title: Postnatal persistence of sex-dependent renal developmental programmed structural and molecular changes in nonhuman primates

city: Winston-Salem

first: Laura

last: Cox

state: NC

address: One Medical Center Blvd

department: Internal Medicine

institute: Wake Forest School of Medicine

summary:
Background: Poor nutrition during development programs kidney function. No studies on postnatal consequences of decreased perinatal nutrition exist in nonhuman primates (NHP) for translation to human renal disease. Our baboon model of moderate maternal nutrient restriction (MNR) produces intrauterine growth restricted (IUGR) and programs renal fetal phenotype. We hypothesized that the IUGR phenotype persists postnatally influencing responses to a high-fat, high-carbohydrate, high-salt (HFCS) diet.
Methods: Pregnant baboons ate chow Control (CON) or 70% of control intake (MNR) from 0.16 gestation through lactation. MNR offspring were IUGR at birth. At weaning, all offspring, (control and IUGR females and males n=3/group) ate chow. At ~3.5 years age, blood, urine, and kidney biopsies were collected before and after a 7-week high HFCS diet challenge. Kidney function, unbiased kidney gene expression, and untargeted urine metabolomics were evaluated.
Results: IUGR female and male kidney transcriptome and urine metabolome differed from CON at 3.5 years, prior to HFCS. After the challenge, we observed sex-specific and fetal exposure-specific responses in urine creatinine, urine metabolites, and renal signaling pathways.
Conclusions: We previously showed mTOR signaling dysregulation in IUGR fetal kidneys. Before HFCS, gene expression analysis indicated that dysregulation persists postnatally in IUGR females. IUGR male offspring response to HFCS showed uncoordinated signaling pathway responses suggestive of proximal tubule injury. To our knowledge, this is the first study comparing CON and IUGR postnatal juvenile NHP and the impact of fetal and postnatal life caloric mismatch. Perinatal history needs to be taken into account when assessing renal disease risk.

overall_design:
Animal Selection and Management:
All animal procedures were approved by the Institutional Animal Care and Use Committee at Texas Biomedical Research Institute and conducted in Association for Assessment and Accreditation of Laboratory Animal Care approved facilities at the Southwest National Primate Research Center. Details of housing, individual feeding cages, training, and environmental enrichment were previously published in [24].
Twelve non-pregnant female baboons (Papio species), 11.5 ± 0.51 (mean ± standard error of the mean [SEM]) years of age and of similar morphometric phenotype, were selected for study and group-housed in outdoor cages with a vasectomized male, thereby providing full social and physical activity. Animals were trained prior to pregnancy to feed in individual cages as described previously [24]. Briefly, at feeding time all baboons passed along a chute and into individual feeding cages. Each baboon's weight was obtained while crossing an electronic scale (GSE 665; GSE Scale Systems, Milwaukee, Wisconsin). Water was continuously available in the feeding cages via individual waterers (Lixit, Napa, California). Following the introduction of a fertile male and starting at 30 days of gestation (dG; equivalent to 0.16G), six females were randomly assigned to the CON group and fed Purina Monkey Diet 5038 (chow; Purina, St Louis, MO) ad libitum. The chow diet contained 12% energy from fat, 18% from protein, and 69% from carbohydrate, consisting of 0.29% glucose and 0.32% fructose. The remaining six females were assigned to the MNR group and fed 70% of the feed eaten by the CON females on a weight-adjusted basis from 0.16G through the rest of pregnancy and lactation [11]. Water was available to all animals, ad libitum.
CON and MNR mothers spontaneously delivered CON and IUGR offspring, respectively, at full term. The offspring were reared with their mothers in group housing until weaning at approximately nine months of age. Then they were moved to a juvenile cage of mixed males and females and maintained on the chow diet.
HFCS Diet Challenge:
At approximately 4.5 years of age (human equivalent of 10.5 years of age), 6 CON offspring ( 3 females and 3 males) and 6 age-matched IUGR offspring (3 females and 3 males) were challenged with a 7-week HFCS diet and given access to a high fructose drink in addition to water, which were all available to the animals throughout the study ad libitum. The HFCS diet contained 73% Purina Monkey Chow 5038 (a grain-based meal), 7% lard, 4% Crisco, 4% coconut oil, 10.5% flavored high fructose corn syrup, and1.5% water. Vitamins and mineral preparations were added to match the micronutrient composition of the chow diet. Palatability was enhanced using non-caloric artificial fruit flavors. Details have been published [25]. The high fructose drink included water, high fructose corn syrup (2.83 Kcal/g, 76% sugar, 41.8% fructose, 34.2% dextrose, ISOSWEET 5500, Staley, Decatuer, IL), and artificial fruit flavoring [25].
During the HFCS diet challenge, all baboons from a single group cage were run once per week into individual feeding cages, passing over an electronic weighing scale. Urine was collected in a pan below each feeding cage with in a  3 hour period. Food and drink were not available during this time to minimize contamination of urine. After the urine was collected during this 3-hour period, animals were given free access to the HFCS diet, high fructose drink, and water for 13.5 hours in the individual feeding cages. They were then returned to the group social area. The high fructose drink was provided in a Lixit waterer (Lixit, Napa, California) that was connected to a gauge to measure sugar drink consumption for each animal. Food consumption was recorded for each animal. Following the 13.5-hour consumption period, animals were returned to the group cage.
RNA Isolation and Microarray Hybridization:
Total RNA was isolated from an approximately 5 mg section of each frozen kidney biopsy using a Power Gen Homogenizer (Omni International, Wilmington, DE) and TRI Reagent™ Solution (Invitrogen™, Carlsbad, CA) as previously described in [12]. RNA integrity was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA), and RNA concentration was determined by UV-Vis spectrophotometry using a Nanodrop™ 2000 (Thermo Fisher Scientific, Wilmington, DE). Complementary RNA (cRNA) was synthesized and biotinylated using the Illumina™ TotalPrep™-96 RNA Amplification Kit (Illumina Inc., San Diego, CA). cRNA samples were then hybridized to HumanHT-12 v3 Expression BeadChips (Illumina, Inc.).
Microarray Data Analysis:
Gene expression data were extracted and log2-transformed using GenomeStudio software (Illumina, Inc.) and subsequently analyzed using Partek® Genomics Suite (Partek®, St. Louis, MO). Signal intensities were all-median normalized, and differentially expressed genes were identified by Analysis of Variance (ANOVA; p < 0.05). Differentially expressed genes were overlaid onto canonical pathways and networks generated using differentially expressed genes in the dataset and the Ingenuity Pathway Analysis (IPA; QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis) Knowledge Base. Right-tailed Fisher's exact test was used to calculate a significant enrichment of differentially expressed genes in pathways, p< 0.01 [29]. Networks were built using the IPA Knowledge Base, requiring direct connections between molecules based on experimental evidence.
We used an end-of-pathway gene expression approach to identify coordinated pathways. We hypothesized that a pathway may only be relevant to the kidney phenotype if gene expression profiles at the end of the pathway were consistent with the overall pathway change. Therefore, pathways meeting this criterion, as well as those downstream of these pathways, were investigated. If there were no differentially expressed genes at the end of a pathway, that pathway was not considered relevant to the phenotype [12].
We performed upstream regulatory network analysis to identify potential causal networks which integrate previously observed cause-effect relationships, leveraging experimental knowledge about the direction of effects to infer upstream regulatory molecules and potential mechanisms explaining observed gene expression changes. The analysis includes prediction of activation or inhibition of the network and consistency of molecules in the network with the predicted activation state. The calculated z-score makes predictions about regulation directions and infers the activation state of a putative regulator (i.e., activated or inhibited) and can be used to determine likely regulators based on statistical significance of the pattern match. The statistical models are provided in detail in Kramer et al., 2014 [30]. Networks were considered significant for p < 0.05.
Statistical Analyses:
PCA and hierarchical clustering showed differences between females and males in morphometrics, clinical measures, kidney transcriptome, and urine metabolome; therefore, we analyzed all data separately for females and males. Initially, normalized transcriptome and metabolome datasets were analyzed by Principal Component Analysis (PCA) to identify sources of variation in each dataset using Partek® Genomics Suite. The greatest variance in each dataset was attributed to sex; therefore, data from females and males were analyzed separately. Pairwise comparisons were performed using two-tailed t-tests. Data from each diet group were analyzed independently for pre- and post-challenge means and then evaluated using two-tailed t-test and two-way ANOVA with significance p < 0.05.

first: Andrew

middle: C

last: Bishop

first: Kimberly

last: Spradling-Reeves

first: Robert

middle: E

last: Shade

first: Kenneth

middle: J

last: Lange

first: Shifra

last: Birnbaum

first: Mark

last: Libardoni

first: Kristin

last: Favela

first: Edward

last: Dick Jr

last: Nijland

first: Cun

last: Li

first: Peter

middle: W

last: Nathanielsz


title: Safeguarding genome integrity during gene-editing therapy of age-related macular degeneration

status: Public on Nov 30 2022

city: Beijing

first: Changchang

last: Xin

state: Beijing

address: No.5 Yiheyuan Road Haidian District,Beijing,P.R.China

country: China

institute: Peking University

summary: Ensuring genome safety during gene editing is crucial for clinical translation of the high-efficient CRISPR-Cas9 toolbox. Therefore, the undesired events including chromosomal translocations, vector integrations, and large deletions arising during therapeutic gene editing remain to be adequately addressed or tackled in vivo. Here, we apply CRISPR-Cas9TX in comparison to CRISPR-Cas9 to target Vegfa for the treatment of age-related macular degeneration (AMD) disease in a mouse model. AAV delivery of both CRISPR-Cas9 and CRISPR-Cas9TX can efficiently inhibit laser-induced neovascularization. Importantly, Cas9TX almost eliminates chromosomal translocations that occur at a frequency of approximately 1% in Cas9-edited mouse retinal cells. Strikingly, the widely observed AAV integration at the target Vegfa site is also greatly dropped from nearly 50% of edited events to the background level during Cas9TX editing. Our findings reveal that chromosomal structural variations routinely occur during in vivo genome editing and highlight Cas9TX as a superior form of Cas9 for in vivo gene disruption.

overall_design: We employed primer-extension-mediated sequencing (PEM-seq) to study the gene-editing outcomes for Cas9 and Cas9TX treated Age-related Macular Degeneration mouse models, and analysed the chromosomal translocations and AAV vector integrations during the genome editing in mouse.

first: Kailun

last: Fang

first: Yanxia

last: Gao

first: Liqiong

last: Ou

first: Shaopeng

last: Yuan

first: Jiazhi

last: Hu

first: Hui

last: Yang

first: Jiaxu

last: Hong


title: Critical quality attributes identify MSCs with potent fitness ranges in response to culture process parameters

city: Toronto

first: Kevin

middle: P

last: Robb

state: Ontario

address: Krembil Research Institute 60 Leonard Avenue, Office 5KD416-K1 / Lab 5KD424-C1

country: Canada

institute: University Health Network

summary: Adipose-derived mesenchymal stromal cells (MSC(AT)) display immunomodulatory and angiogenic properties, but an improved understanding of quantitative critical quality attributes (CQAs) that inform basal MSC(AT) fitness ranges for immunomodulatory and/or angiogenic applications is urgently needed for effective clinical translation. We constructed an in vitro matrix of multivariate readouts to identify putative CQAs that were sensitive enough to discriminate between specific critical processing parameters (CPPs) chosen for their ability to enhance MSC immunomodulatory and angiogenic potencies, with consideration for donor heterogeneity. We compared 3D aggregate culture conditions (3D normoxic, 3D-N) and 2D hypoxic (2D-H) culture as non-genetic CPP conditions that augment immunomodulatory and angiogenic fitness of MSC(AT). We measured multivariate panels of curated genes, soluble factors, and morphometric features for MSC(AT) cultured under varying CPP and licensing conditions, and we benchmarked these against two functional and therapeutically relevant anchor assays – in vitro monocyte/macrophage (MΦ) polarization and in vitro angiogenesis. Our results showed that varying CPP conditions was the primary driver of MSC(AT) immunomodulatory fitness; 3D-N conditions induced greater MSC(AT)-mediated MΦ polarization toward inflammation-resolving subtypes. In contrast, donor heterogeneity was the primary driver of MSC(AT) angiogenic fitness. Our analysis further revealed panels of putative CQAs with minimum and maximum values that consisted of twenty MSC(AT) characteristics that informed immunomodulatory fitness ranges, and ten MSC(AT) characteristics that informed angiogenic fitness ranges. Interestingly, many of the putative CQAs consisted of angiogenic genes or soluble factors that were inversely correlated with immunomodulatory functions (THBS1, CCN2, EDN1, PDGFA, VEGFA, EDIL3, ANGPT1, and ANG genes), and positively correlated to angiogenic functions (VEGF protein), respectively. We applied desirability analysis to empirically rank the putative CQAs for MSC(AT) under varying CPP conditions and donors to numerically identify the desirable CPP conditions or donors with maximal MSC(AT) immunomodulatory and/or angiogenic fitness. Taken together, our approach enabled combinatorial analysis of the matrix of multivariate readouts to provide putative quantitative CQAs that were sensitive to variations in select CPPs that enhance MSC immunomodulatory/angiogenic potency, and donor heterogeneity. These putative CQAs may be used to prospectively screen potent MSC(AT) donors or cell culture conditions to optimize for desired basal MSC(AT) immunomodulatory or angiogenic fitness.

overall_design: MSC(AT) from five human donors were subject to 2D-N, 2D-H, and 3D-N CPP conditions and were licensed with pro-inflammatory cytokines or unlicensed. Samples were run as singlets for a total of 30 samples. One sample was excluded due to QC issues. A 58-gene panel was probed.

first: Sowmya

last: Viswanathan


title: The regional distribution of resident immune cells shapes distinct immunological environments along the murine epididymis

city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

country: Germany

institute: MPI for heart and lung research

type: Expression profiling by high throughput sequencing

summary: Previously, the epididymal regions were demonstrated to react strikingly different to inflammatory stimuli. We therefore aimed at untangling the heterogeneity of extravascular CD45+ cells throughout epididymal regions and identifying their transcriptional profile. Using an unbiased scRNASeq approach, 12 distinct populations were identified in total showing striking differences in their distribution along the epididymal duct. Our data indicate that resident immune cell populations are strategically positioned within the organ to provide distinct immunological environments required to maintain tissue integrity and to adequately tackle invading pathogens ascending through the urogenital tract. This study provides an important common research platform and starting point for future functional studies of distinct populations under physiological and pathological conditions.

overall_design: scNRASeq of extravascular CD45+ cells separately isolated from the four main epididymal regions (Initial segment, caput, corpus, cauda) in mice

first: Christiane

last: Pleuger

last: Guenther

first: S

last: Epelman

first: C

last: Kantores

last: Bhushan

first: A

last: Meinhardt


title: Serotonin-1A receptor, a psychiatric disease risk factor, modulates offspring immunity via sex-dependent genetic nurture

status: Public on Nov 29 2022

city: New York

first: Miklos

last: Toth

address: 525 E. 68th St Whitney Pavilion Floor 5

country: USA

institute: Weill Cornell Medicine

summary: Serotonin-1A receptor (5HT1AR) is highly expressed in corticolimbic regions and its deficit has been associated with anxiety and depression. A similar reduction in 5HT1AR heterozygous knockout (Het) mice results in anxiety-like and increased stress-reactivity phenotypes. Here we describe immunological abnormalities in Het females, characterized by an activated state of innate and adaptive immune cells. Het males showed only limited immune dysregulation. Similar immune abnormalities were present in the genetically WT female (F1) but not in male offspring of Het mothers, indicating sex-specific immune system abnormalities that are unrelated to the individual’s receptor expression but instead dependent on the mother’s 5HT1AR deficit, known as a maternal genetic effect or “genetic nurture”. Studying the maternal-fetal interface by scRNA-seq revealed reduced immune cell invasion to the decidua and accelerated trophoblast migration that was normalized by term. Despite these phenotypes, 5HT1AR is not, or only minimally expressed in the immune system and placenta, raising the possibility of a central regulation of the immune system by highly expressed brain receptors. We conclude that 5HT1AR deficit, by altering the maternal immune system and midgestational in utero environment, leads to sex-biased outcomes, predominantly immune dysregulation in females and anxiety-like behavior in males.

overall_design: Peripheral whole blood (n = 4 females) or the placenta decidua (n = 3 females) was collected from pregnant female mice (Swiss Webster wild type and 5HT1A-R +/- heterozygotes) at GD10.5 and pooled for single cell or single nucleus sequencing, respectively.

first: Rosa

last: Chen

first: Anika

last: Nabila


title: Recruited monocytes/macrophages drive pulmonary neutrophilic inflammation and irreversible lung tissue remodeling in cystic fibrosis.

status: Public on Dec 01 2022

city: New Haven

first: Giulia

last: Biancon

state: Connecticut

address: 300 George Street

department: Internal Medicine - Yale Cancer Center

institute: Yale University

summary:
Controlled recruitment of C-C Motif Chemokine Receptor 2 (CCR2)-positive monocytes into lung tissues is a crucial component of the host immune response to infections. Pathogenesis of cystic fibrosis (CF) lung remodeling, which ultimately leads to respiratory failure, is caused by chronic infections and neutrophilic inflammation, but the immune mechanisms are not fully understood. Here, we show that lung explants from CF patients with severe disease contain abundant CCR2-positive classical monocytes in the submucosal tissues of small airways and areas of tissue remodeling. Recapitulating human CF lung remodeling by repeatedly exposing CF mice to lipopolysaccharide (LPS), and by using genetic ablation or pharmacological inhibition of CCR2, we demonstrate that CCR2-dependent accumulation of classical monocytes and monocyte-derived macrophages drive lung neutrophilia, pathogenic Transforming Growth Factor Beta signaling and irreversible lung tissue scarring. Importantly, six weeks after cessation of LPS exposure, the lungs of CF mice still have elevated numbers of pro-inflammatory monocytes with higher expression of neutrophil chemoattractants, thus perpetuating recruitment of neutrophils and lung tissue damage. Lastly, we demonstrate that the increased accumulation of immune cells to inflamed lungs is driven by dysfunctional CF transmembrane conductance regulator (CFTR) expression in CCR2-positive cells. These studies identify uncontrolled recruitment of CCR2-positive cells as a key driver of lung remodeling, and as a novel therapeutic target for patients with CF for which lung hyper-inflammation and tissue damage remain an issue despite recent advances in CFTR-specific therapeutics.
Keywords: monocytes; macrophages; neutrophils; chronic lung inflammation; lipopolysaccharide; recruitment; CC-motif chemokine receptor 2; transforming growth factor beta; lung remodeling; cystic fibrosis

overall_design: In this study, we performed high-throughput sequencing of RNA isolated from lung monocyte and macrophage populations (classical monocytes (cMon), interstitial macrophages (IM), monocyte-derived alveolar macrophages (moAM) and tissue resident alveolar macrophages (trAM)) of WT and B6.129P2-Cftrtm1Unc/J (CF) mice that were exposed to LPS over 5 weeks (time point  1, T1) and after 6 weeks of recovery from LPS (time point 2, T2). Sequencing was performed performed in 2 biological replicates per genotype, cell type and timepoint. For Recovery time points (T2), lung cells were pooled from two mice for each sample.

first: Hasan

middle: H

last: Öz

first: Toma

last: Tebaldi

first: Stephanie

last: Halene

first: Emanuela

middle: M

last: Bruscia


title: CUT&RUN Sequencing of EZH2 binding sites in HEK293T cells expressing a Dual-specificity Aptamer that specifically increases O-GlcNAcylation on beta-catenin

city: Baltimore

first: Yi

last: Zhu

address: 511 WBSB, 725 N. Wolfe St.

department: Biological Chemistry

institute: Johns Hopkins University, School of Medicine

type: Genome binding/occupancy profiling by high throughput sequencing

summary: O-GlcNAc is a dynamic post-translational modification on thousands of intracellular proteins, it regulates protein functions and is involved in many metabolic diseases. Using a dual-specificity aptamer, we targeted the O-GlcNAc transferase (OGT) to endogenous β-catenin and specifically increased O-GlcNAcylation of β-catenin. We found that O-GlcNAc on β-catenin promoted its interaction with EZH2 regardless of the Wnt signaling status. To study how this interaction regulates the distribution of EZH2 on the genome, we performed Cleavage Under Targets and Release Using Nuclease (CUT&RUN) on HEK293T cells expressing individual (Ctrl.) or dual-specificity aptamers. Under Wnt - and Wnt + conditions, the dual-specificity aptamer increased the binding of EZH2 on 923 and 3473 genomic sites, respectively. These two sets of differential binding sites showed little overlap.

overall_design: 12 samples from 4 groups of HEK293T cells were analyzed: cells expressing Ctrl (T1 ∙ bc339) or Dual-specificity (3JB8F+12) aptamers were exposed to Wnt- or Wnt+ medium. Each group contains 3 replicates.

first: Gerald

last: Hart


title: single cell RNA-sequencing of  diabetic mouse kidney

city: Hamburg

first: Yu

last: Zhao

address: Falkenried 94

department: Institute of Medical Systems Biology

institute: University Medical Center Hamburg-Eppendorf

summary: single-cell RNA sequencing of renal cells from type 2 diabetes mice (BTBR ob/ob) at the early stage of DN.

overall_design: Glomeruli were enriched by depleting the PT segment from the kidneys using Percoll density gradient centrifugation, then the single-cell RNA sequencing was performed to BTBR ob/ob vs. BTBR +/+ in glomerular cells.

first: Shuya

last: Liu

first: Tianran

last: Zhang


title: SESAME-catalyzed H3T11 phosphorylation inhibits Dot1-catalyzed H3K79me3 to regulate autophagy and telomere silencing

city: wuhan

last: Qi

state: Hubei

address: xujiapeng

department: College of life sciences

institute: Hubei university

summary: The glycolytic enzyme, pyruvate kinase Pyk1 maintains telomere heterochromatin by phosphorylating histone H3T11 (H3pT11), which promotes SIR (silent information regulator) complex binding at telomeres and prevents autophagy-mediated Sir2 degradation. However, the exact action mechanism of H3pT11 is poorly understood. Here, we identify Dot1-catalyzed H3K79 tri-methylation (H3K79me3) as the downstream effector of H3pT11 and uncover how this histone crosstalk regulates autophagy and telomere silencing. Mechanistically, Pyk1-catalyzed H3pT11 directly reduces the binding of Dot1 to chromatin and inhibits Dot1-catalyzed H3K79me3, which leads to transcriptional repression of autophagy genes and reduced autophagy. Despite the antagonism between H3pT11 and H3K79me3, they synergically promote the binding of SIR complex at telomeres to maintain telomere silencing. Furthermore, we identify Reb1 as a telomere-associated factor that recruits Pyk1-containing SESAME (Serine-responsive SAM-containing Metabolic Enzyme) complex to telomere regions to phosphorylate H3T11 and prevent the invasion of H3K79me3 from euchromatin into heterochromatin to maintain telomere silencing. Together, these results uncover a novel histone crosstalk and provide insights into dynamic regulation of silent heterochromatin and autophagy in response to cell metabolism.

overall_design: ChIP-Seq examination

first: He

last: Fei


title: The effect of extreme hyperglycemia on the expression program of the human beta cell line EndoC-betaH2

city: Philadelphia

first: Jonathan

last: Schug

state: Pennsylvania

address: 12-156 Smilow Ctr Trans Research

department: School of Medicine

institute: University of Pennsylvania

summary: Our in vivo Insulin-rtTA;TET-DTA mouse model of severe hyperglycemia revealed that proloned exposure to very high glucose levels inhibits cell cycle entry and progression of pancreatic beta cells. To investigate the molecular mechanisms responsible for this observation and the relevance to human beta cells, we performed an in vitro experiment on EndoC-betaH2 mimiking the in vivo conditions and subjected them to RNAseq analysis.

overall_design: We incubated EndoC-betaH2 human beta cell line for 3 days in 5.5mM (basal glucose level), 20mM (360mg/dl) or 35mM (630mg/dl) glucose, simulating normoglycemic, hyperglycemic and extreme hyperglycemic conditions respectively and subjected the cells to RNAseq analysis.

first: Judith

last: Lavi

first: Dana

last: Avrahami

first: Benjamin

last: Glaser


title: Gene expression profiling analysis of RNA-seq data for colorectal cancer organoids and spontaneous metastases mouse models

city: Utrecht

first: Emre

last: Kucukkose

address: Heidelberglaan 100

country: Netherlands

institute: University Medical Center Utrecht

summary: We performed RNA-sequencing to investigate the gene expression profiles of colorectal cancer patient-derived organoids (PDO) and PDO-initiated spontaenous metastases mouse models

last: Küçükköse

first: Onno

last: Kranenburg


title: Whole-transcriptome analyses of sheep embryonic testicular cells infected with bluetongue virus

city: Kunming

first: Zhuoran

last: Li

state: Yunnan

address: Fengyu Road

department: Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory

institute: Yunnan Animal Science and Veterinary Institute

type: Non-coding RNA profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Ribosome profiling and mRNA seq demonstrate that insertion of common resistance cassettes in yeast drives aberrant transcription

city: Berkeley

first: Gloria

middle: Ann

last: Brar

state: CA

address: 628 Barker Hall

department: MCB

institute: UC Berkeley

summary: Our study found that insertion of commonly used selection cassettes in yeast can drive aberrant transcription events and disrupt expression of neighboring genes. We performed ribosome profiling and mRNA-seq on yeast strains where ORFs had been replaced with common selection cassettes and observed aberrant transcripts. Aberrant transcripts positioned near adjacent genomic loci repressed protein synthesis of those genes by transcription interference (ablating normal length transcript production) coincident with translation of competitive uORFs on the long transcript which prevented translation of the main ORF seen by ribosome profiling.

overall_design: We performed ribosome profiling and matched mRNA-seq on wild-type and dbp1∆::KANMX6 yeast of interest and observed the expression of loci near deleted ORFs

first: Emily

last: Powers


title: CD26-negative and CD26-positive tissue-resident fibroblasts contribute to functionally distinct CAF subpopulations in breast cancer [6263_6381]

city: Amsterdam

first: Roebi

middle: de

last: Bruijn

address: Plesmanlaan 121

institute: Netherlands Cancer Institute

summary: Cancer-associated fibroblasts (CAFs) are abundantly present in the microenvironment of virtually all tumors and strongly impact tumor progression. Despite increasing insight into their function and heterogeneity, little is known regarding the origin of CAFs. Understanding the origin of CAF heterogeneity is needed to develop successful CAF-based targeted therapies. Through various transplantation studies in mice we determined that CAFs in both invasive lobular breast cancer and triple negative breast cancer originate from mammary tissue-resident normal fibroblasts (NFs). Single-cell transcriptomics, in vivo tracing and in vitro studies revealed the transition of CD26+ and CD26- NF populations into inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs), respectively. In vitro functional assays showed that CD26+ NFs transition into pro-tumorigenic iCAFs which recruit myeloid cells in a CXCL12-dependent manner and enhance tumor cell invasion via matrix-metalloproteinase (MMP) activity. Together, our data show that CD26+ and CD26- NFs transform into distinct CAF subpopulations in breast cancer.

overall_design: Cdh1F/F;PtenF/F (Eptn) mice (8 weeks old) were intraductally injected with lenti-virus expressing Cre-recombinase to induce tumor formation. Injected mammary glands with tumors were isolated 6, 12 or 18 weeks after intraductal injection (n=2 mice per time point). Together with PBS injected control mice (T=12 weeks after injection) the tissues were processed into a single cell suspension (see manuscript for protocol) and fibroblasts were isolated by FACS sorting (fibroblasts were defined as dapi-/EpCAM-/CD49f/-CD31-/CD45- cells) and processed fro single cells RNA sequencing using the 10X genomics platform (see manuscript for detailed protocol).

first: Julia

last: Houthuijzen

last: de Bruijn

first: Eline

last: van der Burg

first: Anne

last: Drenth

first: Ellen

last: Wientjes

first: Tamara

last: Filipovic

first: Esme

last: Bullock

first: Chiara

middle: S

last: Brambillasca

first: Marja

last: Nieuwland

first: Iris

last: de Rink

first: Frank

last: van Diepen

first: Sjoerd

last: Klarenbeek

first: Ron

last: Kerkhoven

first: Valerie

middle: G

last: Brunton

first: Colinda

middle: L

last: Scheele

first: Mirjam

last: Boelens

first: Jos

last: Jonkers


title: Transplanting rejuvenated blood stem cells extends lifespan of aged immunocompromised mice [scRNA-seq of LSK cells]

city: Hospitalet de Llobregat

first: Maria Carolina

last: Florian

state: Barcelona

address: Gran Via de l’Hospitalet, 199

department: Regenerative Medicine

country: Spain

institute: Bellvitge Biomedical Research Institute (IDIBELL)

summary:
One goal of regenerative medicine is to rejuvenate tissues and extend lifespan by restoring the function of endogenous aged stem cells. However, evidence that somatic stem cells can be targeted in vivo to extend lifespan is still lacking.
Here, we demonstrate that after a short systemic treatment with a specific inhibitor of the small RhoGTPase Cdc42 (CASIN), transplanting aged hematopoietic stem cells (HSCs) from treated mice is sufficient to extend the healthspan and lifespan of aged immunocompromised mice without additional treatment. In detail, we show that systemic CASIN treatment improves strength and endurance of aged mice by increasing the myogenic regenerative potential of aged skeletal muscle stem cells. Further, we show that CASIN modifies niche localization and H4K16ac polarity of HSCs in vivo. Single cell profiling reveals changes in HSC transcriptome, which underlie enhanced lymphoid and regenerative capacity in serial transplantation assays.
Overall, we provide proof-of-concept evidence that a short systemic treatment to decrease Cdc42 activity improves the regenerative capacity of different endogenous aged stem cells in vivo, and that rejuvenated HSCs exert a broad systemic effect sufficient to extend murine health- and lifespan.

overall_design: Aged mice (C57Bl6, >80 weeks old) were treated with CASIN for 4 consecutive days (1 i.p injection every 24h). At day 5, samples were taken from the bone marrow from treated aged mice, as well as young control (C57Bl6, 10-12 weeks old) and aged control mice (controls treated only with cyclodextrin). Lin- cKit+ Sca1+ (LSK) bone marrow cells were sorted to perform scRNA-seq. There are a total of 3 replicates per mice condition.

first: Noelle

last: Ali

first: Sara

last: Montserrat-Vazquez

first: Jan

last: Mallm

first: Maria


title: Sirt6 attenuates chondrocyte senescence and osteoarthritis progression

city: Nanjing

first: Mingliang

last: Ji

state: Jiangsu

address: Dingjiaqiao Road 87

institute: Zhongda Hospital,school of medicine, southeast university

overall_design: 3 chondrocytes from controls and 3 chondrocytes from Sirt6 cKO mice

first: Ming

first: Jun

last: Lu


title: loss of DBP1 during meiosis in yeast

first: Gloria

last: Brar

summary: We used CRISPR/Cas9 to delete the DBP1 ORF in SK1 budding yeast cells. We then used ribosome profiling and mRNA-seq to observe gene expression profiles of wild-type and dbp1∆ cells during meiosis.

overall_design: Cells were grown as described in Powers et al., 2022. Samples were collected 6h post transfer to SPO media. Two biological replicates are included for each genotype.

first: Emily

last: Powers


title: CD26-negative and CD26-positive tissue-resident fibroblasts contribute to functionally distinct CAF subpopulations in breast cancer [6451]

first: Roebi

last: Bruijn

overall_design: primary CD26- and CD26+ normal mammary fibroblasts were isolated from FVB/n mice and cultured in either control conditioned medium (control) or conditioned medium (CM) derived from 6 independent WEPtn-derived ILC tumor cell lines (13/17, 10/221, 18/12, 13/44, 13/90 or 13/138) for 7 days followed by RNA isolation and sequencing.

first: Julia

last: Houthuijzen

last: de Bruijn

first: Eline

last: van der Burg

first: Anne

last: Drenth

first: Ellen

last: Wientjes

first: Tamara

last: Filipovic

first: Esme

last: Bullock

first: Chiara

last: Brambillasca

first: Marja

last: Nieuwland

first: Iris

first: Frank


title: CD26-negative and CD26-positive tissue-resident fibroblasts contribute to functionally distinct CAF subpopulations in breast cancer [6702]

overall_design: CD26- and CD26+ NFs were isolated by FACS from the mammary glands of 8-week old mTmG mice and cultured for one week (2D) before switching the CD26+ NFs to GFP using adeno-Cre. CD26- NFs were expressing Tomato. CD26- and CD26+ NFs were mixed in collagen/BME gels (1:1) and plated alone or in combination with WEPtn (13-MCB-17 or 10-SJK-221)-derived  tumor cells for one week. In addition, tumor cells were also plated in collagen/BME gels without fibroblasts as a control. After 7-day culture the gels were dissolved using collagenase and cells were FACS-sorted to separate CD26- NFs (Tomato+), CD26+ NFs (GFP+) and tumor cells (Tomato-/GFP-) and subsequently sequenced.

last: de Rink

last: van Diepen

first: Sjoerd

last: Klarenbeek

first: Ron

last: Kerkhoven

first: Valerie

last: Brunton

first: Colinda

last: Scheele

first: Mirjam

last: Boelens

first: Jos

last: Jonkers


title: CD26-negative and CD26-positive tissue-resident fibroblasts contribute to functionally distinct CAF subpopulations in breast cancer

first: Roebi

last: Bruijn


title: Single-cell RNA transcriptomics of thymic iNKT cells from BALB/c and Cd80/Cd86 -/- (B7KO) mice

city: Bethesda

first: Masashi

last: Watanabe

address: 10 Center Dr.

institute: NCI

summary: Single-cell RNA-seq analysis of murine thymic iNKT cells from three independent BALB/c mice and three independent Cd80/Cd86 (B7)-deficient mice

overall_design: Processed thymocytes were enriched for PBS57-CD1d reactive cells by incubating thymocyte cell suspensions with PE conjugated PBS57-CD1d tetramers for 45 minutes at 4C, then incubated with anti-PE Ab and magnetic microbeads (STEMCELL) for 15 and 10 minutes at RT, respectively, followed by separation by using an EasySep magnet (STEMCELL) according to manufacturer’s instructions. A 50:50 mix of CD44low and CD44high PBS57-CD1d tetramer+ cells were sorted using a BD FACS Aria. Single-cell RNA-seq was performed using 10X Genomics Chromium isingle-cell controller and 5' gene expression kit.

first: Ricahrd

last: Hodes


title: CD26-negative and CD26-positive tissue-resident fibroblasts contribute to functionally distinct CAF subpopulations in breast cancer [5920]

first: Julia

last: Houthuijzen

last: de Bruijn

first: Eline

last: van der Burg

first: Anne

last: Drenth


title: CD26-negative and CD26-positive tissue-resident fibroblasts contribute to functionally distinct CAF subpopulations in breast cancer [6521]

overall_design: Mouse mammary epithelial cell (MMEC) transplantation of control or WB1P or WB1P-Myc-derived mammary tissue into EN1-Cre;mTmG mice. End-stage tumors or age-matched control mammary tissue was harvested and processed to single cell suspension (see manuscript for detailed protocol). EN1+, CD26+, EN1+/CD26+ (+/+) and En1-/CD26- (-/-) fibroblasts were isolated by FACS sorting and subsequently sequenced.

first: Ellen

last: Wientjes

first: Tamara

last: Filipovic

first: Esme

last: Bullock

last: Brambillasca


title: Protein KIC5 is a novel regulator of artemisinin stress response in the malaria parasite Plasmodium falciparum

city: Tampa

first: Caroline

last: Simmons

state: FL

address: 3720 East Spectrum Blvd St. 404

department: Center for Global Health and Infectious Diseases Research

institute: University of South Florida

summary: RNAseq data profiling the artemisinin-sensitive P. falciparum piggyBac mutant of the KIC5 gene (PF3D7_1138700, Kelch13 Interacting Candidate 5).

sample_organism:
Plasmodium falciparum
Plasmodium falciparum NF54

overall_design: Paired-end fastq data for two complete bioreps of the two parasite lines (NF54 wild-type control; artemisinin-sensitive mutant referred to in sample-names as PB50) collected after 3 rounds of sorbitol synchronization under normal asexual growth conditions. Parasites were at early ring stage (T6), mid ring stage (T12), early trophozoite stage (T24), late trophozoite stage (T36), or schizont stage (T48) as verified by Giemsa-stained microscopy. Each sample was run on an Illumina Nextseq.

first: Justin

last: Gibbons

first: Min

last: Zhang

first: Jenna

last: Oberstaller

first: Camilla

middle: V

last: Pires

first: Chengqi

last: Wang

first: Shulin

last: Xu

first: Debora

last: Casandra

first: Andreas

last: Seyfang

first: Thomas

middle: D

last: Otto

first: Julian

last: Rayner

first: John

last: Adams


title: Comparing gene expression in normal adipose stromal cells and fibro-adipogenic progenitors one day after injury

status: Public on Nov 28 2022

city: Toulouse

first: xavier

last: contreras

address: 4bis avenue hubert curien

country: France

institute: RESTORE, INSERM U1301

summary:
To compare the transcriptome of ASCs and FAPs before and after injury, we performed quadriceps injury using glycerol in mice.
We then isolated subcutaneous adipose tissue and quadriceps muscle and isolated ASCs and FAPs respectively (3 mice per condition). We then extracted RNA and performed RNA-seq.

overall_design: Comparative gene expression profiling analysis in ASCs and FAPs of RNA-seq data from C57BL/6- mice before and after quadriceps injury

first: Xavier

last: Contreras

first: Maxime

last: Mathieu

first: Amandine

last: Girousse

first: Coralie

last: Sengenès


title: CD26-negative and CD26-positive tissue-resident fibroblasts contribute to functionally distinct CAF subpopulations in breast cancer [4880_4954]

first: Roebi

last: Bruijn

overall_design: End-stage mammary tumors were isolated from WB1P and WB1P-Myc mice and processed into a single cell suspension (see manuscript for details). Live single cells (dapi-negative cells) were sorted by FACS and subjected to single-cell RNA sequencing using the Dropseq platform.

first: Julia

last: Houthuijzen

last: de Bruijn

first: Eline

last: van der Burg

first: Anne

last: Drenth

first: Ellen

last: Wientjes

first: Tamara

last: Filipovic

first: Esme

last: Bullock

first: Chiara

last: Brambillasca

first: Marja

last: Nieuwland

first: Iris

last: de Rink

first: Frank

last: van Diepen

first: Sjoerd

last: Klarenbeek

first: Ron

last: Kerkhoven

first: Valerie

last: Brunton

first: Colinda

last: Scheele

first: Mirjam

last: Boelens

first: Jos

last: Jonkers


title: Global transcriptional responses of pneumococcus to human blood components and cerebrospinal fluid

city: Odense M

first: Jens

middle: Sivkær

last: Pettersen

state: Danmark

address: Campusvej 55

country: Denmark

institute: University of Southern Denmark

summary: This study investigates the global transcriptional response of Streptococcus pneumoniae D39V (serotype 2 strain) to human blood components and CSF acquired from discarded and anonymized patient samples.

overall_design: D39V wild type inoculated in triplicates in CDM (control), CDM with 20 % plasma, CDM with 20 % heat-inactivated plasma, CDM with 20 % RBC suspension, and CDM with 20 % CSF, and grown for 15 or 45 minutes before RNA extraction.

first: Frida

middle: F

last: Høg

first: Flemming

last: Nielsen

first: Jakob

last: Møller-Jensen

first: Mikkel

last: Jørgensen


title: Single-cell TCR transcriptomics of thymic iNKT cells from BALB/c and Cd80/Cd86 -/- (B7KO) mice

first: Masashi

last: Watanabe

summary: Single-cell TCR-seq analysis of murine thymic iNKT cells from three independent BALB/c mice and three independent Cd80/Cd86 (B7)-deficient mice

first: Ricahrd

last: Hodes


title: Single-cell transcriptomics of thymic iNKT cells from BALB/c and Cd80/Cd86 -/- (B7KO) mice


title: Establishment of a dopaminergic neuron purification system in mice

city: Suita

first: Kit-yeng

last: Sheng

address: Yamadaoka 2-2

country: Japan

institute: Osaka University

summary: Transcriptomic analysis of enriched of neuron population collected from in-vivo mouse brains has been a challenge in the neuroscience field due to its fragility in withstanding harsh condition during isolation and collection process. We established a fluorescent reporter mouse Ddc-hKO1 to facilitate the identification and collection of Ddc-expressing neurons (dopaminergic, serotonergic, cholinergic and adrenergic neurons) by the detection of red fluorescence signals using a FACs. We utilized an improved isolation protocol that ensure high yield and quality of viable neurons during collection that is suitable for RNA-sequencing. This is the first report of transcriptomic profiling of Ddc expressing (hKO1(+)) neurons. Successful collection of Ddc-expressing neurons were verified by gene markers of dopaminergic, serotonergic, cholinergic neurons in hKO1(+) populations while other neuron types in hKO1(-) population. Furthermore, GSEA analysis were performed on both hKO1(+) and hKO1(-) to further support the neuron types collected in respective population.

overall_design: mRNA profiles of Ddc-hKO1-positive and Ddc-hKO1-negative neuron cells from around 20,000 cells were used for sequencing.

first: Kit-Yeng

first: Shinpei

last: Yamaguchi

first: Toru

last: Nakano


title: Neonatal Intake of Omega-3 Fatty Acids Enhances Lipid Oxidation in Adipocyte Precursors

city: Oklahoma City

first: Michael

middle: C.

last: Rudolph

state: OK

address: 975 NE 10th St. BRC North rm 364

department: Biochemistry and Physiology

institute: OUHSC

overall_design: Refer to individual Series for the bulk and single cell sequencing data


title: Whole-transcriptome analyses of sheep embryonic testicular cells infected with bluetongue virus [RNA-Seq]

first: Zhuoran

last: Li

summary: To comprehensively understand the characterization of bluetongue virus (BTV)-host interactome, and BTV infection and pathogenic mechanisms, RNA-seq was performed with BTV serotype 1 Y863 strain-infected and mock-infected sheep embryonic testicular cells (OA3.Ts) at 24 hours post-infection.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for bluetongue virus serotype 1 (BTV-1)-infected and mock-infected sheep embryonic testicular cells (OA3.Ts).


title: The SR-like protein Npl3 transfers mRNP components from the transcription site onto the mRNA

status: Public on Nov 26 2022

city: Würzburg

first: Kathi

last: Zarnack

address: Biocenter, Am Hubland

department: Theodor Boveri Institute

institute: University Würzburg

summary:
In order to determine specifically the RNA-binding function of proteins involved in nuclear mRNP assembly, we first determined the amino acids involved in RNA-binding by RNPXL. We identified about 100 amino acids cross-linked to RNA in vivo in the nuclear mRNP components Npl3, Nab2, Tho1, Mex67-Mtr2 and the TREX complex. Second, we can now specifically elucidate the function of the RNA-binding activity of these proteins by mutation of the identified amino acids.
Npl3 is an SR-like protein with functions in transcription elongation, splicing, 3’ end processing, mRNP assembly and nuclear mRNA export. The middle part of Npl3 consists of two RNA recognition motif (RRM) domains, RRM1 and RRM2, connected by an eight amino acid long flexible linker. In order to analyze the function of the RNA-binding activity of Npl3, we mutated several amino acids that cross-linked to RNA. We generated three npl3 mutants, one in the RRM1, one in the linker and a third in the RRM2 domain, and elucidated the functional consequences of these mutations. Interestingly, these three npl3 mutants show different phenotypes. Thus, abrogation of mRNA-binding in different regions of Npl3 has different functional outcomes. Furthermore, analysis of the npl3-Linker mutant revealed a novel function of Npl3. Npl3 functions in the transfer of nuclear mRNP components from the site of transcription onto the mRNA.
Taken together, we identify the in vivo RNA-binding sites of nuclear mRNA-binding proteins involved in mRNP assembly and nuclear mRNA export. In addition, we show that abrogation of RNA-binding in different regions of the protein Npl3 has specific and surprisingly different functional consequences. Furthermore, we uncovered a novel function of Npl3 in nuclear mRNP assembly.

overall_design: 24 samples in total. 3 x wildtype (NPL3) as control, 3 x each mutant (npl3-RRM1, npl3-Linker, npl3-RRM2). Each triplicate of samples derived once from 30°C grown S. cerevisiae cells and one set from cells that were shifted to 37°C for 1 hour

first: Philipp

last: Keil

first: Katja

last: Sträßer


title: Next Generation Sequencing Facilitates Quantitative Analysis of normal mice, colitis mice, normal mice receiving berberine and colitis mice receiving berberine colon transcriptomes

city: shanghai

first: li

last: zihao

address: dongchuan road 800

institute: shanghai jiao tong university

summary: We had three goals to perform this study: compare normal mice and colitis mice，compare berberine effect in normal mice and compare berberine effect in colitis mice

overall_design: Comparison of transcriptome of colon samples of normal mice, colitis mice, normal mice receiving berberine and colitis mice receiving berberine after DSS treatment for 7d and berberine treatment for 6d

first: Zihao

first: Zecheng

last: Luo


title: NUDT7 Regulates Total Hepatic CoA Levels and the Composition of the Intestinal Bile Acid Pool in Male Mice Fed a Western Diet

status: Public on Nov 25 2022

city: Morgantown

first: Quincy

middle: Alexander

last: Hathaway

state: WV

address: 1 Medical Center Drive

institute: West Virginia University

summary:
Nudix hydrolase 7 (NUDT7) is a peroxisomal (acyl-)CoA-degrading enzyme that is highly expressed in the liver. We previously showed that liver-specific NUDT7 overexpression affects peroxisomal lipid metabolism, but does not prevent the increase in total liver CoA levels that occurs with fasting. Herein, we show that deletion of Nudt7 alters the composition of the hepatic acyl-CoA pool in mice fed a low fat diet, but only in males fed a western diet does the lack of NUDT7 increase total liver CoA levels. This effect is driven by the accumulation of medium-chain dicarboxylic acyl-CoAs, which are products of the oxidation of dicarboxylic fatty acids in the peroxisomes.
We also show that, under conditions of increased cholesterol intake and elevated bile acid synthesis, Nudt7 deletion increases the production of tauro-muricholic acids, decreasing the hydrophobicity index of the intestinal bile acid pool and increasing fecal cholesterol excretion. Collectively, our findings reveal a key role for NUDT7 in the regulation of the final products of bile acid synthesis and dicarboxylic fatty acid oxidation

overall_design: Comparative gene expression profiling analysis of RNA-seq data for C57BL/6J wildtype and Nudt7-/- male and female mice fed a control and high-fat diet chow

middle: A

first: Roberta

last: Leonardi


title: Unique Ligand and Kinase-Independent Roles of the Insulin Receptor in Regulation of Cell Cycle, Senescence and Apoptosis

city: Boston

first: Jonathan

last: Dreyfuss

state: MA

address: 1 Joslin Pl

department: Bioinformatics & Biostatistics Core

institute: Joslin Diabetes Center

summary: Insulin acts through the insulin receptor (IR) tyrosine kinase to exert its classical metabolic and mitogenic actions. Here, using receptors with either short or long deletion of the β-subunit or mutation of the kinase active site (K1030R), we uncover a second novel IR signaling pathway that is intracellular domain dependent, but ligand and tyrosine kinase-independent (LYK-I). These LYK-I actions of the IR are linked to changes in phosphorylation of a network of proteins involved in the regulation of extracellular matrix organization, cell cycle, ATM signaling and cellular senescence; and result in upregulation of expression of multiple extracellular matrix-related genes and proteins, down-regulation of immune/interferon-related genes and proteins, and increased sensitivity to apoptosis. Thus, in addition to classical ligand and tyrosine kinase-dependent (LYK-D) signaling, the IR regulates a second, novel ligand and tyrosine kinase-independent (LYK-I) pathway which regulates the cellular machinery involved in senescence, matrix interaction and response to extrinsic challenges.

overall_design: To define the receptor-dependent but ligand- and kinase-independent signaling for insulin receptor (IR), we have generated preadipocytes in which the endogenous IR and IGF1R have been genetically inactivated to create DKO cells, and then the cells have been reconstituted with the normal wild-type IR, a kinase-dead mutant of IR (K1030R), a truncated IR lacking 79 amino acids from the C-terminus (∆CT) or a truncated IR lacking almost the entire intracellular domain including both tyrosine-kinase domain and C-terminus, leaving only the intracellular juxtamembrane 36 amino acids (Juxtamembrane-Domain-Only, JMO). We assessed gene expression in the DKO, IR, K1030R, ∆CT and JMO cells using RNAseq after 6 h serum starvation with or without stimulation by 100 nM insulin for 15 min. This was mostly done in triplicate.

first: Hirofumi

last: Nagao

first: C

middle: R

last: Kahn


title: Transcriptionnal signature of Non Small Cell Lung Carcinomas with FHITlow/pHER2high phenotype

status: Public on Nov 24 2022

city: Reims

first: Brisebarre

last: Audrey

address: 45 rue Cognacq-Jay

institute: INSERM

summary:
The tumor suppressor fragile histidine triad (FHIT) is frequently lost in NSCLC. We recently showed that FHIT controls HER2 receptor activity in lung tumor cells and that tumor cells from NSCLC patients harboring an FHITlow/pHER2high phenotype are sensitive to anti-HER2 drugs. Here, we sought to identify the transcriptomic signature of this phenotype and to evaluate its clinical significance.
We performed an RNA sequencing analysis on tumor cells isolated from NSCLC displaying or not an FHITlow/pHER2high status and a functional analysis of differentially regulated genes.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for FHITlow/pHER2high and other tumors

first: Audrey

last: Brisebarre

first: Béatrice

last: Nawrocki-Raby


title: Low n6/n3 Ratio Consumption of Neonates Establishes Lipid Oxidative Metabolism in Adipocyte Precursor Cells [Bulk RNA-seq]

summary: Adipocyte precursor cells were isolated by flow cytometry. CD24+ and CD24- (progenitors and preadipocytes) were sequenced following 10 days of early life high or low n6/n3 exposure.

overall_design: Cross-fostered wildtype pups were exposed postnatally to milk containing either High or Low n6/n3 PUFA ratios


title: Clonal transcriptomics identifies mechanisms of chemoresistance and empowers rational design of combination therapies

city: Cambridge

first: Sophia

middle: Alisa

last: Wild

state: Cambridgshire

address: Robinson Way

department: School of Clinical Medicine

country: United Kingdom

institute: University of Cambridge and Cancer Research UK Cambridge Institute

summary: Intratumour heterogeneity provides a fuel for cancer evolution enabling tumour cell populations to adapt to selective pressures imposed by the tumour microenvironment or therapeutic interventions. To uncover functionally heterogeneous subpopulations of tumour cells with different treatment sensitivity and to understand how clonal lineage-dependent transcriptomic diveristy contributes, we developed WILDseq, a hihg-complexity expressed barcode library for simultanous mapping of each cells' clonal identity and transcriptional states.

overall_design: We developed WILD-seq, a novel barcoding toolkit combining lineage tracing and single-cell RNA sequencing (scRNA-seq). Our WILD-seq toolkit contains our lentiviral barcoding system, experimental workflow and methods, and computational pipelines. Single cells are uniquely labelled with a hertiable WILD-seq barcode which can be captured as an expressed transcript by scRNA-seq on an oligo(dT)-capturing platform. The barcode cassette consists out of two 12 nt barcodes each with a hamming distance of 5 allowing to correct up to 2 errors per barcode. The overall library consists of ~1,600,000 potential barcode combinations. To ensure robust WILD-seq barcode capture, we further developed a one-step PCR enrichment strategy to specifically enrich WILD-seq barcode transcripts out of the pooled amplified cDNA of the transcriptome. We applied our WILD-seq approach to study the nature of clonal response to therapeutic pressure in two syngeneic mammary carcinoma models (4T1 and D2A1-m2) using a taxane-based chemotherapy and a first-in-class small-molecule inhibitor of BET bromodaomines JQ1. In total, we analysed 75,509 transcriptomes including tumour cells and cells from the tumour microenvironment with specific expression of the WILD-seq barcode in the tumour cell clusters. Our work provides a full pipeline from barcoding to data analysis and guidelines on how to customize the protocol allowing researchers to apply the tool for their scientific question.

first: Greg

middle: J

last: Hannon


title: Nucleotide level linkage of transcriptional elongation and polyadenylation

city: Aurora

first: David

last: Bentley

state: Colorado

department: BMG

institute: UCD AMC

summary: Alternative polyadenylation yields many mRNA isoforms whose 3’ termini occur disproportionately in clusters within 3’ UTRs.  Profiles of poly(A) site usage are regulated by the rate of transcriptional elongation by RNA polymerase II (Pol II).  Pol II derivatives with slow elongation rates confer an upstream-shifted poly(A) profile, whereas fast Pol II strains confer a downstream-shifted poly(A) profile.  In yeast, upstream and downstream shifts within isoform clusters occur steadily at the nucleotide level.  In contrast, changes from one isoform to the next are much smaller between clusters, even when the distances between them are relatively large.  Pol II occupancy increases just downstream of the most speed-sensitive poly(A) sites, suggesting a linkage between reduced elongation rate and cluster formation. These observations suggest that 1) Pol II elongation speed affects the nucleotide-level dwell time allowing polyadenylation to occur, 2) poly(A) site clusters are linked to the local elongation rate and hence do not arise simply by intrinsically imprecise cleavage and polyadenylation of the RNA substrate, 3) DNA sequence elements can affect Pol II elongation and poly(A) profiles, and 4) the cleavage/polyadenylation and Pol II elongation complexes are spatially, and perhaps physically, coupled so that polyadenylation occurs rapidly upon emergence of the nascent RNA from the Pol II elongation complex.

overall_design: anti-pol II mNET-seq & RNA-seq

first: Benjamin

last: Erickson


title: Modeling sex differences in humans using isogenic induced pluripotent stem cells

city: Jerusalem

first: Ithai

last: Waldhorn

address: Kiryat Hadassah

department: Goldyne Savad Institute of Gene Therapy

country: Israel

institute: Hebrew University

summary: We developed a cellular model to study sex differences in humans. Using somatic cells from a mosaic Kleinefelter patient, we generated isogenic human induced pluripotent stem cell lines with different sex chromosome complements – 47,XXY / 46,XX / 46,XY and 45,X0.

overall_design: RNA-seq of nine isogenic hiPSC cell lines - XX1, XX2, XX3, XY1, XY2, XY3, XXY1, XXY2, X0.

last: Reubinoff


title: Modeling sex differences using isogenic human neural progenitor cells

summary: We developed a cellular model to study sex differences in humans. Using somatic cells from a mosaic Kleinefelter patient, we generated isogenic human induced pluripotent stem cell lines with different sex chromosome complements – 47,XXY / 46,XX / 46,XY and 45,X0. 46, XX and 46, XY hiPSCs were differentiated to neural progenitor cells and compared.

overall_design: RNA-seq of six cell lines was analyzed: XX1, XX2, XX3, XY1, XY2, XY3. XX samples were compared to XY samples


title: Single-cell sequencing reveals a delicate molecular and cellular interaction during decidualization in humans

last: mingqing

address: NO.419 fangxie Road, Shanghai

institute: Affiliated obstetrics and Gynecology Hospital of Fudan University

summary: Proper decidualization is a critical determinant of pregnancy success. Deficiencies in decidualization are associated with a variety of pregnancy disorders, including female infertility, recurrent implantation failure, and miscarriage. However, the mechanism for triggering decidualization of human endometrium is largely unknown. Here we characterized the transcriptomes of four major cells in human endometrium and decidua at single-cell resolution. We discovered the dynamic change characteristics of six major stromal cells. More importantly, a dialogue between IGF1+ stromal cells and IGF1R+ stromal cells initiates endometrial decidualization under regulation of progesterone and estrogen, and IL1B+ stromal cells trigger the apoptosis of epithelial cells and functional remodeling during decidualization. We defined a unique AREG+ NK cell for accelerating decidualization by interacting with IGF1+ stromal cells, and observed that extravillous trophoblasts promote decidualization possibly by multiply pathways. Additionally, we developed a systematic repository of cell-cell communication for decidualization via the ligand-receptor complexes interactions. Our study provides deeper insights into the molecular and cellular characterizations during decidualization.

overall_design: Endometrium and decidua mRNA profiles were generated by deep sequencing,using 10X Genomics.

first: Mingqing

last: Li

first: Jiawei

last: Shi


title: Regulatory basis for reproductive flexibility in a meningitis-causing fungal pathogen

city: beijing

first: lei

last: chen

address: NO.1 Beichen West  Road, Chaoyang District, Beijing 100101, China

institute: the institute of microbiology chinese academy of sciences

summary: Sexual reproduction facilitates infections and adaptations in Cryptococcus pathogens that cause fatal meningoencephalitis and have two distinct sexual cycles (bisexual and unisexual). Here, by constructing a gene-deletion strains library for transcription factor (TF) genes in Cryptococcus deneoformans, we explore spatiotemporal TF networks in sequential stages of mating community development and identified key regulators that determine processes specific to different reproductive modes. We show that the TFs crucial for bisex-specific syngamy coordinately induce the expression of a specific group of genes centered on the ancient mating determinants, which includes FMP1, a previously unidentified component of the conserved fungal mating pathway. Furthermore, we identify that a recently evolved regulatory cascade mediates the pre-meiotic autodiploidization process that defines Cryptococcus unisex, supporting that unisex is a recent evolutionary innovation. Our findings indicate that genetic circuits with different evolutionary ages govern the hallmark events distinguishing unisex and bisex, enabling reproductive flexibility that benefits Cryptococcus pathogenicity.

overall_design:
18 samples contain wildtype, vea2Δ, CQS2OE and pum1Δ at two time points with 3 repeats, 8 samples contain CQS2 ChIP-Seq data with FLAG and mCherry tag with two repeats.

For RNA-seq analyses, strains were cultured in YPD liquid medium at 30 °C overnight. The cells were washed with ddH2O and spotted on V8 medium to stimulate unisexual reproduction. The level and integrity of RNA in each sample were evaluated using a Qubit RNA Assay Kit on a Qubit 2.0 Flurometer (Life Technologies, CA, USA) and RNA Nano 6000 Assay Kit with the Bioanalyzer 2100 system (Agilent Technologies, CA, USA), respectively. RNA purity was assessed using a Nano Photometer spectrophotometer (IMPLEN, CA, USA). The transcriptome libraries were generated using the VAHTS mRNA-seq v2 Library Prep Kit (Vazyme Biotech Co., Ltd, Nanjing, China) according to the manufacturer?s instructions.

first: Pengjie

last: Hu

first: Hao

last: Ding

first: Huimin

last: Liu

first: Yulin

last: Yang

first: Lei

last: Chen

first: Guang-Jun

last: He

first: Weixin

last: Ke

first: Ping

last: Zhu

first: Xiuyun

last: Tian

first: Yan

last: Peng

first: Zhenghao

last: Shen

first: Xiaoxia

last: Yao

first: Changyu

last: Tao

first: Ence

first: Guojian

last: Liao

first: Xiao

first: Linqi

last: Wang


title: Evi5 is required for Xenopus limb and tail regeneration

city: Shanghai

first: Gufa

last: Lin

address: 1239 Siping road Tongji university

department: Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji Hospital, School of Life Sciences and Technology, Tongji University

institute: Tongji University

summary: Amphibians such as the salamanders and the African clawed frog Xenopus are great models for regeneration studies because they can fully regenerate their lost organs. While axolotl can regenerate damaged organs throughout its lifetime, Xenopus has a limited regeneration capacity after metamorphosis. The ecotropic viral integrative factor 5 (Evi5), a cell-cycle-regulated protein that prevents cells from entering mitosis prematurely, is of great interest for it is highly upregulated in the limb blastema of axolotls, but its expression level remains unchanged in the fibroblastema of postmetamorphic frogs. Yet, its role in regeneration competent context in Xenopus has not been fully analyzed. Here we show that Evi5 is also upregulated in Xenopus tadpoles after limb and tail amputation, as it is in axolotls. Down-regulation of Evi5 with morpholino antisense oligos (Mo) impairs wound healing and blastema formation in limbs and tails in both axolotls and Xenopus tadpoles, suggesting a conserved function for Evi5 in regeneration. Using skin punch as a healing model we show that Evi5 is also involved in cell migration during wound healing. RNA-sequencing analysis shows that in addition to reduced signaling of Lepr, Pdgfa, Gdf5, evi5 Mo also downregulate lysine demethylases kdm6b and kdm7a, which are also required for limb regeneration. Thus, our results demonstrate that Evi5 plays a critical role in the regeneration of multiple systems in amphibians.

overall_design: RNA-seq for Control(XC1, XC2, XC3) and evi5 (XE1, XE2, XE3) morpholino injected stage 53 tadpole 3DPI limb bud

first: Yang

last: Li

first: Youwei


title: ADAR1-dependent editing participates to the human beta-cells transcriptome diversity during inflammation

status: Public on Nov 23 2022

city: Anderlecht

first: Florian

last: Szymczak

state: Brussels

address: Route de Lennik 808 - CP618

department: Faculty of Medicine

country: Belgium

institute: Université Libre de Bruxelles

summary: Enterovirus infection has long been suspected as a possible trigger for type 1 diabetes. Upon infection, viral double-stranded RNA (dsRNA) is recognized by membrane and cytosolic sensors that orchestrate the type I interferon signaling and the recruitment of innate immune cells to the pancreatic islets. In this context, adenosine deaminase acting on RNA 1 (ADAR1) editing plays an important role in dampening the immune response by inducing adenosine mispairing, destabilizing the RNA duplexes and thus preventing an excessive immune activation. Here, we evaluated the role of ADAR1 in human pancreatic b cells and determined the impact of type 1 diabetes pathophysiological environment on ADAR1-dependent RNA editing. We show that both IFNα or IFNɣ/IL1β stimulation promotes ADAR1 expression and increases the A-to-I RNA editing of Alu-Containing mRNAs in EndoC-bH1 cells as well as primary human islets. We demonstrate that ADAR1 overexpression inhibits the type I interferon response signaling, while ADAR1 silencing potentiates IFNα effects. In addition, ADAR1 overexpression triggers the generation of alternative spliced RNAs, highlighting a novel role for ADAR1 as regulator of the b cell transcriptome under inflammatory conditions.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for EndoC-βH1 cells overexpressing, or not, ADAR1

first: Roni

last: Cohen-Fultheim

first: Sofia

last: Thomaidou

first: Arnaud

last: Zaldumbide


title: Single-cell RNA sequencing uncovers dynamic roadmap and cell-cell communication during buffalo spermatogenesis

city: Nanning

first: Liangfeng

last: Huang

state: Guangxi

address: No. 100, East University Road

department: Animal Reproduction Institute

institute: Guangxi University

summary: Spermatogenesis carries the task of precise intergenerational transmission of genetic information from the paternal genome and involves complex developmental processes regulated by the testicular microenvironment. Studies performed mainly in mouse models have established the theoretical basis for spermatogenesis, yet the wide inter-species differences preclude direct translation of the findings, and farm animal studies are progressing slowly. More than 32,000 cells from prepubertal (3-month-old) and pubertal (24-month-old) buffalo testes were analyzed using single-cell RNA sequencing (scRNA-seq), and dynamic gene expression roadmaps of germ cell and somatic cell development were generated. In addition to identifying the dynamic processes of sequential cell fate transitions, the global cell-cell communication essential to maintain regular spermatogenesis in the buffalo testicular microenvironment was uncovered. The findings provide the theoretical basis for establishing buffalo germline stem cells in vitro or culturing organoids and facilitating the expansion of superior livestock breeding.

overall_design: Single-cell transcriptome examination of 3- and 24-month-old buffalo testes.

first: Junjun

last: Zhang

first: Pengfei

first: Xingchen

first: Weihan

last: Yang

first: Runfeng

last: Liu

first: Yangqing

last: Lu

first: Ming

first: Qiang

last: Fu


title: Modeling sex differences using isogenic induced pluripotent stem cells and isogenic human neural progenitor cells

first: Ithai

last: Waldhorn

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

overall_design: Refer to individual Series


title: Low n6/n3 Ratio Consumption of Neonates Establishes Lipid Oxidative Metabolism in Adipocyte Precursor Cells [single-cell RNA-seq]

first: Michael

last: Rudolph

summary: Adipocyte precursor cells from 12-day-old pups were characterized by single cell RNA sequencing

overall_design: Subcutaneous adipose tissue was enzymatically digested producing a stromal vasular pellet that was flow sorted for adipocyte precursor cells

middle: C


title: Gene expression data from primary mouse neocortical cultures

city: Portland

first: stephen

last: smith

state: Oregon

address: 3710 SW U.S. Veterans Hospital Rd., Building 101 Room 525

institute: VAPORHCS

summary:
Primary murine neocortical cultures are a common model for investigating fundamental attributes of neuronal signalling.
Here we utilized an Affymetrix GeneChip platform to measure expression of various genes in vitro in order to chracterize our cortical cultures.

overall_design: We isolated the cortices of p2 mouse pups and dissociated the cells for growth. After 14 days we extracted and hybridized RNA using an Affymetrix microarray and considered expression of target genes between three biological replicates.

first: Stephen

middle: M

last: Smith

first: Salil

last: Rajayer

first: Briana

last: Knight

first: Luke

last: Steiger


title: Global regulation of mRNA and microRNA homeostasis by uridylyltransferases TENT3A/B

city: London

first: Pengcheng

state: Ontario

address: 1151 Richmond St

department: Biochemistry

institute: The University of Western Ontario


title: Haemophilus ducreyi infection induces oxidative stress, central metabolic changes, and a mixed pro- and anti-inflammatory environment in the human host

city: Indianapolis

first: Julie

last: Brothwell

state: IN

address: 635 Barnhill Dr., MS420

department: Department of Microbiology & Immunology

institute: Indiana University

summary: Few studies have investigated host-bacterial interactions at sites of infection in humans using transcriptomics and metabolomics. Haemophilus ducreyi causes cutaneous ulcers in children and the genital ulcer disease chancroid in adults. We developed a human challenge model in which healthy adult volunteers are infected with H. ducreyi on the upper arm until they develop pustules. Here, we characterized host-pathogen interactions in pustules using transcriptomics and metabolomics and examined interactions between the host transcriptome and metabolome using integrated omics. In a previous pilot study, we determined the human and H. ducreyi transcriptomes and the metabolome of pustule and wounded sites of 4 volunteers (B. Griesenauer, et al. mBio 10(3):e01193-19 https://doi.org/10.1128/mBio.01193-19). While we could form provisional transcriptional networks between the host and H. ducreyi, the study was underpowered to integrate the metabolome with the host transcriptome. To better define and integrate the transcriptomes and metabolome, we used samples from both the pilot study (n=4) and new volunteers (n=8) to identify 5,495 human differentially expressed genes (DEGs), 123 H. ducreyi DEGs, 205 differentially abundant positive ions, and 198 differentially abundant negative ions. We identified 42 positively correlated and 29 negatively correlated human-¬H. ducreyi transcriptome clusters. In addition, we defined human transcriptome-metabolome networks consisting of 9 total clusters, which highlighted changes in fatty acid metabolism and mitigation of oxidative damage. Taken together, the data suggests a mixed pro- and anti-inflammatory environment and rewired central metabolism in the host that provides a hostile, nutrient limited environment for H. ducreyi.

sample_organism:
[Haemophilus] ducreyi
Homo sapiens

platform_organism:
[Haemophilus] ducreyi
Homo sapiens

overall_design: Eight human volunteers were infected with H. ducreyi on the upper arm. To control for delivery of the bacteria via puncture wounds in the skin, a wounded control sample that delivered PBS only was also performed for each person. To control for changes in bacterial gene expression, the bacterial cultures (4) used to inoculate the volunteers were collected. All samples were processed for RNA-seq. We compared the differential expression of human genes in pustules v. wounds and the differential expression of in vivo v. cultured bacterial genes.

first: Stanley

last: Spinola


title: Global regulation of mRNA and microRNA homeostasis by uridylyltransferases TENT3A/B [RNA-seq]

summary:
To investigate the regulation of mRNA by 3'end unridylation, we performed RNA sequencing on HEK 293T cells and its TENT3A/B knockout strains (ΔTENT3A/B).
We performed transcriptomic analysis comparing mRNA abundance in HEK 293T cells and ΔTENT3A/B

overall_design: Comparative gene expression profiling analysis of RNA-seq data for HEK 293T cells and its TENT3A/B double knockout strains.

first: Mallory

last: Frederick

first: Ilka

last: Heinemann


title: Genome-wide pooled CRISPR knockout screen for novel regulators of macrophage efferocytosis.

city: New York

first: Hanrui

state: United States

address: 630 West 168th Street

institute: Columbia University Irving Medical Center

summary: We performed a FACS-based genome-wide CRISPR knockout screen in primary murine macrophages to identify regulators of efferocytosis, the phagocytic clearance of dying cells. The screen identified known and novel regulators of macrophage efferocytosis. More broadly, the screen approach can be applied to interrogate complex functional phenotypes in primary macrophages.

overall_design: This is a pooled FACS-based genome-wide CRISPR knockout screening for efferocytosis regulators. Bone marrow (BM) cells were isolated from Rosa26-Cas9 knock-in mice constitutively expressing Cas9 endonuclease (JAX #026179), transduced with lentiviral Brie gRNA libraries and then differentiated to BMDMs for efferocytosis and screening. The success of the screening relies on the effective enrichment of macrophages with high vs. low efferocytosis capacity. Since efferocytosis is a binary event, to facilitate an effective separation and enrichment, we performed two rounds of efferocytosis sequentially. Specifically, human Jurkat cells, an acute T cell leukemia cell line routinely used for in vitro efferocytosis assays, were treated with staurosporine to induce apoptosis, then labeled with fluorescent linkers, PKH67 (Ex/Em: 490/502 nm) or PKH26 (Ex/Em: 551/567 nm). BMDMs were first incubated with PKH67-labeled apoptotic cells (ACs) for efferocytosis, followed by the second round of incubating with PKH26-labeled ACs. BMDMs were then collected for flow cytometry sorting, which separated the BMDMs that engulfed both PKH67+ and PKH26+ ACs, i.e., the efficient eaters (~5%), and BMDMs that did not engulf any ACs, i.e., the non-eaters. We have also collected BMDMs without performing efferocytosis, i.e. the input samples. The non-eaters are expected to enrich for gRNAs targeting positive regulators essential for efferocytosis, i.e., knockout would impair efferocytosis. The efficient eaters are expected to enrich for gRNAs targeting negative regulators, i.e. knockout would enhance efferocytosis. We sequenced the sorted non-eaters, efficient eaters, and the input samples for each of the two replicates and performed MAGeCK analysis to identify the top hits.

first: Jianting

last: Shi


title: An RNA-based platform to neutralize DNMT1 function AptaDiR is an innovative RNA-based platform to neutralize DNMT1 function

first: Mahmoud

middle: Adel

last: Bassal

state: Massachusetts

address: 3 Blackfan Circle

department: Hematology and Oncology

institute: Beth Israel Deaconess Medical Center

summary: DNA methylation is a fundamental epigenetic modification regulating gene expression. Aberrant DNA methylation is the most common molecular lesion in cancer cells. However, medical intervention has been limited to the use of demethylating drugs that act indiscriminately on the genome and come with a toll of high toxicity and low specificity. Aptamers are novel targeting molecules considered high affinity ligands. By conjugating, the RNA inherent ability of inhibiting DNMT1, to an RNA aptamer platform, we generated the first-of-its kind aptamers approach targeting and neutralizing DNMT1 function – the aptaDiR. Molecular modelling of  RNA-DNMT1 interactions’ coupled with biochemical and cellular assays enabled  identification and characterization of the aptaDiR. This novel RNA bio-drug blocks DNA methylation and impairs cancer cell viability.   Collectively, we present an innovative RNA-based approach to modulate DNMT1 activity in cancer or diseases characterized by aberrant DNA methylation and suggest the first alternative strategy to overcome the limitations of currently approved hypomethylating protocols which will greatly improve clinical intervention on DNA methylation.

overall_design: Bisulphite converted DNA from the samples (3 samples in duplicate) were hybridised to the Diagenode EPIC methylation array

first: Carla

last: Esposito

first: I

last: Autiero

first: A

last: Sandomenico

first: S

last: Ummarino

first: M

last: Borchiellini

last: Ruvo

last: Catuogno

last: Ebralidze

first: V

last: de Franciscis

first: Annalisa

last: Di Ruscio


title: A simple but robust approach for isolating chromatin associated RNAs in Arabidopsis thaliana

city: Hangzhou

first: Fengli

last: Zhao

state: Zhejiang

address: No. 28 of Shuidaosuo Road

department: State Key Laboratory of Rice Biology and Breeding

institute: China National Rice Research Institute

summary: In the transcription process, Pol II produces the nascent RNA which associates with chromatin. To acquire co-transcriptional RNA is easy to understand the RNA transcription status. We use a simple and effective way to separate the intact cell nucleus and wash the nucleoplasm, then acquire the chromatin component. With the help of Illumina RNA sequencing, we analyze the nascent RNA extracted from chromatin component in whole genome. We demonstrate the method descripted in this paper can be suitable for analyze co-transcriptional splicing ratio effectively. Interestingly, we find the first intron globally has lower co-transcriptional splicing efficiency. In brief, our method can be used for analyze the nascent RNA in elongating process and the fully transcribed RNA with poly-A tail but not yet released from chromatin.

overall_design: We describe a step-by-step protocol for isolating chromatin-bound RNAs from the pool of cellular RNAs in Arabidopsis thaliana. With this protocal we also can separate the elongating RNAs and full-length polyA RNAs from the chromatin-bound RNAs.

first: Qiqi

last: Zhang

first: Zhe

last: Wu

first: Danling

last: Zhu


title: Global regulation of mRNA and microRNA homeostasis by uridylyltransferases TENT3A/B [miRNA-seq]

first: Pengcheng

overall_design: Comparative gene expression profiling analysis of miRNA-seq data for HEK 293T cells and its TENT3A/B double knockout strains.

last: Frederick

last: Heinemann


title: Functional characterization of TetR-like transcriptional regulator PA3973 from Pseudomonas aeruginosa [RNA-seq]

status: Public on Nov 22 2022

city: Warszawa

first: Adam

last: Kawałek

address: Pawinskiego 5A

department: Department of Microbial Biochemistry

country: Poland

institute: Institute of Biochemistry and Biophysics, PAS

summary: Pseudomonas aeruginosa, a human opportunistic pathogen, is a common cause of nosocomial infections. Its ability to survive under different conditions relies on a complex regulatory network engaging transcriptional regulators controlling metabolic pathways and capabilities to efficiently use the available resources. P. aeruginosa PA3973 encodes a putative TetR family transcriptional regulator, with a helix-turn-helix motif involved in DNA binding. We applied transcriptome profiling (RNA-seq), and genome-wide identification of binding sites using ChIP-seq to unravel the biological role of PA3973.

overall_design: Pseudomonas aeruginosa PAO1161 (leu-, r-, RifR) derivative of PAO1 was used in the experiment (Kawalek et al., 2020), as a control (reference) strain and Pseudomonas aeruginosa PAO1161 PA3973 deletion strain. To determine the impact of an increased PA3973 level on the transcriptome the RNA-seq analysis was performed on RNA isolated from Pseudomonas aeruginosa PAO1161/pKKB3.11 (lacIQ-tacp-PA3973) cultures grown under selection in L broth with 0.05 mM IPTG (hereafter referred to as PA3973+) as well as from Pseudomonas aeruginosa PAO1161 carrying pAMB9.37 (lacIQ-tacp) cells grown under selection with addition of 0.05 mM IPTG (empty vector control, hereafter called EV).Three independent biological replicates of total RNA were isolated for each strain from logarithmic (OD600 0.5) phase of planktonic culture grown on rich medium (L broth) at 37oC.

first: Karolina

last: Kotecka

last: Kawalek

first: Magdalena

last: Modrzejewska

first: Gawor

last: Jan

first: Zuchniewicz

last: Karolina

first: Gromadka

last: Robert

first: Aneta

last: Bartosik


title: Functional characterization of TetR-like transcriptional regulator PA3973 from Pseudomonas aeruginosa [ChIP-seq]

summary:
Pseudomonas aeruginosa PA3973 encodes a putative TetR family transcriptional regulator, with a helix-turn-helix motif involved in DNA binding. We applied phenotype analyses, as well as transcriptome profiling (RNA-seq), and genome-wide identification of binding sites using ChIP-seq to unravel the biological role of PA3973. The ChIP-seq analysis identified more than 300 PA3973 binding sites in the P. aeruginosa genome, among them 139 were located in intergenic regions. The 13 bp sequence was identified as the preferential binding site of PA3973. The PA3973 regulon encompasses genes involved in stress response, including the putative PA3973-PA3970  operon.
Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3973 showed changes in the mRNA level of 648 genes; among them, 374 were down-regulated. Concomitantly, ChIP-seq analysis identified more than 300 PA3973 binding sites in the P. aeruginosa genome, among them 139 were located in intergenic regions. The 13 bp sequence was identified as the preferential binding site of PA3973.

overall_design: Pseudomonas aeruginosa PAO1161 (leu-, r-, RifR) derivative of PAO1, was used in the experiment (Kawalek et al., 2020; BMC Genomics, 21:14). Chromatin immunoprecipitation and sequencing (ChIP-seq) analysis was performed on P. aeruginosa PAO1161 ΔPA3973/pMEB1 (lacIQ-tacp-PA3973-flag) (hereafter referred to as PA3973-F) as well as PAO1161/pABB28.1 (lacIQ-tacp-flag), empty vector control (hereafter called EV-F) strains. Cells were grown under selection in L broth with 0.05 mM IPTG until OD600 = 0.5. For each biological replicate, two independent cultures of each strain were pooled together. The immunoprecipitation was performed with commercially available polyclonal anti-FLAG antibodies (DYKDDDDK Tag polyclonal  antibodies; PA1-985B; Invitrogen).


title: MNX1-AS1, a c-Myc induced lncRNA, promotes the Warburg effect by regulating PKM2 nuclear translocation

city: Hefei

first: Qidong

last: Li

state: Anhui

address: 443 Huangshan Road, Shushan district

institute: Lianxin Liu Lab

summary: Altered glycolysis is the most fundamental metabolic change associated with the Warburg effect. However, the precise mechanisms are not completely understood. Here we dissect how MNX1-AS1 reinforces the Warburg effect through facilitating the non-glycolytic actions of PKM2 in the nucleus. We found that MNX1-AS1 expression was frequently overexpressed in hepatocellular carcinoma (HCC). In the context of HCC, we show MNX1-AS1 acts as a scaffold to promote interactions between PKM2 and importin α5. Upon EGFR activation, the resulting ternary complex drives the translocation of PKM2 into the nucleus. In consequence, glycolytic pathway components including key mediators of the Warburg effect (LDHA, Glut1 and PDK1) are upregulated. Manipulating MNX1-AS1 elicited robust effects on glycolysis associated with marked changes in HCC growth in vitro and in vivo, indicative of the significant contribution of MNX1-AS1 to tumorigenesis. Moreover, while MNX1-AS1 expression is driven by c-Myc, its actions associated with PKM2 were shown to be downstream and independent of c-Myc. Given the status of MNX1-AS1 as a pan-cancer upregulated lncRNA, this implicitly highlights the potential of targeting MNX1-AS1 to selectively counter the Warburg effect in a range of tumor types.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for HepG2 cells with MNX1-AS1 Knockdown

first: Yang

first: Yichun

last: Wang

first: Xiansheng

last: Lin

first: Lianxin

last: Liu


title: The PNUTS-protein phosphatase 1 complex acts as an intrinsic barrier to KSHV replication [ChIP-Seq II]

city: Dallas

first: Nicholas

middle: K

last: Conrad

state: TX

address: 6000 Harry Hines Blvd

department: Microbiology

institute: UT Southwestern Medical Center

summary: In this study, we found that the host protein PNUTS suppressed Kaposi's sarcoma-associated herpesvirus (KSHV) gene expression during lytic reactivation and from heterologous viral reporters. To gain insights into the mechanism, we performed ChIP-seq of the pol II subunit RPB3 in cells with an integrated reporter containing the KSHV ORF59 gene. Our results were consistent with previous reports showing that depletion of PNUTS leads to global loss of pol II slow down and  termination after polyadenylation sites. In contrast, PNUTS depletion appeared to decrease promoter-proximal pausing on our integrated reporter suggesting a distinct mode of gene regulation on viral genes.

overall_design: Pol II ChIP-seq using antibodies against RPB3 was performed on samples treated with either a nontargeting siRNA (siNT) or one of two siRNAs targeting PNUTS (siP1 or siP2). The cells were iSLK cells latently infected with  the BAC16 KSHV bacmid. Pellets and inputs were analyzed for each sample; two biological replicates were perforemd (R1 and 2).

first: Anne

last: Devlin

first: Ashutosh

last: Shukla

first: Ivan

last: D'Orso


title: Targeted systematic evolution of an RNA platform neutralizing DNMT1 function and controlling DNA methylation [MethylationEPIC]

city: Duarte

first: Haiqing

state: CA

address: 1500 E Durate Rd

department: Shared Resources Department

institute: City of Hope National Medical Center

type: Methylation profiling by genome tiling array

summary: DNA methylation is a fundamental epigenetic modification regulating gene expression. Aberrant DNA methylation is the most common molecular lesion in cancer cells. However, medical intervention has been limited to the use of broadly acting, small molecule-based demethylating drugs with significant side-effects and toxicities. To allow for targeted DNA demethylation, we integrated two novel nucleic acid-based approaches: DNMT1 interacting RNA (DiR) and RNA aptamer strategy. By combining the RNA inherent capabilities of inhibiting DNMT1 with an aptamer platform, we generated a first-in-class DNMT1-targeted approach – aptaDiR. Molecular modelling of RNA-DNMT1 complexes coupled with biochemical and cellular assays enabled the identification and characterization of aptaDiR. This novel RNA bio-drug blocked DNA methylation, impaired cancer cell viability and inhibited tumour growth in vivo.  Collectively, we present an innovative RNA-based approach to modulate DNMT1 activity in cancer or diseases characterized by aberrant DNA methylation and suggest the first alternative strategy to overcome the limitations of currently approved hypomethylating protocols, which will greatly improve clinical intervention on DNA methylation

overall_design: Bisulphite converted DNA from the samples (5 samples in duplicate) were hybridised to the Diagenode EPIC methylation array

first: Carla

last: Esposito

first: Ida

last: Autiero

first: Mahmoud

last: Bassal

first: Annamaria

last: Sandomenico

first: Maria

last: Ibba

first: Dongfang

first: Lucrezia

last: Rinaldi

first: Simone

last: Ummarino

first: Giulia

last: Gaggi

first: Marta

last: Borchiellini

first: Piotr

last: Swiderski

first: Menotti

last: Ruvo

first: Silvia

last: Catuogno

first: Alexander

last: Ebralidze

first: Marcin

last: Kortylewski

first: Vittorio

last: de Franciscis

first: Annalisa

last: DiRuscio


title: An injury-associated transient progenitor state in the epicardium mediates heart regeneration

first: Friederike

last: Dündar

state: New York

address: 1300 York Ave

department: Physiology & Biophysics

institute: Weill Cornell Medicine

summary: Adult zebrafish regenerate heart muscle after severe cardiac damage without significant scarring. The epicardium, a mesothelial cell sheet covering the vetebrate heart, is activated by injury and supports muscle regeneration through paracrine effects and as a source of multipotent cells. The understudied cellular heterogeneity of the adult epicardium during heart regeneration has constrained the effort in mobilizing the epicardium for heart repair. To dissect epicardial cell states and the underlying mechanisms that lead to successful heart regeneration in zebrafish, we performed single-cell RNA-sequencing of isolated epicardial cells from the regenerating adult heart and revealed their dynamic cellular heterogeneity. We defined the epithelial and mesenchymal layers of the epicardium and identified a transiently activated epicardial progenitor cell (aEPC) subpopulation that expresses aldh1a2, ptx3a, col12a1b and marcksb. Upon heart amputation injury, aEPCs emerge from the existing epicardial cells, migrate to enclose the wound, and disappear as regeneration progresses. Genetic  lineage tracing combined with modified RNA labelling confirmed an epithelial-mesenchymal transition (EMT) process of aEPCs and their differentiations to pdgfrb+ mural cells and pdgfra+hapln1a+ mesenchymal fibroblast-like cells that support heart regeneration. Genetic ablation of aEPCs blocked wound closure of the injured ventricle, suppressed cardiomyocyte proliferation, and disrupted heart regeneration. Our findings define a transient progenitor state of the adult epicardium that is an indispensable driver of zebrafish heart regeneration and identified ptx3a as a regeneration-specific non-ontogenetic regulator of the epicardium.

overall_design: Single-cell RNA sequencing of isolated epicardial cells from uninjured zebrafish hearts and hearts after amputation injury

first: Yu

last: Xia

first: Sierra

last: Duca

first: Bjoern

last: Perder

first: Paul

last: Zumbo

first: Miaoyan

last: Qiu

first: Yingxi

last: Cao

first: Michael

last: Harrison

first: Lior

last: Zangi

first: Doron

last: Betel

first: Jingli


title: Targeted systematic evolution of an RNA platform neutralizing DNMT1 function and controlling DNA methylation [RNA-seq]

first: Haiqing

last: Li

overall_design: Different gene expresion profiling analysis of RNA-seq data for the theapeutic potential of Ce-49 sh using K562 leukemia xenografts in immunodeficient NSG mice .

first: Carla

last: Esposito

first: Ida

last: Autiero

first: Mahmoud

last: Bassal

first: Annamaria

last: Sandomenico

first: Maria

last: Ibba

first: Dongfang

last: Wang

first: Lucrezia

last: Rinaldi

first: Simone

last: Ummarino

first: Giulia

last: Gaggi

first: Marta

last: Borchiellini

first: Piotr

last: Swiderski

first: Menotti


title: Targeted systematic evolution of an RNA platform neutralizing DNMT1 function and controlling DNA methylation


title: SUMOylation in adrenal cortex homeostasis

city: Clermont-Ferrand

first: Antoine

last: Martinez

address: Faculté de Médecine, CRBC, 28 place Henri Dunant

department: GReD

institute: CNRS, INSERM, Université Clermont Auvergne

summary: Identify genetic programs impacted by increasing protein SUMOylation and controlling adrenal cortex zonation

overall_design: We used adrenal glands from pubescent female and male mice having a genetically induced global SUMOylation. The mutant mice bear adrenal-specific deletion of the Senp2-floxed conditional allele that encodes a deSUMOylase.

first: Damien

last: Dufour


title: Dissecting the fate of Foxl2-expressing cells in ovary development using lineage tracing and Single-Cell Transcriptomics

city: Beijing

first: Xiangxiang

last: Jiang

state: Beijing

address: 1 Beichen West Road, Chaoyang District

institute: Institute of Zoology, Chinese Academy of Sciences

summary: Gonad somatic cells acquire sex-specific fates during sex determination. In XX gonad, a subset of somatic cells expresses Foxl2 after sex determination which are considered as the progenitor of granulosa cells. However, whether these cells also contribute to other cell type at later developmental stage is unknown. In the present study, the cell fate of Foxl2-expressing cells in fetal ovaries was analyzed by lineage tracing and signal-cell transcriptomics. We found that Foxl2-expressing cells gave rise to three cell types at later developmental stage, including granulosa cells, theca-interstitial cells, and stromal cells. Series single-cell RNA sequencing revealed that Foxl2-positive cells were divided into two clusters at P0. One group further differentiated into granulosa cells and theca cells at P14. Another group was classified as stromal cell lineage, then a small portion of them further differentiated into 3β-HSD-positive interstitial cells. We also found that interstitial cells were CYP17a1-positive, whereas theca cells did express this gene. Our data provide an important resource, at single-cell resolution, for better understanding somatic cell differentiation in ovary development.

overall_design: After injection of Tamoxifen to the pregnant mice at E13.5, GFP positive cells were analyzed at E14.5, P0, P7, and P14，which from the gonads of  mT/mG; Foxl2-CreERT2 female mice.

first: Jingjing

last: Zhou

first: Haowei

last: Wu

first: Lianjun

last: Zhang

first: Min

last: Chen

first: Zhiming

last: Shen

first: Xudong

last: Guo

first: Hongmei

first: Fei

last: Gao


title: SFRP4+IGFBP5hi NKT cells induced neural-like cells differentiation to contribute to adenomyosis pain

status: Public on Nov 21 2022

city: Ningbo

first: yichen

last: chen

address: NO.46 Yangshan Road，Ningbo

institute: Ningbo Institute of Medical Science

summary: Adenomyosis is an estrogen-dependent gynecological disease. The pathogenesis of chronic pain, the main clinical symptom of adenomyosis, remains undefined. As a combination lymphocyte with both T-cell and NK-cell properties, NKT cells play a role in immune defense against numerous diseases and modulate cell differentiation. This study analyzed tissue-cell samples from adenomyosis with or without pain by single-cell sequencing. We found a specific population of SFRP4+ NKT cells and a large amount of undifferentiated multipotent stem cells in the adenomyosis pain group. We discovered that high expression of IGFBP5 in SFRP4+NKT cells could promote the differentiation of multipotent stem cells into neural-like cells via the single cell trajectory. Verification by sample, the degree of expression of neuronal marker NEFM correlated with duration of pain in adenomyosis patients. The expression of IGFBP5 was positively correlated with the pain scores of adenomyosis patients. Collectively, these findings suggest SFRP4+IGFBP5hi NKT cells were capable of converting part of the stem cells into neurogenic cells and inducing adenomyosis pain.

overall_design: Single-cell sequencing detects differences between painful and non-painful groups in adenomyosis, validated by immunofluorescence

first: Jing

first: Yichen

first: Jue

last: Zhu

first: Liang


title: Bulk RNA-sequencing of FACS-sorted kidney glomerular cell types from BTBR ob/ob mice and glomeruli isolated from ex vivo perfused kidneys

city: Hamburg

first: Tianran

address: Falkenried 94

institute: University Medical Center Hamburg-Eppendorf

summary:
To investigate the gene expression levels of major glomerular cell types in BTBR ob/ob mice and in glomeruli under hyperfiltration
Bulk RNA-sequencing of glomerular cell types from BTBR ob/ob and BTBR +/+ mice at three time points (6/12/18 weeks). The glomeruli were isolated from ex vivo perfused kidney (hyperfiltration) and unperfused kidney (control) from the mouse.

overall_design: Comparative gene expression profiling analysis of RNA-seq data (BTBR ob/ob vs. BTBR +/+ in each glomerular cell type). Comparative gene expression profiling analysis of RNA-seq data for perfused vs unperfused glomeruli.

first: Yu

last: Zhao

first: Shuya

last: Liu


title: Effects of Wdfy3 knockout on the transcriptomic profile of murine bone-marrow derived macrophages.

first: Hanrui

summary: We determined the transcriptomic profile of bone-marrow derived macrophages (BMDMs) from LysMCre-mediated myeloid-specific Wdfy3 knockout mice and the Cre- littermate control mice.

overall_design: RNA-seq of BMDMs from 9-10 week old male mice (n = 4 Cre- control mice, and 4 Cre+ Wdfy3 knockout mice).

first: Jianting

last: Shi

first: Xun