title: Effect of the CSF1R inhibitor AZD7507 on a relapse mouse model of high-grade serous ovarian cancer

city: London

first: Eleni

last: Maniati

address: John Vane Science Centre, Charterhouse Square

department: Barts Cancer Institute

country: United Kingdom

institute: Queen Mary University of London

summary: We investigated the effect of tumour-associated macrophage (TAM) inhibition on disease relapse through treatment of a high-grade serous ovarian cancer model with the CSF1R inhibitor AZD7507.

overall_design: The transplantable syngeneic high-grade serous mouse model 60577 was established by injecting the 60577 murine cell line intraperitoneally in syngeneic mice. The animals were then treated with Carboplatin for three cycles, once weekly, and then receiced AZD7507 by oral gavage daily for 84 days straight after the end of the three cycles of carboplatin. RNA sequecing was performed on n=3 AZD7507-treated and n=3 vehicle-treated mouse tumours.

first: Chiara

last: Berlato

first: Owen

last: Heath

first: Frances

middle: R

last: Balkwill


title: The transcriptional landscape of the murine middle ear epithelium in vitro

status: Public on Apr 20 2021

city: Dhaka

first: Miraj

middle: Kobad

last: Chowdhury

address: New Science Complex, Ramna

department: Genetic Engineering and Biotechnology

country: Bangladesh

institute: University of Dhaka

summary:
Middle ear epithelium (MEE) is an extended part of the respiratory mucosa and mucociliary differentiation of MEE is crucial for the maintenance of homeostasis and sterility of the middle ear cavity. Previously we developed an in vitro model of murine MEE using air-liquid interface (ALI) culture that replicates aspects of the in vivo middle ear. Defects in MEE function can lead to the development of otitis media (OM). Decoding differential gene expression (DGE) across the MEE during mucociliary differentiation is valuable in understanding the mechanism underpinning the development of OM.
We used ClariomTM S Mouse assay to understand the global gene expression at different time points of murine MEE differentiation at ALI culture and to identify sets of genes up-regulated and down-regulated during this process.

overall_design: Murine MEE cells were isolated from 5-6 middle ears of adult C57/BL6 mice and cultured at the ALI for up to seven days. Total RNA was extracted from freshly isolated MEE cells, and at cells at day 0 (grown at submerged culture and collected prior to ALI culture) and at day 7 ALI culture. 200ng of total RNA from each sample was subjected to analyse on the ClaromTM S Mouse GeneChip (Thermo Fisher) according to the manufacturer protocol.

first: Apoorva

last: Mulay

first: M

middle: M

last: Kobad Chowdhury

first: Cameron

middle: T

last: James

first: Lynne

last: Bingle

first: Colin

middle: D


title: Gadolinium protects Arabidopsis thaliana against  Botrytis cinerea through the activation of JA/ET-induced  defense responses

status: Public on Apr 21 2021

city: Cuernavaca

first: Damien

last: Formey

state: Morelos

address: Avenida de la Universidad

department: Center for Genomic Science

country: Mexico

institute: UNAM

type: Expression profiling by high throughput sequencing

summary: Plant food production is severely affected by fungi; to cope with this problem, farmers use synthetic fungicides. However, the need to reduce fungicide application has led to a search for alternatives, such as biostimulants. Rare-earth elements (REEs) are widely used as biostimulants, but their mode of action and their potential as an alternative to synthetic fungicides have not been fully studied. Here, the biostimulant effect of gadolinium (Gd) is explored using the plant-pathosystem Arabidopsis thaliana–Botrytis cinerea . We determine that Gd induces local, systemic, and long-lasting plant defense responses to B. cinerea, without affecting fungal development. The physiological changes induced by Gd have been related to its structural resemblance to calcium. However, our results show that the calcium-induced defense response is not sufficient to protect plants against B. cinerea, compared to Gd. Furthermore, a genome-wide transcriptomic analysis shows that Gd induces plant defenses and modifies early and late defense responses. However, the resistance to B. cinerea is dependent on JA/ET-induced responses. These data support the conclusion that Gd can be used as a biocontrol agent for B. cinerea. These results are a valuable tool to uncover the molecular mechanisms induced by REEs.

overall_design: RNAseq from Gd-treated Arabidopsis thaliana leaves

first: Juliana

last: Santos Batista-Oliveira

first: Martha

last: Torres

first: Wendy

last: Aragón

first: Yordan Jhovani

last: Romero-Contreras

first: Israel

last: Maruri-López

first: Alexandre

last: Tromas

first: Kátia

last: Freitas Schwan-Estrada

first: Mario

last: Serrano


title: Transcriptomes of tumour-associated macrophages from high grade serous ovarian cancer pre-  and post-platinum-based neoadjuvant chemotherapy.

first: Eleni

last: Maniati

summary: The impact of platinum and taxol based neoadjuvant chemotherapy on tumour-associated macrophages (TAMs)  was examined by analysis of TAMs extracted from high grade serous ovarian cancer (HGSOC) samples pre and post chemotherapy.

overall_design: TAMs were isolated by FACS sorting from high grade serous ovarian cancer, omental metastasis samples, pre and post platinum-based chemotherapy. RNA sequencing was performed on unmatched n = 5 pre and n = 7 post chemotherapy TAM samples.

first: Owen

first: Chiara

last: Berlato


title: ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation

city: Leiden

first: Martijn

middle: S.

last: Luijsterburg

state: zuid-holland

address: Einthovenweg 20

department: Human Genetics

country: Netherlands

institute: LUMC

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Removal of transcription-blocking DNA lesions requires the binding of transcription-coupled repair (TCR) factors to DNA damage-stalled RNA polymerase II (RNAPII). The full repertoire of factors required for TCR remains to be elucidated. Through genome-wide CRISPR screens and strand-specific ChIP-seq, we identify the RNAPII-associated factor ELOF1 as a new core TCR component. Mechanistically, ELOF1 does not affect the association of TCR components CSB and the CRL4CSA ubiquitin ligase, but instead regulates RNAPII ubiquitylation, and subsequent ubiquitin-dependent recruitment of repair factors. This function requires its constitutive interaction with RNAPII close to the K1268 site, revealing ELOF1 as a specificity factor that positions CRL4CSA for optimal RNAPII ubiquitylation to orchestrate transcription-coupled repair. Finally, ELOF1 operates in a second transcription stress-response pathway, regulated by the deubiquitylating enzyme OTUD5.

overall_design: ATAC-seq, BrU-seq,  ChIP-seq and RNA-seq in human RPE1-iCas9 or U2OS cell lines either untreated (mock) or dfferent time points after UV-C treatment with varying dose (as indicated per sample).

first: Yana

last: van der Weegen

first: Klaas

last: de Lint

first: Rob

last: Wolthuis

middle: S


title: Single cell mRNA sequencing on esophageal cells after HH pathway activation

city: Anderlecht

first: Alizee

last: Vercauteren Drubbel

address: 808 route de Lennik

country: Belgium

institute: Université Libre de Bruxelles

summary: In this study we used single cell mRNA sequencing to explore heterogeneity of the fate induced by hedgehog (HH) pathway activation in esophageal cells. We merged the data from the two samples and filtered the merged data to work only with cells expressing the SmoM2-YFP protein (2,413 cells). Using UMAP reduction, we observed 10 clusters with a resolution set at 0.3. Among these clusters, we identified clusters of squamous esophagus epithelial cells but also one cluster enriched for many columnar markers. These results show that activation of the HH pathway in esophageal keratinocytes leads to multiple cell phenotypes including a columnar epithelial one in a minority of keratinocytes.

overall_design: Oesophageal single cell mRNA profiles of Krt5SmoM2 mice by deep sequencing using 10x genomic and Illumina

first: Benjamin

last: Beck

first: Alizée


title: miRNA Alterations in Lung Macrophages after Nitrogen Mustard Exposure

status: Public on Apr 19 2021

city: Gainesville

first: Ley

middle: Cody

last: Smith

state: FL

address: 2187 Mowry Rd

country: USA

institute: University of Florida

summary: Activated macrophages have been implicated in lung injury and fibrosis induced by the cytotoxic alkylating agent, nitrogen mustard (NM). Herein, we determined if macrophage activation is associated with altered miRNA expression. Treatment of rats with NM (0.125 mg/kg, i.t.) resulted in significant increases in miRNA expression 1, 3, 7, and 28 d post exposure as determined by PCR array analysis of miRNAs (miR)s involved in inflammation and fibrosis. Expression of many miRNAs were significantly increased at multiple time-points while one miRNA was uniquely expressed 1 d post-NM, one at 3 d, two at 7 d, and five at 28 d.  An IPA Core Analysis of predicted mRNA targets of differentially expressed miRNAs identified significant enrichment of Diseases and Functions related to cell cycle arrest, apoptosis, cell movement, cell adhesion, and inflammation 1 d and 28 d post NM. Significant enrichment of pathways related to lipid metabolism is consistent with previous studies reporting the development of macrophage foam cells after NM exposure. miRNA-mRNA interaction network analysis identified highly connected miRNAs representing key upstream regulators of mRNAs involved in significantly enriched Diseases and Functions including miR-34c-5p and miR-27a-3p at 1 d post NM and miR-125b-5p, miR-16-5p, miR-30c-5p, miR-19b-3p, and miR-148b-3p at 28 d post NM. Collectively, these data show that NM promotes histone remodeling and alterations in miRNA expression linked to lung macrophage responses during inflammatory injury and fibrosis.

overall_design: Male Wistar rats (8 wk, 225–250 g) were anesthetized with 2.5% isoflurane, and then administered PBS or NM (0.125 mg/kg, mechlorethamine hydrochloride, Sigma-Aldrich, St. Louis, MO) intratracheally. Animals were euthanized by intraperitoneal injection of pentobarbital (Sleepaway, 50 mg/kg, Fort Dodge Animal Health, Fort Dodge, IA) 1 d, 3 d, 7 d and 28 d after administration of PBS or NM.  The lung was removed, and 10 mL of ice-cold PBS slowly instilled and withdrawn through a cannula in the trachea while gently massaging the tissue; this procedure was repeated 4 times.  The cells were then centrifuged (300 x g, 8 min), resuspended in 10 mL PBS and viable cells enumerated using a hemocytometer with trypan blue.  Total mRNA was collected from lung macrophages by phenol-chloroform extraction.  Samples were enriched for miRNAs using an RNeasy MinElute Cleanup kit (QIAGEN Inc., Valencia, CA). Purified miRNA was reverse transcribed using a QIAGEN QuantiTect Reverse Transcription kit and analyzed using a QIAGEN miScript Rat Inflammatory Response and Autoimmunity miRNA PCR Array or by RT-qPCR using a QIAGEN miScript SYBR Green kit according to the manufacturer’s protocol. Amplification was performed using a 7300HT Real Time PCR system (Applied Biosystems, Grand Island, NY). Fold changes were calculated using the ∆∆Ct method. miRNA expression was normalized to Snord72 as its expression not affected by NM administration.

first: Smith

last: Ley


title: Shh is a determinant of gammadelta T-cell development in the thymus

first: Tessa

last: Crompton

address: 30 Guilford Street

department: Immunobiology

institute: UCL

summary: We compared gammadelta T-cell development and subsets in the thymus of mice in which Shh had been conditionally deleted from thymic epithelial cells using FoxN1-Cre to WT mice, and to transgenic mice in which a constitutively activator (Gli2DN2) or constitutuvely repressor (Gli2DC2) form of Gli2 were expressed under the control of the lck-promotor.  We sorted CD27+CD3+TCR-gammadelta+ cells from 4 week old mice and comapred gene expression by RNAsequencing.  All mice were on a C57BL/6 background.

overall_design: Wildtype, Gli2A, Gli2R and Shhfl/fl FoxN1 Cre+ (n=2) 4 week old thymi were sorted for CD3+CD27+gdTCR+ cells. RNA extracted from these cells was sequenced to help understand the transcriptional changes governing gd T cell development.

first: Kostas

last: Mengrelis


title: Healthy peripheral neutrophils total RNA-Seq: Serum Amyloid P component is an essential element of resistance against Aspergillus fumigatus

city: Pieve Emanuele

first: sarah

middle: natalia

last: mapelli

state: Milan

address: Via Rita Levi Montalcini 4

department: Experimental Immunopathology

country: Italy

institute: Humanitas research

summary: Serum amyloid P component (SAP; APCS gene, also known as Pentraxin 2, PTX2), is a component of the humoral arm of innate immunity involved in resistance to bacterial infection and regulation of tissue remodeling. The present study was designed to investigate the role of SAP/PTX2 in antifungal resistance. SAP-deficient Apcs-/- mice showed a dramatic increase in susceptibility to A. fumigatus infection. The defective anti-fungal resistance of gene targeted mice was rescued by in vitro opsonization of A.  fumigatus conidia and in vivo administration of murine SAP. Mouse and human SAP bound conidia, activated the complement cascade and increased phagocytosis by mouse and human neutrophils. Defective resistance of Apcs-/- SAP-deficient mice was associated with defective in vivo recruitment of neutrophils and phagocytosis in the lungs. The opsonic activity of SAP was dependent on the classical pathway of complement activation. In immunosuppressed mice, SAP administration protected hosts against A. fumigatus infection and death. In the context of a study of hematopoietic stem-cell transplantation,  genetic variation in the human APCS gene was associated with susceptibility to invasive pulmonary aspergillosis. Thus, SAP is a fluid phase pattern recognition molecule essential for resistance against A. fumigatus. The results reported here also suggest that SAP has therapeutic potential against an infectious agent which represents a formidable clinical challenge.

overall_design: Whole transcriptome of sorted neutrophils derived from three healthy donors peripheral blood.

first: Sarah

middle: N

last: Mapelli

first: Cecilia

last: Garlanda

first: Alberto

last: Mantovani

first: Andrea

last: Doni


title: Loss of Down syndrome critical region-1 leads to cholesterol metabolic dysfunction that exaggerates hypercholesterolemia in ApoE-null background

city: Kumamoto

first: Takashi

last: Minami

country: Japan

institute: Kumamoto University

summary: Down syndrome critical region (DSCR)-1functions as a feedback modulator for calcineurin- nuclear factor for activated T cell(NFAT) signals, which are crucial for cell proliferation and inflammation. Stable expression of DSCR-1 inhibits pathological angiogenesis and septic inflammation. DSCR-1 also plays a critical role in vascular wall remodeling associated with aneurysm development that occurs primarily in smooth muscle cells. Besides, Dscr-1 deficiency promotes the M1-to M2-like phenotypic switch in macrophages, which correlates to the reduction of denatured cholesterol uptakes. However, the distinct roles of DSCR-1 in cholesterol and lipid metabolism are not well understood. Here, we show that loss of apolipoprotein (Apo) E in mice with chronic hypercholesterolemia induced Dscr-1expression in the liver and aortic atheroma. InDscr-1-null mice fed a high-fat diet, oxidative- and endoplasmic reticulum (ER)-stress was induced and sterol regulatory element-binding protein (SREBP) 2production in hepatocytes was stimulated. This exaggerated ApoE-/--mediated nonalcoholic fatty liver disease (NAFLD) and subsequent hypercholesterolemia. Genome wide screening revealed that loss of both ApoE and Dscr-1 resulted in the induction of immune- and leukocyte activation-related genes in the liver compared to ApoE deficiency alone. However, expression of inflammation-activated markers and levels of monocyte adhesion were suspended upon induction of the Dscr-1 null background in the aortic endothelium. Collectively, our study shows that the combined loss of Dscr-1 and ApoE causes metabolic dysfunction in the liver but reduces atherosclerotic plaques, thereby leading to a dramatic increase in serum cholesterol and the formation of sporadic vasculopathy.

overall_design: We analyzed genome wide gene expression of liver among wild type, Dscr-1 knockout, ApoE knockout and Dscr-1; ApoE double-knockout mice with two independent experiments. Sample number was 8 samples. In these experiments, wild type was used as control. Moreover, we analyzed endothelial cells derived from aorta of Dscr-1 knockout mice or Dscr-1; ApoE double -knockout mice with two independent experiments. Sample number was four. In thees experiments, Dscr-1 was used as control.

first: Masashi

last: Muramatsu

first: Tsuyoshi

last: Osawa

first: Yuri

last: Miyamura

first: Suguru

last: Nakagawa

first: Toshiya

last: Tanaka

first: Tatsuhiko

last: Kodama

first: Hiroyuki

last: Aburatani

first: Juro

last: Sakai

first: Sandra

last: Ryeom


title: Seq-Scope: Submicrometer-resolution spatial barcoding technology that enables microscopic examination of tissue transcriptome at single cell and subcellular levels

city: Ann Arbor

first: Jun Hee

last: Lee

state: MI

address: 109 Zina Pitcher Pl

institute: University of Michigan

summary:
Purpose: we propose Sequence-Scope (Seq-Scope), which can generate ultra-high definition images of sequence-based molecular signatures resolved at a submicrometer scale.
Experimental Methods: Seq-Scope experiment is divided into two consecutive sequencing steps: 1st-Seq and 2nd-Seq. 1st-Seq of Seq-Scope starts with the solid-phase amplification of a single-stranded synthetic oligonucleotide library using an Illumina sequencing-by-synthesis (SBS) platform. 2nd-Seq of Seq-scope begins with overlaying the tissue section slice onto the HDMI-array.    Computational Methods: Tissue boundaries are detected by using a custom python code to draw a smoothed density plot to visualize the density of HiSeq reads in a given XY space of each tile. Digital gene expression (DGE) matrices are  generate using STAR/STARsolo 2.7.5c with Gene,GeneFull, Velocyto, and polyAtrimming options.  Data binning is performed by dividing the imaging space into 100 μm2 square grid with 10 μm simple side or 25 μm2 square grid with 5 μm side and collapsing all HDMI-UMI information into one barcode. Binned DGE matrix was analyzed in the Seurat v4 package for clustering analysis.

sample_organism:
Mus musculus
synthetic construct

platform_organism:
Mus musculus
synthetic construct

overall_design: A total of 2 Seq-Scope experiments (colon and liver) were run. Livers were collected from 8 week-old control (Depdc5F/F/Tsc1F/F, male) and TD (Alb-Cre/Depdc5F/F/Tsc1F/F, female) mice. Colons are from 8-week-old C57BL/6 wild-type male mice.

first: Chun-Seok

last: Cho

first: Jingyue

last: Xi

first: Yichen

last: Si

first: Jun

middle: H

first: Hyun

last: Kang

first: Sung-Rye

last: Park

first: Jer-En

last: Hsu

first: Myungjin

last: Kim

first: Goo

last: Jun


title: Transcriptome Analysis of HMGB1 knock-out iSLK BAC16 cell (2)

city: Daejeon

first: Yun Hee

address: 77 Gyeryong-ro 771 beon-gil, Jung-gu

department: Eulji Biomedical Science Research Institute

country: South Korea

institute: Eulji University School of Mdicine

summary: Purpose: RNA sequencing (RNA-seq) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis. In addition, we have already registered the transcriptome analysis results of HMGB1 KO iSLK BAC16 cell in GSE157275, and this registration is to reproduce it.

overall_design: HMGB1 WT or KO iSLK BAC16 cells transcriptome were analyzed by RNA seq. cells were or were not induced KSHV lytic replication with doxycycline and sodium butyrate.

first: Su-Kyung

first: Myung-Shin


title: mRNA sequencing of esophageal cells and columnar cells from different tissues

first: Alizee

last: Vercauteren Drubbel

summary: In this study, we used RNA sequencing to characterize esophageal progenitors following activation of the hedgehog (HH) pathway in vivo. We observed two main fates following activation of the HH pathway in the squamous epithelium: one population is in an intermediate state between squamous and columnar epithelium, and another achieves a full columnar conversion. We compared these 2 populations to different FACS sorted epithelial cells from different tissues of the gastro-intestinal tract (adult esophagus, embryonic esophagus, transition epithelium from the stomach,  corpus of the stomach and small intestine). Our results suggest a multistep process in which esophageal progenitors first turn on a transcriptomic program that resembles the one from embryonic esophagus, then, a subset of these dedifferentiated cells can turn on a columnar differentiation program that shares similarities with intestinal cells. Collectively, these data demonstrate that some esophageal cells can be reprogrammed to generate columnar cells in vivo.

overall_design: Oesophageal mRNA profiles of wild type (WT), K5SmoM2 and K5SmoM2-K8YFP mice by deep sequencing using Illumina

first: Benjamin

last: Beck

first: Alizée


title: Induction of dorsal mesodermal genes by pinhead and chordin

city: New York

first: Keiji

last: Itoh

state: NY

address: One Gustave L Levy Place, Box 1020, ANBG 25-40

department: Cell, Developmental and Regenerative Biology

institute: Icahn School of Medicine at Mount Sinai

summary: Xenopus embryonic ectodermal cells are responsive to various inducing factors.  Mesoderm is specified and patterned by extracellular factors including FGF, Nodal, BMP and Wnt families.  Pinhead is another secreted protein implicated in mesoderm formation.  We found that Pinhead physically interacts with and antagonizes ADMP (anti-dorsalizing morphogenetic protein) that acts as BMP-like protein to promote ventral mesoderm formation. ADMP and BMPs have been shown to cooperate to activate phospho-Smad1 signaling to lead to ventral mesoderm development.  Since Chordin is a BMP antagonist, we hypothesized that Pinhead together with Chordin can promote downregulation of phospho-Smad1 signaling that leads to dorsal mesoderm development.  RNA sequencing revealed that Pinhead and Chordin synergize in dorsal mesoderm formation in ectodermal explants.

overall_design: Eight samples are collected for RNAseq from four groups with duplicates. One group is control ectodermal explants (Co), the second is Flag-Pnhd expressing explants (Fpinh), the third is Chordin expressing explants (Chor) and the fourth is Pinhd and chordin expressing explants (Chor-Pinh).

first: Olga

last: Ossipova

first: Sergei

middle: Y

last: Sokol


title: A Pig BodyMap Transcriptome Reveals Diverse Tissue Physiologies and Evolutionary Dynamics of Transcription [non-human Hi-C]

city: Chengdu

first: jin

last: long

address: Hui ming

country: China

institute: Sichuan Agricultural University

summary: Comprehensive transcriptomic survey of the pig (Sus scrofa) may lead to a better understanding of mechanisms of tissue specialization that underlie economic traits of this species and accelerate its use as a biomedical model. Here, we characterized four distinct transcript types (lncRNAs, TUCPs, miRNAs and circRNAs) in 31 adult pig tissues and two cell lines, together with protein-coding genes. We dissected their distinct structural and transcriptional features and uncovered transcriptome variability as related to tissue physiology. We discovered extraordinary diversity among 47 anatomically distinct skeletal muscle types, as well as among six adipose depots, which are linked to their diverse origins, metabolic features, cell composition, physical activity and mitochondrial pathways. In particular, transcription of HOX genes across skeletal muscles exhibited a position-dominant pattern, revealing a similar developmental history of these tissues within the same body part. Transcriptional patterns across adipose depots demonstrated a metabolically protective role of subcutaneous adipose tissue and the association of visceral adipose tissue with metabolic dysfunction. Comparative analysis of the transcriptomes of seven tissues of the pig and nine other vertebrates revealed insights into evolutionary divergence of transcription that contributes to lineage-specific tissue biology. We also analyzed long-range regulation of promoters by their enhancers with downstream transcription in subcutaneous adipose tissues of six mammals, showing that evolutionary stability of transcription can mainly be attributed to multiple enhancers buffering gene expression patterns against genetic perturbations, thereby conferring robustness during speciation. Collectively, this study offers a resource for the accelerated use of the pig as a biomedical model for human biology and disease and uncovers molecular bases of its diverse economic traits.

sample_organism:
Canis lupus familiaris
Felis catus
Sus scrofa
Ovis aries
Oryctolagus cuniculus
Rattus norvegicus

overall_design: We generated chromatin conformation capture  sequencing (Hi-C) data of adipose tissue of rat, rabbit, pig, sheep, cat and dog with 1-6 replicates.

first: Long

last: Jin

first: Silu

last: Hu

first: Mingzhou

last: Li

first: Qianzi

last: Tang


title: New genotype of Yersinia pestis found in live rodents in Yunnan Province, China

city: Beijing

first: Jingliang

last: Qin

address: Fengtai East Street

country: Zimbabwe

institute: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology

summary: Yunnan Province, China is thought to be the original source of biovar Orientalis of Yersinia pestis, the causative agent of the third plague pandemic that has spread globally since the end of the 19th century. Although encompassing a large area of natural plague foci, Y. pestis strains have rarely been found in live rodents during surveillance in Yunnan, and most isolates are from rodent corpses and their fleas. In 2017, 10 Y. pestis strains were isolated from seven live rodents and three fleas in Heqing County (HQ) of Yunnan. These strains were supposed to have low virulence to local rodents Eothenomys miletus and Apodemus chevrieri because the rodents were healthy and no dead animals were found in surrounding areas, as had occurred in previous epizootic disease. We performed microscopic and biochemical examinations of the isolates,and compared their whole-genome sequences and transcriptome with those of 10 high virulence Y. pestis strains that were isolated from the adjacent city (Lijiang). We analyzed the phenotypic, genomic, and transcriptomic characteristics of live rodent isolates. The isolates formed a previously undefined monophyletic branch of Y. pestis that was named 1.IN5. Six SNPs, two indels, and one copy number variation were detected between live rodent isolates and the high virulence neighbors. No obvious functional consequence of these variations was found according to the known annotation information. Among the genes that were differentially expressed between the live rodent isolates and their high virulence neighbors, we detected five iron transfer-related genes that were significantly up-regulated in live rodent isolates compared with high virulence isolates (|log2 (FC) | >1, p.adjust <0.05), indicating these genes may be related to the low-virulence phenotype. The novel genotype of Y. pestis reported here provides further insights into the evolution and spread of plague as well as clues that may help to decipher the virulence mechanism of this notorious pathogen.

overall_design: Examination of four Yersinia pestis isolates from two types of natural plague foci

first: Liyuan

last: Shi

first: Peng

last: Wang


title: A single cell atlas of human cornea that defines its development, limbal progenitor cells and their interactions with the immune cell

city: Newcastle Upon Tyne

first: Majlinda

last: Lako

address: International Centre for Life, Central Parkway

department: Institute of Genetic Medicine and Institute for Ageing

institute: Newcastle University

summary: To study the development and composition of human ocular surface, we performed single cell (sc) RNA-Seq at key embryonic, fetal and adult stages and generated the first atlas of the corneal and conjunctival cell types from development to adulthood. The conjunctival epithelium is the first to be specified in the epithelial layer , followed by the corneal epithelium and the establishment of proliferative epithelial progenitors, which predate the formation of limbal niche by a few weeks. Bioinformatic comparison of adult cell clusters identified GPHA2,a novel cell-surface marker for quiescent limbal stem cells (qLSCs), whose function is to maintain qLSCs self-renewal. Combining scRNA- and ATAC-Seq analysis, we identified multiple upstream regulators for qLSCs and transit amplifying (TA) cells and demonstrated a close interaction between the immune cells and epithelial stem and progenitor cells in the cornea. Single cell RNA-Seq analysis indicated loss of qLSCs and acquisition of proliferative limbal epithelial progenitor markers during limbal epithelial cell expansion: this phenomenon was independent of culture method used. Extending the single cell analyses to Keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of TA cells in the corneal epithelium as two key mechanistic changes underlying the disease phenotype. Our scRNA-Seq data comparisons of developing and adult cornea and conjunctiva provide a unique resource for defining pathways/genes that could lead to improvement in ex vivo expansion methods for cell based replacement therapies and better understanding and treatment of ocular surface disorders.

overall_design: scRNA-Seq and scATAC-Seq of 17 human developing cornea/conjunctiva (10-21 post-conception weeks), 4 adult cornea/conjunctiva and 8 human cornea-scleral rings

first: Joseph

last: Collin

first: Rachel

last: Queen

first: Darin

last: Zerti

first: Nicky

last: Moyse

first: Sanja

last: Bojic

first: Marina

last: Molina

first: Gary

last: Reynold

first: Chunbo

last: Yang

first: Rafiqul

last: Hussain

first: Jonathan

last: Coxhead

first: Steven

last: Lisgo

first: Deborah

last: Henderson

first: Agatha

last: Joseph

first: Paul

last: Rooney

first: Saurabh

last: Ghosh

first: Che

last: Connon

first: Muzlifah

last: Haniffa

first: Francisco

last: Figueiredo

first: Lyle

last: Armstrong


title: Specific translation during aging mediated by phosphorylation of Schizosaccharomyces pombe eIF2alpha

city: Cantoblanco

first: Juan

middle: Carlos

last: Oliveros

state: Madrid

address: Darwin 3

country: Spain

institute: CNB, CSIC

summary:
Aging is a highly regulated process in eukaryotes. Many biochemical pathways are affected by aging and, among them, protein homeostasis is one key process.
Our hypothesis is that protein translation may be important to maintain a correct balance of active, properly folded proteins in the cell and, therefore, help supporting a normal longevity.
Phosphorylation of eIF2alpha is one of the most important regulatory steps in protein translation in eukaryotes, reducing total protein translation and promoting the specific production of some proteins (Gcn4, Atf4, etc)
We decided to test whether eIF2alpha phosphorylation has specific roles in protein translation during aging in Schizosaccharomyces pombe

overall_design: We took samples of fission yeast cells at exponential phase (0 hours) or at the early entry into stationary phase (6 hours) and analysed total RNA and mRNAs bound to polysomes in wild type and eIF2alpha.S52A mutant strain

first: Tamara

last: Jimenez-Saucedo

middle: J

last: Berlanga-Chiquero

first: Miguel

middle: A

last: Rodríguez-Gabriel


title: Elucidation of the host bronchial lymph node miRNA transcriptome response to bovine respiratory syncytial virus

city: Dunsany

first: Dayle

last: Johnston

state: Ireland

address: AGRIC

department: Animal and Bioscience Research Department

country: Ireland

institute: Teagasc

summary: Bovine Respiratory Disease (BRD) is responsible for the majority of calf morbidity and mortality. One of the main causes of BRD is initial infection with Bovine Respiratory Syncytial Virus (BRSV). The objective of this study was to elucidate the changes in the host's bronchial lymph node miRNA transcriptome in response to BRSV in a controlled viral challenge. Holstein-Friesian calves were either inoculated with BRSV (103.5 TCID50/ml x 15 ml) (n=12) or mock challenged with phosphate buffered saline (n=6). Clinical signs were scored daily until euthanasia at day 7 post-challenge. Total RNA was extracted and sequenced (75 bp) from bronchial lymph nodes. Sequences were filtered for contaminant short RNA species, and subsequently read counts for known miRNAS were generated using the miRDeep2 package with the UMD 3.1 version of the genome and the bovine mature miRNA sequences from the miRBase database (release 22). EdgeR was used for differential expression analysis and Targetscan was used to identify target genes for the differentially expressed (DE) miRNAs. Pathway and gene ontology analyses on target genes were carried out using Ingenuity Pathway analysis (Qiagen).  There was a clear separation between BRSV challenged and control calves based on miRNA gene expression changes, despite an observed mild clinical manifestation of the disease. There were 119 DE miRNAs (p<0.05, FDR<0.1, fold change>1.5) between the BRSV challenged and control calves. The DE miRNAs were predicted to target 465 genes which were previously found to be DE in bronchial lymph node tissue, between these BRSV challenged and control calves. Out of the DE predicted target genes, 455 genes had inverse fold changes to that of the DE miRNAs. There were 8 enriched pathways among the DE predicted target genes with inverse fold changes to that of the DE miRNA including: granulocyte and agranulocyte adhesion and diapedesis, interferon signaling and role of pathogen recognition receptors in recognition of bacteria and viruses. Functions predicted to be increased included: T cell response, apoptosis of leukocytes, immune response of cells and stimulation of cells. Pathogen recognition and proliferation of cytotoxic T cells are vital for the recognition and subsequent elimination of the virus.

overall_design: Examination of a single tissue type (broncial lymph node) in two different treatment groups, BRSV challenged calves (n=12) and control calves (n=6)

first: Bernadette

last: Earley

first: Matthew

last: McCabe

first: JaeWoo

last: Kim

first: Jeremy

middle: F

last: Taylor

first: Ken

last: Lemon

first: Catherine

last: Duffy

first: Michael

last: McMenamy

first: S

middle: L

last: Cosby

first: Sinead

last: Waters


title: Highly recurrent CBS epimutations in gastric cancer CpG island methylator phenotypes and inflammation

city: Singapore

first: Arnoud

last: Boot

state: Singapore

address: 8 college road

country: Singapore

institute: Duke-NUS Medical School

summary: CIMP (CpG island methylator phenotype) is an epigenetic molecular subtype, observed in multiple malignancies and associated with the epigenetic silencing of tumour suppressors. Currently, for most cancers including gastric cancer [GC], mechanisms underlying CIMP remain poorly understood. We sought to discover molecular contributors to CIMP in GC, by performing global DNA methylation, gene expression, and proteomics profiling across 14 gastric cell lines, followed by similar integrative analysis in 50 GC cell lines and 467 primary GCs. We identify the cystathionine beta-synthase enzyme (CBS) as a highly recurrent target of epigenetic silencing in CIMP GC. Likewise, we show that CBS epimutations are significantly associated with CIMP in various other cancers, occurring even in premalignant gastroesophageal conditions and longitudinally linked to clinical persistence. Of note, CRISPR deletion of CBS in normal gastric epithelial cells induces widespread DNA methylation changes that overlap with primary GC CIMP patterns. Reflecting its metabolic role as a gatekeeper interlinking the methionine and homocysteine cycles, CBS loss in vitro also causes reductions in the anti-inflammatory gasotransmitter hydrogen sulphide (H2S), with concomitant increase in NF-κB activity.  In a murine genetic model of CBS-deficiency, preliminary data indicate upregulated immune-mediated transcriptional signatures in the stomach.  Our results implicate CBS as a bi-faceted modifier of aberrant DNA methylation and inflammation in GC and highlights H2S donors as a potential new therapy for CBS-silenced lesions.

overall_design: Timecourse measurement of DNA methylation changes in CBS knockout cell lines and controls.

first: Nisha

last: Padmanabhan

first: Patrick

last: Tan


title: A novel minor spliceosome component required for splicing of AT-AC introns

city: Helsinki

first: Marco

last: Savarese

address: Topeliuksenkatu 20

department: Samfundet Folkhälsan i svenska Finland r.f.

country: Finland

institute: Folkhälsan Research Center

summary: U12-type introns were originally recognized based on their highly conserved non-consensus AT-AC termini1,2, which are spliced by a separate minor spliceosome3,4. Padgett and Krainer groups later showed that terminal dinucleotides do not differentiate U12-type from U2-type introns, as there are U12-type introns with GT-AG termini and U2-type introns with AT-AC termini5,6. Rather, U12-type introns are recognized by their divergent and highly conserved 5’ splice site (5’ss) and branch point sequences, which both differ from the consensus sequences found in U2-type introns. To date, no functional differences have been ascribed to AT-AC or GT-AG subtypes of U12-type introns, nor have RNAseq analyses of minor spliceosome diseases reported any subtype specificity.  Here, we describe a novel protein component of the minor spliceosome, encoded by the CENATAC locus, that is required for accurate splicing of AT-AC but not GT-AG type minor introns. CENATAC was initially identified in a subset of Mosaic Variegated Aneuploidy (MVA) patients with mutations in CENATAC, which lead to chromosome congression defects during mitosis. Earlier large-scale proteomic analyses tentatively classified CENATAC as a spliceosome component and phylogenetic analyses showed co-segregation of CENATAC with minor spliceosome components. Targeted depletion of CENATAC in HeLa cells, followed by RNAseq revealed global retention of AT-AC minor subtype introns with more than 60% showing statistically significant (up to 90%) intron retention (IR). Additionally, U12-type introns with 5’-AT, but divergent 3’-terminal dinucleotides also showed significant IR. We also detected cryptic U2-type splice site activation near affected AT-AC introns. In contrast, about 10% of GT-AG subtype introns responded to CENATAC depletion. Co-IP experiments revealed that CENATAC is not a U11/U12 di-snRNP component as expected for a specificity factor, but rather associates with the U4atac/U6atac.U5 tri-snRNP via interaction with PRPF3/4, suggesting a role for minor tri-snRNP in initial 5’ss recognition. CENATAC also interacts with TXNL4B, a paralog of TXNL4A in the major tri-snRNP.  Finally, several genes encoding chromosome congression factors harbor U12 AT-AC-type introns that were highly retained in CENATAC depleted cells, potentially explaining the aneuploidy phenotype observed in MVA patients.

overall_design: Targeted depletion of CENATAC gene in HeLa cells, followed by RNAseq.

first: Bas

last: de Wolf

first: Ali

last: Oghabian

first: Sandra

last: Hanks

first: Jolien

last: van Hooff

first: Maureen

last: Akinyi

first: Laura

last: van Rooijen

first: Jens

last: Verbeeren

first: Esther

last: Uijttewaal

first: Lisa

last: van Voorthuijsen

first: Michiel

last: Vermeulen

first: Berend

last: Snel

first: Nazneen

last: Rahman

first: Mikko

last: Frilander

first: Geert

last: Kops


title: RNA-seq and ATAC-sequencing of esophageal cells after HH pathway activation

first: Alizee

last: Vercauteren Drubbel

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

overall_design: Refer to individual Series


title: Phase separation drives aberrant chromatin looping and cancer development [RNA-Seq]

city: Chapel Hill

first: Gang

middle: Greg

last: Wang

state: NC

address: 450 West Drive

department: Dept of Biochemistry and Biophysics

institute: UNC Lineberger Comprehensive Cancer Center

summary: Development of cancer is intimately associated with genetic abnormalities that target proteins with intrinsically disordered regions (IDRs).  In human hematological malignancies, recurrent chromosomal translocation of nucleoporin (NUP98 or NUP214) generates an aberrant chimera that invariably retains nucleoporin?s IDR, tandemly dispersed phenylalanine-and-glycine (FG) repeats. However, it remains elusive how unstructured IDRs contribute to oncogenesis. We show that IDR harbored within NUP98-HOXA9, a homeodomain-containing transcription factor (TF) chimera recurrently detected in leukemias, is essential for establishing liquid-liquid phase separation (LLPS) puncta of chimera and for inducing leukemic transformation. Strikingly, LLPS of NUP98-HOXA9 not only promotes chromatin occupancy of chimera TFs but is also required for formation of a broad, ?super-enhancer?-like binding pattern, typically seen at a battery of leukemogenic genes, potentiating their transcriptional activation. Artificial HOX chimera (FUS-HOXA9), created by replacing NUP98?s FG repeats with an unrelated LLPS-forming IDR of FUS, had similar enhancement effects on chimera?s genome-wide binding and target gene activation. Hi-C mapping further demonstrated that phase-separated NUP98-HOXA9 induces CTCF-independent chromatin looping enriched at proto-oncogenes. Together, this report describes a proof-of-principle example wherein cancer acquires mutation to establish oncogenic TF condensates via phase separation, which simultaneously enhances their genomic targeting and induces organization of aberrant three-dimensional chromatin structure during tumorous transformation. As LLPS-competent molecules are frequently implicated in diseases, this mechanism can potentially be generalized to many malignant and pathological settings.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Differential expression analysis of transcriptome profiles in HEK293 or primary murine hematopoietic stem/progenitor cells (HSPCs) with stable expression of empty vector, wildtype NUP98-HOXA9, FS mutant NUP98-HOXA9, wildtype FUS-HOXA9 and YS mutant FUS-HOXA9.

first: Jeong

last: Ahn

first: Greg

first: Hidetaka

last: Uryu


title: Activation of ERRγ enhances mitochondrial and sarcomere maturation in human induced pluripotent stem cell-derived cardiomyocytes

city: Kyoto

first: Yoshinori

last: Yoshida

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

institute: Kyoto University

summary: We identified two cardiac maturation stimulators from 9,048 compounds using Troponin I1 (TNNI1) and Troponin I3 (TNNI3) double reporter  hiPSC-derived CMs. An estrogen-related receptor gamma (ERRγ)-specific agonist increased the size of hiPSC-CMs and their mitochondrial content and enhanced their mitochondrial function. In addition, combination of this agonist with an inhibitor of S-phase kinase-associated protein 2 (SKP2) increased the TNNI3 expression more highly than previous methods and enhanced cardiac maturation-related gene expression profiles.

overall_design: mRNA profiles of hiPSC-CMs with conpounds were generated by deep sequencing, in triplicate, using Illumina Nextseq 500.

first: K

last: Miki

first: T

last: Monjo

last: Andou

first: M

last: Sasaki

first: A

last: Lucena-Cacace

last: Nishimoto

first: Y


title: ATAC sequencing of esophageal cells from wild type and Krt5SmoM2 mice

institute: Université Libre de Bruxelles

summary: In this study, we used ATAC sequencing to compare the chromatin landscape of esophageal cells in which hedgehog (HH) pathway has been activated (K5SmoM2), to control esophageal cells (WT). We identified more than 50% of the peaks differentially opened in both conditions. Among these differentially opened peaks in EpHI cells, we identified chromatin regions in key columnar markers. Taken together these results show that HH pathway activation triggers chromatin landscape remodeling to facilitate transcommitment.

overall_design: Examination of chromatin modifications in esophageal cells from wild type (WT) and Krt5SmoM2 mice by deep sequencing using Illumina

first: Benjamin

last: Beck

first: Alizée


title: Phase separation drives aberrant chromatin looping and cancer development [ChIP-Seq]

overall_design: We compared the genome-wide binding of wildtype (WT) versus IDR-mutated NUP98-HOXA9 and FUS-HOXA9 fusion in the 293 cells. Please note that the GLE2-binding sequence (GLEBS) was deleted from NUP98-HOXA9 (also termed as N-IDR/A9 in the study) and that FUS-HOXA9 was created by in-frame fusion between an unrelated IDR of FUS (amino acids 1-215 that can phase separate) and the HOXA9 homeodomain (also termed as F-IDR/A9 in the study). Phase-separation-disrupting mutations of NUP98?s IDR and FUS? IDR are Phenylalanine-to-Serine (F->S or FS) substitutions at all NUP98 FG-repeats and Tyrosine-to-Serine (Y->S or YS) substitutions at all FUS YG/S sites, respectively. ChIP-seq was conducted with either HA or GFP antibodies in the independently derived cells that express either 3xHA-3xFLAG-tagged or GFP-tagged NUP98-HOXA9, and cells expressing empty vector (EV) served as a negative control. The binding of both WT and IDR-mutated NUP98-HOXA9 (GFP-tagged) was assessed upon cell treatment with 10% of 1,6-Hexanediol for one minute, in comparison to mock treatment. We also examined the genome-wide H3K27 acetylation (H3K27ac) and CTCF binding in the 293 cells that express WT or IDR-mutated NUP98-HOXA9 (3xHA-3xFLAG-tagged). Genome-wide binding of NUP98-HOXA9 (GFP-tagged) was examined in primary murine hematopoietic stem/progenitor cells (HSPCs) transformed by this chimera.Comparison of the genome-wide binding of the wild type (WT) or phase-separation-defective mutant form of NUP98-HOXA9 and FUS-HOXA9 chimeric fusion in the 293 stable expression cells. Here, cells with empty vector (EV) are used as negative control, and the mutant forms of NUP98-HOXA9 and FUS-HOXA9 fusion are F-to-S (FS) and Y-to-S (YS), respectively, which disrupt phase-separation ability harbored within the intrinsically disorganized region (IDR) of NUP98 and FUS. As for NUP98-HOXA9 fusion, the used WT and mutant forms are termed as N_IDR_WT and N_IDR_FS, respectively; as for FUS-HOXA9 fusion, the used WT and mutant forms are termed as F_IDR_WT and F_IDR_YS, respectively. In order to disrupt phase separation,  cells are treated with 10% of hexanediol for 1 minute. Fusion proteins are tagged with either HA or GFP tag. These cells are  examined by ChIP-seq for their genome-wide patterns of fusion proteins (HA or GFP), H3K27 acetylation (H3K27Ac) or CTCF.


title: OAS1 and OAS3 negatively regulate the expression of chemokines and interferon-responsive genes in human macrophages

city: Seoul

first: Young-Joon

last: Kim

address: Yonsei-ro 50

institute: Yonsei university

summary: We report analysis of mRNA profiles from wild-type and OAS1 and OAS3-KO human cell lines stimulated with PolyI:C.

overall_design: THP1 mRNA profiles of wild type (WT) and OAS1 and OAS3-KO were generated by sequencing, using Illumina HiSeq-2500.

first: Wook-Bin

last: Lee

first: Won

last: Choi


title: H3K9 methyltransferases and demethylases control lung tumor-propagating cells and lung cancer progression (experiment 2)

first: Hideo

last: Watanabe

address: One Gustave L. Levy Place

department: Department of Medicine, Division of Pulmonary, Critical Care and Sleep Medicine

summary: We have identified the histone methyltransferases G9a/Glp as suppressors of aggressive lung tumor-propagating cells (TPCs). Chemically inhibiting G9a/Glp promoted TPC phenotypes in lung adenocarcinoma cells, and caused chromatin changes at genes associated with the differentiation of stem cells. G9a/Glp inhibition in lung progenitor cell organoid cultures disrupted alveolar differentiation. Depleting G9a during tumorigenesis enriched for TPCs, accelerating disease progression and metastasis.  Demethylase inhibition decreased lung adenocarcinoma progression in vivo.

overall_design: Genome-wide maps of histone H3K27 acetylation and H3K9 dimethylation in CK1750 cells treated with either DMSO or a G9a/GLP inhibitor, UNC0638.

first: Sam

last: Rowbotham

first: Carla


title: Transcriptomic analysis for the identification of metabolic pathway genes related to toluene response in Ardisia pusilla

status: Public on Apr 18 2021

city: Wanju-gun

first: Su Young

state: Jeollabuk-do

address: 100, Nongsaengmyeong-ro, Iseo-myeon

department: Floriculture Research Division, National Institute of Horticultural and Herbal Science

institute: Rural Development Administration

summary: Toluene is an industrial raw material and solvent that can be found abundantly in our daily life products. The amount of toluene vapor is one of the most important measurements for evaluating air quality. The evaluation of toluene scavenging ability of different plants has been reported, but the mechanism of plant response to toluene is only partially understood. In this study, we performed RNA sequencing (RNA-seq) analysis to detect differential gene expression in toluene-treated and untreated leaves of Ardisia pusilla. A total of 88,444 unigenes were identified by RNA-seq analysis, of which 49,623 were successfully annotated and 4,101 were differentially expressed. Gene ontology analysis revealed several sub-categories of genes related to toluene response, including cell part, cellular process, organelle, and metabolic processes. We mapped the main metabolic pathways of genes related to toluene response and found that the differentially expressed genes were mainly involved in glycolysis/gluconeogenesis, starch and sucrose metabolism, glycerophospholipid metabolism, carotenoid biosynthesis, phenylpropanoid biosynthesis, and flavonoid biosynthesis. In addition, 53 transcription factors belonging to 13 transcription factor families were identified. We verified 10 differentially expressed genes related to metabolic pathways using quantitative real-time PCR and found that the results of RNA-seq were positively correlated with them, indicating that the transcriptome data were reliable. This study provides insights into the metabolic pathways involved in toluene response in plants.

overall_design: mRNA profiles of toluene-treated and untreated A. pusilla.

first: Su

first: Junping

last: Xu


title: Lung resident memory B cells protect against bacterial pneumonia

city: Boston

first: Kimberly

middle: Alynn

last: Barker

state: MA

address: 72 E Concord St., R205

department: Pulmonary Center

institute: Boston University School of Medicine

summary: Lung resident memory B cells (BRM cells) are elicited after influenza infections of mice, but connections to other pathogens and hosts have yet to be determined, as has their functional significance. We postulate that BRM cells are core components of lung immunity. To test this, we examined whether lung BRM cells are elicited by the respiratory pathogen pneumococcus, are present in humans, and are important in pneumonia defense. Lungs of mice recovered from pneumococcal infections did not contain organized lymphoid structures, but did have non-circulating memory B cells that expressed distinctive surface markers (including CD69, PD-L2, CD80, and CD73) and were poised to secrete antibodies upon stimulation. Human lungs also contained B cells with a resident memory phenotype. In mice recovered from pneumococcal pneumonia, depletion of PD-L2+ B cells, including lung BRM cells, drastically compromised bacterial clearance. These data define lung BRM cells as a common feature of pathogen-experienced lungs and provide direct evidence of a role for these cells in pulmonary anti-bacterial immunity.

overall_design: Whole blood mRNA samples from 10 naïve C57BL/6 mice vs. 10 C57BL/6 mice experienced with serial intranasal exposures to Streptococcus pneumoniae

first: Joseph

middle: P

last: Mizgerd


title: Pro-cachectic factors link experimental and human chronic kidney disease to skeletal muscle wasting programs

status: Public on Apr 17 2021

city: Hamburg

first: Yu

last: Zhao

address: Falkenried 94

department: Institute of Medical Systems Biology

country: Germany

institute: University Medical Center Hamburg-Eppendorf

summary: Skeletal muscle wasting is commonly associated with chronic kidney disease (CKD), resulting in increased morbidity and mortality. However, the link between kidney and muscle function remains poorly understood. Here, we took a complementary interorgan approach to investigate skeletal muscle wasting in CKD. We identified an increased production and elevated blood levels of soluble pro-cachectic factor Activin A, directly linking experimental and human CKD to skeletal muscle wasting programs. Single cell sequencing data identified the expression of Activin A in specific kidney cell populations, namely a subpopulation of fibroblasts and cells of the juxtaglomerular apparatus. Based on our findings, we propose that persistent and increased kidney production of pro-cachectic factors combined with a lack of kidney clearance facilitate a vicious signalling kidney-muscle cycle, leading to exacerbated blood accumulation of Activin A, and thereby skeletal muscle wasting in CKD.

overall_design: Single cell sequencing of 5 week old WT and  Kif3aΔTub mice.

first: Francesca

last: Solagna

first: Caterina

last: Tezze

first: Maja

middle: T

last: Lindenmeyer

first: Shun

last: Lu

first: Guochao

last: Wu

first: Shuya

last: Liu

first: Robert

last: Mitchell

first: Charlotte

last: Mayer

first: Saleh

last: Omairi

first: Temel

last: Kilic

first: Andrea

last: Paolini

first: Olli

last: Ritvos

first: Arja

last: Pasternack

first: Antonios

last: Matsakas

first: Dominik

last: Kylies

first: Julian

last: zur Wiesch

first: Jan-Eric

last: Turner

first: Nicola

last: Wanner

first: Viji

last: Nair

first: Felix

last: Eichinger

first: Rajasree

last: Menon

first: Ina

middle: V

last: Martin

first: Barbara

middle: M

last: Klinkhammer

first: Elion

last: Hoxha

first: Clemens

last: Cohen

first: Pierre-Louis

last: Tharaux

first: Peter

last: Boor

first: Tammo

last: Ostendorf

first: Matthias

last: Kretzler

first: Marco

last: Sandri

first: Oliver

last: Kretz

first: Victor

middle: G

last: Puelles

first: Ketan

last: Patel

first: Tobias

middle: B

last: Huber


title: De novo assembly of macaque gene models

first: Qi

middle: Trinity

last: Peng

state: Beijing

address: No.5 Yiheyuan Road Haidian District

department: Institute of Molecular Medicine

institute: Peking University

summary: We reported an atlas of de novo-defined, full-length macaque gene models on the basis of single molecule long-read transcriptome sequencing (Iso-seq).

sample_organism:
Macaca mulatta
Mus musculus

platform_organism:
Macaca mulatta
Mus musculus

overall_design: We performed Isoform sequencing (Iso-seq) in 13 PacBio Sequel cells to profile the poly(A)-positive RNAs derived from macaque frontal cortex, heart and testis. We also performed Isoform sequencing (Iso-seq) in 1 PacBio Sequel cells to profile the poly(A)-positive RNAs derived from mouse brain.


title: An inter-dependent network of enhancers regulates INK4a/ARF locus

city: Bangalore

first: Bharath

last: Saravanan

state: Karnataka

address: Bellary Road

department: Genetics and Development

country: India

institute: National centre for Biological Sciences

summary: By functionally dissecting  densest enhancer cluster in the gene desert at 9P21 locus, we identified a non-redundant inter-dependent enhancer network that functions over long distances, the perturbation in any enhancer in the network results in the complete collapse of entire enhancer cluster and target genes activity. The enhancer network can be targeted to regulate INK4a/ARF locus in associated pathophysiologies and cancers.

overall_design: The inter-dependent enhancer network was identified by series of deletions of enhancers that interacted with CDKN2A promoters.The RNA-seq upon these deletions displays the GO terms that are directly related to Coronary artery disease and Typ2-diabetes.

first: Dimple

last: Notani

first: Umer

last: Farooq


title: Methylation-induced conformational switch enables ERG oncogenic activation and  prostate cancer progression

status: Public on Apr 16 2021

city: Pieve Emanuele

first: sarah

middle: natalia

last: mapelli

address: Via Rita Levi Montalcini 4

institute: Humanitas research

summary: The TMPRSS2-ERG gene fusion is the most frequent alteration observed in human prostate  cancer but its role in disease progression is still debated. In this study, we uncovered a novel  molecular mechanism promoting progression in ERG-fusion positive prostate cancer. We show  that ERG is methylated by Enhancer of zest homolog 2 (EZH2) at a specific lysine residue    (K362) located within the internal auto-inhibitory domain. Mechanistically, K362 mono-  methylation prevents intra-domain interactions, favors DNA binding and promotes ERG    transcriptional and oncogenic activity in cellular and mouse models. Consistently with the  involvement in ERG oncogenesis, we found that K362 methylation was associated with disease  progression in ERG transgenic mouse models and was enhanced by PTEN deficiency and AKT  activation, which promoted EZH2 substrate switching from histone H3K27 to ERG.  Conversely, EZH2 inhibition blocked ERG methylation along with ERG-induced  transcriptional and phenotypic reprogramming in cell cultures and ERG/PTEN mice. We found  that ERG and EZH2 co-occupy several genomic regions forming prevalently co-activating  complexes. The network of ERG/EZH2 co-regulated target genes was enriched of functionally  aggressive features and was associated preferentially with concomitant ERG gain and PTEN  loss, castration-resistance and adverse clinical outcome in prostate cancer patients. Collectively,  these findings identify ERG methylation as a novel post-translational modification sustaining  disease progression in ERG-positive prostate cancers. Our data also provide an attractive  rationale for developing molecularly targeted therapeutics to antagonize ERG oncogenic  activity.

overall_design: Examination of mERG bindings in TMPRSS2/ERG positive VCaP cells.

first: Giuseppina

last: Carbone

first: Marita

last: Zoma

first: Domenico

last: Albino


title: The g-tubulin meshwork assists to the transport of cytosolic PCNA to chromatin in mammalian cells

city: Lund

first: Maria

last: Alvarado-Kristensson

address: 59 Jan Waldenstr ms street

country: Sweden

institute: Lund University

summary: Changes in the location of g-tubulin ensure cell survival and preserve genome integrity. We investigated whether the nuclear accumulation of g-tubulin facilitates the transport of proliferating cell nuclear antigen (PCNA) between the cytosolic and the nuclear compartment in mammalian cells. We found that the g-tubulin meshwork assists to the recruitment of PCNA to chromatin. Also, decreased levels of g-tubulin reduce the nuclear pool of PCNA. In addition, the g-tubulin C terminus encodes a PCNA-interacting peptide (PIP) motif, and a g-tubulin–PIP-mutant affects the nuclear accumulation of PCNA. In a cell-free system, PCNA and g-tubulin formed a complex. In tumors, there is a significant positive correlation between TUBG1 and PCNA expression. Thus, we report a novel mechanism that constitutes the basis for tumor growth by which the g-tubulin meshwork maintains indefinite proliferation by acting as an opportune scaffold for the transport of PCNA from the cytosol to the chromatin.

overall_design: The experiment was preformed in one cell line and a total number of 22 samples were included.There are two samples for each antibody (gamma tubulin, macrohistone2A and PCNA). For gamma tubulin rhere is 2 samples for G0/G1, 0h, 3h, 5h, 7h, 9h and for MH2A and PCNA the timepoints are 3h and 5h and as control, there are 2 whole cell lysate (Input) samples  included.

last: Kristensson


title: Hoxa10 is a functional positional memory that governs stem cell function in adult skeletal muscle

city: Kashiwa

first: Fumihito

last: Miura

state: Chiba

address: Kashiwanoha 5-1-5

department: Graduate School of Frontier Sciences

institute: The University of Tokyo

type: Methylation profiling by high throughput sequencing

summary: Skeletal muscle is contractile tissue distributed throughout the body with functionally heterogeneous properties that may relate to region-specific pathophysiology of muscle diseases. Recent studies revealed that muscle stem cells (satellite cells) are a functional heterogeneous population in different muscles, dependent not only on fiber-types but also on embryonic origin. Homeobox (Hox) genes are key regulators of the embryonic body plan. Here, we showed that Hox-A cluster genes are robustly maintained in adult muscles and their associated satellite cells of both mice and humans in a body region-specific manner, whose distribution almost recapitulates their embryonic origin. The region-specific expression of Hox genes was linked to DNA hypermethylation status of Hox-A locus. Importantly, Hox gene expression is a functional memory; Hoxa10 inactivation in satellite cells led to genomic instability and mitotic catastrophe, resulting in a decline in the regionally specific regenerative ability of muscles in mice, even though Hox paralogs are known to be often functionally redundant. Thus, our results demonstrate that the topographic Hox gene expression profile could be a geotagging molecular signature that reflects the anatomical location and embryonic history of resident muscle stem cells, potentially contributing to regionally specific regulations of adult skeletal muscles.

overall_design: Genomic DNAs were extracted from MAS and TA muscle tissues and plated satellite cells of adult mice. Libraries for whole-genome bisulfite sequencing was prepared as described in Miura F et al (2019), NAR, 47, e85

first: Kiyoshi

last: Yoshioka

first: Hiroshi

last: Nagahisa

first: Hiromitsu

last: Araki

first: Yasutomi

last: Kamei

first: Yasuo

last: Kitajima

first: Daiki

last: Seko

first: Jumpei

last: Nogami

first: Yoshifumi

last: Tsuchiya

first: Narihiro

last: Okazaki

first: Akihiko

last: Yonekura

first: Seigo

last: Ohba

first: Yoshinori

last: Sumita

first: Ko

last: Chiba

first: Kosei

last: Ito

first: Izumi

last: Asahina

first: Takashi

first: Yasuyuki

last: Ohkawa

first: Yusuke

last: Ono


title: Impact of the bone microenvironment on phenotypic plasticity of ER+ breast cancer cells [exome-seq]

city: HOUSTON

first: Igor

middle: L.

last: Bado

state: Texas

address: One Baylor Plaza, Alkek Building N1130

department: Breast Center

institute: Baylor College of Medicine

summary: Whole Exome Sequencing (WES) was used to evaluate genomic alterations between parental cells, SCPs (Single Cell-derived Subpopulations) and bone-entrained cells.

overall_design: SCP cells were generated from single clones of MCF7 cells (SCP1-4) and expanded in vitro. Bone-entrained cells  derived from IIA-induced bone metastases were passages in vitro. The MDA231-derived SCP21 was also evaluated. Exome sequencing was performed using the Nextera DNA exome Kit.

first: Xiang

last: Zhang


title: Genome architecture in definitive erythroid cells at base-pair resolution

city: Nanjing

first: Peng

last: Hua

state: Jiangsu

address: 101 Longmiandadao

department: State Key Laboratory of Reproductive Medicine and Offspring Health

institute: Nanjing Medical University

summary: By using a novel chromatin conformation capture (3C) method (Micro Capture-C (MCC)), which allows physical contacts to be determined at base-pair resolution. We demonstrate interactions between different classes of regulatory elements in unprecedented detail.

overall_design: Definitive erythroid cells were obtained from phenylhydrazine-treated mice. Spleens were mechanically disrupted to a single cell suspension in ice-cold PBS. To isolate late-stage erythroid cells, cells from a single spleen were resuspended in 5mL of cold PBS/10% FCS and stained with 50uL PE anti-Ter119 antibody (RUO, BD Biosciences) at 4°C for 30 min in the dark. After washing with 40mL of ice-cold PBS/10% FCS, cells were resuspended in 800 μL MACS buffer (PBS, 2 mM EDTA, 0.5% BSA) and 200 μL of anti-PE magnetic beads (Miltenyi Biotec) and incubated for 20 min at 4°C in the dark. Ter119 positive cells were isolated via auto-magnetic-activated cell sorting (MACS) and then processed for downstream applications. Purity of cells was confirmed by flow cytometry.


title: Human stem cell-derived pancreatic acini-like and duct-like organoids demonstrate lineage-specific differences to oncogene activation

city: San Francisco

first: Daniel

middle: N

last: Conrad

state: CA

institute: UCSF

summary: The exocrine compartment of the pancreas consisting of ducts and acini makes up most of the pancreatic tissue and is the site of origin for pancreatitis and pancreatic ductal adenocarcinoma (PDAC). Our understanding of the initiation and progression of human PDAC is limited because of challenges associated with maintaining acinar cells in culture. Here we report the commitment of human pluripotent stem cells towards pancreatic duct-like and acini-like organoids that express properties of the neonatal exocrine pancreas. The expression of PDAC-oncogenes KRasG12D or GNASR201C induced cell lineage-specific effects where GNASR201C was more effective in ductal compared to acinar organoids. KRasG12D was more effective in modeling cancer in vivo when expressed in acinar cells and was associated with acinar to ductal metaplastic changes in culture and in vivo. Thus we demonstrate lineage tropism and plasticity in the human exocrine pancreas and identify a renewable source of ductal and acinar epithelia for understanding exocrine development and diseases.

overall_design: Human embryonic stem cells were differentiated into pancreatic progenitors and then further cultured in conditions designed to generated pancreatic ductal and acinar organoids. Nuclei were isolated from progenitors and organoids at different timepoints, frozen, and then processed for multiplexed single-nucleus RNA-sequencing.

first: Ling

last: Huang

first: Zev

last: Gartner

first: Senthil

middle: K

last: Muthuswamy


title: Impact of the bone microenvironment on phenotypic plasticity of ER+ breast cancer cells

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

overall_design: Refer to individual Series


title: Impact of the bone microenvironment on phenotypic plasticity of ER+ breast cancer cells [ATAC-Seq]

summary: ATAC sequencing was performed to evaluate epigenetic changes induced by the bone microenvironment.  We found a global epigenetic reprogramming of ER+ cancer cells deriving from bone which drives stemness and endocrine resistance

overall_design: SCP2 (Single Cell-derived population #2) cells that derived from MCF7 cells were injected to bone via Intra-iliac artery injection (IIA).  SCP2-Bo cell line was generated from bone lesions and passaged in parallele with the control SCP2 cells in vitro. ATACseq was performed over multiple passages (2 to 12) on both samples.

first: Xiang H.-F


title: Impact of the bone microenvironment on phenotypic plasticity of ER+ breast cancer cells [Evolving Barcode]

summary: Evolving barcode was used to evaluate clonal expansion in bone metastases induced by intra-iliac artery injection. The assocoation of these results with ER expression revealed a non-genomic effect of the bone microenvironment on ER heterogeneity.

overall_design: ER-positive breast cancer cells (MCF7) with hgRNA-A21 and inducible Cas9 expression were inejcted to hind limb of nude mice. Cas9 expression was induced weekly and laser capture microdissection (LCM) was employed to collect independent bone lesions for targeted sequencing at week 3. All samples were processed using the TraceQC package

first: Xiang H.-F.

first: Jingyuan

last: Hu


title: Identification of dormant cells using a novel fluorescent reporter of RNA-polymerase II activity

city: Kiel

first: Seyed Mohammd Mahdi

last: Rasa

state: Schleswig-Holstein

address: Michaelisstrasse 5

department: Institut for Immunology

institute: Universitätsklinikum Schleswig-Holstein

summary: Dormancy is a reversible cellular state in which the cell cycle and metabolic activity of a cell is dramatically decreased, leading to protection from genetic damage and prolonged survival. One example of dormant cells is the somatic stem cell, which is crucial for tissue homeostasis and response to injury or transplantation. However, while dormant cells have been well characterized in some tissues, the ability to identify them irrespective of tissue of origin remains elusive. Moreover, in some tissues (e.g. the intestinal epithelium) dormant stem cells showed heterogeneity in the marker’s profile that makes their identification and isolation challenging. Here, we developed a live cell reporter of dormancy based on our observation that phosphorylation of RNA Polymerase II (RNApII), a hallmark of active mRNA transcription elongation, is largely absent in dormant stem cells from multiple lineages. We show that insertion of a short RNApII kinase target peptide directly into the yellow fluorescent protein Venus generates a live cell Optical Stem Cell Activity Reporter (OSCAR) for detection of dormant cells. We found, using small intestinal crypt cultures, that OSCAR is able to show the dynamics of dormancy induction and cellular differentiation in real time. Generation and analysis of mice ubiquitously expressing OSCAR showed several populations of OSCARhigh and OSCARlow cells. Microscopy, RNA-Seq and single-cell culture confirmed that OSCAR enables direct identification and isolation of cell populations bearing different transcriptional states and provides a useful tool for understanding dormant cell biology.

overall_design: Transcriptional profile of sorted cells using a novel fluorescent reporter of RNA-polymerase II activity

first: Rasmus

last: Freter

first: Paola

last: Falletta

first: Mahdi

first: Katharine

last: Herbert

first: Francesco

last: Annunziata

first: Lisa

last: Adam

first: Sandra

last: Käppel

first: Omid

last: Omrani

first: Zhao-Qi

last: Wang

first: Colin

middle: R

last: Goding

last: Neri


title: Development of Next-generation Tumor-homing Induced Neural Stem Cells to Enhance Treatment of Metastatic Cancers

city: Guangzhou

first: Yuchen

last: Yang

state: Guangdong

address: Xingangxi Road 135

institute: Sun Yat-sen University

summary: Engineered tumor-homing neural stem cells (NSCs) have shown promise in treating cancer. Recently, we transdifferentiated skin fibroblasts into tumor-homing early-stage human induced NSCs (hiNSC) as personalized NSC drug carriers. Here, using a SOX2 and spheroidal culture-based reprogramming strategy, we generated a new hiNSC variant, hiNeuroS, that were genetically distinct from fibroblasts and first-generation hiNSCs and possessed significantly enhanced tumor-homing and anti-tumor properties. To understand the transcriptomic profile of cells during our sphere-based generation of hiNeuroS, we performed single-cell RNA sequencing (scRNA-seq). The results showed that, while some of the hiNeuroS cells shared a similar trajectory states with the fibroblasts, there was a unique cell group at the far end of hiNeuroS trajectory towards the latest pseudotime, and suggested our new sphere-based culture methods generate hiNeuroS that are a genetically distinct NSC sub-populations that expresses tumor-homing pathways at levels greater than hiNSC.

overall_design: Single-cell RNA-seq profiles of hiNeuroS-TRAIL, hiNSC-GFP-TRAIL, and NHF-1 cells were generated by deep sequencing using 10x Genomics Chromium System.

first: Wulin

last: Jiang

first: Shawn

last: Hingtgen


title: The pan-cancer lncRNA PLANE regulates an alternative splicing program and promotes cancer pathogenesis [ONT]

city: Zhengzhou

first: liu

last: Teng

address: No 7,Weiwu Road,Jinshui  District

department: Translational Research Institute

institute: Henan Provincial People’s Hospital and People’s Hospital of Zhengzhou University

summary: Genomic amplification of the distal portion of chromosome 3q that encodes a number of oncogenic proteins is one of the most prevalent chromosomal deformities. Here we functionally characterise a non-protein product of this region, the long noncoding RNA (lncRNA) PLANE, which is upregulated in diverse cancer types through copy number gain as well as transcriptional activation by E2F1. PLANE forms an RNA-RNA duplex with the nuclear receptor co-repressor 2 (NCOR2) pre-mRNA at intron 45, binds to heterogeneous ribonucleoprotein M (hnRNPM) and facilitates the association of hnRNPM with the intron, leading to repression of the alternative splicing (AS) event generating NCOR2-202, a major protein-coding NCOR2 AS variant. As such, PLANE promotes cancer cell proliferation and tumorigenicity and its upregulation is associated with poor outcome of patients. These results uncover the function and regulation of PLANE and suggest that PLANE may constitute a therapeutic target in the pan-cancer context.

overall_design: For long read RNA-seq analysis (Oxford Nanopore Technologies)of PLANE siRNA versus control siRNA, the A549 cells were transfected with siRNAs for 48 hours before harvesting.

first: Liu

first: YuChen

last: Feng

first: Su

last: Guo

first: Pei


title: Resetting proteostasis in aged animals with ISRIB prevents recruitment of profibrotic monocyte-derived alveolar macrophages during pulmonary fibrosis

city: Chicago

first: Alexander

last: Misharin

state: Illinois

address: 303 E Superior

institute: Northwestern University

summary: Aging is among the most important risk factors for the development of pulmonary fibrosis. Inappropriate or prolonged activation of the integrated stress response has been implicated in the pathobiology of both aging and pulmonary fibrosis. We found that a small molecule that relieves translational inhibition induced by activation of the integrated stress response (ISRIB) attenuated the severity pulmonary fibrosis in young and old mice. We demonstrate that severe fibrosis in old mice was associated with increased number of pathogenic monocyte-derived alveolar macrophages. Using transcriptomic profiling we found that ISRIB modulates stress response signaling in alveolar epithelial cells resulting in decreased recruitment of pathogenic monocyte-derived alveolar macrophages. Thus our data suggest that inhibition of the integrated stress response in the lung epithelium can ameliorate pulmonary fibrosis by preventing the recruitment of monocyte-derived alveolar macrophages.

overall_design: Tissue-resident alveolar macrophages and monocyte-derived alveolar macrophages were sorted by flow cytometry from 2 months- and 18 months-old C57BL/6J mice naive and 28 days after administration of asbestos. RNA-seq libraries were prepared using NEBNext Ultra RNA Library Prep Kit with polyA enrichment and sequenced on Illumina NextSeq500 instrument.

first: Satoshi

last: Watanabe

first: Nikolay

middle: S

last: Markov

first: Ziyan

last: Lu

first: Raul

last: Piseaux-Aillon

first: Hiam

last: Abdala-Valencia

first: Yuliya

last: Politanska

first: Mariana

last: Maciel

first: Lango

last: Sichizya

first: G

last: Budinger


title: Resetting proteostasis in aged animals with ISRIB prevents recruitment of profibrotic monocyte-derived alveolar macrophages during pulmonary fibrosis [dataset 2]

overall_design: Monocytes and monocyte-derived alveolar macrophages were sorted by flow cytometry from young adult (3–5 months) and old (18–24 months) C57BL/6J mice treated with ISRIB or vehicle (i.p., every day beginning at day 7) 21 days after administration of bleomycin. RNA-seq libraries were prepared using NEBNext Ultra RNA Library Prep Kit with polyA enrichment and sequenced on Illumina NextSeq 500 instrument.


title: PPAR-y agonists promote the resolution of myelofibrosis in preclinical models

city: Montpellier

first: Mohammad

last: Salma

address: 1919 Route de Mende

country: France

institute: IGMM CNRS

summary: PPAR-g abrogates TGF-β1 signaling by trapping the p300 co-factor in BM stromal cells

overall_design: ChIP analysis was performed on bone marrow MS5 cell line (CVCL_2128) after 5 h of TGF-β1 stimulation (10 ng/mL), with or without pioglitazone (20 ng/mL).

first: Juliette

last: Lambert

first: Joseph

last: Saliba

first: Carolina

last: Calderon

first: Karine

last: Sii-Felice

first: Valérie

last: Edmond

first: Jean-Claude

last: Alvarez

first: Marc

last: Delord

first: Caroline

last: Marty

first: Isabelle

last: Plo

first: Jean-Jacques

last: Kiladjian

first: Eric

last: Soler

first: William

last: Vainchenker

first: Jean-Luc

last: Villeval

first: Philippe

last: Rousselot

first: Stéphane

last: Prost


title: Impact of the bone microenvironment on phenotypic plasticity of ER+ breast cancer cells[Osteogenic cells]

first: Igor

last: Bado

summary: RNA sequencing was used to evaluate the effect of cancer cells on osteogenic cells (FOB 1.19) in presence of estrogen or androgen. Here, we unbiasely evaluated the expression level of fibroblast growth factor receptors (FGFs) in osteogenic cells (FOB) alone.

overall_design: Osteogenic cells (FOB 1.19) were cultured in mono- or co-culture with mouse breast cancer (4T1) or prostate cancer (TRAMP) cells. Total RNA was sequenced and human reads were extracted using in silico sorting before analysis

first: Xiang

middle: H

last: Zhang

first: Hai

last: Wang


title: Normalization of cholesterol metabolism in spinal microglia alleviates neuropathic pain

city: la Jolla

first: Juliana

middle: Maria

last: Navia-Pelaez

state: california

address: 9500 Gilman dr.

department: Medicine

institute: UCSD

summary: Neuroinflammation is a major component in the transition to and perpetuation of neuropathic pain states. Spinal neuroinflammation involves activation of TLR4, localized to enlarged, cholesterol-enriched lipid rafts, designated here as inflammarafts. Conditional deletion of cholesterol transporters ABCA1 and ABCG1 in microglia, leading to inflammaraft formation, induced tactile allodynia in naïve mice. The apoA-I binding protein (AIBP) facilitated cholesterol depletion from inflammarafts and reversed neuropathic pain in a model of chemotherapy-induced peripheral neuropathy (CIPN) in wild-type mice, but AIBP failed to reverse allodynia in mice with ABCA1/ABCG1-deficient microglia, suggesting a cholesterol-dependent mechanism. An AIBP mutant lacking the TLR4-binding domain did not bind microglia nor reversed CIPN allodynia. The long-lasting therapeutic effect of a single AIBP dose in CIPN was associated with anti-inflammatory and cholesterol metabolism reprogramming and reduced accumulation of lipid droplets in microglia. These results suggest a cholesterol-driven mechanism of regulation of neuropathic pain by controlling TLR4 inflammarafts and gene expression program in microglia and blocking the perpetuation of neuroinflammation.

overall_design: RNA-seq of lumbar spinal microglia from wild type or inducible  Abca1 and Abcg1 microglial conditional Knockout mice. Microglia were sorted from naïve and CIPN (chemotherapy-induced peripheral neuropathy) mice treated with Saline or AIBP.

first: Christopher

last: Durant

first: Holger

last: Winkels

first: Yanal

last: Ghosheh

first: Klaus

last: Ley

first: Yury

middle: I

last: Miller


title: The initiation of Otx2 expression in the developing mouse retina requires a unique enhancer and either Ascl1 or Neurog2 activity

status: Public on Apr 15 2021

city: Aurora

first: Michael Kaufman

middle: L

last: Kaufman

state: CO

address: Fitzsimons Building,13001 East 17th Place

department: Department of Biomedical Informatics

institute: CU Cancer Center

summary: We profiled the early initiation of the OTX2 homeobox transcription factor within embryonic E14.5 mouse retinal progenitor cells by electroporation of a GFP-labeled reporter driven by a novel Otx2 enhancer (DHS-4D). This enrichment labeling strategy and subsequent single cell RNA sequencing captured progenitor cells as they become amacrine, horizontal, cone photoreceptor, and ganglion cell types. Using this approach we identified several candidate factors with potential roles in the early regulation of Otx2 expression in the retina, including Ascl1 and Neurog2.

overall_design: Single cell RNA sequencing of E14.5 mouse retinal progenitor cells marked with a GFP reporter for an early Otx2 enhancer.

first: Joseph

middle: A

last: Brzezinski

first: Michael


title: Regulation of FOXA1 protein stability by Polycomb and BUB3/USP7 deubiquitin complexes in prostate cancer [RNA-Seq]

city: Atlanta

first: Jindan

last: Yu

state: GA

address: E330, 1760 Haygood Dr NE

department: Urology

institute: Emory University

summary: FOXA1 transcription factor is essential for the development and maturation of prostate luminal epithelial cells. FOXA1 gene is frequently mutated and its expression is deregulated during prostate cancer progression. We and others have shown that FOXA1 induces androgen-dependent cell growth by transcriptional regulation of target genes related to cell cycle process, DNA replication, cell division, and apoptosis. Despite of its important roles, how FOXA1 expression itself is regulated in prostate cancer cells remains largely unknown. Here we report that EZH2 methylates one of the lysine residues (K295) on FOXA1 protein to reduce its ubiquitination and subsequent degradation. Mechanistically, this EZH2-mediated FOXA1 methylation recruits BUB3/USP7 deubiquitinase complex to remove FOXA1 ubiquitination for increased protein stability.

overall_design: RNA-seq of prostate cancer cells with de-regulation of USP7, BUB3, or FOXA1

first: Kevin

last: Park


title: Extracellular acidity reprograms macrophage metabolism and innate responsiveness

city: Shanghai

first: Wei

last: Jiang

address: No.130, Dong`an Road, XuHui District

department: Department of Immunology, School of Basic Medical Sciences

institute: Fudan University

summary: Although organ hypofunction and immunosuppression are life-threatening features of severe sepsis, the hypofunctioning organs and immune cells usually regain normal functionality if patients survive.   We tested the hypothesis that low extracellular pH (pHe) can induce reversible metabolic and functional changes in tissue macrophages.  When compared with macrophages cultured at normal pHe, macrophages living in an acidic medium used less glucose and exogenous fatty acid to produce ATP.  Lactate, glutamine, and de novo synthesized fatty acids supported ATP production by mitochondria that gained greater mass, maximal oxygen consumption rate, and spare respiratory capacity.  The cells transitioned to a M2-like state, with altered immune responses to LPS and slightly decreased phagocytic ability, yet they regained basal energy production, normal mitochondrial function and pro-inflammatory responsiveness when neutral pHe was restored.  Low pHe induces changes that support macrophage survival while rendering the cells less pro-inflammatory (more ‘tolerant’) and less able to phagocytose bacteria.  Macrophage responses to low interstitial pH may contribute to the reversible organ hypofunction and immunoparalysis noted in many patients with sepsis.

overall_design: Resident peritoneal macrophages harvested from C57BL/6 mice were cultured in the basic DMEM medium at pH 6.8 or 7.4 (buffered with MOPS or HEPES) for 24 h.  After the cells were untreated or treated with 10 ng/ml LPS for 2 h (n = 3/group), total RNA was isolated using Trizol.

first: Mingfang

last: Lu


title: Maintenance DNA methylation in pre-meiotic germ cells regulates meiotic prophase by facilitating homologous chromosome pairing

city: Yokohama

first: Shin-ichi

last: Tomizawa

state: Kanagawa

address: 3-9 Fukuura, Kanazawa-ku

department: School of Medicine

institute: Yokohama City University

summary: During spermatogenesis, mammalian spermatogonia undergo mitotic division, to maintain stem cell pool via self-renewal and generate differentiating progenitor cells for entry into meiotic prophase. During the perinatal stage, de novo DNA methylation occurring in pro-spermatogonia plays a key role to complete meiotic prophase and initiate meiotic division. In contrast, the role of the maintenance DNA methylation pathway for regulation of meiotic prophase, or meiotic division, in the adult is not well understood. Here, by using conditional mutants for Np95 (nuclear protein 95 kDa, also known as Uhrf1) or Dnmt1 [DNA (cytosine-5)-methyltransferase 1], two proteins that are essential for maintenance DNA methylation, we reveal that both NP95 and DNMT1 are co-expressed in spermatogonia and that these factors are necessary for meiosis in male germ cells. We found that Np95- or Dnmt1-deficient spermatocytes exhibited spermatogenic defects involving synaptic failure during meiotic prophase. In addition, assembly of pericentric heterochromatin clusters in early meiotic prophase, a phenomenon that is required for subsequent pairing of homologous chromosomes, is disrupted in Np95-deficient as well as Dnmt1-deficient spermatocytes. Based on these observations, we propose that DNA methylation established in pre-meiotic spermatogonia regulates synapsis of homologous chromosomes, and in turn quality control of male germ cells. Maintenance DNA methylation, therefore, plays a role to ensure faithful transmission of both genetic and epigenetic information to offspring.

overall_design: To investigate the loss of DNA methylation in Np95-deficient spermatocytes in detail, we carried out whole genome bisulfite sequencing (WGBS). Duplicate samples of Np95fl/fl (control) and Np95fl/fl:Stra8-Cre (knockout) male germ cells were analyzed.

first: Yuki

last: Takada

first: Ruken

last: Yaman-Deveci

first: Takayuki

last: Shirakawa

first: Jafar

last: Sharif

first: Fumihito

last: Miura

first: Takashi

last: Ito

first: Michio

last: Ono

first: Kuniko

last: Nakajima

first: Yoko

last: Koseki

first: Fuyuko

last: Shiotani

first: Kei-ichiro

last: Ishiguro

first: Kazuyuki

last: Ohbo

first: Haruhiko


title: Regulation of FOXA1 protein stability by Polycomb and BUB3/USP7 deubiquitin complexes in prostate cancer

first: Jindan

last: Yu


title: Regulation of FOXA1 protein stability by Polycomb and BUB3/USP7 deubiquitin complexes in prostate cancer [ChIP-Seq]

overall_design: ChIP-seq of prostate cancer cells with FOXA1 de-regulation or inhibition

first: Kevin

last: Park


title: Global profiling of cancer cell recovery from therapy-induced stress reveals druggable vulnerabilities

city: London

first: Holger

last: Auner

address: Du Cane Road

department: Department of Immunology and Inflammation

country: United Kingdom

institute: Imperial College london

summary: Cancer cells survive therapy-induced stress, but how they resolve it is not clear. Using an integrated systems level approach we delineate the global mechanisms of cellular recovery from proteotoxic stress. Our findings in proteasome inhibitor-treated myeloma cells provide a layered chart of the protracted processes of stress resolution that encompass extensive metabolic changes. Tumour cells that have survived proteasome inhibition and are recovering from proteotoxic injuries are more vulnerable to certain insults than acutely stressed or unstressed cells. We identify mitochondria and the GCN2-driven cellular response to amino acid depletion as examples of recovery-associated vulnerabilities, and demonstrate that GCN2 is a bona fide target in transcriptional signature-defined subsets of solid cancers irrespective of prior proteasome inhibition. Thus, targeting multi-system vulnerabilities tied to cellular recovery from drug-induced stress has the potential to optimise cancer therapies.

overall_design: Examination of the resolution of proteasome inhibition-induced proteotoxic stress in multiple myeloma cells.

middle: W


title: Smart-RRBS for single cell methylome and transcriptome analysis [scRRBS]

city: Cambridge

first: Ayush

middle: T

last: Raman

address: 75 Ames St

department: Epigenomics Program

institute: Broad Institute of MIT and Harvard

summary: The integration of DNA methylation and transcriptional state within single cells is of broad interest. Several single-cell dual- and multi-omics approaches have been reported recently that will enable further investigation into cellular heterogeneity including the discovery and in depth study of rare cell populations. Such analyses will continue to provide important mechanistic insights into the regulatory consequences of epigenetic modifications. Last year we reported a new method for profiling the DNA methylome and transcriptome from the same single cell. Here we present details of the protocol and provide guidance on its utility. Our Smart-RRBS protocol entails physically separating mRNA and genomic DNA and combines Smart-Seq2 and Reduced Representation Bisulfite Sequencing (RRBS). It generates paired epigenetic promoter and RNA-expression measurements for a substantial fraction (approximately one-fourth) of protein-coding genes in a typical single cell. It also works for micro-dissected tissue samples comprising hundreds of cells. The protocol, excluding sequencing, takes ~3 days to process up to 192 samples manually

overall_design: Technical evaluation of simultaneous  DNA methylation (RRBS) and transcriptome (SMART-Seq2) profiling of 96 single cells

first: Andreas

last: Gnirke

first: Alexander

last: Meissner

first: Hongcang

last: Gu

first: Martin

last: Aryee


title: Protease-mediated processing of Argonaute proteins controls small RNA association [RIP-Seq]

city: Basel

first: Foivos

last: Gypas

address: Maulbeerstrasse 66

country: Switzerland

institute: FMI

summary: Small RNA pathways defend the germlines of animals against selfish genetic elements and help to maintain genomic integrity. At the same time, their activity needs to be well-controlled to prevent silencing of ‘self’ genes. Here, we reveal a proteolytic mechanism that controls endogenous small interfering (22G) RNA activity in the Caenorhabditis elegans germline to protect genome integrity and maintain fertility. We find that WAGO-1 and WAGO-3 Argonaute proteins are produced as pro-proteins that are matured through proteolytic processing of their unusually proline-rich N-termini. In the absence of DPF-3, a P-granule-localized N-terminal dipeptidase orthologous to mammalian DPP8/9, processing fails, causing a change of identity of 22G RNAs bound to these WAGO proteins. Accumulation of repeat- and transposon-derived transcripts, DNA damage and sterility ensue. We propose that DPF-3 acts as a licensing factor for 22G RNA activity.

overall_design: 1xFLAG::WAGO-1. 3 Replicates. IP;   1xFLAG::WAGO-1 dpf-3{delta}. 3 Replicates. IP;   1xFLAG::WAGO-3. 3 Replicates. IP;   1xFLAG::WAGO-1. 3 Replicates. Input;   1xFLAG::WAGO-1 dpf-3{delta}. 3 Replicates. Input;   1xFLAG::WAGO-3. 3 Replicates. Input

first: Helge

last: Grosshans

first: Rajani

last: Gudipatti


title: Protease-mediated processing of Argonaute proteins controls small RNA association

type: Non-coding RNA profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Impact of the bone microenvironment on phenotypic plasticity of ER+ breast cancer cells [MCF7]

first: Igor

last: Bado

summary: Translating Ribosome Affinity Purification (TRAP) sequencing revealed that human osteogenic cells (FOB1.19) strongly alter the transcriptional profile of ER+ breast cancer cells (MCF7) in 3D coculture system.

overall_design: 3D monocultures or cocultures of MCF7 cells with FOB1.19. Translating Ribosome Affinity Purification was performed to purify transcriptome from MCF7 cells, followed by NSG sequencing and data analysis.

first: Xiang

last: Zhang

first: Hai

last: Wang


title: Smart-RRBS for single cell methylome and transcriptome analysis [scRNAseq]


title: Protease-mediated processing of Argonaute proteins controls small RNA association [RNA-Seq]

overall_design: Wild type worms.9 Replicates;   dpf-3{delta}. 3 replicates;   mut-2(ne3370). 3 replicates;   mut-7(ne4255). 3 replicates


title: CNS-endogenous TLR7 and TLR9 induce different immune responses and effects on experimental autoimmune encephalomyelitis

city: Copenhagen N

first: Atul

last: Anand

address: Ole Maaløes Vej 5, building 2, 3 floor

department: Department of Pathology

country: Denmark

institute: Rigshospitalet

summary: Innate receptors, including Toll like receptors (TLRs), are implicated in pathogenesis of CNS inflammatory diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). TLR response to pathogens or endogenous signals includes production of immunoregulatory mediators. One of these, interferon (IFN)β, a Type I IFN, plays a protective role in MS and EAE. We have previously shown that intrathecal administration of selected TLR ligands induced IFNβ and infiltration of blood-derived myeloid cells into the CNS, and suppressed EAE in mice. We have now extended these studies to evaluate a potential therapeutic role for CNS-endogenous TLR7 and TLR9. Intrathecal application of Imiquimod (TLR7 ligand) or CpG oligonucleotide (TLR9 ligand) into CNS of otherwise unmanipulated mice induced IFNβ expression, with greater magnitude in response to CpG. CNS extraparenchymal CD45+ cells were identified as source of IFNβ. Intrathecal CpG induced infiltration of monocytes, neutrophils, CD4+ T cells and NK cells whereas Imiquimod did not recruit blood-derived CD45+ cells. CpG, but not Imiquimod, had a beneficial effect on EAE, when given at time of disease onset. This therapeutic effect of CpG on EAE was not seen in mice lacking the Type I IFN receptor. In mice with EAE treated with CpG, the proportion of monocytes was significantly increased in the CNS. Infiltrating cells were predominantly localized to spinal cord meninges and demyelination was significantly reduced compared to non-treated mice with EAE. Our findings show that TLR7 and TLR9 signaling induce distinct inflammatory responses in the CNS with different outcome in EAE and point to recruitment of blood-derived cells and IFNβ induction as possible mechanistic links between TLR9 stimulation and amelioration of EAE. The protective role of TLR9 signaling in the CNS may have application in treatment of diseases such as MS.

overall_design: Comparison of mice brains after intrathecal PBS and CPG treated mice. Four mice were treated with CPG and three mice were treated with PBS

first: Ruthe

last: Dieu

first: Reza

last: Khorooshi

first: Trevor

last: Owens


title: Protease-mediated processing of Argonaute proteins controls small RNA association [smallRNA-Seq]

overall_design: Wild type worms.9 Replicates;   dpf-3 null worms. 3 replicates;   DPF-3 S784A mutant worms. 3 Replicates;   mut-2(ne3370). 3 replicates;   mut-7(ne4255). 3 replicates


title: Resetting proteostasis in aged animals with ISRIB prevents recruitment of profibrotic monocyte-derived alveolar macrophages during pulmonary fibrosis [AT2]

middle: V

last: Misharin

country: USA

overall_design: Alveolar epithelial type 2 cells were sorted by flow cytometry from 2 months-old C57BL/6J mice 24 hours after the administration of ISRIB or vehicle. RNA-seq libraries were prepared using NEBNext Ultra RNA Library Prep Kit with polyA enrichment.  Additionally, AT2 cells from young mice were sorted 14 days after administration of bleomycin. RNA-seq libraries were prepared with SMARTer Stranded Total RNA-Seq Kit v2 – Pico Input Mammalian kit. All libraries were sequenced on Illumina NextSeq500 instrument.

first: Satoshi

last: Watanabe

first: Nikolay

last: Markov

first: Ziyan

last: Lu

first: Raul

last: Piseaux-Aillon

first: Hiam

last: Abdala-Valencia

first: Yuliya

last: Politanska

first: Mariana

last: Maciel

first: Lango

last: Sichizya

first: G

last: Budinger


title: Protease-mediated processing of Argonaute proteins controls small RNA association [small RNA RIP-seq]

first: Foivos

last: Gypas

overall_design: Checking the differential expression of small noncoding RNAs

first: Helge

last: Grosshans

first: Rajani

last: Gudipatti