title: Effects of chemical in vitro activation versus fragmentation on human ovarian tissue and follicle growth in culture

city: Solna

first: Tianyi

last: Li

address: Bioclinicum NKS J5:20, Solnavägen 30

department: Oncology-Pathology

country: Sweden

institute: Karolinska Institute

summary:
Abstract

STUDY QUESTION
What is the effect of the chemical in vitro activation (cIVA) protocol compared with fragmentation only (Frag, also known as mechanical IVA) on gene expression, follicle activation, and growth in human ovarian tissue in vitro?

SUMMARY ANSWER
Although histological assessment shows that cIVA significantly increases follicle survival and growth compared to Frag, both protocols stimulate extensive and nearly identical transcriptomic changes in cultured tissue compared to freshly collected ovarian tissue, including marked changes in energy metabolism and inflammatory responses.

WHAT IS KNOWN ALREADY
Treatments based on chemical in vitro activation of the phosphatase and tensin homolog (PTEN)- phosphatidylinositol 3-kinase (PI3K) pathway in ovarian tissue followed by auto-transplantation have been administered to patients with refractory premature ovarian insufficiency (POI) and resulted in live births. However, comparable effects with mere tissue fragmentation have been shown, questioning the added value of chemical stimulation that could potentially activate oncogenic responses.

STUDY DESIGN, SIZE, DURATION
Fifty-nine ovarian cortical biopsies were obtained from consenting women undergoing elective caesarean section (C-section). The samples were fragmented for culture studies. Half of the fragments were exposed to bpV (HOpic) + 740Y-P (Frag + cIVA group) during the first 24 h of culture, while the other half were cultured with medium only (Frag group). Subsequently, both groups were cultured with medium only for an additional 6 days. Tissue and media samples were collected for histological, transcriptomic, steroid hormone and cytokine/chemokine analyses at various time points.

PARTICIPANTS/MATERIALS, SETTING, METHODS
Effects on follicles were evaluated by counting and scoring serial sections stained with hematoxylin and eosin before and after the 7-day culture. Follicle function was assessed by quantification of steroids by ultra-performance liquid chromatography tandem-mass spectrometry at different time points. Cytokines and chemokines were measured by multiplex assay. Transcriptomic effects were measured by RNA-sequencing (RNA-seq) of the tissue after the initial 24 h culture. Selected differentially expressed genes (DEGs) were validated by quantitative PCR and immunofluorescence in cultured ovarian tissue as well as in KGN cell (human ovarian granulosa-like tumor cell line) culture experiments.

MAIN RESULTS AND THE ROLE OF CHANCE
Compared to the Frag group, the Frag + cIVA group exhibited a significantly higher follicle survival rate, increased numbers of secondary follicles, and larger follicle sizes. Additionally, the tissue in Frag + cIVA group produced less dehydroepiandrosterone compared to Frag. Cytokine measurement showed a strong inflammatory response at the start of the culture in both groups. The RNA-seq data revealed modest differences between the Frag + cIVA and Frag groups, with only 164 DEGs identified using a relaxed cut-off of false discovery rate (FDR)<0.1. Apart from the expected PI3K-protein kinase B (Akt) pathway, cIVA also regulated pathways related to hypoxia, cytokines, and inflammation. In comparison to freshly collected ovarian tissue, gene expression in general was markedly affected in both Frag + cIVA and Frag groups, with a total of 3,119 and 2,900 DEGs identified (FDR < 0.001), respectively. The top enriched gene sets in both groups included several pathways known to modulate follicle growth such as mammalian target of rapamycin (mTOR)C1 signaling. Significant changes compared to fresh tissue were also observed in the expression of steroidogenesis enzymes and classical granulosa cell markers in both groups. Intriguingly, we discovered a profound upregulation of genes related to glycolysis and its upstream regulator in both Frag and Frag + cIVA groups, and these changes were further boosted by the cIVA treatment. Cell culture experiments confirmed glycolysis-related genes as direct targets of the cIVA drugs. In conclusion, cIVA enhances follicle growth, as expected, but the mechanisms may be more complex than PI3K-Akt-mTOR alone, and the impact on function and quality of the follicles after the culture period remains an open question.

LARGE SCALE DATA
Data was deposited in GEO data base, accession number GSE234765. Code for sequencing analysis can be found in https://github.com/tialiv/IVA_project.

LIMITATIONS, REASONS FOR CAUTION
Similar to the published IVA protocols, the first steps in our study were performed in an in vitro culture model where the ovarian tissue was isolated from the regulation of hypothalamic-pituitary-ovarian axis. Further in vivo experiments will be needed, for example in xeno-transplantation models, to explore the long-term impacts of the discovered effects. The tissue collected from patients undergoing C-section may not be comparable to tissue of patients with POI.

WIDER IMPLICATIONS OF THE FINDINGS
The general impact of fragmentation and short (24 hours) in vitro culture on gene expression in ovarian tissue far exceeded the effects of cIVA. Yet, follicle growth was stimulated by cIVA, which may suggest effects on specific cell populations that may be diluted in bulk RNA-seq. Nevertheless, we confirmed the impact of cIVA on glycolysis using a cell culture model, suggesting impacts on cellular signaling beyond the PI3K pathway. The profound changes in inflammation and glycolysis following fragmentation and culture could contribute to follicle activation and loss in ovarian tissue culture, as well as in clinical applications, such as fertility preservation by ovarian tissue auto-transplantation.

overall_design:
27 samples in total from 11 patients. Tissues were cultured in either matrigel-coated or laminin 221 coated insert. 10 IVA samples were treated with 740Y-P and bpV (HOpic) for 24 h and 6 fragmentation samples were treated with media only.

***Raw data have not been provided due to patient privacy concerns***

first: Jie

last: Hao

first: Manuel

last: Heinzelmann

first: Elisabeth

last: Moussaud-Lamodière

first: Filipa

last: Lebre

first: Kaarel

last: Krjutškov

first: Anastasios

last: Damdimopoulos

first: Catarina

last: Arnelo

first: Karin

last: Pettersson

first: Ernesto

last: Alfaro-Moreno

first: Cecilia

last: Lindskog

first: Majorie

last: van Duursen

first: Pauliina

last: Damdimopoulou


title: Epigenetic and transcriptional regulation of dendritic cell development and anti-tumor function by HDAC1

status: Public on May 27 2024

city: Vienna

first: Cristiano

last: de Sá Fernandes

address: Borschkegasse 8a

department: Center of Cancer Research

country: Austria

institute: MedicalUniversity of Vienna

type: Expression profiling by high throughput sequencing

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Transcriptomic analysis of whole mouse tibia samples

status: Public on May 23 2024

city: Nuthetal

first: Tim

middle: Julius

last: Schulz

address: Arthur-Scheunert-Allee 114-116

country: Germany

institute: German Institute of Human Nutrition

summary: The combined effects of aging and a dietary intervention of reduced energy intake, caloric restriction, were examined by conducting a bulk transcriptomic analysis of mouse whole tibia bone samples. Tissue samples were collected from mice at two different age groups, comparing old and young animals, with caloric restriction in comparison to full food access.

overall_design: Male C57Bl6J mice in two age groups, young mice aged 10 weeks and older mice, aged between 15-18 months, underwent caloric restriction for 8 weeks and were compared to age-matched control mice with free food access. Mice fed with the control diet received the NIH-31 rodent open formula diet, while caloric restriction mice were provided with a modified NIH-31/NIA diet, which is fortified with micronutrients. In the first week of caloric restriction, mice received a 10% restriction, followed by 20% restriction in the second week, and a restriction of 40% from the third week onward until the end of the experiment.

middle: J

first: Charlotte

last: Rinne

first: Masoome

last: Oveisi

first: Marina

last: Leer


title: A high-density microfluidic bioreactor for the automated manufacturing of CAR T cells

status: Public on May 29 2024

city: Singapore

first: Wei-Xiang

last: Sin

address: 1 CREATE Way, #04-13/14, Enterprise Wing

country: Singapore

institute: SMART

summary: The manufacturing of autologous chimaeric antigen receptor (CAR) T cells largely relies either on fed-batch and manual processes that often lack environmental monitoring and control or on bioreactors that cannot be easily scaled out to meet patient demands. Here we show that human primary T cells can be activated, transduced and expanded to high densities in a 2 ml automated closed-system microfluidic bioreactor to produce viable anti-CD19 CAR T cells (specifically, more than 60 million CAR T cells from donor cells derived from patients with lymphoma and more than 200 million CAR T cells from healthy donors). The in vitro secretion of cytokines, the short-term cytotoxic activity and the long-term persistence and proliferation of the cell products, as well as their in vivo anti-leukaemic activity, were comparable to those of T cells produced in a gas-permeable well. The manufacturing-process intensification enabled by the miniaturized perfusable bioreactor may facilitate the analysis of the growth and metabolic states of CAR T cells during ex vivo culture, the high-throughput optimization of cell-manufacturing processes and the scale-out of cell-therapy manufacturing.

overall_design: Comparative gene expression profiling analysis of bulk RNA-seq data for microbioreactor (MBR) cell products versus gas-permeable well plate (GWP) cell products, each from three biological replicates (donors A, B, C).

middle: X

first: N. Suhas

middle: S

last: Jagannathan

first: Denise Bei Lin

last: Teo

first: Faris

last: Kairi

first: Shin Yie

middle: Y

last: Fong

first: Joel Heng Loong

last: Tan

first: Dedy

last: Sandikin

first: Ka-Wai

middle: W

last: Cheung

first: Yen Hoon

middle: H

last: Luah

first: Xiaolin

last: Wu

first: Joshua Jebaraj

last: Raymond

first: Francesca Lorraine Wei Inng

last: Lim

first: Yie Hou

last: Lee

first: Michaela Su-Fern

last: Seng

first: Shui Yen

last: Soh

first: Qingfeng

last: Chen

first: Rajeev

last: Ram

first: Lisa

last: Tucker-Kellogg

first: Michael

middle: E

last: Birnbaum


title: Targeting the intersection of angiopoietin-TIE2 signaling with arachidonic acid metabolism can prevent septic death

city: Burnaby

first: Amy

middle: H.

state: BC

address: 8888 University Drive

department: Molecular Biology and Biochemistry

country: Canada

institute: Simon Fraser University

summary: Neonatal sepsis is a major cause of childhood mortality. Limited diagnostic tools and mechanistic insights have hampered our abilities to develop prophylactic or therapeutic interventions. Biomarkers in human neonatal sepsis have been repeatedly identified as associated with dysregulation of angiopoietin signaling and altered arachidonic acid metabolism. We here show that these two pathways appear to be functionally connected via reactive oxygen species (ROS) with a direct role of nitric oxide (NO). Targeting this intersection with oral administration of arachidonic acid and/or the NO donor L-arginine provided prophylactic as well as therapeutic protection from septic death.

overall_design: The neonatal sepsis model was utilized as previously described. In short, CS was obtained from adult male caeca and resuspended in dextrose 5% water at a concentration of 100mg/mL and then passed through a 70um filter. To provide consistency, our cecal slurry model (PMID: 30741260) employs multiple litters of adult donors of cecal slurry that are pooled, mixed and aliquots were frozen at -80°C until challenge. Following this, each slurry is titred to determine the effective dose (LD50 etc.). Each new cecal slurry lot is directly compared to previous lots to ensure consistency across experiments.  Neonatal mice at day of life (DOL) 6-8 were challenged via intraperitoneal injection at a weight adjusted dose of 1mg/g body weight. Litter-to-litter variation was accounted for by having a balanced number of treated and control mice in each litter. 10 neonatal mice did not receive cecal slurry challeng while 25 neonatal mice received cecal slurry.

first: Nelly

last: Amenyogbe

first: Tobias

middle: R

last: Kollmann

first: Danny

last: Harbeson


title: Spatial transcriptomics in embryonic mouse diaphragm muscle reveals regional gradients and subdomains of developmental gene expression

city: Prague

first: Mehmet

middle: Mahsum

last: Kaplan

address: Vídeňská 1083

department: Department of Developmental Biology

country: Czech Republic

institute: Czech Academy of Sciences

summary: Embryonic mouse diaphragm is a primary model for studying myogenesis and neuro-muscular synaptogenesis, both of which represent processes regulated by spatially organized genetic programs of myonuclei located in distinct myodomains. However, a spatial gene expression pattern of embryonic mouse diaphragm has not been reported. Here we provide spatially resolved gene expression data for horizontally sectioned embryonic mouse diaphragms at E14.5 and E18.5. These data reveal gene signatures for specific muscle regions with distinct maturity and fiber type composition, as well as for a central neuromuscular junction and a peripheral myotendinous junction compartments. Comparing spatial expression patterns of wild type mice with those of mouse mutants lacking either the skeletal muscle calcium channel CaV1.1 or b-catenin, reveals curtailed muscle development and dysregulated expression of genes potentially involved in NMJ formation. Altogether, these datasets provide a powerful recourse for further studies of muscle development and NMJ formation in the mouse embryo.

overall_design: Three E14.5 and two E18.5 diaphragm muscles from control embryos, one E14.5 and one E18.5 diaphragm from Cav1.1 KO and one E18.5 diaphragm from bCat cKO embryo were analyzed by FFPE Visium Spatial Transcriptomics

middle: M

first: Maximilian

last: Zeidler

first: Anabella

last: Knapp

first: Martina

last: Hölzl

first: Michaela

last: Kress

first: Helga

last: Fritsch

first: Anne

last: Krogsdam

first: Bernhard

last: Flucher


title: Genome-wide maps of TAF2 (ChIP-seq) and Gene expression profiles  upon TAF2 depletion (RNA-seq)

status: Public on May 28 2024

city: Taipei

first: Wei-Yi

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

department: Institute of Biochemistry and Molecular Biology

country: Taiwan

institute: National Yang Ming Chiao Tung University

summary: TAF2 is the second largest subunit of general transcription factor TFIID. Although TFIID complex is putatively considered as a general initiation factor, gene-specific functions have been reported for several TAF subunits within it. To gain insights for TAF2 function in transcriptional regulation, genomic occupancy of TAF2 and transcriptiome of cells depleted of TAF2 are examined.

overall_design: Genome-wide binding/occupancy profiling of TAF2, TBP, and histone marks by ChIP-seq in HCT116 cells. Transcriptome profiling by RNA-seq in control and shTAF2-treated HCT116 cells .

first: I-Hsin

last: Cheng


title: Ageing is Associated with an Insufficient Early Inflammatory Response of Lung Endothelial Cells in SARS-CoV-2 Infection

status: Public on May 22 2024

city: Boston

first: Markus

last: Bosmann

state: MA

address: 72 E Concord Street

department: Pulmonary Centre, Department of Medicine

country: USA

institute: Boston University

summary: Advanced age is associated with an increased susceptibility to Coronavirus Disease (COVID)-19 and more severe outcomes, although the underlying mechanisms are understudied. The lung endothelium is located next to infected epithelial cells and bystander inflammation may contribute to thromboinflammation and COVID-19-associated coagulopathy. Here, we investigated age-associated SARS-CoV-2 pathogenesis and endothelial inflammatory responses using humanized K18-hACE2 mice. Survival was reduced to 20% in aged mice (85-112 weeks) versus 50% in young mice (12-15 weeks) at 10 days post infection (dpi). Bulk RNA-sequencing of endothelial cells from mock and infected mice at 2dpi of both age groups (aged: 72-85 weeks; young: 15 weeks) showed substantially lower significant differentially regulated genes in infected aged mice than in young mice (712 versus 2294 genes). Viral recognition and anti-viral pathways such as RIG-I-like receptor signaling, NOD-like receptor signaling and interferon signaling were regulated in response to SARS-CoV-2. Young mice showed several fold higher interferon responses (Ifitm3, Ifit1, Isg15, Stat1) and interferon-induced chemokines (Cxcl10 and Cxcl11) than aged mice. Endothelial cells from infected young mice displayed elevated expression of chemokines (Cxcl9, Ccl2) and leukocyte adhesion markers (Icam1) underscoring that inflammation of lung endothelium during infection could facilitate leukocyte adhesion and thromboinflammation. TREM1 and acute phase response signaling were particularly prominent in endothelial cells from infected young mice. Immunohistochemistry was unable to detect viral protein in pulmonary endothelium. In conclusion, our data demonstrate that the early host response of the endothelium to SARS-CoV-2 infection declines with aging, which could be a potential contributor to disease severity.

overall_design: K18-hACE2 transgenic female mice (aged: 72-85 weeks; young: 15 weeks) were intranasally inoculated with 1x10^6 PFU of SARS-CoV-2 in 50 μL of sterile 1X PBS and mock mice were inoculated with 50 μL of sterile 1X PBS. Inoculations were performed under 1-3% isoflurane anesthesia. Lungs were collected at 2dpi, endothelial cells were isolated, and RNA-seq was performed.

first: Saravanan

last: Subramaniam

first: Archana

last: Jayaraman


title: Transcriptome of activated mouse primary hepatic stellate cells treated with the OGT inhibitor OSMI-1

city: Lille

first: Jérôme

last: Eeckhoute

address: Bâtiment J&K, Faculté de Médecine de Lille-Pôle Recherche, Boulevard du Professeur Leclerc

country: France

institute: CNRS

summary: RNA-seq analysis of mouse primary myofibroblastic hepatic stellate cells (pMF-HSCs) treated or not with OSMI-1 OGT inhibitor (OGTi).

overall_design: Differential gene expression analysis in mouse primary myofibroblastic hepatic stellate cells (pMF-HSCs at 7 d of culture) treated with vehicle (DMSO) or OSMI-1 OGT inhibitor (OGTi used at 50 µM for 24 h).

first: Ninon

last: Very

first: Clémence

last: Boulet

first: Loïc

last: Guille

first: Julie

last: Dubois-Chevalier


title: O-GlcNAcylation controls pro-fibrotic transcriptional regulatory signaling in hepatic stellate cells

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Senescent glia link neuronal mitochondrial dysfunction and lipid droplet accumulation with age [19082_28_MASTER]

status: Public on May 24 2024

city: Philadelphia

first: Nancy

last: Bonini

state: PA

address: 433 S University Ave

department: Biology

institute: University of Pennsylvania

summary: Senescence is a cellular state linked to aging and age-onset disease across many mammalian species. Acutely, senescent cells promote wound healing and prevent tumor formation; but they are also pro-inflammatory, thus chronically exacerbate tissue decline. While senescent cells are active targets for anti-aging therapy, why these cells form in vivo, how they affect tissue aging, and the impact of their elimination remain unclear. Here we identify naturally-occurring senescent glia in aged Drosophila brains and decipher their origin and influence. Using AP1 activity to screen for senescence, we determine that senescent glia can appear in response to neuronal mitochondrial dysfunction. In turn, senescent glia promote lipid accumulation in non-senescent glia; similar effects are seen in senescent human fibroblasts in culture. Targeting AP1 activity in senescent glia mitigates senescence biomarkers, extends fly life and health span, and prevents lipid accumulation. However, these benefits come at the cost of increased oxidative damage in the brain, and neuronal mitochondrial function remains poor. Altogether, our results map the trajectory of naturally-occurring senescent glia in vivo and indicate that these cells link key aging phenomena: mitochondrial dysfunction and lipid accumulation.

overall_design: Through bulk-RNA-seq and a targeted RNAi screen, we determined that loss of select mitochondrial genes in neurons can trigger AP1 activity in glia. To determine if neuronal RNAi-induced AP1+ glia have features of senescence, we FACS-sorted and bulk-RNA-sequenced dsRed+ and dsRedneg cells from brains of 10d old flies with neuronal loss of inner complex genes. For this, an inducible pan-neuronal driver (elav-GeneSwitch) was used to express UAS-RNAi (UAS-ND42-RNAi or UAS-NP15.6-RNAi); cells with AP1 activity were identified using a genomic fluorescent reporter under the control of an AP1 binding motif (dsRed). Whole brains were dissected from 10d flies, dissociated into a cell suspension then dsRed+ and dsRedneg cells were FACS-isolated and bulk RNA-sequenced for downstream gene expression analysis (n=500 cells per biological replicate, 4 replicates).

first: China

middle: N

last: Byrns

first: Alexandra

last: Perlegos


title: Transcriptome of human LX-2 hepatic stellate cells treated with the OGT inhibitor OSMI-1

summary: RNA-seq analysis of human LX-2 cells treated or not with OSMI-1 OGT inhibitor (OGTi).

overall_design: Differential gene expression analysis in human LX-2 cells treated with vehicle (DMSO) or OSMI-1 OGT inhibitor (OGTi used at 50 µM for 24 h).


title: Senescent glia link neuronal mitochondrial dysfunction and lipid droplet accumulation with age [19082R-38_MASTER]

overall_design: Through bulk-RNA-seq and a targeted RNAi screen, we determined that loss of select mitochondrial genes in neurons trigger AP1 activity in glia. To understand the neuronal changes associated with conditions of increased glial AP1 activity (neuronal loss of ND42 loss) vs conditions of reduced glial AP1 activity (neuronal loss of ND30 loss), we dissected whole brains dissected from 10d flies with neuronal knockdown of ND42 or ND30 then performed bulk RNA-sequencing for downstream gene expression analysis.


title: SARS-CoV-2-infected human nasal epithelial air-liquid interface cultures

city: Melbourne

first: Jessie

middle: Jie-Youen

last: Chang

address: 792 Elizabeth Street

country: Australia

institute: The Peter Doherty Institute at The University of Melbourne

summary: Continuous assessment of the impact of SARS-CoV-2 on the host at the cell-type level is crucial for understanding key mechanisms involved in host defense responses to viral infection. We investigated host response to ancestral-strain and Alpha-variant SARS-CoV-2 infections within air-liquid-interface human nasal epithelial cells from younger adults (26-32 Y) and older children (12-14 Y) using single-cell RNA-sequencing. Ciliated and secretory-ciliated cells formed the majority of highly infected cell-types, where the latter derived from ciliated lineages. Strong innate immune responses were observed across lowly-infected and un-infected bystander cells and heightened in Alpha-infection. Alpha highly-infected cells showed increased expression of protein-refolding genes compared with ancestral-strain-infected cells in children. Furthermore, oxidative phosphorylation-related genes were down-regulated in bystander cells versus infected and mock-control, underscoring the importance of these biological functions for viral replication. Overall, this study highlights the complexity of cell-type-, age- and viral strain-dependent host epithelial responses to SARS-CoV-2.

overall_design:
Nasal epithelial ALI-cultures from three human adult and child donors (12-14 years) were infected with one of two strains of SARS-CoV-2 (ancestral strain VIC01, Alpha variant VIC17991).
Single-cell RNA-sequencing with 10X Chromium coupled with Illumina sequencing was carried out with infected samples harvested at 72 hpi with ancestral strain (VIC01) and Alpha variant (VIC17991) and also 48 hpi of the ancestral strain (VIC01). Mock-controls were also sequenced. Count matrices were produced using Cellranger and the cells from the three donors (per age-group) were demultiplexed using genotyping information. Differential expression analysis using pseudo-bulking was carried out using edgeR and limma-voom.

first: Samantha

middle: L

last: Grimley

first: Bang

last: Tran

first: Georgia

last: Deliyannis

first: Carolin

last: Tumpach

first: An

last: Nguyen

first: JianShu

last: Zhang

first: Jan

last: Schröder

first: Leon

last: Caly

last: McAuley

first: Sharon

last: Wong

first: Timothy

middle: P

last: Stinear

first: Miranda

last: Pitt

first: Damian

last: Purcell

first: Elizabeth

last: Vincan

first: Lachlan

last: Coin


title: Metabolic adaptation pilots the differentiation of human hematopoietic cells

city: Paris

first: Laëtitia

last: RACINE

address: 27 rue de Chaligny

institute: EPHE


title: Transcriptional profiling links unique human macrophage phenotypes to the growth of intracellular Salmonella enterica serovar Typhi

status: Public on May 20 2024

city: Stanford

first: Denise

last: Monack

state: CA

address: 299 Campus Drive, Fairchild Building D331

department: Microbiology and Immunology

institute: Stanford University

summary: Macrophages provide a crucial environment for Salmonella enterica serovar Typhi (S. Typhi) to multiply during typhoid fever, yet our understanding of how human macrophages and S. Typhi interact remains limited. In this study, we delve into the dynamics of S. Typhi replication within human macrophages and the resulting heterogeneous transcriptomic responses of macrophages during infection. Our study reveals key factors that influence macrophage diversity, uncovering distinct immune and metabolic pathways associated with different stages of S. Typhi intracellular replication in macrophages. Of note, we found that macrophages harboring replicating S. Typhi are skewed towards an M1 pro-inflammatory state, whereas macrophages containing non-replicating S. Typhi exhibit neither a distinct M1 pro-inflammatory nor M2 anti-inflammatory state. Additionally, macrophages with replicating S. Typhi were characterized by the increased expression of genes associated with STAT3 phosphorylation and the activation of the STAT3 transcription factor. Our results shed light on transcriptomic pathways involved in the susceptibility of human macrophages to intracellular S. Typhi replication, thereby providing crucial insight into host phenotypes that restrict and support S. Typhi infection.

overall_design: To investigate heterogeneity in human macrophage responses to intracellular S. Typhi infection, macrophages were sorted based on intracellular bacterial phenotypes (i.e. replicating, non-replicating, killed by the host), and RNA was isolated from the sorted cells for RNA-Seq.

first: Ruth

last: Schade

first: Daniel

last: Butler

first: Joy

middle: A

last: McKenna

first: Blanda

last: Di Luccia

first: Vida

last: Shokoohi

first: Meagan

last: Hamblin

first: Trung

last: Pham


title: Transcriptome of human LX-2 hepatic stellate cells transfected with siRNA directed against OGT

first: Jérôme

last: Eeckhoute

summary: RNA-seq analysis of human LX-2 cells treated transfected or not with siOGT.

overall_design: Differential gene expression analysis in human LX-2 cells transfected with siControl or siOGT (used at 20 nM for 72 h).

first: Ninon

last: Very

first: Clémence

last: Boulet

first: Loïc

last: Guille

first: Julie

last: Dubois-Chevalier


title: O-GlcNAcylation controls pro-fibrotic transcriptional regulatory signaling in hepatic stellate cells (HSCs)

summary: Human LX-2 cells were used for TEAD4 ChIP-seq

overall_design: Human LX-2 cells were cross-linked, chromatin was sonicated and used for TEAD4 ChIP (chromatin immunoprecipitation with Abcam #ab58310 TEAD4 antibody) assay followed by high-throughput sequencing.


title: Metabolic adaptation pilots the differentiation of human hematopoietic cells (bulk ATAC-Seq)

summary: In order to get insight in the role of metabolic perturbations during the first steps of cell differentiation, the phenotype, gene expression profile and chromatin accessibility of CD34+ cells were examined using bulk- and single-cell techniques during the initial 96 hours of in vitro differentiation. Specific enzyme inhibitors targeting key steps of the energy metabolism were employed to induce perturbations and assess their effect. The results support a model whereby energy requirements play the role of initiator of differentiation. According to it, metabolic perturbations induce rapid and non-specific, enabling a state of multi-primed gene expression. A selective re-compaction of the chromatin constrained by the availability of key metabolites essential for repressive epigenetic modifications and by the action of transcription regulator proteins consolidates the phenotypic response of the cells.

overall_design:
Human hematopoietic CD34+ cells were grown in Xvivo medium supplemented with cytokines with or without energy metabolism inhibitor (DON, 2-DG or AOA) during 03h, 12h or 24h.
For each time point and condition, at least three biological replicates were examined (three different donors). For the control condition without inhibitor (CTRL), five replicates were used.
00h Xvivo time point corresponds to CD34+ cells grown without cytokines in the medium during a short period (less than 03h).
Files containing the read count per detected peak per sample (one sample = merged biological replicates for the same time point) after the union of all peaks list (Reduce function) and DEseq normalization :
DEseq_readcount_union_allsamples.rds (grange object to be used in R)
DEseq_readcount_union_allsamples.csv (table to be read in excel)

last: Racine

first: Romuald

last: Parmentier


title: Effect of OGT inhibition on the epigenome of human LX-2 hepatic stellate cells

summary: Human LX-2 cells treated or not with OSMI-1 OGT inhibitor (OGTi) were used for H3K27ac ChIP-seq as well as for CoP-seq.

overall_design: Human LX-2 cells were treated with vehicle (DMSO) or OSMI-1 OGT inhibitor (OGTi used at 50 µM for 24 h), cross-linked, chromatin was sonicated and used for H3K27ac ChIP (chromatin immunoprecipitation with Active Motif #39685 H3K27ac antibody) or CoP (Column Purified chromatin) assays followed by high-throughput sequencing.


title: Nociceptor-immune interactomes reveal insult-specific immune signatures of pain

city: Los Angeles

first: Riki

last: Kawaguchi

address: 760 Westwood Plaza, Room 37-420

department: Department of Psychiatry and Neurology Department of Psychiatry

institute: University of California, Los Angeles

summary: Inflammatory pain associated with tissue injury and infection largely results from the heightened sensitivity of the peripheral terminals of nociceptor sensory neurons as a consequence of exposure to inflammatory mediators. Targeting immune-derived inflammatory ligands, such as prostaglandin E2, reduces inflammatory pain, but more effective and safer therapies are needed. However, the diversity of immune cell and sensory neuron types, their ligands, and receptors make it challenging to map the immune mechanisms that contribute to inflammatory pain. We, therefore, used single-cell transcriptomics to explore which specific immune cell types affect the development of pain in diverse inflammatory pain models. We found that both the magnitude of macrophage and neutrophil recruitment and the injury-triggered transcriptional program in Cx3cr1 high and MHCII+ dermal macrophages closely mirror the kinetics of inflammatory pain hypersensitivity. We constructed a comprehensive list of potential cell-cell interactions covering receptors, ligands, and metabolites, and generated injury-specific neuroimmune interactomes based on immune cell and sensory neuron single-cell transcriptomic data. This analysis revealed both interactions common to multiple inflammatory pain models and those specific to a particular condition and uncovered immune mediators not previously identified as pain modulators.

overall_design: CD45+ immune cells were FACS sorted cells from mouse (females) plantar skin at time 4h, 24, and 48h post zymosan injection, skin incision and UV burn as well as contra lateral healthy control. Gene expression profiles of immune cells was obtained at single cell level using 10x genomics platform.

first: Aakanksha

last: Jain

first: Clifford

last: Woolf


title: Nextflow Pipeline for Visium and H&E Data from Patient-Derived Xenograft Samples

status: Public on May 21 2024

city: Farmington

first: Jeffrey

last: Chuang

state: CT

address: 10 Discovery Drive

institute: The Jackson Laboratory for Genomic Medicine

summary: We designed a Nextflow DSL2-based pipeline, Spatial Transcriptomics Quantification (STQ), for simultaneous processing of 10x Genomics Visium spatial transcriptomics data and a matched hematoxylin and eosin (H&E)-stained whole slide image (WSI), optimized for Patient-Derived Xenograft (PDX) cancer specimens. Our pipeline enables the classification of sequenced transcripts for deconvolving the mouse and human species and mapping the transcripts to reference transcriptomes. We align the H&E WSI with the spatial layout of the Visium slide and generate imaging and quantitative morphology features for each Visium spot. The pipeline design enables multiple analysis workflows, including single or dual reference genomes input and stand-alone image analysis. We show the utility of our pipeline on a dataset from Visium profiling of four melanoma PDX samples. The clustering of Visium spots and clustering of H&E imaging features reveal similar patterns arising from the two data modalities.

overall_design: The PDX model WISTAR WM4237-1 is a BRAF V600E mutant metastatic melanoma of a subcutaneously implanted solid tumor on NSG mouse #1337, passage 0. The tumor was grown untreated by any therapy. The four samples are near adjacent tissue sections from the tumor tissue block. The samples were analyzed with 10x Visium Spatial Gene Expression.

first: Domanskyi

last: Sergii

first: Srivastava

last: Anuj

first: Kaster

last: Jessica

first: Li

last: Haiyin

first: Herlyn

last: Meenhard

first: Rubinstein

middle: C

last: Jill

first: Chuang

last: Jeffrey


title: AP-1 regulates heterogeneous cellular dormancy in TNBC II

status: Public on May 18 2024

city: Shanghai

first: Yang

last: Dong

state: 上海

address: Shanghai

country: China

institute: Tongji University

summary: Slow-cycling cells (SCCs) are a heterogeneous subpopulation of tumor cells. They pre-exist in tumor tissues and evade killing by chemotherapeutics that target mitosis due to their dormant nature. We here utilized PKH26 retaining method to label and isolate SCCs, and RNA-seq was performed to understand the gene expression characteristics of SCCs. Here we reported that up-regulations of AP-1 subunits regulate triple negative breast cancer (TNBC) dormancy by up-regulating the expression of CDKN1A and GADD45 family as transcription factors.

overall_design: To investigate the gene expression characteristic of slow-cycling cells (SCCs), we first labeled MDA-MB-231 cells with PKH26 and chased in complete DMEM for 12 days. SCCs retain more PKH26 after multiple mitoses due to their slow-cycling nature. We then sorted cells with top 1% PE fluorescence intensity as SCCs by FACS. We also sorted cells with moderate fluorescence intensity as rapid cycling cells (RCCs) / proliferating cells control. Three biological replicates were collected from each group.  We performed gene expression profiling assay using data obtained from RNA-seq of 6 samples (two groups with three biological replicates per group). Comparative gene expression profiling analysis of RNA-seq data for SCCs (dormant) and RCCs (proliferating).

first: Dean

middle: G

last: Tang


title: Metabolic adaptation pilots the differentiation of human hematopoietic cells (scATAC-Seq)

first: Laëtitia

last: RACINE

overall_design:
Human hematopoietic CD34+ cells were grown in Xvivo medium supplemented with cytokines with or without energy metabolism inhibitor (DON, 2-DG or AOA) during 24h.
All samples are derived from an initial pool of six healthy donors.


title: Retinal organoids with X-linked retinoschisis RS1 (E72K) mutation exhibit a photoreceptor developmental delay and are rescued by gene augmentation therapy

status: Public on May 17 2024

city: Changsha

first: chunwen

last: duan

address: No. 932 Lushan South Road

institute: Central South University

summary: Background X-linked juvenile retinoschisis (XLRS) is an inherited disease caused by RS1 gene mutation, which leads to retinal splitting and visual impairment. The mechanism of RS1-associated retinal degeneration is not fully understood. Besides, animal models of XLRS have limitations in the study of XLRS. Here, we used human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) to investigate the disease mechanisms and potential treatments for XLRS. Methods hiPSCs reprogrammed from peripheral blood mononuclear cells of two RS1 mutant (E72K) XLRS patients were differentiated into ROs. Subsequently, we explored whether RS1 mutation could affect RO development and explore the effectiveness of RS1 gene augmentation therapy. Results ROs derived from RS1 (E72K) mutation hiPSCs exhibited a developmental delay in the photoreceptor, retinoschisin (RS1) deficiency, and altered spontaneous activity compared with control ROs. Furthermore, the delays in development were associated with decreased expression of rod-specific precursor markers (NRL) and photoreceptor-specific markers (RCVRN). Adeno-associated virus (AAV)-mediated gene augmentation with RS1 at the photoreceptor immature stage rescued the rod photoreceptor developmental delay in ROs with the RS1 (E72K) mutation.  Conclusions The RS1 (E72K) mutation results in the photoreceptor development delay in ROs and can be partially rescued by the RS1 gene augmentation therapy.

first: Chunwen

last: Duan

first: Jiansu

last: Chen

first: Shibo


title: Targeting neddylation and sumoylation in chemoresistant triple negative breast cancer

city: Houston

last: Chang

address: 6431 Fannin St

institute: University of Texas

summary: Triple negative breast cancer (TNBC) accounts for 15-20% of breast cancer cases in the United States. Systemic neoadjuvant chemotherapy (NACT), with or without immunotherapy, is the current standard of care for patients with early-stage TNBC. However, up to 70% of TNBC patients have significant residual disease once NACT is completed, which is associated with a high risk of developing recurrence within two to three years of surgical resection. To identify targetable vulnerabilities in chemoresistant TNBC, we generated longitudinal patient-derived xenograft (PDX) models from TNBC tumors before and after patients received NACT. We then compiled transcriptomes and drug response profiles for all models. Transcriptomic analysis identified the enrichment of aberrant protein homeostasis pathways in models from post-NACT tumors relative to pre-NACT tumors. This observation correlated with increased sensitivity in vitro to inhibitors targeting the proteasome, heat shock proteins, and neddylation pathways. Pevonedistat, a drug annotated as a NEDD8-activating enzyme (NAE) inhibitor, was prioritized for validation in vivo and demonstrated efficacy as a single agent in multiple PDX models of TNBC. Pharmacotranscriptomic analysis identified a pathway-level correlation between pevonedistat activity and post-translational modification (PTM) machinery, particularly involving neddylation and sumoylation targets. Elevated levels of both NEDD8 and SUMO1 were observed in models exhibiting a favorable response to pevonedistat compared to those with a less favorable response in vivo. Moreover, a correlation emerged between the expression of neddylation-regulated pathways and tumor response to pevonedistat, indicating that targeting these PTM pathways may prove effective in combating chemoresistant TNBC.

overall_design:
As part of the prospective neoadjuvant ARTEMIS trial (NCT02276443) aimed at addressing the issue of chemoresistance in TNBC, we obtained fine needle aspirates or core biopsies at critical clinical decision-making time points including: 1) clinical presentation before NACT (Pre, N=20), 2) Residual tumors after four cycles of Adriamycin (doxorubicin) and cyclophosphamide (Mid, N=8), and 3) at surgical resection of residual tumors after paclitaxel +/- targeted therapy (Post, N=6). This cohort consists of batch normalized (COMBAT) profiles from 34 PDX models wth 10 longitudinal sets.

***************************************************************
These samples are derived from patient tissue, and we do not have consent to share the raw sequence data in a publicly available database.  The raw data is available with a materials transfer agreement with the MD Anderson Cancer Center.
***************************************************************

first: Reid

middle: T

last: Powell

first: Amanda

last: Rinkenbaugh

first: Lei

last: Guo

first: Shirong

last: Cai

last: Shao

first: Xinhui

last: Zhou

first: Xiaomei

last: Zhang

first: Sabrina

last: Jeter-Jones

first: Chunxiao

last: Fu

first: Yuan

last: Qi

first: Faiza

middle: B

last: Hancock

first: Jason

last: White

last: Stephan

first: Peter

middle: J

last: Davies

first: Stacy

last: Moulder

first: W

middle: F

last: Symmans

first: Helen

last: Piwnica-Worms


title: Metabolic adaptation pilots the differentiation of human hematopoietic cells (scRNA-Seq and CITE-Seq)

first: Laëtitia

last: RACINE

overall_design:
Human hematopoietic CD34+ cells were grown in Xvivo medium supplemented with cytokines with or without energy metabolism inhibitor (DON, 2-DG or AOA) during 96h.
Some conditions were supplemented with alpha-ketoglutarate for rescue experiments. Control condition contains two biological replicates (CTRL and CTRL2), they were merged for downstream bioinformatic analysis.
Each sample is derived from a pool of at least seven healthy donors.
The cells were stained with CITE-seq antibodies (ADTs and HTOs) and inserted in a microfluidic chip for single cell analysis.
ADT/HTO barcode sequence hashtagged sample
TotalSeq™ A0251 anti-human hashtag1 GTCAACTCTTTAGCG CTRL, DON, DON+aK
TotalSeq™ A0252 anti-human hashtag2 TGATGGCCTATTGGG 2DG, CTRL+aK, 2DG+aK
TotalSeq™ A0126 anti-human CD133  TGGTAACGACAGTCC
TotalSeq™ A0054 anti-human CD34 GCAGAAATCTCCCTT

last: Racine

first: Romuald

last: Parmentier


title: Cell-type transcriptomes for the choanoflagellate Salpingoeca rosetta

city: San Francisco

first: David

middle: S

last: Booth

department: Biochemistry and Biophysics

institute: UCSF

summary: Transcriptome profiles of four cell types from the choanoflagellate Salpingoeca rosetta : Slow Swimmers, Rosettes, Fast Swimmers, and Thecate

overall_design: Slow Swimmer, Rosette, and Thecate cell type were grown to mid-log phase at 22°C before harvesting mRNA. Rosettes were induced from slow swimmers with outer membrane vesicles from the bacterium Algoriphagus machipongonensis. Fast swimmers were derived from Slow Swimmers by growing to stationary phase and heat shocking at 37°C for 2.75 h.

first: Fredrick

last: Leon


title: Acetylation is not mere a consequence of transcription: histones are acetylated without ongoing transcription [EU-seq]

city: Copenhagen N

first: Chunarum

last: Choudhary

state: --- Select a state ---

address: Blegdamsvej 3B

department: Novo Nordisk Foundation Center for Protein Research

country: Denmark

institute: University of Copenhagen

summary: In all eukaryotes, acetylation of histones lysine residues correlates with transcription activation. Whether histone acetylation is a cause or consequence of transcription is debated. One model suggests that transcription promotes the recruitment and/or activation of acetyltransferases and histone acetylation occurs as a consequence of ongoing transcription. However, the extent to which transcription shapes the global acetylation landscapes is not known. Here we show that global protein acetylation remains virtually unaltered after acute transcription inhibition. Transcription inhibition ablates the co-transcriptionally occurring ubiquitylation of H2BK120 but does not reduce histone acetylation. The combined inhibition of transcription and CBP/p300 further demonstrates that acetyltransferases remain active and continue to acetylate histones independently of transcription. Together, these results show that histone acetylation is not a mere consequence of transcription; acetyltransferase recruitment and activation is uncoupled from the act of transcription and histone and non-histone protein acetylation are sustained in the absence of ongoing transcription.

overall_design: EU-seq experiments in ESC with triptolide treatment. The corresponding EU-seq experiments in ESC without triptolide treatment are publiclly available on GSE146328.

first: Takeo

last: Narita

first: Chunaram


title: The GPI-anchor biosynthesis pathway is critical for syncytiotrophoblast differentiation and placental development

status: Public on May 19 2024

city: Valencia

first: Vicente

middle: Pérez

last: García

address: C/ Eduardo Primo Yúfera

country: Spain

institute: Fundación Centro de Investigación Príncipe Felipe

summary: The glycosylphosphatidylinositol (GPI) biosynthetic pathway in the endoplasmic reticulum (ER) is crucial for generating GPI-anchored proteins (GPI-APs), which are translocated to the cell surface and play a vital role in cell signaling and adhesion. This study focuses on two integral components of the GPI pathway, the PIGL and PIGF proteins, and their significance in trophoblast biology. We show that GPI pathway mutations impact on placental development impairing the development of the syncytiotrophoblast (SynT), and especially the SynT-II layer, which is essential for the establishment of the definitive nutrient exchange area within the placental labyrinth. CRISPR/Cas9 knockout of Pigl and Pigf in mouse trophoblast stem cells (mTSC) confirms the role of these GPI enzymes in syncytiotrophoblast differentiation. Mechanistically, impaired GPI-AP generation induces an excessive unfolded protein response (UPR) in the ER in mTSCs growing in stem cell conditions, akin to what is observed in human preeclampsia. Remarkably, the transcriptomic profile of Pigl- and Pigf-deficient cells separates human patient placental samples into preeclampsia and control groups suggesting an involvement of Pigl and Pigf in establishing a preeclamptic gene signature. Upon differentiation, the impairment of the GPI pathway hinders the induction of WNT signaling for early SynT-II development. Our study unveils the pivotal role of GPI biosynthesis in early placentation and uncovers a new preeclampsia gene expression profile associated with mutations in the GPI biosynthesis pathway, providing novel molecular insights into placental development with implications for enhanced patient stratification and timely interventions.

overall_design: The RNA-seq experiment assesses the transcriptional changes in wild type (Vector) versus Pigl knockout (KO) or Pigf KO mouse trophoblast stem cells at three time points: under stem cell conditions at 0 days (0d), and after 3 days (3d) and 6 days (6d) of differentiation.

first: Andrea

last: Álvarez-Sánchez

first: Johanna

last: Grinat

first: Paula

last: Doria-Borrell

first: Maravillas

last: Mellado-López

first: Érica

last: Pedrera-Alcocer

first: Marta

last: Malenchini

first: Salvador

last: Messeguer

first: Myriam

last: Hemberger

last: Pérez-García


title: Comparative transcriptome analysis reveals molecular damage associated with cryopreservation in Crassostrea angulata D-larvae rather than to cryoprotectant exposure

city: Faro

first: Elsa

last: Cabrita

state: Faro

address: Universidade do Algarve, Campus de Gambelas

department: Aquaculture research group (Aquagroup)

country: Portugal

institute: Centre of Marine Sciences (CCMAR)

summary: The Portuguese oyster Crassostrea angulata, a bivalve of significant economic and ecological importance, has faced a decline in both production and natural populations due to pathologies, climate change, and anthropogenic factors. To safeguard its genetic diversity and improve reproductive management, cryopreservation emerges as a valuable strategy. However, the cryopreservation methodologies lead to some damage in structures and functions of the cells and tissues that can affect post-thaw quality. Transcriptomics may help to understand the molecular consequences related to cryopreservation steps and therefore to identify different freezability biomarkers. This study investigates the molecular damage induced by cryopreservation in C. angulata D-larvae, focusing on two critical steps: exposure to cryoprotectant solution and the freezing/thawing process. Expression analysis revealed 3 differentially expressed genes between larvae exposed to cryoprotectant solution and fresh larvae and 511 differentially expressed genes in cryopreserved larvae against fresh larvae. The most significantly enriched gene ontology terms were “carbohydrate metabolic process”, “integral component of membrane” and “chitin binding” for biological processes, cellular components and molecular functions, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified the “neuroactive ligand receptor interaction”, “endocytosis” and “spliceosome” as the most enriched pathways. RNA sequencing results were validate by quantitative RT-PCR, once both techniques presented the same gene expression tendency and a group of 11 genes were considered important molecular biomarkers to be used in further studies for the evaluation of cryodamage. The current work provided valuable insights into the molecular repercussions of cryopreservation on D-larvae of Crassostrea angulata, revealing that the freezing process had a more pronounced impact on larval quality compared to any potential cryoprotectant-induced toxicity. Additionally, was identify 11 genes serving as biomarkers of freezability for D-larvae quality assessment. This research contributes to the development of more effective cryopreservation protocols and detection methods for cryodamage in this species.

overall_design: To characterize the transcriptional changes related with the different steps of cryopreservation, the whole transcriptome profile of fresh larvae diluted in FSW (fresh larvae), fresh larvae exposed to a CPAs (cryoprotectant exposed larvae) and post-thaw larvae (cryopreserved larvae) was compared. For this purpose, each D-larvae pool was exposed to the three following conditions, one control group and two treatments. As a control group, fresh larvae were concentrated in FSW as previously described. In the first treatment, larvae exposed to CPAs, the concentrated D-larvae were diluted with a 1:1 proportion in a CPAs consisting in a final concentration of 10% (v/v) dimethyl sulfoxide (DMSO) (Sigma-Aldrich), 1% (w/v) Polyvinylpyrrolidone (PVP)-40 40000 MW (Sigma-Aldrich) and 0.2 M Sucrose (Sigma-Aldrich) in milli-Q water, and incubated for 3 min of equilibrium time, at 4 ºC. After incubation, the pools of D-larvae were diluted in a 1:3 proportion in FSW to dissipate the CPAs. The second treatment intended to evaluate the effects of the freezing/thawing process. Following the same procedure as in the first treatment, the pooled larvae were incubated with the same CPAs. During the equilibrium time, the larvae pools diluted in the CPAs were loaded to 0.5 mL French straws (30,000 per straw) and maintained at 4 °C until finishes the 3 min equilibrium time. Subsequently, the straws were frozen in a programable biofreezer (Asymptote Grant EF600, United Kingdom) with the following freezing  protocol: 2.5 °C/min from 0 to -10 °C, hold for 5 min at -10 °C, 0.3 °C/min from -10 °C to -20 °C and 2.5 °C/min down to -35 °C, and finally, plunged into liquid nitrogen (LN) and stored in a LN container. After two months of storage, straws were thawed in a water bath set at 37 °C for 10 s. Afterward, a recovery bath was prepared, by diluting the content of each straw in 2 L of FSW during a period of incubation of 1 h at room temperature. This procedure allowed the removal of cryoprotectants. The post-thaw D-larvae were collected in a 30 µm mesh screen, washed and concentrated in 1.5 mL of FSW. Debris were eliminated, and the larvae quality was evaluated.

first: Catarina

last: Anjos

first: Daniel

last: Duarte

first: Elvira

last: Fatsini

first: Domitilia

last: Matias


title: The role of interleukin-10 receptor alpha (IL10Rα) in Mycobacterium avium subsp. paratuberculosis infection of a mammary epithelial cell line

city: Guelph

first: Christine

middle: Francoise

last: Baes

state: Ontario

address: 50 Stone Road East

department: Animal Biosciences

institute: University of Guelph

summary: Background: Johne’s disease is a chronic wasting disease caused by the bacterium Mycobacterium avium subspecies paratuberculosis (MAP). Johne’s disease is highly contagious and MAP infection in dairy cattle can eventually lead to death. With no available treatment for Johne’s disease, genetic selection and improvements in management practices could help reduce its prevalence. In a previous study, the gene coding interleukin-10 receptor subunit alpha (IL10Rα) was associated with Johne’s disease in dairy cattle. Our objective was to determine how IL10Rα affects the pathogenesis of MAP by examining the effect of a live MAP challenge on a mammary epithelial cell line (MAC-T) that had IL10Rα knocked out using CRISPR/cas9. The wild type and the IL10Rα knockout MAC-T cell lines were exposed to live MAP bacteria for 48 hours. Thereafter, mRNA was extracted from infected and uninfected cells. Differentially expressed genes were compared between the wild type and the IL10Rα knockout cell lines. Gene ontology was performed based on the differentially expressed genes to determine which biological pathways were involved. Results: Immune system processes pathways were targeted to determine the effect of IL10Rα on the response to MAP infection. There was a difference in immune response between the wild type and IL10Rα knockout MAC-T cell lines, and little difference in immune response between infected and not infected IL10Rα knockout MAC-T cells, indicating IL10Rα plays an important role in the progression of MAP infection. Additionally, these comparisons allowed us to identify other genes involved in inflammation-mediated chemokine and cytokine signalling, interleukin signalling and toll-like receptor pathways. Conclusions: Identifying differentially expressed genes in wild type and ILR10α knockout MAC-T cells infected with live MAP bacteria provided further evidence that IL10Rα contributes to mounting an immune response to MAP infection and allowed us to identify additional potential candidate genes involved in this process. We found there was a complex immune response during MAP infection that is controlled by many genes.

overall_design: This study includes RNA-Sequencing paired-end (2 x 150 bp) reads for four treatment groups: unedited bovine mammary epithelial (MAC-T) cells, unedited MAC-T cells infected with Mycobacterium avium subspecies paratuberculosis (MAP) bacteria, IL10Rα knockout cells, and IL10Rα knockout cells infected with MAP bacteria.

first: Aisha

last: Fong

first: Christina

last: Rochus

first: Umesh

middle: K

last: Shandilya

first: Maria

last: Muniz

first: Ankita

last: Sharma

first: Flavio

last: Schenkel

first: Niel

last: Karrow


title: MSA: Scalable DNA methylation BeadChip for human trait screening [EM-seq]

first: Wanding

last: Zhou

address: 3501 Civic Center Blvd

department: Pathology and Lab Medicine

institute: Children's Hospital of Philadelphia

summary: We designed and introduced a new methylation array concentrating on human trait screening and discovery. The new MSA (Methylation Screening Array) leveraged the massive Infinium platform-based data from epigenome-wide association studies, combined with updated knowledge from the latest single cell and cell type-resolution whole genome methylome profiles, to achieve scalable screening of epigenetics-trait association in an ultra-high sample-throughput. Our design encompassed diverse human trait associations, including those with genetic, biological, and demographical variables, environmental exposures, and common human diseases such as neurodegenerative, genetic, cardiovascular, infectious, and immune diseases. We comprehensively evaluated this array's reproducibility, accuracy, and capacity in supporting 5-hydroxymethylation profiling and comprehensive cell-type deconvolution in diverse human tissues. Our first data using this platform uncovered dynamic chromatin and tissue contexts of DNA modification variations and genetic variants with human trait associations.

overall_design: Three cell line (GM12878, K562, HCT116) 5mC profiles are generated using EM-seq. The methylomes are compared to MSA methylome profiles.

first: David

last: Goldberg


title: Profiling the neuroimmune cascade in 3xTg mice exposed to successive mild traumatic brain injuries

city: Atlanta

first: Levi

middle: B.

last: Wood

state: GA

address: 315 Ferst Dr NW Rm 3303

institute: Georgia Institute of Technology

summary: Repetitive mild traumatic brain injuries (rmTBI) sustained within a window of vulnerability can result in long term cognitive deficits, depression, and eventual neurodegeneration associated with tau pathology, amyloid beta plaques, gliosis, and neuronal and functional loss. However, we have limited understanding of how successive injuries affect the brain to result in these devastating long-term consequences. In the current study, we addressed the question of how repeated injuries affect the brain in the acute phase of injury (<24hr) by exposing the 3xTg-AD mouse model of tau and amyloid beta pathology to successive (1x, 3x, 5x) once-daily weight drop closed-head injuries and quantifying transcription at 30min, 4hr, and 24hr after each injury.

overall_design: Left somatomotor cortices of female triple transgenic (3xTg-AD) mice exposed to successive weight drop closed-head injuries. Three injury groups (1x, 3x, 5x) across four time points (pre-injury and 30m, 4h, & 24h post-injury) (n=3-4 per group, 40 total). Sample 30 was removed during analysis due to outlier status from corresponding protein data.

first: Alyssa

last: Pybus

first: Sara

last: Bitarafan

first: Erin

last: Buckley


title: Endothelial ROBO4 suppresses PTGS2/COX-2 expression and inflammatory diseases

status: Public on May 16 2024

city: Kyoto

first: Kazuo

last: Takayama

state: Kyoto

address: Shogoin Kawaharacho 53, Sakyo-ku

department: Center for iPS Cell Research and Application

country: Japan

institute: Kyoto University

summary: Endothelial cells control inflammation in inflammatory and infectious diseases through regulating endothelial gene expression and permeability. Roundabout4 (Robo4) is an endothelial-specific protein that stabilizes endothelial cells. Robo4 has been shown to ameliorate mouse inflammatory and severe infectious diseases, such as sepsis and COVID-19 by reducing vascular permeability. Despite this, it remains uncertain whether these are the only mechanisms by which Robo4 ameliorates the diseases. In this study, we carried out an RNA-seq analysis to investigate the genes regulated by Robo4 in endothelial cells stimulated by TNFα and identified gene, prostaglandin-endoperoxide synthase 2 (PTGS2) , which codes for Cycloocygenase-2 (COX-2). Mechanistic analysis revealed that Robo4 curbs COX-2 expression and endothelial hyperpermeability by preventing prolonged Rac1 activation. Analysis of Robo4 interacting protein identified IQ motif containing GTPase activating protein 1 (IQGAP1) that maintains active Rac1. Robo4 enhanced ubiquitination of IQGAP1 with a ubiquitin E3 ligase, TNF receptor-associated factor 7 (TRAF7), to inactivate Rac1.  Finally, Robo4 deficiency exacerbates COX-2 -associated inflammatory diseases, including arthritis, edema, and pain in mouse models. Taken together, Robo4 inhibits Rac1 activation by interacting with TRAF7 and IQGAP1, thereby suppressing the endothelial expression of COX-2 and reducing hyperpermeability.  Thus, we uncovered further Robo4 capabilities that suppresses COX-2 and inflammatory diseases, as well as their underlying mechanisms, indicating that endothelial Robo4 is a potential therapeutic target for diverse inflammatory diseases.

overall_design: The Robo4 siRNA-transfected or TNFα-treated HUVECs were collected, and then total RNA was collected and RNA-seq was performed.

first: Yoshiaki

last: Okada


title: Catabolite control protein C (CcpC) contributes to virulence and hydrogen peroxide-induced oxidative stress responses in Listeria monocytogenes

city: Mississippi State

first: Hossam

last: Abdelhamed

address: Wise drive

institute: CVM

summary: Listeria monocytogenes causes listeriosis, an infectious and potentially fatal disease of animals and humans. A diverse network of transcriptional regulators, including LysR-type catabolite control protein C (CcpC), is critical for the survival of L. monocytogenes and its ability to transition into the host environment. In this study, we explored the physiological and genetic consequences of deleting ccpC and the effects of such deletion on the ability of L. monocytogenes to cause disease. We found that ccpC deletion did not impact hemolytic activity, whereas it resulted in significant reductions in phospholipase activities. Western blotting revealed that the ΔccpC strain produced significantly reduced levels of the cholesterol-dependent cytolysin LLO relative to the wildtype F2365 strain. However, the ΔccpC mutant displayed no significant intracellular growth defect in macrophages. Furthermore, ΔccpC strain exhibited reduction in plaque numbers in fibroblasts compared to F2365, but  plaque size was not significantly affected by ccpC deletion. In a murine model system, the ΔccpC strain exhibited a significantly reduced bacterial burden in the liver and spleen compared to the wildtype F2365 strain. Interestingly, the deletion of this gene also enhanced the survival of L. monocytogenes under conditions of H2O2-induced oxidative stress. Transcriptomic analyses performed under H2O2-induced oxidative stress conditions revealed that DNA repair, cellular responses to DNA damage and stress, metalloregulatory proteins, and genes involved in the biosynthesis of peptidoglycan and teichoic acids were significantly induced in the ccpC deletion strain relative to F2365. In contrast, genes encoding internalin, 1-phosphatidylinositol phosphodiesterase, and genes associated with sugar-specific phosphotransferase system components, porphyrin, branched-chain amino acids, and pentose phosphate pathway were significantly downregulated in the ccpC deletion strain relative to F2365. This finding highlights CcpC as a key factor that regulates L. monocytogenes physiology and responses to oxidative stress by controlling the expression of important metabolic pathways.

sample_organism: Listeria monocytogenes serotype 4b str. F2365

overall_design:
To further investigate the mechanism underlying the reduced susceptibility of the ΔccpC strain to oxidative stress conditions, we examined the regulatory role of CcpC following exposure to H2O2-induced oxidative stress. RNA-seq was performed on total RNA harvested from wildtype and ΔccpC cultures that had been grown to exponential phase and then exposed to 8 mM H2O2 for 2.5
d1031 is the ccpC mutant strain while d1759 is a gltC mutant strain

first: Seto

last: Ogunleye

first: Shamima

last: Islam

first: Q

middle: M

last: Kader Chowdhury

first: Ozan

last: Ozdemir

first: Mark

last: Lawrence


title: Acetylation is not mere a consequence of transcription: histones are acetylated without ongoing transcription [ChIP-seq]

first: Chunarum

last: Choudhary

overall_design: H3K27ac ChIP-seq experiments in ESC with or without actinomycin D or NVP-2 treatment. EU-seq experiments in ESC with triptolide treatment.

first: Takeo

last: Narita

first: Aihara

last: Hitoshi

first: Maskey

last: Elina

first: Chunaram


title: Investigation of transcript 3' ends in Campylobacter jejuni wildtype and ribonuclease mutants

city: Würzburg

first: Cynthia

last: Sharma

state: Bavaria

address: Josef-Schneider-Straße 2

institute: University of Würzburg

summary: Termination site sequencing (term-seq) (Dar et al., 2016) was used to map transcript 3' ends and potential processing sites in Campylobacter jejuni NCTC11168 wildtype and ribonuclease deletion strains.

overall_design: Total RNA was extracted from C. jejuni NCTC11168 WT and RNase III (rnc, Cj1635c), RNase Y (rny, Cj1209), PNPase (pnp, Cj1253), and RNase R (rnr, Cj0631c) deletion strains grown to exponential phase, genomic DNA was removed with DNase I and rRNA depleted, cDNA libraries were prepared at Vertis Biotechnologie AG, Freising, Germany, deep-sequencing was performed on an Illumina NextSeq 500 instrument

first: Fabian

last: König

first: Sarah

last: Svensson


title: Lycorine attenuated proliferation and induced apoptosis on imatinib-resistant K562 cell by inhibiting autophagy.

city: lanzhou

first: bai

last: jun

institute: Lanzhou University Second Hospital

summary:
Background: Tyrosine kinase inhibitor (TKI) resistance is a significant factor exacerbating the burden on chronic myeloid leukemia (CML) patients and impacting clinical efficacy. The main goal is to offer new insights into overcoming drug resistance in treating CML.
Methods:  (IM) resistant K562/IM cells were generated using gradient induction. Responses to IM, lycorine, and autophagy modulators were assessed using CCK-8. Protein expression of Beclin-1, Atg5, LC3, Caspase-3, P62, Bax, Bcl-2, and P-gp was detected using Western blot. Lycorine-induced apoptosis and cell cycle changes were evaluated through flow cytometry, while autophagy alterations were detected using Monodansylcadaverine(MDC) staining. In the K562/IM mice model, Non-obese diabetic severe combined immunodeficent(NOD-SCID) mice were subcutaneously inoculated with K562/IM cells. After 17 days of lycorine injection, assessments included tumor size, Hematoxylin-eosin(HE) staining, and Ki67 expression.
Results: After 72 hours of IM treatment, K562/IM cells showed a 55.86-fold increase in drug resistance compared to K562 cells. Lycorine treatment for 24 hours inhibited cell proliferation and induced G0/G1 phase cell cycle arrest and apoptosis in both K562 and K562/IM cells. MDC staining indicated reduced autophagy in K562/IM cells, mitigated by lycorine. In vivo experiments demonstrated reduced tumor size and Ki67 proliferation index in the lycorine treatment group (K562+L, K562/IM+L) compared to the control group, particularly in the drug-resistant group. However, no significant change in Ki67 was observed in the K562 group after lycorine treatment.
Conclusion: In summary, K562/IM cells displayed heightened autophagy levels compared to K562 cells. Lycorine effectively impeded the proliferation of K562/IM cells through diverse mechanisms, including reduced autophagy, enhanced apoptosis, and induced cell cycle arrest.

overall_design: We utilized K562, K562+L, K562/IM and K562/IM+L cells for mRNA sequencing, involving three samples for each condition (K562, K562+L, K562/IM and K562/IM+L). In each case, 1 × 10^6 cells were dispersed in 1 mL of Trizol reagent. The mRNA sequencing, along with subsequent analysis, was performed by Shanghai Tianhao Biotechnology Co., Ltd.


title: Acetylation is not mere a consequence of transcription: histones are acetylated without ongoing transcription

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

overall_design: Refer to individual Series


title: Semimethylation is a feature of Diffuse Large B-cell Lymphoma, and subgroups with poor prognosis are characterized by global hypomethylation and short telomere length

city: Umeå

first: Magnus

last: Hultdin

state: Vasterbotten

address: 6M-building, NUS, 2d floor

department: Department of Medical Biosciences, Pathology

institute: Umeå University

summary: We studied the DNA methylation profiles of 93 LBCL cases: Diffuse large B-cell lymphoma not otherwise specified (DLBCL, n=66), High-grade B-cell lymphoma (n=7), Primary CNS lymphoma (n=8), and transformation of indolent B-cell lymphoma (n=12). One sample of normal GC B-cells from tonsils was included as a control. LBCL cases had a particularly aberrant semimethylated pattern with large intertumor variation and overall low hypermethylation. In cases treated with R-CHOP-like regimens, a high percentage of global hypomethylation was independently associated with worse disease-specific survival and progression-free survival.

overall_design: Genomic DNA from fresh frozen tissue samples were extracted using the AllPrep DNA/RNA mini kit (#80204, Qiagen, Hilden, GER). The DNA was bisulfite converted using the EZ DNA Methylation Kit (Zymo Research, Irvine, CA) and the Infinium MethylationEPIC BeadChip (Illumina, San Diego, CA), with coverage of >850 000 CpG sites, were used for genome-wide assessment of DNA methylation profiles. 96 LBCL samples and one normal GC B-cell sample are submitted. 3 LBCL samples (sample 94, sample 95, and sample 96) were excluded from the analysis after the normalization and pre-processing due to low tumor cell content.

first: Olivia

last: Carlund

first: Elina

last: Thörn

first: Pia

last: Osterman

first: Maja

last: Fors

first: Andy

last: Dernstedt

first: Mattias

middle: E

last: Forsell

first: Martin

last: Erlanson

last: Landfors

first: Sofie

last: Degerman


title: Spatial transcriptomics of intraductal carcinoma of the prostate (IDCP)

city: Suita

first: Daisuke

last: Motooka

state: Osaka

address: 3-1, Yamadaoka

institute: NGS core facility, Research Institute for Microbial Diseases, Osaka University

summary: To identify an IDCP-specific gene expression profile effectively, we made an innovative attempt to characterize a typical IDCP case by spatial gene expression analysis.

overall_design: Expression profiling by high throughput sequencing

first: Ryuta

last: Watanabe

first: Noriyoshi

last: Miura

first: Mie

last: Kurata

first: Riko

last: Kitazawa

first: Tadahiko

last: Kikugawa

first: Takashi

last: Saika


title: Premeiotic 24-nt phasiRNAs are present in the Zea genus and unique in biogenesis mechanism and molecular function [TraPR small RNA-seq]

status: Public on May 15 2024

city: Wuhan

first: Junpeng

last: Zhan

state: Hubei

address: 1 Shizishan Street, Hongshan District

institute: Huazhong Agricultural University

type: Non-coding RNA profiling by high throughput sequencing

summary: Reproductive phasiRNAs are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs–named premeiotic 24-nt phasiRNAs–have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 for biogenesis, but premeiotic 24-nt phasiRNAs are unique in that they are likely (i) not triggered by microRNAs, (ii) not loaded by AGO18 proteins, and (iii) not capable of mediating cis-cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass lineage than previously known.

overall_design:
Small RNA and degradome profiling of anther materials from five maize inbred lines, a maize mutant, and three teosinte species or subspecies
Please note that the *mm in sample titles represents the length of the anther used for the corresponding sample (e.g. 0.4-mm anther for 'B73-0.4mm-rep1' sample), and that the 'ago18HM' and 'ago18HT' represents 'ago18 triple homozygous mutant' and 'ago18 triple heterozygote', respectively.

first: Sébastien

last: Bélanger

first: Scott

last: Lewis

first: Chong

last: Teng

first: Madison

last: McGregor

first: Aleksandra

last: Beric

first: Michael

last: Schon

last: Nodine

first: Blake

last: Meyers


title: 1,25-Dihydroxyvitamin D3 Suppresses Prognostic Survival   Biomarkers Associated with Cell Cycle and Actin Organization in a Non-Malignant African American Prostate Cell Line

status: Public on May 14 2024

city: Duarte

first: Yate-Ching

last: Yuan

address: 1500 East Duarte

institute: City of Hope

summary: Vitamin D3 is a steroid hormone that has been shown to prevent tumor growth in prostate cells. Not having enough vitamin D3 in the blood has been linked to advanced prostate cancer and mortality, especially in African American men. We wanted to understand how vitamin D affected pathways that keep prostate cells from becoming cancerous, which could lead to new therapeutic targets and treatments, especially for African American men who tend to be more prone to being vitamin D deficient compared to European men. Here, we studied a non-cancerous African American prostate cell line treated with the active form of vitamin D with a concentration similar to what is found in the body for 24 h. Using RNA whole-transcriptome sequencing, we compared these treated cells with untreated cells to assess genes and pathways significantly changed due to treatment. We found that vitamin D affected the activity of 1600 genes, mainly suppressing pathways linked to prostate cell movement, growth, and viability. Only two genes, ANLN and ECT2, were strongly correlated with prostate cancer prognosis. Downregulation of ANLN and ECT2 was also shown to repress signaling pathways involved in prostate cell movement, growth, malignant transformation, and viability. In further validation utilizing prostate cancer cohorts comprised of African American and European American men, patients tended to have better sur-vival rates when these genes were less active. Our results suggest that vitamin D decreases the activity of these genes and could be important for preventing prostate cancer, especially for African American men. This could lead to the development of new treatments targeting specific genes and pathways involved in prostate cancer growth.

overall_design: A non-malignant African American cell line, RC77N/E, was treated with 10nM of vitamin D metabolite, calcitriol, for 24 hrs (in triplicates). To identify differentially expressed genes regulated by calcitriol, total RNASeq was performed in treated vs untreated group.

first: Jabril

middle: R

last: Johnson

first: Rachel

middle: N

last: Martini

first: Leanne

last: Woods-Burnham

first: Mya

last: Walker

first: Greisha

last: Ortiz-Hernandez

first: Firas

last: Kobeissy

first: Dorothy

last: Galloway

first: Amani

last: Gaddy

first: Chidinma

last: Oguejiofor

last: Allen

first: Deyana

first: Melissa

last: Davis

first: Sean

last: Kimbro

first: Clayton

last: Yates

first: Adam

last: Murphy

first: Rick

last: Kittles


title: Investigation of the regulon and RNA targets of RNase Y in Campylobacter jejuni

first: Cynthia

last: Sharma

summary: RNA-seq was used to study gene expression profiles and potential processing sites in Campylobacter jejuni NCTC11168 wildtype and RNase Y (rny, Cj1209) deletion.

overall_design: Total RNA was extracted from C. jejuni NCTC11168 WT and RNase Y deletion strains grown to exponential phase, genomic DNA was removed with DNase I and rRNA depleted, cDNA libraries were prepared at Vertis Biotechnologie AG, Freising, Germany, deep-sequencing was performed on an Illumina NextSeq 500 instrument

first: Fabian

last: König

first: Sarah

middle: L

last: Svensson


title: Integrating Gene Expression and Splicing Dynamics Across Dose-Response Oxidative Modulators

city: Durham

first: Rasim

last: Barutcu

state: NC

address: 6 Davis Drive

institute: ScitoVation

summary: Toxicological risk assessment increasingly utilizes transcriptomics to derive point of departure (POD) and modes of action (MOA) for chemicals. One essential biological process that allows a single gene to generate several different RNA isoforms is called alternative splicing. To comprehensively assess the role of splicing dysregulation in toxicological evaluation and elucidate its potential as a complementary endpoint, we performed RNA-seq on A549 cells treated with five oxidative stress modulators across a wide dose range. Differential gene expression (DGE) showed limited pathway enrichment except at high concentrations. However, alternative splicing analysis revealed variable intron retention events affecting diverse pathways for all chemicals in the absence of significant expression changes. For instance, diazinon elicited negligible gene expression changes but progressive increase in the number of intron retention events, suggesting splicing alterations precede expression responses. Benchmarkdose modeling of intron retention data highlighted relevant pathways overlooked by expression analysis. Systematic integration of splicing datasets should be a useful addition to the toxicogenomic toolkit. Combining both modalities paint amore complete picture of transcriptomic dose-responses. Overall, evaluating intron retention dynamics afforded by toxicogenomics may provide biomarkers that can enhance chemical risk assessment and regulatory decision making. This work highlights splicing-aware toxicogenomics as a possible additional tool for examining cellular responses.

overall_design: RNA-seq analysis of A549 and MCF7 cells treated with different concentrations of test chemicals, sulforaphane, prochloraz , paraquat , diazinon, and 2,4-dinitrochlorobenzene

first: A

first: Michael

last: Black

first: Raymond

last: Samuel

first: Scott

last: Slattery

first: Patrick

middle: D

last: McMullen

first: Andy

last: Nong


title: Premeiotic 24-nt phasiRNAs are present in the Zea genus and unique in biogenesis mechanism and molecular function [Smart-seq2+nanoPARE]

first: Junpeng

last: Zhan

sample_organism:
Zea mays subsp. mexicana
Zea luxurians
Zea mays subsp. parviglumis
Zea mays subsp. mays

overall_design:
Small RNA and degradome profiling of anther materials from five maize inbred lines, a maize mutant, and three teosinte species or subspecies
Please note that each processed data file was generated from both Smart-seq2 and nanoPARE samples, and is linked to the corresponding Smart-seq2 sample record.
Please note that the *mm in sample titles represents the length of the anther used for the corresponding sample (e.g. 0.4-mm anther for 'B73-0.4mm-rep1' sample), and that the 'ago18HM' and 'ago18HT' represents 'ago18 triple homozygous mutant' and 'ago18 triple heterozygote', respectively.

first: Sébastien

last: Bélanger

last: Lewis

first: Chong

last: Teng

first: Madison

last: McGregor

first: Aleksandra

last: Beric

last: Schon

last: Nodine

first: Blake

middle: C

last: Meyers


title: Mediator Kinase Inhibition Impedes Transcriptional Plasticity and Prevents Resistance to ERK/MAPK-Targeted Therapy in KRAS-Mutant Cancers

first: Daniel

middle: Philip

last: Nussbaum

address: 203 Research Drive, MSRB1, Room 488

institute: Duke University

summary: Acquired resistance remains a major challenge for therapies targeting oncogene activated pathways. KRAS is the most frequently mutated oncogene in human cancers, yet strategies targeting its downstream signaling kinases have failed to produce durable treatment responses. Here, we developed multiple models of acquired resistance to dual-mechanism ERK/MAPK inhibitors across KRAS-mutant pancreatic, colorectal, and lung cancers, and then probed the long-term events enabling survival against this class of drugs. These studies revealed that resistance emerges secondary to large-scale transcriptional adaptations that are diverse and tumor-specific. Transcriptional reprogramming extends beyond the well-established early response, and instead represents a dynamic, evolved population-level process that is refined to attain a stably resistant phenotype. Mechanistic and translational studies reveal that resistance to dual-mechanism ERK/MAPK inhibition is broadly susceptible to manipulation of the epigenetic machinery, and that Mediator kinase, in particular, can be co-targeted at a bottleneck point to prevent diverse, tumor-specific resistance programs.

overall_design: RNA extracted from cell lines treated in each indicated condition were treated with RNA-sequencing. All experiments were performed in biological triplicate.


title: Alternative isoform expression of key thermogenic genes in human beige adipocytes

city: Oslo

first: Philippe

last: Collas

address: PO Box 1112 Blindern

department: Institute of Basic Medical Sciences

country: Norway

institute: University of Oslo


title: Investigation of RNA 5' ends in Campylobacter jejuni wildtype and an RNase III mutant strain

first: Cynthia

last: Sharma

summary: differential RNA sequencing (dRNA-seq) (Sharma et al., 2010; Dugar et al., 2013) was used to differentially map 5' ends in Campylobacter jejuni NCTC11168 wildtype and RNase III (rnc, Cj1635c) deletion.

overall_design: Total RNA was extracted from C. jejuni NCTC11168 WT and RNase III deletion strains grown to exponential phase, genomic DNA was depleted with DNase I, RNA was treated with terminator exonuclease (TEX) to enrich for primary transcripts, cDNA libraries were prepared at Vertis Biotechnologie AG, Freising, Germany, deep-sequencing was performed on an Illumina HiSeq 2000 instrument

first: Fabian

last: König

first: Gaurav

last: Dugar

last: Svensson


title: ZZZ3 protects human embryonic stem cells from nucleolar stress by boosting mTOR/ribosome pathway

city: Turin

first: Chiara

last: Cicconetti

state: (TO)

address: Via Nizza, 52

department: Dept. of Life Sciences and Systems Biol

country: Italy

institute: University of Turin

summary: Embryonic stem cells (ESCs) are defined as stem cells with self-renewing and differentiation capabilities. These unique properties are tightly regulated and controlled by complex genetic and molecular mechanisms whose understanding is essential for both basic and translational research. A large number of existing studies in the broader literature have mostly focused on understanding the molecular mechanisms governing pluripotency and differentiation of ESCs, while the regulation of proliferation has received comparably less attention. Proliferation is a fundamental aspect of stem cell biology and despite its importance, the intricate mechanisms governing proliferation in human ESCs are not fully understood. In mouse ESCs, pluripotency and proliferation can be independent processes meaning that it is possible for mouse ESCs to maintain their pluripotent state without actively proliferating. Ribosome biogenesis and translation have emerged as critical pathways that play essential roles in regulating stem cell homeostasis influencing cell growth and division rates. Here, we aim at investigating the role of ZZZ3 (Zinc Finger ZZ-Type Containing 3) in human ESCs homeostasis. ZZZ3 is expressed in both mouse and human pluripotent stem cells and regulates genes encoding ribosomal proteins (RPGs). However, its precise function in human pluripotent stem cells (PSCs) still remains elusive. Interestingly, we found that knockdown of ZZZ3 strongly decreases ribosome biogenesis, translation, and mTOR signaling leading to nucleolar stress and significant reduction of cell proliferation. This process occurs without affecting pluripotency, suggesting that ZZZ3-depleted ESCs enter a dormant-like state and that proliferation and pluripotency can be uncoupled also in human ESCs. Our study shows that ZZZ3 controls ribosome biogenesis, translation, and proliferation of stem cells and protects hESCs from nucleolar stress.

overall_design:
To investigate the role of Zinc Finger ZZ-Type Containing 3 (ZZZ3) in pluripotent stem cells, we establish embryonic stem cell (ESC) lines in which ZZZ3 has been knocked down by shRNA
We conducted transcriptomics analysis using RNA-seq data obtained from two distinct ZZZ3-silenced ESC lines, along with their corresponding scramble control. Each sample was run in three technical replicates.
We compared gene expression profile derived from RNAseq data of knocked down cell line (ESC1_KD_ZZZ3 and ESC2_KD_ZZZ3) to data of their relative control (ESC1_SCR and ESC2_SCR)

first: Michela

last: Lo Conte

first: Valeria

last: Lucchino

first: Stefania

last: Scalise

first: Clara

last: Zannino

first: Maria

last: Murfuni

first: Luana

last: Scaramuzzino

first: Danilo

last: Matassa

first: Anna

last: Procopio

first: Desiree

last: Valente

first: Graziano

last: Martello

first: Giovanni

last: Cuda

first: Elvira

middle: I

last: Parrotta


title: Reproduction-associated pathways in females of gibel carp (Carassius gibelio) shed light on the molecular mechanisms of the coexistence of asexual and sexual reproduction

city: Brno

first: Andrea

middle: Vetešníková

last: Šimková

address: Kamenice 5

department: Department of Botany and Zoology

institute: Masaryk University

summary: Gibel carp (Carassius gibelio) is a cyprinid fish that originated in eastern Eurasia and is considered as invasive in European freshwater ecosystems. The populations of gibel carp in Europe are mostly composed of asexually reproducing triploid females (i.e., reproducing by gynogenesis) and sexually reproducing diploid females and males. Although some cases of coexisting sexual and asexual reproductive forms are known in vertebrates, the molecular mechanisms maintaining such coexistence are still in question. Both reproduction modes are supposed to exhibit evolutionary and ecological advantages and disadvantages. To better understand the coexistence of these two reproduction strategies, we performed transcriptome profile analysis of gonad tissues (ovaries), and studied the differentially expressed reproduction-associated genes in sexual and asexual females. We used high-throughput RNA sequencing to generate transcriptomic profiles of gonadal tissues of triploid asexual females and males, diploid sexual males and females of gibel carp, as well as diploid individuals from two closely-related species, C. auratus and Cyprinus carpio. Using SNP clustering, we showed the close similarity of C. gibelio and C. auratus with a basal position of C. carpio to both Carassius species. Using transcriptome profile analyses, we showed that many genes and pathways are involved in both gynogenetic and sexual reproduction in C. gibelio; however, we also found that 1500 genes, including 100 genes involved in cell cycle control, meiosis, oogenesis, embryogenesis, fertilization, steroid hormone signaling and biosynthesis were differently expressed in the ovaries of asexual and sexual females. We suggest that the overall downregulation of reproduction-associated pathways in asexual females, and their maintenance in sexual ones, allow for their stable coexistence, integrating the evolutionary and ecological advantages and disadvantages of the two reproductive forms. However, we showed that many sexual-reproduction-related genes are maintained and expressed in asexual females, suggesting that gynogenetic gibel carp retains the genetic toolkits for meiosis and sexual reproduction. These findings shed new light on the evolution of this asexual and sexual complex.

sample_organism:
Carassius auratus
Cyprinus carpio
Carassius gibelio

overall_design: Here, the molecular mechanisms associated with reproduction in C. gibelio were analysed to study the coexistence of asexual and sexual forms. In particular, the expression of reproduction-related genes was expected to differ between asexual and sexual females, since meiosis-related genes are not important for asexually reproducing individuals. To test this hypothesis, transcriptome profile analyses of gonadal tissues (ovaries) from asexual females and sexual females of C. gibelio were performed. In addition, the transcriptomes of the closely-associated species C. carpio and C. auratus were also analysed, with a particular emphasis on the genes contributing to sexual reproduction.

first: Florian

last: Jacques

first: Tomáš

last: Tichopád

first: Martin

last: Demko

first: Vojtěch

last: Bystrý

first: Kristína

last: Civáňová Křížová

first: Mária

last: Seifertová

first: Kristýna

last: Voříšková

first: Md

last: Mehedi Hasan Fuad

first: Lukáš

last: Vetešník


title: Reanalysis of white and brite adipocyte ChIP-Seq of PPARG and MED1 in the hg38 genome

first: Philippe

last: Collas

summary: To compare the binding of PPARG and MED1 at the TSSes of differentially expressed isoforms in white and brite (also known as beige) adipocytes, data from GSE59703 (Loft et al., 2015) was reanalysed on the hg38 genome.  We find that a large majority of the splice variants arise from differential TSS usage, with beige-specific TSSs being enriched for PPARγ and MED1 binding compared to white-specific TSSs.

relation:
SubSeries of: GSE256262
Reanalysis of: GSM1443809
Reanalysis of: GSM1443810
Reanalysis of: GSM1443811
Reanalysis of: GSM1443812
Reanalysis of: GSM1537677
Reanalysis of: GSM1537678
Reanalysis of: GSM1537679
Reanalysis of: GSM1537680
Reanalysis of: GSM1443817
Reanalysis of: GSM1443818

overall_design: PPARgamma, MED1 ChIP-Seq and Input reads for white and brite adipocytes were downloaded from Gene Expression Omnibus (GEO) accession GSE59703 (Loft et al. 2015). These consisted of two replicates per antibody in each condition and an input file per condition to a total of 10 samples.  Reads from replicates were merged and aligned to hg38 using Bowtie2. Peaks were detected using MACS3. ChIP/Input ratio tracks were generated using bamCompare in deepTools. Mean ratios (ChIP/Input) of the PPARgamma and MED1 were calculated for +/- 250 bp windows around the TSS using multiBigwigSummary from deepTools.

first: Sarah

last: Hazell Pickering

first: Mohamed

last: Abdelhalim

first: Nolwenn

last: Briand


title: ChIP-seq of active and repressive histone post-translational modifications in human beige and white adipocytes.

summary: Background: The beneficial effect of thermogenic adipocytes in maintaining body weight and protecting against metabolic disorders has raised interest in understanding the regulatory mechanisms defining white and beige adipocyte identity. Although alternative splicing has been shown to propagate adipose browning signals in mice, this has yet to be thoroughly investigated in human adipocytes.  Methods: We performed parallel white and beige adipogenic differentiation using primary adipose stem cells from 6 unrelated healthy subjects, and assessed differential gene and isoform expression in mature adipocytes by RNA sequencing. Results: We find 693 exon junctions with robust differential usage between white and beige adipocytes in all 6 subjects, mapping to 507 genes. Importantly, only 8% of these differentially spliced genes are also differentially expressed, indicating that alternative splicing constitutes an additional layer of gene expression regulation during beige adipocyte adipogenic differentiation. Functional classification of alternative isoforms point to a gain of function for key thermogenic regulators such as PPARG, CITED1 and PEMT. We find that a large majority of the splice variants arise from differential usage of transcription start sites (TSSs), with beige-specific TSSs being enriched for PPARγ and MED1 binding compared to white-specific TSSs. Finally, we validate beige specific isoform expression at the protein level for two thermogenic regulators, PPARγ and PEMT. Discussion: These results indicate that differential isoform expression through alternative TSS usage is an important regulatory mechanism for human adipocyte thermogenic specification.

overall_design: Primary human adipocyte stem cells from a single subject were differentiated into both beige and white adipocytes in the presence or absence of rosiglitazone, respectively, and harvested for Chromatin Immunoprecipation and sequencing (ChIP-Seq). Four histone post translational modifications were assayed: H3K4me3, H3K27ac, H3K4me1 and H3K27me3.


title: Premeiotic 24-nt phasiRNAs are present in the Zea genus and unique in biogenesis mechanism and molecular function [small_RNA]

first: Junpeng

last: Zhan

platform_organism:
Zea mays subsp. mexicana
Zea luxurians
Zea mays subsp. parviglumis
Zea mays subsp. mays

first: Sébastien

last: Bélanger

first: Scott

last: Lewis

first: Chong

last: Teng

first: Madison

last: McGregor

first: Aleksandra

last: Beric

first: Michael

last: Schon

last: Nodine

first: Blake

last: Meyers


title: Immunologic adaptation following recombinant amphiregulin ( rAREG) exposure in high grade serous ovarian cancer cell lines

status: Public on May 13 2024

city: Providence

first: Nicole

middle: Elizabeth

last: James

state: Rhode Island

address: 200 Chestnut Street

department: OB/GYN

institute: Women and Infants Hospital

summary:
770 genes as well as pathways were deternined in high grade serous ovarian cancer cell lines, OVCAR8WT and PEA1 cells stimulated with either BSA control or recombinant AREG (rAREG)
n/a

overall_design: A total of 12 samples were included in this experiment; OVCAR8WT cells stimulated with BSA control , OVCAR8WT cells stimulated with rAREG, PEA1 cells stimulated with BSA control, PEA1 cells stimulated with rAREG. All treatments were performed at 2 hour timepoints, with 3 biological replicates for each treatment.

first: Jasmine

last: Ebott

first: Christoph

last: Schorl


title: Transcriptomic characterization of lung pericytes in systemic sclerosis associated interstitial lung disease (SSc-ILD)

city: Charleston

first: Carol

last: Feghali-Bostwick

state: SC

address: 96 Jonathan Lucas St room 822

department: Medicine

institute: Medical University of South Carolina

summary: RNA sequencing and differential expression analyses were performed to identify differentially expressed genes in SSc lung pericytes as compared to normal.

overall_design: Gene and systems level analyses revealed the perturbation of major pathways including prostaglandin, PI3K-Akt, calcium and vascular remodeling pathways.

first: Ludivine

last: Renaud

first: Carole

last: Wilson

first: Lynn

last: Schnapp


title: GLP-1 directed NMDA receptor antagonism for obesity treatment [I]

city: Copenhagen

first: Lars

middle: Roed

last: Ingerslev

address: Blegdamsvej 3B

department: NNF Center for Basic Metabolic Research

institute: Copenhagen University

summary: The N-methyl-d-aspartate (NMDA) receptor is a glutamate-activated cation channel critical to many processes in the brain. Genome-wide association studies (GWAS) suggest that glutamatergic neurotransmission and NMDA receptor-mediated synaptic plasticity is important for body weight homeostasis1. Here, we report the engineering and preclinical development of a first-in-class bimodal molecule that integrates NMDA receptor antagonism with glucagon-like peptide-1 (GLP-1) receptor agonism to effectively reverse obesity, hyperglycemia, and dyslipidemia in rodent models of metabolic disease. We demonstrate that GLP-1-directed delivery of the NMDA receptor antagonist MK-801 affects NMDA receptor-mediated synaptic plasticity in the hypothalamus. Importantly, peptide-targeting of MK-801 specifically to GLP-1 receptor-expressing brain regions circumvent adverse physiological and behavioral effects associated with MK-801 monotherapy. In sum, our approach demonstrates the feasibility of cell specific ionotropic receptor-modulation via peptide targeting and highlights the therapeutic potential of unimolecular mixed GLP-1 receptor agonism and NMDA receptor antagonism for obesity treatment.

overall_design: Comparative gene expression profiling analysis of RNA-seq data from brain stem and nucleus accumbens of mice which had been randomized to one of four treatment groups (Vehicle, MK-801, GLP-1 or MK-801/GLP-1). 8 replicates are sequenced for each tissue and treatment combination.

first: Jonas

last: Petersen

first: Mette

middle: Q

last: Ludwig

first: Vaida

last: Juozaityte

first: Pablo

last: Ranea-Robles

first: Charlotte

last: Svendsen

first: Eunsang

last: Hwang

first: Amalie

middle: W

last: Kristensen

last: Fadahunsi

first: Jens

last: Lund

first: Alberte

last: Breum

first: Cecilie

middle: V

last: Mathiesen

first: Luisa

last: Sachs

first: Roger

last: Moreno-Justicia

first: Rebecca

last: Rohlfs

first: James

last: Ford

first: Jon

last: Douros

first: Brian

last: Finan

first: Bryan

last: Portillo

first: Kyle

last: Grose

first: Jacob

last: Trauelsen

first: Annette

last: Feuchtinger

first: Richard

last: DiMarchi

first: Thue

last: Schwartz

first: Atul

last: Desmukh

first: Morten

middle: B

last: Thomsen

first: Kristi

middle: A

last: Kohlmeier

first: Kevin

last: Williams

first: Tune

middle: H

last: Pers

first: Bente

last: Frølund

first: Kristian

last: Strømgaard

first: Anders

last: Klein

first: Christoffer

last: Clemmensen