title: Neuronal differentiation of DFATs

city: fujisawa

first: rei

last: nakano

address: 1866 Kameino

institute: Nihon University

summary: Transplantation of functional neurons improve the functionally recovery in animal models of Alzheimer's disease and other neurological disorders. Although functional neurons can be differentiated from embryonic stem cells or induced-pluripotent stem cells, alternative cell sources of functional neurons may be more available for transplantation. Dedifferentiated fat cells (DFATs) were isolated and cultured from mature adipose tissue, which have advantages in their abundance, ease of isolation and homogeneity compared to other adult stem cells. DFATs are promising for clinical applications such as cell transplation therapy.    Direct reprogramming has been developed in recent years. It is possible to convert mature differentiated cells into other types of functional cells by introducing cell-fate-conversion transcriptional factors. However, the introduction of exogenous transcriptional factors limits their use of clinical application. Previous studies found that in vitro neuronal differentiation follows the processes of in vivo neuronal development, which are related to the surrounding microenvironments. Therefore, the arrangement of a proper microenvironment for the neuronal development process is essential to induce neuronal differentiation.      In this study, we report that DFATs differentiated into neurons by all trans retinoic acid (ATRA) and basic fibroblast growth factor (bFGF). The ATRA/bFGF-treated DFATs expressed the neuron-specific mRNA and pritein expression, exibited neuron-like morphology, generated voltage-dependent sodium current and action potentials and react with neurotransmitters. Transcriptomics resutls suggested that ATRA/bFGF treatment induced a specific differentiatioin into neurons. The lesional analysis revealed that ATRA/bFGF-treated cells are compatible with telemcephalon gabaergic neuron. To investigate the mechanism of ATRA/bFGF-induced neuronal differentiation, we performed the pathway analysis. The pathway analysis suggested that JNK signaling pathway was involved in the neuronal diffeerentiation. Our knockdown experiient further demonstrated that JNK3, a neuronal subtype of JNK, plays a crucial role in ATRA/bFGF-induced neuronal differentiation.    In conclusion, we demonstrated that ATRA/bFGF induced DFATs into functional neurons via the activation of JNK3 signaling pathway. Our study provides an efficient approach for generating functional neurons from adult fat tissue and reveals the functional importance of JNK3 in neuronal differentiation process.

overall_design: Total of 6 samples were analyze. 3 biological replicate for control or ATRA+bFGF treatment.

first: Rei

last: Nakano

first: Hiroshi

last: Sugiya


title: Replication-independent nucleosome turnover confers the functional basis of cis-regulatory elements in adult heart

status: Public on Apr 17 2020

city: Chicago

first: Yong

last: Peng

state: IL

address: 929 E 57th Street

department: Department of Chemistry

country: USA

institute: University of Chicago

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Through integrated H2BGFP ChIP-seq data over multiple conditions and time points, as well as other related ChIP-seq and RNA-seq data in adult mouse heart. We observed genomewide and locus-specific H2B turnover in non-proliferating adult cardiomyocytes (Cms), and a majority of genomic regions with rapid turnover rates distributes on cis-regulatory elements, as well as these regions are associated with heart function, regulatory factors binding and their activities. Moreover, H2B turnover rate is always positively correlated with gene transcription level on enhancers, promoters and exons. The rapid turnover rates at cis-regulatory elements can be partially explained by the binding and co-binding of RNAP II and various transcription factors (TFs).  Our further analysis revealed that not only rapid H2B turnover at cis-regulatory elements responses to heart disease, but also slow turnover at H3K27me3 sites functionally responses to heart disease with a different manner. More generally, we indicate that the binding and co-binding of DNA-binding proteins, such as TFs and epigenetic enzymes, all of them coupled with H2B turnover to modulate gene transcription and chromatin state.

overall_design: Profile H2BGFP occupancy in isolated adult cardiomyocytes from Wildtype (WT), cardiac conditional EED Knockout (CKO), and TAC mice at multiple time points.

first: Shanshan

last: Ai

first: Xianhong

last: Yu

first: Chen

last: Li

first: Yumei

first: Xin

first: Chuanyun

first: Cheng

first: Aibin

last: He


title: Effects of folic acid withdrawal on transcriptomic profiles in murine triple-negative breast cancer cell lines

status: Public on Apr 16 2020

city: chapel hill

first: michael

middle: francis

last: coleman

state: North Carolina

address: 235 dauer drive

institute: UNC

summary: We have previously shown that withdrawal of folic acid led to metabolic reprogramming and a less aggressive phenotype in a cell model of triple-negative breast cancer (TNBC). Herein, we evaluate the effects of folic acid withdrawal on transcriptomic profiles in these cells. Murine cell lines were originally derived from a pool of spontaneous mammary tumors grown in MMTV-Wnt1 transgenic mice. Based on their differential molecular characteristics and metastatic potential, these cell lines were previously characterized as non-metastatic epithelial (E-Wnt), non-metastatic mesenchymal (M-Wnt) and metastatic mesenchymal (metM-Wntliver) cells.

overall_design: Using custom two-color 180K Agilent microarrays, we have determined gene expression profiles for three biological replicates of each subtype kept on standard medium (2.2 mM folic acid) or folic acid-free medium for 72 hours.

first: Dieuwertje

middle: E

last: Kok

first: Ciara

middle: H

last: O'Flanagan

first: Michael

middle: F

last: Coleman

first: Zahra

last: Ashkavand

first: Stephen

middle: D

last: Hursting

first: Sergey

middle: A

last: Krupenko


title: Expression data from mouse splenocytes cocultured with differently treated KLN-205KD cells

city: TAIPEI

first: Li-Han

last: Chen

state: Taipei

address: No. 1, Sec. 4, Roosevelt Rd.

country: Taiwan

institute: National Taiwan University

summary:
Semophorin (SEMA) 6A showed capability to reduce the SEMA3A-derived supression of T cells. However, the effect of SEMA6A on the other immune cells was still unclear. Therefore, we performed gene expression analysis using an Affymetrix mouse genome 430 2.0 array to investigate the potential immune cells regualted by the interaction of SEMA6A and SEMA3A. Thus, we predicted other possible immune cells regulated by SEMA6A using GSEA to analyze the microarray data of co-cultured splenocytes.
The reuslts revealed that SEMA6A decreased the SEMA3A-mediated inhibition of B cells and induced activities of dendritic cells in a SEMA3A-indepenet manner. Moreover, M1 and M2 macrophages were not influenced by SEMA6A and SEMA3A.

overall_design: Mouse splenocytes were cocultured with  (i) SEMA3A-knockdown KLN-205 cells (KLN-205KD, control line), (ii) KLN-205KD cells overexpressing SEMA6A (SEMA6A), (iii) KLN-205KD cells plus exogenous SEMA3A (SEMA3A), and (iv) KLN-205KD cells overexpressing SEMA6A plus exogenous SEMA3A (SEMA6A+SEMA3A) for 9 days.  Interleukin-2 was added at 100 units/mL every three days. Then, The RNA of splenocytes was isoloated to do microarray.

first: Shih-Han

last: Huang

first: Mong-Hsun

last: Tsai

first: Eric

middle: Y

last: Chuang


title: A single-cell transcriptomic atlas of the adult Drosophila ventral nerve cord

city: Oxford

first: Aaron

middle: Munro

last: Allen

address: Tinsley Building, Mansfield Road

department: DPAG

country: United Kingdom

institute: University of Oxford

type: Expression profiling by high throughput sequencing

summary: The Drosophila ventral nerve cord (VNC) receives and processes descending signals from the brain to produce a variety of coordinated locomotor outputs. It also integrates sensory information from the periphery and sends ascending signals to the brain. We used single-cell transcriptomics to generate an unbiased classification of cellular diversity in the VNC of five-day old adult flies. We produced an atlas of 26,000 high-quality cells, representing more than 100 transcriptionally distinct cell types. The predominant gene signatures defining neuronal cell types reflect shared developmental histories based on the neuroblast from which cells were derived, as well as their birth order. Cells could also be assigned to specific neuromeres using adult Hox gene expression. This single-cell transcriptional atlas of the adult fly VNC will be a valuable resource for future studies of neurodevelopment and behavior.

overall_design: scRNA-seq with 10x Genomics Chromium 3' v2 of the Drosophila melanogaster adult ventral nerve cord from females and males

middle: M

first: Megan

middle: C

last: Neville

first: Sebastian

last: Birtles

first: Vincent

last: Croset

first: Christoph

last: Treiber

first: Scott

last: Waddell

last: Goodwin


title: Phosphoribotrap-based profiling of activated hypothalamic cells during acute high-fat diet (HFD)-feeding

city: Cologne

first: Alexander

last: Jais

address: Gleueler Str. 50

department: Neuronal Control of Metabolism

country: Germany

institute: Max Planck Institute for Metabolism Research

summary:
To identify novel neurocircuits activated upon short-term HFD feeding, we employed phosphoribotrap-profiling, which allows for the unbiased identification of alterations in neuronal activation via immunoprecipitation of phosphorylated S6 ribosomal protein-tagged ribosomes from hypothalamic extracts of mice exposed to either 3 days of normal chow diet or HFD-feeding.
We have analyzed mRNA selectively expressed in hypothalamic cells activated by a either normal chow diet (NCD)-feeding for 3 days or high fat diet (HFD)-feeding for 3 days.
10-week-old male C57/BL6N mice were put either on a normal chow diet or a high fat diet for 3 days. Afterwards, mice were sacrificed by cervical dislocation. The hypothalamus was rapidly dissected using a stainless steel brain matrix and immediately frozen in liquid nitrogen. Hypothalamic tissues were pooled (8 per IP).

overall_design: Hypothalamic translatome of activated cells of 10 week old C57/BL6N mice, fed either a normal chow diet (NCD) for 3 days or a high-fat diet (HFD) for 3 days.

first: Jens

last: Brüning


title: BacTRAP-based ribosome profiling of murine hypothalamic PNOC neurons

summary:
We performed a BacTRAP-based ribosome profiling of PNOC-expressing cells in the hypothalamus of mice. Therefore, we employed mice, which express a fusion protein of the ribosomal L10a protein with EGFP (L10a-EGFP) under control of the PNOC promoter (Doyle et al., 2008).
Precipitation of ribosomes of hypothalamic PNOC neurons with anti-GFP antibodies and subsequent mRNA sequencing of associated mRNAs allowed for assessment of an in-depth translational profile of these cells.
Affinity purification of translating ribosomes was performed as described by (Heiman et al., 2014) with minor modifications.

overall_design: Hypothalamic PNOC neuronal translatome of 10-12 week old PNOC-L10a-EGFP mice, fed either a normal chow diet (CD) for 3 days or a high-fat diet (HFD) for 3 days.


title: miRNA expression in breast cancer cell lines

city: Valencia

first: Sara

last: OLTRA SANCHIS

state: Valencia

address: Avda Menéndez Pelayo, 4 accesorio

department: Medical Oncology

country: Spain

institute: INCLIVA

summary: We analyzed the miRNA expression in 6 breast cancer cell lines from young (HCC1500, HCC1937) and old (MCF-7, MDA-MB-231, HCC1806 and MDA-MB-468) patients with breast cancer using the GeneChip® miRNA 2.0 Array (Affymetrix, Santa Clara, CA, USA).

overall_design: Total RNA from BC cell lines was isolated in passage 4 using High Pure RNA Isolation Kit from Roche following the manufacturer’s protocol. RNA concentration was measured using a NanoDrop ND 2000-UV-vis Spectrophotometer (Thermo Fisher Scientific Inc., Wilmington DE, USA). RNA integrity determined by RIN (RNA integrity number) value was assessed with the Agilent 2100 Bioanalyzer using RNA 6000 Nano Assay (Agilent Technologies Inc., Santa Clara, CA, USA). Microarray expression profiling was performed using GeneChip® miRNA 2.0 Array (Affymetrix, Santa Clara, CA, USA).

middle: S

last: Oltra

first: Maria

last: Peña-Chilet

first: M

middle: T

last: Martinez

first: Eduardo

last: Tormo

first: Joan

last: Climent

first: Pilar

last: Eroles

first: Ana

last: Lluch

first: Gloria

last: Ribas


title: The effects of SGLT-2 inhibitors canagliflozin on hepatic transcription

city: Boston

first: Qiong

last: Zhou

state: MA

address: 1 Joslin PLace

institute: Joslin Diabetes Center

summary:
We assessed the change in hepatic transciptional pattern after treatment with SGLT-2 inhibitors canagliflozin in a mice model of diet-induced obesity.
Pharmacologic inhibition of the renal sodium/glucose cotransporter-2 induces glycosuria and reduces glycemia.  Given that SGLT2 inhibitors (SGLT2i) reduce mortality and CV risk in T2D, improved understanding of molecular mechanisms mediating these metabolic effects is required.  Treatment of obese but nondiabetic mice with the SGLT2i canagliflozin (CANA) reduces adiposity, improves glucose tolerance despite reduced plasma insulin, increases plasma ketones, and improves plasma lipid profiles. We utilized an integrated transcriptomic-metabolomics approach to demonstrate that CANA modulates key nutrient-sensing pathways, with activation of AMPK and inhibition of mTOR, independent of insulin or glucagon sensitivity or signaling. Moreover, CANA induces transcriptional reprogramming to activate catabolic pathways, increase fatty acid oxidation, reduce hepatic steatosis and diacylglycerol content, and increase hepatic and plasma levels of FGF21. Taken together, these data demonstrate that SGLT-2 inhibition triggers a fasting-like transcriptional and metabolic paradigm.

overall_design: Diet induced obesity C57Bl/6J were treated with canagliflozin (30mg/kg) for 4 weeks and the effects on hepatic transcription were compared with HFD control and weight-matched control by caloric restriction.

first: Soravis

last: Osataphan

first: Grace

last: Daher

first: Chiara

last: Macchi

first: Vicencia

last: Sales

first: Chisayo

last: Kozuka

first: Mary-Elizabeth

last: Patti


title: Asymmetric demethylation at histone H3 arginine 2 by PRMT6 in gastric cancer progression (AGS cell line)

city: Bunkyo-ku

first: Shu

last: Shimada

state: Tokyo

address: 1-5-45 Yushima

department: Department of Molecular Oncology

country: Japan

institute: Tokyo Medical and Dental University

summary: Purpose: Dysregulation of histone H3 arginine (R) methylation is still unknown in primary cancer including gastric cancer (GC), although PRMT6 plays a role in asymmetric dimethylation at H3R2 (H3R2me2as) in cancer cells. The objective is to clarify biological and molecular roles of H3R2me2as-PRMT6 pathway in GC.  Experimental Design: We assessed H3R2me2as and PRMT6 levels in 133 primary GC tissues by immunohistochemistry. We analyzed biological functions of PRMT6 in GC cell lines using a lentivirus overexpression and CRISPR/Cas9-based knockout of PRMT6 systems.  Results: Increased H3R2me2as was found in 68 GC (51.1%) cases and independently correlated with poor prognosis. PRMT6 was overexpressed in 70 (52.6%) GC, which strongly correlated with the H3R2me2as levels (P<0.001). PRMT6 overexpression in GC cells enhanced global H3R2me2as levels, cell invasiveness in vitro, while PRMT6-knockout GC cells suppressed these effects. PRMT6 knockout also impaired tumorigenicity in vivo. Microarray and ChIP assays demonstrated that PRMT6-knockout GC cells decreased the H3R2me2as levels at the promoter regions of PCDH7, SCD and IGFBP5, resulting in up-reregulation of their gene expression. PRMT6 recruited at the regions of PCDH7 and SCD in the PRMT6-overexpressed cells. Knockdown of tumor suppressor PCDH7 in PRMT6-knockout GC cells elevated cell migration and invasion. PRMT6 expression inversely correlated with PCDH7 expression in primary GC (P=0.021).  Conclusions: H3R2me2as is a strong prognostic indicator of GC patients. Global and gene-specific H3R2me2as are maintained by PRMT6. PRMT6-overexpressed GC cells may acquire invasiveness through direct inhibition of PCDH7 by increasing H3R2me2as activity. Thus, PRMT6-H3R2me2as pathway is a promising new therapeutic target in GC.

overall_design: PRMT6-knockout gastric cancer cells were established by using a lentiviral CRISPR/Cas9 system.

first: Keisuke

last: Okuno

first: Yoshimitsu

last: Akiyama

first: Masatoshi

last: Nakagawa

first: Toshiro

last: Tanioka

first: Mikito

last: Inokuchi

first: Shoji

last: Yamaoka

first: Kazuyuki

last: Kojima

first: Shinji

last: Tanaka


title: Asymmetric demethylation at histone H3 arginine 2 by PRMT6 in gastric cancer progression

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

overall_design: Refer to individual Series


title: Clinically Relevant Biomarker Discovery in High-Risk Recurrent Neuroblastoma

city: Tromso

first: Peter

last: Utnes

address: Hansine Hansens veg 18

country: Norway

institute: UiT - The Arctic University of Norway

summary: Neuroblastoma is a pediatric cancer of the developing sympathetic nervous system. High-risk neuroblastoma patients typically undergo an initial remission in response to treatment, followed by recurrence of aggressive tumors that have become refractory to further treatment. The need for biomarkers that can select patients not responding well to therapy in an early phase is therefore needed. In this study, we used next generation sequencing technology to determine the expression profiles in high-risk neuroblastoma cell lines established before and after therapy. Using partial least squares-discriminant analysis (PLS-DA) with least absolute shrinkage and selection operator (LASSO) and leave-one-out cross-validation, we identified a panel of 55 messenger RNAs and 17 long non-coding RNAs (lncRNAs) which were significantly altered in the expression between cell lines isolated from primary and recurrent tumors. From a neuroblastoma patient cohort, we found 20 of the 55 protein-coding genes to be differentially expressed in patients with unfavorable compared with favorable outcome. We further found a twofold increase or decrease in hazard ratios in these genes when comparing patients with unfavorable and favorable outcome. Gene set enrichment analysis (GSEA) revealed that these genes were involved in proliferation, differentiation and regulated by Polycomb group (PcG) proteins. Of the 17 lncRNAs, 3 upregulated (NEAT1, SH3BP5-AS1, NORAD) and 3 downregulated lncRNAs (DUBR, MEG3, DHRS4-AS1) were also found to be differentially expressed in favorable compared with unfavorable outcome. Moreover, using expression profiles on both miRNAs and mRNAs in the same cohort of cell lines, we found 13 downregulated and 18 upregulated experimentally observed miRNA target genes targeted by miR-21, -424 and -30e, -29b, -138, -494, -181a, -34a, -29b, respectively. The advantage of analyzing biomarkers in a clinically relevant neuroblastoma model system enables further studies on the effect of individual genes upon gene perturbation. In summary, this study identified several genes, which may aid in the prediction of response to therapy and tumor recurrence.

overall_design: Comparison of cell lines isolated from neuroblastoma patients before and after high-dose chemotherapy

first: P


title: The plant-specific COST1 protein balances plant growth and drought tolerance via attenuation of autophagy

city: East Lansing

first: Yan

last: Bao

address: 603 Wilson Road

institute: MIchigan State University

summary: Plants balance their conflicting requirements for growth and stress tolerance via sophisticated pathways and unique genes that control responses to the external environment. We have identified a novel plant-specific gene, COST1(Constitutively Stressed 1), that affects plant growth and negatively regulates drought resistance by manipulating the autophagy pathway. An Arabidopsis cost1 mutant has decreased growth and increased drought tolerance, together with constitutive autophagy and increased expression of drought-response genes. The COST1 protein is degraded upon plant dehydration, and this degradation is blocked by treatment with inhibitors of the 26S proteasome or autophagy pathways. The cost1 mutant drought resistance is dependent on an active autophagy pathway, indicating that COST1 acts through manipulation of autophagy. COST1 co-localizes to autophagosomes with the autophagosome marker ATG8e and the autophagy adaptor NBR1, and physically interacts with ATG8e, indicating a pivotal role in direct regulation of autophagy. We propose a model in which COST1 represses autophagy under optimal conditions, thus allowing plant growth. During drought, COST1 is degraded, enabling activation of autophagy and suppressing growth to enhance drought tolerance.

overall_design: We examined gene expression by RNA-seq in Arabidopsis wide-type and mutant cost1 with and w/o drought

first: Weimeng

last: Song

first: Peipei

last: Wang

first: Hongxia

last: Zhang

first: Diane

last: Bassham


title: Characterizing breast cancer lung metastatic lesions in host environments with or without NK cells by RNA-seq

city: Houston

first: Hin Ching

last: Lo

state: TX

address: 1885 El Paseo St, Apt 1033

institute: Baylor College of Medicine

summary: We demonstrated that natural killer (NK) cells selectively suppress single circulating tumor cells (CTCs) and monoclonal metastasis compared to multicellular CTC clusters and polyclonal metastasis. To better understand how NK cells influence overall metastatic evolution, we generated spontaneous AT3 breast cancer lung metastasis in immunodeficient Rag2-/-IL2rg-/- mice, and treated mice with or without adoptive transfer of NK cells. RNA-seq on individual laser-capture microdissected lesions confirmed the infiltration of NK cells. The expression of epithelial-to-mesenchymal transition (EMT) genes strongly correlated with expression of genes indicating NK cell activation, consistent with experimental data from our lab and others.

overall_design: RNA-seq on AT3 breast cancer lung metastatic lesions from Rag2-/-IL2rg-/- mice (with or without adoptive transfer of NK cells).

first: Hin

first: Zhan

last: Xu

first: Hai

first: Xiang


title: Early fate defines microglia and non-parenchymal brain macrophage development

city: Singapore

first: Nicholas

last: Ang

address: 8a Biomedical Grove, Singapore 138648

country: Singapore

institute: Singapore Immunology Network (SIgN)

summary: Central nervous system (CNS) macrophages comprise parenchymal microglia and border-associated macrophages (BAMs) residing in the meninges, the choroid plexus and the perivascular spaces. With the exception of choroid plexus macrophages, most CNS macrophages emerge during primitive hematopoiesis in the extra-embryonic yolk sac. What remains unknown however, is whether microglia and BAMs share a developmental program or arise from separate predefined lineages. Here, we identified two phenotypically, transcriptionally and locally distinct brain macrophage populations throughout development, giving rise to microglia and BAMs. Two independent macrophage populations were already present in the yolk sac prior to their seeding of the brain. Using fate-mapping system, we demonstrate that in contrast to microglia, the pool of embryonic BAMs in the choroid plexus and the meninges was gradually replaced by precursors emerging later in embryogenesis. The development of microglia was dependent on TGF-β whereas the genesis and maturation of BAMs occurred independently of this cytokine. Collectively, our data show that developing parenchymal and non-parenchymal brain macrophages are separate entities in terms of ontogeny, gene expression signatures and requirement for TGF-β.

overall_design: Gene expression analysis using total RNA from embryonic murine microglia and BAM populations in the brain. C57Bl/6 mice were time-mated and the day of vaginal plug observation was denoted as 0.5 day after conception (E0.5). Pure populations of embryonic microglia and BAM from E10.5 to E18.5 were sorted by FACS.

last: See

first: Bernett

last: Lee

first: Florent

last: Ginhoux


title: The role of  KLF7 in pancreatic adenocarcinoma (PDAC) progression

city: Birmingham

first: Narendra

last: Wajapeyee

state: AL

address: 720 20th Street South, Kaul 540A

department: Department of Biochemistry and Molecular Genetics

institute: University of Alabama at Birmingham

summary: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a dismal prognosis. Currently, there is no effective therapy to treat PDAC, and thus detailed molecular and functional evaluation of PDAC is needed to identify and develop better therapeutic strategies. Here we show that the transcription factor Kruppel-like factor 7 (KLF7) is overexpressed in PDAC and that inhibition of KLF7 blocks PDAC tumor growth and metastasis in cell culture and in mice. KLF7 expression in PDAC can be upregulated due to activation of a MAP kinase pathway or inactivation of the tumor suppressor p53, two alterations that occur in a large majority of PDACs. ShRNA-mediated knockdown of KLF7 inhibits the expression of interferon-stimulated genes (ISGs), which are necessary for KLF7-mediated PDAC tumor growth and metastasis. KLF7 knockdown also results in the downregulation of Discs Large MAGUK Scaffold Protein 3 (DLG3), resulting in Golgi complex fragmentation, and reduced protein glycosylation leading to reduced secretion of cancer-promoting growth factors such as chemokines. Genetic or pharmacological activation of Golgi complex fragmentation blocks PDAC growth and metastasis similar to KLF7 inhibition. Our results demonstrate a therapeutically amenable, KLF7-driven pathway that promotes PDAC growth and metastasis by activating ISGs and maintaining Golgi complex integrity.

overall_design: PANC1 cells stably expressing two individual shRNAs against KLF7 were used to prepare the total RNA, which was then used to analyze for gene expression using RNA-sequencing analysis

first: Parmanand

last: Malvi

first: Romi

last: Gupta

first: Radoslav

last: Janostiak


title: Asymmetric demethylation at histone H3 arginine 2 by PRMT6 in gastric cancer progression (NUGC3 cell line)

first: Shu

last: Shimada

first: Keisuke

last: Okuno

first: Yoshimitsu

last: Akiyama

first: Masatoshi

last: Nakagawa

first: Toshiro

last: Tanioka

first: Mikito

last: Inokuchi

first: Shoji

last: Yamaoka

first: Kazuyuki

last: Kojima

first: Shinji

last: Tanaka


title: Transcriptome analysis after ectopically expressing KAN1 in SAM  epidermis

city: Mohali

first: Hasthi

last: Ram

address: Sector 81

department: Agriculture biotechnology

country: India

institute: NABI (National Agri-Food Biotechnology Institute)

summary: We ectopically expressed KAN1 in epidermis in ap1 cal background by using AtML1 promoter driven inducible KAN1-2GFP for RNA-Seq studies. Expression of this transgenes in ap1 cal background gives phenotype similar to their expression in wild-type (WT) backgrounds. For pML1>>KAN1-2GFP , we sorted the epidermal cells using FACS, after their induction of 6 hours or 16 hours.  As a control we collected the epidermal cells after 6 or 16hr induction of pML1::GR-LHG4 in pML1::BFPer background. Q-PCR analysis on extracted RNA showed the successful sorting and regulation of known target genes of KAN1.

overall_design: 2 different transgenic lines (pML1>>REVgto-2Venus, and pML1::GR-LHG4, pML1::BFPer) were used in ap1cal background. 2 time points (6hr and 16hr) were used for each transgenic line. 3-4 biological replicates were used for each time point. cells were collcted through FACS, and used for RNA-Seq analysis

first: Hathi

first: Sudeep

last: Sahadevan

first: Marcus

last: Heisler


title: Transcriptome analysis after ectopically expressing REV in SAM  epidermis

summary: We ectopically expressed REV in epidermis in ap1 cal background by using AtML1 promoter driven inducible REV(GtoG)-2Venus for RNA-Seq studies. Expression of this transgenes in ap1 cal background gives phenotype similar to their expression in wild-type (WT) backgrounds. For pML1>>REV(GtoG)-2Venus , we sorted the epidermal cells using FACS, after their induction of 6 hours or 16 hours.  As a control we collected the epidermal cells after 6 or 16hr induction of pML1::GR-LHG4 in pML1::BFPer background. Q-PCR analysis on extracted RNA showed the successful sorting and regulation of known target genes of REV.


title: Transcriptome analysis after knocking down class III HD ZIP transcription factors in SAM

summary: We ubiqoutesly expressed mir165a (pUBQ>>mir165a) in ap1 cal background  for RNA-Seq studies. Expression of this transgenes in ap1 cal background gives phenotype similar to their expression in wild-type (WT) backgrounds. We induced the transgenes for 8 hours or 16 hours.  As a control we used pUBQ::GR-LHG4 transgeneic line. Q-PCR analysis on extracted RNA showed the successful down regulation of known target genes of class III HD ZIPs.

overall_design: 2 different transgenic lines (pUBQ>>mir165as, and pUBQ::GR-LHG4) were used in ap1cal background. 2 time points (8hr and 16hr) were used for each transgenic line. 3 biological replicates were used for each time point.


title: Response and resistance of low grade ovarian cancer cells to trametinib

status: Public on Apr 15 2020

first: Kwong-Kwok

last: Wong

address: T4-3900, 1515 Holcombe Boulevard

department: Gynecologic Oncology and Reproductive Medicine

institute: UT M. D. Anderson Cancer Center

summary:
HOC-7 and MPSC1 are low grade serous ovarian cancer cell lines.  These cells was treated with different doses of trametinib in an attempt to identify biomarkers that can be used to predict chemoresponse to this drug.
We used microarrays to identify genes induced by trametinib.

overall_design: HOC-7 and MPSC1 were treated with 10nM trametinib and cells were harvested after 18 hours for gene expression profiling. Each cell type had two samples (untreated control versus trametinib treated test sample)


title: Transcriptomic analysis of dorso-ventral cell-types of Arabidopsis shoot apical meristem

summary: Dorso-ventral (DV) patterning at the Shoot Apical meristem (SAM) is essential for organogenesis in Arabidopsis. Dorsal and ventral gene expressed domains are separated by boundaries from where the new organ initiation starts. Here by using cell-types specific transcriptomics approach, we have identified doral, ventral and boundary specific genes in SAM.

overall_design: 6 different cell-types, and 3 biological replicates for each cell-type were collcted through FACS, and used for RNA-Seq analysis


title: Allosteric HSP70 inhibitors perturb mitochondrial proteostasis and overcome proteasome inhibitor resistance in multiple myeloma

city: Stanford

first: Ian

middle: Daniel

last: Ferguson

state: CA

address: 265 Campus Dr

department: Cancer Biology PhD program

institute: Stanford University

summary: Proteasome inhibitor (PI) resistance remains a central challenge in multiple myeloma. To identify pathways mediating resistance, we first map proteasome-associated genetic co-dependencies. We identify cytosolic heat shock protein 70 (HSP70) chaperones as a potential target, consistent with proposed mechanisms of myeloma tumor cells overcoming PI-induced stress. These results led us to explore allosteric HSP70 inhibitors as myeloma therapeutics. We show these compounds exhibit increased efficacy against both acquired and intrinsic PI-resistant myeloma models, unlike HSP90 inhibition. Surprisingly, shotgun and pulsed-SILAC proteomics reveal that JG’s overcome PI resistance not via the expected mechanism of inhibiting cytosolic HSP70s, but instead through mitochondrial-localized HSP70, HSPA9, destabilizing the 55S mitoribosome. Analysis of myeloma patient data further supports strong effects of global proteostasis capacity, and particularly HSPA9 expression, on PI response. Our results characterize dynamics of myeloma proteostasis networks under therapeutic pressure while further motivating investigation of HSPA9 as a specific target in PI-resistant disease.

overall_design: Data uploaded here: RNA-sequencing of MM.1S myeloma cells were treated with allosteric HSP70 inhibitor JG342 at 350nM or DMSO control for 26 hours in biological duplicate.

first: Arun

last: Wiita


title: Oncogenic ALKF1174L drives tumorigenesis in cutaneous squamous cell carcinoma

city: Zurich

first: giancarlo

last: russo

state: Schweiz

address: winterthurerstrasse 190

department: Functional Genomics Center Zurich

country: Switzerland

institute: ETH/University of Zurich

summary: Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer characterized by increased mortality. Here, we show for the first time that anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase of the insulin receptor superfamily, plays a pivotal role in the pathogenesis of cSCC. Our data demonstrate that the overexpression of the constitutively active, mutated ALK, ALKF1174L is sufficient to initiate the development of cSCC and is 100% penetrant. Moreover, we show that cSCC development upon ALKF1174L overexpression is independent of the cell-of-origin. Molecularly, our data demonstrate that ALKF1174L cooperates with oncogenic KrasG12D and loss of p53, well established events in the biology of cSCC. This cooperation results in a more aggressive cSCC type associated with a higher-grade histological morphology. Finally, we demonstrate that Stat3 is a key downstream effector of ALKF1174L and is indispensable for ALKF1174L-driven cSCC tumorigenesis. In sum, these findings reveal that ALK can exert its tumorigenic potential via cooperation with multiple pathways crucial in the pathogenesis of cSCC. Finally, we show that human cSCCs contain mutations in the ALK gene. Taken together, our data identify ALK as a new key player in the pathogenesis of cSCC and this knowledge suggests that oncogenic ALK signaling can be a target for future clinical trials.

overall_design: mRNA profiles of FACS-sorted tumor cells from ear cSCC from ALKF1174L Lgr5-CreERT2 mice, ALKF1174LKrasG12D Lgr5-CreERT2 mice and from sorted normal ear skin keratinocytes of cre negative siblings were produced in triplicate.

first: Marco

last: Gualandi

first: Maria

last: Iorio

first: Olivia

last: Engeler

first: André

last: Serra-Roma

first: Giuseppe

last: Gasparre

first: Johannes

last: Schulte

first: Daniel

last: Hohl

first: Olga

last: Shakhova


title: Culturing C2C12 myotubes on micromolded gelatin hydrogels accelerates myotube maturation

city: Gainesville

first: Eric

last: Wang

state: FL

address: 2033 Mowry Rd

institute: University of Florida

summary: We report that differentiation of the C2C12 myoblast line on patterened hydrogels results in the increased expression of muscle sarcomere genes and more closely models expression changes that happen in in vivo muscle differentiation than do C2C12 myoblasts cultured on unpatterened substrates.

overall_design: 33 total samples, each individual experimental condition replicated 3 times. Differentiation time course Day 0, Day 4, Day 7 for 3 different culture substrates (plastic, patterned gelatin, unpatterned gelatin) and C2C12 myoblasts versus primary myoblasts

first: Lance

last: Denes


city: New York

first: Gregory

last: Putzel

state: NY

address: 430 East 29th Street

department: Microbiology

institute: NYU Langone Health

summary: Dendritic cells (DCs) are critical mediators of host defense against bacteria. The goal of this microarray study was to understand the global transcriptional response of bone marrow-derived dendritic cells (BMDCs) upon exposure to live bacteria, to better understand how DCs orchestrate a host-protective immune response. We found that BMDCs upregulate a number of critical immune-related genes upon exposure to live E. coli. Most notably, the gene encoding hepcidin, a critical regulator of mammalian iron homeostasis, was significantly upregulated in BMDCs upon exposure to live bacteria.

overall_design:
Microarray of E. coli-infected bone marrow-derived dendritic cells
Bone marrow-derived dendritic cells were infected with E. coli and cells were analyzed on day 4 post-infection
3 replicates were performed

first: G

last: Sonnenberg

first: T

last: Fung

first: N

middle: J

last: Bessman


title: The rRNA m6A methyltransferase METTL5 is involved in pluripotency and developmental programmes

city: Neuherberg

first: Valentina

last: Ignatova

state: Select...

address: Institue of Functional Epigenetics, Ingolstädter Landstr. 1

department: IFE

institute: HMGU

summary: Covalent chemical modifications of cellular RNAs directly impact all biological processes. However, our mechanistic understanding of the enzymes catalysing these modifications, their substrates and biological functions remains vague. Here, we undertook a systematic screen to uncover new RNA methyltransferases. We demonstrate that the methyltransferase-like 5 (METTL5) protein catalyses m6A in 18S rRNA at position A1832. We report that absence of Mettl5 in mouse embryonic stem cells (mESCs) results in a changes gene expression, decrease in translation rate, spontaneous loss of pluripotency and compromised differentiation potential. Mice lacking METTL5 recapitulate symptoms of patients with METTL5 mutations, thereby providing a new mouse disease model. Overall, our work  highlights the importance of m6A in rRNA in stemness, differentiation, development and diseases.

overall_design: polyA RNA profiles of wild type (WT) and Mettl5 -/- mESC cultured in Serum Lif were generated by deep sequencing, in triplicate, using Illumina TruSeq Stranded mRNA protocol

first: Robert

last: Schneider


city: SINGAPORE

first: JIA CHI

last: TAN

address: 8a Biomedical Grove

institute: SIgN

overall_design:
Single cell sequencing of embryonic macrophages from E16.5 C57Bl/6 brain. C57Bl/6 mice were time-mated and the day of vaginal plug observation was denoted as 0.5 day after conception (E0.5). Pure populations of CD45+ F4/80+ CD11b+ Ly6C- cells from E16.5 brain were sorted by FACS, and applied onto 5-10 uM-diameter C1 Integrated Fluidic Circuit (IFC; Fluidigm) to capture single cells.
Please note that the sample characteristics are identical across all samples as we discovered the two distinct macrophage populations only after the data processing steps and this is addressed in the supplementary file.

first: Jinmiao

last: Chen

first: Aymeric

last: Silvin

first: Florent

last: Ginhoux


title: Gene expression profile of human osteoclastoma bone tumor derived giant osteoclast cells

city: Charleston

first: Sakamuri

middle: V

last: Reddy

state: SC

address: 173 Ashley Avenue

department: DCRI/Pediatrics

institute: Medical University of South Carolinia

summary:
Osteoclast is the primary bone resorbing multinucleated cell. However, human osteooclastoma bone tumor derived osteoclat cellsare
However, gene expression profile of giant osteoclat cells is unclear. Here, we performed gene expression profiling of  enriched giant cells to elucidate the neoplastic effects and dynamic changes in  gene expression responsible for abnormal hyperactive osteoclast formation in the tumor bone microenvironment.

overall_design: Gene expression profiling was determined in mouse monoclonal (23C6) antibody against integrin alpha V beta 3 receptor purified or enriched human osteoclastoma bone tumors derived giant osteoclast cells


title: Partial loss of CFIm25 causes aberrant alternative polyadenylation and learning deficits [superseries]

city: Houston,

first: Hari Krishna

last: Yalamanchili

address: 1250 Moursund St #1125,

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Partial loss of CFIm25 causes aberrant alternative polyadenylation and learning deficits [mouse]

summary: We previously showed that NUDT21-spanning copy-number variations (CNVs) are associated with intellectual disability (ID), and that NUDT21-encoded CFIm25 regulates the protein levels of at least one dose-sensitive, ID-associated protein: MeCP2 (Gennarino et al., 2015). However, the patients’ CNVs also spanned multiple other genes raising the possibility that loss or gain of these other genes caused their symptoms. To determine if reduced NUDT21 function alone is sufficient to cause disease, we generated Nudt21 heterozygous null mice to mimic the human state of reduced expression. We found that although these mice have 50% reduced Nudt21 mRNA, they only have 30% less of its cognate protein, CFIm25. Despite this partial protein-level compensation, the Nudt21+/- mice have learning deficits and cortical hyperexcitability. Further, to determine the molecular mechanism driving neural dysfunction, we partially inhibited NUDT21 in human embryonic stem cell-derived neurons to reduce CFIm25 by 30%. This reduction in CFIm25 was sufficient to induce misregulated alternative polyadenylation (APA) and protein levels in hundreds of genes, dozens of which are associated with intellectual disability and whose dysregulation is likely contributing to disease symptoms. Altogether, these results indicate that disruption of NUDT21-regulated APA events in the brain can cause intellectual disability.

overall_design: RNA sequencing profiles of left hippocampus of Nudt21+/- and WT mice.

first: Callison

last: Alcott

first: Hari

middle: K

first: Ping

last: Ji

first: Meike

last: van der Heijden

first: Alexander

middle: B

last: Saltzman

first: Nathan

last: Elrod

first: Mei

last: Leng

first: Bhoomi

last: Bhatt

first: Shuang

last: Hao

first: Qi

first: Afaf

last: Saliba

first: Jianrong

last: Tang

first: Anna

last: Malovannaya

last: Wagner

first: Zhandong

last: Liu

first: Huda

last: Zoghbi


title: Partial loss of CFIm25 causes aberrant alternative polyadenylation and learning deficits [human]

overall_design: Anti-NUDT21 and scramble shRNA-infected WA09 human embryonic stem cell-derived excitatory neuron mRNA 3' end sequencing profiles generated by deep sequencing, in triplicate, using Illumina Nextseq 550.

last: Wang

first: Eric


title: Dendritic cell-derived hepcidin sequesters iron from the microbiota to promote mucosal healing

first: Gregory

last: Putzel

summary: Bleeding and altered iron distribution occur in multiple gastrointestinal diseases, but the significance or regulation of these changes remains unclear. Here we report that hepcidin, the master regulator of systemic iron homeostasis, is required for tissue repair in the intestine following experimental damage. This effect was independent of hepatocyte-derived hepcidin or systemic iron levels. Rather, we identified that conventional dendritic cells (DCs) are a source of hepcidin that is induced by microbial stimulation, prominent in the inflamed intestine of humans, and essential for tissue repair. Mechanistically, DC-derived hepcidin acted on ferroportin-expressing phagocytes to promote local iron sequestration, which regulated the microbiota and subsequently facilitated intestinal repair. Collectively, these results identify a novel pathway whereby DC-derived hepcidin promotes mucosal healing in the intestine via nutritional immunity.

overall_design: Mice were treated with 3.5% DSS in drinking water for 7 days, and colon lamina propria cells were sorted as follows: cDC1 (Live, CD45+, F4/80-, CD64-, CD3-, CD19-, NK1.1-, CD138-, MHCII+, CD11c+, XCR1+, CD172a-); cDC2 (Live, CD45+, F4/80-, CD64-, CD3-, CD19-, NK1.1-, CD138-, MHCII+, CD11c+, XCR1-, CD172a+).

first: Nicholas

last: Bessman

last: Sonnenberg


title: miR‐206 family is important for mitochondrial and muscle function, but not essential for myogenesis in vitro [RNA-Seq]

city: Charlottesville

first: Roza

middle: Kamila

last: Przanowska

state: Virginia

department: Biomedical Engineering

institute: University of Virginia

summary: miR-206, miR-1a-1 and miR-1a-2, foundational members of the myomir family, promote skeletal muscle differentiation and have complete identity in the seed sequence, suggesting functional redundancy. To test if the essential role of these three microRNAs in skeletal myogenesis was masked by redundant function of at least one of the three miRNA left in the cells in previous reports, we generated triple KO (tKO) C2C12 cells by CRISPR/Cas9 (in vitro) and triple KO mice (in vivo model). tKO myoblasts differentiate, producing similar levels of promyogenic mRNAs and normal fusion index. RNAseq analysis confirms tKO clones differentiate but also reveals decrease in mRNA levels of genes related to mitochondrial oxidative phosphorylation. Dimished mitochondria function in tKO cells was confirmed using Seahorse assay. The targets of miR-206/-1a are specifically upregulated during differentiation of tKO myoblasts, when the microRNAs are normally induced, confirming the complete loss of the three microRNA genes. Although we observe partial embryonic lethality of tKO mice, the live mice grow normally to adulthood, have the same size of skeletal muscles as control animals, but perform worse on physical activity tests, which may be partially explained by smaller skeletal muscle fiber diameters. Thus these three myomiRs are not essential for skeletal muscle differentiation but are required for optimal differentiation and provide a clear example of the microRNAs not being a switch but a modulator of differentiation.

overall_design: RNA-seq on 16 samples; control C2C12 cells population 1 and population 2, miR-206, miR-1a-1 and miR-1a-2 triple KO clone 1 and clone 2; proliferation and differentiation conditions

first: Anindya

last: Dutta


title: miR‐206 family is important for mitochondrial and muscle function, but not essential for myogenesis in vitro

type: Non-coding RNA profiling by high throughput sequencing


title: Gene-Specific Control of tRNA expression by RNA Polymerase II

first: Alan

last: Gerber

address: 1230 York Avenue

institute: The Rockefeller University


title: miR‐206 family is important for mitochondrial and muscle function, but not essential for myogenesis in vitro [miRNA-Seq]

overall_design: small RNA-seq on 16 samples; control C2C12 cells population 1 and population 2, miR-206, miR-1a-1 and miR-1a-2 triple KO clone 1 and clone 2; proliferation and differentiation conditions

first: Zhangli

last: Su


title: Gene-Specific Control of tRNA expression by RNA Polymerase II (ChIP-seq)

summary: Increasing evidence suggests that tRNA levels are dynamically and specifically regulated in response to internal and external cues to modulate the cellular translational program. However, the molecular players and the mechanisms regulating the gene-specific expression of tRNAs are still unknown. Using an inducible auxin-degron system to rapidly deplete RPB1 (the largest subunit of RNA Pol II) in living cells, we identified Pol II as a direct gene-specific regulator of tRNA transcription. Our data suggest that Pol II transcription robustly interferes with Pol III function at specific tRNA genes. This activity was further found to be essential for MAF1-mediated repression of a large set of tRNA genes during serum starvation, indicating that repression of tRNA genes by Pol II is dynamically regulated. Hence, Pol II plays a direct and central role in the gene-specific regulation of tRNA expression.

overall_design: Two HEK293 clones knocked out for the endogenous Pol II subunit RPB1 but stably expressing an auxin-degradable RPB1 (tagged-RPB1) were treated either with water (vehicle, control condition) or Doxycycline (Dox), to induce a OsTIR1 ubiquitin ligase from a transgene inserted in the AAVS1 locus, followed by auxin (DA) to rapidly degrade the tagged RPB1 in Dox pretreated cells. RPB3 (Pol II subunit) and RCP62 (Pol III subunit) ChIP-seq was then performed using cross-linked chromatin prepared from these cells

first: Robert

middle: G

last: Roeder


title: Gene expression profile of endothelial cells derived from circulating progenitors issued from patients with rheumatoid arthritis

city: Paris

first: Franck

last: Letourneur

address: 22 rue mechain

country: France

institute: INSERM

summary:
Synovial neoangiogenesis is an early and crucial event to promote the development of the hyperplasic proliferative pathologic synovium in rheumatoid arthritis (RA). Endothelial cells (ECs) are critical for the formation of new blood vessels since they highly contribute to angiogenesis and vasculogenesis.
To better characterize these cells, our group has studied the gene expression profiles of ECs issued from 18 RA patients compared to 11 healthy controls.

overall_design: ECs derived from circulating endothelial progenitor cells (EPCs) were isolated from peripheral blood of RA patients and controls for RNA extraction and hybridization on Affymetrix microarrays. Gene expression profiles of EPC-derived ECs were determined in basal conditions and also after hypoxic exposure.

first: Jérôme

last: Avouac

first: Agathe

last: Leblond

first: Yannick

last: Allanore


title: Gene-Specific Control of tRNA expression by RNA Polymerase II (arrays)

overall_design: Three HEK293 clones knocked out for the endogenous Pol II subunit RPB1 but stably expressing an auxin-degradable RPB1 (tagged-RPB1) were treated either with water (vehicle, control condition) or Doxycycline (Dox), to induce a OsTIR1 ubiquitin ligase from a  transgene inserted in the AAVS1 locus, or Dox followed by auxin (DA) to rapidly degrade the tagged RPB1. Serum starvation and re-stimulation experiments were also performed. Total RNA from all three clones were pooled together for each condition and analyzed by microarray


title: Differentially Expressed tRFs in CD5 Positive Relapsed & Refractory Diffuse Large B Cell Lymphoma and the Bioinformatic Analysis for Their Potential Clinical Use

status: Public on Apr 14 2020

city: Jinan

first: Yujie

last: Jiang

address: No.324, Jingwu Road, Jinan, Shandong 250021, People’s Republic of China

country: China

institute: Shandong Provincial Hospital affiliated to Shandong University

summary:
Background: Patients diagnosed as diffuse large B cell lymphoma (DLBCL) with CD5 positive normally have a worse outcome and poorly respond to the regulatory treatment strategy.
Results: We recently reported differently expressed tRFs and their potential target-genes of tRFs in patients with CD5+ R/R DLBCL. Differently expressed tRFs were detected by Illumina NextSeq instrument and the results were verified by quantitative real-time reverse transcription-PCR. tRF2Cancer database was searched to compared with the results. Further research was performed through bio-informatic analysis including gene ontology (GO) and pathway enrichment analyses, etc. A total of 308 tRFs were identified. Two sequences (AS-tDR-008946, AS-tDR-013492) were chosen for further investigated.
Conclusions: The results of Bioinformatics analysis revealed that the target genes including NEDD4L and UBA52 and several associated pathways including PI3K/AKT and MAPK/ERK might be involved in the development of CD5+ R/R DLBCL. Our preliminary study on the associated tRFs might provide a valuable measure to explore the pathogenesis and progression of CD5+ R/R DLBCL.

overall_design: RNA profiles of three test and three control samples were generated by deep sequencing, using Illumina NextSeq 500.

first: Qingyuan

last: Qu

first: Ying

last: Li

first: Xiaosheng

last: Fang

first: Lingyan

last: Zhang

first: Chao

last: Xue

first: Xueling

last: Ge

first: Xin


title: Next Generation Sequencing Facilitates Analysis of The Mouse Oocyte、Cumulus cells and Blastocyst Transcriptomes From PM2.5 Exposure Group And Filtrated Air Group

city: Shanghai

first: Yi

last: Guo

address: 2699 Western Gaoke Road

department: Shanghai First Maternity and Infant Hospital

institute: Tong ji university

summary:
Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare mouse NGS-derived oocyte、cumulus cell and blastocyst transcriptome profiling (RNA-seq) between PM2.5 exposure group and Filtrated air group
Methods: mouse oocyte、cumulus cell and blastocyst mRNA profiles were generated by deep sequencing, in triplicate for each stage, using Illumina Hi-Seq 2500. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks.
Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9). Genes with adjusted P < 0.05 and fold-change (FC) ≥ 2 were considered differentially expressed genes (DEGs). The oocyte transcriptome analysis showed that 467 DEGs (260 and 207 up- and down-regulated genes, respectively) were identified in the CAP group compared with the FA group. In the cumulus cell analysis, 592 DEGs (358 and 234 up- and down-regulated genes, respectively) were identified in the CAP group compared with the FA group. The blastocyst transcriptome analysis identified 448 DEGs (254 and 194 up- and down-regulated, respectively) in the CAP group compared with the FA group.
Conclusions: Our study represents the first detailed analysis of mouse oocyte、cumulus cell and blastocyst transcriptomes, with biologic replicates, generated by RNA-seq technology between PM2.5 exposure group and Filtrated air group.

overall_design: Mouse oocyte、cumulus cell and blastocyst mRNA profiles of PM2.5 exposure group and Filtrated air group were generated by deep sequencing, in triplicate, using Illumina Hi-Seq 2500


title: Downregulation of miR-192-5p and miR-204-5p in nonclassic apparent mineralocorticoid excess

city: Brisbane

first: Dominic

last: Guanzon

state: Queensland

address: Building 71/918 RBWH Herston

department: UQCCR

country: Australia

institute: University of Queensland

summary: The “nonclassic” apparent mineralocorticoid excess (NC-AME) has been identified in approximately 7% of general population. Our aim was to identify miRNAs within urinary exosomes associated to the NC-AME phenotype. A total of 18 urine samples (9 control and 9 NC-AME) derived from adult and children subjects were analyzed using small RNA sequencing.

overall_design: miRNAs were profiled from urinary exosomes using next generation sequencing (Illumina NextSeq 500 platform).

first: Cristian

last: Carvajal

first: Alejandra

last: Tapia-Castillo

first: Carlos

last: Salomon


title: Gene-Specific Control of tRNA expression by RNA Polymerase II (nascent RNA-seq)

first: Alan

last: Gerber

overall_design: Two HEK293 clones knocked out for the endogenous Pol II subunit RPB1 but stably expressing an auxin-degradable RPB1 (tagged-RPB1) were treated either with water (vehicle, control condition) or Doxycycline (Dox), to induce a OsTIR1 ubiquitin ligase from a transgene inserted in the AAVS1 locus, followed by auxin (DA) to rapidly degrade the tagged RPB1 in Dox pretreated cells. Nascent RNAs were physically isolated and converted to cDNA using Illumina TruSeq stranded protocol, and sequenced on Illumina HiSeq 2500

first: Robert

last: Roeder


title: Analysis of gene expression changes in patient-derived gastric cancer cells after anticancer drug treatment or ALDH1A3 knockdown

city: Tokyo

first: Ryuhei

last: Kawakami

address: 3-8-31, Ariake,Koto-ku

institute: JFCR

summary: Tumors consist of heterogeneous cell population, containing cancer cell subpopulations with anticancer drug-resistant property, called “persister” cells. To reveal the character of the persister cells, we analyzed gene expression profile of patient-derived gastric cells and residual cancer cells after treatment with 5-FU or SN38, an active metabolite of irinotecan. In our study, we identified ALDH1A3 as a marker and a cell proliferation factor of persister cells. To examine molecular pathways regulated by ALDH1A3, we analyzed gene expression profile of patient-derived gastric JSC15-3 in which ALDH1A3 was knocked down by using shRNAs.

overall_design: We treated patient-derived gastric cancer JSC15-3, JSC17-2 and JSC17-7 cells with 5-FU or SN38. We analyzed gene expression of control (DMSO) or drug-treated cells. Meanwhile, we  introduced shRNAs by using lentivirus and selected the infected cells by 1 mg/mL puromycin. We analyzed gene expression in control cells (non-targeting shRNA [NC]) and ALDH1A3 -knocked down cells.

first: Tetsuo

last: Mashima

first: Hiroyuki

last: Seimiya


title: Genomic Sites Ultrasensitive to Ultraviolet Radiation

city: New Haven

first: Douglas

middle: E.

last: Brash

state: CT

address: 333 Cedar St. / HRT213

department: Therapeutic Radiology

institute: Yale School of Medicine

summary:
If the genome contains outlier sequences extraordinarily sensitive to environmental agents, these would be sentinels for monitoring personal carcinogen exposure and might drive direct changes in cell physiology rather than acting through rare mutations. New methods, adductSeq and freqSeq, provided statistical resolution to quantify rare lesions at single-base resolution across the genome. Primary human melanocytes, but not fibroblasts, carried spontaneous apurinic sites and TG sequence lesions more frequently than UV-induced cyclobutane pyrimidine dimers (CPDs). UV exposure revealed hyperhotspots acquiring CPDs up to 170 fold more frequently than the genomic average; these sites were more prevalent in melanocytes. Hyperhotspots were disproportionately located near genes, particularly for RNA-binding proteins, with the most-recurrent hyperhotspots at a fixed position within two motifs: one occurring at ETS1 transcription factor binding sites, known to be UV targets, and at sites of mTOR/TOP-tract translation regulation; the second occurring at A2-15TTCTY, which developed "dark CPDs" after UV exposure, repaired CPDs slowly, and had accumulated CPDs prior to the experiment. Motif locations active as hyperhotspots differed between cell types. Melanocyte CPD hyperhotspots aligned precisely with recurrent UV signature mutations in individual gene promoters of melanomas and with known cancer drivers. At sunburn levels of UV exposure, every cell would have a hyperhotspot CPD in each of the ~20 targeted cell pathways, making hyperhotspots act as epigenetic marks.
Purpose: These experiments searched for genomic sites in human primary fibroblasts and melanocytes that are extraordinarily sensitive to DNA damage, primarily cyclobutane pyrimidine dimers (CPDs) induced by UVC or UVB radiation. They separately detected abasic sites and other spontaneous DNA damage when present.

overall_design:
In most experiments, pre-existing abasic sites were first blocked by nicking with an apurinic endonuclease; in all experiments, shear breaks were blocked using phosphatase. Then DNA was incised with a DNA repair enzyme recognizing CPDs. CPDs were reversed enzymatically and linkers were ligated to the 5' phosphate at the nick. Libraries were constructed and sequenced by paired-end Illumina sequencing to identify linker-ligated sites. Statistical analysis compared +UV data to -UV data and pyrimidine-pyrimidine sites (putative CPD sites) to PuPu sites (controls for copy number and starting cell number), to calculate CPD frequencies at individual bases.

Please note that each processed data file was generated from multiple samples (as indicated in the corresponding sample description field). Several baseline samples were generated to establish the baseline of the technique itself and thus no processed data was generated. Although the baseline samples are not used directly in any of the final conclusions, they are included in the records as discussed heavily in the associated manuscript.

first: Sanjay

last: Premi


title: Expression data of renal cancer cell treated with Oncolytic virus VSVd51 and Kadcyla

city: Ottawa

first: Mohammed

last: Selman

state: ON

address: 501 Smyth Road

department: Cancer Center

country: Canada

institute: Ottawa Hospital

summary:
We propose a strategy to boost the therapeutic efficacy of Oncolytic therapy by combining it with Kadcyla
The mechanism of action was examined by microarray analysis using the Affymetrix Human PrimeView Array.

overall_design: 786-0 cells were plated in 6-well dishes and allowed to adhere overnight. The next day, cells were pretreated for 4hours with Kadcyla, Herceptin, colchicine or the vehicle. Following pre-treatment, the cells were infected with VSVΔ51 at an MOI of 0.01 or left uninfected. Twenty-four hours post infection, RNA was collected using an RNA-easy kit . Biological triplicates were subsequently pooled and RNA quality was measured using Agilent 2100 Bioanalyzer (Agilent Technologies) before hybridization. Hybridized to Affymetrix Human PrimeView Array . Microarray data was processed using Transcriptome Analysis Console (TAC) 3.0 under default parameters of Gene Level Differential Expression Analysis. Fold change in gene expression was calculated for each gene in relation to uninfected, untreated control.

first: Jean-Simon

last: Diallo


title: SAB expression determines severity of sex-dependent acute liver injury

city: Los Angeles

first: Sanda

last: Win

address: 2011 Zonal Ave

department: Medicine

institute: Univeristy of Southern California

summary: miR34a-5p suppresses SAB level in liver.

overall_design: Normal liver samples from 12 weeks old Chow diet (PicoLab Rodent Diet20 #5053) fed C57BL/6N 3 male and 3 female littermates were collected at 11am Janurary 2016.

middle: W

last: Min

first: Christopher

middle: Q

last: Chen

first: Jun

last: Zhang

first: Yibu

first: Meng

last: Li

first: Ayako

last: Suzuki

first: Manal

middle: F

last: Abdelmalek

first: Ying

last: Wang

first: Mariam

last: Aghajan

first: Filbert

last: Aung

first: Anna

last: Diehl

first: Roger

last: Davis

first: Tin

last: Than

first: Neil

last: Kaplowitz


title: Essential Oil from Pinus Koraiensis pinecones Inhibits Gastric Cancer Cells via the HIPPO/YAP Signaling Pathway

city: Harbin

first: yandong

last: zhang

address: 92 xidazhi street

institute: Harbin Institute of Technology

summary: The purpose of this study was to investigate the anti-tumor activity of PEO on MGC-803 cells and its mechanism. Anti-tumor experiments in vitro showed PEO could significantly inhibit the proliferation and migration of MGC-803 cells, and it also could arrest the cell cycle in G2/M phase, decrease the mitochondrial membrane potential and induce apoptosis. Finally, the effects of PEO on genes expression on MGC-803 cells were analyzed by RNA sequencing, and results showed that after treatment with PEO, 100 genes were up-regulated, and 57 genes were down-regulated. According to KEGG pathway and GSEA, FAT4, STK3, LATS2, YAP1 and AJUBA were down-regulated, which are related to HIPPO signaling pathway. Real-time PCR and western blot further confirmed the results of RNA sequencing. These results indicated that PEO may exert anti-tumor activity via the HIPPO/YAP signaling pathway.

overall_design:
The RNA of MGC-803 cells treated and untreated were sequenced by high-throughput sequencing to detect the effect of drugs on the gene of MGC-803 cells.
This research was funded by National Key R&D Program Project "Ecological Protection and Biological Resources Development and Utilization Technology and Demonstration in Northeast Forest Region" (sub-project) (No. 2016YFC0500305-02).

first: Yandong

first: Chao

last: Xin

first: Junqiang

last: Qiu

first: Zhenyu


title: RNA Sequencing of Intratumoral Immune Populations in Pancreatic Ductal Adenocarcinoma Following Radiotherapy and Interleukin 12 Treatment

city: Rochester

first: Bradley

last: Mills

address: 601 Elmwood Avenue

institute: University of Rochester Medical Center

summary: We report differential gene expression in inflammatory monocyte (IM), tumor-associated macrophage (TAM), and CD8 T cell populations following stereotactic body radiation (SBRT), intratumoral interleukin 12 microsphere (IL-12 MS) delivery, and SBRT/IL-12 MS combination. Immune cell populations were sorted from orthotopic murine pancreatic ductal adenocarcinoma (PDA) tumors prior to RNA sequencing.

overall_design: Orthotopic murine PDA tumors (KCKO cell line) were treated with SBRT, IL-12 MS, or SBRT/IL-12 MS combination (duplicate/triplicate); IM, TAM, and CD8 cells were sorted from tumor lysates using a FACSAria II platform; cDNA libraries were constructed using the TruSeq RNA Sample Preparation Kit V2 and sequencing was performed on a HiSeq 2500 platform.

first: Scott

middle: A

last: Gerber

middle: N


title: Root gene expression profiles of two maize inbred lines (Lo5 and T250) showing different nitrogen use efficiency (NUE) in response to the growth without N.

city: Verona

first: Anita

last: Zamboni

address: Strada Le Grazie, 15

department: Biotechnology

country: Italy

institute: University of Verona

summary: Among the mineral elements necessary for plant growth, nitrogen (N) is the macronutrient required in larger amounts. Maize genotypic-specific responses to N shortage could provide more information useful to improve NUE.

overall_design:
Maize (Zea mays L.) seeds of two inbred lines (Lo5 and T250) previously soaked in water for 24 h, were allowed to germinate in the dark at 26°C for 4d. The seedlings were then transferred in pots containing 2.2 L of 0.5 mM
CaSO4 aerated solution for 48h in controlled climatic conditions. After this period, seedlings were subsequently grown for other 4d using a nutrient solution (NS) without N. Three biological replicates were obtained repeating
three independent times the growth experiment. For each experiment and both for Lo5 and T250 lines root samples were obtained pooling the roots of three seedlings collected after 0, 1 and 4d of growth without N.

first: Maria

last: Mascia

first: Davide

last: Sega

first: Zeno

last: Varanini


title: Detection of early-stage Alzheimer’s pathology using blood-based autoantibody biomarkers in elderly hip fracture repair patients

city: Stratford

first: Robert

last: Nagele

state: NJ

address: 2 Medical Center Drive

department: NJISA

institute: UMDNJ-SOM

summary: Human plasma samples from CDR = 0 and CDR ≥ 0.5 patients, and serum samples from non-diseased, non-surgical controls were probed onto human protein microarrays to test the utility of a  previously established panel of MCI-specific autoantibody (aAB) biomarkers to detect prodromal AD presurgically in individuals admitted into the hospital for hip fracture repair (HFR) surgery.

overall_design: In the study presented here, 44 hip fracture repair (HFR) patients and 25 control human serum samples were probed onto human protein microarrays in order to to test the utility of a  previously established panel of MCI-specific autoantibody (aAB) biomarkers to detect prodromal AD presurgically in individuals admitted into the hospital for HFR surgery.  Microarray data was analyzed using several statistical significance algorithms.  Prediction classification analysis using Invitrogen's Prospector v5.2.1 and Random Forest tested the diagnostic efficacy of the MCI-specific aAB biomarkers.

first: Cassandra

last: DeMarshall

first: Esther

last: Oh

first: Rahil

last: Kheikhah

first: Nae-Yuh

first: Frederick

last: Sieber


title: Feng et al 2019 - Microarray Raw Data

city: Kent

first: Manabu

last: Kurokawa

state: OH

address: 222 Cunningham Hall

institute: Kent State

summary: In order to identify mechanisms of drug resistance to HER2-targeted therapy, we performed cDNA microarray analysis on drug naiive BT474 and drug resistant BT474 cells treated with lapatinib for 0, 10, and 20 hrs.

overall_design: 2 samples (sensitve and resistant) with 4 replicates per sample for 3 timepoints. 24 total samples were analyzed

first: Feng

last: William

first: Kurokawa

last: Manabu


title: Gene expression data of naïve wildtype and IL-33 knockout ILC2s isolated from adult male mouse lungs

city: San Francisco

first: Catherine

middle: Anne

last: Steer

state: California

address: 3333 California Street, Suite 385, Box 1348

department: Laboratory Medicine

institute: University of California San Francisco

summary: Group 2 innate lymphoid cells (ILC2s) in mouse lungs are activated by the epithelium-derived alarmin IL-33. Activated ILC2s proliferate and produce IL-5 and IL-13 that drive allergic responses. In neonatal lungs, IL-33 is spontaneously released resulting in activation of lung ILC2s. Here we report that neonatal lung ILC2 activation has significant effects on ILC2 functions in adulthood.  Most neonatal lung ILC2s incorporated bromodeoxyuridine (BrdU) and persisted into adulthood.  BrdU+ ILC2s in adult lungs responded more intensely to IL-33 treatment than BrdU- ILC2s. In IL-33 deficient (KO) mice, lung ILC2s develop normally but they are not activated in the neonatal period. Lung ILC2s in KO mice responded less intensely to IL-33 in adulthood compared to wild type (WT) ILC2s. While there was no difference in the number of lung ILC2s, there were fewer IL-13+ ILC2s in IL-33KO than IL-33WT mice. The impaired responsiveness of ILC2s in KO mice was reversed by intranasal injections of IL-33 in the neonatal period. These results suggest that activation of lung ILC2s by endogenous IL-33 in the neonatal period may “train” ILC2s seeding the lung after birth to become long-lasting resident cells that respond more efficiently to challenges later in life.

overall_design: ILC2s were isolated from adult male mouse lungs from naïve wildtype and IL-33 knock-out mice. RNA was extracted from these ILC2 populations and analyzed for gene expression profiles.

first: Laura

last: Mathä

first: Hanjoo

last: Shim

first: Fumio

last: Takei


title: Gene expression data of naïve postnatal day 10 neonatal, naïve adult, and IL-33-activated adult ILC2s isolated from mouse lungs

overall_design: ILC2s were isolated from naive male and female mouse lungs 10 days (neonate) and 8 weeks (adult) after birth and from adult mouse lungs one day after three consecutive intranasal injections of IL-33 (effector). RNA was extracted from these ILC2 populations and analyzed for gene expression profiles.


title: Hepatic transcriptome phenotypes after mTORC1 activation through Tsc1 and Depdc5 mutations

city: Ann Arbor

first: Jun Hee

last: Lee

state: MI

address: 109 Zina Pitcher Pl

institute: University of Michigan

summary: We report RNA-seq results from liver tissues of control mice, liver-specific Tsc1 knockout mice, liver-specific Depdc5 knockout mice and liver-specific Tsc1/Depdc5 double knockout mice.

overall_design: RNA-seq analyses of liver tissues from four different transgenic mouse strains.

first: Chun-Seok

last: Cho

first: Allison

middle: H

last: Kowalsky

first: Sim

last: Namkoong

first: Jun


title: Multiplexed activation of endogenous genes by CRISPRa elicits potent anti-tumor immunity

city: West Haven

first: Ryan

middle: D

last: Chow

address: 850 West Campus Drive

institute: Yale University

summary: Immunotherapy has transformed cancer treatment. However, current immunotherapy modalities face many limitations. Here, we developed a new form of immunotherapy that elicits anti-tumor immunity through multiplexed activation of endogenous genes. We leveraged the CRISPR activation (CRISPRa) system to directly augment the in situ expression of endogenous genes, and thereby the presentation of tumor antigens, leading to dramatic anti-tumor immune responses. Deploying this as a cell-based vaccination strategy showed efficacy in both prophylactic and therapeutic settings. Intratumoral adeno-associated virus delivery of CRISPRa libraries elicited strong anti-tumor immunity across multiple cancer types. Precision targeting of mutated gene sets eradicated a large fraction of established tumors at both local and distant sites. This treatment modality led to alterations of the tumor microenvironment, marked by enhanced T cell infiltration and anti-tumor immune signatures. Multiplexed endogenous gene activation may serve as a versatile and highly scalable strategy to elicit potent immune responses against cancer.

overall_design: 3 independent mice pooled for each treatment condition

first: Sidi

last: Chen


title: miR-146a is overexpressed and contributes to dampen IL-6 production in cystic fibrosis macrophages

city: Rome

first: Valerio

last: Licursi

address: via degli Apuli, 4

department: c/o Dept. Biology and Biotechnologies "C. Darwin", Sapienza University of Rome

institute: Institute of Molecular Biology and Pathology (IBPM), National Research Council (CNR)

summary: In this study, we investigated whether miRNA deregulation might underlie the functional abnormalities of cystic fibrosis (CF) macrophages. To this aim we performed miRNA profiling in macrophages from CF  and non-CF macrophages. This led to the identification of a panel of differentially expressed miRNAs in CF macrophages compared to non-CF cells.

overall_design: miRNA profiles of monocyte derived macrophages from six CF and six non-CF individuals were generated by microRNA-Seq.

first: Francesco

middle: R

last: Luly

first: Manuella

last: Lévêque

first: Giuseppe

last: Cimino

first: Corinne

last: Martin-Chouly

first: Nathalie

last: Théret

first: Rodolfo

last: Negri

first: Luca

last: Cavinato

first: Fiorentina

last: Ascenzioni

first: Paola

last: Del Porto


title: Identification of differentially expressed lncRNAs and mRNAs in luminal-B breast cancer by RNA-sequencing

city: Deyang

first: Ning

last: Zou

state: Sichuan

address: No. 173, Taishan North Road, Jingyang District

institute: People's Hospital of Deyang City

summary: Background: Luminal B cancers show much worse outcomes compared to luminal A. This present study aims to screen key lncRNAs and mRNAs correlated with luminal-B breast cancer.  Methods: Luminal-B breast cancer tissue samples and adjacent tissue samples were obtained from 4 patients with luminal-B breast cancer. To obtain differentially expressed mRNAs (DEmRNAs) and lncRNAs (DElncRNAs) between luminal-B breast cancer tumor tissues and adjacent tissues, RNA-sequencing and bioinformatics analysis were performed. Functional annotation of DEmRNAs and protein-protein interaction networks (PPI) construction were performed. DEmRNAs transcribed within a 100kb window up- or down-stream of DElncRNAs were searched, which were defined as cis nearby-targeted DEmRNAs of DElncRNAs. DElncRNA-DEmRNA co-expression networks were performed.  Results: A total of 1178 DEmRNAs and 273 DElncRNAs between luminal-B breast cancer tumor tissues and adjacent tissues were obtained. Hematopoietic cell lineage, Cytokine-cytokine receptor interaction, Cell adhesion molecules (CAMs) and Primary immunodeficiency were significantly enriched KEGG pathways in luminal-B breast cancer. FN1, EGFR, JAK3, TUBB3 and PTPRC were five hub proteins of the PPI networks. A total of 99 DElncRNAs-nearby-targeted DEmRNA pairs and 1878 DElncRNA-DEmRNA co-expression pairs were obtained.  Conclusion: This study determined key genes and lncRNAs involved in luminal-B breast cancer, which expected to present a new avenue for the diagnosis and treatment of luminal-B breast cancer.

overall_design: mRNA and lncRNA profiles of cancer tissue and adjacent tissues from 4 patients with luminal-B breast cancer were obtained based on the Illumina Hiseq X-ten platform with PE150 bp sequencing mode.

first: Cheng-Liang

last: Yuan

first: Xiang-Mei

last: Jiang

first: Ying

last: Yi

first: E

last: Jian-Fei

first: Nai-Dan

last: Zhang

first: Xue

last: Luo

first: Wei

last: Wei

first: Ying-Ying

last: Liu


title: Periostin promotes cell cycle in lung fibroblasts

city: Saga

first: Tomohito

last: Yoshihara

state: Saga

institute: Saga Univercity

summary: Idiopathic pulmonary fibrosis (IPF) is a devastating disease with only three to five years of the median survival. Fibroblast proliferation is a hallmark of IPF as well as secretion of extracellular matrix proteins from fibroblasts. However, it is still uncertain how IPF fibroblasts acquire the ability to progressively proliferate. Periostin is a matricellular protein that is highly expressed in the lung tissues of IPF patients and plays a critical role in the pathogenesis of pulmonary fibrosis. However, it remains undetermined whether periostin affects proliferation of lung fibroblasts. In this study, we first comprehensively tried to identify periostin-dependently expressed genes in lung fibroblasts finding that many cell-cycle–related genes are involved in the gene profile. We confirmed that periostin silencing downregulates expression of several cell-cycle–related molecules including the cyclin family, the CDK family, the E2F family, and the transcriptional factors such as B-MYB and FOXM1 in lung fibroblasts. Accordingly, periostin silencing slowed proliferation of lung fibroblasts and affects the distribution of cell cycle particularly at the G1/S checkpoint and drives the cells into G1 arrest. Lung fibroblasts derived from IPF patients also required periostin for maximum proliferation. Moreover, CP4715, a potent inhibitor against integrin V3, a periostin receptor, downregulated proliferation along with expression of cell-cycle–related genes in IPF lung fibroblasts as well as normal lung fibroblasts. These results demonstrate that periostin plays a critical role in proliferation of lung fibroblasts and provide us a beneficial basis to apply the inhibitors against the periostin/integrin V3 interaction to IPF patients.

overall_design: To comprehensively identify genes expressed dependently on periostin in lung fibroblasts, we compared the gene expression profiles with or without knockdown of periostin in lung fibroblasts (MRC-5 cells). MRC-5 cells were transfected with 10 nM periostin siRNA for 48 h.

first: Kenji

last: Izuhara


title: A benchmark of hemoglobin blocking during library preparation for mRNA-Sequencing of human blood samples

city: Kiel

first: Florian

last: Uellendahl-Werth

address: Rosalind-Franklin-Straße 12

department: Institute of Clinical Molecular Biology (IKMB)

institute: Christian-Albrechts Universität zu Kiel

summary:
Background: RNA-Sequencing (RNA-Seq) of peripheral blood can be a valuable source of information for investigating the status and mechanism of diseases. However, blood contains mostly unwanted hemoglobin (Hb) transcripts. A recent method for Illumina RNA-Seq features a ‘Globin Block’ (GB) module that depletes Hb transcripts during library preparation. Here, we aimed to assess GB’s effectiveness, and we checked for technical biases attributable to GB.

Results: We sequenced 91 pairs of GB and non-blocked samples (noGB) and 8 GB and noGB technical replicates from whole blood total RNA samples of 91 healthy individuals. GB reduced the fraction of Hb transcripts from 43 % to 8.0 %. From GB samples we detected 1,397 more expressed genes at approximately 11 million reads per RNA-isolate. Using correlation analyses we showed that GB and noGB samples could be assigned to the same RNA isolate with 89.8 % accuracy. Interestingly, lane and platform bias influenced accuracy stronger than GB. Gene expressions distinguishing between GB and noGB could not be enriched for sequence similarity or molecular function.

Conclusions: Lexogen’s GB RNA-Seq module is a valuable addition during mRNA-Seq library preparation. GB facilitates the detection of low abundant transcripts and yields more non-hemoglobin reads, while preserving biological information.

overall_design: 91 healthy individuals’ whole blood RNA samples were prepared with Lexogen’s 3’ mRNA-Seq Library Prep Kit, once with the optional Globin Block Module, once without. The effect of the Globin Block Module was investigated. 8 samples, once with the Globin Block  were resequenced with Globin Block .

first: Markus

last: Wolfien

first: Andre

last: Franke

first: Olaf

last: Wolkenhauer

first: David

last: Ellinghaus


title: Multiplexed gene editing in the Tmprss11 protease gene cluster reveals a unique role of HAT-like 4 in epidermal barrier formation

city: Bethesda

first: Roman

last: Szabo

state: Maryland

address: 30 Convent Drive, Rm.3A308

institute: National Institute of Dental and Craniofacial Research

summary: The physiological functions of the HAT/DESC sub-family of membrane-anchored serine proteases are poorly defined. The high sequence homology, overlapping pattern of expression, and tight clustering of their corresponding genes have complicated the functional analysis of HAT/DESC proteases by conventional loss-of-function genetics. Here, we exploited the extraordinary efficiency of CRISPR-mediated gene targeting to generate 18 unique congenic mouse strains lacking combinations of HAT/DESC proteases, including a mouse strain deficient in all seven proteases. Surprisingly, mice lacking all HAT/DESC proteases developed to term, were healthy and fertile, and displayed only a modest impairment of epidermal barrier function, as recently described for mice with single deficiency in HAT-like 4. This function of HAT-like 4 in epidermal barrier formation was unique among HAT/DESC proteases. Thus, mice lacking all HAT/DESC proteases displayed transepidermal water loss rates similar to mice lacking only HAT-like 4, and, conversely, mice lacking all HAT/DESC proteases, except HAT-like 4, displayed transepidermal water loss rates identical to wildtype mice. The study yields new insights into the function of HAT/DESC proteases and provides multiple new mouse strains valuable for functional studies of this enigmatic protease sub-family in physiological and in pathophysiological processes.

overall_design: Examination of epidermis and tongue in wild-type mice and mice lacking all seven HAT/DESC proteases

first: LuLu

last: Callies

first: Thomas

last: Bugge


title: RNAseq of prostate from mice submitted to diets containing different fatty acids profile (pork fat, linseed oil and soybean oil)

city: Campinas

first: Danilo

middle: Lopes

last: Ferrucci

state: SP

address: Rua Monteiro Lobato, 255

department: Genetic and Molecular Biology

country: Brazil

institute: Unicamp

summary: To perform mRNA expression analysis through deep sequencing, RNA was isolated from prostate mice treated with normolipidic diets based on linseed oil, soybean oil or lard (varying saturated and unsaturated FA contents and ω-3/ω-6 ratios (ω ratio) for 12 or 32 weeks after weaning

overall_design: Prostate mRNA profiles of mice treated for 12 and 32 weeks with diets containing different lipids profile were generated by RNAseq, five samples per group, using HiSeq® 2500 (Illumina®).

first: D

middle: L

first: H

last: Carvalho


title: PHD inhibition upregulates glycolytic pathway in OGD

city: Tokyo

first: MARIE

last: ITO

address: 7-3-1 Hongo, Bunkyo

department: Department of Nephrology and Endocrinology

institute: the University of Tokyo

summary: To elucidate the mechanism of enarodustat pretreatment-induced protection against oxygen-glucose deprivation (OGD), we have we have employed whole genome microarray expression profiling as a discovery platform to identify genes upregulated by enarodustat exposure. HK2 cells were pretreated with either vehicle (DMSO) or enarodustat for 24 hours prior to OGD for 16 hours. Pathway analysis revealed upregulated glysolysis pathway.

overall_design: A microarray analysis of 3 groups (sham: 24 h DMSO pretreatment + 16 h in normal medium, ctrl: 24 h DMSO pretreatment + 16 h OGD, enarodustat: 24 h enarodustat pretreatment+ 16 h OGD, n=3 for each group) of HK2 cells was conducted.

first: Marie

last: Ito

last: Fukui


title: Effect of BTAF1 downregulation on gene expressions of TBP 3X-WT and TBP 3X-T114A Hela cell lines

city: Baltimore

first: Gerald

last: Hart

state: MD

address: 725 N. Wolfe St.

department: Biological Chemistry

institute: Johns Hopkins School of Medicine

summary: In eukaryotes, gene expression is performed by three RNA polymerases that are targeted to promoters by molecular complexes. A unique common factor, the TATA-box binding protein (TBP), is thought to serve as a platform to assemble pre-initiation complexes competent for transcription. Herein, we describe a novel molecular mechanism of nutrient regulation of gene transcription by dynamic O-GlcNAcylation of TBP. We show that O-GlcNAcylation at T114 of TBP blocks its interaction with BTAF1, hence the formation of the B-TFIID complex, and its dynamic cycling on and off of DNA. Transcriptomic and metabolomic analyses of TBPT114A CRISPR/Cas9 edited cells showed that loss of O-GlcNAcylation at T114 increases TBP binding to BTAF1 and directly impacts expression of 408 genes. Lack of O-GlcNAcylation at T114 is associated with a striking reprograming of cellular metabolism induced by a profound modification of the transcriptome, leading to gross alterations in lipid storage.

overall_design: Samples from each of four cell lines were submitted in triplicate for a total of twelve RNA deep sequencing samples.

first: Stéphan

last: Hardivillé

first: C

middle: C

last: Talbot Jr


title: RNA-deep sequencing (RNA-Seq) analysis of dy2J/dy2J (Lama2-CMD mouse model), mdx (DMD mouse model) and Wild-type skeletal muscles

city: Petah Tikva

first: Nurit

last: Yanay

address: Kaplan 14 St.

department: The Institute of Neurology

country: Israel

institute: Schneider Children's Medical Center of Israel

summary: Congenital muscular dystrophy type-1A (Lama2-CMD) and Duchenne Muscular dystrophy (DMD) result from deficiencies of laminin-α2 and dystrophin proteins, respectively.  Although both proteins strengthen the sarcolemma, they are implicated in clinically distinct phenotypes. We used RNA-deep sequencing (RNA-Seq) of dy2J/dy2J, Lama2-CMD mouse model, skeletal muscle at 8 weeks of age to elucidate disease pathophysiology. This study is the first report of dy2J/dy2J model whole transcriptome profile. RNA-Seq of the mdx mouse model of DMD and WT mouse was carried as well in order to enable a novel comparison of dy2J/dy2J to mdx. A large group of shared differentially expressed genes (DEG) were found in dy2J/dy2J and mdx models (1,834 common DEG, (FDR) < 0.05). Enrichment pathway analysis using Ingenuity Pathway Analysis (IPA) showed enrichment of inflammation, fibrosis, cellular movement, migration and proliferation of cells, apoptosis and necrosis in both mouse models (p-values 3E-10 – 9E-37). Via Canonical pathway analysis; Actin cytoskeleton, Integrin, ILK, NF-kB, Renin-angiotensin, calcium signaling were also enriched and upregulated in both models (FDR<0.05). Interestingly, significant downregulation of Pax7 was detected in dy2J/dy2J compared to upregulation of this key regeneration gene in mdx mice. Pax3 and Mamstr genes were also downregulated in dy2J/dy2J compared to WT mice. These results may explain the distinct disease course and severity in these models. While the mdx model at that stage shows massive regeneration, the dy2J/dy2J shows progressive dystrophic process. Our data deepen our understanding of the molecular pathophysiology and suggest new targets for additional therapies to upregulate regeneration in Lama2-CMD.

overall_design: Quadriceps muscle biopsies from male Wild-type (n=14), dy2J/dy2J (n=11) and mdx (n=15) mice were recruiting at the age of 8 weeks to the experiment in order to analyzed differences in the transcriptome content. Biopsies were taken from each mouse for RNA isolation and subjected for RNA-Seq evaluation. The RNA from WT or mdx genotype was pooled into three batches, 4-6 RNA samples in each pool. Samples from dy2J mice were divided into two batches, 5-6 samples per pool.

first: Yoram

last: Nevo

first: Moran

last: Elbaz

first: Sharona

last: Elgavish

first: Yuval


title: Defining Fallopian-tube Derived miRNA Cancer Signatures

city: Boston

first: Sunita

last: Setlur

address: 221 Longwood Avenue

country: USA

institute: Brigham and Women's Hospital

summary: This study characterizes changes in miRNA signatures in fallopian-tube derived cancer models.

overall_design: miRNAs were profiled using the NanoString nCounter mouse miRNA V1.5 Assay in the following sample types:  Control fallopian tube, doxycycline-induced fallopian tube, peritoneal metastatic sample, Ovarian tumor, plasma, and test serum from individuals with tumors.


title: Gene Expression in wild type and  Atp7b-/- mice

city: Houston

first: Clavia

middle: Ruth

last: Wooton-Kee

state: TX

address: One Baylor Plaza

department: Molecular and Cellular Biology

institute: Baylor College of Medicine

summary: Inactivating mutations in the copper transporter Atp7b result in Wilson’s disease.  The Atp7b-/- mouse develops hallmarks of Wilson’s disease.  The activity of several nuclear receptors is decreased in Atp7b-/- mice, and nuclear receptors are critical for maintaining metabolic homeostasis. Therefore, we anticipated that Atp7b-/- mice would exhibit altered progression of diet-induced obesity, fatty liver, and insulin resistance.  Following 10 weeks on a chow or Western-type diet (40% kcal fat), parameters of glucose and lipid homeostasis were measured.  Hepatic metabolites were measured by LC-MS and correlated with transcriptomic data. Atp7b-/- mice fed a chow diet had lower fat mass and were more glucose tolerant than wild type (WT) littermate controls although body weights did not differ between genotypes.  On Western diet, Atp7b-/- mice exhibited reduced adiposity and hepatic steatosis compared with WT controls.  Atp7b-/- mice fed either diet were more insulin sensitive than WT controls; however, fasted Atp7b-/- mice exhibited hypoglycemia after administration of insulin, due to an impaired glucose counter-regulatory response, as evidenced by reduced hepatic glucose production.  Coupling gene expression with metabolomic analyses, we observed striking changes in hepatic metabolic profiles in Atp7b-/- mice.  In addition, the active phosphorylated form of AMP kinase was significantly increased in Atp7b-/- mice relative with WT controls.  Alterations in hepatic metabolic profiles and nuclear receptor signaling were associated with improved glucose tolerance and insulin sensitivity, as well as impaired fasting glucose production in Atp7b-/- mice.

overall_design: Livers from 3-month old and wild type C57Bl/6 mice were isolated and processed for gene expression with four replicates.

first: Cristian

last: Coarfa

first: Matthew

last: Robertson


title: Comparative transcriptome profiling myometrial and vascular smooth muscle cells

city: Nashville

first: Dhivya

last: Sudhan

state: TN

address: 777 Preston Research Building

department: Hematology/Oncology Division

institute: Vanderbilt University

summary: The goal of this study was to select the optimal myometrial cells for our high-throughput drug discovery assay, as well as determine the similarity or differences of myometrial cells to vascular smooth muscle cells (VSMCs)-the most common off-target of current myometrial therapeutics. RNA-seq was used to: 1) identify which myometrial cells retained the most similar transcriptome profile to native tissue, and 2) compare the uterine myometrial transcriptome to VSMCs in hopes of identifying a uterine-selective transcriptome that was “druggable” for tocolytic or uterotonic use.  Four sources of myometrial cells were examined: 1) term pregnant human primary myometrial cells isolated from tissue biopsies obtained at the time of caesarean sections, 2) term pregnant mouse primary myometrial cells, 3) commercially-available immortalized pregnant human myometrial (PHM1) cells and 4) human telomerase immortalized myometrial (hTERT-HM) cells. Correlation analysis of aligned reads identified that the transcriptome of primary human myometrial and hTERT-HM cells showed 85% and 80% correlation, respectively, to human myometrial tissue and that the transcriptome of hTERT-HM and PHM1 cells is 90% or more correlative to human primary myometrial cells. The expression levels (fold-change) of contraction-associciated transcripts (OXTR, PTGFR, PTGS2 and GJA1) strongly correlated (r=0.93) between RNA sequencing and qRT-PCR analysis. Analysis of aligned reads among myometrial cells revealed the number of differentially expressed transcripts (fold-change≥2.0, adjusted p-value≤0.01) relative to primary human myometrial cells: hTERT-HM (946 upregulated and 2,351 downregulated), PHM1 (1,575 upregulated and 2,415 downregulated) and primary mouse myometrial cells (3,435 upregulated and 2,966 downregulated). Correlation analysis showed that the human primary myometrial cell transcriptome is over 90% similar to the transcriptome of VSMCs examined. A number of genes associated with smooth muscle contractile machinery (TPM1, TPM2, CNN1, CALD1, ACTA2 and PLN)were significantly (p≤0.01) upregulated (≥2-fold) in human primary myometrial compared to vascular SMCs. We identified 498 transcripts were identified as upregulated in human primary myometrial cells compared to all three VSMCs examined. Of these, the drug-gene interaction database identified 142 genes as druggable

overall_design: Myometrial tissue was obtained at the time of scheduled or repeat cesarean section from 5 different women (age 18-45 years) at ≥ 39 gestational weeks . Tissue was used for either RNA isolation for sequencing, or isolation of primary cells. We also isolated primary myometrial cells from 5 different term-pregnant (day 19). PHM1 and adult aorta cells were purchased from ATCC, and fetal aorta cells were purchased from Cell Applications. Immortalized hTERT-HM and fetal DA cells were kindly provided by Drs. Jennifer Condon and Elaine Shelton, respectively. Total RNA was isolated per the Trizol's manufacturer's instructions and then DNase-treated. The RNA was analyzed with an Agilent Bioanalyzer 2100, which showed that all samples had an RIN>8. The coding RNA was enriched using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490) and paired end libraries were generated using the NEBNext Ultra II Directional RNA Library Kit for Illumina (7760). Sequencing was performed at paired-end 100 bp on the Illumina NovaSeq6000 targeting 30M PE reads. Sequencing quality and sample integrity was assessed using the multiQC tools (https://multiqc.info) as well as positive controls and sample yield. Quantitative RT-PCR validation was performed using SYBR green assays. The drug-gene interaction database was used to identify the number of druggable transcripts that were upregulated in human primary myometrial cells compared to VSMCs.

first: Jennifer

last: Herington

first: Shajila

last: Siricilla

first: Quanhu

last: Sheng

first: Jeff

last: Reese

first: Elaine

last: Shelton


title: An Immune Gene Expression Signature Associated With Development of Human Hepatocellular Carcinoma Identifies Mice That Respond to Chemopreventive Agents

city: Barcelona

first: Sara

last: Torrecilla

state: Catalonia

address: Rossello Street, 149

institute: IDIBAPS

summary:
Background & Aims: Cirrhosis and chronic inflammation precede development of hepatocellular carcinoma (HCC) in approximately 80% of cases. We investigated immune-related gene expression patterns in liver tissues surrounding early-stage HCCs and chemopreventive agents that might alter these patterns to prevent liver tumorigenesis.

Methods: We analyzed gene expression profiles of non-tumor liver tissues from 392 patients with early-stage HCC (training set, n=167 and validation set, n=225) and liver tissue from patients with cirrhosis without HCC (n=216, controls) to identify changes in expression of genes that regulate the immune response that could contribute to hepatocarcinogenesis. We defined 172 genes as markers for this deregulated immune response, which we called the immune-mediated cancer field (ICF). We analyzed the expression data of liver tissues from 216 patients with cirrhosis without HCC and investigated the association between this gene expression signature and development of HCC and outcomes of patients (median follow-up 10 years). Human liver tissues were also analyzed by histology. C57BL/6J mice were given a single injection of N-nitrosodiethylamine followed by weekly doses of carbon tetrachloride to induce liver fibrosis and tumorigenesis. Mice were then given orally the multiple tyrosine inhibitor nintedanib or vehicle (controls); liver tissues were collected and histology, transcriptome, and protein analyses were performed. We also analyzed transcriptomes of liver tissues collected from mice on a choline-deficient high-fat diet, which developed chronic liver inflammation and tumors, given orally aspirin and clopidogrel or the anti-inflammatory agent sulindac vs mice on a chow (control) diet.

Results: We found the ICF gene expression pattern in 50% of liver tissues from patients with cirrhosis without HCC and in 60% of non-tumor liver tissues from patients with early-stage HCC. The liver tissues with the ICF gene expression pattern had 3 different features: increased numbers of effector T cells; increased expression of genes that suppress the immune response and activation of transforming growth factor beta signaling; or expression of genes that promote inflammation and activation of interferon gamma signaling. Patients with cirrhosis and liver tissues with the immunosuppressive profile (10% of cases) had a higher risk of HCC (hazard ratio, 2.41; 95% 1.21–4.80). Mice with chemically-induced fibrosis or diet-induced steatohepatitis given nintedanib or aspirin and clopidogrel downregulated the ICF gene expression pattern in liver and developed fewer and smaller tumors than mice given vehicle.

Conclusions: We identified an immune-related gene expression pattern in liver tissues of patients with early-stage HCC, called the ICF, that associates with risk of HCC development in patients with cirrhosis. Administration of nintedanib or aspirin and clopidogrel to mice with chronic liver inflammation caused loss of this gene expression pattern and developed fewer and smaller liver tumors. Agents that alter immune regulatory gene expression patterns associated with carcinogenesis might be tested as chemopreventive agents in patients with cirrhosis.

overall_design: Fresh-frozen liver tissues from 22 mice (3 control, 9 vehicle-treated and 10 nintedanib-treated), were collected and RNA was extracted. Whole-genome transcriptome analysis was obtained using ene Chip HT MG-430 (Affymetrix) according to manufacturer’s protocols. Raw expression data from microarrays were normalized using the robust multiarray algorithm (Irizarry RA et al, Nucleic Acids Res, 2003) with a custom probe set definition that mapped probes to Entrez Gene IDs (HTMG430PM_Mm_ENTREZG) (Dai M et al, Nucleic Acids Res, 2005).

first: Josep

middle: M

last: Llovet

first: Daniela

last: Sia