title: Complete male-to-female sex reversal in mice lacking the miR-17~92 cluster [RNA-seq]

city: Berlin

first: Irene

last: Mota-Gomez

address: Hannoversche strasse 28

institute: Max Delbruck Center

summary:
The deletion of a single miRNA cluster, miR-17~92, is sufficient to induce primary sex reversal in XY mice. The expression of the testis determining gene, Sry, is delayed in embryonic XY miR-17~92 knockout gonads, which immediately activate the ovarian genetic program. Single cell RNA-seq analysis shows that Sertoli cell differentiation is highly reduced, delayed and unable to trigger testis differentiation. Consistent with the well-known role of miRNAs in gene regulation, the expression of target genes of miR-17~92 is not stabilized in XY mutant gonads at E11.5, affecting, in turn, the fine regulation of large gene networks involved in mammalian sex determination. Our results reveal that the miR-17~92 cluster is a novel sex-determining factor that modulates several gene networks required for accurate timing of Sry expression and Sertoli cell differentiation during testis determination and early differentiation.
bulk RNA-seq, single cell RNA-seq as well as IF and other characterizations of a transgenic mice for miR-17~92 cluster

overall_design: Alicia Hurtado

first: Miguel

last: Lao

first: Francisca

last: Real

first: Johanna

last: Jedamzick

last: Burgos

first: Darío

last: Lupiáñez

first: Rafael

last: Jiménez

first: Francisco

last: Barrionuevo


title: Cpxm2 as a novel candidate for cardiac hypertrophy and failure in hypertension [Affymetrix]

status: Public on Jul 10 2023

city: Muenster

first: Anika

last: Witten

address: Domagkstr. 3

department: Institute of Human Genetics, Department Genetic Epidemiology

country: Germany

institute: University of Muenster

summary: We assigned carboxypeptidase X 2 (Cpxm2) to a genetic locus for left ventricular mass. The functional role of Cpxm2 was investigated in Cpxm2-deficient (KO) and wild-type (WT) mice exposed to deoxycorticosterone acetate (DOCA)-salt hypertension and control conditions (SHAM). Both WT and KO animals developed severe and similar systolic hypertension in response to DOCA. WT mice developed severe LV damage. These changes were significantly ameliorated or even normalized (i.e. ejection fraction) in KO-DOCA animals. LV transcriptome analysis in WT, but not in KO mice, showed a molecular cardiac hypertrophy/remodeling signature with significant upregulation of 1234 transcripts including Cpxm2 in response to DOCA.

overall_design: In the mouse model whole transcriptome profiling was performed on LV tissues from male SHAM and DOCA animals of both WT and KO mice (n = 3, respectively). The RNA was processed and hybridized on the GeneChip® Mouse Gene 2.0 ST Array (Affymetrix UK Ltd., High Wycombe, UK). Total RNA for both microarray workflows was prepared for hybridization according to the manufacturer’s instructions.

first: Katja

last: Grabowski

first: Reinhold

last: Kreutz

first: Martin

last: Schädlich

first: Monika

last: Stoll


title: Complete male-to-female sex reversal in mice lacking the miR-17~92 cluster [scRNA-seq]

status: Public on Jul 09 2023

type: Expression profiling by high throughput sequencing

summary: The deletion of a single miRNA cluster, miR-17~92, is sufficient to induce primary sex reversal in XY mice. The expression of the testis determining gene, Sry, is delayed in embryonic XY miR-17~92 knockout gonads, which immediately activate the ovarian genetic program. Single cell RNA-seq analysis shows that Sertoli cell differentiation is highly reduced, delayed and unable to trigger testis differentiation. Consistent with the well-known role of miRNAs in gene regulation, the expression of target genes of miR-17~92 is not stabilized in XY mutant gonads at E11.5, affecting, in turn, the fine regulation of large gene networks involved in mammalian sex determination. Our results reveal that the miR-17~92 cluster is a novel sex-determining factor that modulates several gene networks required for accurate timing of Sry expression and Sertoli cell differentiation during testis determination and early differentiation.

overall_design: bulk RNA-seq, single cell RNA-seq as well as IF and other characterizations of a transgenic mice for miR-17~92 cluster

first: Alicia

last: Hurtado

middle: M

middle: G

middle: J


title: Chemogenetic activation of astrocytes in the hippocampus and cortex changes the transcriptome of microglia

city: Indianapolis

first: Stephanie

last: Philtjens

state: Indiana

address: 320 W. 15th Street, NB Bldg Rm 103

country: USA

institute: Indiana University School of Medicine

summary: Astrocytes are the most common glial cell type in the brain, yet, it is still not clear how their activation affects the transcriptome of neighboring microglia and neurons. Engineered G protein-coupled receptors (GPCRs) called Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) make it possible to selectively activate specific cell types, such as neurons and astrocytes. By combining the selective activation of astrocytes with single cell RNA sequencing, we were able to study transcriptional changes that occur in response to the activation of astrocytes at the single cell level. Interestingly, our data shows that long-term activation of astrocytes in healthy mice results in dramatic alteration in the transcriptome of astrocytes and microglia. Genes that were differentially expressed in these Gq-DREADD-activated astrocytes were involved in neurogenesis and low-density lipoprotein particle biology, while those in the microglia were involved in lipoprotein handling, purinergic receptor activity, and immune cell migration and chemotaxis. Furthermore, network analysis showed that Gq-DREADD-mediated activation in astrocytes resulted in an upregulation of genes involved in the GPCR signaling pathways and calcium ion homeostasis, confirming astrocyte activation through Gq-DREADD expression. Our findings show the importance of considering transcriptomic alterations in microglia after the activation of astrocytes in in vivo models. Therefore, our data will serve as a resource for the broader neuroscience community.

overall_design: Single cells were isolated from the hippocampus and cortex of C57BL/6J mice using ice-cold accutase. Myelin was removed using the Myltenyi's Myelin Depletion Protocol and droplet-based single cell RNA sequencing was performed using the 10x Genomics Chromium Single Cell 3' Library & Gel Bead Kit v2. Libraries were sequenced on an Illumina NovaSeq 6000 SP.

first: Marion

middle: T

last: Turnbull

first: Brian

middle: P

last: Thedy

first: Younghye

last: Moon

first: Jungsu

last: Kim


title: Comparision of gene expression in human nucleus pulposus cells under hypoxia and normoxia condition

city: Shanghai

first: Huajian

last: Zhong

address: 415 Feng Yang Road

country: China

institute: Changzheng Hospital,Second Military Medical University

summary: To investigate the effect of oxygen tension on gene expression of nucleus pulposus cells, human primary nucleus pulposus cells were cultured under condition with low(1%) or normal(21%) concentration of oxygen, respectively. Then, all cells were harvested, and total RNA was extracted using trizol prior to RNA sequencing.

overall_design: Two biological replicates were performed under hypoxia and normoxia, total RNA extracted from normoxia-cultured nucleus pulposus cells were regarded as control

first: Chen

last: Yang

last: Xu

first: Wen

last: Yuan


title: Repression of ARNT2 Maintains Stemness and Limits Differentiation of Glioblastoma Cells

status: Public on Jul 08 2023

city: Dallas

first: Lei

last: Guo

state: TX

address: 5323 Harry Hines Blvd

department: Department of Population and Data Sciences

institute: University of Texas Southwestern Medical Center

summary: To facilitate tumor growth, cancer cells activate pathways that enhance cell proliferation and suppress pathways responsible for cell differentiation. While the pathways involved in metabolic reprogramming and cell cycle progression in tumors have been extensively studied and understood, knowledge regarding the pathways that influence the differentiation status of cancer cells is limited. Our  study  characterizes  a  new  mechanism  regulating  the  balance  between  proliferation  and differentiation involving the repression of the neuronal-specific transcription factor ARNT2 by the MYC oncogene.  We found that MYC represses the transcription of ARNT2 via the activity of the polycomb complex 2. ARNT2 is reduced in glioblastoma and its levels are inversely correlated with patient survival. Using in vitro and in vivo models, we demonstrate that knocking out ARNT2 has no effect on the growth of glioblastoma cells in vitro, but dramatically increases glioblastoma cell growth in vivo, and overexpression of ARNT2 blunts the growth of fully transformed glioblastoma cells in vivo, with no effects in vitro. Mechanistically, we also confirmed that ARNT2 is required for neuronal differentiation including of human pluripotent stem cells. Our findings provide new insights into the complex mechanisms used by oncogenes to limit differentiation in cancer cells and define ARNT2 as a potential tumor suppressor gene in glioblastoma.

overall_design: To investigate the functions of ARNT2 in glioblastoma proliferation and differentiation, we established LN229 cell lines with ARNT2 knocked out by CRISPR.

first: Hao

last: Yi-Heng

first: Conacci-Sorrell

last: Sorrell


title: Cpxm2 as a novel candidate for cardiac hypertrophy and failure in hypertension

first: Anika

last: Witten

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

sample_organism:
Mus musculus
Rattus norvegicus

platform_organism:
Mus musculus
Rattus norvegicus

overall_design: Refer to individual Series


title: the Arraystar Human m6A-mRNA&lncRNA Epitranscriptomic microarray analysis in the neutrophils of ischemic stroke patients and healthy controls

city: Beijing

first: JUNFEN

last: FAN

address: 45 Changchun St, Beijing 100053, China

institute: Xuanwu hospital of Capital Medical University

summary:
The high incidence, mortality, and disability rate of ischemic stroke impose huge economic burdens on patients and social health care systems.N6-methyladenosine (m6A) is one of the most extensive RNA methylation modifications in eukaryotes and participates in the pathogenesis of numerous diseases including ischemic stroke. Peripheral blood neutrophils are forerunners after ischemic brain injury and exert crucial functions.However, the underlying mechanisms of neutrophils in ischemic stroke need to be further clarified. This study aims to explore the transcriptional profiles of m6A modification in neutrophils of patients with ischemic stroke.
The Arraystar Human m6A-mRNA&lncRNA Epitranscriptomic microarray analysis was performed on the peripheral blood neutrophils of 3 patients with ischemic stroke and 3 healthy controls, providing the  clinical significance of m6A modification on ischemic stroke.

overall_design: All  participants  were divided into 2 groups: acute ischemic stroke patients (Trial group) and healthy controls (Control group), each containing 3 participants.

first: Junfen

last: Fan

first: Yumin

last: Luo


title: Complete male-to-female sex reversal in mice lacking the miR-17~92 cluster

first: Irene

last: Mota-Gomez


title: Perturbed epigenetic transcriptional regulation in AML with IDH mutations causes increased susceptibility to NK cells [TT-Seq]

city: Huddinge

first: Anna

last: Palau

address: Hälsovägen 9

country: Sweden

institute: Karolinska Institutet

summary: IDH mutations are found in 20% of acute myeloid leukemia (AML) patients. IDH inhibitors (IDHi) have emerged as a novel treatment option. However, only 30-40% of patients respond to single drug treatment and there is an unmet need for improvement as well as identifying alternative treatment options. The overall aim of this study was therefore to gain deeper insights into the molecular signatures of IDH mutations, the effects of IDHi as well as to identify a molecular vulnerability to tailor novel therapies for AML patients with IDH mutations. Here, we have characterized the transcriptional and epigenetic landscape before and after treatment with IDH2i AG-221, using an IDH2 mutated AML cell line model and AML patient cohorts.    We discovered decreased DNA hydroxymethylation in IDH2 mutated AML cells, particular in enhancers and a perturbed transcriptional regulatory network involving myeloid transcription factors. In addition, hypermethylation of the HLA I cluster caused a dramatic down-regulation of HLA genes, triggering an enhanced natural killer (NK) cell activation, and displayed an increased susceptibility to NK cell mediated killing. These responses were reverted when the AML cells were pre-exposed to IFN-gamma, resulting in up-regulation of cell surface HLA class I. Finally, analyses of DNA methylation data from IDHi-treated patients showed that non-responders continued to harbor hypermethylation in HLA class I genes, suggesting maintained susceptibility to NK cells. In conclusion, this study provides new insights into the perturbed epigenetic and transcriptional regulation in IDH mutated AML and shed light on a potential strategy for personalized medicine in AML.

overall_design: Expression profiling by high throughput sequencing

first: Filip

last: Segerberg

first: Michael

last: Lidschreiber

first: Katja

first: Maria

last: Needhamsen

first: Lisa

last: Jung

first: Maja

last: Jagodic

first: Patrick

last: Cramer

first: Sören

last: Lehmann

first: Mattias

last: Carlsten

first: Andreas

last: Lennartsson


title: H3K4me1 and H3K27ac in Vcap cells

city: Rochester

first: LIGUO

last: WANG

state: MN

address: 200 1st St SW

department: Division of Computational Biology

institute: Mayo Clinic

type: Genome binding/occupancy profiling by high throughput sequencing

summary: To understand the transcription regulation in prostate cancer cell line Vcap, we performed H3K4me1 and H3K27ac ChIP-seq with specific antibody.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for histone H3K4me1 and H3K27ac in Vcap cells.

first: Haojie

last: Huang

first: Alexandra

last: Blee

first: Liguo

last: Wang


title: Perturbed epigenetic transcriptional regulation in AML with IDH mutations causes increased susceptibility to NK cells


title: Super-enhancer switching in early mammalian development involves eRNA mediated SWI/SNF recruitment

city: Houston

first: Junwoo

last: Lee

state: Texas

address: 2120 El Paseo Street, Apt.603

department: Epigenetics and Molecular Carcinogenesis

institute: MD Anderson Cancer Center


title: RNA-seq Analysis of mRNA profiles in β-Gal-overexpressing, and HuR-overexpressing primary hepatocytes.

city: Harbin

first: Zheng

last: Chen

state: Heilongjiang

address: 92 Xidazhijie  Harbin Institute of Technology

institute: Harbin Institute of Technology

summary:
Purpose: The goal of this study is to investigate how HuR regulates hepatocyte death.
Methods: mRNA profiles of β-Gal-overexpressing, and HuR-overexpressing primary hepatocytes were generated by deep sequencing using an Illumina Novaseq 6000 platform. Paired-end clean reads were aligned to the mouse reference genome(GRCm38.p6) with Hisat2 v2.0.5, and the aligned reads were used to quantify mRNA expression by using featureCounts v1.5.0-p3.
Conclusion: Our study represents the detailed analysis of mRNA profiles in HuR-overexpressing primary hepatocytes, generated by RNA-seq technology. Our results show that 2677 genes were upregulated and 2712 genes were downregulated following HuR overexpression. GO analysis indicated that the upregulated genes were primarily related to inflammation response and apoptotic signaling pathway. These data indicate that upregulation of HuR in hepatocytes leads to cell death.

overall_design: mRNA profiles in β-Gal-overexpressing, and HuR-overexpressing primary hepatocytes were generated by deep sequencing using Illumina Novaseq 6000 platform (n=3 for each group).

first: Xinzhi

last: Li


title: Correlation of gene expression changes and cell signalling pathways in insulin-expressing mouse liver cells transduced with an integrating adeno-associated viral vector

city: Ultimo

last: Mahoney

state: NSW

address: Thomas St

country: Australia

institute: University of Technology Sydney

summary: Type 1 Diabetes (T1D) is a chronic, metabolic disorder for which current treatments are unable to prevent the onset of complications. Previously, we used an adeno-associated viral vector (AAV8) to deliver furin-cleavable human insulin (INS-FUR) to the livers of diabetic non-obese diabetic (NOD) mice to reverse T1D. Use of the traditional AAV8-INS-FUR vector was not able to bring about normoglycaemia, however this vector, coupled with a transposon system, in the AAV8/piggyBac-INS-FUR vector, was able to do so. The aim of this study was to investigate the transcriptomic profiles of diabetic, AAV8-INS-FUR transduced, and AAV8/piggyBac-INS-FUR transduced livers and compared these to normal liver to identify genetic differences resulting from delivery of the AAV8/piggyBac-INS-FUR vector which produced normoglycaemia. Differen-tial gene expression was determined by RNA-Seq analysis and the differentially expressed genes from each treatment were mapped on to cellular pathways to determine cellular signalling and downstream effects of the treatments. We observed distinct differences between the piggyBac transduced and diabetic models, particularly in terms of metabolic function and the upregulation of key pancreatic markers in the liver of piggyBac transduced animals. The success of the piggyBac vector in achieving normoglycemia through stable transduction was evident, however further advancements are necessary to achieve complete pancreatic transdifferentiation of liver cells.

overall_design: RNA was extraction from 1) non-diabetic untreated liver 2) diabetic,non-treated liver, 3) AAV8-INS-FUR transduced liver 4) AAV8/piggyBac transduced liver

middle: L

first: Sergio

last: Joshua

first: Najah

last: Nassif

first: Ann

last: Simpson


title: The CUT&Tag sequencing of SiHa cells.

city: Shenzhen

first: sheng

last: zhong

address: The Xinhu Road 1333

institute: The Sourthern Medical University

summary: To analyze the key targets which FOXP1 regarded as a transcriptional regulator might be capable of binding to and transcriptionally activating their expression.

overall_design: The CUt&Tag sequencing is used to analyze that the chromatin protein is bound in situ by anti-FOXP1 specific antibody, which then tethers a protein A-Tn5 transposase fusion protein in SiHa cells .

first: Xia

last: Guo

first: Xiaona


title: Ablation of OCT4 function in cattle embryos by double electroporation of CRISPR-Cas for DNA and RNA targeting (CRISPR-DART)

city: Blacksburg

first: Fernando

middle: H

last: Biase

state: VA

address: 175 West Campus Drive

department: School of Animal Sciences

institute: Virginia Tech

summary: CRISPR-Cas ribonucleoproteins are important tools for gene editing in pre-implantation embryos. However, the inefficient production of biallelic deletions in cattle zygotes has hindered mechanistic studies of gene function. In addition, the presence of maternal RNAs that support embryo development until embryonic genome activation may cause confounding phenotypes. Here, we aimed to improve the efficiency of biallelic deletions and deplete specific maternal RNAs in cattle zygotes by CRISPR-Cas. Two electroporation sessions with Cas9D10A ribonucleoproteins targeting exon1 and the promoter of OCT4 produced biallelic deletions in 91% of the embryos tested. In most cases, the deletions were longer than 1000 nucleotides long. Electroporation of Cas13a ribonucleoproteins prevents the production of the corresponding proteins. We electroporated Cas910A ribonucleoproteins targeting exon 1, including the promoter region, of OCT4 in two sessions with inclusion of Cas13a ribonucleoproteins targeting OCT4 mRNAs in the second session to ablate OCT4 function in cattle embryos. A lack of OCT4 resulted in embryos arresting development prior to blastocyst formation at a greater proportion (7.9%) than controls (30.7%, P<0.001). The few embryos that developed past the morula stage did not form a normal inner cell mass. Transcriptome analysis of single blastocysts, confirmed to lack exon 1 and promoter region of OCT4, revealed a significant (FDR<0.1) reduction in transcript abundance of many genes functionally connected to stemness, including markers of pluripotency (CADHD1, DPPA4, GNL3, RRM2). The results confirm that OCT4 is key regulator of genes that modulate pluripotency and is required to form a functional blastocyst in cattle.

overall_design: We produced transcriptome data from seven in vitro produced blastocysts (day 7.5, controls) and five vitro produced blastocysts (day 7.5) that lack exon 1 from OCT4 gene

first: Biase

last: Fernando


title: Transcriptomic Profiles of Multiple Organ Dysfunction Syndrome Phenotypes in Pediatric Critical Influenza

status: Public on Jul 07 2023

city: Memphis

first: Jeremy

middle: Chase

last: Crawford

state: TN

address: MS 221, 262 Danny Thomas Place

department: Department of Host-Microbe Interactions

institute: St. Jude Children's Research Hospital

summary: Influenza virus is responsible for a large global burden of disease, especially in children. Multiple Organ Dysfunction Syndrome (MODS) is a life-threatening and fatal complication of severe influenza infection. We measured RNA expression of 469 biologically plausible candidate genes in children admitted to North American pediatric intensive care units with severe influenza virus infection with and without MODS. Whole blood samples from 191 influenza-infected children (median age 6.4 years, IQR: 2.2, 11) were collected a median of 27 hours following admission; for 45 children a second blood sample was collected approximately seven days later. Extracted RNA was hybridized to NanoString mRNA probes, counts normalized, and analyzed using linear models controlling for age and bacterial co-infections (FDR q<0.05). Comparing pediatric samples collected near admission, children with Prolonged MODS for ≥7 days (n=38; 9 deaths) had significant upregulation of nine mRNA transcripts associated with neutrophil degranulation (RETN, TCN1, OLFM4, MMP8, LCN2, BPI, LTF, S100A12, GUSB) compared to those who recovered more rapidly from MODS (n=27). These neutrophil transcripts present in early samples predicted Prolonged MODS or death when compared to patients who recovered, however in paired longitudinal samples, they were not differentially expressed over time. Instead, five genes involved in protein metabolism and/or adaptive immunity signaling pathways (RPL3, MRPL3, HLA-DMB, EEF1G, CD8A) were associated with MODS recovery within a week. Thus, early increased expression of neutrophil degranulation genes indicated worse clinical outcomes in children with influenza infection, consistent with reports in adult cohorts with influenza, sepsis, and acute respiratory distress syndrome.

overall_design: We had high quality PAXgene RNA extracts from samples collected early in the PICU course on 191 pediatric patients. Of the final 191 patients included in the analysis, 38 had Prolonged MODS or died, 27 Recovered from MODS, 3 Developed MODS and 126 Never had MODS. Because only 3 patients Developed MODS, we excluded them from further analyses. For a subset of 45 patients, we also had longitudinal samples from the same patients.

first: Tanya

last: Novak

middle: C

first: Paul

last: Thomas

first: Adrienne

last: Randolph


title: 3'DGE-seq analysis of fetal rat testis reveals limited transcriptional changes after exposure to azole fungicides triticonazole and flusilazole despite adverse in vivo effects

city: Rennes

first: Frédéric

last: Chalmel

state: France

address: 9 avenue du Pr. Léon Bernard

department: Physiology and physiopathology of the urogenital tract

country: France

institute: Inserm U1085-Irset

summary:
Purpose: Azoles are widely used antifungal agents. However, many azoles have been shown to have endocrine disrupting properties including triticonazole and flusilazole that we previously showed have adverse effects in vivo. The purpose of this study was to elaborate on how flusilazole and triticonazole can disrupt male reproductive development by different mechanisms, and to investigate whether feminization effects such as short AGD in males can also be detected at the transcript level in fetal testes.
Methods: Testicular mRNA profiles of fetal rats at gestational day 17 or 21 exposed to vehicle, flusilazole, and triticonazole during gestation (from day 7-21) were generated by 3'digital gene expression sequencing, qRT–PCR validation was performed using TaqMan assays and testis histology was assesed by H&E staining and bright field microscopy, protein expression was assesed by immunofluorescence staining and fluorescence microcopy.
Results: We identified 17,350 transcripts in the fetal rat testes. 9 transcripts showed differential expression between control and flusilazole or triticonazole exposed rats, with a fold change ≥1.5 and p value <0.05. Altered expression of 2 genes was confirmed with qRT–PCR. Testis histology was normal.
Conclusions: Sequencing revealed few transcriptional changes in the fetal rat testes exposed to flusilazole or triticonazole. This suggests that flusilazole may be directly targeting steroidogenic enzyme activity in the testis at the protein level, whereas triticonazole may primarily disturbe androgen signaling in androgen-sensitive tissues. Expression of Calb2 and Gsta2 was altered by flusilazole but not triticonazole and may pinpoint novel pathways of disrupted testicular steroid synthesis. Our findings have wider implication for how we integrate omics data in chemical testing frameworks, including selection of non-animal test methods and building of Adverse Outcome Pathways for regulatory purposes.

overall_design: Testicular mRNA profiles of fetal rats at gestational day 17 or 21 exposed to vehicle, flusilazole, and triticonazole during gestation (from day 7-21) were generated by 3'digital gene expression sequencing.

first: Aurélie

last: Lardenois

first: Bertrand

last: Evrard

first: Monica

middle: K

last: Draskau

first: Julie

last: Boberg

first: Terje

last: Svingen


title: Single-cell transcriptome analysis of CD3+ T lymphocytes from prurigo nodularis lesional skin reveals Th17 polarization

city: Paris

first: Jean-David

last: Bouaziz

state: Ile-de-France

address: 1 Avenue Claude Vellefaux

department: Dermatology

institute: Hôpital Saint-Louis

summary: Prurigo nodularis (PN) is a chronic inflammatory skin disease characterized by symmetrically distributed, intensely pruritic nodules of unknown etiology and unmet therapeutic need. The lack of approved therapies and the limited efficacy of off-label treatments reflect the current poor understanding of its pathogenesis. We aimed to comprehensively characterize the phenotypic variation of CD3+ T cells and asses cell-specific gene expression in the lesional skin of patients with PN versus in patients with atopic dermatitis (AD) and in healthy controls (HC).  To this end, single-cell RNA sequencing (scRNA-Seq) using 10x Genomics was carried out to compare CD3+ T cells transcriptomic heterogeneity between lesional samples of PN (n=6), AD (n=5) and HC (n=5).

overall_design: Four millimetres punch skin biopsies were obtained from lesional skin of 11 patients (6 from PN and 5 from AD) and 5 from HC. CD3+ T cells were sorted by flow cytometry from cell suspension obtained after enzymatic and mechanic dissociation of lesional skin biopsy samples or healthy skin samples. Samples with at least 90% viability after lymphocytes sorting were used for further scRNA-Seq analysis. Cell suspensions were loaded onto separate chip inlet to perform cell encapsulation into droplets. Reverse transcription, cDNA generation and library preparation were achieve using the standard 10X Genomics single cell protocol. Raw sequencing data were demultiplexed, aligned to a reference genome (GrCh38) and counted using Cell Ranger (version 3.0.2, 10x Genomics).

first: Andreea

last: Calugareanu

first: Florian

last: Specque

first: Sarah

last: Demouche

first: Chloe

last: Grolleaux

first: Gabor

last: Dobos

first: Marine

last: Merandet

first: David

last: Bergeret

first: Sandy

last: Peltier

first: Marie

last: Jachiet

first: Charles

last: Cassius

first: Thibault

last: Mahevas

first: Anne

last: Saussine

first: Alexandre

last: How-Kit

first: Kevin

last: Serror

first: Mylène

last: Bohec

first: Sylvain

last: Baulande

first: Clemence

last: Lepelletier

first: Marc

last: Mrad

first: Estelle

last: Charvet

first: Adèle

last: de Masson

last: Boccara

first: Maxime

last: Battistella

first: Helene

last: Le Buanec


title: Predicting the metabolomic impact of deregulated microRNAs in lung squamous cell carcinoma

city: Botucatu

first: Natália

last: Bertoni

state: São Paulo

address: Av. Prof. Mário Rubens Guimarães Montenegro

department: Surgery and Orthopedics

country: Brazil

institute: São Paulo State University (UNESP)

summary: We have previously defined key metabolomic changes in Lung Squamous Cell Carcinoma (LSCC) compared to histologically normal tissues (Paes de Araújo et al., 2019). Here our goals were to define microRNA changes in the same tissues, in addition to verifying the detection of deregulated microRNAs in plasma from the same patients.

overall_design: Global microRNA expression was identified in 24 paired samples from eight patients: LSCC, matched histologically normal lung tissues and cell-free plasma using the GeneChip® 4.0 miRNA arrays. Predicted microRNA target-genes were integrated to previously identified metabolites (Paes de Araújo et al., 2019) in the same LSCC patient samples, and networks were created. Computational approaches were used for miRNA target prediction/visualization of networks.

first: Tainara

middle: F

last: Felix

first: Rachel

last: de Araújo

first: Márcio

last: Carvalho

first: Ana

last: Seneda

first: Érica

middle: N

last: Hasimoto

first: Daniele

last: Cataneo

first: Antônio

first: Keir

middle: E

last: Lewis

first: Manfred

last: Beckmann

first: Ran

last: An

first: Bing

last: Ji

first: Sandra

middle: A

last: Drigo

first: Luis

last: Mur

first: Patricia

last: Reis


title: Targeting tumor-intrinsic SLC16A3 to enhance anti-PD-1 efficacy via tumor immune microenvironment reprogramming

city: Jinan

first: Ting

last: Yu

state: Shandong

address: No. 324, Jingwu Weiqi Road, Huaiyin District

institute: Tumor Research and Therapy Centre, Shandong Provincial Hospital Affiliated to Shandong University

summary:
To study the impacts of glycolytic states of tumor cells, we took advantage of different in vitro culture systems and established demanded glycolysis states in B16F10 cells.
B16F10 cells were maintained with either RPMI-1640 or DMEM mediums to maintain different metabolic models.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for B16F10 cells treated in DMEM or RPMI-1640.

first: Meng

last: Wu


title: Vemurafenib induces a noncanonical senescence-associated secretory phenotype in melanoma cells which promotes vemurafenib resistance

city: Dongguan

first: XUERONG

last: SUN

state: Guangdong Province

address: No. 1 Xincheng Avenue, Songshan Lake District

institute: Guangdong Medical University

summary: To explore the characteristics of senescent melanoma cells induced by vemurafenib or cisplatin, melanoma A375 cells were treated with vemurafenib and cisplatin, respectively. The senescent phenotypes were verified by β-gal staining, EdU assay, cell morphology and the senescence-related pathways. RNA-seq was performed to explore the  differentially expressed genes in the senescent cells induced by vemurafenib or cisplatin.

overall_design: Melanoma A375 cells were treated with vemurafenib (10 μM) for 3 days or with cisplatin (2 μM) for 1 day and then returned to normal culture conditions. At day 7 after the treatment, A375 cells were collected and used for RNA-seq and other detections. Normal cultured A375 cells were simultaneously collected and acted as negative control.

first: Xuerong

last: Sun

first: Xinguang

last: Liu

first: Jianyu

last: Peng


title: Adaptive Evolution of a Minimal Organism with a  Synthetic Genome

city: La Jolla

first: Christopher

last: Dalldorf

state: CA

address: 9500 Gilman Dr

department: Bioengineering

institute: UCSD

summary: The bacterial strain JCVI-syn3.0 stands as the first example of a living organism with a  minimized synthetic genome, derived from the Mycoplasma mycoides genome and  chemically synthesized in vitro. Here we report the experimental evolution of a syn3.0- derived strain. Ten independent replicates were evolved for several hundred  generations, leading to growth rate improvements of >15%. Endpoint strains possessed  an average of 8 mutations composed of indels and SNPs, with a pronounced C/G->A/T  transversion bias. Multiple genes were repeated mutational targets across the  independent lineages, including phase variable lipoprotein activation, 5 distinct  nonsynonymous substitutions in the same membrane transporter protein, and  inactivation of an uncharacterized gene. Transcriptomic analysis revealed an overall  tradeoff reflected in upregulated ribosomal proteins and downregulated DNA and RNA  related proteins during adaptation. This work establishes the suitability of synthetic,  minimal strains for laboratory evolution, providing a means to optimize strain growth  characteristics and elucidate gene functionality.

overall_design: Comparitive expression analysis between evolved strains using RNA-seq

first: Troy

last: Sandberg

first: Kim

middle: S

last: Wise

first: Richard

last: Szubin

first: Adam

last: Feist

first: John

middle: I

last: Glass

first: Bernhard

middle: O

last: Palsson


title: Novel Role of Estrogen Receptor-α on Regulating Chondrocyte Phenotype and Response to Mechanical Loading

city: PITTSBURGH

first: Uma

last: Chandran

state: PA

address: 5607 Baum Blvd

department: Biomedical Informatics

institute: University of Pittsburgh

summary: Using bulk RNA-seq for chondrocytes isolated from preserved cartilage and damaged cartilage, 558 differentially expressed genes were identified. Given the shared genetic and biochemical environment of D-C and P-C located within the same knee, the differences in tissue integrity and resulting chondrocyte phenotype could in part be attributed to differences in the mechanical microenvironment; Through ingenuity pathway analysis and other molecular biology method, we identified ESR1 as a promosing up-stream regulator which could regulate chondrocyte phenotype and reponse to mechanical loading.

overall_design: Preserved cartilage and damaged cartilage were identified through outerbridge acore (macroappearance) and OARSI score (histology), and they were isolated from the patients who underwent total knee joint arthroplasty.

first: Hang

last: Lin

first: Ning

last: Wang

first: Alexander

last: Chang


title: The effects of environmentally relevant concentrations of aqueous atorvastatin on Xenopus laevis

city: Ocean Springs

first: Jeremy

last: Johnson

state: Mississippi

address: 103 McIlwain Dr.

institute: University of Southern Mississippi

summary: Numerous studies report active pharmaceutical compounds detected in both wastewater effluent and surface waters.  Exposure to statin drugs in general, and atorvastatin in particular, is likely to be a concern.  We hypothesized that chronic exposure to low concentrations of atorvastatin in water would result in an adverse effect on production of steroids regulating growth and development of the model amphibian Xenopus laevis.  To assess the potential for impacts we used three experimental assays.  The FETAX assay was used to evaluate the effects of a range of doses of atorvastatin on developing embryos.  A 60 day metamorphosis assay assessed the effects of aqueous atorvastatin exposure at environmentally concentrations on metamorphosing tadpoles.  A 60 day chronic flow-through exposure evaluated the effects of chronic low concentrations of atorvastatin on adults.  The data from the FETAX assay confirmed uptake, activity, and effect of atorvastatin in X. laevis tadpoles. The results of the 60-day flow-through exposure on metamorphosing tadpoles showed significant evidence of altered cholesterol biosynthesis.  The dose-dependent increase in cyp19a1 expression also indicated that the steroidogenesis pathway was affected.  The RNAseq analysis confirmed that exposure to environmentally relevant concentrations of atorvastatin does cause significant alterations to global transcriptional profiles in a manner consistent with dysregulation of the cholesterol biosynthesis pathway, both through the downregulation of many genes involved in that pathway, but also in the impacts to other, related pathways.  The qPCR data for both adult males and adult females indicated only slight changes in expression with the exception that hmgcr was significantly downregulated in males, and cyp3a4 expression was significantly downregulated in females.  The data we present here indicated that chronic exposure to environmentally relevant concentrations of atorvastatin does have the potential to impact early life stage frogs, particularly by altering expression of genes involved in critical molecular pathways.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for metamorphosing X. laevis tadpoles exposed to aqueous atorvastation.  Control and exposed treatments in triplicate.

first: Robert

last: Griffitt


title: RNAseq analysis of transcriptome changes in MM27 metastatic melanoma PDX cells upon treatment with AURKA and/or MEK inhibitors

city: Milano

first: Marine

last: Melixetian

address: via Adamello 16

country: Italy

institute: European Institute of Oncology

summary:
Purpose: Study the changes in the transcriptome of metastatic melanoma PDX cells upon combination treatment with AURKA and MEK inhibitors
Methods: RNAseq analysis of melanoma PDX cells treated with AURKA inhibitor Alisertib (500nM) and MEK inhibitor trametinib (100nM) for 72 h

overall_design: Actionable genetic screens unveil the dual targeting of AURKA and MEK as an alternative therapeutic approach for melanoma treatment

first: Federica

last: Marocchi

first: Fernando

last: Palluzzi

first: Paola

last: Nicoli

first: Giovanni

last: Bertalot

first: Salvatore

last: Pece

first: Pier

last: Ferrucci

first: Daniela

last: Bossi

first: Luisa

last: Lanfrancone


title: RNA expression profiles of whole blood cells from a Han Chinese population with or without Type-2 Diabetes Mellitus or/and its complications in nephropathy and retinopathy

city: Taichung

first: Fuu-Jen

last: Tsai

address: No.2, Yude Rd., North Dist.

department: Department of Medical Research

country: Taiwan

institute: China Medical University Hospital

summary:
Our main purpose is to screen specific biomarkers for supporting diagnoses of type-2 diabetic nephropathy (T2DN), type-2 diabetic retinopathy (T2DR), and type-2 diabetes mellitus (T2D) without these complications. The RNA expression database was built to study T2D in humans with the performance of the Clariom™ D Assay, human.
The notable highlight of the analysis results shows there are three main groups which include one control group and two diabetic groups. Interestingly, the diabetic groups share some aging-related pathways, especially fatty acid metabolism. On the other hand, B group with higher BMI coverage has many abundant coding genes in fatty acid degradation, while C group with lower BMI coverage involves in fatty acid elongation. It can be classified into obese and non-obese groups proportionally. In addition, the obese group consists of two T2DN-related cases without an independent T2DR_related case, which is contrary to the non-obese group has all three cases. It means that T2DR can be rarely found in overweight T2D-patients.

overall_design: Although classifications of blood samples were based on doctors-supervised conclusions in medical reports, clinical parameters and group distribution in RNA-expression array analysis supported rearranging in new orders. It means that the analysis reports were divided into two parts, which were influenced by medical doctors and analysts. Specifically, the doctors think that long-term T2D-exposure makes the disease worse, however, the analysts believe that individuals have different conditions and self-treatments, which affect the severe levels of the disease with long durations. Two analysis-ways are helpful to improve the combinational methods of RNA-expression analysis and clinical statistics afterward.

first: Thai

middle: Q

last: Tran

first: Shih-Yin

last: Chen


title: Glucocorticoids sensitive progenitor PDGFRα+ cells switching to white adipocytes cause visceral obese via methylation modification of

city: nanjing

first: yifan

last: lv

state: jiangsu

address: 300 Guangzhou Road

department: Department of Geriatrics

institute: the First Affiliated Hospital to Nanjing Medical University

type: Methylation profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: miRNA expression profiles of whole blood cells from a Han Chinese population with or without Type-2 Diabetes Mellitus or/and its complications in nephropathy and retinopathy

summary:
Our main purpose is to screen specific biomarkers for supporting diagnoses of type-2 diabetic nephropathy (T2DN), type-2 diabetic retinopathy (T2DR), and type-2 diabetes mellitus (T2D) without these complications. The miRNA expression database was built to study T2D in humans with the performance of the GeneChip™ miRNA 4.0 array analysis.
The notable highlight of the analysis results in RNA expression shows there are three main groups which include one control group and two diabetic groups. It is used to apply grouping to the miRNA-expression analysis.

overall_design: Although classifications of blood samples were based on doctors-supervised conclusions in medical reports, clinical parameters and group distribution in RNA-expression array analysis supported rearranging in new orders. It means that the analysis reports were divided into two parts, which were influenced by medical doctors and analysts. Specifically, the doctors think that long-term T2D-exposure makes the disease worse, however, the analysts believe that individuals have different conditions and self-treatments, which affect the severe levels of the disease with long durations. It was highly recommended that the miRNA-expression analysis have to follow the previous classifications of RNA expression.


title: DNA Methylome and 3D Chromatin Landscape of Cell Types Across the Mouse Brain [8C, 8D]

first: Joseph

middle: R

last: Ecker

address: 10010 North Torrey Pines Road

department: Genomic Analysis Laboratory

institute: HHMI-Salk-Institute

summary: Illustrating the cellular architecture of the mammalian brain is critical to understanding its diverse functions and complex animal behaviors. With support from NIMH and NINDS as part of the BRAIN Initiative Cell Census Network (BICCN), the Center for Epigenomics of the Mouse Brain Atlas (CEMBA) applied single nucleus methylation sequencing in 117 brain funtional regions across the whole adult male P56 C57BL/6J mouse brain. In each major brain regions, we identified distinct cell clusters and formed them in a hierarchical taxonomy. These clusters include known primary brain cell types and possible sub-types. We use these data to identify the epigenomic characteristics and to define specific regulatory elements for each cell cluster. We integrate the single cell methylome with single cell gene expression and chromatin accessibility profiles from the same brain region to further identify potential enhancers and their corresponding genes for several neuronal cell types. By brain region spacial comparisons, we found spatial specificity among excitatory neuronal clusters within the same cortical layer. We identify brain-region-specific transcription factors that may regulate cortical region development of excitatory neurons. These data define the landscape of cell type and spatial heterogeneity in the mouse brain and the underlining regulatory epigenomic basis.

overall_design: Apply snmC-seq2 to 117 brain functional regions to identify epigenomic landcape of the adult mouse brain, this subseries consists of one single nucleus methylation sequencing experiment of brain region (8C_191205, 8C_191212, 8D_200707, 8D_200709).

first: Hanqing

last: Liu

first: Anna

last: Bartlett

first: Jacinta

last: Lucero

first: Julia

last: Osteen

first: Jordan

last: Altshul

first: Mia

last: Kenworthy

first: Rosa

middle: G

last: Castanon

last: Nery

first: Peter

last: Berube

first: Huaming

first: Michael

last: Nunn

first: Jesse

last: Dixon

first: M

middle: M

last: Behrens


title: Single-cell RNA-Sequencing data of hPSC-based somite formation model

status: Public on Jul 06 2023

city: Ann Arbor

first: Yue

address: 2350 Hayward St

institute: University of Michigan

summary: We analyzed scRNA-seq data in human pluripotent stem cells derived somite formation models. This in vitro system recapitulates some key aspects of somite formation process along rostral-caudal axis

overall_design: scRNA-seq was performed on dissociated cells from somite formation models at four timepoints (day 2, day 3, day 4, day 6)

first: Jianping

last: Fu


title: Single cell sequencing of Tff2+ cells from mouse pancreas

city: New York

first: Zhengyu

last: Jiang

address: 1130 St Nicolas Ave

institute: CUIMC

summary: The goal of this study is to identify subpopluation of Tff2CreERT2-targeted cells in the mouse pancreas.

overall_design: We isolated by FACS RFP+ cells from panceas of Tff2CreERT2; R26-tdTomato mice and performed scRNAseq using 10x Genomics

first: Timothy

last: Wang

first: Pasquale

last: Laise


title: Diosmetin blocks type Ⅰ interferon  signaling by metabolic control of phosphatidylethanolamine [dataset 2]

city: Shanghai

first: Xiaoyue

state: Shanghai

address: 145 Middle Shandong road

department: Rheumatology

institute: Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University

summary: Type I interferon (IFN-I) is essential in the development of Systemic Lupus Erythematosus (SLE) and many other autoimmune diseases. To explore the metabolic regulations of IFN-I signaling pathway, we conducted a high through-put screening of a small molecule library and identified diosmetin as a potent compound for blocking IFN-I signaling.  Diosmetin can ameliorate lupus-like autoimmune phenotypes in IFNα-accelerated NZB/NZW F1 lupus model and pristane-induced murine lupus model. Of note, diosmetin can block over-activated IFN-I signaling pathway in PBMCs from lupus patients by reducing the expression of CYP1B1. Our findings reveal a novel lipid metabolic regulation of IFN-I signaling and a potent alternative therapeutic target for autoimmune diseases with overactivated  IFN-I signaling pathway.

overall_design: To investigate the function of diosmetin in type I interferon signaling in kidney tissue of NZB/NZW F1 mice.

first: Can

first: Wu

last: Lingling

last: Shen


title: Glucocorticoids sensitive progenitor PDGFRα+ cells switching to white adipocytes cause visceral obese via methylation modification of TEAD1 (RNA-seq)

first: yifan

last: lv

summary: Glucocorticoids have impact on visceral obese which crucial contributor to the burden of obesity complications. Subcutaneous and visceral adipose have the heterogeneity of cellular populations. However, the mechanism of glucocorticoids mediated depot-specific regulation remains unclear. Here, we report PDGFRα+ progenitors, dominated in visceral adipose which can convert to white-typical adipocytes, are glucocorticoid-sensitive progenitors. While CD137+ progenitors, mainly harbored in subcutaneous adipose, which are beige-typical adipocytes, are glucocorticoid-passivated progenitors. Mechanistically, we proved that Glucocorticoids elevate TEAD1, transcriptional coactivator of glucocorticoid receptor, by down-regulating the methylation states in PDGFRα+ progenitors. TEAD1 bonds the accessible binding sites in miR-27b promoter region and transcriptionally increases miR-27b expression which previously proved to be the browning ability inhibitor in human and rodent white adipose. These data provide insight into the mechanism of glucocorticoids mediation of visceral obesity and the pathophysiology of depot-specific variations of high fat diet induced obesity.

overall_design: Differentiated CD137+ and PDGFRα+ cells were treated with PBS or Dexamethasone(DEX,1μM). RNA sequencing.

first: Yifan

last: Lv

first: Guoxian

last: Ding


title: Glucocorticoids sensitive progenitor PDGFRα+ cells switching to white adipocytes cause visceral obese via methylation modification of TEAD1 (ATAC-seq)

overall_design: Differentiated CD137+ and PDGFRα+ cells were treated with Dexamethasone(DEX,1μM). ATAC-seq.


title: Glucocorticoids sensitive progenitor PDGFRα+ cells switching to white adipocytes cause visceral obese via methylation modification of TEAD1 (ChIP-seq)

overall_design: Differentiated CD137+ and PDGFRα+ cells were treated with Dexamethasone(DEX,1μM). GR IP and Input.


title: Diosmetin blocks type Ⅰ interferon 1 signaling by metabolic control of phosphatidylethanolamine [dataset 1]

overall_design: To investigate the function of diosmetin in type I interferon signaling in THP-1 cell lines and primary monocytes.

last: Liu


title: Diosmetin blocks type Ⅰ interferon 1 signaling by metabolic control of phosphatidylethanolamine.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Glucocorticoids sensitive progenitor PDGFRα+ cells switching to white adipocytes cause visceral obese via methylation modification of TEAD1 (Bisultite-seq)

overall_design: Differentiated CD137+ and PDGFRα+ cells.


title: Measuring anti-islet autoimmunity in mouse and human by profiling peripheral blood antigen specific CD4 T cells

first: Luc

last: Teyton

address: 10550 North Torrey Pines Road

department: Immunology and Microbiology

institute: Scripps Research

type:
Expression profiling by RT-PCR
Other


title: The short-acting GABA (A) receptor agonist remimazolam is an inhibitor of T cell activation and can induce phenotype change of regulatory T cells

city: Xinxiang

first: Tianhan

last: Li

state: Henan

address: No.601, Hualan Road

institute: Xinxiang Medical University

summary: Background: GABAA receptor genes are targets for intravenous anesthetics, but their expressions have been observed in multiple lineages of immune cells including T cells. However, knowledge of their agonists on T cells remains very limited, and unbiased system level characterizations of T cells in vivo following GABAA receptor agonist treatment have not been reported.  Methods: The RNA Sequencing, qRT-PCR, CRISPR/Cas9 gene knockout, cell sorting, single cell sequencing and flow cytometry were used. Results: Remimazolam treatment significantly inhibited CD69 expression, ERK phosphorylation following T cell activation, and induced transcriptomic change in Jurkat T cells, such as TGFBI. Knockout of TGFBI significantly altered the inhibition of T cell activation by remimazolam. To assess impact of remimazolam, we first intraperitoneally injected C57BL/6 mice with remimazolam, sorted T cells and performed single-cell RNA sequencing (scRNA-seq) analyses. Single-cell RNA sequencing (scRNA-seq) analysis found that intraperitoneally injection of remimazolam into C57BL/6 mice resulted in significant changes in a Foxp3 expressing sub-cluster among T cells, even though differences in surface expression of T cell activation markers was not found by flow cytometric analyses, which was probably due to pharmacokinetic properties of the ultra-fast acting drug.  Conclusions: Our experimental data have revealed the impact of GABAA receptor agonist on T cells and confirmed inhibitory role of remimazolam on T cell activation, which could provide insights into neuroimmunological links during anesthetic treatment.

overall_design: C57BL/6 female mice were divided into three groups of three mice each injected with saline, 6 mg/kg remimazolam for 4 h or 8 h, respectively. Then  the spleens of mice were collected and gently dissociated in RPMI 1640 medium containing 10% FBS on ice using 1 ml syringe plunger on 100 μm cell strainer (BD), all samples were transferred into 15 mL tubes, respectively. Cell samples from spleens were stained with antibody mixture and sorted for single cell sequencing.

first: Hao

first: Binhui

last: Zhou


title: Measuring anti-islet autoimmunity in mouse and human by profiling peripheral blood antigen specific CD4 T cells [TCR-Seq]

summary: The endocrine pancreas is one of the most inaccessible organs of the human body. Its autoimmune attack leads to type 1 diabetes (T1D) in a genetically susceptible population and a lifelong need for exogenous insulin replacement. Monitoring disease progression by sampling peripheral blood would provide key insights into T1D immune-mediated mechanisms and potentially change preclinical diagnosis and the evaluation of therapeutic interventions. This effort has been limited to the measurement of circulating anti-islet antibodies, which despite a recognized diagnostic value, remain poorly predictive at the individual level for a fundamentally CD4 T cell-dependent disease. Here, peptide-major histocompatibility complex tetramers were used to profile blood anti-insulin CD4 T cells in mice and human. While percentages of these were not directly informative, the state of activation of anti-insulin T cells measured by RNA and protein profiling was able to distinguish normalcy versus disease progression. Activated anti-insulin CD4 T cell were detected at time of diagnosis but also in patients with established disease and in some at-risk individuals. These results support the concept that antigen specific CD4 T cells might be used to monitor autoimmunity in real time. This advance can inform our approach to T1D diagnosis and therapeutic interventions in the pre-clinical phase of anti-islet autoimmunity.

overall_design: Human CD4+ T cells, isolated from blood of donors spanning four cohorts, were stained with either DQ8-INS1220, DQ8-INS1321, DQ8-CP59-70, or DQ8-CP65-75 ; all tetramers were labelled with the fluorscent moiety Phycoerythrin. Cells stained with either tetramer were single cell sorted into single wells of 96 well plates. Single cell cDNA libraries were made according to the Fluidigm BioMark "Two Step Single Cell Gene Expression Using EvaGreen" protocol. Then gene expression of 96 genes was assessed utilizing the StandardBiotools (formerly called Fluidigm) Dynamic Array 96x96 IFC and the "GE Fast 96x96 PCR+Melt v2.pcl" cycling conditions. Data was pre-processed via the SINGuLAR Analysis Toolset and then futhered analyzed using custom R scripts that incorporated UMAP, clustering via k.means and differential gene expression analysis. The protocol for single cell sorting, pre-amplification and analysis via the StandardBiotools Dynamic Array can be found using PMID 29224077 (Holt et al).

first: Siddhartha

last: Sharma


title: DNA Methylome and 3D Chromatin Landscape of Cell Types Across the Mouse Brain [7J, 8A]

first: Joseph

last: Ecker

overall_design: Apply snmC-seq2 to 117 brain functional regions to identify epigenomic landcape of the adult mouse brain, this subseries consists of one single nucleus methylation sequencing experiment of brain region (7J_200520, 8A_191107, 8A_191114).

first: Hanqing

first: Anna

last: Bartlett

first: Jacinta

last: Lucero

first: Julia

last: Osteen

first: Jordan

last: Altshul

first: Mia

last: Kenworthy

first: Rosa

last: Castanon

last: Nery

first: Peter

last: Berube

first: Huaming

last: Chen

first: Michael

last: Nunn

first: Jesse

first: M


title: Recent clinical isolates of enterovirus D68 have increased replication and induce enhanced epithelial immune response compared to the prototype Fermon strain.

city: Madison

first: Daniel

last: Jackson

state: WI

address: 600 highland Ave

institute: University of Wisconsin-Madison

summary: In 2014, enterovirus D68 (EV-D68), previously associated primarily with mild respiratory illness, caused a large outbreak of severe respiratory illness and, in rare instances, paralysis. We compared viral binding and replication of eight recent EV-D68 clinical isolates and the prototype Fermon strain from 1962 in cultured HeLa cells and differentiated human primary bronchial epithelial cells (BEC) to understand the possible reasons for the change in virus pathogenicity. We found no significant differences in binding or replication in HeLa cell cultures between the recent clinical isolates. However, in HeLa cells, Fermon had significantly greater (1.5-2 log) binding and virus progeny yields but a similar level of replication (~2-log increase in viral RNA from 2h to 24h post infection) compared to recent isolates. In differentiated BECs, Fermon and the recent EV-D68 isolates had similar levels of binding; however, the recent isolates produced 1-2-log higher virus progeny yields than Fermon due to increased replication. We then utilized RNA-seq to define the transcriptional responses in BECs infected with four recent EV-D68 isolates, representing major phylogenetic clades, and Fermon strain. All the tested clinical isolates induced similar responses in BECs; however, numerous upregulated genes in antiviral and pro-inflammatory response pathways were identified when comparing the response to clinical isolates versus Fermon. These results indicate that the recent emergence in severe EV-D68 cases could be explained by increased replication efficiency and enhanced inflammatory response induced by newly emerged clinical isolates.

overall_design: Looking at differences in the transcriptional responses elicited by EV-D68 infection in differentiated human primary BECs from five donors using four EV-D68 isolates representing each phylogenetic clade (A: O541, B: C7731, B1: 7788, C: C2386, and Fermon) to control and each other.

first: Mark

last: Devries

first: Yury

last: Bochkov

middle: D

last: Evans

first: James

last: Gern

middle: J


title: iPSC-derived type IV collagen α5-expressing kidney organoids model Alport syndrome

city: Kyoto

first: Ryuichiro

last: Hirayama

address: 53 Kawahara-cho

country: Japan

institute: Kyoto University

summary: Alport syndrome (AS) is a hereditary glomerulonephritis caused by COL4A3, COL4A4 or COL4A5 gene mutations and characterized by abnormalities of glomerular basement membranes (GBMs). Due to a lack of curative treatments, the condition proceeds to end-stage renal disease even in adolescents. Hampering drug discovery is the absence of effective in vitro methods for testing the restoration of normal GBMs. Here, we aimed to develop kidney organoid models from AS patient iPSCs for this purpose. We established iPSC-derived collagen α5(IV)-expressing kidney organoids and confirmed that kidney organoids from COL4A5 mutation-corrected iPSCs restore collagen α5(IV) protein expression. Importantly, our model recapitulates the differences in collagen composition between iPSC-derived kidney organoids from mild and severe AS cases. Furthermore, we demonstrate that a chemical chaperone, 4-phenyl butyric acid, has the potential to correct GBM abnormalities in kidney organoids showing mild AS phenotypes. This iPSC-derived kidney organoid model will contribute to drug discovery for AS.

overall_design: To investigate temporal change of gene expression in kidney organoids, we performed gene expression profiling analysis using data obtained from RNA-seq at five time points.

first: Kenji

last: Osafune


title: Measuring anti-islet autoimmunity in mouse and human by profiling peripheral blood antigen specific CD4 T cells [Fluidigm]

first: Luc

last: Teyton

summary: The endocrine pancreas is one of the most inaccessible organs of the human body. Its autoimmune attack leads to type 1 diabetes (T1D) in a genetically susceptible population and a lifelong need for exogenous insulin replacement. Monitoring disease progression by sampling peripheral blood would provide key insights into T1D immune-mediated mechanisms and potentially change preclinical diagnosis and the evaluation of therapeutic interventions. This effort has been limited to the measurement of circulating anti-islet antibodies, which despite a recognized diagnostic value, remain poorly predictive at the individual level for a fundamentally CD4 T cell-dependent disease. Here, peptide-major histocompatibility complex tetramers were used to profile blood anti-insulin CD4 T cells in mice and human. While percentages of these were not directly informative, the state of activation of anti-insulin T cells measured by RNA and protein profiling was able to distinguish normalcy  versus disease progression. Activated anti-insulin CD4 T cell were detected at time of diagnosis but also in patients with established disease and in some at-risk individuals. These results support the concept that antigen specific CD4 T cells might be used to monitor autoimmunity in real time. This advance can inform our approach to T1D diagnosis and therapeutic interventions in the pre-clinical phase of anti-islet autoimmunity.

overall_design: Human CD4+ T cells, isolated from blood of donors spannign four cohorts, were stained with either DQ8-INS1220, DQ8-INS1321, DQ8-CP59-70, or DQ8-CP65-75 ; all tetramers were labelled with the fluorscent moiety PE. Cells stained with either tetramer were single cell sorted into single wells of 96 well plates. Single cell cDNA libraries were made according to the Fluidigm BioMark "Two Step Single Cell Gene Expression Using EvaGreen" protocol. Then gene expression of 96 genes was assessed utilizing the StandardBiotools (formerly called Fluidigm) Dynamic Array 96x96 IFC and the "GE Fast 96x96 PCR+Melt v2.pcl" cycling conditions. Data was pre-processed via the SINGuLAR Analysis Toolset and then futhered analyzed using custom R scripts that incorporated UMAP, clustering via k.means and differential gene expression analysis.  The protocol for single cell sorting, pre-amplification and analysis via the StandardBiotools Dynamic Array can be found using PMID 29224077 (Holt et al).

first: Siddhartha

last: Sharma

first: Xuqian

last: Tan

first: Josh

last: Boyer

first: Don

last: Clarke

first: Anne

last: Costanzo

first: Brian

last: Abe

first: Lisa

last: Kain

first: Marie

last: Holt

first: Adrienne

last: Armstrong

first: Marynette

last: Rihanek

first: Andrew

last: Su

first: Cate

last: Speake

last: Gottlieb

last: Gottschalk

first: Jeremy

last: Pettus


title: p53 inhibitor iASPP is an unexpected suppressor of KRAS and inflammation driven pancreatic cancer

city: Oxford

first: Xin

last: Lu

state: Oxfordshire

address: Old Road Campus

department: Ludwig Institute for Cancer Research

country: United Kingdom

institute: University of Oxford

summary: To investigate the transcriptomic effects of iASPP deletion or p53 mutation in the KRASG12D background Pdx1-Cre was used to delete iASPP in oncogenic KRASG12D expressing (KC) background to generate KC;iASPPΔ8/Δ8 mice. KC and KC;iASPPΔ8/Δ8 mice were then crossed with mice containing a p53R172H allele (equivalent to human p53 hotspot mutation R175H) to generate KPC and KPC;iASPPΔ8/Δ8 cohorts. Primary cell cultures were then derived from pancreatic ductal adenocarcinoma tissue for RNA-seq.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for KC, KC;iASPPΔ8/Δ8,KC, and KC;iASPPΔ8/Δ8 mouse cell lines.

first: Paul

last: Miller

first: Richard

last: Owen

first: Robert

last: Amess


title: Effects of an aged tissue niche on the immune potency of dendritic cells using simulated microgravity

city: Abu Dhabi

first: Mei

last: ElGindi

address: Saadiyat Island

department: Engineering

country: United Arab Emirates

institute: NYUAD

summary: Microgravity accelerates the aging of various physiological systems, and it is well acknowledged that aged individuals and astronauts both have increased susceptibility to infections and poor response to vaccination. Immunologically, dendritic cells (DCs) are the key players in linking the innate and adaptive immune responses. Their distinct and optimized differentiation and maturation phases play a critical role in presenting antigens and mounting effective lymphocyte responses for long-term immunity. Despite their importance, no studies to date have effectively investigated the effects of microgravity on DCs in their native microenvironment, which is primarily located within tissues. Here, we address a significantly outstanding research gap by examining the effects of simulated microgravity via a random positioning machine on both immature and mature DCs cultured in biomimetic collagen hydrogels, a surrogate for tissue matrices. Furthermore, we explored the effects of loose and dense tissues via differences in collagen concentration. Under these various environmental conditions, the DC phenotype was characterized using surface markers, cytokines, function, and transcriptomic profiles. Our data indicate that aged or loose tissue and exposure to RPM-induced simulated microgravity both independently alter the immunogenicity of immature and mature DCs. Interestingly, cells cultured in denser matrices experience fewer effects of simulated microgravity at the transcriptome level. Our findings are a step forward to better facilitate healthier future space travel and enhance our understanding of the aging immune system on Earth.

overall_design: Comparative gene expression profiling of immature (iDCs) and mature dendritic cells (mDCs) cultured in 3D collagen matrices of different stiffness. Samples were cultured either on ground or on the RPM to simulated an accelerated aging process. Samples (iDC or mDC) cultured in "loose" (1 mg/mL) collagen matrices at static (ground) conditions were used as control groups to compare the other samples.

first: Jiranuwat

last: Sapudom

first: Anna

last: Garcia Sabate

last: Quartey

first: Aseel

last: Alatoom

first: Mohamed

last: Al-Sayegh

first: Rui

last: Li

first: Weiqiang

last: Chen

last: Teo


title: Deubiquitinase JOSD2 improves calcium handling and attenuates cardiac hypertrophy and dysfunction through stabilizing SERCA2a in cardiomyocytes

city: Wenzhou

first: guang

last: liang

state: Wenzhou

address: Wenzhou medical university of  higher education park  chashan town

department: School of Pharmaceutical Sciences

institute: Wenzhou Medical University

summary: Construction of transcriptome sequencing library and transcriptome sequencing was completed by LC-Bio Technology Co., Ltd. (Hangzhou, China). The expression profile of Machado-joseph deubiquitinases (MJDs) family in heart tissues of Ang II mice.

overall_design: We performed RNA sequencing analysis on the heart tissues of Ang II mice.

first: Jibo

last: Han

first: Guang

last: Liang


title: Transcriptomes of human primary skin fibroblasts reveal age-associated mRNAs and long noncoding RNAs

city: Baltimore

first: Supriyo

last: De

state: Maryland

address: 251 Bayview Blvd

department: Laboratory of Genetics and Genomics

institute: NIA-IRP, NIH

type: Non-coding RNA profiling by high throughput sequencing

summary: Changes in the transcriptomes of human tissues with advancing age are poorly cataloged. Here, we set to identify the coding and long noncoding RNAs present in cultured primary skin fibroblasts collected from 82 healthy individuals across a wide age spectrum (22 to 89 years old) who participated in the GESTALT (Genetic and Epigenetic Signatures of Translational Aging Laboratory Testing) study of the National Institute on Aging, NIH. Using high-throughput RNA sequencing and a linear regression model, we identified 1,437 coding RNA (mRNA) and 1,177 linear and circular long noncoding (lncRNA) that were differentially abundant as a function of age. GSEA analysis revealed select transcription factors implicated in coordinating the transcription of subsets of differentially abundant mRNAs, while LncSEA identified RNA-binding proteins predicted to contribute to the age-associated lncRNA profiles. In sum, we report age-associated changes in the global transcriptome, coding and noncoding, from healthy human skin fibroblasts, and propose that they might serve as biomarkers and therapeutic targets in aging skin.

overall_design: Comparative gene expression analysis of RNA-seq data from primary skin fibroblasts across healthy aging (82 individuals spanning from 22 to 89 years old).

first: Dimitrios

last: Tsitsipatis

first: Jennifer

middle: L

last: Martindale

first: Krystyna

last: Mazan-Mamczarz

first: Alison

middle: B

last: Herman

first: Yulan

last: Piao

first: Nirad

last: Banskota

first: Jen

middle: H

last: Yang

first: Linna

last: Cui

first: Carlos

last: Anerillas

first: Ming

middle: W

last: Chang

first: Mary

last: Kaileh

first: Rachel

last: Munk

first: Xiaoling

first: Chee

last: Chia

first: Ajoy

last: Karikkineth

first: Linda

last: Zukley

first: Jarod

last: D’Agostino

first: Kotb

last: Abdelmohsen

first: Nathan

last: Basisty

first: Luigi

last: Ferrucci

first: Myriam

last: Gorospe


title: DNA Methylome and 3D Chromatin Landscape of Cell Types Across the Mouse Brain [6F and 6G]

first: Joseph

last: Ecker

overall_design: Apply snmC-seq2 to 117 brain functional regions to identify epigenomic landcape of the adult mouse brain, this subseries consists of one single nucleus methylation sequencing experiment of brain region (6F_290728, 6G_200714, 6G_200721).

first: Hanqing

last: Liu

first: Anna

last: Bartlett

first: Jacinta

last: Lucero

first: Julia

middle: K

last: Osteen

first: Jordan

last: Altshul

first: Mia

last: Kenworthy

first: Rosa

last: Castanon

last: Nery

first: Peter

last: Berube

first: Huaming

last: Chen

first: Michael

last: Nunn

first: Jesse

last: Dixon

first: M

last: Behrens


title: RRBS of PCK1 knockout in PLC/PRF/5

city: Chongqing

first: Dongmei

last: Gou

state: Chongqing

address: 1 Yixue Yuan Road

institute: Chongqing Medical University

summary: RRBS analysis of Parental and PCK1 knockout groups of PLC/PRF/5 cells.

overall_design: RRBS

first: Ni

last: Tang


title: Effect of Rh(III) on gene expression in HeLa cell line

city: Guangzhou

first: chen

last: Xiao-xiao

state: Guangdong

address: Xiaoguwei

institute: SUN YAT-SEN UNIVERSITY

summary:
We designed a highly emissive rhodium(III) complex ( Rh(III) complex) as the mtDNA intercalator. Rh(III) complex can specifically bind to mtDNA to cause the cytoplasmic release of mtDNA fragments to activate the cGAS-STING pathway. Moreover, Rh(III) complex activates the mitochondrial retrograde signaling by disturbing the key metabolites. Herein,we performed RNA-seq to illustrate the function of Rh(III) complex on HeLa cells.
RNA-seq was performed on HeLa cells, which were treated by 1% DMSO or Rh(III) complex (1 uM, 6h), respectively.

overall_design: We performed RNA-seq to anlyze the effect of Rh(III) on gene expression in HeLa cell lines.

first: Yue

last: Zheng

first: Xiao-xiao


title: Atoh1 drives the heterogeneity of the pontine nuclei neurons and promotes their differentiation

city: Houston

first: Huda

middle: Yahya

last: Zoghbi

state: TX

address: 1250 Moursund St

department: Molecular and Human Genetics

institute: Baylor College of Medicine

summary: Pontine nuclei (PN) neurons mediate the communication between the cerebral cortex and the cerebellum to refine skilled motor functions. Prior studies have shown that PN neurons fall into two subtypes based on their anatomic location and region-specific connectivity, but the extent of their heterogeneity and its molecular drivers remain unknown. Atoh1 encodes a transcription factor that is expressed in the PN precursors. We previously showed that partial loss-of-function of Atoh1 in mice results in delayed PN development and impaired motor learning. In this study, we performed single-cell RNA sequencing to elucidate the cell-state-specific functions of Atoh1 during PN development and discovered that Atoh1 regulates cell cycle exit, differentiation, migration, and survival of the PN neurons. Importantly, our data revealed six previously not known PN subtypes that are molecularly and spatially distinct. Interestingly, we found the PN subtypes exhibit differential vulnerability to partial loss of Atoh1 function, providing insights into the prominence of PN phenotypes in patients with ATOH1 missense mutations.

overall_design: Atoh1-lineage neurons labeled by TdTomato in mouse hindbrain or PN were enriched by FACS, followed by scRNA-seq with 10X chromium 3' v3.1 kit and NovaSeq 6000. Single-cell transcriptomic profiles of control and mice carrying Atoh1 hypomorphic mutation were compared.

first: Sih-Rong

last: Wu

first: Jessica

middle: C

last: Butts

first: Matthew

last: Caudill

first: Jean-Pierre

last: Revelli

first: Ryan

last: Dhindsa

first: Mark

last: Durham

middle: Y


title: The transcription factor Mef2d regulates B:T synapse-dependent GC-Tfh differentiation and IL-21-mediated humoral immunity

city: Seoul

first: YOUN SOO

last: CHOI

address: 103 Daehak-ro Jongro-gu

department: Department of Biomedical Sciences

country: South Korea

institute: Seoul National University College of Medicine

summary: Comparison of transcriptome between PD1+CXCR5+ Tfh and PD1-CXCR5- non-Tfh compartments developed by control and Mef2d-RV OTII CD4 T cells

overall_design: Control and Mef2d-RV CD45.1+ OTII CD4 T cells were adoptively transferred into C57BL/6J mice, which were subsequently immunized with NP-OVA. On day 3 after immunization, CD45.1+ OTII CD4 T cells in the popliteal lymph nodes were enriched and were stained to identify CD4+ CD45.1+ PD1+ CXCR5+ Tfh cells and CD4+ CD45.1+ PD1- CXCR5- non-Tfh cells. Tfh and non-Tfh cells were sorted for RNA-seq analysis.

first: Youn

middle: S

last: Choi

first: Shane

last: Crotty


title: Determination of blood cell gene expression profiles during early life of premature infants who develop bronchopulmonary dysplasia (BPD)

city: Research Triangle Park

first: Douglas

middle: A.

last: Bell

state: NC

address: PO Box 12233

department: Immunity Inflammation and Disease Lab

institute: National Institute of Environmental Health Sciences

summary: To investigate early blood biomarkers of BPD development, RNA from cord blood cells or peripheral blood cells of premature infants was subjected to RNA sequencing (RNA-Seq) and data were analyzed with 9 covariates including gestational age (GA), sex, birth weight (BW), estimated CD4+T cell%, CD8+T cell%, B cell%, monocyte%, granulocyte%, and nucleated red blood cell (NRBC)%. The effect of prolonged oxygen (>14 days O2) treatment in newborn intensive care unit on blood cell transcriptome was determined among nonBPD preterm infants.

overall_design: Differential gene expression profiles were determined by RNA-seq of two independent analyses in an Argentina preterm infant cohort : Analysis 1- Two treatment groups, BPD and nonBPD, at three time points (cord blood, postnatal day 14, and postnatal day 28), Analysis 2 - Two O2 treatment level groups (no-O2 vs High-O2) were compared among nonBPD neonates on postnatal day 14.

first: Hye-Youn

last: Cho

first: Michelle

last: Campbell

first: Vijji

last: Panduri


title: Regulation of oxidative stress in endothelial cells exposed to high glucose by selected microRNA

city: Cheras

first: Nur Syakirah

last: Binti Othman

state: Kuala Lumpur

address: Yaacob Latiff Street

department: Physiology

country: Malaysia

institute: National University of Malaysia

summary: Hyperglycemia is the hallmark of diabetes mellitus that results in oxidative stress, endothelial dysfunction and vascular complications of diabetes. MicroRNAs (miRNAs) play a role in the development of endothelial dysfunction in diabetes, with potential application for the future therapy of diabetic vascular complications. However, there is a limited number of studies that characterize the miRNA profile of endothelial cells exposed to hyperglycemic condition. This study aimed to identify the miRNA profile of human umbilical vein endothelial cells (HUVEC) exposed to hyperglycemic state using RNA sequencing analysis.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for HUVEC

first: Nur

last: Othman

first: Azizah

last: Ugusman

first: Adila

middle: A

last: Hamid

first: Amilia

last: Aminuddin


title: Uncovering the epigenomic determinants of kidney vascular development [scRNA-seq]

city: Charlottesville

first: Jason

middle: Paul

last: Smith

state: VA

address: 409 Lane Rd

department: Pediatrics

institute: University of Virginia

summary: Renin cells are essential for survival. They control the morphogenesis of the kidney arterioles, and the composition and volume of our extracellular fluid, arterial blood pressure, tissue perfusion, and oxygen delivery. Renin cells and associated arteriolar cells descend from FoxD1+ progenitor cells. The chromatin states and transcription factors that determine the differentiation of these cells into those that compose the kidney vasculature are unknown. To answer these questions, we isolated progenitors and their descendants at different embryonic and postnatal stages, and using integrated scRNA-seq and scATAC-seq established the developmental trajectory that leads to the mosaic of cells that compose the kidney arterioles. We constructed a single-cell atlas of chromatin accessibility and gene expression profiles-including the critical transcription factors that determine the identity and fate of the mosaic of cells that occur during kidney vascular development. Furthermore, we identified the factors that determine the elusive, myo-endocrine adult renin-secreting juxtaglomerular cell.

overall_design: FoxD1+ cells, demarcating the renin lineage, were isolated at embryonic (E) day 12, E18, post-natal (P) day 5, and P30 and subjected to independent scRNA-seq and scRNA-seq to uncover the epigenomic determinants of kidney vascular development.

middle: P

first: Alexandre

last: Martini

first: Silvia

last: Medrano

first: Nathan

last: Sheffield

first: Maria

last: Sequeira-Lopez

first: R

last: Gomez


title: Inhibition of MLCK-mediated migration and invasion in human endometriosis stromal cells by NF-kB inhibitor DHMEQ

city: nagakute

first: Yinzhi

last: Lin

state: Aichi

address: 1-1 Yazakokarimata

department: Microbiology and Immunology

institute: Aichi Medical University School of Medicine

summary: To investigate the effect of DHMEQ on gene expression, we have employed whole genomic microarray expression profiling using HESC cells

overall_design: The HESC cells were plated at 80% confluence and cultured for 24 hours in vitro.

first: Sivasundaram

last: Karnan


title: Uncovering the epigenomic determinants of kidney vascular development [scATAC-seq]

middle: G


title: STAT3 signaling in B cells controls germinal center zone organization and recycling [RNA-seq]

city: Chicago

first: Mark

last: Maienschein-Cline

state: IL

address: 900 S Ashland Ave, Rm 1058, M/C 937

department: Research Resources Center

institute: University of Illinois at Chicago

summary: Germinal centers (GCs), sites of antibody affinity maturation, are organized into dark (DZ) and light (LZ) zones. Here, we uncovered a B cell intrinsic role for STAT3 in GC DZ and LZ organization. Altered zonal organization of STAT3-deficient GCs dampened GC output of long-lived plasma cells (LL-PCs) but increased memory B cells (MBCs). Tfh-GC B cell interaction drive STAT3 tyrosine 705 and serine 727 phosphorylation in LZ B cells, facilitating their recycling into the DZ. An inducible system confirmed STAT3 is not involved in initiating or maintaining the GC but sustains GC zonal organization by regulating GC B cell recycling. RNAseq and ChIPseq analysis identified genes regulated by STAT3 that are critical for LZ cell recycling and transiting through the DZ proliferation and differentiation phases of the DZ. Thus, STAT3 signaling in B cells controls GC zone organization and recycling, and GC egress of LL-PCs, but negatively regulates MBC output.

overall_design: CD23creSTAT3fl/fl and CD23cre controls were immunized with 200ug NP-KLH in CFA and boosted with 100ug NP-KLH in IFA on D7. GC B cells were sorted from the spleen at D14 post immunization for RNAseq.

first: Adam

last: Fike

first: Sathi

last: Chodisetti

first: Nathaniel

middle: E

last: Wright

first: Kristen

middle: N

last: Bricker

first: Philip

last: Domeier

first: Aaron

middle: M

last: Rosenfeld

first: Sara

last: Luckenbill

first: Julia

last: Weber

first: Nicholas

last: Chi

first: Eline

middle: T

last: Luning Prak

first: Malay

last: Mandal

first: Marcus

middle: R

last: Clark

first: Ziaur

last: Rahman


title: STAT3 signaling in B cells controls germinal center zone organization and recycling [ChIP-seq]

summary: Germinal centers (GCs), sites of antibody affinity maturation, are organized into dark (DZ) and light (LZ) zones. Here, we uncovered a B cell intrinsic role for STAT3 in GC DZ and LZ organization. Altered zonal organization of STAT3-deficient GCs dampened GC output of long- lived plasma cells (LL-PCs) but increased memory B cells (MBCs). Tfh-GC B cell interaction drive STAT3 tyrosine 705 and serine 727 phosphorylation in LZ B cells, facilitating their recycling into the DZ. An inducible system confirmed STAT3 is not involved in initiating or maintaining the GC but sustains GC zonal organization by regulating GC B cell recycling. RNAseq and ChIPseq analysis identified genes regulated by STAT3 that are critical for LZ cell recycling and transiting through the DZ proliferation and differentiation phases of the DZ. Thus, STAT3 signaling in B cells controls GC zone organization and recycling, and GC egress of LL- PCs, but negatively regulates MBC output.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for STAT3 in mouse splenic germinal center B cells. Two replicates.

middle: J

middle: B

first: Phillip

middle: L

last: Choi


title: Targeted high throughput mutagenesis of the human spliceosome reveals its in vivo operating principles

city: San Francisco

first: Irene

last: Beusch

state: California

address: 600 16th Street

department: Biochemistry and Biophysics

institute: UCSF

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

overall_design: Refer to individual Series


title: Development of an AAV-LK03 variant with improved liver lobular biodistribution in the humanised FRG model II

city: Westmead

first: Suzanne

last: Scott

state: NSW

address: 214 Hawkesbury Rd

institute: Children's Medical Research Institute

summary: AAV variants may differ in their tropism for human vs mouse hepatocytes or portal vs central hepatocytes.  We examined the engraftment of human hepatocytes in the FRG mouse model and the transduction of hepatocytes in this model using single nuclear RNA-seq.

overall_design: Single nuclear RNA-seq of human hepatocytes engrafted into FRG mouse liver, including pre- and 5 months post-engrafted, as well as AAV-treated samples

first: Marti

last: Cabanes-Creus

first: Renina

last: Navarro

first: Rodrigo

last: Carlessi

first: Ramon

last: Roca-Pinilla

first: Elena

last: Denisenko

first: Grober

last: Baltazar

first: Sophia

last: Liao

first: Erhua

last: Zhu

first: Laurence

middle: W

last: Wilson

first: Alistair

last: Forrest

first: Ian

last: Alexander

first: Nina

last: Tirnitz-Parker

first: Leszek

last: Lisowski


title: Uncovering the epigenomic determinants of kidney vascular development

first: Jason

last: Smith


title: Single cell RNA analysis of the left-right organizer transcriptome reveals potential novel heterotaxy genes

city: Indianapolis

first: Stephanie

last: Ware

state: IN

address: 1044 W Walnut Street, R4 W227

institute: Indiana University School of Medicine

summary: Aimed to identify novel node/left right organizer via scRNA

overall_design: Collected 3 wt embryos which were processed via scRNA-seq to identify the node/left-right organizer

first: Helen

last: Bellchambers

first: Maria

last: Padua


title: Bulk RNA analysis of the left-right organizer transcriptome reveals potential novel heterotaxy genes

summary: Aimed to identify novel node/left right organizer via RNA-seq

overall_design: We used a mouse line that expresses GFP in the node/organizer cells. E8.0 embryos were then FACS sorted to separate GFP+ organizer cells and GFP- non organizer cells which were assess via RNA-seq


title: RNA analysis of the left-right organizer transcriptome reveals potential novel heterotaxy genes


title: STAT3 signaling in B cells controls germinal center zone organization and recycling

first: Mark

last: Maienschein-Cline


title: The LIFR-targeting drugs EC330/EC359 are potent inducers of ferroptosis

city: shanghai

first: changzhou

last: feng

address: No. 197, Ruijin 2nd Road

institute: Shanghai Institute of Hematology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine

summary: Ferroptosis is a form of regulated cell death characterized by iron-dependent overaccumulation of lipid peroxides. It can be prevented by cellular mechanisms such as GPX4-mediated elimination of the lipid peroxides at the cost of glutathione (GSH). Since originally being discovered as a property of RAS-mutant cancer cells, ferroptosis has been shown to be regulated by several important oncogenes and tumor suppressors. In particular, LIFR, the receptor of the pleiotropic cytokine LIF, has recently been shown to be required for ferroptosis, as loss of Lifr in mouse liver tumor confers the cells resistance to ferroptosis. In this study, however, we found that the LIFR-targeting drugs, EC330 and EC359, are potent inducers of ferroptosis, thus reflecting an interesting “gain-of-function” effect of EC330/EC359 on LIFR-associated ferroptosis. Treatment of various types of cells with EC330/EC359 causes a rapid nonapoptotic cell death with characteristic morphology of ferroptosis, which can be inhibited by iron chelator (DFO) and lipid ROS scavenger (Fer-1), but not pan-caspase inhibitor (z-VAD-fmk). Consistent with previous observations, the efficiency of EC330/EC359 appears to be correlated with the expression levels of LIF and LIFR. RNA-seq analysis showed that the EC330/EC359 treatment leads to (i) a gene expression profile highly resembling that of RSL3, the GPX4-targeting ferroptosis inducer, and (ii) a specific decrease of expression of a few gene encoding membrane-located proteins, especially those located on the mitochondria (e.g., TOMM6 and COX14), implying defects in cellular/mitochondrial membrane functions. Indeed, transmission electron microscopy imaging of the EC330/EC359-treated cells shows shrunken mitochondria and loss of mitochondrial cristae. EC330/EC359 also decreases the activity of GPX4 in the cells to the level comparable with RSL3; meanwhile, the intracellular level of GSH is also decreased, and thus GPX4 inactivation and GSH depletion may jointly disable the cells to eliminate the toxic lipid peroxides. Lastly, although the STAT3 and AKT signaling pathways downstream of LIFR are inhibited by EC330/EC359, inhibition of these pathways per se can only induce non-ferroptotic cell death, suggesting that the EC330/EC359-induced ferroptosis is independent of these downstream pathways; the LIFR-NFkB-LCN2 axis discovered recently in mouse liver tumor cells might not explain the EC330/EC359-induced ferroptosis either, because LCN2 is not detectable in the herein used cell lines. Taken together, these results reveal an unexpected role of the LIFR-targeting drugs EC330/EC359 as potent inducer of ferroptosis and, in combination with previous studies, suggest that there could be either unknown mechanism for the LIFR-associated ferroptosis or unknown target for EC330/EC359 to effect the ferroptosis induction.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for 293T cells, after treatment with DMSO, EC359, and RSL3 alone, or co-treatment with Fer-1.

first: C

last: Feng

first: X

last: Sun


title: The antidepressant imipramine inhibits breast cancer growth by targeting estrogen receptor signaling and DNA repair events

city: San Antonio

first: Yidong

last: Chen

state: Texas

address: 8403 Floyd Curl Drive, MSC 7784

department: Population Health Sciences

institute: UT Health Science Center at San Antonio

summary: Breast cancer is the most common cancer in women and the leading cause of cancer-related deaths in women worldwide. Although survival of breast cancer patients has improved in the last decade, major hurdles remain. Two problems associated with current therapies include acquired resistance and the debilitating side effects of treatment, especially chemotherapy. Therefore, safer treatment options that effectively suppress cancer progression and reduce treatment-associated side effects are much needed.   Repurposing of clinically approved or investigational drugs could be one type of effective and safe options for treating cancer patients. Imipramine is a tricyclic antidepressant commonly used for many decades. It is a selective serotonin reuptake inhibitor (SSRI) and inhibits other neurotransmitters. Here, we report that imipramine blocks ER+ and TNBC growth and progression by inhibiting key proteins involved in ER-a signaling, cell cycle progression, and DNA repair and replication. Furthermore, imipramine improved the efficacy of PARP inhibitor therapy in TNBC. This study is the first to show that imipramine is a promising therapeutic option for breast cancer and to define imipramine’s mechanism of action and targets in breast cancer. This pre-clinical study is the basis for a currently ongoing clinical trial testing the efficacy of imipramine for treating breast cancer.

overall_design: Total RNA from MCF7 and MDA-MB-231 vehicle or imipramine-treated cells were isolated using RNeasy mini kit (Qiagen). Each sample were replicated and sequenced using Illumina TruSeq RNA Sample preparation and sequencing protocols at UT Health San Antonio sequencing facility.

first: Santosh

last: Timilsina

first: Subapriya

last: Rajamanickam

first: Manjeet

last: Rao

first: Zhao

last: Lai


title: Hepatocytes reprogram liver macrophages involving control of TGF-β activation, influencing liver regeneration and injury

city: Düsseldorf

middle: Daniela

last: Wolf

state: NRW

address: Moorenstrasse 5, Geb. 13.58 Raum 04

department: Gastroenterology, Hepatology and Infectious Disease

institute: Universitätsklinikum Düsseldorf

summary:
We aimed at elucidating the molecular and cellular crosstalk how inflammation controls proper liver regeneration. Therefore populations of liver macrophages were studied in genetically different mouse models after PHx using flow cytometry and single cell sequencing. Intercellular communication was examined in vitro, combining proteomics and transcriptomics. We observed marked changes in the composition of macrophage populations in the liver during the regeneration process. A F4/80+/CD11bhigh/CD14high macrophage population that is recruited in a CCR2 dependent manner increased rapidly after PHx. The polarization of the recruited macrophages differs from that under homeostatic conditions , but reappears during the late phase of the process. Proteomics, secretomics, and transcriptomics show that hepatocyte derived signals reduce the availability of active TGFb and thereby affect macrophage polarization and function towards the aforementioned phenotype. Depleting the TGFb type II receptor in myeloid cells phenocopies the hepatocyte-mediated macrophage polarization in vitro and in vivo. Moreover, disrupting TGFb signal transduction in macrophages  is associated with increased expression of regeneration-relevant cytokines such as IL-6, reduced resection-induced liver damage and prolongated proliferation phase of hepatocytes in mice.   Conclusion:  Upon liver injury, hepatocytes have a major influence on the activation state of recruited liver macrophages by regulating the availability of active TGFb and TGFb induces a macrophage phenotype that aggravates injury. Ultimately, this mechanism influences the extent of intervention-induced liver injury as well as the proliferation phase during liver regeneration.
In this data set, we investigated the influence of hepatocytes on co-cultured macrophages and vice versa.

overall_design: The RNA expression of primary hepatocytes and BMDM were analysed. Therefore 4 different conditions (mono- vs. co-culture, hepatocytes and macrophages) with 5 replicas each were measured to detect the impact of hepatocytes on macrophages and vice versa.

middle: D

first: Christian

last: Ehlting

first: Sophia

last: Mueller-Dott

first: Geron

last: Poschmann

first: Patrick

last: Petzsch

first: Tobias

last: Lautwein

first: Sai

last: Wang

first: Barbara

last: Helm

first: Marcel

last: Schilling

first: Julia

last: Saez-Rodriguez

first: Mihael

last: Vucur

first: Kai

last: Stühler

first: Karl

last: Köhrer

first: Frank

last: Tacke

first: Steven

last: Dooley

first: Ursula

last: Klingmüller

first: Tom

last: Luedde

first: Johannes

last: Bode


title: Genomic alterations drive metastases formation in pancreatic ductal adenocarcinoma cancer: deciphering the role of CDKN2A and CDKN2B in mediating liver tropism

city: Tel Aviv

first: Metsada

last: Pasmanik-Chor

address: Ramat Aviv

department: Biology

country: Israel

institute: Tel Aviv University

summary: ABSTRACT  Metastases are often the direct cause of death of patients with pancreatic ductal adenocarcinoma (PDAC). Yet, the role of genomic alterations in mediating tropism and metastasis formation by pancreatic cancer cells is currently unknown. We aimed to identify genomic alterations predisposing colonization of PDAC cells to distant organs and decipher mechanisms enabling this process.   Methods: Frequency of genomic alterations was assessed in 2668 local-biopsied compared to 2840 metastatic PDAC samples. Tumorigenic phenotype and transcriptomic profile of manipulated PDAC cells was assessed.  Results: The prevalence of genomic alterations was site dependent, with different profiles observed in samples obtained from the pancreas compared to those obtained from the liver, lungs or lymph nodes. Liver metastases were characterized by deletion of the cyclin dependent kinase inhibitors 2A/2B (CDKN2A/B, encoding p16 and p15, respectively). Studies in PDAC cells indicated enhanced growth under liver conditions following p16/15 deletion. The liver is characterized by high ammonia-low glutamine environment, and a transcriptomic assay revealed modulation of glutamine-ammonia pathway-related genes by p15/p16. Accordingly, p15/p16 deletion promoted growth under high ammonia-low glutamine condition, upregulated glutamate-ammonia ligase (GLUL) and downregulated glutaminase 2 (GLS2). Importantly, GLUL-silencing had a more pronounced effect on proliferation of p15/p16-deleted PDAC cells.     Conclusion: These data suggest a unique role for genomic alterations in mediating tropism and metastases formation. Among these alterations, p15/16 loss was identified as a promoter of liver metastases and further studies indicated a novel role for p16/15, namely, regulation of glutamine synthesis. These findings may pave the way for the development of novel therapeutic strategies aiming at the prevention of liver metastases formation.

overall_design: Human PDAC cell line COLO-357 were grown in DMEM containing 10% fetal bovine serum (FBS). Cells were transfected using Jet Pei (Polyplus Transfection, France). p15/p16 silenced cells (COLO-357sh-NS, COLO-357sh-p15, COLO-357sh-p16, and COLO-357sh-p15/p16) were generated. Cells were seeded in triplicates for 24h after which RNAwas extracted.

first: Shani

last: Journo

first: Anat

middle: K

last: Goldberg

first: Ethan

last: Sokol

first: Lotem

last: Zinger

first: Boris

last: Sarvin

first: Dor

last: Simkin

first: Sivan

last: Fuchs

first: Tomer

last: Shlomi

first: Ido

last: Wolf

first: Tami

last: Rubinek


title: CDKN2A-CDK4-E2F3 dependent oxidative skeletal muscle fiber transition in myogenesis, movement, and metabolism

city: Bethesda

first: Youngjae

last: Bahn

address: 10 Center Dr.

institute: NIH

summary: Skeletal muscle function is vital to movement, thermogenesis and metabolism. Muscle fibers differ in contractile ability, mitochondrial content and metabolic properties and muscle fiber transition influences muscle function. However, the molecular mechanisms regulating muscle fiber transition in muscle function are unclear. Here, in over 150 human muscle samples, we observed that markers of oxidative muscle fiber and mitochondria correlate positively with PPARGC1 and CDK4, and, negatively with CDKN2A, a locus significantly associated with type 2 diabetes. Mice expressing an overactive Cdk4 that cannot bind its inhibitor p16INK4a, a product of the CDKN2A locus, are longer, leaner, exhibit increased oxidative myofibers with superior mitochondrial energetics, display enhanced muscle glucose uptake, and are protected from obesity and diabetes. In contrast, Cdk4-deficiency, or skeletal muscle-specific deletion of Cdk4’s transcriptional target, E2F3, reduces oxidative myofiber numbers, deteriorates mitochondrial function and exercise capacity, while increasing diabetes susceptibility. E2F3 activates the PPARGC1 promoter and CDK4/E2F3/PPARGC1 levels correlate positively with exercise and fitness, and negatively with adiposity, insulin resistance and lipid accumulation in muscle. These findings provide insight into oxidative muscle fiber transition and function that is of relevance to metabolic and muscular diseases.

overall_design: Expression profiling by RNA-Seq in Quadriceps or Soleus muscle from Wild type, CDK4RR, CDK4KO, MLC-cre or E2F3-mKO mice.

first: Young

first: Hariom

last: Yadav

first: Sushil

last: Rane