title: Subclonal evolution revealed by single-cell profiles of expressed mutations identifies adaptation mechanisms to immunotherapy in melanoma [scRNA-seq]

city: Bethesda

first: Maxwell

last: Lee

state: MD

address: Building 37, Room 5120

department: CCR

institute: National Cancer Institute, NIH

summary: Intratumoral heterogeneity (ITH) arises from distinct subclonal expansion following genetic or epigenetic alterations, and profoundly influences how tumors response to their immune microenvironment. Tumor progression trees based on single-cell mutational profiles have made it possible to trace subclonal evolution; however, conventional tree-building methods can incorporate only a limited number of cells and mutations, restricting their application to larger single-cell data. To investigate the effect of ITH on the therapeutic response of melanoma, we have developed Trisicell (https://trisicell.rtfd.io), a computational toolkit for scalable inference of mutational ITH through assessment of single-cell genomic variant data. By applying Trisicell to genetically matched mouse melanoma datasets, we found that expressed mutations are sufficient to effectively drive subclonal evolution. On single-cell, full-length RNA sequencing data of mouse melanoma from preclinical immune checkpoint blockade (ICB) studies, the analysis showed that the subtree-seeding mutations in the trees identified distinct subclones associated with a specific developmental state and neural crest lineage markers. Using the tree to trace cell lineages, we found that neoantigens depleted by ICB were predominantly expressed in minor subclones, suggesting that post-treatment recurrence is driven by immunoediting. Moreover, these neoantigens were enriched with those derived from frameshift mutations and mutated nuclear genes. Importantly, recurrently mutated genes in ICB-responding human melanoma exhibited the same features. We next used Trisicell to analyze single-cell, full-length RNA data of brain metastases (BM) from melanoma patients treated with ICB, and discovered that relapsing BM from ICB-responding patients exhibited subclones that also expressed a higher fraction of frameshift mutations and were associated with elevated levels of infiltrated T cells. Notably, they also exhibited more mutated HLA genes and expressed high level of the novel immune checkpoint HLA-G, a putative local immunosuppressive mechanism. In summary, applying Trisicell to single-cell transcriptomic analyses allowed us to identify novel features of ICB-responding melanoma neoantigens and distinct mechanisms for adapting to immunotherapy at different sites. These results have important implications for both melanoma evolution and target identification for immunotherapies, including ICB and cancer vaccines. This is the first study to trace subclonal evolution of neoantigens at single-cell resolution, demonstrating that Trisicell and our datasets represent important resources for the field.

overall_design: 24 clonal sublines were generated from parental mouse melanoma line B2905. For each subline, 8 cells were sorted, and cDNA library was generated from each cell by Smart-seq2 protocol. The library were then submitted for sequencing.

first: Farid

last: Mehrabadi

first: Eva

last: Pérez-Guijarro

first: Kerrie

last: Marie

first: Salem

last: Malikic

first: Erfan

last: Azer

first: Howard

middle: H

last: Yang

first: Charli

last: Gruen

first: Chih

last: Wu

first: Welles

last: Robinson

first: Huaitian

last: Liu

first: Can

last: Kizilkale

first: Michael

last: Kelly

first: Christina

last: Marcelus

first: Cari

last: Smith

first: Sung

last: Chin

first: Jessica

last: Ebersole

first: Sandra

last: Burkett

first: Aydin

last: Buluc

first: Naema

last: Nayyar

first: Philipp

last: Hähnel

first: Magali

last: de Sauvage

first: Albert

last: Kim

first: Samuel

last: Markson

first: Ryan

last: Sullivan

first: Priscilla

last: Brastianos

first: Funda

last: Ergun

first: Glenn

last: Merlino

first: Chi-Ping

last: Day

first: S

last: Sahinalp


title: Effect of overexpression or knockdown of trophoblast-related gene GPRC5A on migration and invasion of esophageal squamous cell carcinoma

status: Public on Oct 24 2024

city: Hong Kong

first: Hongyu

last: Zhou

address: L10-51, No. 21 Sassoon Rd, Hong Kong

department: Clinical Oncology

country: China

institute: The university of Hong Kong

type: Expression profiling by high throughput sequencing

summary: Emerging evidence suggests that cancer cells possess the capacity to disseminate at early stages, preceding the development of conventional macro-metastases. Nevertheless, the mechanisms governing the seeding and transition of these early disseminated cancer cells (DCCs) into metastatic tumors remain largely unknown.  Strikingly, we showed that GPRC5A-overexpressing ESCC cells exhibited an enhanced capacity to effectively implant, survive, and persist, ultimately giving rise to macro-metastases in the lungs in vivo. Therefore, we try to find the potential mechanism of GPRC5A-involved tumor implantation mechanism via overexpressing or silencing GPRC5A in vitro.

overall_design: We established GPRC5A-overexpressing/Control KYSE30 cells and GPRC5A-Shcontrol/GPRC5A knockdown KYSE520 cells.


title: Bone-protective effects of neutralizing angiopoietin-like protein 4 monoclonal antibody in rheumatoid arthritis

city: Shenzhen

first: Wenxiang

last: Cheng

state: Guangdong

institute: Shenzhen Institutes Of Advanced Technology, Chinese Academy Of Sciences,

summary: Despite recent advances, rheumatoid arthritis (RA) patients remain refractory to therapy. Dysregulated overproduction of angiopoietin-like protein 4 (ANGPTL4) is thought to contribute to the disease development. ANGPTL4 was initially identified as a regulator of lipid metabolism, which is hydrolyzed to N-terminal and C-terminal (cANGPTL4) fragments in vivo. cANGPTL4 is involved in several non-lipid-related processes, including angiogenesis and inflammation. This study revealed that the level of ANGPTL4 was markedly elevated in the sera and synovial tissues from patients with RA versus controls. The administration of a neutralizing antibody against cANGPTL4 (anti-cANGPTL4 Ab) resulted in the inhibition of inflammatory processes and bone loss in animal models of collagen-induced arthritis and adjuvant-induced arthritis (AIA). Transcriptomic and proteomic profiling of synovial tissues from an AIA model indicated that the anti-cANGPTL4 Ab inhibited fibroblast-like synoviocyte (FLS) immigration and inflammatory-induced osteoclastogenesis. Mechanistically, the anti-cANGPTL4 Ab has been shown to inhibit TNF-α-induced inflammatory cascades in RA-FLS through the sirtuin 1/nuclear factor-κB signaling pathway. Moreover, the anti-cANGPTL4 Ab was found to block FLS invasion- and immigration-induced osteoclast activation. Collectively, these findings identify ANGPTL4 as a prospective biomarker for the diagnosis of RA, and targeting cANGPTL4 should represent a potential therapeutic strategy.

overall_design: Comparative gene expression profiling analysis on the RNA-seq data of rat synovial tissues of adjuvant-induced arthritis rat model (CON, AIA and Ab, n=5).

first: Liang

last: Li

first: Peng

last: Zhang

first: Liqing

last: Ke


title: SLC2A1-AS1 and CERS6-AS1 mediate the EMT-related gene expression and participate in the tumorigenesis and immune microenvironment response of mucoepidermoid carcinoma

city: Beijing

first: Yan-ping

last: Zou

address: Haidian District

institute: Peking university

type:
Non-coding RNA profiling by array
Expression profiling by array

summary: To further exploration of pathological progress in mucoepidermoid carcinoma, we have employed lncRNA microarray expression profiling and coding transcripts as a discovery platform to identify genes or lncRNAs with the potential to mediate the tumorigenesis of mucopidermoid carcinoma. Normal salivary glands were used as controls.

overall_design: Sample labelling and microarray hybridization were conducted following the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology).Tumor samples were compared to normal salivary gland samples.

first: Ruo-lan

last: Xiang


title: Evaluation of transcriptomic changes in a Neuro2a cell line under extreme stress

city: Tampa

first: Ashutosh

last: Dharap

state: FL

address: 4001 E Fletcher Ave

department: Molecular Medicine

country: USA

institute: University of South Florida

summary: Bulk poly(A) RNA-sequencing was performed to evaluate the transcriptomic profiles of Neuro2a cells exposed to extreme stress following thawing in liquid N2 storage. This RNA-seq experiment was conducted as a part of broader aim in our study to evaluate changes gene expression of stressed cells in the early stages of recovery at Passage 1 post-thaw (P1) and as cells progressed towards a healthy phenotype at Passage 6 post-thaw (P6). Briefly, RNA was extracted and purified from Neuro2a cells at P1 (n=4 for experimental; n=3 for controls) and at P6 (n=3/group); poly(A) RNA was isolated, fragmented, and converted into cDNA libraries. Libraries were sequenced on an Illumina NovaSeq 6000 instrument at an approximate depth of 40 million 150 bp paired-end reads per sample. Sequencing yielded an average of 38.99 million reads per sample with and average of 94.91% of reads having a quality score of at least 30 (Q30) and an average of 96.29% of reads mapping to the mouse reference genome.

overall_design: To evaluate the effects of cryostorage failure on cells, we developed a standardized model to recapitulate a liquid nitrogen storage depletion event and subsequent thawing of Neuro2a cells to temperatures of 4-10°C for approximately 24h and subsequently re-freezing and plating Neuro2a cells using a standard protocol. Gene expression was evaluated using RNA-seq at two timepoints: one at the early stage of recovery shortly after stressed cells regained rapid proliferative capacity (Passage 1) and one at a later stage of recovery when cells appeared phenotypically normal (Passage 6). Cells were collected alongside time-matched controls at each passage for poly(A) RNA-seq and comparative gene expression analysis was performed to identify differentially expressed genes.

first: Ankit

last: Patel


title: CAMKV geneknockown induces transcriptomic changes in neuroblastoma cell line NGP

city: Washington

first: Yang

last: Yu

state: DC

address: 111 Michigan Ave NW

institute: Children's national hospital

summary:
To investigate the effect of CAMKV knockdown on transcriptomic profiling in neuroblastoma  and delineate the role of CAMJV in neuroblastoma growth.
We knockdowned the gene exprssion of CAMKV in NGP cells and performed gene expression profiling analysis using data obtained from RNA-seq.

overall_design:
We compared the transcriptomic change between CAMKV knockdown groups and the control groups.  Differential gene expression (DGE) analysis of RNA-Seq was conducted with the R statistical package DESeq2 (v1.28.1).
We performed gene set analysis on DGEs by over-representation test on the ConsensusPathDB  web-portal. Genes detected in our experiments are selected on the background list.

first: Chunchao

first: Jianhua

last: Yang


title: Method of moments approach for generalized differential gene expression analysis (Perturb-seq)

city: San Francisco

first: Min Cheol

last: Kim

state: CA

address: 505 Parnassus Ave

institute: University of California, San Francisco

summary: Differential expression analysis of scRNA-seq data is central for characterizing how experimental factors affect the distribution of gene expression. However, distinguishing between biological and technical sources of cell-cell variability and assessing the statistical significance of quantitative comparisons between cell groups remains challenging. Here, we introduce memento, a tool designed to address these limitations by providing statistically robust and computationally efficient differential expression analysis of the mean, variability, and gene correlation from scRNA-seq. We applied memento to analyze 70,000 tracheal epithelial cells to identify interferon response genes with distinct variability and correlation patterns, 160,000 T cells perturbed with CRISPR-Cas9 to reconstruct gene-regulatory networks that control T cell activation, 1.2 million PMBCs to map cell-type-specific cis expression quantitative trait loci (eQTLs), and arbitrary cell groups within the entire 50 million cell CELLxGENE Discover data corpus. In all cases, memento identified more significant and reproducible differences in mean expression but also identified differences in variability and gene correlation that suggest distinct transcriptional regulation mechanisms imparted by cytokines, genetic perturbations, and natural genetic variation. These results demonstrate that memento is a first-in-class method for the quantitative analysis of scRNA-seq data, scalable to millions of cells and thousands of samples.

overall_design:
CD4+ T cells were isolated Primary human CD4+ T cells were isolated from peripheral blood mononuclear cells, edited using SLICE protocol and analyzed by scRNA-seq.
Human tracheal epithelial cells were harvested from deceased organ donors according to established protocols (PMID: 1616056), stimulated by 4 different interferons followed by scRNA-seq.


title: RNA-SEQ raw data of apple phlomes at bud breaking stage

city: Qingdao

first: Yanjing

last: Shi

state: Shandong

address: 53# Zhengzhou Road

institute: Qingdao university of Science & Technology

summary: In order to understand the role of phloems of apple dwarfing rootstocks,and investigated the expression differences of dwarfing and vigorous apple stocks in the bud break stage, The phloem tissue at bud break stage(0 DABB(days after buds break) of three apple rootstocks including A1d（a partial GA insensitive mutant of Malus hupehensis ),WT Malus hupehensis and were QZ1(a hybrid of Malus hupensis and a Cylindrical apple variety) were sampled and underwent RNA-Seq analysis.

overall_design: in this study , the phlome of three types of apple rootstocks were sampled at bud break stage: A1d: the partial GA insensitive mutant of WT malus hupehensis；QZ1: it is a hybrid ,the mother was Malus hupehensis Rehd., and the father was a columnar apple line CO (Malus × domestica Borkh.). The QZ1 behaves as columnar and WT malus hupehensis. each with tree replicates.


title: The expression differences of a multibranching mutant (MB)of malus spectabilis"bly114"and its WT plant

summary: in order to  investigate the expression differences of WT and its multibranching mutant of   Malus spectabilis ‘Bly114’,the  young leaves ,new shoot stem and flower (not include petals，only receptacles)were sampled each with 3 replicates.MB is identified to be a strigolactone insensitive mutant and has high contents of cytokinin and abscisic acid.

overall_design: A more-branching mutant (MB) and its wild-type (WT) of ‘Bly114’ apple (Malus spectabilis) were obtained from apple germplasm nursery of the Institute of Agricultural Science of Qingdao (36◦ 240 N, 120◦ 580 E), China. The mutant was originally selected from a branch exhibiting natural variation on a WT plant. The scions of WT, MB,  weregrafted onto 2-year-old shoots of wild apple (Malus robusta) every year from 2014 to 2016. It must be stated that the growth and phenotypes of each genotype-maintained consistency both in Yangling and Qingdao. The grafted plants were cultivated in a field at Yangling (34◦ 520 N, 108◦ 700 E), Shaanxi, China, with periodic water, fertilizer, and pest management treatments. For details, nitrogenous fertilizer was applied at the end of February, and plant nutrient solution containing large and trace elements was applied twice every 15 days in May; imidacloprid was applied to controlled aphid every 10 to 15 days from March to May, and pyrethroids were applied to control leaf pests from May to July.

first: Hongjuan

last: Ge

first: Guofang


title: Tumor-derived small extracellular vesicle PD-L1 impedes macrophage-mediated antibacterial immunity in preexisting malignancy

city: Wuhan

first: He-jing

address: Guangba Rd.

institute: Wuhan university

summary: Tumor-derived small extracellular vesicles (TEVs) were reported to carry various immunosuppressive molecules regulating immune response. We previously reported that TEV could express PD-L1 to systematically inhibit CD8 T cell anti-tumor immunity. Here, we observed that TEV PD-L1 suppressed the phagocytosis and bactericidal capacity of macrophages against E. coli. To investigate the potential mechanisms behind the impaired phagocytosis further, we collected and purified exosomes from the melanoma cell line B16-F10 PD-L1 knockout (KO) or vector culture to challenge RAW264.7 cells. We used RNA sequencing to reveal the key functional associated molecules for inhibiting the phagocytosis-associated pathway.

overall_design: Comparative gene expression profiling analysis of RNA-sequencing (RNA-seq) data for B16 PD-L1 KO and its control exosomes treated LPS induced RAW264.7 cells.

first: Qi-hui

last: Xie


title: The expression differences in different kinds of 60 DABB(Days after buds break) apple rootstock phloem tissue

summary: in order to understand the role of phloems of apple dwarfing rootstocks,and investigated the expression differences of dwarfing and vigorous apple stocks in the phloem tissue at active growing stage. The phloem tissue at active growing stage(60 DABB(days after buds break) of three apple dwarfing rootstocks including M9,B9,A1d（a partial GA insensitive mutant of Malus hupensis）and two vigorous apple rootstock PYTC ( WT of Malus hupensis) and M. sylvestris were sampled and underwent RNA-Seq analysis.

sample_organism:
Malus sylvestris
Malus hupehensis
Malus pumila

overall_design: The phloem tissue at active growing stage(60 DABB(days after buds break) of three apple dwarfing rootstocks including M9,B9,A1d（a partial GA insensitive mutant of Malus hupensis）and two vigorous apple rootstock PYTC( WT of Malus hupensis) and M. sylvestris were sampled and underwent RNA-Seq analysis.


title: Local translation is engaged to sustain synaptic function  in impaired Wallerian degeneration

city: Onna, Kunigami District

first: Marco

last: Terenzio

state: Okinawa

address: 1919-1 Tancha

country: Japan

institute: Okinawa Institute of Science and Technology

summary: After injury, severed axons separated from their somas activate programmed axon degeneration, a conserved pathway to initiate their degeneration within a day. Conversely, severed projections deficient in programmed axon degeneration remain morphologically preserved with functional synapses for weeks to months after injury. How this synaptic function is sustained remains currently unknown. Here, we demonstrate that dNmnat-mediated over-expression attenuates programmed axon degeneration in distinct neuronal populations. Severed projections remain morphologically preserved for weeks. When evoked, they elicit a postsynaptic behavior which is a readout for preserved synaptic function. We used ribosomal pulldown to isolate translatomes from these projections. Transcriptional profiling revealed several enriched biological classes. Identified candidates were validated in a screen using a novel automated system to detect evoked antennal grooming as a proxy for preserved synaptic function. We used RNAi-mediated knockdown to identify mTOR as a mediator of protein synthesis, and candidates involved in protein ubiquitination and calcium homeostasis, required for preserved synaptic function. Our dataset uncovered several uncharacterized genes linked to human diseases. It may offer insights into novel avenues for therapeutic treatments.

overall_design: Ribosomal pulldowns were performed using GFP::Rpl10Ab, in three genotypes (background, wild type and over-expression of dNmnat, respectively) with two conditions (e.g., before and 7 days after injury).

first: Maria

last: Paglione

first: Leonardo

last: Restivo

first: Sarah

last: Zakhia

first: Arnau

middle: L

last: Rosell

first: Lukas

middle: J

last: Neukomm


title: Single-cell transcriptomics of pediatric Burkitt lymphoma

city: New York

first: Riccardo

last: Dalla-Favera

state: NY

address: 1130 St Nicholas Avenue

institute: Columbia University

summary: Burkitt lymphoma (BL) is the most frequent B-cell lymphoma in pediatric patients. While most patients are currently cured, a fraction of them are resistant to therapy. In order to investigate BL heterogeneity and the features distinguishing the non-responders (NR) to therapy, we analyzed pediatric EBV-negative BL specimens collected at diagnosis by single-cell (sc)-transcriptomics. Analysis of the non-tumor component revealed a predominance of immune cell infiltrates and a small representation of fibroblasts that was enriched in NR. Tumor cells displayed patient-specific features, as well as subpopulations that were common to most patients and expressed transcripts related to cell cycle, signaling pathways and cell-of-origin signatures.

overall_design: Paired single-cell transcriptomics and immunoglobulin repertoire analyses were performed on 12 sporadic EBV-negative BL specimens (11 patients), including 8 pleural or abdominal effusions and 4 nodal tumor masses, which were collected at diagnosis and frozen as viable single cell suspensions.

first: Katia

last: Basso

first: Antony

middle: B

last: Holmes

first: Lara

last: Mussolin


title: Single-cell RNA sequencing comparison of CD4+, CD8+ and T-cell receptor γδ+ cutaneous T-cell lymphomas reveals subset-specific molecular phenotypes

city: Vienna

first: Natalia

last: Alkon

address: Währinger Gürtel 18-20

department: Department of Dermatology

country: Austria

institute: Medical University of Vienna

summary:
Background: Malignant clones of primary cutaneous T-cell lymphomas (CTCL) can show a CD4+, CD8+ or T-cell receptor γδ+ phenotype, but their individual impact on tumor biology and skin lesion formation remains ill-defined. We perform a comprehensive molecular characterization of CD4+ vs. CD8+ and TCR-γ/δ+ CTCL lesions.
Methods: We performed scRNA-seq of 18 CTCL skin biopsies to compare classic CD4+ advanced-stage mycosis fungoides (MF) with TCR-γ/δ+ MF and primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma (Berti’s lymphoma).
Results: Malignant clones of TCR-γ/δ+ MF and Berti’s lymphoma showed similar clustering patterns distinct from CD4+ MF, along with increased expression of cytotoxic markers such as NKG7, CTSW, GZMA, and GZMM. Only advanced-stage CD4+MF clones expressed central memory T-cell markers (SELL, CCR7, LEF1), alongside B1/B2 blood involvement, whereas TCR-γ/δ+ MF and Berti’s lymphoma harbored a more tissue-resident phenotype (CD69, CXCR4, NR4A1) without detectable cells in the blood. CD4+ MF and TCR-γ/δ+ MF skin lesions harbored strong type 2 immune activation across myeloid cells, while Berti’s lymphoma was more skewed towards type 1 immune responses. Both CD4+ MF and TCR-γ/δ+ MF lesions showed upregulation of keratinocyte hyperactivation markers such as S100As and KRT16 genes. This increase was entirely absent in Berti’s lymphoma, possibly reflecting an aberrant keratinocyte response to invading tumor cells, that could contribute to the formation of the typical ulcero-necrotic lesions within this entity.
Conclusions: Our scRNAseq profiling study reveals specific molecular patterns associated with distinct CTCL subtypes.

first: Sumanth

last: Chennareddy

first: Katharina

last: Rindler

first: John

middle: R

last: Ruggiero

first: Emry

last: Cohenour

first: Sophia

last: Tran

first: Wolfgang

last: Weninger

first: Johannes

last: Griss

first: Constanze

last: Jonak

first: Patrick

middle: M

last: Brunner


title: CAMKV inhibitor, OTSSP167 induces transcriptomic changes in NGP cells

first: Yang

last: Yu

summary:
To investigate the effect of CAMKV inhibitor, OTSSP167, on NB cell proliferation and delineate the mechanisms of CAMKV-mediated  pathway inhibition in NB.
We treated human neuroblastoma cell line, NGP with OTSSP167 and performed gene expression profiling analysis using data obtained from RNA-seq.

overall_design:
We compared the transcriptomic change between OTSSP167-treated groups and the control groups.  Differential gene expression (DGE) analysis of RNA-Seq was conducted with the R statistical package DESeq2 (v1.28.1).
We performed gene set analysis on DGEs by over-representation test on the ConsensusPathDB  web-portal. Genes detected in our experiments are selected on the background list.

first: Chunchao

last: Zhang

first: Jianhua

last: Yang


title: MBNL splicing factors regulate the microtranscriptome of skeletal muscles [RNA-Seq]

city: Poznan

first: Michał

last: Szcześniak

address: Uniwersytetu Poznanskiego 6

department: Institute of Human Biology and Evolution

country: Poland

institute: Adam Mickiewicz University

summary: Muscleblind-like proteins (MBNLs) regulate various RNA-processing steps, including alternative splicing, polyadenylation, RNA stability, and mRNA intracellular localization. In myotonic dystrophy type 1 (DM1), the most common muscular dystrophy in adults, MBNLs are sequestered on toxic RNA containing expanded CUG repeats, which leads to disruption of MBNL-regulated processes and disease features of DM1. Herein, we showed the significance of MBNLs in the regulation of microtranscriptome dynamics during postnatal development of skeletal muscles and in microRNA (miRNA) misregulation observed in mouse models and patients with DM1. We identified multiple miRNAs sensitive to insufficiency of MBNL proteins and revealed that many of them were postnatally regulated, which was correlated with increases in the activity of these proteins during this process. In adult Mbnl1-knockout mice, miRNA expression exhibited an adult-to-newborn shift. We identified two mechanisms through which MBNLs influence miRNA levels. First, MBNL loss induces transcriptional changes in miRNA precursors. Second, MBNLs affect miRNA biogenesis by regulating the alternative splicing of miRNA primary transcripts.

overall_design: To assess the global impact of MBNLs on the skeletal muscle microtranscriptome, we sequenced a total RNA, with rRNA depletion, isolated from the quadriceps of 12-week-old HSALR mice (3xWT, 3xHSALR)


title: MLKL associated with angiogenesis and osteolysis in periapical lesions

city: Hefei

first: Tingyue

last: Zhou

address: Huangshan road

institute: USTC

summary: OBJECTIVES: To detect the expression levels of MLKL and p-MLKL, and explore its potential roles in inflammatory cell infiltration, angiogenesis, and bone destruction, in human and mouse periapical lesions.   METHODS: Forty-six human periapical tissues, including periapical granulomas (PGs, n =26), radicular cysts (RCs, n =20), and eight healthy control tissues were collected. Samples were fixed and analyzed by HE staining, RNA-Seq, western blot, and immunohistochemical staining. A periapical lesion mouse model was induced by pulp exposure in the first lower molars of 15 C57BL/6J mice. After lesion induction, the mice were sacrificed on days 0, 21, and 35. Mandibles were harvested for microcomputed tomography scanning, histologic observation, immunohistochemistry, enzyme histochemistry, and double immunofluorescence analysis. Double immunofluorescence was utilized to assess the colocalization of phosphorylated MLKL (p-MLKL) with CD34, matrix metalloproteinase-9 (MMP-9), and Cathepsin K (CTSK) in human and mouse periapical lesions.  RESULTS: RNA-Seq analysis showed that, in comparison with healthy gingiva tissues, MLKL was more significantly upregulated in periapical lesions (P-value < 0.05). Immunohistochemistry staining showed that, MLKL and p-MLKL were significantly overexpressed in the RC and PG groups compared with the control group (P-value < 0.05). However, the difference between the RC and PG groups was insignificant (P-value > 0.05). p-MLKL-positive cells were mainly lymphocytes, epithelial cells, and endothelial cells around the vascular wall. In mouse periapical lesions, the expression levels of p-MLKL were positively correlated with the bone defect area and tartrate-resistant acid phosphatase-positive (TRAP+) cell amounts (R2=0.4108, P-value < 0.05; R2=0.5668, P-value < 0.05, respectively). The double-labeling analysis showed that p-MLKL colocalized with CD34 and MMP-9 in human samples, and with CTSK adjacent to the bone in mouse periapical lesions. CONCLUSION: MLKL and p-MLKL were overexpressed in human periapical lesions. p-MLKL exhibited a close relationship with angiogenesis and alveolar bone resorption.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for periapical inflammatory lesions and healthy gingiva tissues

first: Yanqing

last: Wang


title: Expression profiling of fresh, DMSO or UM171 expanded CD34+ cells by high throughput sequencing

city: Montreal

first: Guy

last: Sauvageau

state: QC

address: 2950, Chemin de Polytechnique

country: Canada

institute: Institute for Research in Immunology and Cancer

summary: Ex-vivo expansion of hematopoietic stem cells (HSCs) is gaining in importance for cell and gene therapy and requires a shift from dormancy state to activation and cycling. However, abnormal or excessive HSC activation results in reduced self-renewal ability and increased propensity for myeloid-biased differentiation. To date, the threshold of HSC activation that marks “the point of no return” is undetermined. Identifying the molecular determinants that restrain or control HSC activation is essential for preventing accelerated exhaustion of cultured-HSCs. We now report that activation of the E3 ligase complex CRL3KBTBD4 by UM171 not only induces epigenetic changes through CoREST1 degradation but also control chromatin bound MYC levels to prevent excessive activation and maintain lympho-myeloid potential of expanded populations. Furthermore, reconstitution activity and multipotency of UM171-treated HSC is specifically compromised when MYC levels are experimentally increased despite degradation of CoREST1.

first: Jalila

last: Chagraoui

first: Simon

last: Girard

first: Nadine

last: Mayotte

first: Maria

middle: F

last: Tellechea


title: Long Noncoding VIM-AS1: Biomarker of Breast Fibrosis Susceptibility After Radiation Therapy and Promoter of Transforming Growth Factor Beta1–Driven Fibrosis

city: Evry

first: Nicolas

last: FORTUNEL

address: 2 Rue Gaston Cremieux

country: France

institute: CEA

summary:
Purpose: Fibrosis is a common late complication of radiation therapy. Molecular dysregulations leading to fibrosis have been characterized for the coding part of the genome, notably those involving the TGFB1 gene network. However, because a large part of the human genome encodes RNA transcripts that are not translated into proteins, exploring the involvement of the noncoding part of the genome in fibrosis susceptibility and development was the aim of this work.

Methods and materials: Breast cancer patients having or not having developed severe breast fibrosis after radiation therapy were retrospectively selected from the COPERNIC collection. Exome sequencing and RNA-seq transcriptomic profiling were performed on 19 primary dermal fibroblast strains isolated from the patients' nonirradiated skin. Functional experiments were based on fibrogenic induction by transforming growth factor-Beta1 (TGFB1) and gene knockdown in healthy donor fibroblasts.

Results: Coding and noncoding transcriptomes discriminated fibrosis from nonfibrosis conditions, and a signature of breast fibrosis susceptibility comprising 15 long noncoding RNAs (lncRNAs) was identified. A hazard ratio validation showed that the lncRNA vimentin antisense long noncoding RNA 1 (VIM-AS1) was the best biomarker associated with fibrosis risk. This lncRNA has not been previously associated with any fibrotic disorder, but we found it upregulated in data sets from cardiac fibrosis and scleroderma, suggesting a general role in tissue fibrosis. Functional experiments demonstrated a profibrotic action of VIM-AS1 because its knockdown reduced myofibroblast activation, collagen matrix production, and dermal organoid contraction. RNA-seq data analysis after VIM-AS1 silencing also pointed out the regulation of replication, cell cycle, and DNA repair. Mechanistically, because VIM-AS1 was found coregulated with the vimentin gene, these data support a profibrotic function of the TGFB1/VIM-AS1/vimentin axis, targeting the dynamics of fibroblast-myofibroblast transition.

Conclusions: Noncoding RNA analysis can provide specific biomarkers relevant to the prediction of normal tissue responses after radiation therapy, which opens perspectives of next-generation approaches for treatment, in the frame of the recent developments of RNA-based technologies.

overall_design: 19 breast cancer patients having (10 F) or not  (9NF) developed severe breast fibrosis after RT were retrospectively selected from the COPERNIC collection. RNA-seq transcriptomic profiling were performed on twelve primary fibroblast strains isolated from patient non-irradiated skin. Differential long non-coding RNAS (lncRNAs) were identified by bioinformatics. Functional experiments were based on fibrogenic induction by TGFB1 and gene knockdown in healthy donor fibroblasts. Additionally, to obtain a broader overview of genes affected by VIM-AS1 level, we performed a comparative transcriptomic analysis comparing 4 control (scrambled cells) versus 4 VIM-AS1 knocked-down skin fibroblasts, which had been treated by TGFB1 to initiate the activation of myofibroblasts

first: Tatiana

last: Vinasco-Sandoval

first: Michèle

middle: T

last: Martin

middle: O

last: Fortunel


title: Chromatin dysfunction facilitates stem cells aging in atypical laminopathy-based progeria Mandibuloacral dysplasia type A (MAD)

city: Guangzhou

first: Xinyi

last: Liu

address: No. 74, 2nd Zhongshan Road

institute: Sun Yat-Sen University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Nuclear lamins orchestrate the nuclear envelope architecture to maintain nuclear homeostasis, such as nuclear integrity and chromatin organization. The LMNA mutations lead to a variety of laminopathies, including progeroid syndrome. Other than typical Hutchinson-Gilford progeria syndrome (HGPS), the mechanism of LMNA mutation associated atypical progeria remains largely unexplored. We used patient-specific induced pluripotent stem cells (iPSCs) and their derivatives to remodel an LMNA (c.1579 C>T, p.R527C) mutation that causes Mandibuloacral dysplasia type A (MAD). Vascular lineages, including vascular smooth muscle cells (VSMCs) and endothelial cells (VECs), exhibited premature ageing phenotypes, such as genome instability and nuclear deformations, whereas neural lineages showed no obvious defects. Based on the observation that MAD-iPSCs derived mesenchymal stem cells (MSCs) also recapitulated accelerated senescence, an integrated multi-omics approach was adapted to reveal the underlying mechanisms of stem cells ageing. We found that among available human MSCs ageing models, MAD-MSCs had the highest transcriptional similarity to normal ageing hMSCs. The nuclear lamina-chromatin interaction was dramatically altered for both A and B type lamin-associating domains (LADs) as well as non-LADs binding sites, resulting in ageing-associated gene dysregulations. Chromatin state analysis further showed increased chromatin accessibility, enhancer remodelling, global chromatin compartments loss, topologically associating domains (TADs) and TAD boundaries reorganization in MAD-MSCs. These hierarchical chromatin reorganizations, coupled with gene dysregulation as consequences, expedited the MAD-MSCs ageing. Collectively, this study revealed a multilevel chromatin dysregulation contributed to ageing significantly in a laminopathy-mediated atypical progeroid syndrome.

overall_design: High-throughput profiling of gene expression, Lamins binding, histone modifications, 3D chromatin organizations in iPSC-derived human mesenchymal stem cells (hMSCs) of MAD patient or normal person.

first: Wei

last: Jin

first: Shaoshuai

last: Jiang

first: Junjun

last: Ding

first: Zhongjun


title: Systemic and transcriptional response to intermittent fasting and fasting-mimicking diet in mice

status: Public on Oct 23 2024

city: Graz

first: Jelena

last: Krstic

address: Neue Stiftingtalstrasse 6

department: Department of Cell Biology, Histology and Embryology

institute: Medical University of Graz

summary: Dietary restriction (DR) has multiple beneficial effects on health and longevity and can also improve the efficacy of certain therapies. Diets used to instigate DR are diverse and the corresponding response is not uniformly measured. We compared the systemic and liver-specific transcriptional response to intermittent fasting (IF) and commercially available fasting-mimicking diet (FMD) after short and long-term use in C57BL/6J mice. We show that neither DR regimen causes observable adverse effects in mice. The weight loss was limited to 20% and was quickly compensated during refeeding days. The slightly higher weight loss upon FMD versus IF correlated with stronger fasting response assessed by lower glucose levels and higher ketone body, free fatty acids, and especially FGF21 concentrations in blood. RNA sequencing demonstrated similar transcriptional programs in the liver after both regimens, with PPARα signalling as top enriched pathway, while on individual gene level FMD more potently increased gluconeogenesis-related, and PPARα and p53 target gene expression compared to IF. Repeated IF induced similar transcriptional responses as acute IF. However, repeated cycles of FMD resulted in blunted expression of genes involved in ketogenesis and fatty acid oxidation.  Short-term FMD causes more pronounced changes in blood parameters and slightly higher weight loss than IF, while both activate similar pathways (particularly PPARα signalling) in the liver. On individual gene level FMD induces a stronger transcriptional response, whereas cyclic application blunts transcriptional upregulation of fatty acid oxidation and ketogenesis only in FMD. Hence, our comparative characterization of IF and FMD protocols renders both as effective DR regimens and serves as resource in the fasting research field.

overall_design: organism: mus musculus; tissue type: liver, data type: RNA sequencing data, groups: ad libitum fed standard diet  (FED), intermittent fasted- food was withheld for 24h two times a week (IF), fed fasting-mimicking diet for three days (FMD)

first: Helene

last: Michenthaler

first: Kalina

last: Duszka

first: Isabel

last: Reinisch

first: Markus

last: Galhuber

first: Elisabeth

last: Moyschewitz

first: Sarah

last: Styreck

first: Tobias

last: Madl

first: Andreas

last: Prokesch


title: Effect of a CD28H agonistic monoclonal antibody on NK cells

city: Nantes

first: Richard

last: Danger

address: 30 Boulevard Jean Monnet

institute: CHU Nantes - INSERM- Nantes Universite

summary: Mature NK cell activation relies on a balance of activating, co-stimulatory and inhibitory receptors involved during target recognition. Immunotherapy strategies has been developed to finely tune this balance in order to enhance NK cell cytotoxic functions against cancer cells and secretory functions to enhance adaptative immunity activation. CD28H, a new member of the CD28 family, has been recently studied in T cells and on NK cells as a part of CD28H-CAR-NKL strategy. Given the impressive expression of CD28H molecule on circulating NK cells, we evaluated the use an anti-CD28H mAb on NK cell activation.

overall_design: RNAseq was performed on NK cells incubated overnight with anti-CD28H or the isotype control or untreated, during sixteen hours.

first: Pierre

last: Duplouye

first: Virginie

last: Huchet

first: Fabienne

last: Haspot


title: Single cell sequencing delineates T-cell clonality and pathogenesis of the parapsoriasis disease group

first: Natalia

last: Alkon

summary:
Background:
Mycosis fungoides (MF), the most common cutaneous T-cell lymphoma, is often underdiagnosed in early stages due to similarities with benign dermatoses such as atopic dermatitis (AD). Furthermore, the delineation from so-called “parapsoriasis en plaque,” a disease that can appear either in a small- or large-plaque form, is still controversial. Here, we characterize the parapsoriasis disease spectrum.
Methods:
We performed single-cell RNA sequencing of skin biopsies from patients within the parapsoriasis-to-early-stage MF spectrum, stratified for small and large plaques, and compared them to AD, psoriasis and healthy control skin.
Results:
6 out of 8 large-plaque lesions harbored either an expanded alpha/beta or gamma/delta T-cell clone with downregulation of CD7 expression, consistent with a diagnosis of early-stage MF. By contrast, 6 out of 7 small-plaque lesions were polyclonal in nature thereby lacking a lymphomatous phenotype, and also revealed a less inflammatory microenvironment than early-stage MF or AD. Of note, polyclonal small- and large-plaque lesions characteristically harbored a population of NPY+ innate lymphoid cells and displayed a stromal signature of complement upregulation and antimicrobial hyperresponsiveness in fibroblasts and sweat gland cells, respectively. These conditions were clearly distinct from AD or psoriasis, which uniquely harbored CD3+CRTH2+ IL13-expressing “Th2A” cells or strong type 17 inflammation, respectively.
Conclusion:
These data position polyclonal small- and large-plaque dermatitis lesions as a separate disease entity, that characteristically harbors a so far undescribed ILC population. We thus propose the new term “polyclonal parapsoriasis en plaque” to this kind of lesions, as they can be clearly differentiated from early and advanced-stage MF, psoriasis and AD on several cellular and molecular levels.

overall_design:
Comparison of skin cells obtained from AD, PP and MF patients.
Healthy Control samples from the trunk area 112 – HC1, 115 – HC2, 116 – HC3 and 121 – HC4 used in this manuscript, which are already publicly available via Gene Expression Omnibus GSE173205, were updated using the newer Cell Ranger version (v. 6.1.2; 10x Genomics) for consistency reasons:
AD samples 74 – AD1, 75 –  AD2, 77 –  AD3, 81 – AD4  and 96 – AD5 used in this manuscript are also a part of publicly available dataset (Gene Expression Omnibus GSE222840, manuscript "Single-cell RNA sequencing defines disease-specific differences between chronic nodular prurigo and atopic dermatitis")

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Submitter states that missing raw files are due to patient privacy concerns.
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first: Sumanth

last: Chennareddy

first: Emry

last: Cohenour

first: John

last: Ruggiero

first: Georg

last: Stingl

first: Christine

last: Bangert

first: Katharina

last: Rindler

first: Wolfgang

last: Bauer

last: Weninger

first: Johannes

last: Griss

first: Constanze

last: Jonak

first: Patrick

last: Brunner


title: Single-cell RNA sequencing reveals cellular heterogeneity of spinal microglia in neuropathic pain

city: Shatin

first: Yidan

last: ZOU

state: State

address: Rm902, LKS medical Sciences Bldg, PWH, Shatin , HK, 902

country: Hong Kong

institute: The Chinese university of Hong Kong

summary: Recent studies reveal that microglia modulate synaptic transmission by direct synaptic pruning. Microglia reactivation is a crucial mechanism of central sensitization in neuropathic pain, yet the exact changes of microglia during the development of neuropathic pain and its interaction with the spinal inhibitory circuits remains unclear. In this work, single-cell sequencing delineates temporal changes in spinal microglia and identifies a specific type of microglia as the subpopulation mediating synaptic pruning. We found that peripheral nerve injury induced the transition of spinal microglia from a pro-inflammatory to a “pruning” state,  resulting in pain hypersensitivity by spinal disinhibition.

overall_design: The ipsilateral SCDH portion at the lumbar (L) 4-L6 levels was incised from sham (PID 0) mice or SNI mice on PID 3, 7, and 14. The tissues collected were then fluorescence activated cell sorting (FACS)-sorted using the monocyte/ macrophage cell surface marker Cd11b. The sorted Cd11b+ cells were processed for single-cell RNA sequencing.


title: Beige Fat, Energy, and the Natriuretic Peptide System

city: NASHVILLE

first: Marisol

last: Ramirez

state: TENNESSEE

address: 2220 Pierce Avenue, 571 Preston Research Building

department: Center for Quantitative Sciences, Department of Biostatisticss

institute: Vanderbilt University Medical Center

summary: The purpose of this study is to learn how types of fat profiles differ between lean and obese individuals, and how they differ between racial groups. We are also interested in learning about the relationship of fat profiles with natriuretic peptide hormones, which are hormones produced by the heart. We will measure this by collecting tissue, blood and urine samples, energy expenditure measurements using the metabolic cart, and a DXA to assess the amount of bone, fat and muscle in the body from a single study visit.

overall_design: Blood, urine, and tissue samples will be coded for subject confidentiality.  These samples will be measured for a wide array of cardiometabolic parameters and inflammatory markers. Possible techniques include RNAseq and metabolomics techniques. Additional samples will be frozen and stored for possible future investigation.

first: Mashayekhi

last: Mona

first: Shah

last: Ravi

first: Quanhu

last: Sheng

first: Bachmann

last: Katherine


title: Protective role of aconitate decarboxylase 1 in neuroinflammation-induced dysfunctions of the paraventricular thalamus and sleepiness

city: Shenyang

first: Jianjun

last: Chang

state: Liaoning

address: No. 77 Puhe Road, Shenyang North New Area, China

institute: China Medical University

summary: Sleepiness is commonly associated with neuroinflammation; however, the underlying neuroregulatory mechanisms remain unclear. Previous research suggests that the paraventricular thalamus (PVT) plays a crucial role in regulating sleep‐wake dynamics; thus, neurological abnormalities in the PVT may contribute to neuroinflammation-induced sleepiness. To test this hypothesis, we performed electroencephalography recordings in mice treated with lipopolysaccharide (LPS) and found that the mice exhibited temporary sleepiness lasting for 7 days. Using the Fos-TRAP method, fiber photometry recordings, and immunofluorescence staining, we detected temporary PVT neuron hypoactivation and microglia activation from day 1 to day 7 post-LPS treatment. Combining the results of bulk and single-cell RNA sequencing, we found upregulation of aconitate decarboxylase 1 (Acod1) in PVT microglia post-LPS treatment. To investigate the role of Acod1, we manipulated Acod1 gene expression in PVT microglia via stereotactic injection of short hairpin RNA adenovirus. Knockdown of Acod1 exacerbated inflammation, neuronal hypoactivation, and sleepiness. Itaconate is a metabolite synthesized by the enzyme encoded by Acod1. Finally, we confirmed that exogenous administration of an itaconate derivative, 4-octyl itaconate, could inhibit microglia activation, alleviate neuronal dysfunction, and relieve sleepiness. In summary, our findings highlight PVT’s role in inflammation-induced sleepiness and suggest Acod1 as a potential therapeutic target for neuroinflammation.

overall_design: To assess the transcriptomic alterations induced by LPS in the PVT, we conducted RNA-seq analysis on the PVT tissue of mice. The samples were harvested at two time points: 24 hours post-intraperitoneal administration of PBS or LPS, and at seven days post-intraperitoneal LPS injection.

first: Zijie

last: Li

first: Hui

last: Yuan

first: Xuejiao

last: Wang

first: Jingyi

last: Xu

first: Pingting

last: Yang

first: Ling

last: Qin


title: Fingerprinting tertiary structure in complex RNAs using single-molecule correlated chemical probing

city: Chapel Hill

middle: Stephen

last: Irving

state: NC

address: 250 Bell Tower Dr.

department: Chemistry

institute: The University of North Carolina at Chapel Hill

summary: Single-molecule correlated chemical probing (smCCP) is an experimentally concise strategy for characterizing higher-order structural interactions in RNA. smCCP data yield rich, but complex, structural information on base pairing, conformational ensembles, and tertiary interactions. To date, through-space communication specifically measuring RNA tertiary structure has been difficult to isolate from structural communication reflective of other interactions. Here we introduce mutual information as a filtering metric to isolate tertiary structure communication contained within smCCP data and use this strategy to characterize the structural ensemble of the SAM-III riboswitch. We identified a smCCP fingerprint that is selective for states containing tertiary structure that forms concurrently with cognate ligand binding. We then successfully applied mutual information filters to independent RNAs and isolated through-space tertiary interactions in riboswitches and large RNAs with complex structures. smCCP, coupled with mutual information criteria, can now be used as a tertiary structure discovery tool, including to identify specific states in an ensemble that have higher-order structure. These studies pave the way for use of the straightforward smCCP experiment for discovery and characterization of tertiary structure motifs in complex RNAs.

overall_design: In-vitro transcribed SAM-III riboswitch constructs with structure cassettes, either native sequence or 1 of 4 structure-perturbing mutants, were refolded and structure was probed with DMS (or non-reactive control). Modified RNA was reverse transcribed under MaP conditions using SuperScript II. cDNA sequencing libraries were prepared and sequenced on an Illumina MiSeq instrument.

first: Ana

middle: C

last: Tan

middle: S

first: Jordan

last: Koehn

first: Shouhong

last: Jin

first: David

middle: Y

last: Qiu

first: Kevin

last: Weeks


title: Influence of Myrrh extract on gene expression in humen endometriotic cells.

city: Yangsan

first: Ki-Tae

last: Ha

address: Busandaehak-ro 49

country: South Korea

institute: Pusan National University

summary: To investigate the anti-endometriotic a pathways of Myrrh extract.

overall_design: Total RNAs of 12Z cells were used to identify differential gene expression following Myrrh stimulation

first: Dongryeol

last: Ryu

first: Yunju

last: Jo

first: Bo-sung

last: Kim


title: RNA-seq and m6A-seq analysis of the control-iBAT and M14KO -iBAT, and RNA-seq analysis for the control-hBAT and M14KO-hBAT

city: Chicago

first: Cheng-Wei

last: Ju

state: Illinois

address: 929 East 57th Street, GCIS, Room E313

institute: The University of Chicago

type: Methylation profiling by high throughput sequencing

summary: m6A mRNA methylation in brown adipose tissue regulates systemic insulin sensitivity.  We performed N6-methyladenosine (m6A) profiling and RNA seq of brown adipose tissue (BAT) from control and Mettl14-KO mice. Meanwhile, we performed RNA-seq for control and Mettl14-KO human BAT cell line as well.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design:
The experiment was performed in homozygous, male control, and M14 KO mice. All mice were fed with chow-diet for twenty-four weeks from four week of age. At the end of the experiment, mice were euthanized after overnight fasting, followed by serum and tissue sample collection.
hBAT cells were differentiated for 21 days, washed with PBS and cultured in 1% BSA containing high-glucose DMEM medium for 24 hours, and then collected cell pellet for RNA extraction.

last: Xiao

first: Rohit

middle: N

last: Kulkarni

first: Chuan

last: He


title: Multi-organ Gene Expression Dynamics and Network Modeling Reveals Sex-specific Shifts in Regulatory Control during the Development of Hypertension

city: Albuquerque

first: Rajanikanth

last: Vadigepalli

state: PA

address: 1 University of New Mexico

institute: University of New Mexico

summary: In this study we performed high throughput microfluidic qPCR using BioMark on RNA extracted from 5 organs (adrenal gland, kidney, liver, lung, and spleen) at 5 different time points throughout the development of hypertension (8, 10, 12, 16, and 24 weeks) in Spontaneously Hypertensive Rats (SHR) as compared to Wistar-Kyoto (WKY) control in female subjects (n=3).

overall_design: High throughput microfluidic qPCR using BioMark was performed on RNA extracted from 5 organs at 5 different time points throughout the development of hypertension (n=3). Two 96 x 96 BioMark Arrays were used to assay expression of 92 genes. A serial dilution gene set was included in each chip to confirm reproducibility. Technical replicates were run on each chip to evaluate technical variability.

first: Eden

last: Hornung

first: Sirisha

last: Achanta

first: Alison

last: Moss

first: James

last: Schwaber


title: Hypoxia is linked to acquired resistance to immune checkpoint inhibitors in lung cancer

city: New Haven

first: Camila

last: Robles-Oteiza

state: CT

address: 333 Cedar St

department: Yale Cancer Center

institute: Yale University

summary: Despite the established use of immune checkpoint inhibitors to treat non-small cell lung cancer (NSCLC), only a subset of patients benefit from treatment and approximately 50% of patients whose tumors respond eventually develop acquired resistance. To identify novel drivers of acquired resistance, we generated murine Msh2 knock-out (KO) lung tumors that initially responded but eventually developed acquired resistance to anti-PD-1, alone or in combination with anti-CTLA-4. Resistant tumors harbored decreased infiltrating T cells and reduced cancer cell-intrinsic MHC-I and MHC-II levels, yet remained responsive to IFNɣ. Resistant tumors contained extensive regions of hypoxia, and a hypoxia signature derived from single-cell transcriptional profiling of resistant cancer cells was associated with decreased progression-free survival in a cohort of NSCLC patients treated with anti-PD-1/PD-L1 therapy. Targeting hypoxic tumor regions using a hypoxia activated pro-drug delayed acquired resistance to immune checkpoint inhibitors in murine Msh2 KO tumors. Thus, this work provides rationale for targeting tumor metabolic features, such as hypoxia, in combination with immune checkpoint inhibition.

overall_design: Cells from Msh2 knock-out LKR13-H tumors were isolated by Fluorescence-activated cell sorting (FACS) and analyzed using scRNAseq


title: ANKRD1 promotes breast cancer metastasis by activating NF-κB-MAGE-A6 pathway

city: Bangkok

first: Penchatr

last: Diskul-Na-Ayudthaya

country: Thailand

institute: Chulabhorn Research Institute/Soonchunhyang Institute of MEDI-BIO science

summary: Early detection and surgical excision of tumors have helped improve the survival rate of 24 patients with breast cancer. However, patients with metastatic cancer have a poor prognosis. In 25 this study, we propose that ANKRD1 promotes metastasis of breast cancer. ANKRD1 was found 26 to be highly expressed in the MDA-MB-231 and MDA-LM-2 malignant metastatic breast cancer 27 cell lines compared to the non-metastatic breast cancer cells (MCF-7, ZR-75-30, T47D) and normal 28 breast cancer cells (MCF-10A). Furthermore, high-grade tumors showed increased levels of 29 ANKRD1 compared to low-grade tumors. Both in vitro and in vivo functional studies demon- 30 strated the essential role of ANKRD1 in cancer cell migration and invasion. The previous studies 31 have suggested an important role of NF-κB and MAGE-A6 in breast cancer metastasis, but the 32 upstream regulators of this axis are not well characterized. Our study suggests that ANKRD1 33 promotes metastasis of breast cancer by activating NF-κB as well as MAGE-A6 signaling. Our 34 findings show that ANKRD1 is a potential therapeutic target and a diagnostic marker for breast 35 cancer metastasis.

overall_design:
To elucidate the mechanisms of breast cancer metastasis. We established MDA-LM-2 cell line in which each target gene has been knocked down by shRNA
We then performed gene expression profiling analysis using data obtained from RNA-sequencing of 3 different cells.
Comparative gene expression profiling analysis of RNA-sequencing data for MDA-LM-2 cells and its knock down delivative (shANKRD1-14 and shANKRD1-49)
**Submitters state that raw data has been lost and thus is not included in the records***

first: Seongho


title: Transcriptome analysis of Apis mellifera antennae reveals molecular divergence underlying the division of labor in worker bees

first: Weixing

last: Zhang

address: 135 Xingang west road

institute: SUN YAT-SEN UNIVERSITY

summary: Olfaction system plays a fundamental role in mediating insect behavior. Besides, the division of queen, worker and drone, honeybee also exhibit an age-dependent division of labor. Worker bees perform discrete sets of behaviors throughout their lifespan. These behavioral states rely on the sense of the environments and chemical communications via their olfactory system - antennae. However, the olfactory adaption mechanism of workers in these processes of behavioral development is still unclear. In this study, we conducted a comprehensive and quantitative analysis of gene expression in Apis mellifera antenna of newly emerged workers, nurses, foragers, and defenders using RNA-seq. We found that antennae tissues continue to develop after transformation from pupae to adult. Additionally, we identified both developmental and labor-division specific expressed genes. We validated the unexpected discovery of major royal jelly protein genes, which are highly and specifically expressed in nurse honeybee workers. We further identified and validated that significant alternative splicing events are also involved in the development and division of labor. These findings provided a comprehensive transcriptome profile and new perspective into the molecular mechanism underlying honeybee division of labor.

overall_design: Genes expression profiles of Newly emerged workers, Nurse, Forager,and Defender were generated by RNA-seq using the DNBSEQ platform.

first: Bairu

last: Liu

first: Yicong

last: Xu

first: Jin


title: Targeted DamID Detects Cell Type Specific Histone Modifications in intact tissues or organisms

city: Cambridge

first: Andrea

last: Brand

address: Tennis Court Rd

country: United Kingdom

institute: The Gurdon Institute

summary: Histone modifications play a key role in regulating gene expression and cell fate during development and disease. Current methods for cell-type specific genome-wide profiling of histone modifications require dissociation and isolation of cells and are not compatible with all tissue types. Here we adapt Targeted DamID to recognise specific histone marks, by fusing chromatin binding proteins or single-chain antibodies to Dam, an E. coli DNA adenine methylase. When combined with Targeted DamID (TaDa), this enables cell-type specific chromatin profiling in intact tissues or organisms. We first profiled H3K4me3, H3K9ac, H3K27me3 and H4K20me1 in vivo in neural stem cells of the developing Drosophila brain. Next, we mapped cell-type specific H3K4me3, H3K9ac and H4K20me1 distributions in the developing mouse brain. Finally, we injected RNA encoding DamID constructs into 1-cell stage Xenopus embryos to profile H3K4me3 distribution during gastrulation and neurulation. These results illustrate the versatility of Targeted DamID to profile cell-type specific histone marks throughout the genome in diverse model systems.

sample_organism:
Drosophila melanogaster
Xenopus laevis
Mus musculus

overall_design: Targeted DamID recognising binding profiles of histone modifications was performed in Drosophila melanogaster 3rd instar larval neural stem cells, expressing Dam fusions with the GAL4 system; in mouse embryonic forebrain targeting radial glial cells (using Hes5 promoter), neurons (using Tα1 promoter) and intermediate progenitors (using ND1 promoter) with the use of inter-uterine electroporation to express Dam fusions; and in stage 11 and stage 21 (dorsal and ventral) Xenopus laevis embryos using mRNA injection to express Dam fusions. The histone marks tested were as follows: in Drosophila NSCs, H3K4me3 (with TAF3-phd domain), H4K20me1 (with H4K20me1-specific nanobody 15f11), H3K9ac (with H4K20me1-specific nanobody 19e5) and H3K27me3 (with Cbx7); in mouse RGCs H3K4me3, H4K20me1 and H3K9ac was tested with the same Dam-fusions. In Xenopus and mouse IPCs and neurons, only H3K4me3 was tested.

first: Jelle

last: van den Ameele

first: Manuel

last: Trauner

first: Eva

last: Hörmanseder

first: Alex P.

last: Donovan

first: Oriol

last: Llorà-Batlle

first: Seth W.

last: Cheetham

first: Robert

last: Krautz

first: Rebecca

last: Yakob

first: Anna

last: Malkowska

first: John B.

last: Gurdon

first: Andrea H.


title: Isolation and tracing of matrix-producing notochordal and chondrocyte cells using ACAN-2A-mScarlet reporter human iPSC lines

city: Utrecht

first: Frank

last: Riemers

address: Uppsalalaan 8

country: Netherlands

institute: Utrecht University

summary: The development of human induced pluripotent stem cell (iPSC)–based regenerative therapies is challenged by the lack of specific cell markers to isolate differentiated cell types and improve differentiation protocols. This issue is particularly critical for notochordal-like cells and chondrocytes, which are crucial in treating back pain and osteoarthritis, respectively. Both cell types produce abundant proteoglycan aggrecan (ACAN), crucial for the extracellular matrix. We generated two human iPSC lines containing an ACAN-2A-mScarlet reporter. The reporter cell lines were validated using CRISPR-mediated transactivation and functionally validated during notochord and cartilage differentiation. The ability to isolate differentiated cell populations producing ACAN enables their enrichment even in the absence of specific cell markers and allows for comprehensive studies and protocol refinement. ACAN’s prevalence in various tissues (e.g., cardiac and cerebral) underscores the reporter’s versatility as a valuable tool for tracking matrix protein production in diverse cell types, benefiting developmental biology, matrix pathophysiology, and regenerative medicine.

overall_design: ACAN-2A-mScarlet human iPSC reporter lines were used to investigate the efficiency of chondrogenic and notochordal differentiation. Cells were collected in the end of the differentiation and the mScarlet postive and negative cells were sorted by flowcytometry. Bulk RNA seqeuncing via CEL-seq2 was performed to characterize the different transcriptome of the mScarlet positive and negative cells.

first: Tong

last: Xiaole

first: Deepani

last: Poramba Liyanage

first: Marianna

last: Tryfonidou


title: Machine-learning Directed Conversion of Glioblastoma to Dendritic Cell-like Antigen Presenting Cells as Cancer Immunotherapy

city: Los Angeles

first: David

last: Tran

address: 1441 Eastlake Ave

department: Department of Neurological Surgery

institute: University of Southern California

summary: Here, we report the development and application of a machine-learning precision method to identify cell fate determinants (CFD) that specifically reprogram GBM into induced antigen-presenting cells with DC-like functions (iDC-APC).  In murine GBM models, iDC-APCs acquired DC-like morphology, regulatory gene expression profile and functions, including phagocytosis, direct presentation of endogenous antigens, and, especially, vigorous cross presentation of exogenous antigens comparable to natural DCs to prime naïve CD8+ CTLs, a hallmark DC function critical for anti-tumor immunity.

overall_design: ORF expression clones  were purchased from Genecopoeia. To create mF3, cDNAs encoding mouse IRF8, BATF3, and ID2 were synthesized, linked by P2A and T2A cleavage sequences, and cloned into the lentiviral vector pSF-simple (pSF-simple-mIRF8-P2A-mBATF3-T2A-mID2). For PU.1 constructs, cDNAs encoding mouse PU.1 (Spi1) and PU.1 delta the PEST domain (dP) (16) were cloned into the lentiviral response plasmid pSF-Lenti that contains PGK-puro to generate pSF-Lenti-PU.1 and pSF-lenti-detaPU.1.  Lentiviral  constructs were produced and used to reprogram KR158 cells. On day 5 after transduction, cells were collected and single cell RNAseq libraries were prepared using 10X single cell lib prep kit.   Each sample contained populations from different groups (to reduce the sequencing cost). Each individual group was annotated in downstream analysis by characteristic gene markers.

middle: D

first: Tianyi

last: Liu

first: Dan

last: Jin

first: Son

last: Le

first: Dongjiang

last: Chen

first: Mathew

last: Sebastian

first: Alberto

last: Riva

first: Ruixuan


title: Effect of GCN5 depletion in hepatocytes in NAFLD

city: 南京市

first: Jing

state: 江苏省

address: 南京市江宁区龙眠大道639号中国药科大学

institute: 中国药科大学

summary: Non-alcoholic fatty liver disease (NAFLD) is a burden of global health, yet the mechanism of the disease is not fully elucidated. General control non-repressed protein 5 (GCN5) is histone acetyltransferases. We invesgated the effect of GCN5 deletion on gene expression in a NAFLD cell model.

overall_design: To invesgate the effect of GCN5 depeltion on gene expression in hepatocytes. GCN5 knockout and wild-type HL-7702 cells were separately seeded into 6-well plates, treated with PAOA for 24 h, and harvested for RNA-seq.

first: Zu-Guo

last: Zheng

first: Ping

last: Li


title: 18S rRNA N6-methyladenosine (m6A) modification promotes tumorigenesis and chemoresistance via gain-of-function mutant p53 in nasopharyngeal carcinoma

first: Hao

last: Peng

address: Zhongshan 2nd Road, Guangzhou, 510080, China

department: Department of Breast Cancer

institute: Guangdong General Hospital

summary: Mis-regulated mRNA translation and hyperactivation of ribosome biogenesis are the two main hallmarks of cancer cells, whereas their intrinsic links and molecular mechanisms remain poorly understood. Our current study revealed that METTL5 and its mediated 18S rRNA N6-methyladenosine modification at 1832 position (m6A1832) are elevated in nasopharyngeal carcinoma (NPC) and correlated with disease progression. Gain-of-function and loss-of-function assays showed that METTL5 mediated 18S rRNA m6A1832 modification promotes NPC cells proliferation and metastasis in vitro and in vivo. Mechanistically, loss of 18S rRNA m6A1832 modification impairs the assembly of 80S ribosome and therefore affecting global mRNA translation. Furthermore, polyribosome-bound mRNA sequencing (Polyribosome-RNA-seq), ribosome profiling sequencing (Ribo-seq) and whole exome sequencing (WES) data identified a METTL5/HSF4b/HSP90B1/mutant p53 (mutp53) axis which contributed to NPC tumorigenesis and chemoresistance. Our findings uncovered a novel mechanism underlying rRNA epigenetic modification in regulating mRNA translation and mutp53 pathway in cancer.

overall_design: Polyribosome-bound-RNA-seq and Ribo-seq were used to study the differential translated genes in the METTL5 knockout and control cells.

first: Binbin


title: Microprotein-Encoding RNA Regulation in Cells Treated with Pro-inflammatory and Pro-Fibrotic Stimuli (RNA-Seq Caco2)

city: San Diego

first: April

middle: Elizabeth

last: Williams

state: California

address: 10010 N Torrey Pines Rd

department: IGC

institute: Salk Institute for Biological Studies

summary: Recent analysis of the human proteome via proteogenomics and ribosome profiling of the transcriptome revealed the existence of thousands of previously unannotated microprotein-coding small open reading frames (smORFs). Most functional microproteins were chosen for characterization because of their evolutionary conservation. However, one example of a non-conserved immunomodulatory microprotein in mice suggests that strict sequence conservation misses some intriguing microproteins. Here, we examine the ability of gene regulation to identify human microproteins with potential roles in inflammation or fibrosis of the intestine. To do this, we collected ribosome profiling data of intestinal cell lines and peripheral blood mononuclear cells (PBMCs), and then used gene expression of microprotein-encoding transcripts to identify strongly regulated microproteins, including several examples of microproteins that are only conserved with primates. This approach reveals a number of new microproteins worthy of additional functional characterization, and provides a dataset that can be queried in different ways to find additional gut microproteins of interest.

overall_design: RNA-sequencing of of Caco-2 cells with or without treatment of lipid/non-lipid micelles, and differentiated or undifferentiated

first: Victor

last: Pai

first: Calvin

last: Lau

first: Almudena

last: Garcia-Ruiz

first: Alan

last: Saghatelian


title: CSRP1 in stromal fibroblasts drives high-grade serous ovarian cancer development [ChIP-seq]

first: Qijun

last: Wu

address: No. 36, San Hao Street

institute: Shengjing Hospital of China Medical University

summary: ChIP-seq was performed to identify the target genes of YBX1 regulated by CSRP1 in cancer-associated fibroblasts in HGSOC

overall_design: We performed ChIP-seq analysis of primary human cancer-associated fibroblasts infected with lentiviral vectors expressing CSRP1 and empty vector control using anti-YBX1 antibodies. Cancer-associated fibroblasts were isolated from omentum tissues of HGSOC patients with omentum metastasis.

first: Tingting

last: Gong

first: Xue

last: Qin

first: Xiaobin

last: Wang

first: Yizi

first: Fanghua

first: Yifan

last: Wei

first: Heli

last: Xu

first: Jianfei

last: Pei

first: Ye

last: Kang

first: Yunlong

last: Huo

first: Si

first: Jinchi

first: Yue

last: Han

first: Fan

last: Cao

first: Donghui

last: Huang

first: Xiaoying

first: Lang

first: Meng

last: Niu

first: Huihan

first: Song

last: Gao


title: Microprotein-Encoding RNA Regulation in Cells Treated with Pro-inflammatory and Pro-Fibrotic Stimuli (Ribo-Seq Caco2)

first: April

last: Williams

overall_design: Ribosome profiling RNA-sequencing (ribo-seq) of Caco-2 cells with or without treatment of lipid/non-lipid micelles, and differentiated or undifferentiated

first: Victor

last: Pai

first: Calvin

last: Lau

first: Almudena

first: Alan


title: Microprotein-Encoding RNA Regulation in Cells Treated with Pro-inflammatory and Pro-Fibrotic Stimuli (Ribo-Seq colon cell lines)

overall_design: Ribosome profiling RNA-sequencing (ribo-seq) of four colon-related cell lines (HCEC-1CT, Caco-2, CCD-18Co, PBMC), with samples within each cell line pooled across treatment with individual pro-inflammatory molecules

first: Shane

last: Horman

last: Saghatelian


title: Maternal e-cigarette exposure alters DNA methylome, site-specific CpG and CH methylation, and transcriptomic signatures in the neonatal brain

city: Loma Linda

first: daliao

last: Xiao

address: 11234 Anderson st

department: center for perinatal biology

institute: Loma Linda University

summary: Maternal use of e-cigarette (e-cig) aerosols poses significant risks to fetal brain development,potentially increasing susceptibility to neurodevelopmental disorders in later life. However, the underlying mechanisms remain incompletely understood. This study aimed to understand the effects of fetal e-cig exposure on DNA methylome and transcriptomic changes in the neonatal brain. Pregnant rats were exposed to e-cig aerosols, and neonatal brains (5 males and 5 females/group) from both control and e-cig-exposed groups were used for experimental analysis.Results indicated that prenatal e-cig exposure altered site-specific DNA methylation patterns at both CpG and CH (non-CpG) sites, predominantly in intergenic and intronic regions, with sex dimorphism in methylation and gene expression changes. Gene ontology analysis revealed that e-cig exposure not only affected neuron projection development and axonogenesis but also altered pathways related to neurodegeneration and long-term depression. These findings provide novel insights into the dynamic changes of CpG and CH methylation induced by e-cig exposure,underscoring the susceptibility of the developing brain to maternal e-cig exposure and its potential implications for developmental disorders and neurodegenerative diseases later in life.

overall_design: To investigate the impact of prenatal e-cigarette exposure on the DNA methylome and transcriptomic profiles in the developing brain using a pregnant rat model.

first: Andrew

last: Walayat

first: Maryam

last: Hosseini

first: Chirag

last: Nepal

first: Yong

last: Li

first: Wanqiu

last: Chen

first: Zhong

first: Xiaohui

first: Xuesi

last: Shao

first: Lubo

last: Zhang

first: Charles

first: Daliao


title: Extracellular Vesicles Derived from Inflammatory Cardiomyocytes Mediate Macrophage Polarization and Alleviate Myocarditis via p38 MAPK Pathway

city: Jinan

first: Yingnan

last: You

address: Jingwuweiliu Road

institute: Shandong provincial hospital

summary: EVs derived from lipopolysaccharide （LPS）-stimulated cardiomyocytes （EVLPS） exhibit anti-inflammatory effects on macrophages and reduce cardiac inflammation in mice.To investigate  gene expression changes induced by EVLPS treatment in inflammatory RAW264.7 using next-generation sequencing，we performed RNAseq.

overall_design: mRNA profiles of LPS-stimulated RAW264.7 treated with either EVLPS or PBS were generated by deep sequencing, in quadruplicate

first: Yanjie

last: Jiang

first: Bo

last: Han


title: Dietary supplementation with Bacillus subtilis KC1 alleviates the negative effects of Mycoplasma gallisepticum on growth performance and amino acid metabolism of broiler chickens

city: Jinzhong

first: Jian

last: Wang

address: mingxiannanlu 1

institute: shanxi agricultural university

summary: The aim of this study was to explore whether, and if so, how Bacillus subtilis KC1 can enhance the growth performance of broilers that have been adversely affected by Mycoplasma gallisepticum (MG) infection. A total of 96 1-day-old male broilers were randomly divided into 4 groups: the control group (basal diet), the MG group (basal diet + MG challenge), the Bacillus subtilis KC1 group (basal diet + Bacillus subtilis KC1 supplementation), the Bacillus subtilis KC1 + MG group (basal diet + Bacillus subtilis KC1 supplementation + MG challenge). The trial lasted 42 days, and the results showed that the MG group had significantly reduced body weight and average daily gain, as well as increased feed conversion ratio of broilers, compared to the control group. Dietary supplementation with Bacillus subtilis KC1 significantly improved the growth performance of MG-infected broilers. In addition, dietary supplementation with Bacillus subtilis KC1 significantly improved oxidative stress and inflammatory response markers, characterized by increased superoxide dismutase levels and reduced levels of malondialdehyde, interleukin-1β, and tumor necrosis factor-α. Furthermore, both metabolomics and transcriptomics analyses indicated that MG infection markedly disrupted amino acid metabolism in broilers, whereas Bacillus subtilis KC1 supplementation alleviated the abnormal amino acid metabolism caused by MG infection. These results suggested that Bacillus subtilis KC1 may alleviate the poor growth performance caused by MG infection in broilers by improving amino acid metabolism.

overall_design: A total of 96 one-day-old male AA broilers with an initial average body weight (39.29 ± 0.54 g) were selected and divided into 4 groups: (1) chickens were fed a control diet (Con); (2) chickens were fed a control diet supplemented with 108 CFU/kg of Bacillus subtilis KC1 (KC); (3) chickens were fed a control diet and challenged with MGat day 14 (MG); and (4) chickens were fed a control diet supplemented with 108 CFU/kg of Bacillus subtilis KC1, and further challenged with MG at day 14 (MG_KC). Each treatment was replicated 3 times, with 8 chickens per replicate. All diets were formulated to meet the National Research Council and Chinese chicken feeding standards (NY/T-33–2004) [12,13]. A single-layer, three-dimensional chicken coop was used. Temperature was gradually reduced by 2 °C per week from 33 °C to 23 °C and then kept constant.Lung, liver and ileum samples were collected (3 samples per treatment, 1 per replicate) at day 42. Total RNA was extracted using TRIzol reagent (ET101-01-V2, Beijing TransGen Biotech Co., Ltd, Beijing, China). The RNA concentration and integrity were detected using a Fragment Analyzer (Agilent, California, USA). RNA samples that complied with the criteria of having a concentration of more than 40 ng/μL, an integrity value greater than 7.0, and a total content of 1 μg or more were chosen for further experimentation. Transcriptomic sequencing was completed by BGI.


title: Sall4 and Gata4 Induce Cardiac Fibroblast Transition Towards a Partially Multipotent State with Cardiogenic Potential

city: Houston

first: jerry

last: yang

state: TX

address: 1 Baylor Plaza

institute: Baylor College of Medicine

summary: Cardiac cellular fate transition holds remarkable promise for the treatment of ischemic heart disease. We report that overexpressing two transcription factors, Sall4 and Gata4, which play distinct and overlapping roles in both pluripotent stem cell reprogramming and embryonic heart development, induces a fraction of stem-like cells in rodent cardiac fibroblasts that develop unlimited ex vivo expandability with clonogenicity. Under suitable conditions, these expanding cells demonstrate a high susceptibility to differentiating into various cardiovascular cell types. RNA-Seq was conducted to elucidate the gene expression profiles in these cells with and without specific differentiation.

overall_design: Four groups of cells were designed: GS cell (undifferentiated), GS diff1 (treated with CMM medium), GS diff2 (treated with CMM medium plus EGM2 solution), and untreated parental rat cardiac fibroblasts, which served as controls.Three biological replicates were investigated for each group

first: Hong

last: Gao

first: Saliha

last: Pathan

first: Jianchang


title: NPM1 inhibits tumoral antigen presentation to promote immune evasion and tumor progression

city: Beijing

first: xin

last: wang

address: No.5 DongdanSantiao Road, Dongcheng District

institute: Chinese Academy of Medical Sciences

summary: In this study, we used bulk RNA-seq to to comprehensively compare gene expression in wild-type and Npm1-deficient tumor cells isolated from MC38 tumor tissues.

overall_design: CD45- tumor cells were isolated from subcutaneously inoculated wild type and Npm1-deficient MC38 tumors.

first: Xin

first: Xuetao

last: Cao


title: Epidermal RORα maintains barrier integrity and prevents allergic inflammation by regulating late differentiation and lipid metabolism

city: Madison

first: Jun

last: Dai

state: WI

address: 777 Highland Avenue

department: The School of Pharmacy

institute: University of Wisconsin-Madison

summary: The skin epidermis provides a vital barrier for preventing transepidermal water loss (TEWL) and environmental stimuli. However, the molecular mechanisms ensuring barrier integrity remain not fully understood. RORα is a nuclear receptor highly expressed in the epidermis of normal skin. However, its epidermal expression is significantly reduced in the lesions of multiple inflammatory skin diseases. In this study, using mice with epidermis-specific Rora gene deletion (RoraEKO), we have demonstrated the central roles of RORα in stabilizing skin barrier function. Albeit the lack of spontaneous skin lesion or dermatitis, RoraEKO mice exhibited elevated TEWL rate and skin features indicating barrier dysfunction. The histological and lipidomic analysis uncovered low levels of cornified envelope proteins and aberrant ceramide composition in the RoraEKO epidermis, implying disturbed late epidermal differentiation. In parallel, RNA-seq analysis revealed altered transcription levels of gene clusters related to “keratinization” and “lipid metabolism” in RORα deficient epidermis. Importantly, epidermal Rora ablation greatly amplified percutaneous allergic inflammatory responses to oxazolone in a mouse allergic contact dermatitis (ACD) model. Our results substantiated the essence of epidermal RORα in maintaining late keratinocyte differentiation and normal barrier function while suppressing cutaneous inflammation.

overall_design: To investigate the in vivo role of epidermal RORα in regulating keratinocyte differentiation and skin inflammation, we established a mouse strain with epidermis-specific Rora gene.

first: Xiangmei

last: Hua


title: Lactate prevents Salmonella enterica biofilm formation by inhibiting curli and cellulose expression

city: Philadelphia

first: Francisco

last: Albicoro

address: 3440 N Broad St

institute: Temple University

summary: Salmonella enterica serovar Typhimurium (S. Typhimurium) infection triggers an inflammatory response that changes the concentration of luminal metabolites in the gut, resulting in a distinct environment from a healthy one. We recently demonstrated that S. Typhimurium possesses the ability to form biofilms within the host environment and responds to nitrate as a signaling molecule, enabling it to modulate the transition between sessile and planktonic states. To investigate whether S. Typhimurium utilizes additional metabolites to regulate its behavior, our study delved into the impact of inflammatory metabolites on biofilm formation. Employing a transcriptomic approach we unveiled that lactate enhances the transcription of flagella and invasion genes, highlighting the active role of lactate in modulating the transition of S. Typhimurium from biofilm to motile states. All these findings propose that, as occurring with nitrate, lactate is an inflammatory metabolite being used by S. Typhimurium to support virulence.

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

overall_design: Salmonella enterica samples were incubated with or without the adition of lacate (30mM) in no-salt LB at 28°C with no agitation for 18hs. RNA were extracted from cell pellets in triplicate.

first: Shingo

last: Bessho

first: Kaitlyn

last: Grando

first: Shophia

last: Olubajo

first: Vincent

last: Tam

first: Cagla

last: Tukel


title: Role of TRPV4 in methylation of the CpG sites on miR146a promoter

city: College Park

first: Shaik

middle: Ohidar

last: Rahaman

state: Maryland

address: 4300 Chapel Lane, Skinner Building, Room #3108

department: Nutrition and Food Science

institute: University of Maryland, College Park

summary: Epigenetic modifications, including the role of miRs in various diseases, are key regulators of gene expression. In this study we aimed to explore whether epigenetic alterations could play a role in the TRPV4-mediated regulation of miR-146a. Here we report that there is no significant differences in the methylation status of the examined 10 CpG sites on miR146a promoter between WT and TRPV4 KO cells under LPS treatment, indicating that the regulation of miR-146a by TRPV4 may not be mediated by methylation changes at these specific sites.

sample_organism:
Mus musculus
synthetic construct

platform_organism:
Mus musculus
synthetic construct

overall_design:
To investigate the role of TRPV4 on methylation of CpG sites on miR146a promoter, we isolated Thioglycolate induced peritoneal macrophages from WT and TRPV4KO mice.
Cells were either left untreated or treated with eLPS(100ng/ml). Cell pellets were lysed and bisulphite modification was performed. Bisulphite modified DNA was then amplified using  multiplex or simplex PCRs.
Libraries were next prepared and sequenced on the Ion S5TM sequencer using an Ion 530TM sequencing chip.
FASTQ files from the Ion Torrent S5 server were aligned to a local reference  Methylation levels were calculated.


title: The emergence of the stem cell-associated Sox and POU transcription factors predates animal origins

city: Hong Kong

first: Daisylyn Senna

middle: Young

last: Tan

state: HONG KONG

address: B1202, 9 Lung Wah Strert, Kennedy Town

institute: The University of Hong Kong

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

overall_design: Refer to individual Series


title: The emergence of the stem cell-associated Sox and POU transcription factors predates animal origins (SELEX)

summary: Pluripotent stem cells are a hallmark of animal multicellularity. Sox and POU family transcription factors are pivotal for stemness and were believed to be animal innovations as they were reported absent from the genomes of their unicellular relatives. Here we describe new unicellular holozoan orthologues to Sox and POU families, indicating that they emerged before the appearance of animals. We show that choanoflagellate and filasterean Sox genes have DNA binding specificity similar to Sox2. Choanoflagellate Sox can partner with the POU member Oct4 on DNA elements found in pluripotency enhancers. Consistently, choanoflagellate – but not filasterean – Sox genes can replace Sox2 to reprogram mouse somatic cells into induced pluripotent stem cells (iPSC). In contrast, choanoflagellate POU harbors a unique DNA-binding profile that differs from Oct4 and cannot generate iPSCs. Pluripotency reprogramming with reconstructed ancestral Sox genes shows that their molecular ability to induce stemness was already present in the last common ancestor of animals and their unicellular relatives. Our findings imply that the evolution of stem cells exploited a pre-existing set of transcription factors, where the critical innovation involved an initial change in DNA specificity of POU and the exaptation of the ancestral capacity to interact with Sox transcription factors.

overall_design: To investigate the binding specificity of POU proteins found in unicellular organisms such as Salpingoeca helianthica (Salhel) in comparison to mouse Oct4 (mOct4), the DNA binding domains of the Salhel POU and mOct4 with a 6xHis tag were purified. These proteins were then subjected to Systematic evolution of ligands by exponential enrichment (SELEX) experiments with a 40N randomized library.

first: Daisylyn

first: Xiaomin

last: Zhou

first: Jian

last: Yan

first: Ralf

last: Jauch


title: Temporal regulation of PARP1 acetylation in tumorigenesis

city: New Delhi

first: Sanjeev

last: Das

state: Delhi

address: Aruna Asaf Ali Marg

department: Molecular Oncology Laboratory

country: India

institute: National Institute of Immunology

summary: We examined the impact of PARP1-ATF4 interaction on cellular transcription program. ATF4 plays a pivotal role in stress response by regulating expression of diverse genes. Under genotoxic stress, the acetylation status of PARP1 at lysine 521 influences the expression of diverse genes. Also, co-depletion of ATF4 leads to the downregulation of many genes. These genes are involved in pathways related to cancer risk, such as epithelial-to-mesenchymal transition (EMT), metastasis, cell cycle regulation, and metabolic processes.

overall_design: To investigate the effect of PARP1 K521 deacetylation on the expression of key ATF4-regulated genes, HT29 control, PARP1 knockdown cells expressing PARP1-K521R as well as PARP1 and ATF4 double knockdown cells expressing PARP1-K521R were treated with 5 fluorouracil. We then performed gene expression analysis from data obtained from RNA-seq of these cell lines.

first: Tyagi

last: Witty


title: Microprotein-Encoding RNA Regulation in Cells Treated with Pro-inflammatory and Pro-Fibrotic Stimuli (RNA-Seq colon cell lines)

first: April

last: Williams

overall_design: RNA-sequencing of samples from four colon-related cell lines (HCEC-1CT, Caco-2, CCD-18Co, PBMC), each subjected to treatment with individual pro-inflammatory molecules

first: Victor

last: Pai

first: Calvin

last: Lau

first: Shane

last: Horman

first: Alan

last: Saghatelian


title: Transcriptomics combined with physiology and Metabolomics revealed the responses of potato tuber formation to nitrogen

city: qiqihar city

first: Kaixin

last: Ding

state: heilongjiang province

address: Longsha District

institute: Keshan Branch of Heilongjiang Academy of Agricultural Sciences

summary: Nitrogen (N) can be absorbed by plants, thereby affects plant physiological activity, interferes gene expression, alters metabolite content and influences plant growth. However, the molecular mechanism underlying the potato tuberization response to nitrogen remains unclear. The plants were cultivated in the pots using N-deficient, N-Routine and N-sufficient conditions. Physiological response analysis, transcriptomics and metabolomics were performed on potato stolon exposed to Nitrogen stress. Transcriptomics analysis revealed that 2756 differentially expressed genes (DEGs) responded to nitrogen stress. By using metabolomics analysis, a total of 600 d differentially accumulated metabolites (DAMs) were identified. Further correlation analysis of major DEGs and DAMs showed that 9 key DEGs were involved in alpha-linolenic acid metabolism, 16 key DEGs in starch and sucrose metabolism, 7 key DEGs in nitrogen metabolism, and 16 key DEGs in ABC transporters. Nitrogen deficiency significantly up-regulated the contents of sucrose, GDP-glucose and L-glutamic acid, and promoted the growth of stolon by up-regulating the expression of AMY, SBE, SS, SPS, AGPS and NR-related genes. However, High nitrogen is the opposite. In addition, high nitrogen treatment up-regulated EG, SUS and GDH related genes, accumulated a large number of 9 (S) -HpOTr E, 13 (S) -HpOTr E and L-Glutamine, ultimately affected the balance between plant growth and defense. In general, our study revealed the co-expressed genes and potential pathways related to potato tuber formation under different nitrogen conditions. These comprehensive analysis data provide a better understanding of improving potato tuber traits at the molecular and metabolic levels.

overall_design:
To compare the RNA-seq date gene expression of potato variety Kexin 13 stolons under different nitrogen treatments (AN0, AN1, AN2)，each treatment was repeated three times.

AN0 represents nitrogen deficiency treatment.
AN1 represents conventional nitrogen treatment.
AN2 represents excessive nitrogen treatment.

first: Ying

last: Shan

first: Lichun

last: Wang

first: Yong

last: Zhang


title: Reprogram glioblastoma (GBM) human cell line into DC

first: David

last: Tran

state: CA

summary: 3 human GBM cell lines were reprogrammed into DC by lentiviral vectors containing SPI1, IKZF1

overall_design: ORF expression clones for  human CFDs were purchased from Genecopoeia, and sequence verified. For PU.1 constructs, cDNAs encoding human full-length PU.1 (Spi1)   were cloned into the lentiviral response plasmid pSF-Lenti that contains PGK-puro to generate pSF-Lenti-PU.1 . For lentiviral production, 7.5x106 HEK 293T cells were plated overnight in intact DMEM in a 10cm Poly-D lysine hydrobromide (SIGMA) coated dish, followed by transfection with a 2:1 ratio of total DNA in PEI (µg) together with the viral packaging and envelop plasmids PSPAX2 and PMD2.G, respectively in advanced DMEM medium supplemented with 1.25%FBS, 1X pyruvate, 10mM HEPES and 10mM sodium butyrate. Media were replaced at 16 hrs. after transfection. Viral supernatants were collected every 24hrs and centrifuged for 20hrs. at 25,000g to sediment viral particles, which were resuspended in opti-MEM and viral titers measured.  Human GBM cell lines LN428, LN308, LN827 were cultured in DMEM media with 10% FBS and 1% pen/strep. For transduction of CFD-containing lentiviruses, GBM cells in baseline DMEM media were incubated with indicated lentiviral constructs for 24hrs., followed by media replacement to RPMI 1640 supplemented with 10% FBS, 1% pen/strep and 3µg/µl puromycin for selection. On day 7 after transduction, cells were passaged into RPMI 1640 media supplemented with 10% FBS and 1% pen/strep without puromycin until analysis. For all of the samples from three cell human GBM cell lines, are derived from in vitro reprogramming by indicated factors for 7 days.  Samples with pSF is the empty vector control for reprogramming, cells are collected after reprogramming. Samples with CD45+ are CD45+ positive population sorted by FACS  human monocytes derived DC are purchased from Lonza. "mature"  groups are maturated by KR-luc tumor lysate and TNFalpha, IL-6,IL-1beta

first: Tianyi

last: Liu

first: Dan

last: Jin

first: Son

last: Le

first: Dongjiang

last: Chen

first: Mathew

last: Sebastian

first: Alberto

last: Riva

first: Ruixuan


title: Integrated Single-Cell and Spatial Transcriptomics Reveal Androgen-Driven Disruptions in PCOS Ovarian Microenvironment

city: Zhengzhou

first: Man

last: Luo

address: 195 Tongbai Road, Zhongyuan District

institute: Zhengzhou Central Hospital Affiliated to Zhengzhou University

summary: Polycystic Ovary Syndrome (PCOS) is a widespread reproductive disorder with hyperandrogenemia and impaired follicular development. Granulosa cells (GC) and thecal cells (TC) are crucial during follicular development, but their responses to androgen remain poorly understood at subpopulation levels. Using single-cell RNA sequencing and spatial transcriptomics, we analyzed GC and TC subpopulations in Dehydroepiandrosterone-induced PCOS-like mice to determine the ovarian microenvironment characters. We observed gene expression changes in steroidogenesis and cell-cell communications, with significant landscape shifts in subtypes. GC subsets showed an inflammatory GC5 expansion and loss of luteinized GC7, while TC subsets exhibited an increased TC2 population with enhanced lipid metabolism and androgen response. Intercellular signaling was heightened, particularly with amplified ligand-receptor interactions for Ptn-Ncl and Mdk-Ncl. Steroidogenesis was altered for PCOS's steroid hormone abnormalities. These findings revealed androgen impact in the cell subpopulations of follicular microenvironment and underscored the importance of Ptn-Ncl and Mdk-Ncl in PCOS-associated impaired follicular development.

overall_design: Ovaries were collected from Control mice and PCOS-like mice. PCOS-like mice were generated by subcutaneous administration of Dehydroepiandrosterone (DHEA) into 25-day-old female ICR mice. The DHEA was dissolved in sesame oil at 10% by volume in 95% ethanol, and was administrated in each mouse of model group at 6 mg/100 g of body weight in 0.1 mL once a day over a period of 20 days. The control mice were injected with vehicle in the same way. The mice were housed under a pathogen-free condition with a 12-hour light/dark cycle, free access to food and water.


title: Maximizing HLC production in bioreactors: unveiling the role of oxygen control for enhanced yield and PH1 disease modelling

city: PAMPLONA

first: Juan R

last: Rodriguez-Madoz

address: AV PIO XII 53

country: Spain

institute: CIMA

summary: Hepatocytes-like cells (HLC) derived from human induced pluripotent stem cells show great promise for cell-based liver therapies and disease modelling. However, their application is currently hindered by the low production yields of existing protocols. We aim to develop a bioprocess able to generate high numbers of HLC. We used stirred-tank bioreactors with a rational control of dissolved oxygen concentration (DO) for the optimization of HLC production as 3D aggregates. We evaluated the impact of controlling DO at physiological levels (4%O2) during hepatic progenitors’ stage on cell proliferation and differentiation efficiency. Whole transcriptome analysis and biochemical assays were performed to provide a detailed characterization of HLC quality attributes. When DO was controlled at 4%O2 during the hepatic progenitors’ stage, cells presented an upregulation of genes associated with hypoxia-inducible factor pathway and a downregulation of oxidative stress genes. This condition promoted higher HLC production (maximum cell concentration: 2×106 cell/mL) and improved differentiation efficiencies (80% Albumin-positive cells) when compared to the bioreactor operated under atmospheric oxygen levels (21%O2, 0.6×106 cell/mL, 43% Albumin positive cells). These HLC exhibited functional characteristics of hepatocytes: capacity to metabolize drugs, ability to synthesize hepatic metabolites, and inducible cytochrome P450 activity. Bioprocess robustness was confirmed with HLC derived from different donors, including a primary hyperoxaluria type 1 (PH1) patient. The generated PH1.HLC showed metabolic features of PH1 disease with higher secretion of oxalate compared with HLC generated from healthy individuals. This work reports a reproducible bioprocess, that shows the importance of controlling DO at physiological levels to increase HLC production, and the HLC capability to display PH1 disease features.

first: Pedro

last: Vicente

first: Juan

first: Margarida

last: Serra

first: Maria

middle: E

last: Calleja-Cervantes


title: Clinical and molecular characterization of steatotic liver disease in the setting of immune-mediated inflammatory diseases

city: Santander

first: Enrique

last: García Nieto

state: Cantabria

address: C. Cardenal Herrera Oria, s/n

institute: Instituto de Investigación Marqués de Valdecilla

summary:
Growing evidence suggests an increased prevalence of nonalcoholic fatty liver disease (NAFLD) in the context of immune-mediated inflammatory diseases (IMIDs).
We aimed to clinically and mechanistically characterize a prospective cohort of IMID-NAFLD patients compared to classic-NAFLD patients.

overall_design: Comparative gene expression analysis of RNA-seq data from freshly frozen liver biopsies of IMID-NAFLD and classic-NAFLD patients.

last: García-Nieto

last: Rodriguez-Duque

first: Coral

last: Rivas-Rivas

first: Paula

last: Iruzubieta

first: María

middle: J

last: Garcia

first: Laura

last: Rasines

first: Ana

last: Alvarez-Cancelo

first: Agustín

last: García-Blanco

first: José

middle: I

last: Fortea

first: Angela

last: Puente

first: Beatriz

last: Castro

middle: L

last: Cagigal

first: Javier

last: Rueda-Gotor

first: Ricardo

last: Blanco

first: Montserrat

last: Rivero

first: Susana

last: Armesto

first: Marcos

middle: A

last: González-López

first: Anna

last: Esteve Codina

first: Marta

last: Gut

middle: P

last: Vaque

last: Crespo

last: Arias-Loste