title: Application of a classifier combining bronchial genomics and chest CT features facilitates the diagnostic evaluation of lung cancer in smokers and non-smokers

city: Hangzhou

first: Yang

last: Xia

state: Zhejiang

address: 88 Jiefang Road

department: Department of Respiratory and Critical Care Medicine

institute: Second Affiliated Hospital of Zhejiang University School

summary:
Introduction: Lung cancer screening by computed tomography (CT) reduces mortality but exhibited high false-positive rates. We established a diagnostic classifier combining chest CT features with bronchial genomics.
Materials and Methods: Patients with CT-detected suspected lung cancer were enrolled. The sample collected by bronchial brushing was used for RNA sequencing. R software was applied to build the model.
Results: A total of 283 patients, including 183 with lung cancer and 100 with benign lesions, were included. When incorporating genomic data with radiological characteristics, the advanced model yielded 0.903 AUC with 81.1% NPV. Moreover, the classifier performed well regardless of lesion size, location, stage, histologic type, or smoking status. Pathway analysis showed enhanced epithelial differentiation, tumor metastasis, and impaired immunity were predominant in smokers with cancer, whereas tumorigenesis played a central role in non-smokers with cancer. Apoptosis and oxidative stress contributed critically in metastatic lung cancer; by contrast, immune dysfunction was pivotal in locally advanced lung cancer.
Conclusions: We devised a minimal-to-noninvasive, efficient diagnostic classifier for smokers and non-smokers with lung cancer, which provides evidence for different mechanisms of cancer development and metastasis associated with smoking. A negative classifier result will help the physician make conservative diagnostic decisions.

overall_design:
Establishment of  a model using the combined information of RNA genomics derived from bronchial epithelial cell and CT characteristics to predict the malignancy risk of pulmonary lesions.
*** Due to privacy concerns, submitter declares that raw files will be made available by request upon approval by the Human Genetic Resource Administration of China ***

first: Songmin

last: Ying

first: Rui

last: Jin

first: Hao

last: Wu

first: Ye

last: Shen

first: Tong

last: Yin

first: Fugui

last: Yan

first: Wei

last: Zhang

first: Fen

last: Lan

first: Bin

first: Chen

last: Zhu

last: Li

first: Wen

first: Huahao


title: The Rhesus Macaque serves a model for Human Lateral Branching Nephrogenesis

status: Public on Feb 18 2021

city: Cincinnati

first: Nathan

last: Salomonis

state: OH

address: 3333 Burnet Avenue

department: Biomedical Informatics

country: USA

institute: Cincinnati Children's Hospital

type: Expression profiling by high throughput sequencing

summary: Infants born prematurely are at increased risk for chronic kidney disease and end stage renal disease later in life due to low nephron number. The mechanism of how nephrons are formed during late-gestation human kidney development is not known, and direct study of human fetal kidney development is fraught with moral and technical difficulties. In this study, the rhesus macaque kidney was identified to be morphologically similar to the human kidney during late-gestation. The preliminary findings support that kidney progenitor cells age over time and are different from the progenitor cells early in gestation. The non-human primate model could bridge the gap for molecular study of late-gestation human kidney development to ultimately improve nephron numbers in preterm infants.

overall_design: The morphology of third trimester rhesus kidneys was assessed by immunostaining, clearing, and 3D rendering. Single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq) were performed on four cortically enriched fetal rhesus kidneys 129-131 days gestational age (GA) using cold protease digestion and 10xGenomics platform.

first: Meredith

middle: P

last: Schuh

first: Andrew

last: Potter

first: Lyan

last: Alkhudairy

first: Thakkar

last: Kairavee

first: Kashish

last: Chetal

first: Steven

first: Raphael

last: Kopan


title: Next Generation Sequencing Quantitative Analysis of Wild Type and Mki67-/- 4T1 mouse mammary carcinoma cell transcriptomes

city: Montpellier

first: Daniel

last: Fisher

address: 1919 Route de Mende

department: IGMM

country: France

institute: CNRS

summary:
Purpose: The goals of this study were to compare gene expression profiles of mouse 4T1 cells with intact or disrupted Mki67 gene.
Methods: mRNA profiles of wild-type (WT) and two clones of Ki-67 knockout (Mki67−/−) mouse NIH/3T3 cells, all in duplicate, were generated by deep sequencing using Illumina Hiseq2500 in single-end read mode, 50bp. The sequence reads that passed quality filters were aligned to the mouse genome, quantified, and differential gene expression (DGE) analysis was performed using DEseq2.
Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (GRCm38.p6) and identified differentially expressed genes.  This revealed widespread changes in gene expression, with 2558 genes significantly deregulated in independent clones of Mki67-/- cells (q < 0.05)
Conclusions: Ki-67 knockout causes genome-scale transcriptome alterations

overall_design: mRNA profiles of wild-type (WT) and two clones of Ki-67 knockout (Mki67−/−) mouse 4T1 cells, all in duplicate, were generated by deep sequencing using Illumina Hiseq2500.

first: Priyanka

last: Singh

first: Karim

last: Mrouj

first: Geronimo

last: Dubra

first: Liliana

last: Krasinska


title: Next Generation Sequencing Quantitative Analysis of the transcriptomes of Wild Type and Mki67-/- 4T1 tumors in mice

summary:
Purpose: The goals of this study were to compare gene expression profiles of mouse xenografted 4T1 tumors with intact or disrupted Mki67 gene.
Methods: mRNA profiles of wild-type (WT) and Ki-67 knockout (Mki67−/−) mouse xenografted 4T1 tumors, in triplicate, were generated by deep sequencing using Illumina Hiseq2500 in paired-end read mode, 50bp. The sequence reads that passed quality filters were aligned to the mouse genome, quantified, and differential gene expression (DGE) analysis was performed using DEseq2.
Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (GRCm38.p6) and identified differentially expressed genes.  This revealed widespread changes in gene expression
Conclusions: Ki-67 knockout causes genome-scale transcriptome alterations

first: Alghoul

last: Emile


title: Differential effect of cancer-associated fibroblasts on HNSCC progression: Cancer Promotion or inhibition

city: Daegu

first: SU YOUNG

last: OH

address: Dalgubeol-daero, 2177

country: South Korea

institute: Kyungpook national university

summary:
To study of differential effect on cancer-associated fibroblasts of HNSCC, we have employed  microarray expression profiling.
The critical effect of the tumor microenvironment to caner progression is well recognized. Recent research suggests that the cancer-promoting properties of the tumor stroma may attributed to fibroblasts. However, little is known about fibroblasts that inhibit cancer progression. From the immunohistochemical analysis of cancer tissues from head and neck squamous cell carcinoma (HNSCC) patients, we divided cancer-associated fibroblast (CAF) into two groups depending on whether the boundary between epithelial cancer cells and the surrounding extracellular matrix (ECM) is clear and smooth or not. Therefore, we tried to evaluate whether there is a difference between these two CAFs in HNSCC progression. CAF was primary cultured, followed by co-culture with HNSCC cell to observe the effect on Matrigel invasion. The mRNA expression patterns between these two CAF groups were compared by DNA microarray analysis. FaDu cell and primary CAF from each group was co-transplanted into the oral mucosa of mice, and the tumorigenesis was compared. The CAF group (CAF-Promote, CAF-P) from cancer tissues which have unclear boundary between ECM and epithelial cancer cells showed a tendency to stimulate in vitro Matrigel invasion of HNSCC cell. On the contrary, the CAF group (CAF-Defense, CAF-D) from cancer tissues, which have clear boundary with epithelial cancer cell caused no remarkable increase of Matrigel invasion. In addition, CAF-D reduced the tumorigenicity of FaDu compared to CAF-P in the in vivo mice model. In DNA microarray analysis, COL3A1, COL6A6, COL25A1, and COL26A1 were particularly highly expressed in CAF-D group. In this study, these collagen proteins derived from cancer stroma were found to have a function of inhibiting the progression of HNSCC. It is expected to provide important information for predicting the prognosis of HNSCC and development of drug target in the future.

overall_design: Tumor tissues for CAF culture were obtained by surgical resection from HNSCC patients at Kyungpook National University Hospital. Tissue specimens were used after receiving written, informed consent from the patients and approval from the Institutional Research Ethics Committee of Kyungpook National University Hospital. The stroma adjacent to the cancer was carefully separated by a pathologist, cut into the smallest possible pieces in sterile DMEM, and seeded into 10-cm culture dishes supplemented with 10% FBS. After 2–3 weeks, fibroblast cells were cultured in a culture dish.

first: Su-Hyung

last: Hong

first: Su

last: Oh


title: Kmt2c-mutations superimpose properties of activated and quiescent HSCs to convey a selective advantage after chemotherapy [RNA-seq]

city: St Louis

first: Jeffery

last: Magee

state: Missouri

address: 660 S Euclid Ave.

department: Internal Medicine - Cardiovascular Division

institute: Washington University School of Medicine

summary: Rna-seq analysis of wild type and Kmt2c mutant hematopoietic progenitors

overall_design:
Indicated populations were isolated from Kmt2c conditional knockout mice for RNA-seq
pGM = pre-granulocyte-monocyte

first: Jeffrey


title: Kmt2c-mutations superimpose properties of activated and quiescent HSCs to convey a selective advantage after chemotherapy [ATAC-seq]

type: Genome binding/occupancy profiling by high throughput sequencing

summary: ATAC-seq analysis of wild type and Kmt2c mutant hematopoietic progenitors

overall_design: Indicated populations were isolated from Kmt2c conditional knockout mice for ATAC-seq


title: Characterization and regulation of osteoclast precursors following chronic Porphyromonas gingivalis infection

city: Birmingham

first: Ping

last: Zhang

state: Alabama

address: 1919 7th Ave S

department: Pediatric Dentistry

institute: University of Alabama at Birmingham

summary: Bone destruction in inflammatory osteolytic diseases including periodontitis is related to excessive activity of osteoclasts (OC), which originate from precursor cells of the myeloid lineage, termed osteoclast precursors (OCP). In contrast to ample knowledge that we currently have on mature OC, little is known about OCP and their regulation during bacterial infection. Therefore, this study aimed to identify and characterize OCP following chronic infection with a periodontal bacteria Porphyromonas gingivalis (Pg). We used a micro-osmotic pump to continually release Pg subcutaneously in a murine model. Two weeks after Pg infection, the frequency of CD11b+c-fms+Ly6Chi population is significantly elevated within the bone marrow, spleen and peripheral blood. In vitro and in vivo studies identified these cells as the OCP-containing population and Pg infection significantly enhanced the osteoclastogenic activity of these cells. Furthermore, mRNA sequencing analysis indicated a unique gene and pathway profile in CD11b+c-fms+Ly6Chi population following Pg infection, with changes in genes and pathways related to OC differentiation, cell proliferation and apoptosis, inflammatory response, phagocytosis and immunity, as well as antigen processing and presentation. Moreover, using IL-6 knockout mice, we found that IL-6 is important for Pg-induced accumulation of CD11b+c-fms+Ly6Chi population from the bone marrow and periphery. Our results provide new insights into the characterization and regulation of OCP following a chronic bacterial infection. This knowledge is relevant to the understanding of the pathogenesis of bacteria-induced bone loss, and to the identification of potential therapeutic targets of bone loss diseases.

overall_design: Total RNAs were isolated from SPL CD11b+c-fms+Ly6Chi population of control and Pg-infected mice, and were sent to the GENEWIZ company (South Plainfield, NJ) for sequencing and bioinformatics analysis.

first: Yanfang

last: Zhao

first: Zhaofei

last: Li

first: Lingkai

last: Su

first: Andre

last: Bellesteros-Tato

first: Jannet

last: Katz

first: Suzanne

middle: M

last: Michalek

first: Xu

last: Feng


title: Kmt2c-mutations superimpose properties of activated and quiescent HSCs to convey a selective advantage after chemotherapy

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

overall_design: Refer to individual Series


title: 10x Genomics single-cell RNA-seq of isolated Adult ID4-EGFP-bright mouse spermatogonia treated with Rapamycin

city: San Antonio

first: Brian

middle: P.

last: Hermann

state: TX

address: 1 UTSA Circle

department: Department of Biology

institute: University of Texas at San Antonio

summary: To reveal distinct transcriptome changes among ID4-EGFP-bright adult mouse spermatogonia associated with mTORC1 activity, single-cell transcriptomes were generated from GFP-bright/CD9-bright spermatogonia from adult mice in three groups: control (untreated), 2 days of Rapamycin treatment (Rapamycin) and 2 days Rapamycin plus 1 day washout (Rapamycin_Release). Based on transplantation studies performed previously, ID4-EGFPbright cells are highly enriched for SSCs. We used the 10x Genomics Chromium to perform single-cell RNA-seq.

overall_design: Each cell population (treatment group) was generated from pooled cells from three adult males.

first: Shinnosuke

last: Suzuki

first: John

middle: R

last: McCarrey


title: Aging imparts cell-autonomous dysfunction to regulatory T cells during recovery from influenza pneumonia

city: Chicago

first: Benjamin

middle: D.

last: Singer

state: Illinois

address: 303 E. Superior St., Simpson Querrey 5th floor

institute: Northwestern University Feinberg School of Medicine

type: Methylation profiling by high throughput sequencing


title: Systematic comparison of cellular adaptive stress response activation networks in hepatic stem cell-derived progeny and primary human hepatocytes

city: Leiden

first: Bob

last: van de Water

address: Einsteinweg 55

department: Division of Drug Discovery and Safety

country: Netherlands

institute: Leiden Academic Centre for Drug Research (LACDR), Leiden University

summary: Various adaptive cellular stress response pathways are critical in the pathophysiology of liver disease and drug-induced liver injury. Human-induced pluripotent stem cell (hiPSC)-derived hepatocyte-like cells (HLCs) provide a promising tool to study cellular stress response pathways, but in this context there is limited insight on how HLCs compare to primary human hepatocytes (PHH). Here, we systematically compared the activation of four different stress pathways in PHH, HepG2 liver cancer cells, hiPSCs and different stages within the differentiation towards HLCs (definitive endoderm, hepatoblast, immature hepatocytes and HLCs). We exposed all different cell types in a concentration response to four different compounds that specifically activate the oxidative stress response (diethyl-maleate), unfolded protein response (tunicamycin), DNA damage response (cisplatin) and inflammatory signalling (TNF). We used targeted RNA-sequencing to map concentration response transcriptional similarities and differences using bench-mark concentration modelling for the various stress responses in the different test systems. We observed that HLCs are more sensitive to oxidative stress than PHH showing activation at ten-fold lower concentrations and induce a strong anti-oxidant response. Although similar UPR gene sets were activated in HLCs compared to PHH, PHH were about ten times more sensitive. Furthermore, HLCs were highly sensitive to inflammation conditions similar to HepG2 cells and in sharp contrast to hiPSC, which showed hardly any response. On the contrary, hiPSC and HepG2 were highly sensitive to DNA damage signalling while HLCs resisted a strong p53 DNA damage response upon exposure. Overall, the data indicate that despite limitation in full hepatocyte maturation, HLCs did gain specific adaptive cellular stress response networks that mimic those of PHHs.

overall_design: Targeted transcriptome profile of primary human hepatocytes, HepG2 cells, hiPSCs, hiPSC-derived definitive endoderm, hepatoblasts, immature hepatocytes, and hepatocyte-like-cells exposed to four reference compounds in wide concentration range and solvent controls for three biological replicates.

first: Bas

last: ter Braak

first: Marije

last: Niemeijer

first: Ruben

last: Boon

first: Celine

last: Parmentier

first: Audrey

last: Baze

first: Lysiane

last: Richert

first: Suzanna

last: Huppelschoten

first: Steven

last: Wink

first: Catherine

last: Verfaillie


title: Short-term exposure to intermittent hypoxia leads to changes in gene expression seen in chronic pulmonary disease [single-cell RNA]

first: Yin Yeng

last: Lee

state: Ohio

department: Department of Pediatrics

institute: Cincinnati Children's Hospital Medical Center

summary: Obstructive sleep apnea (OSA) results from episodes of airway collapse and intermittent hypoxia (IH) and is associated with a host of health complications. Although the lung is the first organ to sense changes in oxygen levels, little is known about the consequences of IH to the lung hypoxia-inducible factor- (HIF)-responsive pathways. We hypothesized that exposure to IH would lead to cell-specific up and downregulation of diverse expression pathways. We identified changes in circadian and immune pathways in lungs from mice exposed to IH. Among all cell types, endothelial cells showed the most prominent transcriptional changes. Upregulated genes in myofibroblast cells were enriched for genes associated with pulmonary hypertension and included targets of several drugs currently used to treat chronic pulmonary diseases. A better understanding of the pathophysiologic mechanisms underlying diseases associated with OSA could improve our therapeutic approaches, directing therapies to the most relevant cells and molecular pathways.

overall_design: Overall, 12 mice (6 control and 6 hypoxia) are used for bulk RNA-sequencing and 14 mice (7 control and 7 hypoxia) were divided among 3 separate experiments (biological replicates) for single cell sequencing.

first: G

last: Wu

first: Y

middle: Y

first: E

last: Gulla

first: A

last: Potter

first: J

last: Kitzmille

first: M

middle: D

last: Ruben

first: N

last: Salomonis

middle: A

last: Whitsett

first: L

middle: J

last: Francey

middle: B

last: Hogenesch

first: D

middle: F

last: Smith


title: Short-term exposure to intermittent hypoxia leads to changes in gene expression seen in chronic pulmonary disease [bulk RNA]


title: Short-term exposure to intermittent hypoxia leads to changes in gene expression seen in chronic pulmonary disease


title: Kmt2c-mutations superimpose properties of activated and quiescent HSCs to convey a selective advantage after chemotherapy [ChIP-seq]

first: Jeffery

last: Magee

summary: ChIP-seq analysis of wild type and Kmt2c mutant hematopoietic progenitors

overall_design: Indicated populations were isolated from Kmt2c conditional knockout mice for ChIP-seq

first: Jeffrey


title: RNA sequencing from epidermis of LAMA3-deficient mice

city: Cologne

first: Monique

middle: Marie

last: Aumailley

address: Joseph-Stelzmann-str. 52

country: Germany

institute: University of Cologne

summary:
RNA sequencing data of mouse epidermis isolated from wild type and LAMA3-deficient mice
Genetic, clinical and biochemical studies concurred to establish that integrity of the dermal-epidermal junction requires laminin 332, a particular subset of epithelial laminins. Laminin 332 is composed of three subunits, α3, β3 and γ2, encoded by the Lama3, Lamb3 and Lamc2 genes, respectively. In vivo functional analysis of laminin 332 in skin has been prevented because constitutive mutations of any one of the coding genes, either inherited in human or engineered in mice, cause junctional epidermolysis bullosa and early death. Consequently, it is still unknown whether and how laminin 332 contributes to skin homeostasis. To circumvent the problem, we have generated a mouse model in which disruption of the Lama3 gene is conditional and specifically induced in epidermal keratinocytes after birth. It causes a progressive depletion of laminin 332 in the skin of the mouse, which is compatible with life. To assess how laminin 332 supervises epidermal homeostasis, RNA was prepared from keratinocytes isolated from laminin 332-depleted skin and control animals (three each) to compare their gene expression profiles using RNA sequencing.

overall_design:
Total RNA from 3 biological replicates of wild type and LAMA3-deficient mice. Paired-end sequencing 100bp reads with HiSeq2000.
Lama3flox/flox mice were generated by ARTEMIS Pharmaceuticals GmbH (Cologne, Germany) by flanking exons 50 to 53 of the Lama3 gene with LoxP sites on a C57BL/6 background. Lama3flox/flox mice were crossed with keratin (K) 14 driven Cre deleter mice expressing the Cre recombinase fused to a mutated ligand-binding domain of the human estrogen receptor (K14-CreERT, Jackson laboratories), allowing temporal control of Cre activation by tamoxifen and ablation of the floxed alleles in K14-expressing basal keratinocytes. The Cre recombinase was activated with a tamoxifen-containing chow (400 mg Tamoxifen citrate/1 kg of chow). Consequently, the Lama3 gene was inactivated in the Lama3flox/flox/K14-CreERT, followed by a progressive loss of laminin 332, but not in the Lama3wt/flox / K14-CreERT used as controls. RNA was prepared from keratinocytes isolated from laminin 332-depleted skin and control animals (three mice each). Library preparation and sequencing was performed at the facilities of the Cologne Center for Genomics (University of Cologne).


title: Aging imparts cell-autonomous dysfunction to regulatory T cells during recovery from influenza pneumonia [DNA methylation/RRBS]

first: Benjamin

last: Singer

summary: Regulatory T (Treg) cells promote recovery from lung injury. Aging imparts cell-autonomous dysfunction to regulatory T cells during recovery from influenza pneumonia.

overall_design: Flow cytometry sorted lung Treg cells (CD3e+CD4+CD25hiFR4+) from 2-month-old or 18-month-old C57BL/6 mice at baseline or following challenge with influenza A virus and harvested on day 60 post-infection. n = 5  biological replicates per group.

first: Luisa

last: Morales-Nebreda


title: Aging imparts cell-autonomous dysfunction to regulatory T cells during recovery from influenza pneumonia [RNA-seq]


title: Ribosome profiling reveals sequence-independent post-initiation pausing as a signature of translation

status: Public on Feb 17 2021

first: Yuanhui

last: Mao

address: 1369 West Wenyi Road

country: China

institute: Zhejiang University

summary: The journey of a newly synthesized polypeptide starts in the peptidyltransferase center of the ribosome, from where it traverses the exit tunnel. The interior of the ribosome exit tunnel is neither straight nor smooth. How the ribosome dynamics in vivo is influenced by the exit tunnel is poorly understood. Genome-wide ribosome profiling in mammalian cells reveals elevated ribosome density at the start codon and surprisingly the downstream 5th codon position as well. We found that the highly focused ribosomal pausing shortly after initiation is attributed to the geometry of the exit tunnel, as deletion of the loop region from ribosome protein L4 diminishes translational pausing at the 5th codon position. Unexpectedly, the ribosome variant undergoes translational abandonment shortly after initiation, suggesting that there exists an obligatory step between initiation and elongation commitment. We propose that the post-initiation pausing of ribosomes represents an inherent signature of the translation machinery to ensure productive translation.

overall_design: We performed Ribo-seq in wildtype HEK293 cells and the cells expressed with mutated RPL4.

first: Yan

last: Han

first: Xiangwei

last: Gao

first: Shu-Bing

last: Qian


title: Human engineered meniscus transcriptome after short-term combined hypoxia and dynamic compression

city: Edmonton

first: Adetola

last: Adesida

state: Alberta

address: 3.002E Li Ka Shing Centre for Health Research Innovation

department: Surgery

country: Canada

institute: University of Alberta

summary: This study investigates the transcriptome response of meniscus fibrochondrocytes (MFCs) to the low oxygen and mechanical loading signals experienced in the knee joint using a model system. We hypothesized that short term exposure to the combined treatment would promote a matrix-forming phenotype supportive of inner meniscus tissue formation. Human MFCs on a collagen scaffold were stimulated to form fibrocartilage over 6weeks under normoxic (NRX, 20% O2) conditions with supplemented TGF -β3. Tissues experienced a delayed 24h hypoxia treatment (HYP, 3% O2) and then 5min of dynamic compression (DC) between 30 and 40% strain. Delayed HYP induced an anabolic and anti-catabolic expression profile for hyaline cartilage matrix markers, while DC induced an inflammatory matrix remodeling response along with upregulation of both SOX9 and COL1A1. There were 41 genes regulated by both HYP and DC. Overall, the combined treatment supported a unique gene expression profile favouring the hyaline cartilage aspect of inner meniscus matrix and matrix remodeling.

overall_design: Examination completed using 18 sequenced samples. These came from 3 mensicus donors (6 samples per donor). Each donor had 3 samples treated in hypoxia and 3 in normoxia. In each oxygen tension, the treatments were Dynamic Compression, Static Compression, or Baseline. Each individual sample came from pooled equal masses of RNA from 3-4 biological replicates in the same condition from the same donor.

first: Alexander

last: Szojka

first: Rita

last: de Cassia Marqueti

first: David

middle: X

last: Li

first: Clayton

middle: W

last: Molter

last: Liang

first: Melanie

last: Kunze

first: Aillette

last: Mulet-Sierra

first: Nadr

last: Jomha


title: Gene expression after knockdown of transcription factors in human iPSC-derived cardiac myocytes

city: New York

first: Ian

last: Mellis

state: NY

institute: Columbia University

summary: We report gene expression changes after knockdown of transcription factors in human iPSC-derived cardiac myocytes. In prior experiments we showed that transcription factors known to be important for cardiac development were frequently up-regulated (i.e., "responsive") after small molecule perturbations of cultured iPSC-derived cardiac myocytes. We used RNA sequencing to test whether siRNA-mediated knockdown of transcription factors with different perturbation-responsiveness and tissue-specificity profiles would lead to inappropriate up-regulation of non-myocyte gene sets in cardiac myocytes.

overall_design: Paired-end RNA sequencing of 9 different targeting siRNA conditions, 1 scrambled siRNA control condition, and 1 non-transfected control condition. All conditions in triplicate. 33 total samples.

first: Rajan

last: Jain

first: Arjun

last: Raj


title: MiRNA Transcriptome sequencing of Lubo goat submandibular glands at different ages

city: Tai’an

first: Chao

last: Tianle

state: Shandong

address: Daizong street, no. 61, 6/5000 Shandong Agricultural University

institute: 6/5000 Shandong Agricultural University

type: Non-coding RNA profiling by high throughput sequencing

summary: 9 submandibular gland tissue samples were collected from goats in 1-month-old, 12-month-old and 24-month-old groups (3 samples from each group), and high-throughput transcriptome sequencing was conducted on these samples.

overall_design: Goats were separated into 3 different age groups: 1-month-old, 12-month-old and 24-month-old. Three healthy female goats (Lubo goats) from each group (total, 9 goats) were selected as experimental animals. The animals were obtained from Shandong Fengxiang Animal Husbandry and Seed Industry Technology Co., Ltd. (Laiwu District, Jinan, Shandong Province, China). All goats were raised under the same conditions, including natural lighting and free access to food. And high-throughput transcriptome sequencing was conducted on these samples.

first: Aili

last: Wang

first: Tianle

last: Chao


title: Inhibitory CD161 Receptor Identified in Glioma-infiltrating T cells by Single Cell Analysis

city: Cambridge

first: Orr

last: Ashenberg

state: MA

address: 415 Main Street

department: Klarman Cell Observatory

institute: Broad Institute

summary: T cells are critical effectors of cancer immunotherapies, but little is known about their gene expression programs in diffuse gliomas. Here, we leverage single-cell RNA-seq to chart the gene expression and clonal landscape of tumor-infiltrating T cells across 31 patients with IDH-wildtype glioblastoma and IDH-mutant glioma. We identify potential effectors of anti-tumor immunity in subsets of T cells that co-express cytotoxic programs and several NK cell genes. Analysis of clonally expanded tumor-infiltrating T cells further identifies the NK gene KLRB1 (encoding CD161) as a candidate inhibitory receptor. Accordingly, genetic inactivation of KLRB1 or antibody-mediated CD161 blockade enhances T cell-mediated killing of glioma cells in vitro and their anti-tumor function in vivo. KLRB1 and its associated transcriptional program are also expressed by substantial T cell populations in other human cancers. Our work provides an atlas of T cells in gliomas and highlights CD161 and other NK cell receptors as immunotherapy targets.

overall_design:
We profiled and characterized the types, programs, and states of glioma-infiltrating T cells isolated from fresh tumor samples using full-length or 5’end single-cell RNA-seq (scRNA-seq) in 31 adult patients with either IDH-wildtype glioblastoma (GBM) or IDH-mutant glioma (IDH-G). We use this tumor T cell atlas to identify new pathways regulating T cell function in the tumor microenvironment across GBM and IDH-G cases. We focus on subsets of cytotoxic glioma-infiltrating T cells that co-express several NK cell genes, and may serve as potential effectors of anti-tumor immunity. Analysis of clonally expanded T cells in tumors supports the NK gene KLRB1 (encoding CD161), as a potential inhibitory receptor. We reasoned that CD161 may represent an attractive therapeutic target, as it is a cell surface molecule expressed by both CD8 and CD4 T cell subsets, and a larger fraction of T cells express CD161 than the PD-1 protein. Additionally, CLEC2D, the ligand for CD161, is a surface molecule expressed by myeloid cells and malignant cells, suggesting a ligand-receptor pathway for immunotherapy. Accordingly, functional experiments where KLRB1 was inactivated in primary human T cells using CRISPR/Cas9 showed that CD161 inhibits the anti-tumor function of T cells in vitro and in vivo. Inhibition of ligand binding with a CD161 blocking mAb showed similar anti-tumor efficacy in vitro. Generalizing our observations, we find that KLRB1, and the gene expression signature of a KLRB1 expressing T cell, is shared across diverse cancer types. Our comprehensive atlas of T cell expression programs across the major classes of diffuse gliomas thus identifies the CD161-CLEC2D pathway as a potential target for immunotherapy of diffuse gliomas and other human cancers.
*** Due to patient privacy concerns, the submitter declares that patient data will be submitted to the Data Use Oversight System (https://duos.broadinstitute.org/). ***

first: Nathan

last: Mathewson

first: Itay

last: Tirosh

first: Simon

last: Gritsch

first: Elizabeth

last: Perez

first: Sascha

last: Marx

first: Livnat

last: Jerby-Arnon

first: Rony

last: Chanoch-Myers

first: Toshiro

last: Hara

first: Alyssa

last: Richman

first: Yoshinaga

last: Ito

first: Jason

last: Pyrdol

first: Mirco

last: Friedrich

first: Kathrin

last: Schumann

first: Michael

last: Poitras

first: Prafulla

middle: C

last: Gokhale

first: L

middle: N

last: Gonzalez Castro

first: Marni

middle: E

last: Shore

first: Christine

last: Hebert

first: Brian

last: Shaw

first: Heather

middle: L

last: Cahill

first: Matthew

last: Drummond

first: Wubing

last: Zhang

first: Olamide

last: Olawoyin

first: Hiroaki

last: Wakimoto

first: Orit

last: Rozenblatt-Rosen

first: Priscilla

middle: K

last: Brastianos

first: X

middle: S

last: Liu

first: Pamela

last: Jones

first: Daniel

last: Frosch

last: Louis

first: Keith

last: Ligon

last: Marson

first: E

last: Chiocca

last: Reardon

first: Aviv


title: Gene expression analysis of mouse hair follicle stem cells cultured in different conditions

city: Kobe

first: Makoto

last: Takeo

state: Hyogo

address: 2-2-3 Minatojima-minamimachi, Chuo-ku

department: BDR

country: Japan

institute: RIKEN

summary:
In mammals, organ induction occurs only during embryonic development except for hair follicles (HFs). However, HF-resident epithelial stem cells (HFSCs), which are responsible for repetitive HF regeneration, are not fully characterized.
We establish in vitro culture systems that are capable of controlling the ability of HFSCs to regenerate HFs and perform microarray analysis to compare gene expression profile in different culture condition.

overall_design: Hair follicle epithelial cells we isolated from the bulge region of mouse vibrissa. We separated these cells into two groups and cultured them in different condition (NFFSE vs. NFFS). We repeated same experiment 3 times and subjected to the Affymetrix microarrays.

first: Takashi

last: Tsuji


title: Transcriptional effects of KLF15 deletion in quadriceps muscle

city: Cleveland

first: Ernest

middle: R

last: Chan

address: 10900 Euclid Ave, Robbins E211

department: PQHS

institute: CWRU

summary: We report bulk RNA-sequencing of quadriceps muscle harvested from MyoCre and skeletal muscle specific KLF15 (K15-SKO) mice

overall_design: Quadriceps muscle were isolated from MyoCre or K15-SKO mice (n=4) according to standard protocols (see below) and transcriptional sequencing was performed.

first: Liyan

last: Fan

first: Mukesh

last: Jain

first: Vinesh

last: Vinayachandran


title: Integrated proteomic and transcriptomic analyses reveal the roles of Brucella BI-1 in cell division and membrane homeostasis of Brucella suis S2

city: Yangling, Shanxi

first: Guangdong

last: Zhang

address: No.22 Xinong Road, Yangling, Shanxi, China

department: College of Veterinary Medicine

institute: Northwest A&F University

summary: To explore the role of Brucella BI-1 in Brucella suis S2, we constructed the Brucella BI-1 deletion mutant strain and its complementary strain. We then determined the effect of Brucella BI-1 deletion on the physiological characteristics of Brucella suis S2 and revealed them via integrated transcriptomic and proteomic analyses. Brucella BI-1 deletion altered the membrane properties of Brucella suis S2 and decreased its resistance to acidic pH, H2O2, polymyxin B, and lincomycin. Additionally, deleting Brucella BI-1 led to defective growth, cell division, and viability in Brucella suis S2. In conclusion, our results revealed that Brucella BI-1 is a bacterial cytoprotective protein involved in membrane homeostasis, cell division, and stress resistance in Brucella suis S2.

overall_design: Transcriptomic profiles of the B. suis S2 WT, ΔBI-1, and cBI-1 strains were generated by deep sequencing, in triplicate.

first: Aihua

last: Wang

first: Yaping

last: Jin


title: Single cell transcriptomic analysis reveals cellular diversity of murine esophageal epithelium and age-associated mitochondrial dysfunction.

city: Philadelphia

first: Kelly

last: Whelan

state: PA

address: 3307 N Broad St. PAHB Rm 203

department: Fels Institute

institute: Temple University

summary: We have performed single cell RNA-sequencing to characterize the murine esophageal epithelium in young and aged mice.

overall_design: We used peeled esophageal epithelium from young (≤4 months) and aged (≥18 months) mice to investigate the molecular heterogeneity of the esophageal epithelium at single cell resolution.

first: Adam

last: Karami

first: Mohammad

last: Kabir

first: Kelsey

last: Keith

first: Jozef

last: Madzo

first: Jaroslav

last: Jelinek

first: Kumaraswamy

last: Chitrala


title: Knockdown of Akt1/2 by CRISPR/Cas9 suppresses the metastasis of human prostate cancer cells CWR22rv1 in vitro and in vivo

city: Xinxiang

first: Bing

last: Su

address: East Hualan Avenue

institute: The Third Affiliated Hospital Of Xinxiang Medical University

summary: Our findings will initially map out the downstream signal pathways of Akt and AR mutual feedback to regulate CaP metastasis through the study of these scientific assumptions, which will provide targets for the development of new precise treatments for prostate cancer.

overall_design: we knockout Akt1 and Akt2 genes in CaP CWR22rv1 cells to study effects of Akt1/2 knockout on metastasis in CaP CWR22rv1 cells in vitro and in vivo.

first: Xiaofan

last: Chen

first: Lijuan

first: Wenfang

last: Zhuang

first: Wei


title: Transcriptional effects of KLF15 deletion in soleus muscle

first: Ernest

last: Chan

summary: We report bulk RNA-sequencing of soleus muscle harvested from MyoCre and skeletal muscle specific KLF15 (K15-SKO) mice

overall_design: Soleus muscle were isolated from MyoCre or K15-SKO mice (n=4) according to standard protocols (see below) and transcriptional sequencing was performed.

first: Liyan

first: Mukesh


title: TREM2 sustains macrophage-hepatocyte metabolic coordination in NAFLD and sepsis [Mouse_Kupffer_cell_small_RNA_seq]

city: Hangzhou

first: JinChao

last: Hou

address: Qingchun Road 79

summary: The goals of this study are to assess the NAFLD-induced changes in expression of hepatic Kupffer cells (KCs) miRNAs, we conducted deep sequencing of small RNAs from KCs isolated from 8-week HFD-fed WT and Trem2-/- mice. Over 1000 miRNAs were identified in the hepatic macrophages. A set of miRNAs were upregulated in Trem2-/- versus WT hepatic macrophages, amongh which miR-106b was the most abundant miRNA in KCs.

overall_design: 8-weeks old Trem2-deficient (Trem2–/–) and littermate wild-type (WT) mice were placed on 8 weeks High fat diet feeding, and then their livers Kupffer cells (KCs) were harvest for KCs transcriptome analysis.

first: Jinchao

first: Xiangming

last: Fang

first: Marco

last: Colonna

first: Sicong


title: TREM2 sustains macrophage-hepatocyte metabolic coordination in NAFLD and sepsis [Mouse_Kupffer_cell_RNA_seq]

summary: The goals of this study are to explore the potential mechanism underlying fatty acid oxidation defects in high fat diet (HFD)-fed Trem2-/- livers, we performed RNA-seq and profiled Kupffer cells (KCs) transcriptomes isolated from 8-week HFD-fed Trem2-/- mice and WT littermates. Gene ontology (GO) analysis revealed that antigen processing and presentation, mitochondrial oxidative phosphorylation and ATP synthesis were impaired in Trem2-/- KCs. However, gene links to chemotaxis (Ccr2, Cx3cr1, Spp1), cytokine secretion (Cd36, Acsl1, Mmp19) were upregulated in Trem2-/- KCs. Interestingly, positively regulated genes in RNA polymerase II-mediated pri-miRNA transcription (Bmp2, Klf4) and exsomes (Exos) secretion (Cd63, Cd9) were enriched in Trem2-/- KCs.

first: Ping

last: Cui


title: TREM2 sustains macrophage-hepatocyte metabolic coordination in NAFLD and sepsis [Mouse_liver_RNA_seq]

summary: The goals of this study are to corroborate whether Trem2 has a protective role against metabolic dysfunction and progression in a mouse model of NAFLD. We further performed an unbiased transcriptome analysis from the livers of 8-week HFD-fed WT versus Trem2–/– mice. In this screen, a cluster of 104 transcripts were up-regulated and 57 were down-regulated in response to Trem2 deficiency. Among these differentially expressed genes (adjusted P < 0.05), gene sets that contribute to fatty acid metabolic dysfunction, collagen fibril organization and cytokine secretion were significantly enriched in Trem2–/– livers, whereas long-chain/unsaturated fatty acid metabolic process and monocarboxylic acid metabolic process were overrepresented among WT livers.

overall_design: 8-weeks old Trem2-deficient (Trem2–/–) and littermate wild-type (WT) mice were placed on 8 weeks High fat diet feeding, and then their livers were harvest for liver transcriptome analysis.

first: Jue

last: Zhang


title: TREM2 sustains macrophage-hepatocyte metabolic coordination in NAFLD and sepsis [Human_liver_RNA_seq]

summary: The goals of this study are to investigate whether the defects of energy metabolism and/or mitochondria occurs in Nonalcoholic fatty liver disease (NAFLD), we  performed liver transcriptome analysis from livers of donors who underwent organ donation after cardiac death (DCD; 6 non-NAFLD donors, 5 NAFLD donors). In this screen, a cluster of 462 transcripts were up-regulated and 447 transcripts were down-regulated in response to NAFLD. Among these differentially expressed genes , gene sets that contribute to endoplasmic reticulum stress, intrinsic apoptosis and cytokine production were enriched in NAFLD livers, whereas lipid metabolic process were suppressed in NAFLD livers, accompanied with deficient expression of the mitochondrial matrix enzyme.

overall_design: Liver transcriptome analysis from livers of donors who underwent organ donation after cardiac death (DCD; 6 non-NAFLD donors, 5 NAFLD donors)

first: Xiaoliang

last: Wu


title: TREM2 sustains macrophage-hepatocyte metabolic coordination in NAFLD and sepsis

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: Alterations of 5-hydroxymethylation in circulating cell-free DNA reflect molecular distinctions of subtypes of non-Hodgkin lymphoma

first: Issam

last: Awad

state: IL

address: 5841 S. Maryland Avenue

department: Department of Neurology

institute: The University of Chicago

summary: The 5-methylcytosines (5mC) have been implicated in the pathogenesis of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). However, the role of 5-hydroxymethylcytosines (5hmC) that are generated from 5mC through active demethylation, in lymphomagenesis is unknown. We profiled genome-wide 5hmC in circulating cell-free DNA (cfDNA) from 73 newly diagnosed patients with DLBCL and FL. We identified 294 differentially modified genes between DLBCL and FL. The differential 5hmC in the DLBCL/FL-differentiating genes co-localized with enhancers marks: H3K4me1and H3K27ac. A four-gene panel (CNN2, HMG20B, ACRBP, IZUMO1) robustly represented the overall 5hmC modification pattern that distinguished FL from DLBCL with an area under curve of 88.5% in testing set. The median 5hmC modification levels in signature genes showed potential for separating patients for risk of all-cause mortality. This study provides evidence that genome-wide 5hmC profiles in cfDNA differ between DLBCL and FL and could be exploited as a non-invasive approach.

overall_design: We obtained genome-wide 5hmC profiles using the 5hmC-Seal technique in cfDNA samples from 48 DLBCL patients and 25 FL patients at the University of Chicago Medical Center from 2010 to 2013. Patients were followed through March 18, 2019. The normalized 5hmC-Seal count data of 17,698 gene bodies with non-zero variance from the total 73 samples were used to identify informative genes with differential modification between FL and DLBCL. The elastic net regularization was applied to the multivariate logistic regression models with the 10-fold cross validation using the glmnet package in the R Statistical Environment, to select the most representative genes with differential modifications. To explore the gene regulatory relevance of 5hmC in cfDNA, we also summarized the 5hmC-Seal data to various genomic features from the Roadmap Epigenomics Project B-cell data. Functional relevance of the differentially modified genes to biological pathways and processes were explored using the Database Visualization and Integrated Discovery (DAVID) tool. The prognostic value of a given gene was assessed using the multivariate Cox Proportional Hazards model, controlling age at the time of initial diagnosis and gender.

first: Brian

last: Chiu

first: Chang

first: Qiancheng

last: You


title: Sevoflurane Exposure Alters Long Non-coding RNA and Messenger RNA Profiles in the Neonatal Mouse Hippocampus

city: Milwaukee

first: Thiago

last: Arzua

state: WI

address: 8701 W Watertown Plank Rd

institute: Medical College f Wisconsin

type:
Expression profiling by array
Non-coding RNA profiling by array

summary:
The focus of current study is to investigate the effect of developing sevoflurane exposure on lncRNA profiles and molecular signaling involvement in neurotoxicity.
An Expression Microarray assay (V3.0) and data analysis services were provided by Arraystar Inc. (Rockville, MD) to assess and compare the global abundance of 27,427 lncRNAs and 18,855 mRNAs in the hippocampi of mice exposed to either sevoflurane or normal air controls.

overall_design: Postnatal day 7 (PD7) mice were exposed to either 3% sevoflurane or normal air for 6 hours, n=4. RNA was isolated from the hippocampus, converted to cDNA, synthesized from RNA via reverse transcription, and hybridized to Arraystar’s custom probes.

first: Congshan

last: Jiang

first: Yasheng

last: Yan

first: Xiaowen

last: Bai


title: Arsenic hexoxide has differential effects on cell proliferation and genome-wide gene expression in human primary mammary epithelial and MCF7 cells

city: Cheonan

first: Keunsoo

last: Kang

address: 119 Dandae-ro

institute: Dankook University

summary: Arsenic is reportedly a biphasic inorganic compound for its toxicity and anticancer effects in humans. Recent studies have shown that certain arsenic compounds including arsenic hexoxide_x000D_  (AS4O6; hereafter, AS6) induce programmed cell death and cell cycle arrest in human cancer cells and murine cancer models. However, the mechanisms by which AS6 suppresses cancer cells are incompletely understood. In this study, we show markedly differential effects of AS6 on cytotoxicity and gene expression in human mammary epithelial normal cells (HUMEC) and Michigan Cancer Foundation 7 (MCF7), a human mammary epithelial cancer cell line. AS6 selectively arrests cell growth and induces cell death in MCF7 cells without affecting the growth of HUMEC in a dose-dependent_x000D_  manner. AS6 alters the transcription of a large number of protein-coding genes in MCF7 cells, but much fewer genes in HUMEC. Importantly, we found that the cell proliferation, cell cycle, and DNA repair pathways are significantly suppressed whereas cellular stress response and apoptotic pathways_x000D_  increase in AS6-treated MCF7 cells. Together, we provide the first evidence of differential effects of AS6 on normal and cancerous breast epithelial cells, suggesting that AS6 at moderate concentrations induces cell cycle arrest and apoptosis through modulating genome-wide gene expression, leading to compromised DNA repair and increased genome instability selectively in human breast cancer cells

overall_design: HUMEC and MCF7 cells were treated with 0 or 0.5 μM arsenic hexoxide for 50 h before being processed for RNA-seq analysis.

first: Heeyoun

last: Bunch

first: NaYeon

last: Park

first: Donguk

last: Kim


title: CPEB3 regulates neuron-specific alternative splicing and involves neurogenesis gene expression

city: Wuhan

first: Dong

last: Chen

state: Hubei

address: 388 GaoXin 2nd Road, East Lake Hi-Tech Development Zone

department: Center for Genome Analysis

institute: ABLife, Inc.


title: CPEB3 regulates neuron-specific alternative splicing and involves neurogenesis gene expression [RIP-seq]

summary: In the mammalian brain, alternative pre-mRNA splicing is a fundamental mechanism that modifies neuronal function dynamically where secretion of different splice variants regulates neurogenesis, development, pathfinding, maintenance, migration, and synaptogenesis. Sequence-specific RNA Binding Protein CPEB3 has distinctive isoform-distinct biochemical interactions and neuronal development assembly roles. Nonetheless, the mechanisms moderating splice isoform options remain unclear. To establish the modulatory trend of CPEB3, we cloned and excessively expressed CPEB3 in HT22 cells. We used RNA-seq to analyze CPEB3-regulated alternative splicing on control and CPEB3-overexpressing cells. Moreover, we used iRIP-seq to identify CPEB-binding targets. We additionally validated CPEB3-modulated genes using RT-qPCR. CPEB3 overexpression had insignificant effects on gene expression in HT22 cells. Notably, CPEB3 partially modulated differential gene splicing enhanced in the modulation of transcription, cell cycle, Wnt signaling cascade, neurotrophin, synapse, and specific development pathway. qRT‑PCR verified the CPEB3‑modulated transcription of neurogenesis genes LCN2 and NAV2, synaptogenesis gene CYLD, as well as development gene JADE1. Herein, we established that CPEB3 is a critical modulator of alternative splicing in neurogenesis, which remarkably enhances the current understanding of the CPEB3 mediated alternative pre-mRNA splicing.

overall_design: RIP-seq analysis of CPEB3 IP versus input in HT22 cells

first: Wenrui

last: Qu

first: Heng

last: Tian


title: Global transcriptional profiles of abdominal skin dermis during perinatal period

city: Bunkyo-ku

first: Fumiko

last: Toyoshima

state: Tokyo

address: 1-5-45 Yushima

department: Medical Research Laboratory

institute: Institute of Science Tokyo

summary: We report global transcriptional alteration and transcriptional heterogeneity of dermal cells in remodeling mouse abdominal skin during perinatal period. By single cell RNA sequencing of whole dermal cells isolated from the abdominal skin dermis of non-pregnant (NP) mice, pregnant mice at dpc16, and post-partum mice at dpp42 by using Col1a2creERT2;R26H2B-EGFP mice. By unsupervised evaluation of clustering-based cell identities of total samples on the Seurat platform, we identified 13 main clusters. The second round of unsupervised clustering of each cluster revealed that the vascular cluster exhibits dynamic transcriptome alteration during pregnancy.

overall_design: We treated the mice with tamoxifen during pregnancy and harvested abdominal skin at dpc16 and dpp42. For the control, abdominal skin was harvested from non-pregnant (NP) mice treated with tamoxifen for 3 days and harvested at 48 days after treatment. After dissociation of the dermis into single cells, we generated chromium single cell 3′ v2 libraries from randomly captured dermal cells and sequenced them using Illumina NextSeq500. We profiled 6,020 cells (NP: 1,289cells; Dpc16: 3,650cells; Dpp42: 1,081cells) with a range of 4,500–5,400 mean reads per cell for each sample, whereby approximately 1,200–1,500 median genes per cell for each sample were detected.

first: Ryo

last: Ichijo

first: Mio

last: Kabata

first: Takuya

last: Yamamoto


title: Gene therapy of dominant CRX-Leber congenital amaurosis using patient retinal organoids II

city: Bethesda

first: Zachary

last: Batz

state: MD

address: 6 Center Dr Bldg 6, Rm 303

department: NEI

institute: NIH

summary: Mutations in the cone-rod homeobox (CRX) transcription factor lead to distinct retinopathy phenotypes, including early-onset vision impairment in dominant Leber congenital amaurosis (LCA). Using induced pluripotent stem cells (iPSCs) from a patient with CRX-I138fs mutation, we established an in vitro model of CRX-LCA in retinal organoids that exhibit defective photoreceptor maturation by histology and gene profiling including diminished expression of visual opsins. Gene therapy by delivery of an additional correct CRX allele using an AAV vector partially restored photoreceptor phenotype and expression of phototransduction-related genes as revealed by single cell RNA-sequencing. Retinal organoids derived from iPSCs of a second dominant CRX-LCA patient carrying a K88N mutation revealed loss of opsin expression as a common phenotype, which could also be alleviated by AAV-mediated overexpression of CRX. Our studies provide the proof-of-concept for development of gene therapy for dominant CRX-LCA and other CRX-retinopathies.

overall_design: Single cell RNA-seq profiling of retinal organoids derived from a control line as well as a patient with a CRX mutation with or without AAV rescue treatment.

first: Kamil

last: Kruczek

first: Zepeng

first: Zhijian

last: Wu

first: Anand

last: Swaroop


title: Genome wide H3K27ac status of endometrial stromal cells stimulated by cAMP, and cAMP under C/EBPb-knockdown

city: Ube

last: Maekawa

address: Minami-Kogushi 1-1-1

department: Department of Obstetrics and Gynecology

institute: Yamaguchi University School of Medicine

summary: To know the role of C/EBPb in the changes of H3K27ac during desidualization, we compared the H3K27 profiles between cAMP-stimulation and cAMP-stimulation under C/EBPb-knockdown.

overall_design: ESCs were transfected with control or C/EBPb siRNA and then treated with or without cAMP for 4 days. Cells cultured simultaneously with RNA-sequence experiment were cross-linked by adding formaldehyde to the medium at a final concentration of 1% and incubated for 10 min at 37 °C.  Cross-linking was terminated by addition of glycine (0.125 M, final concentration).  ChIP was performed with H3K27ac antibody.  An unimmunoprecipitated control and immunoprecipitated DNA were used to prepare the ChIP-seq library with NEBNext DNA sample prep master mix set 1 (New England BioLabs, Ipswich, MA) and DNA sample prep kit, oligo only (Illumina) according to the manufacturer’s instructions. After the quality of the library was validated using the Agilent Bioanalyzer 2100 (Agilent Technology), the ChIP-sequence library was sequenced by the Genome Analyzer GAIIx.  The obtained reads from the sequencing platform was first applied for quality checking by the fastx quality stats program in the FASTX-Toolkit package (http://hannonlab.cshl.edu/fastx_toolkit/).  The reads that passed the quality checking were then mapped to the human reference genome sequence (hg19) by using the bowtie program followed by Model-Based Analysis of ChIP-Sequence (MACS) to detect the H3K27ac regions.

first: Isao

last: Tamura


title: CPEB3 regulates neuron-specific alternative splicing and involves neurogenesis gene expression [RNA-seq]

overall_design: transcrptional analysis of CPEB3 overexpression and control in HT22 cells


title: Galectin-8 induces functional disease markers in human osteoarthritis and cooperates with galectins-1 and -3.

city: Vienna

first: Markus

last: Jeitler

address: Lazarettgasse 14

country: Austria

institute: Medical University Vienna

summary: The reading of glycan-encoded signals by tissue lectins is considered a major route of the flow of biological information in many (patho)physiological processes. The arising challenge for current research is to proceed from work on a distinct protein to family-wide testing of lectin function. Having previously identified homodimeric galectin-1 and chimera-type galectin-3 as molecular switches in osteoarthritis progression, we here provide proof-of-principle evidence for an intra-network cooperation of galectins with three types of modular architecture. We show that the presence of tandem-repeat-type galectin-8 significantly correlated with cartilage degeneration and that it is secreted by osteoarthritic chondrocytes. Glycan-inhibitable surface binding of galectin-8 to these cells increased gene transcription and the secretion of functional disease markers. The natural variant galectin-8 (F19Y) was less active than the prevalent form. Genome-wide array analysis revealed induction of a pro-degradative/inflammatory gene signature, largely under control of NF-κB signaling. This signature overlapped with respective gene-expression patterns elicited by galectins-1 and -3, but also presented supplementary features. Functional assays with mixtures of galectins that mimic the pathophysiological status unveiled cooperation between the three galectins. Our findings shape the novel concept to consider individual galectins as part of a so far not realized teamwork in osteoarthritis pathogenesis, with relevance beyond this disease.

overall_design: For microarray experiments, osteoarthritic chondrocytes were isolated from five female patients (59-76 years). Following starvation, cells were incubated in the presence of 10 µg/ml recombinant Galectin-8 for 24 h. For each donor population, untreated cells were included as control. In total, 10 samples were analyzed.

first: Daniela

last: Weinmann

first: Michael

last: Kenn

first: Sebastian

last: Schmidt

first: Katy

first: Sonja

middle: M

last: Walzer

first: Bernd

last: Kubista

first: Reinhard

last: Windhager

first: Wolfgang

last: Schreiner

first: Stefan

last: Toegel

first: Hans-Joachim

last: Gabius


title: Genome wide RNA expression profile of endometrial stromal cells stimulated by cAMP, and cAMP under C/EBPb-knockdown

first: Ryo

summary: To know the role of C/EBPb in the change of mRNA expression during desidualization, we compared the mRNA expression profiles between cAMP-stimulation and cAMP-stimulation under C/EBPb-knockdown.

overall_design: ESCs were transfected with control or C/EBPb siRNA and then treated with or without cAMP for 4 days. The mRNA-seq libraries were generated. After the quality of the library was validated using the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA), the samples were subjected to sequencing with a next-generation sequencer (Genome Analyzer GAIIx, Illumina, San Diego, CA).  Gene expression values were calculated as “reads per kilobase of exon unit per million mapped reads” (RPKM).


title: Genome wide status of endometrial stromal cells stimulated by cAMP, and cAMP under C/EBPb-knockdown


title: MACF1 Promotes Osteoblast Differentiation by Sequestering Wnt Signaling Repressors in Cytoplasm [ChIP-seq]

first: Dong

last: Chen

summary: Osteoblast differentiation leading to bone formation requires a coordinated transcriptional program. We have recently demonstrated that microtubule actin crosslinking factor 1 (MACF1) promotes osteoblast differentiation, suggesting a key role in regulating early-phase osteoblast differentiation. Here, we showed that the early-phase osteoblast differentiation transcriptome dynamics was regulated by MACF1 and the transcription of TCF7/LEF1, key effectors of Wnt signaling that is important for osteoblast differentiation was suppressed by MACF1 knockdown. Co-IP and Protein mass spectrometry revealed that MACF1 interacted with a known and two previously unknown repressors of TCF7/LEF1, DKK1, CDK12 and MEAF6. ChIP-seq analysis of MACF1-associated promoters further revealed that MACF1 interacted with transcription factors TCF12 and E2F6, which also suppressed the transcription of TCF7/LEF1. Furthermore, all these four MACF-interacted proteins inhibited osteoblast differentiation. By studying the underlying mechanism, we found that cytoplasmic-nuclear localization of MACF1 was dependent on its level and the cytoplasmic-nuclear localization of TCF12 and E2F6 was regulated by MACF1 localization. In addition, MACF1 oppositely regulated the transcription activity of TCF12 and TCF7. Current study, for the first time to our knowledge, suggest that MACF1 acts as a sponge of osteoblast differentiation repressors to promote osteoblast differentiation, and indicate a novel mechanism for regulating the cellular location of transcription factors by a protein associated with microtubule and actin.

overall_design: Examinazion of MACF1 binding profile in Osteoblast under control and MACF1 knockdown conditions

first: Yi

last: Zhang

first: Airong

last: Qian


title: Global transcriptional profiles of abdominal skin epidermis during perinatal period

first: Fumiko

last: Toyoshima

summary: We report global transcriptional alteration and transcriptional heterogeneity of epidermal cells in remodeling mouse abdominal skin during perinatal period. By RNA-sequencing of FACS-isolated interfollicular epidermal (IFE) basal cells from abdominal skin of wildtype (WT) non-pregnant (NP) mice, pregnant mice, and Tbx3 cKO pregnant mice, we revealed pregnancy-associated genes and a key role of Tbx3 in regulating the pregnancy-associated transcriptome. To resolve the transcriptional heterogeneity of epidermal cells at single cell resolution, we performed single cell RNA sequencing of epidermal cells isolated from the abdominal skin of NP mice, pregnant mice, and post-partum mice by using Tbx3creERT2;R26H2B-EGFP mice. By unsupervised evaluation of clustering-based cell identities of total samples on the Seurat platform, we identified 14 main clusters, where the IFE1 cluster was appeared to be enriched in Dpc16 samples, and further revealed the pseudotime differentiation of Tbx3cre labeled cells during perinatal period.

overall_design: For RNAseq, mRNA profiles of FACS-isolated interfollicular basal cells (CD49f+, CD34-) from abdominal skin of wildtype (WT) non-pregnant (NP) mice, pregnant mice at dpc16, and K14creERT2;Tbx3flox/flox (Tbx3 cKO) pregnant mice at dpc16 were generated by deep sequencing, in triplicate, using Illumina NextSeq500. For scRNAseq, we treated the mice with tamoxifen during pregnancy and harvested abdominal skin at dpc16, dpp6, and dpp40. For the control, abdominal skin was harvested from non-pregnant (NP) mice treated with tamoxifen for 3 days. After dissociation of the epidermis into single cells, we generated chromium single cell 3′ v2 libraries from randomly captured epidermal cells and sequenced them using Illumina NextSeq500. We profiled 36,912 cells (NP: 6,485 cells; Dpc16: 12,846 cells; Dpp6: 9,029 cells; Dpp40: 8,552 cells) with a range of 20,000–40,000 mean reads per cell for each sample, whereby approximately 1,400–1,600 median genes per cell for each sample were detected.

last: Ichijo

first: Mio

last: Kabata

first: Takuya

last: Yamamoto


title: MACF1 Promotes Osteoblast Differentiation by Sequestering Wnt Signaling Repressors in Cytoplasm [RNA-seq]

overall_design: A total of 14 sequenced samples, including four time points under control and MACF1 knockdown conditions, samples in three time points have two biological replicates


title: Expression profiles in the complete absence of DNA methylation [Agilent-028005]

first: Yuichi

last: Kumaki

department: Center for Developmental Biology

summary: Using all three CpG DNA methyltransferases, Dnmt1, Dnmt3a and Dnmt3b deficient (TKO) mouse ES cells and TKO ntTS cells, we examined three histone modifications H3K4me2, H3K27me3 and H3K9me2, of promoter region in ES, TS and Flk1+ mesodermal cells with WT or TKO background by ChIP-chip analysis. Global profile analysis of gene expression and histone modification showed that the difference of profiles was mainly lineage dependent. Although the effects of DNA methylation loss in three lineages were modest, H3K27me3 is most sensitive to DNA methylation among three modifications. Interestingly, differentially expressed genes between WT and TKO cells were also lineage dependent and small number of genes was overlapped between lineages. Our further analysis showed that the profiles of H3K27me3 modifications were dynamically changed between lineages, especially in TS cells, and the pattern of H3K27me3 modification was defectively established by loss of DNA methylation. Our data suggest that DNA methylation contributes to lineage dependent expression regulation by collaborating with histone modification.

overall_design:
Expression profiles in the complete absence of DNA methylation by using Agilent-028005 SurePrint G3 Mouse GE 8x60K Microarray.
Gene expression was measured in mouse ES cells, Flk+ mesodermal cells and nuclear transferred TS cells with Dnmt1, Dnmt3a and Dnmt3b triple-konockout (TKO) and WT background with duplication, in total 12 samples.

first: Akiko

last: Yamagiwa

first: Chisa

last: Matsuoka

first: Masaki

last: Okano


title: Chip-chip in the complete absence of DNA methylation with H3K4me2, H3K9me2 and H3K27me3

type: Genome binding/occupancy profiling by genome tiling array

overall_design: ChIP-chip experiments were performed with anti-H3K4me2, H3K9me2 and H3K27me3 antibodies in mouse ES cells, Flk+ mesodermal cells and nuclear transferred TS cells with Dnmt1, Dnmt3a and Dnmt3b triple-konockout (TKO) and WT background with duplication, in total 36 samples.


title: Expression profiles in the complete absence of DNA methylation [Agilent-014868]

overall_design:
Expression profiles in the complete absence of DNA methylation by using Agilent-014868 Whole Mouse Genome Microarray 4x44k G4122F.
Gene expression was measured in mouse ES cells, Flk+ mesodermal cells and nuclear transferred TS (ntTS) cells with Dnmt1, Dnmt3a and Dnmt3b triple-konockout (TKO) and WT background with duplication. For each of WT and TKO ntTS cells, two independent cell lines were measured. In total 16 samples.


title: Complete absence of DNA methylation effect on histone modifications H3K4me2, H3K27me3 and H3K9me2

overall_design: Refer to individual Series


title: A cell atlas of chromatin accessibility across 25 adult human tissues

status: Public on Feb 16 2021

city: San Diego

first: Bing

last: Ren

state: California

address: 9500 Gilman Dr

institute: University of California San Diego

summary: Current catalogs of regulatory sequences in the human genome are still incomplete and lack cell type resolution. To profile the activity of human gene regulatory elements in diverse cell types and tissues in the human body, we applied single cell chromatin accessibility assays to 25 distinct human tissue types from multiple donors. The resulting chromatin maps comprising ~500,000 nuclei revealed the status of open chromatin for over 750,000 candidate cis-regulatory elements (cCREs) in 54 distinct cell types. We further delineated cell type-specific and tissue-context dependent gene regulatory programs, and developmental stage specificity by comparing with a recent human fetal chromatin accessibility atlas. We finally used these chromatin maps to interpret the noncoding variants associated with complex human traits and diseases. This rich resource provides a foundation for the analysis of gene regulatory programs in human cell types across tissues and organ systems.

overall_design: Applying single cell chromatin accessibility assays to 25 distinct human tissue types from four donors.

first: Kai

first: James

last: Hocker

first: Michael

last: Miller

first: Xiaomeng

last: Hou

first: Olivier

last: Poirion

first: Allen

last: Wang

first: Sebastian

last: Preissl


title: Expression data from mouse wild type, PRDM14-overexpressing and Dnmts KO embryonic stem cells (ESCs).

summary: Germline cells reprogram extensive epigenetic modifications to ensure the cellular totipotency of the next generation and prevent accumulation of epimutations. Primordial germ cells (PGCs)1, the common source of both oocytes and sperm, erase genome-wide DNA methylation and histone H3 lysine 9 dimethylation (H3K9me2), a process called genome-wide epigenetic reprogramming2,3. However, little is known about the molecular mechanism of DNA demethylation by developing PGCs. Here we show that overexpression of PRDM14, a critical regulator for specification and early differentiation of PGCs, promotes global DNA demethylation in embryonic stem cells (ESCs). PRDM14 directly represses transcription of de novo DNA methyltransferase, Dnmt3b, but its repression is not sufficient for global DNA demethylation. Comparison of global gene expression profiles between PRDM14-overexpressing ESCs and Dnmts triple mutant ESCs clearly demonstrates that overexpression of PRDM14 activates about half of the genes silenced by DNA methylation in ESCs. Furthermore, PRDM14 directly interacts with TET1, which converts 5-methylcytosine to 5-hydroxymethylcytosine, and DNA demethylation by overexpression of PRDM14 is strongly disturbed by pharmacological inhibitors of the base excision repair (BER) pathway. We propose that formation of a PRDM14/TET1 complex triggers the activation of BER-dependent active demethylation across the whole genome of developing PGCs.

overall_design: To determine the effects of DNA demethylation by overexpression of PRDM14 on the global gene expression profile, we exploited microarray analysis using total mRNAs derived from J1 (control), PRDM14-overexpressing, PRDM14-overexpressing + DNMT3B1, Dnmt3b KO, Dnmt1 KO and Dnmts TKO mouse ES cells, in total 10 samples.

first: Kuniaki

last: Ebi

first: Miyuki

last: Nishi

first: Naoki

last: Okashita

first: Osamu

last: Takano

first: Megumi

last: Nakayama

first: Gen

last: Hirano

first: Yoshiyuki

last: Seki


title: scRNAseq of aSMACreER/Ai9+ periosteum cells

city: Farmington

first: William

last: Flynn

state: CT

address: 10 Discovery Drive

institute: The Jackson Laboratory

summary: aSMA identifies osteochondroprogenitors in the periosteum involved in fracture callus formation. Cell surface marker expression, histological analysis, and lineage tracing data indicate that this is a heterogeneous population that contains a subset of self-renewing stem or progenitor cells. The goal of this study was to evaluate the heterogeneity of the initial cell population labeled using plate-based single cell RNAseq, and see if that changed following up to 13 weeks of chase.

overall_design: Periosteal cells were freshly isolated from mice and cell sorts performed directly into a 384-well plate. Animals received 2 doses of tamoxifen (75mg/kg ip) 24h apart, cells were isolated 2 days, 6 weeks, and 13 weeks later. 13 week cohort (H2B-GFP mice) received doxycycline in their diet from P11 for 5 weeks. One sort was performed (all done on the same day) for each cell type (192 cells for 2 days and 13 weeks, 216 cells for 6 weeks)

first: Brya

middle: G

last: Matthews

first: Ivo

last: Kalajzic


title: Single cell RNA-seq analysis of the LLC-OVA tumor-infiltrating T-cell compartment.

city: Brussels

first: Daliya

last: Kancheva

address: Pleinlaan 2

country: Belgium

institute: Vrije Universiteit Brussel

summary: Modulation and depletion of regulatory T cells (Tregs) constitute valid approaches in antitumor immunotherapy but suffer from severe adverse effects due to their lack of selectivity for the tumor-infiltrating (ti-)Treg population. We here employed single-cell RNA-sequencing to discern two ti-Treg populations, one of which is characterized by the unique expression of Ccr8 in conjunction with Treg activation markers. Ccr8 is also expressed by dysfunctional CD4+ and CD8+ T cells, but the CCR8 protein was only prominent on the highly activated and strongly T-cell suppressive ti-Treg subpopulation of mouse and human tumors, with no major CCR8-positivity found on peripheral Tregs. CCR8 expression resulted from TCR-mediated Treg triggering in an NF-kB-dependent fashion, but was not essential for the recruitment, activation nor suppressive capacity of these cells. We generated two CCR8-specific nanobodies (Nbs) that recognize distinct epitopes on the CCR8 extracellular domain. These Nbs were formulated as tetravalent Nb-Fc fusion proteins for optimal CCR8 binding and blocking, containing either an ADCC-deficient or an ADCC-prone Fc region. While treatment of tumor-bearing mice with a blocking ADCC-deficient Nb-Fc did not influence tumor growth, ADCC-prone Nb-Fc elicited antitumor immunity and reduced tumor growth in synergy with anti-PD-1 therapy. Importantly, ADCC-prone Nb-Fc specifically depleted ti-Tregs without affecting peripheral Tregs. Collectively, our findings highlight the efficacy and safety of targeting CCR8 for the depletion of tumor-promoting ti-Tregs in combination with anti-PD-1 therapy.

overall_design: Single cell RNA Seq = Live CD45+CD11b-TCRβ+ cells sorted from LLC-OVA tumors subcutaneously injected in WT C57BL/6 mice.

first: Helena

last: Van Damme

first: Damya

last: Laoui

first: Kiavash

last: Movahedi

first: Isabelle

last: Scheyltjens

first: Niels

last: Vandamme

first: Liesbet

last: Martens


title: Expression profiling by RNA-seq for identifying genes associated with prognosis in non-muscle-invasive bladder cancer.

city: Daejeon

first: Seon-Kyu

last: Kim

address: 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Korea

department: Personalized Genomic Medicine Research Center

institute: Korea Research Institutue of Bioscience & Biotechnology

summary: Non-muscle-invasive bladder cancer (NMIBC) is clinically heterogeneous, and many NMIBC patients fail to respond to treatment and frequently experience disease relapse. We aim to identify a prognostic and predictive signature of the NMIBC heterogeneity. We constructed a transcriptomic data set by RNA-seq experiments from an independent NMIBC patient cohort (n=32). This cohort contains the patients stratified into three prognostic subgroups: Group 1 (patients with non-relapse; n=15), Group 2 (patients experiencing recurrence, n=9), and Group 3 (patients experiencing progression; n=8).

overall_design: We generated a RNA-seq dataset of 32 samples which were stratified into three prognostic subgroups: Group 1 (patients with non-relapse; n=15), Group 2 (patients experiencing recurrence, n=9), and Group 3 (patients experiencing progression; n=8). Total RNA was isolated using the RNeasy Mini Kit (Qiagen, CA, USA) according to the manufacturer’s protocol. The quality and integrity of the RNA were confirmed by agarose gel electrophoresis and ethidium bromide staining, followed by visual examination under ultraviolet light. The sequencing library was prepared using the TruSeq RNA Sample Preparation kit v2 (Illumina, CA, USA) according to the manufacturer’s instructions. In brief, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads, fragmented, and converted into cDNA. Adapters were then ligated to the cDNA, and the fragments were amplified by PCR. Sequencing of paired-end reads (2×150 bp) was performed using the HiSeq 2500 platform (Illumina).

first: SeokJoong

last: Yun

first: Pildu

last: Jung


title: Comprehensive analysis of differentially expressed lncRNA, circRNA and mRNA and their ceRNA networks in mice with severe acute pancreatitis

city: Chengdu

first: bing

last: wang

state: Sichuan

address: No. 270, Tianhuan Road, Rongdu Avenue, Jinniu District, Chengdu City, Sichuan Province

department: General Surgery

institute: The General Hospital of Western Theater Command

summary: Severe acute pancreatitis (SAP) is an acute digestive system disease with high morbidity mortality and hospitalization rate worldwide, due to various causes and unknown pathogenesis. In recent years, a large number of studies have confirmed that non-coding RNAs (ncRNAs) play an important role in many cellular processes and disease occurrence. However, the underlying mechanisms based on the function of ncRNAs, including long noncoding RNA (lncRNA) and circular RNA (circRNA), in SAP remain unclear. In this study, we performed high-throughput sequencing on the pancreatic tissues of 3 normal mice and 3 SAP mice for the first time to describe and analyze the expression profiles of ncRNAs, including lncRNA and circRNA. Our results identified that 49 lncRNAs, 56 circRNAs and 1194 mRNAs were differentially expressed in the SAP group, compared with the control group. Furthermore, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed lncRNAs and circRNAs, and found that the functions of the parental genes are enriched in the calcium-regulated signaling pathway, NF-κB signaling pathway, autophagy and protein digestion and absorption processes, which are closely related to the central events in pathogenesis of SAP. We also constructed lncRNA/circRNA-miRNA-mRNA networks to further explore their underlying mechanism and possible relationships in SAP. We found that in the competitive endogenous RNA (ceRNA) networks, differentially expressed lncRNAs and circRNAs are mainly involved in the apoptosis pathway and calcium signal transduction pathway. In conclusion, we found that lncRNAs and circRNAs play an important role in the pathogenesis of SAP, which may provide new insights in further exploring the pathogenesis of SAP and seek new targets for SAP.

overall_design: Exploring the differentially expressed lncRNA, circRNA and mRNA profile from 3 severe acute pancreatitis mice and 3 control mice

first: Hongyu

last: Sun

first: Bing

last: Wang

first: Jun

last: Wu

first: Qilin

last: Huang


title: ChIP-seq on PfAP2-G2 in trophozoite and gametocyte stages of parasites.

city: University Park

first: Manuel

last: Llinas

address: Millennium Science Complex

department: Biochemistry and Molecular Biology

institute: Pennsylvania State University

summary: Differentiation from asexual blood stages to sexual gametocytes is required for transmission of malaria parasites from the human host to mosquitos, where sexual fertilization occurs to complete the lifecycle. Although preventing gametocyte development would block parasite transmission, the molecular mechanisms underlying sexual commitment and gametocyte maturation are still relatively unknown. Previous studies identified an ApiAP2 protein, AP2-G2, which plays a critical role in gametocyte maturation in rodent malaria parasite Plasmodium berghei by acting as the repressor of asexual stage genes in gametocytes. In this work we characterize the P. falciparum orthologue (PF3D7_1408200) of PbAP2-G2 and report that it plays a critical role in maturation of gametocytes. Disruption of pfap2-g2 did not obstruct commitment to sexual development but the majority of parasites were unable to develop normally beyond stage III gametocytes. In asexual stages PfAP2-G2 binds to the promoters of wide variety of genes expressed in diverse range of parasite’s life cycle stages including gametocytes, mosquito lifecycle stages early ring stage genes and genes encoding for proteins involved in egress and invasion. Surprisingly, we also identify binding of PfAP2-G2 in the gene body of almost 3000 genes. We could also show that PfAP2-G2 interacts with chromatin remodeling proteins and another ApiAP2 protein (PF3D7_1139300) whose motif is also overrepresented in the ChIP-seq data. Overall this work suggests that PfAP2-G2 is a transcriptional regulator that regulates genes possibly by recruiting additional transcription factors and chromatin remodeling machinery and plays a critical role in the development of malaria parasites as they transition from the asexual stage to gametocytes.

overall_design: Three replicates of ChIP-seq were performed during the trophozoite stages of asexual development using GFP taged PfAP2-G2. PfAP2-G2-GFP parasites were highly synchronized for that stage, followed by crosslinking and isolation of the chromatin. Chromatin bound by PfAP2-G2 GFP was isolated via anti-GFP and input material was used as a negative control for each replicate. We also performed two replicates of ChIP-seq using GFP-tagged PfAP2-G2 parasites on gametocyte stage III to define the target genes during sexual development using purified population of gametocytes.

first: Suprita

last: Singh

first: Joana

last: Santos

first: Lindsey

last: Orchard


title: Nuclear Condensation of NUP98-Fusion Proteins drives leukemogenic gene expression

first: Thomas

last: Eder

address: Veterinaerplatz 1

department: Department for Biological Sciences and Pathobiology

institute: University of Veterinary Medicine, Vienna

summary: NUP98-fusion proteins cause acute myeloid leukemia via unknown molecular mechanisms. All NUP98-fusion proteins share an intrinsically disordered region (IDR) featuring >35 repeats of Phenylalanine-Glycine (FG) in the NUP98 N-terminus. Conversely, different C-terminal NUP98-fusion partners are often transcriptional and epigenetic regulators. Given these structural features we hypothesized that mechanisms of oncogenic transformation by NUP98-fusion proteins are hard-wired in their protein interactomes. Affinity purification coupled to mass spectrometry of five distinct NUP98-fusion proteins revealed a conserved set of interactors that was highly enriched for proteins involved in biomolecular condensation. We developed biotinylated isoxazole-mediated condensome mass spectrometry (biCon-MS) to show that NUP98-fusion proteins alter the global composition of biomolecular condensates. In addition, an artificial FG-repeat containing fusion protein was able to phenocopy the induction of leukemic gene expression as mediated by NUP98-KDM5A. Thus, we propose that IDR-containing fusion proteins have evolved to uniquely combine biomolecular condensation with gene control to induce cancer.

overall_design: Retroviral transduction of fetal liver cells with NUP98-KDM5A or two artificial IDR-containing fragments fused to C-KDM5A.

first: Stefan

last: Terlecki-Zaniewicz

first: Theresa

last: Humer

first: Johannes

last: Schmöllerl

first: Elizabeth

last: Heyes

first: Gabriele

last: Manhart

first: Natalie

last: Kuchynka

first: Katja

last: Parapatics

first: Fabio

last: Liberante

first: André

last: Müller

first: Eleni

last: Tomazou

first: Florian

last: Grebien


title: Concordance of inflammatory markers in lymphocytes and prefrontal cortex in schizophrenia

city: Chicago

first: Eleonora

last: Gatta

address: 1601 W Taylor St

department: Psychiatry

institute: University of Illinois at Chicago

summary:
Schizophrenia is a severe neuropsychiatric disorder associated with a wide array of transcriptomic and neurobiochemical changes. Genome-wide transcriptomic profiling conducted in postmortem brain have provided novel insights into the pathophysiology of this disorder, and identified biological processes including immune/inflammatory-related responses, metabolic, endocrine and synaptic function. However, few studies have investigated whether similar changes could be found in peripheral tissue.
Here, we used RNA-sequencing to characterize transcriptomic profiles of lymphocytes in 18 non-psychotic controls and 19 individuals with schizophrenia. We identified 2,819 differentially expressed transcripts (pnominal < 0.05) in the schizophrenia group when compared to controls. Bioinformatic analyses conducted on a subset of 293 genes (pnominal < 0.01 and |log2FC| > 0.5) highlighted immune/inflammatory response as key biological processes in our dataset. Differentially expressed genes in lymphocytes were highly enriched for gene expression profiles from cortex layer 5a & immune cells. Thus, we investigated whether the changes in transcripts levels we observed in lymphocytes could also be detected in the prefrontal cortex (PFC, BA10) in a second validation cohort of schizophrenia subjects. Remarkably, mRNA levels detected in the PFC and lymphocytes were in strong agreement, and measurements obtained using RNA-sequencing were positively correlated with data obtained by RT-qPCR analysis.
Collectively, our work supports a role for immune dysfunction in the pathogenesis of schizophrenia and suggest that peripheral markers can be used as accessible surrogates to investigate putative central nervous system disruptions.

overall_design: Investigation of gene expression profiles in lymphocytes from 18 non-psychotic controls and 19 individuals with schizophrenia.

first: Vikram

last: Saudagar

first: Jenny

last: Drnevich

first: Marc

middle: P

last: Forrest

first: James

last: Auta

first: Lindsay

middle: V

last: Clark

first: Henry

last: Sershen

first: Robert

last: Smith

first: Dennis

last: Grayson

first: John

last: Davis

first: Alessandro

last: Guidotti


title: Transriptome analysis of alternative splicing under heat stress condition in atcyp18-2 mutant compared to Wild type (Col-0) of Arabidopsis

first: Areum

last: Lee

address: 125 Gwahak-ro

department: Plant Systems Engineering Research Center

institute: Korea Research Institute of Bioscience and Biotechnology

summary: To investigate whether the observed changes in pre-mRNA splicing led to AtCYP18-2 depletion, we conducted RNA-sequencing to analyse the quantitative profiling of alternative splicing

overall_design: RNA was extracted from atcyp18-2 and WT rosette leaf of 3 weeks old plant and treated with or without heat stress for 1 h at 47℃

first: Hye

last: Cho


title: Single cell transcriptomc analysis of CD34+ cells from primary myelofibrosis patient

city: Modena

first: Rossella

last: Manfredini

address: Via Gottardi 100

department: Life Sciences

country: Italy

institute: Centre for Regenerative Medicine

summary: We performed single cell RNA sequencing by means of 10X Genomics platform of CD34+ hematopoietic stem/progenitor cells (HSPCs) from primary myelofibrosis (PMF). Cells were isolated through immunomagnetic selection from frozen PBMCs coming from one patient with PMF who evolved to Acute Myeloid Leukemia (AML). Three timepoints were considered: chronic phase (Time1), after 8 months of Ruxolitinib treatment (Time2) and after 11 months of Ruxolitinib treatment at AML diagnosis (Time3). The main purpose of this project was to reconstruct the composition of the neoplastic clone in terms of HSPCs subpopulations at different time-points during disease evolution and to study changes in gene expression within each cell cluster over time.

overall_design: This dataset include 3 samples from the same patient affected by PMF. The first sample correspond to the chronic phase of the disease. The second one is taken 8 month after Ruxolitinib treatment initiation. The third sample was collected 11 month after Ruxolitinib treatment initiation at the time of AML diagnosis.

first: Sandra

last: Parenti

first: Chiara

last: Carretta

first: Selene

last: Mallia

first: Sebastiano

last: Rontauroli

first: Elena

last: Genovese

last: Chiereghin

first: Clelia

last: Peano

first: Elisa

last: Bianchi

first: Sebastian

last: Fantini

first: Stefano

last: Sartini

first: Lara

last: Tavernari

first: Oriana

last: Romano

first: Silvio

last: Bicciato

first: Enrico

last: Tagliafico

first: Matteo

last: Della Porta


title: Transcriptomic analysis of monocytes, M0, M1 and M2 macropahges from H syndrome patients and SAIDs.

city: Birmingham

first: Samuel

middle: J

last: Lara Reyna

address: Edgbaston

country: United Kingdom

institute: University of Birmingham

summary: Biallelic mutations in SLC29A3 cause histiocytosis-lymphadenopathy plus syndrome, also known as H syndrome (HS). HS is a complex disorder, with ~25% of patients developing autoinflammatory complications consisting of unexplained fevers, persistently elevated inflammatory markers and unusual lymphadenopathies, with infiltrating CD68+, S100+ and CD1a– histiocytes, resembling the immunophenotype found in Rosai-Dorfman disease (RDD). We investigated the transcriptomic profiles of monocytes, non-activated (M0), classically-activated (M1) and alternatively-activated macrophages (M2) in two patients with HS, one without autoinflammatory (HS1) and one with autoinflammatory complications (HS2). RNA sequencing revealed a dysregulated transcriptomic profile in both HS patients compared to healthy controls (HC). HS2, when compared to HS1, had several differentially expressed genes, including genes associated with lymphocytic-histiocytic predominance (e.g. NINL) and immunodeficiencies (e.g. B2M). The transcriptomic and cytokine profiles of HS patients were comparable to patients with SAID with high levels of TNF. SERPINA1 gene expression was found to be upregulated in all patients. Moreover, higher levels of IFNg were found in the serum of both HS patients when compared to HC. Gene-ontology (GO) enrichment analysis of the DEGs in HS patients revealed the terms "type I IFN", "IFNg signalling pathway" and "immune responses" as the top 3 most significant terms for monocytes. Finally, the transcriptomic profiles of M0 and M1 macrophages, from patient HS2, were similar to the transcriptomic profile of tissue biopsies taken from patients with RDD-like lymphadenopathies. Monocytes and macrophages from both HS patients showed transcriptomic profiles similar to SAIDs and with a unique dysregulated IFNgsignalling. These findings may help to find better therapeutic options for this rare disorder.

overall_design: To study the transcriptomic profile of monocytes, M0, M1 and M2 macrophages, we obtained CD14+ monocytes from 2 HS, 2 SAID patients and 5 HC, and differentiate the cells into monocyte derived macrophages (MDMs). Isolated human monocytes were cultured in complete RPMI medium supplemented with 20 ng/mL human GM-CSF for macrophage differentiation for 6 days. M0 macrophages were activated with 100 ng/mL human IFN-g and 50 ng/mL LPS, for M1 or 20 ng/mL IL-13 and IL-4, for M2 macrophage polarisation, and incubated for 24 hours.

last: Lara-Reyna

first: James

middle: A

last: Poulter

first: Elton

last: Vasconcelos

first: Mark

last: Kacar

first: Michael

last: McDermott

first: Reuben

last: Tooze

first: Rainer

last: Doffinger

first: Sinisa

last: Savic


title: Secreted phosphoprotein 1 expression in retinal mononuclear phagocytes links murine to human choroidal neovascularization

city: Freiburg

first: Clemens

last: Lange

address: Kilianstraße 5

department: Klinik für Augenheilkunde

institute: Uniklinik Freiburg

summary: Neovascular age-related macular degeneration represents the most common cause of blindness in the western world. Alterations of the outer Blood-retina barrier integrity and a localized inflammatory microenvironment lead to sprouting of choroidal neovascularization in intimate contact with surrounding myeloid cells and ultimately lead to visual impairment. The discovery of novel targets interfering with angiogenesis and inflammation is vital for the future treatments in AMD patients. To identify novel potential targets in the local phagocytes of the retina, microglia, we performed a comprehensive RNA-seq analysis in the mouse model of laser-induced choroidal neovascularization (mCNV). Here, we identified the angiogenic factor Osteopontin (Opn), also known as "secreted phosphoprotein 1” (Spp1), to be one of the most highly expressed genes in retinal microglia in the course of CNV formation. We could confirm the presence of SPP1 at the lesion site in recruited retinal microglia of Cx3cr1CreER:Rosa26-Tomato reporter mice using immunohistochemistry and in whole retinal tissue lysates by ELISA compared to controls highlighting a massive local production of SPP1. Inhibition of SPP1 by intravitreal injection of anti-SPP1 antibody significantly increased the lesion size compared to IgG-treated control eyes. In line with the results in rodents, we found an increased SPP1 mRNA expression in surgically extracted human choroidal neovascular (hCNV) membranes by the quantitative RNA-seq approach of massive analysis of cDNA ends (MACE) and found numerous IBA1+SPP1+ myeloid cells in human CNV membranes. Taken together, these results highlight the importance of SPP1 in the formation of CNV and potentially offer new opportunities for therapeutic intervention by inhibiting the SPP1 pathway.

overall_design: RNA sequencing of 6 laser samples (retinal microglia, mouse, laser CNV) and 6 unlasered controls.

first: Anja

last: Schlecht

first: Peipei

last: Zhang

first: Julian

last: Wolf

first: Adrian

last: Thien

first: Dennis-Dominik

last: Rosmus

first: Stefaniya

last: Boneva

first: Peter

last: Wieghofer


title: DNA methylation patterns identify subgroups of pancreatic neuroendocrine tumors with clinical association

city: Brisbane

first: Katia

last: Nones

state: QLD

address: 300 Herston Road

country: Australia

institute: QIMR Berghofer MEdical Research Institute

type: Methylation profiling by genome tiling array

summary:
This study evaluate the methylation profile of 84 clinically sporadic pancreatic neuroendocrine tumors (PanNETs) with associated clinical and genomic information. We identified three subgroups of PanNETs, termed T1, T2 and T3, with distinct patterns of methylation. The T1 subgroup was enriched for functional tumors and ATRX, DAXX and MEN1 wild-type genotypes. The T2 subgroup contained tumors with mutations in ATRX, DAXX and MEN1 and recurrent patterns of chromosomal losses in half of the genome with no association between regions with recurrent LOH and methylation levels. T2 tumors were larger and had lower methylation in the MGMT gene body, which showed positive correlation with gene expression. The T3 subgroup harboured mutations in MEN1 with recurrent LOH of chromosome 11, was enriched for grade G1 tumors and showed histological parameters associated with better prognosis. Our results suggest a role for methylation in both driving tumorigenesis and potentially stratifying prognosis in PanNETs.
These set of normal adjacent pancreata was compared to ICGC pancreatic neuroendocrine tumour methylation. We identified CpG sites located in promoter regions with low levels of methylation (beta value <0.3) in all normal adjacent pancreata and from those selected the most variable probes with a standard deviation >0.20 of DNA methylation levels across all tumors to identify sub-groups.

overall_design:
84 tumours obtained from International Cancer Genome Consortium (ICGC https://icgc.org/ under projects PAEN-AU and PAEN-IT were compared to 11 normal Pancreata.
contributor: Australian Pancreatic Genome Initiative (APGI)
contributor: Applied Research on Cancer network (ARC-Net)

first: Vanessa

last: Lakis

first: Rita

middle: T

last: Lawlor

first: Felicity

last: Newell

first: Ann-Marie

last: Patch

first: Andrea

last: Mafficini

first: Anguraj

last: Sadanandam

first: Lambros

last: Koufariotis

first: Rebecca

last: Johnston

first: Conrad

last: Leonard

first: Scott

last: Wood

first: Borislav

last: Rusev

first: Vincenzo

last: Corbo

first: Claudio

last: Luchini

first: Sara

last: Cingarlini

first: Luca

last: Landoni

first: Roberto

last: Salvia

first: Michele

last: Milella

first: David

last: Chang

last: Bailey

first: Nigel

middle: B

last: Jamieson

first: Fraser

last: Duthie

first: Marie-Claude

last: Gingras

first: Donna

last: Muzny

last: Wheeler

first: Richard

last: Gibbs

first: Massimo

last: Milione

first: Paolo

last: Pederzoli

first: Jaswinder

last: Samra

first: Anthony

last: Gill

first: Amber

last: Johns

first: John

last: Pearson

first: Andrew

last: Biankin

first: Sean

first: Nicola

last: Waddell

first: Aldo

last: Scarpa


title: TRAFD1 knockdown in THP-1 cell line using siRNA.

city: Groningen

first: Adriaan

last: van der Graaf

state: Groningen

address: Antonius Deusinglaan 1

department: Department of Genetics

institute: University Medical Center Groningen, University of Groningen

summary: RNA-seq dataset of TRAFD1 knockdown in THP-1 cell lines compared to THP1 cell lines treated with non targeting  siRNA (SCR) and untreated cells (WT), under unstimulated and stimulated (LPS) conditions.

overall_design: THP-1 cell lines were grown in triplicate for each condition and treatment. The cells were left untreated  (WT), or treated with non-targeting siRNA (SCR) or siRNA to target TRAFD1 (TRAFD1 KD. 2). Then the cells were left unstimulated (media) or treated with 10ng/ml of LPS. All the treatments were performed in triplicated.

first: Maria

last: Zorro


title: Transcriptome analysis of bisphenol A (BPA) treated Y79 human retinoblastoma cells

city: Seoul

first: Jung-Woong

last: Kim

address: 84 Heuksuk-ro Dongjak-ku

department: Life Science

institute: Chung-Ang University

summary:
Purpose:  finding differential transcripts usage in environmentally harzardous chemical, bisphenol A (BPA) in human retinal cells
Methods: transcriptome analysis by the RNA-seq via mRNA pull-down
Results: We found that BPA increases cellular toxicity by the increasing of early apoptotic pathway in Y79 cells. Also, over thounds of retained intron events were detected from the RNA-seq analysis.
Conclusions: Bisphenol A (BPA) increases early cytotoxic effects in Y79 cells through the early apoptotic pathway and changes the context of trascript through the splicing regulation mechanisms.

overall_design: Y79 cells were treated with various concentration of bisphenol A and all experiments were duplicated.


title: Stimulation of gluten-specific CD4+ T cell lines with aCD3/aCD28

summary: Gluten specific CD4 positive T cell upon anti CD3 and anti CD28 stimulation

overall_design: Cells were stimulated for 3 hours with 2.5 microgram/ml of anti-CD3 (Biolegend, catalog317315) and anti-CD28 (Biolegend, catalog302923) antibodies, Untreated cells were included as control. N=22 samples per condition. Measurements were done at t=0 (untreated) and t=180minutes (stimulated)

first: Serena

last: Sanna

first: Iris

last: Jonkers


title: RNA sequencing derived from intestinal biopsies from healthy controls and celiac disease patients

summary:
Aim: To compare the overall transcriptional profile in healthy controls and celiac disease patients. This dataset, was used to evaluate if our in vitro model (intestinal intraepithelial lymphocytes, desccribed in doi:10.1016/j.jaut.2020.10242 ) is representative of the transcriptional profile in the intestine under healthy or inflammatory conditions.
Samples: Upper colonoscopy biopsies from 5 control and 11 celiac disease patients were taken, total RNA was extracted and RNA-sequencing was performed (without replicates)

overall_design: duodenal biopsies of 5 healthy controls (CTR) and 11 histology proven CeD (PED) for differential expression experimentation

first: Raùl

last: Aguirre


title: Stimulation of Caco-2 cells with IFNg

summary: Caco-2 cells (intestinal human derived cell line) were cultured on transwell plates, and exposed to a stimulus (IFNg) through the bottom compartment of the transwell plates

overall_design: all conditions and treatments in duplicate. Unstimulated cell were used as controls.