title: KRBP72 facilitates ATPase-dependent editing progression through a structural roadblock in mitochondrial A6 mRNA

city: Buffalo

first: Ashutosh

last: Dubey

state: New York

address: 955 Main Street

department: Microbiology & Immunology

institute: University at Buffalo

summary: Uridine insertion/deletion editing of mitochondrial mRNAs in kinetoplastids entails the coordinated action of three complexes. RNA Editing Catalytic Complexes (RECCs) catalyze the enzymatic reactions, while the RNA Editing Substrate Binding Complex (RESC) and RNA Editing Helicase 1 Complex coordinate interactions between RECCs, mRNAs, and hundreds of gRNAs that direct edited sequences. Additionally, numerous auxiliary factors are required for productive editing of specific mRNAs. Here, we elucidate the role of KRBP72, an editing auxiliary factor of the ABC ATPase family that exhibits RNA binding activity. In procyclic form T. brucei, KRBP72 knockdown leads to a pause in editing at the base of a predicted stem loop structure in ATP synthase subunit 6 (A6) mRNA. Enhanced cross-linking and affinity purification revealed KRBP72 binding sites both within and upstream of this stem loop. KRBP72 ATPase activity is essential for its A6 mRNA editing function; however, its RNA binding activity is dispensable. KRBP72 interacts with most RESC proteins in an RNA-dependent manner. By contrast, RESC12A associates with KRBP72 in an RNA-independent fashion, and RESC12A strongly promotes KRBP72’s interaction with RNA. Hence, KRBP72 ATPase activity facilitates the progression of editing through a challenging RNA secondary structure, highlighting this protein’s crucial role in A6 mRNA editing.

overall_design:
To investigate the function of KRBP72 in RNA editing, we generated a KRBP72 RNAi cell line. We then performed RNA-seq analysis on A6 mitochondrial transcripts in two biological replicates (rep1-doxy, rep2-doxy, rep1+doxy, and rep2+doxy). We analyzed these sequences using TREAT to identify the junction end sites in the KRBP72 knockdown cell line. To identify the direct binding sites of KRBP72, we generated a KRBP72-MHT cell line and performed eCLAP analysis in three biological replicates
**Submitters' note: the RNAseq data was submitted to the Sequence Read Archive with accession number PRJNA1133815***

first: Brianna

middle: L

last: Tylec

first: Soon

middle: Y

last: Yi

first: Frank

last: Tedeschi

first: Joseph

last: Smith

first: Laurie

middle: K

last: Read


title: Small molecule-based regulation of gene expression in human astrocytes switching on and off the G-quadruplex control systems

status: Public on Jan 29 2025

city: HOUSTON

first: Vijay Kumar

last: M J Rao

state: Texas

address: 6431 Fannin street

department: Neurology

country: USA

institute: UTHealth Houston

type: Expression profiling by high throughput sequencing

summary: A great deal of attention is being paid to strategies seeking to uncover the biology of the four-stranded nucleic acid structure G-quadruplex (G4) via their stabilization in cells with G4-specific ligands. The conventional definition of chemical biology implies that a complete assessment of G4 biology can only be achieved by implementing a complementary approach involving destabilization of cellular G4s by ad hoc molecular effectors. We report here on an unprecedented comparison of the cellular consequences of G4 chemical stabilization by pyridostatin (PDS) and destabilization by phenylpyrrolocytosine (PhpC) at both transcriptome- and proteome-wide scales in patient-derived primary human astrocytes. Our results show that the stabilization of G4s by PDS triggers the dysregulation of many cellular circuitries, the most drastic effects originating in downregulation of 354 transcripts and 158 proteins primarily involved in RNA transactions. In contrast, destabilization of G4s by PhpC modulates the G4 landscapes in a far more focused manner with upregulation of 295 proteins, mostly involved in RNA transactions as well, thus mirroring the effects of PDS. Our study is the first of its kind to report the extent of G4-associated cellular circuitries in human cells by systematically pitting the effect of G4 stabilization against destabilization in a direct and unbiased manner.

overall_design: Human astrocytes cultured from tissue resections of male age 32 epilepsy patient are used in this study. Three experimental data sets are used in this study. 1. Control astrocytes - age 32. 2. PDS treated astrocytes - age 32. 3. PhpC treated astrocytes. For RNA-seq experiment, N=5 samples are used for each data set. For Mass-spec proteomic analysis, N=3 samples are used for each data set.

first: Vijay

last: Marumulla

first: Jeremie

last: Mitteaux

first: Zi

last: Wang

first: Ellery

last: Wheeler

first: Nitin

last: Tandon

first: Sung

last: Jung

first: Robert

middle: H

last: Hudson

first: David

last: Monchaud

first: Andrey

middle: S

last: Tsvetkov


title: The role of KLF4 in the development of human primordial germ cell

city: Seoul

first: SUN-MIN

last: LEE

state: Other

address: 120, Neungdong-ro, Gwangjin-gu

country: South Korea

institute: Konkuk University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Primordial germ cells (PGCs) are the founder cells that develop into mature gametes. PGCs emerge during weeks 2-3 of human embryo development. Pluripotency genes are reactivated during PGCs specification, including Kruppel-like factor KLF4, but its precise role in PGC development is unknown. Here, we investigated the role of KLF4 in PGC development using our in vitro model for human PGC-like cells (hPGCLCs). We show that depletion of KLF4 using acute protein degradation strategies reduced the efficiency of hPGCLC specification. We conducted transcriptome analysis of KLF4-depleted hPGCLCs, and observed downregulation of genes involved in pattern specification, stem cell differentiation, and epigenetic modifications. Notably, KLF4 depletion led to the derepression of some somatic genes related to endodermal and neuronal differentiation. In PGCLCs, the KLF4 Cut&Run results showed significant co-enrichment with SP and STAT motifs, prominently localized to promoter regions. This represents a distinct binding pattern compared to that observed in naive embryonic stem cells. Our study reveals that KLF4 contributes to the differentiation and maintenance of human PGCLCs by comprehensively regulating both gene activation and repression.

overall_design: To elucidate the role of KLF4 in the differentiation of human PGCLCs, we established an inducible knockdown system using the auxin-degron method in WIS2 NANOS3–tdTomato reporter cell lines. Cells isolated by cell sorting using PGCLC-specific markers underwent gene expression profiling via RNA-seq. Additionally, we performed CUT&RUN using KLF4 antibodies on normal PGCLCs to analyze the KLF4 binding profile across the genome.

first: Sun-Min

last: Lee

first: Merrick

last: Pierson Smela

first: M

last: Azim Surani


title: Combined single-cell RNA-sequencing and single-cell bisulfite-sequencing reveal key regulatory mechanisms associated with oocyte in vitro maturation in pig [scRNA-seq]

city: Murcia

first: Francisco

last: Huertas

address: C/ Campus Universitario de Espinardo Nº7 (Edificio CEEIM)

country: Spain

institute: Marbyt - Smart Solutions for Biotechnology

summary: In vitro maturation (IVM) is a critical step in current animal in vitro embryo production protocols. However, in vitro matured oocytes often exhibit lower developmental competence than their in vivo counterparts, and the molecular mechanisms underlying successful maturation remain unclear. In this study, we compared the developmental competence up to the blastocyst stage of IVM and in vivo matured porcine oocytes (N=397) and also analysed simultaneously their gene expression and DNA methylation profiles at single cell resolution (N=40) using RNA sequencing and bisulfite sequencing. Our final aim was to find the associations between the developmental and molecular differences between the two groups of oocytes. All the oocytes were obtained by aspiration of either ovarian follicles between 3-6 mm diameter, and the subsequent IVM, or ovarian follicles from 8-10 mm diameter, with no need of maturation (in vivo matured oocytes). Significant differences were found at day 2 for cleavage rates (58.2 ± 3.0 and 45.7 ± 4.4, P = 0.010) and at day 7 for the blastocyst rates (31.4 ± 3.7 and 47.5 ± 6.6, P = 0.014) for IVM and in vivo groups, respectively, but not for the total cell number per blastocyst (48.1 ± 1.9 and 48.2 ± 2.7). Using the in vivo group as a reference, 1297 and 476 differentially expressed genes (DEGs) were down-regulated and up-regulated, respectively, in the IVM group. The up-regulated DEGs in IVM oocytes were mainly associated with organelle organization, DNA methylation, and cell cycle processes; down-regulated genes were mainly enriched in ribosomal RNA processing, protein synthesis, oxidative phosphorylation and metabolomic processes such as glycosyl and aldehyde compound pathways. The global percentage of methylation was similar between the IVM and in vivo groups, but 321, 344, and 843 differentially methylated regions (DMRs) were detected (P < 0.05) in CpG islands, promoters and coding regions, respectively. Integrative analysis of RNA-sequencing and DNA methylation data identified the main methylated regions and genes that define each group. A total of 236 loci and 296 genes defined the IVM group, while 856 loci and 688 genes were related to the in vivo group. In addition, in the IVM group, we found a higher number of negative correlations between gene expression and DNA methylation, while the in vivo group showed higher number and stronger positive correlations. Taken together, these results indicate a discrete effect of IVM on the DNA methylation landscape in mature porcine oocytes, but these changes seem to have a greater impact on gene expression regulation.

overall_design: Ovaries from Landrace-Large-White gilts were collected at the slaughterhouse and transported to the laboratory (within 60 minutes) in saline solution containing 100 µg/mL kanamycin sulphate at 38.5°C, washed once in 0.04% cetrimide solution (w/v) and then twice in saline. Then, the IVM protocol was the same as in Romero-Aguirregomezcorta et al. (2024). As for the in vivo matured oocytes, the contents of follicles of 8-10 mm diameter were aspirated and washed twice in Dulbecco's PBS supplemented with 1 mg mL-1 PVA. All IVM and in vivo matured COCs were identified under a stereomicroscope and mechanically detached from the cumulus cells using an automatic pipette set to a volume of 200 microlitres. To do this, the COCs were gently aspirated and repeatedly ejected into the Petri dish until the zona pellucida was visualised free of cumulus cells at 50x magnification. Only oocytes with a visible polar body under the stereomicroscope were selected and snap frozen in LN2 and stored at -80°C until use (n = 20 IVM and 20 in vivo matured oocytes).

first: Pilar

last: Fuster Coy

first: Laura

last: Abril-Parreño

first: Gavin

last: Kelsey

first: Antonio

last: Galvao

last: Huertas-López

first: Emilio

middle: M

last: Serrano-López

last: Martínez-Moreno


title: Downregulation of MLF1 safeguards cardiomyocytes against senescence-associated chromatin opening

city: Shenzhen

first: Zhihua

last: Wang

address: No. 12, Langshan Road, Xili Street, Nanshan District, Shenzhen

department: Shenzhen Key Laboratory of Cardiovascular Disease

country: China

institute: Fuwai Hospital Chinese Academy of Medical Sciences, Shenzhen

summary: Previous studies found that MLF1 mRNA levels tended to decline in aging hearts. Functional studies found that knockdown of MLF1 ameliorated the drug-induced cellular senescence phenotype. Here, we apply CUT&Tag to explore the underlying molecular mechanisms.

overall_design: Cardiomyocyte AC16 cells were cultured in DMEM/F12 . DMEM/F12 supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin, and 100 μg/ml streptomycin. Medium without FBS was replaced before transfection. Negative control siRNA and Mlf1 siRNA or SUZ12 siRNA were transfected into AC16 cells at ~70% confluence using jetPRIME® (Polyplus-transfection, NY, USA). After 12 h of transfection, fresh complete medium was replaced. Utilizing the Hyperactive® Universal CUT&Tag Assay Kit for Illumina (TD903), we performed CUT&Tag experiments as follows: AC16 cells were harvested at room temperature and incubated with ConA Beads for 10 minutes. They were then incubated with anti-MLF1 (ABclonal, A13329), anti-SUZ12 antibody (CST, 3737S, 1:50), anti-EP300 (CST, #57625), anti-H3K27ac (CST, #4353S), or anti-H3K27me3 (CST, #9733) antibodies overnight at 4°C. On the next day, the samples were incubated with a secondary antibody at room temperature for 1h, followed by incubation with pA/G-Tnp for 1h.  After DNA purification with the MinElute kit, and the PCR was performed to amplify the library for 12 cycles and libraries were purified with the MinElute kit. Amplified  libraries were sequenced on the Illumina Novaseq 6000 platform.

first: Jian

last: Lv

first: Qin

last: Chen

first: Junmei

first: Ningning

last: Guo

first: Yu

last: Fang

first: Qiuxiao

first: Jiajie

last: Li

first: Xiao

last: Ma

first: Hongchao

last: Zhan

first: Weihao

first: Li

first: Qingqing

last: Yan

first: Jingjing

last: Tong


title: Circle-seq reveals that eccDNA may be a key blood biomarker for HBV-associated liver cancer (2)

city: Nanning

first: Xiaoke

last: Ran

address: Wuhe Avenue

institute: Guangxi University of Chinese Medicine

summary: To explore the expression of eccDNA in patients with chronic hepatitis B and liver cirrhosis, focusing on their impact on disease development.

overall_design: Five patients were diagnosed with chronic hepatitis B and five patients were diagnosed with liver cirrhosis. We took blood samples from the patients. EccDNA expression profiling and mRNA gene expression profiling was analyzed to determine what extrachromosomal circular DNA in chronic hepatitis B to liver cirrhosis.

first: Xundong

last: Liu


title: C-reactive protein promotes diabetic kidney disease via Smad3 mediated NLRP3 inflammasome activation

city: Hong Kong

first: YIFAN

last: WANG

address: 6/F, 3 Sassoon Road, Pokfulam

department: School of Chinese Medicine

country: Hong Kong

institute: The University of Hong Kong

summary: Diabetic kidney disease (DKD) is the leading cause of end-stage kidney diseases resulting enormous social-economic burden. Accumulated evidence has indicated that C-reactive protein (CRP) exacerbates DKD by enhancing renal inflammation and fibrosis through TGF-β/Smad3 signaling. NLRP3 inflammasome is the key sensor contributing to renal inflammation. However, whether CRP enhances inflammation in DKD via NLRP3 inflammasome related pathway remains unknown. In this study, we demonstrate that CRP promotes DKD via Smad3-mediated NLRP3 inflammasome activation as mice overexpressing human CRP gene exhibits accelerated renal inflammation in diabetic kidneys, which is associated with the activation of Smad3 and NLRP3 inflammasome. In contrast, blockade of CPR signaling with a neutralizing anti-CD32 antibody attenuates CRP-induced activation of Smad3 and NLRP3 in vitro. Importantly, genetic deletion or pharmacological inhibition of Smad3 also mitigates CRP-induced activation of NLRP3 in diabetic kidneys or in high glucose treated cells. Mechanistically, we reveal that Smad3 binds to the NLRP3 gene promoter which is enhanced by CRP. Taken together, we conclude that CRP induces renal inflammation in DKD via to the Smad3-NLRP3 inflammasome-dependent mechanism. Thus, targeting CRP or Smad3-NLRP3 pathways may be a new therapeutic potential for DKD.

overall_design: To investigate the function of human CRP, we constructed human CRP transgenic on db/db mice for RNA sequencing.

first: WANG

last: Yifan

first: YOU

last: Yongke

first: GUO

last: Jianbo

first: CHEN

last: Haiyong


title: Identifying a novel retinal pigment epithelium specific adeno-associated virus vector

city: Guangzou

first: Xiaolai

last: Zhou

state: Guangdong

address: No. 54 Xianlie South Rd

institute: Zhongshan ophthalmic center，Sun Yat-sen university

summary: Retinal Pigment Epithelium (RPE) are crucial for maintaining retinal homeostasis and the visual cycle. Currently, there are no effective treatments for blinding retinal diseases caused by RPE cell degeneration. Gene therapy presents a promising approach, but no gene therapy vectors specifically targeting RPE cells are available. In this study, we utilized an AAV2 random mutation library and high-throughput sequencing to screen AAV vectors （AAV206）capable of specifically targeting RPE cells. Intravitreal injection of the AAV206 vector in mice demonstrated that AAV206 specifically infected RPE cells, whereas AAV2 primarily infects the inner retina. Furthermore, AAV206 exhibited lower immunogenicity and toxicity compared to AAV2. Overexpression of sFLT-1 through AAV206 effectively inhibited the size and leakage of choroidal neovascularization. In summary, AAV206 is an ideal vector for gene therapy targeting RPE cells.

overall_design: To assess retinal changes, mice aged 6-8 weeks were divided into two groups, with five mice per group. The mice received intravitreal injections of AAV2-GFP, and AAV206-GFP, respectively. Fourteen days after intravitreal injection, retinas were harvested for bulk RNA sequencing.

first: Zhou

last: Xiaolai

first: Gong

last: Yajun


title: Novel function of cholecystokinin (CCK): Mediator between feeding and gonadal development

city: wuhan

first: xiangtong

last: zeng

address: shizishan street

institute: Huazhong Agricultural University

summary: We used single cell RNA sequencing (scRNA-seq)to analyze the pituitary cells in Ctenopharyngodon idellus

overall_design: diversity of cells in the pituitary.

first: Hangyu

first: Xiaowen

last: Gao

first: Zhan

last: Yin

first: Guangfu

last: Hu


title: MLKL/USP7/UBA52 Signaling Is Indispensable for Autophagy in Brain through Maintaining Ubiquitin Homeostasis

first: Jie

last: Tu

address: 1068 Xueyuan Boulevard, Nanshan, Shenzhen 518055, China

department: The Brain Cognition and Brain Disease Institute (BCBDI)

institute: Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS)

summary: Individuals with genetic elimination of MLKL exhibit an increased susceptibility to neurodegenerative diseases like Alzheimer’s disease (AD). However, the specific mechanism behind this relationship is not yet fully understood. Here, we initially observed significant compromise in autophagy in the brains of MLKL knockout (KO) mice, as evidenced by the down-regulation of autophagy-initiation proteins Beclin1 and ULK1. To elucidate the mechanism by which MLKL, primarily a cytosolic protein, regulates the protein levels of Beclin1 and ULK1, we performed a proteomic screening of MLKL-association proteins and identified UBA52 as the binding partner under physiological conditions. Loss of MLKL induces a decrease in ubiquitin levels by preventing UBA52 from producing ubiquitin and the C-terminal ribosomal protein L40. Furthermore, we demonstrated that in the brain, the deubiquitinase (DUB) USP7 mediates the processing of UBA52, which is regulated by MLKL. Knock down (KD) of USP7 or UBA52 showed similar effects on autophagy and ubiquitin levels as MLKL KD/KO. Moreover, our results indicated that the reduction of Beclin1 and ULK1 upon MLKL loss is attributed to a decrease in their lysine 63 (K63)-linked polyubiquitination. Additionally, single-nucleus RNA sequencing revealed that the loss of MLKL resulted in the disruption of multiple neurodegenerative disease-related pathways, including those associated with AD. These results were consistent with the observation of cognitive impairment in MLKL KO mice and exacerbation of AD pathologies in an AD mouse model with MLKL deletion. Taken together, our findings demonstrate that MLKL-USP7-UBA52 signaling is required for autophagy in brain through maintaining ubiquitin homeostasis, and highlight the contribution of MLKL loss-induced ubiquitin deficits to the development of neurodegeneration. Thus, the maintenance of adequate levels of ubiquitin may provide a novel perspective to protect individuals from multiple neurodegenerative diseases through regulating autophagy.

overall_design: Nuclei were isolated from the hippocampus of 14-month-old male MLKL KO mice and WT littermates. Briefly, tissues were homogenized by Dounce homogenizer (~25 strokes/sample) in ice-old homogenization buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM tricine-KOH, pH 7.8, 1 mM dithiothreitol, 0.15 mM spermine, 0.5 mM spermidine, protease inhibitors, 5 μg/mL actinomycin, 0.32% Nonidet P-40, and 0.04% bovine serum albumin). The lysate was mixed with equal volume of OptiPrep medium, centrifuged at 10,000 × g for 20 minutes at 4°C, and the nuclei were collected as pellets. The pellets were washed and resuspended in DMEM/F12 medium with 10% FBS to obtain a final concentration of 400 nuclei/μl. The purity of the single-nucleus suspensions was evaluated by flow cytometry, and high purity samples (DAPI+ nuclei > 95%) were used for analysis. RNAsin (Promega) was included in all buffers or solutions at 60 U/ml in nuclei extraction.

first: Zhigang

last: Zhang

first: Shuai

last: Chen

first: Shirui

last: Jun

first: Xirong

last: Xu

first: Yuchuan

last: Hong

first: Xifei

last: Yang

first: Liangyu

last: Zou

first: You-Qiang

last: Song

first: Yu


city: Chongqing

first: Jing

address: Chongqing

institute: Chongqing Medical University

summary:
Candida albicans (C. albicans) is one of the most common pathogens associated with deep fungal infection, which represents a serious threat to human health. Although high mobility group box 1 (HMGB1) plays a key role in C. albicans infection, its mechanism is unclear. We aimed to explore the regulation of small-molecule non-coding RNA (miRNA) for HMGB1 in C. albicans infection.
The miRNA differential expression profiles of mouse primary peritoneal macrophages infected by C. albicans  were screened by 6 miRNA gene chips

overall_design: mouse primary peritoneal macrophages VS C. albicans infection ×3 (total 6 samples)


title: Transcriptomic effect of Fbn1 G234D/G234D mutation on neonatal skin fibroblast

city: Tsukuba

first: ASM SAKHAWAT

last: HOSSAIN

state: Ibaraki

address: 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8577, Japan

department: Graduate School of Comprehensive Human Science

country: Japan

institute: University of Tsukuba

summary: This study contains RNA-seq analysis of neonatal dermal fibroblasts from mice with Fbn1 G234D/G234D mutation to uncover its impact on ECM gene expression, potentially shedding light on the tight skin phenotype. While both wild-type (WT) and mutant fibroblasts showed differences, two of the mutants exhibited more significant changes, aligning with the early onset of the disease. These earlier data was deposited in GSE268383. In this study we added 2 more wild type fibroblasts and 3 more homozygous mutant cells to ascertain the features we obtained prevously. As previously noted, there was no obvious change in ECM and ECM-modifying genes, such as the LOX family, Fbn1, and TGFβ/BMP signaling genes; while the mutants displayed upregulation of muscle developmental genes (e.g., Myod1, Myog, Myh family), immune response, and cell-cell adhesion/cytoskeletal genes. In line with previous data, downregulated genes were still enriched for histone genes and developmental transcription factors, hinting at potential chromatin/transcriptional state modifications underlying the tight skin phenotype. These transcriptomic findings underscore the need for further investigation into the mechanobiological implications of Fbn1G234D/G234D fibroblasts.

overall_design: To investigate whether the mutation induces intrinsic changes in fibroblasts that result in alterations in ECM gene expression, RNA sequencing (RNA-seq) was conducted on primary cultures of neonatal fibroblasts collected from the dermis of 4 WT and 4 mutant mice, and on additional primary cultures of neonatal fibroblasts collected from the dermis of 2 WT and 3 mutant mice

first: ASM Sakhawat

last: Hossain

first: Erna

last: Raja

first: Narenmandula

last: Narenmandula

first: Hiromi

last: Yanagisawa


title: DHX36-mediated G-quadruplexes unwinding is essential for oocyte and early embryo development in mice

city: Hangzhou

first: Heng-yu

last: Fan

state: Zhejiang

address: Yuhangtang Road

institute: Life Sciences  Institute

summary: The role of G-quadruplex (G4) structures and their effects on oocyte and early embryo development remain unclear. We discovered that the G4 helicase DHX36 is essential for oocyte growth and the maternal-to-zygotic transition (MZT). Conditional knockout of DHX36 resulted in DNA G4 accumulation in mouse oocytes, reducing chromatin accessibility, and inhibiting RNA transcription, ultimately disrupting transcriptome homeostasis during oocyte growth and MZT.

overall_design: We performed Smart-seq2 on samples from wild-type (WT) and DHX36 conditional knockout (CKO) female mice, including growing oocytes (2-week-old), fully-grown oocytes (3-week-old and 4-week-old), metaphase II oocytes (3-week-old), and zygotes (3-week-old).

first: Yuxuan

last: Jiao

first: Guowei

last: Bu

first: Hengyu


title: Circle-seq reveals that eccDNA may be a key blood biomarker for HBV-associated liver cancer (4)

first: Xiaoke

last: Ran

summary: To explore the expression of eccDNA in patients with liver cirrhosis and liver cancer, focusing on their impact on disease development.

overall_design: Five patients were diagnosed with liver cancer and five patients were diagnosed with liver cirrhosis. We took blood samples from the patients. EccDNA expression profiling and mRNA gene expression profiling was analyzed to determine what extrachromosomal circular DNA in liver cirrhosis to liver cancer.

first: Xundong

last: Liu


title: RNA N6-methyladenosine-binding protein YTHDFs redundantly attenuate cancer immunity by downregulating IFN-γ signaling in gastric cancer [RIP-Seq]

first: Dongjun

last: Jang

address: 103 Daehak-ro (Yeongeon-dong), Jongno-gu

institute: Seoul National University

summary: Gastric cancer (GC) is a prominent public health issue, especially in East Asia, and holds the fourth rank in terms of cancer mortality. While immunotherapy holds potential as a treatment avenue for GC, resistance to immune checkpoint inhibitors (ICIs) remains an obstacle. One resistance mechanism involves defects in IFN-γ signaling, where IFN-γ is linked to improved responsiveness to ICIs. In this study, we unveiled the role of N6-methyladenosine (m6A) RNA modifications, in the regulation of IFN-γ signaling and the responsiveness to ICIs. The m6A-binding protein YTHDF1 was overexpressed in GC tissues, correlating with the suppression of cancer immunity and poorer survival rates. Overexpression of YTHDF1 impaired the responsiveness to IFN-γ in GC cells, while knockdown studies indicated the redundant effects of YTHDF2 and YTHDF3 with YTHDF1 on IFN-γ responsiveness. RNA immunoprecipitation-sequencing identified that YTHDFs directly targeted the mRNA of IRF1, which is one of master regulators of IFN-γ signaling, leading to reduced RNA stability and consequent downregulation of IFN-γ signaling. Furthermore, in mouse syngeneic tumor models, the depletion of Ythdf1 in cancer cells resulted in reduced tumor growth and heightened tumor infiltrating lymphocytes, which is attributed to the augmentation of IFN-γ signaling. Collectively, our findings highlight how YTHDFs modulate cancer immunity by influencing IFN-γ signaling through IRF1 regulation, thus proposing their viability as therapeutic targets in the realm of cancer immunotherapy.

overall_design:
To investigate the target of YTHDF1 in the regulation of IFN-γ responsiveness, we treated IFN-γ after overexpressing YTHDF1 in SNU638
RNA immunoprecipitation (RIP)-sequencing were performed

first: Chanwoong

last: Hwa

first: Joon-Yong

last: An

first: Sung-Yup

last: Cho


title: RNA N6-methyladenosine-binding protein YTHDFs redundantly attenuate cancer immunity by downregulating IFN-γ signaling in gastric cancer [RNA-Seq]

overall_design: To investigate the role of YTHDF1 in the regulation of IFN-γ responsiveness, we treated IFN-γ after overexpressing YTHDF1 in SNU638


title: The GLP-1R agonist semaglutide reshapes pancreatic cancer associated fibroblasts reducing collagen hydroxyprolination and favoring immune infiltration.

city: Rome

first: Valerio

last: Licursi

address: via degli Apuli, 4

department: c/o Dept. Biology and Biotechnologies "C. Darwin", Sapienza University of Rome

country: Italy

institute: Institute of Molecular Biology and Pathology (IBPM), National Research Council (CNR)

summary: Metabolic syndrome represents a risk factor of pancreatic ductal adenocarcinoma (PDAC), the gastrointestinal cancer with the lowest survival rate and therapeutic outcome. PDAC results bidirectional interconnected with metabolic alteration, which favors its onset and occurs during early phases of its development. Pancreatic neoplastic lesions evolve within a highly dense desmoplastic stroma, characterized by abundant extracellular matrix deposition consequence of a prominent cancer associated fibroblast (CAF) activation. Here, the complex association between dysmetabolism and PDAC has been analyzed with a specific focus on CAFs to unveil novel targets exploitable for diagnostic and therapeutic purposes. Methods: PDAC development upon dysmetabolic conditions was investigated in: 1) wild type immunocompetent syngeneic mice by orthotopic transplantation of PanIN-bearing Pdx1-Cre; Kras+/LSL-G12D; Trp53+/LSL-R172H organoids (KPC mice) fed with high fat diet to recapitulate dysmetabolism-dependent alterations; and 2) primary pancreatic CAFs isolated from chemotherapy naïve PDAC patients with or without an history of metabolic syndrome. Results: The dysmetabolic-associated higher PDAC aggressiveness was paralleled by an enrichment of collagen fibrils due to an increased function of the prolyl 4-hydroxylase subunit alpha 1 (P4HA1). Upon dysmetabolic conditions, P4HA1 boosts collagen hydroxy-prolination, intensifying collagen contraction strength and thus preventing CD3 positive lymphocyte PDAC infiltration. Interestingly, semaglutide, an incretin agonist with anti-diabetic and anti-obesity properties, is able to prevent the higher stromal deposition of PDAC observed in dysmetabolic conditions, preventing CAF contraction, allowing immune infiltration and reducing tumor development. Conclusion: These results shed light on novel therapeutic options for PDAC patients affected by metabolic syndrome aimed at pancreatic cancer stroma reshape

overall_design: RNASeq from OCT sections derived from PDAC preclinical mouse model fed either with low fat diet (LFD) or high fat diet (HFD) to mimic PDAC development in a dysmetabolic environment

first: Chiara

last: Cencioni

first: Silvia

last: Malatesta

first: Francesco

last: Spallotta


summary: The role of G-quadruplex (G4) structures and their effects on meiosis resumption and early embryo development remain unclear. We discovered that the G4 helicase DHX36 is essential for oocyte growth and the maternal-to-zygotic transition (MZT). Conditional knockout of DHX36 resulted in DNA G4 accumulation in mouse oocytes, reducing chromatin accessibility, and inhibiting RNA transcription, ultimately disrupting transcriptome homeostasis during oocyte growth and MZT.

overall_design: ATAC-seq was conducted on growing oocytes (2-week-old) and fully-grown oocytes (3-week-old) from wild-type (WT) and DHX36 conditional knockout (CKO) female mice.


title: Downregulation of NSRP1 confers CDK4/6i resistance in breast cancer via activation of the IFN signaling pathway

city: yangzhou

first: Yu

last: Shiyi

state: Jiangsu

address: hanjiang district jiangyangzhonglu136, Hanjiang district, Jiangyangzhonglu 136

institute: yangzhou university

summary: Via analyzing the expression matrix of palbociclib-sensitive and palbociclib-resistant tumors from patients with breast cancer, it was revealed that NSRP1 is associated with CDK4/6i resistance. Therefore, RNA Sequencing is performed for MCF7 cells transfected with ctr or NSRP1 siRNA to study the molecular mechanism of NSRP1 in mediating CDK4/6i resistance in breast cancer cells.

overall_design: We transfected MCF7 cells with NSRP1 siRNAs or control siRNA.

first: Shiyi

last: Yu

first: Haibo

last: Sun


title: Ribosome profiling of WT vs Cas9-based DBP1 deletion

city: Berkeley

first: Gloria

last: Brar

address: 626 Barker Hall

department: MCB

institute: UC Berkeley

summary: Previous deleitons of the DBP1 locus suffered from an off-target effect due to cassette insertion (Powers et al, eLife, 2022). Here, we made a markerless deletion to assess gene expression in meiosis with and without Dbp1. Although it is normally highly expressed in this context, its deletion does not cause a major defect in meiotic progression or gene expression.

overall_design: We performed ribosome profiling in parallel with mRNA sequencing of yeast cells in sporulation media for 3 or 6 hours, either with or without the DBP1 ORF deleted by a markerless Cas9-based strategy

first: Emily

last: Powers


city: Wuhan

first: Hangyu

last: Li

state: 湖北省

address: No.1,Shizishan Street

summary: We used single cell RNA sequencing (scRNA-seq)to analyze the hypothalamu cells in Ctenopharyngodon idellus

overall_design: diversity of cells in the hypothalamus.

first: Xiaowen

last: Gao

first: Zhan

last: Yin

first: Guangfu

last: Hu


title: Mechanistic Insights into Epithelial-Mesenchymal Transition Mediated Cisplatin Resistance in Ovarian Cancer

city: Jinju

first: Hyonok

last: Yoon

state: Gyeongnam

address: 501 Jinju-Daero

department: Pharmacy

institute: Gyeongsang National University

summary: Epithelial-mesenchymal transition (EMT) is designated as one of the prime causes of chemoresistance in many cancers. In our previous study we established that cisplatin resistance in ovarian cancer (OC) is associated with EMT using sensitive OV90 cells and its resistant counterparts OV90CisR1 and OV90CisR2. In this study, through RNAseq analysis we revealed that ITGA1 can play essential part in EMT mediated cisplatin resistance in OC. We found large number of EMT related terms predominant in the top gene ontologies (GO). We also found ECM and actin cytoskeleton genes highly altered in the resistant cells. This was further confirmed by the protein-protein interaction (PPI) analysis where we identified that the core ECM components e.g., collagen, fibronectin, metalloproteases and integrins possessed most interactions. The pathway analysis revealed the Wnt signaling as the leading pathway. Since integrins have significant interaction with Wnt signaling, we focused our study on integrins among which, ITGA1, ITGA6, ITGA11 and ITGAV were primarily altered. We validated our results by western blotting and found that ITGA1 was highly expressed in resistant cells. Additionally, the high ABCA5 (efflux transporter) expression in resistant cells also supports the EMT proposition. The western blotting also revealed high β-catenin expression in resistant cells confirming the high Wnt signaling activity. Further, we induced xenograft tumors in nude mice. The histopathological analysis confirmed the aggressive nature of resistant tumors and showed the presence of necrotic core which could be implicated to EMT. Finally, the immunohistochemical staining confirmed the high protein expression in resistant tumor.

overall_design: To investigate and identify the genes associated with the cisplatin reistance in ovarian cancer. We established cisplatin resistant counterparts (OV90CisR1 and OV90CisR2) of sensitive OV90 cell lines.  We performed the differential gene expression analysis of the data obtained from the Quantseq-3'mRNA sequencing of 3 cell types (OV90, OV90CisR1 and OV90CisR2). We performed experiments on xenograft mice models to determine the expression of the target proteins. We also validated our results by western blotting and immunohistochemical staining.


title: Integration of dCas9-Based Methylation Editing with GPS Identifies Dynamic Changes of mrDEGs in BRCA

city: Shanghai

first: Baolong

last: Zhang

state: 上海市 — Shanghai Shi

address: 138 Yixueyuan Road, Xuhui District, Shanghai

institute: Fudan University

type: Methylation profiling by high throughput sequencing

summary: Breast carcinoma (BRCA) has emerged as the leading cancer worldwide with the highest incidence. The malignant transformation of normal breast epithelial cells is a crucial prerequisite for the initiation of breast cancer. However, the underlying epigenomic mechanism remains poorly understood. Here, we utilized guide positioning sequencing (GPS) to conduct whole-genome DNA methylation analysis in an MCF10 series of cell lines, a typical model of malignant progression including normal breast epithelial MCF10A, premalignant MCF10AT, low-grade metastatic MCF10CA1h, and high-grade metastatic MCF10CA1a. By integrating mRNA-seq with matched clinical data from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO), six representative methylation-related differentially expressed genes (mrDEGs) were screened, including CAVIN2, ARL4D, DUSP1, TENT5B, P3H2, and MMP28. To mimic tumor progression in vitro, we independently optimized and employed the site-specific dCas9-DNMT3L-DNMT3A system to artificially downregulate TENT5B in MCF10A cells, demonstrating that transcriptional silence of TENT5B accelerates cell proliferation in MCF10A cells owing to the crosstalk between hypermethylation and histone deacetylation. Our data highlight the practical implications of DNA methylation dynamics in reshaping epigenomic features during the BRCA malignant progression. The site-specific methylation technique would help gain a comprehensive understanding of pathogenic mechanisms and facilitate future therapeutic approaches in epigenome editing.

overall_design: In order to analyze the dynamic atlas of DNA methylation patterns in breast cancer progression, we conducted guide positioning sequencing (GPS) to obtain genome-wide methylation data for each CpG site on four cell lines under the same culture conditions during exponential growth phase. We then performed a comprehensive analysis of DNA methylation data using dispersion shrinkage for sequencing data (DSS) to identify DMRs in three distinct comparisons: MCF10A vs. MCF10AT, MCF10A vs. MCF10CA1h, and MCF10A vs. MCF10CA1a.

first: Jin

first: Wenqiang


title: Molecular mechanism of EP300 in AC16 cell line

first: Zhihua

last: Wang

summary: Functional studies found that knockdown of EP300 ameliorated the drug-induced cellular senescence phenotype. Here, we apply transcriptomics to explore the underlying molecular mechanisms.

overall_design: Cardiomyocyte AC16 cells were cultured in DMEM/F12 and DMEM (high glucose), respectively. DMEM supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin, and 100 μg/ml streptomycin. Medium without FBS was replaced before transfection. Negative control siRNA and EP300 siRNA were transfected into AC16 cells at ~70% confluence using jetPRIME® (Polyplus-transfection, NY, USA). After 12h of transfection, AC16 cells were treated with H2O2 (200mM) for 1 h, then fresh complete medium was replaced. After 48 h, RNA  extracted according to the manufacturer's instructions using Trizol reagent. RNA libraries were prepared for sequencing according to manufacturer's instructions of Beijing Genomics Institution (BGI)

first: Jian

last: Lv

first: Qin

last: Chen

first: Ningning

last: Guo

last: Fang

first: Qiuxiao

first: Junmei

first: Dilinaer

last: Maimaiti

first: Jiajie

first: Xiao

last: Ma

first: Weihao

first: Li

first: Qingqing

last: Yan

first: Jingjing

last: Tong


title: A Novel Peptide Encoded by circSRCAP Confers Resistance to Enzalutamide by Inhibiting the Ubiquitin-Dependent Degradation of AR-V7 in Castration-Resistant Prostate Cancer

city: Xuzhou

first: Xiannan

last: Meng

address: Tongshan Road

institute: Xuzhou Medical University

type: Non-coding RNA profiling by high throughput sequencing

summary: Background: The sustained activation of androgen receptor splice variant-7 (AR-V7) is a key factor in the resistance of castration-resistant prostate cancer (CRPC) patients to second-generation anti-androgen (AR) drugs such as enzalutamide (ENZ). The AR/AR-V7 protein is regulated by the E3 ubiquitin ligase STUB1 and a protein complex formed by HSP70. However, the specific mechanism remains elusive. Methods: High-throughput RNA sequencing was employed to detect differentially expressed circular RNAs (circRNAs) in ENZ-resistant and control CRPC cells. The coding potential of circSRCAP was identified through polysome profiling and LC-MS. The function of circSRCAP was validated in vitro and in vivo via gain or loss of function assays. Mechanistic insights were derived from immunoprecipitation analyses. Results: A novel ENZ-resistant circular RNA (circRNA) named circSRCAP has been identified, showing upregulation in ENZ-resistant C4-2B cells (ENZR-C4-2B) and correlation with elevated protein levels of AR-V7. circSRCAP undergoes cleavage into a loop by the splicing factor EIF4A3 and is derived from the nucleus by the RNA helicase DDX39A. Mechanistically, circSRCAP encodes a 75 amino acid peptide, circSRCAP-75aa, which inhibits the ubiquitination of the AR/AR-V7's co-chaperone protein HSP70 by dissociating STUB1, a ubiquitin E3 ligase. This process leads to the upregulation of AR-V7 protein expression, thereby promoting resistance of castration-resistant prostate cancer (CRPC) cells to ENZ. Xenograft tumor models further confirm the role of circSRCAP in CRPC progression and its potential as a therapeutic target for ENZ-resistant CRPC (ENZR-CRPC). Conclusions: circSRCAP presents an epigenetic mechanism that sheds light on the fate determination of AR-V7, offering a promising therapeutic target for the treatment of ENZR-CRPC.

overall_design: The samples utilized for circular RNA sequencing comprised the C4-2B and ENZR-C4-2B cell lines. Sequencing and subsequent analysis were carried out by Annoroad Gene Technology Corporation (Beijing, China). For circular RNA sequencing, total RNAs underwent treatment with a ribo-zero kit and RNase R to eliminate ribosomal RNA and linear RNA, respectively. Subsequently, a cDNA library was constructed, validated using an Agilent 2100 Bioanalyzer, and sequenced via Illumina platform. During the analysis, a criterion of log2 (|fold change|) ≥ 1.5 and p-value < 0.05 was employed to identify differentially expressed circRNAs.

first: Qingxuan

last: Wu

first: Hongmei

last: Yong

first: Wendong

last: Yang

first: Sufang

last: Chu

first: Junnian

last: Zheng

first: Suhua

last: Q-

last: Bai


title: Single-cell RNA seq identifies genes regulating the inflammatory pathways activated by purified Borrelia burgdorferi lipoproteins in murine bone marrow derived macrophages

city: San Antonio

first: Venkatesh

last: Kumaresan

state: TX

address: 3.230, BSE Building, One UTSA circle

department: Molecular Microbiology and Immunology

institute: The University of Texas at San Antonio

summary: Borrelia burgdorferi, the spirochetal agent of Lyme disease, has a large constellation of lipoproteins that play a prominent role in mediating pathogen-specific interactions with tick and vertebrate host cells. Although there is a large body of information on the effects of borrelial lipoproteins on immune modulatory pathways, the application of multi-omics methodologies to decode the transcriptional and proteomic patterns associated with host cell responses induced by lipoproteins in murine bone-marrow macrophages (BMDM) have helped identify additional effectors/pathways. Single-cell RNA seq revealed spatial heterogeneity within BMDM. Gene expression analysis showed that genes involved in pathways such as MAPK, TLR, NOD, chemokines, T-cell receptor signaling are activated in BMDM with unbiased proteomics analysis corroborating these findings.

overall_design: We used single-cell RNA-Seq to study the transcriptional changes in murine bone marrow derived macrophages from C3H/HeN mice exposed to purified borrelial lipoproteins, to uncover the molecular signatures that contribute to inflammatory responses in Lyme disease.

first: Taylor

last: Ingle

first: Brian

middle: P

last: Hermann

first: Janakiram

last: Seshu


title: Targeting CDK7 function in the RNA Polymerase II transcription cycle in inflammatory arthritis

city: New york

first: Yurii

last: Chinenov

address: 535 E 71 str, Hospital for Special Surgery

department: Research Division

institute: Hospital for special surgery

summary: Macrophages are main drivers of inflammation and tissue damage in autoimmune diseases including rheumatoid arthritis1-4. The rate-limiting step for transcription of >70% of inducible genes in macrophages is RNA Polymerase II (Pol II) promoter-proximal pause release5-7, yet, the specific role of Pol II pausing in inflammation or whether it can be modulated therapeutically is unknown. Genetic ablation of a pause-stabilizing negative elongation factor (NELF) in macrophages enhanced the transcriptional response of paused anti-inflammatory genes to LPS, followed by secondary attenuation of inflammatory signaling, and reduced severity of K/BxN-serum transfer (ST) arthritis in mice. To disrupt Pol II pause establishment pharmacologically, we employed covalent inhibitors of the TFIIH-associated cyclin-dependent kinase (CDK) 7, THZ1 and YKL-5-124. Both dramatically reduced Pol II pausing in murine and human macrophages, broadly suppressed induction of pro- but not anti-inflammatory genes, and rapidly reversed pre-established inflammatory macrophage polarization by TNF+IFNγ. In vivo, CDK7i ameliorated the acute K/BxN-ST and chronic progressive hTNF-transgenic arthritis in mice. Finally, CDK7i downregulated a pathogenic gene expression signature in synovial explants from arthritis patients. We propose that interfering with Pol II pausing by targeting CDK7 represents a novel therapeutic opportunity for inflammatory diseases.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: NOT PROVIDED; REQUESTED

first: Xi

last: Chen

first: Gayathri

last: Shibu

first: Dinesh

last: Deochand

first: Baila

last: Sokolsky

first: Laura

middle: T

last: Donlin

first: Inez

last: Rogatsky


title: Chemotactic Signalling Triggers Dental Pulp Fibrosis in the Stem Cell Niches During

city: ChangChun

first: Zhengwen

last: An

state: JiLin

address: HeGuang Road 763

department: Department of Oral Biology

institute: Jilin University

summary: Aging often triggers dental pulp fibrosis, leading to various clinical repercussions, including heightened vulnerability to dental infections, compromised tooth vitality, and reduced responsiveness to dental interventions. Despite its common occurrence in clinical settings, the precise molecular mechanisms driving this condition remain elusive. Leveraging single-cell transcriptome analysis from both our own and publicly available datasets, we identified Ccrl2+ macrophages as particularly prone to susceptibility during the early stages of aging. Notably, dental pulp progenitors exhibiting high expression of Rarres2, a unique ligand for Ccrl2, orchestrate the selective recruitment of a specific macrophage population to the stem cell niches. Subsequently, this results in the presentation of the ligand-receptor complex to Cmklr1, a receptor ubiquitously expressed among all macrophage populations, ultimately leading to macrophage activation and expansion via the Rarres2/Ccrl2/Cmklr1 axis. Our study, supported by rigorous experimental validation, conclusively shows that macrophage activation and expansion in stem cell niches lead to increased secretion of proinflammatory factors, which contribute to dental pulp fibrosis during aging. Our data reveals the complex molecular dynamics of dental pulp aging, particularly focusing on interactions within the immune microenvironment. It offers a novel perspective on potential treatments for age-related pulp diseases, highlighting the role of macrophages and the possibility of modifying the immune microenvironment for therapeutic benefit.

overall_design: Mouse incisors from mandibles were carefully dissected from 15 C57BL/6 mice aged 8-12 months. The incisor pulp, with minimal inclusion of dental epithelium, was isolated and analysed using scRNAseq.

first: Tianmeng

last: Sun

first: Qing

last: Zhong

first: Xiaoyi

last: Yu

first: Huanyu

last: Luo

first: Feilong

last: Ren

first: Cangwei

last: Liu

first: Wei

last: Feng

first: Peng

first: Fabian

last: Flores-Borja

first: Hongchen


title: U6 snRNP inhibits the premature cleavage and polyadenylation of pre-mRNAs (ChIP-Seq)

city: guangzhou

first: chengguo

last: yao

address: No. 74 zhongshan 2rd road

department: school of medicine

institute: sysu

summary:
RNAPII ChIP-seq analysis in U2 and U6 antisense morpholino oligonucleotide (AMO)-treated HeLa cells in comparison to negative control (NC) AMO
To investigate if U2 or U6 AMO treatment caused transcription defect, we carried out RNAPII ChIP-seq analysis in control, U2 and U6 AMO treated HeLa cells

overall_design: ChIP-seq analysis was conducted in HeLa cells using an antibody specific to the RNAPII CTD (C-terminal domain). Two biological replicates were performed for each sample.

first: Qiumin

first: Zejin

last: Lin

first: Danhui

last: Zhao

first: Congting

last: Ye

first: Chengguo

last: Yao


title: Adaptation response to salt stress in the Poecilia reticulata gut

city: Cheongju

first: Kyunghwan

last: Kim

address: Rm #202a, Bldg. S1-5, 1 chungdae-ro

institute: Chungbuk National University

summary: Osmoregulation is essential for the survival of aquatic organisms, particularly teleost fish facing osmotic challenges in variable salinity environments. While gills are traditionally associated with ion exchange, the intestine's role in water and salt absorption is gaining attention. We investigated the adaptive responses of the guppy (Poecilia reticulata) intestine to salinity stress, revealing significant morphological and transcriptomic alterations. Through stepwise salt adaptation experiments, we demonstrated the guppy's remarkable salt tolerance compared to zebrafish. Morphological analysis revealed changes in intestinal epithelial cells, particularly in columnar-shaped enterocytes. Transcriptomic analysis identified key genes involved in osmoregulation, tissue remodeling, and immune modulation. Upregulated genes included SLC transporters (SLC12A1, SLC3A1) facilitating ion and water transport, while Fosl2 and Pik3CB indicated tissue repair and growth responses. Conversely, innate immune system genes (e.g., TNFAIP6) showed downregulation, suggesting a shift towards prioritizing osmoregulatory functions over immune responses. Our findings highlight the intricate mechanisms underlying the guppy's adaptation to salinity stress, emphasizing the adaptive advantage of euryhaline fish and providing insights into osmoregulatory mechanisms in aquatic organisms.

overall_design: To investigate transcriptome changes in the intestine under salinity stress, guppy was exposed to  three salt conditions (0‰, 18‰, 35‰).


title: Liver in humanized-liver mice: Control vs acetoaceto-o-toluidide treatment

city: Osaka

first: Shugo

last: Suzuki

state: Osaka

address: 1-4-3 Asahi-machi, Abeno-ku

department: Molecular Pathology

institute: Osaka Metropolitan University Graduate School of Medicine

summary: The gene expression profiling between Control and acetoaceto-o-toluidide exposure for 4 weeks in liver of NOG-TKm30 female mice transplanted with commercially available human hepatocytes.

overall_design: Female 8-week-old NOG mice expressing HSVtk mutant clone 30 cDNA under the control of mouse transthyretin gene enhancer/promoter (NOG-TKm30 mice) were transplanted with commercially available human hepatocytes. After 10 weeks (18-week-old), mice were treated with 0 or 0.3% acetoaceto-o-toluidide (AAOT) for 4 weeks. After treatment, mice were sacrificed, and removed livers. Control and AAOT treatment: 3 control female mice, 3 AAOT-treated female mice, respectively. Mixed 3 samples from each group for each RNA-seq.


title: Ribosome profiling of mitotic exponential cells expressing either 2XDbp1, 1XDbp1, or Ded1

first: Gloria

last: Brar

summary: We replaced the DED1 ORF with DBP1 and inserted this either twice or four times in a diploid cell. We determined that with twice as much DBP1 expressable, the same amount of protein was made as for DED1, and there were few changes in gene expression. The transcripts that were translated better with DED1 were ones with highly structured 5' leaders, consistent with Dbp1 acting as a less effective initiation helicase than Ded1.

overall_design: We inserted twice as many copies of DBP1 in the genome to match helicase levels and determine helicase specificity

first: Emily

last: Powers


title: Liver in NOG-TKm30 mice: Control vs acetoaceto-o-toluidide treatment

summary: The gene expression profling between Control and acetoaceto-o-toluidide exposure for 4 weeks in liver of NOG-TKm30 female mice.

overall_design: Female 22-week-old NOG mice expressing HSVtk mutant clone 30 cDNA under the control of mouse transthyretin gene enhancer/promoter (NOG-TKm30 mice) were treated with 0 or 0.3% acetoaceto-o-toluidide (AAOT) for 4 weeks. After treatment, mice were sacrificed, and removed livers. Control and AAOT treatment: 3 control female mice, 3 AAOT-treated female mice, respectively. Mixed 3 samples from each group for each RNA-seq.


title: Ribosome profiling of mitotic exponential cells expressing either Dbp1 or Ded1, at 30C or 37C

summary: We replaced the DED1 ORF with DBP1 and tested vegetative growth at 30C or 37C to determine helicase specificity and temperature-dependent effects. We found that DBP1 expressing cells do not downregulate all of the housekeeping genes that DED1 expressing cells normally down regulate at high temperature


title: Somatic mutations and DNA methylation identify a subgroup of poor prognosis within lower risk myelodysplastic syndromes

city: Paris

first: David

last: Rombaut

address: 22 rue Méchain

country: France

institute: Institut Cochin

type: Methylation profiling by genome tiling array

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

overall_design: Refer to individual Series


title: Characterization of the ocular inflammatory response to AAV reveals divergence by sex and age

city: Bristol

first: Alison

middle: Jane

last: Clare

address: University Walk

country: United Kingdom

institute: University of Bristol

summary: Recombinant adeno-associated virus (AAV) is the leading platform for gene therapy in ocular disease. Progress for gene therapy has been hindered by AAV-induced inflammation, which limits dose escalation and long-term efficacy. Characterisation of the ocular immune response to AAV in mice has been restricted to young animals and  demonstrate activation of microglia, antigen presentation and a dominant T cell lymphocyte infiltration. The extent of the inflammatory response alters with age and sex and these factors have not been fully represented in the pre-clinical development of ocular AAV gene therapies. Here, we intravitreally inject a null AAV2 vector in young (3-month), middle aged (9-month) and old (18-month) Cx3cr1-creER:R26tdTomato+/- mice of both sexes. Applying clinical imaging, flow cytometric analyses and bulk-sequencing of sorted resident microglia we interrogate the impact of sex and age on the longitudinal response of both microglia and infiltrating cellular response to AAV. Young animals have a dynamic response, with a peak in inflammation at D10-12 and signs of clinical resolution by D28. Despite similar kinetics in the inflammatory response between young male and females, sex differences are observed in the magnitude of the transcriptional response by microglia and the adaptive component of the infiltrating response. With age, the inflammation increases and persists after AAV2 injection. Based on the microglia transcriptional response to AAV, males maintain similar signature across age, although enhanced with increasing age. Contrary, females have greater divergence in their inflammatory response across age. Of particular note, old females have enriched cellular stress and inflammatory microglia gene signatures, with corresponding retinal degeneration.  These findings inform crucial sex and age differences for therapeutic application of ocular gene therapy. Our dataset highlight the need to further define these differences to appropriately tackle AAV immunogenicity for all populations.

overall_design:
To interrogate the effect of age and sex on the ocular immune response to recombinant AAV, AAV2 was delivered intravitreally (2 µL/eye; 1E10 gc/eye) to male and female Cx3cr1CreER:R26-tdTomato mice on a C57BL/6J background. Mice were aged 3m, 9m or 18m with n = 3 - 4 in each group (single sex).
Retinal microglia were sorted from naïve age and sex-matched samples and at D12 and D28 post AAV injection. Animals were injected accordingly so that samples were collected in the same period.

middle: J

first: Philip

last: Langer

first: A

last: Ward

first: Ying

last: Chan

middle: D

last: Dick

first: D

middle: A

last: Copland


title: Double Negative T Cells Downregulate Intrahepatic Inflammation and Ameliorate Hepatic Ischemia and Reperfusion Injury via CD39-adenosinergic axis [RNA-seq]

city: Beijing

first: Hua

last: Jin

address: Yong an Road 95

institute: Beijing Friendship Hospital

summary: Converted double negative T cells (cDNT) highly express CD39 and hydrolyze ATP into adenosine. ATP/adenosine derived purinergic signaling plays a critical role in immune regulation. However, the role and mechanisms of ATP/adenosine in cDNT is unclear.

overall_design: To investigate the role of purinergic signaling on cDNT, we treated DNT with or without ATPgs (a non-hydrolysable ATP analogue), adenosine, adenosine receptor A2a inhibitor (A2aRi) and performed mRNA transcriptome sequencing.

first: Mingyang

last: Li

first: Guangyong

last: Sun

first: Dong

last: Zhang


title: Exploring the Protective Role of Caffeine against Taraxacum-Induced Ribotoxic Stress: Insights into Autophagy and Mitochondrial Depolarization

city: Taipei

first: Zih-Syuan

last: Wu

address: No.161, Minquan E. Road 6th Sec., Neihu dist.

department: Biochemistry

country: Taiwan

institute: National Defense Medical Center

summary: Background: The ribotoxic stress response (RSR) is a pathway that gets activated when ribosomes get impaired, leading to disruptions in protein synthesis, increased inflammatory signaling, and cell death if left unresolved. Taraxacum can induce apoptosis-associated ribosomal RNA (rRNA) cleavage, however, the exact working mechanism of Taraxacum-induced rRNA cleavage remains unclear. Methods: The RNA integrity (RIN) value, 28S/18S ratio, mRNAs, and proteins were for the drug screening for Taraxacum-induced rRNA cleavage process. Flowcytometry analysis was to examine the effects on necrosis, apoptosis, ROS, and mitochondrial membrane potential of Taraxacum-induced cells. Results:  We first used the RIN value and 28S/18S ratio to confirm the integrity of experiments. Our RNA sequencing data showed that T. formosanum upregulated 893 genes and downregulated 509 genes and triggered hallmark genes of spliceosomes, TNF-α signaling via NF-κB, inflammatory response, and IL6-JAK-STAT3 signaling. Additionally, T. formosanum imbalanced the levels of ribosomal proteins of the large and small subunits. We found that caffeine was the only screening agent that could rescue the cleavage of 28S and 18S rRNA induced by T. formosanum. However, caffeine failed to rescue T. formosanum-targeted mRNAs when the RIN values were relatively lower. T. formosanum induced the N-terminal clipping of histone H3, which was observed not only in human HeLa cervical cancer cells but also in human Huh6 and HepG2 liver cancer cells. Conclusion: Our study revealed that caffeine could reverse the effects of T. formosanum on the reduction of autophagy and the disruption of mitochondrial membrane potential. However, caffeine could only change the populations of necrotic and apoptotic cells but not T. formosanum-induced cell death.

overall_design: In this study, we utilized RNA sequencing analysis to examine the effects of T. formosanum extract on gene expression in HeLa cells. HeLa cells were exposed to either 7 mg/ml of T. formosanum extract alone or in combination with 10 mM caffeine.

first: Chien-Jung

last: Lin

first: Shih-Ming

last: Huang

first: Shu-Ting

last: Liu

first: Teng-Wei

last: Chen


title: Higher-order spatial organization of the Deinococcus radiodurans chromosomes

first: binguang

last: ma

address: No.1,Shizishan Street, Hongshan

institute: Huazhong Agricultural University

summary: Radiation-resistant Deinococcus radiodurans is an extreme microorganism capable of withstanding high levels of ionizing radiation and chemical mutagens. It possesses remarkable DNA repair capabilities and serves as a model organism for studying stress resistance mechanisms. However, our understanding of the relationship between the biological characteristics of this species and its chromosomal 3D structure remains limited. In this study, we employed chromosome conformation capture and sequencing (3C-seq) technology to determine the 3D genome structure of D. radiodurans and to further investigate the changes of chromosome conformation induced by ultraviolet irradiation. We observed that the overall chromatin folding structure of the cells became much looser after UV irradiation, with smaller chromosomal interaction domains (CIDs) merging to form larger CIDs. Integrating transcriptomic data analysis, we found that the majority of upregulated differentially expressed genes were significantly enriched near specific CID boundaries. Additionally, we comprehensively elucidated that Dr_ebfC as a nuclear-associated protein, serves as a global regulatory factor in gene expression processes and may modulate transcriptional regulation by altering chromosomal structure, thereby influencing the physiological state of the bacterium. Overall, our study provides insights into the chromosomal conformational changes of D. radiodurans under different conditions, offering valuable resources for further understanding the molecular mechanisms underlying its extreme resistance.

overall_design: Chromosome conformation capture of Deinococcus radiodurans wt cells, UV irradiated cells and a deletion mutant (dr_0199)

first: Qintian

last: Qiu

first: Caiyun

first: Binguang

last: Ma


title: Targeting CDK7 function in the RNA Polymerase II transcription cycle in inflammatory arthritis [ChIP-seq]

first: Yurii

last: Chinenov

summary: Macrophages are main drivers of inflammation and tissue damage in autoimmune diseases including rheumatoid arthritis. The rate-limiting step for transcription of >70% of inducible genes in macrophages is RNA Polymerase II (Pol II) promoter-proximal pause release, yet, the specific role of Pol II pausing in inflammation or whether it can be modulated therapeutically is unknown. Genetic ablation of a pause-stabilizing negative elongation factor (NELF) in macrophages enhanced the transcriptional response of paused anti-inflammatory genes to LPS, followed by secondary attenuation of inflammatory signaling, and reduced severity of K/BxN-serum transfer (ST) arthritis in mice. To disrupt Pol II pause establishment pharmacologically, we employed covalent inhibitors of the TFIIH-associated cyclin-dependent kinase (CDK) 7, THZ1 and YKL-5-124. Both dramatically reduced Pol II pausing in murine and human macrophages, broadly suppressed induction of pro- but not anti-inflammatory genes, and rapidly reversed pre-established inflammatory macrophage polarization by TNF+IFNγ. In vivo, CDK7i ameliorated the acute K/BxN-ST and chronic progressive hTNF-transgenic arthritis in mice. Finally, CDK7i downregulated a pathogenic gene expression signature in synovial explants from arthritis patients. We propose that interfering with Pol II pausing by targeting CDK7 represents a novel therapeutic opportunity for inflammatory diseases.

overall_design:
Pol II chipSeq in WT and NELF-cKO  LPS-treated macrophages
Pol II and Ser5-P PolII ChIPseq in TNF-IFNg polarized  YKL-treated macrophages
NELF-E ChIPseq in YKL treated mouse macrophages
NELF-E ChIPseq in YKL treated human PBMC-derived macrophages
Pol2 chipseq in mouse macrophages treated with LPS, YKS or both

first: Xi

first: Gayathri

last: Shibu

first: Dinesh

last: Deochand

first: Nicole

last: Soussana

first: Inez

last: Rogatsky


title: A CRISPR/Cas9-based kinome screen identifies ErbB signaling as a new regulator in the regulation of naïve pluripotency and totipotency in humans

first: Chunsheng

last: Han

address: No.5, Yard 1, Beichen West Road, Chaoyang District, Beijing

institute: Institute of Zoology, Chinese Academy of Sciences

summary: Regulation of totipotency and naïve pluripotency in humans is crucial for early human embryo development. However, the mechanisms of naïve pluripotency and totipotency regulation in humans, especially the signaling pathways that are involved in these processes, remains largely unknown. We performed a CRISPR/Cas9-based kinome knockout screen to analyze the effect of disrupting 763 human kinases in regulating human naïve pluripotency. Further validation using small molecules revealed that inhibition of ErbB family promoted the transition of hEPSCs to human naïve pluripotent stem cells. More importantly, chemical inhibition of ErbB family also promoted induction of totipotent signatures in human pluripotent cells under different culturing conditions.

overall_design: A total of 9 samples were analyzed by bulk RNA sequencing, two biological replicates of each sample, which included primed, extended and naïve pluripotent stem cells with or without the treatment of Afatinib or Erlotinib for 7 days under respective culture conditions.

first: Jiayu

first: Jun

last: Xu

first: Xiwen


title: Somatic mutations and DNA methylation identify a subgroup of poor prognosis within lower risk myelodysplastic syndromes [array]

first: David

last: Rombaut

summary: Lower-risk (LR) myelodysplastic syndromes (MDS) are heterogeneous hematopoietic stem and progenitor disorders caused by early accumulation of somatic mutations in epigenetic factors and non-epigenetic factors that may produce convergent DNA methylation patterns driving specific gene expression profiles. The integration of genomic, epigenomic and transcriptomic profiling has the potential to spotlight distinct LR-MDS categories on the basis of pathophysiological mechanisms.

overall_design: DNA methylation profiling of 175 low-risk MDS patients, including 28 with rapid progression to leukemia, and 7 age-matched healthy controls.

first: Sarah

last: Sandmann

first: Tobias

last: Tekath

first: Simon

last: Crouch

first: Aniek

middle: O

last: de Graaf

first: Olivier

last: Kosmider

first: Magnus

last: Tobiasson

first: Andreas

last: Lennartsson

first: Bert

last: van der Reijden

first: Sophie

last: Park

first: Maud

last: D'Aveni

first: Borhane

last: Slama

first: Emmanuelle

last: Clappier

first: Pierre

last: Fenaux

first: Lionel

last: Adès

first: Arjan

last: Van de Loosdrecht

first: Saskia

last: M.C. Langemeijer

first: Argiris

last: Symeonidis

first: Jaroslav

last: Čermák

first: Claude

last: Preudhomme

first: Aleksandar

last: Savic

first: Ulrich

last: Germing

first: Reinhard

last: Stauder

last: Bowen

first: Corine

last: van Marrewijk

first: Elsa

last: Bernard

first: Alexandra

last: Smith

first: Daniel

last: Painter

first: Theo

last: de Witte

first: Julian

last: Varghese

first: Eva

last: Hellström-Lindberg

first: Martin

last: Dugas

first: Joost

last: Martens

first: Luca

last: Malcovati

first: Joop

last: Jansen

first: Michaela

last: Fontenay


title: RNA sequencing to compare gene expression in control and PKMzeta knockdown hepatic stellate cells

city: Austin

first: Ningling

last: Kang

state: MN

address: 801 16th Ave NE

institute: Hormel Institute

summary:
Primary human hepatic stellate cells (HSCs) isolated from healthy donors were purchased from Sciencell. HSC cells were transfected with viruses encoding control or PKMzeta shRNA and stimulated with TGFbeta1 (5 ng/ml) for 24 hours.
Cells were collected for RNA isolation and RNA sequencing. The goal of this study is to identify genes transcriptionally regulated by PKMzeta in HSCs.

overall_design: Comparative gene expression profiling by RNA-sequencing of HSC cells

first: Aurpita

last: Shaha


title: U6 snRNP inhibits the premature cleavage and polyadenylation of pre-mRNAs (RNA-Seq)

first: chengguo

last: yao

summary: To investigate the extent to which U6 AMO affects transcriptome.  we performed mRNA-seq in control, U1 and U6-AMO treated sample

overall_design: we performed polyA+ RNA-seq in control, U1 and U6-AMO treated sample in HeLa cells

first: Qiumin

last: Feng

first: Zejin

last: Lin

first: Danhui

last: Zhao

first: Congting

last: Ye

first: Chengguo

last: Yao


title: Sex specific gene expression differences in mouse pups from obese dams

city: Indianapolis

first: James

last: Ropa

state: IN

address: 950 W Walnut St

institute: Indiana University

summary: Maternal obesity is increasingly common and negatively impact offspring health. Children born to mothers with obesity are at higher risk of developing diseases associated with abnormalities within the hematopoietic system such as atypical immune profiles, hematopoiesis, and metabolic diseases such as type 2 diabetes. Interestingly, disease risks are often dependent on the offspring’s sex, suggesting sex-specific reprogramming effect of maternal obesity on offspring hematopoietic stem and progenitor cell (HSPC) function. However, the impact of maternal obesity exposure on offspring HSPC function is largely unknown, and the capability of HSPC to regulate offspring metabolic health has not been studied.  Here we examined differential transcriptomes of hematopoietic stem and progenitor cells from male and female pups (postnatal day 21) from dams given western diet or control diet. RNA-seq revealed inflammatory gene pathways in female MatOb offspring that potentially protect HSPC function.

overall_design: Bone marrow was harvested from male and female postnatal day 21 pups born to mothers that were fed a western diet or control diet. Lineage negative, Sca positive, Kit positive (LSK) cells were isolated using florescence activated cell sorting (FACS) and RNA was harvested for sequencing.

first: Kok

last: Lim Kua

first: Maegan

last: Capitano


title: The role of MLF1 on accessible chromatin in AC16 cell line

city: Shenzhen

first: Zhihua

last: Wang

address: No. 12, Langshan Road, Xili Street, Nanshan District, Shenzhen

institute: Fuwai Hospital Chinese Academy of Medical Sciences, Shenzhen

summary: Previous studies found that MLF1 mRNA levels tended to decline in aging hearts. Functional studies found that knockdown of MLF1 ameliorated the drug-induced cellular senescence phenotype. Here, we apply transcriptomics to explore the underlying molecular mechanisms.

overall_design: Cardiomyocyte AC16 cells were cultured in DMEM/F12 . DMEM/F12 supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin, and 100 μg/ml streptomycin. Medium without FBS was replaced before transfection. Negative control siRNA and Mlf1 siRNA were transfected into AC16 cells at ~70% confluence using jetPRIME® (Polyplus-transfection, NY, USA). After 12 h of transfection, fresh complete medium was replaced. Accessible chromatin mapping was performed using the ATAC-seq method as previously described. About 50000 AC16 cells were lysed in ATAC-seq lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.5% NP-40) and centrifuged for 10 min at 4°C. Then, nuclei were spun at 1500g for 5 min to remove the supernatant. Nuclei were then incubated with the Tn5 transposome and tagmentation buffer at 37 °C for 30 min using TruePrep® DNA Library Prep Kit V2 for Illumina (Vazyme, TD501-01). After DNA purification with the MinElute kit, and the PCR was performed to amplify the library for 12 cycles and libraries were purified with the MinElute kit. Amplified ATAC libraries were sequenced on the Illumina Novaseq 6000 platform.

first: Jian

last: Lv

first: Ningning

last: Guo

first: Yu

last: Fang

first: Zhenyi

first: Sai

last: Zeng

first: Qiuxiao

first: Lili

last: Li

first: Zhaoyang

last: Chen


title: Early transcriptional similarities between two distinct neural lineages during ascidian embryogenesis

city: Valbonne - Sophia-Antipolis

first: Kevin

last: Lebrigand

address: 660 route des lucioles

institute: IPMC/CNRS

summary: In chordates, the central nervous system arises from precursors that have distinct developmental and transcriptional trajectories. Anterior nervous system is ontogenically associated with ectoderm lineages while posterior nervous systems are associated with mesoderm. Taking advantage of the well-documented cell lineage of ascidian embryos, we addressed how early similarities in the transcriptional states of the different neural lineages become apparent. We performed single-cell RNA sequencing (scRNA-seq) analyses of hand-dissected neural precursor cells of these lineages, together with those of their sister cell lineages, with a high temporal resolution covering five successive cell cycles from 16-cell to neural plate stages. A transcription factor binding site enrichment analysis of neural specific genes at the neural plate stage revealed limited evidence for shared transcriptional control between the two neural lineages, consistent with their different ontogenies. Nevertheless, PCA analysis and hierarchical clustering showed that, by neural plate stages, the two neural lineages cluster together. Consistent with this, we identified a set of genes enriched in both neural lineages at the neural plate stage.

overall_design:
Hand-dissected single-cell neural lineage precursors between 16-cell stage and neural plate (at least 6 replicates per cell type)
Please note that the sample characteristics 'cell type' is based on the nomenclature of Conklin: "The organization and cell-lineage of the ascidian egg". J. Acad. Nat. Sci. Phila. 1905;13:1–119.

first: Richard

middle: R

last: Copley

first: Julia

last: Buttin

first: Marie-Jeanne

last: Arguel

first: Géraldine

last: Williaume

first: Pascal

last: Barbry

first: Clare

last: Hudson

first: Hitoyoshi

last: Yasuo


title: Plasma cell-free DNA hydroxymethylomes define disease state in EGFR-mutant non-small cell lung cancer

city: Chicago

first: Yong

last: Peng

state: IL

address: 929 E 57th Street

department: Department of Chemistry

institute: University of Chicago

summary: The epigenome of plasma cell-free DNA (cfDNA) has demonstrated promise as both a prognostic and predictive cancer biomarker in liquid biopsies. Currently, it remains unknown whether and why cfDNA 5-hydroxymethylcytosine  (5hmC) can identify disease state for non-small cell lung cancer (NSCLC). We profiled 5-hydroxymethylomes using the plasma cfDNA of 302 EGFR-mutant NSCLC patients with different disease states. We found NSCLCs were epigenetically heterogeneous among individuals, especially for cfDNA 5hmC on gene EGFR. The diversity of age, sex, race, smoking status, EGFR-mutation of patients increased the epigenetic heterogeneity of NSCLC, however, only smoking status shaped disease state-associated cfDNA 5hmC. More importantly, disease state-dependent and patients’ characteristics-independent cfDNA 5hmC can be linked to lung function and regulatory elements in human lung cells. Interestingly, although 5-hydroxymethylome heterogeneity of plasma cfDNA among NSCLC patients were detected substantially, cfDNA-5hmC-based machine learning model can accurately predict different disease state in NSCLC.

overall_design: We performed 5hmC-Seal for plasma cfDNA of 302 EGFR-mutant NSCLC samples which included 240 CONTROLLED and 62 UNCONTROLLED samples. “Controlled Disease” was defined as: no change of therapy within 30 days of sample collection. “Uncontrolled Disease” was defined as either untreated disease or requiring a change in therapy within 30 days of sample collection.

first: Jason

last: Karpus

first: Chuan

last: He

first: Christine

last: Bestvian


title: Transcriptome profiling of pathological cardiac hypertrophy

first: Zhihua

last: Wang

summary: Here,  we performed pathological cardiac hypertrophy transcriptome analyses by using heart tissue from Sham or TAC surgery to identify the charecteric in pathological cardiac hypertrophy.  In addition, through combined analysis with the aging-associated transcriptome to identify the similarity and difference between heart ageing and pathological cardiac hypertrophy.

overall_design: Wild-type male mice at 2-month of age  were raised in a stable environment (room temperature, 24 ± 3 °C; room humidity, 55 ± 5%) with a 12-h light/12-h dark cycle, and were fed normal chow, were randomly separated into sham and TAC groups, with at least 6 mice per group. Transverse aortic constriction (TAC) was performed as previously described with 27G needle. The mice were anaesthetized with inhalational anaesthetic and euthanized by cervical dislocation. Hearts were immediately dissected and stored at -80°C. Three independent biological replicate samples from Sham or TAC group were used for RNA extraction. RNA-seq was completed by Beijing Genomics Institution (BGI).

first: Jian

last: Lv

first: Ningning

last: Guo

first: Yu

first: Zhenyi

last: Zhao


title: Double Negative T Cells Downregulate Intrahepatic Inflammation and Ameliorate Hepatic Ischemia and Reperfusion Injury via CD39-adenosinergic axis

first: Hua

last: Jin


title: Double Negative T Cells Downregulate Intrahepatic Inflammation and Ameliorate Hepatic Ischemia and Reperfusion Injury via CD39-adenosinergic axis [ChIP-seq]

summary: Converted double negative T cells (cDNT) highly expressed ATP hydrolytic enzyme CD39 and hydrolyzed extracellular ATP (eATP) into adenosine. We found that adenosine upregulated the transcription factor FOXO1 expression in cDNT. CUT&Tag assay was performed to investigate the FOXO1 binding promoter sites in cDNT.

overall_design: Converted double negative T cells (cDNT) were stimulated with 400µM adenosine with anti-CD3/CD28 antibody treatment in vitro for 6 hours. FOXO1 antibody, CUT & Tag Assay Kit were used to extract the binding site of FOXO1. Then the libraries were sequenced by Illumina Nova Seq 1.5 in Berry Genomics.

first: Mingyang

last: Li

first: Guangyong

last: Sun

first: Dong

last: Zhang


title: Pro-inflammatory Pathways Contribute to Pathogenesis of Clostridioides difficile Infection in a Murine Model - A Spatial Transcriptomics Study

status: Public on Jan 28 2025

city: West Lafayeete

first: Ahmed

middle: AbdelKhalek

last: Hassan

state: Indiana

address: 725 Harrison street, West Lafayette

institute: Purdue University

summary: Clostridioides difficile (C. difficile) is a common cause of antibiotic-induced diarrhea and causes the highest number of nosocomial infections. Only two antibiotics are currently recommended for treating C. difficile infection (CDI), which may contribute to unsatisfactory treatment outcomes and an increased likelihood of recurrence. In this study, we aim to evaluate the difference in gene expression between symptomatic and asymptomatic mice after infection with C. difficile using spatial transcriptomics analysis. We also aim to evaluate the spatial aspect of altered genes between different layers of intestinal mucosa (superficial vs deep) and identify the key pathways. Formalin-fixed paraffin-embedded (FFPE) intestinal sections were utilized for analysis using NanoStringTM platform to evaluate differential gene expressions in the caecum and colon. The IL-17 pathway, including Lcn2, Cxcl2, and S100a8 genes, was significantly upregulated in symptomatic mice. The IL-17 signaling pathway activated downstream signaling through NF-κB and MAPK pathways. Gene expression was significantly altered between the intestinal superficial and deep mucosal layers, highlighting layer-specific differences in gene expression patterns in the intestines of symptomatic and asymptomatic mice. Gene expression patterns in the enteric mucosa explained several clinical signs and lesions in CDI mice.

overall_design: Mice were infected with C. difficile spores and let to develop the disease symptoms. Mice were divided into groups based on the outcome of the infection. Groups included symptomatic and asymptomatic mice. Also, gene expression was measured in different layers of the intestine, superficial, middle and deep.

first: Niloufar

last: Ghahari

last: AbdelKhalek

first: Sanjeev

last: Narayanan

first: Deepti

last: Pillai


title: Effect of depletion of dr_0199 on gene experssion in Deinococcus radiodurans

city: Wuhan

first: binguang

last: ma

summary: The Dr_ebfC protein in D. radiodurans, encoded by the dr_0199 gene, was initially characterized as a NAP with DNA-protective and histone-like properties, involved in safeguarding the DNA from damage. To study the role of this protein in chromosomal organization and transcriptional regulation, we constructed dr_0199 deletion mutant in D. radiodurans. The mutant strain was viable, but compared with wild-type cells, the growth rate was moderately inhibited and the cell density during the stationary phase was lower than that of the wild-type. Through RNA-seq analysis of the mutant strain, we identified a total of 1077 differentially expressed genes, with 636 upregulated genes and 441 downregulated genes compared to the wild-type. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that the upregulated genes in the mutant were mainly associated with environmental stress response, DNA recombination, regulation of metabolic activities, catalytic activities, and DNA binding processes. Notably, several DNA damage repair-related genes (e.g., ddrA, ddrB, ddrC, ddrD, pprA) regulated by PprI/DdrO proteins showed significantly increased expression levels in the mutant. This observation suggests that the absence of the Dr_ebfC protein might lead to cellular stress, implying a potential role of this NAP in DNA protection. Enrichment analysis of the downregulated genes in the mutant revealed their involvement in vital cellular processes closely related to respiration, translation, RNA metabolism, and electron transfer, corroborating the observed slow growth phenotype of the mutant.

sample_organism: Deinococcus radiodurans R1 = ATCC 13939 = DSM 20539

overall_design: Gene expression profiles of D. radiodurans R1 wild type and ΔDr_ebfC strains were generated by deep sequencing using MGISEQ-2000 PE150.

first: Qintian

last: Qiu

first: Binguang

last: Ma


title: Establishment and characterization of a new Pseudomonas aeruginosa infection model using 2D airway organoids and dual RNA sequencing

city: Utrecht

first: Gijs

middle: Jacob Fransiscus

last: van Son

address: Heidelberglaan 25

country: Netherlands

institute: Prinses maxima centrum

summary: Pseudomonas aeruginosa is a Gram-negative bacterium that is notorious for infections in the airway of cystic fibrosis (CF) subjects. Often, these infections become chronic, leading to higher morbidity and mortality rates. Bacterial quorum sensing (QS) coordinates the expression of virulence factors and the formation of biofilms at a population level. QS has become the focus of attention for development of alternatives to antimicrobials targeting P. aeruginosa infections. However, a better understanding of the bacteria-host interaction, and the role of QS in infection, is required. In this study, we set up a new P. aeruginosa infection model, using 2D airway organoids derived from healthy and CF individuals. Using dual RNA-sequencing, we dissected their interaction, focusing on the role of QS. As expected, P. aeruginosa induced epithelial inflammation. However, QS signaling did not affect the epithelial airway cells. The epithelium influenced several infection-related processes of P. aeruginosa, including metabolic changes, induction of type 3 and type 6 secretion systems (T3SS and T6SS), and increased expression of antibiotic resistance genes, including mexXY efflux pump and several porins. Interestingly, the epithelium influenced the regulation by QS of the type 2 (T2SS) and T6SS. Finally, we compared our model with in vivo P. aeruginosa transcriptomic datasets, from samples directly isolated from the airways of CF subjects. This shows that our model recapitulates important aspects of in vivo infection, like enhanced denitrification, betaine/choline metabolism, increased antibiotic resistance, as well as an overall decrease of motility-related genes. This relevant infection model is interesting for future investigations, helping to reduce the burden of P. aeruginosa infections in CF.

overall_design: To investigate the interaction of P.aeruginosa and human airway epithelium, we established a bacteria co-culture system with human airway organoids and characterized it by dual RNA-seq. As part of the study, we included bacteria-only samples used as control. These bacteria-only samples are submitted here.

first: Cayetano

last: Pleguezuelos-Manzano

first: Wouter

last: Beenker

first: Harry

last: Begthel

first: Gimano

last: Amatngalim

first: Jeffrey

last: Beekman

first: Hans

last: Clevers

first: Jeroen

last: den Hertog


title: Serum and Tear Autoantibodies from NOR Mice as Potential Diagnostic Indicators of Local and Systemic Inflammation in Sjögren's Disease [MCF_SH_292_Slide1_532]

city: Dallas

first: Chengsong

last: Zhu

address: 5323 Harry Hines Blvd

department: Immunology

institute: UT Southwestern Medical Center

summary: Male Non-Obese Diabetic (NOD) derived NOR mice develop autoimmune dacryoadenitis by 16 weeks of age including lymphocytic infiltration of the lacrimal glands, reduced tear production, and increased cysteine tear proteases. Unlike the NOD mice, the NOR mice do not develop diabetes and studies designed with the NOR mice are expected to not have diabetes development counfounding the results observed for Sjogren's development. Autoantigens with high levels of reactivity in serum and/or tears of diseased mice may have diagnostic potential.  Serum was collected from mice at 16 weeks old male NOR mice along with age matched male healthy BALBc controls. Approximately 0.8 mL of blood was collected by cardiac puncture into serum separator tubes. Following cold centrifugation, serum was collected. Tears were collected following topical stimulation of the lacrimal gland with carbachol. For tear samples, tears were pooled from 1, 2 or 3 mice, respectively to generate three samples. This was done mainly to optimize the number of mice required to obtain sufficient sample for the assay as well as to assess the sensitivity of mouse tears for the proteinarray. 1 to 10 uL of tear or 10 uL of serum per sample was used to determine reactivity against 94 autoantigens using proteinarray.

sample_organism:
Mus musculus
blank sample

overall_design: Tear: male NOR (16 weeks, N=3), male BALB/c (16 weeks, N=3). Serum: male NOR (16 weeks, N=6), male BALB/c (16 weeks, N=3)

first: Yaping

last: Ju

first: Maria

last: Edman

first: Indu

last: Raman

first: J

last: Mackay

first: Prithvi

last: Raj

first: Sarah

last: Hamm-Alvarez


title: Gene expression profiling of HER2-positive breast cancer samples collected at baseline and at surgery from patients who did not achieve pathological complete response after trastuzumab-based neoadjuvant biochemotherapy.

city: Milan

first: Matteo

last: Dugo

address: via Olgettina 60

department: Department of Medical Oncology

institute: IRCCS Ospedale San Raffaele

summary: Transcriptomic analysis of a cohort of patients with HER2-positive breast cancer who did not achieve a pathological complete response after neoadjuvant treatment with trastuzumab plus chemotherapy. Matched samples were collected before the initiation of therapy (diagnostic biopsy) and at surgery (surgical sample).

overall_design: Gene expression profiling was performed using RNA from FFPE core biopsy from 32 patients with primary HER2+ BC before the initiation of neoadjuvant therapy and at surgery after neoadjuvant treatment

first: Serenella

last: Pupa

first: Lorenzo

last: Castagnoli

first: Loris

last: De Cecco


title: Multi-omic analysis of chronic myelomonocytic leukemia monocytes reveals metabolic and immune dysregulation leading to altered macrophage polarization [ChIPseq]

city: Manchester

first: Kiran

last: Batta

address: Paterson Building

department: Division of cancer Sciences

institute: The University of Manchester

summary: Chronic myelomonocytic leukemia (CMML) is a myelodysplastic/myeloproliferative overlap neoplasm with few treatment options and generally poor prognosis. A defining feature is expansion of classical monocytes, which are responsible for various clinical sequelae and thus represent viable therapeutic targets. Given the near-ubiquity of mutations involving epigenetic regulators, we profiled the epigenome (open chromatin configuration; H3K4-monomethylation; H3K27-acetylation; H3K27/H3K4-trimethylation) and transcriptome of primary CD14+ monocytes from CMML patients and age-matched healthy controls. Compared with healthy monocytes, CMML monocytes displayed heterogeneous but distinct epigenetic and transcriptome signatures. Most epigenetic changes mapped to distal elements, suggesting generalized enhancer dysregulation.  We observed strong epigenetic (but not transcriptional) suppression of the NF-κB pathway, suggesting poised potential to repress downstream targets and modulate inflammatory responses. RNA-seq revealed up-regulation of genes involved in the oxidative phosphorylation (OxPhos) machinery, with significantly heightened OxPhos activity of primary CMML monocytes validated in mitochondrial stress tests, highlighting a potentially tractable therapeutic vulnerability. In line with the observed metabolic phenotype of CMML monocytes, unpolarized CMML monocyte-derived macrophages exhibited transcriptional bias towards an M2 (“anti-inflammatory”) phenotype, reflected by an increased Th2/Th1 ratio in comparison with control monocytes. These observations may be germane to the observed clinical heterogeneity and prominent immune dysfunction characteristic of CMML.

overall_design: CD14-Positive monocyte were sorted from PBMNCs obtained from healthy controls (HV-X) and CMML patients (BC-XXX). Sorted monocytes were subject to ChIP-Seq for four histone modfifications.

first: Hasse

last: Addinsell


title: Multi-omic analysis of chronic myelomonocytic leukemia monocytes reveals metabolic and immune dysregulation leading to altered macrophage polarization [ATACseq]

overall_design: CD14-Positive monocyte were sorted from PBMNCs obtained from healthy controls(HV-X) and CMML patients(BC-XXX). Sorted monocytes were subject to ATAC-Seq


title: Controlling human stem cell-derived islet composition using magnetic sorting

city: St. Louis

middle: Robert

last: Millman

state: MO

address: 660 Euclid

institute: Washington University in St. Louis

summary: Stem cell-derived islets (SC-islets) hold significant promise for treating diabetes, but optimizing their functional maturity remains a challenge. CD49a, an integrin subunit involved in cell adhesion and signaling, has been identified as a potential marker associated with islet cell differentiation. In this study, we employed magnetic-activated cell sorting (MACS) to isolate CD49a-enriched and CD49a-depleted populations from SC-islets. These two distinct populations were evaluated for functional and molecular differences using single-cell RNA sequencing (scRNA-seq), in vitro glucose-stimulated insulin secretion (GSIS), and in vivo transplantation. Our scRNA-seq analysis revealed distinct transcriptional profiles between the CD49a-enriched and CD49a-depleted populations, with the CD49a-enriched population exhibiting higher expression of key β cell lineage markers, including insulin. Functional assays demonstrated that CD49a-enriched SC-islets had significantly improved GSIS, indicative of enhanced β cell functionality. Additionally, when transplanted into mice, the CD49a-enriched SC-islets exhibited superior graft performance, as evidenced by human C-peptide secretion. These findings support that CD49a enrichment through MACS represents a promising approach to enhance the functional maturity of SC-islets. Further investigations into the role of CD49a in islet cell differentiation and function may facilitate the development of more effective stem cell-based therapies for diabetes.

overall_design: Stem cell-derived islets were magnetically sorted using an anti-CD49a antibody using positive selection cell separation method. CD49a-enriched, CD49a-depleted, and unsorted cells were then allowed to aggregate for 7 days. After 7 days cells were single cell dispersed using TrypLE and submitted for single cell RNA sequencing at Washington University Genome Technology Access Center at the McDonnell Genome Institute.

first: Allison

last: Kelley

first: Mira

last: Shunkarova

first: Marlie

last: Maestas

middle: E

last: Gale

first: Nathaniel

last: Hogrebe


title: An ancient gene regulatory network sets the position of the forebrain in chordates

city: Cambridge

first: Elia

last: Benito Gutierrez

address: Downing Street

department: Zoology

institute: University of Cambridge

summary: The evolutionary origin of the vertebrate brain is still a major subject of debate. Its distinctive dorsal position and development from a tubular neuroepithelium are unique to the chordate phylum.  Conversely, apical organs (AO) are larval sensory/neurosecretory centers found in many invertebrate taxa, including in animals without a brain. Previous studies have shown that AOs are specified by a conserved set of genes under the influence of Wnt signalling. Although most of these genes are expressed in chordate nervous systems (including vertebrates), no AOs have ever been described in this group of animals. Here we have exploited single-cell genomic approaches to characterize cells showing AO profiles in sea urchin (ambulacrarian), amphioxus (invertebrate chordate) and zebrafish (vertebrate chordate). This, in combination with co-expression analysis in amphioxus embryos, has allowed us to identify an active and dynamic anterior Gene Regulatory Network (aGRN) in the three deuterostome species. We have further discovered that as well as controlling AO specification in sea urchin, this aGRN is involved in the formation of the hypothalamic region in amphioxus and zebrafish. Using a functional approach, we find that the aGRN is controlled by Wnt signalling in amphioxus, and that suppression of the aGRN by Wnt overactivation leads to a loss of forebrain cell types. The loss of the forebrain does not equate to a reduction of neuronal tissue, but to a loss of identity, suggesting a new role for Wnt in amphioxus in specifically positioning the forebrain. We thus propose that the aGRN is conserved throughout bilaterians and that in the chordate lineage was incorporated into the process of neurulation to position the brain, thereby linking the evolution of the AO to that of the chordate forebrain.

overall_design: Differential gene expression analysis between Azakenpaullone treated and untreated Brachiostoma lanceolatum embryos at 12 and 21 hours post fertilistaion (hpf) - 3 biological replicates per group

last: Benito-Gutiérrez

first: Giacomo

last: Gattoni

first: Daniel

last: Keitley

first: Ashley

last: Sawle


title: Estrogen-induced NETs promote aseptic inflammation via NKCC1 [human]

city: Nanjing

first: Dijun

last: Chen

address: Xianlin

department: Life Sciences

institute: Nanjing University

summary: Although the formation of neutrophil extracellular traps (NETs) is caused by inflammation-related factors, it remains unclear whether endogenous hormones promote NET formation. Here, we investigate NET formation between infection-driven inflammatory endometrium and estrogen-induced hyperplastic endometrium by single-cell multiomics analysis. We identified a unique neutrophil subpopulation (CD24high neutrophil) involved in estrogen-driven NET formation. Estrogen-induced NETs mainly form due to the imbalance of histone caused by estrogen receptors. Inhibition of NETs significantly ameliorated endometrial hyperplasia (EH) in a murine model. Mechanistically, NETs promote cell proliferation by binding to NKCC1 on epithelial cells. Aspirin was screened to inhibit NET formation and alleviated EH in cynomolgus monkey. This study provides a novel nonhormone replacement therapy to treat patients with estrogen abnormalities by targeting NETs.

overall_design:
scRNA-seq of normal endometrium (7); scRNA-seq of endometrial hyperplasia (10); scRNA-seq of endometritis (7); scATAC-seq of normal endometrium (4); scATAC-seq of endometrial hyperplasia (2); scATAC-seq of endometritis (2); ST-seq of normal endometrium (1); ST-seq of endometrial hyperplasia (1).

**The submitter declares that raw data were not submitted due to patient privacy concerns**

first: Yang

last: Sun

first: Jiao

last: Qu

first: Wen

last: Fang


title: Comprehensive RNA-seq analysis in the prostate of benign prostatic hyperplasia (BPH) rats exposed to testosterone and Estradiol

city: GuiYang

first: XiaoHu

last: Tang

address: No. 83 Zhongshan East Road

institute: Guizhou Provincial People's Hospital

summary: Effects of excessive testosterone and estradiol on gene expression of rat prostate

overall_design: Comparative gene expression profiling analysis of RNA-seq data for BPH rat induced with excessive testosterone and estradiol

first: Xiaohu

first: Guangheng

last: Luo