title: Development of circRNAs expression signatures in exosomes derived from different gastric cancer cells

status: Public on May 12 2022

city: Zhengzhou

first: peiming

last: zheng

address: 7 Weiwu Road

institute: People’s Hospital of Zhengzhou University

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

summary: Exosomes from the culture media (CM) of four GC cells (GCCs) and human gastric epithelial cells were isolated. Exosomal RNA was extracted, and circRNA microarray assay was performed to identify GC-specific exosomal circRNAs. A total of 58 exosomal circRNAs were expressed at considerable higher levels in GCCs than those in normal controls.The expression level of several top upregulated circRNAs were detected using qRT-PCR in both GC cells and clinical specimens of GC patients. More interesting, besides the high expression in GC cell derived exosomes, hsa_circ_0015286 was the uniquely upregulated circRNA in the plasma exosomes and tissues of GC patients..The result suggested that exosomal hsa_circ_0015286 may be a potential noninvasive biomarker for the diagnosis and prognosis monitoring of GC.

overall_design: Four human GCC lines (MGC803, BGC823, SGC7901, and AGS) and one normal human gastric (GES-1) cell line were  cultured at 37 °C with 5% CO2 in 1640/DMEM containing 10% exosome-depleted FBS. Exosomal RNA from the CM of four GCCs and GES-1 were purified, five independent samples were collected and circRNA microarray assay was conducted to identify GC-specific exosomal circRNAs.

first: Peiming

last: Zheng

first: Huijie

last: Gao


title: Oncofusion driven de novo enhancer assembly promotes malignancy in Ewing sarcoma via aberrant expression of the stereociliary protein LOXHD1 [enhancer knockdown]

status: Public on Jun 04 2022

city: PHILADELPHIA

first: Irfan

last: Asangani

state: PA

address: 421 Curie Bld.

department: Cancer Biology

country: USA

institute: University of Pennsylvania

type: Expression profiling by high throughput sequencing

summary: Ewing Sarcoma (EwS) is a highly aggressive tumor of bone and soft tissues that mostly affects children and adolescents. The pathognomonic oncofusion EWSR1-ETS (EWS-FLI1/EWSR1-ERG) transcription factors drive EwS by orchestrating an oncogenic transcription program through de novo enhancers. Pharmacological targeting of these oncofusions has been challenged by unstructured prion-like domains and common DNA binding motifs in the EWSR1 and ETS protein, respectively. Alternatively, identification and characterization of mediators and downstream targets of EWS-FLI1 dependent or independent function could offer novel therapeutic options. By integrative analysis of thousands of transcriptome datasets representing pan-cancer cell lines, primary cancer, metastasis, and normal tissues, we have identified a 32 gene signature (ESS32) that could stratify EwS from pan-cancer. Of the ESS32 (Ewing Sarcoma Specific 32), LOXHD1 - coding for a stereociliary protein was the most exquisite gene expressed in EwS. CRISPR-Cas9 mediated deletion or silencing of EWS-FLI1 bound upstream de novo enhancer elements in EwS cells led to the loss of LOXHD1 expression and altered the EWS-FLI1, MYC, and HIF1 pathway genes, resulting in decreased proliferation and invasion in vitro and in vivo. These observations open up new avenues for developing LOXHD1-targeted drugs or cellular therapies for this deadly disease.

overall_design: Gene expression analysis in  control and LOXHD1 enhancer knockdown (eKD2) SK-N-MC  and RD-ES cell lines.

middle: A

first: Qu

last: Deng

first: Ram

last: Natesan


title: Single Cell Analysis of CD8+ T-cells with Aging

status: Public on Jun 28 2022

city: Baltimore

first: Nan-Ping

last: Weng

address: 251 Bayview Blvd

department: LMBI

institute: NIA

summary: CD8+ T cells play essential roles in combatting infection and cancers. The major subsets of CD8+ T cells such as naïve, effector, and memory carry out distinct functions. Although the defined subsets are known to be heterogeneous, the precise composition of these subsets and their functional alterations have not been thoroughly determined. Here, we used single cell RNA sequencing (scRNAseq) and multicolor immunophenotyping to analyze the composition of CD8+ T cells. From scRNAseq analysis of isolated CD8+ T cells from 16 healthy donors, we analyzed 80,775 cells and identified a total of 13 subpopulations of CD8+ T cells including naïve, central and effector memory cells and terminally differentiated effector memory cells (EMRA). In parallel, our multi-color (16 antibody-panel) immunophenotyping analysis by flow cytometry of CD8+ T cells from 165 donors also divided CD8+ T cells into 11 major subpopulations. The majority of the subpopulations identified by these two methods were their age-related changes in percentage. Together, our findings reveal previously unidentified subpopulations of CD8+ T cells, their distinct transcriptional profiles and changes with age.

overall_design: single cell gene expression of CD8+ T-cells from 16 individuals spanning from the newborn to the elderly

first: Jian

last: Lu

first: Raheel

last: Ahmad

first: Thomas

last: Nguyen


title: Epigenetic Divergence during Early Stages of Speciation in an African Crater Lake Cichlid Fish

status: Public on Jun 09 2022

city: Basel

first: Grégoire

middle: M

last: Vernaz

address: Vesalgasse 1

country: Switzerland

institute: University of Basel

type: Methylation profiling by high throughput sequencing

summary: Inheritance and plasticity of epigenetic divergence characterise early stages of speciation in an incipient cichlid species of an African crater lake.

overall_design:
Wild-caught (wild) or common garden (tank-reared) adult male specimens for all datasets.
RNAseq: 4-5 biological replicates per population; WGBS: 2-3 biological replicates per population: RRBS: 11-12 biological replicates per population.
--
Processed files: gene/transcript count matrix (in tpm; RNAseq) and bigwig files (averaged mCG/CG levels per groups across 50bp-long non-sliding windows genome-wide).

first: Eric

last: Miska

first: Martin

middle: J

last: Genner


title: Profiling mRNA, miRNA and lncRNA expression changes in endothelial cells in response to increasing doses of ionizing radiation [RNA-seq]

status: Public on May 07 2022

city: Bethesda

first: Uma

middle: T

last: Shankavaram

state: MD

address: 9000 Rockville Pike

department: NCI

institute: NIH

summary: RNA seq for lncRNA and mRNA differential expression analysis, and small RNA seq for miRNA differential expression analysis were performed in primary human aotic endothelial cells treated with increasing doses of radiation (1 Gy, 2 Gy, 4 Gy, 8 Gy and 10 Gy). Total RNA was collected 24 h and 72 h after radiation. Comparisons were made with unirradiated cells plated for 24 h and 72 h repsectively.

first: Sunita

last: Chopra

first: Michelle

last: Bylicky

first: Juan

last: Dalo

first: Kevin

last: Scott

first: Molykutty

last: Aryankalayil

first: C

middle: N

last: Coleman


title: Profiling mRNA, miRNA and lncRNA expression changes in endothelial cells in response to increasing doses of ionizing radiation [miRNA-seq]

type: Non-coding RNA profiling by high throughput sequencing

summary: small RNA seq for miRNA differential expression analysis were performed in primary human aotic endothelial cells treated with increasing doses of radiation (1 Gy, 2 Gy, 4 Gy, 8 Gy and 10 Gy). Total RNA was collected 24 h and 72 h after radiation. Comparisons were made with unirradiated cells plated for 24 h and 72 h repsectively.


title: CD58 loss in tumor cells confers functional impairment of CAR T cells

status: Public on May 03 2022

city: beijing

first: Xin

last: Yan

address: 28 Fuxing Road

country: China

institute: Chinese PLA General Hospital

summary: Chimeric antigen receptor (CAR) T-cell therapy has achieved significant success in treating a variety of hematologic malignancies, but resistance to this treatment in some patients limited its wider application. Using an unbiased genome-wide CRISPR/Cas9 screening, we identified and validated loss of CD58 conferred immune evasion from CAR T cells in vitro and in vivo. CD58 is a ligand of the T cell costimulatory molecule CD2, and CD58 mutation or downregulated expression is common in hematological tumors. We found that disruption of CD58 in tumor cells induced the formation of suboptimal immunological synapse (IS) with CAR T cells, which conferred functional impairment of CAR T cells, including the attenuation of cell expansion, degranulation, cytokine secretion and cytotoxicity. In summary, we describe a potential mechanism of tumor intrinsic resistance to CAR T-cell therapy and suggest that this mechanism may be leveraged for developing therapeutic strategies to overcome resistance to CAR T-cell therapy in B-cell malignancies.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for CAR T cells co-cultured with WT or CD58KO Nalm6 cells

first: Deyun

last: Chen

first: Zhiqiang

last: Wu

first: Weidong

last: Han


title: Profiling mRNA, miRNA and lncRNA expression changes in endothelial cells in response to increasing doses of ionizing radiation.

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

overall_design: Refer to individual Series


title: YAP promotes cell-autonomous immune responses in vitro to tackle intra-cellular Staphylococcus aureus

city: Saint-Priest-en-Jarez

first: Robin

last: Caire

address: 10 rue de la Marandière

department: Biology

country: France

institute: Université Jean Monnnet Saint-Etienne

summary: Transcriptional cofactors YAP/TAZ have recently been found to support autophagy and inflammation, which are part of cell autonomous immunity and are critical in antibacterial defense. Here, we studied the role of YAP against Staphylococcus aureus using CRISPR/Cas9-mutated HEK293 cells and a primary cell-based organoid model. We found that S. aureus infection increases YAP transcriptional activity, which is required to reduce intracellular S. aureus replication. A 770-gene targeted transcriptomic analysis revealed that YAP upregulates genes involved in autophagy/lysosome and inflammation pathways in both infected and uninfected conditions. The YAP/TEAD transcriptional activity promotes autophagic flux and lysosomal acidification, which are important for defense against intracellular S. aureus. Furthermore, the staphylococcal toxin C3 exoenzyme EDIN-B was found effective in preventing YAP-mediated cell-autonomous immune response. This study provides new insights on the anti-S. aureus activity of YAP, which could be conserved for defense against other intracellular bacteria.

overall_design: HEK293 cells wild type (WT) or YAP knockout (YAP KO or YAP-/-), control (CTRL) or S. aureus (SA) infected  were used. 12 samples were analyzed corresponding to 4 groups: WT CTRL, YAPKO CTRL, WT SA, YAPKO SA with n=3 samples per group. nCounter Nanostring host response panel was used to analyse the expression of 770 genes. 4 comparisions were made between groups : WT CTRL vs WT SA; WT CTRL vs YAPKO CTRL;  YAPKO CTRL vs YAPKO SA; WT SA vs YAPKO SA

first: Paul

middle: O

last: Verhoeven


title: Gene-targeted, CREB-mediated induction of ∆FosB controls distinct downstream transcriptional patterns within D1 and D2 medium spiny neurons

status: Public on Jun 03 2022

city: New York

first: Casey

middle: K

last: Lardner

state: NY

address: 1425 Madison Avenue

department: Nash Family Department of Neuroscience

institute: Icahn School of Medicine at Mount Sinai

summary: ∆FosB, a transcription factor that accumulates in nucleus accumbens (NAc) after exposure to virtually every known rewarding substance, has been shown to directly control gene transcription and behavior downstream of both cocaine and opioid exposure but with potentially different roles in D1 and D2 medium spiny neurons (MSNs). To clarify MSN subtype-specific roles for ∆FosB, and investigate how these subtypes coordinate the actions of distinct classes of addictive drugs, we developed a CRISPR/Cas9-based epigenome editing tool to induce endogenous ∆FosB expression in vivo in the absence of drug exposure and performed RNA-sequencing on bulk male and female NAc tissue.

overall_design: Transcriptional patterns in male and female mouse nucleus accumbens after activating the transcription factor ∆FosB in D1, D2, or D1+D2 medium spiny neurons

first: Yentl

last: van der Zee

first: Molly

middle: S

last: Estill

first: Hope

middle: G

last: Kronman

first: Marine

last: Salery

first: Ashley

last: Cunningham

first: Arthur

last: Godino

last: Parise

first: Jee

middle: H

last: Kim

first: Rachael

middle: L

last: Neve

first: Li

last: Shen

first: Peter

last: Hamilton

last: Nestler


title: Integrative genomics reveals pathogenic mediator of valproate-induced neurodevelopmental outcomes.

status: Public on May 08 2022

city: London

first: Rahel

last: Feleke

address: Ducane Road

country: United Kingdom

institute: Imperial College

summary: Prenatal exposure to the anti-seizure drug sodium valproate (VPA) is associated with an increased risk of adverse postnatal neurodevelopmental outcomes, including lowered intellectual ability, autism spectrum disorder and attention-deficit hyperactivity disorder.  In this study, we aimed to clarify the molecular mechanisms underpinning the neurodevelopmental consequences of gestational VPA exposure using integrative genomics.  First, we assessed the effect of gestational VPA on fetal brain gene expression using a validated rat model of valproate teratogenicity that mimics the human scenario of chronic oral valproate treatment during pregnancy at doses which are therapeutically relevant to the treatment of epilepsy. Two different rat strains were studied - inbred Genetic Absence Epilepsy Rats from Strasbourg (GAERS), a model of genetic generalized epilepsy, and inbred Non-Epileptic Control (NEC) rats.  Female rats were fed standard chow or VPA mixed in standard chow for 2 weeks prior to conception and then mated with same-strain males. In the VPA-exposed rats maternal oral treatment was continued throughout pregnancy. Fetuses were extracted via C-section on gestational day 21 (one day prior to birth) and fetal brains were snap frozen and genome-wide gene expression data generated. We found that gestational VPA exposure via chronic maternal oral dosing was associated with substantial drug-induced differential gene expression in the pup brains, including dysregulated splicing, and observed that this occurred in the absence of evidence for significant neuronal gain or loss.  The set of genes down-regulated by VPA in the pup brains were significantly enriched for pathways related to neurodevelopment and synaptic function, and significantly enriched for heritability to human intelligence, schizophrenia and bipolar disorder. Our results provide a mechanistic link between chronic fetal VPA exposure and adverse neurodevelopmental outcomes mediated by VPA-induced transcriptional dysregulation.

overall_design: Bulk RNA sequencing of brain tissue samples in 30 rat pups from two different strains, inbred Non-Epileptic Controls (NEC) and Genetic Absence Epilepsy Rats from Strasbourg (GAERS). Fourteen  samples are VPA-treated and sixteen samples are controls.


title: RNA sequencing of E15.5 retinal tissue from control and Lhx2 CKO mice

status: Public on Apr 29 2022

city: Nashville

first: EDWARD

last: LEVINE

state: TN

address: 1161 21st Ave S

department: Vanderbilt Eye Institute

institute: Vanderbilt University Medical Center

summary: Lhx2 is a retinal progenitor cell transcription factor critical for eye development. We previously reported that conditional inactivation of Lhx2 at the start of mouse retinal neurogenesis disrupted retinal progenitor cell (RPC) proliferation, greatly reduced the RPC pool and altered neurogenic output as indicated by changes in the production of multiple fated precursor populations. To identify genes whose expression levels are dependent on Lhx2 at this stage of development, Lhx2 conditional inactivation was initiated at E11.5 in RPCs with the progenitor Cre driver Hes1CreERT2 and retinal tissue was collected at E15.5 for RNA sequencing. The gene expression profiles of Lhx2 CKO retinas were compared to control (Lhx2 conditional heterozygotes) were compared. Downregulated and upregulated gene expression was observed, with some likely due to direct and indirect regulation by Lhx2 within RPCs and others due to changes in differentiation and the altered neurogenic output.

overall_design: Lhx2 was inactivated by inducible Cre recombination using CreERT2 knocked into the Hes1 locus. Mice were heterozygous for Hes1CreERT2. Control mice were Lhx2flox/+ and experimental (CKO)mice were Lhx2flox/KO. Tamoxifen was gavage fed at E11.5 and embryos were harvested at E15.5. Each biological replicate contained both retinas from an individual embryo with 4 biological replicates for each condition (genotype).

first: Edward

last: Levine

first: Patrick

last: Gordon


title: Target genes of thyroid hormone (TH) in brown adipose tissue (BAT) and their role during metabolic stressors [ChIP-seq]

city: Lyon

first: Yanis

last: ZEKRI

address: 32-34 Avenue Tony Garnier

institute: IGFL-ENS

type: Genome binding/occupancy profiling by high throughput sequencing

summary: To understand the role of TH target genes in BAT, we developped a murine transgenic model which expressed a tagged-form of the thyroid hormone receptor alpha (TRa1, tagged with GS which is a fusion between protein G and streptavidin) specifically in brown adipocytes. Combined with RNAseq data, it allowed to establish the direct target genes of thyroid hormones (TH) in brown adipocytes

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for TRa in brown adipocytes of murine mice

last: Zekri

first: Romain

last: Guyot

first: Frédéric

last: Flamant


title: Target genes of thyroid hormone (TH) in brown adipose tissue (BAT) and their role during metabolic stressors [RNA-seq]

summary: To understand the role of TH target genes in BAT, we developped a murine transgenic model in which TH signaling is selectively lost in BAT (BATKO mice). After establishing TH target genes in BAT, we used BATKO mice to understand which pathways were controlled by TH in BAT during metabolic stressors, i.e., cold and high-fat diet (HFD)

overall_design: We performed high-throughput gene expression profiling of BAT in BATKO mice and their control (CTRL) after different stimuli


title: Transcriptome analysis for CT mRNA treated MDA-MB-231 cells and negative control cells

status: Public on Apr 28 2022

city: Seoul

first: Hyeon-Cheol

last: Lee

address: 136, Changgyeonggung-ro,Jongro-gu

department: Research center

country: South Korea

institute: BPgene Co., Ltd.

summary: We report the altered metabolite changes by processing CT mRNA in the TNBC cell line, MDA-MB-231. It was confirmed that the treatment of CT mRNA disrupted the synthesis of dTTP during nucleic acid metabolism, increasing the signal related to DNA damage in the cell, and that the energy metabolism and NADPH metabolism in the cell were changed for its recovery. The main energy sources, glucose metabolism and glutamine metabolism, are closely linked to the mitochondrial TCA cycle products. In the CT mRNA treatment group, intracellular nucleic acid depletion was observed to increase glutamine metabolism as a source of N source, and the pathway of aspartate utilization was observed. It was confirmed that it was expanded. As a result, a significant increase in the cysteine influx was observed according to the occurrence of ROS, and an effort to regulate the intracellular NADPH balance was observed through the change of metabolites over time. Finally, it was observed that the oncogenic pathway was significantly reduced in the CT mRNA-treated group compared with all untreated groups or controls with other substrate specificities. This study characterizes changes in intracellular metabolites caused by specific dTTP biosynthesis disturbances.

overall_design: Examination of T001 mRNA, 2 different nucleotidase and untreated cells

first: Hyeon Cheol

first: Bong Seong

last: Koo

first: Jin Sook

first: Dae Ho


title: Real-time quantitative PCR analysis of gene expression in mouse macrophages treated with cottonseed-derived gossypol

status: Public on May 15 2022

city: New Orleans

first: Heping

last: Cao

state: LA

address: 1100 Robert E. Lee Blvd

department: Southern Regional Research Center

institute: USDA-ARS

summary: Relative mRNA levels of 27 genes were evaluated with the SYBR Green qPCR assays after gossypol (Gos) treatment for 2-24 h.

overall_design: Mouse macrophages (RAW264.7 cells, TIB-71) was established from a tumor in a male mouse induced with the Abelson murine leukemia virus (ATCC). The  cells in triplicate were treated with various concentrations of gossypol for 2, 8 and 24 h. RNA isolation and cDNA synthesis were performed as described previously. Relative mRNA levels were evaluated with the SYBR Green qPCR assays using Bio-Rad reagents. Rpl32 mRNA was selected as the internal reference for qPCR analysis for this cell type. 1% DMSO treatment was the sample control. The 2-ΔCT and 2-ΔΔCT method of relative quantification was used to determine the fold change in expression.


title: A cryptic transcription factor regulates Caulobacter crescentus adhesion

status: Public on Apr 27 2022

city: East Lansing

first: Sean

last: Crosson

state: Michigan

address: 567 Wilson Rd

department: Dept. Microbiology and Molecular Genetics

institute: Michigan State University


title: Comparative RNA-sequencing of Brachypodium distachyon wild-type and bdmute developing leaf zones

status: Public on Apr 25 2022

city: Bern

first: Michael

last: Raissig

address: Altenbergrain 12

department: Institute of Plant Sciences

institute: University of Bern

summary: Comparative RNA-sequencing of the developmental leaf zones in Brachypodium distachyon wild type and bdmute mutants that do not form stomatal subsidiary cells was performed. The aim was to identify genes relevant for subsidiary cell formation in B. distachyon.

overall_design: 25 developmental zones (3mm at base of 2nd leaves) were analyzed per replicate and genotype. 3 replicates per genotype were sequenced, WT1, WT2, WT5 and sid1, sid2, sid5. WT corresponds to the reference genotype and sid (=bdmute) corresponds to the mutant genotype.

first: Dominique

middle: C

last: Bergmann


title: LncReader: identification of dual functional long non-coding RNAs using multi-head self-attention mechanism

status: Public on Apr 26 2022

city: Guangzhou

first: Tianyuan

last: Liu

address: Shatai South Road, Baiyun District

department: Bioinformatics Department

institute: Sourthern Medical University


title: Target genes of thyroid hormone (TH) in brown adipose tissue (BAT) and their role during metabolic stressors


title: LncReader: identification of dual functional long non-coding RNAs using multi-head self-attention mechanism [Ribo-seq]

summary: The dual functional lncRNAs have been intensively studied and identified to be involved in various fundamental cellular processes recently. It is essential to understand in which context when a dual functional lncRNA serves as a non-coding RNA or a template for coding peptide, particularly in some pathological conditions. However, apart from time consuming and cell type specific experiments, there is virtually no in-silico method for predicting the identity of dual functional lncRNAs. Here, we developed a deep-learning model with multi-head self-attention mechanism, LncReader, to identify dual functional lncRNAs based on their sequence, physicochemical and secondary structural features. Our data demonstrated that LncReader showed multiple advantage compared to various classical machine learning methods. Moreover, to obtain independent in-house datasets for robust testing, mass spectrometry proteomics combined with RNA-seq were applied in four leukemia cell lines. Remarkably, LncReader achieved the best performance among all these datasets. Therefore, LncReader provides a sophisticated and practical tool that enables fast dual functional lncRNAs identification.

overall_design: Ribosome-seq of four leukemia cell lines (HL60, MOLM13, K562, THP_1), one replicate of each cell line.

first: Dong

last: Wang

first: Bohao

last: Zou


title: Inactivation of the histone H3 K36 methyltransferase NSD1 confers resistance to EZH2 inhibition [ChIP-Seq]

status: Public on May 09 2022

city: Memphis

first: Jacquelyn

last: Myers

address: 262 Danny Thomas Pl

institute: St. Jude Children’s Research Hospital

summary: Disruption of antagonism between SWI/SNF chromatin remodelers and Polycomb repressor complexes drives the formation of numerous cancer types. Recently, an inhibitor of Polycomb protein EZH2 was approved for treatment of sarcomas mutant in SWI/SNF subunit SMARCB1, but resistance occurs.  Here we sought to identify additional contributors to SWI/SNF-Polycomb antagonism and potential resistance mechanisms. We performed CRISPR screens and found that NSD1 loss caused resistance to EZH2 inhibition. NSD1 loss reduced H3K36me2 and impaired transcriptional activation of targets inhibited by EZH2 or activated by SMARCB1. Further, inhibiting the H3K36me2 demethylase KDM2A restored EZH2 inhibition in cells lacking NSD1. Our results expand the mechanistic understanding of SWI/SNF and Polycomb interplay and identify NSD1 as key for coordinating this transcriptional control.

overall_design:
G401 cells were engineered to express either GFP or SMARCB1 (B1) under doxycycline treatment and further transfected with either control (sgC) or NSD1 (sgN) targeting sgRNAs. Cells were then treated with 1ug/mL dox for 24h. For EZH2 inhibition studies, G401 cells stably expressing shCTRL or shNSD1 RNAs were treated for 72h with 1uM GSK126.
In brief, cells were cross-linked directly on the plate using 1% paraformaldehyde for 10 min at room temperature and quenched with glycine. Nuclei were isolated using the Covaris TruCHIP kit and Chromatin extracts were sonicated for 8 min using a Covaris E220 focused ultrasonicator at a peak power of 140, a duty factor of 5 and 200 cycles per burst. Chromatin immunoprecipitation was carried overnight with antibodies bound to dynabeads. Chromatin was de-crosslinked, digested with RNase and Proteinase K and DNA was ourified using a Zymo ChIP DNA kit.

first: Yiannis

last: Drosos

first: Charles

middle: W

last: Roberts


title: Quiescent cancer cells resist T cell attack by forming an immunosuppressive niche (GFP:mCherry 1:1 4T07 mix tumors after Jedi killing Bulk RNA)

status: Public on Apr 20 2022

city: Boston

first: Judith

last: Agudo

state: MA

address: 1 Jimmy fund way

institute: Dana Farber Cancer Institute

summary: Immunotherapy is a promising treatment for Triple-Negative Breast Cancer (TNBC), but patients recur, arising the need to understand mechanisms of resistance. We discovered that in primary breast cancer, tumor cells that survive T-cell attack are in a quiescent state. Quiescent Cancer Cells (QCCs) are found clustering together forming regions with reduced immune infiltration. QCCs display superior tumorigenic capacity and higher expression of stemness genes than their proliferative counterparts. We adapted single-cell-RNA-sequencing with precise spatial resolution to profile infiltrating cells inside and outside QCC niches. This transcriptomic analysis revealed hypoxia-induced programs and identified more exhausted T-cells, tumor-protective fibroblasts, and dysfunctional dendritic cells inside clusters of QCCs. This uncovered differential phenotypes in infiltrating cells based on their specific intra-tumor location. Thus, QCCs constitute immunotherapy-resistant reservoirs by orchestrating a local hypoxic immune-suppressive milieu that blocks T-cell function. Eliminating QCCs holds the promise to counteract resistance to immunotherapy and prevent disease recurrence in TNBC.

overall_design: a 1:1 mix of GFP:mCherry trasduced 4T07 cells were injected into the mammary fat pad of Balb/cJ x Bl6 females. Tumors were grown and Jedi T cells were injected to target the GFP population. mCherry cells were used as non-targeted paired controls for the GFP surviving cells.

first: Pilar

last: Baldominos


title: Single-cell transcriptomics of Caco-2 cells cultured in a human gut-on-a-chip under a mechanodynamic culture condition

status: Public on Apr 01 2022

city: Austin

first: Song

last: Yi

state: TX

address: 1601 Trinity Street, Building B

institute: The University of Texas at Austin

summary: The microphysiological human gut-on-a-chip has demonstrated in vivo-relevant cellular fidelity of intestinal epithelium compared to its cultures in a static condition1, 2. Microfluidic control of morphogen gradients and mechanical cues robustly induced morphological histogenesis with villi-like three-dimensional (3D) microarchitecture, lineage-associated cytodifferentiation, and physiological functions of a human intestinal Caco-2 epithelium3, 4. However, transcriptomic dynamics that orchestrates morphological and functional reprogramming of the epithelium in a microphysiological culture remains elusive. Single-cell transcriptomic analysis revealed that a gut-on-a-chip culture that offers physiological motions and flow drives three distinctive subclusters that offer distinct gene expression and unique spatial representation in 3D epithelial layers. The pseudotemporal trajectory of individual cells visualized the evolutionary transition from ancestral genotypes in static cultures into more heterogeneous phenotypes in physiodynamic cultures on cell cycles, differentiation, and intestinal functions including digestion, absorption, drug transport, and metabolism of xenobiotics. Furthermore, the inversed transcriptomic signature of oncogenes and tumor-suppressor genes of Caco-2 cells verified that a gut-on-a-chip culture drives a postmitotic reprogramming of cancer-associated phenotypes. Thus, we discovered that a physiodynamic on-chip culture is necessary and sufficient for a cancer cell line to be reprogrammed to elicit in vivo-relevant heterogeneous cell populations with restored normal physiological signatures.

overall_design: Caco-2 cells cultured in a gut-on-a-chip were harvested and run for single-cell RNA sequencing. Cells cultured in Transwell inserts were used as a control.

first: Hyun

last: Kim

first: S

first: Woojung

last: Shin

first: Zhe

last: Su


title: Quiescent cancer cells resist T cell attack by forming an immunosuppressive niche


title: Quiescent cancer cells resist T cell attack by forming an immunosuppressive niche (PADMEseq)

overall_design: tdTomato-p27K-expressing 4T07 cells were injected into the mammary fat pad of Kaede x Balb/cJ F1 females. Tumors were embedded in 2% low melting agarose and cut into 300μm slices by the Compresstome VF 310-0Z vibrating microtome (Precisionary Instruments). . Consecutive slices were used to photoconvert cells infiltrating tdTomato-negative areas and then cells in regions with tdTomato-p27KHigh cells.

first: Alex

last: Barbera-Mourelle

first: Olga

last: Barreiro

first: Yu

last: Huang


title: Single cell profiling of primary and paired metastatic lymph node tumors in breast cancer patients(10x RNA and TCR)

status: Public on Apr 30 2022

city: Beijing

first: Zhihua

state: Beijing

address: State Key Laboratory of Molecular Oncology, National Cancer Center, National Clinical Research Center for Cancer, Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China

summary: The microenvironment of lymph node metastasized tumors (LNMT) determines tumor progression and response to therapy, but a systematic study of LNMT is lacking. Here, we generate single-cell maps of primary tumors (PTs) and paired LNMTs in 8 breast cancer patients. We demonstrate that the activation, cytotoxicity, and proliferation of T cells are suppressed in LNMT compared with PT. CD4+CXCL13+ T cells in LNMT are more likely to differentiate into an exhausted state. Interestingly, LAMP3+ dendritic cells in LNMT display lower T cell priming and activating ability than in PT. Additionally, we identify a subtype of PLA2G2A+ cancer-associated fibroblasts enriched in HER2+ breast cancer patients that promotes immune infiltration. We also show that the antigen-presentation pathway is downregulated in malignant cells of the metastatic lymph node. Altogether, we characterize the microenvironment of LNMT and PT, which may shed light on the individualized therapeutic strategies for breast cancer patients with lymph node metastasis.

overall_design: Single cell RNA and TCR sequencing of primary tumor and paired lymph node metastasis in breast cancer

first: Tong

first: Cheng

first: Jing

last: Zhang

first: Xiaofan

last: Wei

first: Hongquan


title: Comparative gene and lncRNA expression profiling in patients with severe community acquired pneumonia and healthy controls.

status: Public on Apr 18 2022

first: Yiming

address: No.9 Dongdan Santiao

institute: Chinese Academy of Medical Sciences & Peking Union Medical College

summary: Investigate the transcriptomic differences between patient with severe community acquired pneumonia (SCAP) and healthy control.

overall_design: Whole blood samples were from patients, diagnosed with SCAP according to the IDSA/ATS2007 guidelines and healthy people. Total RNA was extracted from white blood cells that are isolated from the whole blood. Then, rRNA was removed from total RNA to generate strand-specific libraries. The library were sequenced on a Novaseq 6000 platform and 150 bp strand-specific paired-end reads were generated.

first: Yan

last: Li

last: Sun

first: Jun

last: Xu

first: Chengyu

last: Jiang


title: TTFields treatment effects on newly diagnosed Glioblastoma patients in peripheral T lymphocytes

city: SOUTH PASADENA

first: SON

middle: B

last: LE

state: CA

address: 1645 Amberwood Drive, Unit 11

institute: University of Southern California

summary: We report the application of RNA-based sequencing technology for high-throughput profiling of T cell enriched peripheral blood mononuclear cells. By sequencing in total of 12 pairs GBM patient samples, we extracted TCRαβ V(D)J sequences from the deep RNA-seq of T cells isolated from the 24 PBMCs to determine if TTFields treatment affected TCR diversity, using the Simpson’s diversity index (DI), which is the average proportional abundance of TCR clones based on the weighted arithmetic mean. Of the 12 patients, 9 exhibited negative log fold change (logFC) of TCR DI after TTFields, indicating clonal expansion. Notably, in all but 1 patient, the top 200 most abundant clones post TTFields, which accounted for 38.1% to 100% (median 67%) of detectable clones, showed substantial expansion compared to pre-TTFields T cells, and inversely correlated with the DI. Thus, TTFields treatment is associated with adaptive immune activation as evidenced by clonal expansion of peripheral T cells.

overall_design: Examination of TTFields treatment effects in T cell enriched peripheral blood mononuclear cells.

first: David

middle: D

last: Tran

first: Son

first: Dongjiang

last: Chen


title: The mitochondrial pyruvate carrier regulates antitumor function and memory T cell differentiation

city: Lausanne

first: Tania

last: Wyss

address: Rue du Bugnon 25A

institute: SIB Swiss Institute of Bioinformatics

summary: Effector CD8+ T cells engage glycolysis for both lactate fermentation and pyruvate oxidation, the latter requiring the mitochondrial pyruvate carrier (MPC). How mitochondrial pyruvate metabolism impacts T cell function and fate, remains incompletely understood. We found that genetic deletion of MPC drives CD8+ T cell differentiation towards a memory phenotype. Metabolic flexibility induced by MPC inhibition in a nutrient-rich environment allowed for increased acetyl-coenzyme-A production by glutamine and fatty acid oxidation. This correlated with histone acetylation and chromatin accessibility on pro-memory genes. However, in a nutrient-deprived tumor microenvironment, MPC is essential for sustaining lactate oxidation that limits the metabolic suppression of CD8+ T cell anti-tumor function. To optimally translate these findings, we used a small molecule MPC inhibitor to imprint a stable memory phenotype during chimeric antigen receptor (CAR) T manufacturing, and demonstrated that infusing metabolically conditioned MPC WT CAR T cells resulted in superior and long-lasting anti-tumor activity.

overall_design: We performed a chromatin accessibility analysis by ATAC sequencing on CD8+ T cells that were activated for 3 days in the presence of DMSO or 75 micromolar UK5099 (a mitochondrial pyruvate carrier (MPC) inhibitor) and then cultured for an additional 4 days without MPC inhibition to identify the genomic regions that acquired a stable open conformation, at least over the course of several cell divisions, upon metabolic intervention.

first: Mathias

last: Wenes

first: Pedro

last: Romero


title: Spatial and Temporal Variation in the Skin Transcriptome of Atopic Dermatitis Assessed by 1.5 mm Mini Punch Biopsies

status: Public on Apr 13 2022

city: Copenhagen

first: Tu

last: Hu

address: 5 Artillerivej

department: Infectious Disease Immunology

country: Denmark

institute: Statens Serum Institut

summary: To obtain a detailed molecular understanding of both the temporal and spatial variation in atopic dermatitis (AD), we conducted a longitudinal case-control study, in which we followed a population, the Gentofte AD (GENAD) cohort, of mild-to-moderate AD patients and matched healthy controls for more than a year. By the use of 1.5 mm punch biopsies, we obtained 393 samples from lesional, non-lesional and healthy skin, from multiple anatomic regions at different time points for transcriptomic profiling. After quality control, 339 samples remained for further analysis. We envisage that the application of small biopsies, such as those introduced in this study, combined with omics technologies, will enable future skin research, in which multiple sampling from the same individual will give a more detailed, dynamic picture of how a disease fluctuates in time and space.

overall_design: 30 AD patients and 30 healthy, age- and gender-matched healthy controls (HC) were enrolled in the study. For AD patients, biopsy specimens (1.5 mm) were taken at two lesional (LS) and two non-lesional (NL) skin sites. For HC, two skin biopsies were taken, preferably from the upper inner arm. A subpopulation of patients with AD and HC continued in a longitudinal study, where skin biopsies were taken at 3, 6, 9 and 12 months after the baseline visit. In total, and after quality control, 339 samples were included for transcriptomic profiling.

first: Thomas

last: Litman

first: Lone

last: Skov


title: Hijacking of transcriptional condensates by endogenous retroviruses

status: Public on Mar 30 2022

city: Berlin

first: Sara

last: Hetzel

address: Ihnestraße 63

department: Genome Regulation

country: Germany

institute: Max Planck Institute for Molecular Genetics

summary: Most endogenous retroviruses (ERVs) in mammals are incapable of retrotransposition; therefore, why ERV de-repression is associated with lethality during early development has been a mystery. Here we report that rapid and selective degradation of the TRIM28 heterochromatin adapter protein triggers dissociation of transcriptional condensates from loci encoding super-enhancer -driven pluripotency genes, and their association with transcribed ERV loci in murine embryonic stem cells. Knockdown of ERV RNAs or forced expression of super-enhancer -enriched transcription factors rescued condensate localization at super-enhancers in TRIM28-degraded cells. In a biochemical reconstitution system, ERV RNA facilitated partitioning of RNA Polymerase II, and the Mediator co- activator into phase-separated droplets. In TRIM28 knockout mouse embryos, single-cell RNA-Seq analysis revealed specific depletion of pluripotent lineages. We propose that coding and non-coding nascent RNAs, including those produced by retrotransposons, may facilitate “hijacking” of transcriptional condensates in various developmental and disease contexts.

overall_design: Profiling of RNAseq, TT-SLAM-Seq (nascent transcription) and ChIPseq in Trim28-degraded mESCs in order to study the immediate effects of Trim28 loss. In vivo effects of Trim28 knockout analyzed by scRNAseq of wild-type and Trim28 knockout embryos.

first: Vahid

last: Asimi

first: Abhishek

last: Sampath Kumar

first: Henri

last: Niskanen

first: Christina

last: Riemenschneider

first: Julian

last: Naderi

first: Nina

last: Fasching

first: Niko

last: Popitsch

first: Manyu

last: Du

first: Helene

last: Kretzmer

first: Zachary

last: Smith

first: Raha

last: Weigert

first: Maria

last: Walther

first: Sainath

last: Mamde

last: Meierhofer

first: Lars

last: Wittler

first: Rene

last: Buschow

first: Bernd

last: Timmermann

first: Ibrahim

middle: I

last: Cisse

first: Stefan

last: Ameres

first: Alexander

last: Meissner

first: Denes

last: Hinsz


title: Postoperative pain and dorsal root ganglia transcriptomic signatures in a novel rat model of total knee arthroplasty

status: Public on Mar 23 2022

city: Stony Brook

first: Martin

last: Kaczocha

address: Health Sciences Center, L4-077

institute: Stony Brook University

summary: These data are from a preclinical model of total knee arthroplasty (TKA) performed in healthy rats. One day after surgery, ipsilateral dorsal root ganglia (DRG) from L3 and L4 were collected for RNA extraction and sequencing.

overall_design: Three groups of rats were analyzed: naïve (no procedures), sham (anesthesia, skin incision, sutures), and TKA (anesthesia and complete TKA surgery). Each group consisted of 3 male and 3 female Sprague Dawley rats that weight ~300g. Sham and TKA surgeries were performed on their left knees. Twenty-four hours after surgery, DRGs were collected from the left side at levels L3 and L4. RNA was then extracted and sequenced.

first: David

middle: E

last: Komatsu

first: Sardar

last: Uddin

first: Chris

last: Gordon

first: Martha

middle: P

last: Kanjiya

first: Diane

last: Bogdan

first: Justice

last: Achonu

first: Hira

last: Iftikhar

first: Amanda

last: Ackermann

first: Rohan

last: Shah

first: Jason

last: Shieh

first: Agnieszka

last: Bialkowskac


title: RNA-seq in H1299 cell lines.

status: Public on Mar 09 2022

city: Zhengzhou

first: zhuoyu

last: gu

state: China

address: No. 50, Jianshe East Road, Erqi District

institute: First Affiliated Hospital of Zhengzhou University

summary:
Purpose: The goals of this study are to invsetigate the primary biological process of HNRNPK.
Methods: Briefly, samples (H1299 cells transfected with control or HNRNPK shRNA) were used to extract total RNAs for quality control and RNA-seq loading samples. RNA-seq was performed as DGE on an Illumia HiSeq platform and 50 bp paired-end reads were generated (BGI Co. Ltd.).
Results: Gene Ontology (GO) analysis revealed that cell proliferation and migration were identified as the biological functions of HNRNPK
Conclusions: RNA-seq in H1299 cell lines revealed that HNRNPK mainly regulated cell proliferation and migration.

overall_design: mRNA profiles of H1299  HNRNPK shNC cells and H1299 HNRNPK knockdown cells

first: Zhuoyu

last: Gu


title: Specification and lineage diversification of theca cells in human ovarian follicles is governed by a TCF21/NR5A1 axis

status: Public on Mar 31 2022

city: New York

first: Daylon

last: James

address: 515 East 71st

institute: Weill Cornell Medicine

summary: We isolated cells from human antral follicles of various sizes and sources and subjected them to 10x Chromium single cell sequencing

overall_design: Sequencing libraries prepared from cell populations that were prospectively isolated from antral follicles of patient origin


title: Quiescent cancer cells resist T cell attack by forming an immunosuppressive niche (QCC  vs proliferative cells from tumors Bulk RNA)

first: Judith

last: Agudo

overall_design: mCherry and mVenus-p27k trasduced 4T07, EMt6 and D2A1 cells were injected into the mammary fat pad of Balb/cJ females. Tumors were grown and QCC and proliferative populations from the same tumor were sorted based on the mVenus p27k reporter.

first: Pilar

last: Baldominos


title: Whole Transcriptome analysis of Aurintricarboxylic acid treated NIH3T3 cells

status: Public on Mar 17 2022

city: Cleveland

first: Brian

last: Rubin

state: OH

address: 9620 Carnegie Ave NE2-215

institute: Cleveland Clinic

summary: Aurintricarboxylic acid (ATA) was identified as a potent inhibitor of the TAZ/TEAD transcriptional complex via a Alpha-LISA screen. We have previously demonstrated that a hyperfunctional form of TAZ, TAZ-CAMTA1, is sufficient to induce the development of a rare vascular sarcoma, Epithelioid Hemangioendothelioma. We have demonstrated that TAZ-CAMTA1 can similarly transform NIH3T3 cells to yield anchorage independent growth in vitro and this action requires the formation of a TAZ-CAMTA1/TEAD complex. To identify if ATA is able to inhibit the transcriptional action TAZ-CAMTA1/TEAD complex, whole transcriptomic analysis was performed on TAZ-CAMTA1 stably transfected NIH3T3 cells with or without ATA.

overall_design: Whole Transcriptome analysis was perfomed on Empty vector treated NIH3T3 cells, TAZ-CAMTA1 treated NIH3T3 cells with or without ATA treatment (48 hour treatment)

first: Caleb

last: Seavey


title: Transcriptome analyses for embryonic mouse urethral bilateral region.

city: Wakayama

first: Gen

last: Yamada

address: 811–1 Kimiidera

department: Institute of Advanced Medicine

country: Japan

institute: Wakayama Medical University

summary: Our study presents the first transcriptomic analyses for the urethral bilateral region. Our data shows the sexually dimorphic genes and gene expression behavior by MafB.

overall_design: The urethral bilateral region of the embryonic external genitalia (eExG) were collected for RNA-seq analysis.

first: Daiki

last: Kajioka

first: Tetsuya

last: Sato

first: Mellissa

last: Alcantara

first: Shoko

last: Matsushita

first: Kentaro

last: Suzuki


title: ChIP-seq DNA libraries were constructed by HNRNPK antibody to immunoprecipitate the fragment DNA in H1299 cells

first: li

last: yixin

summary: The typical peak annotation pie chart showed that more than half of the peaks fall into enhancer regions (intergenic and intronic regions), and 6.71% of the peaks are in promoter regions. Moreover, the typical transcription start site (TSS) enrichment plot showed that the IP fragments are mainly enriched at TSS, compared to the input fragments. Importantly, the ChIP-seq data showed that the recruitment of HNRNPK was mostly enriched at upstream and downstream of TSS of the CLCN3 promoter region.

overall_design: HNRNPK antibody was used to immunoprecipitate the fragment DNA in H1299 cells


title: Expression data of quaternary clones from rat lung CD117+ endothelial cells vs. non-clonal control endothelial cells

status: Public on Mar 10 2022

city: Columbus

first: Laszlo

last: Farkas

address: 473 West 12th Avenue

department: Department of Internal Medicine

institute: The Ohio State University - Wexner Medical Center

summary:
One current concept suggests that unchecked proliferation of clonally selected precursors of endothelial cells contribute to severe pulmonary arterial hypertension (pAH). We hypothesized that clonally selected ECs expressing the progenitor marker CD117 promote severe occlusive pulmonary hypertension (PH).
We used microarrays to identify the steady state gene expression profile of quaternary clones derived from CD117+ rat lung ECs vs control ECs derived from rat lung CD117- cells.

overall_design: Following isolation of CD117+ lin- CD31+ cells from the rat lungs, cells were clonally expanded by seeded at limiting dilution (1 cell per well) in 96 well plates. The presence of 1 cell per each well was confirmed by microscopy. After 14 days, the largest colonies with typical endothelial “cobblestone” morphology were identified and the 6 largest endothelial colonies were used for the next round of clonal expansion. This procedure was repeated until fourth generation clonal colonies were obtained. Selection of the largest clonal colonies ensured selection of the clones with the highest proliferation and self-renewal capacity. For experiments, the cells were used after 4 clonal expansions and in the first 5 post-clonal passages. CD117- lin- CD31+ ECs were isolated from rat lungs without clonal expansion and used in passage 1-5.

first: Aneel

middle: R

last: Bhagwani

first: Daniela

first: Brennan

last: Harmon

first: Kayla

last: Authelet

first: Carlyne

last: Cool

first: Martin

last: Kolb

first: Elena

last: Goncharova

first: Mervin

last: Yoder

first: Matthias

last: Clauss

first: Robert

last: Freishtat


title: Loss of circSry leads to decreased γH2AX levels and impairs spermatogenesis

status: Public on Mar 08 2022

city: Chaoyang district

first: Yanze

last: Song

address: Datun Road

department: Regenerative Medicine

institute: The Chinese Academay of Sciences

summary: Purpose:To gain a deeper insight into how circSry regulates spermatogenesis in spermatocytes, RNA-sequencing (RNA-seq) was performed to analyze the genome-wide changes by circSry deletion.

overall_design: Total RNA was extracted from primary spermatocytes of circSry KO mice and Wild-type mice and generated by deep sequencing, in two replicates, using Illumina HiSeq 2500.

first: Haoyi

last: Wang


title: RNA-seq in H1299 cell lines.

status: Public on Mar 06 2022

first: Zhuoyu

last: Gu

address: Jianshe East Road

institute: The First Affiliated Hospital, Zhengzhou University

summary:
Purpose: The goals of this study are to invsetigate the primary biological process of CLCN3.
Methods: Briefly, samples (H1299 cells transfected with control or CLCN3 shRNA) were used to extract total RNAs for quality control and RNA-seq loading samples. RNA-seq was performed as DGE on an Illumia HiSeq platform and 50 bp paired-end reads were generated (BGI Co. Ltd.).
Results: Locomotion and growth were identified as the biological functions of CLCN3
Conclusions: RNA-seq in H1299 cell lines revealed that CLCN3 mainly regulated locomotion and growth.

overall_design: mRNA profiles of H1299 CLCN3 shNC cells and H1299 CLCN3 knockdown cells


title: Androgen-mediated perturbation of hepatic circadian system through epigenetic modulation promotes NAFLD in PCOS mice

status: Public on Mar 05 2022

city: East Lansing

first: Aritro

last: Sen

address: 766 Service Rd., ISTB

department: Animal Science

institute: Michigan State University

summary: This study reports that high androgen level via perturbation of circadian system modulates liver metabolism and contributes to the development of NAFLD in PCOS conditions.

overall_design: RNA-Seq of liver tissue isolated from Placebo and DHT pellet  treated PCOS mice

first: Sambit

last: Roy


title: ER stress sensor PERK drives tumor-promoting myelopoiesis by reprogramming hematopoietic progenitor cells in the spleen

status: Public on Mar 04 2022

city: Guangzhou

first: Mingyu

last: Liu

state: Guangdong

address: NO. 651 Dongfengdong Road, Yuexiu District

department: Collaborative Innovation Center for Cancer Medicine, State Key Laboratory of Oncology in South China

institute: Sun Yat-sen University Cancer Center

summary:
The spleen, as the prominent site for extramedullary myelopoiesis in cancer, generates significant amounts of myeloid cells to promote tumor progression. Cancer-associated splenic myelopoiesis is mediated by a population of functionally distinct hematopoietic stem/progenitor cells (HSPCs) that are committed to immunosuppressive myeloid cell generation, but the molecular mechanism regulating this response remains unclear. Here, we found that HSPCs in the spleen of tumor-bearing mice exhibited accelerated protein synthesis and engaged in a robust endoplasmic reticulum (ER) stress response. Single-cell RNA sequencing showed that the ER stress response was concomitant with the myeloid-biased lineage commitment and dysfunction of HSPCs in the spleen. PKR-like ER resident kinase (PERK), a molecular sensor of ER stress, directed HSPC differentiation into myeloid-derived suppressor cells (MDSCs) via PERK–eIF2α–ATF4–C/EBPβ signaling. Inhibition of this signaling prevented the emergence of granulocyte/macrophage colony-stimulating factor-expressing HSPCs in the spleen and disrupted the positive feedback-driven splenic myelopoiesis. Consequently, a blockade of PERK abated the suppressive function of tumor-infiltrating MDSCs and increased cytotoxic T cell infiltration, reshaping the tumor microenvironment to effectively suppress disease progression and potentiate both immuno- and targeted therapies. In accordance, activation of PERK–ATF4–C/EBPβ signaling was indispensable for the differentiation of human umbilical cord blood-derived HSPCs into functionally competent MDSCs. Together, these results indicate a crucial role for the cellular ER stress sensor PERK in modulating splenic HSPC activities in cancer, suggesting novel therapeutic opportunities for targeting the tumor-promoting myeloid response at its source.

Raw data are available on Genome Sequence Archive, Bioproject accession number: PRJCA003519

overall_design: LSK cells from the BM and spleen of Hepa mice were isolated and purified by FACS. The cells were then processed for scRNA-seq using the Chromium Single Cell 3’ Library & Gel Bead Kit v3 (10x Genomics) following the manufacturer’s protocol. Samples were sequenced at an average of 27,000 reads per cell. The resulting scRNA-seq reads were aligned to the GRCm38 (mm10) reference genome and quantified using cellranger count (Cell Ranger pipeline version 3.1.0, 10x Genomics).

first: Chong

last: Wu

first: Liming

last: Zheng


title: Expression data in muscle cells (myotubes) of motor neuron disorders (ALS, SBMA, SMA-IV) and healthy controls

status: Public on Apr 19 2022

city: Londonderry

first: William

middle: John

last: Duddy

address: C-TRIC building, Altnagelvin Hospital Campus

department: Northern Ireland Centre for Stratified Medicine

institute: Ulster University

summary:
Despite the discovery of many genetic risk factors, the cause of the motor neuron death that drives terminal pathology in Amyotrophic Lateral Sclerosis (ALS) remains unknown. We report that the skeletal muscle of ALS patients secretes exosomal vesicles that are specifically toxic to motor neurons. This could not be attributed to a trivial down-stream consequence of muscle denervation. In a study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) from 67 human subjects, including healthy and disease controls, ALS myotubes had a consistent signature of disrupted exosome biogenesis and RNA-processing, and their exosomes induced shortened, less branched, neurites, greater death, and disrupted localization of RNA and RNA-processing proteins in motor neurons. Toxicity was dependent on presence of the FUS protein, which is highly expressed in recipient motor neurons.
As part of this work, we carried out gene expression analysis of myotubes (differentiated myoblasts) comparing ALS against two other motor neuron disorders as disease controls (SBMA, Spinal and bulbar muscular atrophy; and Spinal Muscular Atrophy Type 4, SMA-IV) and healthy controls.

overall_design: Primary muscle stem cells (myoblasts) were extracted from deltoid muscle biopsies of a total of 23 subjects: 6 ALS, 5 SMA-IV, 6 SBMA, and 6 Healthy controls. Myoblasts were differentiated for 3 days to form myotubes, before RNA extraction and microarray analysis using Affymetrix Human exon array 1.0 ST v2.

middle: J

first: Alexandre

last: Henriques

first: Jose-Luis

last: Gonzales De Aguilar

first: Stephanie

last: Duguez


title: RNA-seq (CAFs stimulated with control or HNRNPK knockdown supernatants)

status: Public on Mar 02 2022

first: zhuoyu

last: gu

summary:
Purpose: The goals of this study are to invsetigate the primary signaling pathway enriched in CAFs after stimulation of LUAD supernatants.
Methods: Briefly, the supernatants (HNRNPK knockdown and control) of H1299 cells were incubated with CAFs for 24h. Then the CAFs were used to extract total RNAs for quality control and RNA-seq loading samples. RNA-seq was performed as DGE on an Illumia HiSeq platform and 50 bp paired-end reads were generated (BGI Co. Ltd.).
Results: Gene Ontology (GO) analysis revealed that cell proliferation and migration were identified as the biological functions of HNRNPK
Conclusions: In CAFs, we confirmed that the PI3K-AKT signaling pathway was significantly enriched after stimulation of H1299 supernatants.

overall_design: mRNA profiles of CAFs stimulated with control or HNRNPK knockdown supernatants


title: OTUB1 promotes gastric cancer proliferation through regulating Hippo signaling pathway

status: Public on Feb 24 2022

city: xinxiang city

first: cheng

last: Yan

address: Jinsui Road 191#

institute: xinxiang univerisity

summary: Gastric cancer is still one of the most common cancer types and third leading cause of cancer deaths worldwide. Recent studies have showed that the Hippo signaling pathway plays a critical role in progression of gastric cancer. It is of great importance to demonstrate the regulation of Hippo signaling pathway and the degradation of YAP protein in gastric cancer. In this study, we found that OTUB1 is a critical factor to facilitate gastric cancer cell stemness and progression, which deubiquitinated and stabilized YAP protein.

overall_design: mRNA expression profiles of AGS cell transfected with si-scramble or si-OTUB1.

first: Cheng

first: Jian

last: Zhu


title: Comparative bulk RNAseq analysis of CXCL12-abundant retiucular (CAR) cells, fetal perichondrium-derived CAR cells and chondrocyte-derived CAR cells.

status: Public on Mar 07 2022

city: Houston

first: Noriaki

last: Ono

state: Texas

department: School of Dentistry

institute: University of Texas Health Science Center at Houston

summary: We revealed the unique molecular signature characterizing fetal perichondrium-derived and chondrocyte-derived CAR cells

overall_design: GFP+tdTomato+ cells were FACS-isolated from postnatal day 21 (P21) femurs of Cxcl12-GFP; Dlx5-creER; R26R-tdTomato and Cxcl12-GFP; Fgfr3-iCreER; R26R-tdTomato mice, both pulsed with a single dose of tamoxifen at embryonic day 12.5 (E12.5).  mRNA were amplifed by SMARTer mRNA-seq kit (Clonetech/Takara) before deep-sequenced by Illumina HiSeq4000.


title: Mitotic bookmarking by CTCF and fast post-mitotic gene reactivation in ES cells

status: Public on Feb 19 2022

city: paris

first: Pablo

last: Navarro

address: 25 rue du Docteur Roux

institute: Institut Pasteur

summary: Mitosis leads to a global downregulation of transcription that then needs to be efficiently restored. We performed high temporal resolution analysis of the transcriptome as cells exit mitosis, in wild-type mouse ES cells as well as in auxin-depleted Ctcf conditions.

overall_design: RNA-seq quantification of ES cells exiting mitosis


title: Hierarchical deployment of Tbx3 dictating the identity of hypothalamic Tac2/Kiss1 neurons

status: Public on Jan 30 2022

city: Beijing

first: Qing-Feng

address: No. 1 West Beichen Road, Chaoyang District

institute: Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences

summary: To investigate how Tbx3 regulates the fate determination of arcuate piptidergic neruons, we performed scRNA-seq, snRNA-seq and CUT&Tag to reveal the function of Tbx3 in fate specification and maintenance of neurons

overall_design:
Lineage tracing experiment: scRNA-seq of Tbx3-CreER::Ai14 mice (E9 > P14); Tbx3 knockout experiment: scRNA-seq and snRNA-seq of Nkx2.1-Cre::Tbx3 (Flox/Flox) mice and its littermate control (P14); CUT&Tag experiment: Nkx2.1-Cre::Tbx3 (Flox/Flox) mice and its littermate control (P0)
Please note that NKO mice is a conditional knockout mice strain that lacking the expression of Tbx3 in hypothalamus and, like the input sample, peak-calling was performed based on reads of the blank control (NKO mice) in the experiment of CUT&Tag.  Therefore no processed data was provided for both 'NKO mice_CUT&Tag_WT_input'  and 'NKO mice_CUT&Tag_NKO_Ab' samples.

first: Xiang

last: Shi


title: ONE-seq: epitranscriptome and gene-specific profiling of NAD-capped RNA

status: Public on Jan 27 2022

city: Shanghai

first: Dean

last: Li

address: Haike Road 100

institute: Chinese Academy of Science

summary: The hub metabolite, nicotinamide adenine dinucleotide (NAD), can be used as an initiating nucleotide in RNA synthesis to result in NAD-capped RNAs (NAD-RNA).  Since NAD has been heightened as one of the most essential modulators in aging and various age-related diseases, its attachment to RNA might indicate a yet-to-be discovered mechanism that impacts adult life-course. However, the unknown identity of NAD-linked RNAs in adult and aging tissues has hindered functional studies.  Here, we introduce ONE-seq method to identify the RNA transcripts that contain NAD cap. ONE-seq has been optimized to use only one-step chemo-enzymatic biotinylation, followed by streptavidin capture and the nudix phosphohydrolase NudC-catalyzed elution, to specifically recover NAD-capped RNAs for epitranscriptome and gene-specific analyses. Our data describes more than a thousand of previously unknown NAD-RNAs in the mouse liver and reveals epitranscriptome-wide dynamics of NAD-RNAs with age.ONE-seq empowers the identification of NAD-capped RNAs that are responsive to distinct physiological states, facilitating functional investigation into this modification.

overall_design: ONE-seq of total RNAs (18-mon-old mouse livers) with spike-in RNA that had different ratio of NAD-RNA (0, 1, 5, 10%); ONE-seq with or without NudC treatment of mouse livers (12-mon-old); NAD-RNA profiles of young (2-mon-old) and aged (18-mon-old) mouse livers were generated by deep sequencing, in quadruplicate, using Illumina Novaseq.

first: Kongyan

last: Niu

first: Jin-yang

last: Zhang

first: Hong

last: Jiang


title: A proximal-to-distal survey of healthy adult human small intestine and colon epithelium by single-cell transcriptomics

status: Public on Feb 21 2022

city: Durham

first: Jarrett

last: Bliton

address: 6 Davis Drive

institute: Altis Biosystems

summary:
Background and Aims: Single-cell transcriptomics offer unprecedented resolution of tissue function at the cellular level, yet studies analyzing healthy adult human small intestine and colon are sparse. Here, we present single-cell transcriptomics covering the duodenum, jejunum, ileum, and ascending, transverse, and descending colon from 3 humans.
Methods: 12,590 single epithelial cells from three independently processed organ donors were evaluated for organ-specific lineage biomarkers, differentially regulated genes, receptors, and drug targets. Analyses focused on intrinsic cell properties and capacity for response to extrinsic signals along the gut axis across different humans.
Results: Cells were assigned to 25 epithelial lineage clusters. Human intestinal stem cells (ISCs) are not specifically marked by many murine ISC markers. Lysozyme expression is not unique to human Paneth cells (PCs), and PCs lack expression of expected niche-factors. BEST4+ cells express NPY and show maturational differences between SI and colon. Tuft cells possess a broad ability to interact with the innate and adaptive immune systems through previously unreported receptors. Some classes of mucins, hormones, cell-junction, and nutrient absorption genes show unappreciated regional expression differences across lineages. Differential expression of receptors and drug targets across lineages reveals biological variation and potential for variegated responses.
Conclusions: Our study identifies novel lineage marker genes; covers regional differences; shows important differences between mouse and human gut epithelium; and reveals insight into how the epithelium responds to the environment and drugs. This comprehensive cell atlas of the healthy adult human intestinal epithelium resolves likely functional differences across anatomical regions along the gastrointestinal tract and advances our understanding of human intestinal physiology.

overall_design: Single cell RNA sequencing libraries from epithelium isolated from Duodenum, Jejunum, Ileum, Ascending Colon, Transverse Colon, and Descending Colon from each of 3 human organ donors were compiled

first: Joseph

last: Burclaff

first: R

first: Keith

middle: A

last: Breau

first: Meryem

middle: T

last: Ok

first: Ismael

last: Gomez-Martinez

first: Jolene

last: Ranek

first: Aadra

last: Bhatt

first: Jeremy

last: Purvis

first: John

last: Woosley

first: Scott

last: Magness


title: Rixosomal RNA degradation contributes to silencing of Polycomb target genes

status: Public on Jan 26 2022

city: boston

first: Danesh

last: Moazed

address: 240 longwood avenue

department: cell biology

institute: Harvard medical school

summary: Polycomb Repressive Complexes 1 and 2 (PRC1 and PRC2) are histone-modifying and -binding complexes that mediate the formation of facultative heterochromatin and are required for silencing of developmental genes and maintenance of cell fate1-3. Multiple pathways of RNA decay work together to establish and maintain heterochromatin in fission yeast, including a recently identified role for a conserved RNA degradation complex called the rixosome or RIX1 complex4,5. Whether RNA degradation also plays a role in the stability of mammalian heterochromatin remains unknown. Here we show that the rixosome contributes to silencing of many Polycomb targets in human cells. The rixosome associates with human PRC complexes and is enriched at promoters of Polycomb target genes. Importantly, depletion of either the rixosome or Polycomb results in accumulation of paused and elongating RNA polymerase at Polycomb-target genes. We identify point mutations in the RING1B subunit of PRC1 that disrupt the interaction between PRC1 and the rixosome and result in diminished silencing, suggesting that direct recruitment of the rixosome to chromatin is required for silencing. Finally, we show that the RNA endonuclease and kinase activities of the rixosome and the downstream XRN2 exoribonuclease, which degrades RNAs with 5’ mono-phosphate groups generated by the rixosome, are required for silencing. Our findings suggest that rixosome-mediated degradation of nascent RNA is conserved from fission yeast to human, although in human cells the rixosomal RNAe degradation activity is primarily associated withes RNA in facultative rather than constitutive heterochromatin.

overall_design: RNA-seq analysis of histone modifications and chromatin associated proteins

first: Haining

last: zhou


title: Amyloid-β and tau aggregates are both necessary to induce novel Alzheimer’s disease stage-specific microglia subtypes

status: Public on Jan 23 2022

city: Baltimore

first: Dong Won

middle: Thomas

last: Kim

state: Maryland

address: 733 N.Broadway, MRB building (Rm 332)

department: Neuroscience

institute: Johns Hopkins University

summary: Purpose: scRNA-Seq was performed to profile changes in microglia in our mouse AD models

overall_design: Tissues (cerebral cortices, hypothalamus, and midbrain) were dissected into Hibernate-A media with a 2% B-27 and GlutaMAX supplement (0.5 mM final). Tissues were dissociated in papain (Worthington) and debris was removed using OptiPrep density gradient media following cell dissociation. Cells were then loaded into 10x Genomics Chromium Single Cell System (10x Genomics) and libraries were generated using v3 or v3.1 chemistry following the manufacturer’s instructions.  Libraries were sequenced on Illumina NovaSeq. Sequencing data were first processed through the Cell Ranger (v.3.10, 10x Genomics) with default parameters, aligned to the mm10 genome (refdata-cellranger-mm10-3.0.0), and matrix files were used for subsequent bioinformatic analysis.

first: Dong

first: Kevin

last: Tu

first: Alice

last: Wei

first: Ashley

last: Lau

first: Anabel

last: Gonzalez-Gil

first: Jonathan

last: Ling

first: Juan

last: Troncoso

first: Philip

last: Blackshaw

first: Ronald

last: Schnaar

first: Tong


title: Engineering A Potent Cancer Vaccine Using Immunoproteasome-Expressing Mesenchymal Stromal Cells

status: Public on Jan 20 2022

city: Montréal

first: Moutih

last: Rafei

state: Québec

address: 2900 Boul. Édouard-Montpetit

country: Canada

institute: Université de Montréal

summary: Raw RNA-seq counts from reads aligned to the mouse genome (mm10 assembly) were generated with Htseq-count (PMID: 25260700). Differentially expressed genes between IPr and Ctl MSCs were calculated by DESeq2 (PMID: 25516281). Pre-ranked gene set enrichment was performed as recommended for RNA-seq data (PMID: 16199517). Custom R scripts were used to filter highly redundant biological processes. A false discovery rate of 0.05 was considered as an acceptable threshold for further investigation. All analyses were conducted in R (v3.6) or Python (v3.7) programming language. The ggplot2, ClusterProfiler and dplyr packages were used for data visualization. Studentâ€™s t-test was performed for normally distributed data to compute the p-value and the Benjamini-Hochberg procedure was used for adjusting the statistical inference of multiple comparisons.

overall_design: We report in this study the effect of IPr subunit expression on the antigen cross-presenting abilities of MSCs.

first: Nehme

last: El-Hachem


title: Single cell RNA analysis of breast cancer bone metastases

status: Public on Feb 08 2022

city: Pittsburgh

first: Kai

last: Ding

address: 204 Craft Ave

institute: University of Pittsburgh

summary: For single cell RNA sequencing analysis, whole cell population isolated from two breast cancer bone metastases and associated organoid models were analyzed by 10X genomic single cell RNA platform. Raw BCL files from Novaseq6000 was converted to FASTQ files with cellranger (Version: 3.1.0) mkfastq function. Cellranger count function was then applied for read alignment, barcode and UMI counting using GRCh38 as reference. scCB2 and DoubletFinder were used to predict and remove empty droplets and doublets of single cell data. Cells with number of features less 300 and more than 9000 were further filtered out based on the distribution of number of features. For bulk RNA sequencing, RNA were extracted from FFPE preserved primary tumor and bone metastasis. Library was prepared with TruSeq Stranded mRNA for FFPE kit, and sequenced by NextSeq 500 with high output 150 cycle kit.

overall_design: Single cell and bulk RNA sequencing of two synchronous bilateral breast cancer bone metastases with associated patient derived organoid models and paired primary tumor.

first: Adrian

middle: V

last: Lee

first: Steffi

last: Oesterreich

first: Kurt

last: Weiss

first: Fangyuan

last: Chen

first: Nolan

last: Priedigkeit


title: CITE-seq of CD4+ T and myeloid cells in MS Twin cohort.

status: Public on Jan 13 2022

city: Planegg-Martinsried

first: Eduardo

last: Beltrán

state: Bayern

address: Großhaderner Str. 9

department: Institute of Clinical Neuroimmunology

institute: University Hospital, LMU Munich

summary: By integrating data on the immune profiles of healthy monozygotic and dizygotic twin pairs we estimated the variance in CD25 expression by naïve Th cells to be largely driven by genetic and shared early environmental influences. Nonetheless, the Th cell subset expanded in MS twins, which was also elevated in non-twin MS patients , emerged independent of the individual genetic makeup. These cells expressed CNS-homing receptors, exhibited a dysregulated CD25-IL-2 axis, and their proliferative capacity positively correlated with MS severity . Together, the pair-matched analysis of the extended twin approach allowed us to discern genetically- and environmentally- determined features of an MS-associated immune signature.

overall_design: Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) of CD4+ T and myeloid cells in monozygotic twin pairs discordant for MS to obtain a comprehensive overview of epitopes, transcriptome and T cell receptor (TCR) clonotypes.

first: Vladyslav

last: Kavaka

first: Klara

middle: M

last: Eglseer


title: ERK-Mediated Phosphorylation of OLA1 Controls Adaptive Mitochondria to Nucleus Communication Regulating Cellular Homeostasis

status: Public on Jan 14 2022

city: Milwaukee

first: Adeleye

middle: J.

last: Afolayan

state: Wisconsin

address: 8701 Watertown Plank Rd., PD278, CRI TBRC, Rm C3278

institute: Medical College of Wisconsin

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

overall_design: Refer to individual Series


title: In vivo partial reprogramming alters manifestation of age-associated molecular changes in aging mice (1 months; sort-term treatment)

status: Public on Jan 11 2022

city: South San Francisco

first: Jason

middle: Anthony

last: Vander Heiden

address: 1 DNA Way

department: Biological Research AI Development

institute: Genentech

summary: Various length of in vivo 4F (four Yamanaka factors) partial reprogramming protocols were performed to evaluate effects of longer-term partial reprogramming in physiologically aging wild-type mice. Here, short-term (1 months) partial reprogramming was induced and transcriptional changes in various tissues were compared to age-matched, untreated mice.

overall_design: Mice carrying one copy of OSKM (Sox2, Oct4, Klf4, and c-Myc) polycistronic cassette and the rtTA transactivator (C57BL/B6 background) were treated with 1mg/ml doxycycline in drinking water for 2 days followed by 5 days of withdrawal per week. Treatment was performed for 1 months started at 25 months of age and ending at 26 months. Untreated 4F, 26-months-old and 3-months-old mice were used as controls. RNA was extracted from skin, kidney, lung, liver, spleen, skeletal muscle and RNA-seq was performed to evaluate transcriptional changes.

first: Kristen

last: Browder

first: Pradeep

last: Reddy

first: Henrich

last: Jasper

last: Belmonte


title: In vivo partial reprogramming alters manifestation of age-associated molecular changes in aging mice (10 months; long-term treatment)

summary: Various length of in vivo 4F (four Yamanaka factors) partial reprogramming protocols were performed to evaluate effects of longer-term partial reprogramming in physiologically aging wild-type mice. Here, long-term (10 months) partial reprogramming was induced and transcriptional changes in various tissues were compared to age-matched, untreated mice.

overall_design: Mice carrying one copy of OSKM (Sox2, Oct4, Klf4, and c-Myc) polycistronic cassette and the rtTA transactivator (C57BL/B6 background) were treated with 1mg/ml doxycycline in drinking water for 2 days followed by 5 days of withdrawal per week. Treatment was performed for 10 months started at 12 months of age and ending at 22 months. Untreated 4F, 22-months-old mice were used as controls. To identify and exclude doxycycline effects, C57BL/B6 mice were treated with doxycycline for 5 months starting at 20-months-old and ending at 25-months-old. Untreated C57BL/B6 mice of 26 months were used as controls. RNA was extracted from skin, kidney, lung, liver, spleen, skeletal muscle and RNA-seq was performed to evaluate transcriptional changes.

middle: C

first: Juan


title: Concurrent delivery of immune checkpoint blockade improves tumor microenvironment for vaccine-generated immunity

status: Public on Jan 10 2022

city: Philadelphia

first: Bo

last: Li

state: PA

address: 3501 Civic Center Blvd

department: Department of Pathology and Laboratory Medicine

institute: The Children’s Hospital of Philadelphia, University of Pennsylvania

summary: Neoantigen vaccines aiming to induce and amplify tumor-specific T cell responses have received promising anti-tumor effect in early clinical trials. However, the underlying mechanism regarding response or resistance to this treatment remains unclear. Here, we observed that neoantigen vaccine-generated T cells could synergize with immune checkpoint blockade (ICB) for effective tumor control. Specifically, we performed single-cell sequencing on over 100,000 T cells and uncovered that combined therapy induced a novel antigen-specific CD8 T cell population with active chemokine signaling (Cxcr3+/Ccl5+), lower co-inhibitory receptor expression (Lag3-/Havcr2-) and higher cytotoxicity (Fasl+/Gzma+). Furthermore, the generation of antigen specific T cells in the drain lymph node is required for combination treatment mediated therapeutic effect. Signature genes of this unique population is associated with T cell clonal frequency and better survival in human. Our study comprehensively profiled the dynamics of tumor infiltrated T cells during neoantigen vaccine and ICB treatments, and high-dimensionally identified neoantigen-reactive T cell signatures for future development of better therapeutic strategies.

overall_design: MC38 tumors, spleen and tumor draining lymph nodes were harvested at day 10 or day 20 post inoculation. CD45+ cells were isolated with CD45 (TIL) MicroBeads (Miltenyi Biotec). Enriched CD45+ cells were washed and re-suspended in FACS buffer (1x PBS with 2% FBS) and stained for CD3e. CD3+ live cells were sorted by BD FACSMelody™.  Spleen and draining lymph node were gently mashed and washed through a 70 μm cell strainer with culture medium. After centrifugation at 500g for 5 minutes, red blood cells in the spleen were removed by ACK lysing buffer. Cell pellets were finally re-suspended in FACS buffer and stained for CD3e. CD3+ live cells were sorted by BD FACSMelody™.  Single-cell RNA-seq were performed by the 10x Genomic single cell 5’ VDJ library platform.  Based on FACS analysis, single cells were sorted into 1.5 ml tubes (Eppendorf) and counted manually under the microscope. The concentration of single cell suspensions was adjusted to 900-1100 cells/μl. Cells were loaded between 10,000 and 17,000 cells/chip position using the Chromium Single cell 5’ VDJ Library, Gel Bead & Multiplex Kit and Chip Kit (10x Genomics, V1 barcoding chemistry). Single-cell gene expression and TCR libraries were generated according to the manufacturer’s instructions. All the subsequent steps were performed following the standard manufacturer’s protocols. Purified libraries were analyzed by Nextseq sequencer with 150-bp paired-end reads at a targeted median read depth of 50,000 reads per cell from total gene expression libraries and 5,000 reads per cell for TCR librarie.

first: Longchao

last: Liu

first: Jiahui

first: Hongyi

last: Zhang

first: Jianfeng

last: Ye

first: Changzheng

last: Lu

first: Yang-Xin

last: Fu


title: In vivo partial reprogramming alters manifestation of age-associated molecular changes in aging mice


title: Bulk and single cell profing of CD45+ LSK cells collected from WT and TET2 deficient mice under normoxia and hypoxia (physioxia) conditions (CITE-SEQ)

status: Public on Jan 07 2022

city: INDIANAPOLIS

first: Xiaoyu

state: IN

address: 410W 10TH ST

department: school of medicine

institute: INDIANA UNIVERSITY

summary: Hematopoietic stem cells (HSCs) manifest impaired recovery and self-renewal with a concomitant increase in progenitor content (HPCs) when exposed to ambient air as opposed to physioxia. Mechanisms behind this distinction are poorly understood but have the potential to enhance HSC function for bone marrow transplantation. High resolution transcriptomic analysis revealed that HSCs under physioxia (HSCs- P) upregulate genes involved in self-renewal and quiescence but downregulate genes involved in apoptosis and differentiation compared to HSCs-A. Remarkably, among several genes, Tet2 was significantly downregulated in HSC-Ps compared to HSC-As, which was associated with reduced 5-hydroxymethylcytosine (5-hmC) levels in HSC-Ps. Interestingly, individuals carrying the TET2 mutation in their HSCs are at a higher risk of developing clonal hematopoiesis, myeloid leukemias and cardiovascular disease. Loss of Tet2 in mice results in more HSCs and enhanced self-renewal. TET enzymes are α-ketoglutarate, iron and oxygen-dependent dioxygenases that convert 5-methylcytosine to 5-hydroxymethylcytosine thereby promoting active transcription. We evaluated whether physioxia affects the numbers and function of HSCs and HPCs from Tet2-/- mice similar to wildtype (WT) mice. In contrast to WT-HSCs, we observed complete non-responsiveness of Tet2-/- HSCs to changes in oxygen tension. Tet2-/- HSCs and HPCs exhibited similar numbers and function under physioxia and ambient air. The lack of functional responsiveness to changes in oxygen tension in Tet2-/- HSCs was associated with similar changes in genes involved in self-renewal and quiescence among WT-HSC-Ps, Tet2-/- HSC-Ps and Tet2-/- HSC-As. Our findings define a novel molecular program involving Tet2 in regulating the self-renewal of HSCs under physioxia.

overall_design: CD45+ cells were extracted under four conditions namely WT and TET2 knockout under normoxia and physioxia conditions

first: Chi

first: Reuben

last: Kapur

first: Hal

last: Broxmeyer


title: ERK-Mediated Phosphorylation of OLA1 Controls Adaptive Mitochondria to Nucleus Communication Regulating Cellular Homeostasis [RNA-seq]

first: Adeleye

summary: We found that changes in mitochondrial membrane potential or ROS generation trigger the translocation of OLA1 from mitochondria and cytoplasm to the nucleus, regulated by ERK1-mediated phosphorylation of OLA1 on Ser232/Tyr236. Subsequent phosphorylation of nuclear-OLA1 on Thre325 by ERK2 activates its function as a genetic factor regulating cellular homeostasis by modulating HDAC9 expression. Disruption of OLA1 phosphorylation blocks its nuclear translocation, compromised the expression of nuclear-encoded mitochondrial proteins, DNA damage response, and multiple stress-response-related-genes, leading to cellular dysfunction. Our findings reveal that OLA1 is a crucial component of mitonuclear response regulatory hubs essential for regulating cellular adaptation to stress.

overall_design: Examination of the effect of OLA1 knockdown and OLA1 nuclear localization on gene expression in pulmonary endothelial cells.

first: Paul

last: Sidlowski


title: Bulk and single cell profiling of CD45+ LSK cells collected from WT and TET2 deficient mice under normoxia and hypoxia (physioxia) conditions (BULK RNA-SEQ)


title: No safe renal warm ischemia time-The molecular network characteristics and pathological features of mild to severe ischemia reperfusion kidney injury

status: Public on Jan 04 2022

first: Jianwen

last: Chen

address: 28th, Fu Xing road, Hai Dian district

institute: First Medical Center of Chinese PLA General Hospital

summary:
Purpose: we performed comparative RNA-seq analyses to identify molecular network characteristics of kidney injury at different ischemia times
Methods:  Unilateral clamping of the left renal pedicle plus contralateral nephrectomy was performed. Briefly, mice were anesthetized, right kidney was excised. The left kidney was clamped for indicated minutes (0min for sham-24h group, 16min for uIRIx16min-24h group, 18minfor uIRIx18min-24h group, 20min for uIRIx20min-24h group, 22min for uIRIx22min-24h group, 24min for uIRIx24min-24h group, 26min for uIRIx26min-24h group, 28min for uIRIx28min-24h group, 30min for uIRIx30min-24h group). Mice were placed on 37°C heating platform while operation, ischemia and before awakening. At 24h after ischemia, mice were anesthetized and blood was taken to detect creatinine, kidney was harvest for PAS staining and mRNA seq.
Results: After sequence, the clean reads rate of all samples were ≥98%.The quality of the assembled transcriptome is good enough for functional annotation and further analysis.
Conclusions: We performed RNA-sequencing (RNA-Seq) analyses to identify molecular network characteristics of kidney injury at different ischemia time.

overall_design:
We performed RNA-sequencing (RNA-Seq) on kidney of 9 groups.

Grant No. : 82100713
Grant title:The role and mechanism of PFKFB3 in promoting fibrosis after acute kidney injury by regulating the metabolic reprogramming of renal tubular epithelial cells.
Name of the funding source: the National Natural Science Foundation of China.

first: Jian-Wen

first: Lei

last: Wang

first: Xin

last: Ma

first: Huai-Kan


title: Systematic Discovery and Therapeutic Targeting of Pro-growth Enhancers in Human Cancer Cells

status: Public on Dec 31 2021

city: Singapore

first: Benson

address: 60 Biopolis Street, Genome, #04-01

department: Genome Institute of Singapore

country: Singapore

institute: A*STAR


title: Identification of gene expression changes in sleep mutants associated with reduced longevity in Drosophila

status: Public on Jan 01 2022

city: New York

first: Zikun

state: New York

address: 1230 York Av

institute: The Rockefeller University

summary: Genome-wide profiling of rhythmic gene expression has offered new avenues for studying the contribution of circadian clock to diverse biological processes. Sleep has been considered one of the most important physiological processes that are regulated by the circadian clock, however, the effects of chronic sleep loss on rhythmic gene expression remain poorly understood. In the present study, we exploited Drosophila sleep mutants insomniac1 (inc1) and wide awakeD2 (wakeD2) as models for chronic sleep loss. We profiled the transcriptomes of heads collected from 4-week-old wild type flies, inc1 and wakeD2 at timepoints around the clock. Analysis of gene oscillation revealed a substantial loss of rhythmicity in inc1 and wakeD2 compared to wild type flies, with most of the affected genes common to both mutants. The disruption of gene oscillation was not due to changes in average gene expression levels. We also identified a subset of genes whose loss of rhythmicity was shared among animals with chronic sleep loss and old flies, suggesting a contribution of aging to chronic, sleep-loss-induced disruption of gene oscillation.

overall_design:
RNA sequencing data was collected around the clock every 4 hours over 2 days from Drosophila melanogaster heads of 27-day-old wild type flies, 49-day-old wild type flies, 27-day-old inc1 flies, and 27-day old wakeD2 flies.

RNA-seq was conducted on the Illumina NextSeq 500 sequencer to generate 75bp single reads, following manufacturer’s protocol. The sequencing depth was about 25 million reads per sample. Reads were aligned to the D. melanogaster reference genome assembly (Release 6.13) (dos Santos et al., 2015) with STAR (Dobin et al., 2013) and read counts were generated with featureCounts (Liao et al., 2014). The raw sequence data are available for samples of all time points except for WT_ZT28 and WT_ZT40. The counts data generated using STAR and featureCounts for all RNA-seq samples are available in supplementary file.

The code used for RNA-seq analysis and intermediary results are available at https://github.com/wangzikun329/Wang2022
RNA-seq expression plots for all genes are available at https://zikunwang.shinyapps.io/rna_seq_gene_plot/


title: Bulk and single cell profing of CD45+ LSK cells collected from WT and TET2 deficient mice under normoxia and hypoxia (physioxia) conditions


title: Enterocyte Superoxide Dismutase 2 Deletion Drives  Hyperinsulinemia and Obesity

city: Lille

first: Lefebvre

last: Philippe

address: Bld Pr Jules Leclerc

institute: Inserm

summary: Compelling evidence support an involvement of oxidative stress and intestinal inflammation as early events in the predisposition and development of obesity and its related comorbidities. Here we show that deficiency of the major mitochondrial antioxidant enzyme superoxide dismutase 2 (SOD2) in the gastrointestinal tract drives spontaneous obesity. Intestinal epithelium-specific Sod2 ablation in mice induced adiposity, inflammation and insulin resistance via phospholipase A2 (PLA2) activation and increased synthesis of omega-6 polyunsaturated fatty acid arachidonic acid. Remarkably, this obese and hyperinsulinemic phenotype was rescued when fed an essential fatty acid deficient diet, which abrogates de novo biosynthesis of arachidonic acid. Data from clinical samples revealed that the negative correlation between intestinal SOD2 mRNA levels and obesity features, such as body mass index and omega-6/omega-3 fatty acid ratio, appears to be conserved between mice and humans. Collectively, our findings suggest a role of intestinal SOD2 levels, PLA2 activity and arachidonic acid in obesity presenting new potential targets of therapeutic interest in the context of this metabolic disorder.

overall_design: To explore correlations between colon transcriptomics and clinical and metabolomics data in human subjects with different degrees of body mass index (BMI), colonic biopsies were obtained and processed for RNA extraction

first: Philippe

last: Lefebvre


title: In vivo partial reprogramming alters manifestation of age-associated molecular changes in aging mice (7 months; long-term treatment)

first: Jason

last: Vander Heiden

summary: Various length of in vivo 4F (four Yamanaka factors) partial reprogramming protocols were performed to evaluate effects of longer-term partial reprogramming in physiologically aging wild-type mice. Here, long-term (7 months) partial reprogramming was induced and transcriptional changes in various tissues were compared to age-matched, untreated mice.

overall_design: Mice carrying one copy of OSKM (Sox2, Oct4, Klf4, and c-Myc) polycistronic cassette and the rtTA transactivator (C57BL/B6 background) were treated with 1mg/ml doxycycline in drinking water for 2 days followed by 5 days of withdrawal per week. Treatment was performed for 7 months started at 15 months of age and ending at 22 months. Untreated C57BL/B6 only, 22-month-old mice were used as controls. RNA was extracted from skin, kidney, lung, liver, spleen, skeletal muscle and RNA-seq was performed to evaluate transcriptional changes.

first: Kristen

last: Browder

first: Pradeep

last: Reddy

first: Henrich

first: Juan


title: Systematic Discovery and Therapeutic Targeting of Pro-growth Enhancers in Human Cancer Cells [ChIP-Seq]

summary: Precision medicine depends critically on the development of treatment strategies that can specifically target cancer cells and have minimal adverse effects on normal cells. Cancer-cell-specific enhancers represent a class of therapeutic targets, however, a lack of comprehensive map of pro-growth enhancers in cancer cells presents a significant hurdle to this strategy. Here we address the knowledge gap by carrying out large-scale CRISPR-based perturbation screens to identify pro-growth enhancers in multiple cancer cell lines. We report the finding of 510 pro-growth enhancers, the majority of which show evidence of accessibility only in cancer cells but not in normal cell types. In addition, 16 pro-growth enhancers are selectively associated with poor clinical outcomes in colorectal cancer. These cancer cell specific pro-growth enhancers  are modulated by lineage-specific transcription factors acting downstream of signaling pathways, and could be pharmacologically targeted in specific cancer cell types. Our results not only uncover the molecular mechanism governing the function of cancer cell-specific pro-growth enhancers but also reveal novel therapeutic targets for cancer treatment.

overall_design: We examined the histone modification in HCT116 and KRAB-dCas9 stably expressed HCT116 cells

first: Bing

last: Ren


title: Gene expression data from  FGFR3 induced mouse bladder tumors

first: Elodie

last: chapeaublanc

address: 26 rue d'ulm

department: UMR 144

institute: Institut Curie

summary: Somatic mutations of the fibroblast growth factor receptor 3 (FGFR3) are one of the most frequent genetic alterations in bladder carcinomas. We report here that human-FGFR3-S249C expression in urothelial cells of transgenic mice induces low-grade papillary tumors presenting genomic instability and resembling human pTa urothelial tumors at the transcriptomic level. Mutated-FGFR3 expression levels impacted the incidence of tumor formation and could account for the tissue specificity of human mutated FGFR3-driven tumors restricted to epithelia presenting high normal expression levels of FGFR3.

overall_design: The expression of a human FGFR3-IIIb carrying the S249C mutation was targeted to the urothelium of mice by using the 5’ regulatory region of the mouse uroplakin II promoter. The UII-FGFR3b-S249C construct was obtained by inserting the 3.6 kb murine uroplakin II promoter (UII) (40) excised with SalI and BamHI into the same restriction sites of the vector containing the β-globin intron 2 and the 3’ polyadenylation sequences of SV40 (41) followed by the insertion of a human S249C mutated FGFR3 cDNA excised with XbaI and HindIII into the SmaI site of this vector. All PCR-generated segments were verified by sequencing both strands. The pUII-hFGFR3b-S249C constructs excised with KpnI were purified and microinjected into fertilized B6D2 oocytes. Genomic DNA was extracted from mouse tails and screened by PCR to identify transgene integration. Two lines were selected, L569 and L538, and mice were back-crossed five times to C57BL/6J mice. Mice were of a mixed background and littermates were used as control. Bladder from mice aged 1 to 24 months were examined for macroscopic lesions followed by a histopathological analysis when required. Mice were then intercrossed to obtain hetero-and homozygous mice for the transgene.

first: Isabelle

last: Bernard-Pierrot

first: François

last: Radvanyi

last: Chapeaublanc


title: Systematic Discovery and Therapeutic Targeting of Pro-growth Enhancers in Human Cancer Cells [PLAC-seq]

overall_design: Examination of chromatin interactions in multiple cancer cell lines


title: RNA-seq for human dermal fibroblasts (HDF), BRAF-V600E positive (MaMel21 and MaMel63a), and BRAF-WT malignant melanoma cell lines (SBcl2 and WM3438) after vemurafenib or DMSO treatment

status: Public on Dec 11 2021

city: Freiburg

first: Eyleen

last: Corrales

address: Breisacherstr. 153

department: Medizinische Fakultät

institute: Institut für Medizinische Bioinformatik und Systemmedizin

summary:
Background: Vemurafenib (PLX4032) is one of the most frequently used treatments for late-stage melanoma patients with the BRAFV600E mutation; however, acquired resistance to the drug remains as a major challenge. It remains to be determined whether off-target effects of the vemurafenib on normal stroma components could reshape the tumor microenvironment in a way that contributes to cancer progression and drug resistance.
Methods: By using temporary-resolved RNA- and ATAC-seq, we studied the early molecular changes induced by vemurafenib in human dermal fibroblast (HDF), a main stromal component in melanoma and other tumors with high prevalence of BRAFV600 mutations.
Results: Transcriptomics analyses revealed a stepwise up-regulation of proliferation signatures, together with a down-regulation of autophagy and proteolytic processes. The gene expression changes in HDF strongly correlated in an inverse way with those in BRAFV600E mutant malignant melanoma (MaMel) cell lines, consistent with the observation of a paradoxical effect of vemurafenib, leading to hyperphosphorylation of MEK1/2 and ERK1/2. The transcriptional changes in HDF were not strongly determined by alterations in chromatin accessibility; rather, an already permissive chromatin landscape seemed to facilitate the early accessibility to MAPK/ERK-regulated transcription factor binding sites. Combinatorial treatment with the MEK inhibitor trametinib did not preclude the paradoxical activation of MAPK/ERK signaling in HDF. When administered together, vemurafenib partially compensated the reduction in cell viability and proliferation induced by trametinib. These paradoxical changes were restrained by using the third generation BRAF inhibitor PLX8394, a so-called paradox breaker compound. However, the advantageous effects on HDF during combination therapies were also lost.
Conclusion: Vemurafenib induces paradoxical changes on HDF, which are allowed by a permissive chromatin landscape. These changes might provide an advantage during combination therapies, by compensating the toxicity induced in stromal cells by less specific MAPK/ERK inhibitors. Our results highlight the relevance of evaluating the effects of the drugs on non-transformed stromal components, carefully considering the implications of their administration either as mono- or combination therapies.

overall_design: Human dermal fibroblasts (HDF), patient derived BRAF-V600E positive malignant melanoma cell lines (MaMel21 and MaMel63a), and BRAF-WT melanoma cell lines (SBcl2 and WM3438), treated  with vemurafenib or an analog volume of vehicle (DMSO) for different durations.

first: Patrick

last: Metzger

first: Melanie

last: Boerries