title: Extracellular vesicles (EVs) secreted by human aneuploid embryos contain a distinct RNA cargo and upregulate MUC1 transcription in decidualised endometrial stromal cells (dESCs)

city: Milan

first: Giulia

middle: Maria

last: Scotti

state: Milan

address: Viale Rita Levi Montalcini, 1

department: National Facility for Data Handling and Analysis

institute: Fondazione Human Technopole

summary:
Study question: Could extracellular vesicles (EVs) secreted by aneuploid embryos a) serve for development of RNA biomarkers for preimplantation genetic testing for aneuploidy (PGT-A) and b) elicit a relevant transcriptomic response in decidualised primary endometrial stromal cells (dESCs)?
Summary answer: Aneuploid embryo EVs a) contain genes PPM1J, LINC00561, ANKRD34C and TMED10 in differential abundance from euploid EVs and b) induce up-regulation of MUC1 transcript in dESCs.
What is known already: PGT-A is popular as it is thought to facilitate transfer of euploid embryos to increase implantation probability but the technology is controversial, as it requires an invasive embryo biopsy with an elusive long-term biosafety. The development of non-invasive methods to screen out aneuploid embryos is paramount. It is also critical to decode the embryo-endometrial dialog underlying implantation failure. We have previously reported that IVF embryos secrete EVs that can be internalised by ESCs, conceptualising that successful implantation to the endometrium is facilitated by EVs, which may additionally serve as biomarkers of ploidy status.
Study design, size, duration: Embryos destined for biopsy on days 5-7 for PGT-A were grown under standard conditions. Spent media (30μl) were collected from euploid (n=175) and aneuploid (n=140)embryos at cleavage (days 1-3) stage and from euploid (n=187) and aneuploid (n=142) embryos at blastocyst (days 3-5) stage. Media samples from n=35 cleavage embryos were pooled in order to obtain five euploid and four aneuploid pools. Similarly, media samples from blastocyst were pooled to create one euploid and one aneuploid pool. ESCs were obtained from five women undergoing diagnostic laparoscopy.
Participants/materials, setting, methods: EVs were isolated from pools of media derived from euploid and aneuploid day 3 embryos with differential centrifugation and EV-RNA sequencing was performed following the SMARTer Stranded Total RNA-Seq approach. ESCs were decidualised (E2:10nM, P4:1uM, cAMP:0.5 mM twice every 48 hours) and treated for 24 hours with 50 ng/ml euploid or aneuploid EVs extracted from blastocyst media. RNA sequencing was performed on ESCs following the Illumina Truseq RNAseq protocol.
Main results and the role of chance: Aneuploid cleavage stage embryos secreted EVs that were less abundant in RNA fragments originating from the genes PPM1J (log2fc=-5.13, p=0.011), LINC00561 (log2fc=-7.87, p=0.010) and ANKRD34C (log2fc=-7.30, p=0.017) and more abundant in TMED10 (log2fc=1.63 p=0.025) compared to EVs from euploid embryos. Decidualisation per se induced downregulation of MUC1 (log2FC=-0.54, p=0.0028) in ESCs as prerequisite for the establishment of receptive endometrium. The expression of MUC1 transcript in decidualised ESCs was significantly increased following treatment with aneuploid compared to euploid embryo-secreted EVs (log2FC=0.85, p=0.0201).
Limitations, reasons for caution: The findings of the study may require validation utilising a second cohort of EVs samples.
Wider implications of the findings: The discovery that the RNA cargo of EVs secreted from aneuploid cleavage stage embryos is diverse from that of euploid embryos potentiates the development of non-invasive methodology for PGT-A. The upregulation of MUC1 in dESCs following aneuploid embryo EV treatment proposes a new mechanism underlying implantation failure.
Funding: The study was supported by a MSCA fellowship awarded to SM by the European Commission (CERVINO grant agreement ID: 79620) and by a BIRTH research grant from Theramex HQ UK Ltd

overall_design: Experiment 1. We collected spent culture medium from undiagnosed embryos (n=105) at the cleavage stage (days 1-3). Media samples from n=35 single cleavage embryos cultures were pooled to create a single pool. Experiment 2. We collected spent culture medium from euploid (n=175) and aneuploid (n=140) embryos at the cleavage stage. Experiment 3. We collected spent culture medium from euploid (n=187) and aneuploidy (n=142) expanded blastocysts (days 3-5). Samples from embryos with the same ploidy were pooled: media samples from n=35 cleavage embryos were pooled in order to obtain five euploid and four aneuploid pool; media samples from blastocyst were pooled to create one euploid and one aneuploid pool.

first: Sofia

last: Makieva

first: Elisa

last: Giacomini

first: Dejan

last: Lazarevic

first: Valentina

last: Pavone

first: Jessica

last: Ottolina

first: Ludovica

last: Bartiromo

first: Matteo

last: Schimberni

first: Marco

last: Morelli

first: Alessandra

last: Alteri

first: Sabrina

last: Minetto

first: Giovanni

last: Tonon

first: Massimo

last: Candiani

first: Enrico

last: Papaleo

first: Paola

last: Viganò


title: Nanostring human miRNA panel: exosome fraction from peripheral blood of trauma patients versus healthy

status: Public on Apr 03 2024

city: Gainesville

first: Rob

last: Maile

state: FL

department: Surgery

country: USA

institute: University of Florida

summary:
827 human miRNA standard human panel
Plasma derived extracellular vesicles (EV)/exosomes can serve as markers of cell damage/disease but can also have therapeutic utility depending on the nature of their cargo, such as miRNA.  Currently there are challenges and lack of innovations regarding early diagnosis and therapeutic options within different aspects of management of trauma patients. We characterized the miRNA cargo of exosomes purified from trauma patients, and compared it to those from healthy participants.   Methods  Plasma was collected from a prospective, cohort study of trauma patients (n = 15; 7 males, 8 females) with hip and/or femur fractures and an injury severity score (ISS) ≥ 15; elective total hip arthroplasty (THA) patients (n = 8; 4 males, 4 females) served as operative controls. Exosomes were isolated from plasma with the Invitrogen Total Exosome Isolation Kit and RNA was isolated using a miRNeasy Mini Kit. Direct quantification of miRNA copies was performed by NanoString Technologies on a human miRNA gene panel and analyzed with nSolver   Results  Trauma plasma-derived exosomes had 60 miRNA that were significantly downregulated and 3 miR upregulated when compared to THA (p<0.05). Thirteen miR have a direct role in hematopoiesis regulation, including miR-223 and miR-451a. Further, male trauma plasma-derived exosomes demonstrated downregulation of 150 miR compared to male THA (p<0.05). While female trauma plasma-derived exosomes demonstrated downregulation of only four miR and upregulation of 2 miR compared to female THA (p<0.05).   Conclusions  Sex-specific differences in the miR from plasma-derived exosomes occurred following severe trauma. These miR changes in plasma-derived exosomes likely contribute to postinjury systemic inflammation, immune system dysregulation, and bone marrow dysfunction and may merit consideration as future potential therapeutic targets.

first: Robert

first: Alicia

last: Mohr


title: NOTCH3 promotes vascular smooth muscle cells progression by targeting RANBP1

city: Lishui City

first: zhongjiao

last: xu

address: No. 289, Gucang Road, Liandu District

country: China

institute: The Fifth Affiliated Hospital of Wenzhou Medical University

type: Expression profiling by high throughput sequencing

summary: Vascular smooth muscle cells participate in the formation and development of fibrous cap in the early stage of atherosclerosis,Notch3 plays an important role in maintaining the phenotypic stability of VSMCs.However,the molecular mechanism between Notch3 and VSMCs is not fully understood

overall_design: Comparative gene expression profiling analysis of RNA-sequencing data，9 groups of vascular smooth muscle cells Samples for RNA-Sequencing , 3 groups of si-NOTCH3-1, 3 groups of si-NOTCH3-2 and 3 groups of normal control (NC) data, in which NOTCH3 was knocked down by different siRNAs.

first: Zhongjiao

last: Xu

first: Jian

first: Tiemin

last: Wei

first: Chunlai

last: Zeng


title: Transcriptome-wide analysis of Agc1 downregulation in mouse oligodendrocyte precursor cells

city: Bologna

first: Federico

middle: Manuel

last: Giorgi

state: Bologna

address: Via Irnerio 49

department: Pharmacy and Biotechnology

country: Italy

institute: Unviersity of Bologna

summary: Aspartate-glutamate carrier isoform 1 (Agc1) is a mitochondrial carrier part of the malate-aspartate shuttle, responsible for the export of aspartate and N-acetyl-aspartate from the mitochondria to the brain. Agc1 deficiency can cause an ultra-rare genetic disease reported as epileptic encephalopathy with global cerebral demyelination often caused by mutation in Slc25a12, the gene from which Agc1 is encoded. Symptoms of the disease include diffused hypomyelination, arrested psychomotor development, severe hypotonia, seizures and are common to other neurological and developmental disorders.  Oligodendrocytes are glial cells responsible for myelination and remyelination and a disruption of acetyl fluxes can severely hamper their ability to form myelin leading to abnormal neuronal functionality. In this work, we explore the transcriptome of mouse oligodendrocyte precursor cells that have been silenced for Agc1 expression, reporting results of both canonical analyses such as differential expression and pathway enrichment analyses which highlight a disruption in fatty acids synthesis from both a regulatory and enzymatic stand. We further explore the transcriptome space with a network and an alternative splicing analysis: these results taken together highlight a severe alteration at the transcriptome level, which brought us to question a possible involvement of Agc1 in transcription regulation.

overall_design: In order to investigate the role of Agc1 (Slc25a12) in oligodendrocyte precursor cells, we established an Oli-Neu cell line in which AGc1 has been knocked down using shRNA. We then performed differential gene expression analysis using data obtained from 3 different replicates per condition (total of 3 wild-type samples and 3 Agc1+/-)

first: Nicola

last: Balboni

first: Giorgia

last: Babini

first: Eleonora

last: Poeta

first: Barbara

last: Monti

middle: M


title: Maternal oxytocin treatment at birth increases epigenetic age in male offspring [RNA-seq]

city: Charlottesville

first: Joshua

last: Danoff

state: VA

address: 485 McCormick Road

institute: University of Virginia

summary: Rates of oxytocin use to induce or augment labor are increasing in the United States with little understanding of the impact on offspring development. Using a prairie vole animal model, we have shown that oxytocin administered to mothers can reach offspring brains with long lasting impacts on the development of social behaviors. Here, we examine the epigenetic and transcriptomic consequences of oxytocin exposure during birth in juvenile male offspring. First, we show that male offspring exposed to oxytocin at birth have increased epigenetic age compared to the saline exposed group. We also find 900 differentially methylated CpG sites (annotated to 589 genes), with 2 CpG sites (2 genes) remaining significant after correction for multiple comparisons. Differentially methylated CpG sites are involved in regulation of gene expression and neurodevelopment. Using RNA-sequencing we find 217 nominally differentially expressed genes (p<0.05) in nucleus accumbens, a brain region involved in reward circuitry and social behavior, including 6 genes that remain significantly differentially expressed after corrections for multiple comparisons. Finally, we show that maternal oxytocin administration leads to widespread alternative splicing in the nucleus accumbens. These results indicate that oxytocin exposure during birth has long lasting epigenetic consequences in the brain and warrant further investigation of how oxytocin administration impacts development and behavior throughout the lifespan.

overall_design: To investigate how perinatal exposure to exogenous oxytocin impacts regulation of gene expression in the juvenile brain, we completed RNA-sequencing on nucleus accumbens from prairie voles born to mothers either injected with oxytocin or saline prior to birth.

middle: S

first: Juozas

last: Gordevičius

first: Milda

last: Milčiūtė

middle: T

last: Brooke

first: C

last: Carter

first: Jessica

middle: J

last: Connelly

first: Allison

last: Perkeybile


title: Claudin-1 as a novel target gene induced in obesity and associated to inflammation, fibrosis and cell differentiation

city: Badalona

first: Lauro

last: Sumoy

state: Barcelona

address: Ctra. Can Ruti, Camí de les escoles s/n

department: High Content Genomics and Bioinformatics

country: Spain

institute: IGTP

summary:
T-lymphocytes from visceral and subcutaneous white adipose tissue (vWAT and sWAT, respectively) can have opposing roles in the systemic metabolic changes associated with obesity. However, few studies have focused on this subject. Claudin-1 (CLDN1) is a protein involved canonically in Tight Junctions (TJs) and tissue paracellular permeability.
We evaluated the gene expression in T lymphocytes from vWAT and sWAT and in the whole adipose depots.

overall_design: A Clariom D-based transcriptomics analysis was performed in T lymphocytes magnetically separated from vWAT and sWAT from patients with obesity (Cohort 1; N = 11). The experimental design for hybridization processing included 4 batches of 9-11 samples each with balancing among different batches between the two compared conditions: vWAT and sWAT.

first: Pablo

last: Fernández-García

first: Siri

middle: D

last: Taxerås

first: Marjorie

last: Reyes-Farias

first: Lorena

last: González

first: Andrea

last: Soria-Gondek

first: Silvia

last: Pellitero

first: Jordi

last: Tarascó

first: Pau

last: Moreno

first: Jacqueline

last: Stephens

first: María

last: Galán

first: Adriana

last: Izquierdo

first: Gema

last: Medina-Gómez

first: Laura

last: Herrero

first: Patricia

last: Corrales

first: Francesc

last: Villarroya

first: Rubén

last: Cereijo

first: David

last: Sánchez-Infantes


title: Genome-wide maps of transcription in Multiple Myeloma cells treated with FHND004 or FHND008

city: China

first: Hongjie

last: Wu

state: Jiangsu

address: Xianlin Road

institute: Nanjing University of Chinese Medicine

summary: FHND004 and FHND008 are newly synthesized epidermal growth factor receptor (EGFR) inhibitor for the treatment of non-small cell lung cancer (NSCLC), they were obtained on the basis of AZD9291. We found that FHND004 and FHND008 had a strong inhibitory effect on the proliferation of multiple myeloma cells in vitro. Genome-wide maps of transcription showed different genes change in Multiple Myeloma cells after 4μM FHND004 or FHND008 intervention.

overall_design: Examination of different genes changes in 2 Multiple Myeloma cell types treated with 4μM FHND004 or FHND008.


title: Asparagine availability controlsgerminal centre B cell homeostasis

city: Oxford

first: Alexander

last: Clarke

address: Roosevelt Drive

department: Kennedy Institute of Rheumatology

country: United Kingdom

institute: University of Oxford

summary: Germinal centre (GC) B cells proliferate at some of the highest rates of any mammalian cell. Yet the metabolic processes which enable this are poorly understood. We performed integrated metabolomic and transcriptomic profiling of GC B cells, and found that asparagine metabolism is highly upregulated. Asparagine is conditionally essential to B cells, and its synthetic enzyme, asparagine synthetase (ASNS) is markedly upregulated following their activation, through the integrated stress response sensor general control non-derepressible 2 (GCN2). When Asns is deleted, B cell survival in low asparagine conditions is severely impaired. Using stable isotope tracing, we found that metabolic adaptation to the absence of asparagine requires ASNS, and that the synthesis of nucleotides is particularly sensitive to asparagine deprivation. Conditional deletion of Asns in B cells selectively impairs GC formation, associated with a reduction in RNA synthesis rates. Finally, removal of environmental asparagine by asparaginase was found to also severely compromise the GC reaction.

overall_design: GC B cells were flow sorted from WT (n=3) and Cd79a-cre × Asns flox mice (n=4) at day 9 following immunisation with SRBC.


title: Effect of depletion of SNHG14 on gene expression  of MDA-MB-231  cells

city: Jinan

first: Ai-yan

last: Xing

address: Wen Hua Xi Road 107

institute: ShanDong University Qilu Hospital

summary: To investigate the function of SNHG14  in breast cancer, we established MDA-MB-231 cell lines in which  this gene has been knocked down by siRNA.

overall_design: We performed gene expression profiling analysis using data obtained from RNA-seq of MDA-MB-231 cells.


title: Acamprosate, Craving and Alcohol Use Disorder: the Role of Inflammation

city: Rochester

first: Hu

last: Li

address: 200 1st St SW

institute: Mayo Clinic

summary: The present study utilized patient-derived “cell-line” model systems treated with anti-craving drugs that are used to treat alcohol use disorder (AUD) as “molecular probes” to help identify molecular mechanisms associated with craving and AUD treatment outcomes.

overall_design: We set out to study the effects of ethanol, and anti-craving drugs on gene expression regulation in iPSC-derived astrocytes. Cells were seeded in T75 flasks treated with EtOH (25 mM), a concentration that is considered physiologically relevant for EtOH use, with 25 mM EtOH being slightly higher than the 0.08% blood alcohol concentration that is often used as a measure of intoxication. The concentrations of acamprosate (5 µM, Sigma, A6981) and naltrexone (30 nM, Selleckchem, S2103) used to perform those experiments were selected to fall within the range of blood concentrations for these drugs observed during clinical therapy. Cells were cultured with the drugs for seven days and the medium was changed daily.

first: Ming

middle: F

last: Ho

first: Richard

last: Weinshilboum

first: Cheng

last: Zhang


title: Nilotinib Induces Endothelial Cell Dysfunction Independent of ABL1 Inhibition

city: Chicago

first: Emily

last: Pinheiro

state: IL

address: 320 East Superior St

institute: Northwestern University

summary: To investigate transcriptome properties of hiPSC-derived endothelial cells compared to primary endotheilal cells and other hiPSC differentiation protocols

overall_design: Comparative gene expression profiling analysis of RNA-seq data for hiPSC lines (19-3, 22-10, 25-3, 26-3, 124-9, 132-2B, 441227, 441228, and 442333) differentiated to ECs, CMs, MECs in comparison to primary EC lines

middle: A

first: Paul

middle: W

last: burridge


title: Chronic hypoxia remodels the tumor microenvironment to support glioma stem cell growth [scRNAseq+Multiome]

city: New York

first: James

last: Nicholson

address: 413 East 69th Street

institute: Weill Cornell Medicine

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Cerebral organoids co-cultured with patient derived glioma stem cells (GLICOs) are an experimentally tractable research tool useful for investigating the role of the human brain tumor microenvironment in glioblastoma. Here we develop long-term GLICOs, a novel model in which COs are minimally seeded with GSCs and tumor development is monitored over extended durations (ltGLICOs). Single-cell profiling of ltGLICOs revealed an unexpectedly long latency period prior to GSC expansion, and that normal organoid development was unimpaired by the presence of low numbers of GSCs. However, as organoids age they experience chronic hypoxia and oxidative stress which remodels the tumor microenvironment to promote GSC expansion. Receptor-ligand modelling identified astrocytes, which secreted various pro-tumorigenic ligands including FGF1, as the primary cell type for GSC crosstalk and single-cell multi-omic analysis revealed these astrocytes were under the control of ischemic regulatory networks. Functional validation confirmed hypoxia as a driver of pro-tumorigenic astrocytic ligand secretion and that GSC expansion was accelerated by pharmacological induction of oxidative stress. When controlled for genotype, the close association between glioma aggressiveness with patient age has very few proposed biological explanations. Our findings indicate that age-associated increases in cerebral vascular insufficiency and associated regional chronic cerebral hypoxia may contribute to this phenomenon.

overall_design: We performed scRNAseq on a cohort of 19 cerebral organoids (COs) seeded with low levels of glioma stem cells (ltGLICOs) spanning 1-24 months in culture, as well as scMultiome (RNA + ATAC) on 9 further ltGLICOs aged 5, 9 & 12months. Next we performed scRNAseq on 2x 7month control COs & 2x 7month COs maintained under hypoxic culture conditions.

first: Stefano

last: Cirigliano

first: Richa

last: Singhania

first: Howard

last: Fine


title: Maternal oxytocin treatment at birth increases epigenetic age in male offspring [array]

first: Joshua

last: Danoff

type: Methylation profiling by genome tiling array

platform_organism:
Homo sapiens
Mus musculus
Rattus norvegicus

first: Juozas

last: Gordevičius

first: Milda

last: Milčiūtė

first: Robert

last: Brooke

first: C

last: Carter

first: Jessica

last: Connelly

first: Allison

last: Perkeybile


title: Whole-transcriptome sequencing analysis of streptozocin-induced diabetic and non-diabetic control rats

city: Fuzhou

first: Jianzhang

last: Hu

state: Fujian

address: No.11 xinquan Road, Gulou District

institute: Fujian Medical University Union Hospital

summary: Diabetes mellitus is a complex metabolic disorder. Long-term hyperglycemia may induce diabetic keratopathy, which is mainly characterized by decreased tear secretion, damaged innervation, weakened cell junctions, and impaired wound healing responses. To investigate the differential expressed genes in the regulation of diabetic keratopathy, we established streptozocin-induced diabetic and non-diabetic control male Brown Norway rats. Total RNA was extracted from the corneal epithelium rats, and were subjected to whole-transcriptome sequencing analysis. Firstly, Cutadapt was used to remove the reads that contained adaptor contamination, low quality bases and undetermined bases. Then sequence quality was verified using FastQC(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). We used Bowtie2 and Hisat2to map reads to the genome from corneal epithelium of streptozocin-induced diabetic and non-diabetic control rats. The mapped reads of each sample were assembled using String Tie. Then, all transcriptomes from corneal epithelium Samples were merged to reconstruct a comprehensive transcriptome using perl scripts. We then performed gene expression profiling analysis using data obtained from RNA-seq of 6 corneas from STZ-induced diabetic rats and 6 corneas from normal controls.

overall_design: mRNA profiles form corneal epithelium of 16-weeks streptozocin-induced diabetic and non-diabetic control rats corneal epithelium

first: Yi

last: Cui

first: Nuo

last: Xu


title: Single-Nucleus Transcriptomic Atlas of Goat (Capra hircus) Ovarian Prolificacy II

city: Nanning

first: Sanbao

last: Zhang

address: No.100,Daxue East Road

institute: Guangxi University

summary: Molecular mechanisms of follicular atresia and prolificacy of mammal remain unclear. We surveyed the single-cell transcriptomic landscape of ovaries from single and prolificacy goat and identified seven ovarian cell types with distinct gene-expression, transcriptional factor networks and reciprocal interactions signatures. In-depth dissection of gene-expression dynamics of granulosa cells (GCs) that displayed development stage-specific expression patterns and specific gene signatures were identified that may reflect developmental competency and ovarian reserve. what’s more, we revealed the origin of theca cells.  Further analysis of cell-type-specific prolificacy-associated transcriptional changes uncovered apoptosis, anabolism and response to hormone stimulation as are crucial factor in dominant follicle development and ovulation. Additionally, differentially expressed genes (DEGs) of SERPINE2 can interact with CYP19A1 to promote cell proliferation, inhibit apoptosis and promoting the anabolism were observed in mouse granulosa cells. Thus, our work provides a comprehensive understanding of the cell-type-specific mechanisms underlying goat ovarian prolificacy at single-cell resolution, provides key insights into offers important clues for improving follicle recruitment in vivo and revealing new diagnostic biomarkers and potential therapeutic targets for ovulation disorder.

overall_design: Ovarian tissue samples from 2 Nubian goats (about 3 years old) and 2 Du’an goat (about 3 years old) were collected and processed for snRNA-seq analysis.

first: Qinyang

last: Jiang

first: Yanna

last: Huang


title: miRNA and mRNA transcriptome profiling of chondrocytes in proliferative zone of acetabular cartilage in developmental dysplasia of the hip

city: Hangzhou

first: Jiahui

last: Liu

state: Zhejiang

address: 3333 Binsheng Rd

institute: Children's Hospital, Zhejiang University School of Medicine

type: Non-coding RNA profiling by high throughput sequencing

summary: Developmental dysplasia of the hip (DDH) is a complex and multifactorial disease with an unclear pathogenesis, and the non-coding RNAs have been found important effect in many diseases. However, their contributions to the pathogenesis of DDH are not well-established. Growth of the acetabular cartilage mainly depends on the regulation of chondrocytes in proliferative zone. Here, we report on some new regulatory mechanisms of gene expression underlying the development of DDH by transcriptome profiling of proliferative zone microdissected from the acetabular cartilage. These results provide original insight into the roles of non-coding RNAs in DDH and provide a valuable resource for further analyses of molecular mechanisms and signaling networks.

overall_design: We explored the mechanism underlying swadding-induced DDH by profiling mRNA and miRNAs expression via RNA-seq. Six neonatal rats were swaddled with use of surgical tape after birth in a manner simulating the human practice as DDH group, and other six neonatal rats in same litter were as normal control. The acetabular cartilage were obtained on five and ten days after birth. Total RNA from microdissected material of the acetabular cartilage was extracted according to the instruction manual of the TRlzol Reagent (Life technologies, California, USA) and the RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 System (Agilent Technologies, CA, USA). A total amount of 1.5 μg RNA per sample was used as input material for Library preparation and sequencing.

first: Qiang

last: Shu


title: The response of splenic macrophages from C57BL/6 and Ch25h-/- mice to infection with Listeria monocytogenes.

city: Seattle

first: Aderem

last: Lab

state: WA

address: 307 Westlake Avenue North, Suite 500

institute: Seattle Children's Research Institute

summary: WT (C57BL/6) and Ch25h-/- mice were infected i.v. with 1e5 CFU of Listeria monocytogenes. At day 3 post-infection, ~100,000 resident splenic macrophages (CD11b+, Ly6G-, Ly6clow) were isolated by flow cytometery and analyzed by RNA-seq

overall_design: Comparative gene expression profiling by RNA-seq of splenic macrophages from C57BL/6 and Ch25h-/- mice infected with Listeria monocytogenes.

first: Alan

last: Diercks

first: Elizabeth

last: Gold

last: Aderem


title: The response to PAM3CSK4 (PAM3) of bone marrow derived macrophages treated with 25-hydroxycholesterol (RNA-Seq).

summary: Bone marrow derived macrophages from C57BL/6 or Ch25h-/- mice (female) were treated with 25-hydroxycholesterol and then stimulated with PAM3CSK4.

overall_design: Comparative gene expression profiling by RNA-seq of bone marrow derived macrophages treated with 25-hydroxycholesterol and then stimulated with PAM3CSK4.


title: Altered plasma microRNA signature in hospitalized COVID-19 patients requiring oxygen support

first: Lauro

last: Sumoy

summary: The lack of available biomarkers for diagnosing and predicting different stages of coronavirus disease 2019 (COVID-19) is currently one of the main challenges that clinicians are facing. Recent evidence indicates that the plasma levels of specific miRNAs may be significantly modified in COVID-19 patients. Large-scale deep sequencing analysis of small RNA expression was performed on plasma samples from 40 patients hospitalized for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (between March and May 2020) (median 13.50 [IQR 9–24] days since symptoms initiation) and 21 healthy noninfected individuals. Patients were categorized as hospitalized not requiring oxygen therapy (n = 6), hospitalized requiring low-flow oxygen (n = 23), and hospitalized requiring high-flow oxygen support (n = 11). A total of 1218 different micro(mi)RNAs were identified. When compared with healthy noninfected donors, SARS-CoV-2 infected patients showed significantly (fold change [FC] >1.2 and adjusted p [padj] <0.05) altered expression of 190 miRNAs. The top 10 differentially expressed (DE) miRNAs were miR-122-5p, let-7b-5p, miR-146a-5p, miR-342-3p, miR-146b-5p, miR-629-5p, miR-24-3p, miR-12136, let-7a-5p, and miR-191-5p, which displayed FC and padj values ranging from 153 to 5 and 2.51 × 10-32 to 2.21 × 10-21, respectively, which unequivocally diagnosed SARS-CoV-2 infection. No differences in blood cell counts and biochemical plasma parameters, including interleukin 6, ferritin and D-dimer, were observed between COVID-19 patients on high-flow oxygen therapy, low-flow oxygen therapy, or not requiring oxygen therapy. Notably, 31 significantly deregulated miRNAs were found when patients on high- and low-flow oxygen therapy were compared. Similarly, 6 DE miRNAs were identified between patients on high flow and those not requiring oxygen therapy. SARS-CoV-2 infection generates a specific miRNA signature in hospitalized patients. Furthermore, specific miRNA profiles are associated with COVID-19 prognosis in severe patients.

overall_design: In this study we aimed at extensively characterizing miRNA profiles in plasma of COVID19 patients. We sequenced and quantified miRNAs by paired-end small RNA-seq and determined their expression pattern  in (n=21) healthy individuals and (n=40) COVID19 patient (hospitalized not requiring oxygen therapy (n = 6), hospitalized requiring low-flow oxygen (n = 23), and hospitalized requiring high-flow oxygen support (n = 11).

first: Sandra

last: Franco

first: Lourdes

last: Mateu

first: Raquel

last: Pluvinet

first: Jose-Francisco

last: Sánchez-Herrero

first: Ruth

last: Toledo

first: Marta

last: Massanella

first: Miguel

last: Martinez


title: The response to lipopolysaccharide (LPS) of bone marrow derived macrophages treated with oxysterol binding protein (OSBP) inhibitors

summary: Bone marrow derived macrophages from C57BL/6 or Ch25h-/- mice (female) were treated with 25-hydroxycholesterol, itraconazole, or Osw-1 and then stimulated with lipopolysaccharide.

overall_design: Comparative gene expression profiling by RNA-seq of bone marrow derived macrophages treated with 25-hydroxycholesterol, itraconazole, or Osw-1 and then stimulated with lipopolysaccharide.


title: Normal human bronchial epithelial cell Next Gen sequencing (RNA-seq) for differential gene expression of untreated versus IL4 or IL4+ALOX15 siRNA treated cells

city: Thousand Oaks

first: Jonathan

last: Phillips

state: CA

address: 1 Amgen center drive

institute: Amgen

summary: Purpose: Identify genes regulated by ALOX15 in Normal human bronchial epithelial (NHBE) cells that were treated with +/- IL4 and +/- ALOX15 siRNA by Next-gen sequencing

overall_design:
mRNA profiles of NHBE cells that were treated with +/- IL4 and +/- ALOX15 siRNA (24 hr pretreatment) for 48 hrs (in triplicate)

Submitter states: Identity of compounds A and B are proprietary.

first: David

last: Fong


title: A549 cell Next Gen sequencing (RNA-seq) for differential gene expression of untreated versus IL4 or IL4+ALOX15 siRNA treated cells

summary: Purpose: Identify genes regulated by ALOX15 in A549 cells that were treated with +/- IL4 and +/- ALOX15 siRNA by Next-gen sequencing

overall_design: mRNA profiles of A549 cells that were treated with +/- IL4 and +/- ALOX15 siRNA for 48 hrs (in triplicate)


title: A Rad50-null mutation in mouse germ cells causes reduced DSB formation, abnormal DSB end resection and complete loss of germ cells

status: Public on Apr 02 2024

city: Shanghai

first: yuefang

last: liu

address: 320 Yueyang Road

institute: Center of Excellence in Molecular and Cellular Science, Chinese Academy of Sciences

summary: Programed DNA double-strand breaks (DSBs) catalyzed by the topoisomerase II-like enzymes, SPO11 and TOPVIBL, initiate meiotic recombination. Following DSB formation, the MRE11-RAD50-NBS1/Xrs2 (MRN/X) complex, along with EXO1 and DNA2, cleave the SPO11-DNA to generate 3′ single-stranded DNA (ssDNA) ends, which are prerequisite for meiotic DSB repair. In both yeast and mammals, MRE11 exhibits endonucleolytic cleavage of the 5′ terminated DNA strand in the vicinity of the DSBs and exonucleolytic resection from 3′ to 5′ towards the DSB ends. RAD50 is a structure maintenance of chromosome (SMC) related protein that contains one ATPase domain at its N- and C- terminal ends, respectively, Zn hook, and anti-parallel coiled coils. RAD50 plays a crucial role in facilitating the MRE11 nuclease activity on DSBs by ATP binding and hydrolysis. However, the in vivo function of Rad50 in mammalian germ cells, particularly its in vivo role in the resection of meiotic DSB ends at the molecular level remains elusive. Here, we performed END-seq with synchronized zygotene spermatocytes from control and germ cell specific Rad50 mutant (Rad50-sKO) mice. We find that the number of formed DSB in the mutant spermatocytes was reduced compared to control spermatocytes (6 636 DSBs in Rad50-sKO spermatocytes vs 8 168 in control spermatocytes) and abnormal DSB end resection occurred in mutant spermatocytes (DSB end resection length: 1 279 nts in Rad50-sKO spermatocytes vs 1 923 nts in control spermatocytes). Thus, RAD50 is essential for DSB formation and end resection during mammalian meiosis.

overall_design: Synchronized zygotene mouse spermatocytes were performed END-seq  for to study meiotic double strand breaks (DSBs) and DSB end resection.

first: Yuefang

first: Zhen

last: Lin

first: Junyi

last: Yan

first: Xi

last: Zhang

first: Minghan

last: Tong


title: The response to PAM3CSK4 (PAM3) of bone marrow derived macrophages treated with 25-hydroxycholesterol (ChIP-Seq).

first: Aderem

last: Lab

summary: Bone marrow derived macrophages from C57BL/6 or Ch25h-/- mice (female) were left untreated or treated with 25-hydroxycholesterol for 18 hours and then were left unstimulated or stimulated with PAM3CSK4 for 18 hours. Chromatin was isolated and immunoprecipiation performed for acetyl-H4.

overall_design: Comparative analysis of histone H4 acetylation between bone marrow derived macrophages treated with 25-hydroxycholesterol and then stimulated with PAM3CSK4.

first: Alan

last: Diercks

first: Elizabeth

last: Gold


title: Genome-wide CPD repair maps of Caenorhabditis elegans

city: College Station

first: Yuchao

last: Jiang

state: TX

address: 3143 TAMU

institute: Texas A&M University

summary: We have adapted the eXcision Repair-sequencing (XR-seq) method to generate single-nucleotide resolution dynamic repair maps of UV-induced cyclobutane pyrimidine dimers (CPD) photoproducts in the Caenorhabditis elegans (C. elegans) genome.

overall_design: We employed XR-seq to evaluate genome-wide excision repair dynamics in WT, csb-1, and xpc-1 C. elegans following UV exposure.

first: Cansu

last: Kose

first: Laura

last: Lindsey-Boltz

first: Aziz

last: Sancar


title: Novel Model of Multiple Sclerosis Induced by EBV-like Virus Generates a Unique B Cell Population

city: Columbus

first: Amy

last: Lovett-Racke

state: Ohio

address: 460 W. 12TH AVE

department: Microbial Infection & Immunity

institute: Ohio State Wexner Medical Center

summary: Objective: Multiple sclerosis (MS) is a chronic, autoimmune disease of the central nervous system. Prior infection with Epstein-Barr virus (EBV), a gammaherpesvirus, is deemed a necessary yet insufficient factor in the development of MS. The objective of this study was to determine whether the EBV-like virus, murid gammaherpesvirus 68, is sufficient to induce inflammatory demyelinating disease in a transgenic TCR mouse strain susceptible to CNS autoimmunity. Methods: B10.PL mice that express the myelin basic protein-specific transgenic T cell receptor (TCRMBP) were intranasally inoculated with murid gammaherpesvirus 68 virus (MHV68), PR8 influenza virus, or PBS. Mice were evaluated for the development of neurologic deficits. Flow cytometry and fluorescent microscopy were used to validate the infiltration of immune cells in the brain and spinal cord of TCRMBP mice. Single cell RNA sequencing of splenic B cells was performed to determine transcriptional changes that resulted from MHV68 infection. Results: MHV68-infected mice developed neurological deficits at a significantly higher frequency (70.8%) than the influenza (7.7%) or mock-infected mice (13.3%). MHV68 infected mice exhibited signs including optic neuritis and ataxia, which are infrequently observed in EAE mice, but common in MS patients. MHV68-infected mice exhibited increased focal immune cell infiltration. Single cell RNA sequencing identified the emergence of a novel, MHV68 infection-associated population of memory-like B cells that highly express genes associated with antigen presentation and costimulation. Conclusion: Infecting myelin basic protein specific TCR transgenic mice with the EBV analogue, MHV68, generates encephalitogenic disease without myelin immunization. MHV68 drives a distinct transcriptional program in B cells capable of enhanced antigen presentation and costimulation to drive an autoreactive T cell response.

overall_design: Splenic B cells from four mice were isolated from wild-type B10.PL mice using negative magnetic bead isolation and subjected to 10X scRNA-seq platform. The first mouse was administered with PBS as a mock infection control. The second mouse was infected with PR8 influzena as an infection control. The final two mice were biological replicates of each other and they were both infected with the gammaherpesvirus MHV68. Splenocytes were collected on day 12 post infection for single-cell RNA sequencing.

first: Joshua

middle: L

last: Deffenbaugh

middle: E


title: Aging aggravates aortic aneurysm and dissection via the miR-1204-MYLK signaling axis

city: Guangzhou

first: Jingsong

last: Ou

state: Please Select

address: 58 Zhong Shan Er Road

department: Surgery/Cardiac Surgery

institute: The First Affiliated Hospital, Sun Yat-sen University

summary: The mechanism by which aging induces aortic aneurysm and dissection (AAD) remains unclear. A total of 430 subjects were recruited for screening of differentially expressed plasma microRNAs. We found that miR-1204 was significantly increased in both plasma and aorta of elder patients with AAD, and was positively correlated with age. Cell senescence induced the expression of miR-1204 through p53 interaction with plasmacytoma variant translocation 1, and miR-1204 induced vascular smooth muscle cell (VSMC) senescence to form a positive feedback loop. miR-1204 aggravated angiotensin II-induced AAD formation, and inhibition of miR-1204 attenuated β-aminopropionitrile monofumarate-induced AAD formation. Mechanistically, miR-1204 directly targeted myosin light chain kinase (MYLK) to promote VSMCs to acquire senescence-associated secretory phenotype (SASP) and lose their contractile phenotype. Overexpression of MYLK reversed miR-1204-induced VSMC senescence, SASP and contractile phenotype changes, and the decrease of transforming growth factor-β signaling pathway. Our findings suggest aging aggravates AAD via 75 miR-1204-MYLK signaling axis.

platform_organism:
Homo sapiens
Human alphaherpesvirus 1
Human alphaherpesvirus 2
Macacine alphaherpesvirus 1
Human betaherpesvirus 5
human gammaherpesvirus 4
Herpesvirus saimiri (strain 11)
JC polyomavirus
Human immunodeficiency virus 1
Human betaherpesvirus 6B
Human gammaherpesvirus 8
Merkel cell polyomavirus
Betapolyomavirus hominis
Betapolyomavirus macacae

overall_design: The plasma samples from young normal (< 50 years old), elder normal (> 50 years old), young patient (< 50 years old) and elder patient (> 50 years old) group (n=3 per group) were used for detecting the miRNA profiles using miRCURY LNA Array (version 19.0) (Exiqon miRCURY LNA™ microRNA Array, 7th gen-hsa, mmu and rno).

first: Zelong

last: Liu


title: Gene expression profiling in old and young  kidneys from wild type mice

city: Madrid

first: Osvaldo

last: Graña

state: Comunidad de Madrid

address: C/ Melchor Fernández Almagro, 3

department: Structural Biology

institute: CNIO

summary: Gene expression by RNA-seq in murine kidneys harvested from 3 month-old and 16 month old wild type mice

overall_design: Using RNA-seq we profiled murine kidneys harvested from 3months old (n=4) and 16 months old wild type mice (n=5)

first: Ana

last: Ortega

last: Graña-Castro

first: Alejo

last: Efeyan


title: Aging aggravates aortic aneurysm and dissection via miR-1204-MYLK signaling axis

summary: The mechanism by which aging induces aortic aneurysm and dissection (AAD) remains unclear.  We found that miR-1204 was significantly increased in both plasma and aorta of elder patients with AAD, and was positively correlated with age. Cell senescence induced the expression of miR-1204 through p53 interaction with plasmacytoma variant translocation 1, and miR-1204 induced vascular smooth muscle cell (VSMC) senescence to form a positive feedback loop.

overall_design: Chromatin immunoprecipitation DNA sequencing for target genes binding to p53 in miR-1204 overexpressing or control vascular smooth muscle cells.


title: Integrative analysis of the lncRNA-miRNA-mRNA Interactions in Smooth Muscle Cell Phenotypic Transitions I

city: Cleveland

first: Olga

last: Cherepanova

address: 9620 Carnegie Avenue

department: Cardiovascular and metabolic sciences

institute: Cleveland Clinic Foundation

summary: To uncover new interactions between OCT4, lncRNA, and miRNA that may eventually help us in understanding not only OCT4-dependent effects, but also general causes of smooth muscle dysfunction associated with AS.

overall_design: Primary mouse aortic smooth muscle cells (MASMC) were isolated and grown as previously described (Cherepanova et al., 2016). For experiments, cells at the 70% confluency were transfected with blocking siOct4 or non-target siRNA (NT siRNA) for 48 hours in serum-free media, followed by the treatment with 10 µg/mL POVPC (Cayman Chem, #10031) or vehicle (DMSO) for 24 hours based on our previous findings (Pidkovka et al., 2007). The samples were used for LncRNA/mRNA expression profiling using Mouse 8 x 60K LncRNA expression array

first: Aatish

last: Mahajan


title: Assessment of myogenic maturation for human iPSC-derived iPAX7 myogenic progenitors in vitro and in vivo

city: Minneapolis

first: Sarah

last: Crist

state: Minnesota

address: 2231 6th St SE

institute: University of Minnesota

summary: Here, we transplanted human iPAX7 myogenic progenitors into skeletal muscles of non-dystrophic and dystrophic mice and compared the transcriptional landscape of human donor-derived myofibers with respective in vitro-differentiated iPAX7 myotubes. Pairing bulk RNA sequencing with computational deconvolution of human reads, we were able to pinpoint key myogenic changes that occur during the in vitro-to-in vivo transition, confirm developmental maturity, and consequently further suggest the utility of cell-based therapies.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: We performed gene expression profiling for data obtained through bulk RNA-sequencing of 14 uniques samples. Samples included non-dystrophic or dystrophic skeletal muscle injected with or without iPAX7 myogenic progenitors, as well as terminally differentiated myotubes in vitro.

middle: B

first: Alessandro

last: Magli

first: Rita

middle: R

last: Perlingeiro


title: TLR-dependent transcriptional reprogramming of LXRα cistrome in bone marrow-derived macrophages [RNA-seq]

first: ANTONIO

last: CASTRILLO

address: Arturo Duperier 4

department: metabolism and cellular signaling

institute: Instituto Investigaciones Biomedicas Alberto Sols CSIC

summary: The Liver X receptors (LXRα and LXRβ) are members of the nuclear receptor superfamily of transcription factors, and play key roles in the coordination of both metabolic and immune responses in macrophages. Both LXRs are expressed in macrophages and they share a high degree of sequence homology but their individual roles in different models of macrophage cultures, particularly in response to inflammation, have not been characterized. Previous studies have shown that ligand pre-treated LXRs repress the induction of pro-inflammatory cytokines in macrophages. Here, we show evidence that the expression of LXRα (but not LXRβ) is transcriptionally regulated at late phases of an LPS-induced inflammatory response in bone marrow-derived macrophages (BMDMs). Regulation of LXRα expression is controlled at the transcriptional levels by inflammatory signaling networks. Transcriptional profiling studies and ChIP-seq data identified a number of innate immune pathways whose expression is substantially controlled by LXRα in BMDMs. In summary, our data shows that LXRα plays a previously unrecognized role in innate immunity by transcriptional regulation of a battery of inflammatory genes through TLR and Type-I IFN-dependent signaling in macrophages.

overall_design: Expression profiling of peritoneal LPS-elicited macrophages (LXR-WT, LXR-aKO, LXR-bKO) treated with lipopolysaccharide 4 mg/Kg 24 hours.

first: Antonio

last: Castrillo

first: Juan

middle: V

last: de la Rosa Medina


title: Characterisation of TET function in drosophila larval central nervous system

city: clermont ferrand

first: yoan

last: renaud

address: place henri dunand

country: France

institute: igred

summary: Enzymes of the Ten Eleven Translocation (TET) family play a key role in the regulation of gene expression in many species by oxidizing 5-methylcytosine (5mC), a prominent epigenetic mark, into 5-hydroxymethylcytosine (5hmC). Yet, TET proteins also have non-canonical modes of action beyond 5mC oxidation, notably in Drosophila, whose genomes is devoid of 5mC. Here, we used a combination of NGS analyses to studied the funciton and mode of action of Tet in the central nervous system of Drosophila larvae.

overall_design: All experiments were performed with biological replicates (duplicates for ChIP-seq and CUT&RUN; quadruplicates for RNA-seq). w1118 (wt)were used as control lines.

first: Lucas

last: Waltzer

first: Yoan

last: Renaud

first: Laurence

last: Vandel

first: Guerric

last: Gilbert


title: A Germline Point Mutation in the MYC-FBW7 Phosphodegron Results in a Tumor-Prone Phenotype

first: Brian

last: Freie

address: 1100 Fairview Ave N

department: Basic Sciences

institute: Fred Hutchinson Cancer Research Center

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

overall_design: Refer to individual Series


title: PARP12 regulates mouse oocyte meiotic maturation

first: Manqi

last: Chen

address: No. 135, Xingang Xi Road

institute: Sun Yat-Sen University

summary: The goal of this study is to identify the differentially expressed genes in PARP12-KD GV-stage fully-grown oocytes (FGO) by comparing their transcriptomes with that of the control-siRNA oocytes via RNA-Seq Analysis.

overall_design: Examination of transcriptome change in  control and PARP12-knockdown GV-stage oocytes

first: Guangyi

last: Cao

first: Chuanbo

last: Sun

first: Jichang

last: Wang


title: Integrative analysis of the lncRNA-miRNA-mRNA Interactions in Smooth Muscle Cell Phenotypic Transitions II

first: Olga

last: Cherepanova

overall_design: Primary mouse aortic smooth muscle cells (MASMC) were isolated and grown as previously described (Cherepanova et al., 2016). For experiments, cells at the 70% confluency were transfected with blocking siOct4 or non-target siRNA (NT siRNA) for 48 hours in serum-free media, followed by the treatment with 10 µg/mL POVPC (Cayman Chem, #10031) or vehicle (DMSO) for 24 hours based on our previous findings (Pidkovka et al., 2007). The samples were used for small RNA expression profiling using Arraystar Mouse small RNA Microarray (8x15K, Arraystar)

first: Aatish

last: Mahajan


title: Novel insights into the role of the histone variant H2A.Z in the plant pathogen Fusarium fujikuroi [RNA-seq]

city: Tulln

first: Harald

last: Berger

state: NOE

address: Konrad-Lorenzstraße 43

department: DAGZ

country: Austria

institute: Universität f. Bodenkultur Wien

summary: Transcriptome in respect to H2A.Z downregulation or overexpression

overall_design: The F. fujikuroi IMI58289 wild-type strain (FfWT) provided by Commonwealth Mycological Institute, Kew, UK, was used as a parental strain to generate the H2A.Z  knock-down strain (Tetoff::FfH2A.Z), the constitutive H2A.Z overexpression (OE::FfH2A.Z) strain as well as the N- and C-terminal  hemagglutinin (HA)-tagged H2A.Z (HA::FfH2A.Z, FfH2A.Z::HA) strains. Submerged fungal growth was performed in 100 mL Darken pre-culture medium (Darken et al., 1959) on a rotary shaker at 180 rpm, 30°C in constant darkness for 3 days.  Cultures inoculated with the TetOff::H2A.Z knock down strain were additionally supplemented  with 0-50 µg/mL DOX to facilitate FfH2A.Z gene silencing. Flasks were incubated on a rotary shaker (180 rpm, 30°C) in the dark. Mycelia were harvested 3-4 days  post inoculation (dpi) for RNA isolation. Isolation of fungal RNA was performed with lyophilized and ground mycelia from cultivation on solid CM or under liquid-standard conditions  (ICI supplemented with 6 or 60 mM glutamine) for 3-4 days. Total RNA was extracted from samples, using the RNA reagent TRIzol (Thermo Fisher Scientific)  as described in the manufacturer’s manual.

first: Anna

last: K Atanasoff-Kardjalieff

first: Slavica

last: Janevska

first: Katharina

last: Steinert

first: Hans-Ulrich

last: Humpf

first: Svetlana

last: Kalinina

first: Joseph

last: Strauss

first: Nadia

last: Ponts

first: Lena

last: Studt-Reinhold


title: Novel insights into the role of the histone variant H2A.Z in the plant pathogen Fusarium fujikuroi


title: Parenchymal damage and myeloid inflammation link the Rag GTPase – mTORC1 axis to mammalian aging [liver]

first: Osvaldo

last: Graña

summary: The mechanistic target of rapamycin complex 1 (mTORC1) controls cellular anabolism in response to nutrient sufficiency and growth factor signaling, and genetic and pharmacological inhibition of mTORC1 extends longevity across eukaryotes. Up to date, mouse models to analyze the mechanisms of aging governed by mTORC1 has been limited by detrimental pleiotropic consequences of genetically-increased mTORC1 activity in mice. RragcS74N/+, RragcS74C/+ and RragcT89N/+ knock-in mice endogenously express active mutant variants of RagC, a GTPase that signaling nutrient sufficiency to mTORC1. Rragcmutant/+ mice show an only moderate increase in nutrient signaling to mTORC1, and after one year of life exhibit multiple features of a premature aging phenotype that include prominent senescence in peripheral organs, parenchymal expression of inflammatory and chemo-attractant molecules, increased myeloid inflammation and extensive features of inflammaging. In vivo transplantation and ex vivo functional experiments with bone marrow-derived cells show that myeloid cells are abnormally activated by parenchymal signals evoked from Rragcmut/+ organs, to where myeloid cells extravasate to inflict additional inflammatory damage. Therapeutic suppression of myeloid inflammation in old Rragcmut/+ attenuates features of premature aging and extends longevity. We provide the first genetic link of elevated nutrient – mTORC1 activity and premature aging in mammals, and provide support for a two-component model in which increased nutrient signaling drives parenchymal damage and myeloid inflammation precipitates organ deterioration and accelerated aging.

overall_design: RNA-seq

first: Ana

last: Ortega

last: Graña-Castro

first: Alejo

last: Efeyan


title: Parenchymal damage and myeloid inflammation link the Rag GTPase – mTORC1 axis to mammalian aging


title: Novel insights into the role of the histone variant H2A.Z in the plant pathogen Fusarium fujikuroi [Mnase-seq]

summary: Localization of nucleosomes in F. fujikuroi

overall_design: Fungal mycelia were cultivated for three days in liquid ICI (6 mm glutamine) prior crosslinking with 1% formaldehyde. Crosslinked and quick-frozen mycelia (3 days, liquid synthetic ICI, 6 mM glutamine) was treated with MNaseI (60 U/µL, Thermo Fisher Scientific) for 4 minutes. The reaction was stopped by the addition of 0.5 M EDTA (final conc. 40 mM EDTA). Reverse cross-linking of digested DNA was performed for at least 30 minutes at 65 °C. Subsequently,  samples were diluted and treated with proteinase K (1h and 40°C, Thermo Fisher Scientific) for protein degradation. DNA fragments were extracted using phenol and separated on an agarose gel to check for successful MNase digestion. Fragmented DNA was cut from the gel at 150 bp and extracted using the Monarch® DNA Gel Extraction Kit (NEB®). Quality control, library preparation and sequencing were performed at the Vienna BioCenter Core Facilities (Vienna, Austria).


title: Novel insights into the role of the histone variant H2A.Z in the plant pathogen Fusarium fujikuroi [ChIP-seq]

summary: ChIP-seq of histone PTM and H2A.Z depositioning in F. fujikuroi

overall_design: The F. fujikuroi IMI58289 wild-type strain (FfWT) provided by Commonwealth Mycological Institute, Kew, UK, was used as a parental strain to generate the H2A.Z  knock-down strain (Tetoff::FfH2A.Z), the constitutive H2A.Z overexpression (OE::FfH2A.Z) strain as well as the N- and C-terminal  hemagglutinin (HA)-tagged H2A.Z (HA::FfH2A.Z, FfH2A.Z::HA) strains. Fungal mycelia were cultivated for three days in liquid ICI (6 mm glutamine) prior crosslinking with 1% formaldehyde. For immunoprecipitation, sonicated mycelia were incubated either with an anti-HA (Abcam, 9110),  an anti-H3K27me3- (Active motif, 39155) or an anti-H3K4me3- (CellSignal, 9751)  specific antibody. The assay was performed in biological duplicates,  including input controls for every strain.


title: Genome-wide maps of chromatin state in MDA-MB-231 cells

city: beijing

first: yan

last: wang

address: 17 panjiayuan nanli, chaoyang district, Beijing, China

country: Zimbabwe

institute: National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences

summary: In order to figure out the genomic binding landscape of CARM1, we analyzed the genome-wide transcriptional targets for CARM1 using the Chromatin immunoprecipitation based-deep sequencing (ChIP-seq) in MDA-MB-231 cells with antibodies against CARM1. Using Illumina instrument, we found lots of CARM1-specific binding peaks and strong  enrichment on the promoters of selected genes involved in classical pathways. This study gave us a new understanding of the role of CARM1 in chromatin status and gene transcription.

overall_design: Examination of  CARM1  target genes in MDA-MB-231 cell line

first: Dandan

last: Feng

first: Yan


title: Detection of newly synthesized RNA reveals transcriptional reprogramming during ZGA and a role of Obox3 in totipotency acquisition

city: Kofu

first: Mizuki

last: Sakamoto

country: Japan

institute: University of Yamanashi

summary: Zygotic genome activation (ZGA) after fertilization is a conserved transcriptional activation process that enables the maternal-to-zygotic transition. However, the global view of ZGA, particularly at the initiation, is not yet completely understood. Here, we develop a method to capture and sequence newly synthesized RNA in the early mouse embryos, which provides a view of transcriptional reprogramming events during ZGA. Our data demonstrate that gene activation associated with major ZGA, previously thought to occur exclusively during the mid-to-late 2-cell stage, is already initiated in zygotes. Furthermore, we identify a set of genes activated during minor ZGA and observe an enrichment of the homeobox-containing Obox factor motif at the promoters of minor ZGA genes. Among several Obox factors, Obox3 overexpression in mouse embryonic stem cells activates ZGA genes. Notably, the expression of Obox3 is severely impaired in somatic cell nuclear transfer (SCNT) embryos, and restoring its expression corrects the ZGA profile and greatly improves SCNT embryo development. Hence, our study reveals the dynamic transcriptional reprogramming during ZGA and underscores the crucial role of Obox3 in facilitating totipotency acquisition.

overall_design: RNA-seq for newly synthesized RNAs, total RNA-seq and CUT&RUN were performed using mouse early embryos or mESCs.

first: Takashi

last: Ishiuchi


title: Parenchymal damage and myeloid inflammation link the Rag GTPase – mTORC1 axis to mammalian aging [old]


title: PDL1 triggered from binding eIF3I contributes to ameliorate diabetes associated wound healing defects by regulating IRS4

first: Bin

last: Li

address: 1200 Cailun Road, Pudong New Area

institute: Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine

summary: We report the application of the transcriptome profiling (RNA-seq) for high-throughput profiling of PD-L1 overexpression in HaCaT cells

overall_design:
Background: Persistent chronic inflammation and delayed epithelialization lead to stalled healing in diabetic ulcers (DU). PDL1, a widely accepted immune checkpoint molecule, shows activities of anti-inflammatory and proliferative in healing defects. However, the role of PDL1 in DU pathogenesis remain unknown.
Methods: Histology and Immunohistochemistry (IHC) staining, Western blot and quantitative real-time polymerase chain reaction (RT-qPCR) were used to examine the expression of eIF3I, PDL1, and IRS4 in DU wounds and HaCaT cell lines. The effects of eIF3I, PDL1, and IRS4 on the proliferation, migration and invasion of HaCaT cells were detected using CCK8, RT-qPCR, wound-healing, and transwell assay. The ability of anti-inflammatory was detected by RT-qPCR. Furthermore, IP-MS, Co-IP, RNA-seq, western blot, and rescue experiments were adopted to explore the regulation relationship among eIF3I, PDL1, and IRS4.
Results: Lower level of PDL1 was found in DU tissues, which might contribute to delayed wound closure. Exogenous PDL1 has therapeutic effects in DU healing process via accelerating re-epithelialization and attenuating prolonged inflammation. In vitro assay demonstrated that overexpression of PDL1 could accelerate HaCaT cells proliferation, migration, invasion, and decrease inflammatory response, whereas silencing of PDL1 could suppress those phenotypes. Mechanistically, PDL1 binded with eIF3I and promoted cutaneous diabetic wound healing through down-regulating IRS4. Relevant to the clinic, we found that human diabetic wounds had elevated IRS4 and reductions in eIF3I and PDL1 compared with normal skin.
Conclusion: Overall, PDL1 ameliorates diabetes associated wound healing defects by interacting with eIF3I involving regulation IRS4. PDL1 can serve as a potential therapeutic target in DU.

first: Yanwei

last: Xiang

first: Le

last: Kuai

first: Yi

last: Ru


title: Parenchymal damage and myeloid inflammation link the Rag GTPase – mTORC1 axis to mammalian aging [young]

overall_design: Expression profiling by high throughput sequencing


title: Catalytic dependent and independent roles of Tet3 in activation of ectodermal and silencing of mesodermal programs during neuroectoderm specification

city: New York

first: Meelad

last: Dawlaty

state: New York

address: 1301 Morris Park Ave, Price 419, Bronx

department: Genetics

institute: Albert Einstein College of Medicine

type: Methylation profiling by high throughput sequencing

summary: In this study: (1) we distinguished Tet3 target genes that are regulated by its catalytic vs. noncatalytic functions in neuroectoderm (NE) cells by transcriptomic profiling of Tet3 wildtype (WT), Tet3 catalytic mutant (Mut), and Tet3 knockout (KO) NE cells by RNA-seq. (2) We mapped genome-wide DNA methylation of Tet3-WT, Tet3-Mut, and Tet3-KO NE cells by WGBS. (3) We determined Tet3 genome-wide occupancy in Tet3-WT NE cells by CUT&Tag. (4) We mapped 5hmC rich regions in Tet3-WT, Tet3-Mut, and Tet3-KO NE cells by hmeDIP. (5) We overexpressed  (OE) the Tet3 target gene Dnmt1 (D) or an empty vector (EV) in Tet3-WT, Tet3-Mut, and Tet3-KO NE cells and assessed gene expression by RNA-seq. (6) We overexpressed (OE) Tet3 (T) or an empty vector (EV) in Tet3-WT, Tet3-Mut, and Tet3-KO NE cells and examined gene expression changes in these cells by RNA-seq.

overall_design: We investigated the catalytic and noncatalytic roles of Tet3 in gene regulation during ESC differentiation toward neuroectoderm (NE). Analysis and integration of RNAseq, WGBS, hmeDIP, and CUT&Tag data revealed novel functions of Tet3 in NE specification. For all omics experiments, Tet3-WT, Tet3-Mut, and Tet3-KO ESCs were differentiated to NE by LIF withdrawal and 1uM retinoic acid (RA) treatment for three days. Cells were collected for each experiment at day 3 (72 hours of differentiation). All RNAseq experiments: RNA was extracted from Tet3-WT, Tet3-Mut, and Tet3-KO NE cell lines (Qiagen RNeasy mini kit) at 72 hours of differentiation. Library preparation and RNAseq were performed by Novogene (https://en.novogene.com/) on an Illumina Novoseq 6000 platform and 20-30 million reads were generated per sample. WGBS: DNA was extracted from one clone of Tet3-WT, Tet3-Mut, and Tet3-KO NE cells (Quick-DNA miniprep kit - Zymo, D3024) and subjected to WGBS analysis at BGI genomics company (https://en.genomics.cn/). Lamda DNA spike-in confirmed the efficiency of bisulfite conversion (>99.0%). CUT&Tag: One Tet3-WT ESC cell line was differentiated to NE (3 days) and used to determine Tet3 genome-wide occupancy in NE cells. Cells were crosslinked, bound to Con-A beads, incubated with primary antibody and treated with pre-loaded pA-Tn5 adapter complex. Libraries were prepared with NEBNext HiFi 2x PCR Master mix and sent for 75-bp paired-end sequencing on an Illumina NextSeq 500 platform at the Einstein Epigenomics core following their protocols. hmeDIP: DNA was extracted from one clone of Tet3-WT, Tet3-Mut, and Tet3-KO NE cells  (Quick-DNA miniprep kit - Zymo, D3024). Pull down of 5hmC enriched regions was perfromed using the Active Motif hmeDIP kit (#55010) and subjected to 75bp paired-end sequencing using Illumina NextSeq 500 platform at the Einstein Epigenomics Core. Details of RNAseq, WGBS, hmeDIP and CUT&Tag procedure and data analyses are described in detail in the methods section of the manuscript.

first: Harmony

middle: C

last: Ketchum


title: Parenchymal damage and myeloid inflammation link the Rag GTPase – mTORC1 axis to mammalian aging [neutrophils]


title: SRSF7-mediated Alternative Splicing of USO1 Promotes Hepatocellular Carcinoma by Activating the MAPK/ERK1-ELK1 Pathway

city: Beijing

first: Gangqiao

last: Zhou

address: 27 Taiping Road, Beijing

institute: Beijing Institute of Radiation Medicine

summary: We conducted a transcriptome-wide survey to identify novel hepatocellular carcinoma (HCC)-associated genes that have undergone aberrant alternative splicing. We revealed that the vesicle transport factor (USO1) is a major alternatively spliced target in HCC, mainly composed of a long wild-type isoform (USO1-L) and a short truncated isoform lacking exons 5 and 15 (USO1-S). A markedly increased isoform switching from USO1-L to USO1-S occurs in approximately 80% of HCCs and predicts poor clinical outcomes. USO1-L suppresses HCC cells growth and metastasis through weakening the activation of MAPK/ERK1-ELK1 signaling by interacting with phosphorylated ERK1 and anchoring it onto the Golgi apparatus to reduce its nuclear translocation; while USO1-S confers a loss-of-function effect. The splicing factor SRSF7, which is shown to be hypomethylated and upregulated in HCC, mediates the splicing of USO1-L to produce USO1-S. Notably, the loss of USO1-L in HCC cells induces their resistance to MEK inhibitors; however, restoring USO1-L through antisense oligodeoxynucleotide (ASO)-mediated blockade of switching from USO1-L to USO1-S can reverse this resistance. In summary, our findings highlight the crucial role of aberrantly alternative splicing of USO1 in HCC, as well as the SRSF7-USO1-MAPK axis as a potential target for this malignancy.

overall_design: To confirm the effects of SRSF7 and antisense oligonucleotides (ASOs) targeting the binding sites of SRSF7 within the USO1 pre-mRNA, MHCC97H cells under overexpression or HepG2 cells under ASO treatment (ASO-5 or ASO-15)  were subjected to RNA extraction and short-read RNA sequencing analyses. Additionally, HepG2 cells under ASO treatment (ASO-5 or ASO-15)  were subjected to RNA extraction and long-read RNA sequencing analyses.

first: Pengbo

last: Cao

first: Qian

last: Jin

first: Hongbo

last: Luo

first: Shizhou


title: The function of USP36  in the ER positive Breast Cancer

status: Public on Apr 01 2024

city: xinxing

first: huijie

last: yang

address: Hongqi District, Xinxiang City

institute: Xinxiang Medical University

summary: To investigate the  function of USP36  in the ER positive Breast Cancer.

overall_design: MCF-7 cell mRNA samples were summarized in six samples, divided into two groups, siControl and siUSP36. Comparative gene expression profiling analysis of RNA-seq data for MCF-7 cells treated with siControl and siUSP36.


title: Next-generation sequencing Facilitates Quantitative Analysis of Wild Type and EED-/- mice cerebellum Transcriptomes and Genome-wide maps of chromatin state  in Wild Type and EED-/- mice cerebellum

first: Peipei

last: liu

address: No. 25-1 West Beisihuan Rd, Haidian District, Beijing

department: State Key Laboratory of Stem cells and Reproductive Biology

institute: institute of zoology,Chinese academy of sciences

summary:
Purpose: The goals of this study are to compare Wild Type and EED-/-  cerebellum transcriptome profiling (RNA-seq) to quantitative reverse transcription polymerase chain reaction (qRT–PCR) differentially expressed genes and find target genes. We performed chromatin-immunoprecipitation coupled to sequencing (ChIP-seq) to dissect the relationship between the differentially expressed genes and the occupancy of H3K27me3, H3K27ac and EED.
Methods: Cerebellum mRNA profiles of 14-day-old(P14) wild-type (WT) and Embryonic Ectoderm Development conditional knockout (EED-/-) mice were generated by deep sequencing,  in triplicate,using  Illumina HiSeq 2500 system.All RNA-Seq data were aligned to mouse genome version mm10 using  Salmon (v.1.1.0) .We use cluster software and Euclidean distance matrix for the hierarchical clustering analysis of the expressed gene and sample program at the same time, the clustering results can be viewed with javaTreeview. qRT–PCR validation was performed using SYBR Green assays. we performed ChIP-seq using freshly isolated P14 from  Wild Type and EED-/-  cerebellar tissues  . DNA libraries generated from ChIP-DNA and input-DNA were deep-sequenced using HiSeq single-end 50bp.
Results:Only transcripts that showed more than 1.5-fold differential expression compared to control were subjected to relevance network analysis. 3149 candidate genes that were differentially expressed with biological functional groups as compared between WT and EED-/- mice .We found that the enrichment of H3K27me3 was decreased H3K27ac was highly increased in EED-/- mice.
Conclusions: Our study represents the first detailed analysis of Wild Type and EED-/  mice cerebellum transcriptomes, with three biologic replicates, generated by RNA-seq technology. We report the application of ChIP-sequencing technology for high-throughput profiling of histone modifications in cerebellar cells.

overall_design: Cerebellum mRNA profiles  and  histone modifications of 14-day-old wild-type (WT) and Embryonic Ectoderm Development conditional knockout (EED-/-) mice

first: Pei-Pei

last: Liu

first: Chang-Mei


title: Integrated transcriptome and Ribosome Profiling analyses elucidate the liver response of rainbow trout (Oncorhynchus mykiss) to heat stress

city: Lanzhou

first: guiyan

last: zhao

address: Yingmen Village 1

institute: Gansu Agricultural University

summary: Rainbow trout (Oncorhynchus mykiss) is highly sensitive to high-temperature stress as an important economic cold-water fish. While previous research has concentrated on the transcriptomic to acute heat stress in rainbow trout, there remains a gap in knowledge regarding the overarching regulatory mechanisms at the translation level. In our research, we utilized a combination of transcriptomic and translatomic analyses to investigate the intricate molecular response mechanisms in the liver of rainbow trout when subjected to heat stress. Through comprehensive multi-omics analysis, we revealed the dynamic translational pattern of rainbow trout liver under heat stress for the first time. Comparative analysis of ribosome analysis data with RNA-seq data showed that the fold changes of gene expression at the transcriptional level were highly correlated (R2 = 0.83) with those at the translational level globally. In total, 2,203 genes exhibited significant alterations exclusively within the translational level. However, the limited overlap in response genes between transcription and translation under heat stress suggests that these two processes may independently modulate the cellular response to thermal challenges. Significant changes in the translation efficiency of 809 genes were observed under heat stress. Further analysis indicated that the translation efficiency of genes were strongly influenced by sequence characteristics such as GC content, coding sequence length and NMFE. Moreover, 3,468 putative uORFs were identified in 2,676 genes, which potentially modulating translation efficiency of mORFs. These findings provide a novel perspective for understanding the physiological adaptations of rainbow trout in response to changes in ambient temperature.

overall_design: mRNA expression profiles in the liver of rainbow trout (Oncorhynchus mykiss) at 18℃ and heat stress（24℃） for 8 h

first: Zhe

first: Gui-yan

last: Zhao


title: Non-genetic differences underlie variability in proliferation among esophageal epithelial clones

city: Philadelphia

first: Sydney

middle: M.

last: Shaffer

state: PA

address: 421 Curie Blvd

department: Department of Pathology

institute: University of Pennsylvania

summary: Individual cells grown in culture exhibit remarkable differences in their growth, with some cells capable of forming large clusters, while others are limited or fail to grow at all. While these differences have been observed across cell lines and human samples, the growth dynamics and associated cell states remain poorly understood. In this study, we performed clonal tracing through imaging and cellular barcoding of an in vitro model of esophageal epithelial cells (EPC2-hTERT). We found that about 10% of clones grow exponentially, while the remaining have cells that become non-proliferative leading to a halt in the growth rate. Using mathematical models, we demonstrate two distinct growth behaviors: exponential and logistic. Further, we discovered that the propensity to grow exponentially is largely heritable through four doublings and that the less proliferative clones can become highly proliferative through increasing plating density. Combining barcoding with single-cell RNA-sequencing (scRNA-seq), we identified the cellular states associated with the highly proliferative clones, which include genes in the WNT and PI3K pathways. Finally, we identified an enrichment of cells resembling the highly proliferative cell state in the proliferating healthy human esophageal epithelium.

overall_design: EPC2-hTERT cells were barcoded and grown in tissue culture, sorted using FACS, and plated sparsely. We then perfomed single-cell RNA sequencing using 10X Genomics sequencing.

first: Raul

last: Reyes Hueros

first: Rodrigo

last: Gier


title: RNA-sequencing analysis of Nrf2 target genes in human pulmonary arterial smooth muscle cells and endothelial cells

city: Yangling

first: Xia

state: Shannxi

address: No.3 Taicheng Road, Yangling Shaanxi, China

institute: northwest a&f university

summary: To investigate the genes regulated by Nrf2 in human PASMC and PAEC, Nrf2 was knockdown by using siRNA. 48h after siRNA transfection， cells were collected for RNA-sequencing.

overall_design: Each cell type were respectively transfected with one Control siRNA and two Nrf2 siRNAs，each treatment has three replicates.

first: Ang

first: Haiyang

last: Tang


title: NEMO Knockout Drives Variations in Gene Expression Regenerative Trajectory and Induces Chromatin Remodeling in  murine muscle satellite cells during healing from traumatic injury

city: Boston

first: Sundari

last: Chetty

state: MA

address: 185 Cambridge Street

department: Harvard Medical School

institute: Massachusetts General Hospital, Harvard University

summary: This study investigates the impact of NEMO knockout on healing from traumatic tibial injury within a murine muscle satellite cell model. NEMO KO  mice were profiled via RNA-seq at 0, 2, 4, 7 days post cold shock injury (DPI). The largest transcriptional response was observed at 2 DPI within the WT model, but NEMO knockouts exhibited delayed myogenesis and muscle regenerative defects. Furthermore, it was observed that new fiber formation is defected upon transplantation of mutant derived MuSCs. mRNA profiling further suggested that loss of NF-kB signaling in MuSC’s in response to NEMO KO alters their ECM and cytokine secretome production. Additional profiling was performed at 2DPI via ATAC-seq within WT and NEMO mouse satellite cells, with observed changes in chromatin remodeling suggesting bidirectional MuSC-transcriptional regulator communication in skeletal muscle regeneration.

overall_design: To investigate the impact of NEMO knockout on healing from muscle injury within murine muscle satellite cells, NEMO KO  mice were profiled via RNA-seq at 0, 2, 4, 7 days post tibial injury. The largest transcriptional response was observed at 2 DPI, so additional profiling was performed at 2DPI via ATAC-seq within WT and NEMO mouse satellite cells.

first: Carlos

last: Aguilar

first: Benjamin

last: Yang

first: Anna

last: Shcherbina

first: Faye

last: Mourkioti


title: RNA-seq analysis of young adult  fed  5mM tryptophan and control  worms

city: Kaifeng

first: Shanqing

last: Zheng

address: 1 Jinming Avenue North, Longting District,  School of Basic Medical Sciences, Henan University

institute: Henan university

summary: RNA-seq analysis was used to identify the gene expression changes of young adults after feeding 5mM tryptophan for 36 hours from the L4 period. Based on these sequencing data, the genes that were up-regulated or down-regulated by at least 2 times (|log2 (folding change) | ≥ 1 and padj ≤ 0.05) were analyzed. The analysis of these differentially expressed genes' pathway and gene ontology (GO) enrichment shows that the influence of different genders after feeding 5mM tryptophan is enriched to some signal pathways, including longevity、ribosome and protein degradation pathway in endoplasmic reticulum, etc.

overall_design: When tryptophan was fed, mRNA profile changes for each group were generated by deep sequencing. Use Illumina Gallx

first: shanqing

last: zheng


title: The Eμ-Ret mouse is a novel model of hyperdiploid B-cell acute lymphoblastic leukemia

city: Vancouver

first: Ali

last: Farrokhi

state: BC

address: BC Childrens Hospital Research Institute, 950 west 28th Ave- Ro0m 3062

country: Canada

institute: University of British Columbia

summary: The presence of supernumerary chromosomes is the only abnormality shared by all patients diagnosed with high-hyperdiploid B cell acute lymphoblastic leukemia (HD-ALL). Despite being the most frequently diagnosed pediatric leukemia, the lack of clonal molecular lesions and complete absence of appropriate experimental models have impeded the elucidation of HD-ALL leukemogenesis. Here, we report that for 23 leukemia samples isolated from moribund Eμ-Ret mice, all were characterized by non-random chromosomal gains, involving combinations of trisomy 9, 12, 14, 15, and 17. With a median gain of three chromosomes, leukemia emerged after a prolonged latency from a preleukemic B cell precursor cell population displaying more diverse aneuploidy. Transition from preleukemia to overt disease in Eμ-Ret mice is associated with acquisition of heterogeneous genomic abnormalities affecting the expression of genes implicated in pediatric B-ALL. The development of abnormal centrosomes in parallel with aneuploidy renders both preleukemic and leukemic cells sensitive to inhibitors of centrosome clustering, enabling targeted in vivo depletion of leukemia-propagating cells. This study reveals the Eμ-Ret mouse to be a novel tool for investigating HD-ALL leukemogenesis, including supervision and selection of preleukemic aneuploid clones by the immune system and identification of vulnerabilities that could be targeted to prevent relapse.

overall_design:
To identify cellular pathways involved in the transition from preleukemia to leukemia, BP-1+ B-cell precursors were harvested from 4 young, healthy Eμ-Ret mice and 3 moribund older leukemic mice.
We then performed comparative gene expression profiliing of data obtained from RNA-seq of all samples

first: Tanmaya

last: Atre

first: Jenna

last: Rever

first: Mario

last: Fidanza

first: Samuel

last: Salitra

first: Junia

last: Myung

first: Meiyun

last: Guo

first: Wendy

last: Duey

first: Sumin

last: Jo

first: Anuli

last: Uzozie

first: Fatemeh

last: Baharvand

first: Nina

last: Rolf

first: Franziska

last: Auer

first: Julia

last: Hauer

first: Stephan

last: Grupp

first: Patrice

last: Eydoux

first: Philipp

last: Lange

first: Alix

last: Seif

first: Christopher

last: Maxwell

first: Gregor

last: Reid


title: SFRP2+ CAFs Hinder Abscopal Effect from Combined Radiotherapy and Immunotherapy by Forming a Hostile Perivascular Niche

city: Guangzhou

first: Yanpei

last: Zhang

state: Guangdong

address: No.1023,South Shatai Road, Baiyun District, Guangzhou, Guangdong, China

institute: Southern Medical University

summary: Despite accumulating cases of radiotherapy-induced abscopal effect in the lung cancer with the introduction of immune checkpoint inhibitors (ICIs), the occurrence of this effect remains infrequent and unpredictable to be a therapeutic goal. Here, we showed that the combination of radiotherapy (8Gy*3F) and ICI alleviated the tumor burden at the irradiated site whereas no discernible benefit was observed in the abscopal tumors. RNA-sequencing data showed that extracellular structure organization pathways were enriched in the abscopal tumors after combined therapy, with Sfrp2 being identified as a central hub. SFRP2 expression was observed in cancer-associated fibroblasts (CAFs) and was elevated in abscopal tumors after combined therapy. Blockade of SFRP2 followed by combined radiotherapy and ICI reinvigorated infiltration and cytotoxicity of CD8+ T cells, and elicited regression of abscopal tumors, which was abrogated by CD8α depletion. Mechanistically, in vitro experiments demonstrated that SFRP2+ CAFs induced apoptosis of CD8+ T cells. The spatial transcriptome analysis showed that SFRP2+ CAFs were located in proximity to the vessels and surrounded by abundant macrophages and limited CD8Tex, thereby creating an immunosuppressive perivascular niche, which was validated in paraffin sections of human lung cancer. Lineage-tracing assays showed SFRP2+ CAFs were derived from pericytes. IGF-1 released by irradiated tumors facilitated the transition of pericytes into fibroblasts and stimulated the expression of SFRP2. In summary, SFRP2+ CAFs hijack the abscopal effect from combined radiotherapy and immunotherapy via inducing apoptosis of CD8+ T cells and orchestrating a hostile perivascular niche in the lung cancer. Targeting SFRP2+ CAF may recondition the TME and promote the abscopal effect.

overall_design: RNA-sequencing of 4T1 murine tumor specimens administered with different treatments:  immune checkpoint inhibitor, or combination of immune checkpoint inhibitor and radiation. 3 replicates for each group.


title: First-in-Class Substrate- and Function-Selective p38alpha Inhibitors with Anti-inflammatory and Endothelial-stabilizing Activities

city: Baltimore

first: Jeffrey

middle: David

last: Hasday

state: MD

address: 20 Penn St

department: Medicine

institute: University of Maryland School of Medicine

summary: We previously identified a small molecule, UM101, predicted to bind to the substrate-binding groove of MAPK14/p38a near the binding site of the proinflammatory substrate, MAPK-activated protein kinase (MK2). UM101 exhibited anti-inflammatory, endothelial-stabilizing, and lung-protective effects. We developed an analog of UM101, GEn-1124, with improved aqueous stability and p38a binding affinity. Compared with UM101, GEn-1124 has 60-fold greater p38a binding affinity as measured by Surface Plasmon Resonance (SPR), 66-fold greater aqueous solubility, enhanced barrier-stabilizing activity in thrombin-stimulated human pulmonary artery endothelial cells (hPAEC) in vitro, and greater lung protection in a mouse models of acute lung injury (ALI). GEn-1124 reduced lung injury and improved survival in a mouse model of ALI induced by intratracheal LPS instillation and exposure to febrile-range hyperthermia (FRH) from 10% to 40% and in a mouse influenza pneumonia model from 0% to 50%. RNASeq and pathway analysis of gene expression in TNFa-treated hPAEC showed that the gene-modifying effects of GEn-1124 were much more restricted to TNFa-inducible genes than SB203580. Gene expression pathway analysis, confocal immunofluorescence analysis of p38a and MK2 subcellular trafficking, and SPR analysis of phosphorylated p38a:MK2 binding affinity supports a novel mechanism of action. GEn-1124 destabilizes the phosphorylated p38a:MK2 complex, dissociates nuclear export of phosphorylated p38a and MK2, thereby promoting intranuclear phosphorylated p38a retention and intranuclear signaling, and accelerating inactivation of p38-free cytosolic MK2 by unopposed phosphatases.

overall_design: Human pulmonary artery endothelial cells (hPAEC) monolayers were pretreated for 60 min with DMSO or 10 µM SB203580, or one of the four novel inhibitors at 25 µM in an 0.1% final DMSO concentration. The cells were then stimulated with 10 ng/ml human TNFa for 3h. Untreated controls received neither pretreatment nor TNFa.  Poly(A)-enriched samples were reverse transcribed and sequenced using the Illumina HiSeq platform. All treatments were done in triplicate.

first: Mohan

last: Tulapurkar

first: Kari

last: Shirey

first: Katerina

last: Lugkey

last: Luo

first: Ritu

last: Lal

first: Adam

last: Galan

first: Omar

last: Mahmoud

first: Nathaniel

last: McClean

first: Steven

last: Fletcher

first: Alexander

last: MacKerell

first: Stefanie

last: Vogel

first: Paul

last: Shapiro


title: STAT6 in mitochondrial outer membrane impairs mitochondrial fusion by inhibiting MFN2 dimerization

city: Suwon

first: Hyunmi

last: Kim

address: 206, World cup-ro, Yeongtong-gu

country: South Korea

institute: Ajou University School of Medicine

summary: Although it is reported that mitochondria-localized nuclear transcription factors (TFs) regulate mitochondrial processes such as apoptosis and mitochondrial transcription/respiration, their functions and mechanisms of mitochondrial dynamics regulated by mitochondria-localized nuclear TFs are yet to be fully characterized. Here, we identify STAT6 as a mitochondrial protein that is localized in the outer membrane of mitochondria (OMM). In addition to regulating mitochondrial gene expression as a nuclear TF, STAT6 in OMM inhibits mitochondrial fusion by blocking MFN2 dimerization. This implies that STAT6 has dual role in overall mitochondrial process. Moreover, mitochondrial accumulation of STAT6 in response to these findings reveal that STAT6 is a new regulator of mitochondrial processes including mitochondrial biogenesis and fusion/fission mechanisms.

overall_design: To further investigate the role of STAT6 in sustaining mitochondrial gene expression, we performed RNA sequencing (RNA-seq) profiling of Mock-mCherry+ and STAT6 WT-mCherry+ 293T cells.

first: Hynumi


title: Interaction between HIV-1 Tat and EBV Zta favours immune escape of B cells by downregulating HLA-ABC expression

city: Villejuif

first: Anna

last: Schwager

address: 114 Rue Edouard Vaillant

institute: Institut Gustave Roussy

summary: Both Human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV) are associated with an increased risk of malignancies. People living with HIV frequently have EBV reactivation and develop EBV-associated B-cell malignancies. In this study, we aimed to uncover the involvement of HIV-1 and EBV co-existence in the development of B-cell malignancies. We studied two viral transcriptional activators (HIV-1 Tat and EBV Zta) and their possible interaction since they both have cell-penetration domains and can be found simultaneously in the blood or cells of people with HIV. We found that Tat and Zta directly bound each other in human B cells, T cells, and blood serum. Using RNA-sequencing, we found that combined Tat and Zta action in B cells differed from a simple combination of two proteins. A subset of genes, activated by Tat or Zta alone, that trigger an immune response and antigen presentation in B cells, remained unchanged when two proteins were combined. B cells, treated or transfected with Tat and Zta, exhibited a substantial decrease in HLA-ABC (MHC class I) expression, a critical component of the antigen processing and presentation pathway. HLA-ABC downregulation induced by Tat and Zta interaction conferred protection against cytotoxic T cell recognition of EBV-infected B cells. Tat and Zta interaction was also observed in serum from an HIV-positive individual. To conclude, we demonstrated for the first time the direct interaction between HIV-1 Tat and EBV Zta; this interaction can bring about immune evasion of EBV-infected or transformed B cells.

overall_design: Purified B cells or BLAS cells were treated with Tat (250 ng/ml ~ 25 μM), Zta (1 µg/ml ~ 37 μM) or Tat and Zta (at 250 ng/ml and 1 µg/ml, respectively) or left untreated. 6 h and 48h later cells were collected and RNA was extracted using a NucleoSpin RNA isolation kit (Macherey-Nagel, Germany) following manufacturer’s instructions. Total RNA from BLAS cell lines was also isolated using the NucleoSpin RNA kit following the manufacturer's instructions. Each condition was performed and analyzed in triplicates. The cDNA libraries were constructed and sequenced in Novogene, China.


title: ZBP1 and TRIF trigger lethal necroptosis in mice lacking caspase-8 and TNFR1

city: South San Francisco

first: Rohit

last: Reja

address: 1 DNA Way

institute: Genentech

summary: Necroptosis is a lytic form of cell death that is mediated by the kinase RIPK3 and the pseudokinase MLKL when caspase-8 is inhibited downstream of death receptors, toll-like receptor 3 (TLR3), TLR4, and the intracellular Z-form nucleic acid sensor ZBP1.  Oligomerization and activation of RIPK3 is driven by interactions with the kinase RIPK1, the TLR adaptor TRIF, or ZBP1.  In this study, we use immunohistochemistry (IHC) and in situ hybridization (ISH) assays to generate a tissue atlas characterizing RIPK1, RIPK3, Mlkl, and ZBP1 expression in mouse tissues.  RIPK1, RIPK3, and Mlkl were co-expressed in most immune cell populations, endothelial cells, and many mucosal epithelia.  ZBP1 was expressed in many immune populations, but had more variable expression in epithelia compared to RIPK1, RIPK3, and Mlkl.  Intriguingly, expression of ZBP1 was elevated in Casp8-/- Tnfr1-/- embryos prior to their succumbing to aberrant necroptosis around embryonic day 15.  ZBP1 contributed to this embryonic lethality because rare Casp8-/- Tnfr1-/- Zbp1-/- mice survived until after birth.  Necroptosis mediated by TRIF contributed to the demise of Casp8-/- Tnfr1-/- Zbp1-/- pups in the perinatal period.  Of note, Casp8-/- Tnfr1-/- Trif-/- Zbp1-/- mice exhibited autoinflammation and morbidity, typically within 5-7 weeks of being born, which is not seen in Casp8-/- Ripk1-/- Trif-/- Zbp1-/-, Casp8-/- Ripk3-/-, or Casp8-/- Mlkl-/- mice.  Therefore, after birth, loss of caspase-8 probably unleashes RIPK1-dependent necroptosis driven by death receptors other than TNFR1.

overall_design: RNAseq on mouse fetal livers, age E15.5 for the following genotypes: WT, Tnfr1 KO, Casp8 Mlkl dKO and Casp8 Tnfr1 dKO

first: R


title: Centromeric chromatin in mitosis reorganizes into a functional structure stabilized by cohesin [CENPA-ChIP-seq]

city: Edinburgh

first: Shaun

middle: Michael

last: Webb

address: 2.21 Swann Building, Kings Buildings

department: Wellcome Trust Centre for Cell Biology

institute: University of Edinburgh

summary: Three-dimensional organization of mitotic chromosomes is established by cohesin and condensin complexes. In the centromere, cohesin maintains sister chromatid pairing until anaphase onset, while condensin provides elastic resistance to spindle forces. However, how condensin and cohesin structure vertebrate centromeres remains unclear. By super-resolution imaging, Capture-C analysis and polymer modeling we show that vertebrate centromeres are partitioned into two condensin-dependent subdomains during mitosis. This bipartite sub-structure is found in human, mouse and chicken centromeres, and is therefore a fundamental feature of vertebrate centromere. Super-resolution imaging and electron tomography reveal that bipartite centromeres assemble bipartite kinetochores with each subdomain capable of binding a distinct microtubule bundle. Cohesin helps to link the centromere subdomains, limiting their separation in response to spindle forces and preventing merotelic attachments. The two-domain structure described here may have implications for avoiding chromosomal instability as uncoupling of centromere subdomains is a common feature of lagging chromosomes in cancer cells.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for Histone veriant CENP-A to identify centromere region.

first: Tetsuya

last: Hori

first: Kumiko

last: Samejima

first: Tatsuo

last: Fukagawa

first: William

last: Earnshaw


title: Centromeric chromatin in mitosis reorganizes into a functional structure stabilized by cohesin [Halo-ChIP-seq]

overall_design: Caliburated Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for condensin I subnit (CAP-H) and condensin II subunit (CAP-H2) at, G2, prophase, and prometaphase.

first: Moonmoon

last: Deb


title: Centromeric chromatin in mitosis reorganizes into a functional structure stabilized by cohesin

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

overall_design: Refer to individual Series


title: Centromeric chromatin in mitosis reorganizes into a functional structure stabilized by cohesin [NG-Capture-C]

overall_design: NG-Capture-C analysis to inveistigate chromatin organisation of non-repetitive centromres such as Z and 5 in chicken DT40 CDK1as cells at G2 and prometaphase.

first: Daniel

last: Robertoson


title: All-Trans-Retinoic Acid Treatment of IDH Mutant Astrocytes

first: Nduka

middle: M

last: Amankulor

address: 3400 Civic Center Boulevard, 15th Floor

department: Department of Neurosurgery

summary: Immortalized human astrocytes were stably transfected to express IDH1 R132H. These cells were treated with 1uM ATRA

overall_design: Astrocytes expressing IDH1 R132H were treated with 1uM ATRA or equal volume DMSO for 48 hrs.

first: Jonathan

last: Sussman

first: Aparna

last: Rao

first: Xiaoran

last: Zhang


title: Dual Barcode is Essential for the Specificity of DSB Repair Capture

city: Stanford

first: Jinglong

last: Wang

state: CA California

address: 1291 Welch Rd, CCSR Room 1240

department: Radiation Oncology

institute: Stanford University

summary: We discovered that PCR-mediated template switching poses a significant challenge in ensemble tagged PCR, particularly in the template sequences with high similarities, which we successfully addressed by introducing a dual barcode. Template switching presents in repair sequencing (repair seq) and severely interfere the interpretation of the association between repair outcomes and the sgRNA in the vicinity. Among the 67 DNA damage response genes, we found that ERCC6L2 was crucial for preventing the DNA end resection.

sample_organism:
Escherichia coli
Homo sapiens

platform_organism:
Escherichia coli
Homo sapiens

overall_design: K562-iCas9-DDR cell line was generated by transfection lentivirus bearing iCas9-blast (includes GFP) and select a K562-iCas9 cell line, followed by transfecting lentivirus bearing pMCB320-DDR bear dual barcodes (includes mCherry) and selected in RPMI-1640 with 5 μM puromycin plus 5 μM blasticidin for two weeks until mCherry-positive cells reached > 99%. To knockout the corresponding DDR genes, 2 μM Doxycycline was added and maintained for more than one week. The DSBs in cycling and G1/G0-arrrest K562-iCas9-DDR cells were generated by Cas12a plus DDR1-crRNA nucleoproteins (63 pmol Cas12a:crRNA per 2 million cells) targeting a region in the dual-barcode DDR cassette. After nucleofection, the K562-Bcl2 cells were maintained in RPMI-1640 plus 5 μM puromycin and 5 μM blasticidin for two days. The genomic DNA were extracted and subjected to standard library prepration with two rounds of PCR.

first: Richard

last: Frock


title: Epidermal Growth Factor Rejuvenates Aged Hematopoietic Stem Cells

city: West Hollywood

first: AGCT

last: Core

address: 8687 Melrose Ave, PDC B230

department: BMS

institute: Cedars Sinai Medical Center

summary: Hematopoietic aging is associated with decreased hematopoietic stem cell (HSC) self-renewal capacity and increased risk for myelodysplasia and leukemia. Deficient DNA repair contributes to the decline in HSC self-renewal capacity during aging and it remains unclear whether extrinsic signals can rejuvenate aged HSCs. Here, we demonstrate that augmentation of non-homologous end-joining (NHEJ) DNA repair in aged HSCs via treatment with epidermal growth factor (EGF) rejuvenates HSC function. Seven day culture of BM CD34-ckit+sca-1+lin- (34-KSL) HSCs from aged C57BL/6 mice with EGF suppressed myeloid skewing and increased production of multipotent CFU-granulocyte, erythroid, monocyte and megakaryocyte (CFU-GEMM) colonies. Aged, EGF-treated HSCs displayed increased donor multilineage engraftment in primary competitively transplanted mice and in secondary mice compared to mice transplanted with aged, control HSCs. Donor cell engraftment within the bone marrow (BM) KSL and SLAM+KSL HSC population was > 2-fold increased in mice transplanted with aged, EGF-treated HSCs. Systemic administration of EGF to aged mice for 6 weeks also increased long term – HSC self-renewal capacity as measured by increased donor bone marrow (BM) competitive repopulation in primary and secondary transplanted mice. Conversely, deletion of EGFR in Scl/Tal1+ hematopoietic cells was associated with increased myeloid skewing and depletion of LT-HSCs in middle aged mice. Mechanistically, EGF treatment decreased DNA damage in aged HSCs through activation of DNA PK-cs, Artemis and NHEJ repair. Inhibition of DNA PK-cs blocked EGF-mediated restoration of multipotent differentiation and suppression of myeloid skewing in aged HSCs, suggesting that the restoration of hematopoietic potential in aged HSCs is dependent on EGF-mediated activation of DNA PK-cs. EGF treatment also converted the transcriptome of aged HSCs from enrichment for genes involved in cell death and survival to genes involved in HSC generation and identity. These data suggest that extrinsic activation of EGFR signaling can restore key functional capacities in aged HSCs.

overall_design: For single-cell RNA sequencing (scRNA-seq), BM CD34-KSL cells were sorted from mice after subcutaneous injection of 1 µg/g EGF or saline in 200 µL daily for 3 days. RNA was extracted using the Chromium Single Cell 3’ V2 Reagents Kits. The libraries were sequenced on the NovaSeq 6000 platform. Sequence reads were aligned and analyzed using the Cell Ranger Count pipeline (v7.0.1) (10X Genomics) to generate cell-associated gene expression matrices. The expression data was then normalized via Scran deconvolution36 and differential expression among samples was measured with DESeq 2 algorithm in Partek Flow. UMAP plots were generated in R v4.2.2 using the Seurat v4.2.1 package following dimensionality reduction using the first ten principal components while density plots were generated using ggplot2 v3.4.0 and the stat_density_2d function. Statistical filters of p < 0.05, FDR < 0.05, and fold change > 1.5X were applied prior to Ingenuity Pathway Analysis

first: John

middle: P

last: Chute

first: Aldi

last: Chan

first: Vivian

middle: Y

last: Chang