title: Comparison of Genetic and Epigenetic Profiles of Periodontitis According to the Presence of Type 2 Diabetes

city: Yangsan

first: Hansong

last: Lee

address: 49 Busandaehak-ro

institute: Pusan National University

type: Methylation profiling by genome tiling array

summary: Type 2 diabetes mellitus (T2DM) and periodontitis (PD) have intricate connections as chronic inflammatory diseases. While the immune response is a key factor that accounts for their association, the underlying mechanisms remain unclear. To gain a deeper understanding of the connection, we conducted research using a multi-omics approach. We generated whole genome and EPIC array data from the periodontium of PD patients with DM (PDDM) and without DM to confirm genetic and epigenetic changes. Independent bulk and single-cell RNA sequencing data were employed to verify the expression levels of hypo-methylated genes. We observed a gradual rise in C>T base substitutions and hypomethylation in PD and PDDM patients compared to healthy participants. Furthermore, specific genetic and epigenetic alterations were prominently associated with the Fc gamma receptor-mediated phagocytosis pathway. The upregulation of these genes was confirmed in both the periodontal tissues of PD patients and the pancreatic tissues of T2DM patients. Through single-cell RNA analysis of peripheral blood mononuclear cells, substantial upregulation of Fc gamma receptors and related genes was particularly identified in monocytes. Our findings suggest that targeting the Fc-gamma signaling pathway in monocytes holds promise as a potential treatment strategy for managing systemic complications associated with diabetes.

overall_design: We aimed to profile methylome of gigival tissue from PD and PDDM patients.

first: Junho

last: Kang

first: Ji-Young

last: Joo

first: Jae-Min

last: Song

first: Hyun-Joo

last: Kim

first: Yun

first: Hae

last: Park


title: ESR1 and p53 Interactome Defines Mechanisms of Therapeutic Response to Tamoxifen Therapy in Luminal Breast Cancer Patients

status: Public on Jun 28 2024

city: BUFFALO

first: Eduardo

last: Cortes

state: NY

address: ELM AND CARLTON STREETS

department: Biostatistics and Bioinformatics

country: USA

institute: ROSWELL PARK CANCER INSTITUTE

type: Expression profiling by high throughput sequencing

summary: The canonical mechanism behind tamoxifen (Tam)’s therapeutic effect on estrogen receptor  /ESR1+ breast cancers is inhibition of ESR1-dependent estrogen signaling. Although ESR1+ tumors expressing wild-type p53 were reported to be more responsive to Tam therapy, p53 has not been factored into choice of this therapy and the mechanism underlying the role of p53 in Tam response remain unclear. In a window of opportunity trial on patients with newly diagnosed stage I-III ESR1+/HER2/ wild-type p53 –breast cancer who were randomized to arms with or without Tam prior to surgery, we reveal that the ESR1-p53 interaction in tumors was inhibited by Tam. This resulted in functional reactivation of p53 leading to transcriptional reprogramming that favors tumor suppressive signaling, as well as downregulation of oncogenic pathways. These findings illustrating the convergence of ESR1 and p53 signaling during Tam therapy contribute to mechanistic understanding of the impact of p53 on the response to Tam therapy.

overall_design: To determine the effect of p53 reactivation by tamoxifen on reprogramming of gene expression in ESR1+/wild-type p53 human breast tumors for the primary purpose of identifying alternative mechanisms that can be targeted for future therapeutic interventions and patient stratification, women with ESR1+ breast cancer were randomized to receive 20 mg of Tam PO daily for 28 days or no intervention between diagnosis and surgery. Transcriptome of surgically resected tumors was analyzed by RNA-Seq.  Differential gene expression in tumors from patients with or without tamoxifen intervention was determined from RNA-Seq data.

first: Gokul

last: Das

first: Swati

last: Kulkarni


title: Acanthamoeba castellanii as a model for unveiling Campylobacter jejuni host-pathogen dynamics

city: LONDON

first: Fauzy

last: Nasher

state: Please select

address: London School Of Hygiene & Tropical Medicine, Keppel Street

country: United Kingdom

institute: London School of Htgiene and Tropical Medicine

summary: The persistence of Campylobacter jejuni in the natural environment, despite its microaerophilic nature, continues to be a subject of active investigation.  Its survival and persistence likely stem from a combination of factors, including interactions with amoebae. C. jejuni transiently interacts with Acanthamoebae and this is thought to provide protection against unfavourable conditions and prime the bacteria for interactions with warm-blooded hosts. Acanthamoebae play vital roles in ecosystems by preying on microorganisms and establishing ecological niches with various bacterial species, some of which are clinically important pathogens. To gain a better understanding on the role of Acanthamoebae in the persistence of C. jejuni in the natural environment, we examined their interactions at the molecular level.

overall_design: RNA was extracted using Qiagen RNA extraction kit after treatment with RNA protect. Samples were subjected to quality control and quantification using Agilent Bioanayzer before being sent for processing and sequencing by Azenta (Genewiz). The paired-end reads were trimmed and filtered using Conda; Kallisto was used to map the reads against the reference sequence, Genome assembly Acastellanii.strNEFF v1 GCA_000313135.1. Kallisto was also used to convert annotations from GFF to GTF format. Library was sequenced by Genewiz (Azenta)- Performer using NovaSeq 6000 system.The paired-end reads were trimmed and filtered using Conda; Kallisto was used to map the reads against the reference sequence, Genome assembly Acastellanii.strNEFF v1 GCA_000313135.1. Kallisto was also used to convert annotations from GFF to GTF format. Statistical analysis was performed in R using the combined data generated from the bioinformatics as well as metadata associated with the study (multifactorial design). Adjusted p-value significance cut-off of <0.05, and log fold change cut-off of >2, was used for multiple comparison.

first: Burhan

last: Lehri


title: Comprehensive single cell transcriptomics analysis of murine osteosarcoma uncovers Skp2 function in metastasis, genomic instability and immune activation and reveals additional target pathways

city: The Bronx

first: Alexander

last: Ferrena

address: 1301 Morris Park Ave

department: Genetics

institute: Albert Einstein College of Medicine

summary: Osteosarcoma (OS) is the most common primary pediatric bone malignancy. One promising new therapeutic target is SKP2, encoding a substrate recognition factor of the SCF E3 ubiquitin ligase responsible for ubiquitination and proteasome degradation of substrate p27, thus driving cellular proliferation. We have shown previously that knockout of Skp2 in an immunocompetent transgenic mouse model of OS improved survival, drove apoptosis, and induced tumor inflammation. Here, we applied single-cell RNA-sequencing (scRNA-seq) to study primary OS tumors derived from Osx-Cre driven conditional knockout of Rb1 and Trp53. We showed that murine OS models recapitulate the tumor heterogeneity and microenvironment complexity observed in patient tumors.  We further compared this model with OS models with functional disruption of Skp2: one with Skp2 knockout and the other with the Skp2-p27 interaction disrupted (resulting in p27 overexpression). We found reduction of T cell exhaustion and upregulation of interferon activation, along with evidence of replicative and endoplasmic reticulum-related stress in the Skp2 disruption models, and showed that interferon induction was correlated with improved survival in OS patients. Additionally, our scRNA-seq analysis uncovered decreased activities of metastasis-related gene signatures in the Skp2-modulated OS, which we validated by observation of a strong reduction in lung metastasis in the Skp2 knockout mice. Finally, we report several potential mechanisms of escape from targeting Skp2 in OS, including upregulation of Myc targets, DNA copy number amplification and overexpression of alternative E3 ligase genes, and potential alternative lineage activation. These mechanistic insights into OS tumor biology and Skp2 function suggest novel targets for new, synergistic therapies, while the data and our comprehensive analysis may serve as a public resource for further big data-driven OS research.

overall_design: One tumor each was isolated from 4 TKO, 3 DKOAA, and 3 DKO animals for scRNAseq.

first: Deyou

last: Zheng


title: ADRA2A is a suppressor of the basal-like/squamous subtype and reduces disease aggressiveness of pancreatic cancer

city: Bethesda

first: Stefan

last: Ambs

state: MD

address: Building 37, Room 3050B

department: CCR

institute: NCI

summary: Pancreatic cancer is a heterogeneous disease and consists of distinct subtypes. Here, we investigated candidate suppressor genes of the basal-like/squamous subtype, a more aggressive molecular subtype of pancreatic ductal adenocarcinoma (PDAC). Through an integrated transcriptome analysis, we identified the ADRA2A, as being downregulated in the basal-like/squamous PDAC using a discovery and validation approach. ADRA2A downregulation is associated with decreased patient survival. The goal of this study was to identify ADRA2A-induced molecular signatures in PDAC.

overall_design: RNA was isolated from human PDAC cell lines (CFPAC-1 with empty vector control; n = 4, CFPAC-1 with ADRA2A transgene overexpression; n = 4) using TRIzol method. The mRNA profiles of the human PDAC cell lines were generated by deep sequencing using Illumina NextSeq.

first: Paloma

last: Moreno

first: Yuuki

last: Ohara

first: Amanda

middle: J

last: Craig

first: Huaitian

last: Liu

first: Shouhui

last: Yang

first: Lin

last: Zhang

first: Gatikrushna

last: Panigrahi

first: Tiffany

middle: H

last: Dorsey

first: Helen

last: Cawley

first: Azadeh

last: Azizian

first: Jochen

last: Gaedcke

first: Michael

last: Ghadimi

first: Nader

last: Hanna

first: S

middle: P

last: Hussain


title: Microglial apolipoprotein E particles contribute to neuronal senescence and synaptotoxicity

city: Chongqing

first: Hongsheng

state: Chongqing

address: No.1, Yixuanyuan Road, Chongqing

country: China

institute: Chongqing Medical University

summary: Apolipoprotein E (apoE) plays a pivotal role in the pathogenesis of Alzheimer’s disease (AD). In the brain, apoE is predominantly expressed and secreted by astrocytes, but is dramatically up-regulated in microglia under AD-associated conditions. Although the function of astrocytic apoE has been widely investigated, whether and how apoE particles derived from different types of glia differ in biological features and function remains elusive. Here, we show that apoE particles derived from astrocytes and microglia exhibit dissimilar sizes. Microglial apoE particles impaired neurite growth and synapses and promoted neuronal senescence, whereas GPNMB-deficient microglial apoE particles abolished these detrimental effects. These findings provide direct evidence supporting that microglia-derived apoE particles contribute to neuronal senescence and toxicity.

overall_design: To explore how apoE particles affect transcriptome of neurons, we treated primary neurons with fresh neurobasal medium containing B27 and penicillin-streptomycin or apoE particle for 24h. We then performed gene expression profiling analysis using data obtained from RNA-Seq of all treatments.

last: Wang

first: Lujian

last: Cai

first: Yingjun

last: Zhao


title: Molecular mechanisms underlying the modulation of T-cell proliferation and cytotoxicity by immobilized CCL21 and ICAM1

city: Rehovot

first: Benjamin

last: Geiger

address: POB 26

department: Department of Immunology

country: Israel

institute: Weizmann Institute of Science

summary: A major challenge in adoptive cancer immunotherapy is the effective balance between the proliferation and cytotoxic capacity of the effector CD8+ T-cells. In this study we explored the molecular mechanisms whereby a “synthetic immune niche” (SIN), composed of immobilized CCL21 and ICAM1, modulates the interplay between these two cellular properties. Temporal phenotypic profiling of OVA-specific murine T-cells revealed major differences in the response to the SIN stimulation following antigen-specific and non-specific activation. Thus, SIN treatment enhanced both the expansion and cytotoxicity of cells activated by OVA-loaded DCs, at day 4, while, similar treatment of cells activated by anti CD3/CD28-conjugated beads dramatically enhanced the cells’ expansion, but unexpectedly blocked their cytotoxicity, which fully recovered only on day 7. Molecular profiling suggest that upregulation of cytotoxic gene signatures drive the SIN effect following DC activation, while down-regulation of exhaustion and pro-apoptotic genes optimize the proliferation-cytotoxicity balance in the beads-activated cells.

overall_design: In the current project, we explored the cellular and molecular mechanisms underlying the differential effects of the CCL21+ICAM1 SIN treatment on cytotoxicity and proliferation of DC and beads-activated cells. Towards that end, we have performed multiparameter immunophenotyping and molecular profiling of untreated or SIN-treated CD8+ T-cells.

first: Sofi

last: Yado

first: Benny


title: Whole transcriptome analysis of murine endothelial cells after treatment with or without nattokinase

city: Taipei

first: Hui-Wen

last: Chiu

address: No.301, Yuantong Rd., Zhonghe Dist., New Taipei City

country: Taiwan

institute: Taipei medical university

summary: Natto is a traditional Japanese food from soybeans fermented with Bacillus subtilis natto. Natto contains a powerful fibrinolytic enzyme called nattokinase (NK), which has thrombolytic, anti-hypertensive, anti-atherosclerotic and lipid-lowering effects. This project aims to study the effects of NK in vascular inflammation.

overall_design: RNA sequencing of murine endothelial cell line (SVEC4-10 cells) after treatment with nattokinase for 24 hours.

first: Yu-Jhe

first: Li-Chin

last: Sung


title: K562-U2AF1_S34F treated with U2AF1-UHM inhibitor, SF1-8

city: Memphis

first: Chao-Yie

last: Yang

state: Tennessee

address: 881 Madison Avenue, Rm559

department: Pharmaceutical Sciences

institute: University of Tennessee HSC

summary: Mutations in RNA splicing factor genes including SF3B1, U2AF1, SRSF2, and ZRSR2 have been reported to contribute to development of myeloid neoplasms including myelodysplastic syndrome (MDS) and secondary acute myeloid leukemia (sAML). Chemical tools targeting cells carrying these mutant genes remain limited and underdeveloped. Among the four proteins, mutant U2AF1 (U2AF1mut) acquires an altered 3’ splice site selection preference and co-operates with the wild-type U2AF1 (U2AF1wt) to change various gene isoform patterns to support MDS cells survival and proliferation. U2AF1 mutations in MDS cells are always heterozygous and the cell viability is reduced when exposed to additional insult affecting U2AF1wt function. To investigate if the pharmacological inhibition of U2AF1wt function can provoke drug-induced vulnerability of cells harboring U2AF1mut, we conducted a fragment-based library screening campaign to discover compounds targeting the U2AF homology domain (UHM) in U2AF1 that is required for the formation of the U2AF1/U2AF2 complex to define the 3' splice site. The most promising hit (SF1-8) selectively inhibited growth of leukemia cell lines overexpressingU2AF1mut and human primary MDS cells carrying U2AF1mut. RNA-seq analysis of K562-U2AF1mut following treatment with SF1-8 further revealed alteration of isoform patterns for a set of proteins that impair or rescue pathways associated with endocytosis, intracellular vesicle transport, and secretion. Our data suggested that further optimization of SF1-8 is warranted to obtain chemical probes that can be used to evaluate the therapeutic concept of inducing lethality to U2AF1mut cells by inhibiting the U2AF1wt protein.

overall_design: To investigate the impact of SF1-8 on the genes and transcripts expression in K562 overexpressed with U2AF1-S34F mutant.

first: Valeria

last: Visconte


title: NOTCH3 Blockade Inhibits Cancer Cell Plasticity through a NOTCH3/PD-L1 Oncogenic Axis in Triple Negative Breast Cancer

city: Rochester

first: Xiaojia

last: Tang

state: MN

address: 200 1st ST SW

institute: Mayo Clinic

summary: Triple negative breast cancer (TNBC) accounts for 15-20% of all breast cancers and mainly affects pre-menopausal and minority women. Therapeutic options are yet limited, in part because of acquisition of tumor cells with cancer stem cell (CSC) properties, leading to intrinsic resistance to chemotherapeutic agents and immune evasion capacity. Dysregulated Notch signaling has been linked to breast cancer and in this study, we demonstrate the pivotal role of NOTCH3 in inducing the enrichment of chemoresistant ALDHhigh CSCs. Significantly, selective targeting of NOTCH3 with AV-353, a novel humanized monoclonal antibody, or by shRNA-mediated knockdown, impaired the enrichment of ALDHhigh CSCs with high self-renewal capacity, induced apoptosis and enhanced chemosensitivity. AV-353 treatment similarly reduced growth of xenografted tumors in vivo. Moreover, blocking  NOTCH3 by AV-353 or shRNA reduced PD-L1 expression in TNBC cells, which  was linked to the inhibition of tumor growth and enhancement of CD8+ T-cell infiltration. Taken together, these findings provide preclinical evidence for NOTCH3 as a novel therapeutic target in TNBC and reveal a novel NOTCH3/PD-L1 oncogenic axis as a platform for the betterment of TNBC therapy.

overall_design: Briefly, several breast cancer cell lines were subjected to different treatments, i.e. lentiviral mediated knockdown of Notch3, PD-L1 or treatment with a N3 blocking antibody. Untreated samples serves as control. Samples were then further processed for RNA-sequencing. Transcriptomes of each condition have been recorded in three independent biological samples.

first: Ellie

last: Babadi

first: Mohammad

last: Jalalirad

first: Minzhi

last: Zhang

first: Eike-Benjamin

last: Braune

first: Esther

last: Rodman

first: Likhita

last: Shaik

first: Roberto

middle: A

last: Leon-Ferre

first: Jann

middle: N

last: Sarkaria

first: Ann

middle: C

last: Tuma

first: Jodi

last: Carter

first: Royal

last: Shrestha

first: Krishna

last: Kalari

first: Andrew

last: Takchi

first: Lucio

last: Miele

first: Alvaro

last: Pena

first: Haidong

last: Dong

first: John

last: Hawse

first: Minetta

last: Liu

first: Evanthia

last: Galanis

first: Liewei

first: Jia

last: Yu

first: Carol

last: Lange

first: Tufia

last: Haddad

first: Robert

middle: B

last: Diasio

first: Larry

middle: R

last: Pease

first: James

last: Ingle

first: Matthew

last: Goetz

first: Urban

last: Lendahl

first: Antonino

last: D’Assoro


title: RNA-Seq of drug-treated and control 4T1 cells

city: xi'an

first: Wei

last: Gaofei

state: Shaanxi

address: beilin

institute: Northwestern Polytechnical University

summary: Cell derived xenografts treated with DMSO, Oxa, APAP or OAP2 were collected and then the total RNA was isolated. After removal of the remaining genomic DNA, mRNA was purified from total RNA using polyT and then fragmented with 10× RNA fragmentation buffer and the RNA-seq library was constructed using Hieff NGS Ultima Dual-mode mRNA Library Prep Kit and was sequenced.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for OAP2 treated cell derived xenograft and other drugs treated cell derived xenograft

first: Renming

last: Fan

first: Gaofei

last: Wei


title: Transcriptomics of pathogen Cronobacter turicensis z3032 in lysogenic broth

city: Zürich

first: Marc

last: Stevens

address: Winterthurerstrasse 272

department: Vetsuisse

country: Switzerland

institute: University of Zurich

summary: Cronobacter (C.) is an important emerging opportunistic foodborne pathogen representing  significant cause of mortality in neonatal patients with bacteremia and meningitis.  Knowledge on the pathobiology of Cronobacter mediated meningitis has to a large extend been explored using in vitro models. To explore the innate immune response against the neonatal sepsis/meningitis causing isolate C. turicensis z3032 in vivo, zebrafish larvae (Danio rerio) were used as infection model. Following establishment of infection in zebrafish larvae with z3032, dual RNA-sequencing of host-pathogen was undertaken to profile RNA expression simultaneously in the pathogen and the head region of the zebrafish host.

overall_design: returned to a petri dish with fresh embryo medium and incubated at 28 °C until development of typical signs of disease such as axis curvature and necrosis which was observed at approx. 36 h dpi. 60 randomly chosen zebrafish larvae from each group (infected, mock-infected) were euthanized by a prolonged immersion in an overdose of 50 mg/L Tricaine solution, followed by decapitation with a sharp blade, their heads collected and transferred into 1.5ml Eppendorf tubes Excess E3 medium was removed and  replaced by RNAlater (Ambion) and stored at 4°C until further processing. For the extraction of total RNA, RNAlater was replaced by 600 μl of Qiazol, and the tissue was homogenized using a pestle motor followed by drawing of sample with a needle until the tissue was completely homogenized. As a control, RNA was isolated from z3032 cultures grown in LB to stationary phase (18 – 24 h). For both the zebrafish and bacterial culture, RNA extractions were performed using the miRNeasy Mini kit (Qiagen) according to the manufacturer’s protocols. An additional DNase treatment was performed with a TURBO DNA-free kit (Ambion) according to the manufacturer’s protocols. The concentration of the extracted RNA was measured using a NanoDrop Spectrophotometer. The total RNA samples were subjected to ribosomal RNA (rRNA) depletion, zebrafish samples mock-infected with PBS were treated with Ribo-Zero Gold rRNA Removal Kit (Human/Mouse/Rat) (Illumina), zebrafish infected with C. turicensis were treated with Ribo-Zero rRNA Removal Kit (Gram-negative Bacteria) (Illumina) followed by Ribo-Zero Gold rRNA Removal Kit (Human/Mouse/Rat) and the C. turicensis sample grown in LB was treated with Ribo-Zero rRNA Removal Kit (Gram-negative Bacteria). The depletion steps and subsequent 125 bp paired-end RNA Sequencing on a HiSeq2500 (Illumina) were performed at the Functional Genomic Center at University Zurich (FGCZ), Zurich, Switzerland.

first: Chiara

last: Cosentino

first: Athmanya

middle: K

last: Eshwar

first: Francis

last: Muchaamba

first: Claudia

last: Guldimann

first: Roger

last: Stephan

first: Angelika

last: Lehner


title: Small RNA signatures of acute ischemic stroke in L1CAM positive extracellular vesicles

city: San Francsico

first: Neel

middle: S

last: Singhal

state: CA

address: 1700 Owens St., Receiving Room 349

department: Neurology

institute: UCSF

type: Non-coding RNA profiling by high throughput sequencing

summary: L1CAM-positive extracellular vesicles (L1EV) are an emerging biomarker that may better reflect ongoing neuronal damage than other blood-based biomarkers. The physiological roles and regulation of L1EVs and their small RNA cargoes following stroke is unknown. We sought to characterize L1EV small RNAs following stroke and assess L1EV RNA signatures for diagnosing stroke using weighted gene co-expression network analysis and random forest (RF) machine learning algorithms. Interestingly, small RNA sequencing of plasma L1EVs from patients with stroke and control patients (n = 28) identified micro(mi)RNAs known to be enriched in the brain. Weighted gene co-expression network analysis (WGCNA) revealed small RNA transcript modules correlated to diagnosis, initial NIH stroke scale, and age. L1EV-derived RNA signatures associated with the diagnosis of AIS were derived from WGCNA and RF classification. These small RNA signatures demonstrated a high degree of accuracy in the diagnosis of AIS with an area under the curve (AUC) of the signatures ranging from 0.833- 0.932. Further work is necessary to understand the role of small RNA L1EV cargoes in the response to brain injury, however, this study supports the utility of L1EV small RNA signatures as a biomarker of stroke.

overall_design: Plasma from humans with acute ischemic stroke within 24 hours or control patients with cardiovascular risk factors was drawn and frozen. Extracellular vesicles (EVs) were isolated from 250 ul of frozen plasma and subjected to co-immunoprecipitation with L1CAM. L1CAM- EVs were lysed and small RNA isolated (Paris mirvana kit, invitrogen) and sequenced on  DNBSeq pipeline (BGI Americas). Reads were aligned with human genome (v38) and small RNA counts generated by the MANATEE pipeline(Handzlik et al. 2020). Data were analyzed with respect to clinical annotations including diagnosis, sex, clinical severity, and comorbidities.

first: Fanny

last: Elahi

first: Bharti

last: Manwani


title: Global gene expression profiling of organoids with various morphologies generated in cerebral organoid differentiation

city: Kyoto

first: Megumi

last: Ikeda

address: 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto, JAPAN

department: Department of Clinical Application

country: Japan

institute: Center for iPS Cell Research and Application, Kyoto University

summary: To characterize organoids with each type of morphology, we performed DNA microarray analysis to identify genes expressed predominantly in each type of organoids on day35 of cortical organoid differentiation protocol (SFBEq).

overall_design: To generate cortical organoids, we employed the differentiation method previously described by Kitahara et al., Stem Cell Reports, 2020. Human induced pluripotent stem cells are cultured in DMEM/F-12 GlutaMAX supplemented with 20% (v/v) KnockOut Serum Replacement (KSR), 5 µM SB431542, and 3 µM IWR1e, using low adheshion 96 well plates.Defining the day on which floating culture of cell aggregates was started as day0, half the medium was changed once every three days until day 15. On day 18, the aggregated cells were transferred to 90-mm non-coated dishes and further cultured in DMEM/F-12 GlutaMAX supplemented with 1% (v/v) N-2 Supplement, 1% (v/v) Chemically Defined Lipid Concentrate (CDLC), 0.25 µg/ml Amphotericin B , 100 U/ml penicillin, and 100 µg/ml streptomycin until day 35. Organoids were cultured on an orbital shaker and maintained at 50 rpm in 20% O2 and 5% CO2 to eliminate the need for cell culture in 40% O2. Complete medium changes were performed once every 3 or 4 days from day 18 to day 35. On day35, partial structures in organoids were classified into 7 groups according to their morphologies. (1) Rosettes, organoids with rosette structures throughout; (2) Potato-like”, those with low transparency and no clear internal structures; (3) Balloon-like, those with balloon-like cystic structures; (4) Cotton-like, organoids with fibrous epithelial-like structures; (5) Transparent, transparent organoids and cyst-like internal structures; (6) Jelly-like, organoids with a transparent periphery and no clear internal structures; and (7) Pigmented, organoids with pigmentation.The tissues with distinct morphological features were isolated by dissection and subjected to microarray analysis. For each morphological groups, 3 samples are analyzed.

first: M

first: J

last: Takahashi


title: Molecular Insights into the Inhibition of Proton-Activated Chloride Channel by Transfer RNA

city: Chengdu

first: pengliang

last: chi

state: Sichuan province

address: No.17 People's South Road

institute: Sichuan University

summary: The proton-activated chloride (PAC) channel (also known as acid-sensitive outwardly rectifying anion channel, ASOR) plays a critical role in acid-induced cell death and endocytosis. However, little is known about the regulatory factors and binding partners of PAC. In this study, we discovered that transfer RNA (tRNA) interacts directly with PAC as an unexpected binding partner. Using cryo-electron microscopy, we determined that two PAC trimers and one co-purified tRNA molecule form a stable complex via a highly conserved KR motif, representing the closed conformation. tRNA is located on the intracellular side of PAC, blocking the channel pores. Furthermore, electrophysiological data showed that tRNA modulates chloride currents and channel open probability of PAC, thus protecting against acid-induced cell death. Our study provides insight into the regulation of PAC activity by cytosolic tRNA and extends the role of tRNAs in pathological and physiological events.

sample_organism:
Spodoptera frugiperda
Homo sapiens

platform_organism:
Spodoptera frugiperda
Homo sapiens

overall_design: RNA profiles of the RNAs co-purified with Xenopus tropicalis PAC (xPAC) and Homo sapiens PAC (hPAC), respectively.

first: Pengliang

last: Chi

first: Xiang

last: Wang

first: Jialu

last: Li

first: Hui

last: Yang

first: Kaiju

first: Yuqi

last: Zhang

first: Shiyi

last: Lin

first: Leiye

last: Yu

first: Shiqi

last: Liu

first: Lu

last: Chen

first: Ruobing

last: Ren

first: Jianping

last: Wu

first: Zhuo

last: Huang

first: Jia

last: Geng

first: Dong

last: Deng


title: Small RNA-seq analysis of exosomes derived from VZV-infected neurons

city: Burlington

first: Seth

last: Frietze

state: VT

address: 149 Beaumont Avenue

department: Biomedical and Health Sciences

institute: University of Vermont

summary: Exosomes have emerged as essential extracellular signaling vesicles that can deliver proteins and nucleic acids to cells during normal and disease states. Herein, we studied the effects of exosomes produced by VZV-infected human sensory neurons on cerebrovascular cells. Exosomes were isolated from mock- and VZV-infected sensory neurons and protein and nucleic acid was determined by mass spectrometry and Next-Generation Sequencing. Subsequently, mock- and VZV-derived exosomes were applied to primary human brain vascular adventitial fibroblasts (HBVAFs). Overall, exosomes from VZV-infected sensory neurons impacted the pro-inflammatory phenotype of HBVAF cells. The presence of pathogenic exosomes that could circulate throughout the body provides insight into clinical infections.

overall_design: Exosomes derived from VZV- or MOCK-infected HD10 neurons were isolated in triplicate per condition and subject to small RNA-seq analysis.

first: Sophie

last: Kogut


title: Col2a1-Cre;Idh1LSL/+ embryonic growth plate chondrocytes - single cell RNA-sequencing

city: Madison

first: jianhong

last: ou

state: WI

address: 330 N Orchard Street

institute: Morgridge institute for research

summary: To determine differences between Idh1 mutant and control growth plates, we used scRNA sequencing. Three E18.5 growth plates from our Idh1 mutant mice and three control mice were studied. Differential gene expression analysis revealed genes that were expressed in the distinct clusters.

overall_design:
In order to assess the Idh1 mutant expressing chondrocytes, we utilized Collagen2a1-Cre males crossed with Idh1 LSL/+ females. Growth plate chondrocytes from E18.5 distal part of femur and proximal part of tibia were collected and chondrocytes were harvested using Pronase I digestion followed by Collagenase IV. Live cells were sorted and GEM were made using 10xgenomics kit. After sequencing and downstream analysis using Seurat, we observed distinct clusters of cells with Idh1 mutation
10X Genomics Single cell RNA-sequencing of growth plate chondrocytes

first: Vijitha

last: Puviindran

first: Ze

middle: Y

first: Yu

last: Xiang

first: Hongyuan

first: Ga

middle: I

last: Ban

first: Jianhong

last: Ou

first: Xiaolin

last: Wei

first: Makoto

last: Nakagawa

first: Xinyi

last: Ma

first: John

last: Martin

first: Yarui

last: Diao

first: Benjamin

last: Alman


title: Influence of GSK3 inhibition and beta Catenin stabilisation on the transcriptome of pre B cells

city: Munich

first: Julia

last: Jellusova

department: Klinikum rechts der Isar

country: Germany

institute: Technical University Munich

summary: The goal of this study was to determine how GSK3 inhibition/beta Catenin stabilisation affects the transcription profile of pre B cells

overall_design: B cell precursors were obtained from hematopoietic stem cell cultures of control or Mb1crex Catnb lox(ex3) mice as described above and sorted based on B220 expression by flow cytometry, after sorting cells were directly processed for RNA isolation. For experiments in which GSK3 was inhibited, B cell precursors were treated with LY2090314 over night and directly processed for RNA isolation without prior sorting. RNA was extracted using Quick-RNA MicroPrep (ZYMO RESEARCH) according to the manufacturer’s instructions. The RNA concentration in RNAse free H2O was analyzed by measuring the absorption at 260 / 280 nm with a NanoDrop (Thermo Scientific/Peqlab). Additionally, unsorted wildtype B cell precursors were incubated with or without 40nM LY2090314 (Sigma) over night and RNA was isolated. RNA was snap frozen in liquid nitrogen, samples were processed and the transcriptome was analyzed by Novogene company.

first: Huda

last: Jumaa


title: Study on the mechanism of TSC22D3 regulating the stability of P53 to promote the quiescence of hematopoietic stem cells

city: Guangzhou

first: Tian

last: Zhang

state: Guangdong

address: 190 Kaiyuan Avenue,Science Park

institute: GIBH

summary: In adults, human hematopoietic stem cells (HSCs) are mostly in a quiescent state (G0 stage) and show multilineage reconstruction. When the cell cycle is activated, HSCs gradually lose the hematopoietic reconstruction ability. It indicates that the maintenance of the quiescent state of HSPCs has very important physiological significance. However, the maintenance and regulation mechanism of the quiescent state of HSCs has not yet been clarified. We found that TSC22D3 was highly enriched in quiescent state HSCs but downregulated in culture with the activation of the cell cycle. We further confirmed that stabilizing TSC22D3 expression in HSCs could promote the maintenance of the quiescent state and provide balanced multilineage reconstitution in primary mouse recipients.We found that overexpression of TSC22D3 can promote the maintenance of the resting state of HSCs and the ability of hematopoietic reconstitution by inhibiting the expression of cell cycle-related kinases. Also we confirmed that TSC22D3 can promote the accumulation of p53 protein in cells and up-regulate the expression of p21, p27 and other proteins by qPCR and western blot. Therefore, we speculate that TSC22D3 can promote the resting state of HSCs by regulating the stability of p53. It remains elusive that how TSC22D3 plays a role in the regulation of the maintenance of HSCs in vitro, therefore, further investigations are required to depict its underlying mechanism. On this basis, We firstly plan to analyze the potential downstream targets of TSC22D3 in the maintenance of HSCs in vitro through RNA-seq based transcriptome comparison; then, we will verify the predicted targets of TSC22D3 through Co-IP, gene overexpression, gene knockdown, and signaling pathway activation/inhibition. Our project could contribute to an in-depth understanding of the quiescent maintenance mechanisms of HSPCs in vitro and provide ideas for the treatment of leukemia and other malignant hematological diseases.

overall_design: We compared the transcriptome of the hematopoitic stem cells with or with culture. Also, we compared the transcriptome of  the eGFP+ and  TSC22D3/eGFP- hematopoietic stem cells by RNA-Seq.

first: Guangjin

last: Pan

first: Ke

last: Huang


title: Bulk RNA sequence and bulk ATAC sequence of treated murine CD8+ T cells

city: Chicago

first: Bin

state: Illinois

address: 300E superior street, Tarry 4-726

institute: Northwestern University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: We identify that GSH maintains the function of CD8+ T cell and GSH metabolism is interacting with A2AR signaling pathway to reshape the metabolism and anti-function in CD8+ T cell. We found A2AR signaling blockade leads to the upregulation of GSH metabolism related genes and loss of the genes abolishes the benefits from A2AR antagonist on CD8+ T cells. Considering to the essential role of GSH metabolism in ferroptosis, we combined the potent ferroptosis inhibitor liproxstatin-1 (Lip-1) and A2AR antagonist (SCH58261) to treat CD8+ T cells. Notably, our combination therapy significantly promotes the anti-tumor immunity of CD8+ T cells with delayed tumor growth in tumor bearing mice.

overall_design: Examination of gene profile in CD8+ T cells from tumor bearing mice treated with or without anti-CD73; or from in vitro 7-days culture with SCH58261 (A2ARi), Lip-1 or A2ARi/Lip-1 treatment

first: Siqi

last: Chen


title: HBVAF cells treated with exosomes from VZV-infected neurons

first: Seth

last: Frietze

overall_design: Exosomes derived from VZV- or MOCK-infected HD10 neurons were isolated and used to treat human brain adventitial fibroblasts (HBVAF) for X days in triplicate per condition. RNA was extracted and used to construct RNA-seq libraries.

first: Sophie

last: Kogut


title: Col2a1-Cre;Idh1LSL/+ embryonic growth plate chondrocytes

first: jianhong

last: ou

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

overall_design: Refer to individual Series


title: Col2a1-Cre;Idh1LSL/+ embryonic growth plate  - Bulk RNA-sequencing

summary: As another method to determine differences between Idh1 mutant and control growth plates, we used Bulk RNA sequencing.  Three E18.5 growth plates from our Idh1 mutant mice and three control mice were studied.  Differential gene expression analysis revealed genes that were  expressed in the distinct clusters, further supporting our single cell RNA data.

overall_design:
In order to assess the Idh1 mutant expressing chondrocytes, we utilized Collagen2a1-Cre males crossed with Idh1 LSL/+ females. Growth plate chondrocytes from E18.5 distal part of femur and proximal part of tibia were collected and snap frozen. Total RNA was isolated and used for RNA-seq analysis.  After sequencing and downstream analysis using Seurat, we observed distinct clusters of cells with Idh1 mutation
Total RNA was isolated using Single Cell RNA Purification Kit and submitted to core facitly for library generation and sequencing

first: Vijitha

last: Puviindran

first: Ze

first: Yu

last: Xiang

first: Hongyuan

first: Ga

last: Ban

first: Jianhong


title: Activation of alveolar epithelial ER stress by b-coronavirus infection disrupts surfactant homeostasis in mice: implications for COVID-19 respiratory failure

status: Public on Jun 27 2024

city: Philadelphia

first: Michael

last: Beers

state: PA

address: 3450 Hamilton Walk

institute: University of Pennsylvania

summary: Covid-19 syndrome is characterized by an acute lung injury phenotype marked by hypoxemic respiratory failure and high mortality. Alveolar Type 2 (AT2) cells are essential for gas exchange, repair, and regeneration of distal lung epithelium. We have shown that the causative agent, SARS-CoV-2 and other members of the b coronavirus genus induces an ER stress response in vitro however the consequences for host AT2 cell function in vivo are less understood.  To study this, two murine models of coronavirus infection were employed– mouse hepatitis virus-1 (MHV-1) in A/J mice and a C57BL6/j adapted SARS-CoV-2 strain.  MHV-1 infected mice exhibited dose-dependent weight loss through Day 8 (8dpi) with histological evidence of distal lung injury accompanied by elevated BALF cell counts and total protein.  AT2 cells isolated at 4dpi and 8dpi showed evidence of both viral infection and increases in Bip /GRP78 expression, consistent with activation of the unfolded protein response (UPR).  AT2 UPR signaling included elevations in spliced XBP-1 indicating activation of IRE1a signaling as well as a biphasic response in PERK signaling mark by phosphorylated eIF2a protein levels and expression of Atf4, Ppp1r15a, and Ddit3. Viral UPR activation was accompanied by marked reductions in AT2 and BALF surfactant protein (SP-B, SP-C) content, increases in surfactant surface tension, and emergence of a re-programmed epithelial cell population (Krt8+, Cldn4+, and Gdf15+) which was attenuated by treatment with the IRE1a inhibitor OPK711.  As proof-of-concept, C57BL6 mice infected with mouse-adapted SARS-CoV-2 demonstrated similar lung injury and evidence of disrupted surfactant homeostasis.  We conclude that hypoxic respiratory failure from b-coronavirus infection results from an aberrant AT2 cell host response activating multiple ER stress pathways, altering surfactant metabolism / function, and changing AT2 endophenotypes offering a mechanistic link between SARS-CoV-2 infection, AT2 cell biology, and hypoxemic respiratory failure.

overall_design: To determine if b-coronavirus infection disrupted functional and phenotypic characteristics of alveolar type 2 (AT2) cells we isolated AT2 and transitional AT2 cells by FACS from lungs of control (n=3)  and 4dpi (n=4) MHV1 infected mice. Lungs were harvested from control and 4 day infected mice, single cells suspension was prepared by mehcanical and enzymatic digetsion.  CD45+ and CD31+ cells were depleted using biotinylated antibodies followed by surface staining with antibody mix to identify At2 and transitional At2 cells. At2 cells were identified as CD45-CD31-Epcam+CD104-MHCII hiCD51- and transitional At2 cells  CD45-CD31-Epcam+CD104-MHCII hiCD51+. Dead cells were excluded by Dapi staining.

first: Aditi

last: Murthy

first: Luis

last: Rodriguez

first: Thalia

last: Dimopoulos

first: Swati

last: Iyer

first: Katrina

last: Chavez

first: Yaniv

last: Tomer

first: Valsamma

last: Abraham

first: David

middle: M

last: Renner

first: Jeremy

last: Katzen

first: Ian

last: Bentley

first: Joshua

last: Englert

first: Susan

last: Weiss

middle: F


title: 25-Hydroxycholesterol inhibits hantavirus infection through reprogramming cholesterol metabolism and suggest statins as candidate hantavirus inhibitors

city: Xi'an

first: Yamei

last: Dang

address: No.169 Changle West Road, Xincheng District,

institute: Fourth Military Medical University

summary: Hantavirus causes two kinds of acute diseases; hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS), and it is still a major health concern due to high mortality and lack of effective treatment. Interferon is only effective to inhibit hantavirus infection at a very early stage. Several interferon-stimulated genes (ISGs) have been indicated to inhibit hantavirus infection. The cholesterol 25-hydroxylase (CH25H) has been indicated as an ISG, which encodes an enzyme that catalyzes the production of 25-hydroxycholesterol (25HC). 25HC plays an important role in regulating cholesterol biosynthesis and inhibits multiple enveloped virus infections. Here, we showed that Hantaan virus (HTNV), the prototype hantavirus, induces CH25H in infected cells. Overexpression of CH25H and treatment of its enzymatic product 25HC both inhibit HTNV infection, possibly through lowering 3-hydroxy-3-methyl-glutaryl coenzyme A reductase regulation (HMG-CoA reductase, HMGCR) that inhibit cholesterol synthesis. Additionally, cholesterol lowering drugs, HMGCR-targeting statins process potent hantavirus inhibition effects. Taken together, our results indicate that the 25HC and statins were potential antivirals against hantavirus infection.

overall_design: We performed gene expression profiling using data obtained from RNA-seq of Huh7 cells treated with EtOH vehicle or 5μM 25HC for 4 or 16 hours

first: Wei

last: Ye

first: Fanglin

last: Zhang


title: Controlling pericellular oxygen tension in cell culture reveals distinct breast cancer responses to low oxygen tensions

city: Boston

first: Zachary

last: Rogers

state: MA

address: 360 Huntington Avenue

department: Chemical Engineering

institute: Northeastern University

summary: In this study, the relationship between pericellular and gas phase oxygen concentration in cell culture was examined. To this end, we examined the influence of cellular oxygen consumption on primary human hepatocyte culture in physioxic (6% O2) and normoxic (18.6% O2) conditions. To understand how pericellular oxygen tension influenced hepatocyte biology in in vitro cell culture, we performed RNA-seq (this dataset).

overall_design: RNA-seq was performed on primary human hepatocytes from the vial (uncultured), and hepatocytes cultured for 36h in normoxia (normoxia) or physioxia (physioxia). More details on methodology can be found in the source publication (https://doi.org/10.1002/advs.202402557)


title: Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence II

city: Acton

last: Greaves

state: act

address: clunies ross street

country: Australia

institute: csiro

summary: Small RNAs (sRNAs) are essential for normal plant development and range in size classes of 21-24 nucleotides.  The 22nt small interfering RNAs (siRNAs) and miRNAs are processed by Dicer-like 2 (DCL2) and DCL1 respectively and can initiate secondary siRNA production from the target transcript amplifying the silencing signal in plants.  22nt siRNAs are under-represented due to competition with DCL4, while only a small number of 22nt miRNAs exist due to the rare occurrence of an asymmetric bulge in the precursor miRNA stem. Here we report a strategy to produce abundant 22nt siRNAs and other desired siRNA size classes using long hairpin RNA (hpRNA) transgenes.  By introducing asymmetric bulges periodically into the antisense strand of hpRNA, we successfully shifted the dominant siRNA size class from 21nt of the traditional hpRNA to 22, 23 and 24nt of the asymmetric hpRNAs.  We showed that the asymmetric hpRNA constructs effectively silenced a β-glucuronidase (GUS) reporter transgene and the endogenous ethylene insensitive-2 (EIN2) and chalcone synthase (CHS) genes. Furthermore, plants containing the asymmetric hpRNA transgenes targeting both GUS and EIN2 showed increased amount of 21nt siRNAs downstream of the hpRNA target site compared to plants with the traditional hpRNA transgenes.  This indicates that these asymmetric hpRNAs are more effective at inducing secondary siRNA production to amplify silencing signals. Consistent with the production of secondary siRNAs, the 22nt asymmetric hpRNA constructs increased gene silencing phenotypes and enhanced virus resistance in plants compared to the traditional hpRNA constructs.

overall_design:
To examine whether hpRNA transgenes containing asymmetric bulges could process different length siRNAs.
Transgenic Arabidopsis thaliana  were transformed with different asymmetric bulge hpRNAtransgenes (Δ22nt, Δ23nt, Δ24nt-1, Δ24nt-2, Δ24nt-3).  3 independent T2 lines were sent for sRNA sequencing per construct

first: Ming

last: Wang

first: Daai

first: Dengwei

last: Jue

first: Neil

last: Smith

first: Jean

last: Finnegan

first: Rob

last: Defeyter

first: Chengcheng

last: Zhong


title: KAT7 promotes malignant phenotypes of colorectal cancer via MRAS-mediated activation of the MAPK/ERK pathway

first: Zhongjie

last: Huang

address: No. 253, Middle Industrial Avenue

institute: Zhuujiang hospital

summary: Histone acetyltransferases (HATs) play a crucial role in transcriptional regulation by acetylating histones. Dysregulation of HAT activity is implicated in developmental disorders and cancer. In this study, we discovered an upregulated KAT7 signaling pathway in colorectal cancer (CRC) and its association with poor patient survival. Knockdown of KAT7 suppressed CRC cell viability, proliferation, migration, and invasion while promoting apoptosis. Conversely, KAT7 overexpression enhanced these cellular processes. In vivo experiments demonstrated that KAT7 knockdown inhibited CRC growth and lung metastasis. Mechanistically, KAT7 acetylated histone H3 at lysine 14 (H3K14) to enhance MRAS transcription, which activated the MAPK/ERK pathway and promoted tumorigenesis. Overexpression of MRAS or treatment with the ERK activator C6 Ceramide rescued the tumor inhibition caused by KAT7 silencing. The acetyltransferase activity of KAT7 is essential for CRC progression. Reexpression of KAT7, but not KAT7 acetyltransferase activity-deficient mutants, rescued MRAS expression, ERK phosphorylation, and CRC tumorigenesis in KAT7 knockdown CRC cells. Taken together, our results define the essential role of KAT7 in CRC tumorigenesis, rationalizing its potential as a therapeutic target for CRC treatment.

overall_design: To investigate the molecular mechanism underlying the contribution of KAT7 to CRC progression, we established HCT116 cell lines in which KAT7 has been knocked down by shRNA. We then performed gene expression profiling analysis using data obtained from RNA-seq. We identified a total of 1708 differentially expressed genes in KAT7 silenced HCT116 cells compared to the control group.

first: Hao

first: Tianwang

last: Guan

first: Rong

last: Hu


title: Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence I

first: Ian

overall_design:
To examine whether hpRNA transgenes containing asymmetric bulges could process different length siRNAs.
Transgenic tobacco individuals containing a GUS transgene were transformed with different asymmetric bulge hpRNA transgenes (Δ22nt, Δ23nt, Δ24nt-1, Δ24nt-2, Δ24nt-3).  3 independent T0 individuals were sent for sRNA sequencing per construct


title: Transcriptional elongation control of hypoxic response and therapeutic approaches

first: Ali

last: Shilatifard

state: IL

address: 320 E Superior St

department: Department of Biochemistry and Molecular Genetics

institute: Northwestern University Feinberg School of Medicine

summary: The release of paused RNA polymerase II (RNAPII) from promoter-proximal regions is tightly controlled to ensure proper regulation of gene expression. The elongation factor PTEF-b is known to release paused RNAPII via phosphorylation of the RNAPII C-terminal domain by its cyclin-dependent kinase component, CDK9. However, the signal and stress-specific roles of the various RNAPII-associated macromolecular complexes containing PTEF-b/CDK9 are not yet clear. Here, we identify and characterize the CDK9 complex required for transcriptional response to hypoxia. Contrary to previous reports, our data indicate that a CDK9 complex containing BRD4 but not AFF1/4 is essential for this hypoxic stress response. We demonstrate that BRD4 bromodomains (BET) are dispensable for the release of paused RNAPII at hypoxia-activated genes and that BET inhibition by JQ1 is insufficient to impair hypoxic gene response. Mechanistically, we demonstrate that the C-terminal region of BRD4 is required for Polymerase-Associated Factor-1 Complex (PAF1C) recruitment to establish an elongation-competent RNAPII complex at hypoxia-responsive genes. PAF1C disruption using a small-molecule inhibitor (iPAF1C) impairs hypoxia-induced, BRD4-mediated RNAPII release. Together, our results provide insight into potentially targetable mechanisms that control the hypoxia-responsive transcriptional elongation.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) and comparative expression profiling of RNA-seq to investigate role of BRD4 and its mutants in Hypoxia.

first: Shimaa

last: Soliman

first: Marta

last: Iwanaszko

first: Bin

last: Zheng


title: Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence [RNA-Seq]

overall_design:
To examine whether hpRNA transgenes containing asymmetric bulges could process different length siRNAs.
Transgenic tobacco individuals containing a GUS transgene were transformed with either a hpGUS[WT], hpGUS[G:U] or hpGUS[Δ22nt] transgenes.  3 independent T0 individuals were sent for sRNA sequencing per construct


title: Microbiota-mediated alteration in the transcriptomes of omental Tregs

city: Birmingham

first: Selene

last: Meza-Perez

address: 1825 Unuversity blvd

institute: UAB

summary: To investigate the effects of microbiota on the function and activities of omental Tregs, we purified Tregs from omenta of specific-pathogen-free (SPF) and germ-free (GF) mice that were either injected with tumors or not, and performed RNA-seq analysis

overall_design: Comparative gene expression profiling analysis of RNA-seq data for omental ST2POS and ST2NEG Tregs from Arginine treated and untreated mice

first: Alex

last: Rosenberg


title: The gut microbiota downregulates renal MAS1 through H3K9bu in high-fat diet-induced obesity-related hypertensive rats

city: Beijing

first: Hui

address: No.2 Yabao Road, Chaoyang District

department: Department of Cardiology

institute: Children’s Hospital, Capital Institute of Pediatrics

summary: Obesity is a leading cause of primary hypertension in children, and a high-fat intake and the gut microbiota may be involved in the pathogenesis of obesity-related hypertension (OrHTN), but the underlying mechanisms are not fully understood. Here, we show that high-fat diet (HFD) feeding alters the gut microbiota composition in OrHTN rats, resulting in a reduced abundance of the butyrate-producing bacteria Ruminococcus and a subsequent decrease in plasma butyrate levels. Histone 3 lysine 9 butyrylation (H3K9bu) levels decreased in the kidneys of OrHTN rats, which downregulates the expression of the hypertension-related MAS1 gene. Furthermore, sodium butyrate affected H3K9bu modification levels in a concentration-dependent manner, with decreased H3K9bu and downregulated MAS1 expression at low concentrations in human proximal tubular epithelial cells. Our results suggest that a HFD contributes to the development of OrHTN by altering the gut microbiota and its metabolites, leading to the downregulation of H3K9bu and hypertension-related gene expression.

overall_design: We first constructed a rat model of obesity-related hypertension induced by high fat diet, investigated the alterations of gut microbiota and plasma metabolites of the hypertensive rats, detected the levels of histone butyrylation modification in the kidneys, and performed ChIP-seq to explore the potential epigenetic mechanism underlying the development of obesity-related hypertension.

first: Yaqi

last: Li

first: Baoling

last: Bai

first: Chen

last: Shen

first: Yuting

first: Xiaodong

last: Sun

first: Haojie

last: Wu

first: Yanyan

last: Liu

first: Qin

last: Zhang

first: Lin

last: Shi


title: Glutamatergic neuronal activity regulates retinal angiogenesis and blood-retinal barrier via Norrin/b-catenin signaling

city: New York

first: Saptarshi Biswas

last: Biswas

state: NY

address: 650 West 168 Street

institute: Columbia University Irving Medical Center

summary: Interactions among neuroglial and vascular components are crucial for retinal angiogenesis and blood-retinal barrier (BRB) maturation. Neuronal synaptic dysfunctions precede vascular abnormalities in many retinal pathologies. However, whether neuronal activity, specifically glutamatergic activity, regulates retinal angiogenesis and BRB maturation remains unclear. Using in vivo genetic studies in mice, single-cell RNA-sequencing and functional validation, we found that deep plexus angiogenesis and paracellular BRB maturation are delayed in Vglut1-/- retinas, where neurons fail to release glutamate. In contrast, deep plexus angiogenesis and paracellular BRB maturation are accelerated in Gnat1-/- retinas, where constitutively depolarized rods release excess glutamate. Mechanistically, Norrin expression and endothelial Norrin/b-catenin signaling are downregulated in Vglut1-/- retinas, and upregulated in Gnat1-/- retinas. Pharmacological activation of endothelial Norrin/ b-catenin signaling in Vglut1-/- retinas rescued both deep plexus angiogenesis and paracellular BRB integrity. Thus, our findings demonstrate that glutamatergic neuronal activity regulates retinal angiogenesis and BRB maturation by modulating Norrin/b-catenin signaling.

overall_design: Retina was isolated from P10 wild-type and Vglut-/- mice in the C57BL/6J background. Following single cell dissociation of the retinal tissue, endothelial cells were labeled with CD31-APC antibody and FACS for single cell RNA sequencing analysis.

first: Saptarshi

first: Sanjid

last: Shahriar

first: Dritan

last: Agalliu


title: Phenolic acids prevent sex-steroid deficiency-induced bone loss and bone marrow adipogenesis in mice

city: New Orleans

first: Jovanny

last: Zabaleta

state: LA

address: 1700 Tulane Ave

department: Interdisciplinary Oncology

institute: LSUHSC

summary: Phenolic acids, such as hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA), are present in the human body throughout life from microbiome digestion of polyphenols. Previous studies showed significant effects of HA and 3-3-PPA on facilitating bone development, however, if these phytochemicals protect bone from degeneration and their mechanism of actions for doing so are unknown. In this report, we present that HA and 3-3-PPA suppression of bone resorption is able to ameliorate bone loss in an ovariectomy (OVX) osteopenic mouse model though not to the extent of Zoledronic acid (ZA). HA and 3-3-PPA treatment were shown to significantly decrease bone marrow adipocyte-like cell formation and inhibited gene expression of key adipogenesis regulator peroxisome proliferator activated receptor gamma (PPARγ) and lipoprotein lipase (Lpl) in bone from OVX mice. In addition, ChIP experiments showed that the association between PPARγ and Lpl promoter region in pre-adipocyte-like cells was significantly suppressed following HA or 3-3-PPA treatment. Contrasting HA and 3-3-PPA, ZA significantly increased TRAPase activity in the area close to growth plate and significantly suppressed bone cell proliferation. This data suggests that phenolics acids such as HA or 3-3-PPA may prevent bone degeneration after OVX through suppression of inflammatory milieu in the bone

overall_design: Wild-type Female C57Bl/6 were subjected to sham surgery or to ovariectomy (OVX) and 2 - 4 d after surgery they received either PBS, HA, 3-3-PPA , or ZA for 8 weeks after which mice were euthanized and the tibias collected for RNA extraction which was later used for the preparation of genomic libraries and those were sequenced to generate 1 x 75 single-end reads. Differentially expressed genes were detected in Partek Flow.

first: Perry

last: Caviness

first: Oxana

last: Lazarenko

first: Michael

middle: L

last: Blackburn

first: Christopher

middle: E

last: Randolph

first: Jin-Ran

last: Chen

first: Jenniffer

first: Fenghuan


first: Selene

last: Meza-Perez

overall_design: Comparative gene expression profiling analysis of RNA-seq data for omental Tregs from SPF and GF mice

first: Mingyong


title: MeRIPseq of m6A in HeLa synchonized cells with total RNA

city: Cerdanyola del Vallés

first: Alicia

last: Roque

state: Barcelona

address: Carrer de la Vall Moronta, C2

department: Bioquímica i biologia molecular

country: Spain

institute: Universitat Autónoma de Barcelona

summary:
MeRIPSeq of HeLa cells synchronized by a double thymidine block to obtain the cell cycle phases and work with total RNA to study m6A mark in mRNA without polyA tail
RNA seq of HeLa cells synchronized by a double thymidine block to obtain the cell cycle phases and work with total RNA to study mRNA without polyA tail

overall_design:
m6A IP of total RNA extraction of HeLa cells synchronized in G1, S and G2M phases of the cell cycle
Total RNA extraction of HeLa cells synchronized in G1, S and G2M phases of the cell cycle

first: Daniel

last: Garcia-Gomis

first: Marta

last: Andres

first: Jordi

last: López

first: Inma

last: Ponte


title: Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence

first: Ian

last: Greaves

sample_organism:
Arabidopsis thaliana
Nicotiana tabacum

platform_organism:
Arabidopsis thaliana
Nicotiana tabacum


title: RNA-seq analysis of cultured VSMCs

city: Cambridge

first: Felix

last: Krueger

address: Granta Park

department: Bioinformatics

institute: Altos Labs

summary: Bulk RNA seq analysis of primary mouse vascular smooth muscle cells

overall_design: Medial cells isolated from mouse aortas were digested to a single cell suspension and cultured in vitro

first: Jennifer

last: Harman

last: Lambert

first: Helle

last: Jørgensen


title: Enhancer engagement sustains oncogenic transformation and progression of B-cell precursor acute lymphoblastic leukemia

city: Rome

first: Stefano

last: Di Giovenale

address: Via Elio Chianesi,  53

country: Italy

institute: IRCCS National Cancer Institute Regina Elena

summary: Numerous evidence report that non-genetic-driven events such as enhancer reprogramming promotes neoplastic transformation and strongly contribute to the phenotypical  heterogeneity of cancers as much as genetic variation. In this study, we investigated the role of enhancers in sustaining oncogenic transformation in Paediatric B-cell Acute Lymphoblastic Leukemia (ALL-B), a type of cancer caused by the accumulation of lymphoid progenitor cells in the bone marrow.

overall_design:
Analysis of ATACseq and RNAseq across the type-B acute lymphoblasitc leukemia cancer progression. Four cancer stages are considered: Healthy, Onset, Remission and Relapse

scMultiomics samples profiled through 10xGenomics

first: Giacomo

last: Corleone

first: Cristina

last: Sorino

first: Matteo

last: Caforio

first: Francesca

last: De Nicola

first: Valentina

last: Bertaina

first: Angela

last: Pitisci

first: Clelia

last: Cortile

first: Franco

last: Locatelli

last: Folgiero

first: Maurizio

last: Fanciulli


title: Analysis of noncoding RNAs in human muscle and their regulation during muscle growth

status: Public on Jun 26 2024

city: Stirling

first: James

last: Timmons

address: Bridge of Allan

institute: Augur Precision Medicine LTD

summary:
A majority of genes code for non-protein-coding RNA (ncRNA). Only a handful of ncRNA have characterised molecular functions, including key epigenetic roles in development and human disease. In human skeletal muscle reliance on suboptimal sequencing-based data has obfuscated characterization of ncRNAs. Herein, using more comprehensive RNA profiling and data-driven quantitative network methods we study regulation of ncRNAs during skeletal muscle hypertrophy. Across five independent supervised exercise-training studies (n=144), 61% of individuals accrued muscle mass beyond established technical variation (lean mass responders, LMR) with the remainder show no measurable lean mass response (NMLMR). The LMR group alone demonstrated differential expression (DE) of 50 ncRNA genes (FDR <1%) and further modelling identified a total of 110 ncRNAs of potential interest. Critically, expression of these ncRNAs was indistinguishable between LMR vs NMLMR at baseline. Notably, <5% of these candidate ncRNAs were detected as being regulated by exercise training using sequencing. A robust co-expression model of the human muscle transcriptome (n=437) used to assign each ncRNA to functional pathways or single-cell types. Candidates, such as the hypertrophy-related ncRNA CYTOR, was leukocyte associated (FDR = 4.9 x10-7; FE = 6.6) and highly expressed in immune-related cells. PPP1CB-DT co-expressed with myofibril assembly genes (FDR = 8.2 x10-8; FE = 47.5), positively associating with type II fibre markers (e.g., TNNT3: r =0.37; FDR = 2.57 x10-14), while others (e.g. EEF1A1P24 and TMSB4XP8) co-expressed angiogenesis pathways. MYREM was positively associated with hypertrophy and we established it has a distinct myonuclear expression in vivo, using merfish probes. We show that single-cell type associations are readily identifiable from bulk transcriptomic data, and that hypertrophy-linked ncRNA genes appear to mediate their association with muscle growth via multiple cell types.
A total of 144 paired sample comparisons were made for the continous (change in lean mass) versus change in gene expression analysis using 137 paris of samples. 7 baseline samples from the HALL study represent a single baseline biopsy and thereafter each leg was subject to a distinct RT protocol with an individual measure of lean mass before and lean mass afterwards from the same leg.

overall_design: Samples marked RT_Study were used the analysis of muscle before and after supervised exercise training (resistance exercise in this case).

first: Jamie

first: Stuart

last: Phillips


title: Microarray analysis reveals potential circRNA biomarkers in human acral lentiginous melanoma

city: Nanjing

first: Cuicui

last: Tian

address: Jiangwangmiao Road 12#

institute: Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College

summary: We have completed the Arraystar Human circRNA Array V2 analysis of the 12 samples that you submitted. Total RNA from each sample was quantified using the NanoDrop ND-1000. The sample preparation and microarray hybridization were performed based on the Arraystar’s standard protocols. Briefly, total RNAs were digested with Rnase R (Epicentre, Inc.) to remove linear RNAs and enrich circular RNAs. Then, the enriched circular RNAs were amplified and transcribed into fluorescent cRNA utilizing a random priming method (Arraystar Super RNA Labeling Kit; Arraystar). The labeled cRNAs were hybridized onto the Arraystar Human circRNA Array V2 (8x15K, Arraystar). After having washed the slides, the arrays were scanned by the Agilent Scanner G2505C.

overall_design: For the present study, 6 ALM and corresponding paired para-cancer tissues samples stored at the Institute of Dermatology, Chinese Academy of Medical Sciences (Nanjing) were randomly selected from January 2016 to May 2018. RNA was then extracted from these tissues for circRNA microarray analysis. In addition, reverse transcription-quantitative PCR was performed to verify the differentially expressed circRNAs in ALM tissues and melanoma cells including A375, A875 and SK-MEL-28. before a competing endogenous RNA (ceRNA) network constructed by integrating predictions from miRcode, miRanda, and TargetScan tools. Furthermore, gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed for these differentially expressed circRNA. Amongst the 192 differentially expressing circRNAs found, 57 up-regulated and 135 down-regulated circRNAs were found according to the P < 0.05 and |fold change (FC)| > 2 thresholds set. According to RT-qPCR results, it was found that circ_0000569 and circ_0005029 were significantly down-regulated, whilst circ_0000094 was up-regulated in ALM tissue samples compared with paired para-cancer tissues, which was consistent with the microarray analysis results.

first: Jing-shu

last: Xiong

first: Cui-cui

first: Wen-bo

last: Bu

first: Li-ming

last: Huang

first: Jian-fang

last: Sun

first: Hao

last: Chen


title: Transcriptomic analysis of mammary tumor biospy from MMTV-ErbB2 transgenic mice [Doxorubicin]

city: Berkeley

first: Jian-Hua

last: Mao

address: One Cyclotron Road

institute: Lawrence Berkeley National Laboratory

summary: The tyrosine kinase ErbB2 positive breast tumors have more aggressive tumor growth, poorer clinical outcome, and more resistance to radiotherapy, chemotherapy and hormone therapy. To identify the biomarkers for predicting the responses to chemotherapy, we treated genetically diverse F1 backcross (F1Bx) mice between C57BL/6J and FVB/N MMTV-Erbb2 transgenic mice with mammary tumor by doxorubicin. Biopsies were taken in sterile conditions and processed for RNA and histological analysis before treatment.  This will help us to have a better understanding of the heterogeneous response to chemotherapy and permit us to design better and more individualized (personalized) treatment strategies for human ErbB2 positive breast cancer.

overall_design: 50 MMTV-ErbB2 mammary tumor biopsy before treatment were analyzed by Affymetrix microarrays.

first: Jesus

last: Perez-Losada


title: Gene expression profiling of lymph node mesenchymal stromal cells in follicular lymphoma.

city: 豊明市

first: Sumie

last: Fujii

state: 愛知県

address: 沓掛町田楽ケ窪1番地98

institute: 藤田医科大学　医学部輸血細胞治療科

summary: This study investigates the gene expression profiles of mesenchymal stromal cells derived from lymph nodes of patients with follicular lymphoma. Using microarray analysis, we identified differentially expressed genes involved in tumor progression and stromal cell differentiation.

overall_design:
F1, F2, F3, F4, F5: Lymph node mesenchymal stromal cells from patients with follicular lymphoma.
MSC: Bone marrow mesenchymal stromal/stem cells from health donor
Gene expression was measured at passage3.

first: Noriko

last: Sugino


title: The Plasmodium falciparum histone methyltransferase PfSET10 is dispensable for the regulation of antigenic variation and gene expression in blood stage parasites

city: Nijmegen

first: Richard

last: Bartfai

state: Gelderland

address: Geert Grooteplein Zuid 28

department: Molecular Biology

country: Netherlands

institute: Radboud Institute for Molecular Life Sciences

summary: The malaria parasite Plasmodium falciparum employs antigenic variation of the virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1) to escape adaptive immune responses during blood infection. Antigenic variation of PfEMP1 occurs through transcriptional switches in the mutually exclusive expression of individual members of the multi-copy var gene family. var genes are located in perinuclear clusters of transcriptionally inactive heterochromatin. Singular var gene activation is linked to locus repositioning into a dedicated zone at the nuclear periphery and deposition of histone 3 lysine 4 di-/trimethylation (H3K4me2/3) and H3K9 acetylation marks in the promoter region. While previous work identified the putative H3K4-specific methyltransferase PfSET10 as an essential enzyme and positive regulator of var gene expression, a recent study reported conflicting data. Here, we used iterative genome editing to engineer a conditional PfSET10 knockout line tailored to study the function of PfSET10 in var gene regulation. We demonstrate that PfSET10 is not required for mutually exclusive var gene expression and switching. We also show that PfSET10 is dispensable not only for asexual parasite proliferation but also for sexual conversion and differentiation of gametocytes. Furthermore, comparative RNA-seq experiments revealed that PfSET10 plays no obvious role in regulating gene expression during asexual parasite development and gametocytogenesis. Interestingly, however, PfSET10 shows different subnuclear localization patterns in asexual and sexual stage parasites and female-specific expression in mature gametocytes. In summary, our work confirms in detail that PfSET10 is not involved in regulating var gene expression and is not required for blood stage parasite viability, at least not when cultured in vitro, thus suggesting PfSET10 may be important for life cycle progression in the mosquito vector or during liver stage development.

overall_design: Synchronized ring stage parasites (0-6 hpi) cultured in the presence of 2.5 ng/μl BSD-S-HCl were split into three 10 ml cultures. Dish 1 (BSD control, baseline reference) was maintained under 2.5 ng/μl BSD-S-HCl selection. Dishes 2 (DMSO control, PfSET10 WT) and 3 (RAPA-treated, PfSET10 KO) were released from BSD-S-HCl pressure and treated with either the DMSO solvent alone or 100 nM RAPA, respectively. Dishes 1-3 were all cultured in parallel for two additional generations to allow for parasite expansion, and sorbitol synchronizations were performed to secure synchronous parasite populations for sample harvest. At the early ring stage (4-10 hpi), the DMSO- and RAPA-treated cultures were split into four equal 10 ml cultures each to be able to harvest four time points across the IDC (TP1, 10-16 hpi; TP2, 22-28 hpi; TP3, 30-36 hpi, TP4, 38-44 hpi), and one separate 10 ml dish each was maintained in culture for ten additional generations. The BSD control ring stage sample (R-WT-G0) was harvested alongside the DMSO- and RAPA-treated ring stage samples (R-WT-G2, R-KO-G2) (TP1), and trophozoite, early schizont and late schizont samples of the DMSO-treated (T-WT-G2, ES-WT-G2, LS-WT-G2) and RAPA-treated (T-KO-G2, ES-KO-G2, LS-KO-G2) cultures were harvested at the corresponding TPs 2-4 in the same IDC. For the DMSO- and RAPA-treated cultures, an additional 10-16 hpi ring stage sample each was harvested ten generations after release from BSD-S-HCl pressure (R-WT-G10, R-KO-G10). For gametocyte samples, DMSO- and RAPA-treatment was performed on synchronous ring stage parasite cultures (0-8 hpi). After two additional invasion cycles, 20 ml parasite culture each was induced using mFA as explained above and the ring stage progeny was split into two 10 ml dishes, cultured in the presence of 50 mM GlcNAc for five days and total RNA samples were harvested on day 6 (D6-WT, D6-KO) and day 12 of gametocytogenesis (D12-WT, D12-KO), respectively. All samples have been harvetsed in biological triplicates.

first: Matthias

last: Wyss

first: Abhishek

last: Kanyal

first: Igor

last: Niederwieser

first: Till

last: Voss


title: Exercise training modifies the whole-blood methylation profile in middle-aged and older women

city: Ribeirão Preto

first: Guilherme

middle: Da Silva

last: Rodrigues

address: Avenida Bandeirantes, 3900

country: Brazil

institute: Universidade de São Paulo

summary: This is a prospective longitudinal study that aimed to investigate whether physical training would modify the whole blood methylation profile in healthy women. A total of 43 individuals were involved in a physical training protocol during a 7-week follow-up, consisting of cardiorespiratory aerobic and muscular strength exercises. Subjects were after 7 weeks of physical training (POST 7). Primary functional outcomes included anthropometric, blood pressure, biochemical measurements, physical tests, and global health assessments. Blood samples were collected at each time point to determine the methylation profile using a DNA methylation array technique to track up to 850,000 different sites. Exercise training lowered blood pressure and triglyceride levels and improved physical performance, including maximal strength in the upper and lower body. Furthermore, physical training improved quality of life markers. By entering the differentially methylated sites into the pathway analysis, we found several pathways involved in the regulation of whole-body metabolism, including AMPK signaling, TGF-beta signaling, and insulin signaling. This study demonstrates that exercise training promotes a robust change in whole blood methylation profile and provides new insights into key regulators of exercise-induced benefits.

overall_design: MethylationBrazil Assay

last: da Silva Rodrigues


title: Patient TSC2 Mutant Cells Exhibit Aberrations in Early Neurodevelopment Accompanied by Changes in the DNA Methylome (Bisulfite-Seq)

city: Nashville

first: Rebecca

last: Ihrie

state: TN

address: 1161 21ST Avenue South

department: Cell & Developmental Biology

institute: Vanderbilt University

type: Methylation profiling by high throughput sequencing

summary: Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. While benign tumors in the heart, lungs, kidney, and brain are all hallmarks of the disease, often the most severe symptoms of TSC are neurological, including seizures, autism, psychiatric disorders, and intellectual disabilities. TSC is caused by a heterozygous loss of function mutation in TSC1 or TSC2 and consequent dysregulation of signaling via mechanistic Target of Rapamycin Complex 1 (mTORC1). While TSC neurological phenotypes are well-documented, it is not yet known how early in neural development TSC1/2-mutant cells diverge from the typical developmental trajectory, and whether such phenotypes are seen in the heterozygous-mutant populations comprising the vast majority of cells in patient tissues. Using TSC patient-derived isogenic induced pluripotent stem cells (iPSCs), we observed aberrant early neurodevelopment in vitro, including misexpression of key transcription factors associated with lineage commitment and premature electrical activity. These alterations in differentiation were coincident with widespread changes in DNA methylation, including at loci associated with key genes in neurodevelopment. Collectively, these data suggest that mutation or loss of TSC2 affects gene regulation and expression at earlier timepoints than previously appreciated, with implications for whether and how prenatal treatment should be pursued.

overall_design: To investigate epigenetic differences between patient heterozygous TSC2 mutant and isogenic wild type controls or homozygous TSC2 mutants at the iPSC and day 10 NPC stages, we extracted gDNA, treated wtih bisulfite, sequenced, and analysed differences between genotypes and timepoints. TSP77 is a patient derived cell line. We used two replicates of iPSCs at least two passages apart and two separate differentiations to day 10 NPCs were used per cell line as replicates.

first: Mary-Bronwen

last: Chalkley

first: Kevin

last: Ess


title: Next Generation Sequencing Analysis of Gene expression of wild-type AGS cells and AGS cells transfected with an siRNA targeting SRSF2

city: Fuzhou

first: Songyi

last: Liu

address: No. 20 Chazhong Road

institute: the First Affiliated Hospital, Fujian Medical University

summary: SRSFs are a subset of RNA binding proteins that exert precise control over RNA splicing, including cancer-associated AS events. Serine/arginine-rich splicing factors 2 (SRSF2), a member of SRSF family, plays a crucial role in regulating splicing events. We profiled gene expression and splicing changes in AGS cells silencing SRSF2.  Our study unveils that SRSF2 binds to the exon 4 region of YTHDF1 pre-mRNA, inducing YTHDF1Δexon 4 skipping and resulting in elevation levels of the oncogenic YTHDF1-S in GC. Overall, we provide evidence that YTHDF1 is an AS target of SRSF2.

overall_design: SRSF2 silencing AGS cells compared to wild-type AGS cells.Total RNA transcriptome from these samples was profiled by RNA-seq

first: Chunlin

last: Lin

first: Jianxin

last: Ye

first: Guangnwei

last: Zhu


title: Evidence of RNA polymerase III recruitment and transcription at protein-coding gene promoters

city: Urbana

first: Sihang

last: Zhou

address: 601 S Goodwin Ave

institute: University of Illinois Urbana-Champaign

summary: RNA polymerase (Pol) I, II, and III are most commonly described as having distinct roles in synthesizing ribosomal RNA (rRNA), messenger RNA (mRNA), and specific small noncoding (nc)RNAs, respectively. This delineation of transcriptional responsibilities is not definitive, however, as evidenced by instances of Pol II recruitment to genes conventionally transcribed by Pol III, including the co-transcription of RPPH1 - the catalytic RNA component of RNase P. A comprehensive understanding of the interplay between RNA polymerase complexes remains lacking, however, due to limited comparative analyses for all three enzymes. To address this gap, we applied a uniform framework for quantifying global Pol I, II, and III occu- pancies that integrates currently available human RNA polymerase ChIP-seq datasets. Occupancy maps are combined with a comprehensive multi-class promoter set that includes protein-coding genes, noncoding genes, and repetitive elements. While our genomic survey appropriately identifies recruitment of Pol I, II, and III to canonical target genes, we unexpectedly discover widespread recruitment of the Pol III machinery to promoters of specific protein-coding genes, supported by colocalization patterns observed for several Pol III-specific subunits. We show that Pol III-occupied Pol II promoters are enriched for small, nascent RNA reads terminating in a run of 4 Ts, a unique hallmark of Pol III transcription termination and evidence of active Pol III activity at these sites. Pol III disruption differentially modulates the expression of Pol III-occupied coding genes, which are functionally enriched for ribosomal proteins and genes broadly linked to unfavorable outcomes in cancer. Our map also identifies additional, currently unannotated genomic elements occupied by Pol III with clear signatures of nascent RNA species that are sensitive to disruption of La (SSB) - a Pol III-related RNA chaperone protein. These findings revise our current understanding of the interplay between Pols II and III and identify potentially novel small ncRNAs with broad implications for gene regulatory paradigms and RNA biology.

overall_design: To investigate the effect of La/SSB on nascent, promoter-derived small RNAs, we performed La/SSB knockdown using siRNA in HEK293T cells. The experiment included three biological replicates for both the control knockdown and the La/SSB knockdown. Promoter-specific raw small RNA signal counts were extracted, and differentially expressed genes were subsequently identified.

first: Ruiying

last: Cheng

first: K

last: Rajendra

last: Van Bortle


title: CCR1 drives immunosuppressive properties of tumor associated macrophages in pancreatic cancer

city: Ann Arbor

first: Marina

last: Pasca di Magliano

state: MI

address: 1500 E Medical Center Dr

institute: University of Michigan Medical School

summary: Immunotherapy approaches have been ineffective in pancreatic ductal adenocarcinoma (PDA), pointing to the need for additional avenues to target in the pancreatic cancer microenvironment. We previously discovered that tumor educated bone marrow derived macrophages express high levels of C-C Motif Chemokine Receptor 1 (CCR1). By single-cell RNA sequencing, we found CCR1 to be expressed predominantly by tumor associated macrophages (TAMs) and granulocytes in both human and mouse PDA. Thus, we sought to investigate the functional role of CCR1 in pancreatic cancer. Using KC; Ccr1-/- mice (Ptf1a-Cre; LSL-KrasG12D; Ccr1-/-), we determined that CCR1 is dispensable during pancreatic cancer initiation, although we observed increased CD8 T cell infiltration in KC; Ccr1-/- pancreata. Using syngeneic orthotopic PDA mouse models we discovered that CCR1 ablation in Ccr1-/- mice or pharmaceutical inhibition of CCR1 both resulted in reduced tumor growth. Further, CCR1 ablation prolonged the overall survival of KPC mice. Through mass cytometry (CyTOF) and co-immunofluorescence staining we showed CCR1 ablation elevated CD8 T cell cytotoxic activity in the orthotopic PDA model. Mechanistically we found TAMs lacking CCR1 expressed less Arginase 1 and CD206 -both immunosuppressive markers of macrophages- compared to wild type TAMs. Further, targeting both CCR1 and Arginase 1 synergized with immune checkpoint blockade anti-PD-L1 to enhance antitumor efficacy in orthotopic model of PDA. Together, our data is consistent with the notion that tumor associated macrophages lacking CCR1 expression are less immunosuppressive, consequently allowing increased CD8 T cell mediated anti-tumor immunity. Targeting CCR1 in combination with immune checkpoint blockade improves antitumor efficacy in pancreatic cancer.

overall_design: We used KC;Ccr1-/- (Ptf1a-Cre;LSL-KrasG12D;Ccr1-/-) to determine the requirement of CCR1 during pancreatic cancer initiation and progression.

first: Yaqing

last: Zhang


title: mRNAseq: Evidence of RNA polymerase III recruitment and transcription at protein-coding gene promoters

address: B520 Chemical & Life Sciences Laboratory

department: School of Molecular & Cellular Biology

institute: The University of Illinois Urbana-Champaign

overall_design: For POLR3A KD Hek293T cells are transfected with either control siRNA or POLR3A siRNA, and cells are collected 2 days after knockdown. For Pol III small molecule inhibition, Hek293T cells are treated with either DMSO (vehicle, 0.1%) or ML60218 (25uM) and cells are collected 4 hours post-treatment. RNA is collected using E.Z.N.A.® Total RNA Kit I. For RNA sequencing, the RNAseq libraries were prepared with the Kapa Hyper Stranded mRNA library kit (Roche) and sequenced on two 10B lanes for 151 cycles from both ends of the fragments on a NovaSeq X Plus with V1.0 sequencing kits, Fastq files were generated and demultiplexed with the bcl2fastq v2.20 Conversion Software (Illumina).

first: Simon

last: Lizarazo

first: Duncan

last: Smith


title: Characteristics of quiescent adult neural stem cells induced by the bFGF/BMP4 combination or BMP4 alone in vitro

city: Shanghai

first: Sutong

last: Xu

address: No. 500 Zhennan Road, Putuo District

institute: Tongji university

summary: Bone morphogenetic protein-4 (BMP4) is involved in regulation of neural stem cells (NSCs) proliferation, differentiation, migration and survival. It was previously thought that the treatment of NSCs with BMP4 alone induces astrocytes, whereas the treatment of NSCs with the bFGF/BMP4 combination induces quiescent neural stem cells (qNSCs). In this study, we performed RNA sequencing (RNA-Seq) to compare the transcriptome profiles of BMP4-treated NSCs and bFGF/BMP4-treated NSCs, and found that both NSCs treated by these two methods were Sox2 positive qNSCs which were able to generate neurospheres. However, NSCs treated by those two methods exhibited different characteristics in state and the potential for neuronal differentiation based on transcriptome analysis and experimental results. We found that BMP4-treated NSCs tended to be in a deeper quiescent state than bFGF/BMP4-treated NSCs as the percentage of ki67-positive cells were lower in BMP4-treated NSCs. And after exposure to differentiated environment, bFGF/BMP4-treated NSCs generated more DCX-positive immature neurons and MAP2-positive neurons than BMP4-treated NSCs. Our study characterized qNSCs treated with BMP4 alone and bFGF/BMP4 combination, which laid a foundation for studying the activation mechanism of qNSCs.

overall_design: NSCs were isolated from the SVZs of adult C57BL/6 mice (2 female mice and 2 male mice were used for one cell isolation experiment) aged 8-9 weeks. The cells were cultured in suspension in proliferation medium (bFGF/EGF). NSCs obtained after at least three passages were cultured in adherent in the medium with bFGF/EGF, after 16-18h, the cells were collected for RNA-seq as bFGF/EGF_0d. Then we replaced bFGF and EGF in the medium with BMP4 as BMP4-treated NSCs, and replaced EGF with BMP4 as bFGF/BMP4-treated NSCs. After 6 days, the cells were collected for RNA-seq as BMP4_6d and bFGF/BMP4_6d. The cells we used 3 biological replicas in every treatment.

first: Xi

first: Zhuoqun

last: Li

first: Chenming

first: Qiulu

first: Huazhen

last: Chai

first: Hongkai

last: Yao

first: Yuping

last: Luo

first: Siguang

first: Chun


title: Patient TSC2 Mutant Cells Exhibit Aberrations in Early Neurodevelopment Accompanied by Changes in the DNA Methylome

first: Rebecca

last: Ihrie

overall_design: To investigate gene expression differences between patient heterozygous TSC2 mutant day 10 NPCs and isogenic wild type controls or homozygous TSC2 mutants, we extracted total RNA, sequenced, and analysed differences between genotypes. Two separate isogenic iPSC lines were used in this study. TSP77 is patient derived while CR39/37 are not. Two separate differentiations were used per cell line as replicates.

first: Mary-Bronwen

last: Chalkley

first: Kevin

last: Ess


title: The DNA damage response of Escherichia coli: differential gene expression after replication inhibition by azidothymidine

city: Waltham

first: Susan

middle: T

last: Lovett

address: 415 South St.

department: Rosenstiel Center

institute: Brandeis University

summary: We examined the DNA damage response of bacterium E. coli using the replication inhibitor azidothyimdine (AZT), and RNA-Seq analysis. We confirm the induction of classic SOS loci by AZT and identify several genes, including many of the pyrimidine pathway, that are induced dependent on LexA cleavage, but have not been previously demonstrated to be DNA damage-inducible. Despite a strong dependence on LexA, these genes lack LexA boxes and their regulation by LexA is likely to be indirect via unknown factors. We show that stringent starvation protein, SspA, is as important as LexA in the regulation of AZT-induced genes. Our experiments identify a new set of LexA-independent DNA damage inducible genes, including 22 small RNA genes, some of which appear to activated by SspA. Motility and chemotaxis genes are strongly down-regulated by AZT, possibly as a result of one of more of the small RNAs or other transcription factors such as AppY and GadE, whose expression is elevated by AZT. Genes controlling the iron siderophore, enterobactin, and iron homeostasis are also strongly induced, independent of LexA. We confirm that IraD anti-adaptor protein and its upstream genes are induced, independent of LexA and that a second anti-adaptor, IraM is also strongly AZT-inducible, independent of LexA, suggesting that RpoS stabilization via these anti-adaptor proteins is an integral part of DNA damage tolerance.

overall_design: We performed RNA seq analysis in 4 different strains backgrounds, wild-type (an rph+ derivative of MG1655), lexA3, sspA∆ mutants and the lexA3 sspA∆ double mutant.  Cells were grown to log phase and treated or not for hr with 100 ng/ml azidothymidine, a chain-terminating replication inhibitor.

first: Thalia

last: Sass


title: Methylation of gDNA from twins with tuberculosis infection or not

city: Tongliao

first: Batudeligen

last: Batudeligen

address: 536 West Hollin River Street

institute: Affiliated Hospital of Inner Mongolia University for the Nationalities

summary:
To search the methylation of genes which were associated with tuberculosis infecion
We used the microarrays to detail the methylation of gDNA between with the tuberculosis infection and without the tuberculosis in monozygous twins

overall_design: Total blood DNA was extracted using the EDTA DNA Blood Mini Kit (Qiagen) according to the manufacturer’s instructions. The amount of DNA was quantified by the spectrophotometric A260 to A280 ratio of 1.8. DNA methylation patterns were analyzed using the Illumina Infinium HumanMethylation850 Bead Chip (Illumina, Inc.), according to the manufacturer’s instructions.

first: Zhi

last: Liu

first: Batu

last: Deligen


title: Tumour-derived Extracellular Vesicle and Particle Reprogramming of Interstitial Macrophages in the Lung Pre-Metastatic Niche Enhances Vascular Permeability and Metastatic Potential

city: new york

first: David

last: Lyden

address: 413 East 69th Street, Room BB1228

department: Pediatrics

institute: Weill Cornell medical College

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

overall_design: Refer to individual Series


title: Single-cell transcriptional analysis of kidney from aging mice with podocyte specific RARRES1 overexpression

city: New York

first: Zeguo

last: Sun

address: 1468 Madison Avenue

institute: Ichan School of Medicine at Mount Sinai

summary: Retinoic acid receptor responder protein-1 (RARRES1) was identified as a podocyte transmembrane protein whose expression correlates with glomerular disease progression. The cytopathic effect of RARRES1 is carried out only when proteolytically cleaved in its ectodomain into a soluble form (sRARRES1) and subsequently endocytosed by podocytes, as a membrane-bound, cleavage site mutant failed to induce any effect. We investigated the effects of long-term podocyte-derived RARRES1 overexpression on aging-induced kidney injury.

overall_design: 18 months of mice with Nphs1-rtTA; TRE-hRARRES1WT transgenes and control littermates were fed with doxycycline-supplemented chow for 16 months. PBS-perfused mouse kidneys were harvested and single-cells were prepared.

first: Ye

last: Feng

first: Jia

last: Fu


title: 3`mRNA sequencing of liver RNA (MACE) at the 30h time point after acetaminophen (APAP) 300mg/kg administration

city: Frankfurt

first: Malte

last: Bachmann

address: Theodor Stern Kai 7

department: General Pharmacology

institute: University Hospital Frankfurt

summary: Hepatic RNA from mice (C57BL/6) of 2 groups (APAP-treated (300mg/kg), n=8; APAP (300mg/kg)-AZD7986 (5mg/kg), n=8) were pooled at equal shares. Thereafter, pooled RNA underwent analysis by 3`mRNA sequencing (MACE, GenXPro, Frankfurt, Germany)

overall_design: Examination of AZD7986 regulated genes under APAP induced liver injury at 30h

first: Jessica

last: Raith

first: Heiko

last: Mühl


title: Integrin αM promotes macrophage alternative M2 polarization in hyperuricemia-related chronic kidney disease

city: Chengdu

first: Jing

state: Sichuan  SC

address: No.17, Renmin South Rd 3rd Section

institute: West China Hospital of Sichuan University

summary: Hyperuricemia is an essential risk factor in chronic kidney disease (CKD), while urate-lowering therapy to prevent or delay CKD is controversial. Alternatively activated macrophages in response to local microenvironment play diverse roles in kidney diseases. Here, we aim to investigate whether and how macrophage integrin αM (ITGAM) contributes to hyperuricemia-related CKD. In vivo, we explored dynamic characteristics of renal tissue in C57BL/6J mice with hyperuricemia-related CKD. By incorporating transcriptomics and phosphoproteomics data, we analyzed gene expression profile, hub genes and potential pathways. In vitro, we validated bioinformatic findings under different conditions with interventions corresponding to core nodes. We found that hyperuricemia-related CKD was characterized by elevated serum uric acid levels, impaired renal function, activation of macrophage alternative (M2) polarization, and kidney fibrosis. Integrated bioinformatic analyses revealed Itgam as the potential core gene and was associated with focal adhesion signaling. Notably, we confirmed the upregulated expression of macrophage ITGAM, activated pathway, and macrophage M2 polarization in injured kidneys. In vitro, through silencing Itgam, inhibiting p-FAK or p-AKT1 phosphorylation, and concurrent inhibiting of p-FAK while activating p-AKT1 all contributed to the modulation of macrophage M2 polarization. Our results indicated targeting macrophage ITGAM might be a promising therapeutic approach for preventing CKD.

overall_design: Seven-week male mice (C57BL/6J, 22-25g) were purchased from Dossy Experimental Animal Co., Ltd (Chengdu, China). These mice were maintained in the controlled environment for 1 week (temperature at 20 ± 2 ℃, humidity at 50–60%, and 12-hour light/dark cycle) and provided with food and water ad libitum. To study dynamic characteristic of uric acid-induced kidney injury, we randomly divided mice into 4 groups: day 0, day 7, day 14, and day 21 groups (n=6 per group). The mice in group day 7, 14 and 21 were administered with a mixture of adenine (0.16 g/kg) and potassium oxonate (2.4 g/kg) by oral gavage once daily.Finally, kidney tissue from day 0 (Control) and day 21 groups were performed mRNA sequencing.

first: Fan

last: Guo

first: Liang

last: Ma

first: Ping


title: Fecal microbiota transfer from autoimmune dry eye disease model mice to conventional mice induces ocular surface inflammation and stereotypic B cell receptor repertoire development in lacrimal gland

city: Seoul

first: Soobin

last: Lee

address: 1, Gwanak-ro, Gwanak-gu, Seoul, Republic of Korea

country: South Korea

institute: Seoul National University

summary: In this study, we transferred gut microbiota of SS-like autoimmune dry eye disease model mice to conventional B6 mice (NOD-FMT). After the transfer, NOD-FMT mice experienced a dramatic change in the gut microbiomes and showed clinicopathological features of SS, including increased corneal fluorescein staining score, decreased tear production, elevated levels of IL-6 mRNA, decreased levels of MUC5AC mRNA encoding mucin. Additionally, we observed that NOD-FMT mice shared stereotypic B cell receptor (BCR) clonotypes with a much higher frequency compared to control group. B cell clones encoding these stereotypic BCR clonotypes developed and expanded locally in the lacrimal gland, and achieved systemic presence in certain clonotypes.

overall_design: Feces were collected from 30 NOD.B10.H2b mice older than 20 weeks with autoimmune dry eye disease, and their microbiomes were transferred to ten mice (NOD-FMT group). In parallel, ten mice were orally gavaged with PBS (PBS-T group), and five mice from the same cohort received no treatment (NC group).

first: Soyeon

last: Ju

first: Sunghoon

last: Kwon

first: Junho

last: Chung

first: Mee Kum

last: Kim


title: Cancer EVP Uptake in Lung Interstitial Macrophages Enhances Vascular Permeability and Metastatic Potential (in vivo)

summary: Enhanced vascular permeability in the pre-metastatic niche facilitates tumor cell extravasation and metastasis. Extracellular vesicles and particles (EVPs) are pivotal mediators of cancer progression, including induction of vascular permeability. We identify a novel mechanism whereby melanoma and osteosarcoma EVPs (B16F10 and K7M2) enhance endothelial cell permeability, tumor extravasation and lung metastasis.In contrast, EVPs from breast tumors (4T1 and 67NR) elicited only mild vascular permeability, tumor cell extravasation, and metastasis. EVP induced vascular leakiness is observed within 48h following tumor implantation and as early one hour following direct injection of the B16F10 or K7M2 derived EVPs in non-tumor bearing mice. Rather than acting directly on endothelial cells, EVPs activated interstitial macrophages (IMs) anddepletion of IM with a CSF1R antibody significantly reduced vascular leakiness and metastatic potential.Subsequent activation of JAK/STAT signaling in IMs induces IL-6 secretion which in turn induces endothelial permeability.B16F10 and K7M2 EVPs highly expressed ITGα5 compared to breast tumors, and knock down ofintegrin-α5impairs IM signaling, vascular permeability and metastasis. Furthermore, in patients Il-6 expression is elevated in tumor-adjacent IMs compared to distant tissues. Our findings identify a key role for IMs in mediating tumor type-specific EVP-driven vascular permeability and metastasis, offering promising targets for therapeutic intervention.

overall_design: To evaluate the effect of B16F10 on interstitial machrophages (IMs), we sorted IMs which uptaken labled EVPs and control IMs. We then performed gene expression profilling anaylsis.

first: Shani

last: Dror