title: Study of the usefulness of human peripheral blood mononuclear cells for the analysis of metabolic recovery after weight loss (METAHEALTH-TEST)

city: Wageningen

first: Evert

middle: M.

last: van Schothorst

address: De Elst 1

country: Netherlands

institute: Wageningen University

summary: The aim of this study is to design and validate a test, METAHEALTH-TEST, based on gene expression analysis in blood cells, to quickly and easily analyse metabolic health. This test will be used to analyse metabolic improvement in overweight/obese individuals and in metabolically obese normal-weight (MONW) individuals after undergoing a weight loss intervention and/or an intervention for improvement in eating habits and lifestyle. Obesity and its medical complications are a serious health problem today. Using peripheral blood mononuclear cells (PBMC) as an easily obtainable source of transcriptomic biomarkers would allow to deepen into the knowledge of adaptations in response to increased adiposity that occur in internal homeostatic tissues, without the need of using invasive biopsies. Moreover, if PBMC were able to reflect lipid metabolism gene expression pattern recovery as a result of weight loss, it would provide valuable information to know the efficacy of therapies aimed at weight loss and, in any case, it would allow to personalize them according to the evolution of obese patients until the desired metabolic recovery is achieved.

overall_design: Apparently healthy subjects aged 18 to 45 years old, including men and women were recruited and classified into two groups depending on their body mass index (BMI). Normal-weight (NW) group (BMI <25 kg/m2) was composed of 20 subjects and overweight-obese (OW-OB) group (BMI ≥25 kg/m2) of 27 subjects. The inclusion criteria were: subjects with no chronic disease who did not take regular medication or drugs. To avoid potential bias, both groups include approx. 50% men/women and there was no difference in their average age. We recruited 6 additional NW individuals presenting 1 metabolic alteration related to MetS (high plasma total or LDL-cholesterol, plasma triglycerides, or plasma C-reactive protein (CRP) concentrations, or hypertension). They were classified as  metabolically obese normal-weight (MONW) individuals. Subjects from the OW-OB group followed a 6-month weight loss program which included a low-calorie food plan (30% reduction in the individual energy requirements) with dietary sessions and exercise counselling. Dietary sessions were offered by a nutritionist every fifteen days who provided face-to-face counselling that was individually adjusted to each subject with the aim of reducing 5% to 10% of initial body weight. Neither dietary supplements nor vitamins were provided and all participants consumed self-selected foods. 20 out of the 27 OW-OB subjects who started the study completed the 6-month weight loss program. All the volunteers underwent what we called the fasting test which consisted of collecting blood samples after 4 and after 6 hours after having had a standard breakfast. The blood extractions were performed by skilled health personnel; once in the NW and MONW groups,and three times (at the baseline point, and after 3 and 6 months of nutritional intervention) in the OW-OB group. Blood was collected using Vacutainer® EDTA tubes. After blood collection, the samples were processed immediately to obtain the PBMC fraction. PBMC were isolated using Ficoll-Paque Plus density gradient media. Total RNA from PBMC samples was extracted using Tripure Reagent and then purified with E.Z.N.A. Total RNA Kit I and precipitated with isopropanol. Isolated RNA was quantified using a NanoDrop ND 1000 spectrophotometer. Its integrity was confirmed using agarose gel electrophoresis and the RIN tool using the Agilent 2100 Bioanalyzer System. For the microarray experiment the following samples were selected: 12 paired samples from the NW group after both 4h and 6h of fasting, 12 paired samples from the OW-OB group after both 4h and 6h of fasting, 12 paired samples from the OW-OB group after the 6-month weight loss programm after both 4h and 6h of fasting, and 6 samples from the MONW group after 6h of fasting at the beginning of the study. For final data analysis, 2 duplicate RNA samples were taken along and confirmed for reproducibility but excluded for overall analyses thereafter: US22502548_257236338304_S01_GE2_1200_Dec17_2_2.txt and US22502548_257236338312_S01_GE2_1200_Dec17_1_2.txt.

first: Andrea

last: Costa

first: Inge

last: van der Stelt

first: Annelies

last: Bunschoten

first: Paula

last: Oliver

middle: M


title: miR-214-3p overexpression inhibits apoptotsis during induction of breast cancer

status: Public on Oct 11 2023

city: Ithica

first: Praveen

last: Sethupathy

state: NY

address: Cornell University College of Veterinary Medicine, Box 17

country: USA

institute: Cornell University

type: Expression profiling by high throughput sequencing

summary: We treated horse mammary derived epithelial cells (MDECs) with either a miR-214-3p mimic or a negative mimic control in the presence of the carcinogenic agent DMBA. Over expression of miR-214-3p suppressed cell apoptosis compared to the control, and suggests a potential oncogenic role in breast cancer.

overall_design: Treatment of horse mammary derived epithelial cell cultures with miR-214 mimic or negative mimic control and DMBA followed by RNAseq.

first: Gerlinde

last: Van de Walle

first: Matt

last: Kanke


title: Rosa26-AML1-ETO 9a knock-in pre-leukemic Lin-cKit+ (LK) single-cell RNA sequencing

city: San Diego

first: Dong-Er

last: Zhang

state: California

address: 3855 Health Sciences Dr

department: Pathology

institute: UCSD

summary: T(8;21), which produces the AML1-ETO (AE) fusion oncoprotein, is one of the most common chromosomal abnormalities in AML patients. Despite its prevalence among patients, it has remained challenging to study the impact of AE on leukemia development in vivo due to limitations of the current t(8;21) mouse models. Here, we devised a germline transmitted transgenic Rosa26-AE9a knock-in mouse model that expresses AE9a protein, a truncated AE variant, at moderately elevated levels compared to endogenous RUNX1. These mice develop myeloid leukemia with longer latency and lower penetrance compared to mice using the AE9a retroviral transduction-transplantation model. Pre-leukemic mice exhibited myeloid progenitor skewing in their bone marrow, with more granulocyte-monocyte progenitors (GMP) at the expense of megakaryocyte-erythroid progenitors (MEP). Importantly, hematopoietic stem and progenitor cells (HSPCs) of pre-leukemic mice had increased clonogenic potential. We performed single cell RNA-sequencing on pre-leukemic HSPCs and confirmed both this granulocytic bias and hindered differentiation. Overall, we have overcome the limitation of uncontrolled AE9a oncogene expression in the retroviral transduction-transplantation mouse model and have devised a new germline transmitted Rosa26 locus AE9a knock-in model that more faithfully represents t(8;21) human disease.

overall_design: Data are single-cell RNA sequencing of Lin-cKit+ (LK) murine bone marrow cells of Rosa26-AE9a mice and control mice.

first: Mengdan

last: Liu

first: Ming

last: Yan

first: Amanda

middle: G

last: Davis


title: scRNAseq on sorted T-cells and CITE-seq on PBMCs

city: Paris

first: Sebastien

last: Mella

address: 28 rue du Dr Roux

department: Immunology

country: France

institute: Institut Pasteur

summary:
Purpose: Analysis at the single cell level of the CD4+ and CD8+ Lymphocytes T extracted and PBMCs from healthy human donor
Methods: Single Cell RNAseq performed using the 10X genomics platform

overall_design: Lymphocytes T mRNA profiles, and PBMC mRNA and cell surface markers profiles from healthy  Human donor

first: Valentina

last: Libri

first: Bernd

last: Jagla


title: Combinatorial interpretation of BMP and WNT allows BMP to act as a morphogen in time but not in concentration

status: Public on Oct 10 2023

city: Houston

first: Aryeh

last: Warmflash

state: TX

address: 6100 Main St

institute: Rice University

summary: We performed bulk RNA sequencing of cells exposed to either 300ng/ml of the ligand WNT3A for 6 or 18 hours, or 8μM of the GSK3b inhibitor CHIR99021 for 6 hours, which is commonly used as a substitute for WNT in differentiation. We observed transcriptional activation of the canonical WNT genes WNT3 and WNT10B confirming that Wnt signaling induces canonical WNTs.

overall_design: hPSCs were seeded at the density 200k per well in a 12-well plate 24 hours before treatment. hPSCs were grown under 4 experimental conditions: untreated pluripotent cells grown in mTeSR1, hPSCs treated with WNT3a (300ng/ml) for 6 hours, hPSCs treated with WNT3a (300ng/ml) for 18 hours or hPSCs treated with CHIR (8uM) for 6 hours. Total RNA was collected with the Invitrogen RNAqueous-Micro Total RNA Isolation Kit. Processed RNA was stored at −80 ̊C, and RNA integrity was checked by Nanodrop, agarose gel electrophoresis, and qPCR and the Agilent 2100 system. Sequencing was performed by Novogene Co. using the Illumina paired-end 150 platform (Novaseq 6000). Another biological repeat was performed using the same protocol, but also adding a new experimental condition: hPSCs treated with CHIR (8uM) for 18 hours, with two replicates.

first: Elena

last: Camacho-Aguilar

first: Sumin

last: Yoon

first: Miguel

middle: A

last: Ortiz-Salazar

first: Siqi

last: Du

first: M

middle: C

last: Guerra


title: Microglia response to allergic airway inflammation during early postnatal period in mice

city: Fukuoka City

first: Ban-yu

last: Saitoh

state: Fukuoka

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

department: Department of Neurology

country: Japan

institute: Kyushu University Graduate School of Medicine

summary: We examined how allergic inflammation in early postnatal period influences microglia using a short- and long-term airway allergy model. Male mice (C57BL/6) were immunized by intraperitoneal injection with aluminum hydroxide and ovalbumin (OVA) or phosphate-buffered saline (PBS) (control) at postnatal days (P) 3, 7, and 11, followed by intranasal challenge with OVA or PBS solution twice a week until P30 or P70. Microglia from whole brain were isolated from PBS- and OVA-treated mice by gating on CD11b+CD45int cells with flowcytometry at P30 and P70 respectively. Results suggest the dysregulation of microglial genes related to synaptic pruning and clearance, aging, and corticosteroid pathway and distinction between short- and long- term allergen challenge.

overall_design: Microglia were isolated from PBS- and OVA- treated mice 1 day after intranasal challenge at P30 and P70. Collected microglia were pooled (4 samples per group) for analysis. Extracted RNA were subjected to RNA array assay. Gene expression of PBS (control group)- and OVA (allergy group)- treated mice at P30 and P70 were compared respectively.

first: Hiroo

last: Yamaguchi

first: Ryo

last: Yamasaki

first: Jun-ichi

last: Kira


title: Molecular Dissection of Disease Heterogeneity in SLE

city: Oklahoma City

first: Joel

last: Guthridge

state: OK

department: Arthritis and Clinical Immunology

institute: Oklahoma Medical Research Foundation

summary: Systemic lupus erythematosus (SLE) affects 1 in 537 of African American (AA) women, which is >2-fold more than European American (EA) women. AA patients also develop the disease at a younger age, have more severe symptoms, and a greater chance of early mortality. We used a multi-omics approach to uncover ancestry-specific immune alterations in SLE patients and healthy controls that may contribute to disease disparities. Cell composition, signaling, and epigenetics were evaluated by mass cytometry; droplet-based single cell transcriptomics and paired proteogenomics (scRNA-Seq/scCITE-Seq).  Soluble mediator levels were measured in plasma and stimulated whole blood. TLR3/4/7/8/9 gene expression pathways in B cells and monocytes were enhanced in AA SLE patients compared to EA patients. TLR7/8/9 and IFN phospho-signaling responses were also heightened in healthy AA versus EA controls. Exposure of AA and EA healthy control cells to TLR7/8/9 agonists or IFN resulted in altered immune cell compositions that recapitulated the ancestry-associated differences in SLE patients. These data support that ancestry-based differences in TLR7/8, TLR9, and IFN responses that can be detected in healthy individuals could influence lupus disease course and severity.

overall_design:
multiplexed scRNA-seq with CITEseq profiles of PBMCs from both healthy controls and SLE patients with high and low disease activity.
Please note that [1] the SUBnnnnn accessions (in the sample title) are deidentified names for the individual demultiplexed samples [2] The "sample.csv" has sample characteristics for each sample [3] Each processed data file was generated from *cite, *gex, and *hto raw data  and is linked to the *cite sample records.

first: Samantha

last: Webb

first: Kevin

last: Thomas

first: Miles

last: Smith


title: RNA-seq profiling of upper tract urothelial carcinoma: bladder cancer consensus classification relevance, molecular heterogeneity and differential immune signatures

first: Guerric

last: Gilbert

address: 11/13 rue Pierre et Marie Curie

institute: Institut Curie

summary: Analyses of large transcriptomics datasets of muscle-invasive bladder cancer (MIBC) has led to a consensus classification. Molecular subtypes of upper tract urothelial carcinomas (UTUC) are less known. Our objective was to determine the relevance of consensus classification in UTUCs by characterizing a novel cohort of surgically treated ≥pT1 tumors.Subtype IHC markers GATA3-CK5/6-TUBB2B in multiplex, CK20, p16 and Ki67, MMR proteins, PD-L1 IHC were evaluated. Heterogeneity was assessed morphologically and/or with subtype IHC. FGFR3 mutations were identified by pyrosequencing. We performed 3’RNA-seq, including with multisampling in heterogeneous cases. Consensus classes, unsupervised groups, and microenvironment cell abundance were determined using gene expression.Most of the 66 patients were men (77.3%), with pT1 (n=23, 34.8%) or pT2-4 stage UTUC (n=43, 65.2%). FGFR3 mutations and dMMR status were identified in 40% and 4.7% of cases, respectively. Consensus subtypes robustly classified UTUCs and reflected intrinsic subgroups. All pT1 tumors were classified as Luminal papillary (LumP). Combining our consensus classification results to that of previously published UTUC cohorts, LumP tumors represented 57.2% of ≥pT2 UTUC, which was significantly higher than in MIBC. Ten patients (15.2%) harbored areas of distinct subtypes. Consensus classes were associated with FGFR3 mutations, stage, morphology and IHC. The majority of LumP tumors were characterized by low immune infiltration and PD-L1 expression, in particular if FGFR3 mutated.

overall_design: 3' Tag RNA-sequencing of 87 upper tract urothelial carcinoma samples from 66 patients

first: Jacqueline

last: Fontugne

first: François

last: Radvanyi

first: Yves

last: Allory

first: Alexandra

last: Masson-Lecomte

first: Fabrice

last: Glibert


title: CD27 costimulation modulates naïve T cell activation through TRAF2/SHP-1 and induces memory-associated gene regulatory networks.

city: Seattle

first: Scott

last: Furlan

state: WA

address: 1241 Eastlake Ave E

institute: Fred Hutchinson Cancer Center

summary: Engagement of CD27 during naïve CD8+ T (TN) cell activation is critical for T cell memory generation through poorly understood mechanisms. To examine the effects of CD27 signaling during TN cell activation we designed a synthetic trimeric CD70 ligand. In conjunction with T cell receptor (TCR) stimulation, ligation of CD27 resulted in immediate receptor internalization, recruitment of TRAF2/SHP-1, and modulation of Lck and ERK/AKT signaling in cells receiving concurrent CD28 costimulation. Independent of this modulatory effect on CD28 costimulated T cells, CD27 signaling enhanced ATF2, FOXO1, and FOXP1 transcription factor circuits, which induced T cell memory rather than the effector associated gene programs observed with CD28 costimulation alone. CD27-costimulated T cells engineered with a chimeric antigen receptor (CAR) exhibited improved tumor control. CD27 signaling during TN cell activation modulates activation strength and directs memory T cell properties that may benefit therapeutic T cells engineered with CARs or T cell receptors.

overall_design: We have performed Muliome-sequencing of in vitro activated human naive CD8+ T cells to assess early changes in the transcriptomic and epigenetic landscape in response to CD28 and/or CD27 costimulation. We have sequenced non-activated (naïve) T cells as well as 24h activated T cells that have been stimulated with  aCD3/aCD28 dynabeads [3:1 ratio of bead:T] or plate-bound aCD3, aCD28 and/or CD70DT (tirmeric ligand) at indicated doses [aCD3/aCD28/CD70DT; ug/mL].

middle: N

first: Olivia

last: Waltner

first: Pranav

middle: V

last: Muthuraman

first: Shruti

middle: S

last: Bhise

first: Sami

middle: B

last: Kanaan

first: Jobelle

last: Peralta

first: Carla

last: Jaeger-Ruckstuhl


title: Concordant B and T Cell Heterogeneity Inferred from the Multiomic Landscape of Peripheral Blood Mononuclear Cells in a Crohn's Disease Cohort

city: Atlanta

first: Greg

last: Gibson

state: GA

address: North Ave NW

institute: Georgia Institute of Technology

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Crohn’s disease is characterized by inflammation in the gastrointestinal tract due to a combination of genetic, immune, and environmental factors. Transcriptomic and epigenomic profiling of intestinal tissue of Crohn’s disease patients have revealed valuable insights into pathology, however have not been conducted jointly on less invasive peripheral blood mononuclear cells (PBMCs). Furthermore, the heterogeneous responses to treatments among individuals with Crohn’s disease imply hidden diversity of pathological mechanisms. We employed single nucleus multiomic analysis, integrating both snRNA-seq and snATAC-seq of PBMCs with a variety of open source bioinformatics applications. Our findings reveal a diverse range of transcriptional signatures among individuals, highlighting the heterogeneity in PBMC profiles. Nevertheless, striking concordance between three heterogeneous groups was observed across B cells and T cells. Differential gene regulatory mechanisms partially explain these profiles, notably including a Crohn’s disease-specific signature involving TGFß signaling in two individuals. A mutation mapped to a transcription factor binding site within a differentially accessible peak associated with the expression of this pathway, with implications fora personalized approach to understanding disease pathology. This study highlights how multiomic analysis can reveal common regulatory mechanisms that underlie heterogeneity of PBMC profiles, one of which may be specific to inflammatory disease.

overall_design: To investigate the heterogeneity of gene regulation in Crohn's disease and healthy  PBMC using multiome snRNA-seq and snATAC-seq.

first: Margaret

last: Brown

first: Anne

last: Dodd

first: Fang

last: Shi

first: Emily

last: Greenwood

first: Sini

last: Nagpal

first: Subra

last: Kugathasan


title: FUST1 is a heat-responsive switch for global translation in plants

city: Beijing

first: Xiaofeng

last: Fang

address: Hai dian

country: China

institute: Tsinghua University

summary: Adequate reprogramming of global translation under stress is of critical importance to cell survival in eukaryotes. The phosphorylation of eukaryotic initiation factor-2α is a major pathway for stress-induced translational arrest in animals. Here we report that eIF2α phosphorylation is not induced by heat in Arabidopsis. Instead, we identify an uncharacterized protein, FUST1, that can directly sense heat and initiate translational shutdown in Arabidopsis. FUST1 exhibits heat-dependent condensation both in vivo and in vitro, which is mainly driven by its prion-like domain (PrLD). Molecular dynamic simulation reveals that PrLD undergoes conformational rearrangements and engages more inter-amino acid interactions as temperature increases. Mutations that block this conformational change also diminish FUST1 condensation in vitro and in vivo and impair heat tolerance. FUST1 condensates preferentially partition mRNAs of greater length via electrostatic interactions, recruit translation repressors and RNA decapping and deadenylation factors. Importantly, disruption of FUST1 condensation dramatically compromises translational arrest under heat. As a result, FUST1 condensation precedes and is necessary for the assembly of heat stress granules. These findings thus uncover FUST1 as a molecular switch for translation under heat stress and shed light on engineering of heat-adaptable crops.

overall_design: Ten-day-old Arabidopsis seedlings with genotype Col-0 and fust1 were either treated with physiological temperature (22 ℃), or subjected to heatshock (37 ℃). For each genotype-temperature combination, 2 biological replicates were profiled with RNA-seq and Ribo-seq.

first: Changxuan

last: Li


title: Transcriptomic and morphological maturation of human astrocytes in cerebral organoids

city: Amsterdam

first: Marloes

last: Verkerke

address: De Boelelaan 1108

institute: Amsterdam UMC

summary: Cerebral organoids (CerOrgs) derived from human induced pluripotent stem cells (iPSCs) are a valuable tool to study human astrocytes and their interaction with neurons and microglia. The timeline of astrocyte development and maturation in this model is currently unknown and this limits the value and applicability of the model. Therefore, we generated CerOrgs from three healthy individuals and assessed astrocyte maturation after 5, 11, 19, and 37 weeks in culture. At these four time points, the astrocyte lineage was isolated based on the expression of integrin subunit alpha 6 (ITGA6). Based on the transcriptome of the isolated ITGA6-positive cells, astrocyte development started between 5 and 11 weeks in culture and astrocyte maturation commenced after 11 weeks in culture. After 19 weeks in culture, the ITGA6-positive astrocytes had the highest expression of human mature astrocyte genes, and the predicted functional properties were related to brain homeostasis. After 37 weeks in culture, a subpopulation of ITGA6-negative astrocytes appeared highlighting the heterogeneity within the astrocytes. The morphology shifted from an elongated progenitor-like morphology to the typical bushy astrocyte morphology. Based on the morphological properties, predicted functional properties, and the similarities with the human mature astrocyte transcriptome, we concluded that ITGA6+ astrocytes have developed optimally in 19-week-old CerOrgs.

overall_design: Cerebral organoids were genererated from 3 healthy iPSC lines (iPSC1, iPSC2, iPSC4). After 5, 11, 19, and 37 weeks in culture, the ITGA6 positive cells, the ITGA6 negative cells, and the complete organoid were processed for bulk RNAseq.

first: Amber

last: Berdenis van Berlekom

first: Vanessa

last: Donega

first: Daniëlle

last: Vonk

first: Jacqueline

last: Sluijs

first: Nayab

middle: F

last: Butt

first: Lois

last: Kistemaker

first: Lot

middle: D

last: de Witte

first: R

middle: J

last: Pasterkamp

first: Jinte

last: Middeldorp

first: Elly

last: Hol


title: Harnessing HetHydrogel: An Approach to Single-Cell Multi-Omics

city: shanghai

first: Guoqiang

last: Zhou

address: Fudan University, Shanghai, 200438, China

institute: Fudan University School of Life Sciences

summary: Developing a single-cell multi-omics technology with mtDNA mutation profiling at its core will enable precise determination of the relationship between mtDNA mutations and cellular phenotypes. To date, the development of permeability-engineered compartmentalization for the isolation and single-cell library preparation of individual cells, which includes Tn5 tagmentation and single-cell barcoding operations in the permeabilized microcapsule format, remains a formidable challenge. Herein, we present a approach for single-cell mitochondrial analysis, harnessing microfluidics to generate heterogeneous hydrogels as artificial membranes. These hydrogels serve to regulate the passage of mitochondrial DNA and nuclear genomes subsequent to the cell lysis step. This approach enables high-throughput mitochondrial DNA genotyping and accessible chromatin profiling at the single-cell level, significantly enhancing the capabilities of permeability-engineered compartmentalization.We conducted a comprehensive evaluation of cell retention and the selective permeability of the capsule reaction system, optimizing conditions for cell lysis, Tn5 tagmentation, and barcode labeling. Validation experiments were performed using Human 293T and Mouse 3T3 cells to assess identification and quantification performance. Our method offers a new strategy for the study of the functions of various mtDNA mutations in biological processes, while also offering a new system for the construction of single-cell multi-omics libraries.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: To assess the impact of different lysis conditions on sequencing data, we performed Tn5 tagmentation-based library preparation on 293T cells encapsulated in hydrogel using fragmentation buffers 1-4.To investigate single-cell library preparation within hydrogel matrices, we conducted scATAC-seq experiments on intact 293T cells and intact 3T3 cells encapsulated in hydrogels.

first: Ting

first: Jingjing

last: Du

first: Mengying

last: Wu

first: Weilin

last: Pu

first: Jingwei

last: Zhang

first: Zhenglong

last: Gu


title: Foxo1 Drives the TGFβ1-Dependent Dichotomy of Th17 Cell Fates

city: TianJin

first: Mengjuan

state: TianJin

address: No.94 Weijin Road, Nankai District, Tianjin, P. R.China

institute: Nankai University

summary: T-helper 17 (Th17) cells play a dual role in immunological responses, serving as essential components in tissue homeostasis and host defense against microbial pathogens, while also contributing to proinflammatory conditions and autoimmunity. While Transforming Growth Factor-beta 1 (TGFβ1) is pivotal for the differentiation of non-pathogenic Th17 cells, the role of TGFβ3 and Activin in steering Th17 cells toward a pathogenic phenotype has been acknowledged. However, the molecular mechanisms governing this dichotomy remain elusive. In this study, we demonstrate that the transcription factor Foxo1 is upregulated in a TGFβ1 dose-dependent manner, serving as a critical regulator that specifically modulates the fate of pathogenic Th17 cells. Analyses in both uveitis patients and an Experimental Autoimmune Uveitis (EAU) mouse model reveal a strong correlation between disease severity and diminished Foxo1 expression levels. Ectopic expression of Foxo1 selectively attenuates IL-17A production under pathogenic Th17-inducing conditions. Moreover, enhanced Foxo1 expression, triggered by TGFβ1 signaling, is implicated in fatty acid metabolism pathways that favor non-pathogenic Th17 differentiation. Our drug screening identifies several FDA-approved compounds capable of upregulating Foxo1. Collectively, our findings offer compelling evidence that Foxo1 serves as a molecular switch to specifically control pathogenic versus non-pathogenic Th17 differentiation in a TGFβ1-dependent manner. These insights suggest that targeting Foxo1 could be a promising therapeutic strategy for autoimmune diseases, offering efficacy without compromising immune homeostasis.

overall_design: To investigate the gene expreesion of differentiated pathogenic Th17 and non-pathogenic Th17, We collect RNA and construct library

first: Yude

last: Guan

first: Meijuan

last: Han

first: Zhihui


title: In depth mechanistic analysis including high-throughput RNA sequencing in the prediction of functional and structural cardiotoxicants using hiPSC cardiomyocytes

city: Göttingen

first: Christopher

last: Bruhn

address: Marie-Curie-Str. 7

country: Germany

institute: Evotec International GmbH

summary:
Background: Cardiotoxicity remains as one of the most reported adverse drug reactions that lead to drug attrition during pre-clinical and clinical drug development. Drug-induced cardiotoxicity can affect all components of the cardiovascular system and may develop as a functional change in cardiac electrophysiology (acute alteration of the mechanical function of the myocardium) and/or as a structural change, resulting in loss of viability and morphological damage to the cardiac tissue.
Research design and methods: Non-clinical models with better predictive value need to be established to improve cardiac safety pharmacology. To this end, high-throughput RNA sequencing (ScreenSeqTM) combined with high-content imaging (HCI) and Ca2+ transience (CaT) was used to analyse compound-treated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).
Results: Analysis of hiPSC-CMs treated with 33 cardiotoxicants and 9 non-cardiotoxicants of mixed therapeutic indications facilitated compound clustering by mechanism of action, scoring of pathway activities related to cardiomyocyte contractility, mitochondrial integrity, metabolic state and diverse stress responses, and the prediction of cardiotoxicity risk. Combination of ScreenSeqTM, HCI and CaT, provided a high cardiotoxicity prediction performance with 89% specificity, 91% sensitivity and 90% accuracy.
Conclusions: Overall, this study introduces a mechanism driven risk assessment approach combining structural, functional and molecular high-throughput methods for the pre-clinical risk assessment of novel compounds.

overall_design: human iPSC-derived cardiomyocytes (hiPSC-CMs) were cultured on 384 well plates and treated with 42 different compounds at 8 concentrations per compound in triplicate for 24h. DMSO wells were included as treatment controls. We applied plate RNA-Seq analysis with well barcode labelling (ScreenSeqTM). Barcodes were stripped from the sequencing files, and one sequencing file per well is provided.

first: Alicia

last: Rosell-Hidalgo

first: Emma

last: Shardlow

first: Ryan

last: Barton

first: Stephanie

last: Ryder

first: Timur

last: Samatov

first: Alexandra

last: Hackmann

first: Gerald

middle: R

last: Aquino

first: Micael

last: Fernandes dos Reis

first: Vladimir

last: Galatenko

first: Ruediger

last: Fritsch

first: Cord

last: Dohrmann

first: Paul

last: Walker


title: Gene expression profile of the impact of Metformin on the gingiva for periodontal disease prevention

city: London

first: Vitor

last: Neves

state: London

address: Great Maze Pond, Floor 17, Guy's Hospital

department: Centre for Host-Microbiome Interactions

country: United Kingdom

institute: King's College London

summary: We used scRNAseq (Mouse) to understand the impact of Metformin on the gingival tissues

overall_design: Gingival cells were isolated from mice by filtering and analysed by scRNAseq.

first: Athul

last: Menon Kallayil


title: Isovalerylspiramycin I suppresses small cell lung cancer growth via ROS-mediated DNA damage and ER Stress

first: yongle

last: xu

state: shanghai

address: shanghaijiaotong university

institute: shanghaijiaotong university

summary: Characterized by rapid progression, early metastasis, and poor prognosis, small cell lung cancer (SCLC) is a highly malignant tumor with limited treatment breakthroughs for nearly 30 years. Exploring alternative approaches is urgent. Tumor cells are more susceptible to ROS triggers for they have a fragile redox balance compared to normal cells. Isovalerylspiramycin I (ISP-I)， purified from a novel macrolide antibiotic carrimycin， inducing accumulation of ROS in several tumor cells, has shown impressive anti-tumor potential. While the specific details and mechanisms of ISP-I's anti-SCLC effects remain unrevealed. We demonstrated that, ISP-I inhibited SCLC growth dose-dependently in vitro and in vivo, with good safety profiles for showing low toxicity to normal cells (MRC-5) and mice at therapeutic doses. Then, we utilized SCLC cell lines H1048 and DMS53 to uncover the anti-SCLC mechanisms of ISP-I. Primarily, ISP-I acted as an inducer of ROS, leading to H1048 and DMS53 cells DNA damage and endoplasmic reticulum (ER) stress through ROS accumulation， ultimately resulting in tumor cells G2/M cell cycle arrest and apoptosis. In conclusion, our study has proven that ISP-I may be a promising therapeutic approach for SCLC with good safety profiles.

overall_design: Total RNA from ISP-I (10μM) or DMSO (control) treated SCLC H1048 cells was extracted.And we usd the RNA to analyze the  differentially expressed genes between the treatment groups and the control groups. Both groups were processed in 3 biological replicates.

first: Yongle

last: Xu


title: ChIP-Seq to identify the enrichement of histone acetylation on the promoter of FASN and ACLY in tumors with BCAA dietary interventions.

city: 陕西省

first: hao

last: wang

address: 长乐西路

institute: xijing hospital

summary: In order to provide more evidence to prove that BCAAs catabolism mediates the expressions of FASN and ACLY by altering histone acetylation in melanoma, ChIP-seq of xenografted A2058 tumors implanted in mice receiving BCAA dietary interventions (normal or high BCAA diet) was employed to idenfity the enrichment of histone acetylation on the promoter of FASN and ACLY.

overall_design: Chromatin immunoprecipitation sequencing(ChIP-seq) for H3ac in  A2058 tumors

first: Chunying

last: Li

first: Weinan

last: Guo

first: Yangzi

last: Tian

first: Jingjing

last: Ma


title: Resolution of Cystic Fibrosis Lung Disease and Inflammation Driven by Mucus Obstruction with Resolvin D1

city: Chieti

first: Domenico

last: Mattoscio

address: Via Luigi Polacchi, 11

country: Italy

institute: University of Chieti

summary: Progressive lung disease remains the major cause of morbidity and mortality of people with cystic fibrosis (CF). CF lung disease evolves from mucus obstruction into non-resolving airway inflammation, bronchiectasis, and damage that impairs respiratory and fitness activity. Therefore, therapeutic strategies that promote resolution of airway inflammation in CF to alleviate the burden of airflow obstruction and improve physical capacity are of wide interest. Here, we report that the proresolving lipid mediator resolvin (Rv) D1 halts mucus-driven inflammation in CF human cells and in vivo. RvD1 dampened migration and pathogenic phenotypes of neutrophils from volunteers with CF as well as inflammatory signaling pathways of CF bronchial epithelial cells triggered by CF mucus. In mice overexpressing the β-subunit of the epithelial Na+ channel (βENaC) RvD1 administration prevented and reverted lung inflammation caused by mucus accumulation and promoted the resolution of pulmonary exacerbation caused by P. aeruginosa. RvD1 also significantly improved physical activity and energy expenditure that are impaired in βENaC mice compared to wild type littermates. These findings demonstrate efficacy of RvD1 in enhancing resolution of lung disease and chronic inflammation associated with mucus obstruction and provide proof of concept for its potential therapeutic approach in FC.

overall_design: To investigate the impact of RvD1 in mucus-derived inflammation in cystic fibrosis models, we carried out RNASeq analyses to evaluate global changes in transcriptome of cystic fibrosis primary epithelial cells CFBEC exposed to airway supernatant (ASN) and vehicle or RvD1. We performed gene expression profiling of CFBEC from three biological replicates, treated with vehicle or RvD1. Comparative gene expression analyses among vehicle- and RvD1-exposed CFBEC

first: G

last: Ferri

first: M

last: Mucci

first: D

first: A

last: Recchiuti


title: Luciferase Expressing Preclinical Model Systems Representing the Different Molecular Subtypes of Colorectal Cancer

city: Halle (Saale)

first: Martin

middle: Sebastian

last: Staege

state: Sachsen-Anhalt

address: Ernst-Grube-Str. 40

department: Universitätsklinik u. Poliklinik für Kinder- u. Jugendmedizin

summary: We established combined 2D/3D, in vitro/in vivo model systems representing the heterogeneity of colorectal cancer (CRC) with regards to different molecular subtypes. Comparative characterization of stable luciferase expressing derivatives of well-established CRC cell lines, derived spheroids and subcutaneous xenograft tumors showed that the 3D-spheroid cultures resembled xenografts more closely than 2D-cultured cells do. The model systems can be used in preclinical research applications to study new therapy approaches and represents the biological heterogeneity of CRC.

overall_design: Luciferase transgenic colorectal cancer cell lines were grown as monolayers, spheroids or xenografts. RNA was extracted and microarray analysis was performed.

first: Arne

last: Rothemund

first: Sarah

last: Brandt

first: Jana

last: Luetzkendorf

first: Henrike

last: Lucas

first: Lutz

middle: P

last: Mueller

first: Thomas


title: RNA-seq and scRNA-seq analysis of the efficient generation of functional human hepatocytes from chemically induced pluripotent stem cells

first: Ziyan

last: Rao

address: 38 Xueyuan Rd, Haidian District

institute: Peking University Health Science Center

summary: Here, we developed a new differentiation protocol by mimicking the two-stage development of hepatoblasts to potentiate their transition to hepatic progenitor cells (hiHPCs), which permits the high-efficiency generation of hiHPCs from chemically induced pluripotent stem cells (hCiPSCs).  Moreover, hCiPSC-derived hiHPCs can further differentiate into mature hepatocytes (hiHEPs).  We use single-cell RNA sequencing (scRNA-seq) to analyze the two-stage of hepatoblasts indicating the similarity between hepatoblasts differentiated in vitro and in vivo.  In addition, we show that the global gene expression profile of hiHPCs was indistinguishable from that of human fetal liver cells (hFLCs) and hCiPSC-derived hiHEPs resembled F-PHHs by using RNA sequencing (RNA-seq).

overall_design: To dissect the two-stage transition of hepatoblasts, the process was analyzed by scRNA-seq. Moreover, hCiPSC-derived hiHPCs and hiHEPs are similar to hFLCs and F-PHHs respectively by RNA-seq.

first: Yun

last: Lv

first: Lulu

last: Liu

first: Jun

last: Jia

first: Dongyu

last: Zhao

first: Hongkui

last: Deng


title: Functional interrogation of twenty Type 2 Diabetes-associated genes using isogenic hESC-derived β-like cells (SNP chip)

city: New York

first: Shuibing

last: Chen

address: A 827B, 1300 York Ave

department: Surgery

institute: Weill Cornell Medical College

summary: Genetic studies have identified numerous loci associated with type 2 diabetes (T2D), but the functional role of many loci has remained unexplored. In this study, we engineered isogenic knockout human embryonic stem cell (hESC) lines for 20 genes associated with T2D risk. We systematically examined β-cell differentiation, insulin production and secretion, and survival. We performed RNA-seq and ATAC-seq on hESC-β cells from each knockout line. Analyses of T2D GWAS signals overlapping with HNF4A-dependent ATAC peaks identified a specific SNP as a likely causal variant. In addition, we performed integrative association analyses and identified four genes (CP, RNASE1, PCSK1N and GSTA2) associated with insulin production, and two genes (TAGLN3 and DHRS2) associated with sensitivity to lipotoxicity. Finally, we leveraged deep ATAC-seq read coverage to assess allele-specific imbalance at variants heterozygous in the parental hESC line, to identify a single likely functional variant at each of 23 T2D GWAS signals.

overall_design: We genotyped the parental MEL1 hESC lines and generated imputed genotypes of all SNPs (r2>0.3) included in the TOPmed panel for the  ATAC-seq allelic imbalance analysis.

first: Dongxiang

last: Xue

first: Narisu

last: Narisu

middle: L

last: Taylor

first: Meili

last: Zhang

first: Caleb

last: Grenko

first: Tingfen

last: Yan

first: Neelam

last: Sinha

first: Michael

last: Erdos

first: Lori

last: Bonnycastle

first: Francis

last: Collins


title: The axolotl kidney: a novel model to study kidney regeneration

city: Guangzhou

first: Liting

state: Guangdong

address: 1838 North Guangzhou Blvd.

department: Division of Nephrology

institute: Nanfang Hospital, Southern Medical University

summary: To investigate whether axolotl could serve as a novel model organism for the study of kidney regeneration after severe renal injury. We established tubular injury model by intraperitoneal injection of gentamicin. We then performed gene expression profiling analysis using data obtained from RNA-seq of 5 different samples from Control or gentamicin-induced injury groups.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for 5 different axolotls from Ctrl or gentamicin-induced injury groups.

first: Jing

last: Li

first: Yanping

last: Ou

first: Meixia

last: Kang


title: Transcriptome analysis in brown adipose tissue of mice lacking the mitochondrial protein optic atrophy 1 (OPA1) in thermogenic adipocytes

city: Iowa City

first: Renata

middle: O

last: Pereira

state: Iowa

address: 169 Newton Rd

department: Internal Medicine

institute: University of Iowa

summary: mRNA expression profiles from interscapular brown adipose (iBAT) tissue of 7-week-old OPA1 BAT KO mice and their WT counterparts.

overall_design: Samples were collected from ad libitum fed mice, reared at room. mRNA was extracted from iBAT of 7-week-old male and female OPA1 BAT KO mice and their WT counterparts.

first: Eric

middle: T

last: Weatherford


title: Xenograft mouse model to investigate tropism to the CNS and retina in primary central nervous system lymphoma

city: Freiburg

first: Geoffroy

last: Andrieux

address: Breisacherstr 153

institute: University clinics Freiburg

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

overall_design: Refer to individual Series


title: Functional interrogation of twenty Type 2 Diabetes-associated genes using isogenic hESC-derived β-like cells

first: Shuibing

last: Chen


title: A role for miR-4734 in conditional suppression of ER stress-associated pro-inflammatory responses

city: Rehovot

first: Ester

last: Feldmesser

state: Rehovot

address: POB 25

department: Life Science Core Facilities

country: Israel

institute: Weizmann Institute


title: A role for miR-4734 in conditional suppression of ER stress-associated pro-inflammatory responses [miR-CTRL or miR-4734-transfected HFF]

summary: Prolonged metabolic stress can lead to severe pathologies. In metabolically challenged primary fibroblasts, we assigned a novel role for the poorly characterized miR-4734 in restricting ATF4 and IRE1-mediated upregulation of a set of pro-inflammatory cytokines and endoplasmic reticulum (ER) stress-associated genes. Conversely, inhibition of this miRNA augmented the expression of those genes. Mechanistically, miR-4734 was found to restrict the expression of NFKBIZ, that is required for optimal expression of the pro-inflammatory genes and whose mRNA is targeted directly by miR-4734. Concordantly, overexpression of NFKBIZ compromised the effects of miR-4734, underscoring the importance of this direct targeting. As the effects of this miR-4734 were evident under stress, but not under basal conditions, it may be considered as a therapeutic agent.

overall_design: For the mRNA expression array analysis, total RNA derived from miR-CTRL or miR-4734-transfected HFF were used.

first: Dan

last: Michael

first: Alexander

last: Maman

first: Moshe

last: Oren


title: RNA-seq Transcriptomic Analysis of 4-octyl itaconate Repressing Myogenic Differentiation

city: Jinan

first: Dawei

last: Wang

state: Shandong

address: No. 324, Road Jing Wu Wei Qi

institute: Shandong Provincial Hospital Affiliated to Shandong First Medical University

summary: The 4-octyl itaconate O1_is a type of cell-permeable itaconate derivative. Studies have shown that with an anti-fibrotic effect in systemic sclerosis, the OI also affects osteoclast differentiation. The aim of this study was to explore the molecular mechanisms underlying the effects of OI on myoblast differentiation by RNA-seq analysis.

overall_design: Comparative gene expression profiling of RNA-seq data for  4-octyl itaconate inhibition of myogenic differentiation in C2C12 cells.

first: Lili

first: Zheng

first: Yu

last: Feng

first: Xiaohui

last: Bai

first: Weihua

last: Liu


title: A role for miR-4734 in conditional suppression of ER stress-associated pro-inflammatory responses [starved vs control HFF]

overall_design: For the mRNA expression array analysis (starved vs control HFF), total RNA derived from either control, 16 or 48 hours starved cells, each from 3 biological repeats, was used.

first: Chagay

last: Gonen

first: Noa

last: Furth

first: Gilgi

last: Friedlander


title: Fisetin suppresses colorectal cancer growth through AKAP12 in the patient-derived organoid xenograft model

city: Seoul

first: Nayun

last: Kim

address: 33, Samyang-ro 144-gil, Dobong-gu, Seoul, Republic of Korea

country: South Korea

institute: Duksung women's university

summary: To investigate the genetic changes induced by fisetin treatment, we established a patient-derived organoid xenograft model (PDOX) of the control group and the group treated with fisetin 200mpk. We then performed gene expression profiling analysis using data from RNA-seq from both groups.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for 33T organoids and fisetin-treated 33T organoids.

first: Joohee

last: Jung


title: Long noncoding RNA NORAD promotes epithelial mesenchymal transition progression in lung adenocarcinoma

city: Wuhan

first: Zhe

last: Dong

state: Hubei

address: Shui guo hu

institute: Zhongnan Hospital of Wuhan University

summary: Non-Coding RNA Activated By DNA Damage (NORAD, also known as LINC00657) is a newly discovered long noncoding RNA and emerging evidences have reported that NORAD had an essential role in various cancers. However, it is not clear about its role in lung adenocarcinoma (LUAD). This study mainly aims to explore the role of NORAD in the occurrence and development of LUAD. In our study, an obvious high expression of NORAD in LUAD was exhibited and we found a strongly positive association between NORAD and epithelial mesenchymal transition (EMT) progression. Moreover, we further predicted that NORAD might upregulate GEMIN2 expression to induce EMT progression in LUAD by acting as a ceRNA to sponge four microRNAs including miR-7-1, miR-33a, miR-590 and miR-4668. In sum, our study demonstrated that NORAD was overexpressed in LUAD and could promote the progression of EMT.

overall_design: To reveal the mechanism of NORAD promoting EMT in lung adenocarcinoma, we performed genetic silence of NORAD in A549 cell line using si-NORAD and examined the effects of NORAD silence on invasion and migration of A549 cells. mRNA profiles of control and si-NORAD cells were generated by deep sequencing, using Illumina HiSeq 4000. In total, six samples were sequenced.

first: Hongjie

last: Shi


title: MiRNA-486-5p Protects Against Kidney Ischemic Injury and Modifies the Transcriptome in Proximal Tubule and Endothelium

city: Ottawa

first: Theodore

last: Perkins

state: ON

address: 501 Smyth Rd.

department: Cellular and Molecular Medicine

country: Canada

institute: Ottawa Hospital Research Institute

summary: Background: Acute kidney injury (AKI) is a common clinical event with high morbidity and mortality. We previously demonstrated that injection of cord blood endothelial colony forming cell (ECFC)-derived exosomes, highly enriched in miRNA(miR)-486-5p, prevented ischemic kidney injury in mice. While preclinical models support involvement of several miRs in AKI, the direct effects of miR-486-5p on injury and the kidney transcriptome are unknown.   Objective: We studied effects of miR-486-5p in mice with kidney ischemia-reperfusion (IR) injury, and compared the impact of miR-486-5p and ECFC-derived exosomes on the transcriptome of proximal tubules (PTs) and renal endothelial cells.  Methods: Adult male mice were subjected to bilateral kidney ischemia (30 min), and injected via tail vein with or without vehicle, miR-486-5p mimic, ECFC-derived exosomes, or scramble miR at the start of reperfusion. Plasma and tissues were collected 24 hrs after reperfusion, and proximal tubular and endothelial cells were isolated for mRNA analysis by RNA-Seq.   Results: In mice with kidney IR, injection of miR-486-5p mimic increased levels of miR-486-5p in proximal tubules and endothelial cells, and significantly improved plasma creatinine, histological injury, neutrophil infiltration, and apoptosis, compared to vehicle treatment. MiR-486-5p inhibited expression of phosphatase and tensin homolog (PTEN), and activated AKT. Similar results were observed with exosomes, but scramble miR had no effect. In proximal tubules, miR-486-5p or exosomes reduced expression of several AKI genes (e.g. Lcn2, Havcr1 and Krt20) and caused alterations in apoptotic genes compared to IR alone. In endothelium, miR-486-5p or exosomes caused milder effect than in PTs but still impacting on expression of injury-related genes.   Conclusion: MiR-486-5p protects mice against ischemic kidney injury. Both miR-486-5p and exosomes reduce expression of apoptotic genes in PTs and endothelium. The results suggest that systemic delivery of miR-486-5p has therapeutic potential in ischemic AKI.

overall_design: Transcriptomic profile of endothelial cells from control (sham), acute kidney injury (AKI), acute kidney injury treated with miR-486-5p mimic (AKI+mimic),acute kidney injury treated with exosomes (AKI+ EXO) and acute kidney injury treated with scrambled miRNA (AKI+SCB)  FVB male mice (n=3 except for AKI+ mimic n=2) The experiment also includes the same experimental groups with samples of proximal tubular cells from sham male mice (n=3), AKI male mice(n=3), AKI+ mimic male mice(n=2) , AKI+ EXO male mice (n=3), and AKI+ SCB male mice (n=3).

first: Jose

last: Vinas

first: Matthew

last: Spence

first: Christopher

last: Porter

first: Adrianna

last: Douvris

first: Alex

last: Gutsol

first: Joseph

last: Zimpelmann

first: Pearl

last: Campbell

first: Kevin

last: Burns


title: RNA sequence (RNA-seq) profiling of SynXI strain

status: Public on Oct 09 2023

first: Jitong

last: Cai

state: NY

address: 435 E 30th street

department: NYU Langone Health

institute: NYU Langone Health

summary: We describe construction of the 660 kilobase synthetic yeast chromosome XI (synXI) and reveal how synthetic redesign of non-coding DNA elements impact the cell. To aid construction from synthesized 5 to 10 kilobase DNA fragments, we implemented CRISPR-based methods for synthetic crossovers in vivo and used these methods in an extensive process of bug discovery, redesign and chromosome repair, including for the precise removal of 200 kilobases of unexpected repeated sequence. In synXI, the underlying causes of several fitness defects were identified as modifications to non-coding DNA, including defects related to centromere function and mitochondrial activity that were subsequently corrected. As part of synthetic yeast chromosome design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show here that targeted insertion of these sites can be used to create extrachromosomal circular DNA on demand, allowing direct study of the effects and propagation of these important molecules. Construction and characterization of synXI has uncovered effects of non-coding and extrachromosomal circular DNA, contributing to better understanding of these elements and informing future synthetic genome design.

overall_design: We used RNAseq analysis to compare the transcriptional profiles of the parental strain to the initial synXI assembly strain ysXIb01 and to ysXIb16, which contains the debugged synXI_9.11. We selected BY4742 as our parental control as its auxotrophic profile most closely matched the synXI strains. For the purposes of our analysis, we eliminated mating-type specific genes to compensate for mating type differences between the parental and synXI strains. This included transcripts of the dubious ORF YKL177W as it almost entirely overlaps STE3, which encodes the receptor for a factor pheromone in MATα cells. The CDS of FLO10 was entirely recoded by an early version of REPEATSMASHER due to the presence of highly repetitive sequence (Richardson et al., 2017).

first: Benjamin

last: Blount

first: Tom

last: Ellis


title: Microprotein knockout in rescue cell lines and control conditions

city: Solna

first: Simon

last: Elsässer

address: Tomtebodavägen 23A

department: Medical Biochemistry and Biophysics

country: Sweden

institute: Karolinska Institutet

summary: RNA-seq sequencing was carried out to investigate which expression alterations were induced by treatment with sgRNAs targeting the sORFs/microproteins of interest. To test   whether any of these changes could be reversed in the microprotein rescue-GFP fusion cells, transcriptional alterations of rescue and parental cells were compared.

overall_design: RNA-Seq was performed in triplicates in cell lines derived from A375. All cell lines carried a constitutively expressed Cas9-T2A-BSD-TagBFP transgene (addgene #196714) and rescue cells additionally contained a GFP-tagged codon-altered microprotein transgene. A375 Cas9 control and microprotein rescue cell lines were treated with either sgRNA targeting the respective microprotein or targeting a control locus (TRAC) before RNA extraction.

first: Dörte

last: Schlesinger

first: Carmen

last: Navarro


title: The coenzyme A precursor pantethine restrains sarcoma growth through promotion of type 1 immunity

city: Marseille

first: Romain

last: Fenouil

address: Parc Scientifique et Technologique de Luminy - Case 906

department: CIML

institute: CNRS

summary: The tumor microenvironment is a dynamic network of stromal, cancer and immune cells that interact and compete for resources. Mitochondria play an essential role in the control of metabolic plasticity and contribute to tumor progression and immune cell functionality. We previously identified the Vanin1 pathway as a tumor suppressor of sarcoma development via vitamin B5 and coenzyme A regeneration. Using an aggressive sarcoma cell line that lacks Vnn1 expression, we showed that administration of pantethine, a vitamin B5 precursor, impairs tumor growth in immunocompetent mice. Pantethine boosts anti-tumor type 1 immunity including polarization of myeloid and dendritic cells towards enhanced IFNγ-driven antigen presentation pathways and improved development of hypermetabolic effector CD8+ T cells endowed with potential anti-tumor activity. At later stages of treatment, the effect of pantethine is limited by the development of immune cell exhaustion. Nevertheless, its activity is comparable to that of anti-PD1 treatment in sensitive tumors. In humans, VNN1 expression correlates with improved survival and immune cell infiltration in soft tissue sarcomas but not osteosarcomas. Pantethine could be a potential therapeutic immunoadjuvant for the development of anti-tumor immunity.

overall_design: 1 subcutaneous MCA205 tumor from D20 and 1 tumor from D28 from PBS- or Pant-treated C57BL/6 mice were harvested, dissociated and processed as mentioned in the extract protocol section. CD45+ cells were enriched by cell-sorting and each condition where discriminated by HTO staining with TotalSeqB before beeing pooled for cDNA libraries construction and sequencing

first: Richard

last: MIALLOT

first: Virginie

last: MILLET

first: Anais

last: ROGER

last: FENOUIL

first: Catherine

last: TARDIVEL

first: Jean-Charles

last: MARTIN

first: Laetitia

last: SHINTU

first: Paul

last: BERCHARD

first: Juliane

last: SOUSA LANZA

first: Bernard

last: MALISSEN

first: Sandrine

last: HENRI

first: Sophie

last: UGOLINI

first: Aurélie

last: DUTOUR

first: Pascal

last: FINETTI

first: François

last: BERTUCCI

first: Jean-Yves

last: BLAY

first: Franck

last: GALLAND

first: Philippe

last: NAQUET


title: Transcriptomic profiling of transplant glomerulopathy to reveal a novel T-cell dominant subclass

city: Rotterdam

first: Iacopo

last: Cristoferi

state: Zuid-Holland

address: Doctor Molewaterplein, 40

institute: Erasmus MC

summary: Kidney transplant biopsies showing transplant glomerulopathy (cg > 0) and microvascular inflammation (MVI) in the absence of C4d staining and DSAs do not fulfill the criteria for chronic active antibody-mediated rejection (CA-AMR) diagnosis or any other Banff category. In this multicenter intercontinental study including 36 cases, we compared, among other types of data, the transcriptomic profiles of 14 KTx biopsies classified as cg+MVI DSA-/C4d- with 22 classified as CA-AMR DSA+/C4d+ through novel transcriptomic analysis using the NanoString B-HOT panel. Due to lack of tissue, one sample was excluded from the transcriptomic analysis. In our analysis, nineteen genes were differentially expressed between the two study groups. Samples diagnosed with CA-AMR DSA+/C4d+ showed a higher transcriptomic cell type scores for macrophages in an environment characterized by increased expression of complement-related genes (i.e., C5AR1) and higher activity of angiogenesis, IFTA, CA-AMR, and DSA-related pathways when compared to samples diagnosed with cg+MVI DSA-/C4d-. Samples diagnosed with cg+MVI DSA-/C4d- displayed a higher activity of the T-cell receptor and B-cell associated transcripts. These results were coherent with those from our 5-plex immunofluorescence orthogonal analyses showing higher abundance of innate immune cells in the interstitium of CA-AMR DSA+/C4d+ samples when compared to cg+MVI DSA-/C4d- samples and a higher glomerular abundance of pan-T-cells in cg+MVI DSA-/C4d- samples when compared to CA-AMR DSA+/C4d+ samples. Here we show that using novel multiomic techniques, KTx biopsies with cg+MVI DSA-/C4d- have a prominent T-cell presence and activity, putting forward the possibility that these represent a more T-cell-dominant phenotype.

overall_design: This retrospective multicenter intercontinental study included a highly selected population of KTx recipients. KTx biopsy samples were retrieved from the archives of the Department of Pathology, ErasmusMC, Rotterdam, the Netherlands (ErasmusMC-cases, n = 19) and the Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, United States of America (MGH-cases, n = 17). Thirty-six transplant recipients who fulfilled the first two diagnostic criteria for CA-AMR were retrospectively included in this study (n = 36). Twenty-two patients were classified as CA-AMR DSA+/C4d+, as they had positive C4d staining of ptcs (Banff C4d lesion score ≥ 1) and showed circulating DSAs. The remaining 14 patients were classified as cg+MVI DSA-/C4d- as they did not have C4d positive staining of ptcs (Banff C4d lesion score = 0) and had no circulating DSAs. Due to lack of tissue, one sample was excluded from the transcriptomic analysis.

first: Marian

last: Clahsen-van Groningen

first: Dana

last: Mustafa

first: Rex

last: Smith

first: Robert

last: Colvin

first: Ivy

last: Rosales


title: BRAFV600E-induced transcriptional changes in cord blood-derived hematopoietic stem and progenitor cells

city: Graz

first: Tommaso

last: Sconocchia

address: Auenbruggerplatz 38

department: Hematology

country: Austria

institute: Medical University of Graz

summary: BRAFV600E is a recurrent mutation in the histiocytoic disorders Langerhans cell histiocytosis (LCH) and Erdheim Chester Disease (ECD).  The mutation can be detected in committed monocytes/macrophages, dendritic cells, and hematopoietic stem and progenitor cells (HSPCs). Although the cell of origin that gives rise to these disorders is currently unknown, it was recently suggested that LCH and ECD arise from BRAFV600E-mutant HSPCs. To understand the underlying molecular mechanisms in HSPCs driving this phenotype, we introduced BRAFV600E into healthy human HSPCs using CRISPR/Cas9.

overall_design: CD34+ HSPCs isolated from three umbilical cord blood were cultured ex vivo and BRAF wildtype (WT) or BRAFV600E cDNA was introduced via CRISPR/Cas9 and AAV6-mediated knock-in. Cells with correct knock-in and cells without knock-in (RNP) were enriched by FACS based on fluoresecent reporter expression and total RNA was isolated for RNA-seq.

first: Andreas

last: Reinisch


title: Deep mutagenesis reveals the distinct mutational landscape of ADan and ABri amyloid nucleation

city: Barcelona

first: Mariano

last: Martín

address: c/ Baldiri Reixac 10-12

country: Spain

institute: IBEC

summary: Different forms of dementia are caused by stop-loss mutations in the ITM2B gene, also known as Bri2, which result in the expression of 34 amino acid long peptides that accumulate as amyloids in human brains. In order to gather mechanistic insights into the formation of amyloids by two of these peptides, ADan and ABri - hallmarks of Danish and British dementia respectively - we employed saturation mutagenesis combined to a massively parallel selection assay that reports on amyloid nucleation. Our results reveal that ADan aggregates into amyloids remarkably faster than both the unextended peptide Bri2 and the extended ABri sequence. The complete mutational landscape of ADan reveals asparagines and charged residues as key players in the nucleation process in addition to aliphatic residues within positions 20-25. What is more, we show that extending Bri2 with just two specific residues is enough to generate a novel amyloid core which we suggest builds the structured core of ADan fibrils. On the other hand, only a handful of mutations can boost the ability of ABri to nucleate amyloids, including a SNV replacing the Bri2 stop codon by a Cys codon. Overall, the remarkably different aggregation profiles and mutational landscapes for the two peptides suggest that different disease mechanisms underlie disease in Danish and British dementia and highlight the importance of accurately measuring the impact of stop extension mutations for these and other sequences across the genome.

overall_design: Systematic measurement of the nucleation of substitutions in ADan and ABri peptides

first: Benedetta

last: Bolognesi


title: Sclerotinia sclerotiorum responses to symptomatic versus asymptomatic host

city: chongqing

first: binnian

last: tian

address: tianshenglu

institute: binniantian

summary:
To exploite S. sclerotiorum to identify differential fungal responses leading to either an endophytic or a pathogenic lifestyle during colonization of both asymptomatic host and symptomatic host
We then performed gene expression profiling analysis using data obtained from RNA-seq of 9 different samples after 2 days.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for 9 samples.

first: Binnian

last: Tian


title: The acRIP-seq  of SiHa and KONAT10SiHa cells

city: Shenzhen

first: sheng

last: zhong

address: The Xinhu Road 1333

institute: The Sourthern Medical University

summary: We investigate genes that the different degrees of  N4-acetylcytidine in the SiHa and Knockout NAT10 SiHa.

overall_design: We then performed gene expression profiling analysis using data obtained from acRIP-seq of 2 different cells.

first: Xia

last: Guo

first: Xiaona

last: Chen


title: CUT&RUN against H3K27ac and H3K27me3 on WT and TKO ESCs (D0), and D2, D4 EpiLCs

city: paris

first: teissandier

last: aurelie

address: 26 rue d'Ulm

institute: institut curie

summary: We investigate  the impact of DNA methylation on chromatin epigenetic marks during epiblast differentiation. To this end, we used in vitro Epiblast-like cell (EpiLC) differentiation on WT and DNA-methylation free TKO murine ESCs. We next performed CUT&RUN against H3K27me3 and H3K27ac at D0, D2 and D4.

overall_design: Probing H3K27me3 and H3K27ac via Cut&Run in WT and TKO from D0 (ESCs), D2 and D4 EpiLCs

first: Mathieu

last: Schulz

first: Mélanie

last: Armand

first: Aurélie

last: Teissandier

first: Déborah

last: Bourc'his


title: WGBS WT J1 ESC at D3 of EpiLC differentiation

type: Methylation profiling by high throughput sequencing

summary: We investigate the dynamic of DNA methylation on epiblast differentiation. To this end, we used in vitro Epiblast-like cell (EpiLC) differentiation on WT murine ESCs during 3 days. We next performed bisulfite conversion and whole-genome sequecing to assess methylation levels.

overall_design: WGB sequencing on WT D3 cells during EpiLC differentiation

first: Marius

last: Walter


title: Single cell SMART RNAseq on WT and TKO E14 EpiLCs

summary: We investigate how DNA methylation absence impact transcriptionnal regulation during epiblast differentiation. More specifically, how synchronous is the naïve to primed transitioning in TKO cells compared to WT, and if there is a "germline-competent" TKO subpopulation of EpiLCs. To this end, we used in vitro Epiblast-like cell (EpiLC) differentiation WT and TKO at D0, D1, D2 and D4. We next performed single-cell SMARTseq2 RNA sequencing.

overall_design: Comparison of Single Cell RNA sequencing results (SMARTseq2 protocol) results of WT and TKO during EpiLC differentiation (D0, D1, D2, D4)

first: Sarah

last: Kinston

first: Berthold

last: Göttgens


title: Comparison of bulk RNAseq results of WT and TKO E14 EpiLCs

summary: We investigate the impact of DNA methylation on epiblast differentiation. To this end, we used in vitro Epiblast-like cell (EpiLC) differentiation on WT and DNA-methylation free TKO murine ESCs. We next performed bulk RNAseq at D0, D2, D4, D6 and D8 of differentiation to study transcriptomic differences.

overall_design: Bulk mRNA sequencing on WT and TKO ESCs (D0), and D2, D4 and D7 cells during EpiLC differentiation


title: Bulk RNAseq on WT and TKO E14 PGCLCs

overall_design: Comparison of bulk mRNA sequencing results of WT and TKO ESCs (D0), and D2, D4 and D7 cells during PGCLC differentiation


title: Effect of NKD1, NKD2 and O2 on Chromatin Accessible Regions

city: Ithaca

first: Hao

last: Wu

address: 236 Tower Rd, Plant Science Building Room #236

institute: Cornell University

summary: NKD1, 2 and O2 are key transcription factors interactively regulating endosperm development. Their mutation combinations may diversely alter the global gene regulatory landscape. To investigate how interactions between NKD1, 2 and O2 affect endosperm regulatome, which is indicated by accessible regions for regulators on chromatins, Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) was performed for 16 DAP endosperms of all 8 genotype combinations (2 biological replicate per genotype). Among 16 sequenced samples, 20,478 to 49,640 (mean: 34,766) peaks were called, and these peaks represent potential chromatin accessible regions. Distribution of the peaks on chromatins varies among samples: 54.35% - 66.73% peaks are located at distal intergenic region (>3kb upstream and >300 bp downstream of a gene) and 6.7% - 12.1% are located at promoter region (within 3 kb flanking transcription starting site), and 4.62% - 8.88% peaks are located within 1kb flanking transcription starting site. Also, in WT background, nkd1 and nkd2 single mutants have overall greater number of opening regions (upregulated peaks) than closing regions (downregulated peaks), whereas nkd1,2 double mutant has similar number of opening and closing regions. In contrast, in o2-background, all nkd1, nkd2 and nkd1,2 mutants have greater number of closing than opening regions. This indicates that o2 may alter differential accessibility of nkd1, nkd2 single or nkd1,2 double mutants.

overall_design: Frozen 16 DAP endosperms of nkd1, 2 and o2 single, double and triple mutants, as well as the wild type counterpart (two biological replicates each) were collected and the crude nuclei were extracted.  The crude nuclei were stained with 4,6-Diamidino-2-phenylindole (DAPI) for nuclear sorting using a flow cytometer. At least 50,000 triploid nuclei (3C) were sorted for ATAC-Seq library construction. The library was constructed using the Active Motif ATAC-Seq Kit. The library was sequenced using NovaSeq 6000 SP flow cell sequencing platform at 2X50 bp paired-end in 50 million bp depth.

first: Philip

middle: W

last: Becraft


title: Genome wide exon trapping

city: Toronto

first: Nicholas

last: Stepankiw

address: 160 College St. Room 1350

institute: University of Toronto

summary: We report the result of testing the splicing potential of random human genomic sequences. We used randomly fragmented HEK293 DNA in a splicing reporter assay to survey the genome for genomic sequences capable of splicing autonomously as a typical internal exon. From ~10 billion sequenced reads we obtained millions of sequences in the human genome capable of splicing autonomously. A read threshold of 100 yields ~1.25 million exons while also capturing most human mRNA exons.

overall_design: Randomly fragmented HEK293 gDNA was assembled into exon trapping reporter assay plasmids. Reporter assays were transiently transfected in HEK293 cells. RNA was extracted and RT-PCR was performed to isolate trapped exons and attach Illumina sequencing platform sequences. Trapped exons were mapped back to the genome to recover the genomic coordinates, and associated read counts, of exons autonomously trapped from the random HEK293 DNA fragments.

first: Timothy

last: Hughes


title: DAP-seq Analysis of Transcription Factors Associated with Maize Endosperm Development

summary: Transcription factors (TFs) play an important role in maize endosperm development regulation. The temporal RNA-seq and co-expression network analysis of maize endosperm reveals a hiararchical regulatory network architechture modulating the endosperm development. In the network, NKD1 and NKD2 are central regulators, and GBF1, HSFTF10, NACTF49 and HB115 are secondary regulators downstream of NKD1 and 2. To further test the relationship between these TFs, a DNA affinity purification and sequencing (DAP-seq) assay was performed. The result shows that the assay on NKD1 and NKD2 called 1,951 and 56,855 peaks, and 93 and 1,692 peaks are located within 3 kb distance to transcription starting site (TSS), respectively. And assays on GBF1, HSFTF10, NACTF49 and HB115 called 15,543; 38,147; 7,388 and 936 peaks, respectively.

overall_design: The open reading frames (ORFs) of genes of interest are cloned into Gateway entry vector, which are then recombined into the pIX-HALO vector using LR clonase II. Proteins are in vitro expressed using the TnT® SP6 High-Yield Wheat Germ Protein Expression System and immoblized on Magne HaloTag beads. The immobleized proteins then bind to maize B73 genomic DNA library, followed by washing and elution. The eluted DNA is PCR amplified and barcoded for sequencing. The barcoded DNA was sequenced on a NextSeq500 with 75 bp SE reads for GBF1, HSFTF10, NACTF49 and HB115 and on Novaseq with 150 bp PE for NKD1 and 2.

first: Mary

last: Galli

first: Andrea

last: Gallavotti


title: Dominant CD4+ T cell receptors remain stable throughout antiretroviral therapymediated immune restoration in people with HIV

city: Chapel Hill

first: Mark

last: Woodcock

state: NC

address: 170 Manning Drive

institute: University of North Carolina at Chapel Hill

summary: In people with HIV (PWH), the post antiretroviral therapy (ART) window is critical for immune restoration and HIV reservoir stabilization. We employ deep immune profiling, T cell receptor (TCR) sequencing and examine proliferation to assess how ART impacts T cell homeostasis. Hierarchies and frequencies of dominant CD4 TCR clonotypes (0.1-11% of all CD4+ T cells) remain stable post-ART, suggesting that clonal homeostasis can be independent of homeostatic processes regulating CD4+T cell absolute number, phenotypes and function. The slow restoration of host-immunity post-ART also has implications for the design of ART interruption studies.

overall_design: Cohort A5248 were people with HIV who had just initiated antiretroviral therapy and were followed for 504 days. In this group (n=4 participants) we examined TCRβ clonotype diversity in CD4 T cells at 5 timepoints between Day 0 and 504.  Cohort LT-ART were people with HIV who had been durably suppressed on antiretroviral therapy for an average of 6.7 years. In this group (n=3 participants) we examined TCRβ clonotype diversity  in CD4 T cells at 3 timepoints over a 1.5 year window.

first: Alexis

last: Sponaugle

first: Ann

middle: K

last: Weideman

first: Jolene

last: Ranek

first: Gatphan

last: Atassi

first: JoAnn

last: Kuruc

first: Adaora

last: Adimora

first: Nancie

middle: M

last: Archin

first: Cynthia

last: Gay

first: Daniel

last: Kuritzkes

first: David

last: Margolis

first: Benjamin

middle: G

last: Vincent

first: Natalie

last: Stanley

first: Michael

last: Hudgens

first: Joseph

last: Eron

first: Nilu

last: Goonetilleke


title: Comparison of bulk RNAseq results of WT and TKO E14 NPCs

first: teissandier

last: aurelie

summary: We investigate the impact of DNA methylation on neural somatic differentiation. To this end, we used in vitro neural progenitor cell (NPC) differentiation on WT and DNA-methylation free TKO murine ESCs. We next performed bulk RNAseq at D0, D2, D4, D6 and D8 of differentiation to study transcriptomic differences.

overall_design: Bulk mRNA sequencing on WT and TKO ESCs (D0), and D2, D4, D6 and D8 cells during NPC differentiation

first: Mathieu

last: Schulz

first: Elena

last: De la mata santaella

first: Aurélie

last: Teissandier

first: Déborah

last: Bourc'his


title: Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems [RIP-seq]

city: Shenyang

first: Teng

last: Fei

state: Liaoning

address: 195 Chuangxin Rd. Life Sciences Building A426. Hunnan District.

department: College of Life and Health Sciences

institute: Northeastern University, China

summary: CRISPR-Cas13 systems have been adapted as versatile toolkits for RNA-related applications. Here we systematically evaluate the performance of several prominent Cas13 family effectors (Cas13a, Cas13b and Cas13d) under lentiviral vectors and reveal surprisingly differential defects and characteristics of these systems. Using RNA immunoprecipitation sequencing, transcriptome profiling, biochemistry analysis and high-throughput CRISPR-Cas13 screening approaches, we determine that each Cas13 system has its intrinsic RNA targets in mammalian cells. Viral process-related host genes can be targeted by Cas13 and affect the production of fertile lentiviral particles, thereby restricting the utility of lentiviral Cas13 systems. Multiple RNase activities of Cas13 are involved in endogenous RNA targeting. Unlike target-induced collateral effect, intrinsic RNA targeting can be specific, target-independent and dynamically tuned by varied states of Cas13 nucleases. Our work not only provides guidance to appropriately utilize lentiviral Cas13 systems, but also raises cautions about intrinsic RNA targeting during Cas13-based basic and therapeutic applications.

overall_design: Cas9 or Cas13 expressing vector transiently transfected 293FT cells were used for RIP-seq library construction or RNA-seq library construction and deep sequencing.  The crRNA lentiviral library-infected cas13d/b-expressing A375 cells harvested in day 5 and day 33 were used for DNA extraction, gRNA enrichment by PCR amplification and deep sequencing.

first: Zexu

last: Li

first: Zihan

first: Xiaolong

last: Cheng

first: Wei


title: CD11b restricts alveolar macrophage activation during pneumococcal pneumonia and prevents excessive lung inflammation

city: Essen

first: Bettina

last: Budeus

address: Virchowstrasse 173

department: Institute for Cell Biology

institute: University Hospital Essen

summary: In a murine lung infection model with Streptococcus pneumoniae, we found significant transcriptomic and proteomic adaptations by resident alveolar macrophages.

overall_design: Our study provides novel insights into the molecular adaptations of alveolar macrophages upon lung infection with Streptococcus pneumoniae.

first: Kristina

last: Zec

first: Stephanie

last: Thiebes

first: Jenny

last: Bottek

first: Devon

last: Siemes

first: Philippa

last: Spangenberg

first: Duc

last: Trieu

first: Nils

last: Kirstein

first: Nirojah

last: Subramaniam

first: Sascha

last: Krauß

first: Camille

last: Soun

first: Robin

last: Christ

first: Francesco

last: Bonella

first: Diana

last: Klein

first: Verena

last: Jendrossek

first: Oliver

last: Pabst

first: Sebastian

last: Reuter

first: Maria

last: Loose

first: Florian

last: Wagenlehner

first: Jadwiga

last: Jablonska

first: Thilo

last: Bracht

first: Barbara

last: Sitek

first: Ludger

last: Klein-Hitpass

first: Matthias

last: Gunzer

first: Dirk

last: Theegarten

first: Olga

last: Shevchuk

first: Daniel

last: Engel


title: Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems [Screen]

first: Teng

last: Fei

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

first: Zexu

last: Li

first: Zihan

first: Xiaolong

last: Cheng

first: Wei


title: Investigation of Gene Network of NKD1, NKD2 and OPAQUE-2 Associated with Maize Endosperm Development

first: Hao

last: Wu

summary: The transcriptomic analysis of nkd1, nkd2 and o2 homozygous mutant set (including 1 WT, 3 single mutants, 3 double mutants and 1 triple mutant) at 8, 12 and 16 DAP revealed temporal regulatory landscape in endosperm grain-filling stage. The data also showed NKD1, NKD2 and O2 interactively regulate gene network overtime in several processes, such as hormone response, cell wall organization, nutrient storage and endoreduplication, etc.

overall_design: Transcriptomes of 8, 12 and 16 DAP endosperm from WT (as reference) and homozygous mutant set of nkd1, nkd2 and o2 were profiled and analyzed.  All were in a W22 inbred genetic background and 4 biological replicates were conducted for each.

first: Philip

last: Becraft


title: Differential gene expression of K. aerogenes during temperature entrainment

city: Lexington

first: Kinga

middle: B.

last: Graniczkowska

state: KY

address: 675 Rose St

department: Biology

institute: University of Kentucky

summary: In this study we investigate and compare DEGs of K. aerogenes macrocolonies during temperature entrainment (12:12, 35°C:36°C) and subsequent free running conditions (35°C). The goal of this experiment was to identify rhythmically expressed genes in K. aerogenes, the first human gut commensal bacterium with described circadian clock (Paulose et al. 2016). K. aerogenes circadian clock is sensitive to temperature entrainment (Paulose et al. 2019). To further investigate this temperature entrainment of K. aerogenes we performed RNA sequencing experiment. We identified 48 differentially expressed genes when comparing timepoints ZT6 to ZT18, 427 differentially expressed genes when comparing timepoints ZT6 to CT6, 6 differentially expressed genes between timepoints CT6 and CT18, and 2 differentially expressed genes between timepoints ZT18 and CT18.

overall_design:
In this study we investigate and compare DGE of K. aerogenes macrocolonies in temperature entrainment and free running conditions
We implemented temperature entrainment using 12:12 cycle of 35°C and 36°C for 3 days. Samples were collected on day 3 at Zeitgeber time 6 (ZT6) and ZT18. Then bacteria were released into constant 35°C and samples were collected on day 4 at circadian time 6 (CT6) and CT18.

first: Vincent

last: Cassone


title: Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

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

overall_design: Refer to individual Series


title: Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems [RNA-seq]


title: Transcriptomic analysis of sorted lung cells revealed a proviral activity of the NF-κB pathway towards SARS-CoV-2 (RNA-Seq total)

status: Public on Oct 08 2023

first: Nolwenn

last: Jouvenet

address: 25 rue du Dr Roux

institute: Institut Pasteur

summary: Investigations of cellular responses to viral infection are commonly performed on mixed populations of infected and uninfected cells or using single-cell RNA sequencing, leading to inaccurate and low-resolution gene expression interpretations. Here, we performed deep polyA+ transcriptome analyses and novel RNA profiling of SARS-CoV-2 infected lung epithelial cells, sorted based on the expression of the viral spike (S) protein. Infection caused a massive reduction in mRNAs and lncRNAs, including transcripts coding for antiviral factors, such as interferons (IFN). This absence of IFN signaling probably explained the poor transcriptomic response of bystander cells co-cultured with S+ ones. NF-κB pathway and the inflammatory response escaped the global shutoff in S+ cells. Functional investigations revealed the proviral function of the NF-κB pathway and the antiviral activity of CYLD, a negative regulator of the pathway. Thus, our transcriptomic analysis on sorted cells revealed additional genes that modulate SARS-CoV-2 replication in lung cells.

overall_design:
Technical triplicates of 2 biological replicates of human alveolar basal epithelial cells transduced with a vector expressing human ACE2 (A549-ACE2) were infected with  SARS-CoV-2 (BetaCoV/France/IDF0372/2020), at MOI of 1 during 24h.Cells were FACS-sorted based on the expression of the viral spike (S) protein to identify infected (S+) and bystander (S-). Total RNA sequencing was performed. Mock infected cells were used as control.
Following transcript assembly, differential expression analysis was performed between mock, bystander and infected cells.

first: Anvita

last: Bhargava

first: Ugo

last: Szachnowski

first: Maxime

last: Chazal

first: Dominika

last: Foretek

last: Caval

first: Sophie-Marie

last: Aicher

first: Juliana

last: Pipoli da Fonseca

first: Patricia

last: Jeannin

first: Guillaume

last: Beauclair

first: Marc

last: Monot

first: Antonin

last: Morillon


title: Transcriptomic analysis of sorted lung cells revealed a proviral activity of the NF-κB pathway towards SARS-CoV-2 (RNA-Seq depeted total)

overall_design:
Technical triplicates of 2 biological replicates of human alveolar basal epithelial cells transduced with a vector expressing human ACE2 (A549-ACE2) were infected with  SARS-CoV-2 (BetaCoV/France/IDF0372/2020), at MOI of 1 during 24h.Cells were FACS-sorted based on the expression of the viral spike (S) protein to identify infected (S+) and bystander (S-). Total RNA sequencing was performed after depletion of the viral reads. Mock infected cells were used as control.
Following transcript assembly, differential expression analysis was performed between mock, bystander and infected cells.


title: Unveiling alterations of epigenetic modifications and chromatin architecture leading to lipid metabolic reprogramming during the evolutionary trastuzumab adaptation of HER2-positive breast cancer [Cut & Tag]

city: Nanjing

first: Nignjun

last: Duan

address: 300 Guangzhou Road

institute: JSPH

summary: Secondary trastuzumab resistance represents an evolutionary adaptation of HER2-positive breast cancer during anti-HER2 treatment. Most current studies have uncovered the alternations of HER2 and associated signaling pathways but pay less attention to broader cellular processes and their relevant genetic and epigenetic mechanisms. Here we have not only characterized transcriptome data but also examined epigenomic and 3D genome architecture information in both trastuzumab-sensitive and secondary-resistant breast cancer cells. Our findings reveal that the global metabolic reprogramming associated with trastuzumab resistance may stem from genome-wide alterations in both histone modifications and chromatin structure. Specifically, the transcriptional activities of key genes involved in lipid metabolism appear to be regulated by variant promoter H3K27me3 and H3K4me3 modifications, as well as promoter-enhancer interactions. These discoveries offer valuable insights into how cancer cells adapt to anti-tumor drugs and have the potential to impact future diagnostic and treatment strategies.

overall_design: To investigate the gene expression changes of HER2 positive breast cancer cells during secondary trastuzumab resistance formation, we treated SKBR3 cells with raising trastuzumab concentration in 30 weeks.

first: Ningjun

first: Yijia

last: Hua

first: Xueqi

last: Yan

first: Yaozhou

last: He

first: Tianyu

last: Zeng

first: Jue

last: Gong

first: Ziyi

last: Fu

last: Li

first: Yongmei

last: Yin


title: Transcriptomic analysis of sorted lung cells revealed a proviral activity of the NF-κB pathway towards SARS-CoV-2

first: Nolwenn

last: Jouvenet


title: Transcriptomic analysis of sorted lung cells revealed a proviral activity of the NF-κB pathway towards SARS-CoV-2 (CUT & Run)

overall_design: Monoplicates of human alveolar basal epithelial cells transduced with a vector expressing human ACE2 (A549-ACE2) were stimulated with TNF-alpha, and CUT & Run targeting p65 was performed.

first: Anvita

last: Bhargava

first: Ugo

last: Szachnowski

first: Maxime

last: Chazal

first: Dominika

last: Foretek

first: Vincent

last: Caval


title: Transcriptomic analysis of sorted lung cells revealed a proviral activity of the NF-κB pathway towards SARS-CoV-2 (RNA-Seq depleted polyA)

overall_design:
Technical triplicates of human alveolar basal epithelial cells transduced with a vector expressing human ACE2 (A549-ACE2) were infected with  SARS-CoV-2 (BetaCoV/France/IDF0372/2020), at MOI of 1 during 24h.Cells were FACS-sorted based on the expression of the viral spike (S) protein to identify infected (S+) and bystander (S-). PolyA+ RNA sequencing was performed after depletion of the viral reads. Mock infected cells were used as control.
Following transcript assembly, differential expression analysis was performed between mock, bystander and infected cells.

first: Sophie-Marie

last: Aicher

first: Juliana

last: Pipoli da Fonseca

first: Patricia

last: Jeannin

first: Guillaume

last: Beauclair

first: Marc

last: Monot


title: Altered expression of circular RNA in patients with cervical artery dissection

first: Li

last: Yudi

address: No. 1098 Xueyuan Avenue, 518060 Shenzhen, Guangdong, China.

institute: Shenzhen Hospital, Southern Medical University

summary: Cervical artery dissection (CeAD), a special cerebrovascular disease and the main cause of stroke in young people, can present with ischemic stroke, headache, subarachnoid hemorrhage, and other symptoms, increasing the possibility of misdiagnosis. As a special class of non-coding RNAs, circRNAs are commonly found in organisms and can play regulatory roles in transcription and post-transcription processes, affecting gene expression.CircRNAs have reported to be associated with neurological diseases; however, their role in CeAD has not been discerned. In this study, we aimed to elucidate the pathophysiological changes in patients with CeAD and identify biomarkers. Peripheral blood mononuclear cells from patients with CeAD and healthy controls were sequenced using high-throughput sequencing. We detected 460 differently \ expressed circRNAs in patients with CeAD (p < 0.5, fold difference ≥ 2), of which 240 were upregulated and 220 were downregulated. Four circRNAs showed significant differences in expression, which were validated using qRT-PCR. These results suggested that three circRNAs were consistent with high-throughput sequencing results. Bioinformatics analysis demonstrated that these differentially expressed circRNAs were involved in protein metabolism, regulation, synapses, and other pathophysiological processes during CeAD-induced stroke. Additionally, various pathways related to inflammation were closely associated with circRNAs. Based on our results, we suggest that the aberrant expression of circRNAs in CeAD may serve as a biomarker for its diagnosis and as a potential therapeutic target.

overall_design:
Peripheral blood mononuclear cells from patients with CeAD and healthy controls were sequenced using high-throughput sequencing.
We performed qRT-PCR to detect circRNA expression levels using the SYBR Green qPCR SuperMix and the 7500 Sequence Detection System (Applied Biosystems).
GO functional annotations and KEGG pathway enrichment analyses were performed using the clusterProfiler package in the Bioconductor/KOBAS 3.0.

first: Yifan

last: Wang

first: Zhaofei

last: Dong

last: Ling

first: Yajie

last: Liu


title: An Amygdalar Local Oscillator Coordinates Cellular and Behavioral Rhythms

city: Baltimore

first: Dong Won

middle: Thomas

last: Kim

state: Maryland

address: 733 N.Broadway, MRB building (Rm 332)

department: Neuroscience

institute: Johns Hopkins University

summary: Purpose: scRNA-Seq and snRNA were performed to characterize mWake-expressing neurons in the lateral amygdala

overall_design: The lateral amygdalae were collected from 7-8 wk old male mWake(Cre/+) mice at ZT0-2 using a previously published protocol.  Briefly, the coronal plane (350 um) containing the LA was sliced using a vibratome in bubbling aCSF.  LA were dissected into Hibernate-A media with a 2% B-27 and GlutaMAX supplement (0.5 mM final concentration).  Both LA from 2 mice were used for scRNA-Seq analysis, which was repeated with 2 additional mice.  This procedure was also performed for snRNA-Seq analysis (in total, 8 mice were used).

first: Qiang

first: Jiali

last: Xiong

first: Dong

first: Sang

last: Lee

first: Benjamin

last: Bell

first: Chloe

last: Alexandre

first: Seth

last: Blackshaw

first: Alban

last: Latremoliere

first: Mark

middle: N

last: Wu


title: Zeb1-knockdown retina endothelial cells

city: Shanghai

first: Qing Cissy

last: Yu

address: 320 Yue Yang Road

department: Institute of Biochemistry and Cell Biology

institute: Shanghai Institute of Biological Sciences


title: Mitochondrial protein import stress augments alpha-synuclein aggregation and neurodegeneration independent of bioenergetics

city: Syracuse

first: Frank

middle: A

last: Middleton

address: 505 Irving Avenue

department: Neuroscience and Physiology

institute: SUNY Upstate Medical Univ

summary: Several genetic and environmental risk factors for Parkinson’s disease have been identified that converge on mitochondria as central elements in the disease process. However, the mechanisms by which mitochondrial dysfunction contributes to neurodegeneration remain incompletely understood. Non-bioenergetic pathways of the mitochondria are increasingly appreciated, but confounding bioenergetic defects are a major barrier to experimental validation. Here, we describe a novel bioenergetics-independent mechanism by which mild mitochondrial protein import stress augments neurodegeneration. We induced this mitochondrial protein import stress in an established mouse model of Parkinson’s disease expressing the A53T mutated form of human alpha-synuclein (SNCA). Mice with import stress in addition to the A53T mutation demonstrated increased size of SNCA aggregates, co-aggregation of mitochondrial preproteins with SNCA protein, worsened neurodegeneration and changes in gene expression, as determined by whole-transcriptome RNA-sequencing analysis of the forebrain (striatum), cerebellum, spinal cord, as well as heart and skeletal muscle.

overall_design: Total RNA from the striatum, cerebellum, and spinal cord in wildtype and transgenic mice and analyzed using RNAseq

first: Liam

last: Coyne

first: Xin

last: Chen

first: Xiaowen

first: Arnav

last: Rana

first: Sanaea

last: Bhagwagar

first: Yumiko

last: Umino

first: Eduardo

middle: C

last: Solessio


title: Chromatin structure profiling of Zeb1-knockdown mouse retina endothelial cells by ATAC-seq

summary: By comparing ATAC-seq from freshly isolated endothelial cells from control or Zeb1-knockdown retina, we observed an increase in the  accessibility of chromatin structure in endothelial cells after Zeb1-knockdown. We used ATAC-seq to study the functional role of Zeb1 and identified genes that exhibited increase of accessibilities to their transcription start sites (TSS).

overall_design: Examination of chromatin accessibility using ATAC-seq in retina endothelial cells isolated from control or Zeb1-knockdown mice.

first: Qing

first: Christopher

middle: B

last: Preusch

first: Guangdun

last: Peng

first: Yi

last: Zeng


title: Transcriptome sequencing analysis of Zeb1-knockdown retina endothelial cells by RNA-seq

summary: To understand the effect of Zeb1 knockdown on retina vasculature, we isolated endothelial cell from P16 control and Zeb1-knockdown mouse retina. RNA-seq results analysis showed that Zeb1 loss leads to enhanced proliferation and failed quiescence entry.

overall_design: The isolated RNA from flow cytrometry isolated primary retina endothelial cells were sequenced using Illumina NextSeq 500 (2 x 150)


title: Role of ZHX2 in VSMCs [RNA-seq]

status: Public on Oct 07 2023

city: WuHan

first: 思源

last: 樊

state: HuBei

address: Address: No.13, Hangkong Road, Wuhan, Hubei, P.R.

institute: Huazhong University of Science and Technology

summary: Primary rat VSMCs were infected with Ad-Control and Ad-ZHX2 and stimulated with PDGF-BB of 24h. RNAseq and differential expression analysis were performed to investigate the role of ZHX2 in VSMCs.

overall_design: mRNA profiles of VSMCs infected with Ad-Control or Ad-ZHX2

first: Siyuan

last: Fan

first: Zhe

last: Zheng

first: Liuye

last: Yang


title: Role of ZHX2 in VSMCs


title: Role of ZHX2 in VSMCs [Chip-seq]

summary: In order to explore the biological function and molecular mechanism of ZHX2 in VSMCs,in this study, we overexpressed ZHX2 in primery VSMCs used chromatin immunoprecipitation sequencing (ChIP-seq) methods to systematically investigate the downstream targets of ZHX2.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for transcription factor ZHX2 .


title: Loss of function of CENH3 causes genome instability in soybean

city: Beijng

first: Jing

last: Wang

address: No. 2, Yard 1, Beichen West Road

institute: Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

type: Genome variation profiling by high throughput sequencing

summary: The centromeric histone H3 (CENH3) is of great importance in centromere identification identity, chromosome mobilization during cell division and thus genome stability. The imbalance of CENH3 loading rate or dosage on parental centromeres often leads to uniparental chromosome elimination in the offspring. A body of studies on the function of CENH3 in genome stability have been reported in Arabidopsis, cotton, and many other monocots, but not in soybean (Glycine max), an important dicot crop. In our study, we identified the a single-copy functional CENH3 in soybean and found its role in genome stability and parent-of-origin effect caused by a conserved glycine site and parental genetic background. This study identified the functional GmCENH3 and would shed light on the future development of CENH3-based haploid induction system and centromere biology in soybean.

overall_design: Genome re-sequencing of sterile soybean plants and chromosome dosage analysis were taken to figure out the karyograms.

first: Xiaofei

first: Weicai

first: Hongju

last: Li


title: Fistula healing in patients with complex perianal fistulising Crohn’s disease

first: Sibo

last: Zhu

state: China

address: songhulu 2005

institute: Fudan University

summary: Background: Perianal fistulising Crohn’s disease (pfCD) is a disabling phenotype of Crohn’s disease (CD) with suboptimal outcomes. We assessed neutrophil extracellular traps (NETs) in perianal fistulas and implicated their roles in pfCD healing. Methods: Patients with complex pfCD who developed preplaced seton drainage were recruited during the verified maintenance of remission in CD. Fistula tracts were sampled during definitive surgery plus seton removal. Patient demographics, CD classification, medication strategy, and healing of pfCD were recorded. RNA sequencing was applied for transcriptomic profile analysis. NETs components, including myeloperoxidase (MPO), neutrophil elastase (NE), and citrullinated histone H3 (CitH3), were identified using immunofluorescence. Serum infliximab (IFX), anti-IFX antibodies, and tissue levels of IFX, adalimumab (ADA), MPO and CitH3 were determined using enzyme-linked immunosorbent assays. Peptidyl arginine deiminase IV (PAD4), tumour necrosis factor (TNF)-α, and NE were detected using immunohistochemistry. Gene expression levels of PAD family members were assessed with qPCR. Results: Twenty-one patients were included, 15 of whom adopted IFX as maintenance treatment. RNA-seq revealed difference in neutrophil associated pathways between unhealed and healed fistulas. NETs components (MPO/NE/CitH3) were detectable in the fistulas and were parallel with the PAD4 levels. Eleven of 21 (52%) patients experienced complete healing of the pfCD 108 weeks post-operatively. Fistula NETs were significantly increased in patients with unhealed pfCD. Increased NETs were associated with abundant TNF-α production and the absence of IFX in fistulas. Conclusions: NETs exist in pfCD fistulas, which are associated with unhealed post-operative fistulas in pfCD, suggesting their prognostic roles in pfCD.

overall_design: A total of 12 perianal tissues of the patients with pfCD are collected for RNAseq, including 6 cases with unhealed and 6 cases with healed fistulas.

last: Cui

first: Dong-xing

last: Cao

first: Ye

last: Zhang