title: RNAseq of mRNA from human NK cells treated with PGE2 or co-cultured with acute myeloid leukemia (AML) blasts

city: München

first: sainitin

last: donakonda

state: Bavaria

address: Ismaningerstr. 22

department: Institute of Immunology

country: Germany

institute: TUM

summary: NK cell effector functions are suppressed by AML-blast derived PGE2. Transcriptional regulation of human NK cells in culture with PGE2 or AML blasts was assessed via mRNA sequencing.

overall_design: NK cells (CD56+ cells) were sort-purified from PBMCs of healthy donors (BR1, BR2, BR3) and treated with either PGE2 (100ng/ml) or co-cultured with AML blasts (both for 6h). In some conditions, NK cells were stimulated by addition of K562 cells for the last two hours of co-culture.

first: Tobias

last: Baumann

first: Bastian

last: Höchst

first: Sainitin

last: Donakonda

first: Rupert

last: Oellinger


title: Bulk RNA transcriptomics of IIM muscle

status: Public on Oct 11 2024

city: Duesseldorf

first: Christopher

last: Nelke

state: NRW

address: UKD, Moorenstraße 5

institute: University Hospital Düsseldorf

type: Expression profiling by high throughput sequencing

summary: We performed bulk RNA transcriptomics of skeletal muscle samples from patients with idiopathic inflammatory myopathies.

overall_design: Total RNA was extracted from tissue samples using Qiagen RNeasy Mini kit following  manufacturer’s instructions (Qiagen, Hilden, Germany). RNA samples were quantified using Qubit 4.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked with RNA Kit on Agilent 5300 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA). RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina following manufacturer’s instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented according to manufacturer’s instruction. First strand and second strand cDNAs were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. Sequencing libraries were validated using NGS Kit on the Agilent 5300 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA). The sequencing libraries were multiplexed and loaded on the flow cell on the Illumina NovaSeq 6000 instrument according to manufacturer’s instructions. The samples were sequenced using a 2x150 Pair-End (PE) configuration v1.5. Image analysis and base calling were conducted by the NovaSeq Control Software v1.7 on the NovaSeq instrument. Raw sequence data (.bcl files) generated from Illumina NovaSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq program version 2.20. One mismatch was allowed for index sequence identification

last: Ruck

first: Anne

last: Schänzer


title: Next Generation Sequencing Facilitates Quantitative Analysis of Human Normal Tissues and Epileptic Tissues

city: Chongqing

first: Zhai

last: Xuan

address: Chongqing 400010, China

department: Department of Neurosurgery

country: China

institute: Children’s Hospital of Chongqing Medical University

type: Non-coding RNA profiling by high throughput sequencing

summary: Epilepsy is a neurological disease that causes serious implications on the quality of life of an individual. Through this study, we aimed to identify regulatory noncoding RNAs and mRNAs involved in Epilepsy. A RNA-seq analysis was conducted to determine epilepsy-related noncoding RNAs and mRNAs. In summary, the newly identified circRNAs in networks and signaling pathways found in this study would provide hints for the development of novel prognostic, diagnostic, and therapeutic strategies for Epilepsy.

overall_design: Brain tissue mRNA, circRNA and lncRNA profiles of normal people and patients with epilepsy.

first: Xuan

last: Zhai

first: Han

last: Xiao

first: Hui

last: Chen

last: Gan


title: Genes downregulated by miR-92a-1-5p overexpression in K562 cells treated with imatinib.

city: Woluwe-Saint-Lambert

first: Joanne

last: Peters

state: Brussels

address: Av. Hippocrate 75

department: MEXP

country: Belgium

institute: De Duve Institute

summary: In order to indetify new target genes of miR-92a-1-5p in chronic myeloid leukemia, K562 cells carrying the BCR::ABL1 fusion gene were transiently transfected with miR-92a mimic or scramble and treated with imatinib. RNA was sequenced to identify genes expressed under imatinib treatment and downregulated after miR-92a-1-5p overexpression.

overall_design: K562 cells (5 million) were nucleoporated using Amaxa Nucleofector Technology (Lonza) with 500 pmol of hsa-miR-92a-1-5p synthetic miRVana mimic (#4464066) or  miRVana miRNA Mimic, Negative Control #1(#4464058) (Thermo Fisher Scientific). 4h after transfection, cells were treated with 1 µM of Imatinib. 24h later, RNA was extracted and sent to Macrogen for RNA- sequencing. A paired-end sequencing was performed with an average sequencing coverage of 62 M. Results obtained were quantified using the Kallisto software.

first: Violaine

last: Havelange

first: Guillaume

last: Dachy


title: Whole genome bisulfite sequencing of arp6-5 and pie1-7 Arabidopsis mutants

city: West Lafayette

first: Jian-Kang

last: Zhu

state: IN

address: 625 Agriculture Mall Dr.

department: Department of Horticulture and Landscape Architecture

country: USA

institute: Purdue University

type: Methylation profiling by high throughput sequencing

summary: Using whole genome bisulfite sequencing to provide single-base resolution of DNA methylation status in arp6-5 and pie1-7 mutant

overall_design: Examination of 2 different mutants' methylome: arp6-5 and pie1-7

first: Wen-Feng

last: Nie

first: Mingguang

last: Lei

first: Kai

last: Tang


title: AtMBD9 ChIP-seq

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Using ChIP-seq to study the enrichment of AtMBD9 in Col-0 control plants and AtMBD9-3xMYC/atmbd9-2 transgenic line

overall_design: Examination of AtMBD9 localization using ChIP-Seq

first: Wenfeng

first: Cui-Jun

last: Zhang

first: Daisuke

last: Miki


title: H2A.Z ChIP-seq of WT, ros1-14, idm1-9, arp6-5 and pie1-7

summary: Using ChIP-seq to study the deposition of H2A.Z in different anti-silencing mutants

overall_design: Examination of H2Z.Z deposition using ChIP-Seq

first: Huan

last: Huang

first: Cui-jun


title: Pervasive off-target activity in CRISPR-interference (CRISPRi) system [ChIP-seq]

city: South San Francisco

first: Rohit

last: Reja

state: CA

address: 1 DNA Way

institute: Genentech

summary: CRISPR/Cas system and its dCas derivatives have been widely and effectively used to alter the intended genomic target. However, the presence of off-targets due to unintended binding of sgRNAs to sequences that closely resemble the target sequence, is still a major challenge. Here, we utilized a genome-wide sgRNA library for the dCas9-KRAB CRISPRi system to investigate the presence of off-target activity and its effects on gene expression. Our study provides strong evidence that CRISPRi off-targets affect the transcriptome of a cell extensively. We also highlight that mis-matches to the target DNA are tolerated while sgRNA binding and the length of the contiguous matches in the PAM-proximal region is one of the most predictive features of binding, along with open chromatin and the DNA-RNA hybrid energy. Additionally, we provide a curated random forest model that can be used to predict the off-target activity in the dCas systems.

overall_design: ChIPseq across two different cell lines, Jurkat and EA.hy926, infected with single guides for ADAT1, HMGB1, GRK4, and LRRC34 respectively and input controls

first: R

first: N

last: Rohatgi


title: Periostin-treated healthy human airway epithelial cell cultures

status: Public on Oct 10 2024

city: Iowa City

first: Alejandro

middle: A.

last: Pezzulo

state: Iowa

address: 6316 Pappajohn Biomedical Discovery Building

department: Department of Internal Medicine

institute: Universty of Iowa

summary: The goal of this study was to examine changes in gene expression in response to periostin treatment.

overall_design: Primary human airway epithelia were exposed to vehicle or periostin, prepared for RNA sequencing, and sequenced on Illumina HiSeq 4000 system using 2x151bp paired end SBS chemistry.

first: Schecter

last: Scott

first: Lorena

middle: A

last: Tran

first: Andrew

middle: L

last: Thurman


title: MethlyC-Seq of  WT (35S-SUC2) and anti-silencing mutants (arp6-5, pie1-7, h2a.z-2,  idm1-9 and ros1-14)

summary: Using MethylC-Seq to provide single-base resolution of DNA methylation status in 35S-SUC2 WT and anti-silencing mutants( arp6-5, pie1-7, h2a.z-2, idm1-9 and ros1-14）

overall_design: MethylC-Seq:  WT and 5 mutants( arp6-5, pie1-7, h2a.z-2, idm1-9 and ros1-14)


title: Anti-silencing mutants

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

overall_design: Refer to individual Series


title: Whole genome bisulfite sequencing of hdp1-1 and hdp2-1 mutants (both with 35S-SUC2 transgene)

summary: Using whole genome bisulfite sequencing to provide single-base resolution of DNA methylation status in hdp1-1 and hdp2-1 mutants

overall_design: 2 samples examined: hdp1-1 and hdp2-1 mutants (with 35S-SUC2 transgene)

first: Cheng-Guo

last: Duan

first: Xingang

last: Wang


title: Co-treatment with melatonin and ortho-topolin riboside exhibits anti-proliferation activity in radioresistant MDA-MB-231 cells by altering metabolic and transcriptomic profiles

city: Seoul

first: Lee

last: Seungcheol

address: Heukseokro 84

country: South Korea

institute: Chungang university

summary: This is the first study to demonstrate that co-treatment with MLT and oTR has a synergistic effect in inhibiting the proliferation of MDA-MB-231/RR cells based on metabolic and transcriptomic analyses. Treatment with either 1 mM MLT or 1 μM oTR was not cytotoxic, while their synergistic activity upon co-treatment was cytotoxic. In our previous study, MDA-MB-231 cells acquired radio resistance as mitochondrial respiration increased; however, co-treatment with MLT and oTR simultaneously inhibited mitochondrial respiration and glycolysis. Furthermore, when integrating the transcriptomic and metabolic profile results, alteration of pyrimidine metabolism (aspartate, uracil, CDA, NME1), steroid hormone biosynthesis (AKR1C1, AKR1C2, AKR1C3), and Wnt/β-catenin signaling (TSNAX-DISC1, CYP1B1) was attributed to the synergistic cytotoxicity by the co-treatment. In future studies, proteomic profiling could be performed to verify the results of the metabolomic and transcriptomic profiling in this study. The results of this study should be validated with in vivo experiment as a prelude to future clinical trials for the treatment of radioresistant TNBC. We expect our findings to provide basic information for the treatment of radioresistant TNBC.

overall_design: We hypothesized that co-treatment of radioresistant MDA-MB-231 (MDA-MB-231/RR) cells with MLT and oTR would result in synergistic cytotoxicity over either treatment alone due to alterations in the metabolome and transcriptome.

first: Soonwook

last: Noh

first: Hyungkyoon

last: Choi


title: Ets1 dampens expression of Tbet and CD8- or NK-like effector programing in postselection iNKT1 cells [scRNA-seq]

city: Chicago

first: Barbara

last: Kee

state: Illinois

address: 924 E57th st

department: Pathology

institute: The University of Chicago

summary: The goal of this study was to compare the gene expression program of thymic iNKT1 cells from Control mice to those with a postselection deletion of the transcription factor Ets1 at the single cell level. Ets1-floxed alleles were deleted in iNKT1 cells using Tbx21Cre bacterial artifical chromosome transgenic mice that also carry a Rosa26-floxed-stop-YFP reporter. Control iNKT1 cells were Tbx21Cre+ Rosa26-floxed-stop-YFP reporter+.  We isolated all thymic YFP+ cells from Control and cKO mice and processed for scRNA-seq using the 10X Genomics Chromium.  Data was processed using the Cell Ranger program for 10X Genomics. .  We found that iNKT1 cells lacking Ets1 upregulated Tbx21 and its protein Tbet, many Tbet target genes, and CD8 effector T cell associated genes.

overall_design: Expression profiling analysis of RNA from thymic iNKT1 cells isolated from Ctrl (Tbx21Cre; Rosa26-YFP+) and cKO (Tbx21Cre; Rosa26-YFP+; Ets1F/F) mice by single cell RNA-sequencing.

first: Roxroy

middle: C

last: Morgan


title: CSRP1 in stromal fibroblasts drives high-grade serous ovarian cancer development

city: Shenyang

first: Qijun

last: Wu

address: No. 36, San Hao Street

institute: Shengjing Hospital of China Medical University

summary: RNA-seq was performed to identify the genes regulated by YBX1 in cancer-associated fibroblasts in HGSOC.

overall_design: We performed RNA-seq analysis of primary human cancer-associated fibroblasts infected with lentiviral vectors carrying YBX1 shRNA and NC shRNA. Cancer-associated fibroblasts were isolated from omentum tissues of HGSOC patients with omentum metastasis.

first: Tingting

last: Gong

first: Xue

last: Qin

first: Xiaobin

first: Yizi

last: Li

first: Fanghua

last: Liu

first: Yifan

last: Wei

first: Heli

last: Xu

first: Jianfei

last: Pei

first: Ye

last: Kang

first: Yunlong

last: Huo

first: Si

first: Jinchi

first: Yue

last: Han

first: Fan

last: Cao

first: Donghui

first: Xiaoying

first: Lang

first: Meng

last: Niu

first: Huihan

first: Song

last: Gao


title: The mitochondrial enzyme pyruvate carboxylase restricts pancreatic β-cell senescence by blocking p53 activation

city: Shanghai

first: Yumei

last: Yang

address: No.180 Fenglin Road

department: Department of Endocrinology and Metabolism

institute: Zhongshan Hospital, Fudan University

summary: Defective glucose-stimulated insulin secretion (GSIS) and β-cell senescence are hallmarks in diabetes. The mitochondrial enzyme pyruvate carboxylase (PC) has been shown to promote GSIS and β-cell proliferation in the clonal β-cell lines, yet its physiological relevance remains unknown. Here, we provide animal and human data showing a role of PC in protecting β-cells against senescence and maintaining GSIS under different physiological and pathological conditions. β-cell-specific deletion of PC impaired GSIS and induced β-cell senescence in the mouse models under either a standard chow diet or prolonged high-fat diet feeding. Transcriptomic analysis indicated that p53-related senescence and cell cycle arrest are activated in PC-deficient islets. Overexpression of PC inhibited hyperglycemia- and aging-induced p53-related senescence in human and mouse islets as well as INS-1E β-cells, whereas knockdown of PC provoked senescence. Mechanistically, PC interacted with MDM2 to prevent its degradation via the MDM2 binding motif, which in turn restricts the p53-dependent senescent program in β-cells. On the contrary, the regulatory effects of PC on GSIS and the tricarboxylic acid (TCA) anaplerotic flux are p53-independent. We illuminate a function of PC in controlling β-cell senescence through the MDM2–p53 axis.

overall_design: Islets isolated from 12-wk-old mice β-PCKO mice and their WT littermates were subjected to RNAseq analysis (n = 3).

first: Baomin

last: Wang

first: Rong

last: Xie

first: Kingyip

last: Cheng

first: Xiaomu


title: Co-cultivating rice plants with Azolla filiculoides modifies root architecture and timing of developmental stages

city: Milano

first: Chiara

last: Paleni

address: Via Celoria, 26

country: Italy

institute: Università degli Studi di Milano

summary: Innovative strategies for increasing the yield of rice, the staple food for more than half of the global population, are needed to keep pace with the expected worldwide population increase, and sustainably forefront the challenges posed by climate change. Traditionally, in Southern-East Asian countries, rice farming benefits from the use of Azolla spp., either as green manure or as co-cultivated plants, for the supply of nitrogen. Azolla spp. are ferns that, in virtue of their symbiosis with the nitrogen-fixing cyanobacterium Trichormus azollae, fix atmospheric nitrogen and release it to the environment upon decomposition of their biomass. However, if and to what extent actively growing Azolla plants impact on the development of co-cultivated rice plantlets remains to be understood. To address this point here we employed an experimental model to follow the growth and development of roots and aerial organs of rice seedlings when co-cultivated with Azolla filiculoides. We show that actively growing A. filiculoides plants alter the architecture of the roots, the transcriptome of the roots, and the hormonal profiles of both roots and leaves.

overall_design: We cultivated rice plants in the presence of actively growing Azolla filiculoides (co-cultivation, R+Af) and in control conditions (rice plants alone, R). At least three boxes per treatment with four rice plants each were employed as replicates for each experiment. RNA from the roots of three biological replicates per treatment at 15 DAT was extracted and sequenced for RNAseq and differential gene expression analysis.

first: Antonietta

last: Saccomanno

first: Martin

middle: M

last: Kater

first: Veronica

last: Gregis

first: Francesco

last: Paolocci

first: Sara

last: Cannavò

first: Alma

last: Costarelli


title: Transcriptional repression by HDAC3 mediates T cell exclusion from Kras mutant lung tumors [KL LJE1 cells p65 KO RNA-seq]

first: Lillian

middle: J

last: Eichner

state: IL

address: 303 E Superior St

department: Biochemistry and Molecular Genetics

institute: Northwestern University

summary: Histone Deacetylase 3 (HDAC3) function in vivo is nuanced and directed in a tissue specific fashion.  The importance of HDAC3 in Kras mutant lung tumors has recently been identified, but HDAC3 function in this context remains to be fully elucidated.  Here, we identified HDAC3 as a lung tumor cell-intrinsic transcriptional regulator of the tumor immune microenvironment.  In Kras mutant lung cancer cells, we found that HDAC3 is a direct transcriptional repressor of a cassette of secreted chemokines, including Cxcl10.  Genetic and pharmacological inhibition of HDAC3 robustly upregulated this gene set in human and mouse Kras, LKB1 (KL) and Kras, p53 (KP) mutant lung cancer cells through an NF-kB/p65-dependent mechanism.  Using genetic engineered mouse models, we found that HDAC3 inactivation in vivo induced expression of this gene set selectively in lung tumors, and resulted in enhanced T-cell recruitment at least in part via Cxcl10.  Furthermore, we found that inhibition of HDAC3 in the presence of Kras pathway inhibitors dissociated Cxcl10 expression from that of immunosuppressive chemokines, and that combination treatment of entinostat with trametinib enhanced T-cell recruitment into lung tumors in vivo.  Finally, we showed that T-cells contribute to in vivo tumor growth control in the presence of entinostat and trametinib combination treatment.  Together, our findings reveal that HDAC3 is a druggable endogenous repressor of T-cell recruitment into Kras mutant lung tumors.

overall_design: RNA-seq in p65 KO and NT KL LJE1 cells

first: Caroline

middle: K

last: McGuire


title: Transcriptional repression by HDAC3 mediates T cell exclusion from Kras mutant lung tumors [KL GEMM Treated Primary Tumors RNA-seq]

overall_design: RNA-seq on primary NSCLC tumors from the KL model treated with trametinib, entinostat, combination, or vehicle


title: Transcriptional repression by HDAC3 mediates T cell exclusion from Kras mutant lung tumors [H3K9Ac ChIP-seq]

overall_design: ChIP-seq for H3K9Ac in HDAC3 KO and NT KL cells


title: RBBP5 is an epigenetic regulator of mammalian hepatic 12h oscillator [RNA-seq]

city: Pittsburgh

first: Bokai

last: Zhu

state: PA

address: 100 Technology Dr, Bridgeside point

institute: University of Pittsburgh

summary: Proteostasis is vital for cellular health, with disruptions leading to pathologies including aging, neurodegeneration and metabolic disorders. Traditionally, proteotoxic stress responses were studied as acute reactions to various noxious factors; however, recent evidence reveals that many stress-response genes exhibit ~12-hour ultradian rhythms under physiological conditions in mammals. These rhythms, driven by an XBP1s-dependent 12h oscillator, are crucial for managing proteostasis. By exploring the chromatin landscape of the murine 12h hepatic oscillator, we identified RBBP5, a key subunit of the COMPASS complex, as an essential epigenetic regulator of proteostasis. RBBP5 is indispensable for regulating both the hepatic 12h oscillator and transcriptional response to acute proteotoxic stress, acting as a co-activator for master proteostasis transcription factor XBP1s. RBBP5 ablation leads to increased sensitivity to proteotoxic stress, chronic inflammation, and hepatic steatosis in mice, along with impaired autophagy and reduced cell survival in vitro. In humans, lower RBBP5 expression is associated with dampened adaptive stress-response gene expression and hepatic steatosis. Our findings establish RBBP5 as a critical regulator of proteostasis, essential for maintaining mammalian organismal health.

overall_design: All mice used for biological rhythm study are in C57BL/6J background, male and between 3 and 4 months of age. Rbbp5 (flox/flox); Alb-CRE (-/-) (n=48) and Rbbp5 (flox/flox); Alb-CRE (+/-) mice (n=48) [Rbbp5 (flox/flox); Alb-CRE (-/-) and Rbbp5 (flox/flox); Alb-CRE (+/-) mice were littermates] were first entrained under LD12:12 conditions for 2 weeks before transferred to constant darkness for 24hrs. Mice were then sacrificed via cervical dislocation at a 2h interval  for a total of 48 hours under constant darkness. Mice were fed ad libitum during the entire experiment. Liver were taken.


title: Epigenetic regulation of global proteostasis dynamics by RBBP5 ensures mammalian organismal health

first: Shuchang

last: Liu

address: 203 Lothrop Street

overall_design: For acute ER stress induction experiment, male littermates WT [Rbbp5 (flox/flox); Alb-CRE (-/-)] and RBBP5 LKO [Rbbp5 (flox/flox); Alb-CRE (+/-) mice] between 5 and 7 months of age were randomly divided into two groups, and intraperitoneally injected with 0.05mg/kg body weight of tunicamycin dissolved in 500ul 3% DMSO in PBS or vehicle control (3% DMSO in PBS), respectively. Mice were injected with Tu between 9~10am in the morning and 8 hours later dissected for transcriptomic analysis for liver.

first: Syeda

last: Kubra

first: Michelle

last: Sun

first: William

last: Dion

first: Ahmet

last: Catak

first: Hannah

last: Luong

first: Haokun

first: Yinghong

last: Pan

first: Jia-Jun

first: Timothey

middle: R

last: Billiar

first: Aishwarya

last: Ponna

first: Ian

last: Sipula

first: Michael

last: Jurczak

first: Silvia


title: Comparison of DNA Methylation Based Classification Models for Precision Diagnostics of Central Nervous System Tumors

city: Memphis

first: Quynh

last: Tran

state: TN

address: 262 Danny Thomas Place, C4029, Mail Stop 250

institute: St. Jude Children's Research Hospital

type: Methylation profiling by genome tiling array

summary: As part of the advancement in therapeutic decision-making for brain tumor patients at St. Jude Children’s Research Hospital (SJCRH), we developed three robust classifiers, a deep learning neural network (NN), k-nearest neighbor (kNN), and random forest (RF), trained on a reference series DNA-methylation profiles to classify central nervous system (CNS) tumor types. The models’ performance was rigorously validated against 2,054 samples from two independent cohorts. In addition to classic metrics of model performance, we compared the robustness of the three models to reduced tumor purity, a critical consideration in the clinical utility of such classifiers. Our findings revealed that the NN model exhibited the highest accuracy and maintained a balance between precision and recall. The NN model was the most resistant to drops in performance associated with a reduction in tumor purity, showing good performance until the purity fell below 50%. Through rigorous validation, our study emphasizes the potential of DNA-methylation-based deep learning methods to improve precision medicine for brain tumor classification in the clinical setting.

overall_design: We validated the performance of these two models and the RFmod with two independent brain tumor cohorts consisting of 1104 samples from GSE109379 and 950 samples from the St. Jude Children’s Research Hospital. Our results showed that although all models performed robustly to missing data, the deep NN model had the highest CNS classification accuracy and the most favorable performance characteristics, especially in minimizing the proportion of subthreshold scores during testing and validation. Average precision and recall of the NNmod started reducing to similar levels of kNNmod and RFmod when tumor purity was less than 50%. This suggests that a deep NN model can be implemented in clinical laboratories as a reliable and essential diagnostic tool to assist in precision therapy for brain tumors.

first: Brent

last: Orr

first: Alex

last: Breuer


title: Epigenetic regulators of clonal hematopoiesis control CD8 T cell stemness during immunotherapy [Bisulfite-Seq]

first: Tian

last: Mi

address: 262 Danny Thomas Pl MS351

department: Immunology

summary: Epigenetic reinforcement of T cell exhaustion is a major barrier limiting durability of T cell responses during immunotherapy, however the central epigenetic regulators restricting therapy-enabling T cell stemness in settings of prolonged antigen exposure remain to be fully resolved. Here we investigated DNMT3A, TET2, and ASXL1, the three most commonly mutated epigenetic regulators promoting clonal hematopoiesis (CH), to determine if they control the cardinal features of T cell stemness. Using a canonical murine model of exhaustion, we show that CD8 T cells lacking Dnmt3a, Tet2, or Asxl1 are able to preserve an immune checkpoint blockade (ICB) responsive progenitor-exhausted (Tpex) population for over a year during chronic antigen exposure without undergoing malignant transformation. Specific investigation into the lesser-studied regulator, Asxl1, revealed that this unprecedented maintenance of Tpex was achieved through preservation of the cell’s self-renewal capacity with less terminal differentiation. Mechanistic investigation into the role of Asxl1 during CD8 T cell differentiation revealed epigenetic modification of the PR-DUB pathway involving H2AK119-ubiquitination. Extending this Tpex-preserving mechanism to immunotherapy, we report synergy between Asxl1 deficient T cells and anti-PDL1, resulting in heightened tumor control in murine tumor models and a survival advantage to the mutated T cells in treated patients. These data collectively define a core set of epigenetic regulators that control longevity of the stem-like T cell population responsible for clinical success during cancer immunotherapy.

overall_design: 2 replicates of Rosa KO samples, 2 replicates of Dnmt3a KO samples, 2 replicates of Tet2 KO samples, 2 replicates of Asxl1 KO samples

first: Tae

middle: G

last: Kang

first: Ben

last: Youngblood

first: Caitlin

last: Zebley


title: Epigenetic regulators of clonal hematopoiesis control CD8 T cell stemness during immunotherapy [RNA-seq]

overall_design: 5 replicates of Rosa KO samples, 4 replicates of Dnmt3a KO samples, 4 replicates of Tet2 KO samples, 4 replicates of Asxl1 KO samples,


title: Epigenetic regulators of clonal hematopoiesis control CD8 T cell stemness during immunotherapy [CUT&Tag]

overall_design: 1 replicate of Naive H3K27me3 sample, 4 replicates of Rosa KO H3K27me3 samples, 5 replicates of Asxl1 KO H3K27me3 samples, 2 replicates of Rosa KO H2AK119Ub samples, 3 replicates of Asxl1 KO H2AK119Ub samples, 4 replicates of Naive H3K4me3 samples, 5 replicates of Rosa KO H3K4me3 samples, 4 replciates of Asxl1 KO H3K4me3 samples, 4 samples of Naive H3K9ac samples, 2 replicates of Rosa H3K9ac samples, 2 replicates of Asxl1 KO H3K9ac samples


title: Epigenetic regulators of clonal hematopoiesis control CD8 T cell stemness during immunotherapy [ATAC-seq]

overall_design: 2 replicates of Rosa KO samples, 2 replicates of Asxl1 KO samples, 2 replicates of Rosa KO Ly108 subset, 2 replicates of Rosa KO Tim3 subset, 4 replicates of Asxl1 KO Ly108 subset, 4 replicates of Asxl1 KO Tim3 subset


title: Gene expression profile of different cell types in the tumor microenvironment of MOC1 tumors treated with Smyd3 anti-sense oligonucleotides (ASOs).

city: ROCKVILLE

first: Vassiliki

last: Saloura

state: MARYLAND

address: 11705 STONEWOOD LN

department: TGMB

institute: NCI

summary: We used scRNA-seq to assess the differential effect of Smyd3 ASOs on the transcriptomic profile of different cell types in the TME of MOC1 tumors.

overall_design: MOC1 tumors were implanted in the flank of C57BL/6 mice were treated with control (n=3) or Smyd3 ASOs (n=3) for 18 days. Tumors were surgically resected, digested into single-cell suspensions and scRNA-seq was conducted.

first: Alexei

last: Lobanov

first: Abdalla

last: Abdelmaksoud


title: Epigenetic regulators of clonal hematopoiesis control CD8 T cell stemness during immunotherapy [scRNA-seq]


title: Epigenetic regulators of clonal hematopoiesis control CD8 T cell stemness during immunotherapy.

overall_design: Refer to individual Series


title: Gene expression profile of different cell types in the tumor microenvironment of MOC1 tumors that responded or not to treatment with Smyd3 ASOs and anti-PD-1 combination therapy.

summary: We used scRNA-seq to assess the transcriptomic differences in the different cell types of the TME of MOC1 tumors that responded or not to Smyd3 ASOs and anti-PD-1 combination therapy.

overall_design: MOC1 tumors were implanted in the flank of C57BL/6 mice and were treated with the combination of Smyd3 or control ASOs plus anti-PD-1 for 29 days. 2 responder and 2 non-responder tumors to the Smyd3 ASO plus anti-PD-1 were surgically resected, digested into single-cell suspensions and scRNA-seq was conducted.


title: Ets1 dampens expression of Tbet and CD8- or NK-like effector programing in postselection iNKT1 cells [RNA-seq]

first: Barbara

last: Kee

summary: The goal of this study was to compare the gene expression program of thymic and liver iNKT1 cells from Control mice to those with a postselection deletion of the transcription factor Ets1. Ets1-floxed alleles were deleted in iNKT1 cells using Tbx21Cre bacterial artifical chromosome transgenic mice that also carry a Rosa26-floxed-stop-YFP reporter. Control iNKT1 cells were Tbx21Cre+ Rosa26-floxed-stop-YFP reporter+.  We examined gene expression by RNA-sequencing of sorted iNKT1 cells (CD1d-Tet-PBS57+ YFP+) from the thymus and liver.  Reads were aligned to the mm10 reference genome using Tophat v 2.1.0.  Reads were assigned to genes using the htseq-count tool from HTSeq v 0.6.1 and gene annotations from Ensembl release 78.   Differential expression was calculated across 3 independent replicates by EdgeR.  We found that iNKT1 cells lacking Ets1 upregulated Tbx21 and its protein Tbet, many Tbet target genes, and CD8 effector T cell associated genes.

overall_design: Expression profiling analysis of RNA from thymic and liver iNKT1 cells isolated from Ctrl (Tbx21Cre; Rosa26-YFP+) and cKO (Tbx21Cre; Rosa26-YFP+; Ets1F/F) mice by RNA-sequencing. 3 independent replicates for each sample.

first: Roxroy

last: Morgan

first: Cameron

last: Frank

first: Mikael

last: Sigvardsson

first: Elizabeth

middle: T

last: Bartom


title: Smyd3-mediated immuno-modulation in HPV-negative head and neck squamous cell carcinoma mouse models.

summary: SET and MYND-domain containing protein 3 (SMYD3) mediates epigenetic repression of type I IFN response genes in human papilloma virus (HPV)-negative HNSCC cells, and Smyd3 depletion using anti-sense oligonucleotides (ASOs) increases the sensitivity of syngeneic mouse oral carcinoma (MOC1) models to anti-PD-1 therapy. In this study, we interrogated the effects ASO-induced Smyd3 depletion in the cancer and immune cell populations, as well as the global changes in the tumor microenvironment (TME) of MOC1 tumors treated with Smyd3 ASOs and anti-PD-1. Single-cell RNA-seq of MOC1 tumors treated with Smyd3 ASOs revealed enrichment of type I IFN response pathways in cancer cells, a shift of CD8+ T-cells towards an activated/memory phenotype and a shift of neutrophils towards an anti-tumorigenic phenotype. While Smyd3 ASOs seemed to also promote a Treg and M2 macrophage phenotype, bulk RNA-seq of MOC1 tumors treated with Smyd3 ASOs and anti-PD-1 revealed enrichment of type I IFN response pathways and upregulation of CD8+ T-cell attracting chemokines, implying the induction of an inflamed TME. Non-responder MOC1 tumors to the Smyd3 ASO and anti-PD-1 combination revealed mechanisms of resistance derived from cancer cells, macrophages and CD8+ T-cells, including neutrophil enrichment through upregulation of Cxcl2, repression of Cxcl9 and defective antigen presentation. This study sheds light in the immunomodulatory functions of Smyd3 in an in vivo setting and provides insight into actionable mechanisms of resistance to improve the therapeutic efficacy of Smyd3 ASO and anti-PD-1 combination treatment.

overall_design: MOC1 tumors were implanted in the flank of C57BL/6 mice and were treated with the combination of Smyd3 or control ASOs plus anti-PD-1 for 32 days. Mice were then sacrificed, tumors were surgically resected, snap-frozen and processed for bulk RNA-seq.


title: IGF2BP1 phosphorylation regulates ribonucleoprotein condensate formation by impairing low-affinity protein and RNA interactions (RNA-Seq)

city: Vienna

first: Aleksandra

middle: S

last: Anisimova

address: Dr. Bohr Gasse 9

country: Austria

institute: Max Perutz Labs, Vienna BioCenter, University of Vienna, Medical University of Vienna

summary: The insulin-like growth factor 2 mRNA binding protein (IGF2BP1) is a conserved RNA-binding protein that regulates RNA stability, localization, and translation. IGF2BP1 is part of various ribonucleoprotein (RNP) condensates. However, the mechanism that regulates its assembly into condensates remains unknown. Here we found, using proteomics, that IGF2BP1 phosphorylation at S181 in a disordered linker is regulated in a stress-dependent manner. Phosphomimetic mutations in two disordered linkers, S181E and Y396E, modulated RNP condensate formation by IGF2BP1 without impacting its binding affinity for RNA. Intriguingly, the S181E mutant, which lies in linker 1, impaired IGF2BP1 condensate formation in vitro and in cells, whereas a Y396E mutant in the second linker increased condensate size and dynamics. Structural approaches showed that the first linker binds RNAs nonspecifically through its RGG/RG motif, an interaction weakened in the S181E mutant. Notably, linker 2 interacts with IGF2BP1’s folded domains and these interactions were partially impaired in the Y396E mutant. Our data reveal how phosphorylation modulates low affinity interaction networks in disordered linkers to regulate RNP condensate formation.

overall_design: RNA-Seq of HCT116 cells expressing mCherry-IGF2BP1 WT, S181E, Y396E and RQ (R167Q, R168Q, R174Q, R178Q) mutants and RNA immunoprecipitation sequencing (RIP-Seq) of mCherry-IGF2BP1 WT, S181E, Y396E and RQ mutants in control and arsenate stress conditions

first: Harald

last: Hornegger

first: Adnan

last: Muratovic

first: Benjamin

last: Burgeois

first: Elena

last: Spinetti

first: Roberto

last: Covino

first: Tobias

last: Madl

first: G

middle: E

last: Karagöz


title: IGF2BP1 phosphorylation regulates ribonucleoprotein condensate formation by impairing low-affinity protein and RNA interactions


title: Sequencing-based study of neural induction of human dental pulp stem cells

city: Tsukuba

first: Shohei

last: Takaoka

state: Ibaraki

address: 2-1-1 Amakubo

department: oral and maxillofacial surgery

country: Japan

institute: Tsukuba university hospital

summary: This study aimed to evaluate the cellular components and differentiation pathways of neural lineage cells obtained via neural induction of human DPSCs. Human DPSCs were induced to neural cells in monolayer culture. The neural lineage cells were subjected to single-cell RNA sequencing (scRNA-seq) to classify cell populations based on gene expression profiles and elucidate their differentiation pathways.  The scRNA-seq and cell type annotations evidenced the presence of neural progenitor cells, astrocytes, neurons, and a small number of non-neural lineage cells. Moreover, trajectory and pseudotime analyses demonstrated that the neural progenitor cells initially engendered neurons, which subsequently differentiated into astrocytes. This result indicates that the aforementioned neural induction strategy generated neural stem/progenitor cells from DPSCs, which might differentiate and proliferate to constitute neural lineage cells. Therefore, neural induction of DPSCs may present an alternative approach to pluripotent stem cell-based therapeutic interventions for nervous system disorder.

overall_design: Human DPSCs were induced to neural lineage cells in monolayer culture, and we evaluated the cellular components and differentiation pathways of neural lineage cells obtained via neural induction of human DPSCs.


title: Transcriptome Cappable-Seq Sequencing Data Set of Gene Expression in Salmonella enterica serovar Typhimurium 14028S inside Acanthamoeba castellanii, and under Oxidative and Starvation Stress Conditions

city: Orenburg

first: Alexander

last: Balkin

address: 11 Pionerskaya st.

country: Russia

institute: Institute for Cellular and Intracellular Symbiosis

summary: Here we present high-resolution Cappable-Seq transcriptomic data on Salmonella gene expression under optimal, starvation and hydrogen peroxide conditions, and after 8 and 15 hours of co-culture with A. Castellanii.

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S

overall_design: To assess changes in global Salmonella gene expression during long-term infection of amoebae, we performed Cappable-Seq sequencing of total Salmonella RNA within A. castellanii at late stages of infection (8 h and 15 h) and also compared three free-living cultures grown under different conditions, including rich medium (LB), poor medium (PAS), and exposure to hydrogen peroxide.

first: Yuri

last: Gogolev


title: RNA-Seq analysis of SUM159 DAPK3 knockout (KO) cells and control cells

city: Houston

first: Charles

last: Foulds

state: TX

address: One Baylor Plaza

department: Lester and Sue Smith Breast Center

institute: Baylor College of Medicine

summary: We conducted RNA sequencing of SUM159 DAPK3 KO and control cells followed by Gene Set Enrichment Analysis (GSEA) to identify altered gene expression pathways by DAPK3 knockout. Epithelial-mesenchymal transition (EMT) pathway was the top significantly down-regulated pathway in SUM159 DAPK3 KO cells as compared to control cells.

overall_design: mRNA profiles of SUM159 cell lines to identify altered gene expression upon DAPK3 knockout

first: Junkai

last: Wang

first: Matthew

last: Ellis


title: Epigenomic, transcriptomic and proteomic characterizations of reference samples

city: Loma Linda

address: 11021 Campus Street

department: Department of Basic Sciences

institute: Loma Linda University

summary: A variety of newly developed next-generation sequencing technologies are making their way rapidly into the research and clinical applications, for which accuracy and cross-lab reproducibility are critical, and reference standards are much needed. Our previous multicenter studies under the SEQC-2 umbrella using a breast cancer cell line with paired B-cell line have produced a large amount of different genomic data including whole genome sequencing (Illumina, PacBio, Nanopore), HiC, and scRNA-seq with detailed analyses on somatic mutations, single-nucleotide variations (SNVs), and structural variations (SVs). However, there is still a lack of well-characterized reference materials which include epigenomic and proteomic data. Here we further performed ATAC-seq, Methyl-seq, RNA-seq, and proteomic analyses and provided a comprehensive catalog of the epigenomic landscape, which overlapped with the transcriptomes and proteomes for the two cell lines. We identified >7,700 peptide isoforms, where the majority (95%) of the genes had a single peptide isoform. Protein expression of the transcripts overlapping CGIs were much higher than the protein expression of the non-CGI transcripts in both cell lines. We further demonstrated the evidence that certain SNVs were incorporated into mutated peptides. We observed that open chromatin regions had low methylation which were largely regulated by CG density, where CG-rich regions had more accessible chromatin, low methylation, and higher gene and protein expression. The CG-poor regions had higher repressive epigenetic regulations (higher DNA methylation) and less open chromatin, resulting in a cell line specific methylation and gene expression patterns. Our studies provide well-defined reference materials consisting of two cell lines with genomic, epigenomic, transcriptomic, scRNA-seq and proteomic characterizations which can serve as standards for validating and benchmarking not only on various omics assays, but also on bioinformatics methods. It will be a valuable resource for both research and clinical communities.

overall_design: HCC1395 and HCC1395BL cell lines were sequenced by RRBS and ATAC-seq


title: IGF2BP1 phosphorylation regulates ribonucleoprotein condensate formation by impairing low-affinity protein and RNA interactions (RIP-Seq)


title: A leiomodin 2 neonatal dilated cardiomyopathy causing mutation results inaltered actin-regulatory gene signatures andcardiomyocyte dysfunction

city: Tucson

first: Jared

last: Churko

state: Arizona

address: 1501 N CAMPBELL AVE LSN 461

department: CMM

institute: University of Arizona

summary: Neonatal dilated cardiomyopathy (DCM) is a poorly understood muscular disease of the heart. Several homozygous biallelic variants inLMOD2, the gene encoding the actin-binding protein Leiomodin 2, have been identified to result in severe DCM. Collectively, LMOD2-related cardiomyopathies present with cardiac dilation and decreased heart contractility, often resulting in neonatal death. Thus, it is evident that Lmod2 is essential to normal human cardiac muscle function. This study aimed to understand the underlying pathophysiology and signaling pathways related to the first reported LMOD2 variant (c.1193G>A, p.Trp398*). Using patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and a mouse model harboring the homologous mutation to the patient, we discovered dysregulated actin-thin filament lengths, altered contractility and calcium handling properties, as well as alterations in the serum response factor (SRF)-dependent signaling pathway. These findings reveal that LMOD2 may be regulating SRF activity in an actin-dependent manner and provide a potential new strategy for the development of biologically active molecules to target LMOD2-related cardiomyopathies.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: To investigate the role of LMOD2 in neonatal dialated cardiomyopathy, RNA-seq was performed on mice and iPSC lines harboring LMOD2 mutations


title: RBBP5 is an epigenetic regulator of mammalian hepatic 12h oscillator [ChIP-seq]

first: Bokai

last: Zhu

overall_design: All mice used for biological rhythm study are in C57BL/6J background, male and between 3 and 4 months of age.Wild-type C57BL/6J mice (n=12) were first entrained under LD12:12 conditions for 2 weeks before transferred to constant darkness for 24hrs. Mice were then sacrificed via cervical dislocation at a 4h interval  for a total of 48 hours under constant darkness. Mice were fed ad libitum during the entire experiment. Liver were taken. ChIP for RBBP5 was performed using anti-RBBP5 antibody (Bethyl, A300-109A) as previously described (Pan et al., 2020). Briefly, mouse liver samples were submerged in PBS + 1% formaldehyde, cut into small (~1 mm3) pieces with a razor blade and incubated at room temperature for 15 minutes. Fixation was stopped by the addition of 0.125 M glycine (final concentration). The tissue pieces were then treated with a TissueTearer and finally spun down and washed twice in PBS. Chromatin was isolated by the addition of lysis buffer, followed by disruption with a Dounce homogenizer. The chromatin was enzymatically digested with MNase. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, Proteinase K and heated for reverse-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. An aliquot of chromatin (30 μg) was precleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 4 μg of antibody. Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinking was reversed by incubation overnight at 65 °C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. The DNA libraries were prepared at the University of Pittsburgh and sequenced at Gene by Gene, Ltd per standard protocols. DNA libraries were prepared with Ovation® Ultralow V2 DNA-Seq library preparation kit (NuGen) using 1ng input DNA. The size selection for libraries was performed using SPRIselect beads (Beckman Coulter) and purity of the libraries were analyzed using the High Sensitivity DNA chip on Bioanalyzer 2100 (Agilent). The prepared libraries pooled and sequenced using Nova-Seq 6000 (Illumina), generating ~30 million 75 bp single-end reads per sample.


title: ADARp150 prohibits whole genome duplication

city: Amsterdam

first: Frank

middle: van

last: Gemert

address: Plesmanlaan 121

country: Netherlands

institute: Netherlands Cancer Institute

summary: Impaired control of the G1/S checkpoint allows initiation of DNA replication under non-permissive conditions. Unscheduled S-phase entry is associated with DNA replication stress, demanding for other checkpoints or cellular pathways to maintain proliferation. Here, we uncovered a requirement for ADARp150 to sustain proliferation of G1/S-checkpoint-defective cells under growth-restricting conditions. Besides its well-established mRNA editing function in inversely oriented short interspersed nuclear elements (SINEs), we found ADARp150 to exert a critical function in mitosis. ADARp150 depletion resulted in tetraploidization, impeding cell proliferation in mitogen-deprived conditions. Mechanistically we show that ADAR1 depletion induced aberrant expression of Cyclin B3, which was causative for mitotic failure and whole-genome duplication. Finally, we find that also in vivo ADAR1-depletion-provoked tetraploidization hampers tumor outgrowth.

overall_design: RNA sequencing data used to generate to Figure 6A, Supplemental Figure 2J and 4. Each sample is cultured and sequenced in triplicate. TBP ARPE transduced with a non-targeting (NT) shRNA is used as a reference in both unperturbed (+FCS) and serum starved (-FCS) conditions. We analyzed the log-fold changes of transcripts upon ADAR1 depletion and ADARp150 reconstitution to identify genes/pathways that could explain the tetraploidization that we observed upon ADAR1 depletion.

last: van Gemert

first: Alexandra

last: Drakaki

first: Isabel

last: Lozano

first: Daniel

last: de Groot

first: Maud

last: Uiterkamp

first: Natalie

last: Proost

first: Cor

last: Lieftink

first: Marieke

last: van de Ven

first: Roderick

last: Beijersbergen

first: Heinz

last: Jacobs

first: Hein

last: te Riele


title: Ribosome collisions trigger subunit splitting in E. coli

city: Baltimore

first: Allen

last: Buskirk

state: MD

address: 725 N. Wolfe St

department: Molecular Biology and Genetics

institute: Johns Hopkins University School of Medicine

summary: Although many clinically important antibiotics inhibit bacterial ribosomes, the mechanisms by which bacterial cells rescue ribosomes stalled by antibiotics remain poorly understood. Ribosome stalling leads to collisions that recruit ribosome quality control (RQC) factors that recycle the ribosome subunits and target nascent proteins for degradation. Surprisingly, loss of known RQC factors in E. coli does not lead to significant antibiotic sensitivity, even though antibiotics stall ribosomes and induce collisions, suggesting the existence of additional, uncharacterized RQC mechanisms. Here we report a novel mechanism for ribosome quality control (RQC) in bacteria in which the DExH-box ATPase HrpA splits stalled ribosomes into subunits. HrpA selectively acts on collided ribosomes and its activity is dependent on ATP hydrolysis. The cryo-EM structure of HrpA bound to collided ribosomes reveals insight into its selectivity and mechanism: the C-terminal domain of HrpA senses the collision and its helicase domain bind mRNA downstream of the ribosomes, where it likely exerts a pulling force that destabilizes the stalled ribosome. These studies highlight the importance of ribosome splitting as a highly conserved RQC mechanism across all three domains of life and identify an important pathway in proteobacteria that allows proteobacteria to tolerate ribosome-targeting antibiotics.

sample_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: Ribosome profiling with two replicates for three strains (wild-type MG1655, delta-hrpA, and the delta-hrpA delta-smrB double) containing the plasmid "IRAGP_reporter".


title: Identification of new therapeutic candidates for Congenital Central Hypoventilation Syndrome

city: Genoa

first: Paolo

last: Uva

address: Via Gerolamo Gaslini, 5

institute: IRCCS Istituto Giannina Gaslini

summary: Congenital central hypoventilation syndrome (CCHS), a rare genetic disease caused by heterozygous PHOX2B mutations, is characterized by life-threatening breathing deficiencies. PHOX2B is a transcription factor required for the specification of the autonomous nervous system that contains respiratory centers. To date, the only treatment offered to patients is mechanical ventilatory support. Therefore, identifying molecules with potential alleviating effects is of prime importance. A transcriptomic dataset from cells transiently expressing both wild type and variant PHOX2B allowed to run the CMap online tool and to identify, using in vitro models, drug compounds capable of relocating PHOX2B mutated proteins in the nucleus and restoring proper regulation of PHOX2B target gene expression. Subsequently, we tested the most efficient molecule ex vivo on hindbrain preparations isolating the principal components of the central respiratory command. Our results provide evidence that the HDAC inhibitor Vorinostat, also known as SAHA, is a promising molecule for the treatment of CCHS

overall_design: To identify drugs of interest for CCHS, SK-N-BE cells were plated in triplicate, transfected with constructs expressing  wild-type or mutant PHOX2B proteins, and treated with vehicle (DMSO) or 17-AAG.

first: Chiara

last: Africano

first: Tiziana

last: Bachetti

first: Gabriel

last: Pitollat

first: Genny

last: Del Zotto

first: Francesca

last: Giacopelli

first: Nicolas

last: Lenfant

first: Amélia

last: Madani

first: Nathan

last: Beckouche

first: Muriel

last: Thoby-Brisson

first: Isabella

last: Ceccherini


title: RNA-Seq in PBS- and mouse αSyn PFF-treated mouse primary cultured neurons

city: Kumamoto

first: Yasushi

last: Yabuki

state: Kumamoto

address: 2-2-1, Honjo, Chuo-ku

department: Genomic neurology

institute: Kumamoto University

summary: The balk RNA-seq in PBS- and αSyn pre-formed fibril (PFF)-treated neurons was done to investigate the expression levels of gene transcripts enriched in both BG4- and pS129-RIP-seq (GSE234238). Analysis of RNA-seq revealed that the expression levels of these common enriched RNAs in mPFF-treated neurons were little changed in comparison to vehicle-treated neurons.

overall_design: Total RNA was extracted from αSyn PFF-treated mouse cultured neurons at DIV 18 using an RNeasy Mini Kit. The library preparation, sequencing, and Fastq file processing and mapping were conducted as described above. RNAseqChef (Etoh K., and Nakao M. (2023). J. Biol. Chem. 299, 104810.) was used for identifying differentially expressed genes between PBS- and αSyn PFF-treated neurons.

first: Norifumi

last: Shioda


title: Loss of PILRB gene at age-related macular degeneration associated PILRB/PILRA locus impairs photoreceptor function in mouse retina

city: Bethesda

first: Zachary

last: Batz

address: 6 Center Dr Bldg 6, Rm 303

department: NEI

institute: NIH

summary: Genome-wide association studies have uncovered mostly non-coding variants at over 60 age- related macular degeneration (AMD) susceptibility loci. To ascertain the target gene at the PILRB/PILRA locus, we used a CRISPR strategy to produce germline deletions in the mouse paired immunoglobin-like type 2 receptor (Pilr) genes that encode highly related activating (PILRB) and inhibitory (PILRA) receptors. We show that a combined loss of Pilrb1 and Pilrb2, but not Pilra, leads to an early but relatively stationary defect in photoreceptor function without any apparent change in other retinal cells. PILRB immunostaining is specifically detected at the proximal part of photoreceptor outer segment. Reduced expression of select calcium-regulated phototransduction and synapse-associated proteins, including GCAP1 and 2, PDE6B, AIPL1, PSD95, and CTBP1, indicate dysregulation of calcium homeostasis as a possible mechanism of retinal phenotype in Pilrb1/2 -/- mice. Our studies suggest an association of PILRB, and not PILRA, with AMD pathogenesis.

overall_design:
Pilra, and Pilrb1/2 genes were deleted in the mouse genome by CRISPR-mediated editing in C57BL/6J zygotes. F0 founder carrying the deleted region was crossed with C57BL/6J mice. F1 founders carrying the deleted region were crossed to C57BL/6J mice for at least two additional generations before heterozygous/homozygous progeny were used in the following experiments. RNA from Pilrb1/2+/+ and Pilrb1/2 -/- mice retinas were isolated using RNeasy Mini Kit (Qiagen, Valencia, CA), with on-column DNA digestion, following the manufacturer's instructions. RNA quality and quantity were evaluated using Agilent 4200 TapeStation (Agilent Technologies, Santa Clara, CA). RNA-seq data was generated using TruSeq Stranded mRNA Sample Prep Kit (Illumina, San Diego, CA), as previously described, and 125 base pair-end reads were generated on NextSeq 2000 platform (Illumina, San Diego, CA). The alignment and quantification pipeline used human reference genome GRCh38.p7 and Ensembl v102 for annotation. Transcript-level counts were performed by kallisto v0.45.0 package and summarized to gene level using tximport v1.30.0. Gene level counts were converted to count per million (CPM) and then TMM normalized using the edgeR v.4.0.3 package in R.
Please note that M12* are samples taken from 12 month old mice and P28* are samples taken from 28 day old mice (28 days post-natal).

first: Partha

middle: N

last: Dey

first: Nivedita

last: Singh

first: Lina

last: Zelinger

first: Jacob

last: Nellissery

first: Noor

middle: D

last: Carreiro

first: Haohua

last: Qian

first: Tiansen

last: Li

first: Robert

last: Fariss

first: Lijin

last: Dong

first: Anand

last: Swaroop


title: Bulk and single-cell transcriptomics identify gene signatures of stem cell-derived NK cell donors with superior anti-tumor activity

city: Oss

first: Monica

last: Raimo

state: Noord-Brabant

address: Kloosterstraat 9

department: Research

institute: Glycostem

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


title: Inhibition of prolyl hydroxylase promotes the reprogramming of Sox9+ renal progenitor cells and renal regeneration

city: Guangzhou

first: Liting

last: Chen

state: Guangdong

address: 1838 North Guangzhou Blvd.

department: Division of Nephrology

institute: Nanfang Hospital, Southern Medical University

summary: Mammalian kidney has very limited ability to repair or regenerate after acute kidney injury (AKI). The maladaptive repair of AKI promotes the progression to chronic kidney disease (CKD). Therefore, it is extremely urgent to explore new strategies to promote the repair/regeneration of injured renal tubules after AKI. It has been shown that hypoxia induces heart regeneration in adult mice. However, it is unknown whether hypoxia can induce kidney regeneration after AKI. In this study, we used a prolyl hydroxylase domain inhibitor (PHDI), MK-8617, to mimic hypoxia condition and found that MK-8617 significantly ameliorates ischemia reperfusion injury (IRI) induced acute kidney injury. We then showed that MK-8617 dramatically facilitates renal regeneration via promoting the proliferation of injured renal proximal tubular cells (RPTCs) after IRI-induced AKI. We then performed bulk mRNA sequencing and discovered that multiple nephrogenesis- related genes were significantly upregulated with MK-8617 pretreatment. Furthermore, we showed that MK-8617 may alleviate proximal tubule injury via stabilizing HIF-1α protein specifically in renal proximal tubular cells. We also demonstrated that MK-8617 promotes the reprogramming of renal proximal tubular cells to Sox9+ renal progenitor cells, and the regeneration of renal proximal tubules. In summary, we discovered that inhibition of prolyl hydroxylase improves renal proximal tubule regeneration after IRI-induced AKI via promoting the reprogramming of renal proximal tubular cells to Sox9+ renal progenitor cells.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for 6 different tumors from IRI or IRI with  MK-8617 groups.

first: Jing


title: Next Generation Sequencing Quantitative Analysis of altered expression of genes in bladder cancer derived EVs educated draining LNs

city: Guang zhou

first: YAN

last: LIN

state: China

address: 72 Zhongshan 2nd Road

institute: Sun Yat-sen University

summary: Metastasis of tumors to LNs predicts disease progression and poor outcomes of patients. Recent studies revealed that tumor cells deliver EVs to particular targeted recipient cells in draining LNs and subsequently alter their gene expression patterns to create a supportive pre-metastatic niche to promote LN metastasis. Yet, the biological role and underlying mechanism of bladder cancer derived EVs (BCa derived EVs) in mediating lymphatic pre-metastatic niche formation remain unclear. The present study aim to explore the differential genes of BCa derived EVs educated draining LNs and underlying mechanism in the lymphatic pre-metastatic niche formation.

overall_design: To detect the precise mechanism of BCa derived EVs in mediating lymphatic pre-metastatic niche formation, the total RNA of draining LNs educated with BCa cell derived EVs or SV-HUC-1 EVs was extracted and the next generation sequencing was conducted to explore the expressing profile difference between BCa derived EVs education group and SV-HUC-1 EVs education group. The results was obtained from three independent experiments.

first: Yan

last: Lin

first: Changhao


title: Unraveling Epigenetic and Immune Mechanisms Underlying Cystogenesis in Autosomal Dominant Polycystic Kidney Disease [ATAC-Seq]

city: Tianjin

first: Jiayuan

state: State...

address: Heping

institute: Tianjin Medical University

summary: Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a prevalent, life-threatening monogenetic disorder that affects millions worldwide. Currently, therapeutic options are limited and often associated with complications. To find more effective treatments, this study aimed to understand the molecular mechanisms driving cystogenesis in ADPKD. By analysing various epigenetic factors, we identified a set of master transcription factors that guided a core regulatory circuitry and remodeled chromatin accessibility in ADPKD. We further explored the role of immune cells, notably macrophages, in the disease’s progression using single-cell RNA sequencing. A subgroup of Lhfpl2+/Fat3+ macrophages, which reactivated the Hgf-Met-Nfkb1-Runx1 pathways, was found essential for cystic cell fate transition. Targeting the identified epigenetic regulators, H3K4me3 and TF Runx1, effectively halted disease progression in a mouse model. These findings may lead to innovative treatments for ADPKD in humans.

overall_design: Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) in primary WT and PKD DBA positive cells.

first: Meijuan

last: Geng


title: Unraveling Epigenetic and Immune Mechanisms Underlying Cystogenesis in Autosomal Dominant Polycystic Kidney Disease [ChIP-Seq]

overall_design: Chromatin immunoprecipitation followed by sequencing (ChIP-seq) in primary WT and PKD DBA positive cells.


title: Whole transcriptomic landscape and regulatory network of High-Altitude Pulmonary Edema

city: Nanjing

first: Qiong

state: Jiangsu

address: Dingjiaqiao Road 87#

institute: Southeast University

summary: High Altitude Pulmonary Edema (HAPE) is a type of idiopathic non-cardiogenic pulmonary oedema at high altitude with a severe clinical course and high mortality rate. Therefore, this study aims to investigate transcriptomic signatures in HAPE based on whole-transcriptome sequencing. We identified the differential expression of mRNAs, miRNAs, circRNAs, lncRNAs, alternative splicing (AS) events, gene fusions and novel transcripts, constructed ceRNA networks and immune cell infiltration landscapes, and annotated the biological functions of target genes based on bioinformatics analysis. This study will clarify the differential expression of coding and non-coding RNAs (ncRNAs) in HAPE, identify novel transcripts and genes, increase our understanding of the transcriptional characteristics of HAPE, elucidate disease-associated pathways and biological processes, and provide underlying mechanisms for HAPE diagnosis and treatment.

overall_design: We identified differentially expressed candidate mRNAs, miRNAs, circRNAs, and lncRNAs in a discovery set (6 HAPE patients and 6 healthy controls) using RNA high-throughput sequencing. All of the participants and health care volunteers were Han Chinese people between the ages of 18 and 65 who had travelled to Tibet.

last: Shen

first: Fujin

last: Fang

first: Chuanli

last: Yang

first: Yu


title: Viruses encode tRNA and anti-retron to evade bacterial immunity [Ec83]

city: Shinjuku-ku

first: Aa

middle: Haeruman

last: Azam

state: Tokyo

address: Toyama 1-23-1

department: Research Center for Therapeutic Drugs and Vaccines Development

institute: The National Institute of Infectious Diseases

summary: Retrons are bacterial genetic retroelements that encode reverse transcriptase capable of producing multicopy single-stranded DNA (msDNA) and function as antiphage defense systems. Phages employ several strategies to counter the host defense systems, but no mechanisms for evading retrons are known. Here, we show that tRNATyr and Rad (retron anti defense) of T5 phage family inhibit the defense activity of retron 78 and a broad range of retrons, respectively. The effector protein of retron 78, ptuAB, specifically degraded tRNATyr leading abortive infection, but phage countervailed this defense by supplying tRNATyr. Rad inhibited retron function by degrading noncoding RNA, the precursor of msDNA. In summary, we demonstrated that viruses encode at least two independent strategies for overcoming bacterial defense systems: anti-defense, such as Rad, and defense canceler, like tRNA.

overall_design: PtuAB from retron Ec83 (Eco4) was cloned under an arabinose inducible promoter pBAD. 0.2% Arabinose and 0.2% Glucose were used to induce and repress the PtuAB expression, respectively.

first: Azam

last: Aa Haeruman

first: Kotaro

last: Chihara

first: Kohei

last: Kondo

first: Tomohiro

last: Nakamura

first: Shinjiro

last: Ojima

first: Azumi

last: Tamura

first: Wakana

last: Yamashita

first: Longzhu

last: Cui

first: Yoshimasa

last: Takahashi

first: Koichi

last: Watashi

first: Kiga

last: Kotaro


title: Whole transcriptome analysis of osteoclasts after treatment with or without etelcalcetide

city: Taipei

first: Hui-Wen

last: Chiu

address: No.301, Yuantong Rd., Zhonghe Dist., New Taipei City

country: Taiwan

institute: Taipei medical university

summary: Phosphate binders, vitamin D analogs, and calcimimetics have recently been used to control the disturbed mineral metabolism in chronic kidney disease-mineral and bone disorder (CKD-MBD). Etelcalcetide , a new calcimimetic agent approved by is considered by patients due to its convenience as an intravenous form which easily affordable during dialysis sessions. In this study, we evaluated the effects of etelcalcetide on osteoclasts.

overall_design: RAW264.7 cells were incubated with RANKL (50 ng/mL) with or without etelcalcetide (4 μM) for 5 days.

first: Yu-Jhe

first: Cai-Mei

last: Zheng


title: P28.ET0213, ET0318, ET0267 Datasets

city: Los Angeles

first: Steve

last: Horvath

state: CA

address: 695 Charles E. Young Drive South, Box 708822

department: Human Genetics

institute: University of California, Los Angeles

summary: Cytosine methylation data generated on mammalian methylation platform

platform_organism:
Macaca mulatta
Homo sapiens
Mus musculus
Rattus

overall_design: All bulk tissue samples and sorted cell populations were harvested from mice. Sorted cells populations were isolated using fluorescence-activated cell sorting. Bulk tissues analyzed in this work include muscle and peripheral blood. Sorted cell populations include muscle stem cells (satellite cells), fibroadipogenic progenitors, endothelial cells, hematopoietic cells (both from muscle and bone marrow), intestinal stem cells, differentiated intestinal epithelial cells, hematopoietic stem & progenitor cells, lymphoid progenitors, myeloid progenitors, B-cells, T-cells, and granulocytes. Pax7-nGFP and Lgr5-eGFP transgenic reporter mouse strains were used to sort satellite cells and intestinal stem cells respectively; all other experiments were conducted with C57BL6 mice. Mice of various pre-specified ages were used.


title: Bulk and single-cell transcriptomics identify gene signatures of stem cell-derived NK cell donors with superior anti-tumor activity [Bulk RNA-seq]

first: Monica

last: Raimo

summary: Allogeneic natural killer (NK) cell therapies are a valuable treatment option for cancer, given their remarkable safety and favorable efficacy profile. Although the use of allogeneic donors allows for off-the-shelf and timely patient treatment, intrinsic interindividual differences put clinical efficacy at risk. The identification of donors with superior anti-tumor activity is essential to ensure the success of adoptive NK cell therapies. Here, we investigated the heterogeneity of 10 umbilical cord blood stem cell-derived NK cell batches. First, we evaluated the donors’ cytotoxic potential against tumor cell lines from solid and hematological cancer indications, to distinguish a group of superior, “excellent” killers (4/10), compared to “good” killers (6/10). Next, bulk and single-cell RNA-Sequencing, performed at different stages of NK differentiation, revealed distinct transcriptomic features of the two groups. Excellent donors showed an enrichment in cytotoxicity pathways and a depletion of myeloid traits, linked to the presence of a larger population of effector-like NK cells early on during differentiation. Consequently, we defined a multi-factorial gene expression signature able to predict the donors’ anti-tumor potential. Our study contributes to the identification of key traits of superior NK cell batches, supporting the development of efficacious NK therapeutics and the achievement of durable anti-tumor responses

overall_design: CD34+ cells from 10 UCB donors were seeded in 6-wells tissue culture treated plates (Corning Incorporated) for expansion culture in Glycostem Basal Growth Medium (GBGM®; Fertipro) supplemented with human serum (Sanquin), thrombopoietin (TPO), interleukin (IL)-7, FMS-like tyrosine kinase 3 ligand (Flt-3L), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, stem cell factor (SCF) (all from Cellgenix) and Neupogen (G-CSF; Amgen) as previously described. After 9 days, TPO was replaced by IL-15 (Cellgenix) and after 14 days of NK progenitor expansion, Flt-3L was replaced by Proleukin (IL-2; Novartis) for the NK cell differentiation phase. Cells were cultured until day 28 and cryopreserved. Upon thawing, cells were directly used for assays, or cultured for an additional 7 days in differentiation medium before use. 10 donors were sequenced at day 35, after a week of recovery.

first: Amanda

last: van Vliet

first: Mirjam

last: van den Hout

first: Danielle

last: Steenmans

first: Adil

last: Duru

first: Anna-Maria

last: Georgoudaki

first: Tanja

last: de Gruij

first: Wilfred

last: van Ijcken

first: Jan

last: Spanholtz


title: HMGA1 promotes the tumorigenesis of colorectal cancer by increasing lipid synthesis

city: Kaifeng

first: yuan

last: zhao

address: Jinming Road

institute: Henan University

summary: Metabolic reprogramming is a typical feature of tumors, in which biological macromolecules and energy produced in abnormal metabolism meet the requirements of highly proliferative tumor cells and participate in multiple stages of tumor development. High mobility group A1 (HMGA1) is a structural transcription factor, which plays a carcinogenic role in regulating the transcription of oncogenes. Bioinformatics analysis showed that HMGA1 was overexpressed in human CRC. However, the mechanisms by which HMGA1 promotes the tumorigenesis of CRC remains unknown. Herein, we applied intestinal epithelium conditional knockout (Hmga1△IEC) and knock-in (Hmga1IEC-OE/+) mice of HMGA1 to induce CRC, and identified that HMGA1 promotes colorectal cancer growth by increasing lipid synthesis. Single-cell sequencing (scRNA seq) and immunohistochemical staining showed that HMGA1 was highly expressed in the epithelial cells of CRC. HMGA1 promoted the CRC cell proliferation and accelerated CRC development in HMGA1-knock-in (Hmga1IEC-OE/+) mice. Knockout of HMGA1 (Hmga1△IEC) in intestinal epithelial cells of mice reduced the lipid accumulation and inhibited the occurrence and development of CRC. We further characterized that HMGA1 upregulated the level of fatty acid synthase (FASN) through enhancing the transcriptional binding of sterol regulatory element-binding protein 1 (SREBP1) to the promoter of FASN, leading to an increase in lipid synthesis in intestinal epithelial cells. High fat diet (HFD) aggravated the malignant progression of CRC in Hmga1△IEC mice and reversed the inhibitory effect of  HMGA1 depletion on CRC. Administration Orlistat (50 mg/kg), an inhibitor of FASN-mediated lipid synthesis, into Hmga1-knock-in (Hmga1IEC-OE/+) mice markedly reduced lipid accumulation in intestinal epithelial cells and decreased the tumorigenesis of CRC. Taken together, our data suggest that HMGA1 promotes the tumorigenesis of colorectal cancer by up-regulating FASN-mediated de novo fatty acid synthesis. Our findings provide strong evidence supporting therapeutic intervention of lipid accumulation for the prevention and treatment of CRC

overall_design: To investigate the regulatory effect of HMGA1 on whole genome gene expression, we analyzed the gene expression profiles of HT29 cells which were stable knockdown of HMGA1 by using RNA seq data. The aim of this study is to reveal that HMGA1 affects tumor malignant progression by regulating lipid synthesis.

first: Yuan

last: Zhao

first: Meng-Jie

last: Liu

first: Lei

last: Zhang

first: Qi

last: Yang

first: Qian-Hui

last: Sun

first: Jin-Rong

last: Guo

first: Xin-Yuan

last: Lei

first: Kai-Yue

last: He

first: Jun-Qi

last: Li

first: Jing-Yu

first: Yong-Ping

last: Jian

first: Zhi-Xiang

last: Xu


title: The Myc-Like Mlx Network Impacts Aging and Metabolism

city: pittsburgh

first: Huabo

last: Wang

address: 4401 Penn Avenue

institute: Children hospital of pittsburgh

summary: The “Mlx” and “Myc” Networks share many common gene targets. Just as Myc’s activity depends upon its heterodimerization with Max, the Mlx Network requires that the Max-like factor Mlx associate with the Myc-like factors MondoA or ChREBP. We show here that body-wide Mlx inactivation, like that of Myc, accelerates numerous aging-related phenotypes pertaining to body habitus and metabolism. The deregulation of numerous aging-related Myc target gene sets is also accelerated. Among other functions, these gene sets often regulate ribosomal and mitochondrial structure and function, genomic stability and aging. Whereas “MycKO” mice have an extended lifespan because of a lower cancer incidence, “MlxKO” mice have normal lifespans and a somewhat higher cancer incidence. Like Myc, Mlx, MondoA and ChREBP expression and that of their target genes, deteriorate with age in both mice and humans, underscoring the importance of life-long and balanced cross-talk between the two Networks to maintain normal aging.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for MlxKO and WT mice at 5 and 20 months of age in omental adipose tissue, liver and skeletal muscle. WT mice RNA-seq data are available in GSE223676.

first: Edward

middle: V

last: Prochownik


title: Next Generation Sequencing  of circRNAs in bladder cancer derived ITGA6+EVs

first: YAN

last: LIN

summary: EVs served as crucial mediators preferably achieve their biological effect in recipient cells to create a supportive pre-metastatic niche via delivering distinct biomolecules, among which circular RNAs (circRNAs) with high stability and abundance in tumor derived EVs were under extensive exploration. Moreover, it is well-established that circRNAs play a significant role in BCa lymphatic metastasis. To explore the crucial circRNAs in BCa derived ITGA6+EVs, the ITGA6+EVs from total UM-UC-3-EVs were isolated by affinity capture using anti-ITGA6 magnetic beads and the circRNA expression profile was investigated using next-generation sequencing (NGS).

overall_design: We isolated ITGA6+EVs from total UM-UC-3-EVs by affinity capture using anti-ITGA6 magnetic beads and degrades the abundant linear RNAs with RNase R. Then, each sample was amplified and transcribed into fluorescent cRNA utilizing a random priming method. Double-stranded cDNA fragments are subjected to end-repair and A-tailing. After amplification by PCR, the reaction system and program for circularization are configured and set up. The concentration and specific activity of circRNAs were measured by DNBSEQ-G400.

first: Yan

last: Lin

first: Changhao

last: Chen


title: Small cell lung cancer responds to inhibition of Jumonji enzyme in vitro and in vivo

city: Dallas

first: Luc

last: Girard

address: 5323 Harry Hines Blvd

department: Hamon Center for Therapeutic Oncology Research

institute: UT Southwestern Medical Center

summary: Small-cell lung cancer (SCLC) arises from neuroendocrine cells within the lung. Along with deletion or loss of function Rb and TP53 mutations, which occur in almost all cases, subsets of SCLC are driven by specific transcription factors including ASCL1, NEUROD1, POU2F3, or YAP1. Various MYC family members provide further inter-tumor heterogeneity. Despite this heterogeneity in SCLC, current treatment for SCLC is subtype agnostic. Although most patients are initially responsive to first line etoposide and platin chemotherapy they soon develop resistance with no effective second line therapy. A recent large NCI screen showed that over 60 SCLC cell line models responded to 500+ investigational drugs in a manner highly correlated with etoposide responses, suggesting none of these drugs would provide benefit for SCLC therapy beyond current chemotherapy. By analyzing publicly available data of shRNA and CRISPR screens in addition to our own RNA-seq expression data we identified KDM4A as a potential candidate to target in SCLC. To underscore the potential of KDM4A as an anti-SCLC target, we tested chemotherapy drug and three JmjC KDM inhibitors across a panel of SCLC cell lines representing all the SCLC transcription factor subtypes and found that SCLC responses to JmjC KDM inhibitors do not correlate to their responses to chemotherapy. Furthermore, we found upon treatment with pan-JmjC KDM inhibitor, JIB-04, many genes in the ER stress signaling pathways are upregulated in SCLC lines, suggesting this is the mechanistic path leading to SCLC slower growth or death. Additionally, analysis found that JIB-04 resistant cells activate pro-survival pathways such as mTOR to perhaps overcome the effects of JIB-04 treatment. Furthermore, the loss of KDM4A in SCLC cell line led to slower proliferation and activity in the ER stress signaling pathway.

overall_design: 8 SCLC cell lines were treated with JIB-04 or DMSO and were analyzed by RNAseq. 2 SCLC cell line xenografts (CLX) were treated with JIB-04,  SD-70, or DMSO, and analyzed by RNAseq.

first: Aiden

last: Nguyen

first: Raghav

last: Chandra

first: Elisabeth

last: Martinez

first: John

last: Minna


title: Viruses encode tRNA and anti-retron to evade bacterial immunity [PrrC170]

first: Aa

last: Azam

overall_design: PrrC170 toxin from PrrC170 anticodon nuclease was cloned under an arabinose inducible promoter pBAD. 0.2% Arabinose and 0.2% Glucose were used to induce and repress the PrrC170 expression, respectively.

first: Azam

last: Aa Haeruman

first: Kotaro

last: Chihara

first: Kohei

last: Kondo

first: Tomohiro

last: Nakamura

first: Shinjiro

last: Ojima

first: Azumi

last: Tamura

first: Wakana

last: Yamashita

first: Longzhu

last: Cui

first: Yoshimasa

last: Takahashi

first: Koichi

last: Watashi

first: Kiga

last: Kotaro


title: Matrix Selection for the Visualization of Small Molecules and Lipids in Brain Tumors Using Untargeted MALDI-TOF Mass Spectrometry Imaging

status: Public on Oct 09 2024

city: Melbourne

first: Saskia

last: Freytag

state: Victoria (V)

address: 1G Royal Parade

country: Australia

institute: WEHI

summary: Matrix-assisted laser desorption/ionization mass spectrometry imaging allows for the study of metabolic activity in the tumor microenvironment of brain cancers. The detectable metabolites within these tumors are contingent upon the choice of matrix, deposition technique, and polarity setting. In this study, we compared the performance of three different matrices, two deposition techniques, and the use of positive and negative polarity in two different brain cancer types and across two species. Optimal combinations were confirmed by a comparative analysis of lipid and small-molecule abundance by using liquid chromatography-mass spectrometry and RNA sequencing to assess differential metabolites and enzymes between normal and tumor regions. Our findings indicate that in the tumor-bearing brain, the recrystallized α-cyano-4-hydroxycinnamic acid matrix with positive polarity offered superior performance for both detected metabolites and consistency with other techniques. Beyond these implications for brain cancer, our work establishes a workflow to identify optimal matrices for spatial metabolomics studies.

overall_design: The extracted normal brain and tumor region were ground in liquid nitrogen, and the RNA was extracted by using the RNeasy RNA extraction kit (Qiagen 74104, Qiagen, Germantown, MD, USA) and then analyzed by using a 2200 Tapestation Analyzer (Agilent, Santa Clara, CA, USA). An input of 500 ng of RNA was used to generate libraries (TruSeq RNA Library Prep v2, Illumina, San Diego, CA, USA). Sequencing was performed on the NextSeq System (Illumina) to produce 132bp single-end reads.

first: Sarah

last: Best

first: James

last: Whittle


title: RNA-seq analysis of differentially expressed genes between stromal vascular fraction (SVF) from inguinal white adipose tissue (iWAT) of mice treated with medicinal herb extract (MHE) and Phosphate-buffered saline (PBS)

city: Suzhou

first: Xiaoqin

last: Yang

address: NO. 199 Renai Road

institute: Soochow University

summary: To assess the influences of a crude medicinal herb extract (MHE) on the immune composition and function in inguinal white adipose tissue (iWAT), we isolated iWAT stromal vascular fractions (SVFs) from control (PBS) and MHE-treated mice and performed RNA-seq analysis. iWAT SVF of mice treated with MHE was defined as the treatment group. Phosphate-buffered saline (PBS) treatment was used as the control group. Then RNA-Seq experiment was performed by OE Biotech Co., Ltd. (Shanghai, China) to analyze gene expression changes in iWAT SVF.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for iWAT SVF of mice treated with MHE and PBS.

first: Qiaoming

last: Long


title: Effect of Bacteroides fragilis administration on microglia in WT and the APP-PS1 model of Alzheimer's disease [Bfrag]

status: Public on Oct 12 2024

city: Boston

first: Caroline

last: Wasen

state: MA

address: 60 Fenwood rd

institute: Harvard Medical School

summary: The gut microbiota and innate immune system play critical roles in Alzheimer’s disease (AD). Bacteroides is elevated in AD patients and correlates with cerebrospinal fluid levels of Aβ and tau. We found that increased amyloid-β (Aβ) plaques in Bacteroides fragilis treated APP/PS1-21 mice were associated with altered cortical expression Aβ processing genes. B. fragilis suppressed peripheral CD4+ T cell production of GM-CSF and IL-4 and transcriptional changes in microglia related to GM-CSF and IL-4 signaling, phagocytosis, and protein degradation. Furthermore, B. fragilis impaired the microglial uptake of intracranially injected Aβ42, whereas Erysipelotrichaceae strains increased uptake. Depleting murine Bacteroidetes with metronidazole decreased amyloid load in aged 5xFAD mice, increased CD4+ T cell GM-CSF production, and activated microglial pathways related to cytokine signaling, phagocytosis and lysosomal degradation. These data suggest that the gut microbiome may contribute to AD pathogenesis by suppressing peripheral cytokines and microglia phagocytic function, leading to impaired immune-mediated Aβ clearance.

overall_design: 200 ul suspension of Bacteroides fragilis with an optical density of 0.7 at 600 nm was administered orallly to  APP/PS1 and wildtype mice for 10 weeks starting at 2.5 months of age.

last: Wasén

first: Julia

last: Vincentini

first: Christian

last: Gauthier

first: Juliana

last: Lopes

first: Oleg

last: Butowsky

first: Laura

last: Cox

first: Howard

last: Weiner


title: Bulk and single-cell transcriptomics identify gene signatures of stem cell-derived NK cell donors with superior anti-tumor activity [scRNA-seq]

first: Monica

last: Raimo

overall_design: CD34+ cells from 10 UCB donors were seeded in 6-wells tissue culture treated plates (Corning Incorporated) for expansion culture in Glycostem Basal Growth Medium (GBGM®; Fertipro) supplemented with human serum (Sanquin), thrombopoietin (TPO), interleukin (IL)-7, FMS-like tyrosine kinase 3 ligand (Flt-3L), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, stem cell factor (SCF) (all from Cellgenix) and Neupogen (G-CSF; Amgen) as previously described. After 9 days, TPO was replaced by IL-15 (Cellgenix) and after 14 days of NK progenitor expansion, Flt-3L was replaced by Proleukin (IL-2; Novartis) for the NK cell differentiation phase. Cells were cultured until day 28 and cryopreserved. Upon thawing, cells were directly used for assays, or cultured for an additional 7 days in differentiation medium before use. 8 donors were selected to sequence at day28 directly after thawing.

first: Amanda

last: van Vliet

first: Mirjam

last: van den Hout

first: Danielle

last: Steenmans

first: Adil

last: Duru

first: Anna-Maria

last: Georgoudaki

first: Tanja

last: de Gruij

first: Wilfred

last: van Ijcken

first: Jan

last: Spanholtz


title: Bile acid synthesis impedes tumor-specific T cell responses during liver cancer [CITE-seq]

city: La Jolla

first: Kathryn

last: Lande

address: 10010 N Torrey Pines Rd

department: IGC

institute: Salk Institute

summary: The tumor's metabolic landscape significantly influences anti-tumor immunity, but it remains unclear how metabolic pathways commonly active in the organ of tumor-origin influence immunosurveillance. This study focused on the liver and identified accumulation of primary conjugated and secondary bile acids (BAs) as metabolic features associated with hepatocellular carcinoma and liver cancer models. Inhibiting conjugated BA synthesis through BAAT-deletion in hepatocytes enhanced tumor-specific T cell responses, reduced growth, and sensitized tumors to immune checkpoint blockade. BAs regulated CD8+ T cells differently; primary BAs like TCDCA induced oxidative stress and secondary BAs like LCA impaired T cell function through ER stress, which was countered by UDCA. Indeed, dietary UDCA provision suppressed tumor progression. These findings demonstrate how manipulating organ-specific metabolites affects antitumor immunity and modifying BA synthesis or dietary intake could enhance immunotherapy in liver cancer.

overall_design: Single-cell RNA CITE-seq of immune cells from tumor brearing livers from naïve (mock infected), AAV-sgSCR and AAV-sgBAAT were performed at Day 30 post infection. Each sample is a pool of 3 tumor samples.

first: Siva Karthik

last: Varanasi

first: Susan

last: Kaech


title: Bile acid synthesis impedes tumor-specific T cell responses during liver cancer.

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

overall_design: Refer to individual Series


title: Bile acid synthesis impedes tumor-specific T cell responses during liver cancer [RNA-seq2]

overall_design:
To investigate the effect of LCA treatment on wild type and Nur77 KO CD8+ T cells
Gene expression profiling analysis using data obtained from RNA-seq of T Cells treated with LCA.


title: Transcriptome insight into lignin utilization by Curvularia clavata and identification of regulators of laccase activity

first: Jinyang

last: Li

address: No. 32, Xiqi Road, Tianjin Airport Economic Park

institute: Chinese Academy of Sciences

summary: Filamentous fungi are promising organisms for lignin degradation and mineralization. However, novel lignin-degrading fungal species are underexplored. Here, we isolated a fungal strain of Curvularia clavata that can utilize lignosulfonate as the carbon source and exhibited a relative high laccase activity during growth on lignosulfonate. Comparative transcriptomic analysis of the WT strain grown on glucose and lignosulfonate indicates that lignosulfonate and/or its metabolites have a significant effect on the gene expression profiles of C. clavata J1. Three regulators of laccase activity were identified, including a methyltransferase CcLaeA and two transcription factors, Rpn-4 and Tah-1. When grown on lignosulfonate, the laccase activity of the CclaeA and rpn-4 disrupted mutants (ΔCclaeA and Δrpn-4) increased by 49.2% and 43.5%, respectively, compared to the wild-type (WT) strain, whereas the tah-1 disrupted mutant (Δtah-1) decreased by 59.2%.

overall_design: The transcriptional response of WT strain to glucose and lignosulfonate was determined by RNA-Seq. And the comparative transcriptomic analysis of ΔCclaeA, Δrpn-4, Δtah-1 mutants and WT strain grown on lignosulfonate were performed.

first: Le

last: Gao

first: Xin

last: Wu


title: Probing the orthogonality and robustness of the mammalian RNA-binding protein Musashi-1 in Escherichia coli [RNA-seq]

city: Paterna

first: Guillermo

last: Rodrigo

address: Calle del Catedrático Agustín Escardino Benlloch, 9

country: Spain

institute: Agencia Estatal Consejo Superior de Investigaciones Científicas

summary: RNA recognition motifs (RRMs) are widespread RNA-binding protein domains in eukaryotes, which represent promising synthetic biology tools due to their compact structure and efficient activity. Yet, their use in prokaryotes is limited and their functionality poorly characterized. Recently, we repurposed a mammalian Musashi protein containing two RRMs as a translation regulator in Escherichia coli. Here, employing high-throughput RNA sequencing, we explored the impact of Musashi expression on the transcriptomic and translatomic profiles of E. coli, revealing certain metabolic interference, induction of post-transcriptional regulatory processes, and spurious protein-RNA interactions. Engineered Musashi protein mutants displayed compromised regulatory activity, emphasizing the importance of both RRMs for specific and sensitive RNA binding. We found that a mutation known to impede allosteric regulation led to similar translation control activity. Evolutionary experiments disclosed a loss of function of the synthetic circuit in about 40 generations, with the gene coding for the Musashi protein showing a stability comparable to other heterologous genes. Overall, this work expands our understanding of RRMs for post-transcriptional regulation in prokaryotes and highlight their potential for biotechnological and biomedical applications.

overall_design: To investigate the effect of Musashi on Escherichia coli gene expression, a vector encoding (or devoid of) MSI gene was transformed into JS006 strain.

first: Raúl

last: Ruiz

first: Roswitha

last: Dolcemascolo

first: Sara

last: Baldanta

first: Lucas

last: Goiriz

first: María

last: Heras-Hernández

first: Roser

last: Montagud-Martínez


title: Probing the orthogonality and robustness of the mammalian RNA-binding protein Musashi-1 in Escherichia coli [Ribo-seq]


title: Identification of host dependency factors shared by multiple SARS-CoV-2 variants of concern

city: Padua

first: Graziano

last: Martello

address: viale Colombo 3

department: Department of Biology

institute: University of Padua

summary: A key feature of RNA viruses, including SARS-CoV-2, is their high mutation rate, which allows them to develop resistance to vaccines and antiviral drugs targeting viral proteins. To overcome this downside, a strategy would be to target host factors, i.e. cell proteins required by the virus for its replication. However, it is still unclear whether cell responses induced by different SARS-CoV-2 variants are conserved and if the same core of host factors is exploited by different variants. We compared 3 variants of concern (VOC) that emerged during the first year of the pandemic and observed that the host transcriptional response was mostly conserved, differing only in the kinetics and magnitude. By CRISPR screening we identified the host genes required for infection by each VOC. These genes were associated with interferon and JAK/STAT pathway, autophagy, mTOR pathway, mitochondrial organisation and activity. Crucially, we failed to identify genes required by only one variant. We further validated our candidates with small molecules and repurposed FDA-approved drugs, which were effective against a novel variant emerged during the course of the study. We observed that SARS-CoV-2 infection induces a rapid spike in Reactive Oxygen Species (ROS) production, which can be targeted to block viral propagation. Our study identifies a core of host genes required for infection, and lays the foundation for general therapeutic strategies aimed at minimising emergence of novel resistant variants.

overall_design: RNA sequencing was performed to assess both viral and cellular transcriptome changes upon infection of Calu-3 cells with different SARS-CoV-2 variants. Samples analysed: Calu-3 cells infected with Wuhan, D614G and Alpha Sars-CoV-2 variants and a Mock sample harvested at the following times post infection: 6, 9, and 12 h.p.i.; 4 replicates for each condition.

first: Frasson

last: Ilaria

first: Zangrossi

last: Manuela

first: Diamante

last: Linda

first: Richter

last: Sara

first: Montagner

last: Marco

first: Martello

last: Graziano


title: Mutant-p53 stimulates Cxcl1 Expression from Distal Enhancers to Suppress Immune Response to Pancreatic Cancer [EXOME-seq]

city: Cambridge

first: Dig

middle: B

last: Mahat

email: mahat@mit.edu, dbm222@cornell.edu

address: Koch Institute, MIT, 500 Main St, Building 76, #417

department: Koch Institute for Integrative Cancer Research

institute: Massachusetts Institute of Technology

summary: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer without effective treatments. It is characterized by activating KRAS mutations and p53 alterations. However, how these mutations alter cell-intrinsic gene programs to influence the immune landscape of the tumor microenvironment (TME) remains poorly understood. Here, we demonstrate that p53R172H enhances tumor growth, establishes a suppressive TME by inducing immune evasion, and blunts the effectiveness of immune checkpoint inhibitors (ICIs). We discovered that the oncogenic function of p53R172H is mediated by upregulation of the immunosuppressive chemokine Cxcl1. Mechanistically, we show that p53R172H binds to the distal enhancers of the Cxcl1 gene and increases enhancer activity and Cxcl1 expression. NF-kB also occupies Cxcl1 enhancers, and p53R172H binds these enhancers in an NF-kB-dependent manner, suggesting a role of NF-kB in commuting p53R172H to the Cxcl1 enhancers. Our findings elucidate how a common mutation in a critical tumor-suppressor gene exploits enhancers to modulate chemokine gene expression and foster an immunosuppressive TME in PDAC that undermines the efficacy of ICI.

overall_design:
To investigate the role of p53R172H in pancreatic ductal adenocarcinoma (PDAC), we generated isogenic Trp53-/- PDAC cells (KrasG12D/+; Trp53-/-) using CRISPR-Cas9. We compared transcriptional programs via RNA-seq and PRO-seq, exonic mutations using Exome-seq, and genome occupancy with CUT&RUN between Trp53R172H/- and isogenic and syngenic Trp53-/- cells, as well as Trp53-/- cells with ectopically expressed p53R172H.
We orthotopically implanted the PDAC cells into the pancreas of mice and evaluated tumor growth, size, and the immune landscape. We further treated the mice with immune checkpoint inhibitors and monitored their survival.
To explore the role of Cxcl1 and its enhancers, we generated Cxcl1-/- cells and Cxcl1 enhancer knockout cells. We then assessed gene expression, immune profiling, and the response to immune checkpoint inhibitors in these models.

first: Heena

last: Kumra

first: Sarah

last: Castro

first: Emily

last: Metcalf

first: Kim

last: Nguyen

first: Ryo

last: Morisue

first: William

middle: W

last: Ho

first: Ivy

last: Chen

first: Brandon

last: Sullivan

first: Leon

last: Yim

first: Arundeep

last: Singh

first: Jiayu

last: Fu

first: Sean

last: Waterton

first: Yu-Chi

last: Cheng

first: Enrico

last: Moiso

first: Vikash

last: Chauhan

first: Hernandez

last: Moura Silva

first: Stefani

last: Spranger

first: Rakesh

last: Jain

first: Phillip

last: Sharp


title: Mutant-p53 stimulates Cxcl1 Expression from Distal Enhancers to Suppress Immune Response to Pancreatic Cancer [CUT&RUN]

first: Dig

last: Mahat

first: Heena

last: Kumra

first: Sarah

middle: A

last: Castro

first: Emily

last: Metcalf


title: Comparative Transcriptomics Identifies Differentially Expressed Genes Regulating endoreplication and trichome branching

city: Zhengzhou

first: han

last: xingzhou

address: 100 Science Avenue

institute: Zhengzhou University

summary:
Purpose:Comparative transcriptome analyses was applied to characterize gene expression after overexpression GaTOP6B in Arabidopsis thaliana.
Methods:The fresh leaves were collected from 4-week-old WT plants and transgenic Arabidopsis lines under drought treatments. The total RNA was first extracted using TRIzol Reagent (TaKaRa, Tokyo, Japan), and treated with RNase-free DNase I (TaKaRa), and then followed with mRNA purification using the Magnetic Oligo (dT) Beads (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol.
Results: In this study,our RNA-Seq analysis suggested overexpression of GaTOP6B could suppress the downstream signal transduction of jasmonic acid, cytokinin and brassinolide. Furthermore, the transcriptome analysis also confirmed the downregulation of B-type cyclins genes related to cell cycle, and TCP, MYB transcription factors, which subsequently regulate downstream signal cascades to modulate trichome development.
Conclusions:  Our results provide evidence on the crucial roles of GaTOP6B for improving trichome branch number by enhancing cell ploidy levels and suppressing the expression of related transcription factors.

overall_design: mRNA profiles of  the leaves of wild-type plants and transgenic lines

first: Xingzhou

last: Han

first: Zhuxuan

last: Fan

first: Fang

last: Wei


title: Mutant-p53 stimulates Cxcl1 Expression from Distal Enhancers to Suppress Immune Response to Pancreatic Cancer [RNA-seq]

first: Kim

last: Nguyen

first: Ryo

last: Morisue

first: William

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