title: Response-Adaptive Surgical Timing in neoadjuvant immunotherapy demonstrates enhanced pathologic treatment response in Head and Neck Squamous Cell Carcinoma

first: Adam

last: Luginbuhl

state: PA

address: 925 chestnut Street

department: Otolaryngology

institute: Thomas Jefferson University

summary: Abstract:  Purpose: We evaluated whether IDO-inhibitor BMS986205 (IDOi) + PD-1 inhibitor nivolumab enhanced T-cell activity and augmented immune-mediated antitumor responses in untreated, resectable HNSCC. We employed response-adaptive surgical timing to identify responders to immunotherapy and enhance their response.  Patients and Methods: Patients with HNSCC were 3:1 randomized to receive nivolumab with or without BMS986205 PO daily (NCT03854032). In the combination arm, BMS986205 was initiated 7 days prior to nivolumab. Patients were stratified by HPV status. Response-adaptive surgical timing involved response assessment by radiographic criteria 4 weeks after nivolumab in both arms. Non-responders underwent surgical resection, while responders received 4 more weeks of randomized therapy before surgery. Biomarker analysis utilized pathologic treatment response (pTR) and RNA sequencing.  Results: Forty-two patients were enrolled, and the addition of IDOi to nivolumab did not result in greater rate of radiographic response (p=0.909).  Treatment was well-tolerated with only 2 (5%) patients experiencing grade 3 immune-related adverse events. The addition of IDO-inhibitor augmented rates of pTR in patients with high baseline IDO RNA expression (p<0.05).  Response-adaptive surgical timing demonstrated reliability in differentiating pathologic responders versus non-responders (p=0.009). A pretreatment NK cell signature, PD-L1 status, and IFN-g expression in the HPV– cohort correlated with response. HPV+ cohort found B-cell and CAF signatures predictive of response/non-response.  Conclusions: Response-adaptive surgical timing enhanced treatment response. IDO-inhibitor BMS986205 augmented pTR in patients with high IDO1-expression in baseline samples, indicating a need for identifying and targeting resistant nodes to immunotherapy. HPV-status-dependent signatures predicting response to immunotherapy in HNSCC warrant further study.

overall_design: Unbiased GSEA

first: Eric

last: Mastrolonardo

first: Alban

last: Linnenbach


title: Single-cell RNA sequencing of human IL-18R supported CAR T cells targeting oncofetal Tenascin C

status: Public on Nov 20 2024

city: Memphis

first: Liqing

last: Tian

state: TN

address: 262 Danny Thomas Pl

country: USA

institute: St. Jude Children's Research Hospital

type: Expression profiling by high throughput sequencing

summary: C.TNC-CAR.Zip18R CAR-T cell of 2 donors were used for 2 in vitro experiments. In the first experiment, we did scRNA-seq for activated CAR-T cells. In the second experiment, the CAR-T cells of each donor were cocultured with tumor cells for 12 hrs, 24 hrs and 48 hrs. The CAR-T cells for each timepoints were extracted for scRNA-seq. Multiplexed 10x Genomics scRNA-seq were performed with a hashtag to label each sample.

overall_design:
The experiments were designed to compare the dynamic of cell population size and enriched pathway in different cell populations.

***************************************************************
Raw files for human/patient samples are being made available in EGA (https://www.ebi.ac.uk/ega/) for controlled access to the personally identifiable sequence data.
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title: Genetic Disruption of Blimp-1 Drastically Augments the Anti-Tumour Efficacy of BCMA-Targeting CAR-T Cells

city: Barcelona

first: David

middle: F.

last: Moreno

state: Barcelona

address: Villarroel 170

department: Hematology

country: Spain

institute: IDIBAPS - Hospital Clínic de Barcelona

summary: Chimeric antigen receptor-T (CAR-T) cells directed against B-cell maturation antigen (BCMA) are an effective treatment for multiple myeloma, but short persistence and frequent relapses are challenges for this immunotherapy. This lack of durability has been attributed to the premature terminal differentiation of CAR-T cells which prevents the formation of long-lived memory cells that maintain anti-tumour responses. To improve long-term efficacy, we used CRISPR/Cas9-mediated gene editing to ablate the expression of the transcription factor Blimp-1 (Prdm1). In a murine model of advanced myeloma, Blimp-1 knockout (KO) CAR-T cells effectively slowed or even prevented disease progression, significantly outperforming control (Mock) CAR-T cells in improving survival. To understand this enhanced in vivo effectiveness, Blimp-1 KO CAR-T cells were characterised after being repeatedly challenged with tumour cells in vitro and subject to gene expression analysis.

overall_design: Human T cells transduced with an anti-BCMA CAR gene (ARI0002h) were expanded in vitro and separated into cultures that acted as a control (Mock) or were subject to CRISPR/Cas9 to ablate the expression of Blimp-1 (Blimp-1 KO). Expanded cells were stimulated four times with the multiple myeloma cell line ARP-1, and RNA was isolated from purified CD8+ cells at the end of the co-cultures. Gene expression profiles were assessed by RNA sequencing.

first: Anthony

last: Battram

first: Joan

last: Mañé


title: Orexin/Hypocretin Receptor 2 Signaling in MCH Neurons Regulates REM sleep and Insulin Sensitivity

status: Public on Nov 19 2024

city: Köln

first: Jens

middle: C

last: Brüning

address: Gleueler Str. 50

department: Neuronal Control of Metabolism

country: Germany

institute: Max Planck Institute for Metabolism Research

summary: Orexin/hypocretin receptor type 2 (Ox2R), which is widely expressed in the brain, receives orexin signals and modulates sleep and metabolism. Ox2R selective agonists are currently under clinical trials for narcolepsy treatment. Nevertheless, it remains unclear whether Ox2R exerts an inhibitory effect via Gi proteins in addition to an excitatory effect via Gq proteins. Here, we focused on Ox2R expression and function in MCH neurons, which have opposite roles to orexin neurons in sleep and metabolism regulation. Ox2R-expressing MCH neurons showed heterogeneity of RNA expression, and orexin B application in brain slices induced both excitatory and inhibitory responses in distinct MCH neuron populations. Ox2R inactivation in MCH neurons reduced transitions from NREM to REM sleep and impaired insulin sensitivity with hyperphagia. In conclusion, Ox2R mediates excitatory and inhibitory responses in MCH neuron subpopulations in vivo, which might regulate sleep and metabolism.

overall_design: Pooled hypothalami from multiple male or female MCH-Cre; lsl-ZsGreen mice were FACS-sorted and subjected to single-nucleus RNA-seq to investigate the molecular subclustering of MCH neurons.

first: Shuntaro

last: Izawa

first: Hong

last: Jiang

first: Lukas

last: Steuernagel


title: Microbial-upcycling of polyamides using engineered Pseudomonas putida

city: Jülich

first: Tom

last: Luthe

address: Wilhelm-Johnen-Strasse

institute: Forschungszentrum Jülich

summary: Metabolic engineering of P. putida to metabolize PA monomers and oligomers to produce polyhydroxybutyrate

overall_design: RNA squencing to reveal metabolic routes

first: Jan

last: de Witt

first: Nick

last: Wierckx


title: A genome-wide screen identifies silencers with distinct chromatin properties and mechanisms of repression [repressed STARR-seq]

city: Vienna

first: Alexander

last: Stark

address: Campus-Vienna-Biocenter 1

country: Austria

institute: The Research Institute of Molecular Pathology (IMP)

summary: Differential gene transcription enables development and homeostasis in all animals and is regulated by two major classes of distal cis-regulatory DNA elements (CREs), enhancers and silencers. While enhancers have been thoroughly characterized, the properties and mechansisms of silencers remain largely unknown. By an unbiased genome-wide functional screen in Drosophila melanogaster S2 cells, we discover a novel class of silencers that bind one of three transcription factors (TFs) and are generally not included in chromatin-defined CRE catalogs, as they mostly lack detectable DNA accessibility. The silencer-binding TF CG11247, which we term Saft, safeguards cell fate decisions in vivo and functions via a novel highly-conserved domain ZAC and the corepressor G9a, independently of G9a’s H3K9-methyltransferase activity. Overall, our identification of silencers with unexpected properties and mechanisms has important implications for the understanding and future study of repressive CREs, as well as the functional annotation of animal genomes.

overall_design: Control and repressed focused STARR-seq experiments, using either a standard DSCP and RpS12 core promoter BAC-scale STARR-seq library mix or an equivalent mix with an invariant DLM3 silencer in the backbone, were perfomed in Drosophila melanogaster Schneider S2 cells. For both conditions two biological replicates were sequenced in paired-end mode, using 1% Drosophila pseudoobscura BAC-scale STARR-seq libraries as spike-in for sample normalization.

first: Lorena

last: Hofbauer

first: Lisa-Marie

last: Pleyer

last: Schleiffer

first: Franziska

last: Reiter

first: Anna

last: Vlasova

first: Leonid

last: Serebreni

first: Annie

last: Huang


title: Type II interferon-dependent antibacterial defense in the lungs is compromised in diabetes

city: Berlin

first: Bastian

last: Opitz

address: Augustenburgerplatz 1

department: Department of Infectious Diseases, Respiratory Medicine and Critical Care

institute: Charité – Universitätsmedizin Berlin

summary: Diabetes mellitus is associated with an increased risk of pneumonia, which is often caused by so-called typical and atypical pathogens including Streptoccocus pneumoniae and Legionella pneumophila, respectively. Here, we employed a varity of mouse models to investigate how diabetes influences pulmonary antibacterial immunity. Following intranasal infection with Streptoccocus pneumoniae or Legionella pneumphila, type 2 diabetic and prediabetic mice had higher bacterial loads in their lungs as compared to control animals. Single cell RNA sequencing, flow cytometry, and functional analyses revealed a compromised type II IFN (IFNg) production by natural killer cells in diabetic and prediabetic mice, which was linked to reduced IL-12 production by CD103+ dendritic cells. Treatment with IFNg rescued antibacterial defense against L. pneumophila but not against S. pneumoniae in diabetic animals, although impaired production of IFNg also contributed to the compromised anti-pneumococcal resistance in diabetic mice. These findings uncover a mechanism that could help to explain why type 2 diabetes predisposes to pneumonia. We establish proof of concept for host-directed treatment strategies to reinforce compromised IFNg-mediated antibacterial defense against atypical lung pathogens.

overall_design: Mice were infected and after 18 hours lungs were digested to single cells and subjected to 10x scRNAseq.  Control_groups and Diabetic_groups.

first: Facundo

last: Fiocca Vernengo

first: Ivo

last: Röwekamp


title: A genome-wide screen identifies silencers with distinct chromatin properties and mechanisms of repression [Silencer-seq]

overall_design: STARR-seq-based silencer screens (= silencer-seq; 500-800bp genomic candidate sequences) were performed in Drosophila melanogaster Schneider S2 cells using two focused BAC-scale reporter libraries (differing in the enhancer driving reporter expression: sgl or ham enhancer) and one genome-wide reporter library (sgl enhancer). All libraries were transfected in two biological replicates and sequenced together with inputs in paired-end mode.


title: Genes expression alterations associated with resistance to combined CDK4/6 inhibitor and endocrine therapy in ER+ breast cancer

city: Odense M

first: Carla

last: Alves

address: Fioniavej 36, 2. sal

country: Denmark

institute: SDU

summary: Combined CDK4/6 inhibitor (CDK4/6i) and endocrine therapy significantly improves the outcome of patients with advanced estrogen receptor-positive (ER+) breast cancer. However, resistance to this treatment and resultant disease progression remains a major clinical challenge. High expression of the receptor tyrosine kinase REarranged during Transfection (RET) has been associated with resistance to endocrine therapy in breast cancer, but the role of RET in CDK4/6i treatment response/resistance remains unexplored. To identify gene expression alterations associated with resistance to combined endocrine therapy and CDK4/6i, we performed global gene expression analysis and RNA sequencing of two ER+ breast cancer cell models resistant to this combined therapy.

overall_design: Cells sensitive, resistant to fulvestrant or resistant to combined fulvestrant and CDK4/6i derived from MCF7 and T47D cell lines were included in the study. Cells were grown to reach 70-80% confluency and RNA was extracted using TRIzol™ Reagent according to manufacturer’s instructions


title: b-Glucan Reprograms Neutrophils to Induce Disease Tolerance Against Influenza A Virus

city: Chicago

first: Veronica

last: Locher

state: IL

institute: University of Chicago

summary: Disease tolerance is an evolutionarily conserved host defence strategy that preserves tissue integrity and physiology without affecting pathogen load. Unlike host resistance, the mechanisms underlying disease tolerance remain poorly understood. In the present study, we investigated whether an adjuvant (β-glucan) can reprogram innate immunity to provide protection against Influenza A virus (IAV) infection. Here we observe that β-glucan treatment reduced the morbidity and mortality against IAV infection, independent of host resistance (viral load). Increased survival of β-glucan treated mice against IA V is associated with the accumulation of neutrophils via RoRgt+ T cells in the lung tissue. Using gain-and-loss-of-function approaches, we demonstrate that β- glucan reprogrammed neutrophils are essential for promoting disease tolerance, limiting pulmonary tissue damage, and enhancing survival against IAV infection. β-glucan treatment promotes granulopoiesis in a type 1 interferon-dependent manner that leads to the generation of a unique subset of neutrophils, which are less mature with higher mitochondrial mass utilizing mitochondrial oxidative (OXPHOS) metabolism. Collectively, our data indicate that β-glucan reprograms hematopoietic stem cells (HSCs) to generate neutrophils with a novel “regulatory” function, which is required for promoting disease tolerance and maintaining lung tissue integrity against viral infection.

overall_design: scRNA-sequencing of lung cell isolates 4 and 7 days following beta glucan treatment


title: Aberrantly Expressed Mitochondrial Lipid Kinase, AGK Activates JAK2-Histone H3 Axis and BCR Signal: A Mechanistic Study with Implication in CLL Therapy

city: Oklahoma City

first: Chao

last: Xu

state: Oklahoma

address: 801 N. E. 13th Street

department: Biostatistics and Epidemiology

institute: University of Oklahoma Health Sciences Center

summary: Purpose: Although the B-cell receptor (BCR) signal plays a critical role in CLL cell survival and a target of current therapies (ibrutinib targets Bruton’s tyrosine kinase; idelalisib targets PI3Kδ), contribution of the cytokine-driven JAK2 pathway to the “CLL cell-survival signaling network” is largely undefined. Experimental Design: CLL patients were enrolled to investigate expression/activation of JAK2 and acylglycerol kinase (AGK), and their functional implication in primary CLL cell survival. A series of biochemical and molecular biology assays were employed to uncover the underlying mechanism. Results: We detected that compared to normal B-cells, CLL cells aberrantly express constitutively active JAK2. Mechanistically, HSP90 forms a chaperoning complex with JAK2, resulting in its aberrant accumulation in CLL cells. We also discovered aberrant upregulation of a novel mitochondrial lipid kinase, AGK, which remains complexed with HSP90 in CLL cells activating JAK2. Although AGK is typically mitochondrial, we detected its nuclear localization in association with JAK2 in some CLL cells. Functionally, JAK2 phosphorylates its non-canonical substrate, histone H3(Y41), but not STAT3, activating transcription of diverse sets of genes in a patient-specific manner. Additionally, JAK2 activates the BCR-signal in CLL cells via LYN/BTK axis. Targeted inhibition of JAK2 as monotherapy, or in combination with the BCR-inhibitors or venetoclax (a BCL2-inhibitor) induced apoptosis synergistically in CLL cells. Conclusions: These findings suggest that aberrantly expressed AGK activates JAK2, independent of cytokine, leading to activation of diverse sets of gene transcription in CLL cells. Combined targeting of JAK2 and BCR signals or BCL2 may be effective in some CLL patients.

first: Asish

middle: K

last: Ghosh


title: A genome-wide screen identifies silencers with distinct chromatin properties and mechanisms of repression [ChIP-seq]

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: ChIP-seq against FLAG was performed in Drosophila melanogaster Schneider S2 cells with endogenously FLAG-AID-tagged transcription factors (CG7372/Phaser and CG11247/Saft). Single-read sequencing of two biological replicates and corresponding input samples was done in each clonal cell line.


title: A genome-wide screen identifies silencers with distinct chromatin properties and mechanisms of repression

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

overall_design: Refer to individual Series


title: A noncanonical role of roX RNAs in autosomal epigenetic repression [RNA-seq]

city: Shenzhen

first: Qing

last: Ma

address: 1068 Xueyuan Avenue, Shenzhen University Town, Shenzhen, P.R.China

country: China

institute: Shenzhen Institutes of Advanced Technology

summary: Long noncoding RNAs known as roX (RNA on X) are crucial for male development in Drosophila, as their loss leads to male lethality from the late larval stages. While roX RNAs are recognized for their role in sex-chromosome dosage compensation, ensuring balanced expression of X-linked genes in both sexes, their potential influence on autosomal gene regulation remains unexplored. Using an integrative multi-omics approach, we revealed that roX RNAs not only govern the X chromosome but also target genes on autosomes that lack male-specific lethal (MSL) complex occupancy, together with Polycomb repressive complexes (PRCs). We observed that roX RNAs colocalize with MSL proteins on the X chromosome and PRC components on autosomes. Intriguingly, loss of roX function reduces X-chromosomal H4K16ac levels and autosomal H3K27me3 levels. Correspondingly, X-linked genes display reduced expression, whereas many autosomal genes exhibit elevated expression upon roX loss. Our findings propose a dual role for roX RNAs: activators of X-linked genes and repressors of autosomal genes, achieved through interactions with MSL and PRC complexes, respectively. This study uncovers the unconventional epigenetic repressive function of roX RNAs.

overall_design: To investigate the impact of roX RNAs in transcriptional regulation, gene expression profiles in roX1 roX2 double knockout (roX-KO) male larvae and their wild type counterparts were generated by RNA-seq, and comparative analysis was performed.

first: Jianjian

last: Li

first: Shuyang

first: Manyin

last: Wu


title: A noncanonical role of roX RNAs in autosomal epigenetic repression [PIRCh-seq]

overall_design: To investigate the association of RNAs with chromatin in Drosophila, histone modifications H4K16ac- and H3K27me3-associated RNAs were identified using PIRCh-seq.

first: Jeffrey

middle: J

last: Quinn


title: A noncanonical role of roX RNAs in autosomal epigenetic repression [ChIP-seq]

overall_design: Genomic binding profiles of H3K27me3 in roX1 roX2 double knockout (roX-KO) male larvae and their wild type counterparts were generated by ChIP-seq.

first: Zicong

last: Liu


title: Acetyl-CoA production by Mediator-bound 2-ketoacid dehydrogenases boosts de novo histone acetylation and is regulated by nitric oxide [Chrom_RNAseq]

city: Milan

first: Francesco

last: Gualdrini

address: Via Adamello, 16

department: Department of Experimental Oncology

country: Italy

institute: European Institute of Oncology

summary: Histone modifying enzymes depend on the availability of cofactors, with acetyl-CoA being required for histone acetyltransferase (HAT) activity. The discovery that mitochondrial acyl-CoA producing enzymes are also delivered to the nucleus suggests that high concentrations of metabolites generated locally may impact acetylation of histones and other nuclear substrates and eventually control gene regulation. Here we show that 2-ketoacid dehydrogenases were stably associated with the Mediator complex in macrophages, thus providing a local supply of acetyl-CoA and increasing the generation of hyper-acetylated histone tails. Nitric oxide (NO), which is produced in large amounts in LPS-stimulated macrophages, inhibited both the activity of 2-ketoacid dehydrogenases and their association with Mediator. Consequently, NO reduced de novo histone acetylation at genomic regions with high acetyl-CoA deposition rates. Our findings indicate that a local supply of acetyl-CoA generated by Mediator-bound 2-ketoacid dehydrogenases is required to maximize acetylation of histone tails at sites of elevated HAT activity.

overall_design: Chromatin-associated RNA-seq in BMDM. Cells were treated with lipopolysaccharide (LPS) from E. coli, serotype EH100(Ra) from Alexis (cat. no. ALX-581-010-L002) at 10 ng/mL (BMDMs). Trichostatin A (TSA, cat. no. S1045, Selleck) was added for 30 min. Spermine Nonoate (NO donor, cat, no. CAY-82150, Cayman Chemical) was added at 0.5 mM for 15 min before TSA or LPS treatment.

first: Marta

last: Russo

last: Vallelonga

first: Elena

last: Prosperini

first: Roberta

last: Noberini

first: Silvia

last: Pedretti

first: Pierluigi

last: DiChiaro

first: Sara

last: Polletti

first: Gabriele

last: Imperato

first: Mattia

last: Marenda

first: Chiara

last: Ghirardi

first: Fabio

last: Bedin

first: Alessandro

last: Cuomo

first: Simona

last: Rodighiero

first: Tiziana

last: Bonaldi

first: Nico

last: Mitro

first: Serena

last: Ghisletti

first: Gioacchino

last: Natoli


title: A noncanonical role of roX RNAs in autosomal epigenetic repression

first: Qing

last: Ma


title: Acetyl-CoA production by Mediator-bound 2-ketoacid dehydrogenases boosts de novo histone acetylation and is regulated by nitric oxide [Chipseq_MED1_RAW2647]

first: Francesco

last: Gualdrini

overall_design: MED1 ChIP-seq in RAW264.7 cells

first: Marta

last: Russo

first: Veronica

last: Vallelonga


title: Acetyl-CoA production by Mediator-bound 2-ketoacid dehydrogenases boosts de novo histone acetylation and is regulated by nitric oxide [Chrom_RNAseq_iNOS1400W]

overall_design: Chromatin-associated RNA-seq in BMDM following 8h and 16h LPS stimulation and in the presence or absence of 100 mM iNOS inhibitor 1400W (1400W, cat. no. CAY-81520, Cayman Chemical)  added 30 min before macrophage activation.

first: Elena

last: Prosperini

first: Roberta

last: Noberini

first: Silvia

last: Pedretti


title: Acetyl-CoA production by Mediator-bound 2-ketoacid dehydrogenases boosts de novo histone acetylation and is regulated by nitric oxide [Chipseq_MED1_HELA]

overall_design: MED1 ChIP-seq in HELA cells

first: Pierluigi

last: DiChiaro

first: Sara

last: Polletti

first: Gabriele

last: Imperato


title: Acetyl-CoA production by Mediator-bound 2-ketoacid dehydrogenases boosts de novo histone acetylation and is regulated by nitric oxide [Chipseq_H3K27ac_spikein]

overall_design: Chip-seq of H3K27ac from BMDM chromatin infected with lentivirus expressing either non-targeting shRNA (shNT) or shRNA targeting MED14. BMDM chromatin was mixed with Drosophila chromatin spike-in. Cells were treated with lipopolysaccharide (LPS) from E. coli, serotype EH100(Ra) from Alexis (cat. no. ALX-581-010-L002) at 10 ng/mL (BMDMs). Trichostatin A (TSA, cat. no. S1045, Selleck) was added for 30 min. Spermine Nonoate (NO donor, cat, no. CAY-82150, Cayman Chemical) was added at 0.5 mM for 15 min before TSA or LPS treatment.

first: Mattia

last: Marenda

first: Chiara

last: Ghirardi

first: Fabio

last: Bedin


title: Cross-species Regulatory Network Analysis Identifies FOXO1 as a Driver of Ovarian Follicular Recruitment

city: Newark

first: Aditya

last: Dutta

state: DE

address: 303 Worrilow, 529 South College Ave

department: Animal & Food Sciences

institute: University of Delaware

summary:
To indentify the master regulators that control the processes between the pre-recruitment and pre-ovulatory F5 follicular stages in laying hens.
RNA was isolated from a group of 3-5 pre-recruitment follicles and the most recently recruiteed pre-ovualtory follicle (F5). After RNA isolation, follicles underwent RNA sequencing and differential gene expression analysis occurred.

relation:
Reanalysis of: GSM7147175
Reanalysis of: GSM7147176
Reanalysis of: GSM7147177
Reanalysis of: GSM7147178
Reanalysis of: GSM7147179
Reanalysis of: GSM7147180
Reanalysis of: GSM7147181
Reanalysis of: GSM7147182
Reanalysis of: GSM7147183
Reanalysis of: GSM7147185
Reanalysis of: GSM7147186
Reanalysis of: GSM7147184

overall_design:
Follicles from two follicular stages from 12 month old hens were used. Total RNA was obtained folliclular tissue samples using TRIZol reagent and RNA-Sequencing occurred.

This study represents a re-analysis of all of the raw data generated for GSE228994.

first: Ashley

last: Kramer

first: Kathryn

last: Ellwood


title: Acetyl-CoA production by Mediator-bound 2-ketoacid dehydrogenases boosts de novo histone acetylation and is regulated by nitric oxide [Chipseq_FLAG_DLST_ODGH]

overall_design: Flag-Tagged Chip-seq  in Hela cells over-expressing  DLST , OGDH or Empty Vehicle.

first: Alessandro

last: Cuomo

first: Simona

last: Rodighiero

first: Tiziana

last: Bonaldi

first: Nico

last: Mitro

first: Serena

last: Ghisletti

first: Gioacchino

last: Natoli


title: Mutations in unfolded protein response regulator ATF6 cause hearing and vision loss syndrome

city: Palo Alto

first: Eun-Jin

last: Lee

address: 3801 Miranda Ave

department: Pathology

institute: Stanford University

summary: Activating transcription factor 6 (Atf6) is a key regulator of the unfolded protein response (UPR) and is important for endoplasmic reticulum (ER) function and protein homeostasis in metazoan cells. Patients carrying loss-of-function ATF6 disease alleles develop the cone dysfunction disorder, achromatopsia.  The impact of loss of ATF6 function on other cell types, organs, and diseases in people remains unclear. Here, we reported that progressive sensorineural hearing loss was a notable complaint in some patients carrying ATF6 disease alleles and that Atf6-/- mice also showed progressive auditory deficits affecting both genders.  In mice with hearing deficits, we found disorganized stereocilia on hair cells and focal loss of outer hair cells.  Transcriptomic analysis of Atf6-/- cochleae revealed marked induction of UPR, especially through the PERK arm.  These findings identify ATF6 as an essential regulator of cochlear health and function.  Furthermore, they supported that ATF6 inactivation in people causes progressive sensorineural hearing loss as part of a blindness-deafness genetic syndrome targeting hair cells and cone photoreceptors.  Lastly, our genetic findings support ER stress as an important pathomechanism underlying cochlear damage and hearing loss with clinical implications for patient lifestyle modifications that minimize environmental/physiologic sources of ER stress to the ear.

overall_design: In light of the broad range of organs impacted by a loss of ATF6 in experimental animal models, we investigated whether patients lacking functional ATF6 had additional diseases or phenotypes beyond the previously reported vision loss. We also characterized the cellular and molecular defects in the ear that underlie the hearing loss in the Atf6-/- mice.

first: Jonathan

last: Lin


title: B-cell Interleukin 1 Receptor 1 modulates the female adipose tissue immune microenvironment during aging

city: Minneapolis

first: Anna

last: Carey

state: Minnesota

address: 312 Church Street SE

department: Biochemistry, Molecular Biology, and Biophysics

institute: University of Minnesota

summary: The Nod like receptor protein 3 (NLRP3) inflammasome and its product interleukin-1β (IL-1β) drive inflammaging in visceral adipose tissue (vWAT) and contribute to the expansion of interleukin-1 receptor 1 (Il1r1) positive aged adipose B-cells (AABs). AABs promote metabolic dysfunction and inflammation under inflammatory challenges. However, it is unclear whether IL-1β contributes to AAB-associated inflammation during aging. Using a B-cell specific knockout of Il1r1 (BKO mice), we characterized old vWAT in the absence of IL-1β – B-cell signaling. In addition to sex-specific metabolic improvements in females, we identified a reduction in the proportion of B-cells and a sex-specific increase in the B1:B2 B-cell ratio in BKO vWAT. Using single cell RNA-sequencing of vWAT immune cells, we observed that BKO differentially affected inflammatory signaling in vWAT immune cells. These data suggest that IL-1β – B-cell signaling supports the inflammatory response in multiple cell types and provides insight into the complex microenvironment in aged vWAT.

overall_design: Single cell RNA sequencing (10x genomics) on sorted CD45+ live cells from the visceral adipose tissue of 3m WT (n=2), 22m WT (n=3), and 22m IL1R1 fl/fl CD19cre+ (n=3) female mice challenged with 0.1 mg/kg LPS for 4 hours.

first: Louise

middle: E

last: Pitcher

first: In

middle: H

last: Jang

first: Katie

last: Nguyen

first: Stephanie

last: Cholensky

first: Paul

middle: D

last: Robbins

first: Christina

last: Camell


title: A genome-wide screen identifies silencers with distinct chromatin properties and mechanisms of repression [MNase-seq]

first: Alexander

last: Stark

overall_design: MNase-seq was performed in Drosophila melanogaster Schneider S2 cells with endogenously FLAG-AID-tagged CG11247/Saft after 48h auxin-induced CG11247/Saft depletion or under mock conditions. For each condition, two biological replicates were sequenced in paired-end mode to elucidate changes in nucleosome distribution upon CG11247/Saft depletion.

first: Lorena

last: Hofbauer

first: Lisa-Marie

last: Pleyer

last: Schleiffer

first: Franziska

last: Reiter

last: Vlasova

first: Leonid

last: Serebreni

first: Annie

last: Huang


title: Mitochondrial NAD+ deficiency in vascular smooth muscle impairs collagen III turnover to trigger thoracic and abdominal aortic aneurysm

city: Shanghai

first: Zhang

last: Weijia

address: Dongan Road 131

institute: Fudan University

summary: Thoracic and abdominal aortic aneurysm poses a substantial mortality risk in adults, yet many of its underlying factors remain unidentified. Here, we identify mitochondrial nicotinamide adenine dinucleotide (NAD)⁺ deficiency as a causal factor for the development of aortic aneurysm. Multiomics analysis of 150 surgical aortic specimens indicated impaired NAD+ salvage and mitochondrial transport in human thoracic aortic aneurysm, with expression of the NAD+ transporter SLC25A51 inversely correlating with disease severity and postoperative progression. Genome-wide gene-based association analysis further linked low SLC25A51 expression to risk of aortic aneurysm and dissection. In mouse models, smooth muscle-specific knockout of Nampt, Nmnat1, Nmnat3, Slc25a51, Nadk2 and Aldh18a1, genes involved in NAD+ salvage and transport, induced aortic aneurysm, with Slc25a51 deletion producing the most severe effects. Using these models, we suggest a mechanism that may explain the disease pathogenesis: the production of type III procollagen during aortic medial matrix turnover imposes a high demand for proline, an essential amino acid component of collagen. Deficiency in the mitochondrial NAD⁺ pool, regulated by NAD⁺ salvage and transport, hinders proline biosynthesis in mitochondria, contributing to thoracic and abdominal aortic aneurysm.

overall_design: Comparative gene expression profiling analysis of RNA-seq data between 32 surgically resected thoracic aorta tissues from patients who underwent aorta replacement surgery and 11 nondiseased aorta from age-matched patients who underwent coronary artery bypass surgery or heart transplant donors without aortic aneurysm, dissection, coarctation, or previous aortic repair.

first: Jingjing

last: Zhang


title: Tumor-associated neutrophils attenuate the immuno-sensitivity of hepatocellular carcinoma and curtail immunotherapy response [RNA-Seq]

city: Hong Kong

first: Nickolas

last: Teo

address: 1/F, Laboratory Block, 21 Sassoon Road

department: SBMS

country: Hong Kong

institute: HKU

summary: Tumor-associated neutrophils (TANs) are heterogeneous; thus, their roles in tumor development could vary depending on the cancer type. Here, we showed that TANs were more detrimental to metabolic dysfunction-associated steatohepatitis hepatocellular carcinoma (MASH-related HCC) than to viral-associated HCC. We attributed this difference to the predominance of SiglecFhi TANs in MASH-related HCC tumors. Linoleic acid and GM-CSF, which are commonly elevated in the MASH-related HCC microenvironment, fostered the development of this c-Myc-driven TAN subset. Through TGFβ secretion, SiglecFhi TANs promoted HCC stemness, proliferation, and migration. Importantly, SiglecFhi TANs supported immune evasion by directly suppressing the antigen presentation machinery of tumor cells. SiglecFhi TAN removal increased the immunogenicity of a MASH-related HCC model and sensitized it to immunotherapy. Likewise, a high SiglecFhi TAN signature was associated with poor prognosis and immunotherapy resistance in HCC patients. Overall, our study highlights the importance of understanding TAN heterogeneity in cancer to improve therapeutic development

overall_design: Single, live, immune (CD45+) and tumor cells (CD45-TER119-) cells were sort-purified from the bone marrow, spleen, and tumor of NRAS/AKT HCC-bearing mice for library preparation with Chromium Next GEM Single Cell 5’ Reagent Kit v2.

first: Jia Ming Nickolas

first: Zhulin

last: Chen

first: Weixin

first: Rachael Julia Yuenyinn

last: Tan

first: Qi

last: Cao

first: Yingming

last: Chu

first: Delin

last: Ma

first: Liting

first: Huajian

last: Yu

first: Ka-Hei

last: Lam

first: Terence

last: Lee

first: Svetoslav

last: Chakarov

first: Burkhard

last: Becher

first: Ning

first: Li

last: Zhao

first: Ruidong

last: Xue

first: Guang Sheng

last: Ling


title: Tumor-associated neutrophils attenuate the immuno-sensitivity of hepatocellular carcinoma and curtail immunotherapy response [scRNA_Seq]

first: Nickolas

first: Jia Ming Nickolas

first: Stephanie


title: The master male sex determinant Gdf6Y of the turquoise killifish arose through allelic neofunctionalization [Mouse]

city: Jena

first: Philipp

last: Koch

address: Beutenbergstraße 11

department: Core Facility Life Science Computing

institute: Leibniz Institute on Aging - Fritz Lipmann Institute

summary: Although sex determination is a fundamental process in vertebrate development, it is very plastic. Diverse genes became major sex determinants in teleost fishes. Deciphering how individual sex-determining genes orchestrate sex determination can reveal new actors in sexual development. Here, we demonstrate that the Y-chromosomal copy of the TGF-β family member gdf6 (gdf6Y) in Nothobranchius furzeri, an emerging model organism in aging research, gained the function of the male sex determinant through allelic diversification while retaining the skeletal developmental function shared with the X-chromosomal gdf6 allele (gdf6X). Concerning sex determination, gdf6Y is expressed by somatic supporting cells of the developing testes. There it induces the male sex in a germ cell-independent manner in contrast to sex determination in zebrafish and the medaka. Looking for downstream effectors of Gdf6Y, we identified besides TGF-β signaling modulators, especially the inhibitor of DNA binding genes id1/2/3, the mRNA decay activator zfp36l2 as a new GDF6 signaling target.

overall_design: Comparative gene expression profiling analysis of murine Sertoli-like TM4 cells that were transfected with a gdf6Y expression plasmid [gdf6Y] or two control plasmids (empty vector [EV] or gdf6Y mutant variant [del9])

first: Annekatrin

last: Richter

first: Hanna

last: Mörl

first: Marco

last: Groth


title: Transcriptomic Data Reveals MYC as an Upstream Transcriptional Regulator in Laying Hen Pre-ovulatory Follicles using Ingenuity Pathway Analysis

first: Aditya

last: Dutta

summary:
To investigate the difference between the pre-ovulatory F5 and pre-recruitment follicular stages in laying hens and identify master regulators that control the processes in these two follicular stages.
RNA was isolated from a group of 3-5 pre-recruitment follicles and the most recently recruited pre-ovulatory follicle (F5). After RNA isolation, follicles underwent RNA sequencing and differential gene expression analysis occurred.

overall_design: Follicles from two follicular stages from 12 month old hens were used. Total RNA was obtained folliclular tissue samples using TRIZol reagent and RNA-Sequencing occurred.

first: Ashley

last: Kramer

first: Kathryn

last: Ellwood


title: The master male sex determinant Gdf6Y of the turquoise killifish arose through allelic neofunctionalization [Human]

overall_design: Comparative gene expression profiling analysis of human HeLa cells that were transfected with a gdf6Y expression plasmid [gdf6Y] or two control plasmids (empty vector [EV] or gdf6Y mutant variant [del9])


title: A genome-wide screen identifies silencers with distinct chromatin properties and mechanisms of repression [RNA-seq]

first: Alexander

last: Stark

overall_design: To investigate the gene-regulatory functions of the CG11247/Saft transcription factor, we profiled gene expression by RNA-seq in the Drosophila melanogaster brain, ovary and Schneider S2 cells in wt or CG11247/Saft knockout/depletion conditions. Fly tissue experiments were performed in two indepedent Saft frame-shift knockout strains or the parent wt strain. Schneider S2 cell experiments were performed in a CG11247/Saft-FLAG-AID-tagged cell line under 24h and 48h auxin-induced CG11247/Saft depletion or no auxin treatment. Two (brain and Schneider S2 cells) or three (ovary) biological replicates were sequenced in single-read mode.

first: Lorena

last: Hofbauer

first: Lisa-Marie

last: Pleyer

first: Franziska

last: Reiter

last: Schleiffer

first: Anna

last: Vlasova

first: Leonid

last: Serebreni

first: Annie

last: Huang


title: Copper chelation redirects neutrophil function to enhance anti-GD2 antibody therapy in neuroblastoma [scRNA-seq]

status: Public on Nov 18 2024

city: Kensington

first: Orazio

last: Vittorio

address: High St

department: School of Biomedical Sciences, Faculty of Medicine

country: Australia

institute: UNSW Sydney

summary: Anti-disialoganglioside (GD2) antibody therapy has provided clinical benefit to patients with neuroblastoma however efficacy is likely impaired by the immunosuppressive tumor microenvironment. We have previously defined a link between intratumoral copper levels and immune evasion. Here, we report that adjuvant copper chelation potentiates anti-GD2 antibody therapy to confer durable tumor control in immunocompetent models of neuroblastoma. Mechanistic studies reveal copper chelation creates an immune-primed tumor microenvironment through enhanced infiltration and activity of Fc-receptor-bearing cells, specifically neutrophils which are emerging as key effectors of antibody therapy. Moreover, we report copper sequestration by neuroblastoma attenuates neutrophil function which can be successfully reversed using copper chelation to increase pro-inflammatory effector functions. Importantly, we repurpose the clinically approved copper chelating agent Cuprior as a non-toxic, efficacious immunomodulatory strategy. Collectively, our findings provide robust evidence for the clinical testing of Cuprior as an adjuvant to enhance the activity of anti-GD2 antibody therapy and improve outcomes for patients with neuroblastoma.

overall_design: To examine changes occuring within the tumor microenvironment with treatment at single-cell resolution, Th-MYCN mice (recruited when tumor diameter was 3-4mm) were treated with saline control or TEPA (400mg/kg) for seven days before tumour collection. Tumours (n=2/condition) were freshly dissociated and subjected to red blood cell lysis before tumor, lymphocyte and myeloid compartments were postitively selected for using flow cytometry. Sample sorting was performed using BD FACSAria III (BD Biosciences) and subjected to downstream analysis using the BD Rhapsody single-cell analysis system according to manufacturer's instructions (BD Biosciences).

first: Jourdin

last: Rouaen

first: Antonietta

last: Salerno


title: Gene Expression analysis of uninfected and infected InsGAS mouse stomachs with and without interleukin 17 receptor A deficiency

city: Nashville

first: Holly

last: Algood

address: 1301 24th Ave South

department: Research

institute: Department of Veterans Affairs

summary: Mice deficient for IL-17RA are more likely to develop lymphoid follicles and gastric cancer and express higher levels of many genes associated with inflammation and cancer in response to H. pylori infection

overall_design: InsGAS mice and InsGAS mice with IL17RA deficiency were infected with H. pylori strain PMSS1 for 3 months.  Gastric tissue was processed for histology, colonization and RNA extraction.  Extracted RNA was processed at the VANTAGE CORE (VUMC) for gene expression analysis using nanostring's nCounter IO 360 mouse panel.  Data was analyzed on the ROSALIND cloud analysis and visualization software.

middle: M

last: Scott Algood

first: Matthew

middle: S

last: Jung


title: Acetyl-CoA production by Mediator-bound 2-ketoacid dehydrogenases boosts de novo histone acetylation and is regulated by nitric oxide [Chipseq_panAc_spikein]

first: Francesco

last: Gualdrini

overall_design: Chip-seq of tetra-acetylated histone H4  from BMDM chromatin with Drosophila chromatin spike-in. Cells were treated with lipopolysaccharide (LPS) from E. coli, serotype EH100(Ra) from Alexis (cat. no. ALX-581-010-L002) at 10 ng/mL (BMDMs). Trichostatin A (TSA, cat. no. S1045, Selleck) was added for 30 min. Spermine Nonoate (NO donor, cat, no. CAY-82150, Cayman Chemical) was added at 0.5 mM for 15 min before TSA or LPS treatment.

first: Marta

last: Russo

first: Veronica

last: Vallelonga

first: Elena

last: Prosperini

first: Roberta

last: Noberini

first: Silvia

last: Pedretti

first: Pierluigi

last: DiChiaro

first: Sara

last: Polletti

first: Gabriele

last: Imperato

first: Mattia

last: Marenda

first: Chiara

last: Ghirardi

first: Fabio

last: Bedin

first: Alessandro

last: Cuomo

first: Simona

last: Rodighiero

first: Tiziana

last: Bonaldi

first: Nico

last: Mitro

first: Serena

last: Ghisletti

first: Gioacchino

last: Natoli


title: Transcriptome regulated by mitochondrial uncouplers in prostate cancer cells

city: Augusta

first: Chunhong

last: Yan

state: GA

address: 1410 Laney Walker Blvd

department: Georgia Cancer Center

institute: Augusta University

summary: We discovered that mitochondrial uncouplers represented by nitazoxanide and its active metabolite tizoxanide inhibited prostate cancer growth. This RNA-seq study aims to elucidate the mechanism by which mitochondrial uncouplers inhibit prostate cancer growth. The results indicate that mitochondrial uncouplers predominantly downregulated E2F1 target genes and thus likely inhibited prostate cancer growth through impeding E2F1-mediated transcription.

overall_design: The prostate cancer cell line C4-2 was treated with DMSO or 20 uM of tizoxanide (TIZ) for 16 h. Total RNAs were prepared from the cells and subjected to RNA-seq. C42_DMSO_1, C42_DMSO_2, and C42_DMSO_3 are the samples treated with DMSO, while C42_Tiz_1, C42_Tiz_2, and C42_Tiz_3 are the samples treated with TIZ.


title: p26 condensates from Pea enation mosaic virus 2 sequester ribosomal components and suppress translation

city: Kansas City

first: Jared

middle: P.

last: May

state: MO

address: 5009 Rockhill Road Spencer Chemistry Building Room 405B

institute: University of Missouri-Kansas City

summary: The p26 movement protein of Pea enation mosaic virus 2 (PEMV2) interacts with the 2'-O-methyltransferase fibrillarin within nuclear and cytoplasmic condensates. This study analyzed global 2'-O-methylation patterns in PEMV2-infected Nicotiana benthamiana using RiboMethSeq, focusing on ribosomal RNAs (rRNAs). The aim was to assess whether PEMV2 disrupts rRNA 2'-O-methylation to interfere with host translation or ribosome assembly.

overall_design: To assess whether PEMV2 infection disrupts rRNA 2'-O-methylation, we conducted RiboMethSeq on Mock- and PEMV2-infected plants. Fib2-silenced plants served as a positive control to confirm that Fib2 knockdown reduces 2'-O-methylation. Leaf tissue was harvested 3 days post-infection for total RNA extraction. RNAs were fragmented under alkaline conditions, followed by end-repair and 5' end phosphorylation. Small RNA sequencing libraries were then prepared and sequenced using the Illumina NovaSeq platform with paired-end 100 base pair reads.

first: Shelby

middle: L

last: Brown

middle: P


title: Exploring the utility of snRNA-seq in profiling human bladder tissue: A comprehensive comparison with scRNA-seq

city: Houston

first: Angela

last: Ting

state: Tx

address: 1881 East Road

department: Epigenetics and Molecular Carcinogenesis

institute: The University of Texas MD Anderson Cancer Center

summary: Single cell sequencing technologies have revolutionized our understanding of biology by mapping cell diversity and gene expression in healthy and diseased tissues. While single-cell RNA sequencing (scRNA-seq) has been widely used, interest in single-nucleus RNA sequencing (snRNA-seq) is growing due to its benefits, including the ability to analyze archival tissues and capture rare cell types that are challenging to dissociate. However, comparative studies across tissues have yielded mixed results, with some reporting enhanced cell type retention using snRNA-seq while others finding cell type identification to be challenging in snRNA-seq data. The GUDMAP consortium aims to construct a molecular atlas of the lower urinary tract (LUT); thus, we set out to determine the strengths and limitations of each approach in characterizing LUT cell types. Using the human bladder, we determined that scRNA-seq offered more discriminative gene sets for identification while snRNA-seq could facilitate capture of previously underrepresented cell types.

overall_design: Eight healthy donor human bladder samples collected from the dome, neck, UO, and UVJ were subjected to RNA sequencing at either single cell or single nucleus resolution on Novaseq using 10X Gemonics library preparation platform. Data was processed using Cellranger v7.1.0 and anayzed using Seurat v4.3.0.1 to characterize various cell types and understand how single cell and single nucleus data contribute to each cell type's identification and gene set enrichment.

first: Briana

middle: A

last: Santo


title: Single cell RNA-seq of zebrafish endothelial cells

city: Uppsala

first: L.

last: He

address: Dag Hammarskjölds Väg 20

country: Sweden

institute: Uppsala University

summary: We performed single-cell RNA sequencing (scRNA-seq) for isolated endothelial cells

overall_design: Tg(kdrl:EGFP) zebrafish embryos were digested into single cell suspension, EGFP-positive cells were isolated using FACS and subjected to 10X Genomics Chromium scRNAseq.

first: Li-Kun

last: Phng


title: A Dual Role of the Senescence Marker P16Ink4a in Liver Endothelial Cell Function

city: Nice

first: Kay-Dietrich

last: Wagner

address: 28 Avenue Valombrose

department: Institute of Biology Valrose

country: France

institute: INSERM

summary: P16Ink4a is a well-established marker of senescence. Although P16Ink4a is expressed in endothelial cells, little is known about its function in these cells. Using isolated liver endothelial cells with silencing or overexpression of P16Ink4a, we show here that dependent on P16Ink4a levels, different pathways and functions are affected. High levels of P16Ink4a reduce proliferation and induce senescence while low levels have the opposite effects. Only high P16Ink4a expression reduces in vitro angiogenesis. Expression profiling reveals an inflammatory phenotype upon silencing of P16Ink4a while P16Ink4a overexpression is associated with a profile associated to DNA damage, repair and senescence. Low levels of P16Ink4a induce reactive oxygen species (ROS) generation and increase endothelial cell leakage. Collectively, P16Ink4a represents an “antagonistic pleiotropy” gene, which is on the one hand required to prevent ROS generation and endothelial damage and on the other hand at high levels inhibits angiogenesis through induction of senescence.

overall_design: Cd31+ or CD146+ endothelial cells were isolated from adult mouse livers and transduced with P16Ink4a small hairpin or overexpression constructs and the respective controls. After 1 week, RNA was isolated and submitted to sequencing.

first: Nicole

first: Hasan

last: Safwan-Zaiter


title: The Farnesoid-X-Receptor activates transcription independently of RXR at non-canonical response elements

city: Innsbruck

first: Jose Miguel

last: Ramos Pittol

address: Innrain 80-82

department: Institute of Biochemistry

institute: University of Innsbruck

summary: The Farnesoid-X-Receptor (FXR) is a nuclear receptor (NR) known to obligately heterodimerize with Retinoid-X-Receptor (RXR). FXR is expressed as four isoforms (α1-α4) that drive transcription from IR-1 (inverted repeat-1) DNA motifs. More recently, FXR isoforms α2/α4 were found to activate transcription predominantly from non-canonical ER-2 (everted repeat-2) DNA motifs, mediating most metabolic effects of general FXR activation. Here, we explored molecular determinants of FXRα2 regulation from ER-2 elements through quantitative interaction proteomics, site-directed mutagenesis, and transcriptomics. We discovered that FXRα2 binds to and activates ER-2 elements in vitro and in reporter assays independently of RXR. Genome-wide analysis in mouse liver revealed higher ER-2 motif enrichment in RXR-lacking FXR binding sites. Abrogation of FXRα2:RXR heterodimerization abolished IR-1, but preserved ER-2 transactivation. Transcriptome-wide, RXR overexpression inhibited 25% of FXRα2 targets in HepG2. These genes were specifically activated by the heterodimerization-deficient mutant FXRα2-L434R, were enriched for ER-2 motifs at their promoters and were involved in lipid metabolism and ammonia detoxification. In conclusion, RXR acts as a molecular switch, promoting FXRα2 activation from IR-1 instead of ER-2 motifs. Our results showcase FXR as the first NR with RXR-dependent and independent modes of activation, highlighting a potential new layer of complexity for other RXR-heterodimerizing NRs.

overall_design:
To investigate the transcriptome-wide effect of RXRA on FXRa2 target genes in liver cells, we generated HepG2 cell lines overexpressing FXRa2 in the presence or absence of ectopic RXRA. In addition, we also analysed the RXR heterodimerization-deficient mutant FXRa2-L434R.
We performed gene expression profiling analysis using data obtained from RNA-seq of 5 cell lines after treatment with an FXR agonist or vehicle.
Comparative gene expression profiling analysis of RNA-seq data to determine (1) the effect of agonist treatment for each line generated and (2) the consequences of FXRa2/RXRA overexpression

first: Jose

first: Nicholas

last: Marchet

first: Suzanne

middle: W

last: van der Veen

first: Saskia

last: van Mil


title: Crosstalk in Skin: Loss of Desmoglein 1 in Keratinocytes Inhibits BRAFV600E-induced Cellular Senescence in Human Melanocytes

city: Chicago

first: Ziyou

last: Ren

address: 303 E Chicago Ave

institute: Northwestern University

summary: Melanoma arises from transformation of melanocytes in the basal layer of the epidermis where they are surrounded by keratinocytes, with which they interact through cell contact and paracrine communication. Although research focuses on how the accumulation of oncogene and tumor suppressor gene mutations in melanocytes drive melanoma development, how alterations in keratinocytes serve as extrinsic drivers of melanoma initiation and progression is poorly understood. We recently identified keratinocyte desmoglein 1 (Dsg1) as an important mediator of keratinocyte:melanoma crosstalk. Here we address the extent to which Dsg1 loss, which occurs in response to acute environmental stress such as ultraviolet radiation, affects early steps in melanomagenesis. RNA-Seq analysis revealed that paracrine signals from Dsg1-deficient keratinocytes mediate a transcriptional switch from a differentiated to undifferentiated cell state in melanocytes expressing BRAFV600E. Of 221 differentially expressed genes in BRAFV600E cells treated with Dsg1-deficient conditioned media (CM), the laminin superfamily member NTN4/Netrin-4, which inhibits senescence in endothelial cells, stood out. Indeed, while BRAFV600E melanocytes treated with Dsg1-deficient CM showed signs of senescence bypass, knockdown of NTN4 reversed these effects. These results suggest that Dsg1 loss in keratinocytes provides an extrinsic signal to push melanocytes towards oncogenic transformation once an initial mutation has been introduced.

overall_design:
Keratinocytes and melanocytes were isolated from neonatal foreskin provided by the Northwestern University Skin Biology and Diseases Research-Based Center as previously described (Roth-Carter et al., 2022). Keratinocytes were grown in M154 medium (Thermo Fisher Scientific) supplemented with human keratinocyte growth supplement (Thermo Fisher Scientific), 1,000 x gentamycin/amphotericin B solution (ThermoFisher Scientific), and 0.07mM CaCl2. Melanocytes were cultured in OptiMEM (Thermo Fisher Scientific) containing 5% fetal bovine serum (MilliporeSigma), 10 ng/ml bFGF (ConnStem), 1 ng/ml heparin (MilliporeSigma), 0.1 mM N6, 2’-O-dibutyryladenosine 3:5-cyclic monophosphate (MilliporeSigma), 0.1 mM 3-isobutyl-1-methyl xanthine (MilliporeSigma), and 1% penicillin/streptomycin (Corning). All cells were maintained at 37 °C with 5% CO2.
Conditioned media (CM) from keratinocytes were prepared by following previously published procedure (Arnette et al., 2020). Melanocytes were transduced with wild type BRAF or mutant BRAFV600E, then treated with CM from keratinocytes expressing shCTL or shDsg1 for 3 days. Total RNA was extracted from cells using the Quick-RNA miniprep kit (Zymo Research) following the manufacturer’s protocol. Novogene Co., LTD conducted preparation of the mRNA library and transcriptome sequencing.

first: Xin

last: Tong

first: Hope

last: Burks

first: Jennifer

last: Koetsier

first: Quinn

middle: R

last: Roth-Carter

first: Kathleen

last: Green


title: Developmentally regulated generation of a systemic signal for long-lasting defence priming in tomato [RNA-seq]

status: Public on Nov 17 2024

city: Birmingham

first: Marco

last: Catoni

address: Edgbaston

department: School of Biosciences

country: United Kingdom

institute: University of Birmingham

summary: Tomato is a major global crop however its production is limited by Botrytis cinerea. Due to the toxicity of post-harvest pesticide application, alternative control methods such as priming are being investigated. Plants were treated with β-aminobutyric (BABA) at two developmental stages and resistance against B. cinerea was tested in fruit tissue and in progenies. DNA methylation and RNA sequencing were conducted to characterise the (epi)genetic changes associated with long-lasting resistance. Grafting experiments were done to assess the systemic nature of this signal, which was further characterised by small RNA (sRNA) sequencing of scions. Only BABA-treated seedlings displayed induced resistance (IR). DNA methylation analysis revealed seedling-specific changes, which occurred in the context of lower basal methylation. BABA-IR was found to be transmissible from primed rootstock to grafted unprimed scions. In these scions, we identified a subset of mobile 24 nt sRNAs associated with primed genes to infection in fruit. Our results demonstrate the functional association of a systemic signal with long lasting IR and priming. Through integrated omics approaches we have identified markers of long-lasting priming in tomato fruit which could also serve as targets for durable resistance in other crops.

overall_design: Examination of trascriptome of leaves and fruits of Solanum lycopersicum (cv. Micro Tom), from plants exposed to β-aminobutyric (BABA) at two different developmental stages, or treated with water, and then infected with Botrytis cinerea, along with mock-treated control plants.

first: Estrella

last: Luna-Diez

first: Katie

last: Stevens


title: Deciphering the function of intrinsic and genomics-driven epigenetic heterogeneity in head and neck cancer progression with single-nucleus CUT&RUN [bulk RNAseq]

city: Singapore

first: Lih Feng

last: Cheow

address: 15 Kent Ridge Cres

department: iHealthtech

country: Singapore

institute: National University of Singapore

summary: Single-cell RNA sequencing (scRNA-seq) has proven to be an invaluable tool for the study of tumor evolution. However, interrogating regulatory epigenetic alterations during cancer progression at the resolution of single-cells has remained a major challenge due to the lack of sensitivity of current assays. Here, we developed the highly-sensitive single-nucleus CUT&RUN (snCUT&RUN) assay to profile histone modifications in primary, metastatic, and cisplatin-resistant head and neck squamous cell carcinoma (HNSCC) patient-derived cell lines. We find that the epigenome can employ diverse modes to contribute towards cancer progression. Notably, we demonstrate that gene expression changes during cancer progression are co-modulated by alterations in both copy number and chromatin activity to drive epigenetic rewiring of cell-states. Furthermore, intratumor epigenetic heterogeneity may predispose sub-clonal populations within the primary tumor to acquire malignant characteristics. In conclusion, snCUT&RUN serves as a valuable addition to existing toolkit of single-cell assays to dissect the function of the epigenome during cancer progression.

overall_design:
Single-nucleus CUT&RUN targeting H3K4me3 and H3K27ac in patient derived head and neck cancer cell lines followed by Miseq sequencing.
bulk RNAseq experiments

first: Howard

last: Womersley

first: Daniel

last: Muliaditan

first: Ramanuj

last: Dasgupta


title: The amniote-conserved DNA-binding domain of CGGBP1 restricts cytosine methylation of transcription factor binding sites in proximal promoters to regulate gene expression (HTS)

city: Gandhinagar

first: Umashankar

last: Singh

state: Gujarat

address: Acad Block 4 Room 317 IIT Gandhinagar Palaj

department: Biological Sciences and Engineering

country: India

institute: IIT Gandhinagar

type: Methylation profiling by high throughput sequencing

summary: Truncated forms of CGGBP1 with (N-term) or without (C-term) the DNA-binding domain (DBD) have been used to to assay global cytosine methylation. HEK293T cells with endogenous CGGBP1 knocked down were used to over-express truncated forms of CGGBP1 followed by MeDIP-Seq analysis. Genome-wide analyses of cytosine methylation and binding of CGGBP1 DBD show that CGGBP1 restricts cytosine methylation in a manner that depends on its DBD and its DNA-binding. Our findings suggest that CGGBP1 protects transcription factor binding sites (TFBS) from cytosine methylation-associated loss. A superimposition of our results and evolution of CGGBP1 suggests that mitigation of cytosine methylation is majorly achieved by its N-terminal DBD. Our results position CGGBP1 DNA-binding as a major evolutionarily acquired mechanism through which it keeps cytosine methylation under check and regulates TFBS retention.

overall_design: MeDIP-seq was performed on genomic DNA from HEK293T cells over-expressing Full-length CGGBP1 and N-term & C-term truncated forms of CGGBP1. ChIP-seq was performed on genomic DNA from HEK293T cells over-expressing N-term truncated form of CGGBP1.

first: Ishani

last: Morbia

first: Praveen

last: Kumar

first: Aditi

last: Satish

first: Subhamoy

last: Datta

first: Akanksha

last: Mudgal


title: The amniote-conserved DNA-binding domain of CGGBP1 restricts cytosine methylation of transcription factor binding sites in proximal promoters to regulate gene expression

summary: Truncated forms of CGGBP1 with (N-term) or without (C-term) the DNA-binding domain (DBD) have been used to to assay global gene expression. HEK293T cells with endogenous CGGBP1 knocked down were used to over-express truncated forms of CGGBP1 followed by RNA extraction and one-coloured global gene expression analysis. The data suggests that while the C-term of CGGBP1 is the major repressor of transcription, just the N-term containing the DBD fails to achieve so. Proximal promoters of CGGBP1-repressed genes, although significantly GC-poor, contain GC-rich transcription factor binding motifs and exhibit base compositions indicative of low C-T transition rates due to targeted prevention of cytosine methylation. Our findings suggest that CGGBP1 protects transcription factor binding sites (TFBS) from cytosine methylation-associated loss and thereby regulates gene expression. By analysing orthologous promoter sequences, we show that protection from cytosine methylation is a function of CGGBP1 progressively acquired during vertebrate evolution.

overall_design: Gene expression changes upon over-expression of truncated forms (N-term and C-term) and full-length CGGBP1 were measured after 48 hours of transfection in HEK293T cells. All the three experiments were performed in triplicates.


title: Human respiratory organoids sustained reproducible propagation of previously uncultivable human rhinovirus C

first: Jie

last: Zhou

address: 102 Pokfulam Road, Pokfulam, Hong Kong

department: Microbiology

institute: The University of Hong Kong

summary: The lack of a robust system to reproducibly propagate HRV-C substantially hampered our understanding of the common respiratory virus. We sought to develop an organoid-based system to reproducibly propagate HRV-C and characterize virus-host interaction using the respiratory organoids established by our team. We demonstrated that airway organoids sustained serial virus passage with the aid of CYT-387-mediated immunosuppression; nasal organoids, an organoid model more closely simulating the human upper airway, achieved this without any intervention. Nasal organoids were more susceptible to HRV-C than airway organoids. Intriguingly, we observed a more intensive innate immune response in airway organoids than nasal organoids upon HRV-C infection, which was reproduced in a Poly (I:C) stimulation assay. Treatment with an anti-CDHR3 and two antivirals significantly reduced HRV-C viral growth in nasal organoids. An organoid-based immunofluorescence assay was established to titrate HRV-C infectious particles. Collectively, we developed an organoid-based system to reproducibly propagate the previously uncultivable HRV-C, which enabled an in-depth elucidation of HRV-C infection and innate immunity unprecedentedly. The organoid-based HRV-C infection model can be extended for developing antiviral strategies. More importantly, our study has paved a new avenue for propagating and studying other uncultivable human and animal viruses.

overall_design: To characterize the cellular response in airway and nasal organoids with HRV-C infection and CYT387 treatment, we infected airway and nasal organoids with or without CYT387 treatment. Two biological replicates of mock-treated, HRV-C infected, HRV-C infected with CYT387 treatment airway and nasal organoids were harvested for RNA extraction using RNeasy Mini Kit (Qiagen, Cas.74106). After a quality check with Bioanalyzer, RNA specimens were applied to library preparation by KAPA mRNA HyperPrep Kit. In brief, Poly-A containing mRNA was collected by using poly-T oligo-attached magnetic beads. The purified mRNA was fragmented to 200 ~ 300 bp by incubating at 94°C for 6 min in the presence of magnesium ions. The fragmented mRNA was then applied as a template to synthesize the first-strand cDNA by using random hexamer-primer and reverse transcriptase. In the second strand cDNA synthesis, the mRNA template was removed and a replacement strand was generated to form the blunt-end double-stranded (ds) cDNA. The ds cDNA underwent 3’ adenylation and indexed adaptor ligation. The adaptor-ligated libraries were enriched by 15 cycles of polymerase chain reaction (PCR). The libraries were denatured and diluted to optimal concentration. Illumina NovaSeq 6000 was used for Pair-End 151bp sequencing. Sequencing reads were assigned to individual samples. Each sample had an average throughput of 4.6 Gb and a total throughput of 54.8 Gb. Quality control of raw fastq data was carried out by FastQC v0.11.7 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and fastp [PMID: 30423086]. Then clean reads were aligned to the UCSC GRCh38 reference and the Rhinovirus C3 strain genome (GenBank: OK161378.1) using Hisat2 v2.2.1 [PMID: 31375807]. HTSeq v0.6.1 [PMID: 25260700] was used to generate raw read count for each gene. Differential expression analysis was performed using DESeq2 [PMID: 25516281]. Genes with log2|fold change| > 1 and adjusted p-value < 0.05 were considered significantly different. Volcano plot was generated using EnhancedVolcano R package (https://github.com/kevinblighe/EnhancedVolcano). Heatmaps of gene expression levels were constructing using the pheatmap R package (https://cran.r-project.org/web/packages/pheatmap/index.html). Gene set enrichment analysis (GSEA) was performed using fgsea R package (https://github.com/ctlab/fgsea).

first: Cun

last: Li

first: Yifei

last: Yu

first: Zhixin

last: Wan

first: Man

last: Chiu

first: Jingjing

last: Huang

first: Shuxin

last: Zhang

first: Xiaoxin

last: Zhu

first: Qiaoshuai

last: Lan

first: Yanlin

last: Deng

first: Ying

first: Wei

last: Xue

first: Ming

last: Yue

first: Cyril

last: Yip

first: Jian

last: Cai

first: Kenneth

last: Wong

first: Xiaojuan

last: Liu

first: Yu

last: Yang

first: Jasper

middle: F

last: Chan

first: Hin

last: Chu

first: Hans

last: Clevers

first: Kwok

middle: Y

last: Yuen


title: PIN1 isomerase promotes the initiation and progression of bladder cancer through SREBP2-mediated cholesterol biosynthesis pathway

city: la jolla

first: Xue

last: Wang

state: CA

address: 10010 N Torry Pines Rd

department: MCBL-H

institute: Salk Institute

summary: Bladder cancer, one of the most diagnosed cancers worldwide, is associated with high morbidity, mortality, and a poor prognosis. The PIN1 phospho-dependent prolyl isomerase is frequently overexpressed in many human cancer types and affects tumor initiation and progression through disrupting the balance of oncoprotein and tumor suppressor protein signaling. However, the functional pSer/Thr.Pro protein substrates of PIN1 and its effects on downstream signaling in bladder carcinogenesis remain largely unknown. Phenotypically, we discovered that ablation of Pin1 in human/mouse bladder cancer cells and organoids formed from mouse primary urothelial cells resulted in decreased cell proliferation, stemness maintenance, cell invasion, migration, urothelium clearance capacity, and reduced free/total cholesterol levels in vitro. Re-expressing Pin1 in Pin1 knockout cells reversed the defects caused by lack of Pin1. Moreover, in vivo subcutaneous xenograft and allograft transplantation mouse models combined with an orthotopic implantation model were utilized to confirm a positive role for Pin1 in controlling tumor growth and potential metastasis. Therapeutically, a combination of the sulfopin PIN1 inhibitor and the simvastatin HMGCR inhibitor was shown to suppress cell proliferation in vitro and tumor growth in vivo synergistically. Mechanistically, we observed a negative enrichment of SREBP2-driven cholesterol metabolism pathways in PIN1 knockout cells via RNA-sequencing and used a suite of diverse molecular and biochemical approaches to show that PIN1 interacts with JNK-dependent phosphorylated SREBP2 (Ser455) and vice versa. These results indicate that active cholesterol biosynthesis pathway in bladder cancer cells is regulated by PIN1-mediated isomerization and JNK-mediated phosphorylation of its key transcriptional factor SREBP2. These findings emphasize that PIN1 can act as a regulator and potential therapeutic target in bladder cancer.

overall_design:
To elucidate the underlying molecular mechanisms by which PIN1 mediated cis-trans isomerization of phosphosites in target proteins induces functional alterations in downstream signaling
We performed bulk RNA-sequencing using T24 and 5637 cells with different PIN1 expression levels.
Comparative gene expression profiling analyses of RNA-seq data for T24 and 5637 cells with different PIN1 expression levels, control, PIN1 knockout and PIN1 re-expression

first: Derrick

last: Lee

first: Tony

last: Hunter


title: Acetyl-CoA production by Mediator-bound 2-ketoacid dehydrogenases boosts de novo histone acetylation and is regulated by nitric oxide [Med1_Chipseq]

city: Milan

first: Francesco

last: Gualdrini

address: Via Adamello, 16

department: Department of Experimental Oncology

institute: European Institute of Oncology

summary:
Histone modifying enzymes depend on the availability of cofactors, with acetyl-CoA being required for histone acetyltransferase (HAT) activity. The discovery that mitochondrial acyl-CoA producing enzymes are also delivered to the nucleus suggests that high concentrations of metabolites generated locally may impact acetylation of histones and other nuclear substrates and eventually control gene regulation. Here we show that 2-ketoacid dehydrogenases were stably associated with the Mediator complex in macrophages, thus providing a local supply of acetyl-CoA and increasing the generation of hyper-acetylated histone tails. Nitric oxide (NO), which is produced in large amounts in LPS-stimulated macrophages, inhibited both the activity of 2-ketoacid dehydrogenases and their association with Mediator. Consequently, NO reduced de novo histone acetylation at genomic regions with high acetyl-CoA deposition rates. Our findings indicate that a local supply of acetyl-CoA generated by Mediator-bound 2-ketoacid dehydrogenases is required to maximize acetylation of histone tails at sites of elevated HAT activity.
Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for the Mediator subunit Med1 (cat.no. A300-793A, Bethyl)

overall_design: Chip-seq of MED1 was performed in RAW 264.7 cells  in either resting or 2h LPS stiumulated conditions.

first: Marta

last: Russo

first: Gualdrini

last: Francesco

first: Elena

last: Prosperini

first: Sara

last: Polletti

first: Serena

last: Ghisletti

first: Gioacchino

last: Natoli


title: Upregulated FoxO1 promotes arrhythmogenesis in mice with heart failure and preserved ejection fraction [scRNA-seq]

city: Los Angeles

first: Thassio

last: Mesquita

state: California

address: 8700 Beverly Boulevard

department: Smidt Heart Institute

institute: Cedars-Sinai Medical Center

summary: Myocardial fibrosis leads to cardiac dysfunction and arrhythmias in heart failure with preserved ejection fraction (HFpEF), but the underlying mechanisms remain poorly understood. Here, RNA sequencing identifies Forkhead Box1 (FoxO1) signaling as abnormal in HFpEF hearts. Genetic suppression of FoxO1 alters the intercellular communication between cardiomyocytes and fibroblasts, alleviates abnormal diastolic relaxation, and reduces arrhythmias. Targeted downregulation of FoxO1 in activated fibroblasts reduces cardiac fibrosis, blunts arrhythmogenesis and improves diastolic function in HFpEF. These results not only implicate FoxO1 in arrhythmogenesis and lusitropy but also demonstrate that pro-fibrotic cardiomyocyte-fibroblast communication can be corrected, constituting a novel therapeutic strategy for HFpEF.

overall_design: single-cell RNA sequencing from isolated left ventricular cells was performed to identify transcriptomic changes in C56Bl6 fed with a standard diet (control), High-Fat + L-NAME diet treated with AAV-scramble-sh (HFpEF-scr), and High-Fat + L-NAME diet treated with AAV-FoxO1-sh (HFpEF-FoxO1-sh).

first: Eugenio

last: Cingolani


title: Upregulated FoxO1 promotes arrhythmogenesis in mice with heart failure and preserved ejection fraction

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

overall_design: Refer to individual Series


title: Copper chelation redirects neutrophil function to enhance anti-GD2 antibody therapy in neuroblastoma [GeoMx]

first: Orazio

last: Vittorio

summary: Anti-disialoganglioside (GD2) antibody therapy has provided clinical benefit to patients with neuroblastoma however efficacy is likely impaired by the immunosuppressive tumor microenvironment. We have previously defined a link between intratumoral copper levels and immune evasion. Here, we report that adjuvant copper chelation potentiates anti-GD2 antibody therapy to confer durable tumor control in immunocompetent models of neuroblastoma. Mechanistic studies reveal copper chelation creates an immune-primed tumor microenvironment through enhanced infiltration and activity of Fc-receptor-bearing cells, specifically neutrophils which are emerging as key effectors of antibody therapy. Moreover, we report copper sequestration by neuroblastoma attenuates neutrophil function which can be successfully reversed using copper chelation to increase pro-inflammatory effector functions. Importantly, we repurpose the clinically approved copper chelating agent Cuprior as a non-toxic, efficacious immunomodulatory strategy. Collectively, our findings provide robust evidence for the clinical testing of Cuprior as an adjuvant to enhance the activity of anti-GD2 antibody therapy and improve outcomes for patients with neuroblastoma.

overall_design: A tissue microarray (TMA) was prepared using 20 Th-MYCN tumor samples (10 control, 10 TEPA-treated) in duplicate, cored at 1mm. Coring sites were chosen at random however areas exhibiting necrosis were excluded prior by an experienced histopathologist using a hematoxylin-eosin stain. The FFPE TMA block was sectioned at 4µm and transferred to a Bond Plus slide (Leica Biosystems, USA; #S21.2113.A) and were processed by the NanoString GeoMx DSP Technology Access Program.

first: Jourdin

last: Rouaen

first: Antonietta

last: Salerno


title: Acetyl-CoA production by Mediator-bound 2-ketoacid dehydrogenases boosts de novo histone acetylation and is regulated by nitric oxide

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Deciphering the function of intrinsic and genomics-driven epigenetic heterogeneity in head and neck cancer progression with single-nucleus CUT&RUN

first: Lih Feng

last: Cheow


title: Short-term polystyrene nanoplastic exposure alters zebrafish male and female germline and reproductive outcomes, unveiling pollutant-impacted molecular pathways

city: Cerdanyola del Vallès

first: Gala

last: Pujol

address: Carrer de la Vall Moronta, Edificio C, despacho C2/109

institute: UAB (Universidad Autónoma de Barcelona)

summary: Nanoplastics pollution is a rising environmental concern whose impacts on biodiversity and human health are far from being understood. This is particularly salient in aquatic ecosystems, where the majority of species depend on external fertilization for reproduction. Here we evaluated the effects of a short-term exposure to engineered polystyrene nanoplastics (NPs) in the zebrafish germ line to further explore their impact on reproduction. To this end, zebrafish (Danio rerio) were exposed to 5 mg/L of 45 nm polystyrene (PS)-NPs via water for 96h. We show that, in males, nanoplastics induced testicular histological alterations with abnormal sperm clustering and chromatin compaction, resulting in viable spermatozoa but with reduced motility. Moreover, in females we observed an alteration in oocyte stages frequencies during oogenesis, possibly reflecting alterations in oocyte growth. RNA-sequencing analysis in male testis links nanoplastic induced alterations in the expression of genes involved in chromatin structure, meiosis and DNA double-strand break formation and repair progression, and gametes recognition. Flow cytometry analysis revealed that the observed effects in males were directly due to nanoplastics penetrating the testicular barrier and being internalized within germline cells. Overall, our results demonstrate that acute exposure to NPs can compromise reproductive fitness, underscoring the environmental and health impacts of NPs pollution.

overall_design: Bulk RNA-seq from adult testis from control (3 samples) and treated animals (3 samples)

first: Laia

last: Marín-Gual

first: Laura

last: González-Rodelas

first: Lucia

last: Álvarez-González

first: François

last: Chauvigné

first: Joan

last: Cerdà

first: Mariana

last: Teles

first: Nerea

last: Roher

first: Aurora

last: Ruiz-Herrera


title: Upregulated FoxO1 promotes arrhythmogenesis in mice with heart failure and preserved ejection fraction [6 wks]

overall_design: RNA sequencing from left ventricular tissue was performed to identify transcriptomic changes in C56Bl6 fed with a standard diet (control group) or High-Fat + L-NAME (HFpEF group) for 6 weeks.


title: Time-resolved gene expression analysis of the astrocyte-derived cell line 1321N1 treated with cadmium, mercury and arsenic.

status: Public on Nov 16 2024

city: Vienna

first: Giulio

last: Superti-Furga

address: Lazarettgasse 14

institute: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences

summary: To get an insight into the time-resolved gene expression response elicited by different heavy metals, we treated the cell line 1321N1 with cadmium, mercury or arsenic for 1.5h, 3h, 6h, 12h and 24h and performed QuantSeq 3'-mRNA sequencing.

overall_design: The 1321N1 cell line was treated with 2.3 µM cadmium chloride, 20 µM mercury chloride or 2.55 µM sodium metaarsenite for 1.5h, 3h, 6h, 12h and 24h. Non-treated (nt) conditions were included for each timepoint. All conditions were performed in technical triplicates. The gene expression analysis was performed via QuantSeq 3'-mRNA sequencing.

first: Andrè

last: Ferdigg

first: Ann-Katrin

last: Hopp

first: Gernot

last: Wolf


title: A novel cooperative therapeutic regimen using gene fusion neoantigen-specific TCR-engineered T cells mediated sustained preclinical efficacy in solid tumors independent on inherent mutation profiling

city: Nanjing

first: Yuncheng

last: Bei

state: Jiangsu

address: Zhongshan Road No.321

department: Oncology

institute: Nanjing Drum Tower Hospital

summary: Neoantigens derived from non-synonymous somatic mutations, especially gene fusion mutation, are restricted to tumor cells and thus considered ideal targets for T-cell receptor-engineering T-cells (TCR-T) immunotherapy. Adoptive transfer of neoantigen-specific TCR-T cells preferentially exhibit potent cytotoxicity preferentially to tumor cells, and has shown promising efficacy in several preclinical human cancer models. However, most neoantigens are unique to individual patient, which hinders the application of TCR-T cell therapy. In this study, we first discovered a paired / TCR repertoire, Tcr-1, that specifically targeted STY-SSX fusion neoantigen shared by almost all synovial sarcomas. Engineered T-cell expressing Tcr-1 (Tcr-T1) exhibited antigen-specific HLA-A*2402-restricted antitumoral activity against synovial sarcomas cell both in vitro and in vivo. Furthermore, to furtherly extend its application beyond synovial sarcomas, we innovatively developed a cooperative therapeutic modality, in which exogenous SYT-SSX fusion neoantigen was loaded into enzyme-responsive nanoparticles (NPs) formed by mPEG-PVGLIG-PCL copolymers (FNeo-AgNPs) for tumor targeting delivery. As expected, FNeo-AgNPs were proven of great tumor penetration, and local release. In situ modification was able to direct engineered Tcr-T1 against to other malignant gastric cancer cell line with significant antigen-specific HLA-A*2402-restricted cytotoxicity resulting in remarkable tumor regression and prolonging survival in both subcutaneous and peritoneally disseminated preclinical models, despite of their inherent mutation profiles. With these favorable data, our established cooperative therapeutic modality has a great potential for further clinical investigation, and provide a now insight for future TCR-T cell therapy development.

overall_design: Preparation of single-cell suspensions: Neoantigen responsive T-cell clones were harvest and CD8+ T cells were furtherly isolated through magnetic beads (Miltenyi Biotec, CD8+ T cell Isolation Kit, Cat no. 130-096-495). Purified CD8+ T cells were subjected to single-cell analysis for TCR sequencing as reported.  Droplet-based single-cell sequencing: Briefly, using a Single Cell 5’ Library and Gel Bead Kit (10X Genomics, Cat no. 1000006) and Chromium Single A Chip Kit (10X Genomics, Cat no. 120236), the single cell suspension (300-600 living cells/ml) was loaded onto a Chromium single cell controller (10X Genomics) to generate single-cell gel beads in the emulsion (GEMs) according to the manufacture’s protocol. Single cells were suspended in PBS containing 0.04% BSA. Approximately 10,000 cells were added to each channel and approximately 6,000 target cells were recovered. Captured cells were lysed and the released RNA was barcoded through reverse transcription in individual GEMs. Reverse transcription was performed on a S1000TM Touch Thermal Cycler (Bio Rad) at 53oC for 45 min, followed by 85oC for 5 min and a hold at 4oC. Complementary DNA was generated and amplified, after which, quality was assessed using an Agilent 4200 (performed by CapitalBio Technology). According to the manufacturer’s introduction, scRNA-seq libraries were constructed using a Single Cell 5’ Library and Gel Bead kit, Human T Cell (Cat no. 1000005). The libraries were sequenced using an Illumina Novaseq6000 sequencer with a paired-end 150-bp (PE150) reading strategy (performed by CapitalBio Technology). TCR sequencing: Full-length TCR V(D)J segments were enriched from amplified cDNA from 5’ libraries via PCR amplification using a Chromium Single-Cell V(D)J Enrichment kit according to the manufacture’s protocol (10X Genomics). For TCR, cells with at least one productive TCR -chain (TRA) and on productive TCR -chain (TRB) were kept for further analysis.

first: Ying

last: Huang

first: Nandie

last: Wu

first: Yishan

last: Li

first: Ruihan

last: Xu

first: Baorui

last: Liu

first: Rutian


title: Gene expression of CD56positive and CD56negative human muscle stem cells from normal and Duchenne Muscular Dystrophy muscles

city: Lyon

first: Benedicte

last: Chazaud

address: 8 Avenue Rockefeller

department: Institut NeuroMyoGène

institute: Université Claude Bernard Lyon 1

summary: In Duchenne Muscular Dystrophy (DMD), the absence of the subsarcolemmal dystrophin protein leads to repeated myofiber damages inducing cycles of muscle regeneration that is driven by muscle stem cells (MuSCs). With time, MuSC regenerative capacities are overwhelmed, leading to fibrosis and muscle atrophy. Whether MuSCs from DMD muscle have intrinsic alterations or are primed by their degenerative/regenerative environment is still debated. We investigated gene expression in human using primary MuSCs derived from DMD or healthy muscles. Cells were isolated from the muscle and bulk expanded then purified as CD56positive cells (CD56 is a myogenic marker that characterizes the myogenic nature of the cells).  Human MuSCs loose the expression of the CD56 with time. We analyzed gene expression by CD56positive and CD56negative cells originating from the same initial CD56positive MuSC population.

overall_design: CD56pos Muscle Stem Cells from healthy control (HC-MuSC) and Duchenne Muscular Dystrophy (DM-MuSC) were cultured in growth medium and passaged until about 50% of the cells have lost CD56 expression. Then the cell culturess were sorted by magnetic cell sorting to obtain CD56positive cells and CD56negative cells, that were analyzed for their gene expression. n=3.

first: Bénédicte


title: Repeated fasting events sensitize chromatin and gene expression to support augmented ketogenesis

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: Mammals evolved to withstand frequent fasting periods due to hepatic production of glucose and ketone bodies. Because the fasting response is transcriptionally-regulated, we asked whether enhancer dynamics impose a transcriptional program during recurrent fasting and whether this produces effects distinct from a single fasting bout. We found that mice undergoing alternate-day fasting (ADF) respond to a following fasting bout profoundly differently from mice first experiencing fasting. Hundreds of genes enabling ketogenesis are ‘sensitized’, i.e. induced more strongly by fasting following ADF. Enhancers regulating these genes are also sensitized and harbor increased binding of the major ketogenic transcription factor. ADF mice show augmented ketogenesis and their sensitized enhancers are covalently marked with ketone body residues. Thus, we found that past fasting events are ‘remembered’ in hepatocytes, sensitizing their enhancers to the next fasting bout and augmenting ketogenesis. Our findings shed light on transcriptional regulation mediating adaptation to repeated environmental signals.

overall_design: Mice were subjected to either a 4-week alternate day fasting (ADF) regimen or unrestricted feeding (URF). At the end of the experiment mice were divided to 3 groups: the first group was euthanized after a 24 h fasting period (ADF_f or URF_f). The second group fasted for 24 h followed by ad libitum refeeding for 24 h at the end of which they were euthanized (ADF_re or URF_re). The third group did not experience any type of fasting (unrestricted feeding). Each group has 3 replicates.

first: Noga

last: Korenfeld

first: Meital

last: Charni-Natan


title: RNA sequencing for human upper tract urothelial carcinoma (UTUC) tissue from Japanese patients

city: Suita

first: Akinaru

last: Yamamoto

state: Outside U.S./Canada

address: Yamadaoka, 2-2

country: Japan

institute: Osaka University Graduate School of Medicine

summary: To know the biological characteristics of UTUC, we conducted the RNA-seq.

overall_design: 74 UTUC tissue and matched pair 73 adjacent normal upper tract urothelial (AN) tissue were conducted.

first: Kosuke

last: Nakano

first: Kotoe

last: Katayama


title: Nanopore Sequencing as a Cutting-Edge Technology for Medulloblastoma Classification

city: Paris

first: Mathilde

last: Filser

address: 26 rue d'Ulm

department: Genetics Department

institute: Institut Curie

summary:
Background: Medulloblastoma (MB) is one of the most prevalent embryonal malignant brain tumors. Current classification organizes these tumors into four molecular groups (WNT-activated, SHH-activated and TP53-wild type, SHH-activated and TP53-mutant, and non-WNT/non-SHH). Recently, a comprehensive classification has been established, identifying numerous subgroups, some of which exhibit a poor prognosis. It is critical to establish effective subgrouping methods for accurate diagnosis and patient’s management that strikes a delicate balance between improving outcomes and minimizing the risk of comorbidities.
Methods: We evaluated the ability of Nanopore sequencing to provide clinically relevant methylation and copy number profiles of MB. Nanopore sequencing was applied to an EPIC discovery cohort of frozen MB, benchmarked against the gold standard EPIC array, and validated further evaluated on an integrated diagnosis cohort of MB.

overall_design: For whole-genome Nanopore sequencing, DNA libraries were prepared using the SQK-RBK004 rapid barcoding kit from Oxford Nanopore Technologies. Two types of flow cells were used: MinION R9.4.1 (FLO-MIN106D) and Flongle R9.4.1 (FLO-FLG001). For MinION flow cells, 400 ng of DNA were tagmented, barcoded, repaired, and pooled in batches of six samples before adaptor ligation. Sequencing involved multiplexing six libraries, with each batch taking 48 to 72 hours per MinION R9.4.1 flow cell. For Flongle flow cells, the same SQK-RBK004 kit (Oxford Nanopore Technologies) was used, but 200 ng of DNA per tumor sample was sequenced individually on a Flongle flow cell for 24 hours. All sequencing was performed on MinION Mk1B or Mk1C devices, and sequencing runs were initiated and monitored using MinKnow software from Oxford Nanopore Technologies.

first: Julien

last: Masliah-Planchon

first: Jacob

last: Torrejon

first: Olivier

last: Ayrault


title: Deciphering the function of intrinsic and genomics-driven epigenetic heterogeneity in head and neck cancer progression with single-nucleus CUT&RUN [single-nucleus CUT&RUN]

first: Lih Feng

last: Cheow

overall_design:
Single-nucleus CUT&RUN targeting H3K4me3 and H3K27ac in patient derived head and neck cancer cell lines followed by Miseq sequencing.
single-nucleus CUT&RUN experiments

first: Howard

last: Womersley

first: Daniel

last: Muliaditan

first: Ramanuj

last: Dasgupta


title: Whole transcriptome sequencing for upper tract urothelial carcinoma (UTUC) of BALB/c female mice treated with N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)

summary: Only BALB/c female developed UTUC treated with BBN. To know the biological features of UTUC, we conducted the RNA-seq.

overall_design: BALB/c female were treated with or without BBN for 18 weeks. The UTUC developed side of ureter, non-UTUC developed side of ureter and bladder from BALB/c female treated with BBN, and normal ureter and bladder from BALB/c female treated without BBN were harvested. mRNA expression of each group was evaluated by RNA-seq and functional analysis

first: Atsunari

last: Kawashima

first: Daisuke

last: Okazaki


title: High-resolution, noninvasive single-cell lineage tracing in mice and humans based on DNA methylation epimutations [LK_RNAseq; transcriptome of single cells]

city: Hangzhou

first: Shou-Wen

last: Wang

address: No. 18 Shilongshan Road, Xihu District

institute: Westlake University

summary: In vivo lineage tracing holds great potential to reveal fundamental principles of tissue development and homeostasis. However, lineage tracing in humans relies on DNA mutations that are extremely rare. In mice, the improved genetic labeling approach has low resolution over cell division histories. Here, we demonstrated for the first time that frequent epimutations on DNA methylation can be exploited to infer lineage histories in normal cells, enabled by our newly developed computational method MethyTree. Using both in-house and public sparse single-cell DNA methylation datasets with known lineage labels, MethyTree reconstructed lineage histories at high resolution and accuracy across different cell types, stages, and species. Applying MethyTree, we identified the first fate decision in human embryo development and pinpointed in total ~230 clones of hematopoietic stem cells in mice. Our study opens the door for high-resolution, noninvasive lineage tracing in mice, humans and beyond.

overall_design: In this study, we carried out an in vitro lineage tracing experiment using blood progenitors extracted from a single adult mouse and generated single-cell multi-omic readouts. Specifically, we extracted Lin-cKit+Sca1- blood progenitors from a single mouse, introduced LARRY lineage barcodes in these cells by lentiviral infection, cultured them in vitro for 6 days in a media that supports cell expansion and pan-myeloid differentiation, and finally profiled these cells with a modified Camellia-seq protocol that we developed recently to obtain LARRY lineage barcode, transcriptome, and DNA methylome simultaneously in single cells. This table only contains data on the transcriptome of single cells.

first: Mengyang

last: Chen

first: Ruijiang

last: Fu

first: Li

last: Li


title: High-resolution, noninvasive single-cell lineage tracing in mice and humans based on DNA methylation epimutations [scBS-seq]

overall_design: In this study, we generate single-cell bisulfite sequence datasets for the HEK 293T cell line to evaluate the performance of MethyTree.


title: High-resolution, noninvasive single-cell lineage tracing in mice and humans based on DNA methylation epimutations [LK_scMultiOmicseq; DNA methylome of single cells]

overall_design: In this study, we carried out an in vitro lineage tracing experiment using blood progenitors extracted from a single adult mouse and generated single-cell multi-omic readouts. Specifically, we extracted Lin−cKit+Sca1- blood progenitors from a single mouse, introduced LARRY lineage barcodes in these cells by lentiviral infection, cultured them in vitro for 6 days in a media that supports cell expansion and pan-myeloid differentiation, and finally profiled these cells with a modified Camellia-seq protocol that we developed recently to obtain LARRY lineage barcode, transcriptome, and DNA methylome simultaneously in single cells. This table only contains data on the DNA methylome of single cells.


title: Developmentally regulated generation of a systemic signal for long-lasting defence priming in tomato [WGBS]

first: Marco

last: Catoni

summary: Tomato is a major global crop however its production is limited by Botrytis cinerea. Due to the toxicity of post-harvest pesticide application, alternative control methods such as priming are being investigated. Plants were treated with β-aminobutyric (BABA) at two developmental stages and resistance against B. cinerea was tested in fruit tissue and in progenies. DNA methylation and RNA sequencing were conducted to characterise the (epi)genetic changes associated with long-lasting resistance. Grafting experiments were done to assess the systemic nature of this signal, which was further characterised by small RNA (sRNA) sequencing of scions. Only BABA-treated seedlings displayed induced resistance (IR). DNA methylation analysis revealed seedling-specific changes, which occurred in the context of lower basal methylation. BABA-IR was found to be transmissible from primed rootstock to grafted unprimed scions. In these scions, we identified a subset of mobile 24 nt sRNAs associated with primed genes to infection in fruit. Our results demonstrate the functional association of a systemic signal with long lasting IR and priming. Through integrated omics approaches we have identified markers of long-lasting priming in tomato fruit which could also serve as targets for durable resistance in other crops.

overall_design: Examination of the DNA methylation profile of leaves and fruits of Solanum lycopersicum (cv. Micro Tom), from plants exposed to β-aminobutyric (BABA) at two different developmental stages, or treated with water.

first: Estrella

last: Luna-Diez

first: Katie

last: Stevens


title: High-resolution, noninvasive single-cell lineage tracing in mice and humans based on DNA methylation epimutations [H9_scBSseq]

overall_design: In this study, we generate single-cell bisulfite sequence datasets for the H9 cell line to evaluate the performance of MethyTree.


title: Itacitinib for Prevention of Graft-Versus-Host Disease and Cytokine Release Syndrome with T-cell Replete Peripheral Blood Haploidentical Transplantation

status: Public on Nov 15 2024

city: Saint Louis

last: Ding

address: 660 S. Euclid Avenue

institute: Washington University in St. Louis

summary: Haploidentical hematopoietic cell transplantation (haplo-HCT) is an increasingly used treatment for hematologic malignancies. Although post-transplant cyclophosphamide (PtCy) has improved graft vs. host disease (GvHD) prophylaxis in haplo-HCT, patients continue to experience life-threatening complications. IFN-γ and IL-6 are central in the pathophysiology of GvHD and cytokine release syndrome (CRS), and both cytokines signal through Janus kinase (JAK)-1. We tested the effect of adding the JAK-1 selective inhibitor, itacitinib, to PtCy-haplo-HCT to mitigate these complications and improve overall survival. This open-label, single-arm study evaluated the safety and efficacy of itacitinib combined with standard GvHD prophylaxis after haplo-HCT. A total of 42 patients were treated with itacitinib 200 mg daily from day -3 through +100 or +180, followed by a taper. Itacitinib resulted in low CRS grades, all patients had grade 0 (22%) or grade 1 (78%) CRS and there were no cases of grade 2-5 CRS. There were no cases of primary graft failure. No patients developed grade 3-4 aGvHD through day +180. The cumulative incidence of grade 2 aGvHD at day +100 was 21.9%. The 1-year cumulative incidence of moderate or severe chronic GvHD was 5%. The cumulative incidence of relapse at 2 years was 14%. Overall survival (OS) at 1 year was 80%. The cumulative incidence of nonrelapse mortality at day 180 was 8%. Itacitinib, when added to standard GvHD prophylaxis, was well tolerated and resulted in low rates of CRS, acute and chronic GvHD, NRM and encouraging rates of GvHD-free relapse-free survival (GRFS) and OS after haplo-HCT.

overall_design:
Peripheral blood and serum samples were collected from patients on study and banked at multiple time points. Control samples were collected from patients undergoing standard-of-care PB haplo-HCT during the same time period; these patients all received the same tac/MMF/PtCy GvHD prophylaxis except did not receive itacitinib.

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Raw files for human/patient samples are being made available in dbGaP (https://www.ncbi.nlm.nih.gov/gap/) for controlled access to the personally identifiable sequence data.
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first: Ramzi

last: Abboud

first: John

last: DiPersio

first: Reyka

middle: G

last: Jayasinghe