Similar Transcriptomic Profiles Research Articles

Next generation yellow fever vaccine induces an equivalent immune and transcriptomic profile to the current vaccine: observations from a phase I randomised clinical trial

Yellow fever (YF), a mosquito-borne acute viral haemorrhagic illness, is endemic to many tropical and subtropical areas of Africa and Central and South America. Vaccination remains the most effective prevention strategy; however, as repeated outbreaks have exhausted vaccine stockpiles, there is a need for improved YF vaccines to meet global demand. A live-attenuated YF vaccine candidate (referred to as vYF) cloned from a YF-17D vaccine (YF-VAX®) sub-strain, adapted for growth in Vero cells cultured in serum-free media, is in clinical development. We report the innate and adaptive immune responses and the transcriptome profile of selected genes induced by vYF. Healthy adults aged 18-60 years were randomised at a 1:1:1:1 ratio to receive one dose of vYF at 4, 5 or 6 Log CCID50 or YF-VAX (reference vaccine), administered subcutaneously in the upper arm (ClinicalTrials.gov identifier: NCT04142086). Blood/serum samples were obtained at scheduled time points through 180 days (D180) post-vaccination. The surrogate endpoints assessed were: serum cytokine/chemokine concentrations, measured by bead-based Multiplex assay; peripheral blood vYF-specific IgG and IgM memory B cell frequencies, measured by FluoroSpot assay; and expression of genes involved in the immune response to YF-17D vaccination by RT-qPCR. There was no increase in any of the cytokine/chemokine concentrations assessed through D14 following vaccination with vYF or YF-VAX, except for a slight increase in IP-10 (CXCL10) levels. The gene expression profiles and kinetics following vaccination with vYF and YF-VAX were similar, inclusive of innate (antiviral responses [type-1 interferon, IFN signal transduction; interferon-stimulated genes], activated dendritic cells, viral sensing pattern recognition receptors) and adaptive (cell division in stimulated CD4+ T cells, B cell and antibody) immune signatures, which peaked at D7 and D14, respectively. Increases in vYF-specific IgG and IgM memory B cell frequencies at D28 and D180 were similar across the study groups. vYF-induced strong innate and adaptive immune responses comparable to those induced by YF-VAX, with similar transcriptomic and kinetic profiles observed. Sanofi.

Investigation of Peroxisome Proliferator-Activated Receptor Genes as Requirements for Visual Startle Response Hyperactivity in Larval Zebrafish Exposed to Structurally Similar Per- and Polyfluoroalkyl Substances (PFAS).

Per- and polyfluoroalkyl Substances (PFAS) are synthetic chemicals widely detected in humans and the environment. Exposure to perfluorooctanesulfonic acid (PFOS) or perfluorohexanesulfonic acid (PFHxS) was previously shown to cause dark-phase hyperactivity in larval zebrafish. The objective of this study was to elucidate the mechanism by which PFOS or PFHxS exposure caused hyperactivity in larval zebrafish. Swimming behavior was assessed in 5-d postfertilization (dpf) larvae following developmental (1-4 dpf) or acute (5 dpf) exposure to PFOS, PFHxS, or 0.4% dimethyl sulfoxide (DMSO). After developmental exposure and chemical washout at 4 dpf, behavior was also assessed at 5-8 dpf. RNA sequencing was used to identify differences in global gene expression to perform transcriptomic benchmark concentration-response () modeling, and predict upstream regulators in PFOS- or PFHxS-exposed larvae. CRISPR/Cas9-based gene editing was used to knockdown peroxisome proliferator-activated receptors (ppars) pparaa/ab, pparda/db, or pparg at day 0. Knockdown crispants were exposed to PFOS or 0.4% DMSO from 1-4 dpf and behavior was assessed at 5 dpf. Coexposure with the ppard antagonist GSK3787 and PFOS was also performed. Transient dark-phase hyperactivity occurred following developmental or acute exposure to PFOS or PFHxS, relative to the DMSO control. In contrast, visual startle response (VSR) hyperactivity only occurred following developmental exposure and was irreversible up to 8 dpf. Similar global transcriptomic profiles, estimates, and enriched functions were observed in PFOS- and PFHxS-exposed larvae, and ppars were identified as putative upstream regulators. Knockdown of pparda/db, but not pparaa/ab or pparg, blunted PFOS-dependent VSR hyperactivity to control levels. This finding was confirmed via antagonism of ppard in PFOS-exposed larvae. This work identifies a novel adverse outcome pathway for VSR hyperactivity in larval zebrafish. We demonstrate that developmental, but not acute, exposure to PFOS triggered persistent VSR hyperactivity that required ppard function. https://doi.org/10.1289/EHP13667.

IRF8 configures enhancer landscape in postnatal microglia and directs microglia specific transcriptional programs.

Microglia are innate immune cells in the brain. Transcription factor IRF8 is highly expressed in microglia. However, its role in postnatal microglia development is unknown. We demonstrate that IRF8 binds stepwise to enhancer regions of postnatal microglia along with Sall1 and PU.1, reaching a maximum after day 14. IRF8 binding correlated with a stepwise increase in chromatin accessibility, which preceded the initiation of microglia-specific transcriptome. Constitutive and postnatal Irf8 deletion led to a loss of microglia identity and gain of disease-associated microglia-like genes. Combined analysis of scRNA-seq and scATAC-seq revealed a correlation between chromatin accessibility and transcriptome at a single-cell level. IRF8 was also required for microglia-specific DNA methylation patterns. Lastly, in the 5xFAD model, constitutive and postnatal Irf8 deletion reduced the interaction of microglia with Aβ plaques and the size of plaques, lessening neuronal loss. Together, IRF8 sets the epigenetic landscape, which is required for postnatal microglia gene expression.

Epicardial cell participation in cardiac repair requires epithelial-to-mesenchymal transition

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Dutch Heart Foundation During development, the epicardium forms as a single cell layer on the outside of the heart. Part of these cells undergo epithelial-to-mesenchymal transition (EMT) and migrate into the myocardium where epicardial-derived cells (EPDCs) participate in the formation of the heart by contributing cells and by producing paracrine factors. In the adult heart the epicardium exists as a dormant layer until there is ischemic injury which leads to a partial reactivation and participation in the repair process. Therefore, the epicardium is considered an interesting cell source for endogenous cardiac repair. Aim to establish if differences between developmental and adult epicardium exist, and in which state (pre- or post-EMT) epicardial cells contribute efficiently to cardiac repair. Methods Primary epicardial cells were isolated from human fetal and adult cardiac tissue samples and cultured as epicardial cells (EPIs) or as EPDCs after inducing EMT with TGFβ3. Cells were subjected to RNA-sequencing or 24hrs of conditioning medium. Myocardial infarction (MI) and intramyocardially injection of EPIs and EPDCs was performed in NOD-SCID mice. Results Fetal and adult EPDCs displayed similar transcriptomic and paracrine profiles. In vitro, the conditioned medium of both cell types equally conveyed cardioprotection to staurosporin-treated cardiomyocytes. Next, we studied the effect of EMT: comparison of adult EPIs with EPDCs revealed that EPDCs have a angiogenic profile. Six weeks post-MI transplantation of adult EPIs or EPDCs resulted in a higher EF (25.4±3.8 vs. 11.1±4.6%, P<0.005), lower ESV (139.6±13.5 vs, 91.37±14.2ul, P<0.05), and a smaller infarct size in EPDC-recipients. EPDC-recipient hearts contained more proliferating cells at 3 days, and a higher vessel density at 6wks post-MI. Interestingly, while cell engraftment was equal in both groups at 3days, no cells were retrieved at 6wks supporting a paracrine effect. Conclusion epicardial EMT is essential to unleash the predominantly paracrine contribution of epicardial cells in cardiac repair.

Dominant dystrophic epidermolysis bullosa is associated with glycolytically active GATA3+ T helper 2 cells which may contribute to pruritus in lesional skin.

Dominant dystrophic epidermolysis bullosa (DDEB) is characterized by trauma-induced blisters and, in some individuals, intense pruritus. Precisely what causes itch in DDEB and optimal ways to reduce it have not been fully determined. To characterize DDEB skin transcriptomes to identify therapeutic targets to reduce pruritus in patients. Using bulk RNA sequencing, we evaluated affected and unaffected skin biopsy samples from six patients with DDEB (all with the very itchy pruriginosa subtype) and four healthy individuals. Single-cell transcriptomes of affected (n = 2) and unaffected (n = 1) DDEB skin and healthy skin (n = 2) were obtained. Dupilumab treatment was provided for three patients. The skin bulk transcriptome showed significant enrichment of T helper (Th)1/2 and Th17 pathways in affected DDEB skin compared with nonlesional DDEB skin and healthy skin. Single-cell transcriptomics showed an association of glycolytically active GATA3+ Th2 cells in affected DDEB skin. Treatment with dupilumab in three people with DDEB led to significantly reduced visual analogue scale (VAS) itch scores after 12 weeks (mean VAS 3.83) compared with pretreatment (mean VAS 7.83). Bulk RNAseq and quantitative polymerase chain reaction showed that healthy skin and dupilumab-treated epidermolysis bullosa (EB) pruriginosa skin have similar transcriptomic profiles and reduced Th1/Th2 and Th17 pathway enrichment. Single-cell RNAseq helps define an enhanced DDEB-associated Th2 profile and rationalizes drug repurposing of anti-Th2 drugs in treating DDEB pruritus.

Impact of heterozygous ALK1 mutations on the transcriptomic response to BMP9 and BMP10 in endothelial cells from hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension donors

Heterozygous activin receptor-like kinase 1 (ALK1) mutations are associated with two vascular diseases: hereditary hemorrhagic telangiectasia (HHT) and more rarely pulmonary arterial hypertension (PAH). Here, we aimed to understand the impact of ALK1 mutations on BMP9 and BMP10 transcriptomic responses in endothelial cells. Endothelial colony-forming cells (ECFCs) and microvascular endothelial cells (HMVECs) carrying loss of function ALK1 mutations were isolated from newborn HHT and adult PAH donors, respectively. RNA-sequencing was performed on each type of cells compared to controls following an 18 h stimulation with BMP9 or BMP10. In control ECFCs, BMP9 and BMP10 stimulations induced similar transcriptomic responses with around 800 differentially expressed genes (DEGs). ALK1-mutated ECFCs unexpectedly revealed highly similar transcriptomic profiles to controls, both at the baseline and upon stimulation, and normal activation of Smad1/5 that could not be explained by a compensation in cell-surface ALK1 level. Conversely, PAH HMVECs revealed strong transcriptional dysregulations compared to controls with > 1200 DEGs at the baseline. Consequently, because our study involved two variables, ALK1 genotype and BMP stimulation, we performed two-factor differential expression analysis and identified 44 BMP9-dysregulated genes in mutated HMVECs, but none in ECFCs. Yet, the impaired regulation of at least one hit, namely lunatic fringe (LFNG), was validated by RT-qPCR in three different ALK1-mutated endothelial models. In conclusion, ALK1 heterozygosity only modified the BMP9/BMP10 regulation of few genes, including LFNG involved in NOTCH signaling. Future studies will uncover whether dysregulations in such hits are enough to promote HHT/PAH pathogenesis, making them potential therapeutic targets, or if second hits are necessary.

High throughput functional profiling of genes at intraocular pressure loci reveals distinct networks for glaucoma.

Primary open angle glaucoma (POAG) is a leading cause of blindness globally. Characterized by progressive retinal ganglion cell degeneration, the precise pathogenesis remains unknown. Genome-wide association studies (GWAS) have uncovered many genetic variants associated with elevated intraocular pressure (IOP), one of the key risk factors for POAG. We aimed to identify genetic and morphological variation that can be attributed to trabecular meshwork cell (TMC) dysfunction and raised IOP in POAG. 62 genes across 55 loci were knocked-out in a primary human TMC line. Each knockout group, including five non-targeting control groups, underwent single-cell RNA-sequencing (scRNA-seq) for differentially-expressed gene (DEG) analysis. Multiplexed fluorescence coupled with CellProfiler image analysis allowed for single-cell morphological profiling. Many gene knockouts invoked DEGs relating to matrix metalloproteinases and interferon-induced proteins. We have prioritized genes at four loci of interest to identify gene knockouts that may contribute to the pathogenesis of POAG, including ANGPTL2, LMX1B, CAV1, and KREMEN1. Three genetic networks of gene knockouts with similar transcriptomic profiles were identified, suggesting a synergistic function in trabecular meshwork cell physiology. TEK knockout caused significant upregulation of nuclear granularity on morphological analysis, while knockout of TRIOBP, TMCO1 and PLEKHA7 increased granularity and intensity of actin and the cell-membrane. High-throughput analysis of cellular structure and function through multiplex fluorescent single-cell analysis and scRNA-seq assays enabled the direct study of genetic perturbations at the single-cell resolution. This work provides a framework for investigating the role of genes in the pathogenesis of glaucoma and heterogenous diseases with a strong genetic basis.

Transcriptomic Profiles of Pectoralis major Muscles Affected by Spaghetti Meat and Woody Breast in Broiler Chickens.

Spaghetti meat (SM) and woody breast (WB) are breast muscle myopathies of broiler chickens, characterized by separation of myofibers and by fibrosis, respectively. This study sought to investigate the transcriptomic profiles of breast muscles affected by SM and WB. Targeted sampling was conducted on a flock to obtain 10 WB, 10 SM, and 10 Normal Pectoralis major muscle samples from 37-day-old male chickens. Total RNA was extracted, cDNA was used for pair-end sequencing, and differentially expressed genes (DEGs) were determined by a false discovery rate of <0.1 and a >1.5-fold change. Principal component and heatmap cluster analyses showed that the SM and WB samples clustered together. No DEGs were observed between SM and WB fillets, while a total of 4018 and 2323 DEGs were found when comparing SM and WB, respectively, against Normal samples. In both the SM and WB samples, Gene Ontology terms associated with extracellular environment and immune response were enriched. The KEGG analysis showed enrichment of cytokine-cytokine receptor interaction and extracellular matrix-receptor interaction pathways in both myopathies. Although SM and WB are macroscopically different, the similar transcriptomic profiles suggest that these conditions may share a common pathogenesis. This is the first study to compare the transcriptomes of SM and WB, and it showed that, while both myopathies had profiles different from the normal breast muscle, SM and WB were similar, with comparable enriched metabolic pathways and processes despite presenting markedly different macroscopic features.

Simple Preservation of Single Cells (SENSE): One-Step Robust Whole Blood Cryopreservation Method Enables the Generation of High-Quality Single-Cell Immune Profiles

Introduction: Single-cell profiling (SCP) has revolutionized our understanding of cellular and molecular states in health and disease. Current multistep methods for cryopreserving single-cell suspensions from blood for single-cell RNA sequencing (scRNA-seq) are time-consuming, require trained personnel and special equipment, limiting their clinical adoption and sample collection/availability. It is critical to develop protocols for efficient capture of precious clinical samples/implementation of SCP in clinics. We developed and validated the Simple prEservatioN of Single cElls (SENSE) method for single-step cryopreservation of whole blood (WB), which, along with granulocyte depletion during single-cell assay, generates high-quality SCPs. Methods: Blood samples (n=6) were split into two parts and cryopreserved using the SENSE and peripheral blood mononuclear cells (PBMC) methods. In the SENSE method, WB was cryopreserved by mixing 1:1 blood and freezing solution (80% FBS, 20% DMSO) and granulocytes removed using EasySep CD15 positive selection kit. For PBMC method, density-gradient method was employed for isolating PBMCs followed by cryopreservation. Single cell assay was performed using 10x Genomics kits. The raw sequencing reads were aligned using 10x Genomics Cell Ranger (Zheng et al., Nat. Comm., 2017) to the reference human genome, and the gene-expression matrices were analyzed using the Seurat package (Hao et al., Cell, 2021). Cell markers were identified by comparing target cell types with others using the Wilcoxon Rank Sum test (adjusted P&amp;lt;.10, average log2FC&amp;gt;0.25, percent cell expression&amp;gt;25%). Potential doublets were marked using the doubletFinder (McGinnis et al., Cell Syst., 2019) algorithm and batch-effect was quantified using Shannon entropy calculated by the CellMixS (Lutge et al., Life Sci Alliance. 2021) package for quality assessment of two methods. Cellular communications were assessed using CellChat, which measures cell-to-cell interactions based on the ligands and receptors expression (Jin et al., Nature Comm., 2021). Results: Highly viable (86.3±1.51%) single-cell suspensions (22,353 cells) were obtained from the SENSE method's WB cryopreserved samples. The captured median gene counts, unique molecular identifiers (UMIs)/cell, and mitochondrial content were comparable between the two methods. Similar proportions of membrane, extracellular, and ribosomal ontology-related genes demonstrated the robustness of the SENSE method in capturing high-quality cells without introducing cellular damage artifacts. Lower doublets (2.4%) were observed with the SENSE method compared to the PBMC method (4.8%). These rigorous quality assessments demonstrated the effectiveness of the SENSE method for obtaining high-quality cells for SCP. Both methods yielded similar transcriptomic profiles with split UMAPs of the integrated scRNA-seq data revealing similar clustering patterns except for some variations in the myeloid cells and T-cells clusters. The SENSE method cryopreserved samples exhibited significantly higher T-cell enrichment, enabling deeper characterization of T-cell subtypes. The functional landscape of T/NK cells was assessed using the CellChat tool. Most pathways showed a similar information flow pattern, indicating the capture of similar signaling networks between the cells processed using either of the two methods. However, the SENSE method captured lower number of myeloid cells, attributable to filtering out of sticky CD15+ myeloid/granulocytes that resulted in the loss of single-cells captured. Nevertheless, the SENSE method recapitulated the myeloid compartment associated with the disease as we observed similar patient-wise differences in cell types from both methods. Finally, we compared the transcriptome profiles of both methods to a publicly available PBMC dataset (Zheng et al., Nat. Comm., 2017). Concordance between the three datasets was observed, with all cell types being consistently identified using key marker gene expression. Conclusions: Comparative analysis of scRNA-seq datasets obtained with the two cryopreservation methods, i.e., SENSE and PBMC methods, yielded similar cellular and molecular profiles, confirming the suitability of the former method's incorporation in clinics/labs for cryopreserving and obtaining high-quality single-cells for conducting critical translational research.

P-366 Unraveling the whole transcriptome profiles of receptive phase endometrium in infertile women

Abstract Study question Are there differential transcriptomic profiles of the receptive phase endometria in infertile women with different infertility diagnoses? Summary answer Endometrium-related pathologies such as endometriosis and RIF seemed to share similar endometrial molecular profiles in mid-secretory phase,while samples from unexplained infertility were similar to controls. What is known already Successful embryo implantation is orchestrated by the combination of a viable blastocyst, a receptive endometrium and a perfectly synchronized molecular dialogue between them. Endometrium-associated pathologies such as endometriosis and recurrent implantation failure (RIF) are suggested to hamper endometrial receptivity, but the molecular mechanisms affected in these and other infertility diagnoses such as unexplained infertility need further investigation. Previous transcriptome studies of the human endometrium have analysed different infertility diagnoses separately, but whether and to what extent receptive-phase endometrial transcriptome profiles vary between distinct infertility diagnoses is underexplored. Study design, size, duration A prospective, cross-sectional study was performed at the Reproductive Unit at the University Hospital between March 2019 and April 2021. Forty-five well-characterized infertile women (age 34.7±3.8 years, BMI 24.5±kg/m2) were recruited before starting any infertility treatment. Four groups according to different infertility diagnoses were established: endometriosis (n = 12), RIF (n = 14), unexplained infertility (n = 10), and male factor infertility as control group (n = 9). Participants/materials, setting, methods Endometrial biopsies were collected using Pipelle curette during mid-secretory phase (LH + 7) of the natural cycle. Total RNA was extracted and sequenced using Illumina NovaSeq 6000 technology. Raw sequencing data was quality assessed, pre-processed and quantified, and differential expression and enrichment analyses were performed using R software. Main results and the role of chance Differential expression analysis revealed 743, 587 and 140 differentially expressed genes (DEGs) in endometriosis, RIF and unexplained infertility, respectively, when compared to control women of the male factor infertility group. Among these, 31 genes exhibited consistent differential expression between all infertility groups. Regarding functional enrichment analyses, the detected DEGs were mainly related to immune response and inflammatory processes, supporting the involvement of these pathways in the impairment of endometrial receptivity. Interestingly, the comparison between different study groups showed similar transcriptome profiles in endometrium-associated pathologies such as endometriosis and RIF, while women with unexplained infertility had similar molecular profiles with the control group of male factor infertility. These study results indicate common molecular pathways in endometrial receptivity between infertile women with endometriosis and RIF, and between unexplained infertility and control male factor infertility patients. Limitations, reasons for caution Further research with bigger sample size is required to confirm these findings. Wider implications of the findings We identified transcriptome profiles associated with infertility diagnoses. Further, women with unexplained infertility tended to have similar molecular profiles with control women, meaning that other factors than endometrial receptivity issues could lead to their infertility. Our study helps to understand the molecular mechanisms underlying female infertility in different infertility diagnoses. Trial registration number not applicable

Paf1 complex subunit Rtf1 stimulates H2B ubiquitylation by interacting with the highly conserved N-terminal helix of Rad6

Histone modifications coupled to transcription elongation play important roles in regulating the accuracy and efficiency of gene expression. The monoubiquitylation of a conserved lysine in H2B (K123 in Saccharomyces cerevisiae; K120 in humans) occurs cotranscriptionally and is required for initiating a histone modification cascade on active genes. H2BK123 ubiquitylation (H2BK123ub) requires the RNA polymerase II (RNAPII)-associated Paf1 transcription elongation complex (Paf1C). Through its histone modification domain (HMD), the Rtf1 subunit of Paf1C directly interacts with the ubiquitin conjugase Rad6, leading to the stimulation of H2BK123ub in vivo and in vitro. To understand the molecular mechanisms that target Rad6 to its histone substrate, we identified the site of interaction for the HMD on Rad6. Using in vitro cross-linking followed by mass spectrometry, we localized the primary contact surface for the HMD to the highly conserved N-terminal helix of Rad6. Using a combination of genetic, biochemical, and in vivo protein cross-linking experiments, we characterized separation-of-function mutations in S. cerevisiae RAD6 that greatly impair the Rad6-HMD interaction and H2BK123 ubiquitylation but not other Rad6 functions. By employing RNA-sequencing as a sensitive approach for comparing mutant phenotypes, we show that mutating either side of the proposed Rad6-HMD interface yields strikingly similar transcriptome profiles that extensively overlap with those of a mutant that lacks the site of ubiquitylation in H2B. Our results fit a model in which a specific interface between a transcription elongation factor and a ubiquitin conjugase guides substrate selection toward a highly conserved chromatin target during active gene expression.

On the correspondence between the transcriptomic response of a compound and its effects on its targets

Better understanding the transcriptomic response produced by a compound perturbing its targets can shed light on the underlying biological processes regulated by the compound. However, establishing the relationship between the induced transcriptomic response and the target of a compound is non-trivial, partly because targets are rarely differentially expressed. Therefore, connecting both modalities requires orthogonal information (e.g., pathway or functional information). Here, we present a comprehensive study aimed at exploring this relationship by leveraging thousands of transcriptomic experiments and target data for over 2000 compounds. Firstly, we confirm that compound-target information does not correlate as expected with the transcriptomic signatures induced by a compound. However, we reveal how the concordance between both modalities increases by connecting pathway and target information. Additionally, we investigate whether compounds that target the same proteins induce a similar transcriptomic response and conversely, whether compounds with similar transcriptomic responses share the same target proteins. While our findings suggest that this is generally not the case, we did observe that compounds with similar transcriptomic profiles are more likely to share at least one protein target and common therapeutic applications. Finally, we demonstrate how to exploit the relationship between both modalities for mechanism of action deconvolution by presenting a case scenario involving a few compound pairs with high similarity.

RSK2 inactivation cooperates with AXIN1 inactivation or β-catenin activation to promote hepatocarcinogenesis

Recurrent somatic mutations of the RPS6KA3 gene encoding for the serine/threonine kinase RSK2 were identified in hepatocellular carcinomas (HCCs), suggesting its tumour-suppressive function. Our goal was to demonstrate the tumour suppressor role of RSK2 in the liver and investigate the functional consequences of its inactivation. We analysed a series of 1,151 human HCCs for RSK2 mutations and 20 other driver genetic alterations. We then modelled RSK2 inactivation in mice in various mutational contexts recapitulating or not those naturally found in human HCC, using transgenic mice and liver-specific carcinogens. These models were monitored for liver tumour appearance and subjected to phenotypic and transcriptomic analyses. Functional consequences of RSK2 rescue were also investigated in a human RSK2-deficient HCC cell line. RSK2-inactivating mutations are specific to human HCC and frequently co-occur with AXIN1-inactivating or β-catenin-activating mutations. Modelling of these co-occurrences in mice showed a cooperative effect in promoting liver tumours with transcriptomic profiles recapitulating those of human HCCs. By contrast, there was no cooperation in liver tumour induction between RSK2 loss and BRAF-activating mutations chemically induced by diethylnitrosamine. In human liver cancer cells, we also showed that RSK2 inactivation confers some dependency to the activation of RAS/MAPK signalling that can be targeted by MEK inhibitors. Our study demonstrates the tumour suppressor role of RSK2 and its specific synergistic effect in hepatocarcinogenesis when its loss of function is specifically combined with AXIN1 inactivation or β-catenin activation. Furthermore, we identified the RAS/MAPK pathway as a potential therapeutic target for RSK2-inactivated liver tumours. This study demonstrated the tumour suppressor role of RSK2 in the liver and showed that its inactivation specifically synergises with AXIN1 inactivation or β-catenin activation to promote the development of HCC with similar transcriptomic profiles as found in humans. Furthermore, this study highlights that activation of the RAS/MAPK pathway is one of the key signalling pathways mediating the oncogenic effect of RSK2 inactivation that can be targeted with already available anti-MEK therapies.

Chondrocyte-like cells in nucleus pulposus and articular chondrocytes have similar transcriptomic profiles and are paracrine-regulated by hedgehog from notochordal cells and subchondral bone.

Objective: The nucleus pulposus (NP) comprises notochordal NP cells (NCs) and chondrocyte-like NP cells (CLCs). Although morphological similarities between CLCs and chondrocytes have been reported, interactions between CLCs and NCs remain unclear. In this study, we aimed to clarify regulatory mechanisms of cells in the NP and chondrocytes. Design: We performed single-cell RNA sequencing (scRNA-seq) analysis of the articular cartilage (AC) and NP of three-year-old cynomolgus monkeys in which NCs were present. We then performed immunohistochemical analysis of NP and distal femur. We added sonic hedgehog (SHH) to primary chondrocyte culture. Results: The scRNA-seq analysis revealed that CLCs and some articular chondrocytes had similar gene expression profiles, particularly related to GLI1, the nuclear mediator of the hedgehog pathway. In the NP, cell-cell interaction analysis revealed SHH expression in NCs, resulting in hedgehog signaling to CLCs. In contrast, no hedgehog ligands were expressed by chondrocytes in AC samples. Immunohistochemical analysis of the distal end of femur indicated that SHH and Indian hedgehog (IHH) were expressed around the subchondral bone that was excluded from our scRNA-seq sample. scRNA-seq data analysis and treatment of primary chondrocytes with SHH revealed that hedgehog proteins mediated an increase in hypoxia-inducible factor 1-alpha (HIF-1α) levels. Conclusion: CLCs and some articular chondrocytes have similar transcriptional profiles, regulated by paracrine hedgehog proteins secreted from NCs in the NP and from the subchondral bone in the AC to promote the HIF-1α pathway.

Transcriptomics and physiological analyses unveil the distinct mechanisms of ATP and glucose-6-phosphate utilization in Phaeodactylum tricornutum

Phosphoesters are a dominant component of marine dissolved organic phosphorus (DOP) and an important source of the phosphorus nutrient for phytoplankton, but the molecular mechanisms of their utilization by phytoplankton are divergent and poorly understood. In this study, we used the model diatom Phaeodactylum tricornutum to investigate and compare the utilization mechanisms of two different phosphoesters, adenosine triphosphate (ATP) and glucose-6-phosphate (G6P). We found that ATP and G6P can both be efficiently used to support the growth of P. tricornutum. Cells grown on ATP or G6P showed lower pigment contents and photosynthetic rates but higher cellular lipids relative to those grown on NaH2PO4 (DIP). Surprisingly, in neither the ATP nor the G6P group were significant increases in whole-cell alkaline phosphatase (AP) activity detected, suggesting that utilization of both DOPs was not reliant on extracellular AP. Yet, ATP-grown cultures released DIP into the medium (i.e., ATP hydrolyzed extracellularly) whereas G6P-grown cultures did not. Furthermore, transcriptomic and RT-qPCR results showed that the gene encoding 5’ nucleotidase (5NT) in the ATP group and PhoD in the G6P group was upregulated. These results indicated that different pathways are involved in the use of these two DOPs, with ATP being hydrolyzed extracellularly likely by 5NT (PHATRDRAFT_44177) to release DIP for uptake, and G6P being directly absorbed and hydrolyzed intracellularly likely by PhoD (PHATRDRAFT_45757). Nevertheless, P. tricornutum under ATP and G6P conditions showed more similar transcriptomic profiles to each other than either compared to DIP-grown cultures, indicating similar metabolic functions of these two DOPs. These findings demonstrate that despite the high similarity in transcriptomic response to ATP and G6P conditions, the utilization mechanisms of these phosphoesters in the same species can be totally different, and the lack of AP activity does not necessarily signal the absence of DIP deficiency or the absence of DOP utilization.

The specialized pro-resolving lipid mediator Protectin D1 affects macrophages differentiation and activity in Adult-onset Still's disease and COVID-19, two hyperinflammatory diseases sharing similar transcriptomic profiles.

COVID-19 and autoinflammatory diseases, such as Adult-onset Still's Disease (AOSD), are characterized by hyperinflammation, in which it is observed massive production and uncontrolled secretion of pro-inflammatory cytokines. The specialized pro-resolving lipid mediators (SPMs) family is one the most important processes counteracting hyperinflammation inducing tissue repair and homeostasis restoration. Among SPMs, Protectin D1 (PD1) is able to exert antiviral features, at least in animal models. The aim of this study was to compare the transcriptome of peripheral blood mononuclear cells (PBMCs) from patients with AOSD and COVID-19 and to evaluate the role of PD1 on those diseases, especially in modulating macrophages polarization. This study enrolled patients with AOSD, COVID-19, and healthy donors HDs, undergoing clinical assessment and blood sample collection. Next-generation deep sequencing was performed to identify differences in PBMCs transcripts profiles. Plasma levels of PD1 were assessed by commercial ELISA kits. Monocyte-derived macrophages were polarized into M1 and M2 phenotypes. We analyzed the effect of PD1 on macrophages differentiation. At 10 days, macrophages were analyzed for surface expression of subtypes markers by flow cytometry. Cytokines production was measured in supernatants by Bio-Plex Assays. In the transcriptomes from AOSD patients and COVID-19 patients, genes involved in inflammation, lipid catabolism, and monocytes activation were specifically dysregulated in AOSD and COVID-19 patients when compared to HDs. Patients affected by COVID-19, hospitalized in intensive care unit (ICU), showed higher levels of PD1 when compared to not-ICU hospitalized patients and HDs (ICU COVID-19 vs not-ICU COVID-19, p= 0.02; HDs vs ICU COVID-19, p= 0.0006). PD1 levels were increased in AOSD patients with SS ≥1 compared to patients with SS=0 (p=0.028) and HDs (p=0.048). In vitro treatment with PD1 of monocytes-derived macrophages from AOSD and COVID-19 patients induced a significant increase of M2 polarization vs control (p<0.05). Furthermore, a significant release of IL-10 and MIP-1β from M2 macrophages was observed when compared to controls (p<0.05). PD1 is able to induce pro-resolutory programs in both AOSD and COVID-19 increasing M2 polarization and inducing their activity. In particular, PD1-treated M2 macrophages from AOSD and COVID-19 patients increased the production of IL-10 and enhanced homeostatic restoration through MIP-1β production.

A robust reprogramming strategy for generating hepatocyte-like cells usable in pharmaco-toxicological studies

BackgroundHigh-throughput pharmaco-toxicological testing frequently relies on the use of established liver-derived cell lines, such as HepG2 cells. However, these cells often display limited hepatic phenotype and features of neoplastic transformation that may bias the interpretation of the results. Alternate models based on primary cultures or differentiated pluripotent stem cells are costly to handle and difficult to implement in high-throughput screening platforms. Thus, cells without malignant traits, optimal differentiation pattern, producible in large and homogeneous amounts and with patient-specific phenotypes would be desirable.MethodsWe have designed and implemented a novel and robust approach to obtain hepatocytes from individuals by direct reprogramming, which is based on a combination of a single doxycycline-inducible polycistronic vector system expressing HNF4A, HNF1A and FOXA3, introduced in human fibroblasts previously transduced with human telomerase reverse transcriptase (hTERT). These cells can be maintained in fibroblast culture media, under standard cell culture conditions.ResultsClonal hTERT-transduced human fibroblast cell lines can be expanded at least to 110 population doublings without signs of transformation or senescence. They can be easily differentiated at any cell passage number to hepatocyte-like cells with the simple addition of doxycycline to culture media. Acquisition of a hepatocyte phenotype is achieved in just 10 days and requires a simple and non-expensive cell culture media and standard 2D culture conditions. Hepatocytes reprogrammed from low and high passage hTERT-transduced fibroblasts display very similar transcriptomic profiles, biotransformation activities and show analogous pattern behavior in toxicometabolomic studies. Results indicate that this cell model outperforms HepG2 in toxicological screening. The procedure also allows generation of hepatocyte-like cells from patients with given pathological phenotypes. In fact, we succeeded in generating hepatocyte-like cells from a patient with alpha-1 antitrypsin deficiency, which recapitulated accumulation of intracellular alpha-1 antitrypsin polymers and deregulation of unfolded protein response and inflammatory networks.ConclusionOur strategy allows the generation of an unlimited source of clonal, homogeneous, non-transformed induced hepatocyte-like cells, capable of performing typical hepatic functions and suitable for pharmaco-toxicological high-throughput testing. Moreover, as far as hepatocyte-like cells derived from fibroblasts isolated from patients suffering hepatic dysfunctions, retain the disease traits, as demonstrated for alpha-1-antitrypsin deficiency, this strategy can be applied to the study of other cases of anomalous hepatocyte functionality.

An Efficient 2D Protocol for Differentiation of iPSCs into Mature Postmitotic Dopaminergic Neurons: Application for Modeling Parkinson's Disease.

About 15% of patients with parkinsonism have a hereditary form of Parkinson's disease (PD). Studies on the early stages of PD pathogenesis are challenging due to the lack of relevant models. The most promising ones are models based on dopaminergic neurons (DAns) differentiated from induced pluripotent stem cells (iPSCs) of patients with hereditary forms of PD. This work describes a highly efficient 2D protocol for obtaining DAns from iPSCs. The protocol is rather simple, comparable in efficiency with previously published protocols, and does not require viral vectors. The resulting neurons have a similar transcriptome profile to previously published data for neurons, and have a high level of maturity marker expression. The proportion of sensitive (SOX6+) DAns in the population calculated from the level of gene expression is higher than resistant (CALB+) DAns. Electrophysiological studies of the DAns confirmed their voltage sensitivity and showed that a mutation in the PARK8 gene is associated with enhanced store-operated calcium entry. The study of high-purity DAns differentiated from the iPSCs of patients with hereditary PD using this differentiation protocol will allow for investigators to combine various research methods, from patch clamp to omics technologies, and maximize information about cell function in normal and pathological conditions.

Parallel recovery of chromatin accessibility and gene expression dynamics from frozen human regulatory T cells

Epigenetic features such as DNA accessibility dictate transcriptional regulation in a cell type- and cell state- specific manner, and mapping this in health vs. disease in clinically relevant material is opening the door to new mechanistic insights and new targets for therapy. Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) allows chromatin accessibility profiling from low cell input, making it tractable on rare cell populations, such as regulatory T (Treg) cells. However, little is known about the compatibility of the assay with cryopreserved rare cell populations. Here we demonstrate the robustness of an ATAC-seq protocol comparing primary Treg cells recovered from fresh or cryopreserved PBMC samples, in the steady state and in response to stimulation. We extend this method to explore the feasibility of conducting simultaneous quantitation of chromatin accessibility and transcriptome from a single aliquot of 50,000 cryopreserved Treg cells. Profiling of chromatin accessibility and gene expression in parallel within the same pool of cells controls for cellular heterogeneity and is particularly beneficial when constrained by limited input material. Overall, we observed a high correlation of accessibility patterns and transcription factor dynamics between fresh and cryopreserved samples. Furthermore, highly similar transcriptomic profiles were obtained from whole cells and from the supernatants recovered from ATAC-seq reactions. We highlight the feasibility of applying these techniques to profile the epigenomic landscape of cells recovered from cryopreservation biorepositories.

Abstract 5729: Imiqualines for pancreatic cancer: first-in-class potent and synergistic inhibitors of microtubule polymerisation

Abstract Background: The survival rate for patients with Pancreatic Ductal Adenocarcinoma (PDAC) is dramatically poor with a five-year survival rate less than 10%. The research of new treatments, which could complement the current therapeutic arsenal constituted by Gemcitabine, FOLFIRINOX (fluorouracile, leucovorin, irinotecan, oxaliplatin) and nab-paclitaxel, is a major challenge. Imiqualines are new original small heterocyclic chemical molecules based on the quinoxalinic moiety. Among these first in class compounds, the lead EAPB02303 (1) displays outstanding nanomolar activities comparable to those of the current best anticancer agents on a panel of human cancer cell lines, notably on poorly sensitive cancer like PDAC and melanoma. We tested here if EAPB02303 could be an attractive first in class molecule in PDAC and we conducted in-deep molecular characterization and bioinformatics studies to decipher its mechanism of action. Methods: We characterized EAPB02303 effect on tumor growth in-vitro by conducting sulforhodamine B assay on a panel of PDAC cells including cells derived from PDX (Patient Derived Xenograft) and 3D models with Cancer Associated Fibroblasts. We assessed in-vivo activity on subcutaneous PDAC xenografts mouse models. We then studied EAPB02303 effect on cell cycle, apoptosis and microtubule polymerisation by flow cytometry and immunofluorescence. We analyzed mRNAseq and Reverse Phase Protein Assay (RPPA) data of PDAC cell lines treated with EAPB02303 at multiple time points and concentrations to identify signaling pathways and key proteins implicated in EAPB02303 effect. We performed differential gene expression and gene set enrichment analysis by using EdgeR, Deseq2 and fgsea packages. We also used PharmacoGx package to seek for similar transcriptomic profiles among the CMAP perturbational database. Results: We showed that EAPB02303 exerts activity at low nanomolar concentrations in-vitro in PDAC cell lines and 3D models, and is able to reduce tumor growth in our xenografts in-vivo mouse models. We also found a potent synergy with Paclitaxel at lower concentrations of both compounds. Furthermore, we found that EAPB02303 induces mitosis arrest and impairment of spindle assembly after 24h treatment. Cells also underwent apoptosis after 48h treatment. mRNAseq and RPPA data showed activation of several signaling pathways including MAPK kinases. CMAP database mining revealed a high connectivity score of transcriptomic signatures between EAPB02303 and inhibitors of microtubule polymerization. All these data suggest that EAPB02303 is a new microtubule-disrupting agent with in-vivo activity in PDAC and in-vitro synergy with Paclitaxel, showing potential for future clinical investigations. (1) Imidazo[1,2a]quinoxalines and derivatives thereof for treating cancers. WO 2009 043934A1. Deleuze-Masqeufa C. et al. Citation Format: Kevin Bigot, Véronique Garambois, Nadia Vie, Marine Bruciamacchie, Pierre-Emmanuel Colombo, Diego Tosi, Cindy Patinote, Yann Maggipinto, Pierre-Antoine Bonnet, Céline Gongora, Carine Deleuze-Masquefa, Christel Larbouret. Imiqualines for pancreatic cancer: first-in-class potent and synergistic inhibitors of microtubule polymerisation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5729.