title: Uncovering the Dynamics of Immune Cell Populations in the First Week of Life: The Interplay of Neutrophils and Breastfeeding [methylation]

city: Boston

first: Ofer

last: Levy

state: MA

address: 300 Longwood Avenue

department: Infectious Disease

institute: Boston Children's Hospital

type: Methylation profiling by genome tiling array

summary: The first few days of life are characterized by rapid external and internal changes that require rapid host adaptations. Despite growing evidence of the impact of this period on an individual’s lifelong health trajectory, this period remains largely uncharted. To decipher key factors associated with changes in peripheral blood cell composition we conducted a stringently standardized, high-throughput immune phenotyping investigation on 796 newborns across diverse settings (The Gambia, West Africa and Papua New Guinea, Australasia) in the framework of a Human Immunology Project Consortium study. Samples were collected twice from each newborn during the first week of life and analyzed via high throughput flow cytometry with an unbiased automated gating algorithm to capture cell compositional changes at an unprecedented level of detail. We found that immune cell composition in peripheral blood changes along patterns highly conserved across populations and environments. Changes with age/day of life were most pronounced in the innate myeloid compartment. While there was minimal impact of sex, season of birth, mother’s age or origin (i.e., environmental and genetic background), breastfeeding and vaccination were strongly associated with increase in cell counts of peripheral blood neutrophils and monocytes. Our results may suggest a possible association of initiation of breastfeeding with changes in immune ontogeny, and thus possibly host immune-mediated protection from infection. These data begin to outline a specific window of opportunity for interventions that can deliberately direct immune ontogeny.

overall_design: longitudinal neonatal cohort study

first: Tobias

last: Kollmann

first: Sebastiano

last: Montante

first: Joann

last: Arce

first: Rym

last: Ben-Othman

first: David

last: Martino

first: EPIC

last: Consortium


title: Role of sex as a biological variable in neonatal alveolar macrophages

status: Public on Aug 05 2024

city: Philadelphia

first: Abiud

last: Cantu

state: Pennsylvania

address: 3615 Civic Center Blvd

department: Neonatology

country: USA

institute: Children's Hospital of Philadelphia

type: Expression profiling by high throughput sequencing

summary: In this study, lung alveolar macrophages were sorted and subjected to bulk RNA-Seq from male and female neonatal lungs.

overall_design: Neonatal male and female pups were exposed to hyperoxia (95% FiO2) from PND1 through PND5. Lungs were homogenized, and alveolar macrophages were sorted using flow cytometry on PND7 and subjected to bulk RNA-Seq.

first: Krithika

last: Lingappan


title: Modeling late-onset Alzheimer's disease neuropathology via direct neuronal reprogramming III

city: St. Louis

first: Zhao

last: Sun

state: MO

address: 4523 Clayton Ave

department: Developmental Biology

institute: Washington University in St. Louis

summary: Late-onset Alzheimer’s disease (LOAD) is the most common form of AD. However, modeling sporadic LOAD, without clear genetic predispositions, to capture hallmark neuronal pathologies such as extracellular amyloid-β (Aβ) plaque deposition, intracellular tau tangles, and neuronal loss, remains an unmet need. Here, we demonstrate that neurons generated by microRNA-based direct reprogramming of fibroblasts from patients affected by autosomal dominant AD (ADAD) and LOAD in a three-dimensional (3D) environment, effectively recapitulate key neuropathological features of AD without additional cellular or genetic insults. These LOAD neurons exhibit Aβ-dependent neurodegeneration, as treatment with β- or γ-secretase inhibitors before (but not subsequent to) Aβ deposit formation mitigated neuronal death. Moreover, inhibiting age-associated retrotransposable elements (RTEs) in LOAD neurons reduced both Ab deposition and neurodegeneration. Our study underscores the efficacy of modeling late-onset neuropathology of LOAD through high-efficiency microRNA-based neuronal reprogramming.

overall_design: By optimizing the potency of microRNA-mediated direct neuronal conversion of patient fibroblasts, we developed a matrigel-based three-dimensional (3D) culture system for generating cortical neurons, a subtype known to be susceptible to develop AD pathologies. For characterizing amyloid β(Aβ) plaques, tau pathology, and neurodegeneration in the reprogrammed cortical neurons, we carried out immunofluorescence staining, RNA-sequencing and other biochemistry-based assays for AD-associated phenotypes.

first: Ji-Sun

last: Kwon

first: Andrew

middle: S

last: Yoo


title: Nucleosome remodeler exclusion by histone deacetylation enforces heterochromatic silencing and epigenetic inheritance

city: Bethesda

first: Shiv

last: Grewal

state: MD

address: 9000 Rockville Pike

department: National Cancer Institute

institute: National Institutes of Health

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Heterochromatin enforces transcriptional gene silencing and can be epigenetically inherited, but the underlying mechanism has remained unclear.Here we show that histone deacetylation, a conserved feature of heterochromatin domains, blocks SWI/SNF subfamily remodelers involved in chromatin unraveling, thereby stabilizing modified nucleosomes that preserve gene silencing. Histone hyperacetylation, resulting from either the loss of a histone deacetylase (HDAC) or the direct targeting of histone acetyltransferase to heterochromatin, permits remodeler access, leading to silencing defects.The requirement for HDAC in heterochromatin silencing can be bypassedby impeding SWI/SNF activity. Highlighting the crucial role of remodelers, merely targeting SWI/SNF to heterochromatin, even in cells with functional HDAC, increases nucleosome turnover, causing defective gene silencing and compromised epigenetic inheritance. This study elucidates a fundamental mechanism whereby histone hypoacetylation, maintained by high HDAC levels in heterochromatic regions, ensures stable gene silencing and epigenetic inheritance,providing significant insights into genome regulation relevant to human diseases.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for the proteins Snf22, Snf21, and the lysine 9 trimethylated histone H3 in fission yeast. Input control and immunoprecipitation samples are provided for each experiment.

first: Rakesh

middle: K

last: Sahu

first: Jothy

last: Dhakshnamoorthy

first: Shweta

last: Jain

first: Hernan

middle: D

last: Folco

last: Wheeler

middle: I


title: Single cell RNA sequencing revealing gene expression profile of thalamus from mice at the age of 2 months

city: Shanghai

first: Howell

last: Cheng

address: 320 Yueyang Road

country: China

institute: Institute of Neuroscience, Chinese Acedemy of Sciences

summary: Single cell RNA transcriptomic expression profile of adult mouse thalamus cells containing astrocytes, oligodendrocytes, mural cells, endothelial cells, etc.

overall_design: The thalamic tissue dissected from two adult wildtype C57BL/6J mice were sequentially digested with papain, protase and dispase in choline chloride solution for preparing single-cell suspensions, and single-cell libraries were generated using 3’-labeling kit (V3, 10X Genomics) and sequenced on the Illumina NovaSeq 6000 sequencer.

first: Xuewen

first: Zhen-Ning

last: Zhou

first: Min

last: Zhang

first: Zhi-Qi

last: Xiong


title: Adipocyte induced distinct gene expression profiling of human endometrioid endometrial cancer cells upon SIRT1 inhibition.

city: VASCO

first: Akanksha

last: Savardekar

state: GOA

address: SANCOALE

department: BIOLOGICAL SCIENCES

country: India

institute: BITS PILANI K K BIRLA GOA CAMPUS

summary: We examined the mechanism by which adiposity regulates endometrioid endometrial cancer progression. Ishikawa EEC cells were co-cultured with mature adipocytes in presence or absence of SIRT1 inhibitor EX527, and total RNA was isolated for RNA-seq analysis and focus on the functional relevance that adipocyte co-culture affected pathways and related biomarkers may have in endometrial cancer response to adiposity.

overall_design: 500ng of total RNA from Ishikawa control, co-cultured EC, Co-cultured EC+EX527 cells was used to enrich the mRNA using NEBNext Poly (A) mRNA magnetic isolation module (Catalog: E7490, New England Biolabs) by following the manufacturers’ protocol. The enriched mRNAs were further taken for the library preparation using the NEBNext® Ultra™ II RNA Library Prep Kit for Illumina (Catalog: E7775S, New England Biolabs). The cDNA was amplified by 12 cycles of PCR with the addition of NEBNext Ultra II Q5 mastermix, and “NEBNext® Multiplex Oligos for Illumina” to facilitate multiplexing while sequencing. The amplified products were then purified using 0.9X AMPure XP beads (Catalog: A63881, Beckman Coulter) and the final DNA library was eluted in 15μl of 0.1X TE buffer. The library concentration was determined in a Qubit.3 Fluorometer (Catalog: Q33216, Life technologies usingThe Qubit dsDNA HS (High Sensitivity) Assay Kit (Catalog: Q32854, ThermoFisher Scientific). The library quality assessment was done using Agilent High Sensitivity D1000 ScreenTape System (Catalog:5067- 5584, Agilent) in a 4150 TapeStation System (Catalog: G2992AA, Agilent) which is designed for analysing DNA molecules from 35 to 1000bp. The sequence data was generated using Illumina HiSeq. The rRNA removed reads mapped onto indexed Human reference genome(GRCh38.p7) using STAR v2 aligner

first: Arnab

last: Banerjee


title: Transcriptional changes in A549 cells after four weeks of exposure to 30 μg/ml diesel exhaust particles measured by Agilent SurePrint G3 Human Gene Expression Microarray.

city: Kanazawa

first: Alexandra

last: Lukyanchuk

state: --Not Applicable--

address: 13-1 Takara-machi

country: Japan

institute: Kanazawa University

summary: Diesel exhaust particles (DEP), which contain hazardous compounds, are emitted during the combustion of diesel. As approximately one-third of the vehicles worldwide use diesel, there are growing concerns on the risks posed by DEP to human health. Long-term exposure to DEP is associated with airway hyperresponsiveness, pulmonary fibrosis, and inflammation; however, the molecular mechanisms behind the effects of DEP on the respiratory tract are poorly understood. Such mechanisms can be addressed by examining transcriptional and DNA methylation changes. In this study, we investigated the effect of 4 weeks exposure to 30 μg/ml DEP on gene expression levels in A549 cells.

overall_design: A549 cells were treated with 30 μg/ml DEP for four weeks, using 2-3 biological replicates. The gene expression leves for control and treated groups were obtained using Agilent SurePrint G3 Human Gene Expression Microarray. The effect of DEP on transcriptome was assesed based on the results of differentially expression analyses compared to the control

first: A

first: N

last: Muraki

first: T

last: Kawai

last: Sato

first: K

last: Hata

last: Ito

last: Tajima


title: Long-term exposure to diesel exhaust particles induces concordant changes in DNA methylation and transcriptome in human adenocarcinoma alveolar basal epithelial cells

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

overall_design: Refer to individual Series


title: DNA methylation changes in A549 cells after four weeks of exposure to 30 μg/ml diesel exhaust particles.

summary: Diesel exhaust particles (DEP), which contain hazardous compounds, are emitted during the combustion of diesel. As approximately one-third of the vehicles worldwide use diesel, there are growing concerns on the risks posed by DEP to human health. Long-term exposure to DEP is associated with airway hyperresponsiveness, pulmonary fibrosis, and inflammation; however, the molecular mechanisms behind the effects of DEP on the respiratory tract are poorly understood. Such mechanisms can be addressed by examining transcriptional and DNA methylation changes. In this study, we investigated the effect of 4 weeks exposure to 30 μg/ml DEP on DNA methylation levels in A549 cells.

overall_design: A549 cells were treated with 30 μg/ml DEP for four weeks, using four biological replicates. The DNA methylation levels of 850K CpG sites were obtained for control and treated groups using the IInfinium MethylationEPIC BeadChips microarray. The effect of DEP on DNA methylame was assesed based on the results of differentially methylated analyses compared to the control


title: Transcriptional changes in A549 cells after four weeks of exposure to 30 μg/ml diesel exhaust particles measured by RNA-seq.

overall_design: A549 cells were treated with 30 μg/ml DEP for four weeks, using four biological replicates. The gene expression leles for control and treated groups were obtained using genome-wide RNA sequensing. The effect of DEP on transcriptome was assesed based on the results of differentially expression analyses compared to the control


title: Identification of a dose of diesel exhaust particles that is sufficient to induce significant DNA methylation changes in A549 cells after four weeks of exposure.

summary: Diesel exhaust particles (DEP), which contain hazardous compounds, are emitted during the combustion of diesel. As approximately one-third of the vehicles worldwide use diesel, there are growing concerns on the risks posed by DEP to human health. Long-term exposure to DEP is associated with airway hyperresponsiveness, pulmonary fibrosis, and inflammation; however, the molecular mechanisms behind the effects of DEP on the respiratory tract are poorly understood. Such mechanisms can be addressed by examining transcriptional and DNA methylation changes. In this study, we investigated DEP dose sufficient to induce significant DNA methylation changes after four weeks of exposure.

overall_design: A549 cells were treated with four doses of DEP (0, 3, 10 and 30 μg/ml ) for four weeks, using 2-3 biological replicates per group. The DNA methylation levels of 850K CpG sites were obtained for each treated group using the IInfinium MethylationEPIC BeadChips microarray. The sufficient dose of DEP was identified based on the results of differentially methylated analyses compared to the control


title: Modeling late-onset Alzheimer's disease neuropathology via direct neuronal reprogramming II

first: Zhao

last: Sun

first: Ji-Sun

last: Kwon

first: Andrew

last: Yoo


title: Revealing the Crucial Roles of Myxoma Cells and CD206+ Macrophages in Cardiac Myxoma Progression Based on Single-Cell Sequencing

city: Chengdu

first: Long

middle: Qian

last: Kang

state: Sichuan Province

address: 新川路2222号

department: Biotherapeutics

institute: Sichuan University

summary: Cardiac myxoma is a commonly encountered, potentially life-threatening tumor within the heart. We used single-cell RNA sequencing (scRNA-seq) to analyze the identification and characterization of cellular subtypes, cell trajectory analysis, and intercellular communication of cardiac myxoma tissues.

overall_design: Human cardiac myxoma cells were analyzed by single-cell sequencing after cardiac myxoma tissue was minced with scissors and digested with collagenase into single-cell suspensions.

first: Zedong

last: Jiang

first: Qianlong

first: Hong

last: Qian

first: Huan

last: Tong

first: Guangqi

last: Li

first: Yunuo

last: Zhao

first: Xuelei

last: Ma


title: Modeling late-onset Alzheimer's disease neuropathology via direct neuronal reprogramming


title: Altered RNA export by SF3B1 mutants confers sensitivity to nuclear export inhibition

city: Miami

first: Felipe

last: Beckedorff

state: FL

address: 1501 NW 10th Avenue room 731.G , Miami, FL

institute: University of Miami

summary: SF3B1 mutations are the most frequent spliceosomal alterations across cancers, yet no successful therapy exists to target this pathway. Previous findings from a phase 2 clinical trial of the XPO1 inhibitor selinexor in patients with high-risk myelodysplastic neoplasms (MDS) relapsed or refractory to hypomethylating agents (HMA) revealed increased activity in patients with SF3B1 mutations. XPO1 (Exportin-1) is responsible for the export of over 200 proteins, but also plays a role in the transport of multiple RNA species, including small nuclear RNAs (snRNAs), ribosomal RNAs (rRNAs), and select messenger RNAs (mRNAs) out of the nucleus. We therefore hypothesized that XPO1 inhibition perturbs RNA export and may preferentially affect SF3B1 mutants via altered splicing, given the role of XPO1 in exporting snRNAs, which form the catalytic portion of the spliceosome.  To evaluate the mechanism underlying preferentially sensitivity of SF3B1-mutants to XPO1 inhibition, we performed nuclear and cytoplasmic fraction followed by RNA sequencing before and after XPO1 inhibition in SF3B1 wildtype and SF3B1 K666N cells (subcellular RNA-seq). Whole transcriptomic analysis of subcellular RNA-seq data revealed more nuclear retention of global RNA transcripts after XPO1 inhibition in the SF3B1 mutant cell line. Similarly, we performed subcellular RNA-seq for small RNAs and found snRNAs to be increased in the nucleus after XPO1 inhibition in the SF3B1 mutant cells. We then performed total cellular RNA sequencing to understand the effect of XPO1 inhibition on global RNA expression and RNA splicing. Differential gene expression analysis identified that XPO1 inhibition had the greatest effect on cell cycle in SF3B1 wildtype cells but in SF3B1-mutant cells differentiation pathways were more significantly affected. Alternative splicing analysis showed increased 3’ alternative splicing events in SF3B1 mutant after XPO1 inhibition. These results signify the mechanistic basis for preferential sensitivity of SF3B1 mutant cells to nuclear export inhibition arises through nuclear retention of spliceosomal snRNAs and select mRNAs that result in perturbation of differentiation pathways.

overall_design:
To determine the effect XPO1 inhibition has on RNA export in SF3B1 mutant cells  we treated K562 SF3B1 wildtype and K562 SF3B1 K666N for 24 hours with vehicle and 200nM selinexor (first-genetation XPO1 inhibitor). We then isolated the RNA to get 1) total RNA 2) nuclear and cytoplasmic fractions of long RNAs/mRNAs 3) nuclear and cytoplasmic fractions small RNAs. All samples were collected in triplicates. Additionally we performed RNA sequencing the combination of XPO1 inhibitors with BCL-family of proteins (venetoclax, A1331852, navitoclax) in duplicates.
Cells were either treated with A) vehicle or B) 200nM selinexor for 24 hours. Followed by one of three RNA sequencing 1) total RNA sequecing 2) total RNA sequecning of nuclear cytoplasmic fractions of long RNAs 3) small RNA sequencing of of nuclear cytoplasmic fractions of short RNAs. The combination treatment RNA sequencing was of PolyA selected RNA sequencing.

first: Sana

last: Chaudhry

first: Justin

last: Taylor


title: RNA-seq of osteogenesis following siRNA depletion of SERPINA3

city: Liverpool

first: David

middle: J

last: Wilkinson

address: William Henry Duncan Building, 6 W Derby St

department: Musculoskeletal & Ageing Science

country: United Kingdom

institute: University of Liverpool

summary: SERPINA3 knockdown was observed to reduce cartilage pellet size following differentiation of mesenchymal stem cells (MSCs) to chondrocytes. To additionally understand any effects on the osteogenesis transcriptome, RNA-sequencing was conducted on primary MSCs depleted of SERPINA3 with siRNA and compared to cells transfected with a siRNA control over a 7 day period.

overall_design: RNA sequencing following mesenchymal stem cells throughout osteogenesis at three different time points with 3 donors as biological replicates with or without SERPINA3 depletion.

first: Jamie

last: Soul


title: Amphiregulin-producing T helper 2 cells have critical role in esophageal fibrosis of EoE

city: chiba city

first: Masahiro

last: Kiuchi

state: chiba

address: cyuoku inohana1-8-1

institute: Chiba univercity

summary:
Background: Massive eosinophil infiltration into the esophagus causes subepithelial fibrosis and esophageal stricture in patients with eosinophilic esophagitis (EoE). However, the pathogenesis of esophageal fibrosis remains unclear.
Objective: The present study aimed to elucidate the cellular and molecular mechanisms underlying the induction of esophageal fibrosis.
Methods: We established a murine model of EoE accompanied by fibrotic responses following long-term intranasal administration of house dust mite (HDM) antigen. Using this murine model, we investigated the characteristics of immune cells infiltrating the fibrotic region of the inflamed esophagus using flow cytometry and histological analyses. We also analyzed the local inflammatory sites in the esophagus of patients with EoE using single-cell RNA sequencing, flow cytometry, and immunohistochemistry.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Murine entire esophagus or human biopsy samples were placed in a C tube  containing digestion buffer (Multi Tissue Dissociation Kit , and tissues were dissociated by a gentleMACS™ octo dissociator with heaters. Esophageal mononuclear cells in cell suspension were separated using Percoll solution.Libraries were then prepared using Chromium Single Cell 3’ Reagent Kits v3.1 following the manufacturer’s protocol. The generated scRNA-seq libraries were sequenced using a total 127 cycle (paired-end reads) with a NovaSeq 6000.Sequence reads from all samples were processed and aggregated using a Cell Ranger. Aggregated data were further analyzed by Seurat. Specifically, we log-normalized the expression matrix, regressed the data against the total number of unique molecular identifier (UMI)’s detected per cell, performed a PCA analysis, and used PCA dimensions 1-50 to find clusters on UMAP. We visualized the single-cell gene expression as UMAP overlays. The single-cell gene expression was also visualized as density plots using the Nebulosa R software package algorithm.

first: Tatsuya

last: Kaneko

first: Chiaki

last: Iwamura

first: Akane

last: Kurosugi

first: Miki

last: Onoue

first: Tomoaki

last: Matsumura

first: Tetsuhiro

last: Chiba

first: Toshinori

last: Nakayama

first: Naoya

last: Kato

first: Kiyoshi

last: Hirahara


title: Systems Biology to Identify Biomarkers of Neonatal Vaccine Immunogenicity - Papua New Guinea (PNG) Validation cohort [methylation]

first: Ofer

last: Levy

summary: Infection is the most common cause of death in early life, especially for newborns and can be reduced by immunization but insufficient knowledge of how vaccines protect the very young limits their optimal use. To gain insight into how vaccines induce protection of the most vulnerable, our project employs two novel approaches studying newborn responses to hepatitis B vaccine (HBV): (a) systems biology that uses technologies which comprehensively measure global changes in molecules such as transcriptomics (RNA) and proteomics (proteins), as well as cell composition of the blood and (b) use of human newborn blood components, collected prior to immunization, to model vaccine responses in vitro (outside the body). Characterizing vaccine-induced molecular patterns (signatures) that correspond to vaccine-mediated protection will accelerate development and optimization of vaccines against early life infections of major global health importance.

first: Tobias

last: Kollmann

first: Robert

last: Hancock

first: Kampmann

last: Beate


title: Transcriptomic analysis of patient-derived resected lung NSCLC cells (part 2)

city: Suita

first: Daisuke

last: Motooka

state: Osaka

address: 3-1, Yamadaoka

institute: NGS core facility, Research Institute for Microbial Diseases, Osaka University

summary: To identify genes encoding cell surface proteins that are preferentially expressed in NSCLC cells, CD45-CD31-EpCAM+ cells were purified by FACS from either tumor regions or unaffected regions in resected lung tissues from patients with NSCLC, then subjected to RNA sequencing analysis.

overall_design: Comparative gene expression profiling analysis of RNA-seq data

first: Naoki

last: Hosen

last: Okuzaki

first: Moto

last: Yaga


title: RNA-seq of chondrogenesis following siRNA depletion of SERPINA3

first: David

last: Wilkinson

summary: SERPINA3 knockdown was observed to reduce cartilage pellet size following differentiation of mesenchymal stem cells (MSCs) to chondrocytes. To understand the global effects on the chondrocyte transcriptome, RNA-sequencing was conducted on primary MSCs depleted of SERPINA3 with siRNA and compared to cells transfected with a siRNA control over a 7 day period.

overall_design: RNA sequencing following mesenchymal stem cells throughout chondrogenesis at three different time points with 3 donors as biological replicates with or without SERPINA3 depletion.

first: Jamie

last: Soul


title: Transcriptomic analysis of patient-derived resected lung NSCLC cells (part 1)


title: Predictive signature of murine and human host response to typical and atypical pneumonia

city: Durham

first: Nicholas

middle: G

last: O'Grady

state: NC

address: 2301 Erwin Rd

department: Department of Medicine, CRU

institute: Duke University

summary: Using gene expression data we developed a predictive signture to discriminate between typical bacterial, atyipcal bacterial, and viral pnuemonia in murine samples and validated it against open source human datasets.

overall_design: Male and Female C57BL/6j mice were exposed to streptococcs pneumoniae, mycoplasma pnuemoniae, and a murine strain of H1N1. Blood was collected for RNA expression analysis at predetermined time point. Amplified RNA was assyed using Affymetrix murine 430A2.0 microarrays containing 22,690 probes. Probe intensities were detected using Axon GenePix 4000B Scanner. These intensities were converted to an eset in the statitical program R. Differential gene expression was  determined using the limma and empirical Bayes tool sets. Pathway analyis was performed using ReactomePA. We use LASSO to develope a predictive sigature to discriminate between these pneumonia types. This signature was validated on open source human datasets.

first: Matthew

last: McCravy

first: Ephraim

last: Tsalik


title: Benchmarking Bulk and Single-cell Variant Calling Approaches on Chromium scRNA-seq and scATAC-seq Libraries

city: Vancouver

first: Tully

middle: Michael

last: Underhill

state: British Columbia

address: 2222 Health Sciences Mall

department: Biomedical Research Centre

country: Canada

institute: University of British Columbia

summary: Single-cell sequencing methodologies such as scRNA-seq and scATAC-seq have become widespread and effective tools to interrogate tissue composition. Increasingly, variant callers are being applied to these methodologies to resolve the genetic heterogeneity of a sample, especially in the case of detecting the clonal architecture of a tumor. Typically, traditional bulk DNA variant callers are applied to the pooled reads of a single-cell library to detect candidate mutations. Recently, multiple studies have applied such callers on reads from individual cells, with some citing the ability to detect rare variants with higher sensitivity. Many studies apply these two approaches to the Chromium (10x Genomics) scRNA-seq and scATAC-seq methodologies. However, Chromium-based libraries may offer additional challenges to variant calling compared to existing single-cell methodologies, raising questions for the validity of variants obtained from such a workflow. To determine the merits and challenges of various variant-calling approaches on Chromium scRNA-seq and scATAC-seq libraries, we use sample libraries with matched bulk whole-genome-sequencing to evaluate the performance of callers. We review caller performance, finding that bulk callers applied on pooled reads significantly outperform individual-cell approaches. We also evaluate variants unique to scRNA-seq and scATAC-seq methodologies, finding patterns of noise but also potential capture of RNA-editing events. Finally, we review the notion that variant calling at the single-cell level can detect rare somatic variants, providing empirical results that suggest resolving such variants is infeasible in single-cell Chromium libraries.

overall_design: Single cell ATAC-seq analyes of epithelioid sarcoma

first: R

middle: W

last: Scott

first: T

middle: M


title: Benchmarking bulk and single-cell variant calling approaches on Chromium scRNA-seq and scATAC-seq libraries

overall_design: Single cell RNA-seq analyes of epithelioid sarcoma


title: Deterioration of multi-level 3D genome organization during breast cancer progression [third-party re-analysis]

status: Public on Aug 06 2024

city: Oslo

first: Roberto

last: Rossini

address: Blindernveien 31

country: Norway

institute: University of Oslo

summary: Breast cancer progression entails intricate, multi-level alterations in genome organization and expression. To gain insights into the modifications in chromatin's three-dimensional (3D) structure during breast cancer progression, we conducted an analysis combining Hi-C data with lamina-associated domains (LADs), epigenomic marks, and gene expression in an in vitro model of breast cancer progression. Our results reveal that while the fundamental properties of topologically associating domains (TADs) remain largely stable, significant changes occur in the organization of compartments and subcompartments. These changes are closely correlated with alterations in the expression of oncogenic genes. We also observed a restructuring of TAD-TAD interactions, coinciding with a loss of spatial compartmentalization and radial positioning of the 3D genome. Notably, we identified a previously unrecognized interchromosomal insertion event, wherein a locus on chromosome 8 housing the MYC oncogene becomes inserted into a highly active region on chromosome 10. This insertion event leads to the formation of de novo enhancer contacts and activation of the oncogene, illustrating how structural variants can interact with the 3D genome to drive oncogenic states. In summary, our findings provide evidence for the degradation of genome organization at multiple scales during breast cancer progression revealing the complex interplay between genomic structure and oncogenic processes.

relation:
Reanalysis of: GSM2258716
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Reanalysis of: GSM2675538
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Reanalysis of: GSM2949413
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Reanalysis of: GSM2949415
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Reanalysis of: GSM3941611
Reanalysis of: GSM4158376
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Reanalysis of: GSM4158379
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Reanalysis of: GSM5504431
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Reanalysis of: GSM5504436
Reanalysis of: GSM5504437
Reanalysis of: GSM5504442
Reanalysis of: GSM5504443
Reanalysis of: GSM5504444
Reanalysis of: GSM5504445

overall_design:
We reanalyzed publicly available ChIP-seq datasets (CTCF, H2AZ, H3K9ac, H3K9me3, H3K27ac, H3K4me3, H3K27me3, H3K79me2, gH2AX, p53) for MCF10A, MCF10AT1 and MCF10Ca1a to compare chromatin states with our subcompartment annotation (GSE246947) (note that not all experiments included dataset for MCF10AT1 and MCF10Ca1a).

The re-analyzed samples are from GSE85158, GSE93757, GSE98551, GSE100234, GSE109721, GSE134297, GSE140252, and GSE181524 Series records.
The sample titles and their GEO, SRA, biosample accessions are provided in the 're-analyzed_sample_accns.txt' and the processed data file associated with each sample is listed in the  'sample_to_processed-data-file.txt'

**Re-analysis data processing step:
nf-core/chipseq v2.0.0
Samples were processed using nf-core/chipseq after collapsing replicates.
hg38 and Gencode v43 were used as reference genome assemby and gene annotation, respectively.
The effective genome size for MACS2 provided to the pipelien through the –macs_gsize option was computed using unique-kmers.py from khmer v2.1.1.
Peaks for transcription factors were called in narrow peak mode, while the rest of the peaks were called in broad peak mode.

**processed data files format and content:
BigWig files contain ChIP coverage scaled to 1 million mapped reads. Files containing coverage for background (control) are suffixed with “_input”.
Files in narrowPeak and broadPeak format contain the peaks called with MACS2.

first: Mohammadsaleh

last: Oshaghi

first: Maxim

last: Nekrasov

first: Aurélie

last: Bellanger

first: Renae

last: Domaschenz

first: Yasmin

last: Dijkwel

first: Philippe

last: Collas

last: Tremethick

first: Jonas

last: Paulsen


title: RNA-Seq profiling of circular RNAs in human colorectal cancer 5-fluorouracil resistance and the potential biomarkers

first: Hanchen

last: Xu

address: No.725, South Wanping Rd

institute: Institute of Digestive, Longhua Hospital, Shanghai University of Traditional Chinese Medicine

type: Non-coding RNA profiling by high throughput sequencing

summary: Colorectal cancer（CRC）is a commonly diagnosed cancer of the digestive system worldwide. Although chemotherapeutic agents and targeted therapeutic drugs are currently available for CRC treatment, drug resistance is a problem that cannot be ignored and needs to be solved. 5-Fluorouracil (5-Fu) is a first-line chemotherapeutic agent for colorectal cancer, to explore the role of circular RNA (circRNA) in 5-Fu resistance, we performed the circRNA expression profile in two CRC cell lines and their homologous 5-Fu resistant cells by high-throughput sequencing. We validated the differentially expressed circRNAs in other two paired CRC cells, confirmed that circ_0002813 and circ_0000236 could have a potential ceRNA mechanism and be involved in the formation of 5-Fu resistance. And we combined the sequencing results of mRNA to construct the regulatory network of circRNA-miRNA-mRNA. Our study revealed that circ_0002813 and circ_0000236 may as the biomarkers to predict the occurrence of 5-Fu resistance in CRC.

overall_design: We performed the circRNA expression profile in two CRC cell lines and their homologous 5-Fu resistant cells by high-throughput sequencing, each group have three samples.

first: Peiqiu

last: Cheng

first: Yujing

last: Liu

first: Shengan

last: Zhang

first: Lu

last: Lu

first: Wenjun

last: Zhou

first: Dan

last: Hu


title: Transcriptome and microbiome-immune changes across preinvasive and invasive anal cancer lesions

status: Public on Aug 04 2024

city: La Plata

first: Martin

middle: Carlos

last: Abba

state: Buenos Aires

country: Argentina

institute: School of Medical Sciences - UNLP

summary: Anal squamous cell carcinoma (ASCC) is a rare gastrointestinal malignancy that is linked to high-risk Human papillomavirus (HPV) infection. It is often preceded by precursor lesions like Low-Grade Squamous Intraepithelial Lesions (LGSIL) and High-Grade Squamous Intraepithelial Lesions (HGSIL). The incidence of ASCC varies across populations, with heightened risk in HIV-positive individuals. In a previous study, we characterized the anal microbiome in high-risk HIV-exposed MSM and TGW subjects. We revealed oncogenic viromes and pertinent bacterial species associated with anal SILs. Our current investigation aimed to delineate transcriptomic and metatranscriptomic changes during the progression from precancerous lesions to ASCC. We collected 70 anal tissue samples across various lesion stages (LGSIL, HGSIL, and ASCC). Our metatranscriptomic analysis revealed that Fusobacterium nucleatum, F. gonidiaformans, Bacteroides fragilis, Campylobacter ureolyticus, and Cribacterium bergeronii were more prevalent in ASCC than in precancerous lesions. These bacterial species contributed with  gene encoding enzymes (e.g.: Acca, glyQ, eno, pgk and por) and oncoproteins (FadA and dnaK) revealing potential new markers for diagnosis or treatment approaches. Our unsupervised transcriptome analysis identified two distinct sample clusters based on histological diagnosis, immune infiltrate, HIV and HPV status, and pathway activities such as immune activation, cell cycle, and antiviral signaling that recapitulate the natural history of anal cancer progression. Mutations were observed affecting KMT2C (30%), PIK3CA (21%), EP300 (21%) and NOTCH1 (13%) cancer driver genes among ASCC but also in precancerous lesions. Our study provides insights into the molecular mechanisms governing anal cancer progression, offering valuable information that may help to stratify HGSIL cases with low- or high-risk progression to the malignant stages.

overall_design: A total of 70 anal samples were collected from patients diagnosed with different stages of anal lesions, including Low-Grade Squamous Intraepithelial Lesions (LGSIL), High-Grade Squamous Intraepithelial Lesions (HGSIL), and Anal Squamous Cell Carcinoma (ASCC).

middle: C

first: Ezequiel

last: Lacunza


title: Heat disrupts host-parasitoid interactions mediated by a viral symbiont

city: Chapel Hill

first: Katherine

last: Malinski

address: 120 South Rd, Coker Hal #108

department: Biology

institute: University of North Carolina

summary: High temperature events can disrupt species interactions, including those among hosts, symbionts, and natural enemies. Understanding the genetic and physiological processes underlying these disruptions is a critical scientific challenge in this era of anthropogenic climate change. We explore how high temperatures disrupt the interactions among an herbivorous insect host, Manduca sexta, its insect parasitoid, Cotesia congregata, and the parasitoid’s symbiotic virus. In this system, high temperatures kill developing parasitoids, but not hosts. We evaluated the physiological and transcriptomic causes of thermal mismatch in ecological interactions using parasitoid egg in vitro experiments, immunological assays, and RNAseq. We found that high temperatures disrupt the capacity of the parasitoid’s symbiotic virus to immunosuppress the host insect, resulting in thermal mismatch and death of the parasitoid.  At the transcriptomic level, key viral genes involved in suppressing host immune pathways showed reduced expression, driven by the virus’s circular genomic structure. This work is among the first to demonstrate the genetic and physiological mechanisms by which a symbiont limits the ecological functioning of host-parasite dynamics, and provides a framework for understanding how molecular processes give rise to ecological outcomes in response to high temperature events caused by climate change.

overall_design: This experiment tested the impact of a heat shock at 40C on the interactions between a host insect (Manduca sexta) and a parasitoid's symbiotic virus (Cotesia congregata bracovirus). The experiment consisted of 2 treatment types (parasitism, temperature) each with 2 levels (parasitized or control; 25C or 40C). Third instar M. sexta were either parasitized by C. congregata wasps (parasitized), or injected with sephadex beads as a control (control), then several hours later placed at either the heat shock temperature (40C) or control (25C, same as rearing temperature) for 24 hours. Insects were then returned to 25C for an additional 24 hours to mitigate acute effects of temperature stress. After this recovery period, insects were flash frozen for transcriptomics. The experiment includes a lab and field population of M. sexta to investigate population effects (indicated as LAB or FLD in the sample name). Because the lab and field populations were run at different times, some lab population individuals were run alongside the field individuals as a control (indicated by LABINFIELD in sample name)

first: Olivia

last: Madalone

first: Joel

last: Kingsolver

first: Christopher

last: Willett


title: Expression data changes induced by 3D conditions in T-ALL cells

city: Madrid

first: Javier

last: Redondo-Muñoz

state: Madrid

address: Ramiro de Maeztu,9

department: Molecular Biomedicine

country: Spain

institute: CIB-CSIC

summary: 3D constrained conditions promoted H3K4 methylation in ALL cells that altered the global chromatin configuration and transcriptional changes related to cell cycle and DNA replication.

overall_design: Jurkat cells (T-ALL cell line) were used for RNA extraction and hybridization on Affymetrix microarrays. Jurkat cells were cultured in suspension or 3D conditions (a type I collagen matrix of 1,7 mg/mL) for 3 hours. Cellsd were lised and mRNA was extracted to compare expressions profiles. We used three replicates of each sample.

first: Raquel

last: González-Novo

first: África

last: González-Murillo

first: Elena

last: Madrazo

first: David

last: Acitores

first: Mario

last: García de Lacoba

first: Manuel

last: Ramírez


title: Bioinformatics Reveal the Potential Molecular Mechanism of Agrimol B against Osteosarcoma

city: 杨浦区

first: Yi-Ping

last: Luo

state: 上海

address: 杨浦区中原路

department: Orthopedics

institute: Department of Orthopedics, Shanghai Tenth People's Hospital, Tongji University School of Medicine

summary: In order to study the molecular mechanism of Agrimol B compound in inhibiting osteosarcoma cells, we established HOS cell line and intervened with Agrimol B for 24 hours.

overall_design: Comparative gene expression profiling of RNA-seq data from HOS cells treated with Agrimol B for 24 hours.

first: YP

first: JF

last: Liu

first: ZF

first: Lei

last: Zhang

first: LP

last: Zheng


title: Brain Plasticity and Neuroinflammatory Protein Biomarkers with Circulating MicroRNAs as Predictors of Acute Brain Injury Recovery – A Prospective Cohort Study

city: Chicago

first: Yan

last: Li

state: IL

address: 900 East 57th St.

department: Center for Research Informatics

institute: The University of Chicago

summary: Introduction: Brain plasticity, synaptic regeneration and neuroinflammation are crucial mechanisms that play important roles in the recovery from acute brain injuries, such as aneurysmal subarachnoid hemorrhage (aSAH), ischemic stroke (IS), and traumatic brain injury (TBI). The p75 neurotrophic receptor (p75NTR) is a significant receptor that modulates brain plasticity, neuroinflammation and apoptosis, making it an interesting target for therapeutic interventions. However, despite the progress made in understanding the molecular mechanisms involved in brain plasticity and neuroinflammation, there are still significant knowledge gaps in identifying protein biomarkers and microRNAs (miRNAs) associated with different types of acute brain injuries. Addressing these gaps will be critical in advancing our understanding of the underlying mechanisms involved in brain injury and developing effective therapies or prognostic strategies for recovery.  Hypotheses: We hypothesized that changes in serum levels of p75NTR and its linked neuroinflammatory signaling proteins could predict recovery from acute brain injuries, regardless of injury type. Differentially expressed (DE) circulating miRNAs following acute brain injuries may regulate studied protein targets and overall injury outcome.  Methods: A prospective cohort of 74 patients with ischemic stroke (n=30), aneurysmal subarachnoid hemorrhage (n=31), and traumatic brain injury (n=13) was evaluated. Serum samples were collected at two time points after the injury (early at 1.9 ± 1.0 days and late at 5.9 ± 2.2 days) and outcome evaluation was performed after three months. Six proteins were measured using ELISAs: p75NTR, NGF, sortilin, IL1, TNF, and cyclophilin A. A 2-tailed 2-sample t-test, univariate, and multivariate linear discriminant analysis (LDA) was used to compare serum molecules between patients with favorable (modified Rankin Scale (mRS) 0-3) and unfavorable outcome (mRS 4-6). Additionally, circulating miRNAs were extracted from the samples (n=48, early and late) and DE miRNAs were identified using DESeq2. The miRNA putative target genes were predicted and LDA was performed using the normalized expression values of identified DE miRNAs.  Results:  In this study, p75NTR (p < 0.01) and IL1 (p < 0.05) were found to be independently associated with outcome among the six circulating protein biomarkers studied. Combinatory LDA analysis of all six biomarkers showed significant predictive value for favorable outcome (OR 2.95 (1.46-5.98); area under the curve (AUC)= 71.2%, 95% CI = (0.590-0.833), p = 0.003). Temporal alterations of miRNA expression were observed in the aSAH, TBI, and IS groups (p<0.05, FDR corrected). Twenty-three common miRNAs were identified that differentiated favorable and unfavorable outcome (p<0.05) in all three groups. LDA of four identified miRNAs with predicted targets to studied proteins showed high predictive accuracy (OR (95% CI) 11.7 (2.39-226)); AUC = 94.1%, 95% CI = (0.849 - 1.00), p = 0.016).  Conclusions This study indicates that assessing serum levels of p75NTR and its mechanistically linked proteins can predict recovery outcome in patients with different types of acute brain injuries. The combined miRNA and protein biomarker models demonstrated accurate outcome prediction across diverse injury types, implying the presence of a common recovery mechanism. DE miRNAs were found to target the studied molecules, suggesting a potential mechanistical role in recovery. The identified common miRNAs may provide new targets for mechanistic validation in disease models. Further investigation is warranted to validate and explore the potential of these proteins and miRNAs in prognostication and as therapeutic targets for enhancing recovery from acute brain injuries.

overall_design: Prospective cohort study in human acute brain insults (aneurysmal subaracnoid hemorrhage, ischemic stroke and traumatic brain injury). To study circulating miRNAs as a prognostic biomarker

first: Antti

last: Sajanti

first: Santtu

last: Hellström

first: Ying

last: Cao

first: Romuald

last: Girard

first: Juzoh

last: Umemori

first: Janek

last: Frantzén

first: Fredrika

last: Koskimäki

first: Seán

middle: B

last: Lyne

first: Johannes

last: Falter

first: Tomi

last: Rantamäki

first: Riikka

last: Takala

first: Jussi

middle: P

last: Posti

first: Susanna

last: Roine

first: Sulo

last: Kolehmainen

first: Miro

last: Jänkälä

first: Jukka

last: Puolitaival

first: Melissa

last: Rahi

first: Jaakko

last: Rinne

first: Eero

last: Castrén

first: Janne


title: The PKCα/ZFP64/CSF1 Axis restricts Tumor Response to Anti-PD1 by driving Macrophage M2 Polarization in Hepatocellular Carcinoma [RNA-seq 1]

first: Chuan

middle: Yuan

last: Wei

state: Shanghai

address: fenglin road 180

department: Liver cancer institute

institute: Zhongshan, Fudan

summary: Despite remarkable achievements, majority of hepatocellular carcinoma (HCC) patients fail to respond to anti-PD1 therapy. Here, we showed that ZFP64 was frequently upregulated in HCC tissues of anti-PD1 resistance patients. Elevated ZFP64 levels triggered tumor progression and induced an immunosuppressive microenvironment. Mechanistically, ZFP64 transcriptionally activated colony-stimulating factor 1 (CSF1) by directly binding to its promoter, and secreted CSF1 driving the shift of macrophages into an alternatively activated phenotype. Importantly, the PKCα was revealed to phosphorylate ZFP64 at S226, resulting in its nuclear translocation to transcribe the CSF1 gene. Particularly, we proposed firstly that the PKCα/ZFP64/CSF1 axis was a critical pathway in fostering immune evasion and anti-PD1 tolerance and inhibiting this axis with lenvatinib or Gö6976 surmounted the anti-PD1 resistance in HCC. Our study indicates that the ZFP64 is an emerging indicator in predicting anti-PD1 efficacy, and reveals the PKCα/ZFP64/CSF1 axis is a suitable target for anti-PD1 combination therapy in HCC.

overall_design: mRNA profiles of tumor tissues in PD and PR HCC patients

first: Chuanyuan

first: Jia

last: Fan


title: Leptin Receptor Signaling Regulates Protein Synthesis Pathways and Neuronal Differentiation in Pluripotent Stem Cells

city: Boston

first: Hui

last: Pan

address: 1 Joslin Place

department: Bioinformatics and Biostatistics Core

institute: Joslin Diabetes Center

summary: The role of leptin receptor (OB-R) in linking pluripotency with growth, development, and the consequences on the progression of metabolic disease is poorly understood. Using a global unbiased proteomics approach we report that embryonic fibroblasts (MEFs) carrying db/db mutation exhibited metabolic abnormalities, while their reprogrammed induced pluripotent stem cells (iPSCs) demonstrated alterations in the expression of proteins involved in embryonic development. An upregulation in the expression of eukaryotic translation initiation factor4e (eIF4e) and Stat3 binding to the eIF4e promoter was supported by enhanced protein synthesis in mutant iPSCs. Directed differentiation of db/db iPSCs towards the neuronal lineage showed defects compared to controls. Gene editing to correct point mutation in db/db iPSCs using CRISPR/Cas9, restored the metabolic defects, expression of neuronal markers, and protein synthesis as corroborated by RNAseq analyses. These data imply a direct role for OB-R in regulating metabolic properties in embryonic fibroblasts and key developmental pathways in iPSCs.

overall_design: Embryonic day 14.5 wild type control (Ctrl) (N=4) and db/db MEFs (N=4) were derived from breeding OB-Rdb/+ heterozygous mice (Jackson Laboratory Inc.). Generation of mouse iPSCs involved infection of primary MEFs with mouse STEMCCA lentivirus vector expressing the reprogramming factors Oct4, Sox2, Klf4 and cMyc. Gene-correction in db/db iPSCs was performed using CRISPR-Cas9. Total RNA was isolated from iPSCs, Ctrl (N=4), db/db (N=4), and db/Corr (N=2)  in duplicates by using RNeasy kit (Qiagen) and RNA sequencing was performed by using Illumina Novaseq6000.

first: Manoj

last: Gupta

first: Rohit

middle: N

last: Kulkarni


title: The PKCα/ZFP64/CSF1 Axis restricts Tumor Response to Anti-PD1 by driving Macrophage M2 Polarization in Hepatocellular Carcinoma

city: Shanghai

overall_design: ChIP-Seq analysis in PLC/PRF/5 cells with ZFP64 overexpression.


title: A pan-KRAS inhibitor and its derived degrader elicit multifaceted anti-tumor efficacy in KRAS-driven cancers [CD45+ scRNA-seq]

status: Public on Aug 03 2024

city: 上海市

first: 新婷

last: 夏

state: 上海市

address: 东川路500号

department: 生命科学学院

institute: 华东师范大学

summary: KRAS is the most frequently mutated oncogene in human cancers, but to date, only KRASG12C inhibitors have been FDA-approved. Small-molecule inhibitors that target other more prevalent KRAS mutations are in urgent clinical need. Here, we report a highly potent pan-KRAS inhibitor, MCB-294, that selectively targets KRAS, but not HRAS or NRAS. Molecular profiling studies show that MCB-294 binds to and inhibits several KRAS mutants, including G12D, G12C, G12V, thereby disabling KRAS oncogenic signaling. MCB-294 is well tolerated in mice and exhibits remarkable therapeutic efficacy against the growth of a comprehensive range of KRAS-driven cancer cell lines, patient-derived organoids, and tumor xenografts and prolongs mouse survival. By employing an MCB-294 derivative as the KRAS binder, we further identify a VHL-based pan-KRAS degrader, MCB-36, that efficiently degrades KRAS in vitro and in vivo through the ubiquitin-proteasome system. Importantly, MCB-294 and MCB-36 not only effectively kill KRASG12C inhibitor-resistant cells, but also reshape the tumor microenvironment by boosting T cell-mediated anti-tumor immunity. Taken together, our findings demonstrate a successful and feasible strategy for targeting pan-KRAS to impair multifaceted features of KRAS-driven cancers.

overall_design: To investigate the  effects of MCB-294 or MCB-36 on the modulation of the tumor microenvironment, CT26 xenografts were treatment with vehicle, MCB-294 or MCB-36 compound for 2 days, and then intratumoral CD45+ cells were isolated using BD FACSAriaIII for sc-RNAseq.

first: juanjuan

last: Feng

first: xuanzheng

last: Xiao

first: xinting

last: Xia


title: A pan-KRAS inhibitor and its derived degrader elicit multifaceted anti-tumor efficacy in KRAS-driven cancers [scRNA-seq]

overall_design: To investigate the  effects of MCB-294 or MCB-36 on the modulation of the tumor microenvironment, CT26 xenografts treatment with vehicle, MCB-294 or MCB-36 compound for 2 days were harvested, and then analyzed using sc-RNAseq.


title: The Role of DNMT1 and C/EBPα in the Regulation of CYP11A1 Expression During Syncytialization of Human Placental Trophoblasts

first: Wangsheng

last: Wang

state: Please Select

address: 845 Lingshan Rd.

institute: Shanghai Jiao Tong University

summary: Progesterone synthesized in the placenta is essential for pregnancy maintenance. CYP11A1 is a key enzyme in progesterone synthesis, and its expression increases greatly during trophoblast syncytialization. However, the underlying mechanism remains elusive. Here, we demonstrated that passive demethylation of CYP11A1 promoter accounted for the upregulation of CYP11A1 expression during syncytialization with the participation of the transcription factor C/EBPα. We found that the methylation rate of a CpG locus in the CYP11A1 promoter was significantly reduced along with decreased DNA methyltransferase 1 (DNMT1) expression and its enrichment at the CYP11A1 promoter during syncytialization. DNMT1 overexpression not only increased the methylation of this CpG locus in the CYP11A1 promoter, but also decreased CYP11A1 expression and progesterone production. In silico analysis disclosed multiple C/EBPα binding sites in both CYP11A1 and DNMT1 promoters. C/EBPα expression and its enrichments at both the DNMT1 and CYP11A1 promoters were significantly increased during syncytialization. Knocking-down C/EBPα expression increased DNMT1 while it decreased CYP11A1 expression during syncytialization. Conclusively, C/EBPα plays a dual role in the regulation of CYP11A1 during syncytialization. C/EBPα not only drives CYP11A1 expression directly, but also indirectly through downregulation of DNMT1, which leads to decreased methylation in the CpG locus of the CYP11A1 promoter, resulting in increased progesterone production during syncytialization.

overall_design: RNA sequecing was used  to indetify the change of genes durning placental trophoblasts syncytialization

first: Wang-sheng


title: The PKCα/ZFP64/CSF1 Axis restricts Tumor Response to Anti-PD1 by driving Macrophage M2 Polarization in Hepatocellular Carcinoma [RNA-seq 2]

overall_design: mRNA profiles of PLC/PRF/5-ZFP64 and PLC/PRF/5-EV cells


title: Transcriptomic analysis of patient-derived resected lung NSCLC cells

first: Daisuke

last: Motooka

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

overall_design: Refer to individual Series


title: Identification of a small protein encoded by PCBP1-AS1 that promotes replication of influenza virus via inducing enhancement of autophagy

city: Fuzhou

first: Song

state: Fujian

address: Cangshan District

institute: Fujian Agriculture and Forestry University

summary: Many annotated long noncoding RNAs (lncRNAs) contain small open reading frames (sORFs), some of which have been demonstrated to encode small proteins or micropeptides with fundamental biological importance. However, the small proteins or micropeptides encoded by lncRNAs and their functions in viral infection remain largely unexplored. Here, we identified and characterized a small 110-amino acid protein named ORF6 encoded by the putative lncRNA PCBP1-AS1. Interestingly, both PCBP1-AS1 and ORF6 were significantly upregulated by influenza virus infection. Furthermore, we observed that they were markedly induced by type I interferon treatment. Overexpression of PCBP1-AS1 or ORF6 enhanced influenza virus replication. Conversely, knockdown of PCBP1-AS1 or knockout of ORF6 inhibited the viral production. In addition, loss-of-function experiment demonstrated that ORF6 is essential for PCBP1-AS1 to facilitate influenza virus replication. Importantly, the overexpression of ORF6 induced enhancement of autophagy by increasing the expression of ATG7, a gene encoding an essential effector enzyme for canonical autophagy. These findings reveal a lncRNA-derived small protein that is exploited by influenza virus, and provide new insights into the virus-host regulatory network.

overall_design: To explore how ORF6 functions in regulating influenza virus infection, we infected A549 cells overexpressing ORF6 or control cells with PR8 influenza virus for 12h, and performed RNA-seq to find differently expressed mRNA.

first: Xiaojuan

last: Chi

first: Ji-Long

last: Chen


title: HDC/histamine signaling axis drives macrophage reprogramming to promote angiogenesis in the hindlimb ischemia mice

status: Public on Aug 02 2024

first: Xiangdong

last: Yang

address: Xietu Road

institute: Zhongshan Hospital Affiliated to Fudan University

summary: Histamine is catalyzed by histidine decarboxylase (HDC), which plays important roles in many physiological and pathological processes, but its role in angiogenesis has not been thoroughly clarified. Here we report that HDC is highly expressed in Ly6C+macrophages, rather than in endothelial cells using Hdc-GFP transgenic mice with hindlimb ischemia (HLI) mouse model. Given the whole-process promoting effect of macrophages on angiogenesis, a cluster of HDC+CXCR2+ macrophages have been identified by single-cell sequencing technology in ischemic tissue. The inactivation of HDC leads to a lack of histamine and pro-angiogenic factor production in macrophages, inducing a harsh inflammatory microenvironment that is not conducive to the interaction between macrophages and endothelial cells. Moreover, HA-DA@histamine hydrogel has been designed and demonstrated to safely treat ischemic injury by modulating inflammation and angiogenesis. These data highlight the critical roles of HDC/histamine signaling in macrophage differentiation, angiogenesis, and muscle regeneration in the early stage of HLI.

overall_design: The total RNA of muscles 3 days after surgery from WT and Hdc-/- mice was extracted using TRIzol reagent (Takara) for RNA-seq analysis. The quality of raw sequencing reads post initial quality control (version 0.20.1) was evaluated using FastQC (version 0.11.9), followed by quantification of gene expression levels through alignment to the reference genome using HISAT2 (version 2.1.0) and analysis with HTSeq-count (version 0.11.2). Clean reads were mapped to the GRCm39 mouse reference genome using STAR (version 2.7.11b) to generate the expression count matrix. The counts matrix was converted into FPKM values to normalize the RNA-Seq data for gene length and sequencing depth. The FPKM matrix was imported into R and analyzed using the DESeq2 package (version 1.22.2)) to normalize the data and estimate dispersion. Determinants of DEG were those with a p-value < 0.05 and a log2 fold change > 1. Ultimately, the analysis of KEGG pathway enrichment was conducted using the cluster Profiler package (version 4.13.0) in R.

first: Dili

last: Sun

first: Jianfu

last: Zhu

first: Gaofeng

last: Zeng


title: An engineered ovarian somatic organoid model exhibits age-dependent changes in cellular organization and function

first: Shweta

last: Dipali

address: 303 E. Superior St., Suite 10-220

institute: Northwestern University

summary: Ovarian somatic cells are essential for tissue function, but there are no ex vivo models which maintain the cellular heterogeneity, in addition to the cell-cell and cell-matrix interactions of this compartment. We engineered a novel ovarian somatic organoid model in which a stroma-enriched fraction of primary mouse ovarian somatic cells was cultured in scaffold-free agarose micromolds. We sought to use this ovarian somatic organoid model to investigate mechanisms of ovarian aging and performed single cell RNA sequencing using primary ovarian somatic cells isolated from reproductively young and old mice, as well as organoids generated from young and old mice at days 1 and 6 of culture to assess age-dependent changes to cellular composition.

overall_design: Primary ovarian somatic cells isolated from reproductively young (6-12 weeks) or old (10-14 months) mice were lifted from 2D culture overnight or dissociated from organoids at day 1 or day 6 of culture and analyzed by scRNAseq

first: Madison

middle: Q

last: Gowett

first: Pratik

last: Kamat

first: Aubrey

last: Converse

first: Emily

last: Zaniker

first: Abigail

last: Fennell

first: Teresa

last: Chou

first: Michele

middle: T

last: Pritchard

first: Mary

last: Zelinski

first: Jude

last: Phillip

first: Francesca

middle: E

last: Duncan


title: A small protein encoded by lncRNA PCBP1-AS1 promotes the replication of influenza virus via regulating the expression of proviral effectors (Ribo-Seq)

first: Song

last: Wang

summary: Many annotated long noncoding RNAs (lncRNAs) contain small open reading frames (sORFs), some of which have been demonstrated to encode small proteins or micropeptides with fundamental biological importance. While the small proteins or micropeptides hidden in lncRNA are gradually being revealed, their functions in influenza virus infection remain largely unexplored. Here, we identified and characterized a small 110-amino acid protein named PAESP that is encoded by the putative lncRNA PCBP1-AS1. We found that both PCBP1-AS1 and PAESP were upregulated by influenza virus infection as well as type I interferon treatment. Overexpression of PCBP1-AS1 or PAESP enhanced influenza virus replication. Conversely, knockdown of PCBP1-AS1 or knockout of PAESP inhibited viral production. In addition, loss-of-function experiment demonstrated that PAESP is essential for PCBP1-AS1 to facilitate influenza virus replication. More importantly, the overexpression of PAESP increases the mRNA level of several genes such as IFIT2 and IFIT3, which have been proved to be retasked by influenza virus from canonical antiviral factors into proviral effectors. Interestingly, when IFIT2 or IFIT3 expression was knocked down in PAESP-overexpressed cells, the virus titers significantly decreased compared to control cells. These findings reveal a novel lncRNA-derived small protein that is exploited by influenza virus and provide new insights into the virus-host regulatory network.

overall_design: To identify putative lncRNAs that can encode functional proteins/peptides during the pathogenesis of  influenza virus infection, we infected A549 cells with or without PR8 influenza virus for 12h, and performed Ribo-seq to find lncRNAs bound by the ribosome.

first: Xiaojuan

last: Chi


title: Genome-wide CRISPR screening identifies the E3 ubiquitin ligase HERC1 as a modulator of the response to nucleoside analogs in acute myeloid leukemia

city: Montreal

first: François

middle: Émile

last: Mercier

state: Quebec

address: 3755 Cote-Sainte-Catherine Road

department: Medicine

institute: McGIll University

summary: We identified the Ubiquitin Ligase HERC1 in the context of cytarabine response in a genome-wide CRISPR screen in murine AML. We hypothesized to detect early transciptomic changes in cytarabine treated murine AML cells genetically targeted by using single guide RNAs targeting HERC1 and  Non-targeting Control.

overall_design: Early Detection of transcriptomic changes in cytarabine treated (200nm)  murine MLL-AF9 transformed progenitor cells which were genetically modified with single guide RNA targeting HERC1 or Non-Targeting Ctrl RNA. AML clones targeted for HERC1 were  sorted by using BD FACSAria II, and clone E5 was chosen for RNA sequencing. sgHERC1 - and sgCtrl MLL/AF9 cells were treated for six hours with 200nM cytarabine or H20 and RNA was subsequently isolated by using TRIzol (Invitrogen) + RNeasy mini kit (Qiagen, #74106).

first: Maja

last: Jankovic

first: Alexandre

last: Orthwein

first: Francois


title: Fat Phagocytosis Promotes Anti-inflammatory Responses of Macrophages in a Mouse Model of Osteonecrosis

city: Dallas

first: Yinshi

last: Ren

state: Texas

address: 2222 Welborn St

institute: Scottish Rite for Children

summary: Osteonecrosis (ON) of the femoral head (ONFH) is a devastating bone disease affecting over 20 million people worldwide. ONFH is caused by a disruption of blood supply, leading to necrotic cell death and increased inflammation. Macrophages are the key cells mediating the inflamma-tory responses in ON. It is unclear what are the dynamic phenotypes of macrophages, and what mechanisms may affect macrophage polarization and therefore the healing process. In our pre-liminary study, we found that there is an invasion of macrophages in the repair tissue during ON healing. Interestingly, in both ONFH patient and the mouse ON model, fat was co-labeled within macrophages using immunofluorescence staining, indicating phagocytosis of fat by mac-rophages. To study the effects of fat phagocytosis on macrophage phenotype, we set up an in vitro macrophage and fat co-culture system. We found that fat phagocytosis significantly de-creased M1 markers expression such as IL1β and iNOS in macrophages. Whereas the expression of M2 marker Arg1 was significantly increased with fat phagocytosis. To investigate if the polar-ization change is indeed mediated by phagocytosis, we treated the cells with Latrunculin A (LA, a phagocytosis inhibitor). LA supplement significantly reversed the polarization marker gene changes induced by fat phagocytosis. To provide an unbiased transcriptional gene analysis, we submitted the RNA for bulk RNA sequencing. Differential gene expression (DGE) analysis re-vealed top up-regulated genes were related to anti-inflammatory responses, while pro-inflammatory genes were significantly downregulated. Additionally, using the pathway en-richment and network analyses (Metascape), we confirmed that gene enriched categories related to pro-inflammatory responses were significantly downregulated in macrophages with fat phagocytosis. Finally, we validated the similar macrophage phenotype changes in vivo. To sum-marize, we discovered that fat phagocytosis occurs in both ONFH patients and the mouse ON model, which inhibits pro-inflammatory responses with increased anabolic gene expressions of macrophages. This fat phagocytosis induced macrophage phenotype is consistent with the in vivo changes shown in the ON mice model. Our study reveals a novel phagocytosis mediated macro-phage polarization mechanism in ON, which fills in our knowledge gaps of macrophage func-tions and provides new concepts in macrophage immunomodulation as a promising treatment for ON.

overall_design: Primary mouse bone marrow macrophages were isolated and co-cultured with necrotic fat supplemented with artifical necrotic bone fluild (aNBF). Fat-con indicate cells cultured in control/low fat medium with aNBF for 24 hours; Fat-high are the cells cultured in fat-rich containing medium with aNBF for 24 hours. The study is designed to investigate the effects of fat phagocytosis on macrophage polarization in the osteonecrotic environment.

first: Zhuo

last: Deng


title: Multimodal analysis reveals cellular diversity and divergent circuits of the zona incerta

city: Cambridge

first: Joshua

last: Levin

state: MA

address: 75 Ames St

department: Stanley Center

institute: Broad Institute

summary: The zona incerta (ZI) plays an important role in diverse behavioral functions and is an emerging clinical target for deep brain stimulation to treat neurological conditions. Despite their importance, the cell type composition of the ZI and the anatomical circuit organization linking specific ZI cell types to brain-wide circuits remain unclear. In this study, we used single-nucleus RNA-sequencing, spatial RNA profiling, ex vivo electrophysiology, and anatomical circuit mapping to generate a multimodal cellular atlas of the ZI and to characterize the brain-wide monosynaptic inputs to specific ZI cell types. We define four genetically distinct populations of ZI GABAergic neurons that display unique spatial distributions, specialized intrinsic electrophysiological properties, divergent efferent projections, and cell-type dependent synaptic input patterns. Finally, using fiber photometry we reveal response divergence between distinct ZI cell types in a novel behavioral assay that centers around approach-avoid conflict produced by innately appetitive and aversive stimuli. This study thus provides a foundational resource for the design and interpretation of future experiments aimed at understanding the organization and function of the ZI.

overall_design: The GEO dataset comprises single-nucleus RNA-sequencing data of neurons of the zona incerta (ZI) from three adult (8-12 weeks old) C57Bl/6J mice.

first: Ryan

last: Kast

first: Sean

last: Simmons

first: Nita

last: Rome

first: Xian

last: Adiconis

first: Lisa

last: Melamed

first: Kate

last: McCabe

first: Dongqing

first: Alexandra

last: Krol

first: Magdalena

last: Krubner

first: Vincent

last: Prevosto

first: Kirsten

last: Levandowski

first: Ubadah

last: Sabbagh

first: Heather

last: Sullivan

first: Ian

last: Wickersham

first: Fan

middle: Z

first: Zhanyan

last: Fu

first: Guoping

last: Feng


title: Transcriptome Responses to Different Nitrogen Levels Reveals Regulatory Networks for Icariin-Flavonoid Metabolism in Epimedium pubescens

city: 北京

first: 尚年

last: 刘

state: 北京

address: 马连洼街道马连洼北路151号

institute: 中国医学科学院药用植物研究所

summary: Nitrogen is a crucial nutrient element for plant growth and productivity, with both excess and deficiency in nitrogen fertilizer application posing adverse effects on plants and the environment. The internal mechanisms by which the medicinal plant Epimedium pubescens (E. pubescens) adapts to varying nitrogen levels remain unclear. This study employed one-year-old E. pubescens as the experimental material to systematically analyze the changes in plant growth traits, carbon metabolites, and Icariin-Flavonoids content under different exogenous nitrogen levels. Furthermore, it examined the transcriptional changes in gene expression within E. pubescens in response to varying nitrogen levels. The results showed that under moderate nitrogen levels (7.5 mmol/L NO3-), E. pubescens exhibited increased biomass accumulation and flavonoid synthesis. However, deficient or excessive nitrogen levels (0, 22.5 mmol/L NO3-) significantly inhibited photosynthesis in E. pubescens, reducing the content of starch, soluble sugars, and Icariin-Flavonoids, leading to decreased biomass and accompanied by changes in leaf color (pale green or browning). Transcriptome analysis revealed the underlying molecular mechanisms of these changes in plants regulated by different nitrogen levels. Nitrogen deficiency and excess triggered distinct transcriptional response patterns, with the number of differentially expressed genes (DEGs) peaking at S2 (36 days) under nitrogen deficiency and significantly declining at S3 (48 days), while the number of DEGs under excess nitrogen continuously increased over time from S1 to S3 (12-48 days). Both conditions significantly affected the expression of genes related to carbon and nitrogen metabolism, flavonoid synthesis, and stress response. Based on the correlation analysis of expression levels of genes related to these pathways, growth traits, and metabolite content indicators, we constructed regulatory network diagrams for carbon-nitrogen metabolism and Icariin-flavonoid metabolism-related genes. Furthermore, we identified hub genes that may be involved in regulating Icariin-flavonoid metabolism in response to nitrogen levels, such as UGT (Ebr06G044290), EpF3H (Ebr04G062950), EpCHS5 (Ebr03G073940) (Fig. 9A), UGT (Ebr06G044660), and EpUGT13_A (Ebr06G044210). Additionally, among the DEGs obtained at the S3 stage, we discovered the transcription factors MYB1_CROXC (Ebr04G001770) and MYB12_ARATH (Ebr01G065030). We constructed their associated gene networks under different nitrogen levels, which may primarily regulate the expression of genes like UGT, CHS, and F3H involved in the flavonoid synthesis stage of E. pubescens under varying nitrogen conditions. In summary, this study has revealed the growth performance and the variation patterns of Icariin-Flavonoid metabolism in E. pubescens in response to different exogenous nitrogen levels, as well as their complex underlying mechanisms. Additionally, key genes involved in regulating the synthesis of flavonol glycosides in E. pubescens by nitrogen levels have been identified. This provides an important basis for a deeper understanding of the mechanisms of nitrogen regulation on the growth and secondary metabolism of medicinal plants. Furthermore, it offers theoretical support and potential genetic resources for the efficient utilization of nitrogen fertilizers in E. pubescens cultivation and the breeding of high-nitrogen-use-efficiency varieties.

overall_design: These seedlings were transplanted into cultivation pots filled with a substrate mixture of river sand and perlite (3:2), and then cultured in a greenhouse. The light intensity was maintained at 2000 luX for 14 hours per day, and the temperature was controlled at 22 ± 2°C. After 20 days of irrigation with 12.5% Hoagland nutrient solution for seedling recovery, seedlings with newly emerged roots and having resumed normal growth were subjected to different nitrogen levels: LN (nitrogen deficiency treatment, 0 mmol/L NO3-), MN (moderate nitrogen treatment, 7.5 mmol/L NO3-), and HN (excessive nitrogen treatment, 22.5 mmol/L NO3-).The microelements H3BO3, MnCl2, ZnSO4, CuSO4, and H2MoO4 were added at concentrations of 2.86, 1.81, 0.22, 0.08, and 0.02 g/L, respectively, while Fe-EDTA was added at a concentration of 0.02 mM. The pH of the nutrient solution was adjusted to 6.5, and the seedlings were irrigated with the nutrient solution every two weeks. Each treatment involved 36 seedlings. On the 24d (day), 36d, and 48d of the experiment, fresh leaves from plants under different treatments were collected, rapidly frozen in liquid nitrogen, and then stored at -80°C for later measurement. On the 48d, plants in the LN (nitrogen deficiency) and HN (excessive nitrogen) treatment groups showed obvious symptoms of damage, and subsequently, all treated plants were measured and harvested.

first: Shangnian

last: Liu

first: Xiaojing

last: An

first: Caixia

last: Chen


title: Longitudinal scRNA-seq analysis of human liver bud organoid (HLBO) with iPS-derived liver sinusoidal endothelial progenitor (iLSEP)

city: Tokyo

first: Norikazu

last: Saiki

address: 1-5-45 Yushima, Bunkyo-ku

department: Institute of Research

institute: Tokyo Medical and Dental University

summary: We directly differentiated human pluripotent stem cells into CD32b+ putative liver sinusoidal progenitors (iLSEP) by dictating developmental pathways. By devising an inverted multilayered air-liquid interface (IMALI) culture, hepatic endoderm, septum mesenchyme, arterial and sinusoidal quadruple progenitors self-organized to generate vascularized liver bud organoid. To uncover cellular characteristics and diversity during organoid development at transcriptomic level, time-series single cell RNA-seq (scRNA-seq) data were acquired.

overall_design: At days 3, 7, and 11 after self-assembly culture, three HLBOs at each time point were subjected to scRNA-seq library preparation.

first: Takanori

last: Takebe


title: Amyloids “at the border”: deep mutagenesis and random sequence extension reveal an incomplete amyloid-forming motif in Bri2 that turns amyloidogenic upon C-terminal extension

city: Barcelona

first: Mariano

last: Martín

address: c/ Baldiri Reixac 10-12

institute: IBEC

summary: Stop-loss mutations cause over twenty different diseases. The effects of stop-loss mutations can have multiple consequences that are, however, hard to predict. Stop-loss in ITM2B/BRI2 results in C-terminal extension of the encoded protein and, upon furin cleavage, in the production of two 34 amino acid long peptides, ADan and ABri, that accumulate as amyloids in the brains of patients affected by familial Danish and British Dementia. To systematically explore the consequences of Bri2 C-terminal extension, here, we measure amyloid formation for 676 ADan substitutions and identify the region that forms the putative amyloid core of ADan fibrils, located between positions 20 and 26, where stop-loss occurs. Moreover, we measure amyloid formation for ~18,000 random C-terminal extensions of Bri2 and find that ~32% of these sequences can nucleate amyloids. We find that the amino acid composition of these nucleating sequences varies with peptide length and that short extensions of 2 specific amino acids (Aliphatics, Aromatics and Cysteines) are sufficient to generate novel amyloid cores. Overall, our results show that the C-terminus of Bri2 contains an incomplete amyloid motif that can turn amyloidogenic upon extension. C-terminal extension with de novo formation of amyloid motifs may thus be a widespread pathogenic mechanism resulting from stop-loss, highlighting the importance of determining the impact of these mutations for other sequences across the genome.

overall_design:
Systematic measurement of the nucleation of Bri2 random extension of 12 amino acids (NNK degenerate codons).
Three independent experiments performed. Illumina sequencing of 1 input sample and 3 output samples (after selection for amyloid nucleation) per experiment.

first: Benedetta

last: Bolognesi


title: Timing and location dictate monocyte fate and their transition to tumor-associated macrophages

city: Villejuif

first: Garett

last: Dunsmore

address: 114 rue Edouard Vaillant

country: France

institute: Gustave Roussy

summary: Tumor-associated macrophages (TAMs) are a heterogeneous population of cells whose phenotypes and functions are shaped by factors that are incompletely understood. Herein, we asked when and where TAMs arise from blood monocytes, and how they evolve during tumor development. We initiated pancreatic ductal adenocarcinoma (PDAC) in inducible monocyte fate-mapping mice and combined single-cell transcriptomics and high-dimensional flow cytometry to profile the monocyte-to-TAM transition. We revealed that monocytes differentiate first into a transient intermediate population of TAMs (IntTAM) that generates two longer-lived lineages of terminally differentiated TAMs with distinct gene expression profiles, phenotypes and intra-tumoral localization. Transcriptome datasets and tumor samples from patients with PDAC evidenced parallel TAM populations in humans and their prognostic associations. These insights will support the design of new therapeutic strategies targeting TAMs in PDAC.

overall_design: tdTomato+/- cell populations were sorted from Ms4a3Cre x RosatdTomato and Ms4a3CreERT2 x RosatdTomato mice using the indicated gating strategies. The cells were processed using the Chromium Single Cell 3’ (v3 Chemistry) platform (10X Genomics). tdTomato+ and tdTomato- cells were sequenced separately using the NovaSeq sequencer (Illumina). Briefly, cells were loaded onto a Chromium Next GEM Chip G, and single cell suspensions in gel beads-in-emulsion (GEMs) were generated using the chromium controller (10X genomics). Reverse transcription (RT) was performed to generate cDNA, which was amplified, cleaned, and fragmented. Finally, libraries were subjected to standard quality control (QC) steps before sequencing.

first: Lai

last: Ng

first: Camille

last: Bleriot

first: Florent

last: Ginhoux


title: Effect of metformin on gene expression in mammary tissues in association with epithelial cell reprogramming in FVB/N mouse model

city: Kannapolis

first: Xiaohe

last: Yang

state: North Carolina

address: 500 Laureate Way

institute: North Carolina Central University

summary: Metformin shows promise in breast cancer prevention, but its underlying mechanisms remain unclear. This study investigated the impact of metformin on the repopulation dynamics of mammary epithelial cells (MECs) and the signaling pathways in non-tumorigenic FVB/N mice. The mice were treated with metformin for 10 weeks, followed by morphogenesis, flow cytometry, analyses of MEC stemness, and RNA sequencing. The study findings indicated that metformin treatment in adult mice reduced mammary gland proliferation, as demonstrated by decreased Ki67+ cells and lateral bud formation. Additionally, metformin significantly reduced both basal and mammary repopulating unit subpopulations, indicating an impact on mammary epithelial cell repopulation. Mammosphere, colony-forming cell, and 3D culture assays revealed that metformin adversely affected mammary epithelial cell stemness. Furthermore, metformin downregulated signaling in key pathways including AMPK/mTOR, MAPK/Erk, PI3K/Akt, and ER, which contribute to its inhibitory effects on mammary proliferation and stemness. Transcriptome analysis with RNA sequencing indicated that metformin induced significant downregulation of genes involved in multiple critical pathways. KEGG-based pathway analysis indicated that genes in PI3K/Akt, focal adhesion, ECM-receptor, small cell lung cancer and immune-modulation pathways were among the top groups of differentially regulated genes. In summary, our research demonstrates that metformin inhibits MEC proliferation and stemness, accompanied by the downregulation of intrinsic signaling. These insights suggest that the regulatory effects of metformin on premalignant mammary tissues could potentially delay or prevent the onset of breast cancer, offering a promising avenue for developing new preventive strategies.

overall_design: Female FVB/N mice were administered 200 mg/kg/day of metformin via intraperitoneal (i.p.) injection from 8 to 18 weeks of age. After treatment, morphogenesis, flow cytometry, analyses of MEC stemness.  Total RNA samples were extracted from mammary tissues of control and treated mice, followed by RNA-seq and bioinformatic analysis to profile genes associated with metformin treatment with associated with phenotypic changes.

first: Minghui

last: Shan

first: Yujie

last: Shi


title: Olanzapine suppresses mPFC-norepinephrine releasing to alleviate CLOCK-induced cancer stemness under chronic stress

city: Dalian

first: jinxin

last: lu

state: Liaoning

address: lvshunkou district lvshun south goad west sector No.9

institute: Dalian Medical University

summary: We identify a new mechanism by which OLZ ameliorates chronic stress-enhanced tumorigenesis and chemoresistance. OLZ reversed chronic stress-enhanced lung tumorigenesis and anxiety-like behaviors by suppressing neuro-activity in the mPFC and reducing norepinephrine (NE) releasing. To investigate the mechanism of NE in cancer stemness we performed RNA-seq of the NCI-H1299 cells under NE treatment and found that NE activated β2-adrenoceptor (ADRB2)-mediated cAMP-PKA-CREB pathway to transactivate CLOCK that further sustains cancer stemness.

overall_design: We then performed gene expression profiling analysis using data obtained from RNA-seq of NCI-H1299 cells under norepinephrine treatment. Comparative gene expression profiling analysis of RNA-seq data for NCI-H1299 treated with DMSO or norepinephrine.

first: Jinxin

last: Lu

first: Bai

last: Cui

first: Quentin


title: Transcriptomic signatures of ZIKV infection in patients and a cell culture model

city: Albany

first: Cara

last: Pager

state: NY

address: 1400 Washington Avenue

institute: University at Albany, State University of New York

summary: Zika virus (ZIKV), a re-emerging flavivirus is associated with devastating developmental and neurological disease outcomes particularly in infants infected in utero. Towards understanding the molecular underpinning of the unique ZIKV disease pathologies, numerous transcriptome-wide studies have been undertaken. Notably, these studies have overlooked the assimilation of RNA-seq analysis from ZIKV-infected patients with cell culture and cell model systems. We determined that ZIKV-infection in human lung adenocarcinoma A549 cells, mirrored both transcriptional and alternative splicing profiles from previously published RNA-seq data of peripheral blood mononuclear cells collected from pediatric patients during early, late acute, and convalescent phases of ZIKV infection. Our analyses show that ZIKV infection in cultured cells correlates with transcriptional changes in patients, while the overlap in alternative splicing profiles was not as extensive. Overall, our data indicate that cell culture model systems support dissection of select molecular changes detected in patients and established the foundation for future studies elucidating the biological implications of alternative splicing during ZIKV infection.

overall_design: Grown A549 cells were incubated with ZIKV (ZIKV-infected cells) or PBS (mock-infected cells) and media. After incubation, samples were sent for RNA extraction and sequencing by GENEWIZ resulting in three ZIKV-infected and three mock-infected samples for further analysis.

first: Gillian

last: Berglund

first: Claudia

middle: D

last: Lennon

first: Pheonah

last: Badu

first: J

middle: A


title: MicroRNA expression profile of different grade pancreatic neuroendocrine tumors

city: Nudapest

first: Bálint

middle: Kende

last: Szeredás

address: Korányi Sándor street 2/a

department: Department of Internal Medicine and Oncology - Department of Endocrinology

country: Hungary

institute: Semmelweis University

summary: The grade of pancreatic neuroendocrine tumors principally influences therapy. Grading is currently done by the histological examination of biopsy samples, which is an invasive and time-consuming method. We performed an NGS on FFPE tumor tissue samples to find microRNAs that show a significant difference in expression according to grade, thus could be suitable for grade determination.

overall_design: Tumor tissue was signed and macrodisected from FFPE blocks by pathologists to make sure the samples we used contain solely tumor tissue. We isolated total RNA from the samples

first: Gábor

last: Nyírő

first: Ábel

last: Decmann

last: Vékony

first: Katalin

last: Borka

last: Dezső

first: Attila

last: Zalatnai

first: Ilona

last: Kovalszky

first: Péter

last: Igaz


title: RNAseq of U251 cell line treated with 100nM of LLY-283 for 24 hours

city: Bethesda

first: Uma

last: Shankavaram

state: MD

address: 9000 Rockville Pike

department: NCI

institute: NIH

summary: RNAseq analysis of U251 cell line treated with 100nM of LLY-283 compared to Vehicle treated cells to analyse the effect of LLY-283 on gene expression and mRNA splicing.

overall_design: Comparative gene expression and splicing analysis between U251 treated with Vehicle versus LLY-283 (4 samples per groups).

first: Charlotte

last: Degorre

first: Steven

last: Lohard

first: Christina

last: Bobrek

first: Komal

last: Rawal

first: Skyler

last: Kuhn

first: Philip

middle: J

last: Tofilon


title: Protein Thermal Stability Changes Induced by the Global Methylation Inhibitor 3-deazaneplanocin A (DZNep)

city: Indianapolis

first: Evan

last: Cornett

state: Indiana

address: 635 Barnhill Drive

institute: Indiana University

summary: DZNep (3-deazaneplanocin A) is commonly used to reduce lysine methylation. DZNep inhibits S-adenosyl-L-homocysteine hydrolase (AHCY), preventing the conversion of S-adenosyl-L-homocysteine (SAH) into L-homocysteine and reducing the level of S-adenosylmethionine (SAM). As a result, the SAM to SAH ratio decreases, an indicator of the methylation potential within a cell. Many studies have characterized the impact of DZNep on histone lysine methylation or in specific cell or disease contexts. Recently, protein thermal stability has provided a new dimension for studying the mechanism of action of small molecule inhibitors. In addition to ligand binding, post-translational modifications and protein-protein interactions impact thermal stability. Here, we sought to characterize protein thermal stability changes induced by DZNep treatment in HEK293T cells using the Protein Integral Solubility Alteration (PISA) assay. DZNep treatment altered the thermal stability of 135 proteins, with over half previously reported to be methylated at lysine residues. In addition to thermal stability, we identify changes in transcript and protein abundance after DZNep treatment to distinguish between direct and indirect impacts on thermal stability. Nearly one-third of the proteins with altered thermal stability had no changes at the transcript or protein level. Of these thermally altered proteins, CDK6 had a stabilized methylated peptide, while its unmethylated counterpart was unaltered. Multiple methyltransferases were among the proteins with thermal stability alteration, including DNMT1, potentially due to changes in SAM/SAH levels. This study systematically evaluates DZNep’s impact on the transcriptome, the proteome, and the thermal stability of proteins.

overall_design: To investigate the impact of DZNep on transcription, global protein, and thermal stability changes, we treated HEK293T cells with either vehicle or 100 uM oF DZNep for 48 hrs.

first: Christine

last: Berryhill

first: Emma

middle: H

last: Doud

first: Jocelyne

last: Hanquier

first: Whitney

middle: R

last: Smith-Kinnaman

first: Devon

middle: L

last: McCourry

first: Amber

last: Mosley


title: A Transcriptomics Analysis of the Regulation of Fiber Cell Differentiation in the Absence of FGFRs and PTEN

city: OXFORD

first: Anil

last: Upreti

state: OH

address: 501 E High St, Oxford, OH 45056

institute: Miami University

summary: The addition of 50% vitreous humor to the media bathing lens epithelial explants results in the induction of both lens fiber cell differentiation and a significant immune/inflammatory response. Although Fgfr loss blocks differentiation in lens epithelial explants, this loss can be partially rescued by deleting Pten, demonstrating an epistatic relationship between FGFR-signaling and PTEN. To investigate the functions of Fgfrs and Pten during lens fiber cell differentiation, we utilized a lens epithelial explant system and conducted RNA sequencing on vitreous humor-exposed explants lacking Fgfrs, or Pten or both Fgfrs and Pten. We found that Fgfr loss impairs both vitreous-induced differentiation and inflammation while the additional loss of Pten restores these responses. Additionally, vitreous exposure triggers a less pronounced immune response in explants lacking both Fgfrs and Pten than in wildtype explants, but a stronger immune response than in explants lacking only Fgfrs. Furthermore, transcriptomic analysis suggested that PDGFR-signaling in FGFR-deficient explants might be required to mediate the rescue of vitreous-induced fiber cell differentiation in explants lacking both Fgfrs and Pten. The blockage of β- crystallin induction in explants lacking both Fgfrs and Pten in the presence of a PDGFR-inhibitor supports this hypothesis. Our findings demonstrate that a wide array of genes associated with fiber cell differentiation are downstream of FGFR-signaling and that the vitreous-induced immune responses also depend on FGFR-signaling. Our data also demonstrates that many of the vitreous-induced gene expression changes Fgfr-deficient explants can be rescued in explants lacking both Fgfrs and Pten.

overall_design: Lens epithelial explants from 8-day oldFVB/N-strainlens epithelia were prepared from mice of four different genotypes. These were 1) wildtype (FVB), 2) homozygous fornull alleles ofFgfr3and loxP- flanked alleles forFgfr1and Fgfr2(TKO), 3) homozygous for loxP-flanked alleles of Pten (PTEN), and TKO explants that also were homozygous for the loxP-flanked alleles of Pten (QKO). Explants were cultured in M-199 media containing 0.1% BSA and a 1% antibiotic-antimycotic solution and incubated at 37°C in a humidified chamber with 5% CO2. Recombinant adenoviral vectors delivered Cre recombinase (used to delete loxP-flanked alleles) to explants at a concentration of 1 X 10 6 PFU per dish containing six explants each. 24 hours after explanting, the media was replaced with fresh media mixed at a 1:1 ratio with bovine vitreous. RNA was extracted from lens epithelial explants and subjected to RNA-seq at four stages as follows:(1) 1 day after Cre addition (2) 5 days after Cre addition (3) 10 days after Cre addition. The differentially expressed genes (DEGs) were identified between different conditions.

first: Michael

last: Robinson


title: Targeting RBM39 for Improved Outcomes as a Novel Therapeutic Strategy in T-cell acute lymphoblastic leukemia

city: Suzhou

first: Zimu

last: Zhang

state: Jiangsu

address: Zhongnan Street

institute: Children’s Hospital of Soochow University

summary: Indisulam demonstrates significant antineoplastic properties in the context of T-cell acute lymphoblastic leukemia (T-ALL). Its effectiveness is primarily due to the degradation of RBM39, attenuating cell proliferation, promoting apoptosis and interfering with cell cycle progression in vitro, while facilitating tumor remission in T-ALL in vivo models.

overall_design: mRNA profiles of J.gamma1 cells treated with 5μM indisulam or DMSO for 16h were generated by RNA sequencing, in double, using Illumina NovaSeq6000.

first: Tongting

last: Ji

first: Jun

last: Lu

first: Jian

last: Pan


title: Characterization of the effect of deleting the Mib1 and Gata6 gene in E (embryonic day) 11.5 OFT (outflow tract).

city: Madrid

first: Jose Luis

last: de la Pompa

state: MADRID

address: Melchor Fernández Almagro 3

institute: Centro Nacional de Investigaciones Cardiovasculares (CNIC)

summary: To investigate the potential interactions between of Gata6 and Mib1 in OFT development, we generated a double heterozygous mice Mib1V943F/+ Gata6STOP/+, harvested OFT at E11.5, and determined gene expression profiles, using RNA-seq.

overall_design: Gene expression profiling with RNA-seq was performed in four pairs of E11.5 OFT control and Mib1V943F/+ Gata6STOP/+ (MUT) samples, each of them obtained by pooling material from three individuals.

first: Rebeca

last: Piñeiro-Sabarís

first: Donal

last: MacGrogan

first: Jose


title: Transcriptomic and proteomic spatial profiling of tertiary lymphoid aggregates in head and neck cancer reveal spatial tissue dynamics and profiles associated with immunotherapy response.

city: Brisbane

first: Habib

last: Sadeghirad

state: Queensland

address: 37 Kent St, Woolloongabba, 37 Kent St, Woolloongabba

country: Australia

institute: The University of Queensland

summary: DSP RNA profling using the whole transcriptome atlas kit was performed on a tissue sample perior to immunotherapy from a patient with head and neck cancer.

overall_design: Differential expression and gene set enrichment analyses were performed on the TLS and GC structures of a whole tissue to identify differences between the TLS and GC structures.

first: Arutha

last: Kulasinghe


title: Impaired B-cell function in ERCC2 deficiency

city: Wien

first: Andreas

last: Diendorfer

address: Leberstraße 20

country: Austria

institute: TAmiRNA GmbH

summary: Trichothiodystrophy-1 (TTD1) is an autosomal-recessive disease and caused by mutations in ERCC2, a gene coding for a subunit of the TFIIH transcription and nucleotide-excision repair (NER) factor. In almost half of these patients infectious susceptibility has been reported but the underlying molecular mechanism leading to immunodeficiency is largely unknown. The aim of this study was to perform extended molecular and immunological phenotyping in patients suffering from TTD1. Cellular immune phenotype was investigated using multicolour flow cytometry. DNA repair efficiency was evaluated in UV-irradiation assays. Furthermore, early BCR activation events and proliferation of TTD1 lymphocytes following DNA damage induction was tested. In addition, we performed differential gene expression analysis in peripheral lymphocytes of TTD1 patients. We investigated three unrelated TTD1 patients who presented with recurrent infections early in life of whom two harboured novel ERCC2 mutations and the third patient is a carrier of previously described pathogenic ERCC2 mutations. Hypogammaglobulinemia and decreased antibody responses following vaccination were found. TTD1 B-cells showed accumulation of g-H2AX levels, decreased proliferation activity and reduced cell viability following UV-irradiation. mRNA sequencing analysis revealed significantly downregulated genes needed for B-cell development and activation. Analysis of B-cell subpopulations showed low numbers of naïve and transitional B-cells in TTD1 patients, indicating abnormal B-cell differentiation in vivo. In summary, our analyses confirmed the pathogenicity of novel ERCC2 mutations and show that ERCC2 deficiency is associated with antibody deficiency most likely due to altered B-cell differentiation resulting from impaired B-cell activation and activation-induced gene transcription.

overall_design: To investigate the differential gene expression in TTD1 patients, we isolated peripheral blood mononuclear cells from 3 unrelated ERCC2 deficient patients as well as 5 healthy controls. Then, RNA was isolated from PBMCs, and total RNA served as input for mRNA sequencing analysis. Bioinformatic analysis following mRNA sequencing should reveal whether impaired or dysregulated transcription in PBMCs from TTD1 patients contributes to impaired antibody response found in these patients.

first: Raphael

last: Rossmanith

first: Kai

last: Sauerwein

first: Christoph

last: Geier

first: Alexander

last: Leiss-Piller

first: Roman

middle: F

last: Stemberger

first: Svetlana

last: Sharapova

first: Robert

middle: W

last: Gruber

first: Helmut

last: Bergler

first: James

last: Verbsky

first: Krisztian

last: Csomos

first: Jolan

last: Walter

first: Herman

middle: M

last: Wolf