title: Nuclear Receptor Corepressors Non-Canonically Drive Glucocorticoid Receptor-Dependent Activation of Hepatic Gluconeogenesis [RNA-Seq]

overall_design: Comparative RNAseq expression analysis of hepatocyte-specific knockout models of NCoR complex members compared to their floxed controls.


title: Spatial transcriptome analysis unveils an innate immune activation signature of acute T cell-mediated rejection in kidney transplants

status: Public on Feb 16 2024

city: Seoul

first: Minji

last: Kang

address: 103 daehak-ro

department: biomedical sciences

country: South Korea

institute: Seoul National University graduate school

summary: To unveil the molecular mechanisms underlying TCMR, we utilized spatial transcriptomics on post-transplant TCMR and non-TCMR patients. Contrary to the prevailing T cell-centric view, our findings reveal uniform inflammatory responses across glomerular and tubulointerstitial regions in TCMR.

overall_design: Kidney biopsy samples

first: Haeyoon

last: Kwon

first: Yong

middle: c

last: Kim

first: Hyun

middle: J


title: Functional activity scores of a DMS library representing coding single residue substitution variants in the transcription factor CRX measured in an engineered reporter cell line

status: Public on Mar 21 2024

city: SAINT LOUIS

first: Barak

middle: Alon

last: Cohen

state: MO

address: 4523 CLAYTON AVE CB #8510

department: Genetics

country: USA

institute: Washington University School of Medicine

summary: Cone-Rod Homeobox, encoded by CRX, is a transcription factor (TF) essential for the terminal differentiation and maintenance of mammalian photoreceptors. Although a handful of human variants in CRX have been shown to cause several different degenerative retinopathies with varying cone and rod predominance, as with most human disease genes the vast majority of observed CRX genetic variants are uncharacterized variants of uncertain significance (VUS). We performed a deep mutational scan (DMS) of nearly all possible single amino acid substitution variants in CRX, using an engineered cell-based transcriptional reporter assay. We measured the ability of each CRX missense variant to transactivate a synthetic fluorescent reporter construct in a pooled fluorescence-activated cell sorting assay and compared the activation strength of each variant to that of wild-type CRX to compute an activity score, identifying thousands of variants with altered transcriptional activity.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: A mutational scanning library was constructed containing all single residue substitution variants of human CRX, each marked by a unique sequence barcode (vBC) and a number of random barcodes (rBC). The library was integrated into a genomic landing pad in a HEK 293-derived cell line engineered to carry a CRX-responsive fluorescent transcriptional reporter. After selecting for successful integrants, cells were sorted into one of four bins based on fluorescence intensity (labeled A–D, lowest to highest fluorescence). Amplicon sequencing libraries were prepared from the sorted cells, and the frequence of each vBC was counted.

first: James

middle: L

last: Shepherdson

first: David

middle: M

last: Granas

first: Jie

last: Li

first: Zara

last: Shariff

first: Stephen

middle: P

last: Plassmeyer

first: Alex

middle: S

last: Holehouse

first: Michael

middle: A

last: White


title: The Peripheral Atf3+ Neuronal Population is Responsible for Nerve Regeneration at Early Stage of Nerve Injury Revealed by Single-Cell RNA Sequencing

status: Public on Feb 09 2024

city: Shanghai

first: Li

last: Liu

state: Please select...

address: Shanghai Pudong New Area Renji Hospital

country: China

institute: shanghai jiaotong university

type: Expression profiling by high throughput sequencing

summary: Peripheral nerve injury (PNI) could transform primary somatosensory neurons into regenerative state. However, details of transcriptomic changes associated to nerve regeneration of somatosensory neurons maintain unclear. 10x single-cell RNA sequencing (scRNA-seq) was conducted on mice dorsal root ganglia (DRG) cells after early stage of nerve injury on day 3 post-constriction chronic injury (CCI). We observed that a novel CCI-induced neuronal population (CIP) emerged and expressed high levels of activating transcription factor (Atf3), a neuronal injury marker. CIP neurons highly expressed regenerative associated genes (RAGs) and were enriched in regeneration-related gene ontology (GO) terms, suggesting these neurons could be a pro-regenerative population. Moreover, intercellular communication networks showed that CIP neurons have a close communication to satellite glia cells (SGCs) and specifically transmitted strong Fgf3-Fgfr1 signaling to SGCs, which could initiate regeneration-associated transcriptional changes of SGCs. This study also confirmed that the regenerative progress occurred at the early stage of nerve injury because immunohistochemistry showed the expression of ATF3 significantly increased since day 3 post-CCI and decreased at 1 month. Our bioinformatics analysis at single-cell resolution advances the knowledge of regenerative dynamic transcriptional changes of DRG cells after injuries and their underlying molecular mechanisms.

overall_design: High-throughput analyses the lumbar4-5 dorsal root ganglia(DRG) from chronic construction injury(CCI) mice and naïve mice to explore genes and targets associated with nerve regeneration in the context of CCI. There are  4 samples, two from the naïve group and two from CCI group.

first: Junhui

last: Chen

first: Wen

last: Yin

first: Po

last: Gao

first: Yinghui

last: Fan

first: Daxiang

last: Wen

first: Yingfu

last: Jiao

first: Weifeng

last: Yu


title: Malassezia responds to environmental pH signals through the conserved Rim/Pal pathway

status: Public on Feb 01 2024

city: Durham

first: J Andrew

last: Alspaugh

state: NC

address: 337 Sands Research Building, 303 Research Drive, DUMC 102359

institute: Duke University

summary: During mammalian colonization and infection, microorganisms must be able to rapidly sense and adapt to changing environmental conditions including alterations in extracellular pH. The fungus-specific Rim/Pal signaling pathway is one process that supports microbial adaptation to alkaline pH. This cascading series of interacting proteins terminates in the proteolytic activation of the highly conserved Rim101/PacC protein, a transcription factor that mediates microbial responses that favor survival in neutral/alkaline pH growth conditions, including many mammalian tissues. We identified the putative Rim pathway proteins Rim101 and Rra1 in the human skin colonizing fungus Malassezia sympodialis. Targeted mutation of these proteins confirmed their role in M. sympodialis growth at higher pH. Additionally, comparative transcriptional analysis of the mutant strains compared to wild-type suggested mechanisms for fungal adaptation to alkaline conditions. These signaling proteins are required for optimal growth in a murine model of atopic dermatitis, a pathological condition associated with increased skin pH. Together these data elucidate both conserved and phylum-specific features of microbial adaptation to extracellular stresses.

overall_design: Comparing the transcriptome of three different strains of ATCC 42132 Malassezia sympodialis (WT, rim101∆, and rra1∆) in DMEM, pH 4, and pH 7.5 conditions. A total of 3 replicates were performed for each strain in each condition, for a total of 27 samples.

first: Kaila

last: Pianalto

first: Calla

last: Telzrow

first: Hannah

last: Brown Harding

first: Jacob

middle: T

last: Brooks

first: Eduardo

last: Gushiken-Ibañez

first: Salomé

last: LeibundGut-Landmann

first: Joseph

last: Heitman

first: Giuseppe

last: Ianiri

first: J


title: Optimization of the Irf8 +32 kb enhancer disrupts dendritic cell lineage segregation [bulk RNA-seq]

city: St. Louis

first: Feiya

last: Ou

address: 4939 Children's Place

department: Pathology & Immunology

institute: Washington University in St. Louis

summary: Autoactivation of lineage-determining transcription factors (TFs) mediates bistable expression to generate distinct cell phenotypes essential for complex body plans. Classical dendritic cells type 1 (cDC1) and type 2 (cDC2) provide non-redundant functions required for defense against distinct immune challenges. Interferon Regulatory Factor 8 (IRF8), the cDC1 lineage-determining TF, undergoes autoactivation in cDC1 progenitors to establish cDC1 identity, yet its expression is downregulated during cDC2 differentiation by an unknown mechanism. This study reveals that the Irf8 +32 kb enhancer, responsible for IRF8 autoactivation, has been tuned to possess low-affinity IRF8 binding sites. Increasing these affinities in vivo induces erroneous IRF8 autoactivation in specified cDC2 progenitors, causing their redirection towards cDC1 and a novel hybrid DC subset with mixed lineage phenotypes. These developmental alterations critically impair both cDC1- and cDC2-dependent arms of immunity. Collectively, our findings underscore the significance of enhancer suboptimization in the developmental segregation of classical dendritic cells required for normal immune function.

overall_design:
Bulk RNA-seq analysis of sort-purified splenic conventional dendritic cell (cDC) populations from mice of different genotypes was performed.

The cell types include type 1 cDC (cDC1, labeled as "dc1"), type 2 cDC (cDC2, labeled as "dc2"), and hybrid DC (labeled as "hybrid").

The genotypes include WT (wt),  Irf8 +32H/+ (h+), Irf8 +32H/H (hh), Irf8 +32H/H Nfil3-/- (hh_nfil3_ko), and Irf8 +32H/H Zeb2 -165D123 (hh_d123).

The samples are from 2 experiments (exp1 and exp2) as indicated in sample titles. Biological duplicates were included in each experiment.

first: Kenneth

last: Murphy


title: Transcriptomic analyses identify pathways linking developmental atrazine exposure to altered intestine development

city: RALEIGH

first: Dereje

last: Jima

address: 850 Main Campus Drive

department: CHHE

institute: NCSU

summary:
Purpose: Identify gene expression changes in whole intestines of Xenopus laevis embryos exposed to atrazine (35 mg/L) compared to DMSO controls
Methods: NF 39 embryos were exposed to 35 mg/L ATR or DMSO control for 24 hours. After 24 hours of exposure, intestines from embryos of each treatment group were dissected from anesthetized embryos with sharpened forceps. The anterior portion of the embryo (including the head, heart, and foregut) were removed and discarded, leaving only the mid-and hindgut portion of the intestine. Intestines from multiple embryos were removed, combined within a microcentrifuge tube containing TRIzol™ Reagent (Invitrogen) such that each replicate contained 10 intestines (n = 4). RNA extraction was performed using the TRIzol™ Reagent (Invitrogen) according to the manufacturer's instructions, followed by lithium chloride precipitation. Quality and quantity of RNA yield was confirmed on a Thermo Fisher Scientific Nanodrop 1000 and an Agilent 2100 Bioanalyzer. RNA-seq was performed by North Carolina State University’s Genomic Sciences Laboratory with an Illumina poly-A enriched directional RNA library and an Illumina NovaSeq 6000 was used for sequencing (SP 150 bp PE flow cell).
Results: We identified 254 significantly differentially expressed genes (p-adj<0.05) between DMSO and atrazine exposed intestines
Conclusions: Gene Ontology analysis of the differentially expressed genes revealed an enrichment for pathways involved in cellular stress, EMT, and the cell cycle, along with pathways involved in cellular (glucose)metabolism.

overall_design: atrazine-exposed mRNA profiles from whole intestines of DMSO control (n=4) and 35 mg/L atrazine-exposed (n=4) Xenopus laevis embryos after 24 hours.

first: Julia

last: Grzymkowski

middle: D

first: Nanette

last: Nascone-Yoder


title: Transcriptional profiling of spleens from mice challenged with Brucella melitensis with or without prior vaccination.

status: Public on Jan 30 2024

city: Columbia

first: Jerod

last: Skyberg

address: Veterinary Pathobiology, 1500 Bouchelle

institute: University of Missouri

summary: We isolated RNA from whole spleens of mice challenged with B. melitensis, some of which were vaccinated prior to infection.

overall_design: Comparative gene expression profiling analysis spleens from C57BL/6J mice after two weeks of challenge with Brucella melitensis.  Mice also received PBS or the S19 vaccine four weeks prior to challenge.

first: Barbara

last: Ponzilacqua-Silva

first: Skyberg

last: Jerod


title: Distinct immune features in the partial remission phase of type 1 diabetes identified through Single-Cell RNA Sequencing and Functional Analysis

status: Public on Jan 18 2024

city: Changsha

first: Ting

last: Zhong

state: Hunan

address: 139 people's Central Road, Furong District

department: The Second Xiangya Hospital

institute: Central South University

summary: Type 1 diabetes is a progressive autoimmune disease with unknown etiology. Although the destruction of β-cells is recognized as an irreversible process, many type 1 diabetes patients experience the partial remission stage characterized by spontaneous and transient recovery of β-cell function. However, a comprehensive understanding of immune disturbances in the progression of type 1 diabetes as well as the immunological mechanisms responsible for the partial remission stage remains to be elucidated.

overall_design: Here, we applied single-cell RNA sequencing to delineate the immune underpinnings of peripheral immune cells from new-onset type 1 diabetes patients and type 1 diabetes patients in the partial remission phase. Combined with cohort validation and functional assays, we identified Treg and CD8+ T cell subsets with highly transcriptional heterogeneity and distinctive functional programs that exhibited compositional changes during the partial remission stage of type 1 diabetes and were significantly associated with β-cell function. Further analysis revealed that TIGIT+ CCR7- Tregs expanded during the partial remission stage and suppressed highly cytotoxic CD226+ CCR7- CD8+ T cells through TGF-β signal, which suggests that this mechanism may have contributed to the restoration of immune balance during the partial remission stage. These findings provide a framework for enriching our knowledge about type 1 diabetes progression and remission, and the cell subsets we identified may offer mechanistic clues for therapeutic intervention.

first: Xinyu

last: Li

first: Kang

last: Lei

first: Rong

last: Tang

first: Qiaolin

last: Deng

first: Bin

last: Zhao

first: Xia


title: Integrative study of mitochondrial dysfunction in murine skeletal muscle during pancreatic cancer cachexia

status: Public on Mar 11 2024

city: Marseille

first: Bianca

middle: Hermine

last: Habermann

address: Avenue de Luminy 163

country: France

institute: IBDM UMR7288

summary: Pancreatic Ductal AdenoCarcinoma (PDAC), the most common pancreatic cancer, is a deadly cancer since it is often diagnosed late and resistant to current therapies. A large proportion of PDAC patients are affected by tumor-induced cachexia. This cachexia, characterized by a loss of muscle mass and strength (sarcopenia), contributes to patient frailty and poor therapeutic response. We have shown that mitochondrial metabolism is reprogrammed in PDAC tumors and constitutes a vulnerability, opening new therapeutic avenues. The objective of this work was to study the molecular mechanisms underlying mitochondrial remodeling in PDAC cachectic skeletal muscle. Our study focused on the gastrocnemius muscle of genetically-engineered mice spontaneously developing an autochthonous pancreatic tumor and cachexia (KIC GEMM). We compared KIC mice developing a pancreatic tumor in 9-11 weeks to control littermates. We performed an integrative study combining in vivo functional analyses by non-invasive Magnetic Resonance, and ex-vivo histology, Seahorse, RNA-sequencing, and proteomic mass spectrometry and Western blotting analyses. KIC cachectic PDAC mice exhibit severe sarcopenia with loss of muscle mass and strength associated with reduced muscle fiber’s size and induction of protein degradation processes. Mitochondrial alterations in skeletal muscle play a central role in PDAC-induced cachexia. Muscle atrophy is associated with strong mitochondrial metabolic defects that are not limited to carbohydrates and protein metabolism, but concern also lipids, ROS and nucleic acids. Our data provide a framework to guide towards the most relevant molecular markers that would be affected early in tumor development and could be targeted in the clinic to limit PDAC-induced cachexia at early stages of the pathology.

overall_design: Comparative gene expression of gastrocnemius skeletal muscle of pancreatic cancer carrying mice versus healthy control using bulk RNA sequencing

first: Fabio

last: Marchiano

first: Tristan

last: Cicquel

first: Alice

last: Carrier


title: PDZ-Binding Kinase, a Novel Regulator of Vascular Remodeling in Pulmonary Arterial Hypertension

status: Public on Jan 11 2024

city: Nanjing

first: Feng

last: Chen

state: Jiangsu Province

address: 101 Longmian Ave

department: Department of forensic medicine

institute: Nanjing Medical University

summary: To investigate the underlying mechanism of pulmonary hypertension, the model of monocrotaline (MCT)-treated pulmonary arterial hypertension (PAH) rats were constructed to detect the differentially expressed profile of genes in lung tissue of PAH rat.

overall_design: The male rats were treated with monocrotaline (MCT) to induce pulmonary arterial hypertension model. One dose of MCT (60 mg/kg) was injected Intraperitoneally into animals. Control rats were injected with the equivalent volume of dissolvent solution according to their weights. After 4 weeks late, the lung tissue was collected to detect the genes expression profile.

first: Huijie

last: Huang

first: David

middle: R

last: Fulton


title: Massive Parallel Sequencing (MPS) of miRNAs in PBMCs

status: Public on Jan 09 2024

city: Prague

first: Aneta

last: Prokopcova

address: Na Slupi 450/4

country: Czech Republic

institute: Institute of Rheumatology

type: Non-coding RNA profiling by high throughput sequencing

summary: We carried out Massive Parallel Sequencing (MPS) to reveal circulating miRNA profiles of 96 patients to distinguish the radiographic and non-radiographic axial spondyloarthritis. The sequencing platform identified 1900 miRNA species, out of which 432 miRNAs were present with the base mean, computed by DESeq2, more than 10. We identified 48 miRNAs, which were different in PBMCs in patients with axial spondyloarthritis compared to healthy controls.

overall_design: We collected PBMCs from 38 nr-axSpA, 38 r-axSpA patients and 20 healthy controls. The statistical analysis was adjusted to potential confounding variables, including sex, age, and CRP, which were identified as relevant factors in our cohort and previous studies.

first: Jiri

last: Baloun

first: Ladislav

last: Senolt


title: Epigenetic dysregulation in Alzheimer s disease peripheral immunity - RNA+TCR+BCR

status: Public on Jan 03 2024

city: Chicago

last: Gate

state: IL

address: 303 E Chicago Ave, Chicago, IL 60611

department: Neurology

institute: Northwestern University

summary: The peripheral immune system in Alzheimer's disease (AD) has not been thoroughly studied with modern sequencing methods. To investigate epigenetic and transcriptional alterations to the AD peripheral immune system, we used single-cell sequencing strategies, including assay for transposase-accessible chromatin and RNA sequencing. We reveal a striking amount of open chromatin in peripheral immune cells in AD. In CD8 T cells, we uncover a cis-regulatory DNA element co-accessible with the CXC motif chemokine receptor 3 gene promoter. In monocytes, we identify a novel AD-specific RELA transcription factor binding site adjacent to an open chromatin region in the nuclear factor kappa B subunit 2 gene. We also demonstrate apolipoprotein E genotype-dependent epigenetic changes in monocytes. Surprisingly, we also identify differentially accessible chromatin regions in genes associated with sporadic AD risk. Our findings provide novel insights into the complex relationship between epigenetics and genetic risk factors in AD peripheral immunity.

overall_design: Peripheral blood mononuclear cells from healthy control and Alzheimer's disease patients, matched for age, sex, and APOE genotype. Samples were thawed, FACS sorted for live cells, and profiled using scRNA-seq and scV(D)J-seq (BCR and TCR).

first: Abhirami

last: Ramakrishnan

first: Natalie

last: Piehl


title: Transcriptome profiles of liver from 7dpf zebrafish larvae from control, sec31anju221 and double mutation of sec31anju221 and UPR effectors by Next Generation Sequencing

status: Public on Jan 01 2024

first: Yuxi

last: Yang

state: Jiangsu

address: Room 402, 12 Xuefu Road, Nanjing University

department: Medical School

institute: Nanjing University

summary: We found that zebrafish larvae bearing a homozygous hypomorphic mutation in the sec31a gene (sec31anju221) exhibited hepatic steatosis accompanied with activation of unfolded protein response (UPR). As systematic efforts to investigate how different branches of UPR impact on the hepatic metabolic maladaptation, critical effectors of the UPR (xbp1, atf4a/4b, atf6, srebf1 and srebf2) were separately knock-outed on sec31anju221 background. In order to access the changes on transcriptome landscape, livers were dissected from the 7 dpf zebrafish larvae and low input RNA-seq was applied.

overall_design: Livers from 7dpf zebrafish larvae were dissected with forceps and dissociated cells with 0.25% trypsin treatment. The libraries were constructed with NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina (NEB, Cat No.6420) and sequenced on Illumina Novaseq platform.

first: Xin

last: Lou


title: Whole-body transcriptome mining for candidate effectors from Diuraphis noxia

status: Public on Dec 31 2023

city: Stellenbosch

first: Anna-Maria

last: Oberholster

state: Western Cape

address: JC Smuts Building, Van der Bijl Street

country: South Africa

institute: University of Stellenbosch

summary: RNA was extracted from a pooled sample of salivary glands that were excised from adult Diuraphis noxia aphids of two biotypes SA1 and SAM2.0. South African D. noxia biotype SA1 is known to be the least virulent aphid, while its offspring, the South African D. noxia biotype SAM2.0 is a newly developed biotype that is capable of feeding on several resistant cultivars. The overall purpose of the experiment was to establish a baseline availability of transcripts contained in the salivary glands of D. noxia aphids as well as help improve on current genome assemblies. In total, 200 glands were excised from both biotypes feeding on various wheat hosts (Tugela, Tugela Dn1, Gamtoss R, SST387). Aphid glands were excised in PBS buffer and stored in RNA-later until RNA extraction with a Qiagen Rneasy kit could commence. Library preparation for sequencing was performed using an Illumina TruSeq Stranded mRNA LT Sample Prep Kit following the TruSeq Stranded mRNA Sample Preparation Guide, Part # 15031047 Rev. E protocol. The single library was then sequenced on a NovaSeq6000 system. De novoassembly was performed making use of the Trinity package.

overall_design: Assembly of combined salivary transcriptome of two Diuraphis noxia biotypes that  fed on wheat cultivars Tugela, Tugela Dn1, Gamtoos R, SST387. Salivary glands were excised from both biotypes feeding on various hosts and pooled together before RNA extraction.

last: Botha-Obertholster

first: Vittorio

last: Nicolis

first: N

middle: V

last: Burger


city: Boston

first: Amy

middle: E

last: Schade

state: MA

address: 77 Avenue Louis Pasteur

department: Medicine

institute: Brigham and Women's Hospital

summary: SUM149PT cell line treated with AKTi (ipatasertib) and EZH2i (tazemetostat).

overall_design: On day -5, SUM149PT cells were seeded at 40% confluency and treated with 5uM tazemetostat or DMSO. On day -3, cells were passaged again at 40% confluency and maintained in DMSO or tazemetostat. On day -1, cells were seeded into 6cm plates at 250,000 cells per plate and maintained in DMSO or tazemetostat. On day 0, cells were treated in media containing 2% FBS with AKTi (ipatasertib) and/or EZH2i (tazemetostat). Cells were collected at 24 hours, RNA was isolated and then sequenced using NGS.

first: Karen

last: Cichowski


title: Transcriptomic profiling of human AML cell lines and primary AML samples

status: Public on Jan 14 2024

city: Charlottesville

first: Kevin

middle: A.

last: Janes

state: VA

address: Box 800759 Health System

department: Department of Biomedical Engineering

institute: University of Virginia

summary:
We sequenced mRNA from both cell lines and primary samples to investigate transcriptomic profiles of human AML cell lines and primary samples.
We then performed gene expression profiling analysis using data obtained from RNA-seq of AML cell lines and primary samples that have sphingolipidomic characterization to identify transcriptional differences between two distinct sphingolipidomic subtypes in AML samples.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for human AML cell lines and primary samples that belonged to two distinct sphingolipidomic subtypes.

first: B

middle: B

last: Paudel

first: Francine

last: Garrett-Bakelman

last: Feith


title: Evaluation of genetic inactivation of lysine deacetylases KDAC6 (HDAC6) and KDAC8 (HDAC8) versus treatment with KDAC-selective inhibitors

status: Public on Feb 15 2024

city: New Orleans

first: Terry

last: Watt

state: LA

address: 1 Drexel Dr.

department: Chemistry

institute: Xavier University of Louisiana

summary: To investigate the specificity and biological impacts of KDAC-selective inhibitors, we treated wild-type HT1080 cells and cell lines containing point mutations that result in endogenous expression of a catalytically-inactive variant with KDAC6 and KDAC8 selective inhibitors Tubastatin A and PCI-34051. Expression profiling analysis was then performed on the treated cells vs. untreated wild HT1080 cells.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for inhibitor-treated HT1080 cells and variants containing inactivated KDACs, with 3 replicates.  WT data is included in GSE228549.

first: Tasha

last: Toro


title: SIRT7 induces cytoskeletal remodelling via RAC1 to enhance host resistance to Mtb

status: Public on Dec 20 2023

city: Shenzhen

first: Fuxiang

last: Li

state: Guangdong

address: Nanhai Avenue 3688

institute: shenzhen university

summary: Mycobacterium tuberculosis (Mtb) secretes several virulence determinants that alter phagosome biogenesis, enabling its survival within the cell. Nevertheless, the mechanism underlying this process remains considerably obscure. Here, we have identified a novel regulatory mechanism whereby SIRT7 mediates Rac1 activation in cytoskeletal remodelling during Mtb infection. We found that SIRT7 are significantly decreased during Mtb infection in both mRNA and protein levels. SIRT7 deficiency impairs macrophage phagocytosis and bacteriacidal activity by disrupts actin cytoskeleton dynamics. In the murine TB model, the deficiency of Sirt7 had an adverse effect on the host's response to Mtb since it led to an increase in bacterial burden and inflammation in the lung. Conversely, the overexpression of Sirt7 impeded bacterial growth. Mechanistically, we have shown that SIRT7 limits Mtb infection by directly interacting with and activating RAC1. Therefore, we conclude that SIRT7 is responsible for driving cytoskeletal remodeling through RAC1, thus providing crucial insight into host response during Mtb infection and offering a potential target for tuberculosis treatment.

overall_design: Total RNA was extracted from either uninfected or H37Rv-infected BMDMs of Sirt7+/+ or Sirt7-/- at 12 hours post-infection, using the mirVana Isolation Kit (Life Technologies). The quality of the RNA was confirmed using an Agilent Bioanalyzer, and only samples with an RNA integrity number >7 were processed. After library construction, library purification, library detection, and library quantification, FastQC was used for sequencing data quality control, and the data was then filtered by Cutadapt. Short reads were aligned using the Hisat2(v2.0.1) software, and gene expression was calculated by the FPKM (Fragments Per Kilo bases per Million reads) (20436464) method using the Htseq software(V0.6.1).

first: Yi

last: Cai

first: Xinchun

last: Chen


title: Loss of p14 reduces immunogenicity in melanoma by affecting the peptide surface density via non-canonical Wnt signaling

status: Public on Dec 13 2023

city: Würzburg

first: Jonas

last: Wohlfarth

state: Bavaria

address: Josef-Schneider-Str. 2

department: Dermatology

country: Germany

institute: University Hospital Würzburg

summary: Immunotherapy has achieved tremendous success in melanoma. However, only around 50 % of advanced melanoma patients benefit from immunotherapy with immunogenicity of melanomas playing a key role. CDKN2A encoding the two tumor-suppressor proteins p14ARF and p16INK4a belongs to the most frequently inactivated gene loci in advanced melanoma where decreased T cell infiltration is accompanied. While the role of p16INK4a has been aberrantly investigated, knowledge about the function of p14ARF in melanoma therapy is scarce. In this study, we elucidate the impact of reduced p14ARF expression on melanoma immunogenicity. Knockdown of p14ARF in melanoma cell lines diminished the recognition and killing by melanoma differentiation antigen (MDA)-specific T cells. Resistance was caused by a reduction of the peptide surface density of presented MDAs. While antigen presentation via HLA-I molecules was enhanced upon p14ARF downregulation in general, absolute and relative expression of cognate peptides was decreased. Limiting WNT5A signaling reverted this phenotype suggesting an involvement of non-canonical Wnt signaling. Taken together, our data indicate a new mechanism limiting MDA-specific T cell responses by decreasing both absolute and relative MDA-peptide presentation in melanoma.

overall_design: To investigate the influence of p14 and p16, melanoma cell line MaMel51 was knocked down for p14, p16 or both of them with respective doxycyclin-inducible shRNAs or a control scramble (scr) shRNA. Three biological triplicates were analyzed for each condition.

first: Bastian

last: Schilling


title: The combination of SLC7A11 inhibitor and oridonin synergistically inhibits cervical cancer cell growth by decreasing the NADPH/NADP+ ratio

city: zhengzhou

first: yichao

last: Xu

state: Henan

address: No.100 Science Avenue

department: School of Pharmaceutical Sciences

institute: Zhengzhou university

summary: Oridonin, as a natural terpenoids isolated from traditional Chinese medicinal herb Rabdosia rubescens, exhibits anticancer activity against multiple cancers with the IC50 ranging from 10 ¦ÌM to 100 ¦ÌM. We observed that SLC7A11 inhibitor Imidazole ketone erastin (1 ¦ÌM) potently increase the anti-proliferative activity of oridonin at low concentrations. Preliminary investigation demonstrated that combination of Imidazole ketone erastin (1 ¦ÌM) and oridonin induced apoptosis and ferroptosis only in a fraction of cells. To explore the synergistic anti-tumor mechanism of combination of oridonin and Imidazole ketone erastin, second-generation transcriptome sequencing(RNA-seq) was utilized to determine the transcriptome change in Hela cells affected by oridonin and Imidazole ketone erastin. PCA analysis showed that the group of oridonin or Imidazole ketone erastin alone located near the control group, while the group of combination gathered far away from the control group, indirectly indicating that combination of oridonin and Imidazole ketone erastin had synergistic biology function.

overall_design: Hela cells were seeded in 6 well paltes, and treated with either 1) DMSO, 2) oridonin, 3) Imidazole ketone erastin, or 4) combination of  oridonin and Imidazole ketone erastin for 24 h. Total mRNAs of treated Hela cells were collected and sequenced on Illumina Novaseq 6000 and 150 bp paired-end reads were generated.

first: Yi-chao

first: Ya

last: Liu

first: Hong

first: Yang

last: Wang


title: Glomerular voom-transformed RNAseq counts from the Neptune Cohort and healthy living donors [re-analysis of GSE197307]

status: Public on Feb 07 2024

city: Ann Arbor

first: Sean

last: Eddy

state: MI

address: 1150 West Medical Center Drive

institute: University of Michigan

summary: Glomerular Transcriptome in the Nephrotic Syndrome study network cohort

overall_design:
Please note that GSE197307 samples were re-analyzed as described below.
Glomerular RNAseq profiles; Raw data (fastq) files are available upon approved ancillary study request from the NEPTUNE consortium (please see https://www.neptune-study.org/ancillary-studies; email contact: NEPTUNE-STUDY@umich.edu
*data processing step:
fastqScreen 0.11.4
cutadapt 1.14 -m 50 -A CTGTCTCTTATA -a CTGTCTCTTATA
STAR 2.5.2.a --bamRemoveDuplicatesType UniqueIdentical --twopassMode Basic --outFilterIntronMotifs RemoveNoncanonical --outSAMtype BAM SortedByCoordinate --outSAMstrandField intronMotif
htseq-count --stranded=no --order=pos --mode=union
genome build/assembly: GRCh 38.86
processed data files format and content: voom transformed data were batch corrected and trimmed to remove transcripts of no detectable abundance.

first: Eichinger

last: Felix

first: Eddy

last: Sean

first: Mariani

last: Laura

first: Menon

last: Rajasree

first: Godfrey

last: Brad

first: Kretzler

last: Matthias


title: MDA-MB-468 and SUM149PT cell lines treated with AKTi (ipatasertib) and EZH2i (tazemetostat)

first: Amy

last: Schade

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

overall_design: Refer to individual Series


title: Machine Learning Reveals Common Transcriptomic Signature Across Brain and Placenta Following Developmental Organophosphate Ester Exposure

first: Dereje

last: Jima

summary:
Purpose: Identify gene expression changes in hippocampus of postnatal day 10 pups whose mothers were exposed to OPFRs during pregnancy (3.3 mg/kg) compared to unexposed controls. Determine if developmental OPFR exposure influences common genes or gene pathways across multiple tissues and developmental time pionts.
Methods: One new RNAseq data set (hippocampus) generated for this study and two extant RNAseq data sets (placenta and cortex) were reanalyzed with a validated machine learning methodology and traditional bioinformatics techniques. Results of differentially expressed genes were compared across tissues and the submitted to pathway analysis.
Results: OPFR effects on gene expression are sex specific, with limited changes in male hippocampus and significant enrichment of genes related to voltage gated ion channels in females. Cross tissue analysis of OPFR exposured P1 cortex and placenta demonstrated common genes identified in each tissue related to mitochondria physiology
Conclusions: Developmental OPFR exposure results in gene expression changes in hippocampus, cortex, and placenta related to proteins underlying mitochondria physiology, particularly oxidative phosphorylation and electron transport chain. These findings support the growing concerns that developmental OPFR exposure may contribute to the etiology of neurodevelopmental disorders.

overall_design: Maternal OPFR exposed mRNA profiles from dorsal hippocampus of 10-day old rats

first: Andrew

last: Newell

first: Benjamin

last: Reading

first: Heather

last: Patisaul


title: Role of myeloid cannabinoid CB1 receptor in atherosclerosis

status: Public on Dec 07 2023

city: Munich

first: Sabine

last: Steffens

address: Pettenkoferstrasse 9b

department: Clinical Pathobiochemistry

institute: Institute for Cardiovascular Prevention-LMU Klinikum

summary: Although the cannabinoid CB1 receptor has been implicated in atherosclerosis, its cellspecific effects in this disease are not well understood. Here, we report that male mice with myeloid-specific Cnr1 deficiency on atherogenic background developed smaller lesions and necrotic cores than controls, while only minor genotype differences were observed in females. Male Cnr1 deficient mice showed reduced arterial monocyte recruitment and macrophage proliferation with less inflammatory phenotype. The sexspecific differences in proliferation were dependent on estrogen receptor (ER)α estradiol signaling. Kinase activity profiling revealed a CB1-dependent regulation of p53 and cyclin-dependent kinases. Transcriptomic profiling further unveiled chromatin modifications, mRNA processing and mitochondrial respiration among the key processes affected by CB1 signaling, which was supported by metabolic flux assays. Chronic administration of the peripherally-restricted CB1 antagonist JD5037 inhibited plaque progression and macrophage proliferation, but only in male mice. Finally, CNR1 expression was detectable in human carotid endarterectomy plaques and inversely correlated with proliferation, oxidative metabolism and inflammatory markers, hinting to a possible implication of CB1-dependent regulation in human pathophysiology. In conclusion, impaired macrophage CB1 signaling is atheroprotective by limiting their arterial recruitment, proliferation and inflammatory reprogramming. The importance of macrophage CB1 signaling seems to be more pronounced in male mice.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for bone marrow-derived macrophages (BMDMs), comparing when CB1 is activated or not.

first: Yong

first: Guo

last: Li

first: Raquel

last: Guillamat-Prats


title: Distinct transcriptomic and epigenomic responses of mature oligodendrocytes during disease progression in a mouse model of multiple sclerosis

city: Solna

first: Bastien

last: Hervé

address: Solnavägen 9

department: MBB

country: Sweden

institute: Karolinska Institutet

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Multiple sclerosis (MS) is a chronic demyelinating autoimmune disease which targets mature oligodendrocytes (MOLs) and their myelin. MOLs are transcriptionally heterogeneous, and can transition to immune-like states in the context of MS. However, the intricacies of their dynamics throughout disease progression remain poorly understood. Here, we employed simultaneous single-cell multiome ATAC and RNA sequencing targeting oligodendroglia (OLGs) from the experimental autoimmune encephalomyelitis (EAE) MS mouse model, at different stages of the disease course. We found that the transition to immune OLGs states occurs already at early stages of EAE and these persist at late stages of the disease, consistent with epigenetic memory of previous neuroinflammation. Interestingly, transcription factor activity suggested immunosuppression activity in MOLs at early stages of EAE and  we also observed a transitory activation of a regenerative programme in MOLs at this stage. Importantly, different MOLs present a differential responsiveness to EAE, with MOL2 exhibiting stronger transcriptional immune response than MOL5/6. Moreover, we observed divergence responses at the epigenetic level of MOL2 and MOL5/6 during disease evolution. Thus, our single-cell multiomic resource highlights dynamic and distinct responses of OLG subpopulations to the evolving environment in EAE, which might modulate their response to regenerative therapeutic interventions in MS.

overall_design: Sox10:Cre-RCE:LoxP mice are a strain of mice obtained originally by crossing mice with Cre recombinase under the control of the Sox10 promoter with reporter mice RCE:loxP-EGFP to label the complete oligodendrocyte (OL) lineage

first: Chao

last: Zheng

first: Mandy

last: Meijer

first: Leslie

last: Ann Rubio Rodriguez-Kirby

first: André

last: Ortlieb Guerreiro Cacais

first: Petra

last: Kukanja

first: Mukund

last: Kabbe

first: Tony

last: Jimenez-Beristain

first: Tomas

last: Olsson

first: Eneritz

last: Agirre

first: Gonçalo

last: Castelo-Branco


title: Genetic determinants of TCF7L2 binding and chromatin availability in mouse liver [RNA-seq]

status: Public on Dec 11 2023

city: Kuopio

first: Juho

middle: Mikael

last: Mononen

address: Yliopistonranta 1E

department: Faculty of Health Sciences

country: Finland

institute: University of Eastern Finland

summary: Most common genetic variants associated with disease are located in non-coding regions of the genome. One mechanism by which they function is through altering transcription factor (TF) binding. In this study, we explore how genetic variation is connected to differences in the regulatory landscape of livers from C57BL/6J and 129S1/SvImJ mice fed either chow or a high-fat diet. To identify sites where regulatory variation affects TF binding and nearby gene expression, we employed an integrative analysis of H3K27ac ChIP-seq (active enhancers), ATAC-seq (chromatin accessibility) and RNA-seq (gene expression). We show that, across all these assays, the genetically driven (i.e. strain-specific) differences in the regulatory landscape are more pronounced than those modified by diet. Most notably, our analysis revealed that differentially accessible regions (DARs, N = 29635, FDR < 0.01 and fold change > 50%) are almost always strain-specific and enriched with genetic variation. Moreover, proximal DARs are highly correlated with differentially expressed genes. We also show that TF binding is affected by genetic variation, which we validate experimentally using ChIP-seq for TCF7L2 and CTCF. This study provides detailed insights into how non-coding genetic variation alters the gene regulatory landscape, and demonstrates how this can be used to study the regulatory variation influencing TF binding.

overall_design: We generated  5-7 RNA-seq libraries from the 4 groups: 129S1/SvImJ and C57BL/6J on either chow or high-fat diet.

first: J

first: M

last: Taipale

first: S

last: Heikkinen


title: Characterizing the Collateral Activity of CRISPR/Cas13 in Mammalian Cells: Implications for RNA Editing and Therapeutic Applications

status: Public on Jan 25 2024

city: Guangzhou

first: Lizhen

last: Huang

address: xaioguwei street

department: School of Biology and Biological Engineering

institute: South China University of technology

summary: The CRISPR/Cas13 system has garnered attention as a potential tool for RNA editing. However, the degree of collateral activity among various Cas13 orthologs and their cytotoxic effects in mammalian cells remain contentious, potentially impacting their applications. In this study, we observed differential collateral activities for LwaCas13a and RfxCas13d in 293T and U87 cells by applying both sensitive dual-fluorescence (mRuby/GFP) reporter and quantifiable dual-luciferase (Fluc/Rluc) reporter, with LwaCas13a displaying notable activity contrary to previous reports. However, significant collateral RNA cleavage exerted only a modest impact on cell viability. Furthermore, the collateral activity of LwaCas13a mildly impeded but did not arrest, porcine embryo development. Our findings reveal that distinct collateral RNA cleavage by Cas13 slightly suppresses mammalian cell proliferation and embryo development. This could account for the lack of reported collateral effects in numerous prior studies and offers new insights into the implications of the collateral activity of Cas13 for clinical application.

overall_design: To investigate the collateral activity of Cas13 in mammalian cells, a series of experiments were conducted involving 293T and U87 cells. Cas13a or Cas13d was employed to target the mRuby or Fluc loci, with the inclusion of non-target crRNA as a control. Through RNA-seq analysis of cells treated with various Cas13/crRNA combinations, our objective is to gain a comprehensive understanding of Cas13's collateral activity in mammalian cells and to uncover alterations in gene expression. Comparative analysis of gene expression profiles between experimental groups and the control will be conducted, with a specific focus on genes exhibiting significant differences compared to the control. This study aims to elucidate the regulatory effects of Cas13a and Cas13d on gene expression under different conditions, providing valuable insights into their roles in gene regulation within mammalian cells.

first: Yanbin

last: Wan

first: Caimei

first: Dongyan

last: Feng

first: Lv

last: Wang

first: Xiding

last: Lin

first: Xuanye

last: Zhao

first: Liya

last: Han

first: Yanyu

last: Zhu

first: Lijing

last: Hao

first: Hongli

last: Du


title: Genetic determinants of TCF7L2 binding and chromatin availability in mouse liver [ChIP-seq]

overall_design: We generated  4 H3K27ac, 2 CTCF and 2 TCF7L2 ChIP-seq libraries from the 4 groups: 129S1/SvImJ and C57BL/6J on either chow or high-fat diet.


title: Mitochondrial Apolipoprotein MIC26 is a metabolic rheostat regulating central cellular fuel pathways

status: Public on Dec 01 2023

city: DUESSELDORF

first: Arun

middle: Kumar

last: Kondadi

address: Universitätsstr. 1

institute: Heinrich Heine University, Dusseldorf

summary: A variety of metabolic disorders are aggravated by obesity. Since mitochondria play key roles in various catabolic and anabolic reactions encompassing lipid metabolism, optimal functioning of mitochondria is critical in preventing various pathologies. Increased amounts of the apolipoprotein O /MIC26 were found in diabetic patients along with lipid accumulation in murine liver upon MIC26 expression.  MIC26 is a MICOS complex constituent present in the mitochondrial inner membrane. The functional interplay between MIC26 and cellular metabolome is not understood. Therefore, we employed a multi-omics approach in WT and MIC26 knockout HepG2 cellular models in normoglycemic and hyperglycemic conditions, along with a variety of functional assays. We found that MIC26 is necessary for maintaining glycolysis and lipid metabolism via CPT1A along with cholesterol synthesis in a nutrient-dependent manner. MIC26 deletion led to metabolic rewiring of glutamine utilisation leading us to propose that mitochondrial ultrastructure and cellular metabolome are functionally interdependent.

overall_design: The study included HepG2 WT and MIC26 KO cells cultured either in normoglycemic (N /LG) or hyperglycemic (H / HG) conditions for 3 weeks. RNA-seq. was performed.

first: Melissa

last: Lubeck

first: Ritam

last: Naha

first: Yulia

last: Schaumkessel

first: Philipp

last: Westhoff

first: Anja

last: Stefanski

first: Patrick

last: Petzsch

first: Kai

last: Stühler

first: Karl

last: Köhrer

first: Andreas

last: Weber

first: Ruchika

last: Anand

last: Reichert

last: Kumar Kondadi


title: Single-cell time-resolved multi-omics reveal macrophage heterogeneity during foam cell formation

city: Shanghai

first: Yiwen

last: Wang

address: 2005, Songhu Road, Yangpu District

institute: Fudan University

summary: The study aims to investigate the macrophage heterogeneity during foam cell formation.

overall_design: One sample of THP-1 derived late stage foam cell.

first: Ling

last: Lin

first: Liang

last: Qiao


title: Genetic determinants of TCF7L2 binding and chromatin availability in mouse liver [ATAC-seq]

first: Juho

last: Mononen

overall_design: We generated  5-7 ATAC-seq libraries from the 4 groups: 129S1/SvImJ and C57BL/6J on either chow or high-fat diet. We also generated 4 replicates of ATAC-seq from F1 liver samples.

first: J

first: M

last: Taipale

first: S

last: Heikkinen


first: Amy

last: Schade

summary: MDA-MB-468 cell line treated with AKTi (ipatasertib) and EZH2i (tazemetostat).

overall_design: On day -5, MDA-MB-468 cells were seeded at 40% confluency and treated with 5uM tazemetostat or DMSO. On day -3, cells were passaged again at 40% confluency and maintained in DMSO or tazemetostat. On day -1, cells were seeded into 6cm plates at 250,000 cells per plate and maintained in DMSO or tazemetostat. On day 0, cells were treated in media containing 2% FBS with AKTi (ipatasertib) and/or EZH2i (tazemetostat). Cells were collected at 24 hours, RNA was isolated and then sequenced using NGS.

first: Karen

last: Cichowski


title: Microinjection of human mesenchymal stem cells to mouse blastocysts

status: Public on Nov 21 2023

city: Macau

first: Ren-He

last: Xu

state: Macau

address: N22 Research Building, University of Macau, Taipa

institute: University of Macau

summary:
Purpose: 1. Compare transcriptomes of hESC-derived MSCs (EMSCs) and somatic MSCs (SMCS); 2. Reveal transriptomic profiles of EMSCs post injection into mouse blastocyst.
Methods: For bulk seq on cultured human MSCs, mRNA was isolated using RNeasy Mini Kit (QIAGEN) and libraries was constructed using an Illumina kit, and then sequenced on NovaSeq platform. For single cell (sc) RNAseq on cultured human MSCs, cells were dissociated and lysed in the Oil-droplet Single Cell Capture System (Nadia Dolomite) and libraries were constructed using Nextera kits (Illumina), and then sequenced on Hiseq 150PE (Illumina). For EMSCs injected into mouse blastocyst, 30~70 embryos were pulled and lyzed in 10uL of Lysis Buffer for SmartSeq.
Results: 1. Both in bulk and in single cell level, all types of MSCs share typical common markers; 2. Unignorable differences exhist among hES-derived and somatic MSCs; 3. Rapid mRNA profile shifts in metabolism, cellular skeleton, and immunoregulatory response happen in hES-derived MSCs post injection to mouse embryos.
Conclusions: These experiments reveal transcriptomic alterations of EMSCs after injection into mouse blastocyst and transcriptomic profiles of human MSCs from different sources.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Examination of transcriptomic profiles of human MSCs maintained in culture and injected into mouse blastocyst

first: Borong

last: Huang

first: Yanan

last: Hao


title: Human hepatocytes can give rise to intrahepatic cholangiocarcinomas

status: Public on Jan 12 2024

city: San Francisco

first: Cindy

middle: K

last: Pino

state: CA

address: 400 Parnassus Ave.

institute: University of California, San Francisco

summary: To determine the transcriptomic similarity of undifferentiated carcinomas (UDCs) and intrahepatic cholangiocarcinomas (iCCAs) generated from primary human hepatocytes (phHeps) by oncogene expression and transplantation into immue-deficient mice with liver injury to iCCAs and hepatocellular carcinomas (HCCs) resected from patients.

overall_design: Comparison of gene expression profile by RNAseq of phHep-derived UDCs 1-3 and phHep-derived iCCAs 4-12 to patient-derived ICCAs A-C and I-O and patient-derived HCCs D-H.

first: Holger

last: Willenbring

first: Bernadette

last: Hsu

first: Eunsun

last: Kim


title: The Ig superfamily ligand B7H6 subjects T cell responses to NK cell surveillance

city: Heidelberg

first: Michael

last: Kilian

address: Im Neuenheimer Feld 280

institute: German Cancer Research Center (DKFZ)

summary: T cells play a critical role in the immune system by recognizing and responding to foreign and tumor antigens. Dysregulation or dysfunction of T cells can lead to autoimmune diseases, infectious diseases and cancer. Understanding the mechanisms that regulate T cell immunity is therefore critical for the development of effective therapies for diseases associated with T cell dysfunction. Co-inhibitory ‘checkpoint molecules’ such as programmed cell death protein 1 (PD-1) are tightly connected to T cell activation and balance excessive or prolonged immune activation by T cell-intrinsic signaling. Here, by screening for mediators of NK cell recognition on T cells, we identify the Ig superfamily ligand B7H6 to be highly expressed by activated T cells, including patient-infused CD19-targeting chimeric antigen receptor (CAR)-T cells. We show that, unlike other checkpoint molecules, B7H6 mediates NKp30-dependent recognition and subsequent cytolysis of activated T cells by NK cells. B7H6+ T cells are prevalent in the tissue and blood of several diseases, and we find that their abundance in tumor tissue positively correlates with clinical response in a cohort of immune checkpoint inhibitor-treated patients with esophageal cancer. In humanized mouse models, we demonstrate that NK cell surveillance via B7H6 is relevant in limiting the persistence and anti-tumor activity of CAR T cells in vivo and that its genetic depletion enhances T cell proliferation and persistence. In summary, we provide evidence of B7H6 protein expression by activated T cells and suggest the B7H6-NKp30 axis as therapeutically actionable NK cell-dependent immune checkpoint that regulates human T cell function.

overall_design:
Glioblastoma-infiltrating CD3 were isolated by fluorescence-activated cell sorting and analyzed using 10x Genomics 5' RNA/TCR sequencing as well as CITE-sequencing of B7H6 surface protein.
**The submitter declares that the raw data have been deposited to EGA (Identifier to follow) due to data protection regulations.**

first: Mirco

middle: J

last: Friedrich

last: Platten


title: Effect of  the  intrinsically disordered region (IDR) within the CLK2 (T343A) mutant in the regulation of intron retention

status: Public on Nov 09 2023

city: Wuhan

first: Wei

last: Wang

state: Hubei

address: 13 Hangkong Road

institute: Huazhong University of Science and Technology Tongji Medical College

summary: We established the HeLa cells expressing the GFP (control) or CLK2 (T343A) with deletion of the IDR [T343A (dIDR)]. These cells were subjected to RNA-seq for the alternative splicing analysis.

overall_design: Two HeLa cell groups (GFP, T343A (dIDR)) were subjected to RNA-seq

first: Bing


title: RNA-seq analysis during GSC differentiation

summary: RNA from the GSC456 cells and  the differentiated GSCs (DGCs) was extracted for the RNA-seq.

overall_design: GSCs and DGCs in triplicate


title: Transcriptomic and epigenetic responses unveil resistance mechanisms to Phytophthora sansomeana in soybean

status: Public on Nov 23 2023

city: Gainesville

first: Meixia

last: Zhao

state: FL

address: 1355 Museum Dr

institute: University of Florida

type: Methylation profiling by high throughput sequencing


title: Plasmodium falciparum MORC protein modulates gene expression through interaction with heterochromatin

status: Public on Nov 15 2023

city: Sao Paulo

first: Maneesh Kumar

last: Singh

state: SP

address: Av. Prof. Lineu Prestes, 580

country: Brazil

institute: University of Sao Paulo

summary: The MORC (microrchidia) family of proteins is highly conserved in all eukaryotic cells and have been shown to play diverse roles by forming protein-protein interactions with immune-responsive proteins, SWI chromatin remodeling complexes, histone deacetylases, and histone tail modifications across metazoans. To dissect the functional roles of MORC in the human malaria parasite, Plasmodium falciparum, we developed a glmS based ribozyme knockdown system to induce PfMORC knockdown upon glucosamine (GlcN) induction. We further conducted a transcriptomic analysis in PfMORC-HA-glmS knockdown parasites at the asexual stage to investigate alterations in global gene expression. Our results indicate an overrepresentation of downregulated genes belonging to the heterochromatin-associated hypervariable gene family proteins. Overall, we found that PfMORC controls the asexual gene expression of the P. falciparum and can be a potential target for malaria disease containment.

overall_design: Investigation of differential gene expression in PfMORC knockdown Plasmodium falciparum transgenic parasite.

first: Israel

last: Tojal da Silva

first: Jack

last: Adderley

first: Christian

last: Doerig

first: Celia Regina

last: da Silva Garcia


title: BLNK negatively regulates C-type lectin receptor-mediated antifungal immunity through inhibiting macrophage migration

status: Public on Nov 07 2023

first: Yiheng

last: Yang

address: 1239 SIPING ROAD

department: School of Medicine

institute: Tongji University

summary: B cell linker protein (BLNK) is a pivotal adaptor protein that interfaces the B cell receptor (BCR)-associated spleen tyrosine kinase (Syk) to regulate B cell function and development. However, it is still not well understood whether BLNK is involved in Syk-coupled C-type lectin receptor (CLR) signaling in myeloid cells, particularly in the context of antifungal immunity. Here, we show that stimulation with fungi-derived β-glucans or α-mannans induced the phosphorylation of BLNK in macrophages, which was significantly impaired due to the deficiency of CLRs including Dectin-1 and Dectin-2, as well as their downstream mediators Syk and Fc-receptor gamma-chain (FcRγ). Furthermore, BLNK is induced to be interacted with casitas B-lineage lymphoma (c-Cbl), which is completely dependent on the engagement of Dectin-1 and Dectin-2. Notably, BLNK deficiency facilitates CLR-mediated recruitment of c-Cbl/phosphatidylinositol 3-kinase (PI3K) complex to F-actin cytoskeleton, thereby promoting macrophage migration. Consequently, mice with monocyte-specific deficiency of BLNK are highly resistant to the infection with Candida albicans, a major human fungal pathogen, through increasing the infiltration of Ly6C+macrophages into kidneys. Together, our data indicate that BLNK functions downstream of Syk-coupled CLRs to negatively regulate antifungal immunity against C. albicans infection. These findings unravel a previously unidentified role of BLNK that negatively regulates macrophage migration through inhibiting CLR-mediated recruitment of c-Cbl/PI3K complex to the cytoskeleton.

overall_design: Total RNA of BMDMs from wt and BLNK KO mice were extracted after stimulation with hyphae (MOI = 0.1) for 3 hours .

first: Yi-heng

last: YANG

first: Xin-ming

last: JIA


title: A critical role for Hepatocyte Nuclear Factor 4 alpha in polymicrobial sepsis-associated metabolic reprogramming and death: effect of IL6 in Hnf4a WT and KO mice

status: Public on Nov 03 2023

city: Gent

first: Steven

last: Timmermans

address: Technologiepark 71

country: Belgium

institute: VIB-UGent

summary: In sepsis, limited food intake and increased energy expenditure induce a starvation response, which is compromised by a quick decline in expression of hepatic PPARα, a transcription factor essential in intracellular catabolism of free fatty acids. The mechanism upstream of this PPARα downregulation is unknown. We found that sepsis causes a progressive hepatic loss-of-function of HNF4α, which has strong impact on the expression of several important nuclear receptors, including PPARα. HNF4α depletion in hepatocytes dramatically increases sepsis lethality, steatosis and organ damage and prevents an adequate response towards IL6, which is critical for liver regeneration and survival. An HNF4α agonist protects against sepsis at all possible levels, irrespectively of bacterial loads, suggesting HNF4α is crucial in disease tolerance to sepsis. In conclusion, hepatic HNF4α fails in sepsis, causing PPARα downregulation and metabolic problems and a disturbed IL6-mediated acute phase response. The data open new insights and therapeutic options in sepsis.

overall_design: RNA-seq in WT mice and Hnf4a KO (inducible: induced by tamoxifen) mice stimulated with IL6 or PBS for 24h and liver were isolated for transcripome profiling, using 4 biological replicates (except KO+PBS: 3 bioloical replicates)

first: Céline

last: Van Dender

first: Ville

last: Paakinaho

first: Tineke

last: Vanderhaeghen

first: Jolien

last: Vandewalle

first: Maarten

last: Claes

first: Bart

last: Roman

first: Jan

last: De Waele

first: Siska

last: Croubels

first: Karolien

last: De Bosscher

first: Jorma

last: Palvimo

first: Claude

last: Libert


title: A Massively Parallel Screen of 5′UTR Mutations Identifies Variants Impacting Translation and Protein Production in Neurodevelopmental Disorder Genes

status: Public on Nov 08 2023

city: St. Louis

first: Joseph

last: Dougherty

state: MO

address: 4370 Duncan Ave

department: Genetics

institute: Washington University in St. Louis

summary: De novo mutations cause a variety of neurodevelopmental disorders including autism. Recent whole genome sequencing has identified hundreds of mutations in untranslated regions (UTRs) of genes from individuals with autism, but it is impossible to predict from sequence alone which are functional, and thus might be causal.  Therefore, we developed a high throughput assay to screen the consequence over 1,000 variants from 5'UTRs mutations on transcript abundance and translation efficiency. This assay successfully enriched for elements that alter reporter translation, identifying over 100 potentially functional mutations.  Studies in patient-derived cell lines further confirmed these mutations alter protein production in individuals in autism, including for multiple genes known to cause of syndromic forms autism, suggesting a diagnosis for these individual patients. Since UTR function varies by cell type, we further optimized this high throughput assay to enable assessment of mutations in neurons of the living brain.  Neurons demonstrate profoundly different principles of regulation by 5'UTRs, consistent with more robust mechanism for reducing impact of 5'UTR RNA structure. Overall our results highlight a new approach for assessing the impact of 5’UTR across cell types and suggest some cases of neurodevelopmental disorder may be caused by such variants.

overall_design: Pooled library screens of synthetic reporter genes containing variable 5' UTR sequences and associated barcode sequences in the 3'UTR were performed either in HEK cells or in mouse brain in vivo in order to measure the effects of 5'UTR elements and their variants on transcript abundance and translation efficiency. HEK cells were transfected with a plasmid library containing approximately 33,000 uniquely barcoded reporters, and in vivo experiments were performed by transcranial delivery of AAV-packaged sub-libraries each containing a third of the total library. RNA and DNA were extracted from each sample, and RNA was subjected to density centrifugation and fraction to isolate mRNA associated with the 43S preinitation complex, monosomes, or polysomes. The barcode-containing 3'UTRs were amplified from each fraction to determine the abundance of each respective 5'UTR-associated reporter. All in vivo mouse experiments were performed in a Vglut2-Cre line so that a LoxP-flanked cassette in the 3'UTR is inverted only in glutamatergic neurons. For each in vivo RNA sample, two reverse transcription primers, one specific to the unrecombined state ("CreOFF") and one specific to the inverted state ("CreON"), were used to prepare libraries to esimtate the reporter abundance in both Cre-expressing glutamatergic neurons and non-Cre-expressing cells. For both HEK and in vivo experiments, six biological replicates were collected.

first: Stephen

last: Plassmeyer

first: Colin

last: Florian

first: Michael

last: Kasper

first: Rebecca

last: Chase

first: Shayna

last: Mueller

first: Kelli

last: McFarland White

first: Courtney

middle: F

last: Jungers

first: Slavica

last: Pavlovic Djuranovic

first: Sergei

last: Djuranovic


title: A critical role for Hepatocyte Nuclear Factor 4 alpha in polymicrobial sepsis-associated metabolic reprogramming and death: HNF4A ChIP-seq in the CLP sepsis model (ChIP-Seq)

status: Public on Oct 31 2023

first: Steven

last: Timmermans

overall_design: Chromatin Immunoprecipitation (ChIP-seq) of HNF4A to determine the changes in chromatin occupancy in the liver of mice after CLP using 4 biological replicates in CLP and SHAM (control) conditions

first: Céline

last: Van Dender

first: Ville

last: Paakinaho

first: Tineke

last: Vanderhaeghen

first: Jolien

last: Vandewalle

first: Maarten

last: Claes


title: Effect of  CLK2 overexpression on alternative splicing

first: Wei

last: Wang

summary:
To investigate the function CLK2 in the regulation of  alternative splicing, we established HeLa cells overexpressing GFP, CLK2 (WT), or (T343A).
We then performed alternative splicing analysis using data obtained from RNA-seq of the three cell lines.

overall_design: HeLa cells expressing GFP, CLK2 (WT) or (T343A) were subjected to RNA-seq

first: Bing


title: Modeling the Progression of Placental Transport from Early‐ to Late‐Stage Pregnancy by Tuning Trophoblast Differentiation and Vascularization

status: Public on Oct 24 2023

city: Hamilton

first: Sonya

last: Kouthouridis

state: ON

address: 1280 Main St W

department: Chemical Engineering

country: Canada

institute: McMaster University

summary: The aim of this work was to compare the syncytiotrophoblast differentiation capacity of placental stem cells (PSCs) to the BeWo b30 trophoblast cell line. Syncytiotrophoblast markers were analyzed, as well as drug, hormone and nutrient transporters.

overall_design: 3 groups were studied: a control group consisting of undifferentiated placental stem cells, differentiated placental stem cells and differentiated BeWo b30 cells. Each group contained N=5 samples.

first: Alexander

last: Sotra

first: Zaim

last: Khan

first: Justin

last: Alvarado

first: Sandeep

last: Raha

first: Boyang

last: Zhang


title: Selective Vulnerability of Parvocellular Oxytocin Neuron in Social Dysfunction

status: Public on Dec 05 2023

city: Kobe

first: Masafumi

last: Tsurutani

state: Hyogo

address: 2-2-3 Minatojima minami, Chuo-ku

department: Center for Biosystems Dynamics Research (BDR)

country: Japan

institute: RIKEN

summary: Despite the rapid progress in dissecting neural circuits for social behaviors, it remains unknown whether specific neural cell types are selectively vulnerable in social dysfunction cases often associated with neurodevelopmental disorders. Here, employing a single-cell transcriptome analysis in mice, we show that an embryonic disturbance known to induce social dysfunction preferentially impairs gene expressions crucial for neural functions in parvocellular oxytocin (OT) neurons—a subtype linked to social rewards—while neighboring cell types experience a lesser impact. Chemogenetic stimulation of OT neurons at the neonatal stage ameliorated social deficits, concomitant with a cell-type-specific sustained recovery of the pivotal gene expressions. Our data illuminates the transcriptomic selective vulnerability within the hypothalamic social behavioral center, offering a potential therapeutic target through specific neonatal neurostimulation.

overall_design: First, to characterize gene expressions in the paraventricular hypothalamus (PVH) of mice that were embryonically exposed to valproic acid and exhibited social dysfunctions, we administrated sodium valproate at a dosage of 500 mg/kg to pregnant female mice on gestation day 12.5. The PVH samples of male OT-Cre; Ai9 mice at 5 weeks of age were dissected based on the fluorescence of tdTomato subjected to 10X Genomics Chromium platform for single nucleus RNAseq. Age-matched animals of the same genotype but prenatally exposed to saline were used as a control. Second, to analyze the effects of neonatal chemogenetic stimulation of oxytocin neurons on gene expressions in the PVH in later young adulthood, we utilized male OT-Cre; stop-hM3 mice at 5–7 weeks of age that were embryonically exposed to VPA and CNO (or saline as a control) at PND 2. We collected nuclei from PVH areas and subjected them to the 10X Genomics Chromium platform.

first: T

last: Masafumi

first: G

last: Teppei

first: I

last: Satsuki

first: H

last: Mitsue

first: M

last: Kazunari


title: Phage-Antibiotic Combinations Against Klebsiella pneumoniae

status: Public on Sep 14 2023

city: Shenzhen

first: xing

last: shi

address: Luohu

department: Department of Respiratory and Critical Care Medicine

institute: Shenzhen Institute of Respiratory Disease

summary: hvKP ATCC43816 and its lytic phage H5 were employed as a phage-antibiotic combination model. Based on the comprehensive characterization of phages, including cryo-electron microscopy, we evaluated the synergic effect of H5 on bacterial killing in vitro when combined with multiple antibiotics, and analyzed the advantages of phage-antibiotic combinations from an evolutionary perspective and proposes a novel PAS mechanism by using ceftazidime as an example.

overall_design: To investigate the underlying resistance mechanisms of Kpn 43816 against single phage stress and phage-antibiotic combination stress, we performed RNA-Seq analysis on four strains under different stress conditions: CP17, which is resistant to the phage-antibiotic combination but shows no mutations in the genome; CP6, which is resistant to the phage-antibiotic combination and contains a wcaJ mutation; P2 and P13, which are resistant to phage only and contain wcaJ mutations; and wild-type strains.

first: Kai

last: Zhou

first: Kunhao

last: Qin

first: Xing

last: Shi


title: Nuclear release of eIF1 restricts start-codon selection during mitosis

status: Public on Dec 02 2023

city: Cambridge

first: Jimmy

last: Ly

state: Massachusetts

address: 455 main street

department: Biology

institute: Whitehead Institute

summary: Regulated start-codon selection has the potential to reshape the proteome through the differential production of upstream open reading frames, canonical proteins, and alternative translational isoforms. However, conditions under which start codon selection is altered remain poorly defined. Here, using transcriptome-wide translation-initiation-site profiling, we reveal a global increase in the stringency of start-codon selection during mammalian mitosis. Low-efficiency initiation sites are preferentially repressed in mitosis, resulting in pervasive changes in the translation of thousands of start sites and their corresponding protein products. This enhanced stringency of start-codon selection during mitosis results from increased association between the 40S ribosome and the key regulator of start-codon selection, eIF1. We find that increased eIF1–40S ribosome interaction during mitosis is mediated by the release of a nuclear pool of eIF1 upon nuclear envelope breakdown. Selectively depleting the nuclear pool of eIF1 eliminates the change to translational stringency during mitosis, resulting in altered synthesis of thousands of protein isoforms. In addition, preventing mitotic translational rewiring results in substantially increased cell death and decreased mitotic slippage in cells that experience a mitotic delay induced by anti-mitotic chemotherapies. Thus, cells globally control stringency of translation initiation, which has critical roles during the mammalian cell cycle in preserving mitotic cell physiology.

overall_design: Paired translation initiation site profiling, elongating ribosome profiling, and RNA sequencing data for synchronized interphase, mitotic arrested, and cycling mitotic HeLa cells. Biological replicates were defined as separate cell synchronization, lysate preparation, and fractionation. N=2 biological replicates were performed each cell cycle stage.

first: Kehui

last: Xiang

first: David

last: Bartel

first: Iain

middle: M

last: Cheeseman


title: The SWI/SNF nucleosome remodeler constrains enhancer activity during Drosophila wing development [anti-GFP Cut & Run]

status: Public on Oct 03 2023

city: Chapel Hill

first: Matthew

last: Niederhuber

state: NC

address: 250 Bell Tower Dr.

institute: UNC-Chapel Hill

summary: Chromatin remodeling is central to the dynamic changes in gene expression that drive cell fate determination. During development, the sets of enhancers that are accessible for use change globally as cells transition between stages. While transcription factors and nucleosome remodelers are known to work together to control enhancer accessibility, it is unclear how the short stretches of DNA that they individually unmask yield the kilobase-sized accessible regions characteristic of active enhancers. Here, we performed a genetic screen to investigate the role of nucleosome remodelers in control of dynamic enhancer activity. We find that the Drosophila SWI/SNF complex, BAP, is required for repression of a temporally dynamic enhancer, brdisc. Contrary to expectations, we find that the BAP-specific subunit Osa is dispensable for mediating changes in chromatin accessibility between early and late stages of wing development. Instead, we find that Osa is required to constrain the levels of brdisc activity when the enhancer is normally active. Genome-wide profiling reveals that Osa directly binds brdisc as well as thousands of other developmentally dynamic regulatory sites, including multiple genes encoding components and targets of the Notch signaling pathway. Transgenic reporter analyses demonstrate that Osa is required for activation and for constraint of different sets of target enhancers in the same cells. Moreover, Osa loss results in hyperactivation of the Notch ligand Delta and development of ectopic sensory structures patterned by Notch signaling early in development. Together, these findings indicate that proper constraint of enhancer activity is necessary for regulation of dose-dependent developmental events.

overall_design:
anti-GFP CUT&RUN from third larval wandering wing discs in Osa-GFP and yw females.

UPDATE: [11-05-2024] The Sample titles were corrected.

last: Leatham-Jensen

first: D

last: McKay


title: Endogenous expression of inactive lysine deacetylases KDAC6 (HDAC6) and KDAC8 (HDAC8)

status: Public on Sep 08 2023

city: New Orleans

first: Terry

last: Watt

address: 1 Drexel Dr.

institute: Xavier University of Louisiana

summary: To investigate the roles of KDAC6 and KDAC8, we generated cell lines containing point mutations in HT1080 cells that result in endogenous expression of a catalytically-inactive variant. Expression profiling analysis was then performed on the resulting cell lines vs. wild HT1080 cells and cells treated with KDAC inhibitors tubastatin A or PCI-34051.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for HT1080 cells, variants containing inactivated KDACs, and inhibitor treatments with 3-4 replicates

first: Tasha

last: Toro

first: Elena

last: Skripnikova


title: Transcriptomic and epigenetic responses unveil resistance mechanisms to Phytophthora sansomeana in soybean [RNA-seq]

status: Public on Nov 24 2023

first: Meixia

last: Zhao

summary: Phytophthora root and stem rot (PRR) caused by oomycete pathogens in the Phytophthora genus poses a significant threat to soybean productivity. While resistance mechanisms against Phytophthora sojae have been extensively studied, the molecular basis underlying immune responses to the newly recognized pathogen, Phytophthora sansomeana, remains largely unknown. In this study, we investigated transcriptomic and epigenetic responses of two resistant (Colfax and NE2701) and two susceptible (Williams 82 and Senaki) soybean lines at four time points (2, 4, 8, and 16 hours post inoculation, hpi) after P. sansomeana inoculation. Through comparative transcriptomic analyses, we identified a greater number of differentially expressed genes (DEGs) upon pathogen inoculation in the resistant lines compared to the susceptible lines, predominantly at 8 and 16 hpi. These DEGs were associated with multiple phytohormones, including ethylene, salicylic acid, and jasmonic acid, along with various transcription factors and signaling cascade proteins. Moreover, DE transposable elements (TEs) were predominantly up-regulated after inoculation, and we found that TEs differentially transcribed in a resistant line were enriched near genes. Notably, we identified a long non-coding RNA (lncRNA) that was significantly differentially transcribed after inoculation exclusively in the resistant lines, potentially regulating two flanking LURP-one-related genes, known as key defense mediators in Arabidopsis against oomycete pathogens. Furthermore, DNA methylation analysis revealed increased CHH (H = A, T, or C) methylation levels in lncRNAs after inoculation, with delayed responses in the resistant line compared to the susceptible line. Overall, our results provide insights into the molecular mechanisms underlying resistance to P. sansomeana in soybean and underscore the potential role of lncRNAs and epigenetic regulation in plant defense responses.

overall_design: We investigated transcriptomic responses of two resistant (Colfax and NE2701) and two susceptible (Williams 82 and Senaki) soybean lines at four time points (2, 4, 8, and 16 hours post inoculation, hpi) after P. sansomeana inoculation.

first: Gwonjin

last: Lee


title: Transcriptomic and epigenetic responses unveil resistance mechanisms to Phytophthora sansomeana in soybean [WGBS]

overall_design: We investigated epigenetic responses of a resistant (Colfax) and susceptible (Williams 82) soybean lines at two time points (4, and 16 hours post inoculation, hpi) after P. sansomeana inoculation.

first: Charlotte

last: N DiBiase2


title: Multimodal gene expression and chromatin accessibility profiles at the single-cell level of synovial tissue from individuals with arthritis

status: Public on Sep 27 2023

first: Anika

last: Gupta

address: 95 Dunster St, 110 Kirkland Mail Center

institute: Harvard University

summary: In this study, we sought to understand the cell states in which autoimmune disease heritability is enriched. To do so, we performed single-nucleus multimodal sequencing of cells in the inflamed synovial tissue of individuals with arthritis (11 with rheumatoid arthritis and 1 with osteoarthritis). We measured both gene expression levels and chromatin peak accessibility levels. We found that dynamically accessible chromatin peaks were disproportionately enriched for the heritability of 14 autoimmune and allergic conditions and pointed to specific RNA-defined cell states where this signal was prevalent.

overall_design:
Synovial tissue samples from 11 RA patients and 1 OA patient were collected from Brigham and Women’s Hospital (BWH) and the Hospital for Special Surgery (HSS) for use in the multimodal single-nucleus RNA + ATAC experiments. Histologic sections of RA synovial tissue were examined, and samples with inflammatory features were selected in both cases.
***Raw sequencing data are available through dbGaP (controlled access) due to patient privacy concerns. Accession number: phs003417.v1***

first: Gupta

last: Anika

first: Weinand

last: Katy

first: Donlin

last: Laura

first: Wei

last: Kevin

first: Raychaudhuri

last: Soumya


title: CRISPR-edited human ES-derived oligodendrocyte progenitor cells improve remyelination in rodents

status: Public on Aug 23 2023

city: Edinburgh

first: Nadine

last: Bestard-Cuche

address: 5 Little France Drive

country: United Kingdom

institute: University of Edinburgh

summary: In Multiple Sclerosis (MS), inflammatory demyelinated lesions in the brain and spinal cord lead to neurodegeneration and progressive disability. Remyelination can restore fast saltatory conduction and neuroprotection but is inefficient in MS especially with increasing age, and is not yet improvable with therapies.  Intrinsic and extrinsic inhibition of oligodendrocyte progenitor cell (OPC) function contributes to remyelination failure, and we hypothesised that the transplantation of ‘improved’ OPCs, genetically edited to overcome these obstacles, could improve remyelination. Here, we edited human(h) embryonic stem cell-derived OPCs to be unresponsive to a chemorepellent released from chronic MS lesions, and transplanted them into rodent models of chronic lesions. Edited hOPCs displayed enhanced migration and remyelination compared to controls, regardless of the host age and length of time post-transplant. This study demonstrates that genetic manipulation and transplantation of hOPCs overcomes the negative environment inhibiting remyelination, with translational implications for therapeutic strategies for people with progressive MS.

overall_design: We deleted Neuropilin-1 (NRP1) from hESC, as the receptor for SEMA3A. We used CRISPR/Cas9 to generate our NRP1 knock out line in GFP+ hESCs with gRNAs targeting exon 6 of NRP1 to generate a complete knock out of NRP1. The control line are the GFP+hESCs cells

first: Laura

last: Wagstaff

first: Maja

last: Kaczmarek

first: Antonella

last: Fidanza

first: Lorraine

last: McNeil

first: Robin

last: Franklin

first: Anna

middle: C

last: Williams


title: SARS-CoV-2 mouse adaptation generates a gain of function that causes TNF driven age-dependent severe disease with human correlates

city: Melbourne

first: Dylan

last: Sheerin

state: VIC

address: 1G Royal Parade

country: Australia

institute: WEHI

summary:
To dissect the nature of the immune dysregulation induced by SARS-CoV-2 infection in mouse lungs using mouse-adapted strains of the virus.
Comparisons were performed using bulk RNA-seq data from mouse (C57BL/6) lung homogenates taken 3 days post-infection with mouse-adapted SARS-CoV-2, both early and late (virulent) passages, or mock-infected mice.

overall_design: Comparative gene expression profiling analysis of murine lung bulk RNA-seq data. Contrasts were made between mice infected with early and late passage virus strains and mock-infected mice. Comparisons were also made between young and aged mice to determine the associated between age and these transcriptional changes.

first: Stefanie

last: Bader


title: SwrA extends DegU over an UP element to activate flagellar gene expression in Bacillus subtilis

status: Public on Aug 15 2023

city: Bloomington

first: Ayushi

last: Mishra

state: Indiana

address: Biology Building, BB432, 1001 E 3rd St

institute: Indiana University

summary: SwrA activates flagellar gene expression in Bacillus subtilis to increase the frequency of motile cells in liquid and further increase flagellar density to swarm over solid surfaces.  Here we perform ChIP-seq and demonstrate that SwrA interacts with many sites on the chromosome indirectly through the response regulator DegU.  We identify a DegU-specific inverted repeat and show that SwrA increased DegU DNA binding affinity in parallel to phosphorylation.  Moreover, we show that SwrA increased the size of the DegU footprint expanding the region of protection towards the promoter.  The remote location of the DegU inverted repeat was critical such that relocating the binding site by full turns of DNA impaired activation by SwrA/DegU and moving the binding site closer to the promoter severely impaired transcription.  We conclude that SwrA/DegU forms a heteromeric complex that enables both remote binding and activator-RNA polymerase interaction in the context of an interceding UP element.  We speculate that multimeric activators that resolve similar cis-element spatial conflicts are common in bacteria such as the diverse master activators of flagellar biosynthesis and those that activate long operons of other multi-subunit complexes.

sample_organism: Bacillus subtilis subsp. subtilis NCIB 3610 = ATCC 6051 = DSM 10

overall_design:
Chromatin Immunoprecipitation Sequencing (ChIP-Seq): Bacillus subtilis cultures were grown to an OD600 of 1.0 at 37°C with constant rotation. 20 ml of cells were cross-linked for 30 minutes at room temperature using 3% formaldehyde (Sigma), quenched with 125 mM glycine, washed with PBS, and then lysed. DNA was sheared to an average fragment size of ~200 bp using Qsonica sonicator (Q8000R), and then incubated overnight at 4ºC with -SwrA or -DegU as indicated. Immunoprecipitation was performed using Protein A Magnetic Sepharose beads (Cytiva #45002511), washed, and DNA was eluted in TES (50mM Tris pH8, 10mM EDTA and 1% SDS). Crosslinks were reversed overnight at 65ºC. DNA samples were treated with a final concentration of 0.2mg/ml RNaseA (Promega #A7973) and 0.2mg/ml Proteinase K (NEB #P8107S) respectively, and subsequently extracted using phenol/chloroform/isoamyl (25:24:1). DNA samples were then used for library preparation using NEBNext UltraII DNA library prep kit (NEB #E7645L). Paired end sequencing of the libraries was performed using Illumina NextSeq 550 platform and atleast 3 million paired end reads were obtained for each sample.  Two or three biological replicates were sequenced for each sample.
          Whole genome sequencing (WGS): Bacillus subtilis cultures were grown to an OD600 of 1.0 at 37°C with constant rotation and 5ml of cells were collected, pelleted and DNA was extracted using Qiagen DNeasy kit (#69504). Genomic DNA was sonicated using Qsonica sonicator (Q8000R) and the sonicated DNA was used to prepare libraries using the NEBNext UltraII DNA library prep kit (NEB #E7645L). Paired end sequencing of the libraries was performed using Illumina NextSeq 550 platform and atleast 3 million paired end reads were obtained for each sample.  Data from WGS was used as input for the ChIP.
          Analysis of ChIP-Seq and WGS data: Sequencing reads for both ChIP and WGS were mapped individually to B. subtilis 3610 genome (CP020102) (Nye et al, 2017) using CLC Genomics Workbench software (Qiagen). The enrichment at ribosomal RNA locations were eliminated and the number of reads mapped to each base pair in the genome was exported into a .csv file. Data was normalized to the total number of reads and fold enrichment was calculated as the ratio of number of reads at each genome location in ChIP-Seq and WGS (ChIP/input). Analysis was performed and graphs were plotted in 1kb bins to show enrichment across the entire genome using custom R-scripts. When required, individual peaks were plotted in 10bp bins across a 4kb range centered around the peak summit. Detailed protocols and scripts are available upon request.

first: Daniel

last: Kearns


title: A critical role for Hepatocyte Nuclear Factor 4 alpha in polymicrobial sepsis-associated metabolic reprogramming and death: ATAC-seq after CLP sepsis model

first: Steven

last: Timmermans

overall_design: Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) tot determine the changes in chromatin accessibility in the liver of mice after CLP using 4 biological replicates in CLP and SHAM (control) conditions

first: Céline

last: Van Dender

first: Ville

last: Paakinaho

first: Tineke

last: Vanderhaeghen

first: Jolien

last: Vandewalle

first: Maarten

last: Claes

first: Bart

last: Roman

first: Jan

last: De Waele

first: Siska

last: Croubels

first: Karolien

last: De Bosscher

first: Jorma

last: Palvimo

first: Claude

last: Libert


title: In situ transcriptomic analysis of spermatocytes in non-obstructive azoospermia reveals senescence-like states in arrested spermatocytes

status: Public on Aug 02 2023

city: Shanghai

first: Wu

last: zhongqin

address: No.800 Dongchuan Road, Minhang District

institute: Shanghai Jiao Tong University

summary: To investigate in situ transcriptome  of spermatocytes in non-obstructive azoospermia , we microdissected spermatocytes in formalin-fixed paraffin-embedded testicular biopsy tissue from two types of non-obstructive azoospermia and obstructive azoospermia as a control, and then constructed cDNA libraries based on Smart-3SEQ method.

overall_design: Comparative gene expression profiling analysis and PCA analysis of RNA-seq data for spermatocytes of  two types of non-obstructive azoospermia (SP_NOA & rST_NOA) and obstructive azoospermia (OA), which had normal spermatogenesis as the case control. SP_NOA group, rST_NOA group and OA group respectively consist of three patients' RNA-seq data of spematocytes from their FFPE testicular biopsy tissues.

first: Zhongqin

last: Wu

first: Xiaolong

first: Zeqian

last: Xu

first: Yan

last: Guo


title: YTHDC1 regulate hematopoietic stem cell survival and regeneration

status: Public on Jul 01 2023

city: hangzhou

first: Hu

last: Wang

state: zhejiang

address: yuhangtang road

institute: Hangzhou normal university

summary: we investigate the role of YTHDC1 in normal hematopoiesis and adult hematopoietic stem/progenitor cell (HSPC) maintenance in vivo. Utilizing conditional Ythdc1 knockout mice ,we show that YTHDC1 is required for hematopoietic stem cell (HSC) maintenance as well as self-renewal of HSC. Transcriptome analysis identified many differentially expressed genes (DEGs) between WT and YTHDC1-KO LT-HSCs.

overall_design: KO HSC

first: Zhenyu

last: Ju


title: Deep dynamical modelling of developmental trajectories with temporal transcriptomics [SCIFATE_PILOT_DATA]

status: Public on Jul 06 2023

city: London

first: Rory

middle: James

last: Maizels

address: 1 Midland Road

institute: The Francis Crick Institute

summary: Developmental cell fate decisions are dynamic processes driven by the complex behaviour of gene regulatory networks. A challenge in studying these processes using single-cell genomics is that the data provides only a static snapshot with no detail of dynamics. Metabolic labelling and splicing can provide time-resolved information, but current methods have limitations. Here, we present experimental and computational methods that overcome these limitations to allow dynamical modelling of gene expression from single-cell data. We developed sci-FATE2, an optimised metabolic labelling method that substantially increases data quality, and profiled approximately 45,000 embryonic stem cells differentiating into multiple neural tube identities. To recover dynamics, we developed velvet, a deep learning framework that extends beyond instantaneous velocity estimation by modelling gene expression dynamics through a neural stochastic differential equation system within a variational autoencoder. Velvet outperforms current velocity tools across quantitative benchmarks, and predicts trajectory distributions that accurately recapitulate underlying dataset distributions while conserving known biology. Velvet trajectory distributions capture dynamical aspects such as decision boundaries between alternative fates and correlative gene regulatory structure. Using velvet to provide a dynamical description of in vitro neural patterning, we highlight a process of sequential decision making and fate-specific patterns of developmental signalling. Together, these experimental and computational methods recast single-cell analyses from  descriptions of observed data distributions to models of the dynamics that generated them, providing a new framework for investigating developmental gene regulation and cell fate decisions.

overall_design: sci-FATE original protocol test, all mES differentiating celld, fixed in PFA, just to test protocol quality

first: Daniel

last: Snell

first: James

last: Briscoe


title: Deep dynamical modelling of developmental trajectories with temporal transcriptomics [BACKGROUND_DATA]

overall_design: here, in vitro differentiation cells collected from D3-D8 in one sample, sequenced using sci-RNA-seq, with no metabolic labelling. This data used to define SNPs and background mutation rate for mutation detection in metabolic labelling experiments


title: Deep dynamical modelling of developmental trajectories with temporal transcriptomics [PROTOCOL_PILOT_DATA]

overall_design: Different protocols (different chemical conversion protocols; different sci-RNA-seq protocols) are multiplexed within one sample and sequenced here using sci-FATE


title: The SWI/SNF nucleosome remodeler constrains enhancer activity during Drosophila wing development

first: Matthew

last: Niederhuber

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

overall_design: Refer to individual Series


title: Synthetic Viscoelastic Activating Cells for T Cell Engineering and Cancer Therapy

status: Public on Sep 12 2023

city: Los Angeles

first: Youcheng

last: Yang

state: California

address: 430 Westwood Plaza, 5121 Engineering V, Box 951600, Attn (PI NAME): Li Lab

department: Bioengineering

institute: UCLA

summary: Immunotherapy shows promise in cancer treatment, but its effectiveness and durability remain limited, and improved technology for engineering immune cells is necessary. Here we develop a scalable platform that creates synthetic viscoelastic activating cells (SynVACs) with programmable mechanical and chemical properties. We demonstrate that the viscoelastic nature of SynVACs significantly impacts T cell functionality. Compared to rigid or elastic microspheres, SynVACs greatly enhance human T cell expansion, achieving approximately 90% chimeric antigen receptor (CAR) transduction efficiency. Additionally, SynVACs promote CD8+ T cell generation while suppressing regulatory T cell formation, resulting in enhanced tumor killing capability. Notably, expanding CAR-T cells with SynVACs leads to a ten-fold increase in T memory stem cells (TMSCs). These engineered CAR-T cells exhibit superior efficacy in eliminating tumor cells in a human lymphoma and ovarian xenograft mouse model, persisting in vivo for longer time period. These findings underscore the crucial role of mechanical signals in T cell engineering and highlight SynVACs' potential in CAR-T therapy and imunoengineering applications.

overall_design: CAR-T cells activated by SynVACs and Dynabeads were cultured and harvested at day 14, followed by sorting with a FACSAria II flow cytometer. The sorted cells were immediately dispatched to the UCLA Technology Center for Genomics and Bioinformatics (TCGB) Core for single cell TCR sequencing. The sequencing was executed with a 10X Genomics Chromium™ Controller Single Cell Sequencing System, as per the manufacturer's guidelines and the TCGB Core's standard protocol. Library preparation was accomplished using the Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001), and the sequencing was performed with 150 bp paired-end reads (5,000 reads/cell) on an Illumina NovaSeq system. Lastly, the reads were mapped to the human T cell receptor reference genome (hg38) using Cell Ranger VDJ. This allowed for the visualization of the frequencies of alpha or beta chain recombination events in CAR-T cells activated by SynVACs and Dynabeads.

first: Zeyang

last: Liu

first: Yan-Ruide

last: Li

first: Yu

last: Zhu

first: Weihao

last: Yuan

first: Tyler

last: Hoffman

first: Yifan

first: Yichen

first: Ying

last: Fang

first: Xinyang

last: Ge

first: Jennifer

last: Soto

first: Lili

first: Song


title: AtDGCR14L contributes to salt-stress tolerance via regulating pre-mRNA splicing in Arabidopsis

status: Public on Sep 06 2023

city: Upton

first: Meng

last: Xie

state: NY

address: 50 Bell Ave

institute: Brookhaven National Laboratory

summary: In plants, pre-mRNA alternative splicing has been demonstrated to be a crucial tier that regulates gene expression in response to salt stress. However, the underlying mechanisms remain elusive. Here, we studied the roles of DIGEORGE-SYNDROME CRITICAL REGION 14-like (AtDGCR14L) in regulating pre-mRNA splicing and salt stress tolerance. We discovered that Arabidopsis AtDGCR14L is required for maintaining plant salt stress tolerance and the constitutively spliced and active isoforms of important stress- and/or abscisic acid (ABA)-responsive genes. We also identified the interaction between AtDGCR14L and splicing factor U1-70k, which needs a highly conserved three amino acid (TWG) motif in DGCR14. Different from wild-type AtDGCR14L, the overexpression of the TWG-substituted AtDGCR14L mutant did not change salt stress tolerance or pre-mRNA splicing of stress/ABA-responsive genes. Additionally, SWITCH3A (SWI3A) is a core subunit of the SWI/SUCROSE NONFERMENTING (SWI/SNF) chromatin-remodeling complexes. We found that SWI3A, whose splicing depends on AtDGCR14L, actively enhances salt stress tolerance. These results revealed that AtDGCR14L may play an essential role in crosstalk between plant salt-stress response and pre-mRNA splicing mechanisms. We also unveiled the potential role of SWI3A in controlling salt stress tolerance. The TWG motif in the intrinsically disordered region of AtDGCR14L is highly conserved and crucial for DGCR14 functions.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for Col-0, atdgcr14l-1 mutant, and its complementation line OE-6 (atdgcr14l-1 35S:AtDGCR14L) 0h and 24h after 200 mM NaCl treatment.

first: Xie

last: Meng


title: Deep dynamical modelling of developmental trajectories with temporal transcriptomics [EXPERIMENT_DATA]

first: Rory

overall_design: Four replicates from in vitro neural differentiation: replicates 1-3 collected on days 3,4,5,6,7,8. Replicate 4 collected between day 3 and 4 on 5 hour intervals (day 3 + 5, 10, 15, 20hr). All differentiations RA + 500nM SAG, ventral neural tube differentiation

first: Daniel

first: James


title: Single-Cell RNA sequencing reveals blood immune features of gout remission patients with monosodium urate crystal deposition.

status: Public on Jun 01 2023

first: Hanqing

last: Yu

state: Shanghai

address: No. 301 Middle Yanchang Road

institute: Shanghai Tenth People's Hospital

summary: As a prominent feature of gout, monosodium urate (MSU) crystal deposition can induce gout flare, yet its impact on blood immune in gout remission patients still remains unclear. In this study, single-cell RNA sequencing (scRNA-seq) is used to compare the gene expression profiling of peripheral blood mononuclear cells (PBMCs) among intercritical gout remission patients, advanced gout remission patients and healthy volunteers. The increase of HLA-DQA1high classical monocytes, and their important role in immune inflammatory responses and osteoclast differentiation are discovered in advanced gout remission patients. Moreover, the differentiation level of CD8+T cells are found to elevate in advanced gout remission patients, which is further validated via flow cytometry. It is also observed that pathways related to bone metabolism and inflammatory responses are overactive in advanced gout remission patients. By analysis on intercellular communication network, immune-related cell-cell interactions among PBMCs are shown to enhance in both intercritical and advanced gout remission patients. The analyses on gene expression and LC-MS/MS together indicate the increased metabolic level of arachidonic acid in gout remission patients with MSU deposition at the intercritical and advanced stage. The study reveals distinctive blood immune characteristics in gout remission with MSU deposition, which provides more sights into its pathogenesis.

overall_design: PBMCs from intercritical gout remission patients, advanced gout remission patients and healthy volunteers were subjected to scRNA-seq with 10x Genomics and validated by flow cytometry and LC-MS/MS.

first: Hongchen

last: Gu

first: Ling

last: Qin

first: Ai

last: Peng