title: MEK inhibition overcomes chemoimmunotherapy resistance by inducing CXCL10 in cancer cells [LLC1]

city: DIJON

first: François

last: Ghiringhelli

address: 1 rue du Professeur Marion

institute: UMR INSERM 1231

summary: Chemotherapy with anti PD-1/PD-L1 mAb has become the standard of care for patients with metastatic non-small cell lung cancer (NSCLC). Using lung tumor models, where pemetrexed-platinum chemotherapy (PEM/CDDP) remains unable to synergize with immune checkpoint inhibitors (ICI), we linked failure of this treatment with its inability to induce CXCL10 expression and CD8+ T cell recruitment. Using drug screening, we showed that combining a MEK inhibitor (MEKi) with PEM/CDDP triggers CXCL10 secretion by cancer cells and CD8+ T cell recruitment, and restores ICI efficacy. PEM/CDDP plus MEKi promotes optineurin (OPTN)-dependent mitophagy, resulting in CXCL10 production in a mitochondrial DNA and TLR9-dependent manner. TLR9 or autophagy/mitophagy processes genetic inactivation of abort the antitumor efficacy of PEM/CDDP plus MEKi/anti PD-L1 therapy. In human NSCLC, high OPTN, TLR9 and CXCL10 expression is associated with better response to ICI. Our results underline the role of TLR9 and OPTN-dependent mitophagy in enhancing chemoimmunotherapy efficacy.

overall_design: All conditions are in triplicat and analyszed at two times after treatment (6 (T6) and 24hrs (T24)). Control LLC1 cells (NT) were treated only with 0,1% DMSO. CP cells were treated with cisplatin (2.5µM) and pemetrexed (2.5 µM). T cells were treated with trametinib (MEK inhibiteur, 10 nM). TCP cells were treated with both cisplatin, pemetrexed and trametinib. Total RNA from tumor cells was extracted with TriReagent (Fisher Scientific), RNA Sequencing was performed by the GenomEast platform, a member of the “France Génomique” consortium (ANR-10-INBS- 0009). Differential expression analysis was performed with the DESeq2 R package (Love et al., 2014). LLC1 cells treated with trametinib, PEM/CDDP or PEM/CDDP plus trametinib were compared to each other and to the control group. Raw p-values associated with each gene were adjusted using Bonferroni correction, as advised. For each comparison, genes with an-adjusted p-value < 0.10 were selected as differential.

first: Emeric

last: Limagne

first: Lisa

last: Nuttin

first: Marion

last: Thibaudin

first: Elise

last: Jacquin

first: Romain

last: Aucagne

first: Marjorie

last: Bon

first: Solène

last: Revy

first: Robby

last: Barnestein

last: Ballot

first: Caroline

last: Truntzer

first: Valentin

last: Derangère

first: Jean-David

last: Fumet

first: Charlène

last: Latour

first: Cédric

last: Rébé

first: Pierre-Simon

last: Bellaye

first: Coureche-Guillaume

last: Kaderbhaï

first: Aodrenn

last: Spill

first: Bertrand

last: Collin

first: Mary

last: Callanan

first: Aurélie

last: Lagrange

first: Laure

last: Favier

first: Bruno

last: Coudert

first: Laurent

last: Arnould

first: Sylvain

last: Ladoire

last: Routy

first: Philippe

last: Joubert


title: MEK inhibition overcomes chemoimmunotherapy resistance by inducing CXCL10 in cancer cells [France3]

status: Public on Dec 08 2021

first: François

last: Ghiringhelli

country: France

type: Expression profiling by high throughput sequencing

overall_design: This advanced non-small cell lung cancer (NSCLC) cohort comprises patients treated with anti-PD1 therapy in first or second line as monotherapy at the Centre Georges-François Leclerc in Dijon (France #3), and is composed of 35 patients. Tumors were collected at diagnosis, stored, and used with the written informed consent of the patients and RNAseq data was generated in our NGS core facility. Raw data were pseudo-aligned and gene counts were quantified using the kallisto software. Clinical data were collected by Prof. F. Ghiringhelli.

first: Emeric

last: Limagne

first: Lisa

last: Nuttin

first: Marion

last: Thibaudin

first: Elise

last: Jacquin

first: Romain

last: Aucagne

first: Marjorie

last: Bon

first: Solène

last: Revy

first: Robby

last: Barnestein

last: Ballot

last: Truntzer

middle: B


title: Mitochondrial variant enrichment from high-throughput single-cell RNA-seq resolves clonal populations

city: Boston

first: Peter

last: van Galen

state: Massachusetts

address: 4 Blackfan Circle

department: Hematology

country: USA

institute: Brigham and Women's Hospital / Broad Institute of MIT and Harvard

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Reconstructing lineage relationships in complex tissues can reveal mechanisms underlying development and disease. Recent methods combine single-cell transcriptomics with mitochondrial DNA variant detection to establish lineage relationships in primary human cells, but are not scalable to interrogate complex tissues. To overcome this limitation, here we develop a technology for high-confidence detection of mitochondrial mutations from high-throughput single-cell RNA-sequencing. We use the new method to identify skewed immune cell expansions in primary human clonal hematopoiesis.

overall_design: This project demonstrates a new technology to enrich mitochondrial DNA (mtDNA) mutations from 3' single-cell RNA-seq (scRNA-seq) libraries. The technology is termed MAESTER, or Mitochondrial Alteration Enrichment from Single-cell Transcriptomes to Establish Relatedness.

first: Tyler

middle: E

last: Miller

first: Caleb

middle: A

last: Lareau

first: Julia

last: Verga

first: Erica

last: DePasquale

first: Vincent

last: Liu

first: Daniel

last: Ssozi

first: Katalin

last: Sandor

first: Yajie

last: Yin

first: Leif

middle: S

last: Ludwig

first: Chadi

last: El Farran

first: Duncan

middle: M

last: Morgan

first: Ansuman

middle: T

last: Satpathy

first: Gabriel

middle: K

last: Griffin

first: Andrew

last: Lane

first: J

middle: C

last: Love

first: Bradley

last: Bernstein

first: Vijay

middle: G

last: Sankaran

last: Van Galen


title: MEK inhibition overcomes chemoimmunotherapy resistance by inducing CXCL10 in cancer cells

first: François

last: Ghiringhelli

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Single-cell RNA sequencing reveals B cell-T cell interactions in vascular adventitia of homocysteinemia-accelerated atherosclerosis

city: Beijing

first: Xiaolong

last: Ma

address: Xueyuan Road 38, Haidian Distrinct, Beijing, China

department: Department of Physiology and Pathophysiology

country: China

institute: Peking University Health Science Center

summary: Hyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis. However, a comprehensive understanding of immune cells in HHcy-accelerated atherosclerotic aortas (HHcy-AA) and the underlying mechanisms are still lacking. In this study, single-cell RNA sequencing (scRNA-seq) analysis of aortas from three atherosclerotic models on ApoE-/- mice revealed 15 distinct immune cell clusters. Among them, B cells showed the most significant increase in the vessels of HHcy ApoE-/- mice compared with those in ApoE-/- mice fed a chow diet or western diet (WD). Further cell-cell contact reanalyses revealed that B cells, but not dendritic cells or macrophages, played a dominant role in the class II major histocompatibility complex (MHCII) signaling in HHcy-AA. Mechanistically, Hcy induced the nuclear translocation of pyruvate kinase M2 (PKM2) in B cells. Nuclear PKM2 interacted with and phosphorylated cyclic AMP-responsive element-binding protein 1 (CREB1), and then PKM2-CREB1 complex bound to promoter III of Ciita, a master transactivator of MHCII, and further induced MHCII expression. Adoptive transfer experiments demonstrated that nuclear PKM2-regulated B cell antigen presentation is essential for B cell-mediated T cell activation and atherogenesis. In summary, our study provides a comprehensive immune cell atlas of HHcy-AA, which is distinct with those in WD-fed ApoE-/- mice. MHCII-related antigen presentation may be the main function of aortic B cells. We also provide a novel perspective for the intervention in early development of atherosclerosis via the PKM2-CREB1-CIITA-MHCII axis in B cells, especially for HHcy-accelerated vascular inflammation.

overall_design: Single-cell RNA sequencing (scRNA-seq) analysis of aortas from three atherosclerotic models on ApoE-/- mice [ApoE-/-, HHcy ApoE-/- and western diet (WD) ApoE-/-] revealed 15 distinct immune cell clusters, including B cells, macrophages/ monocytes, neutrophils, T cells, dendritic cells and mast cells.

first: Jiacheng

last: Deng

first: Yuwei

last: Song

first: Bitao

last: Zhong


title: People critically ill with COVID-19 exhibit peripheral immune profiles predictive of mortality and reflective of SARS-CoV-2 lung viral burden

city: Pittsburgh

first: Anthony

middle: Richard

last: Cillo

state: PA

address: The Assembly, Room 4070C Bay 10, 5051 Centre Ave

department: Immunology

institute: University of Pittsburgh

summary: There remains an urgent need to delinate immune cell states that contribute to mortality in critially ill COVID-19 patients. To better understand determinants of mortality, we performed high dimensional profiling of blood and respiratory samples from critially ill COVID-19 patients. Single-cell RNAseq based characterization of peripheral immune states reveal distinct expression profiles that were predictive of COVID-19 mortality. Temporal analysis revealed a that persistently elevated levels of inflammatory monocyte signatures and persistent interferon signaling preceeded concerted upregulation of inflammatory cytokines. Interrogation of lower respiratory tract saples revelaed that infected myeliod cells upregulated CXCL10, and elevated levels of CXCL10 in plasma were associated with a high risk of death. Overall, our data suggest a pivotal role for myeloid cell states in severe COVID-19 and may faciliate discovery of new diagnostics and therapeutics.

overall_design: We analyzed a total of 86 samples by scRNAseq, including peripheral blood from 10 healthy donors, 5 non-COVID acute respiratory distress syndrome patients (ARDS), and 24 COVID-19 patients. Two samples were typically paired per channel of 10X 5-prime scRNAseq sample using TotalSeq-C antibodies. All COVID-19 and ARDS patients were undergoing treatment in the intensive care units (ICUs) at the University of Pittsburgh Medical Center. Samples were obtained from non-COVID ARDS and COVID-19 at up to 3 timepoints (day 1, day 5 and day 10 post-enrollment in the ICU). On day 1, we obtained 26 COVID-19 PBMC samples and 5 non-COVID ARDS samples; on day 5, we obtained 4 non-COVID ARDS and 20 COVID-19 samples; on day 10 we obtained 2 non-COVID ARDS and 19 COVID_19 samples. In addition to peripheral blood samples, we also obtained 6 endotracheal aspirate samples from COVID-19 patients.

middle: R

first: Tullia

last: Bruno

first: Alison

last: Morris

first: Dario

last: Vignali


title: Role of EHMT1 in Alveolar Rhabdomyosarcoma

city: Singapore

first: Reshma

last: Taneja

address: 2 Medical Drive, MD9

department: Physiology

country: Singapore

institute: National University of Singapore

summary: Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma, wherein the malignant tumors arise from the myoblast like cells due to the perturbations in growth and differentiation. Alveolar rhabdomyosarcoma (ARMS) is one of the two major histological subtypes which exhibits high metastatic potential and tumor recurrence culminating in poor prognosis. In the recent past, understanding the epigenetic modifications in these tumors with less genetic alterations has shed light on the process of tumor pathogenesis and development of potential epi-drugs for the effective treatment strategies. Epigenetic regulator, Euchromatin histone lysine methyltransferase 1 (EHMT1)/G9A-like protein (GLP) and its homolog Euchromatin histone lysine methyltransferase 1 (EHMT2)/G9A has been shown to promote proliferation and inhibit differentiation in the mouse myoblast cells, independently. Also, previous study from our lab reported the overexpression of EHMT2 and molecular mechanism associated with the oncogenic phenotype in ARMS. However, expression pattern and functional role of EHMT1 in ARMS is unknown. Herein, we investigated the role of EHMT1 in ARMS. Our preliminary data indicated the overexpression of EHMT1 and the associated oncogenic phenotypes in ARMS. Although EHMT1 has shown to be upregulated and correlated with the increased disease severity in several cancers including gastric, esophagus, squamous cell carcinoma, ovarian and breast cancer, the mechanism involved in the EHMT1 mediated tumorigenesis was unclear. Therefore, through our current study we aimed to understand the expression pattern and the mechanism through which EHMT1 regulated cancer phenotypes in ARMS, for this we performed whole transcriptomic analysis in ARMS cell lines upon EHMT1 knockdown for the first time. In addition, we also compared the expression levels of EHMT1 between Human skeletal muscle myoblasts (HSMM) and ARMS patient samples (obtained from St Jude cloud database).

overall_design: We examined the differentially expressed genes upon EHMT1/GLP knockdown in ARMS cell line, to identify potential downstream targets of EHMT1. Triplicates for each conditions are used in the current study. Also, RNA expression of EHMT1 is compared between HSMM and ARMS patient tumor samples (n=32) from St. Jude cloud's genomic database (https://platform.stjude.cloud/data/diseases)

first: Alamelu

last: Nachiyappan

first: Joshua

middle: L

last: Soon


title: RNA sequencing to compare gene expression in control and PD-L1 knockdown hepatic stellate cells

city: Austin

first: Ningling

last: Kang

state: MN

address: 801 16th Ave NE

department: The Hormel Institute

institute: University of Minnesota

summary: Primary human hepatic stellate cells (HSCs) isolated from healthy donors were purchased from Sciencell.  They were transduced with viruses encoding control or PD-L1 shRNA and stimulated with TGFbeta1 (5 ng/ml) for 24 hours.  Cells were collected for RNA isolation and RNA sequencing.  The goal of this study is to identify genes transcriptionally regulated by PD-L1.

overall_design: There were 4 cell groups:  NT shRNA, NT shRNA + TGFbeta1, PD-L1 shRNA, PD-L1 shRNA + TGFbeta1. Each group had 3 repeats. So 12 RNA samples were sent to UMN genomic Center for RNA sequencing.  12 RNA samples were converted to Illumina sequencing libraries by using Illumina’s Truseq Stranded mRNA Sample Preparation Kit.  Truseq libraries were then subjected to cluster by using Illumina cBot instrument and sequencing with HiSeq2500


title: Two bHLH Transcription Factors, bHLH48 and bHLH60, Associate with PHYTOCHROME INTERACTING FACTOR 7 to Regulate Hypocotyl Elongation in Arabidopsis thaliana

city: Shanghai

first: Chuanwei

last: Yang

address: 2005 Songhu Road, Yangpu District

department: school of life sciences

institute: Fudan university

summary: A ChIP-Seq was performed using 35S::PIF7-Flash and 35S::bHLH60-Flag transgenic plants after 1 hr shade treatment. Approximately 6374 peaks (5262 genes) were considered as high-confidence of PIF7 binding peaks (Fold change > 2, q-value < 0.05). And 2278 peaks (2143 genes) were considered as high-confidence of bHLH60 binding peaks. The bound genes of PIF7 significantly overlapped with bHLH60-bound genes. And, the transcriptional levels of PIF7 and bHLH60 co-bound genes were also regulated by PIF7 and bHLH48/60. Our results indicated PIF7 and bHLH60 form a heterodimer to bind to promoters of the common target genes and regulate their expressions.

overall_design: 8-day-old white-light-grown seedlings were treated 1 hr shade before harvesting for DNA extraction.

first: Lin

last: Li


title: Identification of genome-wide targets of acetylated NPM1 (AcNPM1) mediated transcriptional regulation.

city: Bangalore

first: Parijat

last: Senapati

address: Jakkur

country: India

institute: Jawaharlal Nehru Centre for Advanced Scientific Research

summary: We report the first genome-wide profile of AcNPM1 occupancy in HeLa S3 cells by performing ChIP-sequencing using a AcNPM1 specific antibody. We show that AcNPM1 is associated with active transcription. AcNPM1 occupies transcriptional regulatory elements such as promoters and enhancers through its interaction with RNA Polymerase II, general transcription factors, transcription factors as well as histone acetyltransferases and chromatin remodeling complexes. Using RNA-sequencing afer NPM1 knockdown in oral cancer cell line AW13516, we also show that AcNPM1 regulates key pathways associated with cell proliferation, migration and invasion.

overall_design: Analysis of AcNPM1 occupancy in cells using ChIP-seq and gene expression changes after NPM1 knocdown in oral cancer cells.

first: Tapas

last: Kundu


title: Tick Adiponectin Receptor-Mediated Phospholipid Metabolism Contributes to   Borrelia burgdorferi Colonization

city: Hangzhou

first: Xiaotian

last: Tang

state: Zhejiang

address: Agro-Bio-Environ Building, Zhejiang University

department: Entomology

institute: Zhejiang University

summary: Adiponectin-mediated pathways contribute to mammalian homeostasis; however, little is known about adiponectin and adiponectin receptor signaling in arthropods. In this study, we demonstrate that Ixodes scapularis ticks have an adiponectin receptor-like protein (ISARL) but lack adiponectin, suggesting activation by alternative pathways. ISARL expression is significantly upregulated in the tick gut after Borrelia burgdorferi infection, suggesting that ISARL signaling may be co-opted by the Lyme disease agent. Consistent with this, RNA interference (RNAi)-mediated silencing of ISARL significantly reduced the B. burgdorferi burden in the tick. RNA-seq-based transcriptomics and RNAi assays demonstrate that ISARL-mediated phospholipid metabolism by phosphatidylserine synthase I is associated with B. burgdorferi survival. Furthermore, the tick complement C1q-like protein 3 interacts with ISARL, and B. burgdorferi facilitates this process. This study identifies a new tick metabolic pathway that is connected to the life cycle of the Lyme disease spirochete.

overall_design:
(1)  Tick gut global gene expression change after silencing adiponectin receptor
Examination of tick gut response by silencing adiponectin receptor after engorgement on Borrelia burgdorferi-infected or uninfected mice, using RNA-seq

(2)  Tick gut global gene expression change after injecting recombinant GFP and adiponectin protein
Examination of tick gut response after injecting recombinant GFP and adiponectin protein, using RNA-seq

first: Erol

last: Fikrig


title: Therapeutic activation of virus-specific resident memory T cells within glioblastoma

city: Minneapolis

first: Sathi

last: Wijeyesinghe

summary: Glioblastoma multiforme (GBM) is among the most aggressive, treatment resistant cancers, and despite standard of care surgery, radiation and chemotherapy, is invariably fatal. GBM is marked by local and systemic immunosuppression, contributing to resistance to existing immunotherapies that have had success in other tumor types. Memory T cells specific for previous infections reside in tissues throughout the host and these tissue resident memory T cells (TRM) are capable of rapid and potent immune activation. Here, we show that virus-specific memory CD8+ T cells expressing tissue resident markers populate the mouse and human glioblastoma microenvironment. Reactivating virus-specific TRM through intra-tumoral delivery of adjuvant-free virus-derived peptide triggered local immune activation. This delivery translated to anti-neoplastic effects, which improved survival in a murine glioblastoma model. Our results indicate that virus-specific TRM are a significant part of the glioblastoma immune microenvironment and may be leveraged to promote anti-tumoral immunity

overall_design: For this study, 5 human GBM tumor samples were acquired. Tumor tissue was sliced into thin sections, and incubated with viral peptides or DMSO alone (control) ex vivo for 9 hours. Sliced tumor sections were treated as technical replicates. RNA was isolated from tumor culture homogenate for RNA-Seq.

first: Pamela

last: Rosato

first: David

last: Masopust


title: Next Generation Sequencing Facilitates Quantitative Analysis of wild type and  Lmna R527C mutant mice spleen Transcriptomes with or without 4Gy irradiation.

status: Public on Dec 07 2021

first: Ming

last: Kuang

address: Xueyuan Road 38 #, Haidian District

institute: Peking University

summary: To induced the atypical progeria symptoms of LMNA R527C mutant mice, mutant and wild type mice were irradiated at 6 weeks of age with doses of 4 Gy. And to examine the pathologic change of mutant mice, mice were sacrificed at day -1and day 7 post-irradiation and spleen was harvested for RNAseq.

overall_design: To elucidate the mechanism by which LMNA R527C mutant mice caused senescence, we performed whole genome wide RNA-seq of mutant and wild type mice with or without 4Gy irradiation, and compared the differentially expressed genes between LMNA mutant and wild type mice.

first: Yingchi

last: Zhao


title: Next Generation Sequencing Facilitates Quantitative Analysis of healthy donor- and  LMNA R527C mutant patient-derived MSCs Transcriptomes

summary: We reprogramed patient derived PBMCs into iPSCs, and differentiated the iPSCs into Mesenchymal stem cells. We performed whole genome wide RNA-seq of patient-derived MSCs and control MSCs. Up-regulated and down-regulated genes of LMNAR527C/R527C patient-derived MSCs were enriched and analyzed.

overall_design:
To elucidate the mechanism by which Lamin A R527C mutation caused senescence, we performed whole genome wide RNA-seq of patient-derived MSCs and control MSCs, and compared the differentially expressed genes between LMNA R527C mutant and wild type.
mRNA profiles of healthy donor- and LMNA R527C mutant patient-derived MSCs


title: MEK inhibition overcomes chemoimmunotherapy resistance by inducing CXCL10 in cancer cells [France4]

first: François

last: Ghiringhelli

overall_design: This advanced non-small cell lung cancer (NSCLC) cohort  comprises patients treated with anti-PD1 therapy in first or second line as monotherapy at the Centre Georges-François Leclerc in Dijon (France #4), and is composed of 71 patients. Tumors were collected at diagnosis, stored, and used with the written informed consent of the patients and RNAseq data was generated in our NGS core facility. Raw data were pseudo-aligned and gene counts were quantified using the kallisto software. Clinical data were collected by Prof. F. Ghiringhelli.

first: Emeric

last: Limagne

first: Lisa

last: Nuttin

first: Marion

last: Thibaudin

first: Elise

last: Jacquin

first: Romain

last: Aucagne

first: Marjorie

last: Bon

first: Solène

last: Revy

first: Robby

last: Barnestein

last: Ballot

first: Caroline

last: Truntzer

first: Valentin

last: Derangère

first: Jean-David

last: Fumet

first: Charlène

last: Latour

first: Cédric

last: Rébé

first: Pierre-Simon

last: Bellaye

first: Coureche-Guillaume

last: Kaderbhaï

first: Aodrenn

last: Spill

first: Bertrand

last: Collin

first: Mary

last: Callanan

first: Aurélie

last: Lagrange

first: Laure

last: Favier

first: Bruno

last: Coudert

first: Laurent

last: Arnould

first: Sylvain

last: Ladoire

last: Routy

first: Philippe

last: Joubert


title: DPPA2 and DPPA4 are dispensable for mouse zygotic genome activation and preimplantation development

status: Public on Dec 06 2021

city: Cincinnati

first: Zhiyuan

last: Chen

state: Ohio

address: 3333 Burnet Avenue

institute: Cincinnati Children's Hospital Medical Center

summary: How maternal factors in oocytes initiate zygotic genome activation (ZGA) remains elusive. Recent studies indicate that DPPA2 and DPPA4 are required for establishing a 2-cell embryo-like (2C-like) state in mouse embryonic stem cells (ESCs) in a DUX-dependent manner. These results suggest that DPPA2 and DPPA4 are essential maternal factors that regulate Dux and ZGA in embryos. By analyzing maternal knockout and maternal-zygotic knockout embryos, we unexpectedly found that Dux activation, ZGA, and preimplantation development are normal in embryos without DPPA2 or DPPA4. Thus, unlike in ESCs/2C-like cells, DPPA2 and DPPA4 are dispensable for ZGA and preimplantation development.

overall_design: Comparion of WT and mutant Dppa2/4 1-cell and 2-cell embryo transcriptome

first: Zhenfei

last: Xie

first: Yi

last: Zhang


title: The specific DNA sequence recognized by the yeast transcription factor Gcn4 is sufficient to predict high-affinity binding on a genome-wide scale

city: Bethesda

first: Christopher

last: Coey

state: MD

address: 6 Center Drive Bldg 6A 2A02

institute: National Institute of Child Health and Human Development

summary: Sequence-specific DNA-binding transcription factors are central to gene regulation. They are often associated with consensus binding sites that predict far more genomic sites than are bound in vivo. One explanation is that most sites are blocked by nucleosomes, such that only sites in nucleosome-depleted regulatory regions are bound. Alternatively, the consensus site may be poorly defined. We compared the binding of the yeast transcription factor Gcn4 in vivo using published ChIP-seq data (546 sites) and in vitro, using a modified SELEX method ("G-SELEX"), which utilizes short genomic DNA fragments to quantify binding at all sites. We confirm that Gcn4 binds strongly to an AP1-like sequence (TGACTCA) and weakly to half-sites. However, Gcn4 binds only some of the 1078 exact matches to this sequence, even in vitro. We show that the high-affinity site is RTGACTCAY (exact match), of which there are only 166 copies in the yeast genome, all occupied in vivo. Generally, RTGACTCAR/YTGACTCAY sites are bound much more weakly and YTGACTCAR sites are unbound, with biological implications for determining induction levels. We conclude that Gcn4 binding can be predicted using the genome sequence. Our study suggests that transcription factor consensus sequences should be defined more precisely using quantitative data.

overall_design: Incubation of yeast genomic DNA fragments with recombinant Gcn4, followed by amplification of bound fragments (3 rounds) and paired-end sequencing

first: David

middle: J

last: Clark


title: Analysis of differential gene expression in Streptococcus sanguinis wild type and ssaACB deletion mutant strains before and after adjustment of the pH from 7.4 to 6.2.

city: Richmond

first: Todd

last: Kitten

state: VA

address: 521 N. 11th Street

department: Philips Institute for Oral Health Research

institute: Virginia Commonwealth University

summary: The purpose of this study was to determine what genes were differentially expressed in a Streptococcus sanguinis mutant deleted for the SsaACB manganese transporter, which underwent growth arrest upon shifting the pH to 6.2 with HCl vs. its wild-type parent, SK36, which continued to grow normally. The WT and mutant strain, JFP169, were grown in a fermenter with minimal aeration in BHI broth. RNA-seq was used to compare gene expression from samples taken 20 minutes prior to addition of HCl to samples taken 10, 25, and 50 min after addition. Samples were treated to deplete ribosomal RNA, then sequenced on a MiSeq sequencer, producing paired-end 75-base reads. Sequences were trimmed and filtered and expression levels determined using the DE-Seq module in the Geneious Prime program.

overall_design: Expression was compared at four time points, one 20 min prior to adjustment of the pH to 6.2 and the other three at 10, 25, and 50 min after pH adjustment. Four biological replicates were analyzed for each time point.

first: Tanya

last: Puccio


title: Melanopsin elevates locomotor activity during the wake state of the diurnal zebrafish

city: Vienna

first: Marcus

last: Dekens

state: Vienna

address: Dr.-Bohr-Gasse 9

country: Austria

institute: Max Perutz Laboratory

summary: Mammalian and fish pineals play a key role in adapting behaviour to the ambient light conditions through the release of melatonin. In mice, light inhibits nocturnal locomotor activity via the nonvisual photoreceptor Melanopsin. In contrast to the extensively studied function of Melanopsin in the indirect regulation of the rodent pineal, its role in the intrinsically photosensitive zebrafish pineal has not been elucidated. Therefore, it is not evident if the light signalling mechanism is conserved between distant vertebrates, and how Melanopsin could affect diurnal behaviour. A double knockout of melanopsins (opn4.1-opn4xb) was generated in the diurnal zebrafish, which manifests attenuated locomotor activity during the wake state. Transcriptome sequencing gave insight into pathways downstream of Melanopsin, implying that sustained repression of the melatonin pathway is required to elevate locomotor activity during the diurnal wake state. Moreover, we show that light induces locomotor activity during the diurnal wake state in an intensity-dependent manner. These observations suggest a common Melanopsin-driven mechanism between zebrafish and mammals, while the diurnal and nocturnal chronotypes are inversely regulated downstream of melatonin.

overall_design: Eye and brain mRNA profiles of 1.5-year old wild type (WT) and opn4.1-/- & opn4xb-/- double knockout (opn4 dko) zebrafish

middle: P

last: Fontinha

first: Miguel

last: Gallach

first: Sandra

last: Pflügler

first: Kristin

last: Tessmar-Raible


title: Alveolar epithelial glycocalyx degradation mediates surfactant dysfunction and contributes to acute respiratory distress syndrome

city: Aurora

first: Christophe

last: Langouët-Astrié

state: CO

department: Pulmonary Sciences and Critical Care

institute: University of Colorado Denver, Anschutz

summary: Acute Respiratory Distress Syndrome (ARDS) is a common cause of respiratory failure yet has few pharmacologic therapies, reflecting the mechanistic heterogeneity of lung injury. We hypothesized that damage to the alveolar epithelial glycocalyx, a layer of glycosaminoglycans interposed between the epithelium and surfactant, contributes to lung injury in ARDS patients. Using mass spectrometry of airspace fluid noninvasively collected from mechanically-ventilated patients, we found that airspace glycosaminoglycan shedding (an index of glycocalyx degradation) occurred predominantly in patients with direct lung injury and was associated with duration of mechanical ventilation. Male patients had increased shedding which correlated with airspace concentrations of matrix metalloproteinases. Selective epithelial glycocalyx degradation in mice was sufficient to induce surfactant dysfunction, a key characteristic of ARDS, leading to microatelectasis and decreased lung compliance. Rapid colorimetric quantification of airspace glycosaminoglycans was feasible and could provide point-of-care prognostic information to clinicians and/or be used for predictive enrichment in clinical trials.

overall_design: We analyzed whole lung RNA from mice intratracheally treated with heparanase (Hep) I/III or heat-inactivated (HI) Hep I/III for 24 hours.

first: Sarah

last: Haeger

first: Yimu

last: Yang

first: Eric

last: Schmidt


title: 10X-single cell seq of IEC and Stroma during recovery after DSS colitis in B cell sufficient/deficient mice

city: Stockholm

first: Eduardo

last: Villablanca

address: visiongatan 18

department: CMM

country: Sweden

institute: Karolinska Institute

summary: B cells expand during the recovery after DSS-induced colonic inflammation and might play a role in influencing tissue repair. To analyze the impact B cells might have on intestinal epithelial cells and stromal cells during recovery after intestinal injury the transcriptional profile of these mice was analysed in mice depleted of B cells and control mice on day 14 after DSS colitis.

overall_design: Female C57BL/6J mice were irradiated and received BM transplants with either CD19cre (ctrl) or CD19cre x iDTR cells. 8 weeks after BM transfer mice were treated with 2%DSS via the drinking water followed by 7 days of recovery. On days 9, 10, 12 and 13 mice received i.p. injections of diphteria toxin (25ng/g BW) in PBS. On day 14 colonic epithelial cells and stromal cells were sorted by flow cytometry and processed according to 10x Genomics protocol for Chromium Single cell 3’ kit (version 2) and submitted for sequencing.

first: Annika

last: Frede

first: Chiara

last: Sorini

first: Kumar

last: Tripathi


title: Expression profiling of miR-142 deficient Treg cells

city: St. Louis

first: Wei-Le

last: Wang

state: MO

address: BJCIH, 8th Floor, Room 8107, 660 S. Euclid Ave

department: Pathology and Immunology

institute: Washington University

summary: miR-142 gene is specifically and abundantly expressed in hematopoietic lineages. Mice with specific deletion of this miRNA gene in Treg develop fatal autoimmunity.  We used RNA-seq to detect whole transcriptome changes in miR-142 null Treg cells.

overall_design: Total RNA from sorted CD4+YFP+ Treg cells (Foxp3Cre-YFP,n=3 and Foxp3Cre-YFPmiR-142fl/fl, n=3) was extracted and subjected to RNA-seq. Samples in Foxp3Cre and Foxp3CremiR-142fl/fl groups are biological replicates and were isolated in age and gender matched mice.

first: Mark

last: Boldin


title: Translocated Legionella pneumophila small RNAs mimic eukaryotic miRNAs to dampen the host immune response

city: Paris

last: Rusniok

address: 25, 28 rue du Docteur Roux

department: Genomes et Genetique

institute: Institut Pasteur

summary: Extracellular vesicles (EVs) produced by bacteria, archaea and eukaryotic cells, are increasingly recognized as important mediators of intercellular communication via transfer of a wide variety of molecular cargoes. To characterize and identify the RNA molecules enriched in Legionella pneumophila extracellular vesicles (Lp-EVs)  we performed RNAseq analyses. Four independent RNAseq libraries were constructed from RNA isolated from purified Lp-EVs and as control from the bacterial pellets, from which the Lp-EVs had been shed and deep sequenced using an Illumina platform. The sequences obtained from the Lp-EVs RNAseq libraries were compared to those obtained from the RNA extracted from the bacterial pellets (Figure 1g). The Lp-EV-sRNA cargo was defined as RNAs for which after normalization at least 1000 reads were sequenced and that showed an enrichment of a log2FC > 5 as compared to the RNAs from the bacterial pellets were. Using these parameters the analysis identified 47 different sRNAs enriched in the Lp-EVs. The 20 highest enriched sRNA that were present in all four biological replicates comprised 8 tRNAs, 7 sRNAs and 5 fragments of mRNA located in the CDS or UTR.

overall_design: For each RNAseq experiment, the RNA extracted from purified Legionella-vesicles was compared to the total RNA extracted from the correspondent bacterial cultures. In total n=4 independent experiments were performed

first: Tobias

last: Sahr

first: Pedro

last: Guerrero

first: Carmen

last: Buchrieser


title: Obesity modulates cell-cell interactions during ovarian folliculogenesis

status: Public on Dec 09 2021

first: Fan

last: Guo

state: Beijing

address: 1 Beichen West Road, Chaoyang District

department: The State Key Laboratory of Stem Cell and Reproductive Biology

institute: Institute of Zoology, Chinese Academy of Sciences

summary: Obesity is known to affect female reproduction, as evidenced by obese patients suffering from subfertility and abnormal oogenesis. However, the underlying mechanisms by which obesity impairs folliculogenesis are poorly documented. Here, we performed comprehensive single-cell transcriptome analysis in both regular diet (RD) and obese mouse models to systematically uncover how obesity affects ovarian follicle cells and their interactions. We found an increased proportion of Inhbb highly expressed granulosa cells (GCs) among all the GC subpopulations in obese mice. Under obese conditions, excessive androgen secreted from endocrine theca cells (ETCs) may contribute to the imbalanced change of GC subtypes through ETCs-GCs interactions. This is alleviated by enzalutamide, an androgen receptor antagonist. We also identified and confirmed typical GC markers, such as Marcks and Prkar2b, for sensitive evaluation of female fertility in obesity. These data represent a resource for studying transcriptional networks and cell-cell interactions during folliculogenesis under physiological and pathological conditions.

overall_design: Somatic cells and oocytes in the ovary were isolated from RD, Lep KO, Lepr KO or HFD mice, and were subjected to scRNA-seq.

first: Xin

last: Long

first: Chan

last: Gu


title: Induction of T cell dysfunction and NK-like T cell differentiation in vitro and in patients after CAR T cell treatment [ATAC-seq]

city: Philadelphia

first: Gregory

last: Donahue

address: 3400 Civic Center Blvd, Bldg 421

department: Cell & Developmental Biology

institute: The University of Pennsylvania

summary: Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies but remains largely ineffective in solid tumors. A major factor leading to the reduced efficacy of CAR T cell therapy is T cell dysfunction, and the mechanisms mediating this dysfunction are under investigation. Here we establish a robust in vitro model to study mesothelin-redirected CAR T cell dysfunction in pancreatic cancer. Continuous antigen exposure results in hallmark features of exhaustion including reduced proliferation capacity and cytotoxicity, and severe defects in cytokine production. Here we identified a transcriptional signature at both population and single-cell levels in CAR T cells after chronic antigen exposure. In addition, TCR lineage tracing revealed a CD8+ T-to-NK-like T cell plasticity that results in reduced antigen- dependent tumor cell killing. The transcription factors SOX4 and ID3 are specifically expressed in the dysfunctional CAR NK-like T cells and are predicted to be master regulators of the dysfunction gene expression signature and of the post-thymic acquisition of an NK-like T cell fate. Finally, we identified the emergence of CAR NK-like T cells in a subset of patients after infusion of CAR T cells. The findings gleaned from this study reveal new approaches to improve the efficacy of CAR T cell therapy in solid tumors by preventing or revitalizing CAR T cell dysfunction and shed light on the plasticity of human CAR T cells.

overall_design: Examines chromatin openness by paired-end ATAC-seq in CD8+ T cells across four biological replicates, with one variable:  antigen exposure (values: control or continuous). Some samples are re-sequenced and were pooled to add coverage; see Data Processing section for details.    Replicates are R2, R3, R4, and R5. Each has a single control day 0 and a continuous day 28 sample.

first: Shunichiro

last: Kuramitsu

first: Charly

last: Good

first: Parisa

last: Samareh

first: Greg

first: Kenichi

last: Ishiyama

first: Yujie

last: Ma

first: Nils

last: Wellhausen

first: Lifeng

last: Tian

first: Sangya

last: Agarwal

first: Sonia

last: Guedan

first: M

last: Aznar

first: Katherine

last: Alexander

first: Zhen

last: Zhang

first: Nathan

last: Singh

first: Max

middle: W

last: Richardson

first: Keisuke

last: Watanabe

first: Janos

last: Tanyi

middle: H

last: O'Hara

first: Marco

last: Ruella

first: Simon

middle: F

last: Lacey

first: Edmund

last: Moon

first: Stephen

last: Schuster

first: Steven

last: Albelda

first: Lewis

last: Lanier

first: Regina

last: Young

first: Shelley

last: Berger

first: Carl

last: June


title: ZNF146/OZF and ZNF507 target LINE-1 sequences

city: Worcester

first: Kevin

last: Creamer

state: MA

address: 55 Lake Ave N

institute: UMMS

summary: Repetitive sequences including transposable elements (TEs) and transposon-derived fragments account for nearly half of the human genome. While transposition-competent TEs must be repressed to maintain genomic stability, mutated and fragmented TE sequences can also contribute to regulation of host gene expression. To gain further insight into the function of LINE-1 targeting proteins ZNF146 and ZNF507, we disrupt their expression using CRISPR/Cas9 and perform RNA sequencing in HEK293 cells.

overall_design: RNA-sequencing of HEK293 cells after CRISPR/Cas9 disruption of ZNF146 and ZNF507 compared to a control, each in biological duplicate.

first: Eric

last: Larsen

first: Jeanne

last: Lawrence


title: Induction of T cell dysfunction and NK-like T cell differentiation in vitro and in patients after CAR T cell treatment [scTCR-seq]

first: Gregory

last: Donahue

overall_design: Examines presence of T cell receptor (TCR) peptides by single-cell sequencing in CD8+ T cells from two donors. One experimental variable: exposure (values: control day 0 and continuous antigen exposure).

first: Shunichiro

last: Kuramitsu

first: Charly

last: Good

first: Parisa

last: Samareh

first: Greg

first: Kenichi

last: Ishiyama

first: Yujie

last: Ma

first: Nils

last: Wellhausen

first: Lifeng

last: Tian

first: Sangya

last: Agarwal

first: Sonia

last: Guedan

first: M

last: Aznar

first: Katherine

last: Alexander

first: Zhen

last: Zhang

last: Singh

first: Mark


title: SHARC-seq: High throughput in vivo determination of 3D RNA structures and dynamics

city: Los Angeles

first: Minjie

state: CA

address: 1985 Zonal Avenue

department: Department of Pharmacology and Pharmaceutical Sciences

institute: University of Southern California

summary: RNA molecules not only carry genetic information like DNA, but also folds into exquisite 3D structures like proteins. Despite strong interests in RNA biology and their medical applications, RNA structure determination in vivo remained a long-standing problem. Here we developed a new technology to directly determine RNA structures in vivo, termed SHARC-seq. Applying SHARC-seq, spatial distances among nucleotides can be measured and used to rebuild in vivo RNA 3D structure and dynamics.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design:
Cells and tissues are crosslinked with 5mM, 12.5mM and 25mM SHARC, and crosslinked RNA is extracted using new developed TNA method. Extracted total RNA were fragmented with RNase III. After fragmentation, proximity ligation and reverse crosslinking, RNA are converted into cDNA library for high-throughput sequencing.
In vitro transcribed 1HR2 are crosslinked with 5mM SHARC. Crosslinked 1HR2 RNA were fragmented with RNase III. After fragmentation, proximity ligation and reverse crosslinking, RNA are converted into cDNA library for high-throughput sequencing.

first: Kongpan

last: Li

first: Zhipeng

last: Lu


title: Gene expression data from bladder cancer patient-derived cells treated with shRNA targeting ALDH1A1

city: Hidaka

first: Kazuhiro

last: Ikeda

state: Saitama

address: 1397-1 Yamane

country: Japan

institute: Saitama Medical University

summary: To examine the role of ALDH1A1 in bladder cancer ｐａｔｉｅｎｔ-derived cells (PDCs), BC-1 and -2 cells were treated with shRNA targeting ALDH1A1 (shALDH1A1) or control shRNA (shControl). ALDH1A1 knockdown study showed that TUBB3 is one of the downregulated genes in PDCs.

overall_design: Total RNA was extracted from bladder cancer PDCs treated with shALDH1A1 or shControl, and then used for Affymetrix microarrays analysis.

first: Takeshi

last: Namekawa

first: Kazuiro

first: Kuniko

last: Horie-Inoue

first: Satoshi

last: Inoue


title: A Point Mutation R122C in RUNX3 Promotes the Expansion of Isthmus Stem Cells and Inhibits Their Differentiation in the Stomach

status: Public on Dec 05 2021

city: Singapore

first: Daisuke

last: Douchi

address: 14 Medical Drive

department: Centre for Translational Medicine

institute: Cancer Science Institute of Singapore

summary:
Background & Aims: RUNX transcription factors play pivotal roles in embryonic development and neoplasia. We previously identified the single missense mutation R122C in RUNX3 from human gastric cancer. However, how RUNX3R122C mutation disrupts stem cell homeostasis and promotes gastric carcinogenesis remained unclear.
Methods: To understand the oncogenic nature of this mutation in vivo, we generated the RUNX3R122C knock-in mice. Stomach tissues were harvested, followed by histological and immunofluorescence staining, organoid culture, flow cytometry to isolate gastric corpus isthmal and non-isthmal epithelial cells, and RNA extraction for transcriptomic analysis.
Results: The corpus tissue of RUNX3R122C/R122C homozygous mice exhibited a precancerous phenotype such as spasmolytic polypeptide-expressing metaplasia (SPEM). We observed mucous neck cell hyperplasia, massive reduction of pit, parietal, and chief cell populations, as well as a dramatic increase in the number of rapidly proliferating isthmal stem/progenitor cells in the corpus of RUNX3R122C/R122C mice. Transcriptomic analyses of the isolated epithelial cells showed that the cell cycle-related MYC target gene signature was enriched in the corpus epithelial cells of RUNX3R122C/R122C mice compared with the wild-type corpus. Mechanistically, RUNX3R122C mutant protein disrupted the regulation of the restriction point where cells decide to enter either proliferative or quiescent state, thereby driving stem cell expansion and limiting the ability of cells to terminally differentiate.
Conclusions: RUNX3R122C missense mutation is associated with the continuous cycling of isthmal stem/progenitor cells, maturation arrest and development of a precancerous state. This work highlights the importance of RUNX3 in prevention of metaplasia and gastric cancer.

overall_design: RNA expression profile of stomach corpus tissue of 6 months old mice. Total RNA was extracted from the corpus tissue.  Triplicate samples are shown.

first: Yoshiaki

last: Ito

first: Akihiro

last: Yamamura

first: Junichi

last: Matsuo

first: Jung-Won

last: Lee

first: Napat

last: Nuttonmanit

first: Yi

last: Lim

first: Kazuto

last: Suda

first: Mitsuhiro

last: Shimura

first: Sabirah

last: Chen

first: ShuChin

last: Pang

first: Kazuyoshi

last: Kohu

first: Mari

last: Kaneko

first: Hiroshi

last: Kiyonari

first: Atsushi

last: Kaneda

first: Hideyuki

last: Yoshida

first: Ichiro

last: Taniuchi

first: Henry

last: Yang

first: Michiaki

last: Unno

first: Jimmy

middle: B

last: So

first: Khay

last: Yeoh

first: Linda

last: Chuang

first: Suk-Chul

last: Bae

first: Motomi

last: Osato


title: mRNA-sequencing analysis of peripheral blood cells from an aggressive refractory pemphigus vulgaris patient

status: Public on Dec 04 2021

first: Siji

address: Qingchun East road

institute: Department of Dermatology and Venereology, Sir Run-Run Shaw Hospital, Zhejiang University School of Medicine

summary: Pemphigus vulgaris (PV) is a kind of IgG-mediated autoimmune blistering disease (AIBD). The peripheral immune system plays a vital role in the pathophysiology of pemphigus vulgaris. This study aimed to explore the changes of peripheral immune cells of the patient before and after medication administration.

overall_design: 3 samples of a pemphigus vulgaris patient obtained from three time points: hospitalization, discharge, and one-week follow-up.

first: Hao

last: Cheng


title: ERK3-MK5 signaling regulates myogenic differentiation and muscle regeneration by promoting FoxO3 degradation

city: Montreal

first: Sylvain

last: Meloche

state: Quebec

address: 2950, Chemin de Polytechnique

country: Canada

institute: Institute of Research in Immunology and Cancer

summary: The physiological functions and downstream effectors of the atypical mitogen-activated protein kinase ERK3 remain to be characterized. We recently reported that mice expressing catalytically-inactive ERK3 (Mapk6KD/KD) exhibit a reduced post-natal growth rate as compared to control mice. Here, we show that genetic inactivation of ERK3 impairs post-natal skeletal muscle growth and adult muscle regeneration after injury. Loss of MK5 phenocopies the muscle phenotypes of Mapk6KD/KD mice. At the cellular level, genetic or pharmacological inactivation of ERK3 or MK5 induces precocious differentiation of C2C12 or primary myoblasts, concomitant with MyoD activation. Reciprocally, ectopic expression of activated MK5 inhibits myogenic differentiation. Mechanistically, we show that MK5 directly phosphorylates FoxO3, promoting its degradation and reducing its association with MyoD. Depletion of FoxO3 rescues in part the premature differentiation of C2C12 myoblasts observed upon inactivation of ERK3 or MK5. Our findings reveal that ERK3 and its substrate MK5 act in a linear signaling pathway to control post-natal myogenic differentiation.

overall_design: We compared the transcriptomes of control C2C12 cells (n=3), ERK3-depleted C2C12 cells (n=3) and MK5-depleted C2C12 cells (n=3) in growth medium (GM) and after 2 days of differentiation in diffrentiation medium (DM2).


title: Quiescent human glioblastoma cancer stem cells mediate tumor initiation, expansion, and recurrence following chemotherapy

city: New York

first: Mungunsarnai

last: Ganbold

state: NYC

address: 417 East 68st

department: Brain Tumor Center

institute: MSKCC

summary: We performed single cell RNA sequencing with eight patient derived xenograft samples to identify quiescent cancer stem cells in glioblastoma. Additionally two samples treated with temozolomide was sequenced to examine the evolution of the cancer stem cells upon chemotherapeutics.

overall_design: whole transcriptome single cell RNA sequencing with 10X method

first: Xuanhua

last: Xie

first: Dan

last: Laks

first: Luis

last: Parada


title: 10X Visium Spatial transcriptomics of murine colon in steady state and during recovery after DSS colitis

city: Solna

first: Ludvig

middle: Ale

last: Larsson

state: Stockholm

address: Tomtebodavägen 23A

department: Gene Technology

institute: SciLifeLab

summary: The limitation of single-cell or bulk transcriptomic profiling is the lack of spatial topographical context. Spatial transcriptomics (ST) allows sequencing of polyadenylated transcripts from a tissue section which can be spatially mapped onto the histological brightfield image using an array of barcoded oligo-dT capturing probes. Using the 10X Visium platform, here, we unbiasedly characterized the spatial transcriptomic landscape of murine colon in steady state and during mucosal healing upon dextran sodium sulfate (DSS) induced injury

overall_design: Wild type C57BL/6J mice were exposed to DSS in drinking water for 7 days and then allowed to recover for 7 more days. Mouse colonic tissues were collected either on day 0 (without any exposure to DSS) or on day14 after exposure to DSS, Swisrolled and frozen in OCT. Mouse colonic tissues (day0 and day14) were cryo-sectioned (10uM) and placed on the already barcoded spots (6.5mm x 6.5mm) on the glass slide. Samples were processed according to 10X Visium spatial transcriptomics protocol and submitted for sequencing.

first: Srustidhar

last: Das

first: Eduardo

last: Villablanca


title: miRNA array of clear cell renal cell carcinoma from patients treated with sunitinib

status: Public on Dec 03 2021

city: Asan-si

first: Dayeon

last: Kang

address: 22, Soonchunhyang-ro, Sinchang-myeon

country: South Korea

institute: Soon Chun Hyang University

summary: A multiple receptor tyrosine kinase inhibitor, sunitinib, is a first-line therapy for clear cell renal cell carcinoma (CCRCC). Unfortunately, it has the major challenges of low initial response rate and resistance after about one year of treatment. Here we evaluated a microRNA (miRNA) and its target responsible for sunitinib resistance. Using miRNA profiling, we identified miR-96-5p upregulation in tumors from sunitinib-resistant CCRCC patients

overall_design: We performed global miRNA profiling using frozen tissues from CCRCC tumors and adjacent noncancerous kidney tissues of six patients who received sunitinib treatment. The mean progression-free survival (PFS) and OS of the resistant group were 4.7 ± 3.1 and 7.3 ± 5.5 months, respectively. The corresponding values in the sensitive group were 41.3 ± 9.3 and 83.3 ± 18.2 months, respectively.

first: Jung

last: Hwang

first: Nayoung

last: Suh

first: Choung-Soo

last: Kim

first: Sang

middle: E

last: Park

first: Wonju


title: RNA-seq of MDA-MB-231/Tet-shHOTAIR and MDA-MB-231/Tet-shCtrl cells

city: Taichung

first: Shao-Chun

last: Wang

address: No. 6 Xueshi Rd

country: Taiwan

institute: China Medical University Hospital

summary: To further understand the cellular function of HOTAIR, we have profiled RNA expression in breast cancer cell lines harboring tetracycline-inducible shRNA of human HOTAIR (Tet-shHOTAIR) or the control shRNA of scrambled sequence (Tet-shCtrl).

overall_design: 3 independent biological repeats of MDA-MB-231/Tet-shHOTAIR and MDA-MB-231/Tet-shCtrl cells were performed with induction by 1 ug/ml doxycycline for 96hr

first: Yuan-Liang


title: TDP-43 represses cryptic exon inclusion in FTD/ALS gene UNC13A

city: Stanford

first: Rosa

last: Ma

address: 279 Campus Dr.

department: Genetics

institute: Stanford University

summary: Through splicing analysis of a publicly available RNA-Seq dataset, we discovered TDP-43 represses a cryptic exon splicing event in UNC13A, a gene that had been associated with FTD/ALS through GWA studies. To confirm the sequences of the cryptic exons, we used shRNA to reduce TDP-43 levels in iPSC-derived motor neurons (iPSC-MNs) and by amplicon sequencing the RT-PCR product, we observed the insertion in cells with TDP-43 depletion but not in control shRNA-treated cells. Through sequence alignment, we verified the sequences of the cryptic exons.

overall_design: iPSC-derived motor neurons incubated fwith lentivirus particles for shScramble or shTDP-43.

first: X

first: Georgiana

last: Miller

first: Katherine

last: Kurylo

first: Shila

last: Mekhoubad

first: Fred

last: Harbinski

first: Beryl

last: Cummings

first: Nolan

last: Nichols

first: Aaron

middle: D

last: Gitler


title: High throughput sequencing of atypical progeria caused by LMNA R527C mutation

city: Beijing

first: Ming

last: Kuang

address: Xueyuan Road 38 #, Haidian District

institute: Peking University

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

overall_design: Refer to individual Series


title: RNA seq analysis of T cells，EpCAM CAR-T cells and rapamycin-pretreated EpCAM CAR-T cells

city: hefei

first: zhigang

last: nian

state: anhui

address: 443 Huang-Shan Road

department: school of life science

institute: university of science and technology of China

summary: Purpose: Compared the differences in the transcriptome of T cells versus EpCAM CAR-T cells and EpCAM CAR-T cells versus rapamycin-pretreated EpCAM CAR-T cells.Methods: Next-generation sequencing (NGS) has revolutionized the systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) between T cells and EpCAM CAR-T cells，and between EpCAM CAR-T cells and rapamycin-pretreated EpCAM CAR-T cells.  Results: RNA-seq showed that CAR-T cells significantly upregulated the expression of exhaustion markers compared to T cells. Gene Set Enrichment Analysis (GSEA) analysis showed that CAR-T cells cells were enriched for signatures of apoptosis. Compared with T cells, Kyoto Encyclopedia of Genes and Genomessignaling(KEGG) pathway analysis showed that the PI3K/AKT/mTOR signaling pathway was significantly enriched. Likewise, GSEA showed that the mTOR signaling pathway was significantly upregulated in CAR-T cells. Next, we analyzed the RNA-seq data between CAR-T cells and rapamycin-pretreated CAR-T cells. RNA-seq showed that rapamycin-treated CAR T down-regulated the expression of exhaustion markers compared with untreated CAR-T cells. GSEA analysis showed that the apoptosis-related gene set was enriched in untreated CAR-T cells.Conclusions:In vitro culture promotes the terminal differentiation of CAR-T cells.CAR-T cells present a highly activated mTOR phenotype. Rapamycin pretreatment prevents CAR-T cells terminal differentiation.

first: Zhigang

last: Nian

first: Xiaohu

last: Zheng


title: mRNA sequence analysis of zebrafish embryos injected with the codon tag reporter library [AnsB]

city: Kita-ku

first: Yuichiro

last: Mishima

state: Kyoto

address: Motoyama, Kamigamo,

department: Department of Frontier Life Sciences

institute: Kyoto Sangyo University

summary: We measured the effect of codons on mRNA stability in zebrafish embryos using reporter mRNAs. We showed that slow decoding of codons by the ribosome promoted mRNA degradation in zebrafish.

overall_design: zebrafish embryonic mRNA profile at 2 different stages (2 hpf and 6 hpf) in control and AnsB-expressing embryos. Experiments are performed as duplicates for AnsB.


title: Transcriptome analysis revealed the symbiosis niche of 3D scaffolds to accelerate bone defect healing

city: Shanghai

first: Ce

last: Ji

address: 197 Ruijin 2nd Road, Shanghai

institute: Shanghai Jiaotong University

summary: 3D printed scaffolds have been shown to be superior in promoting tissue repair, but the cell-level specific regulatory network activated by 3D printing scaffolds with different material components to form a symbiosis niche have not been systematically revealed. Here, three typical 3D printed scaffolds, including natural polymer hydrogel (Gelatin-methacryloyl, GelMA), synthetic polymer material (Polycaprolactone, PCL) and bioceramic (β-tricalcium phosphate, β-TCP), were fabricated to explore the regulating effect of the symbiotic microenvironment during bone healing. Enrichment analysis showed that hydrogel promotes tissue regeneration and reconstruction by improving blood vessel generation by enhancing oxygen transport and red blood cell development. The PCL scaffold regulates cell proliferation and differentiation by promoting cellular senescence, cell cycle and DNA replication pathways, accelerating the process of endochondral ossification and the formation of callus. The β-TCP scaffold can specifically enhance the expression of osteoclast differentiation and extracellular space pathway genes to promote the differentiation of osteoclasts and promote the process of bone remolding. In these processes, specific biomaterial properties can be used to guide cell behavior and regulate molecular network in the symbiotic microenvironment to reduce the barriers of regeneration and repair.

overall_design: mRNA profiles of 2weeks regenerated bone

first: Wenguo

last: Cui

first: Jin

last: Qi

first: Lianfu

last: Deng


title: EndoTime: Non-categorical timing estimates for endometrial tissue during the secretory phase

city: Coventry

first: Jan

last: Brosens

address: University Hospital Coventry and Warwickshire

department: Warwick Medical School

country: United Kingdom

institute: University of Warwick

summary: It is estimated that 1% of women suffer from Recurrent Pregnancy Loss (RPL), a debilitating condition currently defined as three or more consecutive miscarriages with no known preventative treatment. However, as 65% of women are able to successfully bear children following diagnosis the risk of RPL appears to be relatively transient. The foundation for embryo implantation is the endometrium – a complex heterogenous tissue that is also transitory in nature, and which dynamically cycles between phases of proliferation, maintenance and elimination. In order to identify biomarkers for the prediction of pregnancy outcome, we generated RNA-seq libraries of 36 endometrial biopsies from women with a quantified pregnancy history. Each biopsy was timed relative to the pre-ovulatory luteinizing hormone surge to coincide with the midluteal implantation window (reported as LH+ for days following the surge), and later associated with the outcome of pregnancy: ‘live birth’, ‘miscarriage’ or ‘ongoing after 12 weeks’. This sample set was anticipated to represent mid-luteal tissues, but observed temporal signatures suggested that this assignment was incorrect. We developed EndoTime as a result of these findings, utilising RT-qPCR expression data for a panel of six temporal marker genes in order to estimate endometrial biopsy timing along a continuous domain.

overall_design: Endometrial mRNA profiles of 36 independent midluteal biopsies were generated by deep sequencing.

first: Emma

middle: S

last: Lucas

first: Pavle

last: Vrljicak

first: Joanne

last: Muter

first: Sascha

last: Ott


title: Determine the chromatin association change of H3K27me3 and PRC2  in response to PRC2 subtype change or stable CTR9 KD in MCF7 cells

status: Public on Dec 02 2021

city: Madison

first: NGAI TING

last: CHAN

state: WI

address: 4849 Sheboygan Ave

department: Oncology

institute: University of Wisconsin -- Madison

summary: CTR9 is the scaffold subunit in PAFc (Polymerase associated factor complex), a multifunctional complex employed in multiple steps of RNA Pol II-mediated transcription. CTR9/PAFc is well known as an evolutionarily conserved elongation factor regulating gene activation via coupling with histone modifications enzymes. However, little is known about its function to restrain repressive histone markers. Using inducible and stable CTR9 knockdown breast cancer cell lines, we discovered that the amount of H3K27me3 is strictly controlled by CTR9. Quantitative profiling on histone modifications revealed a striking increase of H3K27me3 level upon loss of CTR9. Moreover, loss of CTR9 leads to genome-wide expansion of H3K27me3, as well as increased recruitment of PRC2 on chromatin, which can be reversed by CTR9 restoration. Moreover, CTR9 depletion triggers a PRC2 subtype switch from less active PRC2.2 to PRC2.1 with higher methyltransferase activity. As a consequence, CTR9 depletion generates vulnerability that renders breast cancer cells hypersensitive to PRC2 inhibitors. Our findings that CTR9 demarcates PRC2-mediated H3K27me3 levels and genomic distribution provide a unique mechanism of transition from transcriptionally active to repressive chromatin states and shed light on the biological functions of CTR9 in development and cancer.

overall_design: 1. ChIP-Rx of H3K27me3 and SUZ12 (PRC2 core subunit) in MCF7 cells with Control KD (shControl), PRC2.2 KD (shPRC2.2), PRC2.1 overexpression (PRC2.1-OE) and CTR9 KD (shCTR9) -- 2 replicates (PS: every ChIP reaction was incorportated with same amount of spike-in control).

first: Ngai

middle: T

last: Chan


title: Abnormal exocrine-endocrine cell crosstalk promotes β-cell dysfunction and loss in MODY8

city: Boston

first: Hui

last: Pan

address: 1 Joslin Place

department: Bioinformatics and Biostatistics Core

institute: Joslin Diabetes Center

summary: MODY8 (maturity-onset diabetes of the young, type 8) is a dominantly inherited monogenic form of diabetes associated with frameshift mutations in the carboxyl ester lipase (CEL) gene expressed by pancreatic acinar cells. Patients carrying the mutation develop childhood-onset exocrine pancreas dysfunction followed by the manifestation of diabetes during adulthood. However, it is unclear how CEL mutations cause diabetes. Here we report the transfer of proteins from acinar cells to β-cells as a form of crosstalk between exocrine and endocrine cells. Human β-cells showed a relatively higher propensity for internalizing the mutant versus the wild-type CEL protein. Following internalization, the mutant protein formed stable intracellular aggregates leading to β-cell secretory dysfunction. To investigate the effects in vivo, we transplanted stem cell-derived wild-type or mutant pancreatic progenitors into independent mice and generated insulin-secreting β-like cells and CEL-expressing acinar-like cells. While the β-like cells derived from wild-type stem cells responded to glucose, the β-like cells derived from mutant stem cells were dysfunctional secondary to the accumulation of intracellular mutant CEL protein. Analysis of pancreas sections from a MODY8 patient also revealed the presence of CEL protein in the few extant β-cells. This study provides compelling evidence for the mechanism by which a mutant gene expressed specifically in acinar cells promotes dysfunction and loss of β-cells to cause diabetes.

overall_design: EndoC-βH1 cells were cultured in conditioned media collected from HEK293 cells overexpressing either the empty vector (EV), wild-type CEL gene (WT) or mutant CEL gene (MUT) for 10 days. Conditioned media was refreshed every day. Cells were lysed in RLT buffer and then RNA was extracted using Qiagen RNeasy kit according to the manufacturer’s instructions. RNAseq was performed on an Illumina NovaSeq 6000 according to the manufacturer’s instructions.

first: Sevim

last: Kahraman

first: Rohit

middle: N

last: Kulkarni


title: mRNA sequence analysis of zebrafish embryos injected with the codon tag reporter library

overall_design: zebrafish embryonic mRNA profile at 2 different stages (2 hpf and 6 hpf) in wildtype and znf598 mutant. All experiments are performed as duplicates


title: Effect of FACT depletion on the cell composition of bone marrow and intestine in mice

city: Buffalo

first: Katerina

last: Gurova

state: NY

address: Elm and Carlton Str

department: Cell Stress Biology

institute: Roswell Park Cancer Institute

summary: Preservation of nucleosomes during replication has been extensively studied, while the maintenance of nucleosomes during transcription has gotten less attention. The histone chaperone FACT is involved in transcription elongation, although whether it disassembles or assembles nucleosomes during the passage of RNA polymerase is still unclear. We deleted the FACT subunit in adult mice to clarify the function of FACT in mammals. FACT loss was lethal due to bone marrow and intestinal failure, where the earliest progenitors completely vanished, while several other cell types were increased or decreased. Using cells isolated from several tissues, we showed that FACT loss was lethal only for stem cells but not cells differentiated in vitro. FACT depletion led to increased chromatin accessibility in a transcription-dependent manner, suggesting that nucleosomes are lost from transcribed regions in the absence of FACT. The most prominent response to the loss of chromatin integrity was the activation of interferon signaling and the accumulation of immunocytes in sensitive organs. FACT maintained chromatin integrity during transcription in mammalian adult stem cells, suggesting that chromatin transcription in these cells is different from more differentiated cells.

overall_design: Ssrp1fl/fl; CreERT2+/+ mice of 10-15 weeks of age were treated with vehicle or tamoxifen (2mg, IP, every day for 5 days). 24 hours after the end of treatment bone marrow and small and large intestine (2cm before and after secum, excluding secum) were collected from  mice. Samples from two mice from the same treatement group were pooled together.

first: Jianmin

last: Wang


title: Exploratory study reveals far reaching systemic and cellular effects of verapamil treatment in subjects with type 1 diabetes

city: Ithica

first: Praveen

last: Sethupathy

address: Cornell University College of Veterinary Medicine, Box 17

institute: Cornell University

summary: Currently, no oral medications are available for individuals suffering from type 1 diabetes (T1D). Our randomized placebo-controlled phase 2 trial recently revealed that oral verapamil has short- term beneficial effects in subjects with new-onset type 1 diabetes (T1D) 1. However, what exact biological changes verapamil elicits in humans with T1D, how long they may last, and how to best monitor any associated therapeutic success has remained elusive. We therefore now conducted extended analyses of the effects of continuous verapamil use over a 2-year period, performed unbiased proteomics analysis of serum samples and assessed changes in proinflammatory T-cell markers in subjects receiving verapamil or just standard insulin therapy. In addition, we determined the verapamil-induced changes in human islets using RNA sequencing. Our present results reveal that verapamil regulates the thioredoxin system and promotes an anti-oxidative and anti-apoptotic gene expression profile in human islets, reverses T1D-induced elevations in circulating proinflammatory T-follicular-helper cells and interleukin-21 and normalizes serum levels of chromogranin A (CHGA), a recently identified T1D autoantigen 2,3. In fact, proteomics identified CHGA as the top serum protein altered by verapamil and as a potential therapeutic marker. Moreover, continuous use of oral verapamil delayed T1D progression, promoted endogenous beta cell function and lowered insulin requirements and serum CHGA levels for at least 2 years and these benefits were lost upon discontinuation. Thus, the current studies provide crucial mechanistic and clinical insight into the beneficial effects of verapamil in T1D.

overall_design: Treatment of islet cells with compond (verapamil) followed by transcriptomics.

first: Anath

last: Shalev

first: Matt

last: Kanke


title: RNA Sequencing in COVID-19 patients identifies neutrophil activation biomarkers as promising diagnostic platform for infections.

city: Washington

first: Timothy

last: McCaffrey

state: DC

address: 2300 I St NW, Ross 203

department: Medicine

institute: George Washington University

summary: Infection with the SARS-CoV2 virus can vary from asymptomatic, flu-like with moderate disease, to critically severe.  Severe disease, termed COVID-19, involves acute respiratory deterioration that is frequently fatal.  To understand the highly variable presentation, and identify biomarkers for disease severity, blood RNA from COVID-19 patient in an intensive care unit was analyzed by whole transcriptome RNA sequencing.  Both SARS-CoV2 infection and the severity of COVID-19 syndrome were associated with up to 25-fold increased expression of neutrophil-related transcripts, such as neutrophil defensin 1 (DEFA1), and 3-5-fold reductions in T cell related transcripts such as the T cell receptor (TCR).  The DEFA1 RNA level detected SARSCoV2 viremia with 95.5% sensitivity, when viremia was measured by ddPCR of whole blood RNA.  Purified CD15+ neutrophils from COVID-19 patients were increased in abundance and showed striking increases in nuclear DNA staining by DAPI.  Concurrently, they showed >10-fold higher elastase activity than normal controls, and correcting for their increased abundance, still showed 5-fold higher elastase activity per cell.  Despite higher CD15+ neutrophil elastase activity, elastase activity was extremely low in plasma from the same patients.  Collectively, the data supports the model that increased neutrophil and decreased T cell activity is associated with increased COVID19 severity, and suggests that blood DEFA1 RNA levels and neutrophil elastase activity, both involved in neutrophil extracellular traps (NETs), may be informative biomarkers of host immune activity after viral infection.

overall_design:
38 samples from unique COVID-19+ (SARS-CoV-2+) patients and 4 samples from unique healthy control volunteers were analyzed. Of those groups, 20 COVID-19+ samples and 4 healthy control samples underwent RNA sequencing.
NOTE FROM SUBMITTER: We are not submitting raw files because of concern for our patients' privacy.

first: Richard

last: Wargowsky

first: Philip

last: Dela Cruz

first: John

last: Lafleur

first: David

last: Yamane

first: Justin

last: Kim

first: Ivy

last: Benjenk

first: Eric

last: Heinz

first: Obinna

last: Ome Irondi

first: Katherine

last: Farrar

first: Ian

last: Toma

first: Tristan

last: Jordan

first: Jennifer

last: Goldman


title: Canine DUXC isoforms and functional similarity to human DUX4 [RNA-seq]

city: Seattle

first: Stephen

last: Tapscott

state: WASHINGTON

address: 1100 Fairview N. Ave

department: Human Biology

institute: Fred Hutch Cancer Research Center

summary: Human DUX4 and mouse Dux transcription factors are normally expressed in the germ line and early embryonic cells where they activate the cleavage stage genes. The misexpression of DUX4 in skeletal muscles causes Facioscapulohumeral dystrophy (FSHD). In primates and rodents, DUX4 and Dux arose from retrotransposition of the mRNA from the ancestral intron-containing DUXC, which is not found in these species, and whether it has similar roles in the cleavage stage and FSHD as DUX4 and Dux are unknown. Here, we identified two isoforms of DUXC in canine testis tissues: One encodes the canonical double homeodomain (DUXC) similar to DUX4/Dux, and one contains an extra exon that disrupts the conserved amino-acid sequence of the first homeodomain (DUXC-ALT). We expressed DUXC and DUXC-ALT in canine skeletal muscle and found that the expression of DUXC induced retrotransposons and pluripotent programs similar to DUX4 and Dux, whereas DUXC-ALT did not robustly activate genes in these assays.

overall_design: Determined the transcriptome of DUXC and DUXC-ALT by using a system that parallels tissue culture models of the human DUX4 transcriptome, forced expression of DUX4, DUXC, and DUXC-ALT transcription factors in cultrued canine skeletal muscle cells. We generate three doxycycline-inducible stable canine myoblast cell lines in which addition of doxycycline to the cell culture medium caused expression of three transcription factors and Luciferase (as a negative control).

last: Whiddon

first: Chao-Jen

last: Wong

first: Ashlee

last: Langford

first: Andrea

last: Belleville


title: Virally Encoded Connectivity Transgenic Overlay RNA sequencing (VECTORseq) defines projection neurons involved in sensorimotor integration

city: San Francisco

first: Victoria

last: Cheung

address: 1550 4th Street

institute: University of California, San Francisco

summary:
Purpose: Establish a high-throughput method to transcriptionally define projection neurons, VECTORseq, that reimagines transgenes expressed by widely used retrogradely infecting viruses as multiplexed RNA barcodes that are detected in single-cell sequencing.
Methods: mRNA profiles of adult mouse brains
Conclusions:  Retrograde viruses express mRNA at levels detectable in single-cell sequencing. Different transgenes can be multiplexed in a single sequencing run. VECTORseq identifies both cortical and subcortical projection neurons. VECTORseq defined new superior colliculus and zona incerta projection populations. Established a high-throughput method to transcriptionally define projection neurons, VECTORseq, that reimagines transgenes expressed by widely used retrogradely infecting viruses as multiplexed RNA barcodes that are detected in single-cell sequencing.

overall_design: single cell seq datasets of adult mouse brain regions (V1, SC, Ventral Midbrain)


title: Functional precision medicine pipeline combines comparative transcriptomics and tumor organoid modeling to identify be-spoke treatment strategies for glioblastoma

city: Little Rock

first: Analiz

last: Rodriguez

state: AR

address: 4301 W. Markham St Slot 507

department: College of Medicine, Department of Neurosurgery

institute: University of Arkansas for Medical Sciences

summary: Li-Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome caused by germline mutations in TP53. TP53 is the most common mutated gene in human cancer occurring in 30-50% of glioblastomas (GBM). Here, we highlight a precision medicine platform to identify potential targets for a GBM patient with LFS. We used novel comparative transcriptomics analysis to identify genes that are uniquely overexpressed in a LFS GBM patient relative to a cancer compen-dium of 12,747 tumor RNA sequencing datasets including 200 GBMs. We then used ex vivo pa-tient derived organoid (PDO) viability assays with 4 patient derived cell lines to test efficacy of our identified target. Our comparative transcriptomics bioinformatics pipeline identified that STAT1 and STAT2 were significantly overexpressed in our patient indicating ruxolitinib, a Janus kinase 1 and 2 inhibitor, as a potential therapy. In our institutional high-grade glioma cohort of 45 patients, the LFS patient had the highest level of STAT1 and STAT2 expression.  STAT1 and STAT2 expression levels in 4 cell lines derived from patients (including the LFS patient) corre-lated with levels identified in the respective parent tumors.  Using 2D and 3D assays from pa-tient derived cells, our LFS patient of interest was among the most sensitive to ruxolitinib in comparison to patients with lower STAT1 and STAT2 expression levels. Additionally a sphe-roid-based drug screening assay (3D-PREDICT) was performed and used to identify further therapeutic targets. This manuscript supports the use of comparative transcriptomics to identify personalized therapeutic targets in a functional precision medicine platform for malignant brain tumors.

overall_design: mRNA expression levels derived from RNA-seq for one patient tumor sample and four cell lines derived from patient tumors.

first: Megan

last: Reed

first: Olena

middle: M

last: Vaske


title: Temporal gene expression changes following EWSR1/FLI1 inhibition in EW-8 cells

city: San Antonio

first: Yidong

last: Chen

state: Texas

address: 8403 Floyd Curl Drive, MSC 7784

department: Population Health Sciences

institute: UT Health Science Center at San Antonio

summary: Human Ewing sarcoma cell line EW-8 with different levels of EWSR/FLI1 using siRNA-EWSR1 were collected and profiled using single-cell RNA-seq. The single-cell RNA-sequencing data identified diverse reponses leading to the prediction of two rare cell identities in an unperturbed population (si-Control) with stem-like and dormant-like characterisitics. These newly discovered cell states may play important roles in undrstanding cancer heterogeneity, drug resistance and recurrence in Ewing sarcoma .

overall_design: Three population of EW-8 cells were studied for their gene expression information in a single cell level (Unperturbed EW-8 or si-Control, Dormant 1 population or si-EWSR1-48h, Dormant 2 population or si-EWSR1-120h)

first: Peter

last: Houghton

first: Roxane

last: Khoogar

first: Zhao

last: Lai


title: Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis

first: Leonard

last: Zon

address: 1 Blackfan Circle

department: Oncology/Hematology

institute: Boston Children's Hospital


title: Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I]

summary: Clonal hematopoiesis (CH) results from enhanced fitness of a mutant hematopoietic stem and progenitor cell (HSPC), but how such clones expand is unclear. Here, we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH-associated genes, like asxl1, promoted clonal dominance. Single-cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti-inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator, nr4a1, abrogated the ability of asxl1-mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny.

overall_design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells, we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to adulthood. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.

first: Serine

last: Avagyan

first: Song

last: Yang

middle: I


title: Combined intermittent and sustained hypoxia is a novel and deleterious cardio-metabolic phenotype

status: Public on Dec 01 2021

city: La Jolla

first: Omar

middle: A

last: Mesarwi

state: California

address: 9500 Gilman Drive, MTF 240

institute: University of California San Diego

summary:
Study objectives: Chronic obstructive pulmonary disease and obstructive sleep apnea overlap syndrome is associated with excess mortality, and outcomes are related to the degree of hypoxemia. People at high altitude are susceptible to periodic breathing, and hypoxia at altitude is associated with cardio-metabolic dysfunction. Hypoxemia in these scenarios may be described as superimposed sustained plus intermittent hypoxia, or overlap hypoxia (OH), the effects of which have not been investigated. We aimed to characterize the cardio-metabolic consequences of OH in mice.
Methods: C57BL/6J mice were subjected to either sustained hypoxia (SH, FiO2=0.10), intermittent hypoxia (IH, FiO2=0.21 for 12 hours, and FiO2 oscillating between 0.21 and 0.06, 60 times/hour, for 12 hours), OH (FiO2=0.13 for 12 hours, and FiO2 oscillating between 0.13 and 0.06, 60 times/hour, for 12 hours), or room air (RA), n=8/group. Blood pressure and intraperitoneal glucose tolerance test were measured serially, and right ventricular systolic pressure (RVSP) was assessed.
Results: Systolic blood pressure transiently increased in IH and OH relative to SH and RA. RVSP did not increase in IH, but increased in SH and OH by 52% (p<0.001) and 20% (p=0.001). Glucose disposal worsened in IH and improved in SH, with no change in OH. Serum LDL and VLDL increased in OH and SH, but not in IH. Hepatic oxidative stress increased in all hypoxic groups, with the highest increase in OH.
Conclusions: Overlap hypoxia may represent a unique and deleterious cardio-metabolic stimulus, causing systemic and pulmonary hypertension, and without protective metabolic effects characteristic of sustained hypoxia.

overall_design: Whole liver mRNA profiles of C57BL/6J mice exposed to RA, SH, IH, or OH.

first: Wanjun

last: Gu


title: Canine DUXC isoforms and functional similarity to human DUX4

first: Stephen

last: Tapscott


title: Radiation exposure elicits a neutrophil-driven response in healthy lung tissue that enhances metastatic colonization

city: London

first: Ilaria

last: Malanchi

address: 1 Midland Rd,

institute: The Francis Crick Institute

summary: We used our novel Cherry-niche system to specifically isolate cells from the metastatic niche of 4T1 lung metastasis and compared their profile with the one obtained isolating lungs cells distal from the tumour growth

overall_design: Labelling-4T1 were injected intravenuosly and lungs collected 7 days post-injection. From the dissociated tissue cells were collected via FACS and single cell RNA seqeuncing was performed

first: Emma

last: Nolan

first: Stuart

last: Horswell


title: Signature of lung epithelial and mesenchymal populations from control, irradiated and neutrophil-depleted irradiated mice

summary: We analysed the signature of sorted CD45-CD31-Ter119-EpCAM+ lung epithelial cells and CD45-CD31-Ter119-EpCAM-  lung mesenchymal cells from the control vs. irradiated lungs of BALB/c mice.

overall_design: Female mice received a targeted dose of 13Gy irradiation to the lungs 7 days prior to harvest. Cells were sorted from three groups, each with n=4 mice: (1) control mice (2) irradiated mice  (3) irradiated mice that received daily injections of a neutrophil depletion antibody


title: Catalytic Subunit of Protein Phosphatase 2A (PP2Ac) Influences the Meiosis Initiation During Spermatocyte Meiosis Prophase I

city: Changzhou

first: Wenbin

last: Wang

address: NO.188, Gehu Street, Changzhou

institute: Nanjing Medical University

summary: As a serine/threonine phosphatase, protein phosphatase 2A (PP2A) is essential in numerous physiological processes. Our previously study confirmed PP2A dysfunction can cause azoospermia by generating catalytic subunit of PP2A (Ppp2ca) conditional knockout (CKO) in C57BL/6J mice. Here, we further explored the possible mechanisms by focusing on meiosis initiation and spermatogenesis. The deficiency of Ppp2ca in germ cells conspicuously disturbed spermatogonial differentiation and lead to pachytene arrest, accompanied by defects in programmed double-strand break (DSB) repair and meiotic sex chromosome inactivation (MSCI). Furthermore, Ppp2ca-deficient spermatocytes exhibited abnormal agglutination and cohesion complex degradation of chromosome, probably contributing to pachytene arrest. Our study demonstrates the irreplaceable role of PP2A in spermatogenesis and provide more evidences on azoospermia etiology.

overall_design: Testis mRNA profiles of 56-day old wild type (WT) and Ppp2ca-cKO mice

first: Xia


title: A single-cell transcriptome atlas of the human retinal pigment epithelium

city: Chongqing

first: Zongren

last: Xu

state: Chongqing

address: 1 Youyi Road, Yuzhong District, Chongqing, China

department: ophthalmology department

institute: The First Affiliated Hospital of Chongqing Medical University

summary: The retina could convert light into neurochemical information that is ultimately transmitted to the brain. The macula is a special structure at the back of the retina in humans and primates. The retinal pigment epithelium is a monolayer tissue layer that is fundamentally important for retinal development and function, and RPE dysfunction can lead to a variety of retinal degenerative diseases. Therefore, it is particularly important to study the differences between macular RPE region and peripheral RPE region. Single-cell sequencing technology enables us to analyze the heterogeneity of RPE, and found different molecular functions.So, we constructed a single-cell transcriptome atlas of macular and peripheral cells. The differences of TF, receptor ligand and function between macular RPE and peripheral RPE were analyzed. Finally, some clusters associated with retinal disease was identified. Our results provide data support for exploring the pathogenesis of RPE and retinal diseases, contributing to a deeper understanding of the physiological mechanism of retina and providing new ideas for the treatment of diseases.

overall_design: RPE cells were isolated from the eyes of 3 donors and profiled with single-cell RNA sequencing(10× Genomics).cDNA libraries were generated using the Chromium Single cell 3’ V2 kit.

first: Xingyun

last: Liao

first: Peizeng

first: Shengping

last: Hou


title: Ebola virus delta peptide is an enterotoxin

city: New Orleans

first: Robert

middle: f

last: Garry

state: LA

address: Dept Mic/Imm, 1430 Tulane Avenue

institute: Tulane

summary: Transcriptomic analyses showed that Ebola virus delta peptide triggers damage response and cell survival pathways. Delta peptide also downregulates the expression of transporters and exchangers that mediate absorption of ions, sugars, and small chain fatty acids.

overall_design: Ligated ileal loops instilled with either buffer or a synthetic peptide representing the active portion of Ebola virus delta peptide,  resected at 6 and 9 hours after surgery and subjected to  RNA-Seq analysis

first: Lilia

last: Melnik

first: Shantanu

last: Guha

first: Jenisha

last: Ghimire

first: Allison

middle: R

last: Smither

first: Brandon

last: Beddingfield

first: Andrew

last: Hoffmann

first: Leisheng

last: Sun

first: Nate

last: Ungerleider

first: Melody

last: Baddoo

first: Erik

middle: K

last: Flemington

first: William

last: Gallaher

middle: C

last: Wimley


title: A non-canonical tricarboxylic acid cycle underlies cellular identity

city: New York

first: Lydia

last: Finley

address: 430 E 67th St

department: Cell Biology Program

institute: Memorial Sloan Kettering Cancer Center

summary: The tricarboxylic acid (TCA) cycle is a central hub of cellular metabolism, oxidizing nutrients to generate reducing equivalents for energy production and critical metabolites for biosynthetic reactions. Despite the importance of the products of the TCA cycle for cell viability and proliferation, mammalian cells display diversity in TCA-cycle activity. How this diversity is achieved, and whether it is critical for establishing cell fate, remains poorly understood. Here we identify a non-canonical TCA cycle that is required for changes in cell state. Genetic co-essentiality mapping revealed a cluster of genes that is sufficient to compose a biochemical alternative to the canonical TCA cycle, wherein mitochondrially derived citrate exported to the cytoplasm is metabolized by ATP citrate lyase, ultimately regenerating mitochondrial oxaloacetate to complete this non-canonical TCA cycle. Manipulating the expression of ATP citrate lyase or the canonical TCA-cycle enzyme aconitase 2 in mouse myoblasts and embryonic stem cells revealed that changes in the configuration of the TCA cycle accompany cell fate transitions. During exit from pluripotency, embryonic stem cells switch from canonical to non-canonical TCA-cycle metabolism. Accordingly, blocking the non-canonical TCA cycle prevents cells from exiting pluripotency. These results establish a context-dependent alternative to the traditional TCA cycle and reveal that appropriate TCA-cycle engagement is required for changes in cell state.

overall_design: C2C12s were maintained as myoblasts in growth media (high glucose DMEM supplemented with 10% FBS and 4mM L-glutamine) or switched to differentiation media (high glucose DMEM with 2% horse serum, 4mM L-glutamine, and 100X insulin-transferrin-selenium-ethanolamine) for 5 days to induce formation of myotubes. Cells were harvested in triplicate, followed by extraction of total RNA using TRIzol.

first: Paige

last: Arnold

first: Katrina

last: Paras

middle: W


title: Canine DUXC isoforms and functional similarity to human DUX4 [ChIP-seq]

first: Stephen

last: Tapscott

overall_design: We genereated ChIP-seq datasets of DUX4, DUXC, and DUXC-ALT expressed in cultured canine skeletal muscle and compared to negative control of Luciferase to identify the binding sites of these three transcription factors. The DUX4 and DUXC datasets were generated from  monoclonal cell lines; the DUXC-ALT from polycolnal and luceferase from both monoclonal and polyclonal cell liens.

first: Jennifer

middle: L

last: Whiddon

first: Chao-Jen

last: Wong

first: Ashlee

last: Langford

first: Andrea

last: Belleville


title: Gene expression profiling of SEPHS1 knockdown mouse articular chondrocytes

city: Seoul

first: Jin-Hong

last: Kim

address: 1 Gwanak-ro, Gwanak-gu

institute: Seoul National University

summary: mRNA profiles of primary mouse chondrocytes transfected with control siRNA or Sephs1 siRNA were generated by mRNA sequencing. This study provides insights into the role of SEPHS1 in chondrocytes.

overall_design: Gene expression profiling of primary cultured mouse articular chondrocytes transfected with negative control siRNA or Sephs1 siRNA. Three biological replicates were used for each group.

first: Donghyun

last: Kang

first: Jeeyeon

last: Lee


title: Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [II]

first: Leonard

last: Zon

state: MA

overall_design: To analyze the effect of germline runx1 homozygous mutant hematopoiesis compared to runx1 heterozygous hematopoiesis. Embryos from runx1 heterozygous incross were grown to adulthood. Whole kidney marrow cells from 4 runx1 heterozygous and 4 runx1 homozygous mutant zebrafish were collected for preparation for 10x single cell RNA sequencing.

first: Serine

last: Avagyan

first: Song

last: Yang


title: Gene expression microarray analysis of whole mouse hamstring muscles to identify genes promoting the devlopment of neurogenic heterotopic ossifications following spinal cord injury

city: Brisbane

first: Jean-Pierre

last: Levesque

state: Queensland

address: 37 Kent St

department: Mater Research Institute

country: Australia

institute: Mater Research Institute-University of Queensland

summary: To better understand which genes/pathways are activated in injured muscles following a spinal cord injury (SCI) and how these promote the development of neurogenic heterotopic ossification (NHO), we performed gene expression profiling by microarray of muscles in the early phase following muscle injury.

overall_design: Total RNA was isolated from injured hamstring muscles two days post-injury from four experimental groups: 1) Spinal cord injury (SCI) + cardiotoxin (CDTX): SCI plus intramuscular (i.m.) CDTX injection, the only condition in which NHOs form in the injured muscle; 2) SCI+Saline: SCI surgery plus i.m. injection of saline); 3) Sham+CDTX: Sham surgery plus i.m. injection of CDTX, ; or 4) Sham+Saline.

first: Hsu-Wen

last: Tseng

first: Irina

last: Kulina

first: Gethin

last: Thomas

first: Lawrie

last: Wheeler


title: miR181a is a novel key player in the STAT3 mediated survival network of TCRab CD8+ T-LGL leukemia [RNA-Seq]

city: Rotterdam

first: Anton

last: Langerak

state: Zuid Holland

address: Dr. Molewaterplein 40

department: Immunology

country: Netherlands

institute: ErasmusMC

summary: TCRαβ CD8 large granular lymphocyte (LGL) leukemia is a rare heterogeneous hematological disorder with a chronic disease course that mostly affects elderly. It is generally accepted that LGL cells arise as a consequence of chronic antigenic stimulation and inflammation and thrive because of dysregulation of mainly the STAT3 but also to some extent the ERK pathway, which are constitutively activated. In approximately 40% of patients this is due to STAT3 activating mutations, but for the other 60% it remains to be elucidated, why the STAT3 pathway is chronically activated. miRNAs are one of the most potent regulators in health and disease and are also linked with various leukemias. Therefore, we hypothesized that aberrant miRNA expression could contribute to dysregulation of the STAT3 pathway. miRNA sequencing in LGL leukemia cases and aged-matched healthy control TEMRA cells revealed overexpression of miRNA181a. Furthermore, gene set enrichment analysis (GSEA) of miRNA181a implicated its involvement in the STAT3 and ERK1/2 pathways. Phosphoflow analysis of STAT3, ERK1/2 in miR181a-transfected human CD8 T cells revealed that miR181a overexpression results in STAT3 and ERK1/2 phosphorylation. By using TL1, a human T-LGL cell line model, we have now established that miR181a is the common actor in T-LGL leukemia, driving STAT3 activation by SOCS3 inhibition in LGL leukemia patients. Additionally, miR181a induces ERK1/2 phosphorylation by inhibition of DUSP6. Taken together, our data show that miR181a is the missing link to explain why STAT3-unmutated patients show hyperactive STAT3.

overall_design: mRNA sequencing of T-LGL patients compared to healthy control TEMRA CD8+ T cells

first: Jorn

last: Assmann

first: Leticia

middle: G

last: Leon


title: Small non-coding RNA sequencing of human monocyte-derived macrophages

city: Sheffiled

first: Chiara

last: Niespolo

address: Beech Hill Road

department: Infection, Immunity and Cardiovascular Disease

institute: University of Sheffield

type: Non-coding RNA profiling by high throughput sequencing

summary: The goal of this experiment was to identify the differential expression of microRNAs in human polarised macrophages. We used monocyte-derived macrophages isolated from 8 different healthy invididuals. Differentiated macrophages were polarised towards the pro-inflammatory M1-like phenotype, by using LPS and INF-𝛾. Total RNA was extracted and sent for small-RNA sequencing to the company Novogene.

overall_design: Identification of microRNAs differentially expressed between unpolarised and M1 polarised macrophages

first: Sumeet

last: Deshmukh

first: Ian

last: Sudbery

first: Heather

last: Wilson

first: Endre

last: Kiss-Toth


title: Role of neuronal Xlr4 gene expression in cocaine addiction vulnerability: a microarray study in medial prefrontal cortex and Nucleus Accumbens of C57 and DBA mice.

city: Roma

first: Ivan

last: Arisi

address: viale Regina Elena 295

department: Bioinformatics Facility

country: Italy

institute: European Brain Research Institute

summary: Although several studies have been performed in rodents, non-human primates and humans, the biological basis of vulnerability to develop cocaine addiction remains largely unknown. Exposure to critical early events (as repeated cross fostering (RCF)) has been reported to increase sensitivity to cocaine in adult C57BL/6J female mice. Here we reasoned that by comparing the RCF-induced genes expression alterations in the NAc of adult mice (3 months of age) from two inbred strains (C57 and DBA), we could be able to identify critical genes underlying the adult behavioral phenotype. By a microarray analysis  of gene expression (Agilent paltform)in the NAc of adult animals we found, among the several genes modulated, only 2 genes, the X-linked lymphocyte-regulated 4a and 4b (Xlr4a, Xlr4b), that were simultaneously modulated by the early experience in the two strains, but, notably, in opposite direction. All together, these data strongly indicate a critical role for Xlr4, a new yet unexplored candidate gene, in mediating vulnerability to cocaine addiction in mouse.

overall_design: A total of n=31 samples were processed, with 4 samples/group except one group with only 3 samples (C57_mpFC_ctr). The samples are divided into 8 experimental groups, based on the three factors: strain (2 levels=C57 or DBA) X tissue (2 levels=mpFC or Nac) X treatment (2 levels= control or RCF).

first: Matteo

last: Di Segni

first: Diego

last: Andolina

first: Sebastian

last: D'Addario

first: Lucy

last: Babicola

first: Donald

last: Lelpo

first: Alessandra

last: Luchetti

first: Tiziana

last: Pascucci

first: Camilla

last: Mancini

last: Accoto

first: Luisa

last: Lo Iacono

first: D'Amato

last: Francesca

first: Rossella

last: Brandi

first: Sabrina

last: Turturro

first: Mara

last: D'Onofrio

last: Ventura