title: Glucocorticoid Receptor Suppresses GATA6-Mediated RNA Polymerase II Pause Release to Modulate Classical Subtype Identity in Pancreatic Cancer

city: Stuttgart

first: Steven

last: Johnsen

address: Auerbachstrasse 112

institute: Robert Bosch Center for Tumor Diseases

summary: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with a five-year survival rate of 12%. It has two major molecular subtypes: classical and basal, regulated by the master transcription factors (MTFs) GATA6 and ΔNp63, respectively. This study aims to uncover the transcriptional regulatory mechanisms controlling PDAC subtype identity. We integrated primary tumor single-cell RNA-seq, patient-derived xenograft RNA-seq, and multispectral imaging to identify MTF-dependent, subtype-specific markers. We created subtype-specific fluorescent reporter systems and conducted drug screenings to find actionable targets. We analyzed genome-wide occupancy (ChIP-seq) for epigenetic status (H3K27ac), MTFs (GATA6, ΔNp63), RNA polymerase II (Pol II), nascent RNA capture sequencing (PRO-seq), and H3K4me3-anchored chromatin topology (HiChIP). Additionally, we used nuclease-dead Cas9 (dCas9) to manipulate transcriptional regulatory mechanisms. Our approach identified glucocorticoid receptor (GR) agonists as agents that suppress the classical transcriptional program by modulating GATA6 activity. GATA6 regulates classical-specific transcription through promoter-proximal pause (PPP) release. Depletion of GATA6 increased Pol II occupancy at GATA6-bound enhancers and transcriptional start sites, stabilizing enhancer-promoter interactions. Artificially inducing pausing at GATA6-bound enhancers with dCas9 abrogated target gene expression and induced pausing at both the enhancer and target gene promoter. Conversely, ΔNp63 in basal PDAC promotes Pol II recruitment and stabilizes enhancer-promoter interactions. This study provides new insights into the transcriptional control of PDAC subtypes and suggests potential therapeutic strategies, highlighting the role of glucocorticoids in PDAC chemotherapy.

overall_design: To identify robust biomarkers for the classical subtype of PDAC, we performed single-cell formalin-fixed, paraffin embedded sequencing (scFFPEseq) on primary pancreatic cancer patient tissue.

first: Thomas

last: Ekstrom


status: Public on Jan 07 2025

middle: A

country: Germany

overall_design: To investigate the role of glucocorticoid receptor (GR) agonists and master transcription factors (MTF) GATA6 and deltaNp63 on subtype identity, we performed leChROseq in classical PDX models GöPDX5 and GöPDX8 and basal PDX models GöPDX4 and GöPDX15.

middle: L


overall_design: To investigate the role of glucocorticoid receptor (GR) agonists and master transcription factors (MTF) GATA6 and deltaNp63 on subtype identity, we performed qPROseq in classical cell line AsPC1 and basal cell line L3.6pl after siRNA depletion of MTFs in repsective cell lines or CDK9 inhibition with Enitociclib. We also inculded a 2% spike in by cell number of NIH3T3 cells.


overall_design: To investigate the role of glucocorticoid receptor (GR) agonists and master transcription factors (MTF) GATA6 and deltaNp63 on subtype identity, we performed H3K4me3 HiChIP in classical cell line AsPC1 and basal cell line L3.6pl after siRNA depletion of MTFs in repsective cell lines or CDK9 inhibition with Enitociclib.


title: Inosine Monophosphate Dehydrogenase 2 (IMPDH2) Modulates Response to Therapy and Chemo-Resistance in Triple Negative Breast Cancer

city: Buffalo

first: Spencer

last: Rosario

address: Elm & Carlton Sts

country: USA

institute: Roswell Park Comprehensive Cancer Center

type: Expression profiling by high throughput sequencing

summary: Triple negative breast cancer (TNBC) is one of the deadliest subtypes of breast cancer, whose high frequency of relapse is often due to occurrence of resistance to chemotherapy. Here, we identify inosine monophosphate dehydrogenase 2 (IMPDH2) as a contributor to doxorubicin resistance in different TNBC models. Analysis of public dataset reveals elevated IMPDH2 expression to correlate with worse overall TNBC prognosis in the clinic, including lower recurrence-free survival post adjuvant/neoadjuvant therapy. Importantly, genetic depletion or pharmacological inhibition of IMPDH2 leads to reduction of pro-tumorigenic phenotypes in multiple doxorubicin-resistant TNBC models, both in vitro and in vivo. Overall, we propose IMPDH2 as a novel vulnerability that could be leveraged therapeutically to suppress and/or prevent the growth of chemoresistant lesions.

overall_design: To investigate the effects of IMPDH2, we have established Doxorubicin resistant MDA-MB-231 cells, which we then knocked IMPDH2 down in using short hairpin RNA (shRNA).

first: Anna

last: Bianchi-Smiraglia

first: Tatiane

last: da Silva Fernandes

middle: R


title: Multi-locus CRISPRi targeting with a single truncated guide RNA

city: Cambridge

first: Fadi

middle: John

last: Najm

state: Massachusetts

address: 415 Main Street 6043

department: Epigenomics

institute: Broad Institute

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Multi-locus CRISPRi targeting with a single truncated guide RNA [RNA-seq]

summary: A critical goal in functional genomicsis evaluating which non-coding elements contribute togene expression, cellular function, and disease. Functional characterization remains a challenge due to the abundance and complexity of candidate elements. Here, we develop a CRISPRi-based approach for multi-locus screening of putative transcription factor binding sites with a single truncated guide. A truncated guide with hundreds of sequence match sites can reliably disrupt enhancer activity, which expands the targeting scope of CRISPRi while maintaining repressive efficacy. We screen over 13,000 possible CTCF binding sites with 24 guides at 10nucleotides in spacer length. These truncated guides direct CRISPRi-mediated deposition of repressive H3K9me3 marks and disrupt transcription  factor binding at most sequence match target sites. This approach is valuable for elucidating functional transcription factor binding motifs or other repeated genomic sequences and is easily implementable with existing tools.

overall_design: Jurkat or A375 cell lines treated with KRAB-dCas9 & guides targeting CTCF sites, CD81 promoter, or Safe Harbor control. Guides were 20nt in length or truncated (sg4 and sg8 and g[9nt])

middle: J

first: Siddarth

last: Wekhande

first: Molly

middle: M

last: Moore


title: Whole-transcript expression array analysis of intestinal epithelial cells from vehicle, TNBS-induced colitis BALB/cJ mice treated with vehicle or I3C

status: Public on Jan 06 2025

city: Columbia

first: Prakash

last: Nagarkatti

state: South Carolina

address: 6439 Garners Ferry Road, Basic Science Bldg 1, Rm C-26

department: Department of Pathology, Microbiology, and Immunology

institute: University of South Carolina School of Medicine Columbia

summary:
Whole-transcript expression array analysis of intestinal epithelial cells from  TNBS-induced colitis mice treated with vehicle or I3C demonstrated several differentially expressed genes
Downregulation of AhR expression in colitis mice was associated with downregulation of various antimicrobial peptides such as α-defensin 1, β-defensin1, reg1, reg4, reg3a and reg3b. However, treatment with I3C, an AhR ligand, increased the expression of AhR and AMPs such as α-defensin 1

overall_design: For TNBS-induced colitis model, the animals were randomized into experimental and control groups and divided into 4 groups. These included: 1) naïve 2) TNBS, 3) TNBS+I3C, and 4) I3C alone. Intestinal epitheial cells (IECs) were isolated from ileal tissue of control and experimental animals and identified by EPCAM(+) staining using flow cytometry

first: Manikandan

last: Palrasu

first: Philip

last: Brandon Busbe

first: Mitzi


title: Influenza B virus infection alters regenerative potential of alveolar type 2 pneumocytes (RNA-Seq)

city: Durham

first: Nicholas

last: Heaton

state: NC

address: 213 Research Drive, 426 CARL Building

department: Molecular Genetics and Microbiology

institute: Duke University School of Medicine

summary: Alveolar type 2 cells proliferate after lung injury. It was found that survivor alveolar type 2 cells downregulate genes relating to developmental, proliferation and differentiation pathways.

overall_design: Bystander and survivor alveolar type 2 cells were isolated from mice using FACs based on tdTomato expression

first: Satoko

last: Nakano

middle: S


title: Multi-locus CRISPRi targeting with a single truncated guide RNA [ChIP-seq]

overall_design: Jurkat cell line treated with KRAB-dCas9 & guides targeting CTCF sites or Safe Harbor control. Guides were 20nt in length or truncated (sg4 and [aag]sg4 and sg8)


overall_design: To investigate the role of glucocorticoid receptor (GR) agonists and master transcription factors (MTF) GATA6 and deltaNp63 on subtype identity, we performed ATAC in classical cell lines AsPC1 and TCCPAN2 and basal cell lines L3.6pl and T3M4 after BI 653048 treatment or siGATA6 treatment.


title: Influenza B virus infection alters regenerative potential of alveolar type 2 pneumocytes (scRNA-Seq)

summary: We found that distal epithelial cell types, specifically alveolar type 2 cells can survive infection by influenza B virus.

overall_design: Mock, bystander, infected, or survivor epithelial cells were isolated from mice using FACs based on tdTomato and EpCAM expression

first: Cait

middle: E

last: Hamele


overall_design: To investigate the role of glucocorticoid receptor (GR) agonists and master transcription factors (MTF) GATA6 and deltaNp63 on subtype identity, we performed RNAseq in classical cell lines AsPC1, HPAFII, PaTu8988s, and TCCPAN2 and PDX models (PAX) and basal cell lines L3.6pl and T3M4 under control conditions and after GR agonist treatment or siRNA depletion of MTFs in respective cell lines.


title: Effect of bryostatin-1 on in vitro exhausted CD8+ T cells

city: Rotterdam

first: Peter

middle: D.

last: Katsikis

state: Zuid-Holland

address: Dr. Molewaterplein 40

department: Department of Immunology

country: Netherlands

institute: Erasmus University Medical Center

summary: Bryostatin-1, a pharmacological agent from marine organisms, has been studied for HIV and cancer therapies due to its modulation of protein kinase C. In our previous study (Zhao M et al. Pharmacological research. 2019;139:524-34.), we found that bryostatin-1 downregulated inhibitory receptor PD-1 on activated CD8+ T cells from people with HIV. Since HIV-specific CD8+ T cells become functionally exhausted during chronic HIV infection and PD-1 expression is known to be related to the exhaustion state of these cells, we hypothesized that bryostatin-1 may modulate CD8+ T cell exhaustion. To test this, we generated in vitro exhausted OT-I CD8+ T cells as previously described (Zhao M et al PLoS Pathog. 16(6): e1008555) by repeatedly stimulating cells with OVA(257-264) peptide. Then we treated both exhausted CD8+ T cells (repeat peptide stimulated cells) and non-exhausted CD8+ T cells (single peptide stimulated cells) with bryostatin-1 for 3 days. We found that bryostatin-1 decreased exhaustion-associated markers and improved the functionality of exhausted CD8+ T cells. To investigate the underlying mechanism of how bryostatin-1 exerts its effects on exhausted CD8+ T cells, we performed RNA-sequencing (RNA-seq) on in vitro exhausted OT- I CD8+ T cells which were treated with bryostatin-1 or DMSO, with single peptide stimulated cells as non-exhausted controls. Taken together, our study demonstrate bryostatin-1's effect on exhausted CD8+ T cells and bryostatin-1's  potential in enhancing T cell immunity against chronic infection or cancer.

overall_design: OT-I CD8+ T cells are first exhausted in vitro for 5 days, then treated with either DMSO or bryostatin-1 for 3 days before RNA isolation and sequencing. Non-exhausted OT-I CD8+ T cells (stimulated only once with peptide) were treated with either DMSO or bryostatin-1 are controls.

first: Manzhi

last: Zhao

first: Ling

last: Li

first: Dwin

last: Grashof


title: The LncRNA 3222401L13Rik Is Upregulated in Aging Astrocytes and Regulates Neuronal Support Function Through Interaction with Npas3

city: Gottingen

first: Andre

last: Fischer

state: NS

address: Von Sieboldstr 3a

department: Epigenetic Mechanisms in Dementia

institute: German Center for Neurodegenerative Diseases (DZNE)

summary: Aging is linked to a decline in cognitive functions and significantly increases the risk of neurodegenerative diseases. While molecular changes in all central nervous system (CNS) cell types contribute to aging-related cognitive decline, the mechanisms driving disease development or offering protection remain poorly understood. Long non-coding RNAs (lncRNAs) have emerged as key regulators of cellular functions and gene expression, yet their roles in aging, particularly within glial cells, are not well characterized. In this study, we investigated lncRNA expression profiles in non-neuronal cells from aged mice. We identified 3222401L13Rik, a previously unstudied lncRNA enriched in glial cells, as being specifically upregulated in astrocytes during aging. Knockdown of 3222401L13Rik in primary astrocytes revealed its critical role in regulating genes essential for neuronal support and synapse organization. This function was also conserved in human iPSC-derived astrocytes. Additionally, we found that 3222401L13Rik mediates its cellular effects through interaction with the transcription factor Neuronal PAS Domain Protein 3 (Npas3), and that overexpression of Npas3 effectively rescued the functional deficits observed in astrocytes lacking 3222401L13Rik. Our findings suggest that upregulation of 3222401L13Rik in aging astrocytes acts as a compensatory mechanism to enhance neuronal and synaptic support, potentially delaying the onset of molecular and structural changes in both astrocytes and neurons. Strategies to boost 3222401L13Rik expression earlier in life may help mitigate age-associated loss of neuronal plasticity.

overall_design: To investigate the function of 3222401L13Rik, we performed a knockdown (KD) using Gapmers (Qiagen) in primary mouse astrocytes on DIV12. We harvested the cells on DIV14, isolated RNA, generated libraries and performed total RNA sequencing

first: Sophie

last: Schroeder

first: M

last: Sakib


title: The O-glycosyltransferase C1GALT1 promotes EWSR1::FLI1 expression and is a therapeutic target for Ewing sarcoma

status: Public on Jan 22 2025

city: Worcester

first: Junhui

state: MA

address: 364 Plantation St

institute: University of Massachusetts Medical School

summary: Ewing sarcoma (ES) is an aggressive bone cancer driven by the oncogenic fusion-protein EWSR1::FLI1, which is not present in normal cells and is therefore an attractive therapeutic target. However, as a transcription factor, EWSR1::FLI1 is considered undruggable. Factors that promote EWSR1::FLI1 expression, and thus whose inhibition would reduce EWSR1::FLI1 protein levels and function, are potential drug targets. Here, using genome-scale CRISPR/Cas9 knockout screening, we identify C1GALT1, a galactosyltransferase required for the biosynthesis of many O-glycoproteins, as a factor that promotes EWSR1::FLI1 expression. We show that C1GALT1 acts by O-glycosylating the pivotal Hedgehog (Hh) signaling component Smoothened (SMO), thereby stabilizing SMO and stimulating the Hh pathway, which we find directly activates EWSR1::FLI1 transcription. Itraconazole, an FDA-approved anti-fungal agent that is known to inhibit C1GALT1, reduces EWSR1::FLI1 levels in ES cell lines and suppresses growth of ES xenografts in mice. Our study reveals a therapeutically targetable mechanism that promotes EWSR1::FLI1 expression and ES tumor growth.

overall_design: For the genome-wide CRISPR/Cas9 knockout screen, the A673/EWSR1::FLI1tdTomato/EGFP/Cas9 reporter cell line was transduced with the Human CRISPR Knockout Pooled Library (Brunello) (Addgene, Cat# 73178)10, consisting of ~76,441 sgRNAs targeting 19,114 genes at multiplicity-of-infection (MOI) of 0.3. Cells were selected with 2 µg/ml puromycin for 12 days, followed by 3 days recovery in the absence of puromycin. At least 1x108 cells were FACS sorted using a BD FACSAria II cell sorter, and the population with tdTomatolow EGFPhigh expression (~7% of the total population) was isolated and expanded. Total genomic DNA was extracted from the sorted and unsorted populations, and 20 µg used to prepare a next-generation sequencing (NGS) library.

first: Shahid

last: Banday

first: Alok

middle: K

last: Mishra

first: Romana

last: Rashid

first: Tianyi

last: Ye

first: Amjad

last: Ali

first: Jason

middle: T

last: Yustein

first: Michelle

last: Kelliher

first: Lihua

last: Zhu

first: Sara

last: Deibler

first: Sunil

last: Malonia

first: Michael

last: Green


first: Steven

last: Johnsen

overall_design: To investigate the role of glucocorticoid receptor (GR) agonists and master transcription factors (MTF) GATA6 and deltaNp63 on subtype identity, we performed ChIPseq in classical cell lines AsPC1, HPAFII, PaTu8988s and TCCPAN2 and classical PDX models GöPDX5 and GöPDX8 and basal cell line L3.6pl and T3M4 under control conditions or after GR agonist treatment, siRNA depletion of MTFs in respective cell lines, or CDK9 inhibition with Enitociclib.

first: Thomas

last: Ekstrom


title: Intercellular bridges are essential for transposon repression and meiosis in the male germline

city: Piscataway

first: Matthew

last: Lawlor

state: NJ

address: 604 Allison Road

department: Genetics

institute: Rutgers University

summary: Germ cell connectivity via intercellular bridges is a widely conserved feature but its functional significance is poorly understood. Intercellular bridges are essential for fertility in male mice as genetic ablation of a critical bridge component, TEX14, causes loss of germ cell connectivity and meiotic failure, however the underlying reasons are unknown. A screen for meiosis-defective mouse mutants yielded a sterile mutant harboring a point mutation in Tex14 that leads to a partial loss of germ cell intercellular bridges. We utilized the Tex14 hypomorphs with reduced intercellular bridges, along with Tex14-null mice that completely lack bridges to examine the roles of germ cell connectivity during adult spermatogenesis. Complete or partial loss of intercellular bridges does not severely deplete spermatogonia, but spermatocytes are unable to undergo meiotic DNA replication and enter meiotic prophase. Phenotypic severity correlates with the extent of intercellular bridge depletion, with Tex14 hypomorphs containing a relatively larger number of spermatocytes undergoing meiosis, thus allowing us to assess impacts on meiotic progression. Tex14-deficient germ cells that enter prophase form meiotic breaks and initiate synapsis, but later exhibit synaptic failures and retain unrepaired breaks. Strikingly, Tex14-deficient germ cells derepress LINE1 retrotransposons and accumulate LINE1-encoded proteins during meiosis. Single-cell RNA-sequencing of testes from Tex14 hypomorphs show that intercellular bridge depletion modestly impacts the RNA landscape in early germ cell stages, while mutant cells that progress to late spermatocyte and post-meiotic spermatid stages broadly upregulate retrotransposons and dysregulate gene expression. RNA-sequencing confirms extensive sharing of transcripts between normal spermatids and demonstrates its partial attenuation in Tex14 hypomorphs, indicating that intercellular bridges enable exchange of cytoplasmic material between connected germ cells in testes. Tex14 hypomorphs display a progressive decline in germ cell numbers starting from the spermatocyte stage, with a complete lack of elongating spermatids, and are therefore sterile. Our findings reveal that the regulation of meiosis is non-cell-intrinsic and inform a model in which intercellular bridges orchestrate critical meiotic events and protect germline genome integrity during spermatogenesis, possibly by facilitating the exchange of regulatory factors.

overall_design: Single Cell RNA-seq of Tex14m/m and WT testis. Cells from each animal were counted and tagged with unique cell multiplexing oligo barcodes. Labelled cells from one wild type and one mutant were pooled equally and processed for cell encapsulation targeting 12,000 cells (~6,000 cells per mouse), followed by library preparation. This was done in duplicate to capture ~12,000 cells per mouse. Labelled cells from the second wild type and second mutant were pooled and processed similarly.

first: Matt

first: Chris

last: Ellison

first: Saher

last: Hammoud

first: Devanshi

last: Jain


title: Single-cell RNA-seq reveals immune cell heterogeneity and increased Th17 cells in keloid

city: GuangZhou

first: Yan

last: Zhang

state: GuangDong

address: Road LuJing

country: China

institute: Dermatology Hospital of Southern Medical University

summary: The immune response is postulated to exert a pivotal role in the occurrence and progression of keloid. In recent years, the impact of the peripheral immune microenvironment on keloid has garnered significant attention in scholarly investigations exploring the underlying pathogenesis of this condition. In this study, we isolated CD45+ cells from keloid and normal scar dermis tissues by fluorescence activated cell sorting (FACS) and performed single-cell RNA sequencing analysis.  Our results revealed the intricate cellular landscape of immune cells in keloid.  We found that compared to normal scar tissue, the percentage of Th17 cells was significantly increased in keloid.  Further functional studies revealed that Th17 cell promotes proliferation, migration and collagen expression of keloid fibroblast through IL17a.  These findings will help us more thoroughly understand keloid pathogenesis and provide potential targets for keloid therapies.

overall_design: We isolated CD45+ cells from keloid and normal scar dermis tissues.  After stringent quality control, we obtained transcriptomes of 41084 cells (Keloid(nsample=3): 17402;  Normal scar(nsample=3):23682).

first: ChengCheng

last: Deng


title: Early and Delayed STAT1-Dependent Responses Drive Local Trained Immunity of Macrophages in the Spleen [scRNA_seq_training]

city: Rehovot

first: Roi

last: Avraham

address: Herzl St 234

country: Israel

institute: Weizmann Institute of Science

summary: Trained immunity (TI) is the process wherein innate immune cells gain functional memory upon exposure to specific ligands or pathogens, leading to augmented inflammatory responses and pathogen clearance upon secondary exposure. While the differentiation of hematopoietic stem cells (HSCs) and reprogramming of bone marrow progenitors are well-established mechanisms underpinning durable TI protection, the characteristics of effector differentiated cells within the tissue remains underexplored. In this study, we delve into the nature of TI in splenic myeloid cells, both recruited and local, responsible for tissue mediated protection. Our findings emphasize the centrality of the JAK-STAT1 pathway, activated by the presence of BCG in peripheral immune tissues, in driving TI. Utilizing single cell RNA-sequencing and flow cytometry, we discerned STAT1-regulated genes in TI-associated resident and recruited splenic myeloid populations. The temporal dynamics of TI were further elucidated, revealed both early and late classical and non-classical monocyte subsets with time-dependent, STAT1-specific signatures. Tissue-resident red pulp macrophages (RPM), initially depleted by BCG exposure, are restored from both a pool of tissue-trained, self-renewing macrophages and from bone marrow-derived progenitors, fostering long lasting local defense. Lastly, upon transient application of specific JAK-STAT inhibitors acting upstream of STAT1 activation, we were able to pinpoint a pivotal temporal window soon after BCG vaccination that anticipates the onset of training; the interference of which obstructs the TI phenotype. As a whole, our study unravels the diverse manifestations of TI as it emerges in mature immune cells, demonstrating its role within the functional tissue milieu of the spleen.

overall_design: 2 samples, one treated (BCG), one control (PBS), and sorted single cell

first: Aryeh

last: Solomon

first: Noa

last: Bossel

first: Ben

last: Moshe

first: Dotan

last: Hoffmann

first: Sebastien

last: Trezbanski

first: Dror

last: Yehezkel

first: Leia

last: Vainman

first: Mihai

last: Netea


title: Early and Delayed STAT1-Dependent Responses Drive Local Trained Immunity of Macrophages in the Spleen [bulk_RNA_seq_kinetics]

overall_design: After i.p. inoculation with  BCG or PBS, mice splenoctyes were excised and lysed for bulk RNA libraries. Mice were sacrificed at day 3, 5, 14, 30, 45, and 60 post inoculation. Biological replicates range from 2-6, as indicated in the samples.


title: Early and Delayed STAT1-Dependent Responses Drive Local Trained Immunity of Macrophages in the Spleen [bulk_RNA_seq_type2_inh]

overall_design: Mice were inoculated with BCG or PBS i.p. in conjunction with Fedratinib, injected i.p for five days. After 2-weeks, mice were challenged with Salmonella Typhimuirum or PBS and sacrificed 24-hours post infection. Splenocytes were extracted and bulk sorted for RNA-seq.


title: Early and Delayed STAT1-Dependent Responses Drive Local Trained Immunity of Macrophages in the Spleen

overall_design: Refer to individual Series


title: Early and Delayed STAT1-Dependent Responses Drive Local Trained Immunity of Macrophages in the Spleen [bulk_RNA_seq_inhibitor_exp]

overall_design: Mice were inoculated with BCG or PBS i.p. in conjunction with three different inhibitors, Ruxolitinib, Fedratinib, and Deucravacitinib injected i.p for five days. After 2-weeks, mice were sacrificed, splenocytes were extracted and bulk sorted for RNA-seq.


title: Early and Delayed STAT1-Dependent Responses Drive Local Trained Immunity of Macrophages in the Spleen [bulk_RNA_seq_MS4A3]

overall_design: Ms4a3-TdTomato labeled mice were inoculated with BCG or PBS i.p. After 2-weeks, mice were sacrificed, splenocytes were extracted, and monocyte/macrophage subsets that were Tdtomato high or low were bulk sorted for RNA-seq.


title: Blimp1/Prdm1 is required for uterine remodeling and repair in the mouse following RU486-induced abortion

city: Oxford

first: Arne

middle: Wyndham

last: Mould

state: Oxfordshire

address: Warneford Hospital

department: Psychiatry

country: United Kingdom

institute: Oxford University

summary: Transcriptional profiling of E6.0 wild type and Blimp1 mutant decidua, 14 hours after intraperitoneal injection with RU486 or mineral oil with was performed using RNA-seq to identify the role of Blimp1 in uterine remodelling and repair post abortion.

overall_design: To induce Prdm1 gene deletion in the uterus, Prdm1 BEH/+;PRCre/+ studs (Vincent et al., 2005, Soyal et al., 2005) were crossed with female Prdm1CA/CA mice (Shapiro-Shelef et al., 2003) to generate Prdm1 BEH/CA ;PRCre females, hereinafter referred to as Blimp1 mutant. Female littermates (6–10 weeks of age) were used as the wild type. At E5.5 days post coitum, females Blimp1 mutant and wild type mice were administered 150 micrograms of RU486 in mineral oil or mineral oil alone by intraperitonal injection. The decidual swellings were collected 14 hrs later and analysed by RNA-Seq.

first: Maria-Eleni

last: Xypolita

first: Mubeen

last: Goolam

first: Elizabeth

last: Bikoff

last: Robertson


title: Transcriptomic and chromatin profiling of familial Alzheimer’s disease mutations in PSEN1, PSEN2, APP [RNA-seq]

city: La Jolla

first: Shankar

last: Subramaniam

state: CA

address: 9500 Gilman Drive

department: Department of Bioengineering

institute: University of California, San Diego

summary: Mutations in PSEN1, PSEN2, and APP cause familial Alzheimer’s Disease (FAD) with an early age at onset and progressive cognitive decline. We mechanistically characterize mutations in these three FAD genes using patient-derived neurons by integrating RNA- and ATAC-sequencing. Here, we demonstrate that FAD mutations share common disease endotypes with varying severity, particularly activation of non-ectoderm lineage and loss of neuron mitochondrial energy production, paving the way for potential therapeutic interventions.

overall_design: Gene expression (RNA-seq) profiling of iPSC-derived neurons from healthy donors (NDC) and familial Alzheimer's disease (FAD) patients with APP_V717I, PSEN1_A79V, and PSEN2_N141I mutations

first: Andrew

middle: B

last: Caldwell

first: Phoebe

last: Valdes

first: Qing

last: Liu

first: Michael

middle: Q

last: Fitzgerald

first: Srinivasan

last: Ramachandran

first: Celeste

last: Karch

first: Douglas

last: Galasko

first: Shauna

middle: H

last: Yuan

first: Steven

last: Wagner


title: Transcriptomic and chromatin profiling of familial Alzheimer’s disease mutations in PSEN1, PSEN2, APP [ATAC-seq]

overall_design: Chromatin accessibility (ATAC-seq) profiling of iPSC-derived neurons from healthy donors (NDC) and familial Alzheimer's disease (FAD) patients with APP_V717I, PSEN1_A79V, and PSEN2_N141I mutations


title: Transcriptomic and chromatin profiling of familial Alzheimer’s disease mutations in PSEN1, PSEN2, APP


title: Characterization and effective expansion of CD4-CD8- TCRab+ T cells from individuals living with type 1 diabetes

status: Public on Jan 05 2025

city: Blainville

first: Adam-Nicolas

last: Pelletier

state: Quebec

address: 97, Montcalm

country: Canada

institute: RPM Bioinfo Solutions

summary: CD4-CD8- TCRab+ (double-negative [DN]) T cells represent a rare T cell population that promotes immunological tolerance through various cytotoxic mechanisms. In mice, autologous transfer of DN T cells has shown protective effects against autoimmune diabetes and graft-versus-host disease. Here, we characterized humanDNT cells from people living with type 1 diabetes (PWT1D) and healthy controls. We found that while DN T cells and CD8+ T cells share many similarities, DN T cells are a unique T cell population, both at the transcriptomic and protein levels. We also show that by using various cytokine combinations, human DN T cells can be expanded in vitro up to 1,000- fold (mean >250-fold) and remain functional post-expansion. In addition, we report that DN T cells from PWT1D display a phenotype comparable to that of healthy controls, efficiently expand, and are highly functional. As DN T cells are immunoregulatory and can prevent T1D in various mouse models, these observations suggest that autologous DN T cells may be amenable to therapy for the prevention or treatment of T1D.

overall_design: RNA-seq profiling of human CD4+, CD8+, TCRgd+ and DN T cells from 3 healthy donors, ex vivo and after 14-day expansions in vitro.

first: J. Ernesto

last: Fajardo-Despaigne

first: Félix

last: Lombard-Vadnais

first: Aïnhoa

last: Olazabal

first: Lucie

last: Boutin

first: Sarah

last: Pasquin

first: Valérie

last: Janelle

first: Laurent

last: Legault

first: Jean-Sébastien

last: Delisle

first: Erin

last: Hillhouse

first: Lise

last: Coderre

first: Sylvie

last: Lesage


title: Cut&Tag HD KI Q140 2,6,10 months

status: Public on Jan 08 2025

city: strasbourg

first: Charles

last: DECRAENE

address: 12 rue Goethe

department: UMR 7364 CNRS

country: France

institute: CNRS

summary: Temporal epigenomic analysis of HD KI Q140 mouse striatal neurons using CUT&TAG on purified sorted neuronal nuclei.

overall_design: NeuN+/- nuclei from mixed male and female animals, WT and HD KI Q140 mice striatum were used (50000 nuclei). Two different histone marks were evaluated: H3K27ac and H3K27me3 coming from the same sample. Rabbit IgGs were used as control.

first: Karine

last: Merienne

first: Baptiste

last: Brule

first: S

last: Le Gras

last: Decraene


title: Transcriptome analysis of  vehicle and PDBAG1 treated TNBC cells

city: Nanjing

first: Xingxing

last: Li

state: Jiangsu

address: 123 Tianfei Road

institute: Women’s Hospital of Nanjing Medical University

summary: To investigate the genes affected by a newly discovered functional peptide, PDBAG1, on triple-negative breast cancer cells.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for MDA-MB-231 cells treated with vehicle and PDBAG1

first: Mingming

last: Lv

first: Cheng

last: Lu


title: PBRM1 directs PBAF to pericentromeres and protects centromere integrity [RNA-Seq]

city: London

first: Jessica

last: Downs

address: 237 Fulham Road

institute: The Institute of Cancer Research

summary: The specialised structure of the centromere is critical for effective chromosome segregation, but its repetitive nature makes it vulnerable to rearrangements. Centromere fragility can drive tumorigenesis, but protective mechanisms preventing fragility are still not fully understood. The PBAF chromatin remodelling complex is frequently misregulated in cancer, but its role in cancer is incompletely characterized. Here, we identify PBAF as a protector of centromere and pericentromere structure with profound consequences for genome stability. A conserved feature of isogenic cell lines lacking PBRM1, a subunit of PBAF, is compromised centromere and pericentromere integrity.  PBAF is present at these regions, and binding patterns of PBAF and H3K9 methylation change when PBRM1 is absent. PBRM1 loss creates a dependence on the spindle assembly checkpoint, which represents a therapeutic vulnerability. Importantly, we find that even in the absence of any perturbations, PBRM1 loss leads to centromere fragility, thus identifying a new player in centromere protection.

overall_design: RNA-sequencing of RPE1 and 1BR3 parental cells and those with PBRM1 CRISPR knockout. 3 biological replicates for parental (WT, n=3) and 2 biological replicates for two independent PBRM1 KO clones (which were combined for n=4 in total) samples are included

first: Katheryn

last: Begg

first: Alison

last: Harrod

first: Karen

last: Lane


title: PBRM1 directs PBAF to pericentromeres and protects centromere integrity

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


title: Hydroxytyrosol, a component of olive oil for breast cancer prevention in women at high risk of cancer

status: Public on Jan 03 2025

city: Houston

first: Zheng

last: Yin

state: TX

address: 6565 Fannin St, MC: F6-300

department: Systems Medicine and Bioengineering

institute: Houston Methodist Cancer Center

summary: This study evaluates the effects of hydroxytyrosol (HT), a component of olive oil, on mammographic breast density reduction. We explored effects of HT on Wnt β-catenin and other pathways involved in cancer stem cell renewal, DNA repair, cell proliferation and differentiation. Hydroxytyrosol reduced breast density only in women over 60 years, especially in those with high baseline breast density. HT also reduced proliferation and affected Wnt signaling pathway.  This study lays the foundation for future larger studies in exploring a natural compound with well tolerability and overall nontoxic profile for chemoprevention of breast cancer.

overall_design: The trial was preregistered at http://clinicaltrials.gov/ (NCT02068092). 25mg/day oral dose of HT were given for 12 months in pre- and post-menopausal women at increased risk of breast cancer. Out of 51 patients enrolled, 41 completed the study. The annualized percent decrease in maximum mammographic volumetric breast density (Max VBD%) between baseline (BL) and end-of-treatment (EOT) was analyzed. RNA sequencing (RNA-Seq) and multiplex analysis was performed on the breast biopsies to compare the BL with EOT samples.

first: Akshjot

last: Puri

first: Jenny

middle: C

last: Chang


title: Transcriptomic Analysis of Small Extracellular Vesicles (sEVs) from Mouse Liver Tissue Across Age Groups: Implications for Aging and Lipid Metabolism

city: Beijing

first: XIAODONG

last: LUAN

address: DONGDAN SANTIAO 9, DONGCHENG District

department: Peking Union Medical College Hospital

institute: Peking Union Medical College & Chinese Academy of Medical Science

type: Non-coding RNA profiling by high throughput sequencing

summary:
The present study investigates the transcriptomic profiles of small extracellular vesicles (sEVs) isolated from the liver tissues of mice at different ages to understand the role of sEVs in aging and lipid metabolism. We hypothesize that age-related changes in sEVs could reflect alterations in cellular communication and contribute to the development of age-associated diseases. Through a comprehensive analysis of miRNA expression, we aim to identify biomarkers and potential therapeutic targets for liver aging and metabolic disorders.
The study involved the extraction and characterization of sEVs from the liver tissues of mice at various ages.The age distribution and corresponding numbers of mice included 10 mice at 1 week (1 W), 5 mice at 6 weeks (6 W), 5 mice at 6 months (6 M), 5 mice at 12 months (12 M), and 5 mice at 14 months (14 M). Transcriptome analysis was performed to identify differentially expressed miRNAs.

overall_design: miRNA-seq study involved obtaining sEVs from liver tissues from mice across different age groups: 1 week (1 W), 6 weeks (6 W), 6 months (6 M), 12 months (12 M), and 14 months (14 M). A total of 5 mice were used for each age group, resulting in 25 samples.

first: Ye

last: Jin

first: Gaoge

last: Sun

first: Binxian

last: Chen

first: Siqin

last: Feng

first: Muyun

last: Tang

first: Zhuang

last: Tian

first: Hang

first: Shuyang

last: Zhang

first: Xiaodong

last: Luan


title: Multipronged impact of environmental temperature on Staphylococcus aureus infection by phage Kayvirus rodi: Implications for biofilm control

status: Public on Jan 04 2025

city: Oviedo

first: Lucia

last: Fernandez

address: C/ Francisco Pintado Fe 26

country: Spain

institute: Instituto de Productos Lacteos de Asturias (IPLA-CSIC)

summary: Environmental cues sometimes have a direct impact on phage particle stability, as well as bacterial physiology and metabolism, having a profound effect on phage infection outcome. Here, we explore the impact of temperature on the interplay between phage Kayvirus rodi (phiIPLA-RODI) and its host, Staphylococcus aureus. Our results show that phiIPLA-RODI is a more effective predator at room (25 °C) compared to body temperature (37 °C) against planktonic cultures of several strains with varying degrees of phage susceptibility. This result differs from most known examples of temperature-dependent phage infection, in which optimum infection is correlated with the host growth rate. Further characterization of this phenomenon was carried out with strains IPLA15 and IPLA16, whose respective MICs were 7 log units and a 1-log unit higher at 37 °C than at 25 °C. Our results demonstrated that the phage also had a greater impact at room temperature during biofilm development and for the treatment of preformed biofilms. There was no difference in phage adsorption between the two temperatures for strain IPLA16. Conversely, adsorption of phiIPLA-RODI to IPLA15 was reduced at 37 °C compared to 25 °C. Moreover, confocal microscopy analysis indicated that the biofilm matrix of both strains has a greater content of PIA/PNAG at 37 °C than at 25 °C. Regarding infection parameters, we observed longer duration of the lytic cycle at 25 °C for both strains, and infection of IPLA15 by phiIPLA-RODI resulted in a smaller burst size at 37 °C than at 25 °C. Finally, we also found that the rate of phage resistant mutant selection was higher at 37 °C for both strains. Altogether, this information highlights the impact that bacterial responses to environmental factors have on phage-host interactions. Moreover, phage phiIPLA-RODI appears to be a highly effective candidate for biofilm disinfection at room temperature, while its efficacy in biofilm-related infections will require combination with other antimicrobials.

overall_design: 6 samples were analyzed, correponding to 3 biological replicates of 24 hours biofilms formed by S. aureus IPLA16 at 25 ºC or 37 ºC

first: Lucía

last: Fernández

first: Ana

last: Duarte

first: Andrea

last: Jurado

first: Laura

last: Bueres

last: Rodriguez

first: Pilar

last: Garcia


title: TRAJECTORY ANALYSIS OF HEPATIC STELLATE CELL DIFFERENTIATION REVEALS METABOLIC REGULATION OF CELL COMMITMENT AND FIBROSIS

city: Barcelona

first: Juanjo

last: Lozano

address: C/ Rosselló 153

department: Plataforma de Bioinformatica

institute: CIBEREHD

summary: Defining the trajectory of cells during differentiation and disease is key for uncovering the mechanisms driving cell fate and identity. However, trajectories of human cells remain largely unexplored due to the challenges of studying them with human samples. In this study, we investigated the proteome trajectory of iPSCs differentiation to hepatic stellate cells (diHSCs) and identified RORA as a key transcription factor governing the metabolic reprogramming of HSCs necessary for HSCs’ commitment, identity, and activation. Using RORA deficient iPSCs and pharmacologic interventions, we showed that RORA is required for early differentiation and prevents diHSCs activation by reducing the high energetic state of the cells. While RORA knockout mice had enhanced fibrosis, whereas RORA agonists rescued multi-organ fibrosis in in vivo models. Notably, RORA expression was consistently found to be negatively correlated with liver fibrosis and HSCs activation markers in patients with liver disease. This study reveals that RORA regulates cell metabolic plasticity, crucial for mesoderm differentiation, pericyte quiescence, and fibrosis, influencing cell commitment and disease.

overall_design: RNA expression was determinedby RNAseq in Human Stellate cells (HSC)  from treated with RORA vs untreted   . We performed sequencing using Illumina HiSeq2500 system,  paired-end reads and 101 length.

first: Raquel

last: Martinez-Garcia-de-la-Torre

first: Pau

last: Sancho-Bru


title: Citrullinated IGF2BP1 promotes rheumatoid synovial aggression via increasing the mRNA stability of SEMA3D

city: Wenzhou

first: Xinyu

last: Yang

state: Not applicable

address: Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, 325035, China.

department: School of Pharmaceutical Sciences

institute: Wenzhou Medical University

summary: Protein citrullination modification plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA), and anti-citrullinated protein antibodies (ACPAs) are extensively employed for clinical diagnosis of RA. However, there remains limited understanding regarding specific citrullinated proteins and their implications in the progression of RA. In this study, we screened and verified insulin-like growth factor-2 mRNA binding protein 1 (IGF2BP1) as a novel citrullinated protein and R167 was the primary citrullination site. Functional verification showed that citrullination at the R167 site of IGF2BP1 promoted the proliferation, migration and invasion of RA fibroblast-like synoviocytes (RA-FLSs). To further explore the specific downstream target of citrullinated IGF2BP1, RNA sequencing was conducted in this study.

overall_design: To identified the downstream target gene of citrliinated IGF2BP1, IGF2BP1 was firstly knocked down in RA-FLSs,  then rescued with a mutant IGF2BP1 plasmid  with R167K point mutaion, or a wildtype IGF2BP1 plasmid. (WUT= IGF2BP1 R167K group, NC= IGF2BP1 wildtype group, n=3 per group.)

first: Yang

last: Qin

first: Li

last: Liu

first: Yanwen

first: Xiaowei

first: Sunwang

last: Ling

first: Jianguang

last: Wang


title: Differential RNA expression of peritoneal macrophages infected with antimony-sensitive (LD-S) antimony-unresponsive (LD-R) at 4hrs, 24hrs, and 48hrs post-infection keeping uninfected macrophages as control.

city: Kharagpur

first: Budhaditya

last: Mukherjee

state: West Bengal

address: Life Science Building

department: School of Medical Science and Technology

country: India

institute: Indian Institute of Technology Kharagpur

summary: Recent clinical LD field isolates still showed antimony resistance (LD-R) even after the withdrawal of pentavalent antimonials in treating Visceral leishmaniasis in India decades back due to the emergence of antimony resistance. Clinical infection with LD-R results in aggressive pathogenesis characterized by higher parasite burden, chronic hepatosplenomegaly, and severe anemia, the reason of which is poorly understood. Thus, it is paramount to unveil the underlying signaling contributing to severe anemia and chronic hepatosplenomegaly resulting from parasite overburden observed in LD-R infection. Our data provides insights into how LD-R modulates murine macrophages in-vitro to proliferate rapidly while dodging off the host immune surveillance at the host-parasite interface at early and late time points of infection as compared to its sensitive counterparts.

overall_design: Murine peritoneal macrophages were infected with AG83 (LD-S) and BHU575 (LD-R) at 4hrs, 24hrs, and 48hrs keeping uninfected macrophages as control. Each experimental sets are kept in duplicates. Total RNA was isolated from the experimental samples. The main objective of RNAseq analysis was to check the differential gene expression of LD-S vs LD-R infected macrophages at early and late time points at the host-parasite interface to unveil the differential host modulation by the  semsitive and unresponsive LD isolates.

first: Souradeepa

last: Ghosh

first: Krishna Vamshi

last: Chigicherla

first: Supratim

last: Pradhan

first: Shubhangi

last: Chakraborty


title: Second-generation BRAF inhibitor Encorafenib resistance is regulated by NCOA4-mediated iron trafficking in the drug-resistant malignant melanoma cells

city: Bursa

first: Ceyda

last: Colakoglu Bergel

address: Görükle Kampüsü, Nilüfer

country: Turkey

institute: Bursa Uludag University

summary: The current study established the first in vitro Encorafenib resistance protocol in BRAF-mutated malignant melanoma (MM) cells and investigated the resistance-related mechanisms. RNA-Seq results exhibited altered epigenetic regulation of resistance; particularly ferritin family members, ion transport pathways. Then, increased NCOA4, FTH1, and iron levels detected in A375-R suggest that the iron metabolism-related mechanism, such as ferritinophagy, might be triggered, which was supported by TEM and oxidative stress analysis. Iron storage, transport, and ferritinophagy have the promising potential to be targeted for combining with BRAF-targeted therapy to reverse Encorafenib resistance in MM. Moreover, this is the first study evaluating in vitro Encorafenib resistance mechanisms, and we suggest that our findings contribute to improving new drug combinations targeting BRAF and iron metabolism in different MM cells.

overall_design: To determine the effect of Encorafenib resistance on transcriptional regulation, transcriptome sequencing was performed. Total RNA extraction was carried out using the RNeasy Mini Kit (Hilden, Germany) from resist and sensitive cells. To perform RNA sequencing, mRNA and small RNA libraries were generated using the NGS sequencing Illumina NextSeq 500 platform with the SMARTer smRNA-Seq Kit (Illumina, Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. The libraries were gel-purified and assessed using the D1000 ScreenTape System (Agilent Technologies, Waldbronn, Germany).

first: Isil Ezgi

last: Eryilmaz

first: Gulsah

last: Cecener

first: Unal

last: Egeli


title: PBRM1 directs PBAF to pericentromeres and protects centromere integrity [CUT&RUN]

first: Jessica

last: Downs

overall_design: Cleavage Under Targets and Release Using Nuclease with salt fractionation (CUT&RUN.salt) sequencing for SMARCA4, CENPB, PBRM1, H3K9me2, H3K9me3, IgG in RPE1 WT cells, and for SMARCA4, H3K9me2, H3K9me3, IgG in RPE1 PBRM1KO cells. H3K27me3 in RPE1 WT cells was used as a positive control. Anti-SMARCA4 in a RPE1 SMARCA4KO cell line was used as a negative control for SMARCA4 mapping. 3 biological replicates are included. For  PBRM1KO cells, replicates 1 and 2 were performed using KO#1 (B3), replicate 3 was performed using KO#2 (B16).

first: Lillian

last: Wu

first: Alison

last: Harrod


title: Loss of PARP7 increases IFN-I signalling preventing pancreatic tumour growth by enhancing immune cell infiltration

city: Oslo

first: Jason

last: Matthews

address: Sognsvannsveien 9

department: Nutrition

country: Norway

institute: University of Oslo

summary: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer, and despite low incidence rates, it remains the sixth leading cause of cancer related deaths worldwide. Immunotherapy, which aims to enhance the immune system’s ability to recognize and eliminate cancer cells, has emerged as a promising approach in the battle against PDAC. PARP7, a mono-ADP-ribosyltransferase, is a negative regulator of the type I interferon (IFN-I) pathway, thereby reducing antitumour immunity. By characterizing murine pancreatic cancer cells, we found that loss of PARP7 elevated the levels of interferon stimulated gene factor 3 (ISGF3) and its downstream target genes, even in the absence of STING. We also observed that cancer cells deficient in PARP7 resulted in smaller tumours when injected into wildtype mice. Transcriptomic analyses revealed that tumours knocked out for Parp7 (Parp7KO) had increased expression of genes involved in immunoregulatory interactions and interferon signalling pathways. Characterization of tumour infiltrating leukocyte (TIL) populations showed that Parp7KO tumours had higher proportions of natural killer cells, CD8+ T cells and a lower proportion of anti-inflammatory macrophages (M2). The overall TIL profile of Parp7KO tumours was suggestive of a better response to anti-PD-1 immune checkpoint therapy. Our data show that loss of PARP7 reduces PDAC tumour growth by increasing the infiltration of immune cells and enhancing antitumor immunity. These findings provide support to pursue PARP7 as a therapeutic target for PDAC

overall_design: CR705 cells derived from a spontaneous pancreatic tumour in a LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx1-Cre (KPC) mouse were used in this study. Total RNA was isolated from approximately 100 mg of tumours from CR705 WT or PARP7KO cells using the Aurum™ Total RNA isolation kit (BioRad) according to the manufacturer’s protocol.

first: Vinicius

last: Kannen

first: Marit

last: Rasmussen

first: Siddhartha

last: Das

first: Paolo

last: Giulianna

first: Linnea

last: Erlingsson

first: Ninni

last: Olafsen

first: Paola

last: Cappello

first: Indrek

last: Teino

first: Toivo

last: Maimets

first: Kristaps

last: Jaudzems

first: Antanas

last: Gulbinas

first: Zilvinas

last: Dambrauskas

first: Landon

last: Edgar

first: Denis

last: Grant


title: PTP inhibition improves the macrophage anti-tumor immune response and the efficacy of chemo- and radiotherapy

status: Public on Jan 02 2025

city: Birmingham

first: Nestor

last: Prieto-Dominguez

state: AL

department: Microbiology

institute: The University of Alabama at Birmingham (UAB)

summary: Traditional anti-cancer therapies induce tumor cell death and subsequent release of Damage Associated Molecular Patterns (DAMPs) that activate the innate inflammatory response. Paradoxically, after treatment macrophages often adopt a pro-wound healing phenotype that contributes to cancer progression. We found that tumor cells upregulate the expression of Pros1 in areas proximal to cell damage. Tumor-secreted Pros1 binds to the macrophage Mer receptor, effectively limiting responsiveness to DAMPs by preventing Toll Like Receptor (TLR) signal transduction. Pharmacologically inhibiting PTP1b signaling downstream of Mer rescues the pro-inflammatory response even in the presence of Pros1. Combining PTP inhibition with traditional therapeutics, like chemo- or radiotherapy, rescues the innate immune response to DAMPs, increases immune infiltration, and causes 40-90% reductions in tumor growth in multiple treatment refractory preclinical models. Our findings suggest a novel use for PTP1b inhibitors as a tumor agnostic means of improving the efficacy of some of the most widely used anti-cancer therapeutic agents.

overall_design: scRNA-seq analysis of CD45+ cells obtained from end-stage B16F10 syngeneic murine tumors treated with PBS (ip, qd), two doses of Cisplatin (5mg/kg, ip, once daily for three days), BVT948 (10mg/kg, ip, qd) or the combination

first: Paran

last: Goel

first: Valeriya

last: Kuznetsova

first: Robert

last: Welner

first: Eric

last: Ubil


title: Optimized gene transduction in human lung organoids for advanced research applications

city: toronto

first: Haibo

last: Zhang

state: ontario

address: 209 victoria st

department: Anesthesia

institute: Unityhealth Toronto

summary: Human induced pluripotent stem cell (iPSC)-derived lung organoids, engineered to carry targeted genes, offer a robust platform for investigating mechanistic insights in lung research. Although lentiviral vectors (LVVs) are highly effective for stable expression due to their integrative properties, achieving efficient transduction in human iPSC-derived lung organoids poses a significant technical challenge, likely due to the complex structure of these organoids. In this study, we optimized a method to enhance LVV transduction efficiency by physically disrupting the organoids to increase surface area, followed by spinoculation to apply shear force during cell dissociation. This approach, combined with the use of an optimized culture medium, significantly improved transduction efficiency. The success of this method was validated at both the gene and protein levels using single-cell RNA sequencing (scRNA-seq) and various cellular and molecular assays. Our optimized transduction protocol may provide a valuable tool for investigating specific cellular and molecular mechanisms in development and disease models using human iPSCs-derived lung organoids.

overall_design: Single cell RNA sequencing profiling of naïve and transduced Induced-pluripotent stemcells derived lung organoids (iPSC-LOs) at day 60

first: Jasmin

last: Khateeb

first: Jady

last: Liang

first: Yuchong

last: Li

first: Thenuka

last: Thanabalasingam

first: Julie

last: Khang

first: Mirjana

last: Jerkic

first: Giovanna

last: Pellecchia

first: Bhooma

last: Thiruv

first: Ya-wen

last: Chen

first: Ori

last: Rotstein

first: Arthur

last: Slutsky


title: Ptp1b inhibition improves the macrophage anti-tumor immune response and the efficacy of chemo- and radiotherapy.

first: Nestor

last: Prieto-Dominguez

summary: Traditional anti-cancer therapies induce tumor cell death and subsequent release of Damage Associated Molecular Patterns (DAMPs) that activate the innate inflammatory response. Paradoxically, after treatment macrophages often adopt a pro-wound healing, rather than pro-inflammatory, phenotype and contribute to cancer progression. We found that in areas proximal to DAMP release, tumor cells upregulate the expression of Pros1. Tumor-secreted Pros1 binds to the macrophage Mer receptor, effectively limiting responsiveness to DAMPs by preventing Toll Like Receptor (TLR) signal transduction. Pharmacologically inhibiting PTP1b signaling downstream of Mer rescues the pro-inflammatory response even in the presence of Pros1. Combining PTP inhibition with traditional therapeutics, like chemo- or radiotherapy, rescues the innate immune response to DAMPs, increases immune infiltration, and causes 40-90% reductions in tumor growth in multiple treatment refractory preclinical models. Our findings suggest a novel use for PTP1b inhibitors as a tumor agnostic means of improving the efficacy of some of the most widely used anti-cancer therapeutic agents.

overall_design: RNA-seq profiling of wild-type B16F10 and Pros1-deficient (BdP) syngeneic murine tumors treated with vehicle (PBS) or two rounds of cisplatin (5mg/kg, intraperitoneally, once daily for 3 days)


title: Transcriptome and DNA methylation profiling during the NSN to SN transition in mouse oocytes [ChIP-Seq]

city: Cambridge

first: Soumen

last: Khan

address: Babraham Hall

department: Epigenetics

institute: Babraham Institute

summary: During oocyte maturation, the transition from a non-surrounded nucleolus (NSN) to a surrounded nucleolus (SN) stage coincides with dramatic chromatin reconfiguration and transcriptional silencing and is crucial for developmental competence. To study the transcriptome and DNA methylation dynamics during the NSN to SN transition we used single cell (sc)M&T-seq to generate scRNA-seq and sc bisulphite-seq data from GV oocytes, classified as NSN or SN by Hoechst staining of their nuclei. Transcriptome analysis showed a lower number of detected transcripts in SN oocytes as well as downregulation of 576 genes, which were enriched for processes related to mRNA processing. We used the RNA-seq data to generate a classifier, which can infer chromatin stage in scRNA-seq data sets, based on their transcription profile. The classifier was successfully tested in multiple published datasets of mouse models with a known skew in NSN:SN ratios. DNA methylation analysis showed increased methylation in SN compared to NSN oocytes, which was most pronounced in regions with intermediate levels of DNA methylation. Overlap with ChIP-seq data for the histone modifications H3K36me3, H3K4me3 and H3K27me3 showed that regions gaining methylation in SN oocytes are enriched for overlapping H3K36me3 and H3K27me3, which is unusual as these marks do not typically coincide. We believe that these late-methylating SN regions are in regions with high chromatin plasticity and that the overlap of H3K36me3 and H3K27me3 may indicate a transient switch or heterogeneity on a single-cell level.

overall_design: Ultra-low input native ChIP-sequencing was performed on pooled GV oocytes (~280 oocytes per replicate) from 25-day old C57BL6/Babr mice, to assess the distribution of H3K36me3.

first: Courtney

last: Hanna

first: Hannah

last: Demond

first: Gavin

last: Kelsey


title: Loss of aryl hydrocarbon receptor reduces pancreatic tumor growth by increasing immune cell infiltration [II]

first: Jason

last: Matthews

summary: Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease which remains poorly understood. Increasing evidence suggests that the aryl hydrocarbon receptor (AHR) plays a role in the pathogenesis of several cancers; however, its role in PDAC is unclear because AHR exhibits both pro- and anti-tumor activities. Here we evaluated the role of AHR in CR705 and K8484 murine PDAC cells in vitro and CR705 cells in vivo. Loss of Ahr did not affect cell proliferation compared with Cas9 control cells and no differences in tumor development between CR705Cas9 and CR705AhrKO cells were observed in immunocompromised mice. Conversely, tumors from CR705AhrKO cells grew more slowly than tumors from CR705Cas9 cells in immune competent mice. RNA sequencing identified 1279 genes upregulated and 586 genes downregulated in CR705AhrKO tumors compared with CR705Cas9 tumors. Pathway analysis identified immunoregulatory interactions, interferon signaling, and chemokine signaling among the top upregulated pathways. Increased infiltration of CD45+ cells and higher numbers of CD8+ T cells and F4/80+ cells were observed in CR705AhrKO tumors. Ahr deficiency in macrophages (LysMCre) or lymphocytes (RorcCre) did not alter tumor development of CR705Cas9 cells compared with WT C57BL/6 mice. CR705AhrKO tumors in RorcCre mice, but not in LysMCre mice had significantly lower tumor weights normalized to body weights compared with CR705AhrKO tumors in WT mice. These findings show that Ahr loss in CR705 pancreatic cancer cells is sufficient to induce proinflammatory gene responses that contribute to increased immune cell infiltration and reduced tumor growth.

overall_design: CR705 WT and AHRKO tumor cells  were maintained in RPMI (1.0 g/L glucose; Sigma-Aldrich), supplemented with 10% v/v heatinactivated fetal bovine serum (FBS; Sigma-Aldrich), 1% v/v L-glutamine (Sigma-Aldrich), and 1% v/v penicillin-streptomycin, (P/S; Sigma-Aldrich). Cells were cultured at 37°C, with 100% humidity and 5% CO2. Cells were subcultured when the cells reached 80% confluency.

first: Vinicius

last: Kannen

first: Ninni

last: Olafsen

first: Siddhartha

last: Das

first: Paolo

last: Giulana

first: Fauzia

last: Izzati

first: Hani

last: Choksi

first: Samaneh

last: Åhrling

first: Paola

last: Cappello

first: Indrek

last: Teino

first: Toivo

last: Maimets

first: Kristaps

last: Jaudzems

first: Antanas

last: Gulbinas

first: Zilvinas

last: Dambrauskas

first: Landon

last: Edgar

first: Denis

last: Grant


title: Transcriptome and DNA methylation profiling during the NSN to SN transition in mouse oocytes [Bisulfite-Seq]

first: Soumen

last: Khan

type: Methylation profiling by high throughput sequencing

overall_design: scBS sequencing was performed on oocytes to identify methylation changes during NSN to SN transition

first: Courtney

last: Hanna

first: Hannah

last: Demond

first: Gavin

last: Kelsey


title: Effect of GPRC5B-deficiency on macrophage function [dataset 1]

city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

institute: MPI for heart and lung research

summary: To investigate whether loss of GPRC5B affects macrophage function, we generated myeloid-specific Gprc5b knockout mice and then did RNA-seq using resident peritoneal macrophage to analyze the gene expression profile.

overall_design: Gene expression profiling in resident peritoneal macrophages from control mice (LysCre-negative;Gprc5b(fl/fl)) and myeloid-specific Gprc5b knockout mice (LysCre+;Gprc5b(fl/fl)) without any stimulation (triplicates for each group).

first: Jeonghyeon

last: Kwon

first: Nina

last: Wettschureck


title: Effect of GPRC5B-deficieny on macrophage function [dataset 2]

summary: To investigate whether loss of GPRC5B affects macrophage function, we generated myeloid-specific Gprc5b knockout mice and then did RNA-seq using bone marrow-derived M0 macrophages to analyze the gene expression profile.

overall_design: Gene expression profiling in bone marrow-derived M0 macrophages from control mice (LysCre-negative;Gprc5b(fl/fl)) and myeloid-specific Gprc5b knockout mice (LysCre+;Gprc5b(fl/fl)) treated with or without LPS 1µg/ml for 6h (triplicates for each condition).


title: Transcriptome and DNA methylation profiling during the NSN to SN transition in mouse oocytes

overall_design: RNA-seq was performed for oocytes belonging to either the NSN or SN stage in development to identify transcriptomic differences


title: The effect of A-196 and TMP-269 epigenetic enzyme inhibitor treatments on endothelial cells and fibroblasts

city: Kuopio

first: Tiit

last: Örd

address: Neulaniementie 2

country: Finland

institute: A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland

summary: Epigenetic-based therapy has gained interest with regard to cardiovascular diseases. However, cell-type-specific effects and the limited complexity of cellular interactions in the screening methods causes challenges in finding novel drug candidates. Using a library of epigenetic modulator compounds, we found that A-196 and TMP-269 demonstrated functional effects in a fibrin bead angiogenesis assay. To study how these epigenetic enzyme inhibitors affect genome-wide gene expression profiles, we performed RNA-Seq of human umbilical vein endothelial cells (HUVEC) and human pulmonary fibroblasts (HPF) treated with these compounds.

overall_design: Cells were subjected to 72 h chemical treatment, followed by RNA-Seq using an mRNA 3' tag approach aimed at gene expression quantification.

first: Mustafa

last: Beter

first: Einari

last: Aavik

first: Seppo

last: Ylä-Herttuala

first: Johanna

last: Laakkonen


title: A PARP14/TARG1-Regulated RACK1 MARylation Cycle Drives Stress Granule Dynamics in Cancer Cells [Ribo-seq]

city: Dallas

first: W. Lee

last: Kraus

address: 5323 Harry Hines Blvd.

institute: UT Southwestern Medical Center

summary: In this study we examine the role of RACK1 MARylation in stress granule assembly and translational regulation in ovarian cancer cells. In addition, we describe the role of TARG1, an ADPR hydrolase enzyme in stress granule assembly and translation.

overall_design: Ribo-seq (ribosome profiling) was performed on OVCAR3 cells with dox-mediated knockdown of endogenous RACK1 and re-expression of wildtype or MAR deficit mutant RACK1 proteins. We also performed Ribo-seq on OVCAR3 cells subjected to siRNA-mediated knockdown of TARG1.

first: Sridevi

last: Challa

first: Tulip

last: Nandu

first: Jin

last: Chen

first: W


title: Increased molecular and functional heterogeneity of hematopoietic stem cells contributes to age-related decline of tissue and body function

city: Boston

first: Wenhao

last: Zhang

address: Warren Alpert Bldg, Rm 160, 200 Longwood Ave

institute: Harvard Medical School

summary: Aging is a process accompanied by functional decline in tissues and organs with great social and medical consequences. Previous studies have demonstrated that aged hematopoietic stem cells (HSCs) are functionally compromised, which at least partly contributes to aging-related decline of the body health. However, the underlying mechanism is largely unknown. Here we reveal a clear heterogeneity of old HSCs, which can be marked by the CD150 levels. Comparative molecular and functional analyses revealed that CD150low HSCs from old mice have a younger aging clock, transcriptome, and better repopulation capacity compared to that of the CD150high HSCs. Mechanistically, CD150high HSCs from old mice have greatly compromised differentiation capacity compared to that of the CD150low HSCs. Importantly, decreasing the CD150high HSC ratio in old mice can alleviate aging-related functional decline. Thus, our study not only reveals how HSC heterogeneity contributes to aging, but also points to a potential way for rejuvenation.

overall_design: We have conducted RNA sequencing and single-cell RNA sequencing to invesitgate the transcriptomic heterogenetity in HSCs aging. RRBS profiling is conducted and applied to calculate the epigenetic age.

first: Yuting

last: Wang

first: Chao

first: Yi


title: A PARP14/TARG1-Regulated RACK1 MARylation Cycle Drives Stress Granule Dynamics in Cancer Cells [RNA-seq]

overall_design: RNA-seq was performed on OVCAR3 cells with dox-mediated knockdown of endogenous RACK1 and re-expression of wildtype or MAR deficit mutant RACK1 proteins. We also performed RNA-seq on OVCAR3 cells subjected to siRNA-mediated knockdown of TARG1.


title: RNA-seq data of mammalian cells treated with artificial transcription factors

status: Public on Jan 01 2025

city: Columbia

first: Davis

last: Noblitt

state: SC

address: 631 Sumpter Street

department: IBS

institute: University of South Carolina

summary: Interferon (IFN)-γ is a central regulator of cell-mediated immunity in human health and disease, but reduced expression of the target receptors impairs signaling activity and leads to immunotherapy resistance. Although intracellular expression of IFN-γ restores signaling and downstream functions, we lack tools to activate the IFNG gene instead of cell surface receptors. This work describes the design and characterization of an artificial transcription factor (ATF) protein that recognizes the IFNG gene with six zinc finger domains, which are dovetailed to a VP64 signaling domain that promotes gene transcription and translation. Biological studies with human Jurkat T cells reveal that the ATF treatment induces potent IFN-γ expression and amplifies the gene transcriptome profile for multiple class I HLA alleles and interferon-stimulated genes (ISGs). Biophysical characterization showed the recombinant ATF protein recognizes the human IFNG gene with nanomolar affinity (KD = 5.27 ± 0.3 nM), adopts a protein secondary structure associated with the ββα-fold of zinc finger domains, and is resistant to thermal denaturation. These studies demonstrate that transcriptional targeting of cytokine genes, rather than surface receptors, can activate cytokine expression and show potential for directing immune function.

overall_design: We profiled the gene expresion of human Jurkat T cells after treatment with an anti-IFNG artificial transciption factor (ATF), isotype control (Aart6), and vehicle (media). The cells were transfected with plasmid by electroportation , and supplemented after 2 hours with zinc chloride, PMA/Ionomycin, and Brefeldin A. After 48 hours, the cells were collected and processed to extract the total RNA content with an RNeasy Minikit (Qiagen, 74106) and RNase-free DNase (Qiagen, 79254). The RNA quality was verified by electrophoresis. The sample isolates that passed quality control were sequenced on an Illumina Genome Analyser (Azenta).

first: Nicholas

last: Truex

first: Ashley

last: King

first: Olivia

last: Sherron

first: Clara

last: Kjerfve

first: Lydia

last: Pless


title: Maternal Immune Activation imprints translational dysregulation and differential MAP2 phosphorylation in descendant neural stem cells

city: London

first: Sandra

middle: Maria

last: Martin-Guerrero

address: Charterhouse Square Campus

department: Cancer Genomics and Computational Biology

institute: Barts Cancer Institute, Queen Mary University of London

summary: To undertake a comprehensive examination of the molecular mechanisms governing the alterations induced by maternal immune activation (MIA) during gestation that impacts the subsequent neurodevelopment of progeny, we have devised an in vitro model based on neural stem cells (NSCs) sourced from fetuses carried by animals subjected to Poly I:C treatment. These neural progenitors demonstrate proliferative properties and can be effectively differentiated into both neurons and glial cells. Through transcriptomic, proteomic, and phosphoproteomic analyses conducted on these cellular models, in conjunction with counterparts from control treatments, discernible shifts in the expression levels of a specific subset of proteins implicated in neuronal function were elucidated. Noteworthy is the absence of congruence between these alterations and the transcriptomic findings, suggesting a potential dysregulation at the level of protein translation. Furthermore, a discernible discrepancy in the basal phosphorylation of proteins was observed between differentiated cells from both experimental groups, particularly within proteins associated with cytoskeletal architecture and synaptic functionality, notably those belonging to the MAP family. These observed alterations were found to correlate with changes in neuronal function, particularly with respect to synaptic plasticity and the establishment of neuronal synapses, thereby reaffirming previous assertions regarding the potential impact of MAP2 function modulation via its phosphorylation state on neuronal structure and function.

overall_design: To delve deeper into the biological mechanisms implicated in the MIA effects on neurodevelopment processes, we have generated neural stem cell lines from fetuses carried by pregnant mice treated with Poly I:C with the aim of investigating the alterations that persist in cultured neurons derived from the differentiation of these neural progenitors.

first: Juan F

last: Lopez-Gimenez

first: Pedro R

last: Cutillas

first: Sandra M

first: Maria

last: Martin-Estebane

first: Antonio

last: Lara-Ordóñez

first: Miguel

last: Cánovas

first: David

last: Martín-Oliva

first: Javier

last: González-Maeso


title: The effect of rewarming ischemia on tissue transcriptome signatures: a clinical observational study in lung transplantation

city: Leuven

first: Johan

last: Van Weyenbergh

address: Herestraat 49

department: DMIT

country: Belgium

institute: KU Leuven

summary:
BACKGROUND: In lung transplantation (LuTx), various ischemic phases exist, yet the rewarming ischemia time (RIT) during implantation has often been overlooked. During RIT, lungs are deflated and exposed to the body temperature in the recipient's chest cavity. Our prior clinical findings demonstrated that prolonged RIT increases the risk of primary graft dysfunction. However, the molecular mechanisms of rewarming ischemic injury in this context remain unexplored. We aimed to characterize the rewarming ischemia phase during LuTx by measuring organ temperature and comparing transcriptome and metabolome profiles in tissue obtained at the end versus the start of implantation.
METHODS: In a clinical observational study, 34 double-LuTx with ice preservation were analyzed. Lung core and surface temperature (n=65 and 55 lungs) was measured during implantation. Biopsies (n=59 lungs) were wedged from right middle lobe and left lingula at start and end of implantation. Tissue transcriptomic and metabolomic profiling were performed.
RESULTS: Temperature increased rapidly during implantation, reaching core/surface temperatures of 21.5°C/25.4°C within 30min. Transcriptomics showed increased pro-inflammatory signaling and oxidative stress at the end of implantation. Upregulation of NLRP3 and NFKB1 correlated with RIT. Metabolomics indicated elevated levels of amino acids, hypoxanthine, uric acid, cysteineglutathione disulfide alongside decreased levels of glucose and carnitines. Arginine, tyrosine, and 1-carboxyethylleucine showed correlation with incremental RIT.
CONCLUSIONS: The final rewarming ischemia phase in LuTx involves rapid organ rewarming, accompanied by transcriptomic and metabolomic changes indicating pro-inflammatory signaling and disturbed cell metabolism. Limiting implantation time and lung cooling represent potential interventions to alleviate rewarming ischemic injury.

overall_design:
Lung tissue biopsy pieces preserved at -80°C were homogenized in Total RNA Lysis Solution (Bio-Rad, Cat#7326820, US) with a 3mm tungsten carbide bead (Qiagen, Cat#69997, Netherlands) using the TissueLyser II (Qiagen, Netherlands). The homogenate underwent RNA extraction with TRIzol (Invitrogen, Cat#15596026, US) and RNA purification with the Aurum Total RNA mini Kit (Bio-Rad, Cat#7326820, US). RNA quality was verified using the NanoPhotometer NP80 Touch (Implen, Germany).
We did nCounter (NanoString Technologies, US) digital gene expression analysis with the Immunology V2 panel targeting 579 immune system-associated genes. The workflow was carried out as established before in critical COVID-191, fatal COVID-19 nursing home outbreaks2, and respiratory infections at the emergency department3. In short, transcripts -including 15 housekeeping genes - were quantified by hybridization with specific fluorescent barcodes linked to a 50 bp reporter probe and an adjacent 50 bp capture probe.
Data were normalized for background (negative control probes), internal positive control probes and housekeeping genes using nSolver software version 4.0 (NanoString Technologies, US). Differentially expressed genes and predefined biological pathway scores were determined using nSolver, with correction for multiple testing by the Benjamini-Hochberg method at a 5%  false discovery rate for multiple comparisons cut-off. Comparison of baseline RNA levels for lung side, mechanical ventilation time, donation type and cold ischemia time was done according to the negative binomial distribution with generalized linear models.
References
1.         Menezes SM, Braz M, Llorens-Rico V, Wauters J, Van Weyenbergh J. Endogenous IFNβ expression predicts outcome in critical patients with COVID-19. Lancet Microbe. Jun 2021;2(6):e235-e236. doi:10.1016/s2666-5247(21)00063-x
2.         Cuypers L, Keyaerts E, Hong SL, et al. Immunovirological and environmental screening reveals actionable risk factors for fatal COVID-19 during post-vaccination nursing home outbreaks. Nat Aging. Jun 2023;3(6):722-733. doi:10.1038/s43587-023-00421-1
3.         Fukutani KF, Nascimento-Carvalho CM, Bouzas ML, et al. In situ Immune Signatures and Microbial Load at the Nasopharyngeal Interface in Children With Acute Respiratory Infection. Front Microbiol. 2018;9:2475. doi:10.3389/fmicb.2018.02475

first: Jan

last: Van Slambrouck

first: Laurens

last: Ceulemans


title: Case study of a neuroendocrine tumor of uncertain origin: single-cell transcriptomics unravels potential primary source

city: Moscow

first: Alesya

last: Maluchenko

address: Kulakova str. 20

country: Russia

institute: Moscow Center for Advanced Studies

summary: In this study, we present a clinical case of a patient with a retroperitoneal non-functional non-organ-confined neuroendocrine tumor, as determined by routine testing. The histogenesis of the tumor was assessed based on bulk and single-cell RNA sequencing (bulk RNA-seq and scRNA-seq) data, as standard diagnostic procedures proved ineffective. The use of publicly accessible bulk and single-cell RNA-seq data from pancreatic and small intestinal NETs allowed for the development and validation of gene signatures tailored to each specific localization of origin. Additionally, it enabled the prediction of the primary origin of the previously described NET,  indicating that ectopic pancreatic tissue is the most probable source.

overall_design: Neuroendocrine tumor sample from one patient was analyzed using single-cell RNA-seq

first: Frolova

last: Tatyana

first: Ekaterina

last: Avsievich

first: Diana

last: Salimgereeva

first: Zoia

last: Antysheva

first: Alesia

first: Denis

last: Maksimov

first: Ilia

last: Feidorov

first: Mark

last: Voloshin

first: Olga

last: Glazova

first: Natalia

last: Bodunova

first: Nikolay

last: Karnaukhov

first: Pavel

last: Volchkov

first: Julia

last: Krupinova


title: Myristoylated Eepd1 Enhances Lipolysis and Thermogenesis through PKA Activation to Combat Obesity

city: Shanghai

first: Suzhen

last: Chen

state: -- None --

address: 600 Yishan Road, Shanghai, China

institute: Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine

summary: Middle-aged obesity, characterized by excessive fat accumulation and systemic energy imbalance, often precedes various health complications. Recent research has unveiled a surprising link between DNA damage response and energy metabolism. Here, we explore the role of Eepd1, a DNA repair enzyme, in regulating adipose tissue function and obesity onset. Eepd1 is primarily expressed in adipose tissue, where its downregulation or deletion accelerates obesity development. We show that Eepd1 ablation in vivo hinders PKA activation, thereby inhibiting lipolysis and thermogenesis in adipose tissue. leading to obesity progression and dysregulation of glucose metabolism. This study reveals Eepd1’s unexpected role in promoting adipose lipolysis and thermogenesis, underscoring its potential as a promising therapeutic target to combat obesity.

overall_design: RNA-seq profiling of white adipose tissue from WT and EKO mice after high-fat diet for 3 months.

first: Yanping

last: Wang

first: Qian

last: Zhou

first: Qiqi

last: Qian

first: Quanxin

last: Jiang

first: Chuchu

last: Liu

first: Junli


title: Toll-like receptor adaptor protein TIRAP has specialized roles in signaling, metabolic control and leukocyte migration upon wounding in zebrafish larvae

city: LEIDEN

first: Li

address: Verbeekstraat 29D

institute: Leiden University

summary: The Toll-interleukin-1 Receptor (TIR) domain-containing adaptor protein (TIRAP), also known as MyD88-associated ligand (MAL), is an essential adaptor protein in Toll-like receptor 2 (TLR2) signaling which links TLR2 and TLR4 to the other adaptor protein myeloid differentiation protein 88 (MyD88). Here, we investigate the transcriptomic and metabolomic profiles of zebrafish larvae from a tirap mutant and a myd88 mutant and the corresponding wild type controls under unchallenged normal developmental conditions. Comparing data from tirap and myd88 mutants with published data on tlr2 mutant, we found that Tirap has specialized roles in signaling and metabolic control. Transcriptome analysis revealed the specific involvement of tirap in calcium homeostasis and myosin regulation. Metabolomic profiling showed that the tirap mutation results in lower glucose levels, whereas a tlr2 mutation leads to higher glucose levels.  Previously, tlr2 and myd88 have been indicated to be involved in responses to wounding by regulating leukocyte migration in zebrafish, but the role of tirap in regulating leukocyte migration has still not been investigated. In this study, a tail-wounding zebrafish larval model was used to identify the specific roles of tirap in leukocyte migration to tissue wounding. We found that more neutrophils were recruited to the wounded region in the tirap mutant larvae compared to the wild type controls, whereas there was no difference in macrophage recruitment. In contrast, published data show that tlr2 and myd88 mutants recruit fewer neutrophils and macrophages to the wounds. We visualized neutrophil migration trajectories in tail-wounding tirap mutant and wild type zebrafish larvae. Based on cell tracking analysis, we demonstrated that the neutrophil migration speed is increased in the tirap mutant. This is in contrast to our published results for neutrophil behavior in myd88 and tlr2 mutants. In conclusion, our findings demonstrate that tirap plays specialized roles distinct from tlr2 and myd88 in signaling, metabolic control, and in regulating neutrophil migration speed upon wounding.

overall_design: For the sequencing of tirap mutant zebrafish larvae, zebrafish larvae at 5 dpf from line tirap mutant and wild-type control were collected for RNAseq. For the sequencing of myd88 mutant zebrafish larvae, zebrafish larvae at 5 dpf from line myd88 mutant and wild-type control were collected for RNAseq.

first: Herman

middle: P

last: Spaink

first: Wanbin

last: Hu


title: CD133+PD-L1+ cancer cells confer resistance to adoptively transferred engineered macrophage-based therapy in melanoma.

city: 上海

first: Xu

last: Jiaojiao

state: 上海Shanghai

address: Zhangheng Road

institute: Fudan university

summary: Adoptive transfer of genetically or nanoparticle-engineered macrophages represents a promising cell therapy modality for treatment of solid tumor. However, the therapeutic efficacy is suboptimal without achieving a complete tumor regression, and the underlying mechanism remains elusive. Here, we discover a subpopulation of cancer cells with upregulated CD133 and programmed death-ligand 1 in mouse melanoma, resistant to the phagocytosis by the transferred macrophages. Compared to the CD133-PD-L1- cancer cells, the CD133+PD-L1+ cancer cells express higher transforming growth factor-β signaling molecules to foster a resistant tumor niche, that restricts the trafficking of the transferred macrophages by stiffened extracellular matrix, and inhibits their cell-killing capability by immunosuppressive factors. The CD133+PD-L1+ cancer cells exhibit tumorigenic potential. The CD133+PD-L1+ cells are further identified in the clinically metastatic melanoma. Hyperthermia reverses the resistance of CD133+PD-L1+ cancer cells through upregulating the ‘eat me’ signal calreticulin, significantly improving the efficacy of adoptive macrophage therapy. Our findings demonstrate the mechanism of resistance to adoptive macrophage therapy, and provide a de novo strategy to counteract the resistance.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: The bulk RNA-seq samples in this study included the in vitro cultured NPs-hMΦ and Un-hMΦ; B16F10 tumor tissues following different treatment; the sorted CD133+ and CD133- B16F10 cancer cells; and the sorted GFP+CD133+PD-L1+ and GFP+CD133-PD-L1- cancer cells from the relapsed B16F10 tumors.

first: Jiaojiao

last: Xu

first: Zhe

last: Li

first: Qinli

last: Tong

first: Sihang

last: Zhang

first: Jianchen

last: Fang

first: Aihua

last: Wu

first: Guoguang

last: Wei

first: Chen

first: Sheng

last: Yu

first: Binbin

last: Zheng

first: Hongzheng

last: Lin

first: Xueling

last: Liao

first: Zeyu

last: Xiao

first: Wei

last: Lu


title: Transcriptome and physiological analysis of class III peroxidase family in ice plant seedlings in response to salt-induced oxidative stress

city: Taichung

first: Li-Ching

last: Hsieh

address: 145 Xingda Rd.

department: Graduate Institute of Genomics and Bioinformatics

country: Taiwan

institute: National Chung Hsing University

summary: Ice plant (Mesembryanthemum crystallinum L.) is a halophyte and an inducible CAM plant. Ice plant seedlings exhibit moderate salt tolerance, with root growth unaffected by 200 mM NaCl treatments, while hypocotyl elongation is hindered in salt-stressed etiolated seedlings. Superoxide anion accumulation was prominent in cotyledons and primary leaves but decreased in root tissues over time but was not significantly affected by salt treatment. Hydrogen peroxide (H2O2) levels surged initially in both control and salt-treated seedlings, with higher and more persistent H2O2 levels in salt-treated seedlings, indicating salt-induced ROS accumulation, especially in etiolated seedlings. An RNA-seq analysis of etiolated seedlings revealed 6,326 unigenes (about 8%) showing more than a four-fold change in expression after a 6-h 200 mM NaCl treatment. The top GO terms for 4-fold upregulated DEGs in the Molecular Function category included “cation binding,” “metal ion binding,” “oxidoreductase activity,” “monooxygenase activity,” and “antioxidant activity.” The top GO terms for 4-fold down-regulated DEGs in the Biological Process category included “metabolic process”, “cellular metabolic process”, and “biosynthetic process”. Upregulated genes were primarily linked to ion transport and stress responses and downregulated genes to growth processes like ribosomal protein synthesis and cell wall formation. This indicates that salt stress hinders growth but enhances ion homeostasis and stress response mechanisms. For class III peroxidase family genes, 14 out of 53 identified transcripts met the criteria for differentially expressed genes. Quantitative RT-PCR confirmed that the expression of McPrx4.1, McPrx12.1, and McPrx12.3 increased, while the expression of McPrx60.3 decreased. We suggest distinct roles for individual class III peroxidase members in the trade-offs between plant growth and stress response. Unveiling these responses will advance our understanding of the growth–stress balance in the intrinsic salt tolerance in halophytes.

overall_design: In this study, ice plant seedlings were used to evaluate their initial responses to salt-induced oxidative stress. The accumulation of reactive oxygen species (ROS) and the activities of enzymes involved in ROS detoxification were measured. Transcriptome profiling and DEG analysis revealed the complex molecular mechanisms that ice plant employs to cope with high salinity. This approach provides a functional perspective on how the seedlings cope with oxidative stress under conditions of high salinity.

first: Hungchen

last: Yen

first: Chia-Che

last: Lee

first: Kai-Fu

first: Hui‐Hsien

last: Chang

first: Cheng-Hsun

first: Hsuan-Jung

last: Huang


title: Two-hit mouse model of heart failure with preserved ejection fraction combining diet-induced obesity and renin-mediated hypertension

city: Philadelphia

first: Daniel

last: Kelly

state: PA

address: 3400 Civic Center Blvd

department: Penn Cardiovascular Institute

institute: University of Pennsylvania

summary: Heart failure with preserved ejection fraction (HFpEF) is increasingly common but its pathogenesis is poorly understood. The ability to assess genetic and pharmacologic interventions is hampered by the lack of robust pre-clinical mouse models of human HFpEF. We have developed a novel “2-hit” mouse model, which combines obesity and insulin resistance with chronic pressure overload to recapitulate clinical features of HFpEF. C57Bl6/NJ mice fed a high fat diet for at least 10 weeks were administered an AAV8-driven vector resulting in constitutive overexpression of mouse Renin1d. Control mice, HFD only, Renin only and HFD-Renin (aka “HFpEF”) littermates underwent a battery of cardiac and extracardiac phenotyping. HFD-Renin mice demonstrated obesity and insulin resistance, a 2-3-fold increase in circulating renin levels and resulted in 30-40% increase in LVH hypertrophy, preserved systolic function, and diastolic dysfunction indicated by altered E/e’, IVRT, and strain measurements; increased left atrial mass; elevated natriuretic peptides; and exercise intolerance. The transcriptomic and metabolomic signature of HFpEF indicates upregulation of fibrotic pathways and downregulation of metabolic pathways, in particular branched chain amino acid catabolism, similar to human HFpEF data. Treatment of these mice with the sodium-glucose cotransporter 2 inhibitor empagliflozin, a powerful but incompletely understood HFpEF therapy, showed improvement in exercise tolerance, left heart enlargement, and insulin homeostasis. The HFD-Renin mouse model recapitulates many critical features of human HFpEF and allows for dissection of how individual pathogenic drivers contribute to cardiac and peripheral metabolic changes. Multiple HFpEF models will also allow for orthogonal studies and increase validity in pre-clinical research efforts.

overall_design: To determine the effect of HFpEF on transcriptional changes in heart tissue, control mice were compared to HFpEF (e.g. HFD + Renin) or constituent (e.g. HFD alone or Renin alone) cohorts. Male C57BL/6NJ mice (Jackson Laboratory, Stock 005304) age 8-10 weeks old, were randomized to chow or 60%kcal HFD (D12492i, Research Diets) for 10 weeks. Mice were again randomized to receive AAV-Renin or null vector (control). Samples were collected at 6 weeks post-retroorbital injection. Mice (n=4-6) were euthanized with pentobarbital, and heart tissue was freeze clamped, prior to processing.

first: Justin

last: Berger

first: Yuji

last: Shi

first: Timothy

last: Matsuura

first: Kirill

last: Batmanov

first: Xian

last: Chen

first: Kelly

last: Tam

first: Mackenzie

last: Marshall

first: Richard

last: Kue

first: Jiten

last: Patel

first: Renee

last: Taing

first: Russell

last: Callaway

first: Joanna

last: Griffin

first: Atilla

last: Kovacs

first: Dinesh

last: Shanthappa

last: Miller

first: Bei

first: Rachel

last: Roth Flach


title: A unique genetic signature for cancer-associated fibroblasts in Melanoma metastases

city: Heidelberg

first: Christoph

last: Plass

address: Im Neueneimer Feld 280

department: Division of Cancer Epigenomics

institute: DKFZ (German Cancer Research Center)

summary: Cancer-associated fibroblasts (CAFs) represent a central population of the tumor microenvironment (TME). Recently, single-cell RNA sequencing (scRNA-seq) analyes of primary tumors of different cancer entities yielded different classifications of CAF subsets underscoring heterogeneity of CAFs within the TME. Here, we analyzed the transcriptional signatures of approximately 8,400 CAFs and normal fibroblasts by scRNA-seq and compared genetic profiles of CAFs from murine melanoma primary tumors to CAFs from corresponding melanoma lung metastases. This revealed distinct subsets for primary tumor and metastasis specific CAF populations, respectively. Combined with spatial characterization of metastasis CAFs at the RNA and protein level, scRNA-analyses indicate a tumor-dependent crosstalk between neutrophils and CAFs, mediated via SAA3 and IL1b related signaling pathways, which can be recapitulated in vitro. Importantly, analyzing tissue sections of human patient samples this link was found to be present in human melanoma metastasis. Taken together, our data highlights unique characteristics of metastasis CAFs with potential therapeutic impact for melanoma metastasis.

overall_design: Melanoma cells and cells from the tumor microenvironment were isolated from tumor and metastasis bearing mT/mG mice and FACS sorted based on tdTomato expression and analyzed by scRNA sequencing.

first: Saskia

last: Tauch

first: Joschja

last: Hey

first: Etienne

last: Sollier

first: Bettina

last: Kast


title: SLC29A1 and SLC29A2 are human nicotinamide cell membrane transporters

city: Hangzhou

first: Mingyang

last: Chen

address: Yuhangtang 866

institute: Zhejiang University

summary: Nicotinamide, a main precursor of NAD+, is essential for cellular fuel respiration, energy production, and other cellular processes. Transporters for other precursors of  NAD+ such as nicotinic acid and nicotinamide mononucleotide have been identified, but the cellular transporter of nicotinamide has not been elucidated. Here, we demonstrated that equilibrative nucleoside transporters 1 and 2 (ENT1 and 2, encoded by SLC29A1/2) drove cellular nicotinamide uptake and set nicotinamide metabolism homeostasis. In addition, ENT1/2 exhibited a strong capacity to change the composition of cellular metabolites and the profile of transcripts, especially those related to nicotinamide. Then, we further found that ENT1/2 could regulate cellular respiration and senescence, which are universally acknowledged as nicotinamide biological functions, to which the NAD+ pool level and mitochondrial status influenced by ENT1/2 made important contributions. Together, ENT1 and ENT2 act as both cellular nicotinamide-level keepers and nicotinamide biological regulators through their NAM transport functions.

overall_design: To investigate the effects of SLC29A1 and SLC29A2 to cellualr  behavior and fate, the SLC29A1 and SLC29A2 was knocked down, respectively, in AC16 cell line and MSC  with specific siRNA. We then performed expression profiling analysis using data obtained from RNA-seq of above cell groups. Comparative gene expression profiling analysis of RNA-seq data for negtive control and KD group(siNC, siSLC29A1 and siSLC29A2) in AC16 cell and MSC.

first: Huidi

last: Jiang


title: Corticosteroid resistance is predetermined by early immune response dynamics at acute Graft-versus-Host disease onset

city: Paris

first: David

last: Michonneau

address: 1 avenue Claude Vellefaux

department: Hematology and transplantation

institute: Assistance Publique des Hôpitaux de Paris

summary: Transcriptomic analysis from patient's samples  after hematopoietic stem cell transplantation with or without acute GVHD

overall_design: PBMC samples of patients treated with haematopoietic stem cell transplant, presenting with or without aGVHD

first: Sophie

last: Le Grand


title: A PARP14/TARG1-Regulated RACK1 MARylation Cycle Drives Stress Granule Dynamics in Cancer Cells

first: W. Lee

last: Kraus

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

overall_design: Refer to individual Series


title: Attenuated IL-2 Signaling Rewires Transcriptional and Metabolic Programs in T Cells to Enable Coupling Proliferation and Stemness

city: Lausanne

first: Isaac

last: Crespo

state: Vaud

address: Bugnon 25A

department: Oncology

country: Switzerland

institute: Ludwig Cancer Research,UNIL

summary: Interleukin-2 (IL-2) is a fundamental cytokine that controls the proliferation and differentiation of T cells1. Its direct administration in vivo has conferred durable, complete responses in patients with metastatic melanoma and renal cell cancer, while it is also used to expand tumor-infiltrating lymphocytes (TILs) in vitro for adoptive cell therapy (ACT)2.  Unfavorable properties of IL-2 including terminal differentiation of effector T cells3-6, induction of activation-induced cell death (AICD)2, 7, 8, expansion of regulatory T (Treg) cells, and toxicity at high doses, have driven the development of IL-2 variants with modified biological properties9-13. However, how cytokine modifications alter cell fate decisions is not well understood. Here, taking a systems biology approach we have comprehensively elucidated how a non CD25-binding, IL-2-biased agonist (IL-2v)14 reprograms signaling, transcriptional, and metabolic networks of CD8+ T cells in vitro to favor stemness while simultaneously driving expansion and functionality. Such "expansion-stemness state" defines metabolically fit CD8+ T cells capable of superior persistence and tumor control. These findings broaden our fundamental understanding of the effects of IL-2v on T-cell biology and provide an investigational framework for the rational development and evaluation of cytokine variants with clinical relevance.

overall_design: OT1 T cells cultured for 10 days with 5 and 50 IU/mL of IL-2v, IL-2 and IL‑15

first: Yaquelin

last: Ortiz

first: George

last: Coukos

first: Melita

last: Irving