title: Inhibition of tumor-intrinsic NAT10 enhances antitumor immunity by triggering type I interferon responses via MYC/CDK2/DNMT1 pathway

city: Suzhou

first: Wancheng

last: Liu

state: jiangsu

address: 100 Chongwen Road, Industrial Park

institute: Chinese Academy of Medical Sciences and Peking Union Medical College

summary: Purpose:Posttranscriptional modifications are deeply involved in cancer progression; however, there remains knowledge gap regarding the function and immune regulatory mechanism of newly discovered mRNA acetylation modification.Methods: The expression of NAT10 in human tumor tissues was analyzed based on TCGA data. NAT10 knockout murine cell lines were generated to analyze the NAT10-associated tumor progression and anti-tumor immune response in tumor xenograft models. Immune cells and cytokines in TME were quantified by immunofluorescence, flow cytometry, quantitative reverse transcription-PCR, and ELISPOT assay. NAT10-associated differentially expressed genes were investigated in cancer cells and tissues with RNA-Seq. Results:Loss of tumoral NAT10 significantly stimulated tumor-specific cellular immune responses and suppressed tumor growth. Mechanistically, we identified MYC as a key downstream target of NAT10 via enhancing mRNA stability and translation efficiency. Inhibition of NAT10 blocked the MYC/CDK2/DNMT1 pathway, subsequently enhancing double-stranded RNAs (dsRNA) formation, which triggered type I interferon responses to enhance the in vivo response of tumor specific CD8+ T cells. Conclusions: Inhibition of NAT10 using either small molecule inhibitor (Remodelin) or PEI/PC7A/siNAT10 nanoparticles combined with PD-1 blockade synergistically enhanced the anti-tumor immune response and repressed tumor progression. Our findings uncovered the crucial role of tumor-intrinsic NAT10 in tumor immune microenvironment, representing a promising target for enhancing cancer immunotherapy.

overall_design: mRNA profiles of MCA205 and TC1 wild type (WT) and sgNAT10 cell lines;  mRNA profiles of TC1 wild type (WT) and sgNAT10 tumor tissue.

first: W

first: Y

last: Wei

first: J

first: D

last: Ma


title: ATAC-Seq Analysis Reveals Arid3a-Dependent Chromatin Accessibility Changes in VSMCs

status: Public on Aug 30 2024

city: guiyang

first: Dijie

last: Zheng

address: No.8 beijing road

country: China

institute: guizhou medical universuty

type: Genome binding/occupancy profiling by high throughput sequencing

summary: This study leverages ATAC-seq to reveal Arid3a's chromatin accessibility effects in VSMCs, identifying key gene expression changes and implicating Arid3a in actin filament dynamics linked to AAA pathogenesis.

overall_design: We utilized RNA interference technology to knockdown Arid3a in Vascular Smooth Muscle Cells (VSMCs), successfully establishing several Arid3a knockdown cell lines. In this study, we applied ATAC-seq to dissect the chromatin accessibility dynamics in Vascular Smooth Muscle Cells (VSMCs) following Arid3a knockdown. The ATAC-seq assay was designed to capture the open chromatin regions that are potentially bound and regulated by Arid3a, offering insights into its direct and indirect transcriptional regulatory effects.

first: Yazhu

last: Deng

first: Mo

last: Zhang


title: sciMETv3 for atlas-scale single-cell DNA methylation analysis

city: Portland

first: Andrew

last: Adey

state: OR

address: 3181 S.W. Sam Jackson Park Rd, RHH, L103

department: MMG

country: USA

institute: Oregon Health & Science University

type: Methylation profiling by high throughput sequencing

summary: We describe sciMETv3, a combinatorial indexing-based technique that is capable of producing single-cell DNA methylation datasets with 100's of thousands of cells in a single experiment by one individual over two days. sciMETv3 is compatible with capture techniques to reduce sequencing costs (sciMET-cap) as well as enzymatic conversion methods. sciMETv3 libraries can be designed for compatibility with both Illumina and Ultima sequencing platforms.

overall_design: Four speciems were used in this study, each a BA46 cortex specimen from a healthy donor and processed using sciMETv3.

first: Adey

last: Andrew

first: Nichols

last: Ruth

first: Rylaarsdam

last: Lauren


title: Myokine secretion enhancement by the addition of lavender essential oil in cultured skeletal muscle cells

city: Tsukuba

first: RANDEEP

last: RAKWAL

state: Ibaraki

address: 1-1-1 Tennodai

department: Institute of Health and Sport Sciences

country: Japan

institute: University of Tsukuba

summary: In recent years, aromatherapy for sportspersons termed as sports aromatherapy has been attracting attention in the field of sports, and aromatic baths and aromatherapy treatments are being used as a new conditioning method, mainly by athletes. Among these, aroma treatment is used to improve performance and recovery from fatigue. On the other hand, myokines (muscle-derived cytokines and chemokines) secreted from the skeletal muscles after exercise, have recently been attracting attention. Many types of myokines are known to act on various organelles and through diverse beneficial metabolic actions promote health. One of them is IL-6, which has been reported to act on muscles and promote myogenesis. Previous studies on essential oil treatment effects are thought to be due to aroma or massage stimulation, and there are no reports of direct effects on muscle at the molecular level comparing essential oils with carrier oils alone. Therefore, we aimed to elucidate the molecular mechanism of myokine secretion by the addition of an essential oil (Lavender oil, LO) using an in vivo exercise model of a mouse muscle cell line (C2C12 cells), which are known as exercise cells.

overall_design: Whole genomic DNA microarray was performed in C2C12 cells (total RNA was extracted using RNeasy Micro Kit), suggesting myokines produced by lavender essential oil and EPS(Electric Pulse Stimulation treatment).

first: Fumiko

last: Takenoya

first: Junko

last: Shibato

first: Michio

last: Yamashita

first: Makoto

last: Kanzaki

first: Seiji

last: Shioda

first: Randeep

last: Rakwal


title: Specific pre-plasma cell states and local proliferation at the dark zone – medulla interface characterize germinal center-derived plasma cell differentiation in lymph node (Figure 4 and Figure 5)

city: Marseille

first: chuang

last: dong

address: 163 Av. de Luminy

country: France

institute: CIML

type: Expression profiling by high throughput sequencing

summary: High affinity antibody-producing plasma cell (PC) production in germinal centers (GC) is crucial for antibody-mediated immune protection after vaccination or infection. The selection of high affinity B cells in the GC light zone instructs PC differentiation in a subset of cells, but the phenotype, differentiation trajectory and spatial localization of those prePC intermediates remain to be characterized. Here, we have used a mouse model to track GC-derived B cells with integrative single-cell and spatial analyses in draining lymph node after immunization. We first identified putative prePC in scRNA-seq datasets, then enriched those cells through their specific surface phenotype for further analysis of their gene expression trajectories and BCR repertoire. We found a continuum of actively proliferating transitional states bridging selected LZ GC B cells and recently exported PCs, with gradually increasing levels of endoplasmic reticulum stress-associated genes and immunoglobulin transcripts. Spatial analyses revealed that recently differentiated PC continued their maturation and proliferation at the interface between the DZ and extensions of the lymph node medulla. Our results provide insights into the intermediate stages and microenvironmental factors involved in the differentiation of GC B cells into PC, with implications for vaccine development and understanding antibody responses.

overall_design: C57BL/6 mice were immunized with NP-KLH in Sigma Adjuvant System, and we investigated cells 4 days after tamoxifen gavage, at day 15 of the primary response.

first: Pierre

last: Milpied

first: Laurine

last: Binet

first: Chuang

last: Dong


title: Specific pre-plasma cell states and local proliferation at the dark zone – medulla interface characterize germinal center-derived plasma cell differentiation in lymph node (Figure 6)

overall_design: We collected draining lymph node from mice that were immunized since 14 days and gavaged since 3 days. Freshly dissected draining lymph nodes were dryed on absorbent paper, embedded in OCT, snap frozen in isopentane over dry ice, and stored at -80°C until processing. On the day of the experiment, 10 µm-thick cryosections were prepared from the region of interest using a cryostat. Four sections separated by 50µm in depth were placed on a Visium slide within the designated capture areas. Hematoxylin and eosin (H&E) staining was performed according to the manufacturer's guidelines with the following modifications: hematoxylin incubation for 30 seconds, bluing buffer for 5 seconds, and eosin incubation for 1 minute 30 seconds, all at room temperature. Following washing, brightfield images of the stained sections were acquired before proceeding to subsequent steps of the 10x Genomics Visium Spatial Gene Expression protocol. The tissue was embedded in OCT medium and a 10 µm section was cut on a cryostat and deposited on the capture area of the Visium slide following the guidelines of the 10x Genomics Visium Spatial Gene Expression protocol. Briefly, we performed permeabilization (18 min), reverse transcription, second strand synthesis, denaturation, cDNA amplification (16 cycles of PCR), and library construction according to the manufacturer’s instructions. The resulting libraries were sequenced on an Illumina NextSeq2000, generating an average of 120 million reads per library (Read 1: 28 cycles, Read i7: 10 cycles, Read i5: 10 cycles, Read 2: 79 cycles).

first: Bertrand

last: Escaliere


title: Specific pre-plasma cell states and local proliferation at the dark zone – medulla interface characterize germinal center-derived plasma cell differentiation in lymph node (Figure2) III

overall_design: Mice were immunized with 100µg of NP-KLH at 1µg/µL emulsified with Sigma Adjuvant System (SAS) at a 1:1 (v:v) ratio, subcutaneously at the base of the tail, 50µl on each side. For induction of the Cre-ERT2-mediated labelling, we gavaged the mice once with 5mg of tamoxifen (TS648-1G, Sigma) in 200µL of peanut oil (P2144-250 ML, Sigma), at least 6 days after immunization. Mice were euthanized at day 10, day 16 and day 22 of the primary response. For experiments described in Figure 2a, cells from draining lymph nodes were washed and resuspended in FACS buffer (PBS containing 5% FCS and 2 mM EDTA) at a concentration of 10E8 cells/ml. Samples from different mice and different time points were processed separately using cell hashing as described (DOI : 10.1038/s41592-019-0392-0). Cells were individually stained with distinct barcoded anti-mouse CD45 antibodies (in-house conjugated) in FACS buffer for 30 minutes on ice. Subsequently, cells were washed and stained with a mix of primary antibodies, then Live/Dead Fixable Aqua Dead Cell Stain (Thermofisher). Live cells of interest (IgDneg eYFP+ PC and B cells) from each sample were bulk-sorted using a BD Influx cell sorter. PC and non-PC were captured in distinct wells for droplet-based scRNA-seq to maximize the recovery of BCR sequence information from non-PC. Within each fraction, cells from different samples were pooled and loaded for the 10x Genomics Single Cell 5' v2 workflow. Libraries were prepared according to the manufacturer's instructions with modifications for generating B cell receptor (BCR) sequencing libraries. Following cDNA amplification, SPRI select beads were used to separate the large cDNA fraction derived from cellular mRNAs (retained on beads) from the hashtag oligonucleotide (HTO)-containing fraction (in supernatant). For the mRNA-derived cDNA fraction, 50 ng were used to generate the transcriptome library, and 10-20 ng were used for BCR library construction. Gene expression libraries were prepared according to the manufacturer's instructions. For BCR libraries, heavy and light chain cDNAs were amplified by two rounds of PCR (6 cycles + 8 cycles) using external 12  primers recommended by 10x Genomics. Approximately 800 pg of purified amplified cDNA was tagmented using the Nextera XT DNA Sample Preparation kit (Illumina) and amplified for 12 cycles using the SI-PCR forward primer (10x Genomics) and a Nextera i7 reverse primer (Illumina). For the HTO-containing fraction, 5 ng were used to generate the HTO library. The resulting libraries were pooled and sequenced on an Illumina NextSeq2000 platform with single-indexed paired-end kits following the manufacturer’s guidelines.


title: Specific pre-plasma cell states and local proliferation at the dark zone – medulla interface characterize germinal center-derived plasma cell differentiation in lymph node (Figure2) II

overall_design: Mice were primed with NP-KLH in Sigma Adjuvant System, and we investigated cells 5 days after tamoxifen gavage, at day 16 of the primary response, using droplet-based 5’-end scRNA-seq. For experiments described in Figure 2a, cells from draining lymph nodes were washed and resuspended in FACS buffer (PBS containing 5% FCS and 2 mM EDTA) at a concentration of 10E8 cells/ml. Samples from different mice and different time points were processed separately using cell hashing as described (DOI : 10.1038/s41592-019-0392-0). Cells were individually stained with distinct barcoded anti-mouse CD45 antibodies (in-house conjugated) in FACS buffer for 30 minutes on ice. Subsequently, cells were washed and stained with a mix of primary antibodies, then Live/Dead Fixable Aqua Dead Cell Stain (Thermofisher). Live cells of interest (IgDneg eYFP+ PC and B cells) from each sample were bulk-sorted using a BD Influx cell sorter. PC and non-PC were captured in distinct wells for droplet-based scRNA-seq to maximize the recovery of BCR sequence information from non-PC. Within each fraction, cells from different samples were pooled and loaded for the 10x Genomics Single Cell 5' v2 workflow. Libraries were prepared according to the manufacturer's instructions with modifications for generating B cell receptor (BCR) sequencing libraries. Following cDNA amplification, SPRI select beads were used to separate the large cDNA fraction derived from cellular mRNAs (retained on beads) from the hashtag oligonucleotide (HTO)-containing fraction (in supernatant). For the mRNA-derived cDNA fraction, 50 ng were used to generate the transcriptome library, and 10-20 ng were used for BCR library construction. Gene expression libraries were prepared according to the manufacturer's instructions. For BCR libraries, heavy and light chain cDNAs were amplified by two rounds of PCR (6 cycles + 8 cycles) using external 12  primers recommended by 10x Genomics. Approximately 800 pg of purified amplified cDNA was tagmented using the Nextera XT DNA Sample Preparation kit (Illumina) and amplified for 12 cycles using the SI-PCR forward primer (10x Genomics) and a Nextera i7 reverse primer (Illumina). For the HTO-containing fraction, 5 ng were used to generate the HTO library. The resulting libraries were pooled and sequenced on an Illumina NextSeq2000 platform with single-indexed paired-end kits following the manufacturer’s guidelines.


title: Diverse subpopulations of reactive mouse astrocytes following chronic Toxoplasma infection.

city: Riverside

first: Emma

middle: H

last: Wilson

state: CA

address: 900 University Avenue

institute: UC Riverside

summary: Astrocytes provide physical and metabolic support for neurons, regulate the blood brain barrier, and react to injury, infection, and disease. When astrocytes become reactive, the regulation of the inflammatory state, the forms it takes, and its longevity are all poorly understood. Several models of acute inflammation have revealed astrocyte subpopulations that go beyond a two-activation state model, instead encompassing distinct functional subsets. However, how reactive astrocyte (RA) subsets evolve over time during chronic inflammatory disease or infection have been technically difficult to address. Here we use a prolific human pathogen, Toxoplasma gondii, that causes lifelong infection in the brain alongside a novel Lcn2CreERT2 reporter mouse line to examine astrocyte subsets during chronic inflammation. Single cell RNA sequencing revealed diverse astrocyte populations during chronic inflammation. Furthermore, we observed transcriptionally unique Lcn2CreERT2+ RAs, some of which changed over time in a manner suggestive of potential resolution and overall plasticity of RAs. In addition to an immune regulating Lcn2CreERT2+ astrocyte population enriched with gene transcripts encoding chemokines Ccl5, Cxcl9, Cxcl10 and receptors Ccr7 and Il7r, a specific set of Lcn2CreERT2+ astrocytes highly expressed transthyretin (Ttr), a secreted carrier protein involved in glycolytic enzyme activation and potential vasculature regulation and angiogenesis. These findings provide novel information about the evolution and diversity of reactive astrocyte subtypes and functional signatures at different stages of infection and an undocumented role for transthyretin expressing astrocytes in immune regulation at the central nervous system (CNS) vasculature.

overall_design: We performed scRNAseq on the mixed samples of whole brain taken from brain mononuclear cells collected following 14, 28, and 42 days of Toxoplasma infection and obtained 21,941 high quality cells. We repeated this utilizing ACSA2 for each time point to get 3,736 high quality ACSA2 astrocytes. Lastly, we performed scRNAseq on Lcn2CreErt2;Ai9 transgenic mice following 14 and 42 days of Toxoplasma infection to collect Lipocalin (Lcn2) expressing cells obtaining 5,875 high quality Lcn2+ cells. A tamoxifen rested time point was included as well, and mice were infected with the chronic 42 day infection regimen and collected 2 weeks later to assay any changes to previously labeled chronic Lcn2 astrocytes.

first: Zoe

last: Figueroa


title: SMARCA4 regulates BRD4 nuclear distribution in cellular plasticity

city: Madison

first: Allan

last: Brasier

state: WI

address: 715 Highland

department: ICTR

institute: university of Wissconsin

summary: Inducible cell stress programs in basal epithelial cells shape repair and host defense pathways in mucosal surfaces.  Previously, we discovered that expression of the core SWI/SNF complex ATPase, SMARCA4/Brg1, is required for genomic organization supporting a fully differentiated epithelial state, regulating cellular plasticity, paracrine mesenchymal signaling and anti-viral response.  Here, we observe that SMARCA4 expression is intrinsically engaged with IFN response genes and dynamically engages the atypical histone acetyltransferase, BRD4, upon RSV infection, an association required for intrinsic immunity.  To investigate the mechanisms how the SMARCA4 BRD4 complex functions in intrinsic immunity, we performed Cleavage Under Targets and Release Using Nuclease (CUT&RUN) of BRD4 binding in wild type (WT) and SMARCA4 knockdown (KD) small airway epithelial cells in mock- or RSV-infected states.  In mock-infected cells, 4,017 high confidence BRD4 peaks are identified that localize to the gene bodies controlling motility programs.  By contrast, RSV replication repositions BRD4 to 2,339 regions, upstream of transcription start sites in genes controlling inducible cytokine, cell adherence and anti-viral programs.  RSV redistributes BRD4 into super enhancers regulating immune response-associated long noncoding RNAs.  BRD4 binding is not affected by SMARCA4 KD in mock-infected cells, but BRD4 recruitment is reduced on 739 peaks after RSV infection, including superenhancers. These data extend the complex interrelationship between the SWI/SNF ATPases and that of the BRD4 bromodomain epithelial progenitors linking cell state and innate immunity.

overall_design: wild type or SMARCA4 knockdown cells were mock infected or infected with RSV.  CutNRun assays were using IgG, or antiBRD4 Abs.  Three replicates per condition

first: Xiaofang

last: Xu


title: Effects of Roseoflavin and/or Trametinib Treatment on 3D-Cultured PDAC Cell Lines

city: Sapporo

first: Takako

last: OOshio

address: Kita-15, Nishi-7, Kita-Ku

institute: Hokkaido University

summary: Pancreatic ductal adenocarcinoma (PDAC) remains a formidable challenge with a survival rate of approximately 10%, largely due to inadequate diagnostic and therapeutic options. To identify novel therapeutic targets for PDAC, we have previously developed a Drosophila model, '4-hit' fly, which mimics the four gene alterations characteristic of PDAC: KRAS, TP53, CDKN2A, and SMAD4. Through a whole-body genetic screening using the 4-hit fly, we discovered that riboflavin (RF) pathway represents a novel potential target for PDAC treatment. Expression analysis revealed that four out of five RF pathway-related genes were upregulated in PDAC compared to non-tumor pancreas. Inhibition of RF pathway resulted in metabolic changes, notably decreased mitochondrial respiratory chain activity, and subsequently suppressed PDAC cell proliferation. Furthermore, a combination therapy using the RF pathway inhibitor roseoflavin with MEK inhibitor trametinib significantly suppressed the expansion of human PDAC xenografts and spheroid formation more effectively than either treatment alone. These findings suggest that dual inhibition of RF pathway and MEK could offer a novel therapeutic strategy for PDAC.

overall_design: Pancreatic cancer cells were cultured in three dimensions in a methylcellulose-containing medium for three days before treatment with roseoflavin and/or trametinib. Spheroid samples were collected one day post-treatment and analyzed using RNA sequencing.

last: Ooshio

first: Masahiro

last: Sonoshita

first: Tetsuo

last: Mashima

first: Hiroyuki

last: Seimiya


title: Gene expression profile of iron fractionated and non-iron fractionated macrophages from subcutaneous injected syngeneic murine fibrosarcoma in WT mice.

city: Philadelphia

first: William

last: Molina Arocho

address: 421 Curie Blvd BRB II/III Rm 436

institute: University of Pennsylvania

summary: We used bulk RNA sequencing to study the differences between iron-high and iron-low tumor associated macrophages in murine fibrosarcoma.

overall_design: Cells were isolated from subcutaneous implanted syngeneic fibrosarcoma tumors in C57BL/6 mice. Cells were iron enriched using magnetic columns to obtain an iron positive eluate and an iron negative flowthrough. Both fractions were analyzed with bulk RNAseq.

first: Ian

middle: W

last: Folkert

middle: A

first: Malay

last: Haldar


title: NCBP2 promotes colorectal cancer growth and metastasis by driving lipid droplet accumulation by stabilizing LIPG mRNA

city: Hefei

first: shoudong

last: ye

address: No 111, Jiu Long Road

department: Cell Biology

institute: Anhui University

summary: RNA-binding proteins are critical for RNA processing and aberrantly expressed in colorectal cancer (CRC). In this study, by screening and bioinformatic analyses of the TCGA database and GSE20916 and GSE18105 datasets, NCBP2 was ultimately identified as a potential oncogene in CRC. We observed that NCBP2 was overexpressed in tumour tissues; its overexpression was correlated with CRC invasion and distant metastasis and indicated poorer survival in CRC patients. NCBP2 overexpression promoted the proliferation, migration and invasion of CRC cells in vitro and in vivo. Mechanistically, the NCBP2 protein stabilizes LIPG mRNA and increases LIPG expression via direct binding to m7G in the 5’-cap structure of LIPG mRNA. NCBP2 also facilitates lipid droplet accumulation in CRC cells in a LIPG-dependent manner. This study demonstrated that the NCBP2–LIPG–lipid droplet axis represents a new mechanism for CRC growth and distant metastasis and is a promising target for CRC treatment.

overall_design: HCT116 cells cultured with 10%FSP serum were collected using Trizo. PCDH was used as the control group and overexpressed NCBP2 was used as the experimental group. Each treatment group had 2 replication groups. The samples were sent to MajorBIO (Shanghai, China) for deep sequencing using illumina Hiseq.


title: sciMET-ATAC for simultaneous single-cell DNA methylation and chromatin accessibility analysis

first: Andrew

last: Adey

summary: We describe sciMET-ATAC, a combinatorial indexing-based technique that is capable of producing single-cell DNA methylation plus chromatin accessibility datasets.

overall_design: A single BA46 cortex specimen was used in this study and taken through sciMET-ATAC library preparation and analysis.

first: Ruth

last: Nichols

first: Lauren

last: Rylaarsdam


title: Thioridazine reverses trastuzumab resistance in gastric cancer by inhibiting S-phase kinase associated protein 2-mediated aerobic glycolysis

city: 开封

first: yiwei

last: zhao

address: 河南大学金明校区

institute: 河南大学抗体药物开发技术国家地方联合工程实验室

summary: Trastuzumab constitutes the fundamental component of initial therapy for patients with advanced human epidermal growth factor receptor 2 (HER-2)- positive gastric cancer (GC). However, the efficacy of this treatment is hindered by substantial challenges associated with both primary and acquired drug resistance. While S-phase kinase associated protein 2 (Skp2) overexpression has been implicated in the malignant progression of GC, its role in regulating trastuzumab resistance in this context remains uncertain. Despite the numerous studies investigating Skp2 inhibitors among small molecule compounds and natural products, there has been a lack of successful commercialization of drugs specifically targeting Skp2.To discover a Skp2 blocker among currently available medications and develop a therapeutic strategy for HER2- positive GC patients who have experienced progression following trastuzumab-based treatment.

overall_design: Skp2 exogenous overexpression plasmids and small interfering RNA vectors were utilized to investigate the correlation between Skp2 expression and trastuzumab resistance in GC cells. Q-PCR, western blot, and immunohis- tochemical analyses were conducted to evaluate the regulatory effect of thioridazine on Skp2 expression. A cell counting kit-8 assay, flow cytometry, a amplex red glucose/glucose oxidase assay kit, and a lactate assay kit were utilized to measure the proliferation, apoptosis, and glycolytic activity of GC cells in vitro. A xenograft model established with human GC in nude mice was used to assess thioridazine's effectiveness in vivo


title: Gene expression profile at single cell level of iron positive and iron negative cells from subcutaneous implanted syngeneic fibrosarcoma tumors in C57BL/6 mice.

summary: Iron-rich tumor-associated macrophages (iTAMs) are characterized by high intracellular iron, heme metabolism, and expression of the endothelin receptor B. iTAMs promote angiogenesis and immunosuppression in tumors. Heme-mediated degradation of the transcriptional repressor BACH1 induces the iTAM transcriptional program in macrophages.

overall_design: Cells were isolated from subcutaneous implanted syngeneic fibrosarcoma tumors in C57BL/6 mice. Cells were iron enriched using magnetic columns to obtain an rion positive eluate and an iron negative flowthrough. Both fractions were analyzed with scRNAseq.

last: Arocho


title: Gene expression profile at single cell level of CD45+ cells from synovial sarcoma tumors in SYT-SSX2 conditional mice.

summary: We used scRNAseq to characterize CD45+ cells in synovial sarcoma murine model.

overall_design: SYT-SSX2 conditional mice were injected in gastrocnemius muscle with recombinant TAT-Cre protein to induce synovial sarcoma tumors. Tumors were then harvested and digested into single cell suspensisons for scRNAseq.

first: Tsun

middle: K

last: To


title: Human Early Postnatal Thymus [CITE-seq]

city: Vancouver

first: Fabio

last: Rossi

state: BC

address: 2222 Health Sciences Mall

department: School of Biomedical Engineering

country: Canada

institute: University of British Columbia

summary: Within the thymus, regulation of the cellular cross-talk directing T cell development is dependent on spatial interactions within specialized niches. To create a holistic, spatially defined map of tissue niches guiding postnatal T cell development we employed the multidimensional imaging platform CO-detection by indEXing (CODEX), as well as Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) and Assay for Transposable-Accessible Chromatin (ATAC-seq). We generated age-matched 4–5-month-old postnatal thymus datasets for both male and female donors, and describe signaling and cell-cell interaction networks in sequential thymocyte developmental niches. Together, these data represent a unique age-matched spatial multiomic resource to investigate how sex-based differences in thymus regulation and T cell development arise, and provide an essential resource to understand the mechanisms underlying immune function and dysfunction in males and females.

overall_design: 4-5 month old sex-matched human thymus + a 33-month-old human thymus samples were processed prior to sequencing to enrich for CD45- and CD25+CD8- cells. See Stankiewicz et al. 2023 (BioRxiv) for experimental details.

first: Laura

middle: N

last: Stankiewicz

first: Kevin

last: Salim

first: Megan

last: Levings

first: Peter

last: Zandtsra

middle: M


title: Gene expression profile at single cell level of CD45+ cells from subcutaneous implanted fibrosarcoma tumors in T cell depleted Bach1 deficient mice.

summary: We used scRNAseq to characterize the CD45+ cell population diversity in fibrosarcoma in T cell depleted Bach1-deficient mice.

overall_design: CD45+ cells were isolated from subcutaneous implanted fibrosarcoma tumors in T cell depleted Bach1 knockout mice and analyzed with scRNAseq.


title: Gene expression profile at single cell level of cells from subcutaneous implanted syngeneic fibrosarcoma tumors in C57BL/6 mice injected with autologous erythrocytes suspended in buffered saline

overall_design: Cells were isolated from subcutaneous implanted syngeneic fibrosarcoma tumors in C57BL/6 mice injected with autologous erythrocytes suspended in buffered saline or saline control and analyzed with scRNAseq.


title: Gene expression profiles at single cell level of murine undifferentiated pleomorphic sarcoma (UPS)

summary: We used scRNAseq to characterize the cell population diversity in UPS

overall_design: Mouse tumors were generated via injection of adenovirus-expressing Cre recombinase into the right gastrocnemius muscle of 3-month-old KP (LSL-KrasG12D+; Trp53fl/fl) mice. Tumors were then isolated, and dissociated into single cells, which were analyzed using scRNAseq.

first: Ying

last: Liu


title: Human Early Postnatal Thymus [ATAC-seq]

overall_design: 4-5 month old sex-matched human thymus samples were processed prior to sequencing to enrich for CD45- and CD25+CD8- cells. See Stankiewicz et al. 2023 (BioRxiv) for experimental details.

last: Zandstra


title: Distinct roles of SOX9 in self-renewal of progenitors and mesenchymal transition of the endothelium [RNA-seq]

city: woolloongabba

first: laura

last: sormani

state: QLD

address: 37 kent street

department: Diamantina Institute

country: Australia

institute: The University of Queensland Diamantina Institute

summary: Regenerative capabilities of the human endothelium are governed by vessel-resident progenitors termed endothelial colony forming cells (ECFCs). Endothelial cells acquire mesenchymal phenotypes during atherogenesis, via a process termed endothelial-to-mesenchymal transition (EndMT). This study aimed to investigate the molecular drivers of EndMT in the progenitor population under an atherogenic model. In the presence of oxidised low-density lipoprotein (oxLDL), cultured human ECFCs displayed classical features of EndMT, through reduced endothelial gene and protein expression, function as well as increased mesenchymal genes, contractility, and motility. Importantly, in vitro oxLDL resulted in a dramatic loss in progenitor self-renewal through single-cell colony formation assay and numbers. RNA sequencing of ECFCs exposed or not to oxLDL validated gene expression changes suggesting EndMT but identified SOX9 as one of the highly differentially expressed genes. ATACseq revealed SOX9 binding sites were associated with regions of dynamic chromosome accessibility      resulting from exposure to oxLDL. EndMT phenotype and gene expression changes induced by oxLDL in vitro or high fat diet (HFD) in vivo was reversed by an shRNA knockdown of SOX9 or endothelial specific conditional knockout of Sox9 in murine models respectively. Finally, analysis of single cell RNA sequencing of human atherosclerotic aorta identified endothelial populations with mesenchymal characteristics overexpressing SOX9 when compared to controls. Overall, our findings support that in the atherogenic process, EndMT induced by oxLDL or HFD is driven by SOX9 and strongly affects progenitor function opening new avenues for targeting this process.

overall_design: RNA-sequencing of endothelial colony-forming cells (ECFCs; 4 donors per group) treated with oxLDL (25ug/ul)or vehicle for 5 days.

first: Jilai

last: Zhao

last: Sormani

first: Chenhao

last: Zhou

first: Kiarash

last: Khosrotehrani


title: Distinct roles of SOX9 in self-renewal of progenitors and mesenchymal transition of the endothelium

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

overall_design: Refer to individual Series


title: Gene expression profile at single cell level of human brain metastasis samples

city: Madrid

first: Elena

last: Piñeiro-Yáñez

address: Calle de Melchor Fernández Almagro, 3

country: Spain

institute: Spanish National Cancer Research Centre (CNIO)

summary: We used Chromium Fixed RNA Profiling to analyze the heterogeneity of astrocytes in brain metastasis at the single cell level

overall_design: Fresh patient biopsies were fixed, digested and processed with Chromium Chip/Single Cell Fixed RNA kit for Human Transcriptome (10X Genomics)

first: Neibla

last: Priego


title: METTL3 Facilitates Hypertrophic Scar Fibrosis by Promoting pri-miRNA-31 Maturation and ZBTB20 Silencing in m6A-dependent Manner [RNA-Seq]

city: Shanghai

first: Qirui

last: Wang

state: Shanghai

address: 639 Zhi Zao Ju Road

department: Department of Plastic and Reconstructive Surgery

institute: Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine

summary: To clarify the role of METTL3-mediated m6A modification in hypertrophic scars (HS), we conditionally knocked out the m6A methyltransferase METTL3 in HS-derived dermal fibroblasts (HSFBs). We then performed RNA-seq analysis on normal control and Mettl3 knockdown HSFBs.

overall_design: We performed RNA-seq analysis on normal control and Mettl3 knockdown HSFBs.

first: Jialin

last: Hou

first: Siyi

last: Zeng

first: Xue

first: Danru

first: Renpeng


title: METTL3 Facilitates Hypertrophic Scar Fibrosis by Promoting pri-miRNA-31 Maturation and ZBTB20 Silencing in m6A-dependent Manner


title: Gene expression changes induced by overexpression of PJA2 in SW-480 cells

city: Nanjing

first: Zhihao

last: Chen

address: Jiangsu Province

institute: NANJING MEDICAL UNIVERSITY

summary: To investigate the impact of PJA2 on the progression of colorectal cancer, we employed lentiviral constructs to establish PJA2-overexpressing SW-480 cell lines. Subsequently, we conducted gene expression profiling analysis using RNA-seq data obtained from both vector SW-480 cells and PJA2-overexpressing SW-480 cells.

overall_design: Comparative gene expression profling analysis of RNA-seq data for SW-480 cells and its overexpression derivative (OE PJA2)

first: Peng

last: Yang

first: Chaofan

last: Peng

first: Chi

last: Jin

first: Tuo

first: Ye

first: Yueming

last: Sun


title: Effect on chromatin accessibility of knock down of Scrambled control or Kdm3b on wild-type or Idh2 R140Q TF-1 cells

city: New York

first: Richard

last: Koche

state: New York

institute: Memorial Sloan Kettering Cancer Center

summary: Clonal hematopoiesis (CH) is a common premalignant state in the blood and confers an increased risk of blood cancers and all-cause mortality. Identification of therapeutic targets in CH has been hindered by the lack of an ex vivo platform amenable for studying primary hematopoietic stem and progenitor cells (HSPCs) with and without CH-associated mutations. Here, we utilize an ex vivo co-culture system of primary murine HSPCs with bone marrow endothelial cells to perform CRISPR/Cas9 screens and identify vulnerabilities specific to mutant versus wild-type HSPCs. Our data reveal that loss of the histone demethylase family members KDM3B and JMJD1C specifically reduces the fitness of IDH2- and TET2- mutant HSPCs, but not wild-type HSPCs, including in TET2-mutant human HSPCs and Tet2-mutant murine leukemia cells with cooperating disease alleles. KDM3B loss in mutant cells leads to decreased expression of critical cytokine receptors including MPL, rendering mutant HSPCs preferentially susceptible to inhibition of downstream JAK2 signaling. Our study provides a scalable platform to identify genetic dependencies in mutant HSPCs and reveals an atlas of dependencies in normal and CH-mutant HSPCs. This study nominates an epigenetic regulator and an epigenetically regulated receptor signaling pathway as genotype-specific therapeutic targets in a subset of CH and myeloid malignancies.

overall_design: We cultured TF-1 cells with and without an isogenic IDH2 R140Q mutation, infected them with lentivirus enconding an shRNA against scrambled control or kdmb, and sorted infected cells after 6 days.

first: Michael

last: Waarts

first: Ross

last: Levine


title: Lymphocyte activation gene 3 (Lag3) supports Foxp3+ Treg cell function by restraining c-Myc-dependent aerobic glycolysis

city: Chicago

first: Booki

last: Min

state: IL

address: 300 E. Superior Street

department: Microbiology-Immunology

institute: Northwestern University

summary: Lymphocyte activation gene 3 (Lag3) has emerged as the next-generation immune checkpoint molecule due to its ability to inhibit effector T cell activity. Foxp3+ regulatory T (Treg) cells, a master regulator of immunity and tolerance, also highly express Lag3. While Lag3 is thought to be necessary for Treg cell-mediated regulation of immunity, the precise roles and underlying mechanisms remain largely elusive. In this study, we report that Lag3 is indispensable for Treg cells to control autoimmune inflammation. Utilizing a newly generated Treg cell specific Lag3 mutant mouse model, we found that these animals are highly susceptible to autoimmune diseases, suggesting defective Treg cell function. Genome wide transcriptome analysis further uncovered that Lag3 mutant Treg cells upregulated genes involved in metabolic processes. Mechanistically, we found that Lag3 limits Treg cell expression of Myc, a key regulator of aerobic glycolysis. We further found that Lag3-dependent Myc expression determines Treg cells’ metabolic programming as well as the in vivo function to suppress autoimmune inflammation. Taken together, our results uncovered a novel function of Lag3 in supporting Treg cell suppressive function by regulating Myc-dependent metabolic programming.

overall_design: Comparative gene expression profiling analysis of RNA-seq data of regulatroy T cells and its Lag3 knockout phenotype.

first: Dongkyun

last: Kim

first: Giha

first: Rongzhen

last: Yu

first: Juyeun

last: Lee

first: Sohee

first: Kevin

last: Qiu

first: Elena

last: Montauti

first: Deyu

last: Fang

first: Navdeep

middle: S

last: Chandel

first: Jaehyuk

last: Choi


title: Effect on gene expression of knock down of Scrambled control or Kdm3b on wild-type or Idh2 R140Q TF-1 cells


title: Distinct roles of SOX9 in self-renewal of progenitors and mesenchymal transition of the endothelium [ATAC-seq]

first: laura

last: sormani

overall_design: ATAC-sequencing of endothelial colony-forming cells (ECFCs; 4 donors per group) treated with oxLDL (25ug/ul)or vehicle for 5 days.

first: Jilai

last: Zhao

first: Laura

last: Sormani

first: Chenhao

last: Zhou

first: Kiarash

last: Khosrotehrani


title: Perturb-seq against putative NAFLD target genes in differentiated HepaRG cells

city: Stockholm

first: Peter

middle: Daniel

last: Saliba Gustafsson

state: Stockholm

address: C2:94, Karolinska Universitetssjukhuset Huddinge

department: Medicine, Huddinge

country: Sweden

institute: Karolinska Institutet

summary: We used Perturb-seq to characterize the role of putative NAFLD genes prioritized from molecular human genetic studies in differentiated HepaRG cells.

overall_design: dCas9-expressing HepaRG cells were harvested at Day 42 after transduction with lentiviral vectors carrying sgRNAs targeting multiple putative NAFLD genes in a pooled format. Cells were analysed using the feature barcodes technology from 10X Genomics allowing for pooled CRISPRi screens.

last: Gustafsson

first: Johanne

last: Justesen

first: Joshua

last: Knowles


title: Induction ofsteatosis in primary human hepatocytes manifests into hepatic pathophysiology of metabolic dysfunction-associated liver disease

city: Nuthetal

first: Pascal

last: Gottmann

address: Arthur-Scheunert-Allee 114-116

country: Germany

institute: Deutsches Institut für Ernährungsforschung

summary: Background & Aims:The prevalence of metabolic dysfunction-associated liver disease (MASLD) has been strongly increasing over the last decades. As MASLD is often associated with more severe disease states, such as metabolic dysfunction-associated steato-hepatitis (MASH), insulin resistance and type 2 diabetes, the increasing number of patients will contribute to an epidemic rise in metabolic abnormalities and end stage liver diseases. Despite the high demand, there are still no FDA-approved pharmaceutical treatments for MASLD due to low efficacy and high toxicity of the targets pursued, indicating a lack of appropriate pre-clinical models for selection and validation. To facilitate earlier stop-go decisions for continued target development, we have established and extensively characterized a primary human steatoticin vitrohepatocyte model system that could guide treatment strategies for MASLD. Methods:Cryopreserved primary human hepatocytes of different donors varying in sex and ethnicity were cultured with free fatty acids (FFA) in a 3D collagen sandwich system for 7 days and the development of MASLD was followed by assessing classical hepatocellular functions. As aproof-of-concept, the effects of Firsocostat (GS-0976) onin vitroMASLD phenotypes were evaluated. Results:Incubation with FFA induced known MASLD pathologies, including steatosis, insulin resistance, mitochondrial dysfunction, inflammation and alterations in prominent human gene signatures similar to patients with MASLD/MASH, indicating the recapitulation of human MASLD in this system. As the application of Firsocostat rescued clinically observed fatty liver disease pathologies, it highlights the ability of thein vitrosystem to test drug efficacy and potentially characterize their mode of action. Conclusions:Altogether, our human MASLDin vitromodel system could guide the development and validation of novel targets and drugs for the treatment of MASLD.

overall_design: RNA-Seq of primary hepatocytes from 5 patients (3 reps) cultured with and without FFA for seven days in a 3D collagen sandwich system was performed.

first: Yun

last: Kwon

first: Surui

last: Wang

first: Joel

last: Tissink

first: Karsten

last: Motzler

first: Revathi

last: Sekar

first: Wiebke

last: Albrecht

first: Cristina

last: Cadenas

first: Jan

last: Hengstler

first: Annette

last: Schürmann

first: Anja

last: Zeigerer


title: METTL3 Facilitates Hypertrophic Scar Fibrosis by Promoting pri-miRNA-31 Maturation and ZBTB20 Silencing in m6A-dependent Manner [MeRIP-Seq]

first: Qirui

summary: To clarify the role of METTL3-mediated m6A modification in hypertrophic scars (HS), we conditionally knocked out the m6A methyltransferase METTL3 in HS-derived dermal fibroblasts (HSFBs). We then performed MeRIP-seq analysis on HSFBs.

overall_design: We performed MeRIP-seq analysis to screen for potential m6A-modified pri-miRNAs in HSFBs.

first: Jialin

last: Hou

first: Siyi

last: Zeng

first: Xue

first: Danru

first: Renpeng

last: Zhou


title: Effect on gene expression of knock-out of Rosa or Kdm3b on wild-type or Idh2 R140Q hematopoetic stem and progenitor cells

first: Richard

last: Koche

overall_design: We co-cultured hematopoetic stem and progenitor cells from either wild-type or Idh2 R140Q mutant mice, infected them with lentivirus encoding sgRNAs against Rosa or Kdm3b, and co-cultured infected cells with bone marrow endothelial cells. Infected lineage negative cKIT positive cells were sorted after 7 days and used for RNA-sequencing.

first: Michael

last: Waarts

first: Ross

last: Levine


title: Role of REDD1 in mouse skin response to proinflammatory 12-(O)-tetra-decanoyl-acetate (TPA) and anti-inflammatory glucocorticoid fluocinolone acetonide (FA)

first: Irina

last: Budunova

address: 303 E. Chicago Ave, WARD 9-015

department: Dermatology

summary: We assessed the role of REDD1 gene in skin inflammation using TPA-induced psoriasiform dermatitis in REDD1 KO mice. We also assessed the anti-inflammatory effect of glucocorticoid fluocinolone acetonide in these animals

overall_design: REDD1 KO and wild type (WT) female in the same F1 C57Bl x 129 genetic background mice were shaved on the back, randomly divided in 3 groups and treated topically with 1). Control (Vehicle control, acetone, 200ul/animal) ; 2). 12-O-tetradecanoyl-acetate (TPA, 4 ug in 200 ul of acetone); or 3) TPA+FA. Some TPA-treated animals were pretreated with glucocorticoid fluocinolone acetonide (FA, 2 ug in 200 ul acetone) one hr before TPA application . Skin was harvested in 24 hrs after TPA application, epidermis was mechanically isolated (by scraping), total RNA was isolated and subjected to RNA-sequencing.

first: Gleb

last: Baida


title: SHY2-dose dependent transcriptome analysis

city: Seoul

first: Hee-Seung

last: Choi

state: Seoul

address: 1 Gwanak-ro, Gwanak-gu, Seoul, School of Biological Sciences,, room 506, bdg 502

department: School of Biological Sciences

country: South Korea

institute: Seoul National University

summary: To identify genes involved in auxin respone depending on SHY2 level, we profiled the transcriptome of plants induced differential level of SHY2 by 0.1 nM or 1 μM dexamethosone treatment. For this we used the roots of seedlings expressing non-degradable version of the Aux/IAA3 (shy2-1) fused to the glucocorticoid receptor domain (shy2-1:GR) under the root hair cell specific EXPA7 promoter. The nuclear translocation of the non-degradable Aux/IAA, specifically in root hair cells, that acts as a biomodal switch in regulation of target genes transcription both positively and negatively.

overall_design: Cold treated seeds of the pE7:shy2-1:GR lines were germinated on 0.5 X MS medium with hygromycin (30 μg/ml) and transferred 2.5 d after germination under 16 h light / 8 h dark conditions to plates containing 0.1 nM DEX or 1 μM DEX for 24h. The plates containing EtOH were used for control experiments.

first: Hyung-Taeg

last: Cho

first: Min-Soo

last: Lee


title: Intronic miR342 is a master regulator of cellular glycolysis in Foxp3+ regulatory CD4 T cells

first: Booki

last: Min

summary: Foxp3+ regulatory T (Treg) cells are a subset of CD4 T cells that play a potent and indispensable role in regulating immunity and tolerance. The precise mechanisms by which Treg cells mediate such functions have extensively been explored, and there are many cellular and molecular factors that are instrumental for adequate Treg cell functions. miRNAs, small non-coding RNA molecules, are one of the factors capable of modulating Treg cell functions. In this study, we report that miR-342 is essential for Treg cells to mitigate autoimmune inflammation in the central nervous system. Utilizing novel mouse models with Treg cell-specific miR-342 deficiency or overexpression, we demonstrate that miR-342 expression in Treg cells, while dispensable for immune homeostasis at steady-state conditions, is necessary for Treg cells to control inflammatory responses. Mechanistically, we found that Treg cells deficient in miR-342 display dysregulated metabolic programming, elevated glycolysis and decreased oxidative phosphorylation, a metabolic phenotype associated with functionally defective Treg cells. Collectively, our findings uncover that miR-342 may serve a master regulator of Treg cell metabolism and functions.

overall_design: Comparative gene expression profiling analysis of RNA-seq data of regulatroy T cells and its microRNA mir-342 knockout phenotype.

first: Rongzhen

last: Yu

first: Silei

last: Li

first: Sohee

last: Kim

first: Giha

first: Li-fan

last: Lu

first: Dongkyun


title: Innate protection against intrarectal SIV acquisition by a live Simian HIV vaccine

city: Bethesda

first: Thomas

middle: Joshua

last: Meyer

state: Maryland

address: 41 Center Drive, Building 41, Room B620

department: National Cancer Institute

institute: National Institutes of Health

summary: Identifying immune correlates of protection is a major challenge in AIDS vaccine development. Anti-Envelope antibodies have been considered critical against SIV/HIV acquisition. Here, we evaluated the efficacy of a SHIV vaccine against SIVmac251 challenge, where the role of antibody was excluded as there was no cross-reactivity between SIV and SHIV Envelope antibodies. After eight low-dose rectal challenges with SIVmac251, 12 SHIV-vaccinated animals demonstrated 83% efficacy, compared to six naïve controls, suggesting protection could be achieved in the absence of anti-Envelope antibodies. Interestingly, CD8+ T cells (and some NK cells) were not essential for the preventing viral acquisition, as none of the CD8-depleted macaques was infected by SIVmac251 challenges. Initial investigation of protective innate immunity revealed that protected animals had elevated pathways related to platelet aggregation/activation, and reduced pathways related to interferon and responses to virus. Moreover, higher expression of platelet factor 4 (PF4) on circulating platelet-leukocyte aggregates was associated with reduced viral acquisition. Our data highlighted the importance of innate immunity and may provide new opportunities for novel HIV vaccines or therapeutic strategy development.

overall_design: We then performed gene expression profiling analysis using data obtained from RNA-seq of macaque PBMC samples from three different groups: Naïve-infected group, SHIV vaccinated-infected group and SHIV-vaccinated-protected group.

first: Joshua

first: Yongjun

last: Sui

first: Jay

last: Berzofsky

first: Maggie

last: Cam


title: The role of histone lysine demethylases in a genetically defined subset of clonal hematopoiesis

first: Richard

last: Koche

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Chinmo maintains adult stem cell sex identity by directly regulating Doublesex and Insulin signaling

city: Shenzhen

first: Qing

last: Ma

address: 1068 Xueyuan Avenue, Shenzhen University Town, Shenzhen, P.R.China

institute: Shenzhen Institutes of Advanced Technology


title: Gene expression profile at single cell level of brain metastasis-associated astrocytes

first: Manuel

last: Valiente

address: Melchor Fernández Almagro, 3

institute: CNIO

summary: We used single cell RNA sequencing (scRNA-seq) to analyze the heterogeneity of astrocytes in brain metastasis

overall_design: Astrocytes from naive brains and brains from intracranially injected mice with B16/f10-BrM cell line were enriched in the samples using anti-ACSA2 microbeads and analyzed using scRNAseq

first: Neibla

last: Priego

first: Carolina

last: Hernández-Oliver


title: Effect of STAT3-/+ secretome in CD8+ T cells in vitro culture

summary:
To investigate the effect of the addition of STAT3-/+ astrocyte derived-conditioned medium in CD8+ T cells previously activated in vitro
We then performed gene expression profiling analysis using data obtained from RNA-seq of 3 CD8+ T cells cultures of STAT3- conditon and 3 CD8+ T cells cultures of STAT3+ conditon

overall_design: Comparative gene expression profiling analysis of RNA-seq data for CD8+ T cells + STAT3-/+ conditioned medium


title: RNA-Seq analysis of bladder cancer cells after protodioscin treatment

city: Taichung

first: Wei-Chung

last: Cheng

address: No.91, Hsueh-Shih Road

department: Research Center for Tumor Medical Science

country: Taiwan

institute: China Medical University

summary: We performed RNA-seq analysis using different bladder cancer cell lines (5637 and T24) after protodioscin treatment. We reported that protodioscin regulates MAPK pathway-associated genes in 5637 and T24 cells. Protodioscin induces apoptosis and suppresses cell cycle progression, cell migration and invasion in 5637 and T24 cells.

overall_design: Examination of mRNA profile of different bladder cancer cell lines (5637 and T24) under protodioscin treatment.

first: Chia-Yang


title: Adverse Impact of Female Reproductive Signaling on Age-Dependent Neurodegeneration After Mild Head Trauma in Drosophila

status: Public on Aug 29 2024

city: Atlanta

first: James

last: Zheng

state: GA

address: 615 Michael Street

institute: Emory University

summary: Environmental insults, including mild head trauma, significantly increase the risk of neurodegeneration. However, it remains challenging to establish a causative connection between early-life exposure to mild head trauma and late-life emergence of neurodegenerative deficits, nor do we know how sex and age compound the outcome. Using aDrosophilamodel, we demonstrate that exposure to mild head trauma causes neurodegenerative conditions that emerge late in life and disproportionately affect females. Increasing age-at-injury further exacerbates this effect in a sexually dimorphic manner. We further identify Sex Peptide (SP) signaling as a key factor in female susceptibility to post-injury brain deficits. RNA sequencing highlights changes in innate immune defense transcripts specifically in mated females during late life. Our findings establish a causal relationship between early head trauma and late-life neurodegeneration, emphasizing sex differences in injury response and the impact of age-at-injury. Finally, our findings reveal that reproductive signaling adversely impacts female response to mild head insults and elevates vulnerability to late-life neurodegeneration.

overall_design:
To gain molecular insights into the sexually dimorphic responses to mild head trauma and the progressive development of late-life neurodegeneration, we conducted RNA sequencing on freshly dissected brain tissue from flies with and without vmHT exposure. Here, we focused on flies injured on day 3 after eclosion (D3Inj) since under this injury condition, only females mated with wildtype males exhibited behavioral and pathological deficits late in life (on D45, see Figure 3).  Four distinct D3Inj groups were chosen for RNA extraction and sequencing: wildtype males, females mated with wildtype males, virgin females, and females mated with SP0 males (Figure 8A). We assessed transcriptomic changes in the brain at two timepoints; 1-day post-injury (1dpi) for acute changes and 6-weeks post-injury (6wkpi) for chronic changes. Principal Component Analyses revealed that sex accounted for a large portion of variance in sequencing dataset, but the effects of vmHT were not immediately clear in either timepoints (Figure 8B). This observation reiterated the very mild nature of our injury paradigm. To further identify transcriptomic drivers of neurodegeneration after injury, we grouped the dataset by time post-injury, sex, and reproductive condition.
Injured and sham flies were injured at 3 days post-eclosion. At 1 day and 6 weeks post-injury, brains from males, wildtype-mated females, virgin females, and SP0-mated females were collected, resulting in 16 conditions, each condition containing three independent cohorts of n=40 flies. RNA from brain tissue was extracted by TRIzol®/Chloroform extraction (ThermoFisher, 15596026) and further clean-up with the Zymo DNA Clean & Concentrator-5 kit (D4004) with on column DNase treatment (ThermoFisher, AM1907). Once obtained, RNA samples were transferred on dry ice to Admera Health for library preparation and sequencing. Total RNA integrity was checked by BioAnalyzer. QIAseq FastSelect rRNA Fly + NEB Ultra II Directional kit was used to deplete rRNA and prepare stranded cDNA libraries. Samples were sequenced on NextSeq High Output Flow Cell (Illumina) for 150 cycles to generate a total of 60M paired-ended, 101 base-pair reads.

first: Changtian

last: Ye

first: Ryan

last: Ho

first: Kenneth

last: Moberg

middle: Q


title: Molecular Insights into PCB Neurotoxicity: Comparing Transcriptomic Responses across Dopaminergic Neurons, Population Blood Cells, and Parkinson's Disease Pathology

city: Maastricht

first: Julian

last: Krauskopf

address: Universiteitssingel 50

country: Netherlands

institute: Maastricht University

summary: In this study, we conducted a comprehensive assessment of the transcriptomic responses in iPSC-derived dopaminergic neurons upon exposure to different concentrations (0.01 μM, 0.5 μM, and 10 μM) of PCBs. Specifically, we focused on PCB-180, a congener known for its relatively high accumulation in human brains. The exposure durations were 24 hours and 74 hours, allowing us to capture both short-term and more prolonged effects on gene expression patterns.

overall_design: First, we differentiated human iPSCs into dopaminergic neurons. Cells were examined using temporal transcriptomics analysis at various time-points after 24h of exposure to the potential neurotoxin PCB-180.


title: Novel function of U7 snRNA in the repression of HERV1/LTR12s and lincRNAs in human cells

city: Poznan

first: Michał

last: Szcześniak

address: Uniwersytetu Poznanskiego 6

department: Institute of Human Biology and Evolution

country: Poland

institute: Adam Mickiewicz University

summary: U7 snRNA is part of the U7 snRNP complex, required for the 3' end processing of replication-dependent histone pre-mRNAs in S phase of the cell cycle. Here, we show that U7 snRNA plays another function in inhibiting the expression of a subset of human endogenous retroviruses of HERV1/LTR12 class and LTR12-containing long intergenic noncoding RNAs (lincRNAs), both bearing sequence motifs that perfectly match the 5’ end of U7 snRNA. We demonstrate that U7 snRNA inhibits HERV1/LTR12 and lincRNA transcription and propose a mechanism in which U7 snRNA hampers the binding/activity of the NF-Y transcription factor to CCAAT motifs within LTR12 elements. Thereby, U7 snRNA plays a protective role in maintaining the silencing of deleterious genetic elements in selected types of cells. In turn, the expression of U7-dependent HERV1/LTR12s and lincRNAs seems to be relevant during early spermatogenesis in testis, where their synthesis is highly upregulated.

overall_design: To investigate novel functions of U7 snRNA in human cells, we depleted U7 snRNA using a chimeric antisense oligonucleotide (ASO) in HEK293T cells. We then performed gene expression profiling analysis using data obtained from RNA-seq of 3 biological replicates of HEK293T transfected with the control ASO or ASO targeting U7 snRNA.

first: Katarzyna

middle: D

last: Raczynska

first: Michal

last: Szczesniak


title: The Exserohilum turcicum effector EtEC81 reprograms alternative splicing in maize and activates immunity

city: Beijing

first: Haiyue

last: Yu

address: Yuanmingyuanxilu No.2

institute: Chinese Academy of Agricultural Sciences

summary: Some pathogen-derived effectors reprogram mRNA splicing in their host plant to regulate plant immune responses. The fungus Exserohilum turcicum is the causal agent of northern corn leaf blight, a damaging maize (Zea mays) disease. However, the low efficiency of genetic transformation of E. turcicum has hampered research on its effectors and whether E. turcicum effectors interfere with RNA splicing remained unknown. Here, using an alternative splicing (AS) reporter system, we identified the secreted protein EtEC81 (Exserohilum turcicum effector 81), which modulates the AS of maize pre-mRNAs and negatively regulates the pathogenicity of E. turcicum. EtEC81 physically interacts with EtEC81-interactiNG protein 1 (ZmEIP1), which associates with maize spliceosome components, regulating AS and positively regulating the defense response against E. turcicum. EtEC81 binding further enhanced the effect of ZmEIP1 on AS. Transcriptome analysis revealed 119 common genes with altered AS in maize plants transiently overexpressing ZmEIP1 or EtEC81, suggesting that these factors cause the mis-regulation of cellular activities and thus induce immune responses. We used RT-qPCR to verify representative AS events in the plants transiently overexpressing ZmEIP1 and EtEC81. Together, our results suggest that the EtEC81 effector targets ZmEIP1 to reprogram pre-mRNA splicing in maize.

overall_design: The fourth leaves of transgenic maize plants with virus-induced overexpression of EtEC81 or GFP and T2 transgenic maize plants overexpressing ZmEIP1 and WT plants were collected for total RNA extraction and RNA-seq library construction. The RNA-seq libraries were sequenced on an Illumina HiSeq platform in paired-end mode with a read length of 150 bp (Novogene, Beijing, China).  The raw reads were filtered using ht2-filter in the HTQC package (1.92.1) with default parameters to remove low-quality reads. The filtered reads were compared to the reference maize genome (Zea mays RefGen_V4) using TopHat (2.0.11) to remove host genome sequences. The mapped reads were assembled, and fragments per kilobase of transcript per million mapped reads (FPKM) values were calculated using Cufflinks. The Cufflinks output GTF file and the TopHat output BAM file were loaded into rMATS.3.2.2 to identify differential AS events. AS events with a P value < 0.05 were considered to be differential AS events.

first: Xuetao

last: Shi

last: Ning

first: Hongliang

last: Wu

first: Jie

last: Mei

first: Xiaoyu

last: Gu

first: Hongchun

last: Ruan

first: Mingcai

last: Zhang

first: Zhiqiang

last: Li

first: Wende

last: Liu


title: In vitro generation of CD8 Trm cells

city: Melbourne

first: Luke

middle: C

last: Gandolfo

state: Victoria

address: 1G Royal Parade Parkville

department: Bioinformatics

institute: WEHI

summary: The precise molecular mechanisms controlling tissue resident T cell (Trm cell) development remain unclear. In our studies, we have found that the vitamin A metabolite all-trans retinoic acid (atRA) synergises with TGFβ to promote Trm cells. We further show that polyclonal activation of naive splenic CD8 T cells in the presense of IL-2, IL-15, TGFβ + atRA leads to the development of CD69+CD103+ Trm cells. We have examined the transcriptome of CD8 T cells under IL-2/15, IL-2/15/atRA, IL-2/IL-15/TGFβ and IL-2/IL-15/TGFβ/atRA conditions.

overall_design: RNA isolation was conducted on day 7 from cells cultured in either IL-2 + IL-15, IL-2 + IL 15 + atRA, IL-2 + IL-15 + TGFβ, or IL-2 + IL-15 + TGFβ + atRA

first: Laura

last: Mackay

first: Grace

last: Rodrigues

first: Colby

last: Zaph


title: Transcriptomics-informed pharmacology identifies epigenetic and cell cycle regulators as enhancers of AAV production

city: Berkeley

first: Joshua

last: Tworig

address: Weill Hall, Frank Schlessinger Way

department: Molecular and Cell Biology

institute: University of California, Berkeley

summary: Recombinant adeno-associated virus (rAAV) is a widely used viral vector for gene therapy. Despite its clinical efficacy, the manufacturing of rAAV faces challenges in productivity and quality, leading to limited availability. To address the growing demand, next-generation process development should be informed by a mechanistic understanding of the cellular response to rAAV. In this study, we performed transcriptomic analysis of 5 cell lines with variable capacities for rAAV production. Using an intersectional approach, we assessed the transcriptional response to rAAV production and compared transcriptional profiles between high and baseline producers to identify possible targets for enhancing production. Modulation of cell cycle and nucleosome components suggested a reduction of proliferative capacity and a shift toward DNA replication to support rAAV production. During rAAV production, we observed upregulation of several core functions including transcription, stress response, and Golgi and endoplasmic reticulum organization. Conversely, inhibitors of DNA-binding proteins and mitochondrial components were consistently downregulated during rAAV production. We next performed a drug connectivity analysis of these results and identified 5 classes of drugs predicted to enhance rAAV production. Validation studies confirmed the efficacy of HDAC and microtubule inhibitors. Our data uncover novel and previously identified pathways that may enhance rAAV productivity, potentially enabling a path to engineer improved processes and cell lines for higher yields and better quality rAAV production.

overall_design:
Using a triple plasmid transfection, we produced rAAV in 5 different HEK293 cell lines. We collected total RNA for transcriptome analysis at 3 time points: before transfection (0 hr), 24 hours, and 72 hours after transfection.
We identified differentially expressed genes between time points to determine how AAV production affects gene expression. We also compared gene expression profiles between 2 high and 2 baseline AAV-producing cell lines to identify genes that are associated with enhanced AAV production.

first: Francis

last: Grafton

first: Markus

last: Hörer

first: Christopher

last: Reid

first: Mohammad

last: Mandegar


title: Hepatocyte-specific loss of melanocortin 1 receptor disturbs fatty acid metabolism and promotes adipocyte hypertrophy

city: Helsinki

first: Bishwa

middle: Raj

last: Ghimire

address: Tukholmankatu 8

department: Institute for Molecular Medicine Finland

country: Finland

institute: University of Helsinki

summary: Melanocortins mediate their biological functions via five different melanocortin receptors (MC1R - MC5R). MC1R is expressed in the skin and leukocytes and is thus involved in the regulation of skin pigmentation and inflammatory responses. MC1R is also present in the liver and white adipose tissue, but its functional role in these tissues is unclear. This study aimed at determining the regulatory role of MC1R in fatty acid metabolism. Recessive yellow (Mc1re/e) mice, a model of global MC1R deficiency, and hepatocyte-specific MC1R deficient mice (Mc1r LKO) were fed a chow or Western diet for 12 weeks. The mouse models were characterized for body weight and composition, adipose tissue mass, adipocyte size, glucose metabolism and lipid metabolism. Furthermore, qPCR and RNA sequencing analyses were used to investigate gene expression profiles in the liver and adipose tissue. HepG2 cells and primary mouse hepatocytes were to study the effects of pharmacological MC1R activation. Chow- and Western diet-fed Mc1re/e showed increased liver weight, white adipose tissue mass and plasma triglyceride (TG) concentration compared to wild type mice. This phenotype occurred without significant changes in food intake, body weight, physical activity or glucose metabolism. Mc1r LKO mice displayed a similar phenotype characterized by larger fat depots, increased adipocyte hypertrophy and enhanced accumulation of TGs in the liver and plasma. In terms of gene expression, markers of de novo lipogenesis, inflammation and apoptosis were upregulated in the liver of Mc1r LKO mice, while enzymes regulating lipolysis were downregulated in white adipose tissue of these mice. In cultured hepatocytes, selective activation of MC1R reduced ChREBP expression, which is a central transcription factor for lipogenesis. Hepatocyte-specific loss of MC1R disturbs fatty acid metabolism in the liver and leads to an obesity phenotype characterized by enhanced adipocyte hypertrophy and TG accumulation in the liver and circulation.

overall_design:
In order to study the role of Melanocortin 1 recetpor in lipid metabolism, hepatocyte-specific melanocortin 1 receptor knockout mice (Mc1r LKO) mice were generated by breeding mice homozygous for a floxed Mc1r allele (Mc1rfl/fl) mice with transgenic AlbCre/+ mice
We kept these two different genotypes (Control & Mc1r LKO) in cholesterol-rich Western diet for 12 weeks and liver tissue samples were collected and total RNA was extracted for RNA-seq
We further analyzed the gene expression profiling using data obtained from RNA-seq of Control (n=4) and Mc1r LKO (n=4)


title: Single-cell characterization of differentiated HepaRG cells

first: Peter

last: Saliba Gustafsson

summary: We used scRNA-seq to characterise the differentiation status of HepaRG cells after applying standard differentiation protocols on these cells. For downstream Perturb-seq we also characterised two cell lines of different genotype; Wt and cells transduced with dCas9-KRAB.

overall_design: HepaRG cells were harvested in their proliferative state at Day 5 of culturing, after differentiation at Day 28 and after differentiation at Day 42 to control if cells can be kept in their differentiated state. Both Wt and dCas9 cells were captured. Lastly, dCas9-expressing HepaRG cells were transduced with lentiviral vectors carrying sgRNA in a pooled format, which were analysed using the feature barcodes technology from 10X Genomics allowing for pooled CRISPRi screens.

first: Johanne

last: Justesen

last: Knowles


title: Effect of 8-weeks of chronic jet lag lighting (CJL) on gene expression in the livers of Eu-MYC mice

city: La Jolla

first: Rebecca

last: Mello

state: California

address: 10550 North Torrey Pines Road

institute: Scripps Research

summary: Background: Disruption of natural diurnal light cycles, such as that experienced by shift workers, is linked to enhanced cancer incidence. Several mouse models of cancer have been shown to develop more severe disease when exposed to irregular light/dark cycles, further supporting the connection between circadian disruption and increased cancer risk. Cryptochrome 2 (CRY2), a repressive component of the molecular circadian clock, facilitates the turnover of the oncoprotein c-MYC, one mechanism that may link the molecular clock to tumorigenesis. In Eμ-MYC mice, which express transgenic c-MYC in B cells and develop aggressive lymphomas and leukemia, global Cry2 deletion reduces overall survival and enhances tumor formation. Lighting conditions that mimic the disruption experienced by shift workers dampen Cry2 transcripts in peripheral tissues of C57BL/6J mice. Although it is much milder than homozygous deletion of Cry2, we hypothesized that reduced Cry2 rhythmicity could alter MYC protein accumulation and contribute to enhanced cancer risk caused by circadian disruption. We decided to test this hypothesis in the context of MYC-driven lymphoma. Methods: We housed Eμ-MYC mice in light-tight boxes set to either the control (continuous cycles of 12-hours of light followed by 12-hours of dark, LD12:12) or chronic jetlag (eight-hour light phase advances every two to three days, CJL) lighting conditions and assessed the impact of disrupted light cycles on overall survival and tumor formation in Eμ-MYC mice.  Results: Environmental disruption of circadian rhythms did not alter tumor location, tumor growth, or overall survival in female or male Eμ-MYC mice on the c57BL6 genetic background.  Conclusions: The dampened transcriptional rhythms of Cry2 caused by exposure to disruption of circadian light exposures is very different from the complete loss of Cry2 expression caused by genetic deletion. The lack of any phenotype caused by altered circadian gene expression patterns in contrast to the enhanced tumorigenesis caused by homozygous deletion of Cry2 suggests that the dosage of CRY2 may impact this model. Importantly, these findings indicate that the well-documented increase in cancer risk associated with circadian disruption arises from one or more mechanisms that are not recapitulated in this mouse model, and may be different in distinct tumor types.

overall_design: To confirm changes on the transcriptome in the Eu-MYC mice housed in CJL relative to those housed in normal lighting conditions


title: Time-Restricted Feeding Reduces Atherosclerosis in LDLR KO Mice but Not in ApoE Knockout Mice

first: Terry

last: Lin

address: 10010 N Torrey Pines Rd, RBIO-P

department: RBIO

institute: Salk Institute

summary: Investigating the mechanisms of time-restricted feeding that can prevent weight gain, hypercholesterolemia, and atherosclerosis in LDLR-KO mice.

overall_design: Livers of LDLR-KO mice fed ad libitum or time-restricted were sequenced in fed or fasted state.

first: Amandine

last: Chaix

first: Satchidananda

last: Panda


city: Debrecen

first: Laszlo

last: Nagy

address: Egyetem ter 1.

country: Hungary

institute: University of Debrecen

summary: The alveolar macrophages (AMs) as a component of the innate immunity of the lung play important role in the elimination of inhaled microbes and harmful agents. The transcription factor EGR2 is a known marker transcription factor of AMs but its exact epigenetic and transcriptomic effects have not been examined. In our study, we performed ATAC-seq, RNA-seq, and CUT&RUN in WT and EGR2 deficient alveolar macrophages to describe the mechanism of action and to predict potential direct target genes of EGR2. Clec7a is one of the targets of this transcription factor which is essential in the zymosan-induced inflammatory response. We further analyzed this process by applying in vivo mouse model. Our findings demonstrate that EGR2 is a key transcriptional activator, responsible for the intact protective program against different pathogens, especially fungi in AMs.

overall_design: CUT&RUN in wild type and EGR2 knock-out mouse alveolar macrophages were performed from C57BL6 background control and EGR2 myeloid-specific conditional knock-out mice. The mice were euthanized by isoflurane inhalation then we isolated the cells from bronchoalveolar lavage. We sorted the CD45+ and F4/80+ alveolar macrophages. We did not apply any treatment.

first: Dora

last: Bojcsuk

first: Zsuzsanna

last: Kolostyak


title: Transcriptomic profiling of isolated hepatic macrophage subsets (Kupffer cells, Monocyte-derived Macrophages and Monocytes) in common bile duct ligated (CBDL)-mice, mice with acute colitis (DSS), and combinations (CBDL+DSS)

city: Ghent

first: Lindsey

last: Devisscher

address: Corneel Heymanslaan 10

country: Belgium

institute: Ghent University

summary: In this study, we aimed to unravel the transcriptional profiles of specific hepatic macrophage subsets during cholestatic liver injury, acute colitis and when colitis is induced as a second hit on advanced cholestatic liver injury. Differentially expressed genes (DEGs) and corresponding pathways were determined via DESeq2 in R and STRING-App comparing groups or cell types as outlined in the 'overall design' section below. Our results highlight the heterogeneity of the liver macrophage (MF) pool and describe functional differences between MF subsets. Longitudinally, the CBDL-associated expression profiles of each macrophage subset remained mostly stable. CBDL-induced cholestatic liver injury was characterized by the emergence of Trem2/Spp1 expressing monocyte-derived macrophages (MoMFs). DSS-colitis as a second hit on cholestatic liver injury enhanced this phenotype of MoMFs.

overall_design: This RNA-Seq study investigates 3 cell types (KC, MoMF and Mo) isolated from livers of mice from 5 experimental groups (CBDL2w, CBDL6w, Sham6w, DSS and CBDL+DSS). We aimed to include 4 replicates per group, but for several specific samples, replicates had to be pooled. Differential gene expression was determined for each individual cell type i) comparing each injury group with the control group (Sham6w), ii) comparing cholestatic liver injury at onset with advanced injury (CBDL2w vs CBDL6w), iii) comparing advanced cholestatic liver injury as such with the same injury including acute colitis as a second hit (CBDL6w vs CBDL+DSS). We also determined differential gene expression between cell types within each experimental group.

first: Tim

last: Meese

first: Filip

last: Van Nieuwerburgh

first: Kevin

last: De Muynck


summary: The alveolar macrophages (AMs) as a component of the innate immunity of the lung play important role in the elimination of inhaled microbes and harmful agents. The transcription factor EGR2 is a known marker transcription factor of AMs but its exact epigenetic and transcriptomic effects have not been examined. In our study, we performed ATAC-seq and RNA-seq in WT and EGR2 deficient alveolar macrophages to describe the mechanism of action and to predict potential direct target genes of EGR2. Clec7a is one of the targets of this transcription factor which is essential in the zymosan-induced inflammatory response. We further analyzed this process by applying in vivo mouse model. Our findings demonstrate that EGR2 is a key transcriptional activator, responsible for the intact protective program against different pathogens, especially fungi in AMs.

overall_design: ATAC-seq in wild type and EGR2 knock-out mouse alveolar macrophages were performed from C57BL6 background control and EGR2 myeloid-specific conditional knock-out mice. The mice were euthanized by isoflurane inhalation then we isolated the cells from bronchoalveolar lavage. We sorted the CD45+ and F4/80+ alveolar macrophages. We did not apply any treatment.


title: FJ004: Dopaminergic subset specification as a model for molecular mechanisms of neuronal vulnerability in Parkinson's Disease

city: Utrecht

first: Marian

last: Groot Koerkamp

state: Utrecht

address: Heidelberglaan 25

department: Research

institute: Princess Maxima Center for Pediatric Oncology

summary: Mesodiencephalic dopaminergic (mdDA) neurons critically regulate locomotory behavior and emotion, and dysfunction is associated with psychiatric and neurodegenerative diseases, including Parkinson's Disease (PD). A pathological hallmark of PD is the specific degeneration of Substantia Nigra (SN) neurons, whereas Ventral Tegmental Area (VTA) neurons are relatively unaffected. Differential molecular programming of the developing SN versus VTA may underly this specific vulnerability, and indeed molecularly distinct mdDA subsets have been identified during development. Driven by the hypothesis that the molecular signature of the SN and VTA originates from differential developmental programming and position along the rostral/caudal axis, we performed genome-wide expression profiling of FACS-purified rostral versus caudal mdDA neurons. Here, we present the distinct transcriptomes of these mdDA subsets and demonstrate how, on a genome-wide scale: 1) transcription factor regulation relates to developmental position, 2) rostral and caudal mdDA neurons differ in their functional genome, 3) the molecular signature of rostral versus caudal mdDA neurons relates to the SN versus VTA molecular profile in adult mice and human and 4) human homologues of rostrally enriched genes are mostly downregulated in the SN of PD patients. Our analyses substantiate the relationship of developmental positions with the SN versus VTA distinction in adult, and suggest cross-species conservation of molecular programs. Driven by these observations, we present 69 target genes that are rostral enriched and downregulated in PD, and thus provide new leads for the investigation of both the complex coding of developing mdDA subsets and and the molecular mechanisms underlying subset-specific vulnerability in PD.

overall_design: RNA was  isolated  from FACS-sorted Pitx3(gfp) neurons. Three individual samplepools of Pitx3(gfp/+) were isolated and hybridized to one reference-samplepool of Pitx3(gfp/-). On  each  of  three microarray samples, dye swap was performed to correct dye effects.

first: Jesse

middle: V

last: Veenvliet

first: Lars

last: von Oerthel

first: Frank

last: Jacobs

first: Nina

middle: L

last: Haring

middle: J

first: Martijs

last: Jonker

first: Rob

last: Dekker

first: Marten

middle: P

last: Smidt


title: Single cell transcriptomics of human epidermis shows decreased expression of several structural genes associated with skin barrier development in aged skin

status: Public on Aug 28 2024

city: Vienna

first: Hannes

last: Kühtreiber

state: Vienna

address: Lazarettgasse 14

department: Department of Dermatology

country: Austria

institute: Medical University Vienna

summary: Epidermal keratinocytes are continuously dividing cells which, upon differentiation establish the outer skin barrier, the stratum corneum. In older age the epidermis becomes thinner, papillary protrusions flatten and the capacity for skin barrier repair decreases. Here we performed single-cell RNA sequencing of epidermal keratinocytes obtained by suction blisters from the volar forearm of younger (20-30) and older (65-70) healthy female individuals.

overall_design: Eight healthy women participated in this study – 4 in the younger age group (24, 24, 29,29 yrs) and 4 in the older age group (71,67, 66, 71 yrs).  All women in the older age group were menopaused for at least 10 years and were not taking hormonal substitution therapies. None of the women in the younger age group was taking oral contraceptives. All women in both groups were non-smokers, none had a history of skin disease, none were visiting tanning parlors.

first: Erwin

last: Tschachler

first: Michael

last: Mildner

first: Dragan

last: Copic


title: Acute myeloid leukemia with mixed phenotype is characterized by stemness features with limited lineage plasticity

city: New York

first: Jacob

middle: Lowell

last: Glass

state: NY

address: 1275 York Avenue

institute: Memorial Sloan Kettering Cancer Center

summary: Mixed phenotype (MP) in acute leukemias poses unique classification and management dilemmas and can be seen in entities other than de novo mixed phenotype acute leukemia (MPAL). Although WHO classification empirically recommends excluding AML with myelodysplasia related changes (AML-MRC) and therapy related AML (t-AML) with mixed phenotype (referred to as “AML-MP”) from MPAL, there is lack of studies investigating the clinical, genetic, and biologic features of AML-MP. We report the first cohort of AML-MP integrating their clinical, immunophenotypic, genomic and transcriptomic features with comparison to MPAL and AML without MP. Patients with AML-MP share similar clinical and genetic features to its AML counterpart but differs from MPAL. AML-MP harbors more frequent RUNX1 mutations than AML without MP and MPAL. RUNX1 mutations or complex karyotypes did not impact the survival of MPAL patients. Unsupervised hierarchal clustering based on immunophenotype identified biologically distinct clusters with phenotype/genotype correlation and outcome differences. Furthermore, transcriptomic analysis showed an enrichment for stemness signature in AML-MP and AML without MP as compared to MPAL. Lastly, MPAL but not AML-MP often switched to lymphoid only immunophenotype after treatment. Expression of transcription factors critical for lymphoid differentiation were upregulated only in MPAL, but not in AML-MP. Our study for the first time demonstrates that AML-MP clinically and biologically resembles its AML counterpart without MP and differs from MPAL, supporting the recommendation to exclude these patients from the diagnosis of MPAL. Future studies are needed to elucidate the molecular mechanism of mixed phenotype in AML.

overall_design: Multiparameter flow cytometry was performed on bone marrow aspirates at diagnosis. Briefly, up to 1.5 million cells from freshly drawn bone marrow aspirate were stained with 10-color panels, washed, and acquired on a Canto-10 cytometer (BD Biosciences, San Jose, CA). The results were analyzed with custom Woodlist software (generous gift of B. L. Wood, Children's Hospital Los Angeles). Cryopreserved bone marrow aspirate specimens collected at diagnosis from our institutional biospecimen bank were thawed and flow sorted into myeloid blasts (CD33+, CD7-) and T lymphoblasts (CD33-, CD7+)  using FACS-Aria Fusion cell sorter (BD Biosciences). Samples were prepared for RNA-seq using the SMARTer Stranded Total RNA-Seq Kit (Takara Bio) and sequenced at the MSKCC Integrated Genomics Core. FASTQ files were subsequently processed using the Center for Epigenomics Research pipeline consisting of adapter and quality trimming using TrimGalore! (Version 0.6.4), alignment to hg19 using STAR (version 2.7.10b) in two pass mode, and quantification using HTSeq (version 0.9.1).

first: Deepika

last: Dilip

first: Pallavi

last: Galera

first: Wenbin

last: Xiao


title: NAD+ precursors prolong survival and improve cardiac phenotypes in a mouse model of Friedreich’s Ataxia

city: Philadelphia

first: Joseph

last: Baur

state: PA

address: 3400 Civic Center Blvd

institute: University of Pennsylvania

summary: Friedreich’s ataxia (FRDA) is a progressive disorder caused by insufficient expression of frataxin, which plays a critical role in assembly of iron-sulfur centers in mitochondria. Individuals are cognitively normal, but display a loss of motor coordination and cardiac abnormalities. Many ultimately succumb to heart failure. Administration of nicotinamide adenine dinucleotide (NAD+) precursors has shown promise in mitochondrial myopathy and rodent models of heart failure, including mice lacking frataxin in cardiomyocytes. We studied mice with systemic knockdown of frataxin (shFxn), which display motor deficits and early mortality with cardiac hypertrophy. Hearts in these mice do not “fail” per se but become hyperdynamic with small chamber sizes. Data from an ongoing natural history study indicate that hyperdynamic hearts are observed in young individuals with FRDA, suggesting that the mouse model could reflect early pathology. Administering nicotinamide mononucleotide or riboside to shFxn mice increases survival, modestly improves cardiac hypertrophy, and limits increases in ejection fraction. Mechanistically, most of the transcriptional and metabolic changes induced by frataxin knockdown are insensitive to NAD+ precursor administration, but glutathione levels are increased suggesting improved antioxidant capacity. Overall, our findings indicate that NAD+ precursors are modestly cardioprotective in this model of Friedreich’s Ataxia and warrant further investigation.

overall_design: These are heart sampels from doxycycline-inducible transgenic mice with a short hairpin against frataxin mRNA. Mice were treated with nicotinamide riboside or nicotinamide mononucleotide as indicated for 16-20 weeks after loss of frataxin. There were at least 4 biological replicates per treatment group. We were exploring whether treatment would improve the cardiac health or extend lifespan of mice

first: Caroline

middle: E

last: Perry

first: David

middle: R

last: Lynch


title: Identification of miRNAs Related to Osteoporosis by High-throughput Sequencing

city: Guangzhou

first: Haolin

last: Yang

address: The Third Affiliated Hospital of Guangzhou University of Chinese Medicine

country: China

type: Non-coding RNA profiling by high throughput sequencing

summary: Osteoporosis is a significant health concern, and the role of microRNAs (miRNAs) in cell growth and development regulation is well recognized. High-throughput sequencing technology is widely employed in current research. This study aimed to identify and validate miRNAs associated with osteoporosis. Bone specimens were collected from patients with osteoporosis (n=3) and without osteoporosis (n=3). High-throughput sequencing was utilized to screen for miRNAs, followed by analysis using volcano maps, Wayne maps, gene ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The identified miRNAs were further confirmed using qRT-PCR. Sequencing analysis revealed 12 down-regulated and five upregulated miRNAs in osteoporosis. GO and KEGG analysis indicated the association of these miRNAs with bone metabolism. qRT-PCR results demonstrated a significant decrease in miR-140-5p, miR-127-3p, miR-199b-5p, miR-181a-5p, miR-181d-5p, and miR-542-3p (all P<0.05) in osteoporosis compared to controls, while miR-486-3p and miR-486-5p exhibited a significant increase (P<0.05). This study utilized high-throughput sequencing to identify differential miRNA expression in individuals with osteoporosis. Specifically, six miRNAs (miR-140-5p, miR-127-3p, miR-199b-5p, miR-181a-5p, miR-181d-5p, and miR-542) showed decreased expression, whereas two miRNAs (miR-486-3p and miR-486-5p) exhibited increased expression. The differential expression of these miRNAs may serve as predictive indicators, potentially aiding in the prognosis and management of osteoporosis.

overall_design:
Sample Type: Specimens were obtained from the femoral intertrochanteric area or vertebrae of six orthopedic surgery patients at Guangzhou University of Chinese Medicine Affiliated Orthopedic Hospital. The samples included bone specimens from individuals with a background of persistent pain in the lower back and legs, as well as individuals diagnosed with osteoporosis based on bone mineral density (BMC) or bone mineral density (BMD) levels.
Experimental Conditions: 1. Specimen Preservation: The collected specimens were treated with liquid nitrogen within 15 minutes of harvesting and preserved in an RNA Fixer Reagent at a temperature of -80°C until the RNA extraction process. 2. RNA Isolation: RNA quantity and quality assessment were performed by Novogene Co., Ltd. RNA degradation and contamination were examined on agarose gels, and the purity and concentration of RNA were determined using a Nano Photometer® spectrophotometer and a Qubit® RNA Assay Kit, respectively. 3. Library Preparation: The small RNA library was constructed using 3 μg of total RNA per sample. The NEBNext Multiplex Small RNA Library Prep Set for Illumina was used, and index codes were incorporated to assign sequences to individual samples. The library quality was assessed using the Agilent Bioanalyzer 2100 system. 4. Clustering and Sequencing: Index-coded samples were clustered using the TruSeq SR Cluster Kit, and sequencing was performed on an Illumina Hiseq 2500/2000 platform, generating 50-bp single-end reads.
Study Variables: 1. Differential Expression Analysis: DESeq and DEGseq packages were used to analyze differential expression between two conditions/groups, taking into account samples with and without biological replicates. Adjusted p-values and q-values were calculated to determine significant differential expression. 2. Target Gene Prediction: The psRobot tar in miRanda was utilized to predict the target genes of miRNAs. 3. Quantitative RT-PCR: qRT-PCR was performed using the Gene Amp PCR System 9700, and PCR primers are listed in Table 2. 4. Gene Ontology Enrichment Analysis: The differentially expressed miRNAs were subjected to gene ontology (GO) enrichment analysis to identify enriched biological processes and molecular functions. 5. KEGG Pathway Analysis: KEGG pathway analysis was conducted using the KOBAS program to evaluate the statistical enrichment of potential gene candidates. 6. Statistical Analysis: Statistical analysis was performed using the Mann-Whitney U-test or Wilcoxon signed-rank test. The results were presented as averages ± standard deviations (SD), and two-sided p-values less than 0.05 were considered statistically significant.

first: Jiachun

last: Huang

first: Shuang

last: Chai

first: Yanping

last: Lin

first: Hongxing

first: Zhihai

last: Zhang

first: Lei

last: Wan


title: The RNA-seq data in riboflavin-treated  alcoholic liver disease

city: Harbin

first: Yanan

last: Jiang

state: Heilongjiang Province

address: Health Road Street

institute: Harbin medical university

summary: Alcoholic liver disease (ALD) is a kind serious liver disease, which will develope into the cirrhosis, liver cancer and so on. The study results show that riboflavin has the protective effect against ALD. Then the study divides the C57BL/6 mice into the three groups that were Control (C), Alcohol, Alcohol with riboflavin (AR) groups respectively. And the study makes the mouse liver RNA sequencing (RNA-seq) to find the  differential expression mRNAs among three groups futher and does the related analysis in riboflavin-treated alcoholic liver disease.

overall_design: The study divides the C57BL/6 mice into the three groups that were Control (C), Alcohol, Alcohol with riboflavin (AR) groups respectively. And the study makes the mouse liver RNA sequencing (RNA-seq) to find the  differential expression mRNAs among three groups futher.To investigate the protective effect of riboflavin against ALD futher.

first: Xiuyun

last: Shen

first: Hui

last: Sun


title: Transcriptomics integrated with metabolomics reveals partial molecular mechanisms of nutritional risk and neurodevelopment in children with congenital heart disease

city: Qingdao

first: Minglei

last: Gao

address: No. 217, Liaoyang West Road

institute: Qingdao Women and Children’s Hospital

summary: To explore molecular mechanisms affecting nutritional risk and neurodevelopment in children with congenital heart disease (CHD) by combining transcriptome and metabolome analysis. In this study, transcriptomic and metabolomic analysis of blood and serum samples from 26 children with CHD was performed to search for key DEGs and DEMs, and explore molecular mechanisms affecting nutritional risk and neurodevelopment in children with CHD.

overall_design: A total of 26 blood and serum samples from 3 groups of children with CHD (low nutritional risk combined with normal neurodevelopment (group A), low nutritional risk combined with neurodevelopmental disorders (group B) and high nutritional risk combined with normal neurodevelopment (group C)) were analyzed by transcriptome and metabolomics to search for differentially expressed genes (DEGs) and metabolites (DEMs). Functional analysis was conducted for DEGs and DEMs. Further, the joint pathway analysis and correlation analysis of DEGs and DEMs were performed.

first: Yang

first: Ping

first: Chang

last: Yuan

last: Su

first: Zipu

last: Li


title: RNA-seq in wild type and EGR2 knock-out mouse alveolar macrophages upon vehicle and zymosan treatment

first: Laszlo

last: Nagy

overall_design: RNA-seq in wild-type and EGR2 knock-out mouse alveolar macrophages were performed upon vehicle control and zymosan treatment. The mice were intranasally treated with 300 μg zymosan in 40 μl PBS+2 mM NaN3 for 6 hours and 24 hours or just with 40 μl PBS+2 mM NaN3 as vehicle control. The mice were euthanized by isoflurane inhalation then the cellular components were isolated through bronchoalveolar lavage. We sorted the CD45+ and F4/80+ alveolar macrophages.

first: Dora

last: Bojcsuk

first: Zsuzsanna

last: Kolostyak


title: HIV-induced RSAD2/Viperin supports sustained infection of monocyte-derived macrophages

first: Priyankara

last: Wickramasinghe

address: 3601 Spruce Street

country: USA

institute: The Wistar Institute

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

overall_design: Refer to individual Series


title: In Fanconi Anemia, impaired accumulation of bone marrow neutrophils during emergency granulopoiesis induces hematopoietic stem cell stress

city: Chicago

first: Priyam

last: Patel

address: 750 North Lake Shore Drive, Rubloff 11-146

department: Quantitative Data Science Core

institute: Northwestern University

summary: Fanconi Anemia (FA) is an inherited disorder of DNA-repair caused by mutation in one of 20+ interrelated genes that repair intra-strand DNA crosslinks and rescue collapsed or stalled replication forks. The most common hematologic abnormality in FA is anemia, but progression to bone marrow failure (BMF), clonal hematopoiesis, or acute myeloid leukemia (AML) may also occur. In prior studies, we found that Fanconi DNA-repair is required for successful emergency granulopoiesis; the process for rapid neutrophil production during the innate immune response. Specifically, Fancc-/- mice did not develop neutrophilia in response to emergency granulopoiesis stimuli, but instead exhibited apoptosis of bone marrow hematopoietic stem cells (HSCs) and differentiating neutrophils. Repeated emergency granulopoiesis challenges induced BMF in most Fancc-/- mice, with AML in survivors. In contrast, we found equivalent emergency granulopoiesis-induced neutrophilia in Fancc-/-Tp53+/- mice and wild type (WT) mice, without BMF in either. Since bone marrow neutrophil accumulation triggers termination of emergency granulopoiesis, we hypothesize neutrophilia protects Fancc-/-Tp53+/- bone marrow from the stress of sustained inflammatory physiology, as experienced by Fancc-/- mice.

overall_design: We find blocked neutrophil accumulation during emergency granulopoiesis in Wt, Fancc-/- and Fancc-/-Tp53+/- mice in vivo with Ly6G antibody injections. In these studies, emergency granulopoiesis was induced by injection of Alum (ovalbumin + aluminium chloride) with saline as a steady state control. Mice were assessed for bone marrow failure and leukemia during these treatments. Bone marrow from the mice was harvested at various time points after treatment. LSK cells were separated by flow cytometry and subjected to total RNA sequencing.

first: Elizabeth

last: Eklund

first: Liping

last: Hu

first: Pryiam