title: FTO-dependent inhibition of ferroptosis in gastric cancer through epigenetic regulation of the HOXC8/MDM2/p53 signaling pathway

city: Nanjing

first: yingjie

last: Zhang

address: No.300 Guangzhou Road

institute: Department of Rehabilitation Medicine, the First Affiliated Hospital of Nanjing Medical University

summary: Only a few studies have attempted to explore the potential role of FTO in gastric cancer, with one focusing on mitochondrial metabolism, while others have focused on the association of FTO with cell proliferation, migration, and invasion. To date, no study has comprehensively linked FTO-dependent m6A methylation to any form of cell death. We comprehensively explore the role of FTO-mediated m6A modification in gastric cancer ferroptosis by MeRIP-seq.

overall_design: Examination of FTO-mediated m6A modifications in GC cell.

first: Min

first: Yini

last: Dang

first: Yingjie


title: Androgen Deprivation Alters the Transcriptome of Endothelial Cells More Dramatically in Human Prostate Cancer Tissue than in Benign Prostate Tissue

status: Public on Mar 03 2024

city: Buffalo

first: Yue

last: Wu

state: Ny

address: 665 Elm St

country: USA

institute: Roswell Park Cancer Institute Dept or Urology

type: Expression profiling by high throughput sequencing

summary: Purpose: Examine the cell type specific (Epithelial, Endothelial, and Stromal) transcriptome profile between prostate primary tissue xenografts of prostate tissue tumor tissue and uninvolved tissue (named non-tumor hereafter) in response testosterone (T) deprivation.  Additional cell type specific untreated fresh tissue from both tumor and non-tumor samples is included for additional validation.  Related samples previously uploaded to GEO are found under accession number GSE145641. Method: Nude-Fox1nu mice were castrated and silastic tubing containing dry T was inserted providing “humanized” levels of circulating T.  5-8 pieces of fresh human prostate tissue was then inserted subcutaneously along the flank of the animal.  For samples not subjected to T-deprivation, after 30 days the animal was euthanized, and tissue was harvested.  For samples undergoing T-deprivation, after 30 days with humanized circulating T levels, T-deprivation was performed by surgical removal of silastic tubing after which serum T levels dropped below the level of detection within 24 hours.  Samples were then harvested at Pre T-deprivation time point, an early T-deprivation time point (3-days) or late T-deprivation timepoint (15-17 days) post-surgery.  Tissue from fresh human prostate or tissue from xenograft and was then enriched into endothelial, epithelial and the remaining stromal compartments using magnetic bead enrichment.  RNA-Seq was then preformed on cell types from both origins and the results compared to determine the cell type specific effects of T-deprivation on the human prostate transcriptome.

overall_design: This data set contains 127 samples from 24 individuals across three cell types (Endothelial, Epithelial, and Stroma) across two tissue types (tumor and non-tumor) and in both Fresh prostate Xenografts and fresh prostate tissue.  Samples from fresh porstate xenografts were subjected to T-deprivation and harvested at one of three timepoints (Pre T-deprivation, Early T-deprivation (3 days), or late T-deprivation (15-17 days))

first: Nelson

middle: T

last: Gross

first: Jianmin

last: Wang

first: Eduardo

middle: C

last: Gomez

first: Alejandro

middle: S

last: Godoy

first: Li

last: Tang

first: Gary

last: Smith


title: CFDP1 regulates the stability of pericentric heterochromatin thereby affecting RAN GTPase activity and mitotic spindle formation

status: Public on Mar 02 2024

city: Rochester

first: Thomas

middle: Gustav Heinrich

last: Diekwisch

state: NY

address: 601 Elmwood

department: Oral and Craniofacial Sciences

institute: Eastman Institute for Oral Health

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The densely packed centromeric heterochromatin at minor and major satellites is comprised of H3K9me2/3 histones, the heterochromatin protein HP1α and histone variants.  In the present study we sought to determine the mechanisms by which condensed heterochromatin at major and minor satellites stabilized by the chromatin factor CFDP1 affects the activity of the small GTPase Ran as a requirement for spindle formation.  CFDP1 co-localized with heterochromatin at major and minor satellites and was essential for the structural stability of centromeric heterochromatin.  Loss of CENPA, HP1α and H2A.Z heterochromatin components resulted in decreased binding of the spindle nucleation facilitator RCC1 to minor and major satellite repeats.  Decreased RanGTP levels as a result of diminished RCC1 binding interfered with chromatin-mediated microtubule nucleation at the onset of mitotic spindle formation.  Rescuing chromatin H2A.Z levels in cells and mice lacking CFDP1 through knock-down of the histone chaperone ANP32E not only partially restored RCC1 dependent RanGTP levels but also alleviated CFDP1-knockout related craniofacial defects and increased microtubule nucleation in CFDP1/ANP32E co-silenced cells.  Together, these studies provide evidence for a direct link between condensed heterochromatin at major and minor satellites and microtubule nucleation through the chromatin protein CFDP1.

overall_design: Chromatin immunoprecipitation followed by DNA sequencing (ChIP seq) using anti-FLAG antibody and anti-H2A.Z antibody in control siRNA, CFDP1 siRNA and FLAG-CFDP1 overexpressing NIH3T3 cells.

first: Gokul

last: Gopinathan

first: Qian

last: Xu

first: Luan

last: Xianghong

middle: G


title: Effects of Cryo-thermal therapy  on gene expression in neutrophils and monocytes

city: Shanghai

first: Shicheng

address: 1954 Huashan Road, Xuhui District, Shanghai

country: China

institute: Shanghai Jiao Tong University

summary: The characteristics of the tumor immunosuppressive microenvironment represent a major challenge limiting the efficacy of immunotherapy. Our previous results suggested that cryo-thermal therapy (CTT), a tumor ablation system developed in our laboratory, could promote macrophage M1-type polarization and full maturation of DCs to remodel the immunosuppressive environment. However, it is not clear which cells respond promptly to CTT. CTT can cause extensive cell death and the release of danger-associated molecular patterns (DAMPs) and antigens. Neutrophils are the first white blood cells recruited to sites of damage and acute inflammation. We therefore hypothesized that neutrophils were the initial cells to respond to CTT and were involved in the subsequent establishment of antitumor immunity. We found that CTT led to a rapid and strong proinflammatory neutrophil response, which was essential for the long-term survival of mice. In vivo neutrophil depletion and in vitro coculture experiments demonstrated that CTT-induced neutrophils promoted the differentiation of monocytes toward to mature antigen presenting cells and further upregulated the expression of IFN-γ and cytotoxic molecules in T and NK cells in a CD86-dependent manner. These results reveal the important role of neutrophil-monocyte interactions for the development of anti-tumor immunity and highlight that CTT could be used as an immunotherapy for targeting neutrophils and monocytes to enhance antitumor immunity.

overall_design: Neutrophils in the spleen from tumor-bearing mice and mice at 12 h after CTT were labeled with PE anti-Ly6G and isolated  using EasySep Mouse PE Positive Selection Kit II. Monocytes in the spleen from mice 5 days after CTT  with or without anti-Ly6G treatment were labeld with  PE anti-CCR2 and isolated using EasySep Mouse PE Positive Selection Kit II. The RNA-seq analysis on neutrophils and monocyes was performed.


title: Non-linear transcriptional responses to gradual modulation of transcription factor dosage

city: New York

first: Julia

last: Domingo

address: 101 6th Av

institute: New York Genome Center

summary: Genomic loci associated with common traits and diseases are typically non-coding and likely impact gene expression, sometimes coinciding with rare loss-of-function variants in the target gene. However, our understanding of how gradual changes in gene dosage affect molecular, cellular, and organismal traits is currently limited. To address this gap, we induced gradual changes in gene expression of four genes using CRISPR activation and inactivation. Downstream transcriptional consequences of dosage modulation of three master trans-regulators associated with blood cell traits (GFI1B, NFE2, and MYB) was examined using targeted single-cell multimodal sequencing. We showed that guide tiling around the TSS is the most effective way to modulate cis gene expression across a wide range of fold-changes, with further effects from chromatin accessibility and histone marks that differ between the inhibition and activation systems. Our single-cell data allowed us to precisely detect subtle to large gene expression changes in dozens of trans genes, revealing that many responses to dosage changes of these three TFs are non-linear, including non-monotonic behaviours, even when constraining the fold-changes of the master regulators to a copy number gain or loss. We found that the dosage properties are linked to gene constraint and that some of these non-linear responses are enriched for disease and GWAS genes. Overall, our study provides a straightforward and scalable method to precisely modulate gene expression and gain insights into its downstream consequences at high resolution.

overall_design: ECCITE-seq experiments obtaining three different modalities from a single cell: Protein Hashes (HTO) to identify CRISPRi or CRISPRa cell line of origin, cDNA as measurement of RNA levels and sgRNAs (GDO) to ienditfy the unique loci perturbation

first: Júlia

first: Mariia

last: Minaeva

first: John

middle: A

last: Morris

first: Marcello

last: Ziosi

first: Neville

middle: E

last: Sanjana

first: Tuuli

last: Lappalainen


title: Immune dysfunction mediated by the competitive endogenous RNA network in fetal side placental tissue of polycystic ovary syndrome

city: Hangzhou

first: Ningning

last: Xie

address: 1 Xueshi Road

institute: Women’s Hospital, School of Medicine, Zhejiang University

summary: Polycystic ovary syndrome (PCOS) is a commonly occurring endocrine and metabolic disorder that impacts women in their reproductive years. Emerging evidence suggests that the maternal-fetal immune system is crucial for proper pregnancy. However, whether immune function is altered during pregnancy in PCOS women and the underlying molecular mechanisms is currently unexplored. Herein, the basic maternal immune system was investigated (n = 136 in the control group; n = 103 in the PCOS group), and whole-transcriptome sequencing was carried out to quantify the mRNAs, miRNAs, and lncRNAs expression levels in fetal side placental tissue of women with PCOS. GO, KEGG, and GSEA analysis were employed for functional enrichment analysis. The process of identifying hub genes was conducted utilizing the PPI network. CIBERSORT and Connectivity Map were deployed to determine immune cell infiltration and predict potential drugs, respectively. A network of mRNA-miRNA-lncRNA was constructed and then validated by qRT-PCR.

overall_design: Whole-transcriptome sequencing was carried out to quantify the mRNAs, miRNAs, lncRNAs, and circRNAs expression levels in fetal side placental tissue of women with PCOS.

first: Fangfang

first: Danqing

last: Chen

first: Jue

last: Zhou

first: Jian

first: Fan

last: Qu


title: KDM4 histone lysine demethylases maintain the adrenergic core regulatory circuitry programed by MYCN in neuroblastoma

city: Memphis

first: Hongjian

last: Jin

state: TN

address: 262 Danny Thomas Place

department: Center for Applied Bioinformatics

institute: St Jude Children's Research Hospital

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

overall_design: Refer to individual Series


title: Next generation sequencing facilliates quantitative analysis of Wild type and KitW/Wv Smooth muscle cells transcriptomes

city: Miami

first: Anthony

last: Griswold

state: FL

address: 1501 NW 10th Ave, BRB 318

institute: University of Miami

summary: We extracted RNA from primary smooth muscle cells from five KitW/WV mice and five Wild type mice and used next generation RNA sequenceing to compare the difference in gene expression profiles

overall_design: Examination of differential gene expression in vascular smooth muscle cells when the cells lose c-Kit signaling

first: Zachary

last: Zigmond

first: Laisel

last: Martinez

first: Roberto

last: Vazquez-Padron


title: Cas13bt3 knockdown of VEGFA mRNA in retinal organoid [scRNA-Seq]

status: Public on Mar 01 2024

city: Melbourne

first: Guei-Sheung

last: Liu

address: 32 Gisborne Street

country: Australia

institute: Centre for Eye Research Australia

summary: Purpose: We report expression of a new Cas13 enzyme in retinal cells and evaluate its efficacy in targeting the VEGFA mRNA to establish safety and efficacy of the enzyme got anti-VEGFA therapy. Methods: Three retinal organoids were cultured and treated, (1) Untreated, (2) AAV2.7m8.Cas13bt3.VEGFA.sgRNA, (3) AAV2.7m8.Cas13bt3.NTsgRNA.  Single-cell RNA sequencing was performed to determine expression of Cas13bt3 across retinal cell types, and the corresponding VEGFA mRNA expression. Results: All retinal cell showed observable transduction and RPE cells were found most susceptible to infection, while bipolar cells had the least infection rate. Significant silencing of VEGFA was observed in RPE cells. Conclusions: AAV2.7m8 can be used for retina-wide transduction, and Cas13bt3 may be a potential new tool for control of VEGFA.

overall_design: Examination of Cas13bt3 transduction and anti-VEGFA potential in mature 3D retinal orgnaoids.

first: Liu

last: Guei-Shueng


title: Cas13bt3 knockdown of VEGFA mRNA in mammalian cells and mouse retina [RNA-Seq]

summary: Purpose: We report expression of a new Cas13 enzyme in retinal cells and evaluate its efficacy in targeting the VEGFA mRNA to establish safety and efficacy of the enzyme for anti-VEGFA therapy. Methods: Mammalian cells (HEK293FT) or mouse retina were treated with shRNA, CasRx or Cas13bt3 carrying VEGFA targeting sgRNA. Total RNA was collected for RNA seqeuencing to study VEGFA mRNA expression and well as off-target genes. Results: Significant knockdown of VEGFA mRNA was observed with no effect on predicted off-target genes in mammalina cells and mouse retina. No loss of mouse VEGFA was also detected. Conclusions: AAV2.7m8 can be used for retina-wide transduction, and Cas13bt3 may be a potential new tool for control of VEGFA.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Examination of Cas13bt3 efficiency and transduction and anti-VEGFA potential in mammalian cells and mouse retinae.


title: Histone lysine demethylase 4 family proteins maintain the transcriptional program and adrenergic cellular state of MYCN-amplified neuroblastoma


title: Human serum: Control vs. Lung Cancer

city: Foshan

first: He

middle: Yan

last: Bin

state: GUANGDONG

address: Sino-European Center A, 2 Lingnan Avenue South, Shunde, Foshan, Guangdong, China

institute: BCBIO

summary: The autoantibodies in serum (IgG antibody labeled with Cy3 and IgM antibody labeled with Cy5) were quantified by serum incubation human protein microarray, and the data of normal people and lung cancer patients were compared to find the predictive markers of autoantibodies in lung cancer

overall_design: A two-stage design experiment with two conditions, LungCancer vs. Normal serum sample. Phase I Biological replicates: 8 control, 8 Cancer.Phase II  Biological replicates: 60 Negative control,60 Disease control, 60 Cancer. One replicate per array.

first: Yanbin

last: He

first: Shucai

last: Wu


title: Landscape of TFs binding in mouse early embryos [ChIP-seq]

first: Gran

last: Young

state: --- Select a state ---

address: 1239th, Siping Road, Yangpu district

institute: Tongji University

summary: In mammals, early lineage specification originates from the establishment of cell polarization in early embryos, before providing the foundation for all somatic development. Although there have been heated debates about whether transcription factors (TFs) or epigenetic information plays the dominant role in cell fate decisions, the precise annotation of TFs remains largely elusive in early embryos, restricting the functional study of developmentally important TFs. Here, we investigated the genome-wide landscapes of two known triggers of mouse embryo polarization: transcription factor AP-2 gamma (Tfap2c) and TEA domain transcription factor 4 (Tead4). We found that Tfap2c can bridge the connection between the promoter and the proximal enhancer, and further facilitate the functions of downstream Tead4 and Klf5 in boosting polarization. Our study also implicated that Rarg-initiated active demethylation might be a prerequisite for proper Tfap2c functioning. Furthermore, the results demonstrated that both genomic imprinting and nucleotide polymorphism can instruct TF binding, leading to allele-specific expression in early embryos. Overall, our study characterized TF dynamics during the peri-implantation stage and revealed their spatiotemporal interactions with the epigenetic environment in driving polarization and lineage specification.

overall_design: Histone modifications comparison in 8-cell stage among samples w/ or w/o Tfap2c interference.

first: Guang

last: Yang

first: Rui

last: Gao

first: Shaorong

first: Cizhong

last: Jiang

first: Jiayu


title: Loss-of-function mutation of Plastid Terminal Oxidase (ZmPTOX) cause zebra leaf and white kernel by affecting plastid development in maize

city: Chengdu

first: Qiang

last: Huang

state: SC

address: No.211, Huimin Road, Wenjiang District

institute: sichuan agriculture university

summary: Zebra leaf is a special phenotype of variegated leaf in monocotyledonous plant. Here, we characterized a mutant zebra10 (zb10) in maize, which displays defective chloroplast development in white section of leaves at the seedling stage and pale-white kernels. Map based cloning revealed that ZB10 encodes plastid terminal oxidase (ZmPTOX) which is localized in chloroplasts. ZmPTOX protein is highest expressed in leaves and also highly expressed in endosperm. Expression and metabolism analysis showed that function-loss of PTOX interferes with carotenoid synthesis and chloroplast biogenesis by affecting phytoene desaturase activity. In-depth observation showed that crossband formation of zebra leaf could be related to photoperiodic rhythm, and expression of alternative oxidases 2 (ZmAOX2) could be regulated by circadian rhythm and light intensity in zb10. Further studies revealed ectopic expression of ZmAOX2 protein can function in chloroplasts and rescue the defective phenotype of im. We conclude that ZmPTOX plays an vital role in carotenoid synthesis and plastid biogenesis in maize. ZmAOX2 involve in formation of zebra crossbands with diurnal rhythm and green-revertible process of leaves in zb10.

overall_design: Leaves of WT and zb10 at 4th-leaf stage [WT_V4, zb10_V4G (green section),  zb10_V4W (white section)] and 9th-leaf stage (WT_V9, zb10_V9) and 16DAP endosperms (WT_16d endo,  zb10_16d endo) were selected for transcriptome sequencing samples (three biological replicates per tissue).  To compare the effect of ZB10 gene mutation on the expression of carotenoid synthesis pathway genes and AOX family genes in maize.

first: Zhuofan

last: Zhao

first: Xiaowei

first: Moju

last: Cao


title: Transcriptome profiling of T24 cells treated with Momordica Charantia MAP30

city: su zhou shi

first: Jin

last: Wang

state: None Selected

address: 199 Renai road, Suzhou industrial park, Dushu Lake Campus of Soochow University

institute: Suzhou University

summary: Bladder cancer (BC) is notably prevalent, particularly among men. Emerging evidence highlights that traditional Chinese medicine (TCM) has distinct anticancer properties. This study focuses on recombinant MAP30, a key active constituent of bitter melon extract, recognized for its antiviral, immunomodulatory, and antitumor effects. We discovered that MAP30 suppresses BC cell proliferation and induces apoptosis in a dose-responsive manner. It also hinders cell migration, as evidenced by Transwell and wound healing assays. In nude mice, MAP30 injections adjacent to tumors significantly curtail tumor growth. Molecular analyses after MAP30 exposure show elevated SA-β-Gal activity and increased expression of cell cycle inhibitors p21 and p16 in BC cells. Integrating TCGA BC data, MAP30 appears to correct dysregulated gene expression associated with the cell cycle, senescence, apoptosis, and key pathways such as FOXO, TNF, and MAPK. Proteomic analysis identifies CENPA as a central molecule inhibited by MAP30. Correlation studies reveal CENPA’s significant association with oncogenic and immune-modulatory gene expression and immune cell infiltration. CENPA knockdown diminishes BC cell growth and motility, while its overexpression in MAP30-treated cells reverses these effects, suggesting CENPA’s pivotal role in MAP30’s anticancer activity and its potential as a therapeutic target. In conclusion, recombinant MAP30 effectively hampers bladder cancer cell proliferation and migration via CENPA downregulation and induces apoptosis and senescence, offering therapeutic potential against bladder cancer.

overall_design: Bladder cancer cell T24 were treated with MAP30 at a concentration of 10 μg/ml and collected at 48 hours. There are three replicates for the control group and the treatment group, respectively

first: Shaoqi

last: Zhang

first: Kun

last: Lu

last: Chao

first: Xiangyu

first: Longjun

last: Cai

first: Jianjun

first: Conghui

last: Han


title: FTO suppresses cardiac fibrosis after myocardial infarction via m6A-mediated epigenetic modification

first: Jian

address: Pujian Load 160th, Pudong New Aera

institute: Shanghai Jiao Tong University School of Medicine

summary: Cardiac fibrosis is common in cardiovascular diseases. N6-methyladenosine (m6A) is one of the most common modifications in eukaryotic mRNAs. Previous research has suggested that m6A modification is vital in cardiovascular diseases. The underlying targets of FTO were selected through transcriptome sequencing (RNA-seq) combined with methylated RNA immunoprecipitation sequencing (MeRIP-seq). According to MeRIP-seq and RNA-seq, FTO inhibited collagen synthesis in CFs.

overall_design: Neonatal rat cardiac fibroblasts (NRCFs) were collected from healthy Sprague-Dawley rats within 3 days post-birth. NRCFs were transfected with the plasmid of FTO and Vector and performed comparative gene expression profiling between FTO and Vector groups.

first: Yanyan

last: Li

first: Lijie

last: Deng

first: Yafang

last: Zha

first: Song


title: Differential transcriptomic responses to heat in wild-type and heat-evolved Symbiodiniaceae in corals

city: Parkville

first: Hugo

last: Scharfenstein

state: VIC

address: Grattan Street

institute: The University of Melbourne

summary: Heat-evolved Symbiodiniaceae can improve the physiological performances of their coral host under heat stress, but their gene expression responses to heat remained unknown. We explore here the transcriptomic basis of differential thermal stress responses between in hospite wild-type and heat-evolved Cladocopium proliferum strains and their coral host Platygyra daedealea.

overall_design: Three P. daedalea colonies were fragmented into nubbins. Once recovered, the vast majority of native symbionts was removed through chemical bleaching (exposure to menthol and diuron). Once bleached, the nubbins – kept in separate tanks – were inoculated with either a wild-type C. proliferum strain (WT10), heat-evolved C. proliferum strains (SS1 or SS8) or native symbionts (RIC, controlling for the effect of chemical bleaching). Additionally, unbleached and uninoculated nubbins (C) were maintained in parallel as experimental controls. After symbiosis re-establishment, half of the corals were subjected to heat stress (ramp up to 32.2°C), while the remaining half were at ambient temperature (27°C). After 4.9 eDHWs, ten nubbins were sampled from each colony at each temperature. For each colony, the ten nubbins were taken across the five treatments (i.e., two samples per treatment per colony per temperature treatment). In some cases, we were only able to recover one nubbin per colony per treatment.

first: Patrick

last: Buerger

first: Matthew

last: Nitschke

first: Lesa

last: Peplow

first: Wing

middle: Y

last: Chan

first: Madeleine

last: van Oppen


title: Comparative cofactor screens reveal the influence of transactivation domains and core promoters on the mechanisms of transcription [SLAM-seq]

first: Charles

last: Bell

state: VICTORIA

address: VCCC, Gate 1, 14 Flemington Road

department: Cancer Research

institute: Peter MacCallum Cancer Centre

summary: Eukaryotic transcription factors (TFs) activate gene expression by recruiting cofactors to promoters. However, the relationships between TFs, promoters and their associated cofactors remain poorly understood. Here, we combine GAL4-transactivation assays with comparative CRISPR-Cas9 screens to identify the cofactors required by nine different TFs in human cells. Using this dataset, we associate key TFs with their cofactors, classify cofactors as ubiquitous or specific, discover novel transcriptional co-dependencies and demonstrate that certain TFs use the tail 2 and kinase submodules of Mediator to potentiate transcriptional elongation. By employing a reductionistic and comparative approach, we demonstrate that TFs do not display discrete mechanisms of activation. Instead, each TF is dependent on a unique combination of cofactors, which influences distinct steps in the transcriptional process. We also extend our screens to nine different core promoters to explore how core promoter elements influence cofactor dependence. Our data suggest that different classes of promoter are constrained by either initiation or pause release, which influences their dynamic range of gene expression and compatibility with specific cofactors. Overall, our comparative cofactor screens uncover the interplay between TFs, cofactors and core promoters and reveal general principles by which they influence transcription.

overall_design: A total of 10 million K562 cells were treated with dTAGV-1 (Tocris) for a total of 4 hours. In the final hour of treatment, cells were labelled with 200uM of 4-thioruidine (4sU; Cayman Chemical) to capture nascent mRNA transcripts. RNA extraction was performed using TRIzol (Ambion) following the manufacturer's instructions, with the addition of 1mM DTT to the isopropanol precipitation and ethanol wash steps. Total RNA was eluted in nuclease-free H2O containing 0.1mM DTT. 10ug of total RNA was treated with fresh 10mM iodoacetamide (Pierce) in reaction buffer containing 50mM Tris, pH 8.0 and 50% DMSO at 50oC for 15 minutes, light-protected and shaking at 1000rpm. The alkylation reaction was quenched with 20mM of fresh DTT, followed by adding 1ug of unlabelled Drosophila S2 RNA. Total RNA was cleaned up using the RNeasy MinElute Cleanup Kit (Qiagen) and DNAse treated. Libraries were prepared using 500ng of material using the QuantSeq 3’ mRNA-Seq Library Prep Kit FWD (Lexogen; V1 kit with single 6nt i7 indexes) according to the standard manufacturer’s protocol. Sequencing of cDNA libraries was performed on the Illumina NextSeq2000 with 100bp single-end configuration. SLAM-seq was performed in biological triplicate.

first: Mark

last: Dawson

first: Laure

last: Talarmain

first: Andrea

last: Gillespie


title: Comparative cofactor screens reveal the influence of transactivation domains and core promoters on the mechanisms of transcription [ChIP-nexus]

overall_design: ChIP nexus for RNA-POL2 at a reporter activated with different TADs or different promoters. Cells were harvested 4hrs after addition of degron or relevant drug (CDK8i). For all ChIP experiments, at least 20 million cells were crosslinked for 15 mins with 1% formaldehyde. Crosslinked material was sonicated to approximately 200-1000bp using the Covaris Ultrasonicator e220. Sonicated material was incubated overnight with each antibody, then incubated for 3hrs with Protein A magnetic beads. Beads were washed with low and high salt wash buffers, LiCl buffer and TE, before being eluted and de-crosslinked overnight. DNA was purified using Qiagen Minelute columns. All ChIP antibodies were used at ~10ug per IP and are listed under the antibodies section. Sequencing libraries were prepared from eluted DNA using Rubicon ThruPLEX DNA-seq kit. Libraries were size selected between 200-500bps and sequenced on the NextSeq500 using the 75bp single-end chemistry.


title: Self-organization of pluripotent stem cells into reproducible early embryonic developmental patterns through epigenome editing

city: Santa Cruz

first: Ali

last: Shariati

state: CA

address: 1156 High St

department: Biomolecular Engineering

institute: University of California Santa Cruz

summary: During embryonic development, different embryonic cell types co-develop together where each cell type expresses specific fate-inducing transcription factors through activation of developmental non-coding regulatory elements and interactions with neighboring cells. Here, we hypothesized that cell-intrinsic activation of a minimum number of such endogenous regulatory elements is sufficient to self-organize PSCs into early embryonic models. Our results show that CRISPR activation of only two regulatory elements in the genome of pluripotent stem cells is sufficient to generate embryonic patterns that show spatial and molecular resemblance to that of pre-gastrulation mouse embryonic development. Quantitative single-cell live fluorescent imaging showed that emergence of the spatially-ordered embryonic pattern happens through intrinsic induction of cell fate that leads to an orchestrated collective cellular motion. Based on these results, we propose a straightforward approach to efficiently form 3D embryo models through intrinsic CRISPRa-based epigenome editing and independent of external signaling cues. CRISPRa-induced embryo models show highly consistent composition of major embryonic cell types that are spatially organized, with nearly 80% of the structures forming an embryonic cavity. Single cell transcriptomics confirmed the presence of main embryonic cell types in CRISRPa-induced embryo models with transcriptional similarity to pre-gastrulation mouse embryos and revealed novel signaling communication links between different embryonic cell types. Our findings demonstrate that self-organization of embryonic patterns through minimum intrinsic epigenome editing presents a promising approach for efficient production of stem cell-based embryo models with high consistency and will pave the way for future straightforward systematic CRISPR-based screening of embryo models

overall_design: Single-cell RNA-seq analysis was performed on 3D organoids grown using the protocol displayed below. We compared the generation of organoids either assembled from individual cell lines inducing either a control, Cdx2 or Gata6 sgRNA, or when assembling the 3 cell lines together, to assess self-organizing properties of embryonic cell types

first: Gerrald

last: Lodewijk

first: Sayaka

last: Kozuki

first: Seungho

last: Lee

first: Ben

last: Topacio

first: Abolfazl

last: Zargari

first: Clara

first: Gavin

last: Knight

first: Randolph

last: Ashton


title: Polystyrene nano- and microplastic particles induce an inflammatory gene expression profile in neural stem cell-derived astrocytes

city: Ottawa

first: Santokh

last: Gill

state: Ontario

address: 251 SIR FREDERICK BANTING

department: 251 Sir Frederick Banting Driveway

country: Canada

institute: Health Canada

summary: Cellular uptake and cytotoxicity data from neural cells treated with microplastics were compared and contrasted. Transcriptomic data obtained by RNA-seq from astrocytes treated with microplastics was assessed further.

overall_design: Compare and contrast cellular uptake and cytotoxicity data for various neural cells and compare trascriptomics data between control- and microplastic-treated astrocytes using RNA-seq.

first: Kristen

last: Marcellus

first: Steven

last: Bugiel

first: Andrée

last: Nunnikhoven

first: Ivan

middle: H

last: Curran


title: Comparative cofactor screens reveal the influence of transactivation domains and core promoters on the mechanisms of transcription [Degron ChIP]

first: Charles

last: Bell

overall_design: ChIP-seq for RNA-POL2 assess the effect of degredation of candidate Mediator subunits. Cells were harvested 4hrs after addition of degron or relevant drug (CDK8i). For all ChIP experiments, at least 20 million cells were crosslinked for 15 mins with 1% formaldehyde. Crosslinked material was sonicated to approximately 200-1000bp using the Covaris Ultrasonicator e220. Sonicated material was incubated overnight with each antibody, then incubated for 3hrs with Protein A magnetic beads. Beads were washed with low and high salt wash buffers, LiCl buffer and TE, before being eluted and de-crosslinked overnight. DNA was purified using Qiagen Minelute columns. All ChIP antibodies were used at ~10ug per IP and are listed under the antibodies section. Sequencing libraries were prepared from eluted DNA using Rubicon ThruPLEX DNA-seq kit. Libraries were size selected between 200-500bps and sequenced on the NextSeq500 using the 75bp single-end chemistry.

first: Mark


title: Comparative cofactor screens reveal the influence of transactivation domains and core promoters on the mechanisms of transcription [CRISPR promoter]

overall_design: GFP negative cells were isolated on D5, D6 and D7 after guide infection to identify the genes required for the activity of nine different core promoters. Sufficient cells were used to maintain 1000-fold representation at all stages of the screening process. The cells were transduced with an appropriate volume of viral supernatant to ensure only a single guide was present in most cells (MOI=0.3). At day 5, 6 and 7 day after guide infection, guide positive, GFP negative cells (< 25% of the MFI of the entire population) were sorted. Guide positive cells were also sorted as a library control at each time point to provide a library control reference to calculate enrichment. Genomic DNA was extracted using Monarch Genomic DNA purification kit (New England Biolabs), according to the manufacturer’s instructions. PCR was conducted to maintain guide representation, using Q5 High Fidelity DNA Polymerase (New England Biolabs). PCR products were pooled and sequenced on the NextSeq500 using 75bp paired-end chemistry.

last: Dawson

first: Laure

first: Andrea


title: CRISPR Screen identifies regulators for T cells activities in pancreatic cancer

city: Stony Brook

first: Richard

last: Lin

address: 1 Circle Road

institute: Stony Brook University

summary: Most human pancreatic ductal adenocarcinoma  (PDAC) are not infiltrated with cytotoxic T cells and are highly resistant to immunotherapy. Over 90% of PDAC have oncogenic KRAS mutations, and phosphoinositide 3-kinases (PI3Ks) are direct effectors of KRAS. Our previous study demonstrated that ablation of Pik3ca in KPC (KrasG12D; Trp53R172H; Pdx1-Cre) pancreatic cancer cells induced host T cells to infiltrate and completely eliminate the tumors in a syngeneic orthotopic implantation mouse model. This study now shows that implantation of Pik3ca-/- KPC (named αKO) cancer cells induces clonal expansion of cytotoxic T cells infiltrating the pancreatic tumors in this mouse model. To identify potential molecules that can regulate the activity of these anti-tumor T cells, we conducted an in vivo genome-wide gene-deletion screen using αKO cells implanted in the mouse pancreas. The result shows that deletion of propionyl-CoA carboxylase subunit B gene (Pccb) in αKO cells (named p-αKO) leads to immune evasion, tumor progression and death of host mice. Surprisingly, p-αKO tumors are still infiltrated with T cells but they are inactive against tumor cells. However, blockade of PD-L1/PD1 interaction reactivated clonally expanded T cells infiltrating p-αKO tumors, leading slower tumor progression and improve survival of host mice. These results indicate that Pccb can modulate the activity of cytotoxic T cells infiltrating some pancreatic tumors and this understanding may help to improve immunotherapy for PDAC.

overall_design: aKO cells were transfected with the genome-wide CRISPR screen library (GeCKOv2) that target 20,611 genes in the mouse genome with 3 sgRNA for each gene, resulting in 1 gene KO in each cell. After impanting the transfected aKO cells to the pancreas of B6 mice, tumor progression was monitored. Tumor tissues were harvested from mice at week 7 and week 13, followed by genomic DNA extraction and PCR amplification of the sgRNAs. The amplified library was then sequenced with amplicon seq.

first: Han

last: Han


first: Hongjian

last: Jin

overall_design: Refer to individual Series


title: Analysis of time-resolved immunological phases in the NIF mouse model for liver fibrosis.

city: Umeå

first: Dan

last: Holmberg

address: Building 6M

department: Medical Biosciences

country: Sweden

institute: Umeå university

summary: A time-course, bulk RNA-sequencing analysis of liver and kidney tissue from NIF mice was performed to identify the dynamics of key processes contributing to the pathogenesis. Transcriptomics profiling of the NIF mouse revealed a gradual transition from an initially reactive inflammatory response to a regenerative, pro-fibrotic inflammatory response suggesting new avenues for treatment strategies that focus on immunological targets. Despite the lack of metabolic stress induced liver phenotype, a large similarity between the NIF mouse and the immunological phase of human MASH was detected. The translational value was further confirmed through comparative analyses with MASH patients and additional animal models.

overall_design: Comparative gene expression analysis was performed using data obtained from RNA-seq of NIF and control 24abNOD mice at three different ages, 3 weeks, 6 weeks and 18 weeks.

first: Julia

last: Nilsson

first: Anja

last: Scmidt-Christensen

first: Gustaw

last: Eriksson

first: Sofia

last: Mayans


title: Long-Read Single-Cell RNA Sequencing Enables Cell Genotyping to Refine Subclone Detection in Chronic Lymphocytic Leukemia

city: Salt Lake City

first: Gabor

last: Marth

state: Utah

department: Human Genetics

institute: University of Utah

summary: Bruton's tyrosine kinase (BTK) inhibitors are effective treatments for chronic lymphocytic leukemia (CLL) due to BTK’s role in B-cell survival and proliferation. Though effective, resistance occurs most commonly due to a BTKC481S mutation that inhibits drug binding. Here, we sought to understand the impact of co-occurring BTK resistance mutations with known CLL driver mutations and the differential transcriptomic behavior of BTK-resistant CLL subclones in six patients who acquired BTKC481S mutations. We utilize MAS-seq, a long-read scRNAseq technology, to increase transcript coverage and expand the set of mutations that can be used to link cells to tumor subclones.

overall_design:
B-cells from pre- and post-treatment patient samples were isolated and sequenced using long-read scRNA-seq.

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Submitter states that missing raw data are being made available for controlled access in dbGaP.
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first: Gage

last: Black

first: Xiaomeng

last: Huang

first: Yi

last: Qiao

first: Deborah

middle: M

last: Stephens

first: Jennifer

last: Woyach


title: Comparative cofactor screens reveal the influence of transactivation domains and core promoters on the mechanisms of transcription [ZFP-TAD]

first: Charles

overall_design: GFP negative cells were isolated on D5, D6 and D7 after guide infection to identify the genes required for the activity of nine different transcription factors.. Sufficient cells were used to maintain 1000-fold representation at all stages of the screening process. The cells were transduced with an appropriate volume of viral supernatant to ensure only a single guide was present in most cells (MOI=0.3). At day 5, 6 and 7 day after guide infection, guide positive, GFP negative cells (< 25% of the MFI of the entire population) were sorted. Guide positive cells were also sorted as a library control at each time point to provide a library control reference to calculate enrichment. Four of the Gal4-TAD cell lines from the comparative CRISPR screens were also maintained until day 14 after guide infection to test for genes required for cell growth. These four TAD lines were used as independent replicates for the dropout analysis. Genomic DNA was extracted using Monarch Genomic DNA purification kit (New England Biolabs), according to the manufacturer’s instructions. PCR was conducted to maintain guide representation, using Q5 High Fidelity DNA Polymerase (New England Biolabs). PCR products were pooled and sequenced on the NextSeq500 using 75bp paired-end chemistry.

first: Mark

last: Talarmain

last: Gillespie


title: Glucocorticoid receptor-dependent therapeutic efficacy of tauroursodeoxycholic acid in preclinical models of Spinocerebellar ataxia type 3

city: Braga

first: Sara

last: Duarte-silva

address: Campus de Gualtar

department: School of Medicine, ICVS

country: Portugal

institute: University of Minho

summary: Spinocerebellar ataxia type 3 (SCA3) is an adult-onset neurodegenerative disease caused by a polyglutamine expansion in the ataxin-3 (ATXN3) gene.  No effective treatment is available for this disorder, other than symptomatic approaches. Bile acids have shown therapeutic efficacy in neurodegenerative disease models. Here, we pinpointed tauroursodeoxycholic acid (TUDCA) as an efficient therapeutic, improving the motor and neuropathological phenotype of SCA3 nematode and mouse models. Surprisingly, transcriptomic and functional in vivo data showed that TUDCA acts in neuronal tissue through the glucocorticoid receptor (GR), but independently of its canonical receptor, the FXR. TUDCA was also predicted to bind to the GR, similarly to corticosteroid molecules. GR levels were decreased in disease-affected brain regions, likely due to increased protein degradation as a consequence of ATXN3 dysfunction, being restored by TUDCA treatment. Lastly, based on results of a cohort of pre-symptomatic and symptomatic SCA3 patients, we demonstrated that peripheral expression of GR and FKBP5 might be indicators of clinical conversion and disease progression, respectively. We have established a novel in vivo mechanism for the neuroprotective effects of TUDCA in SCA3, and propose this readily available drug for a clinical trial in SCA3 patients.

overall_design: RNA was extracted from flash-frozen brainstem of 34-week-old mice using the AllPrep® DNA/RNA/miRNA kit (Qiagen, Hilden, Germany), following the manufacturer's instructions. A total of 3 mice per group (WT, TG and 7 day TUDCA-treated TG mice) was evaluated. Library preparation was carried out by targeted amplicon amplification for AmpliSeq, as previously described (https://doi.org/10.1186/s12864-015-2270-1). The quality of assembled libraries was assessed using the Agilent Bioanalyzer High Sensitivity chip. Library amplification was performed by emulsion PCR in an Ion Chef Instrument (Thermo Scientific, Waltham, Massachusetts), and cDNA quality was assessed with the Agilent TapeStation High Sensitivity tape. Sequencing was performed in the Ion S5TM XL sequencer (Thermo Scientific, Waltham, Massachusetts), and reads were aligned to the AmpliSeq Mouse Transcriptome v1, obtaining a total of 23,930 transcripts.

last: Duarte-Silva

first: Jorge

middle: D

last: Da Silva

first: Daniela

last: Monteiro-Fernandes

first: Marta

last: Costa

first: Andreia

last: Neves-Carvalho

first: Mafalda

last: Raposo

first: Carina

last: Soares-Cunha

first: Joana

last: Correia

first: Gonçalo

last: Nogueira-Goncalves

first: Henrique

last: Fernandes

first: Stephanie

last: Oliveira

first: Ana

middle: R

last: Ferreira-Fernandes

first: Fernando

last: Rodrigues

last: Pereira-Sousa

last: Vilasboas-Campos

last: Guerreiro

first: Jonas

last: Campos

first: Liliana

last: Meireles-Costa

first: Cecilia

last: Cabantous

first: Sergio

middle: F

last: Sousa

first: Manuela

last: Lima

last: Teixeira-Castro

first: Patricia

last: Maciel


title: Comparative Transcriptomic Analysis of aKO and p-aKO in Pancreatic Cancer Cells

first: Richard

last: Lin

overall_design: aKO cells were generated by knocking out p110a from KPC, and Pccb was knocked out from aKO cells and generated p-aKO cells. Total RNA was extracted from the two cell lines (aKO vs p-aKO) and after polyA enrichment and library prep, samples were sequenced by illumina PE150.

first: Han

last: Han


title: Consequence of knock down of CBX7 in multiple cell lines

city: nanjing

first: Wu

last: Zhichong

state: china

institute: Nanjing university

summary: We conduct an experiment where CBX7 is knocked down in 4 different cell lines: A549, H1299, K562 and THP-1. After culturing these cell lines for three days with or without knockdown of CBX7, we then perform RNA sequencing (RNA-seq) to assess the impact.

overall_design: 3 controls vs. 3 cases in each cell lines

first: Xu

last: Leilei

first: Yi

last: long


title: Gene expression data of ODH-08-treated Western diet-fed murine liver

city: Seoul

first: Haena

last: Choi

address: , Gwanak_1 Gwanak-ro, Gwanak-gu

country: South Korea

institute: Seoul National University

summary:
Hepatic fibrosis is a dynamic process characterized by the net accumulation of extracellular matrix resulting from chronic liver injury such as nonalcoholic steatohepatitis. During the pathogenesis of hepatic fibrosis, activation of hepatic stellate cells (HSCs) causes transdifferentiation of quiescent cells into proliferative and fibrogenic myofibroblasts. In the present study, we developed a novel RORα-selective ligand, ODH-08, based on the modification of JC1-40, a previously reported N-methylthiourea analog. Administration of ODH-08 to Western diet (WD)-fed mice improved the signs of hepatic fibrosis: decreased hepatic collagen deposition and suppression of the expression of fibrogenic markers. ODH-08 inhibits the TGF1-induced fibrogenic activation of HSCs through suppression of the TGFβ1–SMAD signaling pathway, which represents a novel mechanism for the antifibrogenic effect of RORα. Thus, ODH-08 appears to be a promising antifibrotic agent to treat hepatic fibrosis.
We performed a microarray analysis in the liver tissue of ODH-08-treated WD-fed mice to anlyse differentially expressed genes under ODH-08 administration. The control group was vehicle-treated WD-fed mice.

overall_design: WD feeding was done to 7-week-old C57BL/6 mice for 21 weeks. After 16 weeks of the diet feeding, ODH-08, a RORα agonist, was administrated orally for 5 weeks from 17th week to 21th week of WD. RNA was extracted from the liver of each group (n=2). Whole transcriptome analysis by microarray was performed with Affymetrix Mouse 2.0ST.

first: Hyeon-Ji

last: Kim

first: Mi-Ock

last: Lee


title: Dux promotes metabolism-lactylation-MET cascaded network during early iPSC reprogramming with Brg1 as the histone lactylation reader [Cut & Tag]

city: Harbin

first: Xinglin

last: Hu

state: Heilongjiang

address: Harbin Medical University, 194 Xuefu Rd, Nangang District, Harbin, Heilongjiang Province, P.R. China 150081

department: Histology and Embryology

institute: Harbin Medical University

overall_design: Comparative gene expression profiling analysis of CUT&Tag data for MEF, D4, D4-OE-DUX, and iPSC .

first: Xingwei

last: Huang

first: Lei

last: Lei


title: RNA Sequencing Reveals Transcriptomic Changes in HEK293 Cells Following Introduction of rs16851030 DNA Variant

city: Kuala Lumpur

first: Eng Wee

last: Chua

address: Jalan Raja Muda Abdul Aziz

country: Malaysia

institute: The National University of Malaysia

summary: rs16851030, a single-nucleotide variant located in the 3’-untranslated region of the ADORA1 gene, has been proposed as a potential marker of caffeine sensitivity in apnea of prematurity, aspirin-induced asthma, and the development of acute chest syndrome. However, its functional significance is still unconfirmed. This study aimed to elucidate the functional impact of rs16851030 by using CRISPR/Cas9 approach to induce physiological changes associated with the DNA variant. rs16851030 was introduced into HEK293 cells through homology-directed repair. Edited cells were then fluorescence-enriched, sorted, isolated, and grown into single cell-derived clones. The single-base edit was confirmed by Sanger sequencing. Finally, RNA sequencing was performed to elucidate the pathways affected by rs16851030. Our study provides valuable information about key pathways associated with rs16851030 DNA variant.

overall_design: Transcriptome analysis was used to detect the mRNA expression profiles of rs16851030-mutant HEK293 cells and wild-type cells. A total of six RNA libraries were constructed from the total RNA isolated from the following two groups of cells: rs16851030-mutant group and wild-type group (three biological replicates in each group). The total RNA of each sample was extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions, and the residual DNA was removed using the RapidOut DNA Removal Kit (Thermo Fisher Scientific, USA). After RNA quantification and purity checks, the samples were shipped to GENEWIZ (Suzhou, China) for RNA sequencing and pre-processing of raw sequencing data.

first: Poh

middle: K

last: Wong

first: Eng

middle: W


title: Differential susceptibility to colonic ulceration in mice with genetic deletion of Ptges

city: Farmington

first: Masako

last: Nakanishi

state: CT

address: 263 Farmington Ave

department: Center for Molecular Oncology

institute: UConn Health Center

summary: Prostaglandin E2(PGE2) exerts pleiotropic effects on inflammation, cancer, and maintenance of mucosal homeostasis.  A global knockout (KO) of the PGE2 synthase, Ptges, results in the spontaneous development of colonic ulcer in strain A mice (A/D:KO), a phenotype that is largely absent in C57BL/6 mice (B6D:KO) with the same genetic deletion.  Gene expression profiling of normal colonic mucosa several weeks prior to the onset of active disease identified a number of signaling pathways that may contribute to mucosal injury in the strain A mice, including the activation of inflammatory networks and antibacterial responses.  Alterations toseveral microbial species includeAkkermansia muciniphilaandBacteroides vulgatus, likely associated with dysregulation of short chain fatty acid (SCFA) synthesis.  These results suggest that absence ofPtgescauses a dramatic shift in the inflammatory milieu in strain A mice, an effect thatmay result from unremitting bacterial infections, triggered in part by dysregulation of epithelial homeostasis and a subtle defect in intrinsic immune regulation.

overall_design: To identify differentially expressed genes that may be associated with the divergent susceptibility to colonic ulceration observed in the two mouse lines, we performed bulk RNA sequencing (RNAseq) analysis on the proximal colons of 10-week-old A/D and B6D mice, with or without Ptges inactivation

first: Michael

middle: J

last: Martinez

first: Marion

last: Leclerc

first: Neranjan

last: Perera

first: Daniel

last: Rosenberg


title: Dux promotes metabolism-lactylation-MET cascaded network during early iPSC reprogramming with Brg1 as the histone lactylation reader

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


title: Impaired innate and adaptive immune responses to BNT162b2 SARS-CoV-2 vaccination in systemic lupus erythematosus

city: Stanford

first: Hong

last: Zheng

state: California

address: 240 Pasteur Dr

institute: Stanford university

summary: Understanding the immune responses to SARS-CoV-2 vaccination is critical to optimizing vaccination strategies for individuals with autoimmune diseases, such as systemic lupus erythematosus (SLE). Here, we comprehensively analyzed innate and adaptive immune responses in 19 patients with SLE receiving a complete 2-dose Pfizer-BioNTech mRNA vaccine (BNT162b2) regimen compared with a control cohort of 56 healthy control (HC) volunteers. Patients with SLE exhibited impaired neutralizing antibody production and antigen-specific CD4+ and CD8+ T cell responses relative to HC. Interestingly, antibody responses were only altered in patients with SLE treated with immunosuppressive therapies, whereas impairment of antigen-specific CD4+ and CD8+ T cell numbers was independent of medication. Patients with SLE also displayed reduced levels of circulating CXC motif chemokine ligands, CXCL9, CXCL10, CXCL11, and IFN-γ after secondary vaccination as well as downregulation of gene expression pathways indicative of compromised innate immune responses. Single-cell RNA-Seq analysis reveals that patients with SLE showed reduced levels of a vaccine-inducible monocyte population characterized by overexpression of IFN-response transcription factors. Thus, although 2 doses of BNT162b2 induced relatively robust immune responses in patients with SLE, our data demonstrate impairment of both innate and adaptive immune responses relative to HC, highlighting a need for population-specific vaccination studies.

overall_design: We performed bulk mRNA sequencing (RNA-seq) of whole blood from 18 patients with SLE at four time points after Pfizer-BioNTech (BNT162b2) COVID-19 mRNA vaccination.


title: Gene expression profile of hair follicle stem cells (HFSCs) isolated from wild type and homozygous Dab2 cKO (Dab2 Fl/Fl) mice at postnatal day 35

city: Navi Mumbai

first: SANJEEV

last: WAGHMARE

state: MAHARASHTRA

address: Sector 22, Kharghar

department: STEM CELL BIOLOGY GROUP

country: India

institute: ACTREC, TATA MEMORIAL CENTRE

summary: Disabled 2 is an endocytic adaptor protein known to regulate various signaling pathways such Wnt , BMP, TGF and Ras/MAPK. In the present study, we observed that Dab2 is expressed in all the compartments of the hair follicle. K14 Cre specific homozygous Dab2 knockout resulted in delayed activation of HFSCs and delayed Ist hair follicle cycle initiation. At PD35, while WT progresses to anagen II, the Dab2 cKO mice still remains at telogen. In addition, loss of Dab2 alos led to reduced HFSCs pool extended quiescence, and reduced self renewal capacity of HFSCs at PD35. We performed expression profiling of HFSCs isolated from WT and Dab2 cKO mice at PD35 to check for the differentially regulated genes involved in HFSCs activation. The expression analysis helped us to elucidate the detailed molecular mechanisms involved in Dab2 mediated regulation of HFSCs activation and quiescence.

overall_design: To understand the detailed molecular mechanisms involved in Dab2 mediated regulation of HFSCs, we performed expression profiling on HFSCs isolated from dorsal skin sections of WT and Dab2 cKO mice at PD35.

first: S

last: waghmare


overall_design: We sequenced peripheral blood mononuclear cell (PBMC) samples from 3 patients with SLE at three time points after Pfizer-BioNTech (BNT162b2) COVID-19 mRNA vaccination.


title: A deep mutational scanning platform to characterize the fitness landscape of anti-CRISPR proteins

city: Heidelberg

first: Dominik

last: Niopek

state: Baden-Württemberg

address: Im Neuenheimer Feld 364

department: Institute of Pharmacy and Molecular Biotechnology (IPMB), Faculty of Engineering Sciences

country: Germany

institute: Heidelberg University

summary: Deep mutational scanning is a powerful method for exploring the mutational fitness landscape of proteins. Its adaptation to anti-CRISPR proteins, which are natural CRISPR-Cas inhibitors and key players in the co-evolution of microbes and phages, facilitates their characterization and optimization. Here, we developed a robust anti-CRISPR deep mutational scanning pipeline in Escherichia coli that combines synthetic gene circuits based on CRISPR interference with flow cytometry coupled sequencing and mathematical modeling. Using this pipeline, we characterized comprehensive single point mutation libraries for AcrIIA4 and AcrIIA5, two potent inhibitors of CRISPR-Cas9. The resulting mutational fitness landscapes revealed considerable mutational tolerance for both Acrs, suggesting an intrinsic redundancy with respect to Cas9 inhibitory features, and – for AcrIIA5 – indicated mutations that boost Cas9 inhibition. Subsequent in vitro characterization suggested that the observed differences in inhibitory potency between mutant inhibitors were mostly due to changes in binding affinity rather than protein expression levels. Finally, to demonstrate that our pipeline can inform Acrs-based genome editing applications, we employed a selected subset of mutant inhibitors to increase CRISPR-Cas9 target specificity by modulating Cas9 activity. Taken together, our work establishes deep mutational scanning as a powerful method for anti-CRISPR protein characterization and optimization.

overall_design: To determine the activity of AcrIIA4 and -5 mutants, E. coli libraries expressing either AcrIIA4 or -5 single point mutants were FACS sorted into 4 fractions according to Acr activity (1st fraction corresponds to low Acr activity, 4th fraction corresponds to high Acr activity). FACS-sorted E. coli cell fractions were then lysed and DNA extracted for subsequent first-stage PCR amplification of the AcrIIA4 or AcrIIA5 genes using primers with Nextera XT index overhangs. After a second stage barcoding PCR using the TG Nextera XT Index Kit v2 Set A (Illumina), the libraries were sequenced on the Illumina MiSeq system using 2x250 paired-end sequencing reagents (MiSeq Reagent Kit v2, 500 cycles).

first: Stadelmann

last: Tobias

first: Heid

last: Daniel

first: Jendrusch

last: Michael

first: Mathony

last: Jan

first: Aschenbrenner

last: Sabine

first: Rosset

last: Stéphane

first: Correia

last: Bruno E.

first: Niopek

last: Dominik


title: Effect of EPCR knockdown by siRNA in human umbilical vein endothelial cells

city: 上海/上海市/杨浦区

first: 青

last: 李

address: 长海路168号

institute: 中国人民解放军海军军医大学第一附属医院

summary: Endothelial protein C receptor (EPCR, PROCR) is a single-pass transmembrane glycoprotein that is detected on the surface of vascular endothelial cells (ECs) and stem cells. Besides the anti-coagulation role, EPCR can mediate intracellular signaling of activated protein C (aPC)[7]. The activation of downstream protein kinase B (AKT) promotes cell growth, improves cardiac function, and favors tumor progression. However, the role of EPCR in pathological retinal neovascularization remains unclear.To investigate the role of EPCR in endothelial cell function, we knockdown EPCR with siRNA and compared with siCtrl. We then performed gene expression profiling analysis using data obtained from RNA-seq of 4 different samples each condition.

overall_design: 8 samples were analyzed: siCrt (4 replicates), siEPCR (4 replicates)

first: hongyuan

last: song

first: wei

last: shen

first: qing

last: li

first: xiao

last: gui


title: An MMSET inhibitor-induced changes in gene expression profile of multiple myeloma cells

city: Shimotsuke

first: Sae

last: Matsuoka

state: Tochigi

department: Center for Molecular Medicine

country: Japan

institute: Jichi  Medical  University

summary: We performed gene expression profiling to understand the mechansims of cytotoxic effects of an MMSET inhibitor on t(4;14)-positive multiple myeloma cells.

overall_design: We cultured the human myeloma cell line KMS34 with the MMSET inhibitor RK-552 and performed gene expression profiling using an Agilent GeneChip Array.

first: Jiro

last: Kikuchi


first: Hong

last: Zheng

overall_design: Refer to individual Series


title: Corpora cavernosa fibroblasts mediate penile erection

city: Solna

first: Anais

last: Julien

state: non-US Resident

address: Solnavagen 9

department: CMB

institute: Karolinska Institutet

summary: Penile erection is mediated by the corpora cavernosa, a trabecular-like vascular bed, which enlarges upon vasodilation, but its regulation is not completely understood. Here, we show that perivascular fibroblasts in the corpora cavernosa support vasodilation by reducing norepinephrine availability. We used scRNAseq to investigate fibroblast heterogeneity in the penis.

overall_design: Slc1a3-derived cells were isolated from adult mouse penises using the Slc1a3CreERT;tdTomato line. Cells were sorted based on tdTomato expression in 384 well plates containing lysis buffer and subject to scRNAseq.

first: Eduardo

last: Guimaraes

first: Christian

last: Göritz


title: Transcriptome analysis of Sodium-Glucose Cotransporter-2 Inhibitors in Acute Myocardial Infarction

city: Chongqing

first: Huiling

last: Chen

address: Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China

institute: Chongqing Medical University

summary: Acute myocardial infarction (AMI) is the leading cause of mortality worldwide. Dapagliflozin (DAPA), a sodium-glucose cotransporter-2 inhibitor (SGLT2i), is a new type of oral antidiabetic agent. Here, we examined the effects of DAPA on AMI and investigated the potential mechanisms.Heart tissue specimens were collected from C57BL/6J mice induced by ligation of the left anterior descending coronary artery and treated with DAPA at 1 mg/kg/day dose for 4 weeks after surgery. Echocardiography, histological staining, and RNA sequencing (RNA-seq) of these specimens were performed. The differentially expressed genes of interest were confirmed by qualitative RT-PCR (RT-qPCR) and western blotting (WB). In vitro experiments were performed to evaluate the effect of DAPA on myosin light-chain 4 (Myl4) upregulation and reduction of autophagy following hypoxic treatment. As a result, DAPA could improve cardiac function post MI by upregulating Myl4 and reducing autophagy; this finding suggests that the molecular regulation associated with Myl4 might be an important mechanism of AMI treatment by SGLT2i.

overall_design: RNA sequencing (RNA-Seq) of the cardiac tissues from C57BL/6J mice, which was induced by left anterior descending (LAD) coronary artery ligation, treated with DAPA at a dose of 1mg/kg/day for 4 weeks after surgery was conducted. Differentially expressed genes (DEGs) enriched in the mice heart after DAPA treatment were identified at the transcriptome level.


title: Two metazoan osmosensors encoded by GPCR genes in C. elegans

city: Oklahoma City

first: Xinxing

last: Zhang

state: Oklahoma

address: 940 Stanton L. Young Blvd, BMSB Rm538

department: Department of Cell Biology

institute: University of Oklahoma Health Sciences Center

summary: Osmosensors play essential roles in maintaining body fluidic balance in the animal kingdom. However, the molecular identities of osmosensors in mammals are still elusive. TRP channels have been proposed to be candidate osmosensors, but their roles in regulating body fluid homeostasis are still controversial. The major difficulty in identifying osmosensors in animals, is in part because of the lack of efficient screening methods. Here, we designed and conducted a neuronal activity-based genetic screen to identify the first osmosensors in animal kingdom using C. elegans. Previous studies show G proteins are required for C. elegans sensing osmotic stimuli. Thus, we proposed that the osmosensors are GPCRs. To identify the GPCRs enriched in ASH neurons, which are the major osmosensing neurons, we did RNA-seq after FACS sorting for ASH. After screening the enriched GPCRs in ASH, we successfully found one GPCR, OSGR-1, functions as an osmosensor in C. elegans. We also found, its closest paralog, OSGR-2, is also an osmosensor. This is also the first shown that GPCRs are capable of sensing osmolarity, which will facilitate the identification of osmosensors in other animals.

overall_design: To get enriched genes in C. elegans ASH neurons, we used FACS sorting to sort out YFP labeled ASH neurons together with non-YFP cells as controls. RNA-seq was used to map the transcriptome and both Cufflinks/CuffDiff and HTseq/DESeq2 were used to calculate DEG genes.


title: Cognate antigen-independent differentiation of resident memory t cells in chronic kidney disease

city: Birmingham

first: Kyle

middle: Hartley

last: Moore

state: Alabama

department: Division of Nephrology

institute: University of Alabama at Birmingham

summary: Although resident memory T cells (TRMs), which are memory T cells that are retained locally within tissues, have recently been described as antigen-specific frontline defenders against pathogens in barrier and non-barrier epithelial tissues, they have also been noted for perpetuating chronic inflammation. Furthermore, the conditions responsible for TRM differentiation are still poorly understood, and their contributions, if any, to sterile models of chronic kidney disease (CKD) remain a mystery. In this study we identify a substantial population of TRMs in the kidneys of mice with aristolochic acid (AA)-induced CKD. Flow cytometry of injured kidneys showed T cells bearing TRM surface markers and single cell RNA sequencing revealed these cells as expressing well-known TRM transcription factors and receptors responsible for TRM differentiation and maintenance. While kidney TRMs expressed Cd44, a marker of antigen experience and T cell activation, their derivation was surprisingly independent of cognate antigen-T cell receptor interactions, as the kidneys of transgenic OT-1 mice still harbored considerable proportions of TRMs after injury. Our results suggest a non-antigen-specific or antigen-independent mechanism capable of generating TRMs in the kidney and highlight the need to better understand TRMs and their involvement in CKD.

overall_design: C57BL/6J mice were administered aristolochic acid 2 mg/kg body weight i.p. for 5 consecutive days and kidneys were harvested two and six weeks later for single cell RNA sequencing. Quiescent kidney immune cell sequencing from GSE200115 was used for controls.

first: Elise

middle: N

last: Erman

first: Amie

last: Traylor

first: Stephanie

last: Esman

first: Yanlin

last: Jiang

first: Jennifer

last: LaFontaine

first: Anna

last: Zmijewska

first: Yan

last: Lu

first: Reham

last: Soliman

first: James

last: George

first: Anupam

last: Agarwal


title: A biomimetic calcium phosphate nanowire coating on titanium surface enhances osteoimmunomodulation and osteointegration

city: Chengdu

first: Chengdong

address: Second Ring Road

institute: Southwest Jiaotong University

summary: The appropriate immune microenvironment plays crucial roles in bone regeneration. Surface structure and chemisty are key factors affecting immune cells behaivor and subsequently regulating the activity of bone-related cells. To achieve rapid osteointegration, this study prepared a biomimetic calcium phosphate (CaP) coating on Ti-based substrates (THCaP) with the help of oleic acid under hydrothermal condition. The coating was composed of hydroxyapatite nanowires and further self-assembled into macropores. Also, the effect of the biomimetic CaP coating on the immune response of macrophages and the impact on angiogenesis and osteogenesis were investigated. In vitro cell culture results showed that compared with pristine Ti and similar titanate nanowire structure (TH), THCaP not only stimulated the polarization of macrophages towards to pro-healing M2 phenotype, but also enhanced the angiogenic and osteogenic differentiation of endothelial cells and preosteoblastic cells, respectively. Especially, macrophages on THCaP induced a favorable immune microenvironment and further facilitated the osteogenic diffferention of preosteoblastic cells. In vivo tests confirmed the aforementioned results, which proved that the implanted THCaP could decrease the inflammatory response and facilitate new bone formation when compared with pristine Ti and TH implants. These findings indicate that preparation of biomimetic CaP network structure is a promising strategy to surface design of Ti-based substrates, which is beneficial to generate a favorable osteoimmunomodulatory microenvironment for enhancing osteointegration.

overall_design: To investigate the immunomodulatory effects of titanium surface bionic calcium phosphate nanowire coating (THCaP), we used the Raw 264.7 cells line with titanate nanowires (TH) obtained by alkaline heat treatment as control samples. The total RNAs from cells on the samples were extracted after culture of 1 d, then we  performed gene expression profiling analysis using data obtained from RNA-seq.

first: Yu

last: Pan

first: Guowen

last: Duan

first: Dongqin

last: Xiao


title: DNA methylation analysis of liver samples from healthy individuals and patients with advanced metabolic dysfunction associated steatohepatitis (MASH)

city: Wilrijk

first: Claudia

last: Theys

state: Antwerpen (nl)

department: FBD

country: Belgium

institute: Universiteit Antwerpen

summary: To investigate MASH-associated changes in DNA-methylation, a genome-wide DNA methylation profiling of livers from healthy individuals compared to those with MASH was performed using the 850K MethylationEPIC BeadChip Array. Further differential methylation analysis allowed us to identify a MASH epigenetic signature. Both males and females were included in this cohort, consisting of 4 MASH patients and 3 healthy individuals.

overall_design: methylation profiles of liver biopsies of healthy and MASLD patients

first: Evelien

last: Van Dijck


title: TGF-β specifies TFH versus TH17 cell fates in murine CD4+ T cells through c-Maf (Seq05, bulk-RNAseq)

city: Bonn

first: Dirk

last: Baumjohann

state: NRW

address: Venusberg-Campus 1

department: Medical Clinic III

institute: University Hospital Bonn

summary: We have shown that TGFb in combination with IL-6 potently induces a  T follicular helper cell (Tfh) cell phenotype in activated murine CD4+ T cells in vitro. The transcription factor c-Maf critically contributes to the differentiation of Tfh cells in vitro and in vivo.

overall_design: Here we cultured naive CD4+ T cells from control and T cell-specific Maf knockout mice with increasing doses of TGF-β (blocking/anti-TGFb (TfhA), 1 ng/ml TGF-β (Tfh1), and 5 ng/ml TGF-β (Tfh5); all conditions included anti-IFNg, anti-IL4, IL-6, IL-21)

first: Yinshui

last: Chang


title: RNA-seq of aortic dissection animal model induced by co-administration of BAPN and Fasudil and control groups

city: Shanghai

first: Wei

last: Zhang

address: No.180 Fengling Road, Xuhui

department: Vascular Surgery

institute: Zhongshan Hospital, Fudan University

summary: Rho-kinase signaling and YAP signaling are related to aortic dissection (AD) formation. However, as important biomechanical signaling pathways, their relationships with aortic smooth muscle cell (AoSMC) mechanics have not been investigated in AD formation. This study aims to explore the correlation between RhoA/ROCK1 and YAP, as well as their relationship with intrinsic AoSMC stiffness during AD formation. The expression of RhoA/ROCK1 and YAP as well as F-actin polymerization were analyzed in AoSMCs isolated from normal and AD human aortas. The intrinsic cell stiffness of normal and AD AoSMCs was measured using atomic force microscopy (AFM). The correlations among RhoA/ROCK1, YAP, and intrinsic AoSMC stiffness were explored by manipulating the activity of RhoA and ROCK1, and YAP expression. Finally, the role of RhoA/ROCK1/YAP/F-actin and AoSMC stiffness during AD formation was studied in pharmaceutically induced AD mouse models.Compared to normal human AoSMCs, the expression of RhoA and ROCK1 was downregulated, while the phosphorylation of YAP was increased in AD human AoSMCs, coupled with impaired F-actin polymerization and decreased intrinsic cell stiffness. Pharmaceutical inhibition of RhoA or ROCK1 activities and depletion of YAP all led to impaired F-actin polymerization and decreased intrinsic AoSMC stiffness. Moreover, abnormal collagen deposition and impaired cell-ECM interactions were found when RhoA/ROCK1/YAP/F-actin signaling was inhibited in normal human AoSMCs. We further analyzed the normal human AoSMC treated by Y27632 or not using RNA sequencing to investigate the impact of RhoA/ROCK1/YAP/F-actin on AoSMCs. GO analysis showed that differentially expressed genes (DEGs) related to cAMP-mediated signaling, cytoskeleton organization, cell-matrix adhesion, and extracellular matrix (ECM) organization were affected when ROCK1 activity was inhibited by Y27632 in normal AoSMCs. KEGG analysis showed differences in PI3K-Akt signaling, focal adhesion, MAPK, actin cytoskeleton, and ECM-receptor interaction. Consistently, cAMP, actin cytoskeleton organization, and ECM structural constituents were all downregulated in AoSMCs treated with Y27632 according to GSEA. We also analyzed the downregulated genes in AoSMCs treated with Y27632. Results demonstrated that the downregulated genes were mainly related to the cell-ECM unit, PI3K, MAPK, focal adhesion, and cAMP signaling pathways.

overall_design: Experiement group: mouse co-administered with BAPN and Fasudil; Control: mouse administered with BAPN; mouse administered with Fasudil; mouse administered with vehicle

first: Mengxiao

last: Wang

first: Enci

first: Zengxia

last: Li

first: Gaofei

last: Hu

first: Qi

first: Wenxin

last: Shan

first: Yongjun

last: Dang

first: Lemin

last: Zheng

first: Lixin


title: Zfp462 regulates the pluripotency of embryonic stem cells by cooperating with the core transcriptional network

city: chongqing

first: yu

last: meng

address: xinqiao street

institute: Army medical university

summary: Zfp462 is a vertebrate-specific C2H2-type transcription factor that has been found to play a crucial role in chromatin assembly and heterochromatin-mediated transcriptional silencing. However, its exact function in regulating the pluripotency of embryonic stem cells (ESCs) remains poorly understood. In this study, we observe that Zfp462 is highly expressed in undifferentiated ESCs, gradually decreases upon pluripotency exit, but then increases again during lineage differentiation. Knocking out of Zfp462 leads to reduced self-renewal of ESCs, abnormal pluripotency exit, and imbalance of lineage specification/differentiation. Multi-omics integrative analysis suggests that Zfp462 may function as a component of the ESC CORE module and collaborate with Oct4/Sox2/Nanog (OSN) to regulate the expression of pluripotency and developmental genes. Collectively, our research not only reveals the functions of Zfp462 in ESC self-renewal and differentiation but also proposes Zfp462 as a novel player in the pluripotency gene regulatory network (PGRN).

overall_design: To investigate the impact of Zfp462 knockout (KO) on the self-renewal and differentiation of embryonic stem cells (ESCs), we generated stable ESC lines with Zfp462 KO. Subsequently, we obtained Zfp462 KO ESCs and control (CTL) mESCs in the undifferentiated state (D0), as well as differentiated cells at embryoid body  differentiation after 3 days (EB_D3), for RNA-seq analysis.

first: Meng

last: Yu

first: Jiaqi

first: Ran

last: Yang

first: Yan

last: Ruan


title: Glycolysis-stimulated Esrrb Lactylation Promotes the Self-renewal and XEN Differentiation of Embryonic Stem Cells

city: New York

first: XIANLE

last: SHI

state: NY

address: 168 street

institute: CUMC

summary: Embryonic stem cells (ESCs) favor glycolysis over oxidative phosphorylation for energy production, and glycolytic metabolism is critical for pluripotency establishment, maintenance and exit. However, how glycolysis regulates the self-renewal and differentiation of ESCs remains elusive. Here, we demonstrated that protein lactylation, regulated by intracellular lactate, contributes to the self-renewal of ESCs. Next, the lactylome profiles of ESCs with and without a lactate dehydrogenase (Ldh) inhibitor, which suppresses the conversion of pyruvate to lactate, were depicted. It was notable that many lactylated proteins are involved in the self-renewal and differentiation of ESCs. We further showed that Esrrb, an orphan nuclear receptor involved in pluripotency maintenance and extraembryonic endoderm stem cell (XEN) differentiation, is lactylated on K228 and K232. Lactylation of Esrrb enhances its activity in promoting ESC self-renewal in the absence of LIF and XEN differentiation of ESCs, through increasing its binding at target genes. Our studies reveal the importance of protein lactation in the self-renewal and XEN differentiation of ESCs, and the underlying mechanism for glycolytic metabolism regulating cell fate choice.

overall_design: Chromatin immunoprecipitation followed by sequencing (ChIP–seq) for Esrrb in XEN differentiation of embryonic stem cells

first: Qiman

last: Dong

first: Lingyi

last: Chen


title: Single-cell trasncripional regulation by Vascular Endothelial Cell Growth Factor (VEGF) treatment in human endothelial cells

city: Kumamoto

first: Shunsuke

last: Kamei

address: 2-2-1 Honjo

institute: Kumamoto University

summary: HUVEC cells were treated with human recombinant VEGF-A165 for either 0h or 18h. Whole cells were used for scRNA-seq analysis.

first: Yuri

last: Miyamura

first: Takashi

last: Minami


title: Identification of prognostic biomarkers in early stage squamous cell lung cancer (LUSC) by transcriptomic and miRNA profiling [sRNA-Seq]

city: Zagreb

first: Maja

last: Šutić

address: Bijenicka street 54

department: Division of Molecular Medicine

country: Croatia

institute: Ruder Boskovic Institute

type: Non-coding RNA profiling by high throughput sequencing

summary: Squamous cell lung cancer (LUSC) is a frequently diagnosed histological subtype of lung malignancy associated with high mortality due to limited number of treatment options. Identification of potential targets suitable for drug development using high-throughput methods is still lacking. Therefore, the purpose of this research is to analyze expression profiles of mRNA and miRNA in LUSC, aiming to identify the key molecules associated with tumorigenesis and prognosis (overall survival; OS). We performed differential gene expression analysis, pathway enrichment analysis and gene ontology analysis on RNA-seq data obtained from 23 FFPE tumor samples and 3 FFPE healthy individuals. TCGA LUSC and GTEx healthy donors’ cohorts were used for validation. We identified 1133 up-regulated and 644 down-regulated genes, common for both cohorts. The most significant up-regulated genes were involved in cell-cycle regulation pathways, while down-regulated genes predominately belonged to immune-related pathways. Survival analysis performed on selected genes, commonly dysregulated in both cohorts and performed only on non-metastatic patients, identified novel prognostic biomarkers associated with OS in early-stage LUSC patients (HOXC4-worse OS, p=0.0001; LILRA5-better OS, p=0.0086). In total, 50 differentially expressed genes are correlated with patients’ OS. Aiming to get better insight into the profile of the tumor microenvironment, we estimated levels of immune-cells infiltration in LUSC and identified 4 subtypes based on immune-cell infiltration levels. Protein-protein interaction (PPI) network was built based on differentially expressed genes, and PPI modules and hub-genes were identified. Weighted gene co-expression network analysis was conducted to identify significant modules and hub-genes in correlation to clinical traits. Differential analysis on miRNA-Seq data, obtained from 16 tumor and 4 healthy tissue samples, was conducted as well. 75 up-regulated and 47 down-regulated miRNAs were identified. Further, targets of differentially expressed miRNA were identified, following gene ontology analysis and miRNA-mRNA networks construction.

overall_design: Expression profiling of miRNA from 14 patients diagnosed with squamous cell lung cancer (LUSC), and 4 healthy controls. Profiling was done on FFPE tissue samples, for both LUSC and control samples.

first: Jelena

last: Knežević


title: Differential gene expression of gene during media induced aggregation

city: Aberdeen

first: Centre for Genome-Enabled Biology and Medicine

last: on behalf of listed contributors

state: Aberdeenshire

address: 23 St Machar Drive

department: CGEBM

country: United Kingdom

institute: University of Aberdeen

summary: This study aimed to characterise the transcriptional response of two strains of Candida auris (UACa11 and UACa20) during media-induced aggregation due to differences in phenotype. RNA was isolated from each strain under both aggregating and non-aggregating culture conditions.

overall_design: Non-aggregative strain UACa11 and aggregative strains UACa20 were grown under two different culture media. Sabouraud's Dextrose induced aggregation only in aggregative strains while RPMI-1640 saw all strains grow as a single-celled morphology. Non-aggregative strain was added to remove noise from the media change in data. After incubation for 16 hours in the indicated media. Cells were harvested and RNA purified to preform RNA-seq to examine differential gene expression. Three replicates from each condition were collected, totaling 12 samples.

first: Chloe

last: Pelletier

first: Sophie

last: Shaw

first: Alistair

last: Brown

first: Alexander

last: Lorenz


title: Identification of differential expression of miRNA profile in serum samples of WHO CNS5  adult-type diffuse glioma

city: Bialystok

first: Magdalena

last: Niemira

address: Kilinskiego 1

country: Poland

institute: Medical University of Bialystok

summary: Developing a classification model based on differential expression miRNAs profile in serum WHO CNS5 adult-type diffuse glioma samples to improve of diagnosis of grade 4 glioma.

overall_design: In the study presented here, well-defined cohort of 32 grade 2-3 glioma and 78 grade 4 patients were used to acquire expression profiles of 798 miRNAs using NanoString platform.


title: Isolation of biologically active small peptide from KSHV LANA protein sequence, which induces CHD4 degradation to promote cell differentiation and inhibits leukemic cell growth

city: Sacramento

first: Clifford

middle: G.

last: Tepper

state: CA

address: 4645 2nd Avenue, Room 2300A

department: Biochemistry and Molecular Medicine

institute: UC Davis School of Medicine

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


title: Effect of Pterostilbene (PTE) treatment on gene expression after intracerebral hemorrhage injury

city: Shijiazhuang

first: gen

middle: shen

last: zhang

state: Hebei

address: Hepingxi Road

institute: HEBEIMEDICAL UNIVERSITY

summary: To investigate the effect of Pterostilbene(PTE) on gene expression after intracerebral hemorrhage injury, we employed RNA sequencing (RNA-seq) to profile the mRNA expression ensuing PTE treatment after ICH. Different gene distribution could be clearly seen on the volcano plot between diferent comparisions among Sham, ICH, and ICH+PTE group. Finally, we showed that 1021 shared genes overlapping between those differentially expressed genes, which was reversed by PTE treatment

overall_design: Experimental mice were randomly and equally divided into three groups: sham group, ICH group, ICH + PTE group. At 3 days  after diferent treatments,  3 mice were randomly selected from each group and used to preform RNA sequencing.

first: Yang

last: Wu

first: Xun

first: Xiaoliang

last: Wang

first: Genshen

last: Zhang


title: Isolation of biologically active small peptide from KSHV LANA protein sequence, which induces CHD4 degradation to promote cell differentiation and inhibits leukemic cell growth (SLAM-seq)

summary: Kaposi's sarcoma-associated herpesvirus (KSHV) establishes a latent infection, in which most viral genes are silenced except a few latent proteins such as latency-associated nuclear antigen (LANA). In latent viral chromatin, viral genes are poised to be transcribed, and KSHV LANA plays a major role in maintaining such transcription status. In the poised chromatins, LANA recruits cellular CHD4 (Chromodomain Helicase DNA binding protein 4) and suppresses inducible viral gene promoters. The CHD4 is known to regulate cell differentiation by restricting enhancer-promoter interactions and its mutation or overexpression deregulates the host cell transcription program and therefore associates with tumorigenesis. Here, we identified a putative CHD4 inhibitor from the LANA amino acid sequence from the LANA-CHD4 interaction surface. The small peptide interacts with CHD4 at its PHD domain with 14 nM KD (dissociation constant). The introduction of the peptide into the primary effusion lymphomas induces caspase-mediated CHD4 cleavage and subsequently triggered cell apoptosis and autophagy.  A series of MTT assays demonstrated that the peptide preferentially killed lymphoma and leukemia cell lines at low micromolar concentrations, while peripheral mononuclear cells or adhesion cell lines were found to be more resistant to the peptide treatment. A monocyte cell differentiation model demonstrated that pre-treatment with the peptide at a low dose substantially enhanced transitioning into M2 macrophage (by reducing CHD4 occupancies from gene promoters), and globally altered the repertories of phorbol myristate acetate target genes in U937 cells. Finally, the PEL xenograft mouse model inhibited tumor growth without measurable side effects, and PEL cells isolated from xenograft tumors showed reduced CHD4 and LANA expression with terminally differentiated phenotype. We propose that the peptide isolated from the KSHV LANA sequence is biologically active and may function as a CHD4 inhibitor.

overall_design: The goal of these studies was to utilize thiol(SH )-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) in order to define the direct transcriptional effects of treatment with the VGN73 peptide. This was performed in the context of the BC3, BCBL-1, and Raji cell line models. Replicate cultures from each cell line were treated with either vehicle control, wild-type VGN73 peptide (at the IC50 for each cell line), or mutant VGN73 peptide (at the equivalent concentrations to the IC50 of wild type peptide for each cell line) for 30 minutes, and then incubated for an additional 1.5 hours in the presence of 4-Thiouridine (s4U; 300 uM) in order to label nascent transcripts. Following treatment, the cells were harvested for isolation of total RNA (containing s4Uracil-labeled transcripts) and followed by library preparation and next-generation sequencing (NGS).

first: Yoshihiro

last: Izumiya

first: Hiroki

last: Miura

middle: G


title: Isolation of biologically active small peptide from KSHV LANA protein sequence, which induces CHD4 degradation to promote cell differentiation and inhibits leukemic cell growth (RNA-seq)

overall_design: The goal of this study was to define the effects of VGN73 on the regulation of phorbol-12-myristate-13-acetate (PMA)-mediated differentiation of U937 monocyte-macrophage cells. U937 cells were pretreated with VGN73 (4 or 8 μM) for 24 hours, and then stimulated with PMA (10 ng/ml final concentration) for an additional 24 hours. Controls groups included PMA stimulation (10 ng/ml, 24 hrs) without pretreatment and VGN treatment alone (4 μM, 48 hrs). All treatments were performed in triplicate. Cells were harvested at the completion of the treatments and followed by total RNA isolation and RNA-sequencing.


title: MCP-1 production regulated by ATG5 in inflammatory monocytes selectively modulates anti-malarial CD4+ Th1 response

first: taiping

last: Liu

address: Gaotanyan zhengjie 30, Shapingba district, Chongqing

institute: Department of Pathogenic Biology, Army Medical University (Third Military Medical University)

summary:
A promising strategy to design effective malaria blood-stage vaccines is to induce parasite-specific CD4+ Th1 cell responses, but the regulatory mechanism of which is still largely unknown. Here, we showed that the ATG5-deficiency in myeloid cells significantly inhibit the growth of rodent blood-stage malaria parasites through specifically enhancing parasite-specific CD4+ Th1 cell responses. This effect was independent of ATG5-mediated canonical autophagy and non-canonical autophagy.
Mechanistically, the deficiency of ATG5 promoted the survival of inflammatory monocytes (CD11b+F4/80- cells), and sequentially increased the generation of chemokine MCP-1 in parasite-infected mice. The interaction of CD11b+F4/80- cells-derived MCP-1 with CCR2 on CD4+ Th1 cells for its optimized differentiation dependent on Jak2/stat4 pathway. Therefore, we highlight the previously unrecognized role of ATG5 in the modulation of the survival of inflammatory monocytes to produce MCP-1, and the selective promoting effect of MCP-1 on CD4+ Th1 cell differentiation, which shed new lights on effective malaria vaccine designation.

overall_design: RNASeq analysis of CD11b+ cells isolated from splenocytes of P. yoelii 17XNL-infected mice at Day 0, Day 3 and Day 6 post-infection.

first: Wenyue

last: Xu

first: Taiping


title: The nucleoskeleton protein NuMA maintains local chromatin architecture partially through promoting linker histone H1’ binding to chromatin and nucleosome stacking [RNA-Seq]

city: Beijing

first: Wenxue

last: Zhao

address: Yiheyuan Road

institute: Peking University

summary: Nucleoskeleton is generally considered to be the grid structure involved in regulation of genome expression and maintenance in the eukaryotic nucleus. However, its actual biological functions and mechanisms remain controversial. Here, we show the nuclear mitotic apparatus protein (NuMA), an essential regulator of the spindle, serve as one of the components of nucleoskeleton to regulates genome high-order architecture. In the interphase, fast depletion of NuMA leads to changes in chromatin organization, including increased accessibility of the genome, remodeling of nucleosome fibers and changes of transcription levels in certain heterochromatin regions. These effects are probably due to NuMA and linker histone H1’s interplay. We prove that NuMA interacts with linker histone H1 and linker DNA through its C-terminal domain. Such interaction facilitates H1’s binding to DNA and promotes nucleosome stacking, and might be part of the reason that NuMA maintains the local condensed state of heterochromatin. Collectively, our results reveal new biological functions of NuMA in the interphase, and also associates nucleoskeleton with genome organization at the mono-nucleosome level for the first time. This in-depth study on the mechanism of NuMA’s regulation on H1 and nucleosomes shed new light on how nucleoskeleton regulates genome architecture and gene expression.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for wildtype and NuMA-depleted HCT116 cells

first: Yao


title: The nucleoskeleton protein NuMA maintains local chromatin architecture partially through promoting linker histone H1’ binding to chromatin and nucleosome stacking

overall_design: Refer to individual Series


title: BRG1 establishes the neuroectodermal chromatin landscape to restrict dorsal cell fates

city: Research Triangle Park

first: Jackson

middle: Andrew

last: Hoffman

state: North Carolina

address: 111 TW Alexander Dr, Building 101 Room C432

department: DIR

institute: NIEHS

summary: Cell fate decisions are achieved with gene expression changes driven by lineage-specific transcription factors (TFs). These TFs depend on chromatin remodelers including the BAF complex to activate target genes. BAF complex subunits are essential for development and frequently mutated in cancer. Thus, interrogating how BAF complexes contribute to cell fate decisions is critical for human health. We examined the requirement for the catalytic BAF subunit BRG1 in neural progenitor cell (NPC) specification from human embryonic stem cells. During the earliest stages of differentiation, BRG1 was required to establish chromatin accessibility at neuroectoderm-specific enhancers. BRG1 depletion resulted in abnormal NPC populations that differentially expressed neuroectodermal TFs, were more prone to neuronal differentiation, and precociously formed neural crest lineages. These findings demonstrate that BRG1 mediates NPC specification by ensuring proper expression of lineage-specific TFs and appropriate activation of their transcriptional programs.

overall_design: RNAseq, single cell RNAseq, ATAC-seq, and Cut&Tag in H1 human embryonic stem cells and H1 cell during NPC differentiation. Cells were treated with Doxycycline to induce expression of BRG1 shRNA or treated with PROTAC to degrade BRG1

middle: A

first: Ginger

last: Muse

first: Lee

last: Langer

first: Isabella

last: Patterson

last: Gandara

first: James

last: Ward

first: Trevor

last: Archer


title: Recombinant GM-CSF enhances the bactericidal ability of PMNs by increasing intracellular IL-1β and improves the prognosis of secondary Pseudomonas aeruginosa pneumonia in sepsis

city: Fuzhou

first: Fuquan

last: Tu

state: Fujian

address: Xinquan road NO. 29

institute: Fujian Medical University

summary: This study tested the hypothesis that recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) enhances polymorphonuclear neutrophils (PMNs) via IL-1β to improve the prognosis of secondary infection in sepsis. The latter stage of sepsis is prone to induce immunosuppression, resulting in secondary fatal infections. rGM-CSF has become a way for sepsis-induced immunosuppression due to its immunomodulatory effect. However, the functional impact of GM-CSF on PMNs in sepsis remains obscure. This study aimed to study the role of rGM-CSF on the bactericidal ability of PMNs in septic mice, assessing its effect on the prognosis of secondary pneumonia, and explore the mechanism of rGM-CSF by intervening PMNs in patients with sepsis. The C57BL/6J sepsis mouse model was induced by cecal ligation and puncture (CLP). rmGM-CSF was used in vivo when mice developed immunosuppression, which was characterized by abnormal bactericidal function of PMNs in peripheral blood. rmGM-CSF improved the prognosis of secondary pneumonia and reversed the function of PMNs. PMNs isolated by Percoll from septic patients were treated by rhGM-CSF in vitro. The expression of CD11b, reactive oxygen species (ROS), phagocytosis and neutrophil extracellular traps (NETs) release in PMNs were enhanced by rhGM-CSF treatments. Whole-transcriptomic sequencing of mouse PMNs indicated that recombinant GM-CSF increased the expression of il1b gene in PMNs. Blocking and inhibiting IL-1β release effectively counteracted the enhancing effect of GM-CSF on the bactericidal function of PMNs. RmGM-CSF enhances the bactericidal function of PMNs in vivo and improves the prognosis of secondary pneumonia in septic mice, and recombinant GM-CSF increases IL-1β precursor reserves, which, if stimulated, can rapidly enhance the bactericidal capacity of PMNs.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for mice peripheral blood PMNs from three groups (sham operation group, CLP 5days group and CLP5days +GM-CSF groups).

first: Lili

last: Pan

first: Yuanhua

last: Cai


title: The nucleoskeleton protein NuMA maintains local chromatin architecture partially through promoting linker histone H1’ binding to chromatin and nucleosome stacking [Cut & Tag]

overall_design: CUT&Tag (Cleavage Under Targets and Tagmentation) analysis targetting NuMA C terminal and NuMA N terminal in wildtype HCT116. CUT&Tag analysis targetting histone H1 in wildtype and NuMA-depleted HCT116 cells.

last: Wang


title: PRC2 is a tumor suppressor in malignant peripheral nerve sheath tumor (MPNST)

first: Ping

last: Chi

address: 417 East 68th Street

department: Human Oncology and Pathogenesis Program

institute: Memorial Sloan Kettering Cancer Center

summary: Using mouse models, we show that PRC2 restrains MPNST development in the peripheral nervous system in the absence of both Nf1 and Cdkn2a tumor suppressor genes. Here we conducted bulk RNA-sequencing of tumors generated in mice.

overall_design: RNA isolated from MPNST tumors in mice with Nf1, Cdkn2a, and Eed flox allele inactivation in the Schwann cell lineage (via tamoxifen inducible Plp-CreERT activation).

first: Amish

last: Patel


title: Transcriptome sequencing of postharvest melon fruit infected by Alternaria alternata

city: Urumqi

first: Bai

middle: Yu

last: jia

address: Nong Da Dong Lu No. 311, Urumqi, Xinjiang，China

institute: Xinjiang Agricultural University, College of Food Science and Pharmacy

summary: Jiashi melon and 86-1 melon were inoculated with Alternaria alternata, and the difference of gene expression was analyzed after 0, 6, 12, 18 and 24 days storage.

overall_design: Jiashi melon and 86-1 melon were inoculated with Alternaria alternata (control inoculated with sterile water), and samples were collected at 0d, 6d, 12d, 18d and 24d after storage, respectively, and transcriptome sequencing was performed to compare gene expression differences. The experiment was set up with 3 replicates.

last: Yujia