title: Basophils play a protective role in the recovery of skin barrier function from mechanical injury in mice

city: Boston

first: Manuel

last: Leyva-Castillo

state: MA

address: 300 Longwood Ave

institute: Boston Children's Hospital

summary: Mechanical skin injury promote an acute inflammation that impact the skin barrier function. The mechanisms to restore skin barrier function are not fully understood

overall_design: After shaving the back of Balb/c mice was tape stripped 6 times with the help of a tegaderm. After  24h skin biopsy were collected and samples were stored in RNAlater. Total skin RNA was isolated with RNeasy Mini Kit (Qiagen), followed by cDNA synthesis using the SuperScript VILO cDNA Synthesis Kit (ThermoFisher Scientific). The Ion AmpliSeq Transcriptome Mouse Gene Expression Kit was used to prepare bar-coded libraries and sequenced by using an Ion S5 next-generation sequencer. The AmpliSeqRNA plug-in (ThermoFisher Scientific) was used to calculate differential gene expression analysis

first: Juan

last: Leyva Castillo


title: Gene expression profiling of malignant peripheral nerve sheath tumors (MPNSTs) and benign neurofibroma tissue samples.

status: Public on Feb 13 2024

city: Oslo

first: Anita

last: Sveen

address: P.O.Box 4953, Nydalen

department: Department of Molecular Oncology

country: Norway

institute: Institute for Cancer Research, Oslo University Hospital

summary: We have performed gene expression analyses as part of a European multicentre study on MPNST. The data was used for transcriptomic subtyping, gene set enrichment analyses and integration with DNA copy number data.

overall_design: This series consists of tissue samples from 64 MPNSTs and 15 neurofibromas analyzed on Affymetrix Human Transcriptome 2.0 Arrays.

first: Maren

last: Høland

first: Ina

middle: A

last: Eilertsen

first: Ragnhild

last: Lothe


title: RNA-seq of mouse tissue samples from animal model：Effects of MMP9 on gene expression.

city: Kaifeng

first: ruizhen

last: wang

address: Jinming Avenue, Kaifeng City, Henan Province

country: China

institute: Henan University

type: Expression profiling by high throughput sequencing

summary: In the present study, RNA-seq technique were performed To explore biological significance of MMP9 in the neuro-cutaneous signaling network of chronic itch. We used MMP-9-IN-1, a specific inhibitor of MMP9, or its vehicle to treat ACD model mice, and conducted high-throughput sequencing on ear tissue samples of mice. It was found that inhibition of MMP9 expression affected the expression of a variety of itching factors and skin inflammatory factors related to the skin pathology of chronic dermatitis. These changes may indicate that MMP9 is closely related to inflammation and pruritus. This finding might help us to better understand the immune-neuro-modulatory mechanism in itch circuits.

overall_design: Mouse ear tissue were from MMP-9-IN-1-injected ACD model mice (10mg/Kg ,mice intraperitoneal injection) or vehicle control. The gene expression of these cells were investigated utilizing DNBSeq platform.

first: Ruizhen

last: Wang

first: Jianghui

last: Meng


title: N1E-115 cells and sorted NOS1+ mouse enteric neurons

status: Public on Feb 14 2024

city: Rochester

first: Tamas

last: Ordog

state: MN

address: 200 1st St SW

country: USA

institute: Mayo Clinic

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Resting CD4+ T cells exposed to oleic acid upregulate genes involved in immunometabolism resulting in increased IL-9+ frequencies post-activation [DNA methylation]

city: Leiden

first: Thomas

middle: B

last: Kuipers

address: Einthovenweg 20

country: Netherlands

institute: LUMC

type: Methylation profiling by genome tiling array

summary: T cells are the most common immune cell in atherosclerotic plaques. Furthermore, T cell function is influenced by fatty acids that are abundant in the circulation and atherosclerotic plaques. Here, we define the effect of oleic acid, one of the most abundant fatty acids in the human circulation, on the transcriptome of resting CD4+ T cells and subsequently the polarization of the cells into T cell subsets post-activation. We first performed RNA sequencing of resting CD4+ T cells from 9 donors after 0.5, 3, 24, 48, and 72 hours exposure to 30µg/mL oleic acid. This revealed the upregulation of genes involved in cholesterol biosynthesis (HMGCR, SQLE, MVD and MVK) and de novo fatty acid biosynthesis (ACACA and FASN). Both pathways point to a metabolic reprogramming of the cells towards a pro-inflammatory state. Subsequently, a 26-marker spectral flow cytometry panel showed increased frequencies of IL-9+ and IL-17A+ cells among activated CD4+ T cells pre-exposed to oleic acid. IL-9 and IL-17A are primarily produced by the respective T helper (TH) subsets TH9 and TH17, where, especially TH9, has been implicated in the aggravation of atherosclerosis. Our data shows that oleic acid may induce a shift in immunometabolism in resting CD4+ T cells towards an activated profile that leads to more pro-inflammatory and pro-atherogenic polarization of these cells post-activation. Taken together, our study signals a potential role for the interaction between circulating fatty acids and CD4+ T cells in the development and pathogenesis of atherosclerosis.

overall_design: Comparative DNA methylation analysis of EPIC array data (850k DNA methylation array) for resting human buffy coat derived CD4+ T cells exposed to control or 30μg/mL oleic acid for either 0.5, 3, 24, 48, or 72 hours. RNA and DNA were simultaneously extracted from the cell samples and DNA was sent for EPIC array measurements. There are 90 samples in total from 9 different donors, 45 exposed to the control (EtOH) and 45 exposed to 30μg/mL oleic acid. There are no replicates.

first: Nathalie

last: Reilly

first: Koen

middle: F

last: Dekkers

first: Friederike

last: Sonnet

first: Joanneke

middle: C

last: Kwekkeboom

first: Lucy

last: Sinke

first: Stan

last: Hilt

first: Hayat

last: Suleiman

first: Marten

last: Hoeksema

first: Hailiang

last: Mei

first: Erik

middle: W

last: van Zwet

first: Bart

last: Everts

first: Andreea

last: Ioan-Facsinay

first: J

last: Jukema

first: Bastiaan

middle: T

last: Heijmans


title: Chromosome conformation analysis of N1E115 cells by HiC

summary: Neuronal nitric oxide synthase 1 (NOS1) produces the gaseous signaling molecule nitric oxide (NO), which plays important roles in the development and function of the nervous system. The regulation of Nos1 gene expression is incompletely understood. Here, we explored the role of physiological hypoxia in the control of Nos1 transcription and the underlying mechanisms using N1E115 mouse neuroblastoma cells as a model. N1E115 cells were cultured for 3 days at high (20%) and low (4%) oxygen levels. Following the verification of upregulation of Nos1 mRNA and protein levels in response to 4% oxygen, we analyzed 3-dimensional chromosome conformation by HiC.

overall_design: N1E115 cells were cultured in the presence of 20% or 4% oxygen (at ambient barometric pressure). Cells were cross-linked with 1% formaldehyde and quenched by 2.5 mol/L glycine and processed using the Arima, Inc. HiC kit (Cat# A510008) or using NcoI followed by established protocol. Sequencing libraries were prepared with the KAPA Hyper Prep kit (Roche, Cat# KK8500) and sequenced using an Illumina HiSeq 4000 instrument.

first: Fei

last: Gao


title: Convergence of distinct RNA-silencing pathways on GW182/Tnrc6 [RIP-seq II]

city: Basel

first: Helge

last: Grosshans

address: Maulbeerstrasse 66

country: Switzerland

institute: Friedriche Miescher Institute for Biomedical Research (FMI)

summary: RIP-sequencing in mouse embryonic stem cells. Experiment 3 (Samples 2-24) & 4 (Samples 25-48): Base cell line (untagged control cells): GFP_5xT71BS (mES [129S6/SvEvTac], yGlob [GFP_5xT71BS], stable transfection FLAG-Cas9::T2A::Blasticidin). HA-Tnrc6a: endogenously HA-AVI-tagged Tnrc6a. HA-Tnrc6a_Trim71KO: HA-AVI-tagged Tnrc6a, Trim71-/-. Experiment 2 (Samples 19-42): Ctrl: base cell line GFP_5xT71BS. Tnrc6a: endogenously HA-AVI-tagged Tnrc6a. Tnrc6a_Trim71KO: HA-AVI-tagged Tnrc6a, Trim71-/-. Tnrc6a_Ago2KO: HA-AVI-tagged Tnrc6a, Ago2-/-.

overall_design: Cells were grown in triplicates to confluency in 10 cm dishes. Cells were washed 1x with PBS and lysed with lysis buffer (25 mM Tris-HCL at pH 7.5, 100 mM KCl, 0.5% [v/v] NP-40, 1 mM EDTA, EDTA-free cOmplete miniprotease inhibitor [Roche], PhosSTOP [Roche]. RNAse inhibitors [SUPERase•In™ RNase Inhibitor, Invitrogen, AM2694] was added to all buffers. Lysates were cleared by centrifugation. Equal amounts of protein were incubated with 20 µL of Pierce™ HA magnetic beads (Thermo Scientific, 88836) for 2 h at 4°C. Beads were washed three times with 200 µL of washing buffer (50 mM Tris-HCl at pH 7.5, 150 mM KCl, 5 mM MgCl2, 0.05% NP-40, EDTA-free cOmplete miniprotease inhibitor [Roche, 11836153001], PhosSTOP [Roche]). RNA was isolated using the Norgen single-cell RNA purification kit (Norgen, 51800) with on-column DNase digestion (Norgen, 25710).

last: Welte

first: Alison

last: Goulois

first: Michael

last: Stadler

first: Daniel

last: Hess

first: Charlotte

last: Soneson

first: Anca

last: Neagu

first: Chiara

last: Azzi

first: Marlena

last: Lübke

first: Jan

last: Seebacher

first: Isabel

last: Schmidt

first: David

last: Estoppey

first: Florian

last: Nigsch

first: John

last: Reece-Hoyes

first: Dominic

last: Hoepfner

last: Großhans


title: Convergence of distinct RNA-silencing pathways on GW182/Tnrc6 II

summary: RNA-sequencing in mouse embryonic stem cells: Ctrl: GFP_5xT71BS (mES [129S6/SvEvTac], yGlob [GFP_5xT71BS]. Trim71-/-: GFP_5xT71BS (mES [129S6/SvEvTac], yGlob [GFP_5xT71BS] Trim71 -/-. Note pervasive gene expression changes in samples with TNRC6A overexpression in comparison with samples with CAS9 overexpression.

overall_design: Cells were grown in triplicates 24-well dishes. Cells were washed 1x with PBS and lysed using the norgen single-cell RNA kit according to manifacturers instructions.

first: Thomas


title: Profile of protein-coding and long non-coding RNA expression and their clinical importance in T-cell acute lymphoblastic leukemia

city: NEW DELHI

first: Anita

last: Chopra

state: DELHI

address: Dr BRA-IRCH AIIMS

department: Laboratory Oncology

country: India

institute: All India Institute of Medical Science

summary: We analyzed the prognostic relevance of immunophenotype,expression pattern of protein coding genes, epigenetic modifiers and long non-coding RNAs in T-ALL.

overall_design: A total of 134 T-ALL cases diagnosed by morphology, cytochemistry and immunophenotyping, were enrolled in this study. The T-ALL patients were divided into 2 cohorts: Discovery (n=35) and Validation (n=99). Total RNA seq was performed in the Discovery cohort.

first: Deepak

last: Verma

first: Rajive

last: Kumar


title: SNRPD3 preserves balanced MYCN-driven alternate splicing and represents a therapeutic vulnerability in neuroblastoma

city: Randwick

last: Carter

address: Lowy Cancer Research Centre

country: Australia

institute: Children's Cancer Institute

summary: Many of the pro-tumorigenic functions of the oncogene MYCN are attributed to its regulation of global gene expression programs. Alternative splicing is another important regulator of gene expression and has been implicated in neuroblastoma development, however, the molecular mechanisms behind this remain unknown.  We found that MYCN up-regulated the core spliceosomal protein, SNRPD3, in models of neuroblastoma initiation and progression. MYCN directly bound SNRPD3, and the protein arginine methyltransferase, PRMT5. RNA-sequencing revealed a global increase in the number of genes being differentially spliced when MYCN was overexpressed, while depletion of SNPRD3 in the presence of overexpressed MYCN was sufficient to induce additional increases in differential splicing, particularly genes involved in the cell cycle. SNRPD3 knockdown in the presence of MYCN was accompanied with marked loss of cell viability, suggesting that SNRPD3 maintains splicing balance for MYCN, and prevents detrimental differential splicing of cell cycle genes in neuroblastoma. Further analysis revealed BIRC5 and CDK10 to be among the topmost differentially spliced cell cycle genes, both of which play a role in the G2/M phase of the cell cycle. Flow cytometry analysis revealed that depletion of SNRPD3 resulted in G2/M cell cycle arrest. Furthermore, SNRPD3 was an oncogenic co-factor for MYCN in vitro and in vivo. As such, SNRPD3 represents a therapeutic vulnerability for neuroblastoma cells in the presence of high MYCN expression. Indeed, the PRMT5 inhibitor, JNJ-64619178, that prevents SNRPD3 methylation was cytotoxic in neuroblastoma cell lines with high SNRPD3 and MYCN expression. Overall, our findings demonstrate a selective relationship exhibited between MYCN and SNRPD3 and suggest that SNRPD3 plays a vital role in MYCN-driven tumorigenesis and could serve as a novel therapeutic target.

overall_design: Leafcutter comparison of differential splicing in neuroblastoma cell line SHEP21N for 6 conditions (3 replicates per condition): i) SHEP21N cells + DOX + siControl (+SNRPD3-MYCN), ii) SHEP21N cells + DOX + siSNRPD3#1 (-SNRPD3-MYCN)_1, iii) SHEP21N cells + DOX + siSNRPD3#2 (-SNRPD3-MYCN)_2, iv) SHEP21N cells + DMSO + siControl (+SNRPD3+MYCN), v) SHEP21N cells + DMSO + siSNRPD3#1 (-SNRPD3+MYCN)_1, vi) SHEP21N cells + DMSO + siSNRPD3#2 (-SNRPD3+MYCN)_2

first: Alice

last: Salib

first: Nisitha

last: Jayatilleke

first: Chelsea

last: Mayoh


title: Effect of Beta-Hydroxybutyrate on Gene Expression in Immunotherapy and Castration Resistant Prostate Cancer Cells

city: Notre Dame

first: Xin

last: Lu

state: IN

address: 109 Galvin Life Science Center

department: Biological Sciences

institute: University of Notre Dame

summary: Attempt to determine the diffences of gene expression in castration and immunotherapy resistant prostate cancer by means of incubating some samples with the ketone beta-hydroxybutyrate. We wanted to see if there were any hallmarks of immune sensitization

overall_design: 2 samples of the PTEN/P53/SMAD4 castration and immunotherapy resistant prostate cancer cell line (PPS-ICBR ) were incubated in DMEM and two samples were incubated in DMEM with 3mM of Beta-hydroxybutyrate

first: Sean

last: Murphy

first: Sharif

last: Rahmy


title: Vorinostat treatment of PPS-ICBR cells

summary:
We generated immune checkpoint blockade resistant cell lines from a prostate tumor driven by Probasin-Cre Pten/P53/Smad4 loxp deletion, through serial implantation and ICB treatment. We then wanted to explore whether gene expression changes associated with resistance to immune checkpoint blockade could be reversed by treating ICB resistant cells with the pan-HDAC inhibitor Vorinostat.
We used a microarray to determine gene expression changes caused by Vorinostat treatment in these cells with biological duplicates.

overall_design: Cells were seeded at 60% confluency and treated overnight with either DMSO or Vorinostat dissolved in DMSO. RNA was extracted and converted into cDNA and sent out for analysis.


title: A ketogenic diet can revert acquired resistance to immunotherapy in prostate cancer through β-hydroxybutyrate-mediated inhibition of histone deacetylases

summary: Prostate cancer is the second leading cause of cancer deaths in men in the US. In the past decade, immune checkpoint blockade (ICB) drugs targeting programmed death 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) have revolutionized the field of cancer therapeutics, but the objective response rate in prostate cancers is in the low single digits. A major complicating issue is that immunotherapies can have significant adverse effects, so there is a clinical need for novel combinatorial strategies that do not involve additional or compounding adverse effects. We developed a model system of advanced prostate cancer that has acquired resistance to ICB therapy. The PD-1 resistant sublines showed strong downregulation of the mouse major histocompatibility complex (MHC-I), which was reversed by treating cells with either the pan-histone deacetylase (HDAC) inhibitor Vorinostat or β-hydroxybutyrate. When mice bearing these ICB resistant tumor are treated with either Vorinostat, with a cyclical ketogenic diet (CKD), or with a 1,3-butanediol diet (BD), tumors are resensitized to ICB therapy. We analyzed changes in the tumor immune microenvironment and showed that this effect is driven by a shift in monocyte differentiation, changing from M2 polarized immunosuppressive macrophages into iNOS+ dendritic cells instead. This differentiation is dependent on an ICB-induced increase in CD40L+ CD8+ T-cells in the tumor immune microenvironment. Our results indicate a novel mechanism in which a ketogenic diet can be used to increase therapeutic efficacy of ICB therapy.

overall_design: Samples consist of single cell suspensions, derived from 3 different tumors per treatment group (standard diet (SD), butanediol diet (BD), immune checkpoint blockade (ICB), and BD + ICB.

first: Dailin

last: Gan


title: Short-term effects of Elastase inhibition on human islets protein phosphorylation

first: Jonathan

middle: M

last: Dreyfuss

address: 1 Joslin Pl

department: Bioinformatics & Biostatistics Core

institute: Joslin Diabetes Center

summary: To evaluate the short-term effect of elastase inhibition using telaprevir or sivelestat or DMSO on human islets

overall_design: Human islets (N=3) were treated for 10 or 30 minutes with DMSO, telaprevir (1 and 100 µg/ml) or sivelestat (100 µg/ml) to evaluate the changes in phosphorylation levels of proteins using the KAM-1325 phospho antibody microarray (Kinexus).

first: Giorgio

last: Basile

first: Rohit

last: Kulkarni


title: Connective Tissue Growth Factor, a Potential Prognostic Marker of Triple Negative Breast Cancer treatment

city: Suwon-si

first: Jongsu

last: Kim

address: 2066 Seobu-ro, Jangan-gu, Suwon-si, Gyeonggi-do, Republic of Korea (16419)

country: South Korea

institute: Sungkyunkwan university

summary: Triple negative breast cancer (TNBC) patient with high level of connective tissue growth factor (CTGF) show poor survival and prognosis. Identification of novel target contributes to the development of TNBC treatment. Kahweol, a coffee diterpene molecule, is a natural compound with pharmacological activities such as anti-inflammatory, anti-angiogenetic, and apoptotic effect in several cancer cells. In this study, we demonstrated the involvement of CTGF expression level in TNBC treatment. Experiments were performed on MDA-MB 231, a TNBC cell line. Firstly, functional annotation and gene set enrichment analysis found that the CTGF involves in migratory pathways in TNBC treatment. We also investigated that kahweol regulates CTGF expression and functions as antitumor compound resulting in inhibition of cell migration and colony formation in TNBC. Next, CTGF knockdown synergically suppressed cell motility with kahweol. Migratory ability and colony conformation were also performed in vitro to confirm the results from functional annotation assay. Overall, our results suggest that CTGF is a potential target as a prognostic marker predicting the prognosis of TNBC treatment, and kahweol is a potential antitumor compound to regulate CTGF expression for TNBC.

overall_design: Examination of Kahweol treated MDA-MB-231 cell line transfected with control or CTGF siRNA

first: Jeong

middle: H

last: Lee

first: Hong

middle: S


title: Single-cell sequencing technology demonstrates cellular and functional heterogeneity within the prostate and between the transitional and peripheral zones

city: Beijijng

first: Xingbo

last: Long

address: No 1, Dongdan Dahua Road, Dongcheng District

department: Urology

institute: Beijing Hospital

summary: The identification of cellular and functional heterogeneity within the prostate and between the transitional zone (TZ) and peripheral zone (PZ) is critical for understanding the spatial distribution of prostate diseases. Three paired human prostate PZ and TZ tissue samples  were used for scRNA-seq.

first: X


title: Analyses of barcode repertoire in hematopoietic cells

city: Chiba

first: Ichiro

last: Manabe

address: 1-8-1 Inohana, Chuo

country: Japan

institute: Chiba University

summary: We compared the barcode repertoire in circulating leukocytes, and cardiac and renal macrophages.

overall_design: Amplicon sequencing.

first: Manabe

last: Ichiro

first: Nakayama

last: Yukiteru

first: Fujiu

last: Katsuhito


title: The -KTS splice variant of Wt1 is required for ovarian differentiation and sex determination

city: Rennes

first: Frédéric

last: Chalmel

state: France

address: 9 avenue du Pr. Léon Bernard

department: Physiology and physiopathology of the urogenital tract

country: France

institute: Inserm U1085-Irset

summary: Here we identify -KTS, a major alternatively spliced isoform of the Wilms’ tumor suppressor WT1, as a key determinant of female sex determination.

overall_design: E12 (23±3ts) gonads from wild-type, -KTS KO and +KTS KO mice were enzymatically dissociated and single cells were loaded on a 10× Chromium instrument. The libraries were sequenced using an Illumina HiSeq4000. Each genotype was performed in two independent replicates.

first: Marie-Christine

last: Chaboissier

first: Elodie

middle: P

last: Grégoire

first: Paul

last: Rivaud


title: Epigenetic analyses of hematopoietic stem cells

summary: We analyzed genome-wide chromatin accessibility in hematopoietic stem cells (HSCs) using ATAC-seq in mice after sham operation and transverse aortic constriction (TAC).

overall_design: ATAC-seq of HSCs.


title: Long-term effects of Elastase inhibition on human islets at single-cell resolution

summary: To evaluate the long-term effect of elastase inhibition using telaprevir, sivelestat, or DMSO on human islet cells.

overall_design: Human islets (N=3) were treated for 24 hours with DMSO, telaprevir (1 and 100 µg/ml), or sivelestat (100 µg/ml) to evaluate the changes in gene expression levels in endocrine and exocrine cells using single-cell RNA-seq (scRNA-seq) procedures.


title: Transcriptomic analyses of hematopoietic stem cells

summary: We analyzed transcriptomes of hematopoietic stem cells (HSCs) using RNA-seq in mice after sham operation and transverse aortic constriction (TAC).

overall_design: RNA-seq profiling of HSCs.


title: Expression analyses of circulating monocytes and cardiac macrophages in hematopoietic stem cell (HSC)-transplanted mice

summary: We analyzed transcriptomes of blood and cardiac macrophages in the mice transplanted with the mixture of HSCs from control and four-weeks post-transverse aortic constriction (TAC) mice.

overall_design: RNA-seq profiling of sorted monocytes and cardiac macrophages.


title: Transcriptomic and epigenetic analyses of hematopoietic stem cells

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

overall_design: Refer to individual Series


title: Gene expression analysis in platelets of septic patients

status: Public on Feb 12 2024

city: Hirakata

first: Natsuki

last: Anada

state: Osaka

address: 5-1 Shinmachi2

department: Anesthesiology

institute: Kansai Medical University

summary: Patients with severe sepsis are often associated with thrombocytopenia. This study comprehensively analyzes gene expression in platelets of patients with sepsis to explore the mechanisms by which septic patients develop thrombocytopenia.

overall_design: Three group comparison: Control patients, septic patients without thrombocytopenia and septic patients with thrombocytopenia

first: Yoshinobu

last: Nakayama

first: Yasufumi

last: Nakajima


title: Rewiring endothelial cell sphingolipid metabolism to favor S1P over ceramide signaling protects from coronary atherosclerosis

city: New York

first: Olivier

last: Elemento

state: NY

address: 1305 York Avenue

institute: WEILL MEDICAL COLLEGE OF CORNELL UNIV

summary: Growing evidence correlated changes in bioactive sphingolipids, particularly sphingosine-1-phosphate (S1P) and ceramides, with coronary artery diseases. Furthermore, specific plasma ceramide species can predict major cardiovascular events. Dysfunction of the endothelium lining  lesion-prone areas plays a pivotal role in the initiation and progression of atherosclerosis. Yet, how sphingolipid metabolism and signaling change and contribute to endothelial dysfunction and atherosclerosis remain poorly understood.  By using a mouse model of coronary atherosclerosis, we demonstrated that hemodynamic stress induces an early metabolic rewiring of endothelial sphingolipid de novo biosynthesis favoring S1P signaling over ceramide as protective response. Furthermore, our data are paradigm shift from  the current believe that ceramide accrual contributes to endothelial dysfunction.  The de novo biosynthesis of sphingolipids is commenced by serine palmitoyltransferase (SPT), and is downregulated by NOGO-B, an ER membrane protein. We showed that Nogo-B is upregulated by hemodynamic stress in myocardial endothelial cells (EC) of ApoE-/- mice and is expressed in the endothelium lining coronary lesions in mice and human. We demonstrated that mice lacking Nogo-B specifically in EC (Nogo-A/BECKOApoE-/-) were resistant to coronary atherosclerosis development and progression, and mortality. Fibrous cap thickness was significantly increased in Nogo-A/BECKOApoE-/- mice and correlated with reduced necrotic core and macrophage infiltration. Mechanistically, the deletion of Nogo-B in EC sustained the rewiring of sphingolipid metabolism towards S1P, imparting an atheroprotective transcriptional signature that refrain coronary atherogenesis and its progression.  These findings also set forth the foundation for sphingolipid-based therapeutics to reduce the treat this condition.

overall_design: FACS sorted ECs were isolated from hearts of ApoE-/- and Nogo-A/B(ECKO)ApoE-/- mice after 8 weeks post-TAC and were then processed for single cell RNAseq using standard 10x Chromium.

first: Annarita

last: Di Lorenzo

first: Yang

last: Hu


title: Gut microbiota-derived butyrate robustly facilitates prenatal development and neurogenesis of enteric nervous system.

city: Guangzhou

first: Weiqiang

last: Li

address: Zhongshan Road 2

institute: Sun Yat-sen university

summary: Our study demonstrates that the short-chain fatty acid, butyrate, greatly enhances the in utero development of ENCSCs and adult enteric neurogenesis, which was partially mediated through the receptors MCT1 and Ras signaling pathway. In conclusion, butyrate treatment may represent a potential therapy for various gastrointestinal motility disorders.

overall_design: 3 samples, including undifferentiated enteric neural crest stem cells (D0), neuronal committed ENCSCs under the treatment of NaB on D14 and D28 (D14-NaB, D28-NaB) were analyzed.

first: Qi

last: Zhao

first: Andy

last: Xiang


title: The effect of microcurrent-stimulation on MCF-7 and MDA-MB-231

city: 福岡市

first: Yuya

last: Yoshida

state: 福岡県

address: 東区馬出3-1-1 九州大学薬学部棟本館3F 薬剤学教室, 薬物動態学分野

institute: Kyushu-university

summary: Microcurrent-stimulation often uses wound healing, its effect on cancer cells is unknown.

overall_design: Differences in gene expression in MCF-7 and MDA-MB-231 with and without microcurrent-stimulation were measured.

first: Naoya

last: Matsunaga

first: Tomohito

last: Tanihara

first: Kaori

last: Yasuda

first: Atsushi

last: Doi


title: gRNA-seq from Ishikawa after medroxyprogesterone treatment

city: Jinan

first: Yao

last: Liu

state: Shandong

address: Wenhuaxi Road 107#

institute: Qilu Hospital of Shandong University

summary: Hormone therapy serves as a primary choice for fertility preservation and is also considered for advanced and recurrent cases with endometrial cancer. While a number of patients fail to respond favorably to hormone treatments. In order to explore the mechanisms underpinning hormone resistance, Here we constructed a whole-genome library for EAC cell lines using the CRISPR-Cas9 system and performed high-throughput sequencing after medroxyprogesterone treatment in this study. For CRISPR screening, ishikawa cells were infected pooled GeCkov2 lentiviral library with functional MOI of 0.3. Genomic DNA from each group was isolated and amplified by PCR. The PCR products were purified and subjected to NGS by using the Novaseq 6000-PE150 platform. After 48h of puromycin selection, the cells were divided into three groups, the baseline group cells were collected and frozen at -80 degree centigrade, the control group cells were treated with DMSO for 10 days while the MPA group cells were treated with 15 μM MPA for 10 days.

overall_design: Genome-wild lentiviral Human_GeCKOv2_Library _B was purchased from AZENTA LIFE SCIENCES . For CRISPR screening, 1×10^8 ishikawa cells were infected with pooled GeCKOv2 lentiviral library with functional MOI of 0.3. After 48h of puromycin selection, the cells were divided into three groups, the "baseline" group cells were collected and frozen at -80C, the "control" group cells were treated with DMSO for 10 days while the "MPA" group cells were treated with 15 μM MPA for 10 days. Genomic DNA from each group was isolated using HMV DNA Kit and amplified by PCR. The PCR products were purified and subjected to NGS by using the Novaseq 6000-PE150 platform.

first: Shourong

last: Wang

first: Jie

last: Jiang


title: A novel bystander effect in tamoxifen treatment: PPIB derived from ER+ cells attenuates ER- cells via endoplasmic reticulum stress-induced apoptosis

city: Wuhan

first: Tinglin

last: Yang

state: Hubei

address: Jiefang Avenue 1277, Wuhan, Hubei Province, P. R. China

department: Department of Breast and Thyroid Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology

summary: Tamoxifen (TAM) is the frontline therapy for estrogen receptor-positive (ER+) breast cancer in premenopausal women that interrupts ER signaling. As tumors with elevated heterogeneity, amounts of ER-negative (ER-) cells are present in ER+ breast cancer that cannot be directly killed by TAM. Despite complete remissions have been achieved in clinical practice, the mechanism underlying the elimination of ER- cells during TAM treatment remains an open issue. Herein, we deciphered the elimination of ER- cells in TAM treatment from the perspective of the bystander effect. Markable reductions were observed in tumorigenesis of ER- breast cancer cells by applying both supernatants from TAM-treated ER+ cells and a transwell co-culture system, validating the presence of a TAM-induced bystander effect. The major antitumor protein derived from ER- cells, peptidyl-prolyl cis-trans isomerase B (PPIB), is the mediator of the TAM-induced bystander effect identified by quantitative proteomics. The attenuation of ER- cells was attributed to activated BiP/eIF2α/CHOP axis and promoted endoplasmic reticulum stress (ERS)-induced apoptosis, which can also be triggered by PPIB independently. Altogether, our study revealed a novel TAM-induced bystander effect in TAM treatment of ER+ breast cancer, raising the possibility to develop PPIB as a synergistic antitumor agent or even substitute endocrine therapy.

overall_design: ER- breast cancer cell lines MDA-MB-231 and BT-549 were treated with the conditioned medium generated from TAM-treated ER+ cell line MCF7. MDA-MB-231 and BT-549 cells treated with the conditioned medium from DMSO-treated MCF7 was used as control groups. RNA sequencing was conducted to analyze the diffrentially expressed genes.

first: Wenhui

last: Ming

first: Tao

last: Huang


title: Effect of MKP5 deletion in bone-marrow-derived macrophages on gene expression during oxidized-LDL stimulation

city: Philadelphia

first: Jun

last: Yu

state: PA

address: 1058 MERB, CMDR, 3500 N Broad St

department: CMDR

institute: Temple University

summary: MKP-5 is a MAP kinase phosphatase that is responsible for the dephosphorylation of MAP kinases p-38 MAPK, JNK, and sometimes ERK. MKP-5 has been implicated in many diseases and its deletion results in increased inflammation. However, MKP-5 expression and regulation have not been studied in atherosclerosis, and our project aims to identify MKP-5 as a relevant gene in this disease. Specifically, we aim to identify the role of MKP-5 in the regulation of plaque stability. In atherosclerotic diseased states, oxidized LDL particles promote inflammatory mechanisms and form macrophage foam cells. We show that comparing wild-type and MKP-5-/- bone-marrow-derived macrophages stimulated with oxidized-LDL, results in the identification of significant gene expressions and major up- and down-regulated targeted pathways via bulk-RNA sequencing. Taken together, our data provides an unbiased way to test the mechanistic role of MKP-5 in an atherogenic environment. Furthermore, our data confirms and contributes to the current knowledge of gene expression influenced by MKP-5, as well as the influence of MKP-5 deletion on pathways critical to plaque stability including inflammation and collagen synthesis and deposition.

overall_design: To investigate the effect of MKP5 deletion on gene expression and major targeted pathways, bone marrow-derived macrophages (BMDMs) were isolated from the femurs of WT and MKP-5-/- mice and differentiated during a 7-day culture period with BMDM growth media containing 15% of L-929 supernatant. WT and MKP-5-/- BMDMs were then stimulated with ox-LDL (20µg/mL) for 0hr and 24hrs and collected at the respective end time point. Samples were then processed for RNA isolation and sent for bulk RNA seq using 1ug of RNA per sample.

first: Baldini

last: Margaret

first: Azuma

last: Masashi

first: Yu

last: Jun


title: Expression data from healthy C57BL/6 mice treated with BAMLET

city: Lund

first: Ines

last: Ambite

address: Klinikgatan 28

department: Laboratory Medicine

country: Sweden

institute: Lund University

summary: Tumor surveillance is essential to stop tumor progenitors in their tracks. Clearly, endogenous protective mechanisms other than immune surveillance are important, but their molecular nature and mechanisms of action remain unclear. In the newborn intestine, the immature cells need to be surveilled to promote differentiation and tumor development prevented. This study addressed if molecules in milk may be relevant for tumor surveillance. Alpha-lactalbumin, which is the most abundant protein in human milk and crucial for the survival of mammals, due to its role in lactose synthesis, also forms tumoricidal oleic-acid complexes with documented therapeutic efficacy. This study identifies BAMLET, a complex formed by bovine alpha-lactalbumin and oleic acid, as a tumor surveillance molecule. BAMLET substitution of the drinking water protected tumor-prone ApcMin/+ mice from developing intestinal cancer and prolonged their survival, with pronounced inhibition of genes regulating the tumor environment, metastasis, Wnt/β-catenin signaling and cancer related gene networks. Unexpected extra-intestinal effects on lungs, livers, kidneys and spleens further suggested a protective effect of BAMLET beyond the intestine. These effects were specific for the cancer-prone ApcMin/+ mice.  Healthy C57BL/6 mice were largely unaffected by peroral BAMLET administration, except for improved metabolic functions such as lipid and glucose metabolism and insulin resistance and there was no evidence of toxicity. The results suggest that molecules, which have evolved to support tissue differentiation, may identify and remove tumor progenitors. The findings support the potential of BAMLET as a tumor surveillance molecule with potential for clinical use in cancer-prone patients.

overall_design: Female C57BL/6 mice received 20 mg of BAMLET in 2 x 200 μl PBS (BAMLET) or 2 x 200 μl of PBS alone (sham) by daily gavage for ten days. The mice were sacrificed two or five weeks after the end of treatment. Alternatively, mice received 20 mg of BAMLET in 5 ml PBS or PBS alone, daily in the drinking water until sacrifice after eight weeks. RNA isolated from intestine was subjected to Affymetrix whole genome transcriptomic analysis.

first: Hien

last: Tran

first: Murphy

last: Wan

first: Catharina

last: Svanborg


title: Effect of HORMAD1 and CT83 Cancer/Testis Gene expression on human mammary epithelial cell line (HMLE)

city: Paris

first: Pierre-Antoine

last: Defossez

address: 35 rue Hélène Brion

institute: Epigenetics and Cell Fate Unit

summary: Breast cancer is the most prevalent type of cancer in women worldwide. Within breast tumors, the basal-like subtype has the worst prognosis and no dedicated therapy, prompting the need for new tools to understand, detect, and treat these tumors. Certain germline-restricted genes show aberrant expression in tumors and are known as Cancer/Testis genes; their misexpression has diagnostic and therapeutic applications. Here we designed a new approach to examine Cancer/Testis gene misexpression in breast tumors. We identify several new markers in Luminal and HER-2 positive tumors, some of which predict response to chemotherapy. We then use machine learning to identify the two Cancer/Testis genes most associated with basal-like breast tumors: HORMAD1 and CT83. We show that these genes are expressed by tumor cells and not by the microenvironment, and that they are not expressed by normal breast progenitors; in other words, their activation occurs de novo. We find these genes are epigenetically repressed by DNA methylation, and that their activation upon DNA demethylation is irreversible, providing a memory of past epigenetic disturbances. Basal-like tumors expressing both genes have a poorer outcome than tumors expressing either gene alone or neither gene. Simultaneous expression of both genes in breast cells in vitro has a synergistic effect that increases stemness and activates a transcriptional profile also observed in double-positive tumors. Therefore, we reveal a functional cooperation between Cancer/Testis genes in basal breast tumors; these findings have consequences for the understanding, diagnosis, and therapy of the breast tumors with the worst outcomes.

overall_design: To investigate the function HORMAD1 and CT83 in HMLE cell line, and their eventual cooperation, we established 4 HMLE-derived cell lines overexpressing Control, HORMAD1, CT83 or HORMAD1 and CT83 transgenes. Cell lines were selected using puroResistance gene.

first: Laisné

last: Marthe

first: Gupta

last: Nikhil

first: Defossez

last: Pierre-Antoine


title: LET-381/FoxF and its target UNC-30/Pitx2 specify and maintain the molecular identity of C. elegans mesodermal glia that regulate motor behavior

first: Nikolaos

last: Stefanakis

address: 1230 York Avenue

department: Laboratory of Developmental Genetics

institute: The Rockefeller University

summary: Differential RNAseq analysis to identify transcripts enriched in the GLR  glia of C. elegans

overall_design: GLR glia were isolated from C. elegans L4 larvae, strain OS11715,  by FACS, based on expression of a nuclear YFP marker under control of the GLR glia-specific promoter nep-2prom7. All other cells, which are not expressing YFP, were collected separately for control. RNA reads from YFP-positive and YFP-negative samples were compared to identify GLR glia-enriched genes.

first: Shai

last: Shaham


title: Differential expression of paralog RNA binding proteins establishes a dynamic splicing program required for normal cerebral cortex development

first: Pierre

last: de la Grange

address: 29, rue du Faubourg Saint-Jacques

department: Paris Biotech Santé

institute: GenoSplice technology

summary: The paralog RNA binding proteins (RBPs) Sam68 and SLM2 are co-expressed in the cerebral cortex and display very similar splicing activity. However, their relative function(s) in this context is unknown. By performing a time-course analysis, we found that these RBPs exhibit an opposite expression pattern during development. Sam68 expression declines postnatally while SLM2 increases after birth, and this developmental pattern is reinforced by hierarchical control of Sam68 expression by SLM2. Analysis of Sam68:Slm2 double knockout (Sam68:Slm2dko) mice revealed hundreds of exons that are sensitive to concomitant ablation of these proteins. Moreover, parallel analysis of single and double knockout cortices indicated that exons regulated mainly by SLM2 are characterized by a dynamic splicing pattern during development, whereas Sam68-dependent exons are spliced at relatively constant rates. Dynamic splicing of SLM2-sensitive exons is completely suppressed in the Sam68:Slm2dko developing cortex. Sam68:Slm2dko mice die perinatally with defects in neurogenesis and in neuronal differentiation, and the development of a hydrocephalus, consistent with splicing alterations in genes related to these biological processes. Thus, our study reveals that maintenance of the Sam68 and Slm2 paralog genes encoding homologous RBPs enables the orchestration of a dynamic splicing program while ensuring a robust redundant mechanism that supports proper cortical development.

overall_design: 3 cortex of Wild type mice at the embrional day of 18.5 and 3 cortex of Sam68:Slm2ko mice at the embrional day of 18.5

first: Eleonora

last: Cesari

first: Donatella

last: Farini

first: Vanessa

middle: N

last: Medici

first: Ingrid

last: Ehrmann

first: Chiara

last: Naro

first: Marika

last: Guerra

first: Maria

last: Geloso

first: Livia

last: Labarbera

first: Marcello

last: D'Amelio

first: Tiziana

last: Orsini

first: Stefano

last: Vecchioli

first: Erika

last: Testa

last: Viscomi

first: Luca

last: Tamagnone

first: David

middle: J

last: Elliot

first: Claudio

last: Sette


title: The RNA helicase DDX1 associates with the nuclear RNA exosome and modulates R-loops [DRIP-seq]

city: Atlanta

first: Bing

last: Yao

address: 615 Michael Street

department: Department of Human Genetics

institute: Emory University

summary: The RNA exosome is a ten-subunit complex that mediates both RNA processing and degradation. This complex is evolutionarily conserved, ubiquitously expressed, and required for fundamental cellular functions, including rRNA processing. The RNA exosome plays many roles in regulating gene expression and protecting the genome, including modulating the accumulation of R-loops at sites of transcription. The RNA exosome interacts with specific target RNAs for decay or processing by interacting proteins termed cofactors. Attributing to the essential function of the RNA exosome for processes critical in all cells, such as production of mature rRNA, missense mutations in structural subunit genes of this complex cause neurological diseases. One possibility for why point mutations cause neurological diseases is that the RNA exosome partners with cell- or tissue-specific protein cofactors. Here, we employed a murine neuronal cell line (N2A) and performed immunoprecipitation of the RNA exosome subunit, EXOSC3, followed by mass spectrometry to obtain a snapshot of the RNA exosome interactome. We validated an interaction with DDX1, a putative RNA helicase. DDX1 plays roles in double-strand break repair, rRNA processing, and RNA metabolism. To explore shared functions of EXOSC3 and DDX1, we investigated the interaction after inducing DNA damage, and performed DNA/RNA immunoprecipitation followed by sequencing (DRIP-Seq) and RNA-seq on N2A cells depleted of either EXOSC3 or DDX1. These findings suggest that EXOSC3 and DDX1 function together in the absence of severe DNA damage to modulate spontaneous events such as RNA-DNA hybrid (R-loop) formation.

overall_design: R-loop profiles of DDX1 or EXOSC3 knockdown N2a cells

first: Julia

middle: L

last: de Amorim

first: Sara

last: Leung

first: Ramona

last: Haji-Seyed-Javadi

first: Yingzi

last: Hou

last: Yu

first: Homa

last: Ghalei

first: Sohail

last: Khoshnevis

first: Anita

last: Corbett


title: The RNA helicase DDX1 associates with the nuclear RNA exosome and modulates R-loops


title: UBTF tandem duplication acute myeloid leukemias are sensitive to menin inhibition

city: Memphis

first: Jeffery

last: Klco

state: TN

address: 262 Danny Thomas Place

department: Pathology

institute: St Jude Children's Research Hospital


title: Expression data from Apcmin/+ mice treated with BAMLET

first: Ines

last: Ambite

overall_design: Ten-week-old female ApcMin/+ mice received 20 mg of BAMLET in 2 x 200 μl PBS (BAMLET) or 2 x 200 μl of PBS alone (sham) by daily gavage for ten days. The mice were sacrificed two or five weeks after the end of treatment. Alternatively, mice received 20 mg of BAMLET in 5 ml PBS or PBS alone, daily in the drinking water until sacrifice after eight weeks or long-term follow-up. Isolated RNA from intestine, liver, spleen, kidney was subjected to Affymetrix whole genome transcriptomic analysis.

first: Hien

last: Tran

first: Murphy

last: Wan

first: Catharina

last: Svanborg


title: The RNA helicase DDX1 associates with the nuclear RNA exosome and modulates R-loops [RNA-seq]

overall_design: RNA profiles of DDX1 or EXOSC3 knockdown N2a cells


title: The translational protein 4E-T regulates the balance between differentiation and establishment of postnatal neural stem cell pools

city: Toronto

first: Adelaida

last: Kolaj

state: Ontario

address: 27 King's College Circle

country: Canada

institute: University of Toronto

summary:
Microarrays were performed (N=3) on postnatal precursors grown as neurospheres to determine level of gene expression
To determine the transcript targets of the translational regulator 4E-T and whether these targets are repressed, we performed RIPs (GSE224966) for these targets as well as Ribotag (GSE224853) in addition to the following microarray as control to determine gene expression in posntatal precursors and ensure the differences observed in Ribotag were not simply due to differences in gene expression levels

overall_design: Postnatal neural precursors were obtained by dissecting the V-SVZ of P8 pups and culturing the NPCs as neurospheres for ~7 days

first: Dennis

last: Gallagher


title: Transcriptomics study on HepG2 cells treated with 0.001 μM 2,4-D for 0, 10, 30, 360 min

city: Beijing

first: Yuan

last: Li

address: 38 Science Park Road, National Center for Protein Sciences (Beijing), Changping District, Beijing

department: Post-translational modification

institute: Beijing Proteome Research Center

summary: With the general adoption of new approach methodologies, the omic-based technologies are of particular importance for chemical hazard characterization owning to the premise that any apical endpoint change indicative of impaired health must be underpinned by some alterations at the omic level. In this work we studied cellular response to coumarin by measuring transcriptomics experiments. The HepG2 cells were treated with 0.001 μM 2,4-D for 0, 10, 30, 360 min. Each group has 3 biological replicates.

overall_design: The HepG2 cells were treated with 0.001 μM 2,4-D for 0, 10, 30, 360 min. Each group has 3 biological replicates (12 samples in total).

first: Ping

last: Xu

first: Tao

last: Zuo

first: Qi

last: Long


title: PC4 is a post-transcriptional manipulator of D-type cyclins and centromere proteins (eCLIP-Seq).

city: Chongqing

first: Qimei

last: Pan

address: No.30, Gaotanyan Stright Street, ShaPingba District

institute: Army Medical University

summary:
To investigate the binding site in which PC4 protein association with its target RNAs.
eCLIP-seq was performed to profile PC4-binding RNA targets in Huh7 cells.

overall_design: The PC4-RNA complex was immunoprecipitated by anti-PC4 antibody with eCLIP assay protocol. Then all the PC4-binding RNAs were analyzed by deep sequencing by using an Hiseq Xten platform.

first: Chunmeng

last: Shi


title: PC4 is a post-transcriptional manipulator of D-type cyclins and centromere proteins (RIP-Seq).

summary:
To investigate the mRNA targets of PC4 that worked as an RNA binding protein to control cell cycle progression.
RIP-seq was displayed to profile PC4-binding RNA targets in Huh7 cells.

overall_design: The RNA-protein complexes were enriched using anti-PC4 antibody conjugated with protein A/G beads. Then all the PC4-binding RNAs were analyzed by deep sequencing by using an Illumina HiSeq 2500 platform.


title: Adult and larval tracheal systems exhibit different molecular architectures in Drosophila

city: Kiel

first: Judith

last: Bossen

address: Am Botanischen Garten 3-9

department: Molecular Physiology

country: Germany

institute: CAU Kiel, Zoological Institute

summary: We analysed the RNA-seq profile of larval and adult trachea of the fruitfly Drosophila melanogaster, as well as from whole flies.

overall_design: We dissected the tissue manually and synthezised cDNA. Low amounts of cDNA were amplified via PCR and used for RNA sequencing.

first: Thomas

last: Roeder

first: Ruben

last: Prange


title: Glutaminase inhibition in combination with azacytidine in myelodysplastic syndromes: Clinical efficacy and correlative analyses

city: Houston

first: Xiaoping

last: Su

state: Texas

address: 7300 Brompton St, Apt 5633

institute: MD Anderson Cancer Center

summary: Malignancies can become reliant on glutamine as an alternative energy source and as a facilitator of aberrant DNA methylation, thus implicating glutaminase (GLS) as a potential therapeutic target. We demonstrate preclinical synergy of telaglenastat (CB-839), a selective GLS inhibitor, when combined with azacytidine (AZA), in vitro and in vivo, followed by a phase Ib/II study of the combination in patients with advanced MDS. Treatment with telaglenastat/AZA led to an ORR of 70% with CR/mCRs in 53% patients and a median overall survival of 11.6 months. scRNAseq and flow cytometry demonstrated a myeloid differentiation program at the stem cell level in clinical responders. Expression of non-canonical glutamine transporter, SLC38A1, was found to be overexpressed in MDS stem cells; was associated with clinical responses to telaglenastat/AZA and predictive of worse prognosis in a large MDS cohort. These data demonstrate the safety and efficacy of a combined metabolic and epigenetic approach in MDS.

overall_design: Bone marrow samples were harvested from MDS patients at the time of enrollment to clinical trial and after treatemnt completion and submitted to scRNA  analysis.

first: Marina

last: Konopleva

first: Natalia

last: Baran


title: PC4 is a post-transcriptional manipulator of D-type cyclins and centromere proteins (RNA-Seq).

summary:
To investigate the downstream targets of PC4 in controlling cell cycle, we knockdowned PC4 with two different siRNAs in Huh7 cells.
We performed RNA-seq in Huh7 cells transfected with NC, siPC4-1 or siPC4-2.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for contrl and PC4 knockdown (siPC4-1.siPC4-2) in Huh7 cells.


title: A Biodistribution Study of OTV vs. ASO by Intravenous or Intrathecal Administration in Cynomolgus Monkeys

city: South San Francisco

last: Sandmann

email: genomics@dnli.com, sandmann@dnli.com

state: California

address: 161 Oyster Point Blvd

institute: Denali Therapeutics

summary: The objective of this study is to evaluate ASO biodistribution and pharmacodynamic response following intravenous delivery of conjugated OTV, which utilizes transferrin-receptor binding to deliver ASO, vs. intrathecal delivery of naked ASO in cynomolgus monkeys, the current gold standard for ASO delivery. Three groups were included for comparison: 1) Negative control cohort = Four biweekly intravenous doses of saline (n=3); 2) IT cohort = Single intrathecal dose of 4mg MALAT1 ASO (n=3); 3)  OTV multi dose cohort = Four biweekly intravenous doses of 30mpk OTV:MALAT1 (n=3). Tissues were collected two weeks after the final dose to allow sufficient time for target knockdown. Brain, spinal cord, and peripheral tissues were dissected and frozen for molecular analysis. Compared to an intrathecal injection of ASO, systemic administration of OTV resulted in significantly more homogenous biodistribution of ASO in the central nervous system as measured by a probe-based hybridization assay as well as staining with an anti-ASO antibody. One potential consequence of heterogeneous ASO distribution is heterogeneous target knockdown throughout the CNS. We observed more consistent target knockdown throughout the cortex, subcortical areas, and spinal cord of monkeys dosed peripherally with OTV. By contrast, monkeys dosed intrathecally with naked ASO showed much more knockdown in the spinal cord compared to the brian, consistent with ASO distribution.

overall_design: Single dose of 4mg MALAT1 ASO intrathecally (“ASO IT”), n=3 2)    Four biweekly doses of saline intravenously (“Saline” or “Vehicle”), n=3 3)    Four biweekly doses of 30mpk OTV:MALAT1 intravenously (“OTV IV”), n=3 Tissues were collected two weeks after the final dose to allow sufficient time for target knockdown. Brain tissue was dissected and frozen for pharmacodynamic analysis. Compared to an intrathecal injection of ASO, systemic administration of OTV resulted in significantly more widespread and homogenous biodistribution of ASO in the central nervous system as measured by staining with an anti-ASO antibody

first: Scarlett

last: Barker

first: David

last: Tatarakis

first: Connie

last: Ha

first: Sarah

last: DeVos


title: Post-transcriptional regulation shapes the transcriptome of quiescent yeast

city: Seattle

first: Alison

last: Greenlaw

state: WA

address: 1100 Fairview Ave N

department: Basic Sciences

institute: Fred Hutch Cancer Center

summary: To survive in nutrient-poor conditions, cells must exit the cell cycle and enter a reversible non-replicative state known as quiescence. Yeast cells reprogram their gene expression during quiescence entry to silence transcription, but how the nascent transcriptome changes in quiescence has not been determined. By investigating the nascent transcriptome in quiescent yeast cells, we found noncoding transcription represented a larger portion of the quiescent transcriptome than in G1. To enable our nascent transcriptome analyses, we annotated over a thousand noncoding RNAs (ncRNAs) in quiescence and G1. Our work revealed that both mRNA and ncRNA are subject to increased post-transcriptional regulation in quiescence compared to G1. We found that the nuclear exosome-NNS pathway suppresses over one thousand mRNAs, in addition to canonical noncoding RNAs, in quiescence. In quiescence, a minority of the mRNAs affected by the NNS-nuclear exosome pathway are the same as those identified when glucose was removed, demonstrating a previously unidentified role for the pathway. RNA sequencing through the diauxic shift revealed at least two distinct time points at which the nuclear exosome controls the abundance of mRNAs involved in protein production, cellular organization, and metabolism, for optimal quiescence entry. Both transcription and post-transcriptional regulation are dramatically reprogrammed in quiescence, shifting the balance of noncoding and coding transcripts. The nuclear exosome and NNS are crucial for proper regulation of both mRNAs and ncRNAs in quiescence and through quiescence entry.

overall_design: To understand how the nascent transcriptome changes in quiescence, we measured both nascent and steady-state transcription in wild-type, RRP6-deleted, and Nab3-depleted yeast cells in both quiescent and G1 cell states. RNA-seq experiments were performed to obtain measures of steady-state transcription for the different cell states and genetic backgrounds. To obtain measures of both nascent and steady-state transcription, 4tU-seq experiments were conducted for the same cell states and genetic backgrounds. RNA-seq was performed for RRP6-deleted and wild-type control cells in the hours surrounding the diauxic shift. All experiments were performed with two biological replicates, and technical replicates were generated for some samples.

first: Kris

middle: G

last: Alavattam

first: Toshio

last: Tsukiyama


title: Transcriptomics study on HepG2 cells treated with different doses of coumarin for 6 h.

first: Yuan

last: Li

summary: With the general adoption of new approach methodologies, the omic-based technologies are of particular importance for chemical hazard characterization owning to the premise that any apical endpoint change indicative of impaired health must be underpinned by some alterations at the omic level. In this work we studied cellular response to coumarin by measuring transcriptomics experiments. The HepG2 cells were treated with 6 doses of coumarin for 6 h. 1 control group was set (0 uM of coumarin). Each group has 3 biological replicates.

overall_design: The HepG2 cells were treated with 6 doses of coumarin for 6 h. 1 control group was set (0 uM of coumarin). Each group has 3 biological replicates (21 samples in total).

first: Ping

last: Xu

first: Tao

last: Zuo


title: Head to head comparison of target knockdown following OTV vs. anti-TfR:ASO administration in mice

email: tatarakis@dnli.com, genomics@dnli.com

department: Discovery Genomics

summary: Using single nucleus RNA sequencing, we determined that peripheral administration of OTV:Malat1 results in much more widespread and robust target knockdown throughout the mouse brain compared to anti-TfR:Malat1.

overall_design: TfR.KI mice received four weekly doses of 50mpk OTV:Malat1, anti-TfR:Malat1, Malat1 ASO (at an ASO-equivalent dose), or saline. Tissues were harvested 72 hours after the final dose and snap frozen for molecuar analysis.


title: Glioblastoma stem cells after METTL8 knockdown.

city: Singapore

first: Oleg

last: Grinchuk

address: 61 Biopolis Dr, Proteos

country: Singapore

institute: IMCB

summary: This project investigates the role METTL8 in human glioblastoma stem cells. In order to understand the transcriptomic changes upon deletion of METTL8, we performed this RNA seq and further downstream analyses to determine the pathways changed. From this, we hope to identify novel methods to target glioblastoma stem cells via manipulation of METTL8.

overall_design: RNA-seq from glioblastoma stem cell specimens (TS-543) following treatment with either non-targeting RNA (shNT) , targeting RNA sh515  or targeting RNA sh841.

first: Bernice

last: Lee

first: Derrick

last: Ong


title: Circulating exosomal microRNAs contribute to the antimigraine effects of acupuncture

city: Chengdu

first: Lu

last: Liu

state: Sichuan Province

address: No.37 Shier Qiao Road

institute: Chengdu University of Traditional Chinese Medicine

type: Non-coding RNA profiling by high throughput sequencing

summary: We conducted this study to determine whether exosome regulation underlies the antimigraine mechanisms of acupuncture. By comparing serum samples from patients with migraine and healthy controls using high-throughput small RNA sequencing technology , we identified 705 exosomal microRNAs that are differentially expressed in patients with migraine, and this set of 705 microRNAs included five that are particularly well characterised (hsa-miR-369-5p, hsa-miR-1268b, hsa-miR-145-5p, hsa-miR-222-5p, and hsa-miR-4488). By comparing serum samples collected from patients with migraine before and after acupuncture treatment, we showed that acupuncture normalised the expression levels of those five well-characterised exosomal microRNAs.

overall_design: We compared differential expressed serum-derived exosomal miRNAs between the migraine patients group and healthy controls. And we compared differential expressed serum-derived exosomal miRNAs in migraine patients before and after acupuncture

first: Wenchuan

last: Qi

first: Yanan

last: Wang

first: Xixiu

last: Ni

first: Shan

last: Gao

first: Ziyang

last: Zhou

first: Zhenxi

last: He

first: Daohong

last: Chen

first: Dingjun

last: Cai

first: Ling

last: Zhao


title: Transcriptomics study on HepG2 cells treated with different doses of coumarin for 24 h

summary: With the general adoption of new approach methodologies, the omic-based technologies are of particular importance for chemical hazard characterization owning to the premise that any apical endpoint change indicative of impaired health must be underpinned by some alterations at the omic level. In this work we studied cellular response to coumarin by measuring transcriptomics experiments. The HepG2 cells were treated with 6 doses of coumarin for 24 h. 1 control group was set (0 uM of coumarin). Each group has 3 biological replicates.

overall_design: The HepG2 cells were treated with 6 doses of coumarin for 24 h. 1 control group was set (0 uM of coumarin). Each group has 3 biological replicates (21 samples in total).


title: Evaluating genome-wide expression following the disruption of CDK12 reveals a bimodal pattern of gene regulation

city: Boston

first: Cherubin

last: Manokaran

state: Massachusetts

address: 450 Brookline Ave

department: Cancer Biology

institute: Dana Farber Cancer Institute

summary: We demonstrate that the loss of CDK12 protein or activity, unlike CDK9 or CDK7, significantly upregulates the expression of certain genes, including genes whose activation contributes to the anti-proliferative effects of CDK12 inhibition. In HER2+ breast cancer, a malignancy where CDK12 is almost invariably co-amplified with HER2, CDK12 inhibition drives up the expression of MYC, which is toxic to HER2+ cancer cells. This upregulation of c-myc largely mediates the killing effect of CDK12 inhibition. Therefore, we report a novel mode of regulation of gene expression via CDK12, which represents a promising target for cancer driven by HER2 gene amplification.

overall_design: Examination of the effects of inhibting three transcriptional kinases (CDK12, CDK9, CDK7) on genome-wide expression

first: Yubao

first: Thomas

last: Roberts


title: Microarray-analysis of primary lung fibroblasts cell lines (established from 3-4weeks old mice)

first: Kalluri

last: Lab

address: 1901 East Rd

institute: UT MD Anderson Cancer Center

summary:
Purpose:  The goal of this analysis is to compare gene expression profiling of activated-fibroblasts lines (expressing K-RasG12D) to non-activated-fibroblast expressing WT K-Ras.
Methods: Fibroblasts lines were obtained after lung-tissue digestion with collagenase. Cells were homogenized in TRIzol reagent and total RNA was extracted according to manufacturer’s protocol. Hybridization of RNA was performed using Affymetrix Mouse-Gene 1.0ST platform by Dana Farber Microarray Core Facility and data analysis was performed using dChip software. Gene expression analysis was conducted using Transcriptome Analysis Console Software (TAC) using comparing method (SKY vs WT).
Results: Gene expression analysis revealed deregulation of several canonical genes involved in different signaling pathways, as Wnt, Notch and Hedgehog signaling pathways. Deregulations of ECM remodeling and focal adhesion expression were reported as well. In addition the secreted molecule Hgf was increased in activated fibroblasts compared to WT line.

overall_design: Examination of 2 different mRNA profiles of 3-4weeks old wild type (WT) and SKY (K-RasG12D) lung fibroblasts cell lines

first: Mariangela

last: Natale

first: Raghu

last: Kalluri


title: Annexin-A1-mediated regulation of the efferocytosis in tumor-associated macrophage promotes anti-tumor immune response by activating the cGAS/STING pathway in pancreatic cancer

status: Public on Feb 11 2024

city: Fuzhou

first: Changgan

state: --- Select One ---

address: #29 Xinquan Road, Fuzhou 350001, People’s Republic of China

institute: Fujian Medical University Union Hospital

summary: To explore the mechanism of macrophage Anxa1 deficiency to induce anti-tumor responses, we  preformed RNA sequencing of pancreatic tumors from Anxa1ΔMɸ and Anxa1WT mice.

overall_design: Panc02 cells were subcutaneously implanted into Anxa1flox/flox and Anxa1ΔMɸ mice. After 4 weeks, the mice were sacrificed and the tumors were obtained for RNA sequence.

first: Zelin

last: Hou


title: E2F1 mediates competition, proliferation and drug sensitivity of co-cultured resistant and sensitive ovarian cancer cells.

city: Chicago

first: Daniela

middle: Elena

last: Matei

state: IL

address: 303 E Superior St

department: Obstetrics and Gynecology

institute: Northwestern University

summary: Background: Tumor heterogeneity is one of the key factors leading to chemo-resistance relapse. It remains unknown how resistant cancer cells influence sensitive cells during cohabitation and growth within a heterogenous tumors. The goal of our study was to identify driving factors that mediate the interactions between resistant and sensitive cancer cells and to determine the effects of cohabitation on both phenotypes. Methods: We used isogenic ovarian cancer cell (OC) lines pairs, sensitive and resistant to platinum: OVCAR5 vs. OVCAR5 CisR and PEO1 vs. PEO4, respectively, to perform long term direct culture and to study the phenotypical changes of the interaction of these cells. Results: Long term direct co-culture of sensitive and resistant OC cells promoted proliferation (p < 0.001) of sensitive cells and increased the proportion of cells in the G1 and S cell cycle phase in both PEO1 and OVCAR5 cells. Direct co-culture led to a decrease in the IC50 to platinum in the cisplatin-sensitive cells (5.92 µM to 2.79 µM for PEO1, and from 2.05 µM to 1.51 µM for OVCAR5). RNAseq analysis of co-cultured cells showed enrichment of Cell Cycle Control, Cyclins and Cell Cycle Regulation pathways. The transcription factor E2F1 was predicted as the main effector responsible for the transcriptomic changes in sensitive cells. Western blot and qRT-PCR confirmed upregulation of E2F1 in co-cultured vs monoculture. Furthermore, an E2F1 inhibitor reverted the increase in proliferation rate induced by co-culture to baseline levels. Conclusions: Our data suggest that long term cohabitation of platinum-sensitive and -resistant cancer cells drive sensitive cells to a higher proliferative state, more responsive to platinum. Our results reveal an unexpected effect caused by direct interactions between cancer cells with different proliferative rates and levels of platinum resistance, modelling competition between cells in heterogeneous tumors.

overall_design: To study the interaction between platinum resistant (PEO4) and platinum sensitive (PEO1) ovarian cancer cells, we perform RNAseq analysis of co-cultures. We co-culture PEO1 cells with PEO4 cells in a proportion of 1:7 respectively for 14 days. Cells then were separated using FACS sorting, and RNA was extracted for RNAseq analysis. We compared PEO1 co-cultured vs PEO1 on monoculture (n=2). Later we compared PEO1 cultured on regular media vs PEO1 cultured with conditioned media of PEO4 (n=2) to study the influence of the conditioned media on the sensitive cells.

first: Andres

last: Valdivia

first: Matthew

last: Cowan

first: Horacio

last: Cardenas

first: Ana

last: Isac

first: Guangyuan

first: Hao

last: Huang


title: Crosslinked elastin-like polypeptides mediate direct cardiac differentiation of mouse embryonic and induced pluripotent stem cells without use of cardiac differentiation factors

city: RTP

first: NIEHS

last: Microarray Core

state: NC

address: 111 T.W. Alexander Drive

department: DIR

institute: NIEHS

summary: Elastin-like polypeptides (ELPs) are promising for biomedical applications due to their unique thermos-responsive and elastic properties. ELP-based biomaterial has been produced through enzymatic crosslinking of modified ELPs. We investigated the role of elastin like polypeptide in cardiomyocyte proliferation and differentiation in mouse ES cells

overall_design: RNA from mESCs, Cardiomyocyte differentiation at 9 days and 14 days will be compared.  Each treatment group has four replicates.

first: Chang

last: Lee

first: Kilsoo

last: Jeon

first: Akhilesh

last: Kumar

first: Sara

last: Grimm

first: Anton

last: Jetten

first: Harvinder

last: Gill


title: Effect of depletion of Elovl6 on gene expression of hematopoietic stem cells

city: Tsukuba

first: Yusuke

last: Kiyoki

state: Ibaraki

address: 1-1-1 Tennodai

department: Hematology

institute: University of Tsukuba

summary: To investigate the function of Elovl6 in hematopoietic stem cells

overall_design: Comparative gene expression profiling analysis using data obtained from RNA-seq of 2 different cells.

first: Takayasu

last: Kato

first: Shigeru

last: Chiba


title: Effect of depletion of Elovl6 on gene expression of MLL-AF9 transduced cells

summary: To investigate the function of Elovl6 in MLL-AF9 transduced cells


title: CMKLR1 in Vascular Smooth Muscle Cells: A Therapeutic Focus for Cardiovascular and Cerebrovascular Diseases

status: Public on Feb 10 2024

city: north york

first: michael

last: dong

state: ON

address: 114-38 hollywood ave

institute: Shenzhen Children's Hospital

summary: Background and Aims: Atherosclerosis, a major contributor to cardiovascular and cerebrovascular diseases, remains a leading cause of global mortality and morbidity. This study focuses on the role of chemokine-like receptor 1 (CMKLR1) in VSMCs and its potential impact on atherosclerotic plaque formation and remodelling. Methods: Cmklr1-/-Apoe-/- mice, generated using CRISPR/Cas9 technology, were fed a high-fat western diet for 16 weeks to model atherosclerosis. Primary VSMCs were isolated from Cmklr1-/- and Cmklr1+/+ mice for in vitro experiments. Cell viability, senescence-associated β-galactosidase staining, and RNA interference were performed to assess the effect of CMKLR1 deficiency on VSMCs. Various assays, including flow cytometry, immunocytochemistry, and RNA-seq analysis, were employed to investigate the molecular mechanisms and downstream signals associated with CMKLR1 deficiency. Results: CMKLR1 deficiency was found to disrupt the cell cycle, leading to VSMC senescence both in vitro and in vivo. Cmklr1-/-Apoe-/- mice exhibited increased atherosclerotic plaque burden, emphasizing the role of CMKLR1 in plaque stability. Transcriptome analysis revealed significant changes in cellular components and biological processes, with a particular impact on the extracellular matrix and cellular response to stimulus. The TGFβ signalling pathway emerged as a potential downstream target of CMKLR1, affecting VSMC differentiation, vessel development, and extracellular matrix synthesis. The deficiency of CMKLR1 led to a reduction in TGFβ1 expression and downstream molecule SM22α, influencing plaque composition and stability. Conclusion: This study demonstrates that CMKLR1 deficiency promotes VSMC senescence and exacerbates atherosclerosis. The disrupted TGFβ signalling pathway, identified as a potential molecular mechanism, provides insights into the complex regulatory network associated with CMKLR1 in atherosclerotic plaque formation and remodelling. These findings highlight the importance of CMKLR1 in atherosclerosis and its potential as a therapeutic target for plaque stability.

overall_design: Total RNA was extracted from the VSMCs of Cmklr1-/- and Cmklr1+/+ mice following standard protocols of RNA isolation. Quantification and quality of RNA was assessed. The library preparation and sequencing were performed by Wenzheng Biotechnology Co., Ltd. on an Illumina NovaSeq platform following the manufacturer’s instructions, and the paired-end reads were generated. The raw data were stored as FASTQ files. The clean data was obtained by removing adapters and low-quality reads. All downstream analyses were based on clean, high-quality data. The RPKM (reads per kilobase per million reads) of each gene was calculated based on the read counts mapped to this gene and the length of the gene. Differential expression analysis between the two groups was performed using the DESeq R package (1.16.1). Genes with the P <0.05 and abs (log2 (fold change)) >2 were defined as differentially expressed. Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.kegg.jp) and Gene Ontology (GO) (http://www.geneontology.org) analyses of differentially expressed genes were performed using the OmicShare tools, a free online platform for data analysis (https://www.omicshare.com/tools). Statistical enrichment in gene functions and biological pathways was obtained. The transcription factor enrichment analysis was conducted with ChEA3 (ChIP-X Enrichment Analysis Version 3, https://maayanlab.cloud/chea3/). The STRING database (http://string-db.org) was used for assessing the interactions of protein-protein, including direct (physical) and indirect (functional) associations.

first: Rongning

last: Wang


title: Efficient Non-integrating and Growth Factors-Free Culture System for Generation of Human Induced Pluripotent Stem Cells via Activation of Glycolysis

city: shanghai

first: zhicheng

last: shao

address: dongan road 130

institute: fudan

summary: we found that a dual-specificity tyrosine phosphorylation-regulated kinase inhibitor ID-8 robustly enhanced human somatic cell reprogramming by upregulation of PDK4 and activation of glycolysis. Furthermore, we identified a novel growth factor-free hiPSC generation system using ID-8/KGN (IK). IK combined with Low-dose bFGF supported the long-term expansion of human pluripotent stem cells. Also, IK-iPSCs showed high pluripotency and normal karyotype. Our studies provide a novel growth factor-free culture system to facilitate generation of hiPSCs for multiple application in regenerative medicine.

overall_design: Examination of ID-8 promote reprogramming in fibroblast and control group E8 and E6 (E8 without bFGF,TGFb)

first: Jinhong

last: Xu


title: Structural insights into histone exchange by human SRCAP complex

city: Shanghai

first: Fengrui

last: Sui

address: Dong'an Road

institute: Fudan University

summary: Histone variant H2A.Z is found at promoters and regulates transcription. The ATP-dependent chromatin remodeler SRCAP complex (SRCAP-C) promotes the replacement of canonical histone H2A-H2B dimer with H2A.Z-H2B dimer. Here, we determined structures of human SRCAP-C bound to H2A-containing nucleosome at near-atomic resolution. The SRCAP subunit integrates a 6-subunit actin-related protein (ARP) module and an ATPase-containing motor module. The ATPase-associated ARP module encircles half of the nucleosome along the DNA and may restrain net DNA translocation, a unique feature of SRCAP-C. The motor module adopts distinct nucleosome-binding modes in the apo (nucleotide-free), ADP-bound, and ADP-BeFx-bound states, suggesting that ATPase-driven movement destabilizes H2A-H2B by unwrapping the entry DNA and pulls H2A-H2B out of nucleosome through the ZNHIT1 subunit. Structure-guided chromatin immunoprecipitation (ChIP) sequencing analysis confirmed the requirement of H2A-contacting ZNHIT1 in maintaining H2A.Z occupancy on the genome. Our study provides structural insights into the mechanism of H2A-H2A.Z exchange mediated by SRCAP-C.

overall_design: This experiment investigates the role of ZNHIT1's N-terminal in the SRCAP complex's function of swapping H2A with H2AZ. Utilizing human DLD-1 cells, ZNHIT1 is degraded via the dTAG system, and the changes in H2AZ distribution are analyzed at various time points (6 hours, 12 hours, 24 hours). The study compares cells overexpressing wild-type ZNHIT1 and its mutants post ZNHIT1 degradation, assessing the impact on H2AZ distribution and exploring the restoration to the normal phenotype.


title: Effect of depletion of Elovl6 on gene expression

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

overall_design: Refer to individual Series


title: PC4 is a post-transcriptional manipulator of D-type cyclins and centromere proteins

first: Qimei

last: Pan


title: Expression data from meningioma cell after DSF and Cu treatment

city: Taipei

first: Ying

last: Kao

address: No.145, Zhengzhou Rd

country: Taiwan

institute: Taipei City Hospital

summary:
We demonstrate that DSF/Cu treatment reduced meningioma cell viability and increased intracellular ER stress.
We used microarrays to analyze the DSF/Cu regulated gene expression.

overall_design: Meningioma cells (P9) treated with DSF/Cu were selected for RNA extraction and hybridization on Affymetrix microarrays.

first: Yu-Chan

last: Chang

first: Micheal

last: Hsiao


title: Genetic Screen Identified Prmt5 as a Neuroprotection Target against Cerebral Ischemia [RNA-seq]

city: Durham

first: Brian

last: Bennett

summary: Epigenetic regulators present novel opportunities for both ischemic stroke research and therapeutic interventions. While previous work has implicated that they may provide neuroprotection by potentially influencing coordinated sets of genes and pathways, most of them remains largely uncharacterized in ischemic conditions. In this study, we used the oxygen-glucose deprivation (OGD) model in the immortalized mouse hippocampal neuronal cell line HT-22 and carried out an RNAi screen on epigenetic regulators. We identified Prmt5 as a novel negative regulator of neuronal cell survival after OGD, which presented a phenotype of translocation from the cytosol to the nucleus upon oxygen and energy depletion both in vitro and in vivo. Prmt5 bound to the chromatin and a large number of promoter regions to repress downstream gene expression. Silencing Prmt5 significantly dampened the OGD-induced changes for a large-scale of genes, and gene ontology analysis showed that Prmt5-target genes were highly enriched for Hedgehog signaling. Encouraged by the above observation, we treated mice with middle cerebral artery occlusion (MCAO) with the Prmt5 inhibitor EPZ015666 and found that Prmt5 inhibition sustain protection against neuronal death in vivo. Together, our findings revealed a novel epigenetic mechanism of Prmt5 in cerebral ischemia and uncovered a potential target for neuroprotection.

overall_design: HT-22 cells were kindly provided by Dr. Richard Dargusch (Salk). Cells were routinely cultured in Dulbecco’s Modified Eagle’s Medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), and the cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2. The cultured neurons were used for experiments within 8-10 days. All experimental protocols and animal handling procedures were performed in accordance with the National Institutes of Health (NIH) guidelines for the use of experimental animals and were approved by the Animal Care and Use Committee of The Second Affiliated Hospital of Air Force Medical University.  Oxygen-glucose deprivation (OGD) treatment was carried out as previously described. Briefly, cells were rinsed and cultured in the glucose-free Earle’s balanced salt solution (BSS) with the following composition (in mmol/L): 116 NaCl, 5.4 KCl, 0.8 MgSO4·7H2O, 1 NaH2PO4·2H2O, 26.2 NaHCO3, 0.01 glycine, 1.8 CaCl2·2H2O, and pH 7.4. The culture was then placed in an Hypoxia Incubator Chamber (StemCell Technology) and were equilibrated for 15 minutes with a continuous flux of 95% N2/5% CO2 gas. The chamber was then sealed and placed into a humidified incubator at 37°C for 8 hours (HT-22) or 60 minutes (primary cortical neuron). OGD was terminated by removing the cultures from the chamber, replacing BSS with normal culture medium, and returning cells to the normoxic conditions for 24 hours. Cells cultured under normal conditions during the experimental period were used as controls. For Prmt5 inhibitor treatment, EPZ015666 (Selleckchem Company) was prepared in dimethyl sulfoxide (DMSO) at a concentration of 100 mM and stored at -20 °C before use. EPZ (10μM final concentration) or DMSO was added to cells 20 minutes before the initiation of OGD and to the BSS during the OGD treatment. For siRNA transfection of the primary neurons, the culture medium was refreshed at day 7. Cells were transfected with siRNAs using Lipofectamine 3000 according to manufacturer’s instructions. The medium was replaced the next day, and OGD experiments were performed 48 h after transfection. For pLKO.1 shRNAs, complementary single-stranded oligos were annealed and cloned as suggested by the RNAi consortium. pGIPZ shRNAs were ordered from Dharmacon. All plasmids were confirmed by sequencing. The shRNA library used for the screen was as previous describe. shRNA lentivirus was prepared by transfecting 293T cells using TransIT-293 (Mirus) using a standard protocol from Addgene. Total RNA was isolated from cells using the GeneJet RNA purification kit (Thermo Scientific), and 0.5 μg total RNA was reverse transcribed to generate cDNA using the iScriptTM cDNA Synthesis Kit (Bio-Rad) according to manufacturer’s instructions. RT-qPCRs were performed using the SsoFastTM EvaGreen Supermix (Bio-Rad) on the Bio-Rad CFX-384 Real-Time PCR System. Actin was used for normalization. All experiments were performed at least three times, and representative results were shown in the figures. For RNA-seq, libraries were prepared from two biological replicates using the TruSeq RNA Sample Prep Kit and sequenced on the NextSeq (Illumina).

first: Jiaji

last: Lin

first: Guang

last: Hu


title: Starfysh reveals heterogeneous spatial dynamics in the breast tumor microenvironment

city: New York

first: SIYU

last: HE

state: NY

institute: Columbia University

summary: Integrative analysis of primary estrogen receptor-positive (ER+) breast cancer, triple-negative breast cancer (TNBC), and metaplastic breast cancer (MBC) tumors using Starfysh.

overall_design:
spatial transcriptomics, 8 samples
Submitter will deposit the raw data to dbGap based on the requirement of the R00 grant.


title: Transcriptomic profiles of liver tissue from hindlimb unloaded mice treated with 4-phenylbutyrate

city: Sharjah

first: Amir

middle: Ali

last: Khan

address: Emirates of Sharjah

department: Department of Applied Biology

country: United Arab Emirates

institute: University of Sharjah

summary: Bulk transcriptomic profiles of liver tissues from male, 18 weeks old c57Bl/6j mice, including ground-based controls, hindlimb-unloaded mice treated with vehicle, and hindlimb unloaded mice treated wit 4-phenylbutyrate (4-PBA)

overall_design: Liver tissues were taken from three goups: Group1: 18 weeks old, male C57Bl6/J mice that were grounf-based controls. Group 2: The 18 weeks old, male C57bl6/J mice that were hindlimb-uploaded and treated with vehicle.  Group 3: The liver tissue from The 18 weeks old, male C57bl6/J mice that were hindlimb-uploaded and treated with 4-phenylbutyrate (4-PBA).

first: Rizwan

last: Qaisar

first: Asima

last: Kareem


title: Slam-seq reveals that miR-430 regulates zygotic mRNA during zebrafish embryogenesis [lna_data]

city: Kansas City

first: Ariel

middle: Alejandro

last: Bazzini

state: Missouri

institute: Stowers Institute for Medical Research

summary: Background: Early embryonic developmental programs are guided by the coordinated interplay between maternally inherited and zygotically manufactured RNAs and proteins. Although these processes happen concomitantly and affecting gene function during this period is bound to affect both pools of mRNAs, it has been challenging to study their expression dynamics separately. Results: By employing Slam-seq, a nascent mRNA labeling transcriptomic approach, in a developmental time series we observe that over half of the early zebrafish embryo transcriptome consists of maternal-zygotic genes, emphasizing their pivotal role in early embryogenesis. We provide an hourly resolution of de novo transcriptional waves and follow nascent mRNA trajectories, finding that most de novo transcriptional events are stable throughout this period. Additionally, by blocking microRNA430 function, a key post-transcriptional regulator during zebrafish embryogenesis, we directly show that it destabilizes hundreds of de novo transcribed mRNAs from pure zygotic as well as maternal-zygotic genes. This unveils a novel miR-430 function during embryogenesis, fine-tuning zygotic gene expression, which highlights that Slam-seq can be used to disentangle transcriptional and post-transcriptional regulation of mRNA levels.  Conclusion: Such valuable insights into zebrafish early embryo transcriptome dynamics emphasize the significance of post-transcriptional regulators in zygotic genome activation. These findings pave the way for future investigations into the coordinated interplay between transcriptional and post-transcriptional landscapes required for the establishment of animal cell identities and functions.

overall_design: Zebrafish embryos were dechorionated and injected at single cell stage with 50mM s4-UTP, plus 20µM of locked nucleic acid against either miR-430 or mismatched sequence. Embryos were kept in the dark until collection time, shield stage (~6 hours post-injection). Total RNA was extracted, and kept away from direct light,  alkylated, used for library preparation with QuantSeq 3′ mRNA‐Seq Library Prep Kit for Illumina (FWD).

first: Danielson

last: Baia Amaral

first: Rhonda

last: Egidy

first: Anoja

last: Perera


title: Slam-seq reveals that miR-430 regulates zygotic mRNA during zebrafish embryogenesis [timecourse_data]

overall_design: Zebrafish embryos were dechorionated and injected at single cell stage with ~1000pL of 75mM s4-UTP. Embryos were kept in the dark until collection time. Total RNA was extracted, and kept away from direct light,  alkylated, used for library preparation with QuantSeq 3′ mRNA‐Seq Library Prep Kit for Illumina (FWD).

middle: A


title: Effect of depletion of Anxa2 on gene expression during Oxygen-glucose deprivation and reoxygenation of BV2 microglia cells

city: Changsha

first: Xibin

last: Tian

address: Tianma Road

institute: Hunan University

summary: To investigate the  function Anxa2  in the regulation of immune inflammation after Oxygen-glucose deprivation and reoxygenation, we established BV2 microglia cells in Anxa2 has been knocked down by shRNA.

overall_design:
We then performed gene expression profiling analysis using data obtained from RNA-seq of shCtrl-CTRL, shCtrl-OGDR,  shAnxa2-CTRL, shAnxa2-OGDR
Comparative gene expression profiling analysis of RNA-seq data for shAnxa2-OGDR  and shCtrl-OGDR in BV2 microglia cells

first: Haijun

last: Tu


title: Quantitative Analysis of sham and Cerebral Ischemia 1.5 h Reperfusion 24 h Transcriptomes in mouse hippocampus

first: Wei

last: Jiang

state: Hunan

address: Lushan Road (S), Yuelu District,Changsha, Hunan Province

department: College of Biology

summary: The goals of this study are to compare hippocampus transcriptome profiling (RNA-seq) after Cerebral Ischemia 1.5 h Reperfusion 24 h in mouse stroke model

overall_design: Hippocampus mRNA profiles of 56-day old wild type mice for Cerebral Ischemia 1.5 h Reperfusion 24 h and sham operation.


title: CSTF2 is correlated with prognosis and progression in hepatocellular carcinoma

city: Shenzhen

first: Qiangnu

last: Zhang

state: None Selected

address: 1017 Dongmen Bei Lu, 518020Shenzhen, China

department: Department of General Surgery

institute: Shenzhen people's hospital

summary: The role and mechanism of CSTF2 in hepatocellular carcinoma are unclear. We constructed CSTF2 knockout HUH7 and PLC/PRF/5 cells to analyze the function of CSTF2 in liver cancer and the downstream gene changes it induces.

overall_design: We established CSTF2-knockout HUH7 and PLC/PRF/5 cells and used high-throughput sequencing to analyze gene changes induced by CSTF2 downregulation.

first: Liping

last: Liu


title: Genetic Screen Identified Prmt5 as a Neuroprotection Target against Cerebral Ischemia


title: RNA sequencing of sputum cells from a phase IIa placebo controlled clinical trial of Risankizumb in severe asthma

city: Leicester

first: David

middle: John

last: Cousins

address: NIHR BRC Respiratory, Glenfield Hospital

department: Respiratory Sciences

country: United Kingdom

institute: University of Leicester

summary: Interleukin (IL)-23 is implicated in T2 and T17-mediated airway inflammation, supporting a role in asthma. We undertook a Phase II, randomized, double-blind, placebo-controlled, 24-week, parallel-group, multicenter trial to assess the efficacy and safety of risankizumab, an IL23p19 monoclonal antibody, administered subcutaneously (90 mg 4 weekly) in adults with severe asthma. Sputum samples were collected at several timepoints: visit 1B (week -3), visit 2 (week 0 proir to treatment), visit 7 (week 20), visit 8 (week 24, end of treatment), visit 12 (week 40, end of observation). RNA sequencing of sputum cells.

overall_design: Comparison of sputum cells from asthma patients treated with either placebo or Risankizumab.


title: Heightened turnover and failed maturation of monocyte-derived macrophages in Chronic Granulomatous Disease

city: Denver

first: Donna

middle: Leslie

last: Bratton

state: CO

address: 1400 Jackson St. Rm A540

department: Pediatrics

institute: National Jewish Health

summary: Loss of NADPH oxidase activity in phagocytes leads to altered cellular responses and exaggerated inflammation in Chronic Granulomatous Disease (CGD). We sought to assess the effects of Nox2 absence on monocyte-derived macrophages (MoMacs) in gp91phox-/y mice during zymosan-induced peritonitis. MoMacs from CGD and wild type (WT) peritonea were lavaged and characterized over time. Though the numbers harvested from both genotypes were virtually identical, there were marked differences in maturation: newly recruited WT MoMacs rapidly enlarged and matured with loss of Ly6C and gain of MHCII, CD206 and CD36, while MoMacs in CGD remained small and were mostly Ly6C+MHCII-. RNAseq analyses showed few intrinsic differences between genotypes in newly recruited MoMacs, but significant differences over time. WT MoMacs demonstrated changes in metabolism, adhesion and reparative functions, while CGD MoMacs remained inflammatory. Labeling with PKH dye demonstrated that while WT MoMacs were mostly recruited within the first 24 hours and remained in the peritoneum while maturing and enlarging, CGD monocytes continued to stream into the peritoneum for days with many migrating to the diaphragm where they were found in fibrin(ogen) clots surrounding clusters of neutrophils in what appeared to be nascent granulomata. Importantly, these observations appeared to be entirely driven by the milieu: adoptive transfer of CGD MoMacs into inflamed peritonea of WT mice resulted in immunophenotypic maturation and behavior, and conversely, altered maturation/behavior of WT MoMacs was seen after adoptive transfer into inflamed peritonea of CGD mice. These data demonstrate heightened recruitment and fundamental failure of MoMac maturation in CGD.

overall_design: Monocytes and monocyte-derived macrophages from C57BL/6 (WT) and gp91 phox -/y (CGD) mice were collected from peritoneal lavage at 3 timepoints after intraperitoneal zymosan injection. Peritoneal lavage cells were FACS sorted to purify monocyte/macropahges with differential expression of the phenotypic markers Ly6C and MHCII, yielding populations of Ly6C+MHCII-, Ly6C+MHCII+, Ly6C-MHCII- or Ly6C-MHCII+ monocyte/macrophages over time. Three replicate cohorts of mice were collected for each treatment condition, resulting in a total of 42 samples analyzed.

first: Sophie

last: Gibbings

first: Thomas

last: Danhorn

first: Heidi

last: Nicks

first: S

last: Frasch


title: Genetic Screen Identified Prmt5 as a Neuroprotection Target against Cerebral Ischemia [ChIP-seq]

first: Brian

last: Bennett

overall_design: HT-22 cells were subjected to oxygen-glucose deprivation (OGD) treatment and crosslinked with 1% formaldehyde for 10 min at room temperature. Formaldehyde was quenched by 200 mM glycine and cells were rinsed twice with ice-cold PBS. Cells were transferred to 15 ml conical tubes and collected by centrifugation. Cells were lysed with lysis buffer A (50 mM HEPES-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA, 0.5% NP-40, 0.25% Triton X-100, 10% Glycerol and protease inhibitor cocktail (Roche)), incubated at 4°C for 10 min and collected by spinning at 1300 × g for 5 min at 4°C. Cells were then resuspended in lysis buffer B (10 mM Tris-Cl (pH 8), 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and protease inhibitor cocktail), incubated at room temperature for 10 min. Nuclei were pelleted by spinning at 1300 × g for 5 min at 4°C. The pellet was suspended with lysis buffer B (10 mM Tris-Cl (pH 8), 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine and protease inhibitor cocktail) and incubated for 15 min on ice. Chromatin shearing was conducted with cells on ice, using a microtip attached to Misonix 3000 sonicator. Sonicate 8-12 cycles of 30 s ON and 90 s OFF around 30-watt power-output. A final concentration of 1% Triton X-100 was added and gently mixed by pipetting. The chromatin solution was clarified by spinning at 20 000 g at 4°C for 30 min. Chromatin immunoprecipitation was performed with 50 ul Dynabeads protein G (Life technology) conjugated preliminary antibodies antibody overnight at 4°C. The immunoprecipitated material was washed five times with wash buffer (10 mM Tris-Cl (pH 8), 1 mM EDTA, 0.5% NP40, 0.5M LiCl, 0.5% Na-Deoxycholate) and once with TE buffer (PH 8.0), then, eluted by heating for 30 min at 65°C with elution buffer (50 mM Tris-Cl (pH 7.5), 10 mM EDTA, 1% sodium dodecyl sulphate). To reverse the crosslinks, samples were incubated at 65°C overnight, then the eluted was digested with a final concentration of 0.5 μg/ml RNasesA at 37°C, followed with a final concentrated of 0.5 μg/ml Proteinase at 55°C for 2 h. The immunoprecipitated DNA were then purified using the DNA clean and concentrator 5 column (Zymo Research). The ChIP DNA was used for qPCRs using indicated primers and data were plotted as the percentage of input. All experiments were performed three or more times, and representative results were shown in the figures. For ChIP-seq, 1 ng precipitated DNA or input was used to generate sequencing libraries using the Nextera XT DNA sample preparation Kit (Illumina). The resulting libraries were sequenced on Next-Seq (Illumina). Two biological replicates were performed, and combined reads were used for further analysis.

first: Jiaji

last: Lin

first: Guang

last: Hu


title: Transcriptome and DNA methylation cell-type deconvolutions produce similar estimates of bulk differential gene expression and differential methylation [bead array]

status: Public on Feb 09 2024

city: Cleveland

first: Emily

middle: Rae

last: Hannon

state: Ohio

address: 10900 Euclid Ave

department: The Center for Global Health and Diseases

institute: Case Western Reserve University

summary: Motivation: Changing cell-type proportions can confound studies of differential gene expression or DNA methylation (DNAm) from peripheral blood mononuclear cells (PBMCs). We examined how cell-type proportions derived from the transcriptome versus the methylome (DNAm) influence estimates of differentially expressed genes (DEGs) and differentially methylated positions (DMPs). Methods: Transcriptome and DNAm data were obtained from PBMC RNA and DNA of Kenyan children before, during, and 6 weeks following uncomplicated malaria. DEGs and DMPs between time points were detected using cell-type adjusted modelling with Cibersortx or IDOL, respectively. Results: Most major cell types and principal components had moderate to high correlation between the two deconvolution methods (r = 0.60-0.96). Estimates of cell-type proportions and DEGs or DMPs were largely unaffected by the method, with the greatest discrepancy in the estimation of neutrophils. Conclusion: Variation in cell-type proportions is captured similarly by both transcriptomic and methylome deconvolution methods for most major cell types.

overall_design: 24 Bulk DNA methylation EPIC array from PBMC in 8 Kenyan children before, during and after uncomplicated malaria.

middle: R

first: Carmen

last: Marsit

first: Arlene

middle: E

last: Dent

first: Paula

last: Embury

first: Sidney

last: Ogolla

last: Midem

first: Scott

last: Williams

first: James

last: Kazura


title: Transcriptome and DNA methylation cell-type deconvolutions produce similar estimates of bulk differential gene expression and differential methylation [RNA-Seq]

overall_design: Bulk RNA-seq from PBMC in Kenyan children before, during and after uncomplicated malaria.

middle: M

middle: W


title: Transcriptome and DNA methylation cell-type deconvolutions produce similar estimates of bulk differential gene expression and differential methylation


title: A common regulatory switch controls a suite of C4 traits in multiple cell types

city: Irapuato

first: Carlos

last: Ortiz-Ramírez

state: Guanajuato

address: Km 9.6 Libramiento Norte Carretera Irapuato-León.

department: UGA

country: Mexico

institute: Cinvestav

summary: The C4 photosynthetic pathway provided a major advantage to plants growing in hot, dry environments, including the ancestors of our most productive crops. Two traits were essential for the evolution of this pathway: increased vein density and the functionalization of bundle sheath cells for photosynthesis. Although GRAS transcriptional regulators, including SHORT ROOT (SHR), have been implicated in mediating leaf patterning in both C3 and C4 species1-4, little is known about what controls the specialized features of the cells that mediate C4 metabolism and physiology. We show in the model monocot, Setaria viridis, that SHR regulates components of multiple cell identities, including chloroplast biogenesis and photosynthetic gene expression in bundle sheath cells, a central feature of C4 plants. Furthermore, we found that it also contributes to the two-cell compartmentalization of the characteristic four-carbon shuttle pathway. Disruption of SHR function clearly reduced photosynthetic capacity and seed yield in mutant plants under heat stress. Together, these results show how cell identities are remodeled by SHR to host the suite of traits characteristic of C4 regulation, which are a main engineering target in non-C4 crops to improve climate resilience.

overall_design: To investigate the function of the transcription factor SHORT ROOT in establishing multiple cell identities in C4 plants, we generated CRISPR loss-of- function mutants, and performed expression profiling at single-cell resolution using high-throughput droplet-based technology.

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