title: Vegfc lineage tracing identifies progenitor cells that change cell fate after musculoskeletal injury

city: Dallas

first: Sneha

last: Korlakunta

state: TX

address: 6000 Harry Hines Blvd

institute: UT Southwestern Medical Center

summary: Aberrant repair after musculoskeletal injury is one of the most costly and common clinical scenarios in health care. While advances in our understanding of the key cells and pathways during the repair process have improved, there remains a gap in knowledge of the key progenitor cells and their interaction with the surrounding microenvironment. Heterotopic ossification (HO) is a pathological process caused by aberrant cell-fate determination resulting in the formation of bone in non-skeletal tissues after musculoskeletal injury. While the involvement of blood vessels in musculoskeletal repair is well-documented, the role of lymphatic vessels and the cells responsible for their ingrowth in musculoskeletal tissues during both homeostasis and after injury remains poorly understood. In this study, we employed a mouse model of traumatic HO to investigate lymphangiogenesis during musculoskeletal injury and repair and to uncover the key progenitor cells responsible to stimulate lymphangiogenesis and undergo aberrant cell fate differentiation. Our findings demonstrated that musculoskeletal injury triggered lymphangiogenesis at the injury site, including invasion of lymphatic vessels into the tendon proper. This increased lymphangiogenesis was driven by elevated levels of active Vascular Endothelial Growth Factor C (VEGF-C) one-week post-injury. Through single-cell transcriptomic analyses, we identified mesenchymal progenitor cells (MPCs) as a source of Vegfc following injury as well as the source of enzymes responsible for activating VEGF-C. These Vegfc-expressing MPCs underwent osteochondral differentiation and actively contributed to the formation of HO following injury.  Notably, Vegfc haploinsufficiency resulted in a near 50% reduction in lymphangiogenesis and HO formation. Collectively, these findings suggest that MPC expression of VEGF-C and its activating enzymes in response to soft tissue trauma play a critical role in stimulating pathologic lymphatic vessel growth. Furthermore, Vegfc expressing MPCs undergo aberrant osteochondral differentiation responsible for HO. Targeting Vegfc expression and Vegfc-expressing progenitors therapeutically could potentially mitigate pathologic lymphangiogenesis, preventing aberrant tissue repair and subsequent HO formation.

overall_design: The uninjured Achilles tendon served as the control group compared with the 7 and 21 days B/T Achilles tendon as our experimental group. Cells from five Achilles tendons in each group were pooled together for single-cell analysis

first: Neda

last: Vishlaghi

first: Michael

last: Dellinger

last: Levi


title: A Conserved Transcription Factor Regulatory Program Promotes Tendon Fate

status: Public on Jun 13 2024

city: Boston

first: April

last: Craft

address: 300 Longwood Ave

country: USA

institute: Boston Children's Hospital

type: Expression profiling by high throughput sequencing

summary: Tendons, which transmit force from muscles to bones, are highly prone to injury. Understanding the mechanisms driving tendon fate would impact efforts to improve tendon healing, yet this knowledge is limited. To find direct regulators of tendon progenitor emergence, we performed a zebrafish high-throughput chemical screen. We established Forskolin as a tenogenic inducer across vertebrates, functioning through Creb1a, which is required and sufficient for tendon fate. Putative enhancers containing cAMP response elements (CRE) in human, mouse and fish, drove specific expression in zebrafish cranial and fin tendons. Analysis of these genomic regions identified motifs for Ebf/EBF transcription factors. Mutation of CRE or Ebf/EBF motifs significantly disrupted enhancer activity and specificity in tendons. Zebrafish ebf1a/ebf3a mutants displayed defects in tendon formation. Notably, Creb1a/CREB1 and Ebf1a/Ebf3a/EBF1 overexpression facilitated tenogenic induction in zebrafish and human pluripotent stem cells. Together, our work reveals functional conservation of two novel transcription factors in promoting tendon fate.

overall_design: Col2a1-egfp or Prg4-tdtomato cells were isolated from the 3xR mouse ESC line and subjected to bulk RNA sequencing

first: Xubo

last: Niu

first: Delmy

last: Melendez

first: Andrew

last: Zhu

first: Lick

last: Lai

last: McMahon

first: Jenna

last: Galloway


title: Notch induces transcription by stimulating release of paused RNA Pol II without increasing chromatin accessibility [ATAC-seq]

first: Julia

middle: M

last: Rogers

state: Massachusetts

address: 45 Shattuck Street

department: BCMP

institute: Harvard Medical School

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Notch proteins undergo ligand-induced proteolysis to release a nuclear effector that influences a wide range of cell fate decisions by regulating gene activity. However, despite years of study, how Notch induces the transcription of its target genes remains unclear. Here, we comprehensively examined the response to human Notch1 across a time course of activation, using genomic assays of nascent RNA and chromatin accessibility. These data revealed that Notch induces target gene transcription primarily by releasing paused RNA polymerase II (RNAPII), in contrast to prevailing models suggesting that Notch acts by promoting chromatin accessibility. Indeed, we found that open chromatin is established at Notch-responsive regulatory elements prior to Notch signaling, through SWI/SNF-mediated remodeling. Notch activation, however, elicited no further chromatin opening at these loci. Together, these studies reveal that the nuclear response to Notch signaling is dictated by the pre-existing chromatin state and RNAPII distribution at the time of signal activation.

overall_design: To determine how Notch signaling affects chromatin accessibility, and how SWI/SNF inhibition affects chromatin accessibility in the Notch response, Notch signaling was induced in SC2 cells by GSI washout. Cells were harvested at 1, 2, and 4 hours post Notch activation, and immediately after mock GSI washout, and harvested for ATAC-seq sample preparation. Mock washout control samples were also harvested at 1, 2, and 4 hours. For each of these time points, cells were either treated with SWI/SNF inhibitor (1 µM BRM014) or 0.01% DMSO at the time of washout or mock-washout. There are 14 total conditions, and two replicates of the full experiment.

first: Claudia

middle: A

last: Mimoso

first: Benjamin

middle: J

last: Martin

first: Karen

last: Adelman

first: Stephen

middle: C

last: Blacklow


title: Single cell gene expression analysis of hematopoietic progenitors in Ddx41-heterozygous mice

city: Cincinnati

first: Timothy

last: Chlon

state: Ohio

address: 3333 BURNET AVENUE

department: Pediatrics

institute: Cincinnaty Childrens Hospital Medical Center

summary: To determine the effect of heterozygosity of Ddx41 on hematopoiesis, we conducted single cell transcriptome analysis on hematopoietic progenitor cells from Ddx41-heterozygous and control bone marrow. We found that heterozygoisty of Ddx41 did not signficantly alter the distribution of progenitor cell types, and changes in gene expression within the clusters were minimal.

overall_design: Bone marrow mononuclear cells from Ddx41 flox/+ or control mice were transplanted into lethally-irradiated recipient mice, and then the recipients were treated with tamoxifen to excise the floxed alleles. The bone marrow of these mice was harvested 8-weeks post-transplant and the Lineage-negative, cKit-psotive progenitor cells were sorted. After cell hashing to enable multiplexing, these cells were processed for 10X Chromium analysis.

first: Emily

last: Stepanchick

last: Wilson

first: Mario

last: Pujato


title: An in situ method for identification of transcriptome-wide protein-RNA interactions in cells [isSTAMP]

city: La Jolla

first: Brian

last: Yee

state: CA

address: 9500 Gilman Dr

department: Health

institute: UCSD

summary: RNA-binding proteins (RBPs) play important roles in RNA metabolism including splicing, stability, localization, and translation. RBP-RNA interaction profiles are indicative of many diseases. Existing methods for mapping RBP-RNA interactions transcriptome-wide have notable limitations: immunoprecipitation (IP)-based technologies require large quantities of input materials, and RNA shearing during processing prevents identification of RNA isoforms. Meanwhile, profiling methods using RNA-modifying enzymes require ectopic expression of fusion proteins in cells of interest, potentially distorting interaction profiles. Here we report in situ STAMP, an RBP-RNA profiling method that overcomes the limitations of existing methods. In situ STAMP utilizes a chimeric fusion of the cytosine deaminase APOBEC1 and an IgG-targeting single-domain antibody (nanobody). We demonstrate that this fusion protein can be specifically targeted to proteins of interest including the RBPs RBFOX2 and TDP-43 when combined with primary antibodies targeting these proteins, enabling identification of their binding sites in un-engineered HEK293T cells. The canonical binding motifs of both RBFOX2 (UGCAUG) and TDP43 (UGUGUG) could be identified by de novo motif analysis from in situ STAMP data, demonstrating the method’s high specificity. In situ STAMP preserves intact RNAs and is therefore compatible with direct cDNA PacBio long-read sequencing, enabling the method to distinguish between RNA isoforms. Importantly, in situ STAMP is compatible with multiple fixation methods including methanol and formaldehyde fixation, enabling its application to tissue samples collected in research or clinical settings. Thus, in situ STAMP enables the profiling of authentic RBP-RNA interactions using small quantities of primary cells or tissues, thereby bridging a critical gap in uncovering the roles of RBPs in RNA-related disease mechanisms in authentic biological contexts.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: INSCRIBE (C-to-U RNA editing) in fixed cells

first: Qishan

last: Liang

first: Tao

last: Yu

first: Eric

last: Kofman

first: Pratibha

last: Jagannatha

first: Kevin

last: Rhine

middle: D

last: Corbett

first: Gene

middle: W

last: Yeo


title: DDX41 heterozygosity impairs hematopoietic progenitor cell fitness and cooperates with p53 mutation to drive malignancy [MOLM13]

first: Kwangmin

last: Choi

address: 333 Burnet Avenue

department: Experimental Hematology & Cancer Biology

institute: Cincinnati Children's Hospital Medical Center

summary: Germline heterozygous mutations in DDX41 predispose individuals to hematologic malignancies in adulthood. Most of these DDX41 mutations result in a truncated protein, leading to loss of protein function. To investigate the impact of these mutations on hematopoiesis, we generated mice with hematopoietic-specific knockout of one Ddx41 allele. Under normal steady-state conditions, there was minimal effect on lifelong hematopoiesis, resulting in a mild yet persistent reduction in red blood cell counts. However, stress induced by transplantation of the Ddx41+/- BM resulted in hematopoietic stem/progenitor cell (HSPC) defects and onset of hematopoietic failure upon aging. Transcriptomic analysis of HSPC subsets from the transplanted BM revealed activation of cellular stress responses, including upregulation of p53 target genes in erythroid progenitors. To understand how the loss of p53 affects the phenotype of Ddx41+/- HSPCs, we generated mice with combined Ddx41 and Trp53 heterozygous deletions. The reduction in p53 expression rescued the fitness defects in HSPC caused by Ddx41 heterozygosity. However, the combined Ddx41 and Trp53 mutant mice were prone to developing hematologic malignancies that resemble human myelodysplastic syndrome and acute myeloid leukemia. In conclusion, DDX41 heterozygosity causes dysregulation of the response to hematopoietic stress, which increases the risk of transformation with a p53 mutation.

overall_design:
We transduced MOLM13 cells with pLKO-GFP viruses expressing control or DDX41-targeting shRNAs
Cells were sorted for GFP by flow cytometry 4-days post-transduction
RNA was isolated from 1M GFP+ cells and prepared for RNA-Seq

last: Choin


title: DDX41 heterozygosity impairs hematopoietic progenitor cell fitness and cooperates with p53 mutation to drive malignancy

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

overall_design: Refer to individual Series


title: DDX41 heterozygosity impairs hematopoietic progenitor cell fitness and cooperates with p53 mutation to drive malignancy [LSK]

overall_design:
We transplanted lethally-irradiated BoyJ mice with bone marrow cells from Ddx41+/flox or Ddx41+/+ RosaCreERT2 mice and immediately treated the mice with tamoxifen to excise the floxed alleles.
At 8 weeks post-transplant, we harvested the bone marrow from these mice and then sorted Lin-Sca1+cKit+ cells by flow cytometry.
We harvested RNA from approximately 20K LSK cells from each mouse, and then isolated the RNA. We conducted gene expression analysis by RNA-seq on these samples.


title: DDX41 heterozygosity impairs hematopoietic progenitor cell fitness and cooperates with p53 mutation to drive malignancy [CD71]

overall_design:
We transplanted lethally-irradiated BoyJ mice with bone marrow cells from Ddx41+/flox;Trp53+/- or Trp53+/+ or Ddx41+/+; Trp53+/- or Trp53+/+; RosaCreERT2+ mice and immediately treated the mice with tamoxifen to excise the floxed alleles.
At 8 weeks post-transplant, we harvested the bone marrow from these mice and then sorted CD71+Ter119+ R2 erythroid precursor cells by flow cytometry.
We isolated RNA from approximately 100K R2 cells from each mouse, and then isolated the RNA. We conducted gene expression analysis by RNA-seq on these samples.


title: An in situ method for identification of transcriptome-wide protein-RNA interactions in cells [eCLIP-seq ]

overall_design: eCLIP-seq with RBFOX2 specific antibody in mouse whole brain samples

first: Yan

last: Song


title: MYCN is an RNA-binding accessory protein of the nuclear exosome

city: Wuerzburg

first: Martin

last: Eilers

address: Am Hubland

department: Chair for Biochemistry and Molecular Biology

country: Germany

institute: University of Wuerzburg

summary:
The MYCN oncoprotein broadly binds promoters in a heterodimer with its partner protein MAX. MYCN also interacts with the nuclear exosome, a 3’-5’exoribonuclease complex, to facilitate progression through the S phase hinting at an RNA-centric function. Here we show that MYCN forms stable high molecular weight complexes with multiple RNA-binding proteins including the nuclear exosome. RNA-binding assays reveal that MYCN directly binds to thousands of predominantly intronic RNA sites via a short, highly conserved sequence motif termed MYCBoxI.
Exosome depletion results in MYCN globally re-localizing from promoters to intronic RNAs. The vacant promoters are then occupied by the MXD transcription repressor protein MNT, which inhibits MYCN-dependent transcription. MYCN fosters the degradation of its bound introns via tight association with the nuclear exosome targeting complex (NEXT). Our data demonstrate that MYCN is an RNA-binding protein that controls RNA turnover and argue that the competition between MYCN’s RNA- and DNA-bound states links the dynamics of the MYCN/MAX/MXD network to mRNA processing.

overall_design: CUT&RUN, ChIP and ChIP-Rx DNA-sequencing was done with SH-EP NMYC-ER cells where the NMYC expression can be induced with 4-OHT. For all CUT&Run sequencing and some ChIP-seq and ChIP-Rx sequencing experiments, SH-EP NMYC-ER cells were used that carry a doxycycline inducible shRNA against TF3C5. For CUT&Run sequencing samples, the immunoprecipitation of digested DNA was done for EXOSC5 with or without NMYC expression and with or without expression of shTF3C5. As control, DNA was sequenced following immunoprecipitation with an unspecific IgG antibody. For ChIP-seq and ChIP-Rx sequencing experiments, the immunopresipitated antibodies are listed below. All experiments were done with or without NMYC expression and where indicated, a shRNA targeting TF3C5 was induced via doxycycline or one of the below listed inhibitors was applied.

first: Dimitrios

last: Papadopoulos

first: Stefanie

last: Ha

first: Daniel

last: Fleischhauer

first: Leonie

last: Uhl

first: Katharina

last: Schneider

first: Ivan

last: Mikicic

last: Russell

first: Annika

last: Brem

first: Omkar

middle: R

last: Valanju

first: Christina

last: Schülein-Völk

first: Hans

last: Maric

first: Petra

last: Beli

first: Gabriele

last: Büchel

first: Seychelle

last: Vos


title: Inhibitory effect of curcumin on colon cancer cells

city: Xi'an

first: Weilongli

last: Li

address: No.1 Changle East Road

country: China

institute: Air Force Engineering University

summary:
To investigate the effect of curcumin on the proliferation and migration of colon cancer cells, we used curcumin treated with HCT116 cells
We then performed gene expression profiling using RNA-seq data from HCT116 cells.

overall_design: Comparative gene expression profiling of RNA-seq data from HCT116 cells in the treated and non-treated groups.

first: Dan

last: Wu

first: Ying

last: Liang


title: The Mohawk homeobox gene represents a marker and osteo-inhibitory factor in cranial suture stem cells [Mkx siRNA]

city: Baltimore

first: Aaron

last: James

state: Maryland

address: 720 Rutland Avenue, Ross Bldg., Room 529

department: Pathology

institute: Johns Hopkins University

summary: The regeneration of craniofacial bones of the mammalian skeleton necessitates the action of both intrinsic and extrinsic inductive factors from multiple cell types, which function in a hierarchical and temporal fashion to control the differentiation of osteogenic progenitors. Single-cell transcriptomics of developing mouse cranial suture recently identified a suture mesenchymal progenitor population with tendon- or ligament-associated gene expression profile previously uncharacterized. Here, we developed a Mohawk homeobox (MkxCG;R26RtdT) reporter mouse, finding that this teno-ligamentous gene identifies a cranial suture resident cell population within the adult mouse that gives rise to calvarial osteoblasts and osteocytes overtime during homeostatic conditions.  Single cell RNA-Sequencing (scRNA-Seq) demonstrated that Mkx+ suture cells demonstrate a stem-like phenotype with expression of teno-ligamentous genes.  Bone injury with Mkx+ cell ablation showed delayed bone healing.  Remarkably, Mkx gene played a critical role as an osteo-inhibitory factor in cranial suture cells, as knockdown or knockout resulted in increased osteogenic differentiation.  Furthermore, in vivo local deletion of Mkx in Mkx floxed mice resulted in robustly increased calvarial defect repair.  Finally, we observed that mechanical stretch dynamically regulates Mkx expression in turn regulating calvarial cell osteogenesis. Overall, we identify Mkx+ cells within the suture mesenchyme as a progenitor cell population for adult craniofacial bones required for bone repair and Mkx itself as mechanical stretch responsive gene which functions to prevent osteogenic differentiation within the stem cell niche.

overall_design: There are 6 samples in total with 3 biological replicates each of  Mkx siRNA knockdown compared to scramble controls.

first: Yiyun

last: Wang

first: Qizhi

last: Qin

first: Ziyi


title: An in situ method for identification of transcriptome-wide protein-RNA interactions in cells [in_situ_STAMP]

first: Brian

last: Yee

overall_design: in situ STAMP (C-to-U RNA editing) in fixed cells and mouse brain tissue

first: Qishan

first: Tao

last: Yu

first: Eric

last: Kofman

first: Pratibha

last: Jagannatha

first: Kevin

last: Rhine

last: Corbett

first: Gene

last: Yeo


title: Transcriptomics and proteomics approach for the identification of altered blood microRNAs and plasma proteins in Parkinson’s disease by Custom Brain Specific miRNA OpenArray Real-time PCR panel

city: Raebareli

first: SANJAY

last: YADAV

state: UTTAR PRADESH

address: All India Institute of Medical Sciences Dalmau Road, Munshiganj,

country: India

institute: AIIMS RAEBARLI

summary: The blood samples of PD patients and Healthy controls were collected from the general OPD of Department of Neurology, KGMU, Lucknow. RNA (including small RNAs) was extracted by Ribopure Blood RNA Isolation Kit by Thermo Fisher as suggested protocol by manufacturer. A custom Taqman OpenArray panel was used to profile the miRNAs expression.

overall_design: RNA isolated from the PD patients and Healthy control group blood samples were quantified and performed reverse transcription reaction using custom primer pool for cDNA synthesis. Further preamplification of cDNA samples were also performed using preamplification primer pool. The preamplification products then diluted in TE buffer and mixed with Taqman Open Array universal master mix and run the Open array.

first: Sanjeev

last: Kumar Yadav

first: Abhishek

last: Jauhari

first: Anuj

last: Pandey

first: Nishant

last: Singh

first: Sana

last: Sarkar

first: Shweta

first: Ravindra

last: Kumar Garg

first: Devendra

last: Parmar

first: Sanjay

last: Yadav


title: Targeting the Spindle Assembly Checkpoint Pathway Synergizes with Taxanes to Inhibit Anaplastic Thyroid Cancer

city: Aurora

first: Nikita

last: Pozdeyev

state: CO

address: 1890 N. Revere Court | Mailstop F600

department: Biomedical Informatics

institute: University of Colorado

summary: We used cancer cell line synthetic lethal functional genomics screen to identify pathways to target in combination with taxane therapy.

overall_design: Pooled kinome-wide shRNA-based dropout screen was used to identify kinases and pathways that are synthetic lethal in combination with docetaxel in C643 ATC cells. C643 cells were transduced with MISSION TRC lentiviral pooled kinome shRNA library obtained from the University of Colorado Functional Genomics Shared Resource. This library consisted of 3075 shRNAs targeting 654 kinases as well as two control libraries aimed at essential human genes and control mouse genes. C643 cells were treated for 14 days with 0.35 nM of docetaxel (Selleckchem, Cat #S1148) or vehicle. After treatment, DNA was extracted, hairpin DNA was amplified, barcoded, and sequenced (Illumina HiSeq 2500). Three biological replicates were performed. Raw sequencing data was analyzed  using a bioinformatics pipeline based on the edgeR package in R. Up to six shRNA per gene were used.

first: Bryan

last: Haugen

first: Vibha

last: Sharma


title: A chronic signaling TGFb zebrafish reporter identifies immune response in melanoma [SORT-seq]

first: Leonard

last: Zon

state: MA

address: 1 Blackfan Circle

department: Oncology/Hematology

summary: Developmental signaling pathways associated with growth factors such as TGFb are commonly dysregulated in melanoma. Here we identified a human TGFb enhancer that was specifically activated in melanoma cells treated with TGFB1 ligand. We generated stable transgenic zebrafish with this enhancer driving green fluorescent protein (TIE:EGFP). EGFP was not expressed in normal melanocytes or early melanomas but was expressed in spatially distinct regions of mature melanomas. Single cell RNA-sequencing revealed that TIE:EGFP+ melanoma cells down-regulated interferon response, while up-regulating a novel set of chronic TGFb target genes. AP-1 factor binding is required for activation of this chronic TGFb reporter. Overexpression of the chromatin remodeler, SATB2, which is associated with tumor spreading shows activation of TGFb signaling in melanoma precursor zones and early melanomas. Confocal imaging and flow cytometric analysis showed that macrophages are recruited to EGFP positive regions and preferentially phagocytose TIE:EGFP+ cells. This work identifies a TGFb induced immune response and demonstrates the need for the development of chronic TGFb biomarkers to predict patient response to TGFb inhibitors.

overall_design:
SORT-seq TIE:EGFP+ melanomas
scRNA-seq of zebrafish melanomas expressing TIE:EGFP
2 MCR:MCS tumors expressing TIE:EGFP were processed, 1 MCR:SATB2 tumor expressing TIE:EGFP was processed

first: Haley

last: Noonan

first: Julia

last: Barbano

first: Michael

last: Xifaras

first: Chloe

middle: S

last: Baron

first: Song

last: Yang

first: Katherine

last: Koczirka

first: Alicia

last: McConnell


title: A chronic signaling TGFb zebrafish reporter identifies immune response in melanoma [RNA-seq, ZMEL]

overall_design:
RNA-seq +/- TGFB1
Bulk RNA-seq on ZMEL1 treated with 10ng/mL TGFB1 ligand or vehicle control
ZMEL1 cells were serum starved for 2 hours, then treated with 10ng/mL human recombinant TGFB1 or 4mM HCl containing 1 mg/mL BSA vehicle control for an additional two hours. Experiment performed in technical triplicate.


title: A chronic signaling TGFb zebrafish reporter identifies immune response in melanoma [RNA-seq, A375]

overall_design:
RNA-seq +/- TGFB1
Bulk RNA-seq on A375s treated with TGFB1 ligand or vehicle control
A375 cells were serum starved for 2 hours, then treated with 10ng/mL human recombinant TGFB1 or 4mM HCl containing 1 mg/mL BSA vehicle control for an additional two hours. Experiment performed in technical triplicate.


title: A chronic signaling TGFb zebrafish reporter identifies immune response in melanoma

sample_organism:
Danio rerio
Homo sapiens

platform_organism:
Danio rerio
Homo sapiens


title: A chronic signaling TGFb zebrafish reporter identifies immune response in melanoma [ChIP-seq]

overall_design:
ChIP-seq +/- TGFB1
ChIP-seq on A375s treated with 10ng/mL TGFB1 ligand or vehicle control
A375 cells were serum starved for 2 hours, then treated with 10ng/mL human recombinant TGFB1 or 4mM HCl containing 1 mg/mL BSA vehicle control for an additional two hours. Experiment performed in technical triplicate.


title: A long-term high fat diet induces differential gene expression changes in spatially distinct adipose tissues.

status: Public on Jun 12 2024

city: Newark

first: Shawn

middle: William

last: Polson

state: DE

department: Center for Bioinformatics and Computational Biology

institute: University of Delaware

summary: The accumulation of visceral adipose tissue (VAT) is strongly associated with cardiovascular disease and diabetes. In contrast, individuals with increased subcutaneous adipose tissue (SAT) without corresponding increases in VAT are associated with a metabolic healthy obese phenotype. These observations implicate dysfunctional VAT as a driver of disease processes, warranting investigation into obesity-induced alterations of distinct adipose depots. To determine the effects of obesity on adipose gene expression, mice were fed either a high fat or normal laboratory diet for 12-14 months. Mesenteric VAT and hindlimb SAT were isolated from four lean controls and four obese mice for bulk RNA- sequencing. AT from lean controls served as a reference to obesity-induced changes. The long-term high fat diet induced the expression of 169 and 814 unique genes in SAT and VAT, respectively. SAT from obese mice exhibited a total of 308 differentially expressed genes (164 upregulated, 144 downregulated). VAT from obese mice exhibited 690 differentially expressed genes (262 genes upregulated, 428 downregulated). KEGG pathway and GO analyses revealed that metabolic pathways were upregulated in SAT vs. downregulated in VAT while inflammatory signaling was upregulated in VAT. We next determined common genes that were differentially regulated between SAT and VAT in response to obesity and identified four genes that exhibited this profile: elovl6 and kcnj15 were upregulated in SAT/downregulated in VAT while trdn and hspb7 were downregulated in SAT/ upregulated in VAT. We propose that these genes in particular should be further pursued to determine their roles in SAT vs. VAT with obesity.

overall_design:
Hindlimb subcutaneous adipose tissue and mestenteric visceral adipose tissue were isolated from lean and diet-induced obese WT C57Bl/6 mice. Obesity was induced by feeding a high fat (42% kcall saturated fat, 0.2% cholesterol) diet for 12-14 months. Lean controls were maintained on a normal rodent laboratory diet for the same duration. Diets were started at 10 weeks of age.
We did gene expression profiling using 4 different adipose tissues samples (subcutaneous adipose tissue from lean mice, visceral adipose tissue from lean mice, subcutaneous adipose tissue from obese mice and visceral adipose tissue from obese mice). High fat diet-induced changes in VAT vs. SAT were evaluated via pair-wise analyses relative to corresponding lean mouse gene expression values.

first: Malak

last: Alradi

first: Hassan

last: Askari

first: Mark

last: Shaw

first: Jaysheel

last: Bhavsar

first: Brewster

middle: F

last: Kingham

first: Ibra

last: Fancher


title: Human organoid systems reveal in vitro correlates of fitness for SARS-CoV-2 B.1.1.7

city: Rotterdam

first: Mart

middle: Matthias

last: Lamers

address: Wytemaweg 80

department: Viroscience

country: Netherlands

institute: Erasmus MC

summary: A new phase of the COVID-19 pandemic has started as SARS-CoV-2 variants with increased transmissibility are emerging globally, calling for the development of reliable platforms to rapidly phenotype viruses. Currently, no rapid phenotyping system exists that both accurately models human biology and can be standardized properly. Organoids accurately model viral target cells in vivo, can be established from many tissues, passaged indefinitely, biobanked and shared. We found that the British variant (clade B.1.1.7), compared to an ancestral 614G-containing clade B virus, produced higher levels of infectious virus late in infection and had a higher replicative fitness in human airway, alveolar and intestinal organoid models. Our findings unveil extended shedding as a correlate of fitness for SARS-CoV-2, possibly explaining the rapid emergence of SARS-CoV-2 B.1.1.7. Combined with genomic surveillance systems, virus phenotyping using standardized organoid models may be implemented into public health decision making.

overall_design: AT2 cells were grown in 3D in alveolar medium, mechanically disrupted, infected with Bavpat-1 or B.1.1.7

first: Bart

last: Haagmans


title: Intermittent MEK inhibition with GITR co-stimulation rescues T cell function for increased efficacy with CTLA-4 blockade in solid tumor models

city: NEW YORK

first: LEVI

last: MANGARIN

state: NY

address: 413 E 69TH STREET

department: MEYER CANCER CENTER

institute: WEILL CORNELL MEDICINE

summary: MEK inhibitors (MEKis) have limited success with MAPK/ERK pathway dependent cancers due to various resistance mechanisms tumor cells can employ. CH5126766 (CKI27) is a novel inhibitor that binds to MEK and prevents release of RAF, reducing the relief of negative feedback commonly observed with other MEKis. We observed that CKI27 increased MHC expression on tumor cells and improved T cell mediated killing. Yet, CKI27 also decreased T cell proliferation, activation, and cytolytic activity by inhibiting the MAPK/ERK pathway that is activated downstream of T cell receptor signaling. Therefore, we aimed to balance the positive and negative immunomodulatory effects of MEKis for optimal combination with immunotherapy. Intermittent administration of CKI27 allowed T cells to partially recover and co-stimulation via GITR and OX-40 agonist antibodies completely alleviated inhibition of function. In Kras mutant lung and colon tumor mouse models, intermittent CKI27 and anti-GITR significantly decreased tumor growth and prolonged survival when further combined with CTLA-4 immune checkpoint blockade. Moreover, this triple combination increased CD8+ and CD4+ T cell proliferation, activation, and effector/memory subsets in the tumor draining lymph nodes and tumors and led to intra-tumoral Treg destabilization. These data, collectively, will allow for more informed decisions when optimizing combination regimens by overcoming resistance, reducing toxicity, and generating long-term immune responses.

overall_design: Tumor draining lymph nodes (TDLNs) were isolated from LLC tumor bearing mice treated with different drug regimens. Single cell suspensions of the TDLNs were individually hash-tagged, pooled into treatment groups, labeled with the TotalSeq-C mouse universal cocktail, and fluorescence-activated cell sorting (FACS) was used to sort live CD45+ cells for scRNA-seq.

first: Lauren

last: Dong

first: Levi

last: Mangarin

first: Jedd

last: Wolchok

first: Taha

last: Merghoub


title: Functional Schizophrenia-associated genetic variant near the TSNARE1 and ADGRB1 genes

first: Dimitrios

last: Avramopoulos

address: 733 North Broadway

department: Genetic Medicine

summary: Recent collaborative genome wide association studies (GWAS) have identified >200 independent loci contributing to risk for schizophrenia (SCZ). The genes closest to these loci have diverse functions, supporting the potential involvement of multiple relevant biological processes; yet there is no direct evidence that individual variants are functional or directly linked to specific genes. Nevertheless, overlap with certain epigenetic marks suggest that most GWAS-implicated variants are regulatory. Based on the strength of association with SCZ and the presence of regulatory epigenetic marks, we chose one such variant near TSNARE1 and ADGRB1, rs4129585, to test for functional potential and assay differences that may drive the pathogenicity of the risk allele. We observed that the variant-containing sequence drives reporter expression in relevant neuronal populations in zebrafish. Next, we introduced each allele into human induced pluripotent cells and differentiated 4 isogenic clones homozygous for the risk allele and 5 clones homozygous for the non-risk allele into neural precursor cells. Employing RNA-seq, we found that the two alleles yield significant transcriptional differences in the expression of 109 genes at FDR <0.05 and 259 genes at FDR <0.1. We demonstrate that these genes are highly interconnected in pathways enriched for synaptic proteins, axon guidance, and regulation of synapse assembly. Exploration of genes near rs4129585 suggests that this variant does not regulate TSNARE1 transcripts, as previously thought, but may regulate the neighboring ADGRB1, a regulator of synaptogenesis. Our results suggest that rs4129585 is a functional common variant that functions in specific pathways likely involved in SCZ risk.

overall_design: We introduced a schizophrenia associated allele in human iPSCs and differentiated 4 isogenic homozygous risk clones  and 5 homozygous non-risk allele clones into neural precursor cells. RNA-seq was performed on these clones to identify transcriptomic differences.

first: Avramopopoulos

last: Dimitrios


title: Cytokine defined human B cell subsets

city: Philadelphia

first: Diego

last: Espinoza

state: PA

address: 3400 Civic Center Boulevard

institute: University of Pennsylvania

summary: To explore the potential mechanisms that modulate the differentiation of cytokine defined human B cell subsets

overall_design: RNA-sequencing on cytokine positive human B cells.

first: Rui

last: Li

first: Ayman

last: Rezk

first: Amit

last: Bar-Or


title: Gene expression data from the hippocampus of mice treated with oleacein in an in vivo model of depression induced by lipopolysaccharide (LPS)-mediated inflammation.

city: Tsukuba

first: Farhana

last: Ferdousi

state: Ibaraki

address: Tennodai 1-1-1

country: Japan

institute: University of Tsukuba

summary:
Gene expression profiling reveals potential effects of Oleacein in promoting neurogenesis and mitigating neuroinflammation in an in vivo model of depression induced by lipopolysaccharide (LPS)-mediated inflammation
We evaluated the neuroprotective effects of oleacein (OC), a rare secoiridoid derivative found in extra virgin olive oil. our goal was to explore the BDNF/TrkB neurotrophic activity of OC and subsequently assess its potential for modulating neuroinflammatory response in vivo.

overall_design: According to the manufacturer's guide, the RNA was extracted using Isogen kit (Nip-pon Gene Co. Ltd., Japan). Then, RNA quantity and quality were determined using the NanoDrop 2000 spectrophotometer (ThermoScientific, USA). DNA microarray analysis was conducted on control, oleacein-treated, LPS induced, and oleacein+LPS treated SH-SY5Y cells using the GeneChip WT PLUS Reagent Kit (ThermoFisher Scientific) and GeneChip™ Hybridization, Wash and Stain Kit (ThermoFisher Scientific) following the manufacturer's instructions. In brief, complementary DNA (cDNA) was synthesized from 100 ng of RNA solutions. cRNA was synthesized from in vitro transcription of cDNA and then purified and reverse transcribed. Finally, single-stranded cDNA (ss-cDNA) was synthesized, purified, fragmented, and labeled following the manufacturer's instructions. Cartridge Array Hybridization was performed using the Clariom S array (Human; ThermoFisher Scientific) on the GeneChip™ Fluidics Station (ThermoFisher Scientific). Scanning was performed using GeneChip Scanner (ThermoFisher Scientific). The raw image data obtained after scanning were analyzed using the Transcriptome Analysis Console (TAC) software (ver. 4.0.2, ThermoFisher Scientific). The raw data were normalized following the signal space transformation robust multi-chip analysis (SST-RMA) algorithm. Further, gene-level analysis was performed using the Limma Bioconductor package. For differential expression analysis, a One-Way ANOVA followed by an empirical Bayes correction was performed. The detected above back-ground (DABG) cutoff was set to 0.05. The positive vs negative area under the curve (AUC) value was set at greater than or equal to 0.7. Finally, genes that passed the filter criteria of p value < 0.05 (one-way between-subjects ANOVA) and fold change > 2 (in linear space) were considered as differentially expressed genes (DEGs).


title: Gene expression data from oleacein-treated SH-SY5Y cells

summary:
Gene expression profiling reveals potential effects of Oleacein in promoting neurogenesis and mitigating neuroinflammation in SH-SY5Y cells under normal and lipopolysaccharide (LPS)-induced conditions.
We evaluated the neuroprotective effects of oleacein (OC), a rare secoiridoid derivative found in extra virgin olive oil. our goal was to explore the BDNF/TrkB neurotrophic activity of OC and subsequently assess its potential for modulating neuroinflammatory response in vitro using human neuroblastoma cells (SH-SY5Y cells).


title: miRNAseq of extracellular vesicles from immortalized dental pulp mesenchymal stem cells from three different biopsies

city: Valencia

first: Imelda

last: Ontoria-Oviedo

address: Avda Fernando Abril Martorell 106

department: Regeneration and Heart transplantation Unit

country: Spain

institute: Instituto de Investigación Sanitaria La Fe

type: Non-coding RNA profiling by high throughput sequencing

summary: Extracellular vesicles (EVs) derived from three different biopsies of dental pulp mesenchymal stem cells (MSC-EVs) were isolated and characterized by different omic analysis. The goal was to characterized the EVs cargo to study their implication in an animal model of chronic cardiac inflammation

overall_design: MSC-EVs were isolated form immortalized dental pulp MSC by tangential flow-filtration followed by size exclusion chromatography (TFF-SEC). The MSC-EVs were injected intramyocardially in a model of acute myocardial infarction (AMI) in nude rats, and their therapeutic effect was assessed 7 and 21 days post-infarction

first: Elena

last: Amaro-Prellezo

first: Marta

last: Gómez

first: Pilar

last: Sepúlveda


title: The penetrant chordoid glioma PRKCA mutation is an oncogenic gain-of-function kinase inactivation eliciting early onset chondrosarcoma in mice

city: DERIO

first: VERONIQUE

middle: FRANÇOISE

last: CALLEJA

state: Vizcaya

address: ASTONDO BIDEA 612

institute: Hawk Biosystems

summary: A highly penetrant heterozygous mutation in PRKCA was found recently in chordoid glioma. To establish whether this mutant of PKCapha was a driver of tumour formation or maintenance in vivo, knock-in mice were generated. The constitutive, heterozygous expression of the Prkca D463H mutant in mice provoked the development of bilateral hindlimb chondrosarcomas and early neonatal mortality. The lack of activity of the D463H mutant was not the trigger of the phenotype as heterozygous knock-in of the related inactivating mutation D463N produced offspring indistinguishable from parental strains. Moreover, this surprising distinction between D463H and D463N mutations did not reflect a selective lack of dominance for the D463N mutation over the wild type (WT) allele since homozygous expression of this mutation was phenotypically WT. In cell culture, the D463H mutant expression, localisation and downregulation behaviours closely mimicked that of the WT, with the D463N mutant displaying some subtle differences. Biochemically, both the D463H and D463N mutants were shown to be inactive, although based on thermal shift assays the D463N mutant had reduced ATP affinity compared to WT or D463H proteins. Using induced transcriptional changes as a functional readout, altered mRNA expression indicates that the D463H acts as a dominant gain-of-function PKC mutant, despite a complete lack of intrinsic kinase activity; transcriptional changes induced by the D463N mutant were distinct from both the WT and D463H mutant. These findings suggest a model in which there is a non-catalytic effector function of PKCalpha which it is presumed operates through protein-protein interactions, as evidenced here by TurboID proximity labelling. The penetrant selection of the specific inactivating D463H mutation in chordoid glioma and the distinction with the D463N mutation indicates that this is conformationally sensitive. The properties characterised suggest this is driven by ATP binding and by inference, in this tissue context, the catalytic activity of this kinase acts as an autoregulatory negative feedback control.

overall_design: RNA sequencing was done using U87MG gliomablastoma cell line stably expressing WT, D463H or D463N mutant of Myc-PKCalpha. The parental U87MG cells were used as control. The condition were done in triplicate.

first: Véronique

last: Calleja

first: Stefan

last: Boeing

first: Jack

last: Henry


title: Correction of age-associated defects in dendritic cell functions enables CD4+T cells to eradicate tumors in the elderly

city: Cambridge

first: Ramnik

last: Xavier

address: 415 Main Street

institute: Broad Institute of MIT and Harvard

summary: Defective host defenses later in life are associated with changes in immune system activity. The means to correct immune defects to ensure immunity in the elderly are undefined. In this study, we found that CD8+T cells, which are necessary for anti-tumor immunity in young mice, are not required to eradicate the same cancers later in life. Rather, CD4+T cells drive anti-tumor immunity in elderly mice. The generation of anti-tumor CD4+T cells requires multiple dendritic cell (DC) activities that are elicited by immune agonists known as hyperactivators, but not by adjuvants that model those used clinically. DC hyperactivators correct age-associated defects in DC migration and T cell co-stimulation while enabling NLRP3 inflammasome activities within living cells. These combined activities enable DCs to induce TH1-skewed T cells that persist into old age and eliminate implantable tumors. These results raise the possibility of correcting age-associated immune defects through DC manipulation.

overall_design: 8 weeks or 92 weeks old mice were immunized with either PBS or OVA+LPS+PGPC. Mice received two boost injections every 7 days. 21 days post immunization, spleen samples were subjected to CD3e T cell enrichment to achieve highly pure (>85%) of CD3+ T cells. We then generated scRNA-seq data from splenic T cells using the NovaSeqX platform to explore the transcriptomic differences between the age groups.

first: Joshua

last: Park

first: Orr

last: Ashenberg

first: Jonathan

last: Kagan

first: Dania

last: Zhivaki


title: Capmatinib shows superior efficacy for MET-fusion driven pediatric high-grade glioma and synergizes with radiotherapy (mouse methylation array)

city: Memphis

first: Jared

last: Andrews

state: Tennessee

address: 262 Danny Thomas Pl

department: Developmental Neurobiology

institute: St. Jude Children's Research Hospital

summary: Background: Pediatric high-grade glioma (pHGG) is the most frequent malignant brain tumor in children and can be subclassified into multiple entities. Fusion genes activating the MET receptor tyrosine kinase often occur in infant-type hemispheric glioma (IHG) but also in other pHGGs, and are associated with devastating morbidity and mortality.  Methods: To identify new treatment options, we established and characterized two novel orthotopic mouse models harboring distinct MET fusions. These included an immunocompetent, murine allograft model and patient-derived orthotopic xenografts (PDOX) from a MET-fusion iHG patient who failed conventional therapy and targeted therapy with cabozantinib. With these models, we analyzed the efficacy and pharmacokinetic properties of three MET inhibitors, capmatinib, crizotinib and cabozantinib, alone or combined with radiotherapy (RT).  Results: The PDOX models recapitulated the poor efficacy of cabozantinib experienced by the patient. In contrast, capmatinib showed superior brain pharmacokinetic properties and greater in vitro and in vivo efficacy than cabozantinib or crizotinib. Strikingly, capmatinib treated mice displayed long-term progression-free survival (PFS) when combined with radiotherapy in two complementary mouse models. Molecular analysis of the treated tumors revealed impaired DNA repair after MET inhibition as a plausible mechanism of radiosensitization  Conclusions: We comprehensively investigated the combination of MET inhibition and radiotherapy as a novel treatment option for MET-driven pHGG. Our seminal preclinical data package includes pharmacokinetic characterization, recapitulation of clinical outcomes, coinciding results from multiple complementing in vivo studies and a plausible molecular mechanism. We thereby demonstrate the groundbreaking efficacy of capmatinib and radiation, as a highly promising concept for future clinical trials.

overall_design: The Illumina Infinium MouseMethylation array (Illumina, San Diego, USA) data from allograft murine brain tumors with a human TFG-MET fusion were used to obtain a genome-wide assessment of DNA methylation in accordance with manufacturer's instructions.

first: Marc

last: Zuckermann

first: David

last: Jones

first: Suzanne

last: Baker


title: Substrate elasticity does not impact on DNA methylation changes during differentiation of pluripotent stem cells.

city: Aachen

first: Wolfgang

last: Wagner

address: Pauwelsstrasse 20

department: Helmholtz Institute for Biomedical Engineering

institute: RWTH Aachen University

summary: Substrate elasticity may direct cell-fate decisions of stem cells. However, it is largely unclear how matrix stiffness impacts on differentiation of induced pluripotent stem cells (iPSCs) and if this is also reflected by epigenetic modifications. We have therefore cultured iPSCs on tissue culture plastic (TCP) and polydimethylsiloxane (PDMS) with different Young´s modulus (0.2 kPa, 16 kPa, or 64 kPa) to investigate the sequel on growth and differentiation towards endoderm, mesoderm, and ectoderm. Immunofluorescence and gene expression of canonical differentiation markers was hardly affected by the substrates. Notably, when we analyzed DNA methylation profiles of iPSCs or after three-lineage differentiation, we did not see any significant differences on the three different PDMS elasticities. Only when we compared DNA methylation profiles on PDMS-substrates versus TCP, we observed clear epigenetic differences, particularly upon mesodermal differentiation. Taken together, stiffness of PDMS-substrates did not impact on directed differentiation of iPSCs, whereas the moderate epigenetic differences on TCP might also be attributed to other chemical parameters.

overall_design: Genomic DNA was isolated using the NucleoSpin Tissue Kit (Macherey-Nagel) and quantified on a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). 1.2 µg of DNA were bisulfite converted and analyzed using the EPIC BeadChip microarray (Illumina, San Diego, USA) by Life & Brain company (Bonn, Germany).

first: Mohamed

middle: H

last: Mabrouk

first: Kira

last: Zeevaert

first: Ann-Christine

last: Henneke

first: Catharina

last: Maaßen


title: The HMG-box module in FACT is critical for suppressing epigenetic variegation of heterochromatin in fission yeast

city: Kobe

first: Atsushi

last: Matsuda

address: 588-2, Iwaoka, Iwaoka-cho, Nishi-ku

institute: National Institute of Information and Communications Technology

summary: Chromatin condensation state is the key for retrieving genetic information. High-mobility group protein (HMG) proteins exhibit DNA-binding and bending activities, playing an important role in the regulation of chromatin structure. We have shown that nucleosomes tightly packaged into heterochromatin undergo considerable dynamic histone H2A-H2B maintenance via the direct interaction between HP1/Swi6 and facilitate chromatin transcription (FACT), which is composed of the Spt16/Pob3 heterodimer and Nhp6. In this study, we analyzed the role of Nhp6, an HMG box protein, in the FACT at heterochromatin. Pob3 mutant strains showed derepressed heterochromatin-dependent gene silencing, whereas Nhp6 mutant strains did not show significant defects in chromatin regulation or gene expression, suggesting that these two modules play different roles in chromatin regulation. We expressed a protein fusing Nhp6 to the C-terminus of Pob3, which mimics the multicellular FACT component Ssrp1. The chromatin-binding activity of FACT increased with the number of Nhp6 fused to Pob3, and the heterochromatin formation rate was promoted more strongly. Furthermore, we demonstrated that this promotion of heterochromatinization inhibited the heterochromatic variegation caused by epe1+ disruption. Heterochromatic variegation can be observed in a variety of regulatory steps; however, when it is caused by fluctuations in chromatin arrangement, it can be eliminated through the strong recruitment of the FACT complex.

overall_design: Gene expression profile at exponentially-growing phase in the fission yeast deletion mutants of rhp6,pob3 and spt4.

first: Shinya

last: Takahata

first: Asahi

last: Taguchi

first: Ayaka

last: Takenaka

first: Miyuki

last: Mori

first: Yuji

last: Chikashige

first: Chihiro

last: Tsutsumi

first: Yasushi

last: Hiraoka

first: Yota

last: Murakami


title: Capmatinib shows superior efficacy for MET-fusion driven pediatric high-grade glioma and synergizes with radiotherapy [human methylation array]

first: Jared

last: Andrews

type: Methylation profiling by genome tiling array

overall_design: The Illumina Infinium MethylationEPIC BeadChip array (Illumina, San Diego, USA) data from an initial pre-treatment and recurrent post-chemotherapy tumors from a single HGG patient with a TRIM24-MET fusion were used to obtain a genome-wide assessment of DNA methylation in accordance with manufacturer's instructions.

first: Chen

last: He

first: Jason

last: Chiang


title: Transcriptional portrait of M. bovis BCGΔBCG1419c during in vitro planktonic culture

city: Seattle

first: Eliza

last: Peterson

state: WA

address: 401 Terry Ave N

institute: Institute for Systems Biology

summary: We have developed the vaccine candidate BCGΔBCG1419c, by deletion of BCG1419c in BCG Pasteur ATCC 35734, which improved control of tuberculosis (TB) in preclinical models. Here, we compared the transcriptomes of BCG and BCGΔBCG1419c during planktonic growth.

sample_organism: Mycobacterium tuberculosis variant bovis BCG

overall_design: To gain insight into the molecular differences occcuring between planktonic cells of BCG Pasteur and the vaccine candidate BCGDBCG1419c, we compared the transcriptome to wildtype BCG Pasteur to that of the mutant with deletion of BCG1419c, when both strains were grown in Middlebrook 7H9 media with 10% OADC, 0.2% glycerol, 0.05% Tween 80, grown in tissue cultured flasks with vented caps, at 100 rpm, 37ºC up to an OD600nm 0.8.

first: Nitin

last: Baliga

first: Mario

last: Flores-Valdez


title: Heat shock protein 72 supports extracellular matrix production in metastatic mammary tumors

city: Boston

first: Benjamin

last: Lang

address: 330 Brookline Ave

institute: BIDMC

summary: This study identified tumorigenic processes most dependent on murine HSP72 in the MMTV-PyMT mammary tumor model, which give rise to spontaneous mammary tumors that exhibit HSP72-dependent metastasis to the lung. RNA-seq expression profiling of Hspa1a/Hspa1b (Hsp72) WT and Hsp72 -/- primary mammary tumors discovered significantly lower expression of genes encoding components of the extracellular matrix (ECM) in Hsp72 knockout mammary tumors compared to WT controls. In vitro studies found that genetic or chemical inhibition of HSP72 activity in cultured collagen-expressing human or murine cells also reduces mRNA and protein levels of COL1A1 and several other ECM-encoding genes. In search of a possible mechanistic basis for this relationship, we found HSP72 to support the activation of the TGF-β – SMAD3 signaling pathway and evidence of SMAD3 and HSP72 co-precipitation, suggesting potential complex formation. Human COL1A1 mRNA expression was found to have prognostic value for HER2+ breast tumors over other breast cancer subtypes, suggesting a possible human disease context where targeting HSP72 may have a therapeutic rationale. Analysis of human HER2+ breast tumor gene expression data using a gene set comprising ECM and protein folding-related genes as an input to the statistical learning algorithm, Galgo, found a subset of these genes that can collectively stratify patients by relapse-free survival, further suggesting a potential interplay between ECM and protein-folding genes may contribute to tumor progression.

overall_design: To investigate tumorigenic processes dependent on murine HSP72, we performed expression profiling on spontaneous murine mammary tumors of HSP72 WT and HSP72 knockout mice expressing the PyMT oncogene under the control of the MMTV promoter.  Differential expression analysis was performed to identify mRNAs differentially expressed between HSP72 WT and HSP72 knockout mammary tumors.


title: Single-cell RNAseq of murine Peyer's patches after one dose of anti-α4β7 antibody or PBS

city: New York

first: Darwin

last: D'Souza

address: 1470 Madison Avenue

department: ImmunoBio

institute: Human Immune Monitoring Center

summary: Targeting the α4β7-MAdCAM-1 axis with vedolizumab (VDZ) is a first-line treatment in ulcerative colitis (UC). However, mechanism(s) of action (MOA) of VDZ remain relatively undefined. Here, we examined five distinct cohorts of patients with UC (n=83, n=60, n=21, n=31, n=401), to determine the effect of VDZ on the mucosal and peripheral immune system. We found a significant decrease in colonic and ileal naïve B and T cells and circulating gut- homing plasmablasts (β7+) in VDZ-treated patients, pointing to gut-associated lymphoid tissue (GALT) targeting by VDZ. Murine Peyer’s patches (PP) demonstrated a significant loss cellularity associated with reduction in follicular B cells, including a unique population of epithelium-associated B cells, following anti-α4β7 antibody (mAb) administration. Photoconvertible (KikGR) mice demonstrated impaired cellular entry into PPs in anti-α4β7 mAb treated mice. In VDZ-treated, but not anti-tumor necrosis factor-treated UC patients, lymphoid aggregate size was significantly reduced in treatment responders compared to non- responders, with an independent validation cohort further confirming these data. Response to VDZ was associated with a significant decrease of naïve B cells and a reduction in B cell follicle organization in the GALT. Responders had a significant reduction of β7+IgG+ plasmablasts in circulation, as well as IgG+ plasma cells and FcgR-dependent signaling in the intestine. GALT targeting represents a previously unappreciated MOA of α4β7-targeted therapies, with major implications for this therapeutic paradigm in UC, and for the development of new therapeutic strategies.

overall_design: Peyer's patches were isolated from the small inthestine of treated mice and incubated with collagenase D, before mechanical homogenization to obtain a single-cell suspension.

first: Pablo

last: Canales-Herrerias

first: Mathieu

last: Uzzan

first: Akihiro

last: Seki

first: Saurabh

last: Mehandru


title: A light signaling factor, ELONGATED HYPOCOTYL 5 (HY5) interacts with Histone deacetylase 9 (HDA9) to play a suppressive role in the glucosinolate biosynthesis in Arabidopsis

city: Anseong-si

first: Dasom

last: Choi

state: Gyeonggi-do

address: 4726, Seodong-daero

country: South Korea

institute: Chung-Ang University

summary: In this study, we found that a light signaling factor, Long Hypocotyl 5 (HY5) is closely involved in the regulation of GSLs contents in light condition. In addition, HY5 was shown to physically interact with a histone deacetylase HDA9 and bind to proximal promoter region of MYB29 and IMD1 to suppress aliphatic GSL biosynthetic process. These results demonstrate that HY5 acts to suppress GSL accumulation at daytime, thus properly modulating the GSL contents on a daily basis of Arabidopsis plant.

overall_design: Performed transcriptome analysis and targeted metabolite profiling of seedlings from the Arabidopsis loss-of-function mutants hy5-215 and hda9-1 compared to Col-0 wild-type plants.

first: Seong-Hyeon

last: Kim

first: Da-Min

first: Jeong-Il

first: Enamul

last: Huq

first: Dong-Hwan


title: Capmatinib shows superior efficacy for MET-fusion driven pediatric high-grade glioma and synergizes with radiotherapy.

first: Jared

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

overall_design: Refer to individual Series


title: ARID1A regulates DSB repair through chromatin organization and its deficiency triggers DNA damage-mediated immune response [ACT-seq]

city: Heidelberg

first: Christoph

last: Plass

address: Im Neueneimer Feld 280

department: Division of Cancer Epigenomics

institute: DKFZ (German Cancer Research Center)

summary: AT-rich interaction domain protein 1A (ARID1A), a SWI/SNF chromatin remodeling complex subunit, is frequently mutated across various cancer entities. Loss of ARID1A leads to DNA repair defects. Here we show that ARID1A plays epigenetic roles to promote both DNA double-strand breaks (DSBs) repair pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). ARID1A is accumulated at DSBs after DNA damage and regulates chromatin loops formation by recruiting RAD21 and CTCF to DSBs. Simultaneously, ARID1A binds to and promotes the recruitment of HDAC1 and RSF1 to DSBs to control the distribution of activating histone marks and chromatin accessibility leading to eviction of RNAPII and silencing of transcription in transcriptionally active chromatin. ARID1A depletion resulted in enhanced accumulation of micronuclei, activation of cGAS-STING pathway, and an increased expression of immunomodulatory cytokines upon ionizing radiation. Furthermore, low ARID1A expression in cancer patients receiving radiotherapy was associated with higher infiltration of several immune cells. The high mutation rate of ARID1A in various cancer types highlights its clinical relevance as a promising biomarker that correlates with the level of immune regulatory cytokines and estimates the levels of tumor-infiltrating immune cells, which can predict the response to the combination of radio- and immunotherapy.

overall_design: We used both WT AID-DivA (Auxin-Inducible Degron DSB Inducible via AsiSI) cells and AID-DivA cells with an ARID1A knock-out. We used 3 different treatments: untreated (UT), treated with 300 nM 4-hydroxytamoxifen (4OHT) and incubated for 4h, then treated with Auxin to stop the enzymatic generation of the double-strand breaks, and cell collection either immediately after auxin treatment (T0) or 4h after addition of auxin (T4). We used 3 different antibodies against H2AK118ac, H3K27ac or IgG. Two or three replicates were used for each condition.

first: Ali

last: Bakr

first: Peter

last: Schmezer


title: ARID1A regulates DSB repair through chromatin organization and its deficiency triggers DNA damage-mediated immune response [4C-seq]

overall_design: We used both WT AID-DivA (Auxin-Inducible Degron DSB Inducible via AsiSI) cells and AID-DivA cells with an ARID1A knock-out. We used 2 different treatment types. For the first treatment, we used 3 different treatments: untreated (UT), treated with 300 nM 4-hydroxytamoxifen (4OHT) and incubated for 4h, then treated with Auxin to stop the enzymatic generation of the double-strand breaks, and cell collection either immediately after auxin treatment (T0) or 4h (T4) after addition of auxin. For the second treatment, we either used BRG1 inhibitors (brg1i) or not (nobrg1i). We used three viewpoints: one close to HR-prone DSB (DSB2), one close to NHEJ-prone DSB (DSB3), and one with no adjacent DSB (ctrl). A variable number of replicates was used for each condition.


title: ARID1A regulates DSB repair through chromatin organization and its deficiency triggers DNA damage-mediated immune response [ATAC-seq]

overall_design: We used both WT AID-DivA (Auxin-Inducible Degron DSB Inducible via AsiSI) cells and AID-DivA cells with an ARID1A knock-out. We used 2 different treatment types. For the first treatment, we used 6 different conditions: untreated (UT), untreated and after waiting 12h (UT12), treated with 300 nM 4-hydroxytamoxifen (4OHT) and incubated for 4h, then treated with Auxin to stop the enzymatic generation of the double-strand breaks, and cell collection either immediately after auxin treatment (T0), 2h (T2), 4h (T4) or 12h (T12) after addition of auxin. The second treatment was the addition of a BRG1 inhibitor (BRM014). Three replicates were used for each condition.


title: RNA sequencing of U937 cell clones carrying 0,1 or 2 copies of FURIN gene

city: Singapore

first: Sujoy

last: Ghosh

address: 8 College Rd

country: Singapore

institute: National University of Singapore

summary: Pro-monocytic U937 cells were subjected to FURIN gene editing via an optimized CRISPR technology, resulting in clones with 1 or both copies of FURIN deleted. Paired-end RNA sequencing was performed on these cells, in addition to wild-type cells, to determine the effects of FURIN depletion on the transcriptome.

overall_design: 3 cell-types (WT, hemizygous (1 copy of FURIN present), and nullizygous (0 copies of FURIN present) x 3 replicates, subjected to paired-end RNA sequencing.

first: Ruiming

last: Chua

first: Lijin

last: Wang


title: ARID1A regulates DSB repair through chromatin organization and its deficiency triggers DNA damage-mediated immune response [RNA-seq]

overall_design: We used both WT AID-DivA (Auxin-Inducible Degron DSB Inducible via AsiSI) cells and AID-DivA cells with an ARID1A knock-out. Cells were untreated. For each genotype, 3 replicates were performed.


title: Unique vulnerability of RAC1-mutant melanoma to combined inhibition of CDK9 and immune checkpoints

city: 3601 Spruce Street

first: Yan

last: Zhou

address: 333 Cottman Ave

department: Bioinformatics

institute: Fox Chase Cancer Center

summary: RAC1P29S is the third most prevalent hotspot mutation in sun-exposed melanoma. RAC1 alterations in cancer are correlated with poor prognosis, resistance to standard chemotherapy, and insensitivity to targeted inhibitors. Although RAC1P29S mutations in melanoma and RAC1 alterations in several other cancers are increasingly evident, the RAC1-driven biological mechanisms contributing to tumorigenesis remain unclear. Lack of rigorous signaling analysis has prevented identification of alternative therapeutic targets for RAC1P29S-harboring melanomas. To investigate the RAC1P29S-driven effect on downstream molecular signaling pathways, we generated an inducible RAC1P29S expression melanocytic cell line and performed RNA-sequencing (RNA-seq) coupled with multiplexed kinase inhibitor beads and mass spectrometry (MIBs/MS) to establish enriched pathways from the genomic to proteomic level. Our proteogenomic analysis identified CDK9 as a potential new and specific target in RAC1P29S-mutant melanoma cells.In vitro, CDK9 inhibition impeded the proliferation of in RAC1P29S-mutant melanoma cells and increased surface expression of PD-L1 and MHC Class I proteins.In vivo, combining CDK9 inhibition with anti-PD-1 immune checkpoint blockade significantly inhibited tumor growthonly in melanomas that expressed the RAC1P29Smutation. Collectively, these results establish CDK9 as a novel target in RAC1-driven melanoma that can further sensitize the tumor to anti-PD-1 immunotherapy.

overall_design: Gene expression profiles from mutatant RAC1 melanocytes were compared with mutatnt RAC1 melanocytes with induced RAC1 expression were compared. Duplicate samples under each condition were used.

first: Alexa

last: Cannon

first: Jonathan

last: Chernoff


title: Precise editing of pathogenic nucleotide repeat expansion in iPSCs without inducing DNA double-strand breaks using a prime editor [RNA-Seq]

city: Suwon

first: Dongmin

last: Gim

address: 2066 Sueobu-Ro

department: School of Pharmacy

institute: Sungkyunkwan University

summary: Nucleotide repeat expansion disorders, a group of genetic diseases characterized by the expansion of specific DNA sequences, pose significant challenges to treatment and therapy development. Here, we present a precise and programmable method called prime editor–mediated correction of nucleotide repeat expansion (PE-CORE) for correcting pathogenic nucleotide repeat expansion. PE-CORE leverages a prime editor and paired pegRNAs to achieve targeted correction of repeat sequences. We demonstrate the effectiveness of PE-CORE in HEK293T cells and patient-derived induced pluripotent stem cells (iPSCs). Specifically, we focus on spinal and bulbar muscular atrophy and spinocerebellar ataxia type, two diseases associated with nucleotide repeat expansion. Our results demonstrate the successful correction of pathogenic expansions in iPSCs and subsequent differentiation into motor neurons. Specifically, we detect distinct downshifts in the size of both the mRNA and protein, confirming the functional correction of the iPSC-derived motor neurons. These findings highlight PE-CORE as a precision tool for addressing the intricate challenges of nucleotide repeat expansion disorders, paving the way for targeted therapies and potential clinical applications.

overall_design: Using PE-CORE, we transfected the iPSC-derived from patient, Spinal and Bulbar Muscular Atrophy (SBMA) and Spinocerebellar Ataxia Type 1 (SCA1), and generated gene-corrected disease iPSCs. We differentiate the Motor Neurons (MNs) from the iPSCs and treat 30 nM dihydrotestosterone (DHT) for 1 week to induce androgen activation. Next, we performed gene expression profiling analysis using data obtained from RNA-seq of 4 different cells with biological duplication. Comparative gene expression profiling analysis of RNA-seq data for disease iPSCs and gene-corrected disease iPSCs.

first: Hye-Yeon

last: Hwang

first: Hwalin

last: Yi

first: Hyewon

last: Jung

first: Jaecheol

last: Lee

first: Daesik


title: The effect of LAO1 in mouse breast milk on the offspring hippocampal brain

city: Fuchu-shi

first: Jun

last: Kambe

state: Tokyo

address: -5-8 Saiwai-cho

department: Agriculture

institute: Kentaro Nagaoka

summary: L-amino acid oxidase 1 (LAO1) is an enzyme that catalyzes the conversion of L-amino acids to α-keto acids, ammonia, and hydrogen peroxide. Previous studies have shown that offspring fed breast milk from LAO1 knockout (KO) mice show clear differences in gut microbiota composition compared to both wild-type (WT) and LAO1 KO mice. LAO1 KO mice also show impaired hippocampal memory function. The primary objective of this study is to determine how changes in the gut microbiota induced by breast milk in LAO1 KO mice affect the brain development of their offspring. To account for potential effects of endogenous LAO1, foster parents were switched on the day the pups were born, with the mother mice being either WT or LAO1 KO and the pups themselves being LAO1 KO. Hippocampi were collected from these mice at postnatal 10 days of age and microarray analysis was performed to reveal altered gene expression associated with the myelin.

overall_design: We compared hippocampal gene expression in LAO1 KO (konckout) offspring at P10 nourished WT and LAO1 KO dams.


title: The m6A reader HNRNPC promotes glioma progression by enhancing the stability of IRAK1 mRNA through the MAPK pathway

city: Nanchang

first: Junjun

last: Chen

address: No.461, Bayi Avenue, Nanchang City, Jiangxi Province

institute: Nanchang University

summary: Glioma is the most common and aggressive primary malignant brain tumor. N6-methyladenosine (m6A) modification widely exists in eukaryotic cells and plays an important role in the occurrence and development of human tumors. Here, we show that the m6A reader HNRNPC was overexpression and related to poor prognosis in glioma patients. HNRNPC plays crucial role in glioma cell proliferation, invasion and tumorigenesis. HNRNPC augments m6A-dependent mRNA stability of IRAK1, which impacted poor survival for glioma patients, further activated the downstream MAPK pathway. HNRNPC promotes glioma cells progression largely through the upregulation of IRAK1. Together, our findings demonstrate the novel HNRNPC-IRAK1-MAPK axis critical for glioma tumorigenesis and extend the reason for the upregulation of IRAK1.

overall_design: To investigate the mechanism whereby HNRNPC affects the malignant behavior of gliomas, we performed RNA-seq on the U251 cell line after HNRNPC knockdown by siRNA.


title: ARID1A regulates DSB repair through chromatin organization and its deficiency triggers DNA damage-mediated immune response

first: Christoph


title: Capmatinib shows superior efficacy for MET-fusion driven pediatric high-grade glioma and synergizes with radiotherapy [RNA-seq]

first: Jared

last: Andrews

summary: Pediatric high-grade glioma (pHGG) is the most frequent malignant brain tumor in children and can be subclassified into multiple entities. Fusion genes activating the MET receptor tyrosine kinase often occur in infant-type hemispheric glioma (iHG) but also in other pHGGs, and are associated with devastating morbidity and mortality. To identify new treatment options, we established and characterized two novel orthotopic mouse models harboring distinct MET fusions. These included an immunocompetent, murine allograft model and patient-derived orthotopic xenografts (PDOX) from a MET-fusion iHG patient who failed conventional therapy and targeted therapy with cabozantinib. With these models, we analyzed the efficacy and pharmacokinetic properties of three MET inhibitors, capmatinib, crizotinib and cabozantinib, alone or combined with radiotherapy (RT). The PDOX models recapitulated the poor efficacy of cabozantinib experienced by the patient. In contrast, capmatinib showed superior brain pharmacokinetic properties and greater in vitro and in vivo efficacy than cabozantinib or crizotinib. Strikingly, capmatinib treated mice displayed long-term progression-free survival (PFS) when combined with radiotherapy in two complementary mouse models. Molecular analysis of the treated tumors revealed impaired DNA repair after MET inhibition as a plausible mechanism of radiosensitization. We comprehensively investigated the combination of MET inhibition and radiotherapy as a novel treatment option for MET-driven pHGG. Our seminal preclinical data package includes pharmacokinetic characterization, recapitulation of clinical outcomes, coinciding results from multiple complementing in vivo studies and a plausible molecular mechanism. We thereby demonstrate the groundbreaking efficacy of capmatinib and radiation, as a highly promising concept for future clinical trials.

overall_design: RNA seq of tumors from a somatic TFG-MET driven high-grade glioma model, treated in 6 different treatment arms: capmatinib, crizotinib, vehicle +/- radiation. 4 Mice each were sacrificed after two days of treatment (PD cohort), the remainig mice were monitored until onset of symptoms (Survival cohort)

first: Suzanne

last: Baker

first: Marc

last: Zuckermann


title: Macrophages Coated Tumor Cluster (MCTC) Aggravated Hepatoma Invasion and Immunotherapy Resistance via Entrapping Cytotoxic T Cells to Generate Local Immune Deprivation in a M2BP-dependent manner

city: Tianjin

first: Junya

last: Ning

state: -- None --

address: Huanhuxi Road, Hexi District, Tianjin

institute: Tianjin Medical University Cancer Institute & Hospital

summary: Immune checkpoint inhibitors (ICIs) represent promising treatment for hepatocellular carcinoma (HCC) due to abundant lymphocyte infiltration. However, some HCC patients respond poorly to ICI therapy due to the presence of various immunosuppressive factors in tumor microenvironment. Our research revealed that macrophage-coated tumor clusters (MCTCs) signified a unique spatial structural organization in HCC, correlating with diminished recurrence-free survival (RFS) and overall survival (OS) in a total of 572 HCC cases from 3 internal cohorts and 2 external validation cohort independently. Mechanistically, Tumor-derived M2BP induced MCTCs formation, and entrapped immunocompetent cells at the edge of MCTCs to induce intratumoral cytotoxic T cells exclusion and local immune deprivation. Combined treatment of anti-PD-1 antibody and Mac-2 antagonist GB1107 before MCTC formation could significantly attenuated HCC growth and inhibited HCC metastasis in vivo by recovering intratumoral infiltration of cytotoxic T cells, offering a potential strategy to enhance ICI efficacy in HCC.

overall_design: we employed single-cell RNA sequencing to analyze tissues from 3 MCTC+ and 3 MCTC¯ cases.


title: Untangling the Dynamics of Lysine Acetylation and Phosphorylation in Adipogenesis in the Established Human and Mouse Adipocyte Cell Lines SGBS and 3T3L1

city: Leipzig

first: Jana

last: Schor

state: Saxony

address: Permoserstr. 15

department: Molecular Systems Biology

institute: Helmholtz Centre for Environmental Research

summary: Obesity is one of the most pressing global public health challenges of our time and prevalence continues to increase. It is characterized by enlarged and dysfunctional adipose tissue and is often associated with the development of metabolic or cardiovascular comorbidities. Post-translational protein modifications (PTMs) are important determinants of health and disease and enable vibrant cellular signalling. However, sound knowledge about how PTMs affect adipogenesis is still scarce. In addition to protein phosphorylation, which plays a crucial role in insulin signalling, lysine acetylation provides a direct link between cellular metabolism and signal transduction and is therefore of particular importance for the study of metabolic diseases. Stable cell lines are an integral part of research into the development and physiology of adipocytes in health and disease and various models have been used to date.    Here, we investigate the temporal dynamics of the transcriptome, proteome, the levels of central carbon metabolites as well as the acetyl and phosphoproteome in adipogenesis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in combination with PTM enrichment strategies on the established human (SGBS) and mouse (3T3-L1) adipocyte cell culture models.   Both cell lines showed unique temporal PTM profiles during adipogenesis, with a large number of acetylated and phosphorylated sites being significantly regulated. Enriched pathways of acetylated proteins were predominantly associated with central energy metabolism, while phosphorylated proteins were enriched in the areas of insulin/glucagon signalling, cell adhesion processes and organization of cellular structure. We identified several key driver candidates with strong correlation to the time course of adipogenesis, including CD44 and the acetylation site FASN K673 (negative) or IDH K272 (positive). The general agreement between results from SGBS and 3T3L1 cells was high, however details appeared cell line specific.   We provide a detailed data resource on the transcriptome, proteome, the levels of central carbon metabolites, the acetylome and phosphoproteome during adipogenesis of the established human and mouse adipocyte cell lines SGBS and 3T3L1, accessible for further mining.

overall_design:
Human SGBS cells were provided by the laboratory of Prof. Dr. Wabitsch at the University Clinic Ulm. Cells were differentiated according to the standard protocol described previously (Wabitsch et al., 2001).
The cell line was maintained under 5 % CO2 at 37°C and 95 % humidity. Cell culture experiments for transcriptomic (SGBS) data analysis were prepared in four replicates.
Differential expression was determined with the R package DESeq2 (Love et al., 2014).

first: Alix

last: Aldehoff

first: Isabel

last: Karkossa

first: Cornelius

last: Goerdeler

first: Laura

last: Krieg

first: Beatrice

last: Engelmann

first: Martin

last: Wabitsch

first: Kathrin

last: Landgraf

first: Jörg

last: Hackermüller

first: Ante

last: Körner

first: Ulrike

last: Rolle-Kampczyk

first: Kristin

last: Schubert

last: von Bergen


title: RNA-seq in SCLC transformation

city: Shenyang

first: cheng

last: yang

address: wenhua street  number 103

institute: Shenyang Pharmaceutical University

summary: SCLC transformation is one of the mechanisms of TKI resistance in lung cancer. To clarify its epigenetic changes, we used RNA-seq to detect the changes of genes in pre-LUAD and post-SCLC transformation cells.

overall_design: To detect the changes of genes in pre-LUAD NCI-H1650 cells and post-SCLC transformation NCI-H1650Re cells

first: Cheng

last: Yang


title: Single cell analysis uncovers striking cellular heterogeneity of lung infiltrating Tregs during steroid-responsive vs. steroid-resistant allergic airway inflammation

city: Chicago

first: Booki

last: Min

state: IL

address: 300 E. Superior Street

department: Microbiology-Immunology

institute: Northwestern University

summary: Allergic airway inflammation is a chronic inflammatory condition resulting from uncontrolled immune responses to environmental antigens. While it is well-established that allergic immune responses exhibit a high degree of diversity, driven by primary effector cell types like eosinophils, neutrophils, or CD4 T cells with distinct effector signatures, the exact mechanisms responsible for such pathogenesis remain largely elusive. Foxp3+ regulatory T cells (Treg) is an indispensable immune regulator during chronic inflammation including allergic airway inflammation. Emerging evidence suggests that Tregs infiltrating inflamed tissues exhibit distinct phenotypes dependent on the specific tissue sites and can display significant heterogeneity and tissue residency. Whether diverse allergic inflammatory responses in the lung influences infiltrating Treg heterogeneity or Treg lung residency has not previously been explored. We employed an unbiased single-cell RNAseq approach to study lung-infiltrating Tregs in models of eosinophilic and neutrophilic airway inflammation models, in which Tregs are critical immune regulators of inflammation. We found that lung-infiltrating Tregs are highly heterogeneous and that Tregs displaying lung residency phenotypes are significantly different depending on the types of inflammation. Tregs expression of ST2, a receptor for alarmin cytokine IL-33, was predominantly induced by eosinophilic inflammation and by tissue residency. However, Treg-specific ST2 deficiency did not affect the development of eosinophilic allergic inflammation nor the generation of lung resident Tregs. These results uncover a striking heterogeneity among Tregs infiltrating the lungs during allergic airway inflammation. The results also indicate that varying types of inflammation may give rise to phenotypically distinct lung resident Tregs. Thus, the present study underscores a novel mechanism by which inflammatory environments may shape the composition of infiltrating Tregs, allowing them to regulate inflammatory responses through tissue-adapted mechanisms.

overall_design: The goal is to evaluate the cellular heterogeneity of Tregs that infiltrate the lung tissues during eosinophilic and neutrophilic airway inflammation. Experimental allergic airway inflammation was induced and lung infiltrating Tregs were FACS sorted for single cell analylsis.

first: Supinya

last: Iamsawat

first: Rongzhen

last: Yu

first: Sohee

last: Kim

first: Kevin

last: Liu

first: Jaehyuk

last: Choi


title: A CARM1 inhibitor potently suppresses breast cancer both in vitro and in vivo

city: Xiamen

first: Jiancheng

last: Ding

state: Fujian

address: School of Pharmaceutical Sciences, Xiamen University Xiang`an South Road

institute: Xiamen University

summary: CARM1, a member of the protein arginine methyltransferase (PRMT) family, regulates a variety of cellular processes. CARM1 overexpression and/or aberrant activation are tightly linked to cancer development, and it is considered as a promising therapeutic target for cancer diagnosis and therapy. However, the number of specific and potent CARM1 inhibitors is limited to date. We herein discovered a CARM1 inhibitor, iCARM1, showed better specificity and stronger activity towards CARM1 both in vitro and in vivo compared to the known CARM1 inhibitors EZM2302 and TP-064. Similar as CARM1 knockdown, iCARM1 suppressed the expression of oncogenic estrogen/ER-target genes, whereas activated type I interferon (IFN) and IFN-induced genes (ISGs) in breast cancer cells. Consequently, iCARM1 potently suppressed tumor growth in multiple mouse breast tumor models. Based on the gene programs iCARM1-regulated, combination therapy with iCARM1 and endocrine therapy drugs or etoposide exhibited synergetic effects in suppressing breast tumor growth. Taken together, we demonstrated that targeting CARM1 by iCARM1 is effective in suppressing CARM1-regulated gene program and breast cancer development, providing a therapeutic avenue for treating breast cancers in the clinic.

overall_design: RNA-seq performed in this study was designed to reveal the genes that regulated by iCARM1 and CARM1 knockdown

first: Wen

first: Bing-ling

last: Peng

first: Ting

last: Ran

first: Xue

last: Chen


title: microarray in SCLC transformation

summary:
The histological transformation of lung adenocarcinoma (LUAD) to an aggressive neuroendocrine (NE) derivative resembling SCLC is a signature example of lineage plasticity in cancer.But the changes of gene expression need to be studied.
We used microarrays to detail the global programme of gene expression under transformation and reversed transformation cells and identified distinct classes of up-regulated genes during this process.

overall_design: we performed Affymetrix microarrays in Transformation-cells (T), Reversed cells (RT) and Parental cells (P)

first: Yuchen

last: Han


title: ChIP-seq in SCLC transformation

summary: SCLC transformation is one of the mechanisms of TKI resistance in lung cancer. To clarify its epigenetic changes, we used Chip-seq to detect the changes of H3K9me2 in pre-LUAD and post-SCLC transformation cells.

overall_design: Chromatin immunoprecipitation sequencing(ChIP-seq) for H3K9me2 in pre-LUAD (lung adenocarcinoma) PC9 cells and post-SCLC (small cell lung cancer) transformation  PC9 cells


title: Hunk as aregulator of TNBC genes involve in tumor immunity

city: Indianapolis

first: Elizabeth

last: Yeh

state: Indiana

address: 635 Barnhill Drive MS A418

department: Pharmacology and Toxicology

institute: Indiana University School of Medicine

summary:
The goal of this study was to target tumor immune specific genes and allowed us to specifically  immune profiling  what genes are change in HUNK or HUNK knockdown groups.
A main objective was to determine if genes related to the citokine signaling pathway were changed in HUNK control compared to  HUNK knockdown groups.

overall_design: Total of 6 samples: HUNK control (n=3) and HUNK sh (n=3). CTL is the HUNK control references sample

first: Adrian

middle: L

last: Oblak

first: Nicole

last: Solis


title: Effect of SPNS1 on lipid metabolism in mice

first: Thi Thuy Hoa

last: Ha

address: 28 Medical Drive

summary: Accumulation of sphingolipids, especially sphingosines, in the lysosomes is attributed to the pathogenesis of several lysosomal storage diseases. In searching for a lysosomal protein that mediates the release of sphingosines, we identified SPNS1 which shares the highest homology to SPNS2, a sphingosine-1-phosphate (S1P) transporter. We generated knockout cells and mice for Spns1 and employed lipidomics and metabolomics to identify SPNS1 ligands. We found that knockouts of Spns1 resulted in the accumulation of sphingolipids including sphingosines in embryonic brains and cell lines. These results suggest that deficiency of SPNS1 affects the clearance of sphingolipids in lysosomes. Biochemical assays demonstrated that sphingosines released from lysosomes required SPNS1. Postnatal deletion of Spns1 in mice causes lipid accumulation in the lysosomes and pathological conditions that are reminiscent of sphingolipid lysosomal storage diseases. These results reveal a critical molecular role of SPNS1 as a transporter for lysosphingolipids and lysoglyerophospholipids from the lysosomes and link its physiological roles with lysosomal storage diseases.

overall_design: To investigate the role of SPNS1 in lipid metabolism, SPNS1 has been knocked out in mice.

first: Thi

middle: T

first: Nam

last: Nguyen


title: Effect of SARS-CoV-2 infection on breast cancer cell lines (24h)

city: Milan

first: Loris

last: De Cecco

address: via Venezian 1

country: Italy

institute: IRCSS Istituto Nazionale Tumori

summary: COVID-19 pandemic, caused by SARS-CoV-2 virus, had significant consequences for individuals already dealing with various pathological conditions, including breast cancer. Emerging evidence suggests that SARS-CoV-2 infection may directly affect breast cancer cell biology, even though a deeper understanding of the virus's effects on tumor cells is still not completely achieved. The present study aimed at examining the molecular events taking place in three different breast cancer cell lines of distinct molecular subtypes upon SARS-CoV-2 virus infection. Specifically, MCF7, MDAMB231, and HCC1937 cell lines, representing luminal A, basal B/claudin low, and basal A molecular subtypes, respectively, were subjected to SARS-CoV-2 infection and gene expression profile analysis was conducted at 24 hours and 7 days post infection. Obtained data revealed that MCF7 cells exhibited the highest susceptibility to viral replication. Interestingly, genes upregulated by SARS-CoV-2 virus in all three cell lines were able to identify a subgroup of breast cancer patients characterized by poor prognosis. Given the ongoing spread of the SARS-CoV-2 virus within the population, it is crucial to gain a better comprehension of its direct impact on breast cancer and to assess the long-term consequences of COVID-19 on breast cancer outcomes.

overall_design: MCF7, MDAMB231 and HCC1937 cell lines were seeded at a density of 700000 cell/well in a 6-well culture plate in quadruplicates in complete RPMI 1640 medium at 37°C, in a humidified atmosphere containing 5% CO2. The following day, cells were infected with SARS-CoV-2, lineage B.1 (SARS-CoV-2/human/ITA/Milan-UNIMI-1/2020, GenBank MT748758.1). Cell infection was carried out by viral adsorption in complete medium for 2 hours, using a multiplicity of infection (MOI) of 0.1 (8.78x108 copies/mL) at 37°C in a 5% CO2 atmosphere. All studies with SARS-CoV-2 were performed in a Biosafety Level 3 laboratory. Total RNA was extracted 24 hours and 7 days post infection with QIAzol Lysis Reagent (Qiagen, Hilden, Germany) and RNeasy Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions.


title: Association between gut microbiota and CpG island methylator phenotype in colorectal cancer

city: Camden

first: Jean-Pierre

last: Issa

state: NJ

address: 403 Haddon Avenue

department: Research Labs

institute: Coriell Institute for Medical Research

type: Methylation profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Effect of SARS-CoV-2 infection on breast cancer cell lines (7days)


title: Association between gut microbiota and CpG island methylator phenotype in colorectal cancer [16S rRNA]

summary: We report the association between CpG islander methylator phenotype (CIMP) and the gut microbiome in human colorectal cancer tumor and adjacent normal tissue.

overall_design: DNA was isolated from human colorectal cancer tumor and adjacent normal tissue samples and performed 16S rRNA gene sequencing for microbial taxa profiling

first: Pyoung Hwa

last: Park

first: Kelsey

last: Keith

first: Gennaro

last: Calendo

first: Jaroslav

last: Jelinek

first: Jozef

last: Madzo

first: Raad

middle: Z

last: Gharaibeh

first: Jayashri

last: Ghosh

first: Carmen

last: Sapienza

first: Christian

last: Jobin


title: Compartmentalisation proteomics revealed endolysosomal protein network changes in goat model of atrial fibrillation

city: Liverpool

first: Rebecca

last: Burton

address: Nuffield Red Block, Sherrington Building

department: Pharmacology and Therapeutics

country: United Kingdom

institute: University of Liverpool

summary: To identify the role of the endolysosomal (EL) protein network in atrial fibrillation (AF), we applied a newly developed organelle isolation method to the AF goat model. To accomplish the goal, we used proteomics, transcriptomics, integrated analysis, enzyme assays, electron tomography, and western blotting and identified several regulating EL proteins, related protein networks and pathways.

overall_design: In order to understand the endolysosomal protein level changes in AF goat model we conducted transcriptomics,proteomics, and integrated analysis. Furthermore lysosomal enzymatic activities and structural changes were examined using techniques such as, enzyme assays, western blotting and electron tomography. AF was induced and maintained in female goats (C. hircus) for 6 months and left atrial tissue were obtained by an open chest sacrifice experiment in (N=4 AF and N=4 sham controls without induced AF).

first: Thamali

last: Ayagama

first: Adam

middle: P

last: Cribbs

first: Philip

last: Charles


title: Integration of Metabolomics and Transcriptomics Reveals HDL-Driven Biliary Reverse Cholesterol Transport Involved in the Antiatherogenic Effect of San-wei-tan-xiang Capsule

first: Binbin

last: Liu

address: Jinshan Road

department: Xiamen Cardiovascular Hospital

summary: Biliary reverse cholesterol transport (RCT) plays a crucial role in cholesterol clearance and regulation of atherogenesis. San-wei-tan-xiang capsule (SWTX), a traditional Chinese medicine, has shown potential in inhibiting atherogenesis by increasing high-density lipoprotein (HDL) cholesterol levels and promoting macrophage-mediated cholesterol efflux. However, the specific role of HDL-driven cholesterol metabolism in the anti-atherogenic effects of SWTX remains unclear. In this study, liquid chromatography coupled with tandem mass spectrometry was used to analyze the circulating metabolic profile, and RNA sequencing was performed on liver samples from ApoE−/− mice fed a cholesterol-enriched diet. We found that SWTX treatment induced significantly differential expression of metabolites and genes involved in cholesterol and lipid metabolism, as well as bile secretion pathways, which are critical for HDL-driven biliary RCT. Furthermore, alterations in L-carnitine and choline metabolism induced by SWTX treatment was involved in the atheroprotective effects of SWTX. Notably, SWTX treatment led to a significant increase in the expression of cholesterol 7α-hydroxylase (CYP7A1), a key enzyme involved in bile acid synthesis during atherogenesis. Additionally, the expression of CYP7A1 and CYP7A1-mediated bile acid secretion were enhanced by the addition of choline in hepatic cells, suggesting that SWTX-induced elevation of choline metabolic products may contribute to the upregulation of CYP7A1 and CYP7A1-mediated biliary RCT. Overall, SWTX demonstrated its ability to attenuate atherosclerotic plaque formation, which can be attributed to alterations in carnitine and choline metabolism, as well as the modulation of CYP7A1-mediated HDL-driven biliary RCT.

overall_design: The SWTX used in this study was obtained from Kaimeng Pharmaceutical Group Co., Ltd. (Hohhot, China) and had the following lot numbers: 20051401.1571, 20051901.1814, and 20051401.1555. The quality control of SWTX was performed according to the standards outlined in the Chinese Pharmacopoeia. The chemical fingerprint analysis of SWTX was conducted as described in our previous report (Ye et al., 2023). For administration, SWTX particles were dissolved in normal saline (NS) using ultrasonication for 40 minutes to prepare a suspension for treatment.  ApoE−/− male mice with a C57BL/6J background (Gempharmatech, Guanzhou, China) at 8 weeks of age were used in this study. The mice were housed in specific pathogen-free facilities maintained at a temperature of 22-23°C with a 12-hour light:12-hour dark circadian cycle. They were fed a Paigen atherogenic diet (PAD, Dyets, Wuxi, China) containing 12.5 g/kg cholesterol and 5 g/kg sodium cholic acid for a duration of 8 weeks. The mice were randomly divided into two groups: the model group, which received NS administered by gavage, and the SWTX group, which received a dosage of 3.5 g/kg/d administered by gavage.

first: Heng

last: Ye

first: Gang

last: Wang


title: SENP1 Regulates Germinal Center B Cell Responses through PAX5-deSUMOylation mediated AID expression

city: Shanghai

first: Jingjing

last: Qi

address: South Chongqing Road No.280

institute: Shanghai Jiao Tong University School of Medicine

summary: Humoral immunity depends on germinal center reaction where B cells are tightly controlled for class switch recombination (CSR) and somatic hypermutation, and finally generated into plasma and memory B cells. However, how protein SUMOylation control the key events of GC B cells remains to be understood. Here, we show that SUMO-specific protease 1 (SENP1) is upregulated in GC B cells. Selective ablation of Senp1 within GC B cells led to defective dark zone versus light zone GC B organization, impaired IgG1-switched GC B cells, and compromised antibody response. In addition, the formation of antigen-specific plasma and memory B cells were also diminished when SENP1 was deleted in GC B cells. Mechanistically, SENP1 was indispensable for expression of activation-induced cytidine deaminase (AID). SENP1-mediated Paired box protein 5 deSUMOylation suppressed the transcription to AID, which might be responsible for defective CSR in vivo. Our study not only established the importance of protein SUMOylation for the maintenance of GC B cell function but also clarified a novel mechanism to the modulation of AID expression.

overall_design: To understand how SUMOylation modification regulate the response of germinal center B cells, we used the  SUMO-specific protease 1-conditional deletion mouse model. We checked the immunce response used model antigen NP-CGG,  and checked the overall RNA changes when Senp1 is deleted.


title: Rhytidome- and cork- type barks of holm oak, cork oak and their hybrids highlight processes leading to cork formation

city: 17003 Girona

first: Mercè

last: Figueras

state: Girona

address: C/ Mª Aurèlia Campmany, 41 (campus Montilivi)

institute: Universitat de Girona

summary: The periderm is basic for land plants due to its protective role during radial growth, which is achieved by the polymers deposited in the cell walls. Despite the research on the topic has unravelled the role of several enzymes and transcription factors, many questions remain open, especially those regarding cell development. Here we use the outer bark of cork oak (cork), holm oak (rhytidome), and their natural hybrids’ to further understand the mechanisms underlying periderm development. Cork is an outstanding model as it consists of a thick and very homogeneous periderm produced by a permanent mother-cell layer (phellogen). Conversely, holm oak contains a more heterogeneous bark including several thin periderms mixed with phloem, also known as a rhytidome. The inclusion of hybrid samples showing rhytidome-type and cork-type barks is valuable to approach cork development, allowing an accurate identification of candidate genes and processes. The present study underscores that biotic stress and cell death signalling are enhanced in rhytidome-type barks while lipid metabolism and cell cycle are enriched in cork-type barks. Based on the DEGs most expressed related to development, we highlight that cell division, cell expansion, and cell differentiation could account for the differences found between cork and rhytidome-type barks.

sample_organism:
Quercus suber
Quercus ilex
Quercus x morisii

overall_design: Comparative gene expression profile analysis of RNA-seq data of outer bark from cork oak, holm oak and the hybrids. Sample: Outer barks of tree trunks from four adult cork oaks (Quercus suber L.), four holm oaks (Quercus ilex L.) and six Q. ilex x Q. suber hybrids. Trees were naturally grown in a mixed holm oak-cork oak forest in Fregenal de la Sierra (Extremadura, Spain). Outer bark of cork oak is named as cork, outer bark of holm oak is named as rhytidome, outer of the hybrid similar to that of cork oak is named as cork-like and outer bark of the hybrids similar to holm oak are named as rhytidome-like.  Replicates: 4 south orientation biological replicates from the cork oak, holm oak, and rhytidome-like bark hybrid and 4 samples coming from the north, south, west, and east of the cork-like bark hybrid

first: Iker

last: Armendariz

first: Unai

last: López de Heredia

first: Marçal

last: Soler

first: Adrià

last: Puigdemont

first: Maria

middle: M

last: Ruiz

first: Patricia

last: Jové

first: Álvaro

last: Soto

first: Olga

last: Serra