title: Transcriptomic analysis of neuroblastoma cells in response to stable over-expression of GATA2 antisense RNA 1 (GATA2-AS1)

first: Qiangsong

last: Tong

address: 1277 Jiefang Avenue

department: Department of Surgery

institute: Union Hospital of Tongji Medical College

summary: Long noncoding RNAs (lncRNAs), one type of endogenous RNA longer than 200 nucleotides, play emerging roles in tumorigenesis and aggressiveness. However, the functions and underlying mechanisms of lncRNAs in regulating neuroblastoma progression still remain elusive. We identify GATA2 antisense RNA 1 (GATA2-AS1), a lncRNA derived from GATA binding protein 2 (GATA2), as a novel suppressor of neuroblastoma progression. To investigate the mechanisms underlying the oncogenic functions of GATA2-AS1, we employed the Illumina HiSeq X Ten as a discovery platform to analyze the transcriptome profiling changes of human neuroblastoma BE(2)-C cells in response to stable over-expression of GATA2-AS1. The results showed that stable over-expression of GATA2-AS1 led to altered expression of 1933 human mRNAs, including 857 up-regulated genes and 1076 down-regulated genes. Furthermore, we validated the RNA-seq results by real-time RT-PCR with high identity. Overall, our results provided fundamental information about the transcriptomic changes in response to GATA2-AS1 over-expression in human tumor cells, and these findings will help us understand the pathogenesis of tumor progression.

overall_design: Total RNA of cells stably transfected with empty vector or GATA2-AS1 was extracted using the TRIzol® reagent according to the manufacturer's instructions. RNA concentration was measured using a Qubit® RNA Assay Kit with a Qubit® 2.0 Fluorometer (Life Technologies, Inc.), and integrity was assessed using the RNA Nano 6000 Assay Kit with a Bioanalyzer 2100 system (Agilent Technologies, CA). Library preparation and transcriptome sequencing on an Illumina HiSeq X Ten platform were performed by Wuhan SeqHealth Technology Co., Ltd. (Wuhan, China), and 100 bp paired-end reads were generated. HTSeq v0.6.0 was used to count the reads numbers mapped to each gene.

first: Liduan

last: Zheng


title: Hyperactive STAT5 Hijacks T-Cell Receptor Signaling and Drives Immature T-Cell Acute Lymphoblastic Leukemia

status: Public on Apr 15 2024

city: Vienna

first: Tobias

last: Suske

state: Vienna

address: Veterinärplatz 1

department: Functional Cancer Genomics

country: Austria

institute: University of Veterinary Medicine

type: Expression profiling by high throughput sequencing

summary:
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive immature T-cell cancer.
Mutations in IL7RA were analyzed genetically, but downstream effector functions such as
STAT5A/B hyperactivation are poorly understood. Here, we studied the most frequent and
clinically challenging STAT5BN642H driver in T-cell development and immature T-cell cancer
onset and compared it to STAT5A hyperactive variants in transgenic mice. Enhanced STAT5
activity caused disrupted T-cell development and promoted an early T-cell progenitor (ETP)-
ALL phenotype with upregulation of genes involved in T-cell receptor (TCR) signaling, even in
absence of surface TCR. Importantly, TCR pathway genes were overexpressed in human T-
ALL and mature T-cell cancers and activation of TCR pathway kinases was STAT5-dependent.
We confirmed STAT5-binding to these genes using ChIP-seq analysis in human T-ALL cells,
which were sensitive to pharmacologic inhibition by dual STAT3/5 degraders or ZAP70
tyrosine kinase blockers in vitro and in vivo. We provide first genetic and biochemical proof
that STAT5A and STAT5B hyperactivation can initiate T-ALL through TCR pathway hijacking
and suggest similar mechanisms for other T-cell cancers. Thus, STAT5 or TCR component
blockade are targeted therapy options, particularly in patients with chemo-resistant clones
carrying STAT5BN642H.

overall_design: Comparative gene expression profiling analysis of RNA-seq data from murine thymocytes in wildtype, Rag deficient, Stat5a and Stat5b gain of function mutations in 3 sates of T-cell differentiation (determined by CD4 and CD8 cell surface markers)

first: Helena

last: Sorger

first: Frank

last: Ruge

first: Nicole

last: Prutsch

first: Mark

middle: W

last: Zimmerman

first: Thomas

last: Eder

first: Barbara

last: Maurer

first: Christina

last: Wagner

first: Susann

last: Schönefeldt

first: Katrin

last: Spirk

first: Daniel

last: Pölöske

first: Dennis

last: Jungherz

first: Tony

middle: A

last: Müller

first: Myint

middle: M

last: Aung

first: Ha

middle: T

last: Pham

first: Kerstin

last: Zimmel

last: Krausgruber

first: Christoph

last: Bock

first: Mathias

first: Maik

last: Dahlhoff

first: Auke

last: Boersma

last: Rülicke

first: Roman

last: Fleck

first: Patrick

last: Gunning

first: Tero

last: Aittokallio

first: Satu

last: Sanda

first: Sylvia

last: Hartmann

first: Florian

last: Grebien

first: Gregor

last: Hoermann

first: Torsten

last: Haferlach

first: Philipp

middle: B

last: Staber

first: Heidi

last: Neubauer

first: Alfred

last: Look

first: Marco

last: Herling

first: Richard

last: Moriggl

first: Gabriele

last: Manhart

first: Diaa

last: Abdallah

first: Emina

last: Hubanic

first: Elvin

middle: D

last: de Araujo


title: Comprehensive transcriptomic profiling of glioblastoma patient-derived primary tumor cell lines using bulk RNA-sequencing

status: Public on Apr 14 2024

city: Seoul

first: Sun Ha

last: Paek

address: 101, Daehak-ro, Jongno-gu

department: Department of Neurosurgery

country: South Korea

institute: Seoul National University College of Medicine, Seoul National University Hospital

summary: Glioblastoma multiforme (GBM) is a highly aggressive primary brain cancer with significant transcriptomic heterogeneity among patients. Our study aims to contribute to the field of personalized medicine for GBM patients by providing comprehensive transcriptomic profiles of GBM patients (N = 45) who underwent concurrent chemoradiotherapy with temozolomide at Seoul National University Hospital. Additionally, we include transcriptomic profiles of normal brain tissue obtained from GBM patients. This dataset may add depth to existing knowledge by uncovering distinct transcriptomic signatures related to aggressiveness in GBM, and may inform future research on the molecular mechanisms underlying transcriptomic features of GBM and personalized treatment strategies.

overall_design: We performed gene expression profiling analysis of RNA-seq data obtained from patient-derived GBM cells at the time of diagnosis and recurrence. Total RNA was isolated from these cells using the RNeasy Mini Kit (74106, QIAGEN) following the manufacturer's protocol. Libraries for bulk RNA-seq were prepared using the TruSeq Stranded Total RNA Library Prep Plant Kit. Primary GBM cell lines were cultured in Dulbecco’s modified Eagle’s high glucose medium (SH30243.01, Hyclone) supplemented with 10% heat-inactivated fetal bovine serum (2357664RP, Gibco) and 1% antibiotic-antimycotic (15240062, Gibco). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2 and tested regularly for mycoplasma contamination by mycoplasma detection kit (rep-mys-20, InvivoGen).

first: Sun

middle: H

first: Sanghyuk

last: Lee

first: Jong

middle: E

first: Yona

last: Kim


title: ChIP-seq analysis of G3BP1 in KGN cells

city: Jinan

first: Nianyu

last: Li

state: ShanDong

address: 44 wenhua xi road

country: China

institute: Shandong University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: To investigate whether G3BP1 binds to telomeric DNA, we performed the ChIP-seq analysis of G3BP1 in KGN cells.

overall_design: ChIP-seq using the G3BP1 antibody in KGN cells.

first: Xue

last: Jiao

first: Ting

last: Dong

first: Huirui

last: Wang


title: Effects of a humanized ectopic niche (human ossicle or hOss) on human hematopoiesis

city: Paris

first: Chloé

last: Friedrich

address: 8 rue Méchain

country: France

institute: Cochin Institute

summary: Human hematopoiesis takes place in the bone marrow (BM) where a multitude of interactions regulate differentiation and preservation of hematopoietic stem/progenitor cells (HSPC). Well-studied in mouse models, human BM microenvironment still contain unexplored features. Development of immune-deficient mouse models have considerably improved our knowledge of the molecular and cellular regulation of human HSPC, but remain limited by the numerous non-cross-reactive signals provided by the mouse BM to human HSPC. Here, we thoroughly characterized a humanized BM model, based on Mesenchymal stromal cells (MSC) and called human ossicles (hOss), and the different hematopoietic cell compartments generated 3 months post-transplant of human CD34+ cells using scRNA sequencing.

overall_design:
Human CD34+ HSPCs from a cord blood source were injected intravenously into NSG mice. Mice were either hOss-bearing or hOss-free. Three months after HSPC injection, mice were sacrificed and mononuclear cells (MNC) that had engrafted into hOss and murine BM were pooled for each condition. In total, we had 1 sample of murine BM (mBM W/o hOss, and 2 samples of pooled ossicles (F/hOss and P-N/hOss). To enrich samples in CD34+ fraction, immunomagnetic sorting was performed. The CD34+ cell fraction was then used to prepare scRNA-seq libraries.
F hOss: hematopoietic stem cells (HSC) derived from organoids of fetal origin
P-N hOss: hematopoietic stem cells (HSC) derived from organoids of postnatal origin

first: Laurent

last: Renou

first: Wenjie

last: Sun

first: Klaudia

last: Galant

first: Cécile

last: Conrad

first: Evelia

last: Plantier

first: Katharina

last: Schallmoser

first: Linda

last: Krisch

first: Vilma

last: Barroca

first: Saryami

last: Devanand

first: Nathalie

last: Déchamp

first: Andréas

last: Reinisch

first: Jéléna

last: Martinovitch

first: Alessandra

last: Magnani

first: Lionel

last: Faivre

first: Julien

last: Calvo

first: Leila

last: Perié

first: Olivier

last: Kosmider

first: Françoise

last: Pflumio


title: Gene expression changes in primary human endometrial stromal cells following METTL3 knockdown during in vitro decidualization

city: Guangzhou

first: Ji-Long

last: Liu

address: 483 Wushan Rd

institute: South China Agricultural University

summary: We developed an in vitro model in which primary human endometrial stromal cells (HESCs) were induced to differentiate through treatment with MPA and 8-Br-cAMP. SiRNA-mediated knockdown of METTL3 was performed on HESCs, 6 h prior to treatment of 8-Br-cAMP and MPA.

overall_design: To investigate the effect of METTL3 on decidualization, HESCs were subjected to decidualization for 4 days after transfection of siRNA targeting METTL3 (RiboBio, Guangzhou, China; #siG151008053209-1-5; siN0000001-1-5 serving as negtive control) for 6 hours.


title: Global m6A-modified mRNAs in undecidualized and decidualized primary human endometrial stromal cells [meRIP-seq]

summary: Global m6A-modified mRNAs in undecidualized and decidualized primary human endometrial stromal cells were analyzed by using methylated RNA immunoprecipitation sequencing (MeRIP-seq)

overall_design: We investigated global m6A-modified mRNAs in undecidualized and decidualized primary human endometrial stromal cells by using methylated RNA immunoprecipitation sequencing (MeRIP-seq)


title: Gene expression changes in conditional METTL3 overexpression mouse uterus compared to control mouse uterus on gestational day 8

summary: We specifically over-expressed Mettl3 in mouse uterus using the Pgr-Cre driver. To portray the molecular mechanism for Mettl3 function in mouse uterus during decidualization, uterine tissues were collected from METTL3-OE and control mice on gestational day 8 and subjected to RNA-seq analysis.

overall_design: Uterine tissues were collected from METTL3-OE and control mice on gestational day 8 and subjected to RNA-seq analysis.


title: Alternate high fat diet aggravates atherosclerosis through an IL1b-dependent reprogramming of neutrophil progenitors

city: Nijmegen

first: Mumin

last: Ozturk

address: Geert Groote Plein Zuid 30

department: Cell Biology

country: Netherlands

institute: Radboud University

summary: Systemic immune responses induced by chronic hypercholesterolemia contribute to the initiation, progression and complications of atherosclerosis. However, the consequences of an alternate high fat diet and the associated variations in plasma cholesterol levels on atherosclerosis are unknown. To address this relevant issue concerning common situations where dietary habits change over time, we developed a novel protocol in athero-prone mice that allowed us to compare the effects of alternate versus continuous high fat diet (HFD) on atherosclerosis, the overall period of time of exposure to HFD being similar between groups. We showed that alternate HFD lead to accelerated atherosclerosis in Ldlr-/-  and Apoe-/- mice compared to continuous HFD. The pro-atherogenic effect of alternate HFD was also found in Apoe-/-Rag2-/- mice lacking T, B, and NKT cells, ruling out a role for the adaptive immune system in the observed phenotype. Using different complementary in vivo approaches, we found that lipid-rich diet discontinuation in alternate group downregulated Runx1, a negative regulator downstream of TLR-4 pathways. As a result, after re-exposure to HFD, myeloid progenitors were more sensitive to hypercholesterolemia leading to acute differentiation into IL-1b- producing immature neutrophils, which caused a state of emergency myelopoiesis. Consecutively, neutrophils markedly increased in the peripheral blood, infiltrated atherosclerotic lesions and locally released neutrophil extracellular traps. Anti-Ly6G neutrophil depletion abolished the pro-atherogenic effects of alternate HFD. Specific deletion of Il1b and Il1b receptor confirmed that the IL-1b signaling pathway was a major driver of the pro-inflammatory and pro-atherogenic neutrophil signature. Finally, treatment with anti-IL-1b neutralizing antibody or inflammasome inhibitor during re-exposure to HFD reversed the pro-atherogenic effects of alternate HFD. In summary, we showed that alternate HFD accelerates atherosclerosis through IL-1b-dependent neutrophil progenitor reprogramming.

overall_design: Lin- cells from femur bones of HFD exposed and non-exposed Ldlr-/- mice were dissected and single cell RNA Seq libraries were prepared with ParseBio Evercode WT Mega v2 kit.

first: Yutaka

last: Negishi

first: Musa

last: Mhlanga

first: Hafid

last: Ait-Oufella


title: Expression of miRNA in exosomes from transudative pleural effusion and tuberculous pleural effusions

city: Wuhan

first: Li-Mei

last: Liang

state: Hubei

address: 1277 JieFang Avenue

department: Department of Respiratory and Critical Care Medicine, Union Hospital

institute: Huazhong University of Science and Technology

type: Non-coding RNA profiling by high throughput sequencing

summary: Pleural fibrosis is defined as an excessive deposition of extracellular matrix (ECM) that results in destruction of the normal pleural tissue architecture and compromised function. However there is currently no effective medication for pleural fibrosis. Understanding the detailed mechanisms of pleural fibrosis is an important unmet need which could lead to the identification of new targets for treatment of this condition.  microRNAs (miRNAs) play an important role in the posttranscriptional control of gene expression. In our study, cellular fractions from TBPE contained activities capable of promoting fibrosis-like behavior in pleural mesothelial cells (PMCs), the goal of this study is to compare the exosomal miRNA composition of TBPE and TPE.   We isolated exosomes from transudative pleural effusion and tuberculous pleural effusions and performed miRNA sequencing.  Our study represents the first detailed analysis of exosomal miRNA composition of TBPE and TPE with biologic replicates, generated by miRNA-Seq technology.

overall_design: Three independent  preparations of exosomes from transudative pleural effusion or tuberculous pleural effusions

first: Meng

last: Wang

first: Hong

last: Ye

first: Wan-Li

last: Ma


title: The p21+ Perinecrotic Hepatocytes Produce the Chemokine Cxcl14 After a Severe Acetaminophen Overdose Promoting Hepatocyte Injury and Delaying Regeneration

city: Kansas City

first: David

middle: S

last: Umbaugh

email: dumbaug@gmail.com, hjaeschke@kumc.edu

state: KS

address: 3901 Rainbow Blvd

country: USA

institute: University of Kansas Medical Center

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Investigating the phenotypic plasticity of two red grapevine varieties: an integrated study of transcriptome and environmentome by WGCNA

city: Catania

first: Angela Roberta

last: Lo Piero

state: CT

address: Via S. Sofia 98

country: Italy

institute: University of Catania

summary: Grapevine (Vitis spp., family Vitaceae) is characterized by a marked phenotypic plasticity and its ability to withstand various environmental conditions depends on the activation of highly coordinated responses, resulting from the interaction among genotypes (G) and environmental factors (E). Understanding genotype by environment (GxE) interplays is highly complex and challenging, and often the outcome of the genetic responses to field performance is not straightforward also due to the difficulties to reach a precise definition of the E component in field studies. In this work, the transcriptome of commercially ripe berries of Cabernet sauvignon and Aglianico genotypes grown in open-field at three different sites of central-southern Italy (Campania, Molise and Sicily) was analyzed by RNAseq. These transcriptomic data were integrated with a comprehensive set of climatic indices, forming an “environmentome”, through Weighted Gene Co-expression Network Analysis (WGCNA). A total of 11,887 differentially expressed genes (DEGs) have been retrieved, most of them found in the Aglianico genotype, suggesting that Aglianico transcriptome is largely influenced by the environment. Furthermore, the comparisons involving the Sicilian site exhibited the highest number of DEGs for both genotypes. WGCNA suggested that most of the climatic data (such as daily maximum air temperature, relative humidity, air pressure, dew point, hours of sun radiation) significantly correlated with “lightcyan1” module. Among these eingengenes, the low expression of ACA10 cation transporter is suggested to be indirectly related to the low anthocyanin content in Cabernet sauvignon in Campania, thus confirming WGCNA as powerful approach to identify gene of high biological interest. Similarly, transcriptome was also correlated with quality trait, such as total soluble solids and polyphenol content, leading to the discovery of hub genes that might function as markers of wine quality.

overall_design:
In order to explored the gene-environment (GxE) interactions of two red grape varieties, samples of fully ripe berries of Aglianico and Cabernet Sauvignon varieties grown in open-field conditions at three different sites in southern Italy (Sicily, Molise and Campania) were collected.
We then performed RNAseq and gene expression analysis considering three biological replicates for each sample above described
Differential expression analysis of each variety in the trhee sites was performed
WGCNA was applied to correlate the RNAseq results with several climatic parameters, as well as and with quality traits including total soluble solid, anthocyanin and polyphenol content.

first: Angelo

last: Sicilia

first: Clizia

last: Villano

first: Riccardo

last: Aversano

first: Ermanno

last: Di Serio

first: Elisabetta

last: Nicolosi

first: Filippo

last: Ferlito

first: Angela

middle: R


title: Hnrnpk is essential for embryonic limb bud development as a transcription activator and insulator collaborator

first: Yuyu

last: Chen

state: Guangdong

address: Zhongshan II road, 74th

institute: Sun Yat-sen University

summary: Proper development of limb bud relies on the concordance of various signals, otherwise limb deformities occur. We report that heterogeneous nuclear ribonucleoprotein K (hnRNPK) is essential for limb bud development. here, we knock out Hnrnpk in limb bud and exert the RNA-seq, ATAC-seq, CUT&RUN-seq, and Hi-C assay using primary limb bud cells to explore the function of Hnrnpk in limb bud development.

overall_design: ATAC-seq, mRNA, Ctcf binding, Hnrnpk binding, H3K4me1 binding, H3K4me3 binding and HiC profile of mice limb bud cells with or without Hnrnpk

first: Taifeng

last: Zhou


title: Transcriptome sequencing of liver tissue in hepatocyte-specific IRF8-deficient mice (IRF8-cKO) mice and wild type (WT) mice that were subjected to warm ischemia/reperfusion(I/R) injury

status: Public on Apr 13 2024

city: Nanjing

first: Hongxi

last: Wu

state: Jiangsu

address: No. 639, LongMian Road, Jiangning District, Nanjing

institute: China Pharmaceutical University

summary: Interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence-binding protein, is an essential lineage-specific transcriptional regulator in the IRF family, controls diverse cellular processes and is has been implicated in innate immunity, immune cell differentiation. IRF8 has been shown to have both oncogenic and tumor suppressor activities, in myeloid cells and nonhematopoietic cells. As a transcription factor, other functions which may contribute to its oncogenic and anti tumor potential are not clear. So it is meaningful to study the role of IRF8 in different diseases. Our recent study demonstrated the protective effects of IRF8 on the liver cancer through the regulation of immune response. In addition, studies have shown that IRF8 has protective effects on ischemia-reperfusion injury of heart and brain and we wondered. However, the role of IRF8 in hepatic ischemia-reperfusion injury has not been reported.

overall_design: mRNA profile of injured liver tissues were generated by deep sequencing in hepatocyte-specific IRF8-deficient mice(IRF8-cKO) mice and wild type mice(WT) that were subjected to warm ischemia/reperfusion injury (I/R)

first: Wu

last: Hongxi

first: Shi

last: Guangjiang


title: Effect of electroacupuncture  on gene expression of adipose tissue in HFD-induced obese mice

city: Chengdu

first: Hongyan

last: Gao

address: 1166 Liutai Avenue

institute: Chengdu University of Traditional Chinese Medicine

summary:
To investigate whether electroacupuncture (EA) promotes adipose tissue browning by regulating the expression of  angiogenesis genes through EA, we sequenced the adipose tissue of mice fed high fat diet with and without electroacupuncture, as well as those fed normally.
We then performed gene expression profiling analysis using data obtained from RNA-seq of 9 mice in three groups.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for ND group,HFD goup and HFD+EAgroup.

first: Yanhui

last: Li


title: Recognition of a pediatric glioblastoma-associated histone mutation by chromatin reader ZMYND8 promotes tumorigenesis

city: Shanghai

first: Yang

last: Shi

address: NO. 138, Yi-Xue-Yuan Road

institute: Fudan university

summary: Histone H3 point mutations have recently been identified in pediatric brain cancers, including missense mutations on histone H3 at positions 27 (lysine to methionine; K27M) and 34 (glycine to arginine, G34R), respectively. H3K27M mutations are associated with a majority of pediatric Diffuse Intrinsic Pontine Pediatric Glioma (DIPG) cases, while H3.3G34R is found in pediatric glioblastoma (GBM) arising in the cerebral cortex1,2. While the mechanisms of H3K27M-driven tumorigenesis are beginning to unravel, much less is known about the H3.3G34R mutation. Here, we provide both biochemical and structural evidence that the PHD (plant homedomain) domain of the enhancer-binding protein, RACK7 (ZMYND8), recognizes H3.3G34R mutant histone via a mechanism distinct from other PHD domains. In an H3.3G34R-expressing neuronal precursor cell line, we demonstrate that RACK7 is aberrantly recruited to enhancers bound by H3.3G34R, resulting in the down-regulation of many neuronal genes, and the up-regulation of oncogenes such as MYC. Neural precursor cells expressing H3.3G34R showed enhanced tumor growth when injected subcutaneously. Inhibiting RACK7 binding to H3.3G34R reduces tumor size and alters gene expression, suggesting that H3.3G34R regulation of enhancers via RACK7 promotes tumorigenesis. Specifically, we show that both the gene expression and tumor size regulation are critically dependent on the physical interaction of H3.3G34R with RACK7. Both regulations are compromised in the absence of RACK7, and can be rescued by wildtype, but not an H3.3G34R-binding defective, RACK7. Taken together, we have identified a reader for H3.3G34R and demonstrate that RACK7 binding to H3.3G34R alters enhancer activity to directly suppress neural gene transcription and to increase oncogenic propensity of neural precursor cells, thus providing a potential molecular basis for pediatric GBM driven by the H3.3G34R mutation.

overall_design: H3.3A knockout cells were generated by CRISPR-Cas9 (gRNA-pX335-hSpCas9) system. H3.3WT and H3.3G34R cells were generated by transfection of either a HA-tagged wildtype or G34R mutant H3.3 transgene into the H3.3A KO cells. Cells were selected by puromycine. ChIP-seq of RACK7,HA tagged H3.3wt and H3.3G34R, H3K4me1, H3K27ac were performed with two cells seperately.

first: Rui

last: Guo


title: CRISPR-based gene disruption coupled to targeted integration of novel, high-avidity, natural T cell receptors to WT1 for acute leukemia

city: Segrate

first: Ivan

last: Merelli

state: Milano

address: via F.lli Cervi 93

department: Istituto di Tecnologie Biomediche

institute: Consiglio Nazionale delle Ricerche

summary: To better characterize and identify the candidate TCR among the receptors that we have isolated, we dissected the gene expression profiles and biological pathways of HD1- and HD3-TCR edited T cells by single cell sequencing. When challenged with target cells (either WT137-45- pulsed T2 cell line or primary AML blasts), results highlighted an immune response gene signature in each stimulated T cell culture, compared to resting counterparts. Interestingly, upregulation of genes mediating IFNa and g response, T cell proliferation and CD4+ T cell activation was observed in HD1- but not in HD3-TCR T cells stimulated with primary leukemic blasts.

overall_design: T cells harvested from 3 independent donors (DN1, DN2, DN3) were genetically engineered with the TCR gene editing protocol and transduced with the HD1- or HD3-TCR. 14 days upon stimulation with the anti-CD3 and anti-CD28 magnetic beads (ClinExVivo CD3/CD28; Invitrogen), engineered T cells from each donor were co-cultured with WT137-45- pulsed HLA-A*02:01+ T2 cells or with primary leukemic blasts (pAML1) at an effector to target ratio of 2:1. As control we included in the experimental setting HD1- and HD3-edited T cells  without further addition of target cells. 24 hours later T cells were enriched by magnetically sorting using the PE-fluorescently labeled Vb8 (for HD1-TCR cultures) and the Vb7.2 (for the HD3-TCR cultures) antibodies and the anti-PE microbeads (Miltenyi Biotec). In total, we  collected T cells from 18 different conditions, 6 for each DN included in the analysis: HD1-TCR T cells and HD3-TCR T cells, each left unstimulated, co-cultured with WT137-45-pulsed T2 cells, or co-cultured with pAML1 blasts. Cell hashing (74,75) with anti-human CD45, anti-human CD3 and anti-human β2-microglobulin barcoded antibodies (TotalSeq-C, Biolegend) was performed in order to multiplex samples from each donor in an individual single cell reaction following the manufacturer’s instruction. Afterwards, 5x104 cells/sample were washed and pooled in 3 different Eppendorfs (1 for each donor) in PBS supplemented with 0.04% Bovine Serum Albumine (BSA).

first: Eliana

last: Ruggiero

first: Chiara

last: Bonini


title: Targeting metabolism to improve immunotherapy in GSNOR-deficient colorectal cancer

city: Córdoba

first: Antonio

last: Rodriguez Ariza

state: Córdoba

address: Av. Meléndez Pidal S/N

department: Medical Oncology

country: Spain

institute: Maimonides Institute of Biomedical Research of Cordoba

summary: Colorectal cancer (CRC) is the third most diagnosed type of cancer and the second leading cause of cancer death worldwide. Despite the increasing knowledge of CRC molecular biology and the development of new targeted therapies, its high heterogeneity hampers the efficacy of current treatments. Thus, there is a pressing need to identify new effective therapeutic targets and improve immune therapies for these patients. In this regard, S-nitrosoglutathione reductase (GSNOR) is a denitrosylase enzyme that has been suggested to play a tumour suppressor role, although the mechanisms responsible are still largely unclear. Therefore, the main objective of this project was to understand the role of GSNOR in CRC tumorigenesis and its therapeutic implications. Firstly, we classified CRC tumours as GSNOR-high or low according to their GSNOR expression as assessed by immunohistochemistry (IHC). Accordingly, we found that GSNOR deficiency was associated with worse prognosis factors such as a larger tumour size at diagnosis, higher TNM stage, higher grade of tumour budding (TB), the CMS4 subtype, lower expression of the intestinal differentiation markers CDX2 and AE1/AE3 cytokeratin and a worse progression free survival (PFS) and overall survival (OS). We next investigated the differences in gene expression between CRC GSNOR-high and low tumours, uncovering significant alterations in metabolism and immune system pathways. Hence, GSNOR-deficient tumours were characterized by immune suppressive features and a dysregulation of their metabolism, favouring other metabolic pathways than OXPHOS.

overall_design: Total RNA from human CRC samples was extracted using the commercial RNeasy Mini Kit (Ref.: 74004, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Briefly, RNA from fresh‐frozen CRC tissues were obtained by mechanical disruption of 100 mg of tissue using gentleMACS™ M Tubes (Ref.: 130-096-335, Miltenyi Biotec, Bergisch Gladbach, Germany) and then lysed with 600 µL RLT buffer at 3,000 rpm for 10 min. at 4°C. Then, supernatants were centrifuged with 700µL of 70% ethanol using gDNA removal columns at 10,000 rpm for 1 minute at RT. Next, samples were washed with ethanol 70%. Samples were then washed with 700 µL RW buffer in RNA columns at 10,000 rpm for 1 minute at RT. Samples were washed twice with 500µL RPE buffer columns at 10,000 rpm for 1 minute at RT. Finally, RNA was collected using 30 µL RNase-free water at 10,000 rpm for 1 minute at RT. Finally, total RNA was quantified using the NanoDrop® 1000 spectrophotometer (NanoDrop® ND-1000 UV-Vis Spectrophotometer, NanoDrop Technology, Thermo Fisher Scientific, MA, USA), and RNA integrity Number (RIN) was measured using the Agilent 2200 TapeStation equipment (Agilent, CA, USA). After RNA was extracted as described above, a quality check was performed using TapeStation Analysis Software A.02.02 (SR1) (Agilent Technologies, CA, USA). A series of 34 CRC human samples with an RNA RIN ≥ 7 were selected and 100 ng were sent to Centre for Genomic Regulation (CRG, Barcelona, Spain) to obtain RNA libraries. The RNA libraries obtained were composed of short fragments of 50bp with paired ends and sequenced by using the NextSeq2000 sequencing system (100 cycles), performing 40 million reads/sample.

first: Ana

last: Soldado

last: Ariza


title: Transcriptome comparison of monocytes, activated CD4 T cells, and activated CD8 T cells in healthy human peripheral blood

first: Wang

last: Yafeng

address: Puzhuzhonglu

institute: Nanjing Drum Tower Hospital, Medical School, Nanjing University

summary: Molecular tether-mediated extracellular targeted protein degradation (ePTD) emerges as a promising drug modality. The existing strategies for ePTD have exploited several membrane degrader proteins. However, the current panel of ePTD degraders clearly needs to be expanded, as a given degrader may present tissue-dependent expression and activity. Based on screening of >50 receptors on endocytic rates and on their tissue distribution, we established a resource of potential endocytic carriers that may mediate effective ePTD. We subsequently introduced an adaptable design to assemble “Selected endocytic carrier-targeting chimera (SecTAC)” based on bispecific antibody constructs. The resultant modular chimeras, by co-opting newly identified degraders, directed efficient internalization of extracellular protein cargoes (or nucleic acids). Importantly, shaped by the availability of the corresponding endocytic carrier, the output of a SecTAC could present cell subset selectivity. We also validated the therapeutic potential of SecTAC agents for targeting several cell membrane proteins in tumor cells and primary T cells. Furthermore, we adapted the platform to establish multi-cargo tethers that triggered simultaneous degradation of two cell surface proteins in primary T cells. Taken together, our SecTAC platform has laid the foundation for development of preclinical drug candidates capable of inducing extracellular and membrane target degradation in desirable population of cells.

overall_design: Monocytes were directly isolated from human whole blood by immunomagnetic negative selection using EasySep™ Direct Human Monocyte Isolation Kit (STEMCELL). CD3+ T cells were isolated from PBMCs and activated and proliferated in vitro for 3 days. Total RNA was isolated and perform RNAseq services.The data were mainly used to compare gene expression differences between different cell populations

first: Yafeng

last: Wang

first: Guiquan

last: Zhang


title: Global m6A-modified mRNAs in mouse uterus on gestational day 4 [meRIP-seq]

status: Public on Apr 12 2024

first: Ji-Long

last: Liu

summary: Global m6A-modified mRNAs in wild-type mouse uterus on gestational day 4  were analyzed by using methylated RNA immunoprecipitation sequencing (MeRIP-seq)

overall_design: We investigated global m6A-modified mRNAs in wild-type mouse uterus on gestational day 4 by using methylated RNA immunoprecipitation sequencing (MeRIP-seq)


title: Gene expression changes in Mettl14d/d mouse uterus compared to Mettl14f/f mouse uterus on gestational day 4 [RNA-seq]

summary: We specifically deleted Mettl14 in mouse uterus using the Pgr-Cre driver. To portray the molecular mechanism for Mettl14 function in mouse uterus, uterine tissues were collected from Mettl14f/f and Mettl14d/d mice on gestational day 4 and subjected to RNA-seq analysis.

overall_design: Uterine tissues were collected from Mettl14f/f and Mettl14d/d mice on gestational day 4 and subjected to RNA-seq analysis.


title: Enhancing Cell-Based Regenerative Therapies with Peptide-Modified Thermo-responsive Citrate-Based Biomaterials for Treatment of Critical Limb Ischemia

city: Chicago

first: Bin

last: Jiang

state: IL

address: 676 N St Clair St, Suite 650

institute: Northwestern University

summary: Critical limb ischemia (CLI) poses a significant health challenge, marked by severe morbidity and limited treatment options leading to high mortality rates. Despite the promise of cell-based therapy, challenges such as poor cell survival and engraftment during and after cell delivery hinder its efficacy. This study explores the potential of peptide-modified thermo-responsive citrate-based biomaterials as carriers for endothelial cell delivery to promote vascular regeneration in CLI. Specifically, various pro-survival peptides were covalently tethered to poly(polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN) to investigate their effect on the delivery of vascular endothelial cells to skeletal muscle tissue. After screening with in vitro and in vivo experiments, laminin-derived peptide A5G81 and VEGF-derived peptide QK were identified to promote endothelial cell spreading, proliferation, and prolonged cell survival when tethered onto PPCN  The viscoelastic properties of PPCN provided protection against shear stress induced cell death during injection, while the peptides regulated endothelial cell behavior via distinct molecular pathways. Importantly, intramuscular delivery of endothelial cells with PPCN-A5G81 and PPCN-QK in a murine hindlimb ischemia model resulted in significant improvements in limb perfusion, tissue preservation and functional outcomes. Furthermore, endothelial cell delivery with PPCN-A5G81 and PPCN-QK also promoted positive skeletal muscle remodeling following ischemic injury. These findings underscore the potential of bioactive materials as novel cell delivery carriers for CLI therapy, with implications for advancing regenerative therapeutics and revolutionizing CLI treatment strategies.

overall_design: Extraction of RNA was carried out utilizing the Aurum™ Total RNA Mini Kit (Bio-rad), following the manufacturer's protocol. The quality of the extracted RNA was assessed using the Agilent Bioanalyzer 2100, with acceptance criteria for RNA integrity numbers (RIN) set at > 7. Quantification was performed using Qubit. To construct RNA sequencing libraries, Illumina TruSeq mRNA Sample Preparation Kits were employed, adhering to the manufacturer's instructions. The procedure involved the capture of polyadenylated mRNAs from total RNA through oligo-dT selection. Subsequently, cDNA synthesis occurred through reverse transcription, and each sample was ligated to Illumina sequencing adapters featuring unique barcode sequences. Following this, barcoded samples underwent PCR amplification, and the resulting cDNA libraries were quantified using qPCR. To achieve a comprehensive overview, equimolar concentrations of each cDNA library were pooled and sequenced on the Illumina HiSeq 4000. Subsequently, the quality of reads in FASTQ format was evaluated using FastQC. Trimming of reads, specifically removing Illumina adapters from the 3’ ends, was executed using cutadapt. Trimmed reads were aligned to the human genome (hg38) using STAR. Read counts per gene were computed utilizing htseq-count alongside a gene annotation file for hg38 obtained from Ensembl. Normalization and differential expression analyses were conducted using DESeq2 1.42.0, employing the Wald test. The criterion for identifying significantly differentially expressed genes was an FDR-adjusted p-value less than 0.05, employing the Benjamini-Hochberg method. Genes were annotated with biomaRt 2.58.0 using GRCh38.p14 human genome. Gene set enrichment analysis was done using clusterProfiler 4.10.0 with gene sets c2.all.v2023.2.Hs.symbols, h.all.v2023.2.Hs.symbols, and c5.all.v2023.2.Hs.symbols.

first: Huifeng

first: Xinlong


title: Histone modifications of Monocytes in the context of thrombocytopenia

city: Rochester

first: Tyler

last: Stahl

state: NY

address: 601 Elmwood Ave

institute: University of Rochester Medical Center School of Medicine and Dentistry

summary: We sought to define changes in open and closed chromatin in monocytes isolated from control and thrombocytopenic mice

overall_design: Mice were made thrombocytopenic or not, and monocytes isolated for ATAC-seq

first: Craig

last: Morrell

first: Chen


title: The Nociceptive Activity of Peripheral Sensory Neurons is Modulated by the Neuronal Membrane Proteasome

city: Baltimore

first: Eric

last: Villalon

state: Maryland

address: 725 N. Wolfe St

department: Biological Chemistry

institute: Johns Hopkins University

summary: Proteasomes are critical for peripheral nervous system (PNS) function. Here, we investigate mammalian PNS proteasomes and reveal the presence of the neuronal membrane proteasome (NMP). We show that specific inhibition of the NMP on distal nerve fibers innervating the mouse hind paw leads to reduction in mechanical and pain sensitivity. Investigating PNS NMPs we demonstrate their presence on the somata, proximal, and distal axons of a subset of dorsal root ganglion (DRG) neurons. Single-cell RNA sequencing experiments reveal that the NMP expressing DRGs are primarily MrgprA3+ and Cysltr2+. NMP inhibition in DRG cultures leads to cell autonomous and non-cell autonomous changes in Ca2+ signaling induced by KCl depolarization, ab-meATP, or the pruritogen histamine. Taken together, these data support a model whereby NMPs are expressed on a subset of somatosensory DRGs to modulate signaling between neurons of distinct sensory modalities and indicate the NMP as a potential target for controlling pain

overall_design: Primary mouse DRG nuerons isolated from 21 - 30 day old C57Bl/6 mice. All DRGs from 4 mice were combined together for each sample. Antibody feeding on live cells was performed immediately after dissociation of cells to label neurons positive for the NMP. Cells were then FACS sorted to separate into NMP-positive and NMP-negative neurons. The cells were then submitted for single cell RNA-sequencing analyisis.

middle: V

last: Landeros

first: Seth

last: Margolis


title: RNA expression in Monocytes from thrombocytopenic mice

summary: We sought to define changes in gene expression in monocytes in the context of thrombocytopenia

overall_design: Mice were made thrombocytopenic or not and monocytes isolated from the blood for RNA-seq

last: Li


title: Distinctive Transcriptional and Microbial Signature in Cutaneous Acute Graft-vs-Host-Disease

city: Woodbury

first: Ashraf

middle: Sameh

last: Shabaneh

address: 2768 Liberty Trail

department: Institute of Health Informatics

institute: University of Minnesota

summary: Skin acute graft-vs-host disease (aGVHD) is often the first manifestation of GVHD, yet very few preclinical and clinical studies have focused on this target organ, leaving a critical information gap in the pathophysiology of GVHD. We hypothesized that analysis of host gene expression and microbiome profiling could yield novel insights into the molecular and immunologic mechanisms underlying skin GVHD. Our objectives were to determine the differential host gene expression and microbiome profile of human skin aGVHD samples compared to normal skin, and aGVHD corticosteroid responders to non-responders. We performed RNA-Sequencing on lower arm biopsies from 45 patients compared to 10 healthy controls. Our findings suggest a distinctive transcriptional signature of cutaneous aGVHD, that could identify potentially actionable targets for prevention or treatment of corticosteroid refractory disease. Our analysis suggests a key role of dendritic cells and macrophages, potentially mediated by differential expression of MIF, in the development of cutaneous aGVHD and corticosteroid responsiveness. Additionally, we describe a unique microbial signature in cutaneous aGVHD that includes skin microbes not previously described in this population.

overall_design:
Performed RNA-Sequencing (RNA-seq) on 20 µm slices of formalin-fixed paraffin embedded (FFPE) diagnostic lower arm skin biopsies from 45 adult patients with histologic grade 2-2.5 acute GVHD (aGVHD).
Compared these to 10 healthy controls from discarded surgical lower arm skin tissue.
Assessed aGVHD treatment response at day 28 and identified 35 complete or partial responders (CR or PR) and 10 non-responders (NR) based upon their response to systemic immunosuppression with corticosteroids at day 28 of aGVHD therapy.
extracted RNA from the FFPE biopsies using PureLink FFPE RNA Isolation Kit. FFPE samples yielded an adequate quantity of RNA for sequencing (median = 98717524.5 reads, range = 74316820 to 229178960).
After extraction, we prepared the libraries with the SMARTer Stranded Total RNA Kit v2 and performed sequencing on a single lane of a NovaSeq S4 2x150-bp.
Aligned and assembled using hisat2 and Cufflinks. Single gene analysis performed and pathway analysis via Enrichr.
Unaligned reads alignment via Pathseq - microbiome analysis.


title: Notch1 regulates hepatic thrombopoietin production

city: Xuzhou

first: Jianlin

last: Qiao

state: --- Select One ---

address: 84 West Huaihai Road

institute: Xuzhou Medical University

summary: Notch signaling regulates cell-fate decisions in several developmental processes and cell functions. However, a role for Notch in hepatic thrombopoietin (TPO) production remains unclear. We noted thrombocytopenia in mice with hepatic Notch1 deficiency, and so investigated TPO production and other features of platelets in these mice. We found that the liver ultrastructure and hepatocyte function were comparable between control mice and Notch1-deficient mice. However, the Notch1-deficient mice had significantly lower plasma TPO and hepatic TPO mRNA levels, concomitant with lower numbers of platelets and impaired megakaryocyte differentiation and maturation, which were rescued by addition of exogenous TPO. Additionally, JAK2/STAT3 phosphorylation was significantly inhibited in Notch1-deficient hepatocytes, consistent with the RNA-seq analysis. JAK2/STAT3 phosphorylation and TPO production was also impaired in cultured Notch1-deficient hepatocytes after treatment with desialylated platelets. Consistently, hepatocyte-specific Notch1 deletion inhibited JAK2/STAT3 phosphorylation and hepatic TPO production induced by administration of desialylated platelets in vivo. Interestingly, Notch1 deficiency downregulated the expression of HES5 but not HES1. Moreover, desialylated platelets promoted the binding of HES5 to JAK2/STAT3, leading to JAK2/STAT3 phosphorylation and pathway activation in hepatocytes. Hepatocyte Ashwell-Morell receptor (AMR) (asialoglycoprotein receptor 1, ASGR1) physically associates with Notch1 and inhibition of AMR impaired Notch1 signaling activation and hepatic TPO production. Furthermore, blockage of Dll4 on desialylated platelets inhibited hepatocyte Notch1 activation and HES5 expression, JAK2/STAT3 phosphorylation and subsequent TPO production. In conclusion, our study identifies a novel regulatory role of Notch1 in hepatic TPO production, indicating that it might be a target for modulating TPO level.

overall_design: Hepatocyte-specific Notch1 knockout mice were established to evaluate the role of Notch1 in hepatic thrombopoietin production. We then isolated livers from control mice (n=3) and knockout mice (n=3) and performed RNA-seq analysis.

first: Yueyue

last: Sun

first: Huan

last: Tong

first: Xiang

last: Chu


title: IL-6/gp130 signaling in CD4+ T cells drives the pathogenesis of pulmonary hypertension

city: Suita

first: Tadakatsu

last: Inagaki

state: Osaka

address: 6-1, Kishibesinnmachi

department: Vascular Physiology

country: Japan

institute: National Cerebral and Cardiovascular Center

summary: Pulmonary arterial hypertension (PAH) is characterized by stenosis and occlusions of small pulmonary arteries, leading to elevated pulmonary arterial pressure and right heart failure. Although accumulating evidence shows the importance of interleukin (IL)-6 in the pathogenesis of PAH, the target cells of IL-6 are poorly understood. Using mice harboring the floxed allele of gp130, a subunit of IL-6 receptor, we found substantial Cre recombination in all hematopoietic cell lineages from the primitive hematopoietic stem cell level in SM22α-Cre mice. We also revealed that a CD4+ cell-specific gp130 deletion ameliorated the phenotype of hypoxia-induced pulmonary hypertension in mice. Disruption of IL-6 signaling via deletion of gp130 in CD4+ T cells inhibited phosphorylation of signal transducer and activator of transcription 3 (STAT3) and suppressed the hypoxia-induced increase in T helper 17 cells. To further examine the role of IL-6/gp130 signaling in more severe PH models, we developed Il6 knockout (KO) rats using the CRISPR/Cas9 system and showed that IL-6 deficiency could improve the pathophysiology in hypoxia-, monocrotaline-, and Sugen5416/hypoxia (SuHx)-induced rat PH models. Phosphorylation of STAT3 in CD4+ cells was also observed around the vascular lesions in the lungs of SuHx rat model, but not in Il6 KO rats. Blockade of IL-6 signaling had an additive effect on conventional PAH therapeutics, such as endothelin receptor antagonist (macitentan) and soluble guanylyl cyclase stimulator (BAY41-2272). These findings suggest that IL-6/gp130 signaling in CD4+ cells plays a critical role in the pathogenesis of PAH.

sample_organism:
Mus musculus
Rattus norvegicus

platform_organism:
Mus musculus
Rattus norvegicus

overall_design: mRNA profiles of total lung tissue and CD4-positive cells of wild type (WT) and IL6-/- SuHx rats,  and of CD4-positive cells of splenocytes from Ai14;SM22alpha-Cre mice

first: Tomohiko

last: Ishibashi

first: Makoto

last: Okazawa

first: Manabu

last: Shirai

first: Yoshikazu

last: Nakaoka


title: Mitochondrial ACSS1-K-635 acetylation mimic knock-in mice exhibit altered lipid metabolism, hepatic cell senescence, and acute non-alcoholic fatty liver [1stSet_analysis]

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: We generated an ACSS1-acetylation (Ac) mimic mouse, where lysine 635 was mutated to glutamine (K635Q). Male Acss1K635Q/K635Q mice were smaller with higher metabolic rate and blood acetate, and decreased liver/serum ATP and lactate levels. After a 48-hour fast, Acss1K635Q/K635Q mice presented hypothermia and liver aberrations, including enlargement, discoloration, lipid droplet accumulation, and microsteatosis – consistent with nonalcoholic fatty liver disease (NAFLD). RNAseq analysis suggested dysregulation of fatty acid metabolism, cellular senescence, and hepatic steatosis networks, consistent with NAFLD.

overall_design: Fasted and fed C57BL/6J WT and ACSS1K635Q mice were studied in the context of non-alcoholic fatty liver disease. The liver tissue was collected and prepared for RNAseq as well as for Western blotting and qPCR analysis.

first: David

last: Gius

first: Guogang

last: Xu

first: Joseph

last: Schell

first: Nobuo

last: Horikoshi

first: Zhao

last: Lai


title: Neuropilin-1 high monocytes protect against neonatal inflammation

city: Guangzhou

first: Wen

last: Lei

address: Bioisland

institute: Pediatric Immunity and Healthcare Biomedical Co., Ltd.

summary: Neonates are susceptible to inflammatory disorders such as necrotizing enterocolitis (NEC) due to their immature immune system. The timely appearance of regulatory immune cells in early life contributes to the control of inflammation in neonates, the underlying mechanisms of which remain poorly understood. In this study, we identified a subset of neonatal monocytes characterized with high levels of neuropilin-1 (Nrp1), termed Nrp1 high monocytes. Nrp1high monocytes displayed potent immunosuppressive activity as compared with their Nrp1low counterpart. Nrp1 deficiency in myeloid cells aggravated the severity of NEC, whereas adoptive transfer of Nrp1 highmonocytes led to remission of NEC. Mechanistic studies showed that Nrp1, via binding to its ligand Sema4a, induced intracellular p38-MAPK/mTOR signaling and activated the transcription factor KLF4. KLF4 transactivated Nos2 and enhanced the production of nitric oxide (NO), a key mediator of immunosuppression in monocytes. These observations uncover an important immunosuppressive axis in neonatal monocytes and provide a potential therapeutic strategy for inflammatory disorders in neonates.

overall_design: In this study, we investigated whether Nrp1 contributes to the immunosuppression in neonates. By profiling Nrp1 expression in distinct type of immune cells fromneonatal mice, we identified a subset of monocytes with high Nrp1 expression named Nrp1high monocytes. Nrp1high monocytes displayed potent immunosuppressive activity and declined in age-dependent manner. Mechanistic studies showed that upon ligation with its receptor Sema4a, Nrp1 induced intracellular p38-MAPK/mTORC1 signaling and the activation of the transcription factor Klf4. KLF4 transactivated the expression of Nos2 and enhanced the production of NO. Combination of conditional knockout mice and adoptive transfer experiments, we demonstrated that Nrp1high monocytes  protected against inflammatory disease NEC in neonatal mice. These observations provide insights into the immunosuppressive function of neonatal monocytes, which may have therapeutic value in the control of inflammation in early life.

first: Xiaoqing

last: Zheng

first: Jie

last: Zhou


title: Bub1 negatively regulates osteoclast activation through NF-κB suppression in inflammatory arthritis

city: Toon

first: Yuuki

last: Imai

state: Ehime

address: Shituskawa

department: Proteo-Science Center

institute: Ehime University

summary: Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by synovitis and, bone and cartilage destruction. Although disease-modifying anti-rheumatic drugs (DMARDs) have dramatically improved clinical outcomes, these therapies are not universally effective in all patients due to the heterogeneity of RA pathogenesis. Therefore, there is still a need to elucidate molecular mechanisms underlying RA pathogenesis to identify novel therapeutic targets. In this study, we identified Budding uninhibited by benzimidazoles 1 (BUB1) was highly expressed in RA patients’ synovium and murine ankle tissue with arthritis. Since we revealed CD45+CD11b+ myeloid cells as Bub1 highly expressing population among synovial cells, myeloid cell-specific Bub1 conditional knockout (cKO) mice were generated. Obtained cKO mice showed no significant phenotypes in healthy conditions while they exhibited reduced bone mineral density under K/BxN serum-transfer arthritis. Moreover, histomorphometric analysis suggested that the reduced bone mass was caused by increased osteoclast differentiation and activation. RNA-seq and subsequent Gene set enrichment analysis (GSEA) demonstrated significantly enriched NF-κB pathway among up-regulated genes in RANKL-stimulated bone marrow macrophages obtained from Bub1 cKO, suggesting Bub1 KO macrophages are sensitive to inflammatory conditions. Indeed, osteoclastogenesis in Bub1 KO macrophages was enhanced by RANKL and TNFα treatment and localization of NF-κB component p65 was observed. Finally, osteoclastogenesis was increased by Bub1 inhibitor BAY1816032 treatment. These data suggested that BUB1 in myeloid cells plays a protective role against bone loss under inflammatory arthritis.

overall_design: To investigate the functions of Bub1 in myeloid cells, bone marrow-derived macrophages (BMMs) were isolated from Ctrl or myeloid specific Bub1cKO mice. We then induced osteoclast differentiation and performed gene expression profiling analysis using RNA-seq data of BMMs.

first: Shuhei

last: Yoshida


title: Single cell RNA sequencing of healthy human airway epithelia with IL-13 treatment time course

city: Iowa City

first: Alejandro

middle: A.

last: Pezzulo

state: Iowa

address: 6316 Pappajohn Biomedical Discovery Building

department: Department of Internal Medicine

institute: Universty of Iowa

summary: The goal of this study was to examine changes in cell composition and gene expression with IL-13 treatment over time within different cell types from air-liquid interface cultures from healthy primary human airway epithelia.

overall_design: Well differentiated, primary human airway epithelia at the air liquid interface were dissociated and prepared for single cell sequencing via 10X Genomics Chromium controller, and sequenced on Illumina HiSeq4000

first: Andrew

middle: L

last: Thurman


title: Transcriptional profiling of THP-1 monocytes conditioned with Lacticaseibacilli rhamnosus R0011 secretome

city: Oshawa

first: Michael

middle: P

last: Jeffrey

state: Ontario

address: 2000 Simcoe St N

department: Faculty of Science

country: Canada

institute: Ontario Tech University

summary: Macrophage responses to activation are fluid and dynamic in their ability to respond appropriately to challenges, a role integral to host defence. While bacteria can influence macrophage differentiation and polarization into pro-inflammatory and alternatively activated phenotypes through direct interactions, many questions surround indirect communication mechanisms mediated through secretomes derived from gut bacteria, such as lactobacilli. We examined effects of secretome-mediated conditioning on THP-1 human monocytes, focusing on the ability of the Lacticaseibacillus rhamnosus R0011 secretome (LrS) to drive macrophage differentiation and polarization and prime immune responses to subsequent challenge with lipopolysaccharide (LPS). Genome-wide transcriptional profiling revealed increased M2-associated gene transcription in response to LrS conditioning in THP-1 cells. Cytokine and chemokine profiling confirmed these results, indicating increased M2-associated chemokine and cytokine production (IL-1Ra, IL-10). These cells had increased cell-surface marker expression of CD11b, CD86, and CX3CR1, coupled with reduced expression of the M1 macrophage-associated marker CD64. Mitochondrial substrate utilization assays indicated diminished reliance on glycolytic substrates, coupled with increased utilization of citric acid cycle intermediates, characteristics of functional M2 activity. LPS challenge of LrS-conditioned THP-1s revealed heightened responsiveness, indicative of innate immune priming. Resting stage THP-1 macrophages co-conditioned with LrS and retinoic acid also displayed an immunoregulatory phenotype with expression of CD83, CD11c and CD103 and production of regulatory cytokines. Secretome-mediated conditioning of macrophages into an immunoregulatory phenotype is an uncharacterized and potentially important route through which lactic acid bacteria and the gut microbiota may train and shape innate immunity at the gut-mucosal interface.

overall_design: THP-1 human monocytes were challenged with 8 different challenges. Briefly, THP-1 cells were incubated for 24-, 48-, or 72-hrs with either the Lacticaseibacillus rhamnosus R0011 secretome (LrS) (20% v/v) or a matched L-lactic acid control (LA). For some experiments, THP-1 cells were challenged with LPS (125ng/mL) alone for 6 hours or after 72-hrs of conditioning with the LrS. There were 4 biological replicates for each challenge and the response to each challenge was compared to unchallenged controls.

first: Chad

last: MacPherson

first: Lin

last: Saleem

first: Sandra

last: Clarke

first: Thomas

last: Tompkins

first: Julia

last: Green-Johnson


title: Tissue-specific non-heritable influence drive uterine immune system variation (RNA-seq)

city: Huddinge

first: Dan

last: Sun

address: Alfred Nobels allé 8

department: MedH

country: Sweden

institute: Karolinska Institutet

summary: Human twin studies have revealed the combined contribution of heritable and non-heritable (environmental) factors in shaping peripheral blood immune system variability. However, the heterogeneity within tissue immune compartments and the contribution of tissue-specific external factors remain unexplored. The human uterus is a tissue under constant regeneration exposed to distinct environmental factors. To assess uterine immune system variation, we have performed a transcriptomic and epigenetic analysis of uterine and peripheral blood immune cells in monozygotic twins and longitudinal samples in relation to heritable versus non-heritable factors.

overall_design: The objective of this study was to investigate immune cell variation in the human uterus and relate the findings to matched peripheral blood immune cells. In collaboration with the Swedish Twin Registry, peripheral and menstrual blood was collected from monozygotic twin pairs (N=3). In addition, we collected a longitudinal cohort of healthy women (N=3). For the longitudinal cohort, we collected samples from two time points, with the long follow-up being four to six years after the initial sampling. All women included in the longitudinal cohort were of fertile age, healthy, not taking any medications, not using contraceptives, and were nulligravida at the first sampling. All human blood and tissue samples were collected after written and oral informed consent was obtained, and the study was approved by the Regional Ethics Review Board, Stockholm, Sweden.

first: Jonna

last: Bister

first: Iva

last: Filipovic

first: Ylva

last: Crona-Guterstam

first: Martin

last: Cornillet

first: Andrea

last: Ponzetta

first: Jakob

last: Michaëlsson

first: Sebastian

last: Brusell Gidlöf

last: Ivarsson

first: Benedikt

last: Strunz

first: Niklas

last: Björkström


title: Tissue-specific non-heritable influence drive uterine immune system variation

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

overall_design: Refer to individual Series


title: Tissue-specific non-heritable influence drive uterine immune system variation (ATAC-seq)


title: Differential CSF1R signaling enables widespread integration of genetically unmodified human microglia into the mouse brain

status: Public on Apr 11 2024

city: Stanford

first: Yongjin

last: Yoo

address: 265 Campus Drive

department: Institute for Stem Cell Biology and Regenerative Medicine

institute: Stanford University

summary: Microglia are long-lived myeloid cells in the central nervous system that are implicated in many neurological diseases. The differentiation of pluripotent stem cells provides an opportunity to develop in vitro human cellular models carrying disease gene mutations complementing existing animal models of disease. Microglia are particularly sensitive to their cellular environment and can adopt a variety of reactive states depending on different pathological conditions which may be difficult to mimic in vitro. Therefore, to best investigate human microglia in vivo, it would be helpful to generate mice whose endogenous microglia are exchanged with human cells without the need of genetic manipulation of donor cells which could alter microglia function. Colony stimulating factor 1 receptor (CSF1R) signaling is critical for microglial survival in mice, and humans with CSF1R mutations are born with fewer microglia. We made the surprising discovery that transplanted human pluripotent stem cell-derived microglia (hMG) survive pharmacological CSF1R inhibition unlike endogenous mouse microglia. Cellular assays confirmed that CSF1R signaling is necessary for human microglia survival and revealed differential CSF1R signaling with species-specific ligands. Moreover, receptor ligands and small molecule inhibitors acted in a competitive fashion. Based on these insights, we found that transient CSF1R inhibition after cell transplantation led to near-complete and wide-spread repopulation of hMGs into the mouse brain of immunodeficient mice with humanized CSF1 ligand. This approach allows the facile generation of mice whose brain microglia are replaced with genetically unmodified human cells.

overall_design: Xenografted human embryonic stem (hES) cell derived microglia

first: Marius

last: Mader

first: Alexa

last: Scavetti

first: Tamara

last: Chan

first: Takeshi

last: Uenaka

first: Kevin

middle: K

last: Kumar

first: Yohei

last: Shibuya

first: Kelly

last: Mahaney

first: Vivek

last: Buch

first: Gerald

last: Grant

first: Chris

last: Bennett

first: Frank

last: Heppner

last: Wernig


title: The inhibitory effect of EGLN3 on proliferation in gastric cancer cells

city: Tianjin

first: Cai

middle: feng

last: lin

state: China

address: Tianjin Medical University Cancer Institute and Hospital

summary:
Background: Egl-9 family hypoxia inducible factor 3 (EGLN3)  belongs to the family of 2-oxoglutarate (2-OG)- and ferrous iron (Fe 2+)-dependent dioxygenases, which  play critical roles in all steps of tumorigenesis. Previous studies have found that EGLN3 regulates the malignant biological behavior of malignant tumors. However, its role and mechanism in the occurrence and development of gastric cancer (GC) are still unclear. This study aimed to investigate the role of EGLN3 in gastric carcinogenesis
Methods:Bioinformatics analysis, qRT-PCR, western blot, CCK-8, and in vivo xenograft tumor model were used to examine the expression and function of EGLN3.  Survival analysis was performed to determine the correlation between EGLN3 expression and prognosis of patients with GC. Genes modulated by EGLN3 in NCI-N87 cells were identified through RNA next generation sequence screening and further verified by qRT-PCR in  NCI-N87 cells.
Results: EGLN3 expression was markedly reduced in GC tissues as compared with that in normal tissues. Likewise, EGLN3 expression was also significantly reduced in a panel of GC cell lines . Forced expression of EGLN3 inhibited proliferation in NCI-N87 cells in vitro. Additionally, EGLN3 impaired growth of NCI-N87 cells in immunodeficient mice. Survival analysis showed that EGLN3 expression was positively associated with favorable outcome in patients with GC.

overall_design: Conclusions: Collectively, this study unveils the anti-cancer role of EGLN3 in GC, providing a promising diagnostic and therapeutic target.

middle: f

first: Ma

last: gang

first: Deng

middle: j

last: yu


title: ChIP-Seq analysis of H3K4me3 and H3K27ac histone marks without and with METTL8 knockdown

city: Singapore

first: Oleg

last: Grinchuk

address: 61 Biopolis Dr, Proteos

country: Singapore

institute: IMCB

summary: This project investigates the role METTL8 in human glioblastoma stem cells. In order to understand the transcriptomic changes upon deletion of METTL8, we performed this ChIP-seq and further downstream analyses. From this, we hope to identify novel methods to target glioblastoma stem cells via manipulation of METTL8.

overall_design: ChIP-seq analysis of H3K4me3 and H3K27ac histone marks from glioblastoma stem cell specimens (TS-576) following treatment with either non-targeting RNA (shNT) , targeting RNA sh515 or targeting RNA sh516.

first: Bernice

middle: W

last: Li Lee

first: Jeehyun

last: Yoon

first: Derrick

last: Tong Ong


title: A body-brain circuit regulating body inflammatory responses

city: New York

first: Hao

last: Jin

state: New York

address: 3227 Broadway

institute: Columbia University

summary: The body-brain axis is emerging as a principal conductor of organismal physiology.  It senses and controls organ function, metabolism and nutritional state.  Here, we show that a peripheral immune insult powerfully activates the body-brain axis to regulate immune responses.  We demonstrate that pro- and anti-inflammatory cytokines communicate with distinct populations of vagal neurons to inform the brain of an emerging inflammatory response.  In turn, the brain tightly modulates the course of the peripheral immune response.  Genetic silencing of this body-to-brain circuit produced unregulated and out-of-control inflammatory responses.  By contrast, activating, rather than silencing, this circuit affords exceptional neural control of immune responses.  We used single-cell RNA sequencing, combined with functional imaging, to identify the circuit components of this neuro-immune axis, and showed that its selective manipulation can effectively suppress the pro-inflammatory response while enhancing an anti-inflammatory state.  The brain-evoked transformation of the course of an immune response offers new possibilities in the modulation of a wide range of immune disorders, from autoimmune diseases to cytokine storm and shock.

overall_design: cells from the nucleus of the solitary tract were isolated by enzymatic digestion and fluorescence-activated cell sorting based on the presence or absence of tdTom reporter expression and analyzed using scRNA-seq

first: Koral

last: Goltseker

first: Charles

last: Zuker


city: Jinan

first: Guiping

last: Wang

address: 202 Gongye North Road, Licheng District, Jinan City, Shandong Province

institute: Shandong Academy of Agricultural Sciences

summary: This study aims to investigate genes regulated by Water Immersion

overall_design: Water immersion significantly improved decocooning of Osmia excavata. Transcriptome Analysis Provides Insights into Water Immersion Promoting the Decocooning of Osmia excavata Alfken

first: Guangzhao

first: Jiale

last: Li

first: Yixiang

last: Ma

first: Yinwei

last: You

first: Zizhang

last: Zhou

first: Yunhe

last: Zhao

first: Xingyuan

last: Men

first: Yingying

last: Song

first: Yi

last: Yu


title: The Single-Cell Landscape of Spermioteleosis in Mouse and Pig

city: Qingdao

first: JunLin

address: Changcheng Road

institute: JunLin Song

summary: Spermatozoa acquired motility and matured in epididymis after production in testis; however, the characteristics of sperm development between species remain poorly understood. In this study, we constructed a single-cell RNA dataset to investigate spermatogenesis characteristics between mouse and pig. This approach enabled us to analyze cell compositions in both testicular and epididymal tissues, providing insights into changes occurring during meiosis and spermiogenesis in mouse and pig. Additionally, distinct gene expression signatures associated with various spermatogenic cell types were identified. The findings revealed differences in testicular cell clusters between these two species. Furthermore, different regions of the epididymis displayed unique gene expression patterns among its epithelial cells. Notably, regional gene expression patterns were observed within principal cell clusters of mouse epididymis as well. Moreover, we analyzed specific characteristics of differentially expressed genes related to the epididymis in mouse and pig, identifying potential marker genes associated with sperm development and maturation for each species studied.

sample_organism:
Sus scrofa
Mus musculus

platform_organism:
Sus scrofa
Mus musculus

overall_design: Testicular and epididymal tissues were collected from three sexually mature male C57BL/6J mice and three sexually mature Landrace pigs for single cell sequencing to compare the differences in testicular spermatogonesis and epididymal maturation between these two species


title: Single-cell profiling reveals transcriptome dynamics during bovine oocyte growth

city: Celbridge

first: Lais

last: Barbosa Latorraca

state: Kildare

address: UCD Lyons Farm

department: Agriculture and Food Science

country: Ireland

institute: University College Dublin

summary: Mammalian follicle development is characterised by extensive changes in morphology, endocrine responsiveness, and function, providing the optimum environment for oocyte growth, development, and resumption of meiosis. In cattle, the first signs of transcription activation in the oocyte are observed in the secondary follicle, later than during mouse and human oogenesis. While many studies have generated extensive datasets characterising gene expression in bovine oocytes, they are mostly limited to the analysis of fully grown and matured oocytes. The aim of the present study was to apply single-cell RNA sequencing to interrogate the transcriptome of the growing bovine oocyte from the secondary follicle stage through to the mid-antral follicle stage. Single-cell RNA-seq libraries were generated from oocytes of known diameters (<60 to >120 um), and datasets were binned into non-overlapping size groups for downstream analysis. Combining the results of weighted gene co-expression network and Trendy analyses, and differently expressed genes (DEGs) between size groups, we identified a decrease in oxidative phosphorylation and an increase in maternal -genes and transcription regulators across the bovine oocyte growth phase. In addition, around 5,000 genes did not change in expression, revealing a cohort of stable genes. An interesting switch in gene expression profile was noted in oocytes greater than 100 um in diameter, when the expression of genes related to cytoplasmic activities was replaced by genes related to nuclear activities (e.g., chromosome segregation). The highest number of DEGs were detected in the comparison of oocytes 100-109 versus 110-119 um in diameter, revealing a profound change in the molecular profile of oocytes at the end of their growth phase. The current study provides a unique dataset of the key genes and pathways characteristic of each stage of oocyte development, contributing an important resource for a greater understanding of bovine oogenesis.

overall_design: Bovine (Bos taurus) ovaries were collected at a local abattoir and returned to the laboratory in cold PBS. Fully-grown oocytes (>130 µm diameter) were removed first by aspirating 3-8 mm surface visible follicles. Subsequently, the ovaries were butterflied open, submerged in cold Dissection medium (TCM199 supplemented with 0.4% BSA fraction V, 0.164 mM penicillin, 0.048 mM streptomycin, 1,790 units/L heparin, and 5 μM cilostamide (Barros et al., 2020)), and growing oocytes from late secondary to early antral follicles were liberated by slicing the ovarian cortex. Subsequently, the medium containing the oocytes was filtered through a descending series of mesh filters, including 260 mm mesh, 100 and 40 mm PluriStrainer® filters. Recovered oocytes were washed in cold Dissection medium, denuded by pipetting, and allocated singly to drops of cold PBS, where their diameter was measured under a Nikon Eclipse TE2000-S microscope and NIS Elements BR 5.02.00 64-bit software. Measured oocytes were snap-frozen in 4 mL PBS + 4 mL RTL-Plus buffer (Qiagen) in 0.2 mL PCR microtubes, and kept at -80°C until processed from RNA sequencing.

first: Antônio

last: Galvão

first: Maria Belem

last: Rabaglino

first: Julieta

middle: M

last: D'augero

first: Gavin

last: Kelsey

first: Trudee

last: Fair


title: Gene perturbation in human arterial smooth muscle cells under treatments promoting synthetic or contractile phenotype

city: Kuopio

first: Tiit

last: Örd

address: Neulaniementie 2

country: Finland

institute: A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland

summary: Smooth muscle cell (SMC) gene regulatory networks are thought to play a role in atherosclerosis, a disease characterized by alterations to SMC phenotype. Based on analysis of multiple bulk and single-cell RNA-Seq atherosclerosis data sets, FRZB and ALCAM were prioritized as candidate regulators of a disease-associated gene expression program in SMC-s. The current RNA-Seq experiment was devised to study the contribution of these genes to SMC transcriptional phenotype under different treatment conditions.

overall_design: RNA-Seq was used to profile gene expression in cultured human coronary artery smooth muscle cells. The cells were transduced with lentivirus overexpressing FRZB or transfected with siRNA silencing ALCAM to study the effect of these genes. Empty vector lentivirus and negative control siRNA were used as controls, respectively. Cells were treated with TGFbeta or cholesterol (or mock-treated for no treatment control) to induce atherosclerosis-relevant cellular phenotypes.

first: Prosanta

last: Singha

first: Uma

middle: T

last: Arasu

first: Minna

middle: U

last: Kaikkonen


title: Gene expression profile in the pons-medulla (brainstem) of male mice during Chronic Intermittent Hypoxia and Normoxia

city: Seattle

first: Hemalatha

last: Bhagavan

state: WA

address: 1900 9th Ave

department: Center for Integrative Brain Research

institute: Seattle Childrens Hospital

summary: The goal of this project is to gain first insights into the impact of CIH on three major cell classes of the brainstem - excitatory neurons, inhibitory neurons, and oligodendrocytes.

overall_design: C57BL6/J male mice were weaned at PND21 and exposed to CIH or maintained at room air from PND22 to PND43. The CIH group was subjected to intermittent episodes of hypoxia. Firstly, nitrogen (N2) was continuously injected into the chamber for 60 seconds, reducing the percentage of inspired oxygen (O2) within the chamber to a range of 4.5 to 5%. Subsequently, compressed air was injected for up to 5 minutes to restore the percentage of O2 to 21% before the initiation of a new hypoxia cycle. This protocol was repeated 80 times per day over 8 hours for 21 days. Mice were euthanized and the brainstem was frozen. Single nuclei suspensions were prepared for each of two groups: CIH and Normoxia. The Chromium Next GEM Single Cell 3’ Kit v3.1 (10x Genomics, Pleasanton, CA) was used to encapsulate single nuclei, cDNA preparation, and library construction according to the manufacturer’s protocol.

first: Aguan

middle: D

last: Wei

first: Luiz

last: Oliveira

first: Kimberly

middle: A

last: Aldinger

first: Jan-Marino

last: Ramirez


title: Nutrient-Delivery and Metabolism Reactivation Therapy for Melanoma

city: shanghai

first: yang

last: chen

address: siping road

institute: tongji university

summary: To fulfil the demands of rapid proliferation, tumour cells, including those in melanoma, undergo significant metabolic alterations, with some pathways being upregulated while others are downregulated. Suppression of hyperactivated metabolism has been proven to counteract tumour growth. However, whether the reactivation of inversely downregulated metabolic pathways has therapeutic effects remains unexplored. Here, we report a nutrient-based metabolic reactivation strategy for effective melanoma treatment. Briefly, L-tyrosine nanomicelles (MTyr-OANPs) were constructed for targeted supplementation of tyrosine to reactivate melanogenesis in melanoma cells. We found that reactivation of melanogenesis using MTyr-OANPs significantly impedes the proliferation of melanoma cells, primaryly through the inhibition of glycolysis. We discovered that reduced glycolysis was a consequence of pyruvate kinase activity inhibition by melanin intermediates indole-5,6-quinone. Furthermore, leaveraging melanin as a natural photothermal reagent for photothermal therapy (PTT), we demonstrated the complete eradication of tumours in B16F10 melanoma-bearing mice through treatment with MTyr-OANPs and PTT. To the best of our knowledge, this is the first study of metabolism activation-based tumour treatment, suggesting specific nutrients as potent activators of metabolic pathways.

overall_design: The B16F10 cells were treated with MTy-OANPs for 48hrs and the total RNA were collected for analysis. The untreated cells were used as controls. (n=3 biological repicates)

first: Yang

last: Chen

first: Chaochao

last: Wang

first: Yelin

last: Wu


title: Single-cell 5-formylcytosine Landscapes of Mammalian Early Embryos at Single-base Resolution

city: Beijing

first: Jiansen

last: Lu

address: No. 5 Yiheyuan Road, Haidian District

department: Biomedical Pioneering Innovation Center (BIOPIC)

institute: Peking University

summary: Active DNA demethylation in mammals involves Ten-eleven translocation (TET) family proteins-mediated oxidation of 5-methylcytosine (5mC). However, base-resolution landscapes of 5-formylcytosine (5fC, an oxidized derivative of 5mC) at the single-cell level remains unexplored. Here we present “CLEVER-seq”, which is a single-cell, single-base resolution 5fC sequencing technology, based on biocompatible, selective chemical labeling of 5fC and subsequent C-to-T conversion during amplification and sequencing. CLEVER-seq of mouse early embryos reveals the highly patterned genomic distribution and parental specific dynamics of 5fC during mouse early pre-implantation development and synergistic 5fC production in paternal and maternal genomes. Integrated analysis demonstrates that promoter 5fC production precedes the expression upregulation of a clear set of developmentally and metabolically critical genes. Collectively, our work reveals the dynamics of active DNA demethylation during early mouse pre-implantation development and provides an important resource for further functional studies of epigenetic reprogramming in single cells.

overall_design: We developed a novel single-cell 5fC sequencing technique, termed “CLEVER-seq” (chemical labeling enabled C-to-T conversion sequencing), to map the genome-wide base resolution 5fC landscapes in mouse early embryos at single-cell level. We applied CLEVER-seq to mESCs (2i), mouse sperm, metaphase II oocytes, zygotes, 2-cell embryos, 4-cell embryos, inner cell mass (ICM) and trophectoderm (TE). The male and female pronuclei within each individual PN3-PN4 stage zygote were physically isolated by micromanipulation; the individual blasotomeres of 2-cell and 4-cell embryos were also separated. In addition to mESCs (2i), we also performed CLEVER-seq to mESCs (serum), mEpiSCs, and hESCs. In total, we sequenced more than 1,500 GB data for 139 single cells, and identified 3,216-41,347 5fCpG sites from these samples.

first: Chenxu

last: Zhu

first: Yun

last: Gao

first: Hongshan

last: Guo

first: Bo

last: Xia

first: Fuchou

last: Tang

first: Chengqi

last: Yi


title: An interplay between extracellular matrix proteins and the cancer cell promotes the multistep process of in vitro vasculogenic mimicry formation.

city: Santiago

first: Gareth

last: Owen

state: Region Metropolitana

address: Avenida Libertador Bernardo O'Higgins 340

department: Department of Physiology

country: Chile

institute: Pontificia Universidad Catolica De Chile

summary: Cancer vasculogenic mimicry (VM) is the formation of vasculature structures in the absence of endothelial cells. We previously established an in vitro model that facilitates the formation of a lumen-containing and fluid-conducting tubular structures after 4 days cancer cell growth on Matrigel. Herein, we mechanistically characterize this model in breast and ovarian cancer cell lines demonstrating distinct phases of VM formation and the dependence of specific extracellular matrix proteins. We report that VM occurs in four distinct stages. Firstly, alignment, migration then clustering delineate the area of the future tubular structure. Secondly, contraction of aligned structures followed by loss of attachment of some cells and cellular blebbing. Thirdly, a phase of mass proliferation followed by the raising of specific areas of the cancer cell mass above the Matrigel (bridge). Finally, the formation of a cell monolayer closes the tubular structure, forms a glycoprotein-rich luminal lining, then elevates the structure. Only later stages of VM require AKT and FAK signaling. We demonstrate that the lining of the tubular lumen is rich in laminin. Laminin 111 (but not collagen I) must be presence in the extracellular matrix (Matrigel) for VM to occur and integrin β1, but not integrin β3, is required. RNASeq demonstrates that formation is principally post-transcriptional regulation, however, this technique together with siRNA knockdown identified the first and essential biological function for the protein SMIM11A. VM is associated with poor patient survival and thus an understanding of the mechanism of VM may bring to light novel anticancer targets.

overall_design: To understand if they were changes on the levels of transcripts during vasculogenic mimicry formation, RNA was obtained from HEY-A8 cell lines that were seeded on either Matrigel, which allows the formation of vasculogenic mimicry, or on denatured Matrigel, which only forms a monolayer of cells. RNA samples were obtained at different timepoints of formation: 2, 8, 24 and 48 hours after the cells were seeded on either of both conditions.

first: Gabriel

last: Mingo

first: Andres

last: Valdivia

first: Varina

last: Aldana


title: Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens in a dual fluorescence readthrough reporter (2)

city: Toronto

first: Jack

last: Greenblatt

address: 160 College St

department: Donnelly Center

institute: University of Toronto

summary: The majority of messenger RNA precursor (pre-mRNA) undergoes 3' end cleavage and polyadenylation (CPA), termed as to 3’ end processing. This processing is specified by a polyadenylation site (PAS) and adjacent RNA sequences and regulated by a large variety of core and auxiliary CPA factors. To date, most of the CPA factors have been discovered through biochemical and proteomic studies. However, genome-wide genetic identification of CPA factors has been hampered by the lack of a reliable high-throughput method. Here, we developed a dual fluorescence readthrough reporter system with a PAS inserted between two fluorescent reporters, GFP and mCherry. This system enables the measurement of 3’ end processing in living cells via flow cytometry analysis. Using this system in combination with a human CRISPR library, we conducted a genome-wide screen for CPA factors. The screens identified most of the well-known CPA factors, including most components of the core CPA machineries (i.e. CPSF, CSTF, CFI, and CFII complexes), the CPA scaffold protein SYMPK, PPP1R10 in PNUTS-PP1 complex, as well as CBLL1 (a subunit of m6A RNA methyltransferase complex). Importantly, the screens also uncovered CCNK/CDK12 as a functional core CPA machinery. Furthermore, the screens identified RPRD1B, an RNA polymerase II (RNAP II) binding protein, as a CPA factor. Further characterization showed that RPRD1B binds RNA and regulates the release of RNAP II at the 3’ end of genes. Thus, this dual fluorescence reporter coupled with CRISPR screen provides a reliable tool for identifying bona fide CAP factors, offering a platform for investigating 3’ end processing in various contexts.

overall_design: RNA polymerase II (RNAP II) ChIP-seq data of RPRD1B knockout and Scramble control, as well as overexpression of GFP-tagged RPRD1B and GFP control in HEK293 cells (Treated with 1µg/ml doxycycline for 4 days), each with 2 biological replicates and input sample.

first: Zuyao

last: Ni

first: Nujhat

last: Ahmed

first: Syed

last: Nabeel-Shah

first: Xinghua

first: Shuye

last: Pu

first: Jingwen

last: Song

first: Edyta

last: Macron

first: Giovanni

last: Burke

first: Amy

middle: H

last: Yan Tong

first: Katherine

last: Chan

first: Kevin

middle: C

last: Ha

first: Benjamin

middle: J

last: Blencowe

first: Jason

last: Moffat

middle: F


title: T cell-derived IL-22 directs mature colonocyte fate and upregulates host defense genes during Citrobacter rodentium infection.

city: Birmingham

first: Min

last: Gao

state: AL

address: 1900 University Blvd, THT Building Suite #130G

institute: University of Alabama at Birmingham

summary: We identified two distinct subsets of absorptive epithelial cells in the mid-distal colons of mice that are regionally divided and differentially respond to IL-22 signals.

overall_design: Cells from the epithelial layer of mid-distal colons of naïve BL/6, d9 C.r-infected Il22/hCD4 floxed (control), and d9 C.r-infected Il22/hCD4.CD4/Cre (T cell-specific deficiency of IL-22; T cKO) mice were isolated with DTT, followd by EDTA, and then sorted on all live cells (L/D dye-).

first: Carlene

last: Zindl

first: David

last: Figge

first: Robin

last: Hatton

first: Casey

last: Weaver


title: Transcriptomics coupled proteomics reveals the critical signaling pathways regulating OVA antigen-specific CD8+ T cells in mouse spleen and liver

status: Public on Apr 10 2024

city: Chongqing

first: Qihuan

last: Fu

state: Chongqing

address: 181 Hanyu Road, Shapingba District

department: Chongqing University Cancer Hospital

institute: Chongqing University

summary: Background: Antigen-specific T cells are particularly important for eliminating bacterial infection, and the dynamics in this process help uncover the mechanisms by which bacteria are eliminated.Results: In this study, we sorted the antigen-specific CD8+ T cell in the mouse liver and spleen at 5, 7, and 14 days after infection of Listeria monocytogenes expressing the OVA protein, finding that antigen-specific CD8+ T cell expansion after infection peaked on Day 7. Through the RNAseq analysis, we identified 355 genes whose expression peaked on 7 days post-infection shared in both of spleen and liver. For these shared genes, the KEGG pathway analysis showed a notable enrichment in the metabolic pathway. We also discovered 1621 proteins shared in the mouse liver and spleen that peaked seven days after infection by the LC-MS/MS analysis. The KEGG pathway analysis for these proteins revealed a significant enrichment in Splicesome, RNA transport, Ubiquitin mediated proteolysis, and Huntington disease. Transcriptomics coupled proteomics reveals 45 genes/proteins that were elevated on day 7 post listeria infection both in the spleen and liver. These 45 molecules were enriched in Human T-cell leukemia virus 1 infection and T cell activation, which played an important role in the activation of T cells and the anti-infection process.Conclusion: Transcriptomics coupled proteomics reveals the key signaling pathways to regulate antigen-specific CD8+ T cells by infection of Listeria monocytogenes expressing the OVA protein in mouse spleen and liver.

overall_design: This study used MHC class I tetramers to recognize the antigen OVA antigen-specific CD8+ T cells in mouse spleen and liver. Then combined the RNAseq and LC-MS/MS analysis to track the changes of the OVA antigen-specific CD8+ T cells at different time points (5 days,7 days, and 14 days post listeria infection).

first: Fang

last: Liu

first: Ying

last: Wan

first: Jiangbei

last: Yuan


title: Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens in a dual fluorescence readthrough reporter

first: Jack

last: Greenblatt


title: The electrophysiological functions of macrophages are involved in the development of atrial fibrillation

city: Hangzhou

first: Wenjing

last: Chen

address: No.79 Qingchun Road

institute: Zhejiang University

summary: Background: Inflammation is closely related to atrial fibrillation (AF), in which macrophages play an important role as immune cells. Recent studies have shown that macrophages participate in the electron conduction in the heart, indicating that they have electrophysiological characteristics. However, whether the electrophysiology of macrophages is associated with AF remained unclear.  Methods: In the present study, we investigated the electrophysiological changes in macrophages using patch-clamping after tachypacing to mimic AF. RNA-seq was performed to investigate the expression change of atrial myocytes after tachypacing. Rapid atrial stimulation was performed to measure the AF incidence in macrophages-specific CX43 knockout mice. Results: We found that the APD90 and the ICa,L were reduced. Further, we found that tachypacing atrial myocytes led to the secretion of Wnt 7a, further inhibiting the expression of CACNA1C in macrophages. Moreover, the knockout of CX43 in macrophages decreased the incidence of AF in a chronic inflammation mice model.  Conclusion: Altogether, these results demonstrated that the electrophysiology of macrophages was related to the development of AF and might be a potential therapeutic target for AF.

first: Jieqiong

last: Wang

first: Zewei

last: Sun


title: Single cell RNA sequencing profile of unique mesenchymal progenitor subsets from the murine brain

city: Vancouver

first: Tully

middle: Michael

last: Underhill

state: British Columbia

address: 2222 Health Sciences Mall

department: Biomedical Research Centre

institute: University of British Columbia

summary: The sensitive central nervous system is protected from potentially toxic components in the circulation by a semi-permeable interface, the blood brain barrier (BBB). This barrier is tightly regulated by several stromal cell types, however in certain regions of the brain it requires distinct properties. In particular, two of these regions, the choroid plexus and the pituitary gland are considered differentially permeable compared to other regions. Perivascular mesenchymal progenitors are intimately associated with endothelium and are therefore key participants in the regulation of the BBB. Using a mesenchymal progenitor specific lineage tracing tool, gene expression in this population from the choroid plexus and pituitary gland was profiled at the single cell level to investigate the genetic programs that allow these regions to maintain their respective specific pervious properties.

overall_design: Single cell RNA-seq analyes of mesenchymal progenitors from the choroid plexus and pituitary gland. A lower proportion of other nucleated cell types were included for comparison.

first: W

last: Bastedo

first: R

last: Scott

first: T


title: A hominoid-specific signaling axis setting the tempo of synaptic maturation II

first: Dong

last: Jian

address: 393 Middle Huaxia  Road

institute: shanghaitech university

summary: Compared to that of other mammals, human cortical neurons exhibit higher dendritic complexity and synaptic density, and the maturation process is much protracted. However, the molecular mechanism governing these specific features is unclear. Here, we report that the hominoid-specific gene TBC1D3, a core duplicon in the human genome, promotes dendritic arborization and maintains the slow pace of synaptogenesis. Furthermore, to gain insights into the mechanism by which the TBC1D3-MICAL1-ATRX complex determines the slow pace of spinogenesis, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) experiment using an ATRX antibody targeting the C-terminal region in cultured mouse cortical neurons.

overall_design: Contol neurons (DIV14) and MICAL-FL neurons (DIV14)  were  conducted ChIP-seq experiment using an ATRX antibody.

first: Jian

last: Dong

first: Zhen-ge

last: Luo


title: Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens in a dual fluorescence readthrough reporter (1)

overall_design: Flp-In T-Rex HEK293 cells were infected with the pooled lentiviral genome-wide TKOv3 gRNA library at a MOI of 0.3, and subjected to Fluorescence-activated cell sorting (FACS). The gRNA in mCherry/GFP ratio increased cells was compared to that of unsorted cells each with 4 biological replicates.

first: Zuyao

last: Ni

first: Nujhat

last: Ahmed

first: Syed

last: Nabeel-Shah

first: Xinghua

last: Guo

first: Shuye

last: Pu

first: Jingwen

last: Song

first: Edyta

last: Macron

first: Giovanni

middle: L

last: Burke

first: Amy

middle: Y

last: Tong

first: Katherine

last: Chan

first: Kevin

last: Ha

first: Benjamin

last: Blencowe

first: Jason

last: Moffat


title: Hysteresis of IL-10R primes solid tumour microenvironment for bacterial immunotherapy

city: Shang Hai

first: Yan

last: Wang

address: Yue yang Road 320

institute: Chinese Academy of Science

summary: Hysteresis, a ubiquitous phenomenon in nonlinear systems, plays a vital role in diverse scientific disciplines. Here, we demonstrated the presence of hysteresis in the expression of interleukin-10 receptor (IL-10R) within the tumour microenvironment (TME) during bacterial immunotherapy. The engineered Salmonella enterica, administered intravenously, exhibited remarkable efficacy in multiple tumour types, effectively eliminating tumours and preventing recurrence and metastasis. We found that the hysteretic response of IL-10R can prime TME for bacterial homing and reinvigorate intratumoural CD8+ T cells. Furthermore, the differential IL-10R levels observed in different human tumour types suggest a potential correlation with clinical outcomes. Overall, our study not only uncovers the central role of hysteresis in driving the therapeutic effects of bacterial immunotherapy, but also provides a framework for intratumoural immunomodulation in solid tumour treatment.

overall_design: The intratumoural CD8+ T cells were sorted from B16 melanoma-bearing mice after treatment with PBS or DB1, followed by total RNA extraction and bulk RNA-seq.

first: Yichuan

last: Xiao

first: Chenli

last: Liu

first: Zhiguang

last: Chang


title: Chemogenomic screening of acute myeloid leukemia samples

city: Montreal

first: Brian

last: Wilhelm

state: Quebec

address: 2950 Chem. de Polytechnique

department: Institut de recherche en immunologie et en cancérologie

institute: Université de Montréal

summary: Acute myeloid leukemia (AML) represents 20% of pediatric leukemia, but accounts for most of disease-related mortality in children, due to chemo-resistance or relapse. Treatment modalities tailored according to specific AML genetic subgroups are critically needed to improve survival and decrease morbidity. To address this need we report here the results of a small molecule screen that was performed using 12,000 compounds using human leukemia models, matched patient diagnostic and relapse samples, and cell lines. As part of this work, expression profiling was performed in order to correlate drug response to gene expression differences between samples.

overall_design: Expression profiling of AML samples from patients, established cell lines, and synthetic models

first: Safia

last: Safa-Tahar-Henni

first: Verena

last: Greß

first: Karla

last: Paez Martinez

first: Élodie

last: Roques

first: Mélanie

last: Bilodeau

first: Kolja

last: Eppert

first: Sonia

last: Cellot

first: Frédéric

last: Barabé


title: Systemic comparison of molecular characteristics in different skin fibroblast senescent models

city: Nanjing

first: Xiaokai

last: Fang

state: Jiangsu

address: No.12, Jiangwangmiao Street, Xuanwu District, Nanjing City, Jiangsu Province

institute: Dermatology Hospital of Chinese Academy of Medical Sciences

summary: Each senescent FB model exhibited different characteristics. Besides the upregulated expression of natural senescence features, FB-UVB and FB-ATV expressed high levels of senescence-related genes including SASP, and FB-P30 had the greatest similarity with FB-E. However, D-galactose-stimulated FB did not clearly present aging characteristics. It provides references for choosing senescent FB model in studying aging and related skin disease.

overall_design: To explore and compare their signatures among senescence-induced FB models, human skin primary FBs were obtained from children (FB-C) and elderly healthy individuals (FB-E). Further senescent models were induced by ultraviolet B irradiation (FB-UVB), D-galactose stimulation(FB-D-gal), atazanavir treatment(FB-ATV), and replication exhaustion induction (FB-P30), respectively.