title: Trancsriptomic analyses reveal aberrant differentiation trajectory with WNT1 Loss-of-Function in type XV osteogenesis imperfecta

city: Shenzhen

first: Peikai

last: Chen

address: 1 Haiyuan Rd

institute: The University of Hong Kong - Shenzhen Hospital

summary: Osteogenesis imperfecta (OI) is a serious genetic bone disorder characterized by congenital low bone mass, deformity and frequent fractures. Type XV OI is a moderate to severe form of skeletal dysplasia caused by WNT1 mutations. In this cohort study from southern China, we summarized the clinical phenotypes of patients with WNT1 mutations and found the proportion of type XV patients was around 10.3% (25 out of 243) with diverse phenotypic spectrums. Functional assays indicated that mutations of WNT1 significantly impaired its secretion and effective activity, leading to moderate to severe clinical manifestations, porous bone structure and enhanced osteoclastic activities. Analysis of proteomic data from human skeleton indicated that the expression of SOST was dramatically reduced in type XV patients. Single-cell transcriptome data generated from human tibia samples revealed aberrant differentiation trajectory of skeletal progenitors and impaired maturation of osteocytes, resulting in excessive CXCL12+ progenitors and abnormal cell populations with adipogenic characteristics. The integration of multi-omics data from human skeleton delineates how WNT1 regulates the differentiation and maturation of skeletal progenitors, which will provide a new direction for the treatment strategy of type XV osteogenesis imperfecta and relative low bone mass diseases such as early onset osteoporosis.

overall_design: Bone marrow MSCs were transfected with pcDNA3.1-WNT1 or empty vectors. Two duplicates per group (transfected or not transfected groups) were used for transcriptomics.

first: Zhijia

last: Tan

first: Michael

last: To


title: Genomic and Transcriptomic Analyses of Chemical Hepatocarcinogenesis Aggravated by Loss of Oncoproteins in Hepatocytes [RNA-Seq]

status: Public on Aug 13 2024

city: SAN DIEGO

first: Xinyi

state: California

address: 9500 Gilman Dr, La Jolla

department: Pathology

country: USA

institute: University of California, San Diego

type: Expression profiling by high throughput sequencing

summary:
Background and Aims: The chemical carcinogen diethylnitrosamine (DEN) is often used to induce HCC in mice. Curiously, several labs have reported that the removal of oncoproteins from hepatocytes exacerbated DEN induced HCC, with mechanisms unknown. This study aimed at deciphering molecular mechanisms underlying the tumor suppressive effect of oncoproteins.

Approach and Results: We generated mutant mouse lines with hepatocyte-specific deletions of Met, Ptpn11/Shp2, Ikkβ, or ctnnb1/β-catenin and assessed DEN-induced tumorigenesis in the wild-type and mutant mice. To systematically examine genetic and molecular signaling alternations, we performed whole exome and RNA-sequencing on liver samples collected at the pre-cancer and established cancer stages. Although the mutational profiles of DEN-induced tumors were barely different in wild-type and mutant mice, oncoprotein ablation increased DEN-induced mutational burdens, especially in Shp2-deficient tumors. RNA-sequencing revealed multiple changes in signaling pathways, in particular, upregulated epithelial-mesenchymal transition, cell migration, and tumor metastasis, as well as downregulated small molecule metabolism that was affected by oncoprotein ablation. We identified key molecules and pathways that are associated with hepatic innate immunity and implicated in liver tumorigenesis. In addition, we unveiled markedly changed expression of a few miRNAs in the human HCC database.

Conclusions: The aggravation of DEN-induced HCC progression seen on oncoprotein ablation could be caused by common and distinct genomic and signaling alterations. This study reveals a new level of complexity in hepatocarcinogenesis and elucidates molecular mechanisms underlying tumor evolution and recurrence.

overall_design: Bulk RNA seq on liver samples collected from KO models and WT from various months ages

last: Wang

first: Yingluo

last: Liu

first: Shuo

last: Zhang

first: Jiemeng

first: Xiaoxue

last: Lin

first: Yan

last: Liang

first: Min

last: Zong

first: Kaisa

middle: L

last: Hanley

first: Jin

last: Lee

last: Karin

first: Gen-Sheng

last: Feng


title: Multi-Omic analyses reveal aberrant differentiation trajectory with WNT1 Loss-of-Function in type XV osteogenesis imperfecta

country: China

overall_design: With patients’ consents, the skeletal tissues were collected after tibia osteotomies from a patient (male, 8-year-old) carrying compound heterozygous mutations on WNT1 (c.C371T, p.T124M and c.G620A, p.R207H), and a patient with leg-length-discrepancy (male, 7-year-old) (Figure S1). The control patient was diagnosed with Proteus syndrome, with genetic testing confirming a pathogenic mutation (c.49G>A, p.E17K, sample frequency 13.3%) on AKT1. Single-cell RNA-seq reads happened to cover this region, where it showed a frequency of 6.3% (c.49G>A, 5 out of 79). Although it is not a perfect control, given the relatively normal bone density and histology, we assumed that it could be a useful reference for type XV OI.

middle: K


title: 50-year fire legacy regulates soil microbial carbon and nutrient cycling responses to new fire

status: Public on Aug 12 2024

city: Sevilla

first: Daniel

last: Revillini

address: Avenida Reina Mercedes 10

country: Spain

institute: IRNAS-CSIC

summary: Fire disturbances are becoming more common, more intense, and further-reaching across the globe, with consequences for ecosystem functioning. Importantly, fire can have strong effects on the soil microbiome, including community and functional changes after fire, but surprisingly little is known regarding the role of soil fire legacy in shaping responses to recent fire. To address this gap, we conducted a manipulative field experiment administering fire across 32 soils with varying fire legacies, including combinations of 1-7 historic fires and 1-33 years since most recent fire. We analyzed soil metatranscriptomes, determining for the first time how fire and fire legacy interactively affect metabolically-active soil taxa, the microbial regulation of important carbon (C), nitrogen (N) and phosphorus (P) cycling, expression of carbohydrate-cycling enzyme pathways, and functional gene co-expression networks. Experimental fire strongly downregulated fungal activity while upregulating many bacterial and archaeal phyla. Further, fire decreased soil capacity for microbial C and N cycling and P transport, and drastically rewired functional gene co-expression. Perhaps most importantly, we highlight a novel role of soil fire legacy in regulation of microbial C, N, and P responses to recent fire. We observed a greater number of functional genes responsive to the interactive effects of fire and fire legacy than those affected solely by recent fire, indicating that many functional genes respond to fire only under certain fire legacy contexts. Therefore, without incorporating fire legacy of soils, studies will miss important ways that fire shapes microbial roles in ecosystem functioning. Finally, we showed that fire caused significant downregulation of carbon metabolism and nutrient cycling genes in microbiomes under abnormal soil fire histories, producing a novel warning for the future: human manipulation of fire legacies, either indirectly through global change-induced fire intensification or directly through fire suppression, can negatively impact soil microbiome functional responses to new fires.

overall_design: Total RNA was extracted from soils that experienced Fire or did not (Control) using the Rneasy PowerSoil Total RNA Kit (Qiagen). Forty-eight samples that met the sequencing facility standards were then sent to the Joint Genome Institute (Walnut Creek, California, USA) for further quality control and sequencing on Illumina NovaSeq 6000 (S4 PE). Libraries were prepared using Qiagen FastSelect using a minimum of 100 ng RNA after ribo-depletion.

first: Michelle

last: Afkhami

first: Cristopher

last: Searcy


title: Identification of differentially expressed genes associated with dimethyl sulfoxide-enhanced mineralization in mouse MC3T3-E1 preosteoblast cells.

city: Toyama

first: Yoshiaki

last: Tabuchi

state: Toyama

address: 2630 Sugitani

department: Life Science Research Center

country: Japan

institute: University of Toyama

summary:
Dimethyl sulfoxide (DMSO) is widely used as a solvent for water-insoluble substances and has biological properties such as inducing cellular differentiation, scavenging free radicals, and acting as a radioprotectant. DMSO has also been reported to enhance mineralization in osteoblastic cells. However, its molecular mechanisms have not yet been fully elucidated.
In this study, we identified the differentially expressed genes associated with dimethyl sulfoxide-enhanced mineralization in mouse MC3T3-E1 preosteoblast cells using GeneChip® oligonucleotide microarrays.

overall_design: Mouse MC3T3-E1 preosteoblast cells were cultured in osteogenic medium with or without 0.6% DMSO at 37°C for 3 days. For control experiments, cells were cultured in normal medium at 37°C for 3 days. Total RNA samples were prepared from the cells, and quality of the RNA was analyzed using a Bioanalyzer 2100. Gene expression was analyzed by an Affymetrix GeneChip® system with a GeneChip Mouse Genome 430 2.0 array. Sample preparation for array hybridization was carried out as described in the manufacturer’s instructions.

first: Shiro

last: Watanabe


title: Investigation of differentially expressed genes in subpopulations of adherent human umbilical vein endothelial cells (HUVECS) isolated after fluid shear stress compared to the whole population of endothelial cells

city: Milwaukee

first: Brandon

middle: J

last: Tefft

state: WI

address: 8701 W Watertown Plank Rd

department: Biomedical Engineering

institute: Medical College of Wisconsin & Marquette University

summary: The goal of this study is to determine whether EC loss is a random process or whether it is possible to predict which cells are more likely to remain adherent upon exposure to fluid SS. We used fluid SS to isolate a subpopulation of ECs that resist detachment and remain adherent. We used RNA-seq to examine differential gene expression of adherent subpopulation ECs compared to the whole population to identify targets for improving retention

overall_design: Primary HUVECs were cultured overnight on fibronectin-coated 100mm dishes in the flow path area using a rectangular gasket and then subjected to 15 dyn/cm2 fluid SS using a rectangular parallel pate flow chamber. HUVECs were exposed to fluid SS for 0, 5, 10, or 30 min to generate the whole population, 5 min adherent, 10 min adherent, and 30 min adherent subpopulations, respectively. Plates from each time point were pooled together to generate sufficient RNA for analysis. A total of n=3 replicates from independent experiments were used for each time point.

first: Jayne

middle: T

last: Wolfe

first: Vaya

last: Chen

first: Yiliang


title: Pulmonary osteoclast-like cells in silica induced pulmonary fibrosis

city: Seattle

first: Cole

last: Trapnell

state: WA

address: 3720 15th Ave NE

institute: University of Washington

summary: The pathophysiology of silicosis is poorly understood, limiting development of therapies for those who have been exposed to the respirable particle. We explored mechanisms of silica-induced pulmonary fibrosis in human lung samples collected from patients with occupational exposure to silica and in a longitudinal mouse model of silicosis using multiple modalities including whole-lung single-cell RNA sequencing and histological, biochemical, and physiologic assessments. In addition to pulmonary inflammation and fibrosis, intratracheal silica challenge induced osteoclast-like differentiation of alveolar macrophages and recruited monocytes, driven by induction of the osteoclastogenic cytokine, receptor activator of nuclear factor κΒ ligand (RANKL) in pulmonary lymphocytes, and alveolar type II cells. Anti-RANKL monoclonal antibody treatment suppressed silica-induced osteoclast-like differentiation in the lung and attenuated pulmonary fibrosis. We conclude that silica induces differentiation of pulmonary osteoclast-like cells leading to progressive lung injury, likely due to sustained elaboration of bone-resorbing proteases and hydrochloric acid. Interrupting osteoclast-like differentiation may therefore constitute a promising avenue for moderating lung damage in silicosis.

overall_design: Snap-frozen whole mouse lung tissue was collected from twelve samples spanning four timepoints. Lung tissue was dissociated using a gentleMACS tissue dissociator. Nuclei suspension were snap-frozen in liquid nitrogen and stored in liquid nitrogen for single-cell combinatorial indexing RNA sequencing (sci RNA-seq) library preparation. Libraries were sequenced twice.

first: Francis

last: McCormack

first: Jennifer

last: Franks

first: Yoshihiro

last: Hasegawa


title: Engineering potent chimeric antigen receptor T cells by programming signaling during T-cell activation [RNA-seq]

city: South San Francisco

first: Jia

last: Lu

state: CA

address: 201 Haskins Way, Suite 101

institute: Lyell Immunopharma

summary: Programming cell signaling during T-cell activation represents a simple strategy for improving the potency of therapeutic T-cell products. Stim-R™ technology (Lyell Immunopharma) is a customizable, degradable synthetic cell biomimetic that emulates physiologic, cell-like presentation of signal molecules to control T-cell activation. A breadth of Stim-R formulations with different anti-CD3/anti-CD28 (αCD3/αCD28) antibody densities and stoichiometries were screened for their effects on multiple metrics of T-cell function. We identified an optimized formulation that produced receptor tyrosine kinase-like orphan receptor 1 (ROR1)-targeted chimeric antigen receptor (CAR) T cells with enhanced persistence and polyfunctionality in vitro, as assessed in repeat-stimulation assays, compared with a benchmark product generated using a conventional T-cell–activating reagent. In transcriptomic analyses, CAR T cells activated with Stim-R technology showed downregulation of exhaustion-associated gene sets and retained a unique subset of stem-like cells with effector-associated gene signatures following repeated exposure to tumor cells.  Compared with the benchmark product, CAR T cells activated using the optimized Stim-R technology formulation exhibited higher peak expansion, prolonged persistence, and improved tumor control in a solid tumor xenograft model. Enhancing T-cell products with Stim-R technology during T-cell activation may help improve therapeutic efficacy against solid tumors.

overall_design: Human CAR T cells produced using the optimized Stim-R formulation or the benchmark reagent were collected at the end of the third stimulation with H1975 tumor cells (Day 10) in the normalized repeat-stimulation assay. Live CD45+EGFR+CD8+ T cells were isolated by flow sorting for bulk RNA-Seq profiling.

first: Aileen

middle: W

last: Li

first: Jessica

middle: D

last: Briones

first: Quinn

last: Walker

first: Rowena

last: Martinez

first: Hajime

last: Hiraragi

first: Bijan

middle: A

last: Boldajipour

first: Purnima

last: Sundar

first: Shobha

last: Potluri

first: Gary

first: Omar

last: Ali

first: Alexander

middle: S

last: Cheung


title: Identification of differentially expressed genes associated with osteogenic differentiation in mouse MC3T3-E1 preosteoblast cells

first: Yoshiaki

last: Tabuchi

summary:
It has been reported that many kinds of genes are involved in the differentiation process from undifferentiated osteoblasts to differentiated osteoblasts. However, the genes involved in the differentiation process of osteoblasts have not yet been fully elucidated.
In this study, we identified the differentially expressed genes related to the osteogenic differentiation of mouse MC3T3-E1 preosteoblast cells using GeneChip® oligonucleotide microarrays.

overall_design: Mouse MC3T3-E1 preosteoblast cells were cultured in osteogenic medium at 37°C for 3 or 6 days. For control experiments, cells were cultured in normal medium at 37°C for 3 or 6 days. Total RNA samples were prepared from the cells, and quality of the RNA was analyzed using a Bioanalyzer 2100. Gene expression was analyzed by an Affymetrix GeneChip® system with a GeneChip Mouse Genome 430 2.0 array. Sample preparation for array hybridization was carried out as described in the manufacturer’s instructions.

first: Shiro

last: Watanabe


title: HPV induced R-loop formation represses innate immune gene expression while activating DNA damage repair pathways [H3K9me3 ChIP seq]

city: Chicago

first: Conor

last: Templeton

address: 300 E Superior Street

institute: Northwestern University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: R-loops are trimeric nucleic acid structures that form when an RNA molecule hybridizes with its complementary DNA strand, displacing the opposite strand. These structures regulate transcription as well as replication, but aberrant R-loops can form, leading to DNA breaks and genomic instability if unresolved. R-loop levels are elevated in many cancers as well as cells that maintain high-risk human papillomaviruses. We investigated how the distribution as well as function of R-loops changed between normal keratinocytes and HPV positive cells derived from a precancerous lesion of the cervix (CIN I). The levels of R-loops associated with cellular genes were found to be up to 10-fold higher in HPV positive cells than in normal keratinocytes while increases at ALU1 elements increased by up to 500-fold. The presence of enhanced R-loops resulted in altered levels of gene transcription, with equal numbers increased as decreased. While no uniform global effects on transcription due to the enhanced levels of R-loops were detected, genes in several pathways were coordinately increased or decreased in expression only in the HPV positive cells. This included the downregulation of genes in the innate immune pathway, such as DDX58, IL-6, STAT1, IFN-b, and NLRP3. All differentially expressed innate immune genes dependent on R-loops were also associated with H3K36me3 modified histones. Genes that were upregulated by the presence of R-loops in HPV positive cells included those in the DNA damage repair such as ATM, ATRX, and members of the Fanconi Anemia pathway. These genes exhibited a linkage between R-loops and H3K36me3 as well as gH2AX histone marks only in HPV positive cells. These studies identify a potential link in HPV positive cells between DNA damage repair as well as innate immune regulatory pathways with R-loops and gH2AX/H3K36me3 histone marks that may contribute to regulating important functions for HPV pathogenesis

overall_design: Formaldehyde was added to 1 × 107 cells to a final concentration of 1% for 10 min at room temperature. Excess formaldehyde was quenched upon adding 0.125M glycine before then washing samples with PBS. Cells were then incubated in collection buffer (0.1M TrisHCl pH 9.4 and 10mM DTT containing Roche Protease Inhibitor Cocktail)  for 10min on ice. Cells were then collected and spun down before being sequentially washed and incubated with NCP1 (10mM EDTA, 0.5mM EGTA, 10mM HEPES pH 6.5, 0.25% Triton X100) and NCP2 (1mM EDTA, 0.5mM EGTA, 10mM HEPES, and 200mM NaCl) before being lysed in 0.5% Empigen BB, 1% SDS, 10mM EDTA, 50mM Tris HCl pH 8.0 containing Roche Protease Inhibitor Cocktail for 30 min on ice. Samples were then sonicated using a Bioruptor (Diagenode) on high power, 30 s on/90 s off cycles for 20 min. After sonication, Input DNA was removed before loading the samples into preblocked magnetic beads in IP buffer containing 2 ug of the H3K36me3 antibody. Immunoprecipitations were allowed to incubate overnight at 4 °C while rotating. The next day, samples were washed 8 times with RIPA buffer for 5 min while rotating. One wash in TE buffer was performed before samples were eluted for 10 min at 65 °C in 10% sodium dodecyl sulfate (SDS), 10 mM Tris pH 7.4, 50 mM ethylenediaminetetraacetic acid (EDTA). DNA was purified from these elutions using a PCR purification kit (Qiagen) and stored at −20 °C.

first: Laimonis

last: Laimins


title: HPV induced R-loop formation represses innate immune gene expression while activating DNA damage repair pathways [HFK RNA seq]

overall_design: Gene expression was examined in CIN 612 cells (keratinocytes isolated from a cervical biopsy that stably maintain the HPV 31 genome) - samples 3 and 4 - and HPV negative human foreskin keratinocytes -samples 1 and 2- through RNA sequencing and differential gene expression was reported.


title: HPV induced R-loop formation represses innate immune gene expression while activating DNA damage repair pathways [gH2AX ChIP seq]

overall_design: Formaldehyde was added to 1 × 107 cells to a final concentration of 1% for 10 min at room temperature. Excess formaldehyde was quenched upon adding 0.125M glycine before then washing samples with PBS. Cells were then incubated in collection buffer (0.1M TrisHCl pH 9.4 and 10mM DTT containing Roche Protease Inhibitor Cocktail)  for 10min on ice. Cells were then collected and spun down before being sequentially washed and incubated with NCP1 (10mM EDTA, 0.5mM EGTA, 10mM HEPES pH 6.5, 0.25% Triton X100) and NCP2 (1mM EDTA, 0.5mM EGTA, 10mM HEPES, and 200mM NaCl) before being lysed in 0.5% Empigen BB, 1% SDS, 10mM EDTA, 50mM Tris HCl pH 8.0 containing Roche Protease Inhibitor Cocktail for 30 min on ice. Samples were then sonicated using a Bioruptor (Diagenode) on high power, 30 s on/90 s off cycles for 20 min. After sonication, Input DNA was removed before loading the samples into preblocked magnetic beads in IP buffer containing 2 ug of the gH2AX antibody. Immunoprecipitations were allowed to incubate overnight at 4 °C while rotating. The next day, samples were washed 8 times with RIPA buffer for 5 min while rotating. One wash in TE buffer was performed before samples were eluted for 10 min at 65 °C in 10% sodium dodecyl sulfate (SDS), 10 mM Tris pH 7.4, 50 mM ethylenediaminetetraacetic acid (EDTA). DNA was purified from these elutions using a PCR purification kit (Qiagen) and stored at −20 °C.


title: N-acetyltransferase 10 mediates cognitive dysfunction through the acetylation of GABABR1 mRNA in sepsis-associated encephalopathy.

city: Shanghai

first: Shenjia

last: Gao

address: 130 Dong 'an Road, Xuhui District, Shanghai

institute: Fudan university

summary: Sepsis-associated encephalopathy (SAE) is a critical neurological complication of sepsis and represents a crucial factor contributing to high mortality and adverse prognosis in septic patients. This study explore the influence of NAT10-mediated mRNA acetylation on SAE and the mechanisms underlying cognitive dysfunction in this context. SAE was induced in C57BL/6 J male mice through cecal ligation and puncture (CLP) surgery. In vivo, we observed a significant increase in NAT10 expression in the excitatory neurons of the hippocampal the dentate gyrus (DG) in CLP mice, and this change was accompanied by a trend toward an increase in mRNA acetylation. Specific knockout of Nat10 in neurons led to an improvement in cognitive dysfunction in septic mice. Proteomic analysis and RNA immunoprecipitation and real-time quantitative polymerase chain reaction (RIP-qPCR) revealed GABABR1 as a crucial downstream target of NAT10. Nat10 deletion reduced GABABR1 expression, subsequently weakening inhibitory postsynaptic currents in hippocampal DG neurons. Additionally, RNA sequencing (RNA-seq) revealed an increase in hippocampal inflammation post-CLP, characterized by the excessive activation of microglia and the release of inflammatory mediators. These factors were linked to the increased NAT10 expression in neurons. Treatment with PLX3397, a microglia depleting agent, resulted in a reduction in NAT10 and GABABR1 expression in neurons, which correlated with improvements in cognitive dysfunction induced by SAE. Taken together, our findings revealed that after CLP, NAT10 in neurons of the hippocampal DG region promotes GABABR1 expression by acetylating its mRNA, leading to cognitive dysfunction. These findings provide new insights into the molecular pathogenesis of SAE and suggest potential therapeutic targets for addressing cognitive deficits in septic patients.

overall_design: To investigate the changes in the gene expression profile in the mouse hippocampus after CLP modeling, we collected hippocampal tissue for RNA sequencing on the first day following CLP modeling in wild-type mice.

first: Ruling

last: Shen

first: Jie


title: Engineering potent chimeric antigen receptor T cells by programming signaling during T-cell activation [CITE-seq]

first: Jia

last: Lu

overall_design: Human CAR T cells produced using the optimized Stim-R formulation or the benchmark reagent were collected at the end of the third stimulation with H1975 tumor cells (Day 10) in the normalized repeat-stimulation assay. The cells were stained with CITE-Seq antibodies (ADTs and HTOs), and live CD45+EGFR+ T cells were isolated by flow sorting for single cell CITE-Seq profiling.

first: Aileen

first: Jessica

last: Lee


title: Reduction of chromosomal instability and inflammation is a common aspect of adaptation to aneuploidy

city: Amsterdam

first: Dorine

last: Hintzen

address: Plesmanlaan 121

department: Cell Biology

country: Netherlands

institute: Netherlands Cancer Institute

summary: Aneuploidy, while detrimental to untransformed cells, is notably prevalent in cancer. Aneuploidy is found as an early event during tumorigenesis which indicates that cancer cells have the ability to surmount the initial stress responses associated with aneuploidy, enabling rapid proliferation despite aberrant karyotypes. To generate more insight into key cellular processes and requirements underlying adaptation to aneuploidy, we generated a panel of aneuploid clones in p53-deficient RPE-1 cells and studied their behavior over time. As expected, de novo generated aneuploid clones initially displayed reduced fitness, enhanced levels of chromosomal instability and an upregulated inflammatory response. Intriguingly, after a prolonged period of culturing, aneuploid clones exhibited increased proliferation rates while maintaining aberrant karyotypes, indicative of an adaptive response to the aneuploid state. Interestingly, all adapted clones displayed reduced chromosomal instability (CIN) and reduced inflammatory signaling, suggesting that these are common aspects of adaptation to aneuploidy. Collectively, our data suggests that CIN and concomitant inflammation are key processes that require correction to allow for fast proliferation in vitro. Finally, we provide evidence that amplification of oncogenic KRAS can promote adaptation.

overall_design: To investigate how cells can adapt to aneuploidy we generated a panel of aneuploid clones in RPE-1 p53KD cells and exposed them to long-term culturing in the lab.


title: HPV induced R-loop formation represses innate immune gene expression while activating DNA damage repair pathways

overall_design: 1 × 107 cells were harvested and collected in Southern lysis buffer before being treated with RNase A (5 ng/mL) and Proteinase K (7.5 ng/mL) at 37 °C overnight. DNA was purified from these samples using phenol-chloroform extractions, and 25 to 50 mg of DNA was used for each sample. DNA was sheared using a Bioruptor (Diagenode) on high power, 30 s on/90 s off cycles for 20 min or digested using 1U of mung bean nuclease for 1 h at 37 °C. Input DNA was removed before loading the samples into preblocked magnetic beads in IP buffer containing 2 mg of the RNA:DNA hybrid antibody. Immunoprecipitations were allowed to incubate overnight at 4 °C while rotating. The next day, samples were washed 8 times with RIPA buffer for 5 min while rotating. One wash in TE buffer was performed before samples were eluted for 10 min at 65 °C in 10% sodium dodecyl sulfate (SDS), 10 mM Tris pH 7.4, 50 mM ethylenediaminetetraacetic acid (EDTA). DNA was purified from these elutions using a PCR purification kit (Qiagen) and stored at −20 °C. Samples were stored at -80 C until being shipped to Admera Biosciences (NJ), who performed the sequencing experiments. Briefly, the library was prepared using a KAPA HyperPrep Kit (Kapa Biosystems) following the manufacturer’s recommendation. Input DNA was end-repaired and 3’-dA tailed. Adapter was then ligated to the DNA, and the ligated product was PCR amplified and cleaned up using the SPRIselect Reagent (Beckman Coulter). Quality control was then performed for the final library, followed by sequencing.


title: B cell-stromal cell cross talk drives mesenteric lymph node eosinophilia during intestinal helminth infection

city: London

first: Lalit

last: Dubey

address: Charterhouse Square

country: United Kingdom

institute: QMUL

summary: We hypothesised that eosinophil activation and gene expression were linked to their interaction with the activated stroma within the lymph node. However, the role of eosinophil stromal crosstalk and how this modulate eosinophil function is unknown. To directly assess this hypothesis, we activated lymph node derived stromal cells using an agonist antibody for LTR (Clone 4H8WH2). Considering eosinophils have long been associated with providing B cell proliferative and survival factors, we mimicked a cell-based activation model using purified naïve B cells given that B cell interactions with stroma can produce factors that can regulate eosinophil activation and migration. B cell-based activation as well as agonist antibody-based activation resulted in an activated stromal phenotype and were later used for co-culture with bone marrow-derived eosinophils. After 8 hours of co-culture with activated stroma, the eosinophils were purified, and bulk RNA sequencing was performed to identify global changes in their transcriptomes.

overall_design: To investigate the impact of stromal cells on eosinophils gene expression. Bone marrow-derived eosinophils were generated in vitro and used for the co-culture experiment. Experiments were performed in triplicate wells and eosinophils were pooled post stimulation to create a single sample. RNAseq was performed using BGI Genomics DNBSEQ sequencing technology.

first: Emily

last: Bessell


title: Immunomodulated Bone Regeneration Using a Conductive Micro-hydrogel System Through Mitochondrial Transfer and Mitochondrial Metabolic Regulation

status: Public on Aug 11 2024

city: Shenyang

first: Ziyi

last: Feng

address: Puhe road

institute: China medical university

summary: Biomaterial-based bone tissue engineering offers a promising prospect for the treatment of bone defects. In particular, the ability of biomaterials to regulate the immune microenvironment of the defect site is essential for effective bone regeneration. Electro-biomaterials have been confirmed to induce macrophage M2 polarization through metabolic pathways, thereby enhancing bone regeneration. Considering the central role of mitochondria in cellular metabolism and their ability to influence the function of neighboring cells through intercellular transfer, and inspired by the fact that tumor cells can uptake mitochondria from immune cells to generate energy, we hypothesize that the metabolic activation of immune cells by electro-materials can be transmitted to preosteoblasts through mitochondria to promote bone repair. Therefore, this study proposed a conductive micro-hydrogel (CMH) system composed of conductive hydrogel microspheres made from GelMA and poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS), which served as scaffolds for defect filling, and a biomimetic periosteum made from poly-l-lactic acid (PLLA) and polydopamine (PDA) for microsphere immobilization and isolation of soft tissue. The microspheres exhibited excellent tissue support and degradation properties, their high specific surface area enhanced tissue remodeling, and their good conductivity eliminated free radicals and induced macrophage M2 polarization, which were confirmed by tests of mechanical property, swelling and degradation, conductivity and assays of cellular biocompatibility, ROS generation, and macrophage phenotype. In vivo experiments using a rat mandibular defect model confirmed the excellent bone repair capabilities of the CMH system, and transcriptomics, metabolomics, and metabolic testing revealed that the CMH system upregulated the oxidative phosphorylation pathway of macrophage, enhancing mitochondrial respiration and ATP production. Mitochondrial tracing experiments demonstrated the transfer of macrophage mitochondria to preosteoblasts, resulting in enhanced metabolic activity and osteogenic differentiation of preosteoblasts. This study may be the first suggest that conductive biomaterials facilitate osteogenic immunomodulation through mitochondrial transfer, which provides a promising method for regulating the immune microenvironment and reveals a novel pathway by which M2 macrophages enhance osteogenesis.

overall_design: RAW 264.7 macrophages were cultured with CMH system group or control GelMA group under static and dynamic conditions for 3 d as aforementioned. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA sequencing was performed by Shanghai Genechem Co., Ltd (China). The thresholds for differentially expressed transcripts were P<0.05 and an absolute fold change≥1. Differentially expressed transcripts were subjected to function and signaling pathway enrichment analysis using the GO (Gene Oncology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases. GSEA (v4.1.0) was used to analyze the differences in a pre-defined gene set.

first: Meiqi

last: Jin

first: Minhao

last: Li

first: Shu

last: Guo

first: Xiaoting

last: Sun

first: Huazhe

last: Yang


title: Bronchial Epithelial Cell RNASeq Pilot

city: Boston

first: Joanne

last: Sordillo

state: MA

address: 181 Longwood Avenue

department: Channing Division of Network Medicine

institute: Mass General Brigham

summary: In this study, we tested two environmental exposure stimuli, bacterial lipopolysaccharide (LPS) and house dust mite (HDM), in an in vitro model of primary human bronchial epithelial cells (HBECs) from 6 individuals, to identify differentially expressed genes using RNAseq analysis in response to these exposures both individually and in combination.  Our findings illustrate that exposure mixtures increase the strength and complexity of transcriptomic responses in HBECs as compared to individual stimuli alone, and that asthmatic’s cells are more sensitive to these stimuli, eliciting a greater number of differentially expressed genes vs. controls.  Our results provide a basis for future studies in this area, such as the expansion of this in vitro model to test multiple exposure combinations (i.e. the exposome) and their influence on molecular signaling in the bronchial epithelium.

overall_design: Well-differentiated cells in ALI culture were stimulated with lipopolysaccharide (LPS) and/or house dust mite (HDM) extract. LPS was purchased from sigma (L2654) and dissolved in PBS. Low endotoxin HDM extract (product no. XPB70D3A2.5, lot no. 279020, 1590 endotoxin units/2.5 mL sized vial) were purchased from Greer Laboratories (Lenoir, NC) and dissolved in PBS. Prior to exposure to stimuli, cells were maintained for 20 hours in a minimal medium, which was depleted of hydrocortisone, epidermal growth factor, and bovine pituitary extract. Cells were apically stimulated with 250ul of minimal media containing either LPS (at 10 μg/ml), HDM (at 25 μg/ml), or the combination of LPS and HDM. As a vehicle treatment, minimal media containing PBS was used. After four hours, cells were used for total RNA isolation. For RNA sequencing analysis total RNA was isolated using miRNeasy Kit (Qiagen). From the validation experiment using additional donors, total RNA was isolated using RNeasy Mini Kit (Qiagen, Germantown, MD) for qPCR analysis.

first: Jin-ah

last: Park


title: Corepressors NCOR1 and SMRT regulate metabolism via intestinal regulation of carbohydrate transport

first: Translational

last: Bioinformatics

address: 72 East Concord Street

institute: Clinical and Translational Science Institute

summary: Objective: Nuclear receptor action is mediated in part by the nuclear receptor corepressor 1 (NCOR1) and the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT; also known as NCOR2). NCOR1 and SMRT regulate metabolic pathways that govern body mass, insulin sensitivity and energy expenditure and represent an understudied area in the realm of metabolic health and disease. Previously, we found that NCOR1 and SMRT are essential for maintaining metabolic homeostasis and their knockout (KO) leads to rapid weight loss and hypoglycemia, which is not survivable. Because of a potential defect in glucose absorption, we sought to determine the role of NCOR1 and SMRT specifically in intestinal epithelial cells (IECs).  Methods: We used a post-natal strategy to disrupt NCOR1 and SMRT throughout IECs in adult mice. These mice were characterized metabolically by assessing body weight, glucose levels and subjecting the mice to metabolic phenotyping, body composition analysis and glucose tolerance testing. IECs were isolated from the jejunum of the small intestine and profiled by bulk RNA sequencing. Results: We found that the post-natal KO of NCOR1 and SMRT from IECs leads to rapid weight loss and hypoglycemia with a significant reduction in survival. This was accompanied by alterations in glucose metabolism and activation of fatty acid oxidation in IECs. Metabolic phenotyping confirmed a reduction in body mass driven by a loss of body fat without any difference in food intake. This appeared to be driven by a reduction of key intestinal carbohydrate transporters, including SGLT1, GLUT2 and GLUT5.   Conclusions: Intestinal NCOR1 and SMRT act in tandem to regulate glucose levels and body weight. This in part may be mediated by regulation of intestinal carbohydrate transporters.

overall_design: A tamoxifen-inducible Cre approach was used to disrupt NCoR1 and SMRT in the intestinal epithelial cells (IECs) of adult mice. IECs from control and IEC-specific double knockout (I-DKO) mice were collected and used to perform bulk RNA sequencing.

first: Megan

last: Ritter

first: Anthony

middle: N

last: Hollenberg


title: Voluntary exercise sensitizes cancer immunotherapy via collagen degradation-orchestrated inflammatory tumor immune microenvironment

city: Nanjing

first: Jie

last: Mei

state: Jiangsu

address: No. 101, Longmian Road

institute: Nanjing Medical University

summary: Physical activity reduces cancer-associated mortality through multiple mechanisms, including tumor immune microenvironment (TIME) reprogramming. However, whether and how physiological interventions promote anti-tumor immunity and immunotherapy remains elusive. Here we report that clinically relevant voluntary exercise promotes muscle-derived exosomal miR-29a-3p for extracellular matrix (ECM) degradation in patients and mouse models with multiple types of cancer, thereby permitting immune cell infiltration and immunotherapy. Mechanistically, an unbiased screening identified exercise-responsive exosomal miR-29a-3p that targets tumor cell and cancer associated fibroblasts to downregulate COL1A1 and reduce ECM. State-of-the-art techniques including cytometry by time-of-flight (CyTOF) demonstrated miR-29a-3p-induced TIME remodeling and immune cell infiltration. Combining immunotherapy with voluntary exercise or miR-29a-3p further increased the anti-tumor efficacy in preclinical models. Clinically, miR-29a-3p was correlated with degraded ECM and T-cell infiltration in various cancer types in patients, and correlated with immunotherapy responders. Our work reveals the novel predictive value of miR-29a-3p for immunotherapy, provides mechanistic insights into exercise-promoted anti-cancer immunity, and highlights the therapeutic potential of voluntary exercise in immunotherapy.

overall_design: To identity the potential target genes of miR-29a-3p in lung cancer cells, total RNA of A549 and NCI-H1299 cells transfected with miR-ctrl and miR-29a-3p were submitted for RNA-seq.

first: Yaying


title: TFEB/3 govern repair Schwann cell generation and function following peripheral nerve injury

city: Newark

first: Haesun

last: Kim

address: 195 University Ave

institute: Rutgers University

summary: After peripheral nerve injury, adult Schwann cells convert to progenitor cell-like repair Schwann cells, which are pivotal for nerve regeneration. We show that Schwann cell-specific deletion of TFEB/3 disrupts the transcriptomic reprogramming necessary for injury-induced repair Schwann cell generation in mice. The mutant mice fail to generate proliferating repair Schwann cells to populate the injured nerves. Distal Schwann cells fail to express injury-responsive genes and continue to maintain the expression of myelin-associated genes. TFEB/3 binding motifs are enriched in injury-induced enhancers, suggesting their role in repair gene activation. Autophagy-dependent myelin breakdown is not impaired in the mutant mice despite the known function of TFEB/3 as autophagy activators. However, the mutant mice exhibit defects in axon regeneration, target reinnervation, and functional recovery. Therefore, TFEB/3 play a critical role in orchestrating transcriptional changes essential for repair Schwann cell generation and function necessary for peripheral nerve regeneration.

overall_design: To investigate the role of TFEB and TFE3 in the generation of repair Schwann cells after sciatic nerve injury we developed Tfeb/3 SC-dKO mice with a Schwann cell specific Dhh-Cre driven Tfeb knockout on a global Tfe3 knockout background (Dhh-Cre+:Tfebflox/flox;Tfe3KO) and control mice (Dhh-Cre-:Tfebflox/flox;Tfe3+). Intact sciatic nerves without nerve injury and distal segments following 5 days post transection injury (5DPI) were examined by RNA-seq analysis. Transcriptomic analysis was performed with 4 groups: Control Intact (100), Tfeb/3 SC-dKO Intact (200), Control 5DPI (300), and Tfeb/3 SC-dKO 5DPI (400), with 3 biological replicates per group.

first: Akash

last: Patel


title: Genetically- and spatially-defined basolateral amygdala neurons control food consumption and social interaction

city: Martinsried

first: Tobias

last: Straub

address: Großhadener Str. 9

department: Biomedical Center, Bioinformatics

country: Germany

institute: LMU Munich

summary: The basolateral amygdala (BLA) contains discrete neuronal circuits that integrate positive or negative emotional information and drive the appropriate innate and learned behaviors.  Whether how these circuits consist of genetically-identifiable and anatomically segregated neuron types, is currently poorly understood.  Also, our understanding of the response patterns and behavioral spectra of genetically-identifiable BLA neurons is limited. Here, we classified 11 glutamatergic BLA cell types having topography in BLA. Several clusters were enriched in lateral versus basal amygdala, others were enriched in either anterior or posterior regions of the BLA. Two of these BLA subpopulations innately responded to valence-specific stimuli, whereas one represented to both aversive and social cues. Positive-valence BLA neurons promoted normal feeding, while mixed selectivity neurons promoted fear learning and social interactions. These findings enhance our understanding of cell type diversity and spatial organization of the BLA and the role of distinct BLA populations in representing valence-specific and mixed stimuli.

overall_design: Single-nucleus RNA sequencing was focused on the basolateral amygdala (BLA). For visually guided dissection of the BLA, we practiced by using the fluorescent expression in basal amygdala (BA) of Rspo2-cre; tdTomato mice 3 and dense fiber tracks surrounding BLA boundaries to facilitate complete microdissection of BLA. Each single nucleus sequencing dataset includes BLA tissues from 4 male brains (both hemispheres). To reduce potential batch effects, brains were always from the same litter, collected and processed in parallel at the same time.

first: Hansol

last: Lim

first: Yue

last: Zhang

first: Christian

last: Peters

first: Johanna

last: Mayer

first: Rüdiger

last: Klein


title: Effect of TGF-β1 treatment on gene expression of HK2 cells

city: Wuhan

first: Ben

last: Tang

address: JieFang Avenue

department: Department of Nephrology

institute: Union Hospital, Tongji Medical College, Huazhong University of Science and Technology

summary:
To elucidate the expression file under TGF-β1 treatment , we performed RNA-seq on HK2 cells treated with TGF-β or not
We then performed gene expression profiling analysis using data obtained from RNA-seq of HK2 cells untreated or treated with TGF-β1 (5ng/ml) for 24 h

overall_design: Comparative gene expression profiling analysis of RNA-seq data for HK2 cells and TGF-β1-treated HK2 cells.

first: Qian

last: Yuan

first: Beng


title: Effect of PRDM16 tubular specific knock out  on gene expression of UIRI mice model

summary:
To elucidate the expression file under PRDM16 tubular specific knock out , we performed RNA-seq on kidney sample of UIRI model  Ksp-cre/PRDM16flox/flox mice kidney with Cre activity or not
We then performed gene expression profiling analysis using data obtained from RNA-seq of these kidney samples.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for UIRI kidney sample and UIRI kidney sample with PRDM16 tubular specific knock out.


title: Infection history imprints prolonged changes to the Kupffer cell epigenome, transcriptome and function of Kupffer cells

city: Brussels

first: Daliya

last: Kancheva

address: Laarbeeklaan 103, Building E

country: Belgium

institute: Vrije Universiteit Brussel - VIB

summary: Background and aims: Macrophages are highly versatile innate phagocytes present in most organs of the body and perform various homeostatic and immunological functions. Macrophages are classically known for their important roles in compacting pathogens. Whereas, increasing evidence supports that exposure to infections invokes prolonged changes to tissue-resident macrophages, it remains unclear whether this occurs to liver macrophages. Methods: Here, we utilized a model of parasitic infection invoked by the protozoan parasite Trypanosoma brucei brucei to investigate whether infection history can durably reshape hepatic macrophage identity and function. Employing a combination of genetic lineage-tracing, single cell transcriptomics, epigenomic analysis, and functional assays, we studied the alterations to the liver macrophage compartment during and after the resolution of infection. Results: We show that the infection alters the composition of liver-resident macrophages, leading to the infiltration of monocytes that differentiate into various infection-associated macrophage populations with divergent transcriptomic profiles. Whereas infection-associated macrophages disappear post-resolution of infection, monocyte-derived macrophages assuming the Kupffer cell (KC)-like profile engraft in the liver and co-existed with embryonic KCs in the long-term. Remarkably, the prior exposure to infection imprinted a specific transcriptional program on post-resolution KCs that was independent of cell ontogeny. This reprogramming of KCs was driven by an epigenetic remodeling of KC chromatin landscapes and associated with altered KC function and was associated with increased resiliance to subsequent bacterial infection. Conclusion: Our study demonstrates that prior exposure to a parasitic infection can reshape the identity and function of the liver macrophage pool in the long-term.

overall_design: CITE-seq: Macrophages were FACS sorted ( 7AAD-negative/low CD45-positive CD11b-intermediate F4/80-high) from liver samples from non-infected, day 28-infected with Trypanosoma brucei brucei, 2 weeks and 8 weeks post-curing of the infection of Flt3-Cre Rosa26-Yfp mice. The cells were stained with antibody panel containing 158 oligo-conjugated antibodies and RNA and ADT libraries were prepared using the 10x Genomics platform. Multiome-seq: Single nuclei were extracted from non-infected mice and mice 8 weeks post-curing of Trypanosoma brucei brucei infection. Mice were from Flt3-Cre Rosa26-Yfp background. Joint snRNA- and scATAC libraries were prepared using the 10x Genomics platform. Bulk ATAC-seq: Macrophages were FACS sorted ( 7AAD-negative/low CD45-positive CD11b-intermediate F4/80-high TIM4-positive) from liver samples from non-infected (2 weeks  post melarsoprol), 2 weeks and 8 weeks post-curing of the infection  with Trypanosoma brucei brucei.


title: PRDM16 deficiency triggered by TGF-β1 signaling promotes aberrant mitochondrial homeostasis and kidney fibrosis

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Lamin C supplementation as therapeutic target for treatment of Lmna Dilated Cardiomyopathy (DCM) and risk of Lamin A toxicity

city: Singapore

first: Jian Ming

last: Jiang

address: 10 Medical Drive, MD6

department: Cardiovascular Research Institute

country: Singapore

institute: National University of SIngapore

summary: Dilated cardiomyopathy (DCM) is a severe, non-ischemic heart disease, which ultimately results in heart failure (HF). Pathological genetic variants in LMNA cause DCM, which currently lacks specific treatment. Perturbing candidates related to dysregulated pathways have shown to ameliorate LMNA DCM, but their long-term efficacy as potential therapeutic targets is unknown. Here, we evaluated 14 potential candidates including Lmna gene products, key signaling pathways, calcium handling, proliferation regulators and Lamin interacting proteins, in a cardiac-specific Lmna DCM model. The candidates with improved cardiac function were further assessed through survival analysis. After comparing cardiac function, marker gene expression, Tgfβ signaling pathway activation, fibrosis, inflammation, proliferation, and DNA damage, we uncovered that restored cardiac function significantly correlated with suppression of HF/fibrosis marker expression and cardiac fibrosis in Lmna DCM. Interestingly, Lamin C or Sun1 shRNA administration achieved consistent, prolonged survival which highly correlated with reduced heart inflammation and DNA damage. In addition to Sun1 shRNA, perturbing the interaction between the nucleoskeleton and cytoskeleton via the KASH domain of Nesprin1 also effectively suppressed Lmna DCM. In contrast, Lamin A supplementation did not rescue long term survival and may impart a detrimental cardiotoxicity risk.  Furthermore, transcriptome profiling was used to compare the differences between Lamin A and Lamin C treatment. Mechanistically, we identified that this lapse was attributed to a dose-dependent toxicity of Lamin A, which was independent of its maturation. This study highlights the potential for advancing Lamin C and Sun1 as therapeutic targets for the treatment of LMNA DCM.

overall_design: To investigate the effect of Lamin A or Lamin C upregulation in treatment of Lmna DCM (generated by knockdown of Lmna) using rAAV9 delivery platform

first: ChiaYee

last: Tan

first: PuiShi

last: Chan

first: Jianming


title: Phosphorylation of 53BP1 by ATM enforce neurodevelopmental programs in cortical organoids

city: Memphis

first: Wenjie

last: Qi

state: TN

address: 262 Danny Thomas Place

institute: St.Jude Children's Research Hospital

summary: 53BP1 is a well-established DNA damage repair factor that has recently been shown to regulate gene expression and critically influence tumor suppression and neural development. For gene regulation, how 53BP1 is regulated remains unclear. Here, we showed that 53BP1-serine 25 phosphorylation by ATM is required for neural progenitor cell proliferation and neuronal differentiation in cortical organoids. 53BP1-serine 25 phosphorylation dynamics controls 53BP1 target genes for neuronal differentiation and function, cellular response to stress, and apoptosis. Beyond 53BP1, ATM is required for phosphorylation of factors in neuronal differentiation, cytoskeleton, p53 regulation, and ATM, BNDF, and WNT signaling pathways for cortical organoid differentiation. Overall, our data suggest that 53BP1 and ATM control key genetic programs required for human cortical development.

overall_design: We used RNA-seq to profile the transcriptomes of  cortical organoids derived from WT and ATM-KO hESCs at D35 and D37 after differentiation. We next examined ATM-dependent phosphorylation

first: Bitna

first: Mohamed

last: Djekidel

first: Yurika

last: Matsui

first: Seunghyun

last: Jung

first: Zuo-Fei

first: Xusheng

last: Wang

first: Xiaoyang

last: Yang

first: Abbas

last: Pilehroud

first: Haitao

last: Pan

first: Fang

first: Shondra

last: Pruett-Miller

first: Kanisha

last: Kavdia

first: Vishwajeeth

last: Pagala

first: Yiping

last: Fan

first: Junmin

last: Peng

first: Beisi

last: Xu

first: Jamy

middle: C


title: H3K27ac HiChIP analysis in BALL cells

first: Tze King

address: 14 Medical Drive

department: Cancer Science Institute

institute: National University of Singapore

summary: H3K27ac HiChIP analysis was performed in an B-ALL cell line to analyze active chromatin-chromatin interactions in B-ALL cells.

overall_design: B-ALL cells were crosslinked with formaldehyde for 10 min.  DNA was enriched by chromatin-immunoprecipitation (ChIP), tagmented with Tn5 and analyzed by DNBseq.  HiChIP was performed using an antibody against H3K27ac Antibody  (ab4729).

first: Wang

last: Lu

first: Tze

last: King Tan

first: Takaomi

last: Sanda


title: Transcriptomes of normal aging brains in female nonhuman primates.

city: Winston-Salem

first: Jeannie

last: Chan

state: NC

address: Medical Center Boulevard

department: Internal Medicine

institute: Wake Forest School of Medicine

summary:
The objective was to study gene expression in the prefrontal cortex across the adult age using baboons as a nonhuman primate model.
RNA-Seq data was analyzed by Weighted Gene Coexpression Network Analysis (WGCNA), and two modules containing 587 transcripts negatively correlated with age were identified.

overall_design:
Brain tissues from the prefrontal cortex were collected at necropsy, snap-frozen in liquid nitrogen and stored at -80ºC until use.
Total RNA was extracted from tissues to construct cDNA libraries, which were sequenced (2 x 150 bp) on the NovaSeq to profile the brain transcriptomes of these animals.

first: Laura

last: Cox

first: Sobha

last: Puppala

first: Peter

last: Nathanielsz


title: Gene expression profiling of pancreatic neuroendocrine carcinoma and mixed neuroendocrine-non-neuroendocrine neoplasm

city: Bunkyo-ku

first: Yuko

last: Kinowaki

state: TOKYO

address: 1-5-45 Yushima

department: Comprehensive Pathology

institute: Tokyo Medical and Dental University

summary: Pancreatic neuroendocrine carcinoma (NEC) and mixed neuroendocrine-non-neuroendocrine neoplasm (MiNEN) are rare pancreatic malignant tumors, and comprehensive gene analyses are scarce. In this study, six NECs and six MiNENs were collected, immunohistochemistry for synaptophysin, chromogranin A, INSM1, Ki-67, and Rb was conducted, and KRAS mutational status was examined. Among these cases, comprehensive gene expression analysis of oncogene pathways using nCounter® were performed with six NECs and four MiNENs, and those data were compared with that of three pancreatic ductal adenocarcinomas (PDACs), with that of three normal pancreatic ducts, and with each other. By dividing NEC and MiNEN cases into KRAS-mutated group and KRAS-wild group, the difference of clinicopathological data and gene expression profiling data were examined between the two groups. Compared to the data of normal pancreatic epithelium, all 13 cancer-related pathways were upregulated in PDAC, MiNEN, and NEC group with more upregulation in this order. Compared to the data of PDAC, genes of DNA Damage repair pathway was most upregulated both in NECs and MiNENs. Regarding the difference between KRAS-mutated and KRAS-wild groups, several genes were differentially expressed between the two, where MMP7 was the upregulated gene with highest p-value and NKD1 was the downregulated gene with highest p-value in KRAS-mutated group. From the extent of upregulation of 13 pathways, MiNEN was considered more progressed stage than PDAC, and NEC was considered more progressed than MiNEN. From the comparison of KRAS-mutated and KRAS-wild NECs and MiNENs, several differentially expressed genes were identified in this study.

overall_design: We extracted mRNA from formalin-fixed paraffin-embedded (FFPE) specimens obtained from surgical resected specimens of six cases of pancreatic primary neuroendocrine carcinoma (NEC) and four cases of mixed neuroendocrine-non-neuroendocrine neoplasm (MiNEN). Subsequently, we quantified the mRNA expression levels using the nCounter platform from NanoString Technologies. We performed comparative expression analysis between these samples and used two normal pancreatic epithelial samples as controls, as well as three tissue samples from pancreatic ductal adenocarcinoma.

first: Yuki

last: Fukumura

first: Genji

last: Kawade

first: Keiichi

last: Akahoshi

first: Masanori

last: Kobayashi

first: Hiroaki

last: Ono

first: Atsushi

last: Kudo

first: Minoru

last: Tanabe

first: Takumi

last: Akashi

first: Kenichi

last: Ohashi

first: Morito

last: Kurata


title: Effect of maternal nutrient reduction (MNR) during pregnancy on fetal liver gene expression in baboons (Papio anubis).

summary:
The objective was to determine changes in gene expression in baboon fetal livers at 90, 120, 140 and 165 days gestation (dG) due to reduction of feed consumed by dams.
Control dams were fed ad libitum throughout the study, while 30% feed reduction (based on feed consumed by controls at the same gestation stage and adujsted for body weight) started at 30 dG for MNR dams.
RNA-seq data was analyzed to identify differentially expressed genes and transcript variants across four time points.

overall_design:
8 control and 8 MNR livers were collected at the 90 and 165 dG time points, and 6 control and 6 MNR livers were collected at the 120 and 140 dG time points.
RNA-seq was performed. Gene expression in MNR vs. control livers at different gestation time points was analyzed.

first: Kip

last: Zimmerman


title: WT/Usp5-MKO PM cells were stimulated with LPS or not

city: Suzhou

first: zigang

last: qiao

address: Chongwen Road

department: Suzhou Institute of Systems Medicine

institute: Chinese Academy of MedicalSciences & Peking Union Medical College

summary: Test transcripts in mock and LPS stimulated PM cells from WT or Usp5 myeloid knockout mice

overall_design: Gene expression profiling analysis of RNA-seq data for mock and LPS stinulated PM cells from WT or Usp5 myeloid knockout mice

first: Zigang

last: Qiao


title: The significance of CD16+ monocytes in the occurrence and development of Chronic Thromboembolic Pulmonary Hypertension: insights from single-cell RNA sequencing

status: Public on Aug 10 2024

city: Fuzhou

first: Chaosheng

last: Deng

state: Fujian

address: No. 20, ChaZhong Road

institute: First Affiliated Hospital of Fujian Medical University

summary: Our current understanding of the pathobiology of chronic thromboembolic pulmonary hypertension (CTEPH) is primarily based on studies using specimens obtained through pulmonary endarterectomy (PEA). However, there is a significant gap in our knowledge regarding the characteristics and functions of circulating immune cells in CTEPH patients. To address this gap, our study aims to characterize the peripheral blood circulating immune cells in CTEPH patients, with a particular focus on monocytes. In this study, we performed single-cell RNA sequencing (scRNA-seq) on peripheral blood mononuclear cells (PBMCs) extracted from five CTEPH patients. This approach allowed us to identify various immune cell types present in the peripheral blood, including monocytes, T cells, and B cells. Notably, we observed an increased proportion of CD16+ monocytes in CTEPH patients. This monocyte subset exhibited distinct transcriptional profiles, indicating their involvement in critical biological processes such as cell adhesion, T cell activation, coagulation, and platelet activation. Our findings suggest that CD16+ monocytes may play a significant role in the pathogenesis of CTEPH, particularly in pulmonary arterial thrombosis and intimal remodeling. Furthermore, we observed that this subset of monocytes might be recruited into the pulmonary artery intima and differentiate into macrophages characterized by high IL-1β expression, potentially contributing to disease progression. This study provides new insights into the immune landscape of CTEPH and highlights the potential role of CD16+ monocytes in the disease's pathophysiology.

overall_design: The study focuses on the characterization of circulating immune cells in patients with chronic thromboembolic pulmonary hypertension (CTEPH). We utilized single-cell RNA sequencing (scRNA-seq) to analyze peripheral blood mononuclear cells (PBMCs) from five CTEPH patients. The primary aim was to identify and profile various immune cell types, with a specific focus on the monocyte population.

first: Maohe

last: Chen

first: Qiuxia

last: Wu

last: Shao

first: Xingyue

last: Lai


title: The expression profiles of CLDN11-overexpressing and control human induced pluripotent stem cell (iPSC)-derived oligodendrocytes

city: Beijing

first: Yibo

last: Wang

state: Beijin

address: 167 Beilishi Road, Xicheng District, Beijing

institute: Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences & Peking Union Medical College

summary: Claudin 11 (CLDN11) is a member of the claudin family. Claudins are cell-cell adhesion molecules situated at tight junctions. The claudin family in mammals comprises 27 four-transmembrane domain proteins. Therein, cerebral CLDN11 is concentrated in oligodendrocytes and its dysregulation has been documented in several CNS disorders. We found that the expression of CLDN11 was significantly upregulated in oligodendrocytes of epileptic patients. To clarify the role of CLDN11 in oligodendrocytes in epileptic pathology, we transfected the human iPSC-derived oligodendrocytes with CLDN11 plasmid or vector control plasmid for 48 hours, respectively. Subsequently, three CLDN11-overexpressing and three vector control human iPSC-derived oligodendrocyte samples were subject to RNA sequencing.

first: Penghu

last: Wei

first: Ruofei

last: Li

first: Yushan

last: Tang

first: Cheng

last: Lv

first: Tao

last: Yu

first: Yongzhi

last: Shan

first: Quanlei

last: Liu

first: Yang

last: Dai

first: Yu

last: Zhang

first: GuoGuang

last: Zhao


title: RNA-Seq Analysis of Paclitaxel-Resistant PDX Models in Mice

city: zhengzhou

first: liting

last: zhou

address: 100 Science Avenue

institute: Zhengzhou University

summary: In this study, we aimed to investigate the gene expression changes associated with the development of paclitaxel resistance in patient-derived xenograft (PDX) models. We established paclitaxel-resistant PDX models in mice by administering 10 mg/kg of paclitaxel via intraperitoneal injection once a week. The treatment continued until the sixth generation of the PDX models was successfully established. RNA sequencing was performed on samples from the first generation (control and treated groups) and the sixth generation (control and treated groups) to identify differentially expressed genes and pathways involved in drug resistance. Our findings provide insights into the molecular mechanisms underlying paclitaxel resistance and may inform the development of more effective therapeutic strategies.

overall_design: We constructed paclitaxel-resistant PDX models using two PDX lines. Mice were treated with 10 mg/kg of paclitaxel via intraperitoneal injection once a week. The treatment was continued until the sixth generation of the PDX models was established. RNA sequencing was performed on four groups of samples: the first generation control group (Ctrl1), the first generation treated group (Treated1), the sixth generation control group (Ctrl6), and the sixth generation treated group (Treated6). Each group consisted of biological replicates to ensure robust statistical analysis. Sequencing libraries were prepared using standard protocols and sequenced on an Illumina platform. The resulting data were analyzed to identify differentially expressed genes and pathways associated with paclitaxel resistance.

last: Dong

first: Xiang

first: Mee-Hyun

last: Lee

first: Liting

last: Zhou

first: Jie

last: Tian


title: Multi-omics characterization of human Embryonic Stem Cells-derived Pharyngeal Endoderm cells (Single-cell RNA-Seq Batch_2)

city: Roma

first: Alessio

last: Colantoni

state: RM

address: Piazzale Aldo Moro, 5

department: Dipartimento di Biologia e Biotecnologie "Charles Darwin"

country: Italy

institute: "Sapienza" University of Rome

summary: We have developed a novel in vitro protocol for the derivation of bona fide Pharyngeal Endoderm (PE) cells from hESCs. We demonstrated that our PE cells robustly express Pharyngeal Endoderm markers, they are transcriptionally similar to PE cells isolated from in vivo mouse development and represent a transcriptionally homogeneous population. Importantly, we elucidated the contribution of Retinoic Acid in promoting a transcriptional and epigenetic rewiring of PE cells. In addition, we defined the epigenetic landscape of PE cells by combining ATAC-Seq and ChIP-Seq of histone marks.

overall_design: We performed a scRNA-Seq analysis of Definitive Endoderm (Day 2), Anterior Foregut Endoderm (Day 3 and Day 5) and Pharyngeal Endoderm (Day 5 + RA) cells.

first: Andrea

last: Cipriano

first: Danielle

last: Gomes

first: Alexander

last: Parker

first: Mahdi

last: Moqri

first: Matthew

last: Caldwell

first: Francesca

last: Briganti

first: Jonathan

last: Fiorentino

first: Maria

middle: G

last: Roncarolo

first: Antonio

last: Baldini

first: Katja

last: Weinacht

first: Gian

last: Tartaglia

first: Vittorio

last: Sebastiano


title: Treg insufficiency in autoimmune diabetes is driven by selective loss of Neuropilin-1 on intra-islet regulatory T cells

city: Pittsburgh

first: Jian

last: Cui

state: PA

address: 5051 Centre Avenue

department: Immunology

institute: University of Pittsburgh

summary: Strategies targeting Treg insufficiency are vital for treating autoimmune diseases like T1D. Our findings highlight the critical role of Neuropilin 1 (Nrp1) in stabilizing Tregs within pancreatic islets. Boosting Nrp1 levels delays diabetes onset by enhancing Treg function. This underscores Nrp1 as a potential target for bolstering Treg activity and combating autoimmune diseases.

overall_design: Our experimental design aimed to explore the influence of Neuropilin 1 (Nrp1) expression on regulatory T cell (Treg) function within NOD mouse islets, a model of autoimmune diabetes. We first identified Tregs lacking Nrp1 expression and observed diminished function. Augmenting Nrp1 levels delayed disease onset, suggesting its importance in Treg function and stability. Comparing Nrp1-induced Tregs to wild-type counterparts, we conducted single-cell RNA sequencing (scRNAseq) experiments to elucidate transcriptional changes. Our findings revealed improved Treg activity in response to Nrp1 expression within the islets, indicating potential therapeutic implications for autoimmune diseases.

first: Stephanie

last: Grebinoski

first: Dario

last: Vignali


title: Bacterial mRNA pseudouridylation by RluA [Rend-seq]

city: New Haven

first: Wendy

last: Gilbert

state: CT

address: 333 Cedar Street, P.O. Box 208024

institute: Yale University

summary: Pseudouridine (Ψ) is an ubiquitous RNA modification, present in the tRNAs and rRNAs of species across all domains of life. Conserved pseudouridine synthases modify the mRNAs of diverse eukaryotes, but the modification has yet to be identified in bacterial mRNAs. Here, we report the discovery of pseudouridines in mRNA from E. coli. By testing the mRNA modification capacity of all 11 known pseudouridine synthases, we identify RluA as the predominant mRNA-modifying enzyme, modifying the majority of high-confidence sites.

overall_design: RNA expression and transcript end detection by Rend-Seq

first: Cassandra

last: Burgos

middle: V

first: Gene-Wei

last: Li


title: Gene expression profiling of glucocorticoid receptor signaling in primray prostate cancer associated fibroblasts

city: Innsbruck

first: Martin

last: Puhr

state: Tyrol

address: Anich street 35

department: Department of Urology

country: Austria

institute: Medical University of Innsbruck

summary:
Glucocorticoid receptor (GR) has been recently identified as a candidate for acquired anti-androgen and chemotherapy resistance.
In order to identify glucocorticoid receptor (GR) targets and studying stromal GR signaling gene expression profiling was performed in primary prostate cancer associated fibroblasts using different treatments.

overall_design: Primary cancer associated fibroblasts (PCAF) from 4 prostate cancer patients were treated for 24h in 3 different treatment groups (1 DMSO=Control treatment, 2 Dexamthasone, 3 RU486+Dexamethasone).

first: Hubert

last: Hackl


title: Mapping the heterogeneity of pDCs and Monocytes of systemic sclerosis at single cell level

city: New York

first: David

last: Oliver

state: New York

address: 535 E 70th St

department: Research

institute: Hospital for Special Surgery


title: Carfilzomib Suppresses SENP1 to Trigger Cardiotoxicity by DDX17 Destabilization

city: Tianjin

first: Liu

last: Zhiqiang

address: 22 Qxiangtai Rd

institute: Tianjin Medical University

summary: Carfilzomib has a good effect in treating multiple myeloma, but more and more clinical studies have shown that the drug can cause cardiac toxicity during use, but the mechanism of action is unclear. This study aimed to comprehensively elucidate the mechanism of CFZ-induced cardiomyopathy by constructing a C57BL/6 mouse model and combining cell experiments. The research results showed that the hearts of mice treated with CFZ appeared significantly enlarged, and the cardiac function indicators decreased. The serum enzyme LDH, AST, and CK-MB levels significantly increased, suggesting severe myocardial injury. RNA extraction and QPCR detection of the heart showed that the expression of heart hypertrophy and fibrosis markers increased. Meanwhile, histological staining found that there were fewer cardiomyocytes, damaged myofibrils, and cardiac fibrosis. In the cell experiments, CFZ treatment caused the release of more LDH from rat cardiomyocytes and H9C2 cells, increased CASPASE 3/7 activity, abnormal cell morphology, enlarged cell size, myocardial hypertrophy and fibrosis marker gene expression in cardiomyocytes. Through the study of the SUMOYLATION pathway, it was found that CFZ's effects on cardiomyocyte structure and function were related to the expression of SENP genes, especially SENP1, which showed a significant downregulation in cardiomyocytes and mouse heart tissue, and was positively correlated with the levels of cardiac injury marker genes. This study further explored the mechanism of action by specifically altering the expression of SENP1 in cardiomyocytes. The results show that the knockdown of SENP1 in cardiomyocytes leads to more severe myocardial damage after CFZ treatment, suggesting that the expression of SENP1 can potentially affect the cardiotoxicity caused by CFZ. Further, by using silver staining and mass spectrometry to identify the interacting proteins with SENP1 after CFZ treatment, we found that the knockdown of DDX17 had a similar myocardial injury effect as SENP1. Subsequently, through Co-IP, protein stability experiments, and ubiquitination analysis experiments, we determined that SENP1 can maintain the protein stability of DDX17 by inhibiting its SUMO modification, thereby preventing cardiomyocyte injury and apoptosis. We hope to provide a new perspective and potential cardioprotective strategy for clinical use.

overall_design:
In order to investigate the mechanism of cardiotoxicity caused by carfilzomib,RNA-sequencing and analysis was performed using the CFZ-and vehicle-treated NRVMs (n=2 per group) at 24h.
We then performed gene expression profiling analysis and differentially expressed gene analysis using data obtained from RNA-seq.

first: Sheng

last: Wang

first: Xin


title: Mapping the heterogeneity of pDCs and Monocytes of systemic sclerosis at single cell level [scRNA-seq]

summary: Systemic sclerosis (SSc) is an uncommon disease characterized by elevated autoantibody production, vasculopathy and fibrosis of the skin and internal organs. pDCs are the core cell type to produce type I IFN and contributes to the ISG signature and SSc progression. Using single-cell RNA sequencing, we profiled 32529 pDCs enriched and T cell depleted peripheral blood mononuclear cells (PBMCs) from 4 patients with SSc and 4 matched controls. Increased CD16+ monocytes and decreased cDCs are the major changes in the cell composition between SSc and heathy controls. Increased expression of interferon-stimulated genes (ISGs) and apoptotic genes distinguished cells from patients with SSc from healthy control cells. The high ISG expression signature (ISGhi) derived from a small number of transcriptionally defined subpopulations within major cell types, including monocytes, natural killer cells, conventional and plasmacytoid dendritic cells, B cells. Profiling of 10976 pDCs revealed a newly identified PTGDS+ population and a clear increased ISG hi clusters in SSc pDCs. Profiling of 13317 Monocytes revealed increased CD16+ Monocytes and a clear increased ISG hi clusters in SSc monocytes. We then compared the TLRs-IFN response of the pDCs from SSc and Healthy control. RNASeq revealed SSc pDCs enables an unexpected TLR8-IFN signaling to increase the IFN signature. This study lays the groundwork for resolving the origin of the SSc transcriptional signatures and the disease heterogeneity towards precision medicine applications.

overall_design: Frozen PBMCs from 4 healthy and 4 SSc patient donors were sorted to obtain enriched pDCs (CD3-CD14-CD123+BDCA4+) and T/pDC cell depleted PBMCs (CD3-CD123-BDCA4-) fractions, the two fractions were then remerged at 1:2 ratio. Cell viability of all donors are above 85%. 8000 cells from each donor were loaded to 10x single cell microfluidic chip to obtain around 5000 recovery cells. Libraries were constructed by following the instructions of 10x company (Chromium Single Cell 3’ Reagent kits v3). The 8 libraries were quantified by NEBNext Library Quant Kit (E7630S) and equally merged and sequenced by Novaseq. The sequenced data were analyzed in Cellranger to align reads, generate feature-barcode matrices. A merged dataset were obtained and batch corrected using FastMNN17 in a base of Seurat pipeline18 (https://satijalab.org/seurat/articles/get_started.html). Doublets were removed manually by excluding cells with more than two cell type markers. The trimmed dataset are visualized by UMAP plot.

first: Yong

last: Du

first: Franck

last: Barrat


title: Mapping the heterogeneity of pDCs and Monocytes of systemic sclerosis at single cell level [Bulk RNA-seq]

overall_design: 40 ml blood were collected from 4 SSc patients and pDCs were purified from PBMCs. 1-3 x 104 purified pDCs were cultured in 96 U-bottom well with tissue culture medium or TLR8L (200 μg/ml) for 6h. Total RNA was isolated using RNeasy Plus Mini kit. NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (NEB E6420S) were used to prepare Illumina-compatible sequencing libraries. Quality of all RNA were evaluated with BioAnalyser 2100 (Agilent). Pair-end reads were obtained on an Illumina HiSeq 4000 in the Weill Cornell Epigenomics Core Facility at the depth of 16-27 million fragments per sample. Sequencing quality was measured with fastp 19. Reads were then mapped reads in genes counted against the human genome (hg38) with STAR aligner and Gencode v21 20, 21. Differential gene (DEGs) expression analysis was performed in R 22 using the edgeR package 23. Genes with low expression levels (< 3 cpm) were filtered from all downstream analyses. The Benjamini–Hochberg false discovery rate procedure was used to calculate the FDR.


title: Bacterial mRNA pseudouridylation by RluA [pseudoseq]

overall_design: Pseudouridine profiling by Pseudo-Seq in knockout strains of all 11 pseudouridine synthases in E. coli MG1655

last: Schaening Burgos


title: Genome-wide transcriptome analysis of VC and VHL-KO Caki-1 cells.

city: Durham

first: Meng

last: Jiao

address: 905 S. LaSalle Street

institute: Duke University

summary: To compare the transcriptomic changes mediated by VHL knockout in Caki-1 cells, we carried out the next-generation sequencing (NGS)-derived transcriptomic profiling. We found significant enrichment of gene signatures involving IFNa/b signaling in VHL-KO Caki-1 cells compared to control cells. Our analysis also demonstrated that VHL deficiency induced the expression of genes associated with the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway involved in sensing the cytoplasmic presence of double-stranded DNA (dsDNA).

overall_design: mRNA expression profiles of control and VHL-KO Caki-1 cells.

first: Xuhui

last: Bao

first: Chuan-Yuan


title: Chromatin-remodeling factor OsINO80 regulate H2A.Z incorporation [anti-H2A.Z & H3 ChIP-seq]

city: Shanghai

first: Liufan

last: Yin

state: Shanghai

address: No. 2005, Songhu Road

department: School of Life Science

institute: Fudan University

summary: INO80, one member of ATP-ase remodeler factor, play a role on chromatin dynamic modulation. OsINO80 (LOC_Os03g22900) is identified as a homologue of INO80 in rice. To study the function of OsINO80 on H2A.Z, we analyzed the distribution of OsINO80 and H2A.Z in 14-week-old seedling of Oryza stative Japanese rice plant through ChIP assay following by high through sequencing (ChIP-seq).Integrated analysis showed that OsINO80 regulate the H2A.Z level.

overall_design: ChIP-seq was applied to comprehensively profile the H2A.Z and H3 landscape in NIP and osino80 mutant

first: Kangxi

first: Chao

first: Yuhao

last: Liu

first: Aiwu

last: Dong


title: Bacterial mRNA pseudouridylation by RluA

first: Wendy

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

overall_design: Refer to individual Series


title: Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Cd226-/- BMDM Transcriptomes

city: xi'an

first: Yun

last: Song

address: NO.169 changle west road

institute: The fourth military medical university

summary: Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare WT-BMDM vs. cd226-BMDM transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis

overall_design: BMDM mRNA profiles of 56-day old wild type (WT) and Cd226-/- mice


title: Chromatin-remodeling factor OsINO80 regulate H2A.Z incorporation [OsINO80 ChIP-seq]

overall_design: ChIP-seq was applied to comprehensively profile the OsINO80 binding landscape in OsINO80 overexpressed plant.


title: Plasma microRNA interpersonal variability in healthy individuals, pregnant women, and an individual with a stably-altered neuroendocrine phenotype

first: Klaas

middle: E. A.

last: Max

state: NY

address: 1230 York Avenue

department: RNA Molecular Biology

institute: Rockefeller University

type: Non-coding RNA profiling by high throughput sequencing

summary: Large cohort: exRNA composition of platelet-depleted plasma from a cohort of 173 female and 63 male healthy volunteers of diverse races and ages, collected at two visits spaced two weeks apart; 4 formerly-collected aliquots from study subject P12, who displayed a stable neuroendocrine miRNA signature in plasma; 20 plasma samples of pregnant women spanning all trimesters T1 to T3 of pregnancy, with an expected placenta miRNA contribution. P12 Family: exRNA from two replicates of platelet-depleted plasma from three unstudied P12 family members and a sample from P12 collected four years after the initial discovery of the phenotype, along with samples of two healthy volunteers. Plasma subfractions: exRNA of samples from P12 and his family, two healthy male volunteers, two non-pregnant, and nine pregnant women, separated by differential ultracentrifugation into 10,000 × g and 100,000 × g pellets, and corresponding 10,000 × g and 100,000 × g supernatants.

overall_design: sRNA-derived cDNA profiling of exRNA isolated from platelet-depleted EDTA plasma and 10,000 x g and 100,000 x g plasma subfractions. Pellet and supernatant fractions were generated by differential ultracentrifugation. Automated RNA isolation and multiplexed library preparation were done using propriatory protocols (KEA Max et al., PNAS, 2018).

middle: E

first: Victoria

middle: R

first: Michael

last: Chang

first: Jonathan

last: Liau

first: Pavel

last: Morozov

first: Zev

last: Williams

first: Thomas

last: Tuschl


title: RNA-sequencing assay to Identify Genes Promoting Lung Metastasis of Esophageal Squamous Cell Carcinoma (ESCC) Cells

first: Zhihua

address: No.17, Panjiayuannanli, Chaoyang District

department: Cancer Institute & Hospital

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

summary:
Purpose: To elucidate the molecular basis governing lung metastasis in ESCC cells, we established two subpopulations of ESCC cells with enhanced pulmonary metastatic potential, namely K30LM3 and K450LM2 respectively. Then, transcriptional profiling of highly metastatic cells (K30LM3 and K450LM2) and their parental cells (K30 and K450) were performed to characterize differentially expressed genes (DEG).
Methods: When parental and highly metastatic cells grew to approximately 80% confluence, these cells were washed twice using pre-chilled PBS. Total RNAs were harvested using Trizol reagents (Thermo Fisher Scientific).  Poly(A) mRNA was isolated and enriched using NEBNext Poly(A) mRNA Magnetic Isolation Module. The mRNA was then recovered for library generation with NEBNext ® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, E7420S) following the manufacturer’s instructions. The cDNA libraries were sequenced at WuXiNextCODE (China). FastQC was used to examine the quality of raw reads. Read alignment was conducted using STAR (v2.5.1b),  and R package edgeR (v3.8.5) was used to determine relative transcript abundances and differentially expressed genes (DEGs) between sample pairs.
Results: Generally, 1502 genes (FDR<0.05, Fold Change>2) between K30 and K30LM3 cells and 650 genes (FDR<0.05, Fold Change>2) between K450 and K450LM2 cells were detected. Among thses DEGs, 52 genes were co-upregulated while 32 genes were co-downregulated in K30LM3/K450LM2 cells. GSEA showed that these commonly-changed genes were significantly enriched in epithelial-mesenchymal transition (EMT), cell adhesion, and anoikis process.
Conclusions: In vivo selection approach enriched highly metastatic subpopulations with acquired essential  molecular traits.

overall_design: Three biological replicates for each group - K30, K450, K30LM3, and K450LM2 - were subjected to RNA-sequencing analysis respectively, among which K30 acted as the reference sample for K30LM3 and K450 for K450LM2.

first: Xuantong

last: Zhou

first: Furong

last: Huang

first: Gang

last: Ma


title: B cells targeting parasites capture spatially linked antigens to secure T cell help

status: Public on Aug 09 2024

city: Canberra

first: Xin

last: Gao

state: ACT

address: 131 Garran Rd

country: Australia

institute: The Australian National University

summary: Our understanding of T-cell-dependent humoral responses has been largely shaped by studies involving model antigens such as recombinant proteins and viruses. In these contexts, B cells internalize the entire antigen or pathogen, and present a range of antigens to helper CD4+ T cells to initiate the humoral response. However, this model does not account for large pathogens (such as parasites) that are too large to be taken up by individual B cells, and the mechanisms by which B cells acquire and present antigens from large complex pathogens to T cells remain poorly understood. Here we used Plasmodium, the causative parasite of malaria, as a model to investigate the requirements for T cell help for B cells targeting the Plasmodium surface circumsporozoite protein (CSP). Upon Plasmodium sporozoite (SPZ) immunization, CSP-specific B cells can form a synapse-like structure with SPZs and take up CSP and non-CSP surface antigens. As a result, CSP-specific B cells can receive help from CD4+ T cells specific to antigens that are located on the surface but not cytosol of the Plasmodium SPZ. Therefore, B cells can obtain help, not only from T cells with the same protein specificity, but also from T cells specific for spatially linked antigens. This flexibility in T cell help may enhance the initiation and maintenance of humoral immune responses to complex pathogens.

overall_design: MD4 mice were transferred with/without Ighg2A10 B cells, followed by rPfCSP-alum, PfCSP-SPZ immunization or left untreated, and spleens were collected 10 days post immunization. Tfh cells (CD4+CD44+PD-1+CXCR5+) were FACS-purified, hashtaged, counted and pooled (around 2000 cells per mice) for 10X scRNA-seq. Each sample has cells from 5 individual mice that are Hashtagged as 1-5. For sample 1, Hashtag1,2,3 : CSP1,2,3; Hashtag4,5: NoB1, NoB2. For sample2, Hashtag1,2,3: SPZ1,2,3; Hashtag4,5: UT1,2. More detailed information can be found at the R Markdown file associated with the paper at https://doi.org/10.6084/m9.figshare.26526313

first: Ian

last: Cockburn


title: CUT&Tag analysis of H3K9la in Cal27 and HN30 cells

first: Hailong

address: Zhizaoju Rd

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

summary: To identify the genes directly regulated by H3K9la on a genome-wide scale, we performed CUT&Tag assays using a H3K9la specific antibody in CAL27 and HN30 with or without the lactic acid treatment, and performed CUT&Tag assays using a H3K9la specific antibody in HN30 under hypoxia condition.

overall_design: Refer to individual Series.

first: Wang

last: Ruijie

first: Li

last: Chuwen

last: Mingyu


title: PRDM3/16 Regulate Chromatin Accessibility Required for NKX2-1 Mediated Alveolar Epithelial Differentiations and Function [Cut&Run H3K4me3]

city: Cincinnati

first: Yan

last: Xu

state: OH

address: 3333 Burnet Ave

institute: Cincinnati Children's Hospital Medical Center

summary: Differential chromatin accessibility accompanies and mediates transcriptional control of diverse cell fates and their differentiation during embryogenesis. While the critical role of NKX2-1 and its transcriptional targets in lung morphogenesis and pulmonary epithelial cell differentiation is increasingly known, mechanisms by which chromatin accessibility alters the epigenetic landscape and how NKX2-1 interacts with other co-activators required for alveolar epithelial cell differentiation and function are not well understood. Here, we demonstrate that the paired domain zinc finger transcriptional regulators PRDM3 and PRDM16 regulate chromatin accessibility to mediate cell differentiation decisions during lung morphogenesis. Combined deletion of Prdm3 and Prdm16 in early lung endoderm caused perinatal lethality due to respiratory failure from loss of AT2 cell function. Prdm3/16 deletion led to the accumulation of partially differentiated AT1 cells and loss of AT2 cells. A combination of single cell RNA-seq, bulk ATAC-seq, and CUT&RUN demonstrated that PRDM3 and PRDM16 enhanced chromatin accessibility at NKX2-1 transcriptional targets in peripheral epithelial cells, with all three factors binding together at a multitude of cell-type specific cis-active DNA elements. Network analysis demonstrated that PRDM3/16 regulated genes critical for perinatal AT2 cell differentiation, surfactant homeostasis, and innate host defense. Lineage specific deletion of PRDM3/16 in AT2 cells led to lineage infidelity, with PRDM3/16 null cells acquiring a partial AT1 fate. Together, these data demonstrate that NKX2-1-dependent regulation of alveolar epithelial cell differentiations is mediated by epigenetic modulation via PRDM3/16.

overall_design:
Lungs were harvested from fetal wild type and PRDM3.16 double knockout C57Bl6 mice at E17.5 and the epithelial cells were isolated using EpCAM+ microbeads.
We investigated methylation activation marks via Cut&Run with an H3K4me3 antibody in C57BL6 and PRDM3.16 double knockout EpCAM+ sorted lung epithelial cells at E17.5 timepoint

first: Hua

last: He

first: Shelia

middle: M

last: Bell

first: Ashley

last: Davis

first: Shuyang

last: Zhao

first: Anusha

last: Sridharan

first: Cheng-Lun

first: Minzhe

last: Guo

first: John

last: Snowball

first: William

last: Zacharias

first: Daniel

last: Swarr

first: Mineo

last: Kurokawa

first: Susumu

last: Goyama

first: Jeffery

last: Whitsett


title: Epigenetic and transcriptional alterations with Kmt2c and Kmt2d knockout

first: Naitao

last: Wang

address: 1275 York Avenue

department: HOPP

institute: Memorial Sloan Kettering Cancer Center

summary: KMT2C and KMT2D are two of the most frequently mutated genes in bladder cancer and in histologically normal urothelium. In this study, we developed mouse models to investigate the molecular mechanism of Kmt2c/d loss in urothelial tumorigenesis.

overall_design: Comparision of epigenome and transcriptome between WT and Kmt2c/d knockout (dKO) mouse bladder urothelail cells

first: Yu

last: Chen


title: Single cell RNA Squencing of Human Eosinophils in Allergc Inflammation in the Esophagus

city: cincinnati

first: Marc

last: Rothenberg

state: OHIO

address: 3333 burnet avenue

institute: cincinnati children's hospital mc

summary: Eosinophils are elusive cells involved in allergic inflammation. Herein, we profiled 586 human eosinophils from the circulation and an allergic inflammatory site, the esophagus, of patients with eosinophilic esophagitis by Seq-Well–based single-cell RNA sequencing. The esophageal eosinophils were composed of a population of activated eosinophils (enriched in 659 genes compared with peripheral blood-associated eosinophils) and a small population of eosinophils resembling peripheral blood eosinophils (enriched in 62 genes compared with esophageal eosinophils). Esophageal eosinophils expressed genes involved in sensing and responding to diverse stimuli, most notably interferon-Ɣ(IFN-Ɣ), interleukin 10 (IL-10), histamine and leukotrienes, and succinate metabolite signaling. Esophageal eosinophils were most distinguished from other esophageal populations by gene expression of the receptors CCR3, HRH4, SUCNR1, and VSTM1; transcription factors CEBPE, OLIG1, and OLIG2; protease PRSS33; and hallmark eosinophil gene CLC. A web of bidirectional eosinophil interactions with other myeloid cells, T cells, fibroblasts, and the epithelium and vasculature was derived. Comparing esophageal eosinophils and mast cells revealed that esophageal eosinophils expressed genes involved in DAP12 interactions, IgG receptor-triggered events, immunoregulation, and IL-10 signaling, whereas esophageal mast cells expressed genes involved in arachidonic acid metabolism and response to unfolded proteins. These findings indicate that esophageal eosinophils exist as two populations, a minority population resembling blood eosinophils and the other population characterized by high de novo transcription of diverse sensing receptors and inflammatory mediators readying them to intersect with diverse cell types.

overall_design: Blood samples from EoE patients and control individuals were subject to single-cell suspensions and  following red blood cell lysis. The cells were subject to a commercial seq-well platform, the Hive CLX gravity based scRNA-sequencing

first: Netali

middle: B

last: Morgenstern


title: Transcriptome sequencing of sheep hypothalamus tissue reveals the regulatory role of lncRNA in the mechanism of pubertal estrus initiation

city: Alar

first: Rong

last: Xuan

state: XinJiang

address: No. 1487, Tarim Avenue East, Alar City, Xinjiang

department: Tarim University

institute: College of Animal Science and Technology

summary: Improving the fertility of sheep is an important goal in sheep breeding as it greatly increases the productivity. Dolang sheep is a typical representative breed of lamb in Xinjiang and is the main local sheep breed and meat source in the region. To explore the genes associated with the initiation of puberty in Dolang sheep, the hypothalamic tissues of Dolang sheep prepubertal, pubertal, and postpubertal periods were collected for RNA-seq analysis on the Illumina platform.

overall_design: Dolang sheep maintained in the Tarim University experimental station were used as the model in this study. The ewes were observed at 10 o ‘clock, 14 o ‘clock, and 18 o ‘clock every day. The criteria for judging puberty in ewes were mental restlessness, the tendency to walk, the acceptance of ram riding, and the presence of mucus in the vulva. All sheep were in good health. Hypothalamus samples were collected from ewes that were first found to be undergoing puberty, immediately frozen in liquid nitrogen, and stored at −80°C for further analysis. The hypothalami were further collected from prepubertal and postpubertal ewes maintained in the same manner. A total of nine sheep’s hypothalamic tissues were collected. Three biological replicates were performed for each period and analyzed.

first: Feng

last: Xing

first: Zhishuai

last: Zhang

first: Zhiyuan

last: Sui


title: PARK7/DJ-1 deficiency impairs microglial activation in response to LPS-induced inflammation

city: Strassen

first: Tony

last: KAOMA

address: 1 A-B Rue Thomas Edison

department: Quantitative Biology Unit

country: Luxembourg

institute: Luxembourg Institute of Health

summary: Background: Specific microglia responses are thought to contribute to the development and progression of neurodegenerative diseases, including Parkinson’s disease (PD). However, the phenotypic acquisition of microglial cells and their role during the underlying neuroinflammatory processes remain largely elusive. Here, according to the multiple-hit hypothesis, which stipulates that PD etiology is determined by a combination of genetics and various environmental risk factors, we investigate microglial transcriptional programs and morphological adaptations under PARK7/DJ-1 deficiency, a genetic cause of PD, during lipopolysaccharide (LPS)-induced inflammation. Methods: Using a combination of single-cell RNA-sequencing, bulk RNA-sequencing, multicolor flow cytometry and immunofluorescence analyses, we comprehensively compared microglial cell phenotypic characteristics in PARK7/DJ-1 knock-out (KO) with wildtype littermate mice following 6- or 24-hour intraperitoneal injection with LPS. For translational perspectives, we conducted corresponding analyses in human PARK7/DJ-1 mutant induced pluripotent stem cell (iPSC)-derived microglia and murine bone marrow-derived macrophages (BMDMs). Results: By excluding the contribution of other immune brain resident and peripheral cells, we show that microglia acutely isolated from PARK7/DJ-1 KO mice display a distinct phenotype, specially related to type II interferon and DNA damage response signaling, when compared with wildtype microglia, in response to LPS. We also detected discrete signatures in human PARK7/DJ-1 mutant iPSC-derived microglia and BMDMs from PARK7/DJ-1 KO mice. These specific transcriptional signatures were reflected at the morphological level, with microglia in LPS-treated PARK7/DJ-1 KO mice showing a less amoeboid cell shape compared to wildtype mice, both at 6 and 24 hours after acute inflammation, as also observed in BMDMs.  Conclusions: Taken together, our results show that, under inflammatory conditions, PARK7/DJ-1 deficiency skews microglia towards a distinct phenotype characterized by downregulation of genes involved in type II interferon signaling and a less prominent amoeboid morphology compared to wildtype microglia. These findings suggest that the underlying oxidative stress associated with the lack of PARK7/DJ-1 affects microglia neuroinflammatory responses, which may play a causative role in PD onset and progression.

overall_design:
Comparison of sorted microglia (CD11b+CD45int cells) between PARK7/DJ-1 KO and wildtype peripherally LPS-injected mice at single-cell resolution. One condition: LPS 24h in PARK7/DJ-1 KO  (from 2 mice per group; 1 female and 1 male). Comparison conducted with LPS 24h wildtype (from 2 mice per group; 1 female and 1 male)

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Submitter states that they were not provided raw data by the sequencing platform for submission.
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first: Carole

last: Sousa

first: Suresh

last: Poovathingal

last: Kaoma

first: Alexander

last: Skupin

first: Alessandro

last: Michelucci