title: Sodium fluoride exposure leads to ATP depletion and altered RNA decay in Escherichia coli under anaerobic conditions

city: Taipei

first: Sue

last: Lin-Chao

address: 128 Academia Road, Section 2, Nankang,

department: Institute of Molecular Biology

country: Taiwan

institute: Academia Sinica

summary:
We report genome-wide transcriptome profiles of E. coli obtained in the absence (control) and presence of 20 mM and 70 mM sodium fluoride (NaF) under anaerobic conditions, and assess the impact of fluoride-dependent ATP depletion on RNA turnover. We found that transcripts with increased abundance in response to NaF treatment correspond to genes that control cell envelope and osmotic stress adaptation, signal transduction systems, lipid biosynthesis, amine and polyamine degradation as well as acquisition of iron and iron homeostasis. In contrast, downregulated genes are involved in glycolysis, fatty acid metabolism, amino acid biosynthesis, energy production, cytochrome c biogenesis, protein translocation, translation, translation factors, protein folding/processing factors, transport for amino acid, sugar, or ion, and RNA metabolism.
By using a quantile-based K-means clustering approach to identify gene clusters with similar expression profiles, we identified subset (100 genes) of transcriptome whose gene expression was up- and down-regulated under fluoride and diluted fluoride conditions, respectively. In addition, we found that about 40% of the highly abundant transcripts carry repetitive extragenic palindromes (REPs). By determining the mRNA stability of osmC as well as yghA, and addressing their ribonucleases/enzymes required for RNA degradation under anaerobic conditions, we found that fluoride ions slow down RNA degradation by increasing RNA stability, in turn increasing the steady-state level of RNA. Furthermore, our results show that turnover of these REP-containing transcripts is dependent on RNase E. Collectively, our study not only reveal the effects of NaF at the whole transcriptome level under hypoxic growth conditions, but also shows that fluoride can affect gene expression post-transcriptionally by slowing down the ATP-dependent degradation of structured RNAs.

platform_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: To compare the tiling array-derived transcriptome profiles of Escherichia coli with and without NaF treatment under anaerobic growth conditions

first: Oleg

last: Murashko

first: Chen-Hsin

last: Yu


title: Mapping the landscape of genetic dependencies in chordoma

status: Public on Mar 09 2023

city: Cambridge

first: Tanaz

last: Sharifnia

state: MA

address: 415 Main Street

department: Chemical Biology and Therapeutics Science Program

country: USA

institute: Broad Institute

type: Expression profiling by high throughput sequencing

summary: Identifying the spectrum of genes required for cancer cell survival can reveal essential cancer circuitry and therapeutic targets, but such a map remains incomplete for many cancer types. We apply genome-scale CRISPR-Cas9 loss-of-function screens to map the landscape of selectively essential genes in chordoma, a bone cancer with few validated targets. This approach confirms a known chordoma dependency, TBXT (T; brachyury), and identifies a range of additional dependencies, including PTPN11, ADAR, PRKRA, LUC7L2, SRRM2, SLC2A1, SLC7A5, FANCM, and THAP1. CDK6, SOX9, and EGFR, genes previously implicated in chordoma biology, are also recovered. We find genomic and transcriptomic features that predict specific dependencies, including interferon-stimulated gene expression, which correlates with ADAR dependence and is elevated in chordoma. Validating the therapeutic relevance of dependencies, small-molecule inhibitors of SHP2, encoded by PTPN11, have potent preclinical efficacy against chordoma. Our results generate an emerging map of chordoma dependencies to enable biological and therapeutic hypotheses.

overall_design: RNA-sequencing of sg-ADAR- and sg-EGFP-treated UM-Chor1 chordoma cells. Two independent sgRNAs targeting ADAR were used.

first: Stuart

middle: L

last: Schreiber


title: The Isolation and In vitro Differentiation of Primary Fetal Baboon Tracheal Epithelial Cells for the Study of SARS-CoV-2 Host-Virus Interactions

status: Public on Mar 14 2023

city: Oklahoma City

first: Jonathan

middle: Daniel

last: Wren

state: OK

address: 825 NE 13th St

department: GHD

institute: OMRF

summary: The mucociliary airway epithelium lines the human airways and is the primary site of host-environmental interactions in the lung. Following virus infection, airway epithelial cells initiate an innate immune response to suppress virus replication. Therefore, defining the virus-host interactions of the mucociliary airway epithelium is critical for understanding the mechanisms that regulate virus infection, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Non-human primates (NHP) are closely related to humans; and provide a model to study human disease. However, ethical considerations and high costs can restrict the use of in vivo NHP models. Therefore, there is a need to develop in vitro NHP models of human respiratory virus infection that would allow for rapidly characterizing virus tropism and suitability of specific NHP species to model human infection. Using the olive baboon (Papio anubis), we have developed methodologies for the isolation, in vitro expansion, cryopreservation, and mucociliary differentiation of primary fetal baboon tracheal epithelial cells (FBTECs). Furthermore, we demonstrate that in vitro differentiated FBTECs are permissive to SARS-CoV-2 infection and produce a potent host innate-immune response. In summary, we have developed an in vitro NHP model that provides a platform for the study of SARS-CoV-2 infection and other human respiratory viruses.

overall_design: mRNA profiling of primary fetal baboon tracheal epithelial cells (FBTECs) differentiated on air-liquid interface (ALI) culture for 28 days.

first: Matthew

middle: S

last: Walters


title: Gene expression correlates of HIV-1 susceptibility and gene expression changes following HIV-1 infection

status: Public on Mar 10 2023

city: Seattle

first: Romel

middle: D

last: Mackelprang

state: WA

address: 1959 NE Pacific Street  Box 356460

institute: University of Washington

summary: We sought to determine how gene expression changes during the first two years of HIV-1 infection among participants from HIV-1 serodiscordant couple cohorts from sub-Saharan Africa. This study included whole blood samples from 26 participants who did not have HIV-1 at study enrollment, had a steady sexual relationship with a partner with HIV-1 and acquired HIV-1 during follow-up. Most participants had samples from before and after infection.

overall_design: Gene expression was measured by Illumina HumanHT-12 v4 Expression BeadChips. Whole blood samples were preserved at -80 degrees C in PAXgene tubes. Samples were not replicated on the arrays.

first: Jairam

middle: R

last: Lingappa


title: Characterizing expression changes in noncoding RNAs during aging and heterochronic parabiosis across mouse tissues

city: Saarbruecken

first: Viktoria

last: Wagner

country: Germany

institute: Universität des Saarlandes

type: Non-coding RNA profiling by high throughput sequencing

summary:
Molecular mechanisms of organismal and cell aging remain incompletely
understood. We, therefore, generated a body-wide map of noncoding
RNA (ncRNA) expression in aging (16 organs at ten timepoints from 1 to
27 months) and rejuvenated mice. We found molecular aging trajectories
are largely tissue-specific except for eight broadly deregulated microRNAs
(miRNAs). Their individual abundance mirrors their presence in circulating
plasma and extracellular vesicles (EVs) whereas tissue-specific ncRNAs
were less present. For miR-29c-3p, we observe the largest correlation with
aging in solid organs, plasma and EVs. In mice rejuvenated by heterochronic
parabiosis, miR-29c-3p was the most prominent miRNA restored to similar
levels found in young liver. miR-29c-3p targets the extracellular matrix
and secretion pathways, known to be implicated in aging. We provide a
map of organism-wide expression of ncRNAs with aging and rejuvenation
and identify a set of broadly deregulated miRNAs, which may function as
systemic regulators of aging via plasma and EVs.

overall_design: This study contains the following two independent experimental setups, also denoted as experimental batches:  1. small non-coding RNAseq of all major organs of mice over the lifespan   2. small non-coding RNAseq of mice of a rejuvenation model (heterochronic parabiosis)

first: Oliver

last: Hahn

first: Nicholas

last: Schaum

first: Fabian

last: Kern

first: Nicole

last: Ludwig

first: Tobias

last: Fehlmann

first: Annika

last: Engel

first: Dominic

last: Henn

first: Shusruto

last: Rishik

first: Alina

last: Isakova

first: Michelle

last: Tan

first: Rene

last: Sit

first: Norma

last: Neff

first: Martin

last: Hart

first: Eckart

last: Meese

first: Steve

last: Quake

first: Andreas

last: Keller

first: Tony

last: Wyss-Coray


title: CRISPR/Cas9-mediated repair of oncogenic V560G-KIT in HMC-1.2 renders a novel human mast cell line with a single KIT mutation (D816V): an improved preclinical model for therapeutic research on mastocytosis

city: Bethesda

first: Ana

last: Olivera

state: MD

address: 10 Center Dr, Bldg 10

institute: NIAID (National Institutes of Health)

summary: The HMC-1.1 and 1.2 human mast cell (huMC) lines are often employed in the study of attributes of neoplastic huMCs as found in patients with mastocytosis and their sensitivity to interventional drugs in vitro and in vivo. Both cell lines express mutated and constitutively active KIT, an essential growth factor receptor for huMC survival and function, with one KIT mutation in HMC-1.1 cells (V560G-KIT) and two in HMC-1.2 cells (D816V-KIT and V560G-KIT). D816V-KIT is commonly associated with systemic mastocytosis, while V560G-KIT appears more frequently in other human neoplasms. The functional consequences of the coexisting KIT mutations in HMC-1.2 cells are unknown. We used CRISPR/Cas9 engineering to reverse the V560G-KIT mutation in HMC-1.2 cells, resulting in a subline (HMC-1.3) with a single monoallelic D816V-KIT mutation. Transcriptome analyses of differentially expressed genes in HMC1.3 compared to HMC-1.2 predicted reduced activity in pathways involved in survival, cell-to-cell adhesion, and neoplasia, with differences in expression of molecular components and cell surface markers. Consistently, subcutaneous inoculation of HMC-1.3 into mice produced significantly smaller tumors than HMC-1.2 cells, and in colony assays, HMC-1.3 formed less numerous and smaller colonies than HMC-1.2 cells. However, in liquid culture conditions, the growth of HMC-1.2 and HMC-1.3 cells was comparable, albeit increased compared to HMC-1.1 cells. Phosphorylation levels of ERK1/2, AKT and STAT5, representing pathways associated with constitutive oncogenic KIT signaling, were also similar between HMC-1.2 and HMC-1.3 cells but increased compared to HMC-1.1 cells. Despite these similarities, survival of HMC-1.3 cells in liquid culture was diminished in response to various pharmacological inhibitors, including tyrosine kinase inhibitors used clinically for treatment of advanced systemic mastocytosis, and JAK2 and BCL2 inhibitors, making HMC-1.3 more susceptible to these drugs than HMC-1.2 cells. Our study reveals that the additional V560G-KIT variant in HMC-1.2 cells modifies transcriptional programs induced by D816V-KIT, confers a survival advantage, alters sensitivity to interventional drugs, and increases the tumorigenicity, suggesting that engineered huMCs with a single D816V-KIT variant may represent an improved preclinical model for mastocytosis.

overall_design: mRNAseq from 3 human mast cell lines (huMC) HMC-1.1, HMC-1.2, and HMC-1.3. These have one KIT mutation in HMC-1.1 cells (V560G-KIT) and two in HMC-1.2 cells (D816V-KIT and V560G-KIT). We used CRISPR/Cas9 engineering to reverse the V560G-KIT mutation in HMC-1.2 cells, resulting in a subline (HMC-1.3) with a single monoallelic D816V-KIT mutation. Each cell line was sequenced in triplicate, with 2 subclones of the HMC-1.3 sequenced in triplicate, resulting in 12 total libraries.

first: Geethani

last: Bandara

first: Guido

middle: H

last: Falduto

first: Andrea

last: Luker

first: Yun

last: Bai

last: Pfeiffer

first: Justin

last: Lack

first: Dean

last: Metcalfe


title: Spontaneous tumor regression mediated by human T cells in a humanized immune system mouse model

status: Public on Mar 06 2023

city: Tarrytown

first: Wei Keat

last: Lim

address: 777 Old Saw Mill River Rd

institute: Regeneron Pharmaceuticals

summary: Immunodeficient mice reconstituted with a human immune system (HIS mice) give rise to human T cells, which make them an attractive system to study human immune responses to tumors. However, such HIS mice typically exhibit sub-optimal responses to immune challenges as well as fail to develop antigen-specific B or T cell memory. Here we report HIS mice mediate spontaneous regression of human B cell lymphoma Raji. Tumor regression was dependent on CD4+ and CD8+ T cell responses and resulted in T cell memory. The T cell memory elicited was mainly Raji-specific, however some level of cross-protection was also elicited to a related B cell lymphoma cell line Ramos. Analysis of regressing Raji tumor indicated human T cell infiltration. We isolated T cells from regressing Raji tumors as well as concomitant spleens to assess the transcriptional profile of T cells as well as specific TCR sequences that may be expanded in infiltrating T cells.

overall_design: We isolated human T cells from regressing Raji tumors, outgrowing (uncontrolled) Raji tumors as well as concomitant spleens. Additional controls were T cells isolated from non-tumor challenged HIS mice spleens. T cells were isolated by FACS sorting for hCD45+/mCD45- and CD3+/CD19- cells from prepared tissues. We performed high throughput transcriptomic (scRNAseq) and TCR repertoire (scTCRseq) characterization of isolated human T cells.

first: Aditi

last: Patel

first: Ankur

last: Dhanik

first: Christina

last: Adler

first: Min

last: Ni

first: Yi

last: Wei

first: Maggie

last: Zhong

first: Cindy

last: Nguyen

first: Jun

first: Yi-Fen

last: Lu

first: Gavin

last: Thurston

first: Lynn

last: Macdonald

first: Andrew

middle: J

last: Murphy

first: Cagan

last: Gurer

first: Davor

last: Frleta


title: Single-cell transcriptomic analyses of pancreatic islets reveals islet cell-type-specific adaptations to pregnancy and the postpartum

status: Public on Mar 07 2023

city: Birmingham

first: Robert

last: Welner

state: AL

address: WTI 510D Zip 3300

department: Department of Medicine

institute: University of Alabama at Birmingham

summary: Pancreatic β-cell mass expands during pregnancy and regresses in the postpartum period in conjunction with dynamic metabolic demands on maternal glucose homeostasis. To understand transcriptional changes driving these adaptations in β-cells and other islet cell types, we performed single-cell RNA sequencing on islets from virgin, late gestation, and early postpartum mice. We identified transcriptional signatures unique to gestation and the postpartum in β-cells, including induction of the AP-1 transcription factor subunits and other genes involved in the immediate-early response (IEGs). Additionally, we found pregnancy and postpartum-induced changes differed within each endocrine cell type, and in endothelial cells and macrophages within islets. Together, our data reveal novel insights into cell-type specific transcriptional changes responsible for adaptations by islet cells to pregnancy and their resolution postpartum.

overall_design: Comparing pregnant and postpartum islets to those of non-pregnant females

first: Jin-Yong

last: Chung

first: Yongjie

last: Ma

first: Hubert

last: M. Tse

first: Rob

first: Ronadip

last: Banerjee


title: Starvation-induced changes in somatic Insulin/IGF-1R signaling drive metabolic programming across generations

city: New YorK

first: Columbia

middle: Carlotta

last: University

state: NEW YORK

address: 1212 Amsterdam Avenue

department: Biological Sciences

institute: HHMI/Columbia University

summary: Exposure to adverse nutritional and metabolic environments during critical periods of development can exert long-lasting effects on health outcomes of an individual and its descendants. Although such metabolic programming has been observed in multiple species and in response to distinct nutritional stressors, conclusive insights into signaling pathways and mechanisms responsible for initiating, mediating and manifesting changes to metabolism and behavior across generations remain scarce. By employing a multigenerational starvation paradigm in C. elegans, we show that starvation-induced changes in DAF-16/FoxO activity, the main downstream target of insulin/IGF-1 receptor signaling, are responsible for metabolic programming phenotypes. Tissue-specific depletion of DAF-16/FoxO during distinct developmental time points further demonstrates that DAF-16/FoxO acts in somatic tissues, but not directly in the germline, to both initiate and manifest metabolic programming. In conclusion, our study deciphers multifaceted and critical roles of highly conserved insulin/IGF-1 receptor signaling in determining health outcomes and behavior across generations.

overall_design: Differential gene expression analysis of effects of L1 starvation on adult transcriptome (F4), as well as on the transcriptome of great-grandchilden (F4+3)

first: Merly

middle: C

last: Vogt

first: Oliver

last: Hobert


title: PI3Kβ controls immune evasion in PTEN-deficient breast tumors

city: Boston

first: Jean

last: Zhao

address: 450 Brookline Ave

institute: Dana Farber Cancer Institute

summary: Loss of the PTEN tumor suppressor is one of the most common oncogenic drivers across all cancer types. PTEN is the major negative regulator of phosphoinositide-3 kinase (PI3K) signaling. Notably, the PI3Kβ isoform has been shown to play an important role in PTEN-deficient tumors, but the mechanisms underlying the importance of PI3Kβ activity remain elusive. Using a syngeneic genetically-engineered mouse (GEM) model of invasive breast cancer driven by concurrent ablation of Pten and Trp53 (p53), we showed that genetic inactivation of PI3Kβ led to a robust anti-tumor immune response that abrogated tumor growth in syngeneic immunocompetent mice, but not in immunodeficient mice. Mechanistically, PI3Kβ inactivation in the PTEN-null setting led to reduced STAT3 signaling and increased expression of immune stimulatory molecules, thereby promoting anti-tumor immune responses. Pharmacological PI3Kβ inhibition also elicited anti-tumor immunity, and synergized with immunotherapy to inhibit tumor growth. Mice with complete responses to the combined treatment displayed immune memory and rejected tumors upon re-challenge. Our findings demonstrate a molecular mechanism linking PTEN loss and STAT3 activation in cancer and suggest that PI3Kβ controls immune escape in PTEN-null tumors, providing a rationale for combining PI3Kβ inhibitors with immunotherapy for the treatment of PTEN-deficient breast cancer.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Comparative gene expression profiling analysis of RNA-seq data for genetic or pharmacological PI3Kβ blockade, STAT3 silencing, or STAT3 activation in PTEN-deficient breast/mammary tumors.

first: Johann

last: Bergholz

first: Qiwei

last: Wang

first: Qi


title: Analysis of donor pancreata defines the transcriptomic signature and microenvironment of early pre-neoplastic pancreatic lesions

status: Public on Mar 08 2023

city: Ann Arbor

first: Marina

last: Pasca di Magliano

state: MI

address: 1500 E Medical Center Dr

institute: University of Michigan Medical School

summary: The adult healthy human pancreas has been poorly studied given lack of indication to obtain tissue from the pancreas in the absence of disease and rapid postmortem degradation. We obtained pancreata from brain dead donors thus avoiding any warm ischemia time. The 30 donors were diverse in age and race and had no known pancreas disease. Histopathological analysis of the samples revealed PanIN lesions in most individuals irrespective of age. Using a combination of multiplex immunohistochemistry, single cell RNA sequencing, and spatial transcriptomics, we provide the first ever characterization of the unique microenvironment of the adult human pancreas and of sporadic PanIN lesions. We compared healthy pancreata to pancreatic cancer and peritumoral tissue and observed distinct transcriptomic signatures in fibroblasts, and, to a lesser extent, macrophages. PanIN epithelial cells from healthy pancreata were remarkably transcriptionally similar to cancer cells, suggesting that neoplastic pathways are initiated early in tumorigenesis.

overall_design: A total of 4 slides containing 6 tissue sections (2 healthy pancreas, 4 tumor) was submitted for spatial transcriptomics.  The Solid Tumor TME Morphology Kit was utilized according to manufacturer instructions to detect epithelial cells (pan-cytokeratin [CK]) and immune cells (CD45), together with DAPI for nuclei detection. 277 ROIs were captured for whole transcriptome profiling.


title: Bombyx Vasa sequesters transposon mRNAs in nuage via phase separation requiring RNA binding and self-association [RIP-Seq]

city: Tokyo

first: Yurika

last: Namba

state: --- Select a state ---

address: 2-11-16 Yayoi, Bunkyo-ku

department: Department of Biological Sciences, Graduate School of Science

country: Japan

institute: The University of Tokyo

summary: Bombyx Vasa (BmVasa) assembles non-membranous organelle, nuage or Vasa bodies, in germ cells, known as the center for Siwi-dependent transposon silencing and concomitant Ago3-piRISC biogenesis. However, details of the body assembly remain unclear. Here, we show that the N-terminal intrinsically disordered region (N-IDR) and RNA helicase domain of BmVasa are responsible for self-association and RNA binding, respectively, but N-IDR is also required for full RNA-binding activity. Both domains are essential for Vasa body assembly in vivo and droplet formation in vitro via phase separation. FAST-iCLIP reveals that BmVasa preferentially binds transposon mRNAs. Loss of Siwi function derepresses transposons but has marginal effects on BmVasa-RNA binding. This study shows that BmVasa assembles nuage by phase separation via its ability to self-associate and bind newly exported transposon mRNAs. This unique property of BmVasa allows transposon mRNAs to be sequestered and enriched in nuage, resulting in effective Siwi-dependent transposon repression and Ago3-piRISC biogenesis.

overall_design: BmVasa binding RNAs (BmVasa FAST-iCLIP) in BmN4 cells

first: Hiroya

last: Yamazaki

first: Mikiko

last: Siomi


title: Bombyx Vasa sequesters transposon mRNAs in nuage via phase separation requiring RNA binding and self-association

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

overall_design: Refer to individual Series


title: Bombyx Vasa sequesters transposon mRNAs in nuage via phase separation requiring RNA binding and self-association [RNA-Seq]

overall_design: Total RNAs (total RNA sequencing) in BmN4 cells


title: Treatment of TT1 mice with transposon-based gene therapy

city: Szeged

first: Lajos

last: Mates

address: Temesvari krt. 62.

department: Institute of Genetics

country: Hungary

institute: Biological Research Centre

summary: DNA transposon-based gene delivery vectors represent a promising new branch of randomly integrating vector development for gene therapy. For the side-by-side evaluation of the piggyBac and Sleeping Beauty systems - the only DNA transposons currently employed in clinical trials - during therapeutic intervention, we treated the mouse model of Tyrosinemia type I. with liver-targeted gene delivery using both transposon vectors. For genome-wide mapping of transposon insertion sites we developed a new Next Generation Sequencing procedure called Streptavidin-Based Enrichment Sequencing, which allowed us to identify approximately 1 million integration sites for both systems. We revealed that a high proportion of piggyBac integrations are clustered in hot regions and found that they are frequently recurring at the same genomic positions among treated animals, indicating that the genome-wide distribution of Sleeping Beauty-generated integrations is closer to random. We also revealed that the piggyBac transposase protein exhibits prolonged activity, which predicts the risk of oncogenesis by generating chromosomal double-strand breaks. Safety concerns associated with prolonged transpositional activity draw attention to the importance of squeezing the active state of the transposase enzymes into a narrower time window.

overall_design: In vivo transposon-based gene therapy was applied to treat Fah KO mice using either the PB or the SB vector system.  At the end of the monitoring periods, genomic DNA was isolated from the PB- and the SB-treated livers. Streptavidin-Based Enrichment Sequencing (SBE-seq) was applied to identify genomic vector insertion sites.

first: Gábor

last: Jaksa

first: Bertalan

last: Takács

first: Gergely

last: Imre

last: Mátés


title: Regulation of alternative splicing by the C* complex

city: Berlin

first: Marco

last: Preussner

address: Takustrasse 6

institute: FU berlin

summary: Knockdown and overexpression of C* proteins in Hela and HEK293 cells was investigated by RNA-Seq.

overall_design: HeLa cells were transfected either with the indicated siRNAs and analyzed by RNA-Seq.

first: Florian

last: Heyd

first: Reinhard

last: Luehrmann


title: Ichthyosis associated with sphingosine 1-phosphate lyase insufficiency syndrome is due to aberrant calcium and sphingolipid regulation

city: London

first: Lou

last: Metherell

address: JVSC, QMUL, Charterhouse Square

department: Centre for Endocrinology

country: United Kingdom

institute: William Harvey Research Institute

summary: Sphingosine 1-phosphate lyase (SGPL1) insufficiency (SPLIS) is a syndrome which presents with adrenal insufficiency, steroid resistant nephrotic syndrome, hypothyroidism, neurological disease, and ichthyosis. Where a skin phenotype is reported, 94% presented with abnormalities such as ichthyosis, acanthosis, and hyperpigmentation. To elucidate the disease mechanism and the role SGPL1 plays in the skin barrier we established CRISPR-Cas9 SGPL1 knock-out and a lentiviral induced SGPL1 overexpression in nTERT immortalised keratinocytes and thereafter organotypic skin equivalents.  Loss of SGPL1 caused an accumulation of S1P, sphingosine and ceramide while its overexpression caused a reduction of these species. RNAseq analysis showed perturbations in sphingolipid pathway genes, particularly in SGPL1_KO and GSEA revealed polar opposite differential gene expression between SGPL1_KO and _OE in keratinocyte differentiation and Ca2+ signalling genesets. SGPL1_KO upregulated differentiation markers while SGPL1_OE upregulated basal and proliferative markers. The advanced differentiation of SGPL1_KO was confirmed by 3D organotypic models that also presented with a thickened and retained stratum corneum, sphingolipid accumulation in the spinous layer and a breakdown of E-cadherin junctions. We hypothesise that SPLIS associated ichthyosis is a multi-faceted disease caused by sphingolipid imbalance and excessive S1P signalling.

overall_design: We generated SGPL1 CRISPR KO nTERT cell lines as well as SGPL1 overexpressing cell lines and compared their transcriptomes using RNAseq. We used 3 biological replicates per group.

first: Chris

last: Smith

first: Jack

last: Williams

first: Charlotte

last: Hall

first: Josefina

last: Casa

first: Matthew

last: Caley

first: Edel

last: O'Toole

first: Rathi

last: Prasad


title: Analysis of IGH repertoire in MRL/lpr SLE prone mice treated with either control SMO or Long isoform of PRLR SMO

city: Monrovia

first: Srividya

last: Swaminathan

state: CA

address: 1218 fifth Ave

department: Systems biology

institute: Beckman Research Institute, City of Hope

summary:
Purpose: IGH sequencing has revolutionized analysis of structural and functional signatures of B cell clonality. The goals of this study are to compare IGH repertoire of splenic B cells before and after knockdown of long isoform of PRLR.
Methods: After amplifying the IGH repertoire of splenic B cells using PCR primers for VHJ558 variable and Cμ constant regions. For next generation sequencing (NGS), a TrueSeq library was prepared by adapter ligation to full-length products and sequenced on the MiSeq Illumina 2x150 bp platform.
Results: In this project, Unique nucleotide sequences were identified, their abundances were calculated, and their complementary determining region 3 (CDR3) length, CDR3 spectrum, VDJ usage, and diversity were analyzed using the International Immunogenetics Information System (IMGT) HighV-QUEST software.
Controls are included C3,C4,C5,C7,C8 and C9. LFPRLR SMO treated mice samples are: A3,A4,A5,A6,A7 and A8

first: Adeleh

last: Taghi Khani


title: The evolutionarily conserved gene Arrdc5 is required for male fertility in mice

city: Pullman

first: Jon

last: Oatley

address: 202 Biotechnology Life Science Building  PO Box 647520,

department: School of Molecular Biosciences

institute: Washington State University

summary: In sexually reproducing organisms, sperm contribute half the genomic material required for creation of offspring and natural fertility relies on males producing millions by way of spermatogenesis. This process is essential for continuity and diversity of a species, yet evolutionarily conserved core molecular regulators are undefined. Here we used single cell RNA-sequencing of testicular tissue from mice, pigs, and cattle to generate a multispecies integrated transcriptome database. Through bioinformatic analysis, the gene arrestin-domain containing 5 (Arrdc5) which encodes for an α-arrestin molecule was identified as a potential evolutionarily conserved regulator of spermatogenesis. Expression of Arrdc5 is testis specific in mice, pigs, cattle, and humans. Knockout of Arrdc5 in mice leads to male specific sterility due to production of low numbers of sperm that are immotile and malformed, resembling oligoasthenoteratospermia (OAT) which is a common diagnosis of infertility in men. Spermiogenesis, the final phase of spermatogenesis when round spermatids morphologically transform to spermatozoa, is defective in testes of Arrdc5 deficient mice. Also, epididymal sperm in Arrdc5 knockouts are unable to capacitate and fertilize oocytes. These findings establish Arrdc5 as a core regulator of mammalian spermatogenesis. Considering the role of arrestin molecules as modulators of G-protein coupled signaling and ubiquitination, Arrdc5 is a potential male contraceptive target.

sample_organism:
Sus scrofa
Bos taurus

platform_organism:
Sus scrofa
Bos taurus

overall_design: Expression profiling by high throughput sequencing of prepubertal bovine (n=3) and porcine (n=3) testicular cells

first: M

middle: I

last: Giassetti

first: J

middle: M

first: N

last: Law


title: Single cell analysis of  iPSC-derived midbrain organoids (GBA1 mutant and controls)

city: Paris

first: Michela

last: Deleidi

address: 24 boulevard du Montparnasse

country: France

institute: Institut Imagine, INSERM UMR1163

summary: Mutations in the lysosomal enzyme β-glucocerebrosidase (GCase), which cause Gaucher´s disease, are the most frequent genetic risk factor for Parkinson’s disease (PD). Here, we employed single-cell genomic approaches in induced pluripotent stem cell (iPSC)-derived midbrain organoids to dissect the mechanisms underlying GCase-related neurodegeneration.

overall_design: Midbran organoids generated from L444P-GBA1 and one E326K-GBA1 human iPSCs lines and corresponding gene corrected lines. For each condition, we prepared single-cell suspensions with a pool of 4 organoids each, all with similar sizes (Day 40).

first: Pascale

last: Baden

first: Maria

last: Perez

first: Hariam

last: Raji

first: Federico

last: Bertoli


title: TEAD1 trapping by the Q353R-Lamin A/C causes dilated cardiomyopathy.

first: Shintaro

last: Yamada

address: 7-3-1, Hongo, Bunkyo-ku

department: Department of Cardiovascular Medicine

type: Genome binding/occupancy profiling by high throughput sequencing

summary: •	Mutations in the LMNA gene encoding Lamin A and C (Lamin A/C), major components of the nuclear lamina, cause laminopathies including dilated cardiomyopathy (DCM), but the underlying molecular mechanisms have not been fully elucidated. Here, by leveraging single-cell RNA-seq, ATAC-seq, protein array, and electron microscopy analysis, we show that insufficient structural maturation of cardiomyocytes owing to trapping of transcription factor TEAD1 by mutant Lamin A/C at the nuclear membrane underlies the pathogenesis of Q353R- LMNA-related DCM. Inhibition of the Hippo pathway rescued the dysregulation of cardiac developmental genes by TEAD1 in LMNA-mutant cardiomyocytes. Single-cell RNA-seq of cardiac tissues from DCM patients with the LMNA mutation confirmed the dysregulated expression of TEAD1 target genes. Our results propose an intervention for transcriptional dysregulation as a potential treatment of LMNA-related DCM.

overall_design: Integrative analysis of single cell RNA-seq and ATAC-seq in knock-in mice with Lmna Q353R mutation

first: Tohiyuki

last: Ko

first: Masamichi

last: Ito

first: Tatsuro

last: Sassa

first: Seitaro

last: Nomura

first: Kiminori

last: Toyooka

first: Akihiro

last: Umezawa

first: Ryo

last: Nitta

first: Hiroyuki

last: Aburatani

first: Issei

last: Komuro


title: Transcriptome RNA Sequencing Data Sets of Francisella tularensis tularensis SchuS4 upon treatment with various concentrations of Doxycycline or Ciprofloxacin antibiotics

status: Public on Mar 05 2023

city: Ness Ziona

first: Galia

last: Zaide

address: Reuven St.

department: Biochemistry and Molecular Genetics

country: Israel

institute: Israel Institute for Biological Research

summary: Francisella tularensis, the etiological agnet of Tularemia is a Category A select agent. Preparedness means for prompt proper antibiotic treatment are of need to prevent morbidity and mortality. While standard antimicrobial susceptibility tests are time consuming, the quantification of the changes in the expression levlels of specific mRNA markers folowing antibiotic exposure may enable a rapid determination of antimicrobial susceptibility.  We submit transcriptomic data sets detailing global gene expression of F. tularensis following exposure to Doxycycline or Ciprofloxacin antibiotics.

sample_organism: Francisella tularensis subsp. tularensis SCHU S4

overall_design: F. tularensis SchuS4 colonies isolated on Cyctine Heart Agar (CHA) plates at 37oC were suspended in cation- adjusted Muller-Hinton broth, supplemented with 2% IsoVitaleX and 3 µM hematin (HLMHI) growth medium, to 5x105 CFU/ml.  Suspended bacteria (200 ml) were grown for 2h (37oC, 200 rpm) to adjust the culture to the growth media.  50 ml of the adjusted bacterial culture were transferred into each of three 250 ml Erlenmeyer flasks and incubated in the presence of 0.5/1 x Minimal Inhibitory Concentration (MIC) of Ciprofloxacin/Doxycycline for 2 hours, followed by centrifugation (20 min., 4oC, 4,000xg). Bacterial pellets were resuspended in 1 ml cold PBS, transferred into Eppendorf tubes, centrifuged (5 min., 10K rpm at 400C), and pellets were frozen by liquid nitrogen and kept at -700C until RNA extraction. Growth control samples were grown under same conditions w/o antibiotic. The Ciprofloxacin / Doxycycline exposure experiments were done each by independent duplicated biological experiment. Total RNA purification and residual DNA elimination were done by the Rneasy Mini kit and Rnase-free Dnase kit (QUIGEN) according to the manufacturer's instructions. Experiments were performed using biosafety level 3 (BSL-3) containment and procedures. Sterile RNA samples were transferred to BSL-2 lab for following procedures. RNA concentration was determined by the NanoDrop ND-1000 spectrophotometer and RNA quality was determined by using the Agilent 2100 Bioanalyzer with the "Prokaryote Total RNA Pico" chip. All RNA samples showed RNA Integrity Number (RIN value) of >9.  RNA samples were kept at -700C until used. Transcriptomic analysis was carried out by the RNA-seq method, at the Genome center, Columbia University.

last: Zaida

first: Inbar

last: Cohen-Gihon

first: Ohad

last: Shifman

first: Ofir

last: Israeli

first: Moshe

last: Aftalion

first: Sharon

last: Maoz

first: Raphael

last: Ber

first: Anat

last: Zvi

first: Ida

last: Steinberger-Levy


title: SATB1 Regulates 3D Genome Architecture in T cells by Constraining Chromatin Interactions surrounding CTCF-binding sites [RNA-seq]

city: shanghai

first: bao

last: wang

address: jin zun road

country: China

institute: shanghai jiao tong university school of medicine

summary: Spatial genome organization is critical for precise gene regulation during development. Special AT-rich sequence binding protein 1 (SATB1) has long been proposed to act as a global chromatin loop organizer in T cells. However, the exact functions of SATB1 in genome organization remain elusive. Here we show that the depletion of SATB1 in human and murine T cells led to transcriptional dysregulation for genes involved in T cell activation, as well as alterations of 3D genome architecture at multiple scales, including the A/B compartment, topologically associating domains (TADs), and loops. Importantly, SATB1 extensively colocalizes with CTCF throughout the genome. Depletion of SATB1 led to increased chromatin contacts among and across the SATB1/CTCF co-occupied sites, thereby affecting the transcription of critical genes involved in T cell activation. The loss of SATB1 did not affect the genome-wide occupancy of CTCF, but significantly reduced the retention of CTCF in the nuclear matrix. Collectively, our data reveal that SATB1 constrains chromatin topology surrounding CTCF-binding sites by tethering CTCF to the nuclear matrix, and suggest that the functional interplay between SATB1 and CTCF contributes to 3D genome organization.

overall_design: Naive CD4+ T cells were isolated from spleens of control and Satb1 flox/flox CD4-Cre mice and subsequently subjected to RNA-seq experiments.

first: Bao

last: Wang

first: Luzhang

last: Ji

first: Qian

last: Bian


title: Spatial transcriptome sequencing of primary nasopharyngeal carcinoma tissues

city: Hong Kong

first: Xin-Yuan

last: Guan

address: L10-56, Laboratory Block, LKS Faculty of Medicine

department: Clinical Oncology

country: Hong Kong

institute: The University of Hong Kong

summary: Understanding of the spatial architecture of the primary NPC microenvironment as well as the co-localization and cell-cell communication between tumor and immune cells

overall_design: Spatial transcriptome analysis of the NPC microenvironment

first: Lanqi

last: Gong


title: Single-cell sequencing of human peripheral blood mononuclear cells

summary: Understanding of the downstream mechanism of CD70-CD27 signaling in Tregs

overall_design: Human PBMCs co-cultured with CD70-NC-C666 and CD70-KO-C666 cells


title: Aryl hydrocarbon receptor activity downstream of IL-10 signaling is required to promote regulatory functions in human dendritic cells [RNA_in_vitro_DC10_iDC_DC10CH]

status: Public on Mar 04 2023

city: Milan

first: Francesca

middle: R.

last: Santoni de Sio

address: via Olgettina 58

department: SR-TIGET

country: Italy

institute: San Raffaele Scientific Institute

summary: Peripheral immune tolerance is a key physiological mechanism sustained by the activity of regulatory cells that impedes immune reaction to self and non-dangerous antigens. Interleukin 10 (IL-10) plays a key immunosuppressive role in this system by inhibiting effector cells and instructing regulatory cells. While the molecular mechanisms and gene expression patterns defining identity and function of T regulatory cells have been deeply studied, the molecular pattern driving tolerogenicity in myeloid antigen presenting cells remain less defined.  We have investigated the molecular mechanisms underlying IL-10 tolerogenic activity in human myeloid cells. By performing chromatin and transcriptomic studies in IL-10-induced monocyte-derived DC, we have found that IL-10 imprinted a pattern of accessible enhancers, which were exploited by Aryl Hydrocarbon Receptor (AHR) to promote the expression of a set of core genes. Functional studies demonstrated that AHR activation and core gene expression were necessary for IL-10-mediated induction of tolerogenic functions in in vitro differentiated DC. AHR exerted its role down-stream to IL-10 inducing tolerogenic and repressing inflammatory genes. Analyses of naturally occurring tolerogenic DC from peripheral blood showed that the IL-10-induced AHR module was active also in vivo in healthy conditions. In multiple sclerosis patients, instead, we observed significant alterations in the IL-10-induced AHR module that correlated with functional defects and reduced frequencies of in vitro differentiated and in vivo occurring IL-10-induced tolerogenic DC, respectively.  This study unveiled a previously undescribed IL-10-induced molecular mechanism required for establishing tolerogenic functions in human myeloid cells.

overall_design: Transcriptome analysis of of 2 subsets of in vitro differentiated dendritic cells

first: Alessandro

last: Testori


title: Aryl hydrocarbon receptor activity downstream of IL-10 signaling is required to promote regulatory functions in human dendritic cells [RNA_in_vitro_DC10_MS]

overall_design: Transcriptome analysis of in vitro differentiated tolDC from healthy and multiple sclerosis monocytes


title: SATB1 Regulates 3D Genome Architecture in T cells by Constraining Chromatin Interactions surrounding CTCF-binding sites

summary: SATB1, a nuclear matrix-associated protein, has long been proposed to function as a global chromatin loop organizer in T cells. However, the precise roles of SATB1 in chromatin organization remain elusive. Here we show that the depletion of SATB1 in immortalized T cells led to pronounced changes in gene expression, particularly for genes involved in cell proliferation and T cell activation, as well as 3D genome architecture at multiple scales, including the A/B compartment, topologically associating domains (TADs), and loops. Importantly, SATB1 extensively colocalizes with CTCF throughout the genome. Depletion of SATB1 led to increased association among the SATB1/CTCF co-occupied sites, as well as increased chromatin contacts across these sites, thereby altering the genome-wide chromatin loop landscape. SATB1 does not regulate genome architecture by modulating CTCF occupancy. Rather, the topological effects imposed by SATB1 may be attributed to SATB1-dependent anchoring of CTCF to the salt extraction-resistant nuclear matrix. Together, our findings suggest that the functional interplay between nuclear matrix and CTCF plays a critical role in orchestrating 3D genome organization.


title: Aryl hydrocarbon receptor activity downstream of IL-10 signaling is required to promote regulatory functions in human dendritic cells


title: Aryl hydrocarbon receptor activity downstream of IL-10 signaling is required to promote regulatory functions in human dendritic cells [RNA_ex_vivo_DC10_MS]

overall_design: Transcriptome analysis of circulating tolDC in healthy and multiple sclerosis donors


title: Aryl hydrocarbon receptor activity downstream of IL-10 signaling is required to promote regulatory functions in human dendritic cells [ATAC_in_vitro_DC10_iDC_DC10CH]

overall_design: Chromatin accessibility analysis of 2 subsets of in vitro differentiated dendritic cells.


title: Aryl hydrocarbon receptor activity downstream of IL-10 signaling is required to promote regulatory functions in human dendritic cells [ATAC_ex_vivo_DC10_cDC]

overall_design: Chromatin accessibility analysis of 2 subsets of ex vivo isolated dendritic cells.


title: Identifying Molecular and Functional Similarities and Differences between Adult Human Primary Valve Interstitial Cells and Ventricular Cardiac Fibroblasts

status: Public on Mar 03 2023

city: Madison

first: Fathima

last: Shabnam

state: WI

address: 1415 Engineering Hall

institute: University of Wisconsin-Madison

summary: Fibroblasts are mesenchymal cells that predominantly produce and maintain the extracellular matrix (ECM) and are critical mediators of injury response. In the heart, valvular interstitial cells (VICs) are a population of fibroblasts responsible for maintaining the structure and function of heart valves. These cells are regionally distinct from myocardial fibroblasts, including left ventricular cardiac fibroblasts (LVCFBs), which are located in the myocardium in close vicinity to cardiomyocytes. Here, we hypothesize these subpopulations of fibroblasts are transcriptionally and functionally distinct. To compare these fibroblast subtypes, we collected patient-matched samples of human primary VICs and LVCFBs and performed bulk RNA sequencing, extracellular matrix profiling, and functional contraction and calcification assays. Here, we identified combined expression of SUSD2 on a protein-level, and MEOX2, EBF2 and RHOU at a transcript-level to be differentially expressed in VICs compared to LVCFBs and demonstrated that expression of these genes can be used to distinguish between the two subpopulations. We found both VICs and LVCFBs expressed similar activation and contraction potential in vitro, but VICs showed an increase in ALP activity when activated and higher expression in matricellular proteins, including cartilage oligomeric protein and alpha 2-Heremans-Schmid glycoprotein, both of which are reported to be linked to calcification, compared to LVCFBs. These comparative transcriptomic, proteomic, and functional studies shed novel insight into the similarities and differences between valvular interstitial cells and left ventricular cardiac fibroblasts and will aid in understanding region-specific cardiac pathologies, distinguishing between primary subpopulations of fibroblasts, and generating region-specific stem-cell derived cardiac fibroblasts.

overall_design: We collected patient-matched human primary valve interstitial cells and left ventricular cardiac fibroblasts from three donors to compare transcriptional profiles between these two cell types.

first: Martha

middle: E

last: Floy

first: Sean

middle: P

last: Palecek


title: Gene expression profile of HCT116 and HT29 cells treated with D/L2HG

status: Public on Mar 02 2023

city: Suita

first: Sho

last: Tabata

state: Osaka

address: 3-2 Yamadaoka

department: Institute for Protein Research,ces

institute: Osaka University

summary: The oncometabolite, 2-hydroxyglutarate (2HG), has been directly implicated in carcinogenesis; however, the underlying molecular mechanisms remain poorly understood. To elucidate the molecular mechanisms through which 2HG contributes to cancer progression, we performed transcriptome analysis in 2HG-treated colorectal cancer HCT116 and HT29 cells.

overall_design: Gene expression in HCT116 and HT29 cells treated with membrane-permeable octyl esters of D2HG (OC-D2HG: 500 μM) or L2HG (OC-L2HG: 500 μM) for 24 h was measured.


title: Coordinated Activation of c-Src and FOXM1 Drives Tumor Cell Proliferation and Breast Cancer Progression

city: Montreal

first: Harvey

middle: Wilmore

last: Smith

state: Quebec

address: Life Sciences Complex, Room 602, 1160 Pine Avenue West

department: Goodman Cancer Research Centre

country: Canada

institute: McGill University

summary: Activation of the tyrosine kinase c-Src promotes breast cancer progression and poor outcome, yet the underlying mechanisms are incompletely understood. Here, we show that deleting c-Src abrogates the activity of Forkhead Box M1 (FOXM1), a master transcriptional regulator of the cell cycle, in a genetically engineered model mimicking the Luminal B molecular subtype of breast cancer. By phosphorylating it on two tyrosine residues, c-Src stimulates the nuclear localization of FOXM1 and the expression of its target genes, including key regulators of G2-M cell cycle progression as well as c-Src itself. This positive feedback loop drives proliferation in genetically engineered and patient-derived models of Luminal B-like breast cancer. Targeting this mechanism, including through novel compounds that destabilize the FOXM1 protein, induces G2-M cell cycle arrest and apoptosis, blocking tumor progression and impairing metastasis. We identify positive correlation of FOXM1 and c-Src expression in human breast cancer and show that the expression of FOXM1 target genes predicts poor outcome and associates with the Luminal B subtype, which responds poorly to approved therapies. These findings indicate that a regulatory network centered on c-Src and FOXM1 is a targetable vulnerability in aggressive luminal breast cancers.

overall_design: Mammary epithelial organoids from a doxycycline-inducible transgenic model of breast cancer (MMTV-rtTA/TetO-PyVmT-IRES-CRE) were used to study the effects of c-Src (Src) deletion on early events in tumorigenesis. Transcriptomic analysis (RNA-Seq) was performed on organoid preparations from three independent groups of mice (n=5) per genotype (wild-type and conditional c-Src knockout), with and without doxycycline treatment to induce expression of the transforming oncogene (polyomavirus middle-T antigen) and Cre recombinase.

first: Ipshita

last: Nandi

middle: W

first: Virginie

last: Sanguin-Gendreau

first: Linjia

first: Alain

last: Pacis

first: Vasilios

last: Papavasiliou

first: Dongmei

last: Zuo

first: Stella

last: Nam

first: Sherif

last: Attala

first: Sung

last: Kim

first: Sierra

last: Lusson

first: Hellen

last: Kuasne

first: Anne-Marie

last: Fortier

first: Paul

last: Savage

first: Constanza

last: Martinez Ramirez

first: Morag

last: Park

first: John

middle: A

last: Katzenellenbogen

first: Benita

first: William

last: Muller


title: Maintenance of pluripotency signature in the entire ectoderm enables potential for neural crest formation [bulk_RNASeq]

city: Bethesda

first: Laura

last: Kerosuo-Pahlberg

address: 30 Convent Dr

institute: NIDCR

summary: The ability of the pluripotent epiblast to contribute progeny to all three germ layers is thought to be lost after gastrulation. The later-forming neural crest (NC) rises from ectoderm and it remains poorly understood how its exceptionally high stem-cell potential to generate mesodermal- and endodermal-like cells is obtained. We monitored transcriptional changes from gastrulation to neurulation using single-cell-Multiplex-Spatial-Transcriptomics (scMST) complemented with RNA-sequencing. Here we show maintenance of pluripotency-signature (Nanog/Oct4-PouV/Klf4-positive) in undecided pan-ectodermal stem-cells spanning the entire ectoderm late during neurulation with ectodermal patterning completed only at the end of neurulation when the pluripotency-signature becomes restricted to NC, challenging our understanding of gastrulation. Furthermore, broad ectodermal pluripotency-signature is found at all axial levels unrelated to the NC lineage the cells later commit to, suggesting a general role in stemness enhancement and proposing a mechanism by which the NC acquires its ability to form derivatives beyond “ectodermal-capacity” in chick and mouse embryos.

overall_design: Samples from the neural crest domain of chick embryos at the midbrain level were collected for bulk RNAseq (HH5, HH6,  HH7 (1ss), 2,ss,  3ss, 4ss, 5ss, 6ss, 7ss, 8ss, 9ss, 10ss)

first: Ceren

last: Pajanoja

first: Jenny

last: Hsin

first: Bradley

last: Olinger

first: Andrew

last: Schiffmacher

first: Rita

last: Yazejian

first: Shaun

last: Abrams

first: Arvydas

last: Dapkunas

first: Zarin

last: Zainul

last: Doyle

first: Daniel

last: Martin

last: Kerosuo


title: Sertoli cells are the unique source of stem cell factor for spermatogenesis

city: Shanghai

first: Bo

middle: O

last: Zhou

address: 320 Yueyang Road

institute: State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, CAS

summary: To systematically investigate the expression patterns of all potential niche factors in testis, we performed single-cell RNA sequencing (scRNA-seq) of all testicular somatic cell types. To enrich somatic cells, we depleted Tomato+ cells from the testes of 2-month-old Ddx4-creER; R26tdTomato mice at 4 weeks after tamoxifen treatment. Then the cells were further captured with 10x Genomics platform. After analysis of the integrated data, we mapped the expression patterns of all known niche factors in testicular somatic cells. We also performed scRNA-seq of testicular cells from 6-week-old control and Amh-cre;Scf fl/fl mice to study the effect of Scf conditional knockout from Sertoli cells on spermatogenesis. By scRNA-seq data analysis, we found that conditional knockout of Scf from Sertoli cells blocks spermatogenesis by depleting differentiating spermatogonia

overall_design: We analyzed single cell transcriptome profile of testicular cells from 8-week-old DDX4-creER;R26tdTomato mice and 6-week-old control and Amh-cre;Scf fl/fl mice

first: Yi

last: Peng

first: Xinyu

last: Tang


title: SOCS1 regulates a subset of NFκB-target genes through direct chromatin binding and defines macrophage functional phenotypes (scRNA-Seq).

status: Public on Mar 01 2023

city: Evanston

first: Brian

last: Wray

state: IL

address: 633 Clark Street

department: Feinberg School of Medicine

institute: Northwestern University

summary: Suppressor of cytokine signaling-1 (SOCS1) exerts control over inflammation by targeting p65 NFκB for degradation in addition to its canonical role regulating cytokine signaling. We report here that SOCS1 does not operate on all p65 targets equally, instead localizing to a select subset of pro-inflammatory genes. Promoter-specific interactions of SOCS1 and p65 determine the subset of genes activated by NFκB during systemic inflammation, with profound consequences for cytokine responses, immune cell mobilization, and tissue injury. Nitric oxide synthase-1 (NOS1)-derived NO is required and sufficient for displacement of SOCS1 from chromatin, permitting full transcriptional activation of these genes. Single cell transcriptomic analysis of NOS1-deficient animals led to the detection of a regulatory macrophage subset, which exerts potent suppression on inflammatory cytokine responses and tissue remodeling. These results provide the first example of a redox-sensitive, gene-specific mechanism for converting macrophages from cells that regulate inflammation to cells licensed to promote aggressive and potentially injurious inflammation.

overall_design: Comparative single cell gene expression profiling of RNA-seq data for CD45+ pulmonary cells from wild type (C65BL6) or NOS1 KO lungs after treatment with LPS.

first: Diego

last: Coelho

first: Flavio

last: Palma

first: Veronica

last: Paviani

first: Katy

last: LaFond

first: Yunping

last: Huang

first: Dongmei

last: Wang

first: Sridhar

last: Rao

first: Feng

last: Yue

first: Marcelo

middle: G

last: Bonini

first: Benjamin

middle: N

last: Gantner


title: SOCS1 regulates a subset of NFκB-target genes through direct chromatin binding and defines macrophage functional phenotypes (ChIP-Seq).

overall_design: Comparative chromatin binding profiling of p65 and SOCS1  for wild type (C65BL6) or NOS1 KO BMDM after treatment with LPS.


title: SOCS1 regulates a subset of NFκB-target genes through direct chromatin binding and defines macrophage functional phenotypes (RNA-Seq).

overall_design: Comparative gene expression profiling analysis of RNA-seq data for wild type (C65BL6) or NOS1 KO BMDM after treatment with LPS.


title: Single-cell transcriptomic and epigenetic profiling of mouse myenteric plexus glial cells

city: Boston

first: Allan

last: Goldstein

address: 185 Cambridge St

department: Pediatric Surgery

institute: Massachusetts General Hospital

summary: This study was undertaken to define the molecular subtypes of myenteric plexus glial cells in mice, and to understand the molecular basis for glial cells’ capacity to become neurons. Methods: We performed single-cell RNA sequencing and single-nucleus ATAC sequencing of enteric neurons from small intestine at the adolescent mice (on or near postnatal day of life 14). We also performed both single-cell RNA sequencing and single-nucleus ATAC sequencing on 3-dimensional neurosphere cultures. Results: We identify numerous distinct transcriptional subgroups of myenteric plexus glial cells, including cells expressing genes associated with neuronal differentiation. Epigenetic analysis shows distinct chromatin accessibility profiles that correlate with gene expression patterns. Glial cells maintain open chromatin at gene loci associated with neuronal fate. 3-dimensional cultures provide a niche for active neurogenesis. Chromatin closes at glial-associate loci during neurogenesis. Conclusion: Utilizing single-cell RNA sequencing and single-nucleus ATAC sequencing, we identify myenteric glial cell subtypes and uncover a molecular basis for a glial-to-neuronal fate transition.

overall_design: Use of 10X Genomics technology to analyze single-cell transcriptomics and single-cell epigenetics of myenteric plexus glial cells, as well as 3-dimensional neurosphere cultures derived from these cells.


title: Genomic profiles of IDH-mutant gliomas: MYCN amplified IDH-mutant astrocytoma had the worst prognosis and true mixed oligoastrocytoma does not exist

city: Seoul

first: Kwanghoon

last: Lee

address: 103, Daehak-ro, Jongno-gu

department: Pathology

country: South Korea

institute: Seoul National University

type: Methylation profiling by genome tiling array

summary:
Background: This study aims to find any ambiguous genetic outlier for “oligodendroglioma, IDH-mutant and 1p/19q-codeleted (O_IDH_mut)” and “astrocytoma, IDH-mutant (A_IDH_mut)” and to redefine the genetic landscape of IDH-mutant gliomas.
Methods: The next-generation sequencing (NGS) using a brain tumor-targeted gene panel, methylation profiles, and clinicopathological features were analyzed in O_IDH_mut (n=74) and A_IDH_mut (n=95).
Results: Three had ambiguous genetic profiles for O_IDH_mut or A_IDH_mut. Two were unusual TP53-mutant O_IDH_mut, but classified into O_IDH_mut by DKFZ methylation classifier (MC) (score: 0.98). The remaining one was a 1p/19q-codeleted and TERTp-mutant A_IDH_mut, which also had TP53 and CIC mutations. This case matched to O_IDH_mut (Score: 0.75) by the DKFZ-MC v.11b4 algorithm but was corrected to A_IDH_mut_HG (high-grade) (Score: 0.84) using the updated DKFZ-MC v.12.5. The remaining 97.3% and 98.9% of O_IDH_mut and A_IDH_mut had a classic genomic landscape. The patients with MYCN amplified and/or CDKN2A/2B homozygously deleted A_IDH_mut had a worse prognosis than those without these genes alterations
Conclusion: CIC and/or FUBP1 mutations were detected in 93.2% and MGMTp methylation were detected in 95.9% of O_IDH_mut patients. Accepting the fact that 1p/19q codeletion and TP53 mutations are not 100% mutually exclusive, as in the three exceptional cases mentioned above, would be of great help in diagnosing the two subtypes of IDH-mutant diffuse glioma. In histopathologically or genetically ambiguous cases, MC can be an objective tool to avoid a diagnosis of NOS (not otherwise specified) or NEC (not elsewhere classified) as well as tumour classification. The authors have not encountered true mixed oligoastrocytomas using genetic and methylation profiles-integrated diagnosis.

overall_design: Bisulfite converted genomic DNA extracted from the 3 tumor samples was hybridized to Illumina Infinium EPIC 850k Human DNA Methylation Beadchip

first: Sung-Hye

last: Park


title: GHBES1-OE-FIBER ChIP-Seq

city: Xian

first: Liping

last: Zhu

address: Guodu street

institute: Shaanxi Normal University

summary:
ChIP-seq data of 5 dpa fibers from GhBES1-Overexpression cotton plants
GHBES1-OE-FIBER-CHIP-SEQ

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for 5 day developed  fibers from GhBES1 overexpression cotton plants

first: Guanghui

last: Xiao


title: p53 dimers elicit unique tumor suppressive activities through an altered metabolic program [ATAC-seq]

city: Houston

first: Yuan

last: Qi

state: TX

address: 1400 Pressler St

department: Bioinformatics & Computational Biology

institute: University of Texas M.D. Anderson Cancer Center

summary: Cancer-related alterations of the p53 Tetramerization Domain (TD) abrogate wild-type (WT) p53 function. They result in a protein that preferentially forms monomers or dimers. These are also normal p53 states under basal cellular conditions. However, their physiological relevance is not well understood. We have established in vivo models for monomeric and dimeric p53 which model Li-Fraumeni Syndrome patients with germline p53 TD alterations. p53 monomers are inactive forms of the protein. Unexpectedly, p53 dimers conferred some tumor suppression that is not mediated by canonical WT p53 activities. p53 dimers upregulate the PPAR pathway. These activities are associated with lower prevalence of thymic lymphomas and inhibition of CD8+ T-cell accumulation. Lymphomas derived from dimeric p53 mice show cooperating alterations in the PPAR pathway, further implicating a role for these activities in tumor suppression. Our data reveal novel functions for p53 dimers and support the exploration of PPAR agonists as therapies.

overall_design: Comparison of differentially open regions in thymus derived from dimeric vs. p53-null  mice

first: J

last: Gencel-Augusto

first: G

last: Lozano


title: Influenza-specific effector memory B cells predict long-lived antibody responses to vaccination in humans [scRNA-Seq]

city: Atlanta

first: Chris

last: Scharer

state: GA

address: 1510 Clifton Rd, Suite 3086A

department: Microbiology and Immunology

institute: Emory University

summary: BCR repertoire sequencing data from influenza vaccinated subjects

overall_design: Three healthy subjects had blood drawn at various time points following IM influenza vaccine.  Cells were sort purified to obtain naive B cells, plasmablasts, flu (HA) specific memory B cells (FcRL5+ and FcRL5neg) and IgD negative memory B cells.  Donor 1 had HA-specific memory B cells for 7 and 14 days post vaccine; Donor 2 had HA-specific memory B cells 7 days post vaccine; Donor 3 had HA-specific memory B cells 14 and 28 days post vaccine.  All donors had IgD negative memory B cells 120 days post vaccine, naive B cells for either 7 or 14 days post vaccine and plasmablasts 7 days post vaccine in years 1 and 2.  These samples were used for repertoire analysis by deep sequencing.

first: Anoma

last: Nellore

first: Esther

last: Zumaquero

first: Christopher

middle: D

middle: F

last: Fucile

last: Tipton

first: Rodney

last: King

first: Tian

last: Mi

first: Betty

last: Mousseau

first: John

last: Bradley

first: Fen

last: Zhou

first: Stuti

last: Mutneja

first: Paul

last: Goepfert

first: Jeremy

last: Boss

first: Troy

last: Randall

first: Ignacio

last: Sanz

first: Alexander

last: Rosenberg

first: Frances

last: Lund


title: Collagen-anchored interleukin-2 and interleukin-12 safely reprogram the tumor microenvironment in soft tissue sarcomas

city: Cambridge

first: K

middle: Dane

last: Wittrup

state: Massachusetts

department: Koch Institute for Integrative Cancer Research

institute: Massachusetts Institute of Technology

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

sample_organism:
Canis lupus familiaris
Mus musculus

platform_organism:
Canis lupus familiaris
Mus musculus

overall_design: Refer to individual Series


title: Prolonged Interferon-Stimulated Gene and Protein  Signatures in Multiple Sclerosis Induced by PEGylated  IFN-beta-1a Compared to Non-PEGylated IFN-beta-1a

city: Chicago

first: Anthony

last: Reder

address: 5841 S Maryland Ave

department: Neurology

institute: University of Chicago Medicine

summary:
Transcriptome analysis of RNA samples from human PBMCs of IFN-beta treated multiple sclerosis patients.
Interferon (IFN)-b-1a (Avonex) and longer half-life, polyethylene glycol-conjugated IFN-b-1a (PEG-IFN-b-1a,  Plegridy), may generate different molecular responses.  At 6 h, non-PEGylated IFN-b-1a injection upregulated expression of 136 genes and PEG-IFN-b-1a upregulated 85. At 24 h, induction was maximal; IFN-b-1a upregulated476 genes and PEG-IFN-b-1a now upregulated 598. Long-term PEG-IFN-b-1a therapy increased expression of antiviral and immune-regulatory genes (IFIH1, TLR8, IRF5, TNFSF10 [TRAIL], STAT3, JAK2, IL15, and RB1) and IFN signaling pathways (IFNB1, IFNA2, IFNG, IRF7), but downregulated expression of inflammatory genes (TNF, IL1B, and SMAD7). Long-term PEG-IFN-b-1a induced longer and stronger expression of Th1,  Th2, Th17, chemokine, and antiviral proteins than long-term IFN-b-1a. Long-term therapy also primed the immune system, evoking higher gene and protein induction after IFN reinjection at 7 months than at 1 month of PEG-IFN-b-1a treatment. Both forms of IFN-b balanced correlations of expression among these genes and proteins, with positive correlations between Th1 and Th2 families, quelling the ‘‘cytokine storm’’ of untreated  MS. Both IFNs induced long-term, potentially beneficial, molecular effects on immune and possibly neuroprotective pathways in MS.

overall_design: We identified different short-term and long-term in vivo global RNA signatures of IFN-stimulated genes in multiple sclerosis (MS) peripheral blood mononuclear cells  and in selected paired serum immune proteins at 6, 24, 48 hours, and baseline.

first: Kristi

last: Nguyen

first: Maya

last: Olcer

first: Quentin

last: Howlett-Prieto

first: Xuan

last: Feng

middle: T


title: Influenza-specific effector memory B cells predict long-lived antibody responses to vaccination in humans

first: Chris

last: Scharer


title: Collagen-anchored interleukin-2 and interleukin-12 safely reprogram the tumor microenvironment in mouse melanomas

summary: Mouse B16F10 melanoma tumors were analyzed for gene expression changes following intratumoral treatment with collagen-anchored IL-2 and IL-12 therapy for comparative analysis to expression changes observed in canine soft tissue sarcomas. Tumors were collected at different intervals following treatment to assess persistence of any therapy-driven gene expression changes.

overall_design: Twelve C57BL/6 mice (Taconic) bearing day 6 B16F10 flank tumors were assigned to cohorts to receive intratumoral doses of interleukin-2 and interleukin-12 collagen-binding fusion proteins or saline (n=3/group). Cohorts were designed to mimic the treatment groups from the canine portion of this work. The first cohort (n=3) had tumors collected 2 days after treatment; the second cohort (n=3) had tumors collected 8 days after treatment; and the third cohort (n=4) received a second cytokine dose 6 days after the first dose, with tumors collected two days later (day 8 post-first dose). Tumor tissue was formalin-fixed and paraffin-embedded prior to RNA extraction (Qiagen FFPE RNEasy) and QC on Bioanalyzer (Agilent). Gene expression was analyzed through hybridization to the Nanostring Mouse PanCancer Immune Profiler nCounter panel set probes, with quantification by Nanostring Digital Analyzer. Expression data was analyzed using Nanostring nSolver software.

first: Jordan

last: Stinson

first: Allison

last: Sheen

first: Timothy

last: Fan


title: Canine peripheral blood TCRαβ T cell atlas: Identification of diverse subsets including CD8+ MAIT-like cells by combined single-cell transcriptome and V(D)J repertoire analysis

city: Davis

first: Stefan

middle: Matthias

last: Keller

state: California

address: One Shields Avenue

department: Pathology, Immunology, Microbiology

institute: University of California, Davis

summary: The diversity of canine T cells is poorly understood. We used 5’ scRNA-seq coupled with immune repertoire sequencing to generate a highly detailed atlas of peripheral blood TCRαβ+ T cells of healthy dogs.

overall_design: FACS-purification of TCRab T cells from PBMC of 4 healthy Beagle dogs based on TCRab expression followed by 5' scRNAseq with V(D)J analysis

first: Maria

last: Eschke

first: Peter

last: Moore

first: Haiyang

last: Chang

first: Gottfried

last: Alber


title: Collagen-anchored interleukin-2 and interleukin-12 safely reprogram the tumor microenvironment in canine soft tissue sarcomas

summary: Canine soft tissue sarcomas were analyzed for gene expression changes following intratumoral treatment with collagen-anchored IL-2 and IL-12 therapy. Tumors were surgically resected at different intervals following treatment to assess persistence of any therapy-driven gene expression changes.

overall_design: Ten client-owned dogs with soft tissue sarcomas were recruited to three treatment cohorts to receive intratumoral doses of interleukin-2 and interleukin-12 collagen-binding fusion proteins. The first cohort (n=3) had tumors surgically excised 2 days after treatment; the second cohort (n=3) had tumors excised 8 days after treatment; and the third cohort (n=4) received a second cytokine dose 6 days after the first dose, with tumors excised two days later (day 8 post-first dose). Tumor tissue was formalin-fixed and paraffin-embedded prior to RNA extraction (Qiagen FFPE RNEasy) and QC on Bioanalyzer (Agilent). Gene expression was analyzed through hybridization to the Nanostring Canine IO nCounter panel set probes, with quantification by Nanostring Digital Analyzer. Expression data was analyzed using Nanostring nSolver software.

first: Noor

last: Momin

last: Hampel

first: Rebecca

last: Bernstein

last: Kamerer

first: Bahaa

last: Fadl-Alla

first: Jonathan

last: Samuelson

first: Elizabeth

last: Fink


title: Cytotoxic T cells targeting spike glycoprotein are associated with hybrid immunity to SARS-CoV-2

city: Seattle

first: Chetan

last: Seshadri

address: 750 Republican Street

institute: University of Washington

summary: mRNA vaccination of individuals with prior SARS-CoV-2 infection provides superior protection against breakthrough infections with variants of concern compared to vaccination in the absence of prior infection. However, the immune mechanisms by which this ‘hybrid immunity’ is generated and maintained are unknown. While genetic variation in spike glycoprotein effectively subverts neutralizing antibodies, spike-specific T cells are generally maintained against SARS-CoV-2 variants. Thus, we comprehensively profiled T cell responses against the S1 and S2 domains of spike glycoprotein in a cohort of SARS-CoV-2-naive or convalescent individuals who received two-dose mRNA vaccine series and were matched by age, sex, and vaccine type. Using flow cytometry, we observed that the overall functional breadth of CD4 T cells as well as polyfunctional Th1 responses were similar between the two groups. However, polyfunctional cytotoxic CD4 T cell responses against both S1 and S2 domains trended higher among convalescent subjects. Multi-modal single-cell RNA sequencing revealed diverse functional programs in spike-specific CD4 and CD8 T cells in both groups. However, convalescent individuals displayed enhanced cytotoxic and antiviral CD8 T cell responses to both S1 and S2 in the absence of cytokine production. Taken together, our data suggest that cytotoxic CD4 and CD8 T cells targeting spike glycoprotein may partially account for hybrid immunity and protection against breakthrough infections with SARS-CoV-2.

overall_design: We performed multi-modal (surface protein, gene expression, and TCR) single-cell RNA sequencing on PBMC collected from SARS-CoV-2-naive (n = 4) and convalescent (n = 4) individuals following two-dose mRNA vaccination.

first: Jolie

last: Phan

first: Krystle

middle: K

last: Yu

first: Melissa

middle: S

last: Aguilar

first: Inah

last: Golez

first: Michael

last: Gale Jr.


title: Molecular Dissection of Polycomb Group Protein-mediated Fetal Hemoglobin Repression [RNA-seq]

city: Philadelphia

first: KUNHUA

last: QIN

email: qink@chop.edu, kunhua.qin@gmail.com

state: PA

address: 3615 Civic Center Blvd, 315H

institute: Children's Hospital of Philadelphia

summary: The fetal (HbF)-to-adult (HbA) hemoglobin switch is a paradigm for developmental gene expression control with relevance to sickle cell disease and beta-thalassemia. Polycomb repressive complex (PRC) proteins regulate this switch, and an inhibitor of the EED subunit of PRC2 entered clinical trials for fetal hemoglobin activation. Yet, how PcG complexes function in this process, their target genes and relevant subunit composition are unknown. Here, we identified the PRC1 subunit BMI1 as a novel HbF repressor. We uncovered LIN28B, IGF2BP1, and IGF2BP3 as direct BMI1 targets and demonstrate that they account for the BMI1 effects. BMI1 functions as part of the canonical PRC1 (cPRC1) subcomplex as revealed by the physical and functional dissection of BMI1 protein partners. Lastly, we demonstrate that BMI1/cPRC1 acts in concert with the PRC2 to repress HbF through the same target genes. Our study illuminates how PRC silences HbF, highlighting an epigenetic mechanism involved in hemoglobin switching.

overall_design: Total RNA was isolated from HUDEP2 cells of 6 or 7 days differentiation or CD34+ HSPCs of day 8 differentiation targeted with CRISPR control sgRNA or BMI1/EZH2/CBX2,4,8  sgRNAs; Transcriptome changes were analyzed by bulk RNA-seq;

first: Kunhua

last: Qin

first: Gerd

last: Blobel


title: Upper respiratory infection drives clinical signs and inflammatory responses in heterologous challenge of SARS-CoV-2 variants of concern

city: Memphis

first: Colleen

middle: Beth

last: Jonsson

state: TN

address: 858 Madison Avenue

institute: University of Tennessee Health Science Center

summary: During the COVID-19 pandemic, three variants of concern (VOC) of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) emerged globally with increasingly greater ability to evade the immune response and enhanced transmissibility. A critical question we have faced with each new VOC is how well the vaccine based on the ancestral strain will protect. Given vaccine hesitancy, it is also important to address how prior live infections may protect against new variants.  To address this later question, K18-hACE2 mice were infected with a low, partially lethal dose of WA1/2020 (WA1), the prototype strain on which the mRNA vaccines are based. On 24 days post-infection (dpi), surviving mice were challenged with WA1, Alpha or Delta. Nasal turbinates and lungs showed active replication on 1- and 3-days post-challenge (dpc) in Alpha- and Delta-, but not WA1- challenge groups. The rate of recovery of Alpha- and Delta-challenged mice lagged behind WA1 with mice showing weight loss despite a greatly dampened proinflammatory response in the lung. In contrast, nasal turbinates had a heightened proinflammatory response for Alpha- and Delta- but not WA1-challenged groups. Inflammatory responses were limited to the upper respiratory tract. Despite this, we did not observe significant difference among the groups in fold change in the neutralizing titer. In summary, infection with a low dose of SARS-CoV-2 confers protection from challenge, but mice in the heterologous challenge group differed in having greater weight loss, viral replication, and a heightened proinflammatory response in the upper respiratory tract.

overall_design: Mice were intranasally infected with SARS-CoV-2 WA1/2020 isolate and challenged with either WA1/2020 or the VOCs, Alpha, or Delta 24 days post-infection. Nasal turbinates and lung samples were collected 1 and 3 days post-infection and post-challenge.

first: Jacob

middle: H

last: Nichols

first: Evan

last: Williams

middle: B


title: Lineage plasticity enables low-ER luminal tumors to evolve and gain basal-like traits

city: Lebanon

first: Diwakar

last: Pattabiraman

state: New Hampshire

address: 1 Medical Center Drive

department: Molecular systems biology

institute: Geisel School of Medicine at Dartmouth

summary: Breast tumors expressing lower levels of Estrogen Receptor (ER) represent a distinct subset of breast cancer characterized by the emergence of basal-like characteristics within tumors that are traditionally considered luminal-like. Lineage tracing of these low-ER tumors in the MMTV-PyMT mouse mammary tumor model revealed that the basal-like tumor cells within these tumors arose from normal luminal epithelial cells, suggesting that luminal-to-basal plasticity is responsible for the evolution and emergence of basal-like characteristics within these tumors. This study aims to identify the potential drivers of this plasticity by using single-cell RNA sequencing to identify populations of lineage-restricted luminal cells and luminal-derived basal cells within the tumor, and comparing their gene expression profiles.

overall_design: Single-cell RNA-sequencing of mouse MMTV-PyMT tumors labelled with GFP on the luminal epithelial lineage

first: Gadisti

last: Mohamed

first: Owen

middle: M

last: Wilkins

middle: R


title: iPSC-derived cells lack immune tolerance to autologous NK-cells due to imbalance in ligands for activating and inhibitory NK-cell receptors

first: Ksenia

last: Anufrieva

state: MA

address: 60 Fenwood Rd

institute: Brigham and Women's Hospital

summary: Dozens of transplants generated from pluripotent stem cells are currently in clinical trials. The creation of patient-specific iPSCs makes personalized therapy possible due to their main advantage of immunotolerance. However, some reports have claimed recently that aberrant gene expression followed by proteome alterations and neoantigen formation can result in iPSCs recognition by autologous T-cells. Meanwhile, the possibility of NK-cell activation has not been previously considered. This study focused on the comparison of autologous and allogeneic immune response to iPSC-derived cells and isogeneic parental somatic cells used for reprogramming. Here we report that cells differentiated from iPSCs can be recognized by NK-cells rather than by autologous T-cells. We observed that iPS-fibro elicited a high level of NK-cell degranulation and cytotoxicity, while isogeneic parental skin fibroblasts used to obtain iPSCs barely triggered an NK-cell response. iPSC-derivatives with B2M knockout did not cause an additional increase in NK-cell activation, although they were devoid of HLA-I, the major inhibitory molecules for NK-cells. Transcriptome analysis revealed a significant imbalance of ligands for activating and inhibitory NK-cell receptors in iPS-fibro. Compared to parental fibroblasts, iPSC-derivatives had a reduced expression of HLA-I simultaneously with an increased gene expression of major activating ligands, such as MICA, NECTIN2, and PVR. The lack of inhibitory signals might be due to insufficient maturity of cells differentiated from iPSCs. In addition, we showed that pretreatment of iPS-fibro with proinflammatory cytokine IFNγ restored the ligand imbalance, thereby reducing the degranulation and cytotoxicity of NK-cells. In summary, we showed that iPSC-derived cells can be sensitive to the cytotoxic potential of autologous NK-cells regardless of HLA-I status. Thus, the balance of ligands for NK-cell receptors should be considered prior to iPSC-based cell therapies.

overall_design: We established three isogenic cell models consisting of parental dermal fibroblasts used for reprogramming, iPSCs-derived fibroblast-like cells (iPS-fibro), and iPS-fibro with the knockout of the B2M gene (ΔiPS-fibro). Total RNA was isolated from intact and IFNγ-stimulated cells of early passages. Transcriptome profiles for the obtained samples were generated using NextSeq 500.

first: Margarita

middle: E

last: Bogomiakova

first: Tatiana

middle: V

last: Grigoryeva


title: Accessible chromatin differences in FCRL5 positive and negative influenza-specific memory B cells

first: Chris

last: Scharer

summary: We examined the accessible chromatin differences between circulating B cell subsets post-vaccination. Using FCRL5 as a marker of Tbet expressing B cells, we sorted FCRL5+ (Tbet+) and FCRL5- (Tbet-) memory B cells, and antibody secreting plasmablasts at day 7 and day 14 post-vaccination. Additionally, we used H1 B cell tetramers to track cells responding to the influenza vaccine in a subset of samples. These data provide insights into the accessible chromatin landscape of distinct B cell subsets that respond to influenza vaccination.

overall_design: ATAC-seq on 4 B cell subsets from patients 7 or 14 days post influenza vaccination.

first: C

first: A

last: Nellore

first: F

middle: L

last: Lund


title: HyperTRIBE uncovers SYNCRIP RNA binding activity in hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs)

city: new york

first: Michael

last: Kharas

state: New York

address: 417 68th street

department: Molecular Pharmacology Program

institute: Mamorial Sloan Kettering Cancer Center

summary: Identifying SYNCRIP RNA-binding targets in HSC and MPP populations, in order to uncover which direct targets influence SYNCRIP's role in maintaining HSCs self renewal capacity.

overall_design: HSCs (Lin-, Sca1+, cKit+, CD150+ CD48-) and MPPs (Lin-, Sca1+, cKit+, all remaining CD150 CD48 cells) from C57BL/6 mice (The Jackson Laboratory, USA) were FACS sorted and transduced with virus expressing either empty vector (MSCV-IRES-GFP) or SYNCRIP-ADAR. 48hrs post transduction cells were sorted for GFP+ expression and used for RNA-seq.

first: Ly

middle: P

last: Vu

first: Florisela

last: Herrejon Chavez

first: Karen

last: Chu

middle: G


title: single cell RNA-seq in transplanted bone marrow cells from SYNCRIP conditional mouse model

summary: SYNCRIP depletion results in HSCs losing their self renewal abilities. Single cell sequencing was conducted in SYNCRIP WT and KO bone marrow LK (lineage -, cKit+) cells to decipher the effect of Syncrip deletion on cellular identities along the hematopoietic hierarchy, and to gain an in-depth assessment of the transcriptomic changes in different cell types upon SYNCRIP loss.

overall_design:
SYNCRIP flox/flox conditional knockout mice were developed for this study. Bone marrow cells from these conditional knockout mice were harvested and transplanted into CD45.1 congenic mice, 6 weeks post transplant mice were intraperitoneal injected with polyinosinic:polycytidylic acid (pIpC). 3 weeks following injections, the transplanted LK (lineage-, cKit+) cells were FACS sorted and used for single cell RNA-seq.
10x Genomics

first: Alli

last: Pine


title: Transcription profiling of HSCs from SYCNRIP WT and KO mice

summary: SYNCRIP depletion results in HSCs losing their self renewal abilities. Next generation sequencing was conducted in SYNCRIP WT and KO HSCs to investigate the transcriptional changes that occur, and result in the loss of self-renewal.

overall_design: SYNCRIP flox/flox conditional knockout mice were developed for this study. Bone marrow cells from these conditional knockout mice were harvested and transplanted into CD45.1 congenic mice, 6 weeks post transplant mice were intraperitoneally injected with polyinosinic:polycytidylic acid (pIpC). 3 weeks following injections, the transplanted HSCs (CD45.2+, Lin-, Sca1+, cKit+, CD150+, CD48-) were FACS sorted and used for RNA-seq.


title: Next Generation Sequencing Facilitates Quantitative Analysis of control, SETD6 Knock-out, SETD3 knock-out and SETD6+SETD3 knock-out HeLa cells Transcriptomes

city: Be'er Sheva

first: Dan

last: Levy

address: David Ben Gurion Blvd 1

institute: Ben-Gurion University

summary: The goal of this study was to identifiy shared target genes for SETD3 and SETD6.

overall_design: mRNA of HeLa control, SETD6 KO, SETD3 KO and SETD3+SETD6 double KO cells

first: Lee

last: Admoni-Elisha


title: Functional change of mouse skeletal stem cells correlated with the progression of osteoporosis

city: wuhan

first: Jianfei

last: Liang

address: No. 237, Luoyu Road, Hongshan District,

institute: wuhan university

summary: To investigate the role of osteoporosis in mSSC, we sorted mSSC from metaphysis of 8w-sham mice and 8w-ovx mice. We then performed gene expression profiling analysis using data obtained from RNA-seq of mSSC.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for mSSC (8w-sham and 8w-ovx).


title: HuR modulation impairs LPS-response in murine macrophages

city: Trieste

first: Silvano

last: Piazza

address: localita' Padriciano 99

institute: ICGEB

summary: Lipopolysaccharide exposure to macrophages induces an inflammatory response that is heavily regulated at the post-transcriptional level. HuR, ELAVL1, is an RNA binding protein that binds and regulate the maturation and half-life of AU/U rich elements (ARE) containing cytokines and chemokines transcripts, mediating the LPS induced response. Here we investigated to what extent small molecules inhibiting HuR-RNA interaction, called tanshinone mimics, counteract the LPS induced macrophage response. We show that tanshinone mimics are present in solution in a keto-enolic tautomerism and that, by molecular dynamic calculations, the ortho quinone form is the preferred species interacting with HuR and favoring the closure of its conformation to a no-binding mode. The protection of the enolic status with diacetate caused the loss of activity of tanshinone mimics in vitro but active in vivo. Murine macrophages cell line RAW264.7 was treated with LPS and tanshinone mimics and the modulation of the LPS induced response was monitored by RNA and Ribonucleoprotein immunoprecipitation sequencing. Correlation analyses indicated that LPS induced a strong coupling between differentially expressed genes and HuR-bound genes and that tanshinone mimics reduced the interaction. Functional annotation addressed a specific set of genes, as Cxcl10, Il1b, Cd40, Fas, involved in chemotaxis and immune response whose association with HuR decreased and led to a reduction of their protein level and secretion. The same effect was observed in primary murine bone marrow derived macrophages and in vivo in a LPS induced peritonitis model, in which the serum level of Cxcl10 and Il1b was strongly reduced, endowing tanshinone mimics with anti-inflammatory properties in vivo.

first: Isabelle

last: Bonomo

first: Giulia

last: Assoni

first: Mariachiara

last: Micaelli

first: Chiara

last: Zucal

first: Elisa

last: Facen

first: Sergio

last: Robbiati

first: Alessandro

last: Provenzani


title: RNA sequencing analysis of TNBC PDX treated with MLK3 and pan MLK's inhibitor

city: CHICAGO

first: Ajay

last: Rana

state: ILLINOIS

address: 840 SOUTH WOOD ST.

department: SURGERY

institute: UNIV. OF ILLINOIS AT CHICAGO

summary: Mixed Lineage Kinase 3 (MLK3) is a viable emerging target for neoplastic diseases; however, it is uncertain whether its activators or inhibitors can act as anti-neoplastic agents. Previously, we reported that the kinase activity of MLK3 was significantly higher in triple-negative breast cancer (TNBC) than hormone receptor-positive human breast tumors where estrogen inhibits MLK3 kinase activity and provides a survival advantage to ER+ breast cancer cells. Here, we identified that in TNBC, the higher MLK3 kinase activity paradoxically promotes cell survival via activating the downstream PAK1-NF-kB axis. The MLKs/MLK3 inhibitors, CEP-1347, and URMC-099 induced cell death in TNBC but not in hormone receptor-positive breast cancer cells. The MLKs/MLK3 inhibitors reduced TNBC cell line and patient-derived xenografts’ tumor burden. The MLKs/MLK3 inhibitors also decreased MLK3, PAK1, and NF-kB protein expression and activation, prompting cell death in breast PDXs. The RNA-seq analyses indicated several genes downregulated upon MLKs/MLK3 inhibition in tumors. Significantly NGF/TrkA MAPK pathway was enriched in tumors undergoing tumor reduction by CEP-1347 and URMC-099. The tumors or cell lines that did respond to MLKs/MLK3 inhibitors had higher TrkA expression, and TrkA overexpression in the presence of MLK3 sensitized the TNBC cell line that initially did not respond to MLKs/MLK3 inhibitors. These results suggest that the functions of MLK3 in cancer cells are dependent on downstream targets/signaling, and rationalized decision needs to be made before using its inhibitor in different breast cancer subtypes. Our data also highlight that MLK3 inhibitor warrants further investigation for treating TNBC.

overall_design: 12 samples of 3' RNA-seq. 3 experimental groups, 4 biological replicates per group.

first: RAKESH

last: NAIR

first: AJAY

last: RANA


title: Biglycan regulates bone development and regeneration

city: Bethesda

first: Marian

last: Young

state: MD

address: Building 30, Room 5A509

institute: NIH

summary: Endochondral bone development and regeneration relies on activation and proliferation of periosteum derived-cells (PDCs). Biglycan (Bgn), a small proteoglycan found in extracellular matrix , is known to be expressed in bone and cartilage, however little is known about its influence during bone development. Here we link Bgn with osteoblast maturation starting during embryonic development that with aging affects bone integrity and strength. Bgn gene deletion reduced the inflammatory response after fracture, leading to impaired periostal expansion and callus formation. Using novel 3D scaffold and analyzing newborn bones, we found that Bgn is required for the cartilage-bone transition of PDCs during endochondral bone development. The absence of Bgn led to accelerated bone development with high levels of osteopontin which appeared to be detrimental to the structural integrity of the bone. Collectively, our study identifies Bgn as an important factor in PDCs activation during bone development and bone regeneration after fracture.

overall_design:
In this study, we isolated cells from WT and Bgn KO embryos (E16.5) and newborn (P-3) bones

Femurs from  6-7 week-old WT or Bgn KO male mice were fractured and collected  for bulk RNAseq 3 days after fracture. For each mouse one femure was fractured and the seconed femure sereved as the control un-fractured bone.
contributor: NIDCD/NIDCR Genomics and Computational Biology Core

first: Reut

last: Shainer

first: Vardit

last: Kram

first: Tina

last: Kilts

first: Li

last: Li

first: Andrew

middle: D

last: Doyle

first: Inbal

first: Daniel

last: Martin

first: Carl

last: Simon

first: Jinyang

last: Zeng-Brouwers

first: Liliana

last: Schaefer


title: Systematic comparison of tools used for m6A mapping from nanopore direct RNA sequencing

city: Guangzhou

first: Zhendong

last: Zhong

state: Guangdong

address: Xingangxi Roda 135#

department: School of Life Sciences

institute: Sun Yat-sen University

summary: State-of-the-art algorithms for m6A detection and quantification via nanopore direct RNA sequencing have been continuously developed, little is known about their capacities and limitations, which makes a comprehensive assessment in urgent need. Therefore, we performed comprehensive benchmarking of 10 computational tools relying on current-based and base-calling “errors” strategies for m6A detection by nanopore sequencing.

overall_design: m6A detection of wild type and Mettl3 knock-out mouse embryonic stem cells.

first: Yingyuan

last: Xie

first: Zhang

last: Zhang

first: Guanzheng

last: Luo


title: SOCS1 regulates a subset of NFκB-target genes through direct chromatin binding and defines macrophage functional phenotypes

city: Evanston

first: Brian

last: Wray

address: 633 Clark Street

institute: Northwestern University

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

overall_design: Refer to individual Series


title: Graded BMP signaling within intestinal crypt architecture directs self-organization of the Wnt-secreting stem cell niche

city: New York

first: Timothy

middle: Cragin

last: Wang

address: 1130 Saint Nicholas Avenue, ICRC

institute: Columbia University

summary: Signals from the surrounding niche drive proliferation and suppress differentiation of intestinal stem cells (ISCs) at the bottom of intestinal crypts. Among sub-epithelial support cells, deep sub-cryptal CD81+ PDGFRAlo trophocytes alone sustain ISC functions ex vivo. Here we show that mRNA and chromatin profiles of abundant CD81- PDGFRAlo stromal cells resemble those of trophocytes and that both populations provide crucial canonical Wnt ligands. Mesenchymal expression of key ISC-supportive factors extends along a spatial and molecular continuum from trophocytes into peri-cryptal CD81- CD55hi cells, which mimic trophocyte activity in organoid co-cultures. Graded expression of essential niche factors is not cell-autonomous but dictated by the distance from bone morphogenetic protein (BMP)-secreting PDGFRAhi myofibroblast aggregates. BMP signaling inhibits ISC-trophic genes in PDGFRAlo cells near high crypt tiers and that suppression is relieved in stromal cells near and below the crypt base, including trophocytes. Cell distances thus underlie a self-organized and polar ISC niche.

overall_design: Pdgfra+ cells were isolated from the small intestine of an adult male c57b6/j mouse using antibody (135905, Biolegend) based sorting. The present dataset has donor matched EpCAM+ cells from GSE205229 (GSM6208863).

first: Ermanno

last: Malagola

first: Judith

last: Kraiczy

first: Ramesh

middle: A

last: Shivdasani

middle: C


title: Defining cardiac functional recovery in end-stage heart failure at single cell resolution

status: Public on Feb 28 2023

city: Saint Louis

first: Kory

last: Lavine

state: Missouri

address: 660 S. Euclid Ave

department: Internal Medicine - Cardiology

institute: Washington University School of Medicine

summary: Recovery of cardiac function is the ultimate goal of heart failure therapy yet is infrequently observed and remains poorly understood. Here we characterize the cellular landscape and predictors of cardiac recovery by performing single nucleus RNA-sequencing (snRNA-seq) from 40 heart failure patients who recovered LV systolic function following left ventricular assist device (LVAD) implantation and patients who did not recover and non-diseased donors. We identify cell type specific transcriptional signatures of recovery in all cell types, most prominently in macrophages and fibroblasts. Pro-inflammatory macrophages and inflammatory signalling in fibroblasts were negative predictors of recovery, while downregulation of RUNX1 transcriptional activity in macrophages and fibroblasts was associated with recovery. In silico perturbation of RUNX1 in macrophages and fibroblasts recapitulated the transcriptional state of recovery. In a mouse model of cardiac recovery mediated by BRD4 inhibition, we observed a decrease in macrophage and fibroblast Runx1 expression, diminished chromatin accessibility within a Runx1 intronic peak, and acquisition of human recovery signatures. These findings suggest that cardiac recovery is a unique biological state and identify RUNX1 as a possible therapeutic target to facilitate cardiac recovery.

overall_design: snRNA-seq in donor and HF (pre-post LVAD split by responders and non-responders)

first: Junedh

last: Amrute

first: Lulu

last: Lai

first: Pan

last: Ma

last: Koenig

first: Kenji

last: Kamimoto

first: Andrea

last: Bredemeyer

first: Thirupura

last: Shankar

first: Christoph

last: Kuppe

first: Farid

last: Kadyrov

first: Linda

last: Schulte

first: Dylan

last: Stoutenburg

first: Benjamin

last: Kopecky

first: Sutip

last: Navankasattusas

first: Joseph

last: Visker

first: Samantha

last: Morris

first: Rafael

last: Kramann

first: Florian

last: Leuschner

first: Douglas

last: Mann

first: Stavros

last: Drakos


title: Transcriptome differences in FCRL5 positive and negative Influenza-specific memory B cells

city: Atlanta

first: Chris

last: Scharer

address: 1510 Clifton Rd, Suite 3086A

institute: Emory University

summary: We examined the transcriptional differences between circulating B cell subsets post-vaccination.  Using FCRL5 as a marker of Tbet expressing B cells, we sorted FCRL5+ (Tbet+) and FCRL5- (Tbet-) memory B cells, naïve B cells, and antibody secreting plasmablasts at day 7 and day 14 post-vaccination.  Additionally, we used H1 B cell tetramers to track cells responding to the influenza vaccine.  These data provide insights into the transcriptional programming of distinct B cell subsets that respond to influenza vaccination.

overall_design: RNA-seq on 4 B cell subsets from patients 7 or 14 days post influenza vaccination.

first: C

first: A

last: Nellore

first: F

last: Lund


title: Chromatin accessibility of human memory T cell subsets from blood before and after ex-vivo CD3/CD28 stimulation

summary: Naive, central memory (TCM), effector memory (TEM), and terminally differentiated effector memory RA (TEMRA, CD8+ only) T cell subsets were FACS separated from PBMC samples of four human donors using CCR7 and CD45RA as distinguishing cell surface markers. Samples were split and either immediately isolated, or incubated for 42-48 hours with anti-CD3/CD28 beads for ex-vivo stimulation and processed for ATAC-seq.

overall_design: ATAC-seq on naive and memory subsets isolated from human blood samples with and without stimulation.

first: J

last: Boss

last: Rose


title: Transcriptome analysis of human memory T cell subsets from blood before and after ex-vivo CD3/CD28 stimulation

summary: Naive, central memory (TCM), effector memory (TEM), and terminally differentiated effector memory RA (TEMRA, CD8+ only) T cell subsets were FACS separated from PBMC samples of four human donors using CCR7 and CD45RA as distinguishing cell surface markers. Samples were split and either immediately isolated, or incubated for 42-48 hours with anti-CD3/CD28 beads for ex-vivo stimulation.

overall_design: RNA-seq on naive and memory subsets isolated from human blood samples