zuanshi Road, Zhangjiakou, China

institute: Hebei North University

summary: YL-0919 can alleviate the inflammatory response, reduce the area of cerebral infarction, decrease the degree of cell apoptosis on the injured side, hasten the regeneration of neurons, improve nerve function injury, and accelerate the recovery of motor function after MCAO surgery.

overall_design: Transcriptome sequencing of mice brain tissues from 3 different groups

last: Ma

first: Yue

first: Yafan

last: Bai


title: Transcriptome Response and Protein Abundance are Correlated with Split Formation and Cell Detachment as Secondary Effects to Autoantibodies in Human Skin Organ Culture Models for Pemphgus Vulgaris

summary: Pemphigus vulgaris (PV) is an autoimmune disease caused by autoantibodies (AAbs) targeting Desmoglein 1 (DSG1) or Desmoglein 3 (DSG3) on keratinocytes, leading to disrupted cell-cell adhesion and epidermal blistering. To investigate the early signaling events triggered by AAb binding, we examined transcriptomic and proteomic responses in a human skin organ culture (HSOC) model for PV. After injecting the single-chain variable fragment (scFv) PX43, which targets DSG1 and DSG3, or the mouse-derived DSG3 AAb AK23, we quantified transcriptome changes at 0, 5, 10, and 24 hours and proteome responses at 0 and 24 hours, using human or murine IgG as controls. Differential pathway regulation was analyzed over time using gene set variation and enrichment analysis, with pathway responses compared to in vitro and in situ responses of normal human epithelial keratinocytes (NHEKs) under conditions such as stretching, differentiation, migration, detachment, and acute wounding. Only PX43 induced split formation in the HSOC model, which was associated with significant transcriptomic and proteomic changes, while AK23 did not alter gene expression or protein levels. Enriched pathways in response to split formation included TNFα, Interferon α/γ, IL2-STAT5, and IL5-STAT3 signaling across all time points. The transcriptomic response closely resembled that of acute skin wounding and keratinocyte detachment, with an inverse correlation to cell stretching and differentiation and no correlation to keratinocyte migration. These findings indicate that AAbs targeting DSG1 and/or DSG3 alone do not drive broad transcriptomic or proteomic changes; rather, observed changes result from split formation, resembling the response to wounding and inflammation.

overall_design: Human skin organ cultures were injected with DSG-targeting PX43 (DSG1 and DSG3) or AK23 (DSG3) antibodies, with transcriptome data collected by RNA-seq at 0, 5, 10, and 24 hours post-stimulation, using human or murine IgG as controls.


title: Transcriptional profiling of striatum and cortex from a mouse model of Huntington's disease (Q140) crossed with Pms1 heterozygous and homozygous mice

city: Princeton

first: Jeff

last: Aaronson

state: New Jersey

address: 155 Village Boulevard, Suite 200

department: Discovery & Systems Biology

institute: CHDI

summary: Cerebral cortex and striatum were isolated from 6-month old Pms1+/- and Pms1-/- mice crossed with the Q140 knock-in (KI) mice and their respective controls. Transcriptomic analysis (RNASeq) was performed on 6 genotypes: WT, Q140, Psm1+/-, Q140 X Psm1+/-, Psm1-/-, and Q140 X Psm1-/-, 8 replicates per genotype.

overall_design: RNASeq analysis was performed on samples from the cerebral cortex and striatum tissues of 6 months old wild type, Q140, Psm1+/-, Q140 X Psm1+/-, Psm1-/-, and Q140 X Psm1-/- mice. N = 16 per group (8M/8F)

first: William

last: Yang

last: Wang


title: CRTC2 Forms Co-condensates with YTHDF2 that Enhance Translational Efficiency of m6A-Modified mRNAs to Drive Hepatocarcinogenesis and Lenvatinib Resistance (RNA-seq)

city: tianjin

first: meixi

last: wang

address: Tianjin Sports Institute North Road Huanhu West Road

institute: Tianjin Medical University Cancer Hospital

summary: As the third most common cause of cancer-related mortality, hepatocellular carcinoma (HCC) is a global health concern. Despite its prevalence, treatment options are limited, underscoring the need to identify potential therapeutic targets and strategies. Here, we identified amplification of CRTC2, situated in the 1q21.3 region, due to copy number alterations in HCC. In a cohort of patients with HCC, CRTC2 protein levels were frequently elevated and correlated with poor prognosis. Genetic depletion of CRTC2 significantly impeded the onset and progression of HCC in mouse models. CRTC2 formed cytoplasmic condensates that recruited the m6A reader YTHDF2. Furthermore, CRTC2 promoted the translocation of m6A-modified mRNAs from decay sites to polyribosomes by interacting with PABP1. The activities of CRTC2 counteracted YTHDF2-mediated mRNA degradation to enhance the translational efficiency of specific mRNAs, including those encoding LRP5 and c-Jun. Targeting CRTC2 in hepatocytes using AAV8.sgCRTC2 elicited substantial therapeutic benefits in HCC mouse model and significantly enhanced the sensitivity to lenvatinib. Together, this research elucidates the pivotal role and underlying molecular mechanisms of CRTC2 in hepatocarcinogenesis and lenvatinib-resistance, highlighting its potential clinical and therapeutic applications.

overall_design: To investigate the function of CRTC2 in HCC, we established stable HCCLM3 cell lines overexpressing CRTC2-WT or CRTC2-R>A. For RNA-seq, comparative gene expression profiling analysis of RNA-seq data for Control, CRTC2-WT and CRTC2-R>A in HCCLM3 cells.


title: Integrative Gene and Isoform Co-expression Networks Reveal Regulatory Rewiring in Stress-related Psychiatric Disorders [whole blood]

city: Munich

first: Janine

last: Knauer-Arloth

address: Kraepelinstr. 2-10

institute: Max Planck Institute of Psychiatry

summary: Isoform-specific expression patterns have been implicated in stress-related psychiatric disorders like major  depressive disorder (MDD), yet the extent of their involvement and their interrelationships remain unclear. We constructed co-expression networks for individuals affected (n=210, 81% with depressive symptoms) and unaffected (n=95) by stress-related psychiatric disorders. We incorporated total gene expression (TE) and isoform ratio (IR) data and validated the inferred networks using advanced graph generation techniques. Our analysis revealed distinct network topology and structure between the two groups. Investigation of the 127 shared hubs (degree >= 10) found that these hubs exhibit co-regulatory patterns unique to each network. The affected individuals’ network also contained 61 hub nodes with a minimum absolute fold increase of two in connectivity compared to the unaffected individuals’ network. Notably, 49% of these hubs showed evidence for association with psychiatric disorders. Gene Ontology enrichment analysis revealed distinct biological processes associated with hubs, such as mRNA processing for affected and immune response and cell adhesion for unaffected individuals. Enrichment analysis of GWAS loci further supported these findings. Analysis of the isoform-specific nodes showed distinct protein-protein interactions compared to gene-level analysis.

overall_design: The total RNA-seq of whole blood from 37 participants with no stress-related mental disorders (controls) was determined.

first: Ghalia

last: Rehawi

first: Elisabeth

last: Binder


overall_design: To investigate the function of CHRONO, we constructed CHRONO knock out cell lines,and performed gene expression profiling analysis using data obtained from RNA-seq of U2OS-WT and U2OS-CHRONO-/- cells with three replications respectively.


title: Integrative Gene and Isoform Co-expression Networks Reveal Regulatory Rewiring in Stress-related Psychiatric Disorders [PBMC]

overall_design: The total RNA-seq of PBMCs from 229 participants with stress-related mental disorders and 70 healthy controls was determined.


title: Enterochromaffin cells act as intestinal signal integration hubs for microbial metabolites

city: Rochester

first: Aditya

last: Bhagwate

state: MN

address: 200 1st Street SW

institute: Mayo Clinic

summary: The intestinal epithelium senses and responds to the myriad of signals from gut microbiota, but it remains unclear how these signals are integrated to drive physiological responses. In this work, we found that enterochromaffin (EC) cells in the gut serve as signal integration hubs for microbial metabolites. EC cells coordinate responses to combinations of microbial metabolites, resulting in complex alterations in serotonin signaling that drive changes in GI physiology. We found that microbial metabolites either directly trigger responses or alter the expression of receptors for other microbial metabolites in EC cells. The microbiota-derived purine derivative hypoxanthine triggers a signaling pathway by activating G-protein coupled receptor A1R, which in turn activates the calcium channel TRPC4, resulting in increased serotonin release and accelerated GI transit. On the other hand, bacteria-derived butyrate does not evoke EC cell calcium influx by itself, but drives epigenetic changes that upregulate TRPC4 expression, thereby enhancing response to metabolites like hypoxanthine and norepinephrine that act via TRPC4. Since the expression TRPC4 is limited to EC cells, these cells function as specialized epithelial sensors that integrate signals from regulatory (butyrate) and effector metabolites like hypoxanthine and norepinephrine. These findings offer new microbiota-driven therapeutic avenues for conditions associated with altered GI function.

overall_design: The experiment was done in QGP-1 cell line, including 2 treatment groups--the control group, where cells were grown in regular medium, and butyrate group, where cells were maintained in medium containing 5 mM butyrate for 24h. Each group had 3 replicates, each containing 4*10^6 cells.

first: Yang

last: Xiao

first: Tijs

last: Louwies

first: Ruben

last: Mars

first: Lisa

middle: M

last: Till

first: Yash

last: Gupta

first: Arnaldo

last: Mercado-Perez

middle: V

first: Shreya

middle: S

last: Bellampalli

first: Alejandro

last: Stark Quiroz

first: Rongfang

last: Liu

first: Laura

last: Heitman

first: Dennis

last: Tienter

first: Michael

last: Thompson

first: Eugene

middle: W

last: Krueger

first: Krishna

last: Kalari

first: Kaitlyn

last: Hawkins

first: Jeong-Heon

last: Lee

first: Brian

last: Edwards

first: Prakasha

last: Kempaiah

first: Daan

last: van der Es

first: Julia

middle: L

last: Willett

first: Chun-Jun

first: Y

last: Prakash

first: Brooke

last: Druliner

first: Tamas

last: Ordog

first: Gianrico

last: Farrugia

first: Arthur

last: Beyder

first: Kristen

last: Smith-Edwards

first: Purna

middle: C

last: Kashyap


title: Regulation of LEAP2 by insulin and glucagon in mice and humans

city: Copenhagen

first: Andreas

last: Constantinou

address: Malov

department: Diabetes Pharmacology, Liver & Gut Biology

country: Denmark

institute: Novo Nordisk

summary: Liver-expressed antimicrobial peptide 2 (LEAP2) is an endogenous antagonist and inverse agonist of the growth hormone secretagogue receptor 1a (GHS-R1a), exhibiting opposing effects on cell signaling, feeding, and hormonal secretion compared to ghrelin. However, despite an emerging interest in LEAP2’s physiology and pharmacology, its endocrine regulation remains unclear. Here, we show that plasma LEAP2 levels decrease significantly upon supraphysiological glucagon infusions during pancreatic somatostatin clamps in humans and that a hypercaloric diet and a sedentary lifestyle for two weeks impair this effect. Moreover, in patients with obesity and type 2 diabetes, plasma LEAP2 levels decrease significantly following glucagon infusion. Additionally, we find that postprandial upregulation of LEAP2 in mice is insulin-dependent. In the postprandial state, insulin receptor antagonism offsets the upregulation of hepatic Leap2expression and plasma LEAP2 levels. Finally, we show that insulin and glucagon receptor-expressing hepatocytes are the primary source of hepatic LEAP2 expression. This coincides with a putative enhancer-like signature bound by insulin- and glucagon-regulated transcription factors at the LEAP2 locus in the liver. Collectively, these findings implicate insulin and glucagonin the regulation of LEAP2 and highlight the need for further investigations into the endocrine mechanisms both upstream and downstream of LEAP2.

overall_design: RNA-seq profiling of the insulin response in HepG2-IGF1R-KO cells in response to insulin in normal or insulin resistant media.


title: Mechanism of initial favourable response to decitabine in TP53 mutated MDS/AML and potential mechanisms of subsequent relapses

first: Ying

last: NI

address: Tat Chee Avenue

institute: City University of Hong Kong

summary: Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) with complex and monosomy karyotype (CK/MK) show high prevalence of TP53 mutations, poor response to induction chemotherapy and adverse patient outcome. These diseases may respond to decitabine but the mechanisms are presently unclear. MDS/AML patients were treated with decitabine for 10 days in a Phase II clinical study. In this study, we collected serial samples from patients before and at completion of decitabine treatment, morphologic remission and relapse. The samples were interrogated with targeted myeloid panel sequencing, nanopore DNA cytosine methylation sequencing and single-cell transcriptomics to investigate potential interactions between leukemic and immune populations. The integrative analysis allowed characterization of shifting dynamics within leukemic and immune cell populations in individual patients. Comparison of these trends between TP53 mutated MDS/AML patients who responded to treatment versus TP53 wildtype patients who were refractory to treatment highlighted the complex interplay of leukemic and immune compartments. Single cell transcriptomic analyses confirmed immune activation in TP53m responders after decitabine treatment. At relapse, leukemic populations showed up-regulation of MYC signaling and heat shock response while T-cells showed exhaustion signature. Our work highlighted the complex interplay between leukemic and immune populations in TP53m patients upon decitabine treatment that might account for clinical responses and subsequent relapses.

overall_design: Adult patients (age 18 to 65 years old) with newly diagnosed CK-AML were enrolled in a Phase 2 open-labelled clinical trial (ClinicalTrials.gov Identifier: NCT03080766) and received decitabine at 20 mg/m2 for 10 days in 28 day-cycle. Bone marrow (BM) aspiration was performed before treatment and on Day 10 and 28 of the first cycle. Patients with marrow blasts ≤ 5% on Day 28 received 5-day treatment while those with marrow blasts > 5% received 10-day treatment in subsequent cycles until blasts ≤ 5%. Subsequent aspiration was performed at physicians’ discretion. Eligible patients with suitable donors could receive allogeneic HSCT.

first: Nelson

last: Ng

last: Ni

first: Runsheng

last: Li

first: Anskar

last: Leung


title: Targeted selection of experimental replicate groups is the likely driver of differential expression artifacts

city: Adelaide

first: Lachlan

last: Baer

address: North Terrace

institute: The University of Adelaide

summary: The design of a scientific experiment forms the foundation for rigorous hypothesis testing and robust interpretations. In RNA-seq analyses where common model organisms are used to study the effects of gene perturbations, breeding schemes are typically designed to produce siblings with either mutant or wildtype alleles of a gene. Siblings are expected to possess reduced genetic variability, allowing the biological responses of a gene perturbation to be observed accurately, and above that of minimal background noise. However, as previously reported (Baer et al. 2024), inter- and intra-genetic variability of the parental subjects allows for the possibility of such between the progeny. What matters is not so much the genetic diversity, but more specifically the differences in polymorphic loci represented between replicate sample groups, which are constructed to perform comparative statistical tests. This is largely due to chance when selecting a subset of progeny at random to ultimately represent the RNA-sequencing samples. For many RNA-sequencing experiments this subset is often small due to minimise costs, and therefore the opportunity of skewing is likely. Additionally, when the subset of samples is not selected at random, particularly when defined based on genotype at a genetic locus, further skewing may artificially and unexpectedly be introduced. The end result is expression differences due to unequal representations of genetic features that influence gene expression, i.e. expression quantitative trai loci (eQTLs), which manifest as noise obscuring the desired signal and therefore confound interpretations. In this study we intentionally implemented an experimental design that opposes our advice from previous findings. We analyse the impacts of strain-specific differences in zebrafish, by outcrossing two different strains of zebrafish. Strain-specific differences were quantified by performing Differential Allelic Representation (DAR) analysis, consequently providing additional evidence that DAR contributes to false positives in differential expression analysis. We first assess the impact of DAR in isolation, where functional differences due to mutation are mitigated by equal representation within each group subject to comparison. We then extend our investigation by rearranging the same set of samples to form additional distinct experimental groups, allowing functional comparisons similar to our previous approach, but in the presence of elevated DAR.

overall_design: mRNA sequencing of 3 month old sibling zebrafish whole brains, consisting of 27 samples that were genotyped at two loci on chromosomes 14 and 17, with n=3 for each genotype combination. Genotypes at chromosome 14 are homozygous for Tubingen (TU) strain, homozygous for PK strain, and heterozygous TU/PK. Genotypes at chromosome 17 are heterozygous for the psen1 T428del allele modelling early onset familial Alzheimer’s disease, heterozygous for the psen1 W233fs allele modelling familial Acne Inversa, and wild type.

first: Ewan

last: Gerken

first: Karissa

last: Barthelson

first: Stevie

last: Pederson

first: Michael

last: Lardelli


title: Impact of oxygen availability on the organelle-specific redox potentials and stress in recombinant protein producing Komagataella phaffii

city: Vienna

first: Nadine

middle: Elpida

last: Tatto

address: Muthgasse 18

department: Department of Biotechnology

country: Austria

institute: University of Natural Resources and Life Sciences

summary: The yeast Komagataella phaffii (syn. Pichia pastoris) is a highly effective and well-established host for the production of recombinant proteins. The redox balance of its secretory pathway, which is multi-organelle dependent, is of high importance for producing secretory proteins. Redox imbalance and oxidative stress  an significantly influence protein folding and secretion. Glutathione serves as the main redox buffer of the cell and cellular redox conditions can be assessed through the status of the glutathione redox couple (GSH-GSSG). Previous research often focused on the redox potential of the endoplasmic reticulum (ER), where  oxidative protein folding and disulfide bond formation occur. In this study, in vivo measurements of the glutathione redox potential were extended to different subcellular compartments by targeting genetically encoded redox sensitive fluorescent proteins (roGFPs) to the cytosol, ER, mitochondria and peroxisomes. Using these biosensors, the impact of oxygen availability on the redox potentials of the different organelles was investigated in non-producing and producing K. phaffii strains in glucose-limited chemostat cultures. It was found that the transition from normoxic to hypoxic conditions affected the redox potentials of all investigated organelles, while the exposure to hyperoxic conditions did not impact them. Also, as reported previously, hypoxic conditions led to increased recombinant protein secretion. Finally, transcriptome and proteome analyses provided novel insights into the short-term adaptation of the cells from normoxic to hypoxic conditions.

overall_design: Differential gene expression analysis of the Komagataella phaffii CBS7435 wildtype strain. The 4 samples are comprised of 4 biological replicates each, grown under two oxygen concentrations, hyperoxia (30% oxygen) and hypoxia (0% oxygen), by controlling the oxygen concentration of the inlet air and at various volume changes (5.0, 0.4, 4.0). All conditions were compared to each other.

first: Aliki

last: Kostopoulou

first: Corinna

last: Rebnegger

first: Borja

last: Bordera

first: Matthias

last: Mattanovich

first: Sandra

last: Maaß

first: Dörte

last: Becher

first: Brigitte

last: Gasser

first: Diethard


title: Reactivation of mTOR signaling slows neurodegeneration in a lysosomal storage disease

city: Bethesda

first: Richard

last: Proia

address: 10 Center Drive Room 9D11

department: National Institute of Diabetes and Digestive and Kidney Diseases

institute: National Institutes of Health

summary: Sandhoff disease, a lysosomal storage disorder, is caused by pathogenic variants in the HEXB gene, resulting in the loss of β-hexosaminidase activity and accumulation of GM2 ganglioside and GA2 glycolipid. This accumulation occurs primarily in neurons, and leads to progressive neurodegeneration through a largely unknown process. Lysosomal storage diseases often exhibit dysfunctional mTOR signaling, a pathway crucial for proper neuronal development and function. In this study, Sandhoff disease model mice exhibited reduced mTOR signaling in the brain. To test if restoring mTOR signaling could improve the disease phenotype, we genetically reduced expression of the mTOR inhibitor Tsc2 in these mice. Sandhoff disease mice with reactivated mTOR signaling displayed increased survival rates and motor function, especially in females, increased dendritic-spine density, and reduced neurodegeneration. Tsc2 reduction also partially rescued aberrant synaptic function–related gene expression. These findings imply that enhancing mTOR signaling could be a potential therapeutic strategy for lysosomal-based neurodegenerative diseases

overall_design: Compare gene expression level from WT, HexbKO, KOTsc, Tsc brain with six replicates from each group.

first: Hongling

last: Zhu

first: Y

middle: T

last: Lee

first: Colleen

last: Byrnes

first: Jabili

last: Angina

first: Danielle

last: Springer

first: Galina

last: Tuymetova

first: Mari

last: Kono

first: Cynthia

last: Tifft


title: Transcriptional profiling of cerebral cortex and striatum from a mouse model of Huntington's disease (Q140) crossed with Msh6 heterozygous and homozygous mice

first: Jeff

last: Aaronson

summary: Cerebral cortex and striatum were isolated from 6-month old Msh6+/- and Msh6-/- mice crossed with the Q140 knock-in (KI) HD mice and their respective controls. Transcriptomic analysis (RNASeq) was performed on 6 genotypes: WT, Q140, Msh6+/-, Q140 X Msh6+/-, Msh6-/-, and Q140 X Msh6-/-, 8 replicates per genotype.

overall_design: RNASeq analysis was performed on samples from the cerebral cortex and striatum tissues of 6 months old wild type, Q140, Msh6+/-, Q140 X Msh6+/-, Msh6-/-, and Q140 X Msh6-/- mice. N = 8 per group (4M/4F)

first: X

last: Yang

last: Wang


title: Transcriptional profiling of piriform cortex from a mouse model of Huntington's disease (Q140)

summary: Piriform cortex were isolated from 12-month old wild type and Q140 knock-in (KI) HD mice. Transcriptomic analysis (RNASeq) was performed on 2 genotypes: WT and Q140, 6 replicates per genotype.

overall_design: RNASeq analysis was performed on samples from the piriform cortex tissues of 12 months old wild type and Q140 mice. N = 6 per group (3M/3F)


title: Regulation of NK cell development, maturation, and antitumor responses by the nuclear receptor NR2F6

city: Innsbruck

first: Johannes

last: Woelk

address: Peter-Mayr-Str. 1a

department: Cell Genetics

institute: Medical University of Innsbruck

summary: Natural killer (NK) cell development and functionality rely on precise regulation by specific transcription factors (TFs). Our study demonstrates that the nuclear orphan receptor NR2F6 represses the expression of the activating receptor NKp46, an established key player in NK cell-mediated cytotoxicity during infection and tumor rejection. Despite normal NK cell development in the bone marrow, germline Nr2f6-deficient mice exhibit impaired terminal maturation of NK cells in the periphery. Short-term NK cell responses to lipopolysaccharide (LPS) activation, independent of NKp46, are subsequently reduced in Nr2f6-deficient mice. Conventional type 1 dendritic cells (cDC1) and macrophage populations are decreased in spleens of Nr2f6-deficient mice, subsequently, IL-15-dependent NK cell priming is limited. Administration of exogenous IL-15 in vitro and as IL-15 complex in vivo can compensate for these deficits, promoting terminal maturation of NK cells in Nr2f6-deficient mice. Subsequent transcriptome analysis reveals significant changes in gene expression profiles of NK cells from IL-15 complex treated Nr2f6-deficient mice, with notable alterations in essential NK genes such as Klrg1, Prdm1, Stat5a, Zeb2, and Prf1. Consequently, Nr2f6-deficient IL-15 complex treated NK cells raise enhanced effector responses of IFNγ, Perforin, and Granzyme B upon ex vivo activation. Of importance, Nr2f6-deficient mice are protected against MHC-I negative B16-F10 melanoma lung metastasis formation, especially with IL-15 complex treatment, indicating the potential of NR2F6 to affect NKp46-dependent NK cell-mediated tumor surveillance. The therapeutic targeting of NR2F6 may be a promising strategy for boosting NKp46-dependent NK-cell-mediated tumor surveillance and metastasis.

overall_design: NK cells were sorted from the spleen of untreated or IL15C treated (3 x i.v. injected with 2 µg IL 15 complex ) wild-type or Nr2f6-deficient mice. NK cells were identified  as live/Lin-/CD45+/NK1.1+/NKp46+/CD49b+ on a FACS Aria III. We submitted lysated from 3 wild-type and 3 Nr2f6-deficient untreated mice. In addition we submitted lysated from 4 wild-type and 4 Nr2f6-deficient IL-15C treated mice.

first: Florian

last: Hornsteiner

first: Stephanie

last: Aschauer-Wallner

first: Patrizia

last: Stoitzner

first: Gottfried

last: Baier

first: Natascha

last: Hermann-Kleiter


title: Th1-poised naïve CD4 T cell subpopulation reflects anti-tumor immunity and autoimmune disease

city: Ulsan

first: Seonjun

last: Park

address: UNIST-gil 50, Ulju-gun, Ulsan

institute: UNIST

summary: Naïve CD4 T cells are traditionally viewed as a quiescent, homogeneous, resting population, but merging evidence reveals their heterogeneity, which can be crucial for understanding disease contexts and therapeutic outcomes. In this study, we identify distinct subpopulations within both murine and human naïve CD4 T cells by single cell-RNA-sequencing (scRNA-seq), particularly focusing on a subpopulation that expresses super-high levels of interleukin-7 receptor (IL-7Rsup-hi), along with CD97, IL-18R, and Ly6C. This subpopulation, absent in the thymus and peripherally induced, exhibits type 1 helper T cell (Th1)-poised characteristics and plays a role in inhibiting cancer progression in mouse B16F10 tumor model. In humans, this IL-7Rsup-hi subpopulation expressing CD97 correlates with responsiveness to anti-PD-1 therapy in cancer patients and disease state of multiple sclerosis. By elucidating the heterogeneity of naive CD4 T cells, including a Th1-poised subpopulation capable of robust type 1 responses, we highlight the importance of this heterogeneity in inflammatory conditions for defining the disease states and predicting drug responsiveness.

overall_design: RNA-seq profiling of IL-7Rsup-hi and IL-7Rlow naïve CD4 T cells.

first: Jae-Won

last: Yoon

first: Sookyung

last: Cho

first: Sung Ho

first: Je-Min

last: Choi


title: Mechanistic Investigation of Five Small-Molecule Inhibitors in the Treatment of Pemphigus Vulgaris via RNA-Seq Analysis

city: Lübeck

first: Hauke

last: Busch

address: Ratzeburger Allee 160

institute: University of Lübeck

summary: Pemphigus vulgaris (PV) is an autoimmune disorder characterized by autoantibodies (AAbs) against Desmoglein 1 (DSG1) and Desmoglein 3 (DSG3) on keratinocytes, resulting in compromised cell-cell adhesion and epidermal blistering. To explore potential therapeutic targets, five small-molecule inhibitors, A66 (PI3Kα inhibitor), BIRB796 (p38 MAPK inhibitor), GW441756 (TrkA inhibitor), Selumetinib (MEK1 inhibitor), and Vandetanib (VEGFR2 inhibitor) were selected through a screen identifying compounds that reduce split formation in a human skin organ culture (HSOC) model. Each inhibitor exhibits distinct transcriptomic profiles that contribute to gene expression modulation, yet all share a common downregulation of chemokine signaling pathways associated with split formation. These inhibitors, capable of mitigating skin blistering in PV, present potential as therapeutic agents for this challenging autoimmune condition.

overall_design: Human skin samples were treated by injection with anti-Dsg1/3 scFv without the addition of pemphigus vulgaris (PV) antibodies (negative control, NC), with PV antibodies alone (positive control, PC), or with PV antibodies in the presence of the indicated inhibitors (A66, BIRB796, GW441756, Selumetinib, and Vandetanib). After 24 hours of culture, samples were subjected to RNA-Seq analysis to assess transcriptomic changes associated with each treatment condition.

first: Imke

last: Burmester

first: Sen

last: Guo

first: Veronika

last: Hartmann

first: Jennnifer

last: Hundt

first: Uta

last: Radine


title: Bone marrow breakout lesions act as key sites for tumor-immune cell diversification in multiple myeloma

city: Heidelberg

first: Alexandra

last: Poos

address: Im Neuenheimer Feld 410

institute: University Hospital Heidelberg

summary: The bone marrow microenvironment plays a crucial role in the development of multiple myeloma. As the disease progresses, malignant myeloma cells can evolve to survive outside the bone marrow. However, the processes underlying bone marrow independence and their consequences for immune control remain poorly understood. Here, we conducted single-cell and spatial multi-omics analyses of bone marrow-confined intramedullary disease and paired breakout lesions that disrupt the cortical bone. These analyses revealed a distinct cellular microenvironment and architectural features of breakout lesions, characterized by extensive areas of malignant plasma cells interspersed with lesion-specific solitary NK and macrophage populations, as well as focal accumulations of immune cell agglomerates. Within these agglomerates, spatially confined T cell clones expanded alongside various immune cells, coinciding with the local genomic evolution of tumor cells. These analyses identify breakout lesions as a hotspot for tumor-immune cell interactions and diversification, representing a key event in myeloma pathogenesis.

overall_design:
Bone marrow samples from 5 myeloma patients with breakout lesions were analyzed using 10X Genomics CITE-seq and VDJT-sequencing.

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Raw files for human/patient samples are being made available in EGA (https://www.ebi.ac.uk/ega/) for controlled access to the personally identifiable sequence data.
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first: Llorenç

last: Solé-Boldo

first: Simon

last: Haas

first: Niels

last: Weinhold


title: DNA Damage Response Deficiency Enhances Neuroblastoma Progression and Sensitivity to Combination PARP and ATR Inhibition

city: Toronto

first: Madeline

middle: Nicole

last: Hayes

state: Ontario

address: 72 Elm St

department: DSCB

country: Canada

institute: Sickkids Research

summary: Next generation sequencing of neuroblastoma (NB) tumors have revealed frequent somatic and germline genetic alterations in genes encoding proteins involved in DNA damage response (DDR) pathways. Despite being well-studied in many adult cancers, roles for DDR disruption in pediatric solid tumors have not been fully elucidated. To address this, patient-relevant loss-of-function mutations in DDR pathway components including Brca2, Atm, and Palb2 were incorporated into an established zebrafish MYCN transgenic model (Tg(dbh:EGFP-MYCN)). These mutations were found to enhance NB formation and metastasis in vivo, and result in upregulation of proliferation, cell cycle checkpoint and DNA damage repair transcriptional signatures, revealing novel molecular vulnerabilities in DDR-deficient NB. Zebrafish DDR-deficient NB and human NB cells with DDR protein knock-down were sensitive to the poly(ADP-ribose)-polymerase (PARP) inhibitor olaparib, and this effect was further enhanced by inhibition of the ataxia telangiectasia and rad3 related (ATR) kinase. Altogether, our data supports a functional role for DDR-deficiency in NB in vivo and therapeutic potential for combination PARP + ATR inhibition in NB patients with alterations in DDR genes.

overall_design: To capture broad transcriptomic differences associated with HDR/DDR-deficiency in primary and transplanted neuroblastoma, we performed RNA sequencing of GFP+ sorted tumor cells from zebrafish MYCN-driven neuroblastoma from various engineered genetic backgrounds

first: Sarah

last: Cohen-Gogo

first: Lynn

last: Kee

first: Alex

last: Weiss

first: Mehdi

last: Layeghifard

first: Yagnesh

last: Ladumor

first: Anisha

last: Rajaselvam

first: David

middle: R

last: Kaplan

first: Anita

last: Villani

first: Adam

last: Shlien

first: Daniel

last: Morgenstern

first: Meredith

last: Irwin


title: BGN stimulates retinal pathological angiogenesis via up-regulation of CXCL12 expression in pericytes (mouse)

city: Shanghai

first: Miao

middle: miao

last: Liu

address: Kongjiang Road

institute: liumiaobiotech

summary: Retinal pathological angiogenesis (PA) is a common hallmark in proliferative retinopathies, including age-related macular degeneration (AMD), proliferative diabetic retinopathy (PDR), and retinopathy of prematurity (ROP). The mechanisms underlying PA is complex and incompletely understood. Using oxygen-induced retinopathy (OIR) mouse as a model, the role of extracellular matrix (ECM) protein biglycan (BGN) in PA was studied. Significant upregulation of BGN in the retina of OIR mice and in neovascular proliferative membranes of PDR patients was found. The reduction of BGN expression by Bgn-specific small interfering RNA (siRNA) effectively diminished retinal PA in OIR mouse. Subsequent analysis of the mouse OIR retinal single-cell RNA sequencing data from the Gene Expression Omnibus dataset (GSE150703) suggested pericyte as a source of BGN. This was verified using cultured retinal capillary pericytes. BGN expression in pericytes was highly sensitive to hypoxic stimulation. BGN stimulated retinal PA via the upregulation of C-X-C motif chemokine ligand 12 (CXCL12). Inhibition of the CXCL12-CXCR4 axis effectively diminished PA in OIR mouse. In conclusion, this study demonstrated the stimulatory role of BGN in retinal PA, identified the link between BGN and CXCL12 expression, and further highlighted the role of pericytes in retinal PA.

overall_design: C57BL/6 mice were used to established the OIR model. P17 retinas from normoxia control mice and OIR mice were collected to investigated the gene expression changes related to pathological angiogenesis.

first: Miaomiao

first: Ping

last: Fei

first: Jia

last: Luo

first: Peiquan

last: Zhao