title: Limb connective tissue is organized in a continuum of promiscuous fibroblast identities during development

city: Paris

first: Estelle

last: Hirsinger

address: 9 quai Saint Bernard

department: LBD

institute: IBPS

summary: Although connective tissues play critical roles in development, our understanding of connective tissue fibroblast developmental programs lag behind that of other components of the musculoskeletal system mainly because fibroblasts are highly heterogeneous and poorly characterized. Combining single-cell RNA-sequencing-based strategies, including trajectory inference, and in situ hybridization analyses, we address the diversity of connective tissue fibroblasts and their developmental trajectories during chicken limb foetal development. We show that fibroblasts switch from a positional information to a lineage diversification program during their development. Muscle connective tissue and tendon contain several fibroblast populations that emerge asynchronously. In fine, these populations map to distinct layers, prefiguring the adult fibroblast layers. Populations that are close in transcriptional identity are found in neighboring layers. Altogether, we propose that the limb connective tissue is organised in a continuum of promiscuous fibroblasts identities, allowing for the robust and efficient connection of highly different tissues such as muscle, bone and skin.

overall_design: Single cell mRNA profiles of whole forelimbs in wild type chicken embryos at two developmental stages (E7 and E9)

first: Cédrine

last: Blavet

first: Marie-Ange

last: Bonnin

first: Léa

last: Bellenger

first: Tarek

last: Gharsalli

first: Delphine

last: Duprez


title: Transcriptomic changes caused by mutation in xylanase regulator 1 (xyr1) in Trichoderma reesei

status: Public on Jul 19 2024

city: Espoo

first: Emmi

last: Sveholm

address: Tekniikantie 21

country: Finland

institute: VTT Technical Research Centre of Finland

type: Expression profiling by high throughput sequencing

summary: Trichoderma reesei is known for its ability to produce large amounts of extracellular proteins and is one of the most important industrially used filamentous fungus. Xylanase regulator 1 (XYR1) as the master regulator is responsible for the activation of cellulase and hemicellulase gene expression, normally under inducing conditions. It has been reported that strains with point mutations in certain areas of xyr1 bypass the carbon catabolite repression, allowing cellulase and hemicellulase production even in the presence of glucose. These mutations also change the profile of produced proteins, shifting it more towards xylanase production, and increase the overall protein production in inducing conditions. However, how these mutations alter the metabolism and other cellular processes to cause these changes remains unclear.  In the present study, our aim was to explore changes caused by a point mutation in xyr1 on transcriptomic and metabolic level to better understand the reasons behind the increased protein production in both repressing glucose and inducing lactose conditions. We observed that the xyr1 mutant strain built more biomass and produced more extracellular proteins during growth on lactose compared to the wild type xyr1 strain. Genes involved in oxidoreductive D-galactose catabolism pathway were upregulated in the xyr1 mutant strain, potentially contributing to the more efficient utilization of lactose. The shift from utilizing lactose to using glucose as carbon source seemed faster in the xyr1 mutant strain. Clustering and enrichment analysis showed over-representation of mitochondria-related Gene Ontology terms in clusters where gene expression was higher in the xyr1 mutant, indicating that mitochondria play a role in the altered metabolic state associated with the xyr1 mutation. Metabolomics revealed that free tyrosine was more abundant in the xyr1 mutant strain in all measured timepoints, whereas multiple fatty acids were less abundant in the mutant strain on glucose.  The results contribute to more in-depth knowledge on T. reesei physiology and aid in finding new targets for improved protein production.

overall_design: Strains QM9414 wtXYR1 (cbh1 under synthetic promoter SES, wild type xyr1 under pdc1 promoter, control strain) and mutXYR1 (cbh1 under synthetic promoter SES, mutated (V821F xyr1 under pdc1 promoter) were grown in 250 mL Ambr bioreactors first on batch lactose for about 72 hours until most lactose was depleted and then on glucose feed for about 27 hours. Samples were collected during batch lactose phase at around 52 hours, then during glucose feed at 78 hours and 99 hours from the start of the cultivation. Four biological replicates were included per strain and sample.

first: Tanja

last: Paasela

first: Tiina

last: Pakula


title: The osteocytic actions of glucocorticoids on bone mass, mechanical properties, or perilacunar remodeling outcomes are not rescued by PTH

status: Public on Jul 18 2024

city: San Francisco

first: Cristal

last: Yee

state: CA

address: 35 Medical Center Way, 923A

department: Orthopaedic Surgery Department

country: USA

institute: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO

summary: Glucocorticoids (GC) and parathyroid hormone (PTH) are widely used therapeutic endocrine hormones where their effects on bone and joint arise from actions on multiple skeletal cell types. In osteocytes, GC and PTH exert opposing effects on perilacunar canalicular remodeling (PLR). Suppressed PLR can impair bone quality and joint homeostasis, including in GC-induced osteonecrosis. However, combined effects of GC and PTH on PLR are unknown. Focusing on subchondral bone and joint homeostasis, we hypothesize that PTH, a PLR agonist, could rescue GC-suppressed PLR. The skeletal effects of GC and PTH, alone or combined, were examined in male and female mice by micro-computed tomography, mechanical testing, histology, and gene expression analysis. For each outcome, females were more responsive to GC and PTH than males. GC and PTH exerted regional differences, with GC increasing trabecular bone volume but reducing cortical bone thickness, stiffness, and ultimate force. Despite PTH’s anabolic effects on trabecular bone, it did not rescue GC’s catabolic effects on cortical bone. Likewise, cartilage integrity and subchondral bone apoptosis, alkaline phosphatase activity, and osteocyte lacunocanalicular networks showed no evidence that PTH could offset GC-dependent effects. Rather, GC and PTH each increased cortical bone gene expression implicated in bone resorption by osteoclasts and osteocytes, including Acp5, Mmp13, Atp6v0d2, Ctsk, differences maintained when GC and PTH were combined.  Since PTH is insufficient to rescue GC’s effects on young female mouse bone, future studies are needed to determine if osteocyte PLR suppression, due to GC, aging, or other factors, can be offset by a PLR agonist.

overall_design: Differential expression of humeri bones from male and female mice implanted with placebo or gluococoritoid (prednisolone) pellet and treated with saline or parathyroid hormone 1-34.

middle: S

first: Serra

last: Kaya


title: Transcriptional dynamics orchestrating the development and integration of neurons born in the adult hippocampus [ds2]

city: Buenos Aires

first: Ariel

last: Chernomoretz

address: Av Patricias Argentinas 435

country: Argentina

institute: Fundacion Instituto Leloir

summary: To profile neurogenesis in the adult mouse hippocampus, we utilized a strategy to isolate developing cells derived from RGLs expressing the proneural gene Achaete-scute homolog 1 (Ascl1). Tamoxifen administration to young-adult Ascl1CreERT2;CAGfloxStopSun1sfGFP mice were used to induce the expression of Sun1-sfGFP in the nuclear membrane of RGLs and permanently label their progeny. This birthdating tag enabled a comprehensive analysis of the entire process encompassing neuronal differentiation, development, and functional maturation. This dataset was built using FACS-sorted nuclei obtained from dentate gyri microdissected at 2-, 3-, 4-, 5- and 8 weeks after TAM induction (cohorts w2 through w8)

overall_design: 10 samples

first: Natalí

middle: B

last: Rasetto

first: Damiana

last: Giacomini

middle: A

last: Berardino

first: Tomás

last: Vega Waichman

first: Maximiliano

last: Beckel

first: Daniela

middle: J

last: Di Bella

first: Juliana

last: Brown

first: M

middle: G

last: Davies-Sala

first: Chiara

last: Gerhardinger

first: Dieter

last: Chichung Lie

first: Paola

last: Arlotta

first: Alejandro

middle: F

last: Schinder


title: SUMOylation inhibition potentiates CAR T activity against Multiple Myeloma

city: Duarte

first: Hsun

middle: Teresa

last: Ku

address: 1500 E. Duarte Road

department: Translational Research and Cellular Therapeutics

institute: City of Hope

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

overall_design: Refer to individual Series


title: Disinhibition of Early CAR-T cell activation via CD5 Knock Out Enhances the Anti-Tumor Activity of Adoptive T Cell Therapies against Cancer

city: Philadelphia

first: Marco

last: Ruella

state: PA

address: 3400 Civic Center Blvd.

institute: University of Pennsylvania

summary: The majority of patients treated with currently approved adoptive T-cell therapies (ACT) and, specifically, chimeric antigen receptor (CAR) T cells will eventually fail treatment. Furthermore, ACT has not been successful against solid cancers and several hematological malignancies, including T-cell lymphomas which have extremely poor prognosis. One of the main barriers to the effective activity of adoptively transferred T cells is their inhibition when they reach the tumor bed. In this study, we discover that CD5 inhibits CAR T activation and that the knockout (KO) of CD5 using CRISPR-Cas9 enhances the anti-tumor effect of CAR and TCR T cells in multiple hematological and solid cancer models. As a consequence, CD5 KO T cells display increased in vivo expansion and persistence. Despite this increased activity, no clear increased toxicity was observed in preclinical models. These findings support the development of CD5 KO adoptive T-cell therapies in early-phase clinical trials for relapsed and refractory cancer.

overall_design: Membrane protein CD5 was CRISPR Cas9 knocked out of primary human T cells to compare to an electroporated Mock control.

first: Ki

middle: H

last: Kim

first: Inkook

middle: R

last: Chun

first: Ruchi

middle: P

last: Patel


title: MMP-9-dependent proteolysis of the histone H3 N-terminal tail, a critical epigenetic step in driving oncogenic transcription and colon tumorigenesis

city: Los Angeles

first: Yonghwan

last: Shin

address: 1450 Biggy St

department: Biochemistry and Molecular Medicine

institute: University of Southern California

summary: The matrix metalloproteinase 9 (MMP-9) is a member of the MMP family and has been recently identified as a nuclear protease capable of clipping histone H3 N-terminal tails (H3NT). This MMP-9-dependent H3NT proteolysis is critical for establishing active state of gene transcription during osteoclast differentiation and melanoma development. However, whether H3NT cleavage by MMP-9 plays a similar role in other cellular events has not been explored. Here we dissect the functional contribution of MMP-9-dependent H3NT clipping to colonic tumorigenesis by using a combination of genome-wide transcriptome data, ChIP/ChIPac-qPCR, CRISPR/dCas9 gene-targeting system, and in vivo xenograft models. We show that MMP-9 is overexpressed in colon cancer cells and catalyzes H3NT proteolysis to drive transcriptional activation of growth stimulatory genes. Our studies using knockdown and inhibition approaches clearly indicate that MMP-9 mediates transcriptional activation and promote colonic tumorigenesis in a manner dependent on its protease activity toward H3NT. Remarkably, artificial H3NT proteolysis at target gene promoters with dCAS9-MMP-9 is sufficient for establishing their transcriptional competence in colon cancer cells, underscoring the importance of MMP-9-dependent H3NT proteolysis per se in transactivation process. Our data establish new functions and mechanisms for MMP-9 in driving oncogenic transcription program in colon cancer through H3NT proteolysis and demonstrate how this epigenetic pathway can be exploited as a potential therapeutic target for cancer treatment.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for SW620 cells  (shCont, and shMMP9).

first: Woojin

last: An


title: Transcriptional dynamics orchestrating the development and integration of neurons born in the adult hippocampus [ds1]

first: Ariel

last: Chernomoretz

summary: To profile neurogenesis in the adult mouse hippocampus, we utilized a strategy to isolate developing cells derived from RGLs expressing the proneural gene Achaete-scute homolog 1 (Ascl1). Tamoxifen administration to young-adult Ascl1CreERT2;CAGfloxStopSun1sfGFP mice were used to induce the expression of Sun1-sfGFP in the nuclear membrane of RGLs and permanently label their progeny. This birthdating tag enabled a comprehensive analysis of the entire process encompassing neuronal differentiation, development, and functional maturation. This dataset was built using FACS-sorted nuclei obtained from dentate gyri microdissected at 1-, 2-, 4-, and 8 weeks after TAM induction (cohorts w1 through w8)

overall_design: 4 samples

first: Natalí

last: Rasetto

first: Damiana

last: Giacomini

last: Berardino


title: Transcriptional dynamics orchestrating the development and integration of neurons born in the adult hippocampus


title: Identifications of vernalization regulatory elements by epigenomic approaches in winter wheat (RNA-Seq)

status: Public on Jul 22 2024

city: Beijing

first: Zefu

last: Lu

state: Haidian

address: No.12 Zhongguancun South St.,Haidian District, Beijing, P.R.China

country: China

institute: Institute of Crop Sciences

summary: Epigenetic modification plays a key role in the vernalization process and maintains gene expression states after low temperature. In this study, the Chinese main cultivar Aikang58 was used to construct the chromatin accessibility maps, expression profile and histone modification (including H3K27me3, H3K27ac, H3K36me3 and H3K4me3) maps of winter wheat before and after vernalization.  We provided a crucial data resource for deep understanding of epigenome in vernalization. What’s more, important core regulatory elements and corresponding transcription factors were discovered, and the complex regulatory network of wheat vernalization was analyzed.

overall_design: RNA-seq of leaf, axillary bud and shoot apex before and after vernalization


title: Identifications of vernalization regulatory elements by epigenomic approaches in winter wheat

type: Genome binding/occupancy profiling by high throughput sequencing


title: RNA-seq right ventricle congenital heart disease paediatric patients

status: Public on Jul 17 2024

city: BRISTOL

first: Francecsa

last: Bartoli-Leonard

address: Mauldin Street

department: Bristol Medical School

country: United Kingdom

institute: University of Bristol

summary: RV samples were collected from discarded myocardial tissue from paediatric patients under 16 years of age undergoing their first cardiac surgery at Bristol Royal Hospital for Children. Fresh biopsies (10 mg net weight) were collected from surgery from the apex of the RV immediately after institution of cardiac pulmonary bypass and snap frozen upon harvesting. Strand-specific bulk RNA sequencing was performed on whole tissue snap frozen RV biopsies via Genewiz (Azenta). Briefly, total RNA from RV was isolated from 10mg of tissue, with poly-A selection. Sequencing was conducted on Illumina NovaSeq, yielding 2x150bp paired end reads, 30 million read pairs per sample, with >80% of bases with Q30 or higher.

overall_design: To investigate the RNA profile of right ventricle in congenital heart diesease patients taken during their first corrective surgery, RNA sequencing was conducted.

first: F

last: Caputo


title: Defective Integrator activity shapes the transcriptome of patients with multiple sclerosis: patient data

city: Aarhus

first: Mikkel

last: Carstensen

address: Palle Juul-Jensens Boulevard 5

country: Denmark

institute: Aarhus University

summary: While the role of the immune system in multiple sclerosis (MS) is widely acknowledged, our comprehension of the transcriptional foundations of this prevalent neurological disorder remains largely incomplete. Here, we conducted high-depth RNA sequencing on monocytes from a cohort of patients with MS to explore rare RNA species associated with the disease. In a subset of the patients, a markedly altered transcriptome was observed, coinciding with a decreased expression of the epigenetic silencer HP1α/CBX5. These patients also exhibited diminished expression of multiple genes encoding subunits of the Integrator complex. Alongside, there were clear indications of decreased Integrator activity, including impaired U snRNA and enhancer-RNA maturation/degradation and dysregulated RNA polymerase II (RNAPII) pause-release. Inactivation of Cbx5 in the mouse recapitulated the defects in Integrator activity and resulted in hypersensitivity to Experimental Autoimmune Encephalomyelitis (EAE). While these data implied an unexpected function for HP1α in regulating RNAPII pause-release, they also showed that the dysregulation of numerous genes in MS patients could be attributed to either the biased distribution of transcription toward the 5' end of genes or the heightened elongation and stability of enhancer RNAs. An impaired Integrator activity, which has been hinted at by mutations within Integrator subunits previously linked to an increased risk of MS, provides a well-defined mechanistic understanding of the transcriptional anomalies associated with the disease, while introducing new criteria to categorize MS patients.

overall_design: With the objective of obtaining high quality RNA-seq allowing detection of rare RNA species, we collected monocytes from a cohort of 18 MS patients and 7 control patients. To explore eventual changes in the RNA populations induced by the disease progression, we selected a very heterogeneous set including patients with familial MS (#68, #125, and #130 – one family, 3 generations), under immune modulating treatment (#125, #127, #135, #136, and #139), or with comorbidities (psoriasis, IDDM, Graves, Crohn and ulcerative colitis). The control group, designated as symptomatic controls (SCs), comprised individuals who had sought medical assistance for symptoms suggestive of MS, but who, upon examination, exhibited no objective clinical or paraclinical findings conclusive for a specific neurological disease at the time of sample collection (Figure 1A). The RNA-seq analysis was conducted using a stranded paired-end protocol, yielding an output of approximately 110 million reads per sample.

last: CARSTENSEN

first: Thor

last: PETERSEN

first: Tove

last: CHRISTENSEN

first: Christophe

last: RACHEZ

first: Mickaël

last: COSTALLAT

first: Christian

last: MUCHARDT


title: RNA sequencing of nasal airway epithelial cells from preterm birth survivors at one year of age

status: Public on Jul 16 2024

city: Perth

first: Patricia

last: Agudelo-Romero

state: WA

address: Northern Entrance, Perth Children's Hospital, 15 Hospital Ave, Nedlands

department: Chronic and Severe Diseases

country: Australia

institute: Telethon Kids Institute

summary: To investigate potential drivers of respiratory disease in survivors of preterm birth by examining the transcriptomic profile of airway epithelial cells at approximately one year of age. Nasal airway epithelial brushings were collected, and primary cell cultures established from 8 term (GA>37w, 3.0 ± 1.1 years at collection) and 11 preterm participants (GA 25-31.2 weeks, 1.4 ± 0.1 years at collection). RNA was extracted from cultured cells and bulk RNA sequencing performed on both the ex vivo and in vitro samples.

overall_design: Comparative gene expression profiling analysis of RNA-seq data from airway epithelial cells collected from infants born either at term or very preterm (<32 weeks).

first: Denby

last: Evans

first: Jessica

middle: K

last: Hillas

first: Thomas

last: Iosifidis

first: Shannon

last: Simpson

first: Anthony

last: Kicic


title: C/EBPβ deletion in macrophages impairs alveolar budding by altering the Wnt landscape during mammary gland development

city: New Orleans

first: Heather

middle: L

last: Machado

state: LA

address: Louisiana Cancer Research Consortium

institute: Tulane Cancer Center

summary: Macrophages have important roles in mammary gland development and tissue homeostasis, but the specific mechanisms that regulate macrophage function need further elucidation. We have identified C/EBPβ as an important macrophage factor that is present in multiple populations of the normal mammary gland.  Mammary glands from mice with C/EBPβ-deficient macrophages showed a significant decrease in alveolar budding during the estrus cycle. Deletion of C/EBPβ from macrophages resulted in changes in cytokine production, and an inability of both epithelial cells and macrophages to respond progesterone. RNA sequencing showed significant changes in PR-responsive genes and alterations in the Wnt landscape of epithelial cells in mammary glands that lacked C/EBPbeta in macrophages, which have been shown to mediate stem cell expansion during diestrus. These studies suggest that C/EBPbeta is a critical macrophage factor that facilitates macrophage-epithelial crosstalk during a key stage of mammary gland tissue homeostasis, and have critical implications for how disruption of tissue homeostasis can lead to tumorigenesis.

overall_design: RNA-seq of mammary gland epithelial cell and macrophages deleted for C/EBPβ.

first: Kathryn

last: Schwertfeger


title: Thymic epithelial organoids mediate T cell development

city: Lausanne

first: Tania

last: Hübscher

address: Station 15

department: SV IBI

country: Switzerland

institute: EPFL


title: Single-cell RNA-seq of telencephalic organoids from healthy CTRL and HD (Huntington’s disease) hESC (RUES2) grown as mono-culture or co-culture of chimeric organoids

city: Melbourne

first: Maria Rosaria

last: Nucera

state: VICTORIA

address: 50, Flemington road

department: Paediatrics

institute: University of Melbourne

summary: Huntington's disease (HD) causes selective degeneration of striatal and cortical neurons, resulting in cell mosaicism of coexisting still functional and dysfunctional cells. The impact of non-cell autonomous mechanisms between these cellular states is poorly understood. Here we generated telencephalic organoids with healthy or HD cells, grown separately or as mosaics of the two genotypes. We used single-cell transcriptomics to test early HD selective cellular phenotypes by comparing healthy (CTRL) and pathologic (HD) telencephalic organoids at days 45 and 120 of differentiation. To test the influence and the interactions between healthy and HD cells, mosaic organoids composed of CTRL and HD cells juxtaposed within the same organoid were grown and analyzed by scRNAseq at day 120. Single-cell RNA sequencing revealed neurodevelopmental abnormalities in the ventral fate acquisition of HD organoids, confirmed by cytoarchitectural and transcriptional defects leading to fewer GABAergic neurons, while dorsal populations showed milder phenotypes mainly in the maturation trajectory. Healthy cells in mosaic organoids restored HD cell identity, trajectories, synaptic density, and communication pathways upon cell-cell contact while showing no significant alterations when grown with HD cells. These findings highlight cell-type-specific alterations in HD and beneficial non-cell autonomous effects of healthy cells, emphasizing the therapeutic potential of modulating cell-cell communication in disease progression and treatment.

overall_design: At DIV 45, 6 individual organoids were independently sequenced for scRNAseq (3 per genotype: the CTRL with 20cag and the HD with 56cag). At DIV 120, 4 pools of 10 mosaic organoids each were collected for FACS sorting to separate the GFP and the TOM cell lines. The 4 pools derived from mono-culture organoids where GFP and TOM cells of the same genotype were grown together (20CAG-GFP with 20CAG-TOM and 56CAG-GFP with 56CAG-TOM) and co-culture organoids by co-culturing cells of different genotypes together (20CAG-GFP with 56CAG-TOM and 20CAG-TOM with 56CAG-GFP). After FACS we obtained and sequenced 8 independent samples.  We then merged them as 4 biological conditions: “CTRL_mono” (CTRL cells grown alone); “CTRL_co” (CTRL cells that were grown in the presence of HD cells and then isolated); “HD_co” (HD cells that were grown in the presence of CTRL cells and then isolated); and “HD_mono” (HD cells grown alone, as for CTRL_mono).

first: Maura

last: Galimberti

first: Maria

first: Vittoria

middle: D

last: Bocchi

first: Paola

last: Conforti

first: Elena

last: Vezzoli

first: Matteo

last: Cereda

first: Camilla

last: Maffezzini

first: Raffaele

last: Iennaco

first: Andrea

last: Scolz

last: Falqui

first: Chiara

last: Cordiglieri

first: Martina

last: Cremona

first: Ira

last: Espuny-Camacho

last: Faedo

first: Dan

last: Felsenfeld

last: Vogt

first: Valeria

last: Ranzani

last: Zuccato

first: Dario

last: Besusso

last: Cattaneo


title: Effect of PIM447 treatment on gene expression of M07e cells transduced with JAK3 Q988P

status: Public on Jul 15 2024

city: Madrid

last: Villa-Morales

email: maria.villa@uam.es, mvilla@cbm.csic.es

state: Madrid

address: Nicolas Cabrera 1

department: Biology

country: Spain

institute: UNIVERSIDAD AUTONOMA DE MADRID

summary: To test whether the activity of PIM proteins promotes an aberrant activation of the molecular cascades that are essential for the leukemogenic effects mediated by JAK/STAT pathway mutations, we analyzed RNA-seq data from M07e cells expressing JAK3Q988P that were treated with PIM447 or left untreated.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for M07e cells expressing JAK3Q988P that were treated with PIM447 or left untreated. Three replicates were used for each sample.

first: María

first: José

last: Fernández-Piqueras

first: Antonio

last: Lahera


title: In vivo single-cell CRISPR uncovers distinct TNF-α programs in tumor evolution

city: Basel

first: Simona

last: Baghai Sain

address: Schanzenstrasse 44

department: Department of Biosystems Science and Engineering

institute: ETH Zurich

summary: The tumor evolution model posits that malignant transformation is preceded by randomly distributed driver mutations in cancer genes, which cause clonal expansions in phenotypically normal tissues. Although clonal expansions can remodel entire tissues1-3, the mechanisms behind why only a small number of clones transform into malignant tumors remain enigmatic. Here, we develop an in vivo single-cell CRISPR strategy to systematically investigate tissue-wide clonal dynamics of the 150 most frequently mutated squamous cell carcinoma genes. We couple ultrasound-guided in utero lentiviral microinjections, single-cell RNA sequencing and guide capture to longitudinally monitor clonal expansions and document their underlying gene programs at single-cell transcriptomic resolution. We uncover a TNF-α signaling module, dependent on TNF receptor 1 and involving macrophages, that acts as a generalizable driver of clonal expansions in epithelial tissues. Conversely, during tumorigenesis, the TNF-α signaling module is downregulated. Instead, we identify a subpopulation of invasive cancer cells that switch to an autocrine TNF-α gene program, associated with epithelial-mesenchymal transition. Finally, we provide in vivo evidence that the autocrine TNF-α gene program is sufficient to mediate invasive properties and show that the TNF-α signature correlates with shorter overall survival in human squamous cell carcinoma patients. Collectively, our study demonstrates the power of applying in vivo single-cell CRISPR screening to mammalian tissues, unveils distinct TNF-α programs in tumor evolution and highlights the importance of understanding the relationship between clonal expansions in epithelia and tumorigenesis.

overall_design: To longitudinally monitor the function of cancer driver genes, we chose the 150 most frequently altered genes in human head and neck (HNSCC) and skin squamous cell carcinomas (sSCC), comprising 134 mutated genes and 16 copy number variations (CNVs). We selected 3 sgRNAs per gene and included 50 non-targeting control sgRNAs, resulting in a CROP-seq library of 500 sgRNAs used for high-titer lentivirus production. Lentiviral library was delivered into E9.5 embryos using ultrasound-guided in utero microinjection to infect the single-layered surface ectoderm, resident immune cells and melanoblasts of constitutively Cas9-expressing mouse embryos and introduce loss-of-function mutations in each of the selected cancer genes. At postnatal day 4 (P4) and 60 (P60), epidermal tissues are collected, sorted for mCherry-positive infected cells, and subjected them to single-cell RNA sequencing (scRNA-seq) to assess gene expression profiles and read out the corresponding sgRNA identity (via dial-out PCR).  At P60, chemical carcinogenesis was induced by DMBA/TPA for 12 weeks, then from 30 of thus obtained skin tumors (from 2 animals) single-cell suspensions were sorted for mCherry-positive and negative cells by FACS, and finally sequenced for single-cell transcriptomes. Visium spatial transcriptomics was additionally performed on 4 other DMBA/TPA-induced tumors.

first: Peter

last: Renz

first: Umesh

last: Ghoshdastider

first: Fabiola

last: Valdivia-Francia

first: Ameya

last: Khandekar

first: Mark

last: Ormiston

first: Martino

last: Bernasconi

first: Clara

last: Duré

first: Jonas

last: Kretz

first: Minkyoung

last: Lee

first: Katie

last: Hyams

first: Merima

last: Forny

first: Marcel

last: Pohly

first: Xenia

last: Ficht

first: Stephanie

last: Ellis

first: Andreas

middle: E

last: Moor

first: Ataman

last: Sendoel


title: Transcriptomic Profile of Breast Tissue of Premenopausal Women Following Treatment with Progesterone Receptor Modulator: Secondary Outcomes of a Randomized Controlled Trial

city: Solna

first: Nageswara Rao

last: Boggavarapu

state: stockholm

address: Visionsgatan 4

department: Women and Children health

country: Sweden

institute: Karolinska Institutet

summary: Progesterone receptor antagonism is gaining attention due to progesterone's recognized role as a major mitogen in breast tissue. Limited but promising data suggest the potential efficacy of antiprogestins in breast cancer prevention. The present study presents secondary outcomes from a randomized controlled trial examining changes in breast mRNA expression following mifepristone treatment in healthy women. We analyzed 32 paired breast biopsies from 16 healthy premenopausal women at baseline and after two months of mifepristone treatment. In total, twenty-seven differentially expressed genes were identified, with enriched biological functions related to extracellular matrix remodeling. Notably, the altered gene signature induced by mifepristone in vivo was rather similar to the in vitro signature. Furthermore, this expression gene signature was associated with breast carcinogenesis and significantly correlated with progesterone receptor expression status in breast cancer, as validated in The Cancer Genome Atlas dataset using the R2 platform. The present study is the first to explore the breast transciptome following mifepristone treatment in healthy breast tissue in vivo, enhancing the understanding of progesterone receptor modulator and its potential protective effects against breast cancer by investigating its action in healthy breast tissue.

overall_design: Study subjects were randomized into two treatment groups, the randomization process is described in the original study. One group was treated with 50 mg mifepristone (one quarter of 200 mg Mifegyne®, Exelgyn) every other day for two months (56 days) starting on the first day of the menstrual cycle. The comparator group received B-vitamin tablets (TrioBe® Recip). For the purpose of the present study, only paired breast samples from the mifepristone treated group were analyzed.

first: Nageswara


title: USP11 promotes prostate cancer progression by upregulating AR and c-Myc activity [CUT&RUN]

status: Public on Jul 13 2024

city: Baltimore

first: Majid

last: Pornour

state: MD

address: 655 W Baltimore street

institute: University of Maryland

summary: Androgen receptor (AR) is a main driver for castration-resistant prostate cancer (CRPC). c-Myc is an oncogene underlying prostate tumorigenesis. Here we find that the deubiquitinase USP11 targets both AR and c-Myc in prostate cancer. USP11 expression was upregulated in metastatic prostate cancer and CRPC. USP11 knockdown significantly inhibited prostate cancer cell growth. Our RNA-seq studies revealed AR and c-Myc as the top transcription factors altered after USP11 knockdown. ChIP-seq analysis showed that either USP11 knockdown or replacement of endogenous USP11 with a catalytic-inactive USP11 mutant significantly decreased chromatin binding by AR and c-Myc. We find that USP11 employs two mechanisms to upregulate AR and c-Myc levels: namely, deubiquitination of AR and c-Myc proteins to increase their stability; deubiquitination of H2A-K119Ub, a repressive histone mark, on promoters of AR and c-Myc genes to increase their transcription. AR and c-Myc re-expression in USP11-knockdown prostate cancer cells partly rescued cell growth defects. Thus, our studies reveal a tumor-promoting role for USP11 in aggressive prostate cancer through upregulation of AR and c-Myc activities and support USP11 as a potential target against prostate cancer.

overall_design: Chromatin immunoprecipitation DNA sequencing (ChIP-seq) was performed for AR and c-Myc in control (pLKO.1) and USP11-KD Rv1 cells. Additionally, ChIP-seq was conducted for HA-USP11, AR, and c-Myc in Rv1 cells with wild-type USP11 and Rv1 cells with a catalytically inactive USP11 mutant.

first: Jianfei

last: Qi


title: Multi-omics characterization of human Embryonic Stem Cells-derived Pharyngeal Endoderm cells (RARα ChIP-Seq)

status: Public on Jul 14 2024

city: Roma

first: Alessio

last: Colantoni

state: RM

address: Piazzale Aldo Moro, 5

department: Dipartimento di Biologia e Biotecnologie "Charles Darwin"

country: Italy

institute: "Sapienza" University of Rome

summary: We have developed a novel in vitro protocol for the derivation of bona fide Pharyngeal Endoderm (PE) cells from hESCs. We demonstrated that our PE cells robustly express Pharyngeal Endoderm markers, they are transcriptionally similar to PE cells isolated from in vivo mouse development and represent a transcriptionally homogeneous population. Importantly, we elucidated the contribution of Retinoic Acid in promoting a transcriptional and epigenetic rewiring of PE cells. In addition, we defined the epigenetic landscape of PE cells by combining ATAC-Seq and ChIP-Seq of histone marks.

overall_design: We performed ChIP-Seq experiments for RARα on Anterior Foregut Endoderm (d5) and Pharyngeal Endoderm (d5 +RA) cells, and we identified the DNA regions bound by this transcription factor in the two cell types.

first: Andrea

last: Cipriano

first: Danielle

last: Gomes

first: Alexander

last: Parker

first: Mahdi

last: Moqri

first: Matthew

last: Caldwell

first: Francesca

last: Briganti

first: Jonathan

last: Fiorentino

first: Maria

last: Roncarolo

first: Antonio

last: Baldini

first: Katja

last: Weinacht

first: Gian

last: Tartaglia

first: Vittorio

last: Sebastiano


title: Endothelial and systemic upregulation of miR-34a-5p fine-tunes senescence in progeria

city: Vienna

first: Roland

last: Foisner

address: Dr.-Bohr-Gasse 9

department: Center for Medical Biochemistry

country: Austria

institute: Max Perutz Labs, Medical University of Vienna

type: Non-coding RNA profiling by high throughput sequencing

summary:
Endothelial defects significantly contribute to cardiovascular pathology in the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). Using an endothelium-specific progeria mouse model, we identify a novel, endothelium-specific microRNA (miR) signature linked to the p53-senescence pathway and a senescence-associated secretory phenotype (SASP). Progerin-expressing endothelial cells exert profound cell-non-autonomous effects initiating senescence in non-endothelial cell populations and causing immune cell infiltrates around blood vessels. Comparative miR expression analyses revealed unique upregulation of senescence-associated miR34a-5p in endothelial cells with strong accumulation at atheroprone aortic arch regions but also, in whole cardiac- and lung tissues as well as in the circulation of progeria mice. Mechanistically, miR34a-5p knockdown reduced not only p53 levels but also late-stage senescence regulator p16 with no effect on p21 levels, while p53 knockdown reduced miR34a-5p and partially rescued p21-mediated cell cycle inhibition with a moderate effect on SASP. These data demonstrate that miR34a-5p reinforces two separate senescence regulating branches in progerin-expressing endothelial cells, the p53- and p16-associated pathways, which synergistically maintain a senescence phenotype that contributes to cardiovascular pathology. Thus, the key function of circulatory miR34a-5p in endothelial dysfunction-linked cardiovascular pathology offers novel routes for diagnosis, prognosis and treatment for cardiovascular aging in HGPS and potentially geriatric patients.

This research was supported by by the Austrian Science Fund grant [P 32595 /Grant DOI: 10.55776/P32595] to Selma Osmanagic-Myers, and (I 4694-B) to Roland Foisner, the latter under the frame of EJP RD, the European Joint Programme on Rare Diseases. In addition, this project has received funding from the European Union’s Horizon 2020 research and innovation programme under the EJP RD COFUND-EJP N 825575.

https://www.doi.org/10.55776/P32595

overall_design: To assess molecular pathways and changes in microRNAs underlying endothelial dysfunction in  Hutchinson-Gilford progeria syndrome (HGPS) and possibly aging population, we isolated endothelial cells as well as their conditioned media and collected plasma from endothelial-specific progerin-transgenic (Prog-Tg), control wild-type lamin A transgenic (LA-Tg) mice and corresponding wild-type control animals (see DOI: 10.18632/aging.203820).  We then performed gene expression and microRNA profiling analysis of endothelial cells, conditioned media (supernatants; Sn) and plasma samples from Prog-Tg, LA-Tg and Wt mice. Subsequently we performed  comparative gene expression profiling analysis of RNA-Seq data for Prog-Tg and LA-Tg with Wt, respectively in endothelial cells (ECs), Sns and plasma samples (each 3 replicates).

first: Selma

last: Osmanagic-Myers

first: Fatih

last: Sarigol

first: Christina

last: Manakanatas


title: CUT&Tag, ATAC-seq, bulk RNA-seq and single-nucleus RNA-seq analysis of histone variant H2BE in cortical neurons

first: Emily

last: Feierman

address: 3400 Civic Center Blvd


title: Disinhibition of Early T cell Activation via CD5 Knock Out Enhances the Anti-Tumor Activity of Adoptive T Cell Therapies

first: Marco

last: Ruella

overall_design: Membrane protein CD5 was CRISPR Cas9 knocked out of primary human T cells to compare to an electroporated Mock control in vivo

first: Ruchi

last: Patel

first: Mathew

last: Angelos

first: Yunlin

last: Zhang

first: Puneeth

last: Guruprasad


title: Prunus dulcis response to novel defense elicitor peptides and control of Xylella fastidiosa infections

city: Girona

first: Luis

middle: Alejandro

last: Moll Dos Santos

state: Catalonia

address: Pic de Peguera 15, Edifici Jaume Casademont

department: Department of Chemical and Agricultural Engineering and Agrifood Technology

institute: University of Girona

summary: Xylella fastidiosa is a plant pathogenic bacterium that has been introduced in the European Union (EU), threatening the agricultural economy of relevant Mediterranean crops such as almond (Prunus dulcis). Plant defense elicitor peptides would be promising to manage diseases such as almond leaf scorch but their effect on the host has not been fully studied. In this work, the response of almond plants to the defense elicitor peptide flg22-NH2 was studied in-depth using RNA-seq, confirming the activation of the salicylic acid and abscisic acid pathways. Marker genes related to the response triggered by flg22-NH2 were used to study the effect of the application strategy of the peptide on almond plants and to depict its time course. The application of flg22-NH2 by endotherapy triggered the highest number of upregulated genes, especially at 6 hours after the treatment. A library of peptides that include BP100-flg15, HpaG23, FV7, RIJK2, PIP-1, Pep13, BP16-Pep13, flg15-BP100 and BP16 triggered a stronger defense response in almond plants than flg22-NH2. The best candidate, FV7, when applied by endotherapy on almond plants inoculated with X. fastidiosa, significantly reduced levels of the pathogen and decreased disease symptoms. Therefore, these novel plant defense elicitors are suitable candidates to manage diseases caused by X. fastidiosa, in particular almond leaf scorch.

overall_design: The transcriptome of Prunus dulcis cv Avijor plants treated by a combination of spray and endotherapy applications of the peptide flg22-NH2 were compared to non-treated ones 6 and 24h post-treatment. Four biological replicates were used. Each biological replicate consisted of 5 plants.

last: Moll

first: Núria

last: Giralt

first: Marta

last: Planas

first: Lidia

last: Feliu

first: Emilio

last: Montesinos

first: Anna

last: Bonaterra

first: Esther

last: Badosa


title: USP11 promotes prostate cancer progression by upregulating AR and c-Myc activity [RNA-seq]

first: Majid

last: Pornour

overall_design: Comparative gene expression profiling analysis of RNA-seq data for pLKO.1 and sh-USP11 in RV1 cells

first: Jianfei

last: Qi


title: SUMOylation inhibition potentiates CAR T activity against Multiple Myeloma (scRNA-Seq)

city: Duarte

first: Hsun

last: Ku

address: 1500 E. Duarte Road

institute: City of Hope

summary: Chimeric antigen receptor (CAR)-T cell therapy has shown remarkable clinical efficacy against hematologic malignancies; however, tumor relapse occurs commonly due to T cell dysfunction presenting as exhaustion and loss of effector function. Here, we identified SUMOylation inhibition promoted T cell effector function and prevented T cell exhaustion. Combination of SUMO E1 inhibitor TAK-981, significantly potentiated CAR T cell anti-tumor activity and improved persistence of CAR-presenting T cells in vivo, presenting a potent novel therapeutic approach.

overall_design: MM1S-Luc cells were intravenously (i.v.) injected to 6-10-week old NSG mice via tail vein (2x106 cells/mouse). After 5 days, tumor xenografts were confirmed by bioluminescence imaging. Mice were injected intravenously with dosed 2.5 × 105BCMA-CAR T cells followed by treatment with TAK-981 (7.5 mg/kg, intraperitoneally (i.p.) twice per week, BIW) or Vehicle (Veh) for 2 weeks. Bone marrow mononuclear cells were isolated and stained with human CD45 antibody. Total human T cells were sorted by gating GFP-/DAPI-/human CD45+.

first: Li

last: Du

first: Steven

middle: T

last: Rosen


title: A human autoimmune organoid model reveals IL-7 function in celiac disease

status: Public on Jul 12 2024

city: Stanford

first: Antonio

last: Santos

state: California

address: 265 Campus Dr.

department: Hematology

institute: Stanford University

summary: In vitro models of autoimmunity are constrained by an inability to culture affected epithelium alongside the complex tissue-resident immune microenvironment. Celiac disease (CeD) is an autoimmune disease where dietary gluten-derived peptides bind the MHC- II molecules HLA-DQ2 or -DQ8 to initiate immune-mediated duodenal mucosal injury. Here, we generated air-liquid interface (ALI) duodenal organoids from endoscopic biopsies that preserve epithelium alongside native mesenchyme and tissue-resident immune cells as a unit without requiring reconstitution. The ALI organoid immune diversity spanned T, B, plasma, NK and myeloid cells with extensive T and B cell receptor repertoires. HLA-DQ2.5-restricted gluten peptides selectively instigated epithelial destruction in HLA-DQ2.5-expressing CeD patient organoids, which was antagonized by MHC-II or NKG2C/D blockade. Gluten epitopes stimulated a CeD organoid network response in lymphoid and myeloid subsets alongside anti-TG2 autoantibody production. Functional studies in CeD organoids revealed IL-7 as a novel gluten-inducible pathogenic modulator which regulated CD8+ T cell-NKG2C/D expression and was necessary and sufficient for epithelial destruction. Further, endogenous IL-7 was markedly induced in patient biopsies from active CeD versus remission disease, predominantly in lamina propria mesenchyme. By preserving epithelium alongside diverse immune populations, this human in vitro CeD model recapitulates gluten-dependent pathology, facilitates mechanistic investigation, and establishes proof-of-principle for organoid modeling of autoimmunity.

overall_design: CeD organoid cultures were dissociated (as described above) 2 days after gliadin or CLIP treatment. Fresh tissues were dissociated on the same day of receiving the sample. Single live CD45+ or EPCAM+ cells from dissociated organoids or fresh tissues were sorted by FACS into WENR media and subjected to droplet based scRNA-seq with the 10X Genomics Chromium single cell 5? platform, with or without single-cell TCR/Ig V(D)J library, per the manufacturer?s protocol using Chromium Next GEM Single Cell 5' Kit v2 (PN-1000263), Library Construction Kit (PN-1000190), Chromium Single Cell Human TCR Amplification Kit (PN-1000252) and Chromium Single Cell Human BCR Amplification Kit (PN-1000253). Cell capture, library construction, and sequencing were performed as previously described


title: Parallel Single B Cell Transcriptomics to Elucidate Pig B Cell Repertoire

city: Urbana

first: Angad

middle: P.

last: Mehta

state: Illinois

address: 600 S Mathews Ave

institute: University of Illinois at Urbana-Champaign

summary: Develop methods to elucidate the pig antibody repertoire using single B cell sequencing

overall_design: Isolate live B cells from pig blood, identify oligonucletoides for amplification and sequencing, perform single cell sequencing and data analysis

first: Stanley

last: Bram

first: Graeme

last: Lindsey

first: Jenny

last: Drnevich

first: Fangxiu

last: Xu

first: Marcin

last: Wozniak

first: Gisselle

middle: N

last: Medina


title: Differential methylation patterns in paternally imprinted gene promoter regions in sperm from hepatitis B virus infected individuals

status: Public on Jul 11 2024

city: Hefei, Anhui Province

first: Baoyan

last: Wu

address: Science and Education Building, 8th floor, the First Affiliated Hospital of Anhui Medical University

type: Methylation profiling by high throughput sequencing

summary: Background：Hepatitis B virus (HBV) infection poses a substantial threat to human health, impacting not only infected individuals but also potentially exerting adverse effects on the health of their offspring. The underlying mechanisms driving this phenomenon remain elusive. This study aims to shed light on this issue by examining alterations in paternally imprinted genes within sperm. Methods: A cohort of 35 individuals with normal semen analysis, comprising 17 hepatitis B surface antigen (HBsAg)-positive and 18 negative individuals, was recruited. Based on the previous research and  the Online Mendelian Inheritance in Man database (OMIM, https://www.omim.org/), targeted promoter methylation sequencing was employed to investigate 28 paternally imprinted genes associated with various diseases. Results: Bioinformatic analyses revealed 42 differentially methylated sites across 29 CpG islands within 19 genes and four differentially methylated CpG islands within four genes. At the gene level, an increase in methylation of DNMT1 and a decrease in methylation of CUL7, PRKAG2, and TP53 were observed. DNA methylation haplotype analysis identified 51 differentially methylated haplotypes within 36 CpG islands across 22 genes.  Conclusions: This is the first study to explore the effects of HBV infection on sperm DNA methylation and the potential underlying mechanisms of intergenerational influence of paternal HBV infection.

overall_design: A total of 35 subjects from the Reproductive Center of the First Affiliated Hospital of Anhui Medical University (Hefei, China) were enrolled in this study. All participants underwent in vitro fertilization due to female causes of infertility at this center. Of the 35 patients , 17 were positive for hepatitis B surface antigen (HBsAg) and constituted the case group, while the remaining 18 individuals, negative for HBsAg, were recruited as controls.

first: Yuying

last: Sheng

first: Wenwei

last: Yu

first: Lewen

last: Ruan


title: Papillary Tumor of the Pineal Region: analysis of DNA methylation profiles and clinical outcomes in 76 cases

city: BETHESDA

first: Zhichao

address: BG 10 RM 4A31 MSC 1500, 10 CENTER DR

department: Center for Cancer Research

institute: NIH/NCI

type: Methylation profiling by genome tiling array

summary: Genomewide DNA methylation array profiling and analysis of 76 new and public published Papillary Tumor of the Pineal Region (PTPR). The methylation idat profiles of 39 newly profiled tumors were provided here.

overall_design: Bisulfite converted genomic DNA extracted from tumor samples was hybridized to Illumina Infinium EPIC and EPIC V2 Human DNA Methylation Beadchips

first: Kenneth

last: Aldape


title: RNA-sequencing of pig oocyte throughout meiotic maturation

city: Adelaide

first: Katja

last: Hummitzsch

state: South Australia

address: Level 5, North Tce/George Street

department: Obstetrics & Gynaecology

institute: The University of Adelaide

summary: Oocyte maturation refers to oocytes at the germinal vesicle stage progressing into metaphase II (MII) stage of development. Even though numerous studies have shown key genes and potential important signalling cascades, which drive the GV to MII transition, a system-wide analysis of underlying differences at gene level and especially at transcript level between the two developmental stages of the oocyte is still lacking. For this, we profiled and analysed RNA from pig oocytes across meiotic maturation (GV, MII and damaged, n=15). We detected 22,516 genes for each sample across meiotic maturation. Principal Component analysis of the data clustered the samples in three stages of development (GV, MII and damaged). Differential expression of genes between the three stages will then be used to delineate the pathways which are up-/down-regulated during these developmental stages. Besides, differential transcript usage will be used to identify the difference of oocytes at distinct developmental stages at isoform level, which might be ignored by traditional differential gene expression analysis.

overall_design: Pig oocyte mRNA profiles from three developmental stages, GV  (germinal vesicle, n=5), MII (metaphase II, n=5) and 'damaged' (oocytes without visible polar body after in vitro maturation/IVM, n = 5)

first: Feng

last: Tang

first: Raymond

last: Rodgers


title: Differential but complementary roles of HIF-1α and HIF-2α in the regulation of bone homeostasis

status: Public on Jul 10 2024

city: Gwangju

first: Su-Jin

last: Kim

address: 77, Yongbong-ro, Buk-gu, Gwangju, Republic of Korea

country: South Korea

institute: Chonnam National University

summary: Bone is a highly dynamic tissue undergoing continuous formation and resorption. Here, we investigated differential but complementary roles of hypoxia-inducible factor (HIF)-1α and HIF-2α in regulating bone remodeling. Using RNA-seq analysis, we identified that specific genes involved in regulating osteoblast differentiation were similarly but slightly differently governed by HIF-1α and HIF-2α. We found that increased HIF-1α expression inhibited osteoblast differentiation via inhibiting RUNX2 function by upregulation of Twist2, confirmed using Hif1a conditional knockout (KO) mouse. Ectopic expression of HIF-1α via adenovirus transduction resulted in the increased expression and activity of RANKL, while knockdown of Hif1a expression via siRNA or osteoblast-specific depletion of Hif1a in conditional KO mice had no discernible effect on osteoblast-mediated osteoclast activation. The unexpected outcome was elucidated by the upregulation of HIF-2α upon Hif1a overexpression, providing evidence that Hif2a is a transcriptional target of HIF-1α in regulating RANKL expression, verified through an experiment of HIF-2α knockdown after HIF-1α overexpression. The above results were validated in an ovariectomized- and aging-induced osteoporosis model using Hif1a conditional KO mice. Our findings conclude that HIF-1α plays an important role in regulating bone homeostasis by controlling osteoblast differentiation, and in influencing osteoclast formation through the regulation of RANKL secretion via HIF-2α modulation.

overall_design: RNA-seq analysis was performed to determine the gene expression profile caused by overexpression of HIF-1α and HIF-2α during primary culture calvarial preosteoblast differentiation.

first: Je-Hwang

last: Ryu

first: Yun Hyun

last: Huh

first: Sun Young

last: Lee


title: DNA aptamers for detection of pyoverdine pf5

city: Ames

first: Marit

last: Nilsen-Hamilton

state: Iowa

address: 2437 Pammel Dr

department: BBMB

institute: Iowa State University

summary: The rhizosphere is a small region surrounding plant roots that is enriched in biochemicals from root exudates and populated with fungi, nematode, and bacteria. Interaction of rhizosphere organisms with plants is mainly promoted by exudates from the roots. Root exudates contain biochemicals that come from primary and secondary metabolisms of plants. These biochemicals attract microbes, which influence plant nutrition. The rhizosphere bacteria (microbiome) are vital to plant nutrient uptake and influence biotic and abiotic stress and pathogenesis. Pseudomonas is a genus of gammaproteobacteria known for its ubiquitous presence in natural habitats and its striking ecological, metabolic, and biochemical diversity. Within the genus, members of the Pseudomonas fluorescens group are common inhabitants of soil and plant surfaces, and certain strains function in the biological control of plant disease, protecting plants from infection by soilborne and aerial plant pathogens. The soil bacterium Pseudomonas protegens Pf-5 (also known as Pseudomonas fluorescens Pf-5) is a well-characterized biological strain, which is distinguished by its prolific production of the secondary metabolite, pyoverdine. Knowledge of the distribution of P. fluorescens secretory activity around plant roots is very important for understanding the interaction between P. fluorescens and plants and can be achieved by real time tracking of pyoverdine. To achieve the capability of real-time tracking in soil, we have used a structure-switching SELEX strategy to select high affinity ssDNA aptamers with specificity for pyoverdine over other siderophores. Two DNA aptamers were isolated, and their features compared.  The aptamers were applied to a nanoporous aluminum oxide biosensor and demonstrated to successfully detect PYO-Pf5. This sensor provides a future opportunity to track the locations around plant roots of P. protegens and to monitor PYO-Pf5 production and movement through the soil.

overall_design: SELEX was performed with PYD-Pf5 as target to select DNA aptamers.

first: Sharif

last: Anisuzzaman


title: RNA-seq in EndoCbetaH5 under high glucose, treated or not with naltrindole

city: Lille

first: Souhila

last: amanzougarene

address: 1 place de Verdun

department: Nord

country: France

institute: CNRS

summary: In human EndoCβH5 cells, i.e. post-mitotic mature pancreatic beta cells that endogenously express OPRD1 encoding delta opioid receptor (transcripts per million [TPM] of 11 according to our RNA-seq data), a specific antagonist for delta opioid receptor (naltrindole [NTI]) significantly increased insulin secretion in high glucose condition. We performed RNA-seq in these cells to decipher signalling pathways under delta opioid receptor antagonism.

overall_design: RNA-seq in EndoCβH5 cells. For this experiment, we used the high glucose condition (16.7 mM glucose) supplemented or not with 10 µM naltrindole (naltrindole hydrochloride, Sigma-Aldrich). RNA was extracted from EndoCβH5 cells using the NucleoSpin RNA kit (Macherey-Nagel, Düren, Germany), following manufacturer’s recommendations. 100 ng total RNA was used to perform library preparation using the NextFlex Poly(A) Bead 2.0 (v21.01; PerkinElmer Waltham, USA). Sequencing was done on the NovaSeq6000 system (flowcell SP, Illumina) using a paired-end 2×100 bp protocol. Raw data were demultiplexed using bcl2fastq v2.20.0.422 (Illumina). An adapter trimming step was done using trimmomatic version 0.39 (MINLEN:35 AVGQUAL:20). Mapping of reads on the human genome (Hg38) was performed using STAR (v2.7.3a). On average, 49 million reads accurately mapped against the human genome. Raw and normalized counting steps were done using RSEM (v1.3.1) using a GTF from Encode (version 39), and Ensembl (version 105) for gene name annotations. Differential analysis was performed using DESeq2 (v1.24.0). Gene-set enrichment analysis was done using Metascape (accessed in April 2022)

first: Amélie

last: Bonnefond

first: Philippe

last: Froguel

first: Sarah

last: Meulebrouck

first: Mehdi

last: Derhourhi

last: Amanzougarene


title: Lamivudine modulates the expression of neurological impairment-related genes and LINE-1 retrotransposons in brain tissues of a down syndrome mouse model

city: Badalona

first: Alessandra

last: Borgognone

state: Barcelona

address: Carretera de Canyet, s/n

institute: IrsiCaixa

summary: Elevated activity of retrotransposons is increasingly recognized to be implicated in a wide range of neurodegenerative and neurodevelopmental diseases, including Down syndrome (DS), which is the most common chromosomal condition causing intellectual disability globally. Previous research by our group has revealed that treatment with lamivudine, a reverse transcriptase inhibitor, improved neurobehavioral phenotypes and completely rescued hippocampaldependent recognition memory in a DS mouse model, Ts65Dn. We hypothesized that retrotransposition rates would increase in the Ts65Dn mouse model, and lamivudine could block retrotransposons. We analyzed the differentially expressed long interspersed element-1 (LINE-1 or L1) mapping on MMU16 and 17, and showed for the first time that retrotransposition could be associated with Ts65Dn’s pathology, as misregulation of L1 was found in brain tissues associated with trisomy. In the cerebral cortex, 6 out of 26 upregulated L1s in trisomic treated mice were located in the telomeric region of MMU16 near Ttc3, Kcnj6, and Dscam genes. In the hippocampus, one upregulated L1 element in trisomic treated mice was located near the Fgd4 gene on MMU16. Moreover, two downregulated L1s rescued after treatment with lamivudine were located in the intronic region of Nrxn1 (MMU17) and Snhg14 (MMU7), implicated in a variety of neurodegenerative disorders. To gain further insight into the mechanism of this improvement, we here analyzed the gene expression profile in the hippocampus and cerebral cortex of trisomic mice treated and no-treated with lamivudine compared to their wild-type littermates. We found that treatment with lamivudine rescued the expression of 24% of trisomic genes in the cortex (located on mouse chromosome (MMU) 16 and 17) and 15% in the hippocampus (located in the human chromosome 21 orthologous regions), with important DS candidate genes such as App and Ets2, rescued in both regions.

overall_design:
To gain insight into the mechanism by which lamivudine improved the neurobehavioral profile in trisomic mice we analyzed the gene expression profile in the hippocampus and cerebral cortex of trisomic treated and no-treated mice compared to their wild type littermates.
We performed bulk RNA-seq profiling

first: Maria

last: Casadellà

first: María

middle: M

last: de Lagran

first: Roger

last: Paredes

first: Bonaventura

last: Clotet

first: Mara

last: Dierssen

first: Aleix

last: Elizalde-Torrent


title: Accurate isoform quantification by joint short- and long-read RNA-sequencing [short reads]

city: Montreal

first: Hamed

middle: S

last: Najafabadi

state: Quebec

address: 740 Dr. Penfield Avenue

department: Human Genetics

country: Canada

institute: McGill University

summary: Accurate quantification of transcript isoforms is crucial for understanding gene regulation, functional diversity, and cellular behavior. Existing methods using either short-read (SR) or long-read (LR) RNA sequencing have significant limitations: SR sequencing provides high depth but struggles with isoform deconvolution, while LR sequencing offers isoform resolution at the cost of lower depth, higher noise, and technical biases. Addressing this gap, we introduce Multi-Platform Aggregation and Quantification of Transcripts (MPAQT), a generative model that combines the complementary strengths of different sequencing platforms to achieve state-of-the-art isoform-resolved transcript quantification, as demonstrated by extensive simulations and experimental benchmarks. Applying MPAQT to an in vitro model of human embryonic stem cell differentiation into cortical neurons, followed by machine learning-based modeling of mRNA abundance determinants, reveals the role of untranslated regions (UTRs) in isoform regulation through isoform-specific interactions with RNA-binding proteins that modulate mRNA stability. These findings highlight MPAQT's potential to enhance our understanding of transcriptomic complexity and underline the role of splicing-independent post-transcriptional mechanisms in shaping the isoform and exon usage landscape of the cell.

overall_design: Gene expression profiling of human embryonic stem cells (HESC) before (day 0) and after undergoing Cortical neuron differentiation (day 41 and 61). Two replicates per condition.

first: Michael

last: Apostolides

first: Benedict

last: Choi

first: Albertas

last: Navickas

first: Ali

last: Saberi

first: Larisa

last: Soto

first: Hani

last: Goodarzi


title: Accurate isoform quantification by joint short- and long-read RNA-sequencing

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

overall_design: Refer to individual Series


title: Accurate isoform quantification by joint short- and long-read RNA-sequencing [long reads]


title: Paternal obesity-induced changes in sperm chromatin accessibility and have mild effect on offspring metabolic health

city: Barcelona

first: Arnaud

last: Obri

address: C/ del Rosselló, 149

institute: IDIBAPS

summary: The increasing global burden of metabolic disorders including obesity and diabetes necessitates a comprehensive understanding of their etiology, which not only encompasses genetic and environmental factors but also parental influence. Recent evidence has unveiled paternal obesity as a contributing factor to offspring's metabolic health via sperm epigenetic modifications. In this study, we investigated the impact of a Western diet-induced obesity in C57BL/6 male mice on sperm chromatin accessibility and the subsequent metabolic health of their progeny. Utilizing Assay for Transposase-Accessible Chromatin with sequencing, we discovered 450 regions with differential accessibility in sperm from obese fathers, implicating key developmental and metabolic pathways. Contrary to expectations, these epigenetic alterations in sperm were not predictive of long-term metabolic disorders in offspring, who exhibited only mild transient metabolic changes early in life. Both male and female F1 progeny showed no enduring predisposition to obesity or diabetes. These results underscore the biological resilience of offspring to paternal epigenetic inheritance, suggesting a complex interplay between inherited epigenetic modifications and the offspring's own developmental compensatory mechanisms. This study calls for further research into the biological processes that confer this resilience, which could inform interventional strategies to combat the heritability of metabolic diseases.

overall_design: Using a diet-induced obesity model in male mice, we examined changes in sperm chromatin accessibility through Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) between F0 Western diet fed male mice (6 months duration) and a control group of F0 Chow diet fed male male. We then assessed the effects of these epigenetic alterations on the metabolic health of F1 offspring through various physiological metabolic tests.

first: Iasim

last: Tahiri

first: Sergio

last: Llana

first: Julia

last: Fos-Domenech

last: Mila-Guash

first: Roberta

last: Haddad-Tóvolli

first: Marc

last: Claret


title: The SaeRS two-component system dynamically regulates cellular adhesion and invasion during group B Streptococcus infection

city: Paris

first: Rachel

last: Legendre

address: 28, rue du docteur Roux

department: Research and Resource Center for Scientific Informatics

institute: Institut Pasteur

summary: Group B Streptococcus (GBS) is a pathobiont responsible for invasive infections in neonates and in the elderly. The transition from a commensal to an invasive pathogen relies on the timely regulation of a set of virulence factors. In this study, we characterized the role of the SaeRS two-component system in GBS pathogenesis. Loss-of-function mutations in the SaeR response regulator decrease virulence in mouse models of invasive infection . Transcriptome and in vivo analysis reveal a regulatory system specifically activated during infection to control the expression of only two virulence factors: the PbsP adhesin and the BvaP secreted protein. The in vivo surge in SaeRS-regulated genes is complemented by fine-tuning mediated by the repressor of virulence CovRS system to establish a coordinated response. Constitutive activation of the SaeRS regulatory pathway increases adhesion and invasion of epithelial and endothelial barriers, though at the cost of reduced virulence. In conclusion, SaeRS is a specialized and dynamic system that balances the invasion of host barriers with disease progression.

overall_design:
To characterize the SaeR-regulated genes, we generated a saeR deletion mutants (∆saeR), a mutant expressing an inactived SaeR variant (SaeR D53A), and a constitutive activated mutant having a specific mutation in SaeS specifically abolishing the phosphatase activity of the histidine kinase (SaeS T133A) in the wild-type strain NEM316 (capsular serotype III, CC-23)
We performed RNA-sequencing of the three mutants against the WT strain.  RNA are purified from exponentially growing cultures (OD600 = 0.5) in rich media (THY) incubated at 37°C in static condition. Biological triplicate (Replicate 1, 2, and 3) are done on different days to take into account batch effect.

first: Giuseppe

middle: V

last: DE GAETANO

first: Francesco

last: COPPOLINO

first: Odile

last: SISMEIRO

first: Hugo

last: VARET

last: LEGENDRE

last: FIRON


title: RBM7 regulates breast cancer progression

city: Dalian

first: Wang

last: Yang

address: 9 Lvshun South Rd West Section, Lüshunkou District, Dalian, Liaoning, China

department: Institute of Cancer Stem Cell

institute: Dalian Medical University

summary: In order to profile the alternative splicing events that were regulated by RBM7, RNA-seq was performed on MDA-MB-231 cells which was used to generate RBM7 stably depleted breast cancer cells. And to explore how RBM7 regulated splicing target MFGE8, MDA-MB-231 cells stably overexpressed respectively MFGE8 long isoform (MFGE8-L) and short isoform (MFGE8-S) that were used for RNA-sequence.

overall_design: This study includes a total of 6 profiling samples, MDA-MB-231_sh1 and MDA-MB-231_sh2 were generated as RBM7 stably depleted MDA-MB-231 breast cancer cells. MDA-MB-231-MFGE8_L and MDA-MB-231-MFGE8_S were generated as MFGE8_L and MFGE8_S stably overexpressed breast cancer cells.


title: LXR-dependent enhancer activation regulates the temporal organization of the liver's response to refeeding leading to lipogenic gene overshoot

city: Rehovot

first: Ido

last: Goldstein

address: POB 12

department: Institute of Biochemistry, Food Science and Nutrition

country: Israel

institute: The Hebrew University of Jerusalem

summary: Transitions between the fed and fasted state are common in mammals. The liver orchestrates adaptive responses to feeding/fasting by transcriptionally regulating metabolic pathways of energy usage and storage. Transcriptional and enhancer dynamics following cessation of fasting (refeeding) have not been explored.  We examined the transcriptional and chromatin events occurring upon refeeding in mice, including kinetic behavior and molecular drivers. We found that the refeeding response is temporally-organized with the early response focused on ramping up protein translation while the later stages of refeeding drive a bifurcated lipid synthesis program. While both the cholesterol biosynthesis and lipogenesis pathways were inhibited during fasting, most cholesterol biosynthesis genes returned to their basal levels upon refeeding while most lipogenesis genes markedly overshoot above pre-fasting levels. Gene knockout, enhancer dynamics and ChIP-seq analyses revealed that lipogenic gene overshoot is dictated by LXRα. These findings from unbiased analyses unravel the mechanism behind the long-known phenomenon of refeeding fat overshoot.

overall_design: Males, 8 weeks-old mice (C57BL/6J) were randomly assigned to either group (6 mice per group). The experiment started after 1 week of acclimation. The Adlib group had ad libitum access to food (Teklad TD2018) and water throughout the experiment. For the rest of the groups, food was removed at the beginning of the inactive phase (shortly after lights on, zeitgeber time 1). For the Refed groups, food was put back in the cage 24 h later while the fasted group was anesthetized and euthanized. The Refed groups were euthanized 3 h, 10 h and 24 h after food reintroduction. At each group endpoint, mice were anesthetized and euthanized (ketamine:xylazine 30:6 mg/ml) and the liver was excised.

first: Noga

last: Korenfeld


title: TET1 Displays Catalytic and Non-catalytic Functions in the Adult Mouse Cortex [RNA-seq]

status: Public on Jul 09 2024

city: Philadelphia

first: Yee Hoon

last: Foong

address: 3400 Civic Center Blvd

institute: University of Pennsylvania

summary: TET1 modulates distinct cortican functions and genomic locations through its catalytic and non-catalytic functions

overall_design: RNAs from 12 mouse cortical samples of 3 genotypes  (Tet1 WT=4, Tet1 HxD = 4, Tet1 KO=4) were sequenced on Illumina NovaSeq 6000

first: Y

first: B

last: Caldwell

first: J

last: Thorvaldsen

first: C

last: Krapp

middle: A

last: Mesaros

first: W

last: Zhou

first: R

last: Kohli

first: M

last: Bartolomei


title: TET1 Displays Catalytic and Non-catalytic Functions in the Adult Mouse Cortex [Methylation array]

overall_design: Bisulfite and bACE converted DNAs from 24 mouse cortical samples of 3 genotypes  (Tet1 WT=8, Tet1 HxD = 8, Tet1 KO=8) were sequenced on Illumina Infinium Mouse Methylation 12v1-0 BeadChip array


title: Effects of depletion of Irf8 and Mafb on gene expression of adult microglia and CNS border associtated macrophages (CAMs)

city: Fukuoka

first: Yasuyuki

last: Ohkawa

address: 3-1-1 Maidashi

country: Japan

institute: Medical Institute of Bioregulation

summary: The central nervous system (CNS) includes anatomically distinct macrophage populations including parenchyma microglia and CNS-associated macrophages (CAMs). The stepwise development and specification of macrophage populations in the CNS rely on several key transcription factors, such as IRF8 and MAFB. In order to better understand the roles of IRF8 and MAFB for phenotypic determination of homeostatic microglia and CAMs, we performed quantitative bulk RNA sequencing of microglia and CAMs isolated from mouse models with myeloid cell-specific deletion of IRF8 or MAFB transcription factors. We found the distinct transcriptional machinery, mediated by IRF8 and MAFB, which underlies the diversity of macrophages in the CNS.

overall_design: Transcriptome profiles of microglia and CAMs from whole brains of the mouse models with myeloid cell-specific deletion of IRF8 or MAFB transcription factors were generated by deep sequencing.

first: Takahiro

last: Masuda

first: Ayato

last: Yamasaki

first: Ohkawa

last: Yasuyuki

first: Kaori

last: Tanaka


title: The molecular basis of tRNA selectivity by human pseudouridine synthase 3 (PUS3)

city: Lausanne

first: Leon

last: Kleemann

address: Station 19

institute: EPFL

summary: Pseudouridine (Ψ), the isomer of uridine, is ubiquitous in most RNA families, including tRNA, rRNA and mRNA. Pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs, but whether it can modify other RNA types, including mRNA, remains elusive. Here, we determine the single particle cryo-EM structure of apo and tRNA-bound human PUS3, showing how it forms a symmetric homodimer that recognizes the characteristic L-shape of tRNA across two distinct interaction interfaces. Structure-guided and patient-derived mutations validate our structural findings in complementary biochemical assays. Furthermore, we deleted PUS1 and PUS3 in HEK293 cells and used Pseudo-seq to detect Ψ sites in the transcriptome. Although PUS1-dependent sites were detectable in tRNA and mRNA, we did not find any evidence for PUS3 modifying other RNA classes than tRNA. In summary, our work provides the molecular basis for PUS3-mediated tRNA modification in humans and aids in understanding how impairments in its activity lead to intellectual disabilities through defects in tRNA modifications.

overall_design: Pseudo-seq analysis to detect pseudouridine sites in the human transcriptome using HEK293T cells. Detection of PUS1 and PUS3 dependent pseudouridine sites in the transcriptome using PUS1-/- and PUS3-/- mutant cell lines.

first: Ting-Yu

last: Lin

first: Sebastian

last: Leidel

last: Glatt


title: Effect of the overexpression of hepcidin in the skin

first: Carole

last: Peyssonnaux

address: 24 rue du Faubourg saint Jacques

institute: INSERM

summary: To investigate the role of hepcidin in the skin, we generated a transgenic murine model overexpressing hepcidin in the skin under the control of keratin14 expressed by keratinocytes (Hamp1-KI Ker)

overall_design: We then performed gene expression profiling analysis using data obtained from RNA-seq of 5 WT mice and 5 Hamp1-KI ker

first: Elise

last: Abboud

first: Nadia

last: Boussetta

first: Doha

last: Chrayteh

first: Zoubida

last: Karim


title: Central role of SUMOylation in the regulation of chromatin interactions and transcriptional outputs of androgen receptor in prostate cancer cells (RNA-seq)

city: Kuopio

first: Ville

last: Paakinaho

address: Yliopistonranta 8

department: School of Medicine

country: Finland

institute: University of Eastern Finland

summary: The androgen receptor (AR) is pivotal in prostate cancer (PCa) progression and represents a critical therapeutic target. AR-mediated gene regulation involves intricate interactions with nuclear proteins, with many mediating and undergoing post-translational modifications that present alternative therapeutic avenues. Through chromatin proteomics in PCa cells, we identified SUMO ligases together with nuclear receptor coregulators and pioneer transcription factors within the AR’s protein interaction network. Intriguingly, this network displayed a significant association with SUMO2/3. To elucidate the influence of SUMOylation on AR chromatin interactions and subsequent gene regulation, we inhibited SUMOylation using ML-792 (SUMOi). While androgens generally facilitated the co-occupancy of SUMO2/3 and AR on chromatin, SUMOi induced divergent effects dependent on the enhancer type. SUMOi augmented AR’s pioneer-like binding on inaccessible chromatin regions abundant in androgen response elements (AREs) and diminished its interaction with accessible chromatin regions sparse in AREs yet rich in pioneer transcription factor motifs. The SUMOi-impacted AR-binding sites divergently influenced AR-regulated genes; those associated with AR-mediated activation played roles in negative regulation of cell proliferation, while those with AR-mediated repression were involved in pattern formation. In conclusion, our findings underscore the pervasive influence of SUMOylation in shaping AR's role in PCa cells, potentially unveiling new therapeutic strategies.

overall_design: Gene expression profiling by RNA-seq from VCaP, LNCaP and RWPE1 cells treated with or without DHT in the presence or absence of SUMOylation inhibitor ML-792. All sequencing was done with Illumina NextSeq 500.

first: Kaisa-Mari

last: Launonen

first: Jorma

middle: J

last: Palvimo


title: Central role of SUMOylation in the regulation of chromatin interactions and transcriptional outputs of androgen receptor in prostate cancer cells


title: Central role of SUMOylation in the regulation of chromatin interactions and transcriptional outputs of androgen receptor in prostate cancer cells (ChIP-seq)

overall_design: Examination of AR, SUMO2, H3K27ac, FOXA1, and HOXB13 chromatin binding by ChIP-seq from VCaP prostate cancer cells in the presence or absence of SUMOylation inhibitor ML-792. The sequencing was done with Illumina NextSeq 500 or Illumina NextSeq 2000.


title: Central role of SUMOylation in the regulation of chromatin interactions and transcriptional outputs of androgen receptor in prostate cancer cells (ATAC-seq)

overall_design: Examination of chromatin accessibility by ATAC-seq from VCaP prostate cancer cells treated with or without DHT in the presence or absence of SUMOylation inhibitor ML-792. All sequencing was done with Illumina NextSeq 500.


title: Central role of SUMOylation in the regulation of chromatin interactions and transcriptional outputs of androgen receptor in prostate cancer cells (LNCaP RNA-seq)

overall_design: Gene expression profiling by RNA-seq from LNCaP and RWPE1 cells treated with or without DHT in the presence or absence of SUMOylation inhibitor ML-792. All sequencing was done with Illumina NextSeq 500.

first: V

first: K

first: J


title: Data-driven identification of small molecules inducing Brown Adipocyte differentiation

status: Public on Jul 08 2024

city: Cambridge

first: Davide

last: Chiarugi

address: Box 289, Addenbrooke’s Hospital

institute: Institute of Metabolic Science

summary: Promoting the recruitment and function of human brown adipose tissue (BAT) holds significant promise for preventing and treating obesity and related metabolic complications. While identifying genes to enhance BAT recruitment and thermogenic activation is crucial, traditional upregulation methods often lack efficiency. Small molecules, however, can effectively facilitate this process through safe and rapid mechanisms. In our study, we employed the DECCODE method, an unbiased drug-induced transcriptomics approach, to identify small molecules that facilitated cell conversion. We began by establishing a transcriptomic profile of BAT cells as our target and then assessed small molecules from the LINCS collection for their similarity to this target, thus identifying a pool of candidates. Our validation in human thermogenic adipocytes revealed that three drugs—Saxagliptin, Tolvaptan, and Targinine—already approved for other uses significantly enhance BAT recruitment and activation in a human thermogenic adipocyte cellular model.

overall_design: Cell cultures from KOLF2 C1 human induced pluripotent stem (iPS)  were differentiated according to a chemically-defined differentiation protocol specifically designed to generate human brown adipocytes (BAs) from PSCs. RNA-seq was performed on samples isolated on Day 50 (D50, three replicates) and Day 60 (D60, three replicates). The transcriptomic profile obtained for D60 was used as an input for the DECCODE method to identify small molecules that could facilitate the differentiation from mesodermal progenitors to mature BAs. The transcriptomic profile obtained for D50 was used to comparative purposes

first: Stefania

last: Carobbio

first: Antonio

last: Vidal-Puig


title: Multimodal profiling of human postnatal thymocytes using CITE-seq

city: Ghent

first: Tom

last: Taghon

address: Corneel Heymanslaan 10

country: Belgium

institute: Ghent University

summary: T cell development in the human thymus has primarily been studied using antibody-based approaches, such as flow cytometry, which can create difficulties in translating phenotypic findings to scRNA-seq data. In order to bridge this gap and obtain paired surface protein and RNA information for individual cells, we carried out CITE-seq with a 143-plex customised antibody panel on human postnatal thymocytes. This was further combined with TCR-seq for TRA and TRB loci to gain insights into the V(D)J recombination progress in developing cells. Using information from all three modalities, we annotated over 30 different stages of human T cell development, which align with known surface marker profiles. This data was utilised in the context of the human thymus spatial atlas for high-resolution spatial mapping of developing T cells, which revealed differences in the migration and maturation kinetics of CD4 and CD8 lineage single positive thymocytes.

overall_design: Thymocytes from 5 paediatric donors were stained with a TotalSeq-C panel containing antibodies against 143 surface markers and 7 isotype controls. Cells were sorted into CD3+ and CD3- subsets to enrich for immature stages and profiled using the 10X Genomics 5’ kit v2.

first: Lena

last: Boehme

first: Jolien

last: Van Hulle


title: LRIG1 controls proliferation of adult neural stem cells by facilitating Tgfß and BMP signalling pathways

city: Toronto

first: Scott

last: Yuzwa

state: Ontario

address: 1 King's College Circle, Medical Sciences Building, Rm 6316

department: Laboratory Medicine and Pathobiology

institute: University of Toronto

summary: Adult Neural Stem Cells (aNSCs) in the ventricular-subventricular zone (V-SVZ) are largely quiescent. Here, we characterize the mechanism underlying the functional role of a cell-signalling inhibitory protein, LRIG1, in the control of aNSCs proliferation. Using constitutive Lrig1 knockout models, we show that Lrig1 ablation results in increased aNSCs proliferation with no change in neuronal progeny and that this hyperproliferation likely does not result from activation of the epidermal growth factor receptor (EGFR). Loss of LRIG1, however, does lead to impaired activation of canonical transforming growth factor beta (TGFβ) and bone morphogenic protein (BMP) signalling pathways. Biochemically, we show in vitro that LRIG1 is able to bind TGFβ/BMP receptors and the TGFβ1 ligand. Finally, we show that the consequences of these interactions are to facilitate SMAD phosphorylation and pathway activation. Collectively, these data suggest that, unlike in embryonic neural stem cells where EGFR is the primary mechanism of action, LRIG1 and TGFβ pathways positively work together to fulfill their inhibitory roles in aNSCs.

overall_design:
To identify putative signalling pathways disrupted by loss of LRIG1 we compated whole transcriptomes of Lrig1 knockout (KO) and wild-type (WT) mice.
We performed mRNA-seq on mRNA isolated from V-SVZ tissue dissected from 8-week old Lrig1 KO and WT mice.

first: Stephanie

last: Ouzikov

first: Kyshona

last: Edwards

first: Tanvi

last: Anandampillai

first: Samuel

last: Watanabe

first: Daniela

last: Lozano Casasbuenas

first: Karen

last: Siu

first: Danyon

last: Harkins

first: Aaron

last: Dou

first: Danielle

last: Jeong

first: Jeffrey

last: Lee


title: Targeting Fatty Acid Oxidation Enhances Response to HER2-targeted Therapy

first: Sherif

middle: Samer

last: Attalla

address: 1160 Ave. Pine W

department: Goodman Cancer Institute

institute: McGill

summary: Metabolic reprogramming is a hallmark of tumorigenesis and includes alterations in glucose and fatty acid metabolism. In this study, we investigated the role of Carnitine palmitoyl transferase 1A (CPT1A), a key enzyme in fatty acid oxidation (FAO), in the induction of HER2+ (Human Epidermal growth factor 2, ErbB2) breast cancer. Using an ErbB2+ genetically engineered mouse models, we found that ablation of CPT1A delayed tumor onset and reduced tumor growth, angiogenesis, and metastatic capacity. CPT1A-deficient ErbB2+ cells exhibited impaired mitochondrial function, leading to a reliance on the tricarboxylic acid cycle to reduce NAD+/FAD for energy production. Consequently, loss of CPT1A resulted in glucose dependency and an inability to metabolize fats. CPT1A-deficient ErbB2+ tumor cells exhibited increased oxidative stress and upregulated nuclear factor erythroid 2-related factor 2 (NRF2) activity. Inhibiting NRF2 or silencing its expression reduced proliferation and glucose consumption in CPT1A-deficient cells. In pre-clinical models of ErbB2+ breast cancer, combining a ketogenic diet with an anti-ErbB2 monoclonal antibody in the context of CPT1A deficiency significantly reduced tumor growth and increased survival. Furthermore, combining the ketogenic diet with CPT1A ablation suppressed tumor growth, enhanced apoptosis, and reduced lung metastasis. Additionally, using an immunocompetent model, we provide evidence that CPT1A inhibition attenuated tumor growth and proliferation by promoting an antitumor immune microenvironment that enhanced the efficacy of ErbB2 targeted therapy. These findings provide insight into the metabolic rewiring in HER2+ breast cancer and highlight the potential of targeting fatty acid oxidation and employing metabolic interventions as a combination therapy strategy for HER2+ breast cancer patients, including those resistant to standard treatment regimes.

overall_design: Mammary epithelial cells from transgenic model of breast cancer (MMTV-Neu NDL 2.5-IRES-CRE) were used to study the effects of CPT1A deletion on HER2-driven tumorigenesis. Transcriptomic analysis (RNA-Seq) was performed on cell line preparations from n = two (NIC/wild-type) and n = 4 (NIC/conditional Cpt1a KO) independent tumor-derived cell lines per genotype,

first: Ipshita

last: Nandi

first: Linjia

last: Ji

first: Harvey

middle: W

last: Smith

first: Daina

last: Avizon

first: Cynthia

last: Lavoie

first: Vasilios

last: Papavasiliou

first: William

last: Muller


title: CPEB2-activated axonal translation of VGLUT2 mRNA promotes glutamatergic transmission and presynaptic plasticity

city: Taipei

first: Yi-Shuian

last: Huang

address: 128 Sec. 2, Academia Rd. Nankang

country: Taiwan

institute: Institute of Biomedical Sciences, Academia Sinica

summary:
Background
Local translation at synapses is important for rapidly remodeling the synaptic proteome to sustain long-term plasticity and memory. While the regulatory mechanisms underlying memory-associated local translation have been widely elucidated in the postsynaptic/dendritic region, there is no direct evidence for which RNA-binding protein (RBP) in axons controls target-specific mRNA translation to promote long-term potentiation (LTP) and memory. We previously reported that translation controlled by cytoplasmic polyadenylation element binding protein 2 (CPEB2) is important for postsynaptic plasticity and memory. Here, we investigated whether CPEB2 regulates axonal translation to support presynaptic plasticity.

Methods
Behavioral and electrophysiological assessments were conducted in mice with pan neuron/glia- or AQ2glutamatergic neuron-specific knockout of CPEB2. Hippocampal Schaffer collateral (SC)-CA1 and temporoammonic (TA)-CA1 pathways were electro-recorded to monitor synaptic transmission and LTP evoked by 4 trains of high-frequency stimulation. RNA immunoprecipitation, coupled with bioinformatics analysis, were used to unveil CPEB2-binding axonal RNA candidates associated with learning, which were further validated by Western blotting and luciferase reporter assays. Adeno-associated viruses expressing Cre recombinase were stereotaxically delivered to the pre- or post-synaptic region of the TA circuit to ablate Cpeb2 for further electrophysiological investigation. Biochemically isolated synaptosomes and axotomized neurons cultured on a microfluidic platform were applied to measure axonal protein synthesis and FM4-64FX-loaded synaptic vesicles.

Results
Electrophysiological analysis of hippocampal CA1 neurons detected abnormal excitability and vesicle release probability in CPEB2-depleted SC and TA afferents, so we cross-compared the CPEB2-immunoprecipitated transcriptome with a learning-induced axonal translatome in the adult cortex to identify axonal targets possibly regulated by CPEB2. We validated that Slc17a6, encoding vesicular glutamate transporter 2 (VGLUT2), is translationally upregulated by CPEB2. Conditional knockout of CPEB2 in VGLUT2-expressing glutamatergic neurons impaired consolidation of hippocampus-dependent memory in mice. Presynaptic-specific ablation of Cpeb2 in VGLUT2-dominated TA afferents was sufficient to attenuate protein synthesis-dependent LTP. Moreover, blocking activity-induced axonal Slc17a6 translation by CPEB2 deficiency or cycloheximide diminished the releasable pool of VGLUT2-containing synaptic vesicles.

Conclusions
We identified 272 CPEB2-binding transcripts with altered axonal translation post-learning and established a causal link between CPEB2-driven axonal synthesis of VGLUT2 and presynaptic translation-dependent LTP. These findings extend our understanding of memory-related translational control mechanisms in the presynaptic compartment.

overall_design: RNAs precipitated by CPEB2 IgG and control IgG

first: Wen-Hsin

last: Lu

first: Hsiu-Chen

last: Lin


title: Transcriptomic profiling of long non-coding RNAs identifies circRmst as a regulator of midbrain dopamine neuron migration

city: Utrecht

first: Ronald

middle: Jeroen

last: Pasterkamp

state: Utrecht

address: Universiteitsweg 100

department: Translational Neuroscience

country: Netherlands

institute: University Medical Center Utrecht

overall_design: Refer to individual Series


title: Transcriptomic profiling of long non-coding RNAs identifies circRmst as a regulator of midbrain dopamine neuron migration [circRMSTKD]

summary: Midbrain dopamine (mDA) neurons play an essential role in cognitive and motor behaviours and are linked to different brain disorders. However, the molecular mechanisms underlying their development, and in particular the role of long non-coding RNAs (lncRNAs), remain incompletely understood.

overall_design: To provide experimental evidence for functional roles of circRMST, we investigated the effect of  circRmst Knockdown on neuron morphology and neuron migration together with the effect of circRmst KD on gene expression.

first: Mateja

last: Rybiczka-Tešulov

first: Oxana

last: Garritsen

first: Morten

last: Venø

first: Roland

last: van Dijk

first: Karim

last: Rahimi

first: Andreia

last: Gomes Duarte

first: Marina

last: de Wit

first: Lieke

last: van de Haar

first: Lars

last: Michels

first: Nicky

last: van Kronenburg

first: Christiaan

last: van der Meer

first: Jørgen

last: Kjems

first: Vamshidhar

middle: R

last: Vangoor

first: R


city: Aarhus N

last: Veno

state: DK

address: Aabogade 15

institute: Omiics ApS

summary:
Midbrain dopamine (mDA) neurons play an essential role in cognitive and motor behaviours and are linked to different brain disorders. However, the molecular mechanisms underlying their development, and in particular the role of long non-coding RNAs (lncRNAs), remain incompletely understood.
Here, we establish the transcriptomic landscape and alternative splicing patterns of linear and circular ncRNAs (lncRNAs and circRNAs) at key developmental timepoints in mouse mDA neurons using short and long-read RNAseq. Spatial analysis shows strong and subregion-specific expression of circRmst in mDA neurons and its post-transcriptional silencing affects soma size, TH+ neuron number, and mDA neuron positioning. Time-laps imaging indeed reveals that circRmst inhibits migration speed in developing mDA neurons. Together, these data for the first time establish a functional role for circRNAs in mDA neurons, further characterize poorly defined aspects of mDA neuron development, and provide a unique framework for future interrogation of lncRNAs in developing mDA neurons.

overall_design: Midbrain dopamine neurons from Pitx3-GFP mice were extracted and purified by FACS at E14 and P0. Midbrains from three litters were used for sample preparation. Three replicates were performed per group.


title: Intratumoral NKT cell accumulation promotes antitumor immunity in pancreatic cancer

status: Public on Jul 07 2024

city: Cold Spring Harbor

first: Jiayun

last: Li

state: NY

address: 1 Bungtown Rd

department: Cancer Center

country: USA

institute: Cold Spring Harbor Laboratory

summary: Here, using the mouse KRT19-deficient (sgKRT19-edited) PDA model, we find that intratumoral accumulation of natural killer T (NKT) cells are required to establish an immunologically active TME. Mechanistically, intratumoral NKT cells facilitate type I interferon (IFN) production to initiate an anti-tumor adaptive immune response, and orchestrate the intratumoral infiltration of T cells, dendritic cells, natural killer cells and myeloid-derived suppressor cells. At the molecular level, NKT cells promote the production of type I IFN through the interaction of their CD40L with CD40 on myeloid cells. To evaluate the theraputical potential of these observations, we find that administration of folinic acid to mice bearing PDA increases NKT cells in the TME and improves their response to anti-PD-1 antibody treatment. In conclusion, NKT cells have an essential role in the immune response to mouse PDA and are potential targets for immunotherapy.

overall_design: To investigate the impact of folinic acid (FA) treatment on tumor microenvironment, we performed gene expression profiling analysis by using bulk-RNAseq data of s.c. PDA tumors islodated from FA- and PBS-treated mice.

first: Douglas

last: Fearon


title: Generation of intestinal organoids from human urine cells

city: Guangzhou

first: Ruifang

last: Zhang

state: Guangdong

address: Haizhu District

country: China

institute: Guangzhou Laboratory

summary: Intestinal organoids are ideal models for studying human intestinal diseases. We report functional intestinal organoid models (U-iIOs) generated by reprogramming human urinary epithelial cells (hUCs). RNA-seq reveals the intestinal lineage identity of U-iIOs.

overall_design: Human urine cells are induced as intestinal organs. We analyzed the transcriptome expression profiles (RNA-seq).


title: Transcriptomic profiling of long non-coding RNAs identifies circRmst as a regulator of midbrain dopamine neuron migration [time series]

first: Ronald

last: Pasterkamp

summary:
Midbrain dopamine (mDA) neurons play an essential role in cognitive and motor behaviours and are linked to different brain disorders. However, the molecular mechanisms underlying their development, and in particular the role of long non-coding RNAs (lncRNAs), remain incompletely understood.
Here, we establish the transcriptomic landscape and alternative splicing patterns of linear and circular ncRNAs (lncRNAs and circRNAs) at key developmental timepoints in mouse mDA neurons using RNAseq at different time points during early development. Spatial analysis shows strong and subregion-specific expression of circRmst in mDA neurons and its post-transcriptional silencing affects soma size, TH+ neuron number, and mDA neuron positioning. Time-laps imaging indeed reveals that circRmst inhibits migration speed in developing mDA neurons. Together, these data for the first time establish a functional role for circRNAs in mDA neurons, further characterize poorly defined aspects of mDA neuron development, and provide a unique framework for future interrogation of lncRNAs in developing mDA neurons.

overall_design: Midbrain dopamine neurons from Pitx3-GFP mice were extracted and purified by FACS at E14, E16 and P0. Midbrains from three litters were used for sample preparation. Three replicates were performed per group.

first: Mateja

last: Rybiczka-Tešulov