A comparison of anatomic and cellular transcriptome structures across 40 human brain diseases.

Genes associated with risk for brain disease exhibit characteristic expression patterns that reflect both anatomical and cell type relationships. Brain-wide transcriptomic patterns of disease risk genes provide a molecular-based signature, based on differential co-expression, that is often unique to that disease. Brain diseases can be compared and aggregated based on the similarity of their signatures which often associates diseases from diverse phenotypic classes. Analysis of 40 common human brain diseases identifies 5 major transcriptional patterns, representing tumor-related, neurodegenerative, psychiatric and substance abuse, and 2 mixed groups of diseases affecting basal ganglia and hypothalamus. Further, for diseases with enriched expression in cortex, single-nucleus data in the middle temporal gyrus (MTG) exhibits a cell type expression gradient separating neurodegenerative, psychiatric, and substance abuse diseases, with unique excitatory cell type expression differentiating psychiatric diseases. Through mapping of homologous cell types between mouse and human, most disease risk genes are found to act in common cell types, while having species-specific expression in those types and preserving similar phenotypic classification within species. These results describe structural and cellular transcriptomic relationships of disease risk genes in the adult brain and provide a molecular-based strategy for classifying and comparing diseases, potentially identifying novel disease relationships.

Molecular divergence of mammalian astrocyte progenitor cells at early gliogenesis.

During mammalian brain development, how different astrocytes are specified from progenitor cells is not well understood. In particular, whether astrocyte progenitor cells (APCs) start as a relatively homogenous population or whether there is early heterogeneity remains unclear. Here, we have dissected subpopulations of embryonic mouse forebrain progenitors using single-cell transcriptome analyses. Our sequencing data revealed two molecularly distinct APC subgroups at the start of gliogenesis from both dorsal and ventral forebrains. The two APC subgroups were marked, respectively, by specific expression of Sparc and Sparcl1, which are known to function in mature astrocytes with opposing activities for regulating synapse formation. Expression analyses showed that SPARC and SPARCL1 mark APC subgroups that display distinct temporal and spatial patterns, correlating with major waves of astrogliogenesis during development. Our results uncover an early molecular divergence of APCs in the mammalian brain and provide a useful transcriptome resource for the study of glial cell specification.

Vitamin D and the risk for cancer: A molecular analysis.

Uncontrolled overgrowth of cells, such as in cancer, is an unavoidable risk in life that affects nearly every second individual in industrialized countries. However, in part this risk can be controlled through lifestyle adjustments, such as the avoidance of smoking, unhealthy diet, obesity, physical inactivity and other cancer risk factors. A low vitaminD status is a risk in particular for cancers of colon, prostate, breast and leukocytes. VitaminD3 is produced non-enzymatically, when the cholesterol precursor 7-dehydrocholesterol is exposed to UV-B from sunlight, i.e., all cholesterol synthesizing species, including humans, can make vitaminD3. VitaminD endocrinology started some 550million years ago, when the metabolite 1α,25-dihydroxyvitaminD3 and the transcription factor vitaminD receptor teamed up for regulating the expression of hundreds of target genes in a multitude of different tissues and cell types. Initially, these genes were focused on the control of energy homeostasis, which later also involved energy-demanding innate and adaptive immunity. Rapidly growing cells of the immune system as well as those of malignant tumors rely on comparable genes and pathways, some of which are modulated by vitaminD. Accordingly, vitaminD has anti-cancer effects both directly via controling the differentiation, proliferation and apoptosis of neoplastic cells as well as indirectly through regulating immune cells that belong to the microenvironment of malignant tumors. This review discusses effects of vitaminD on the epigenome and transcriptome of stromal and tumor cells, inter-individual variations in vitaminD responsiveness and their relation to the prevention and possible therapy of cancer.

Targeted RNA Sequencing of VZV-Infected Brain Vascular Adventitial Fibroblasts Indicates That Amyloid May Be Involved in VZV Vasculopathy.

BACKGROUND AND OBJECTIVES: Compared with stroke controls, patients with varicella zoster virus (VZV) vasculopathy have increased amyloid in CSF, along with increased amylin (islet amyloid polypeptide [IAPP]) and anti-VZV antibodies. Thus, we examined the gene expression profiles of VZV-infected primary human brain vascular adventitial fibroblasts (HBVAFs), one of the initial arterial cells infected in VZV vasculopathy, to determine whether they are a potential source of amyloid that can disrupt vasculature and potentiate inflammation. METHODS: Mock- and VZV-infected quiescent HBVAFs were harvested at 3 days postinfection. Targeted RNA sequencing of the whole-human transcriptome (BioSpyder Technologies, TempO-Seq) was conducted followed by gene set enrichment and pathway analysis. Selected pathways unique to VZV-infected cells were confirmed by enzyme-linked immunoassays, migration assays, and immunofluorescence analysis (IFA) that included antibodies against amylin and amyloid-beta, as well as amyloid staining by Thioflavin-T. RESULTS: Compared with mock, VZV-infected HBVAFs had significantly enriched gene expression pathways involved in vascular remodeling and vascular diseases; confirmatory studies showed secretion of matrix metalloproteinase-3 and -10, as well increased migration of infected cells and uninfected cells when exposed to conditioned media from VZV-infected cells. In addition, significantly enriched pathways involved in amyloid-associated diseases (diabetes mellitus, amyloidosis, and Alzheimer disease), tauopathy, and progressive neurologic disorder were identified; predicted upstream regulators included amyloid precursor protein, apolipoprotein E, microtubule-associated protein tau, presenilin 1, and IAPP. Confirmatory IFA showed that VZV-infected HBVAFs contained amyloidogenic peptides (amyloid-beta and amylin) and intracellular amyloid. DISCUSSION: Gene expression profiles and pathway enrichment analysis of VZV-infected HBVAFs, as well as phenotypic studies, reveal features of pathologic vascular remodeling (e.g., increased cell migration and changes in the extracellular matrix) that can contribute to cerebrovascular disease. Furthermore, the discovery of amyloid-associated transcriptional pathways and intracellular amyloid deposition in HBVAFs raise the possibility that VZV vasculopathy is an amyloid disease. Amyloid deposition may contribute to cell death and loss of vascular wall integrity, as well as potentiate chronic inflammation in VZV vasculopathy, with disease severity and recurrence determined by the host's ability to clear virus infection and amyloid deposition and by the coexistence of other amyloid-associated diseases (i.e., Alzheimer disease and diabetes mellitus).

Radially Aligned Porous Silk Fibroin Scaffolds as Functional Templates for Engineering Human Biomimetic Hepatic Lobules.

Bioengineering functional hepatic tissue constructs that physiologically replicate the human native liver tissue in vitro is sought for clinical research and drug discovery. However, the intricate architecture and specific biofunctionality possessed by the native liver tissue remain challenging to mimic in vitro. In the present study, a versatile strategy to fabricate lobular-like silk protein scaffolds with radially aligned lamellar sheets, interconnected channels, and a converging central cavity was designed and implemented. A proof-of-concept study to bioengineer biomimetic hepatic lobules was conducted through coculturing human hepatocytes and primary endothelial cells on these lobular-like scaffolds. Relatively long-term viability of hepatocyte/endothelial cells was found along with cell alignment and organization in vitro. The hepatocytes showed special epithelial polarity and bile duct formation, similar to the liver plate, while the aligned endothelial cells generated endothelial networks, similar to natural hepatic sinuses. This endowed the three-dimensional (3D) tissue constructs with the capability to recapitulate hepatic-like parenchymal-mesenchymal growth patterns in vitro. More importantly, the cocultured hepatocytes outperformed monocultures or monolayer cultures, displaying significantly enhanced hepatocyte functions, including functional gene expression, albumin (ALB) secretion, urea synthesis, and metabolic activity. Thus, this functional unit with a biomimetic phenotype provides a novel technology for bioengineering biomimetic hepatic lobules in vitro, with potential utility as a building block for bioartificial liver (BAL) engineering or as a robust tool for drug metabolism investigation.

Unique Transcriptomic Changes Underlie Hormonal Interactions During Mammary Histomorphogenesis in Female Pigs.

Successful lactation and the risk for developing breast cancer depend on growth and differentiation of the mammary gland (MG) epithelium that is regulated by ovarian steroids (17ß-estradiol [E] and progesterone [P]) and pituitary-derived prolactin (PRL). Given that the MG of pigs share histomorphogenic features present in the normal human breast, we sought to define the transcriptional responses within the MG of pigs following exposure to all combinations of these hormones. Hormone-ablated female pigs were administered combinations of E, medroxyprogesterone 17-acetate (source of P), and either haloperidol (to induce PRL) or 2-bromo-α-ergocryptine. We subsequently monitored phenotypic changes in the MG including mitosis, receptors for E and P (ESR1 and PGR), level of phosphorylated STAT5 (pSTAT5), and the frequency of terminal ductal lobular unit (TDLU) subtypes; these changes were then associated with all transcriptomic changes. Estrogen altered the expression of approximately 20% of all genes that were mostly associated with mitosis, whereas PRL stimulated elements of fatty acid metabolism and an inflammatory response. Several outcomes, including increased pSTAT5, highlighted the ability of E to enhance PRL action. Regression of transcriptomic changes against several MG phenotypes revealed 1669 genes correlated with proliferation, among which 29 were E inducible. Additional gene expression signatures were associated with TDLU formation and the frequency of ESR1 or PGR. These data provide a link between the hormone-regulated genome and phenome of the MG in a species having a complex histoarchitecture like that in the human breast, and highlight an underexplored synergy between the actions of E and PRL during MG development.

Systems biomarkers for papillary thyroid cancer prognosis and treatment through multi-omics networks.

The identification of biomolecules associated with papillary thyroid cancer (PTC) has upmost importance for the elucidation of the disease mechanism and the development of effective diagnostic and treatment strategies. Despite particular findings in this regard, a holistic analysis encompassing molecular data from different biological levels has been lacking. In the present study, a meta-analysis of four transcriptome datasets was performed to identify gene expression signatures in PTC, and reporter molecules were determined by mapping gene expression data onto three major cellular networks, i.e., transcriptional regulatory, protein-protein interaction, and metabolic networks. We identified 282 common genes that were differentially expressed in all PTC datasets. In addition, six proteins (FYN, JUN, LYN, PML, SIN3A, and RARA), two Erb-B2 receptors (ERBB2 and ERBB4), two cyclin-dependent receptors (CDK1 and CDK2), and three histone deacetylase receptors (HDAC1, HDAC2, and HDAC3) came into prominence as proteomic signatures in addition to several metabolites including lactaldehyde and proline at the metabolome level. Significant associations with calcium and MAPK signaling pathways and transcriptional and post-transcriptional activities of 12 TFs and 110 miRNAs were also observed at the regulatory level. Among them, six miRNAs (miR-30b-3p, miR-15b-5p, let-7a-5p, miR-130b-3p, miR-424-5p, and miR-193b-3p) were associated with PTC for the first time in the literature, and the expression levels of miR-30b-3p, miR-15b-5p, and let-7a-5p were found to be predictive of disease prognosis. Drug repositioning and molecular docking simulations revealed that 5 drugs (prochlorperazine, meclizine, rottlerin, cephaeline, and tretinoin) may be useful in the treatment of PTC. Consequently, we report here biomolecule candidates that may be considered as prognostic biomarkers or potential therapeutic targets for further experimental and clinical trials for PTC.

Stress Responses as Master Keys to Epigenomic Changes in Transcriptome and Metabolome for Cancer Etiology and Therapeutics.

From initiation through progression, cancer cells are subjected to a magnitude of endogenous and exogenous stresses, which aid in their neoplastic transformation. Exposure to these classes of stress induces imbalance in cellular homeostasis and, in response, cancer cells employ informative adaptive mechanisms to rebalance biochemical processes that facilitate survival and maintain their existence. Different kinds of stress stimuli trigger epigenetic alterations in cancer cells, which leads to changes in their transcriptome and metabolome, ultimately resulting in suppression of growth inhibition or induction of apoptosis. Whether cancer cells show a protective response to stress or succumb to cell death depends on the type of stress and duration of exposure. A thorough understanding of epigenetic and molecular architecture of cancer cell stress response pathways can unveil a plethora of information required to develop novel anticancer therapeutics. The present view highlights current knowledge about alterations in epigenome and transcriptome of cancer cells as a consequence of exposure to different physicochemical stressful stimuli such as reactive oxygen species (ROS), hypoxia, radiation, hyperthermia, genotoxic agents, and nutrient deprivation. Currently, an anticancer treatment scenario involving the imposition of stress to target cancer cells is gaining traction to augment or even replace conventional therapeutic regimens. Therefore, a comprehensive understanding of stress response pathways is crucial for devising and implementing novel therapeutic strategies.

Modulation of gene expression on a transcriptome-wide level following human neural stem cell transplantation in aged mouse stroke brains.

INTRODUCTION: Neural stem cell (NSC) transplantation offers great potential for treating ischemic stroke. Clinically, ischemia followed by reperfusion results in robust cerebrovascular injury that upregulates proinflammatory factors, disrupts neurovascular units, and causes brain cell death. NSCs possess multiple actions that can be exploited for reducing the severity of neurovascular injury. Our previous studies in young adult mice showed that human NSC transplantation during the subacute stage diminishes stroke pathophysiology and improves behavioral outcome. METHODS: We employed a well-established and commonly used stroke model, middle cerebral artery occlusion with subsequent reperfusion (MCAO/R). Here, we assessed the outcomes of hNSC transplantation 48 h post-MCAO (24 h post-transplant) in aged mouse brains in response to stroke because aging is a crucial risk factor for cerebral ischemia. Next, we tested whether administration of the integrin α5ß1 inhibitor, ATN-161, prior to hNSC transplantation further affects stoke outcome as compared with NSCs alone. RNA sequencing (RNA-seq) was used to assess the impact of hNSC transplantation on differentially expressed genes (DEGs) on a transcriptome-wide level. RESULTS: Here, we report that hNSC-engrafted brains with or without ATN-161 showed significantly reduced infarct size, and attenuated the induction of proinflammatory factors and matrix metalloproteases. RNA-seq analysis revealed DEGs and molecular pathways by which hNSCs induce a beneficial post-stroke outcome in aged stroke brains. 811 genes were differentially expressed (651 downregulated and 160 upregulated) in hNSC-engrafted stroke brains. Functional pathway analysis identified enriched and depleted pathways in hNSC-engrafted aged mouse stroke brains. Depletion of pathways following hNSC-engraftment included signaling involving neuroinflammation, acute phase response, leukocyte extravasation, and phagosome formation. On the other hand, enrichment of pathways in hNSC-engrafted brains was associated with PPAR signaling, LXR/RXR activation, and inhibition of matrix metalloproteases. Hierarchical cluster analysis of DEGs in hNSC-engrafted brains indicate decreased expression of genes encoding TNF receptors, proinflammatory factors, apoptosis factors, adhesion and leukocyte extravasation, and Toll-like receptors. CONCLUSIONS: Our study is the first to show global transcripts differentially expressed following hNSC transplantation in the subacute phase of stroke in aged mice. The outcome of our transcriptome study would be useful to develop new therapies ameliorating early-stage stroke injury.

An airway organoid-based screen identifies a role for the HIF1α-glycolysis axis in SARS-CoV-2 infection.

It is urgent to develop disease models to dissect mechanisms regulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we derive airway organoids from human pluripotent stem cells (hPSC-AOs). The hPSC-AOs, particularly ciliated-like cells, are permissive to SARS-CoV-2 infection. Using this platform, we perform a high content screen and identify GW6471, which blocks SARS-CoV-2 infection. GW6471 can also block infection of the B.1.351 SARS-CoV-2 variant. RNA sequencing (RNA-seq) analysis suggests that GW6471 blocks SARS-CoV-2 infection at least in part by inhibiting hypoxia inducible factor 1 subunit alpha (HIF1α), which is further validated by chemical inhibitor and genetic perturbation targeting HIF1α. Metabolic profiling identifies decreased rates of glycolysis upon GW6471 treatment, consistent with transcriptome profiling. Finally, xanthohumol, 5-(tetradecyloxy)-2-furoic acid, and ND-646, three compounds that suppress fatty acid biosynthesis, also block SARS-CoV-2 infection. Together, a high content screen coupled with transcriptome and metabolic profiling reveals a key role of the HIF1α-glycolysis axis in mediating SARS-CoV-2 infection of human airway epithelium.

Deciphering mouse uterine receptivity for embryo implantation at single-cell resolution.

OBJECTIVES: Mice are widely used as an animal model for studying human uterine receptivity for embryo implantation. Although transcriptional changes related to mouse uterine receptivity have been determined by using bulk RNA-seq, the data are of limited value because the uterus is a complex organ consisting of many cell types. Here, we aimed to decipher mouse uterine receptivity for embryo implantation at single-cell resolution. MATERIALS AND METHODS: Single-cell RNA sequencing was performed for the pre-receptive and the receptive mouse uterus. Gene expression profiles in luminal epithelium and glandular epithelium were validated by comparing against a published laser capture microdissection (LCM)-coupled microarray dataset. RESULTS: We revealed 19 distinct cell clusters, including 3 stromal cell clusters, 2 epithelial cell clusters, 1 smooth muscle cell cluster, 4 endothelial cell clusters and 8 immune cell clusters. We identified global gene expression changes associated with uterine receptivity in each cell type. Additionally, we predicted signalling interactions for distinct cell types to understand the crosstalk between the blastocyst and the receptive uterus. CONCLUSION: Our data provide a valuable resource for deciphering the molecular mechanism underlying uterine receptivity in mice.

A targeted transcriptomics approach for the determination of mixture effects of pesticides.

While real-life exposure occurs to complex chemical mixtures, toxicological risk assessment mostly focuses on individual compounds. There is an increasing demand for in vitro tools and strategies for mixture toxicity analysis. Based on a previously established set of hepatotoxicity marker genes, we analyzed mixture effects of non-cytotoxic concentrations of different pesticides in exposure-relevant binary mixtures in human HepaRG hepatocarcinoma cells using targeted transcriptomics. An approach for mixture analysis at the level of a complex endpoint such as a transcript pattern is presented, including mixture design based on relative transcriptomic potencies and similarities. From a mechanistic point of view, goal of the study was to evaluate combinations of chemicals with varying degrees of similarity in order to determine whether differences in mechanisms of action lead to different mixtures effects. Using a model deviation ratio-based approach for assessing mixture effects, it was revealed that most data points are consistent with the assumption of dose addition. A tendency for synergistic effects was only observed at high concentrations of some combinations of the test compounds azoxystrobin, cyproconazole, difenoconazole, propiconazole and thiacloprid, which may not be representative of human real-life exposure. In summary, the findings of our study suggest that, for the pesticide mixtures investigated, risk assessment based on the general assumption of dose addition can be considered sufficiently protective for consumers. The way of data analysis presented in this paper can pave the way for a more comprehensive use of multi-gene expression data in experimental studies related to mixture toxicity.

Molecular characteristics and spatial distribution of adult human corneal cell subtypes.

Bulk RNA sequencing of a tissue captures the gene expression profile from all cell types combined. Single-cell RNA sequencing identifies discrete cell-signatures based on transcriptomic identities. Six adult human corneas were processed for single-cell RNAseq and 16 cell clusters were bioinformatically identified. Based on their transcriptomic signatures and RNAscope results using representative cluster marker genes on human cornea cross-sections, these clusters were confirmed to be stromal keratocytes, endothelium, several subtypes of corneal epithelium, conjunctival epithelium, and supportive cells in the limbal stem cell niche. The complexity of the epithelial cell layer was captured by eight distinct corneal clusters and three conjunctival clusters. These were further characterized by enriched biological pathways and molecular characteristics which revealed novel groupings related to development, function, and location within the epithelial layer. Moreover, epithelial subtypes were found to reflect their initial generation in the limbal region, differentiation, and migration through to mature epithelial cells. The single-cell map of the human cornea deepens the knowledge of the cellular subsets of the cornea on a whole genome transcriptional level. This information can be applied to better understand normal corneal biology, serve as a reference to understand corneal disease pathology, and provide potential insights into therapeutic approaches.

Trophoblast derived extracellular vesicles specifically alter the transcriptome of endometrial cells and may constitute a critical component of embryo-maternal communication.

BACKGROUND: The period of time when the embryo and the endometrium undergo significant morphological alterations to facilitate a successful implantation-known as "window of implantation"-is a critical moment in human reproduction. Embryo and the endometrium communicate extensively during this period, and lipid bilayer bound nanoscale extracellular vesicles (EVs) are purported to be integral to this communication. METHODS: To investigate the nature of the EV-mediated embryo-maternal communication, we have supplemented trophoblast analogue spheroid (JAr) derived EVs to an endometrial analogue (RL 95-2) cell layer and characterized the transcriptomic alterations using RNA sequencing. EVs derived from non-trophoblast cells (HEK293) were used as a negative control. The cargo of the EVs were also investigated through mRNA and miRNA sequencing. RESULTS: Trophoblast spheroid derived EVs induced drastic transcriptomic alterations in the endometrial cells while the non-trophoblast cell derived EVs failed to induce such changes demonstrating functional specificity in terms of EV origin. Through gene set enrichment analysis (GSEA), we found that the response in endometrial cells was focused on extracellular matrix remodelling and G protein-coupled receptors' signalling, both of which are of known functional relevance to endometrial receptivity. Approximately 9% of genes downregulated in endometrial cells were high-confidence predicted targets of miRNAs detected exclusively in trophoblast analogue-derived EVs, suggesting that only a small proportion of reduced expression in endometrial cells can be attributed directly to gene silencing by miRNAs carried as cargo in the EVs. CONCLUSION: Our study reveals that trophoblast derived EVs have the ability to modify the endometrial gene expression, potentially with functional importance for embryo-maternal communication during implantation, although the exact underlying signalling mechanisms remain to be elucidated.

Pro- and anti-inflammatory macrophages express a sub-type specific purinergic receptor profile.

Extracellular nucleotides act as danger signals that orchestrate inflammation by purinergic receptor activation. The expression pattern of different purinergic receptors may correlate with a pro- or anti-inflammatory phenotype. Macrophages function as pro-inflammatory M1 macrophages (M1) or anti-inflammatory M2 macrophages (M2). The present study found that murine bone marrow-derived macrophages express a unique purinergic receptor profile during in vitro polarization. As assessed by real-time polymerase chain reaction (PCR), Gαs-coupled P1 receptors A2A and A2B are upregulated in M1 and M2 compared to M0, but A2A 15 times higher in M1. The ionotropic P2 receptor P2X5 is selectively upregulated in M1- and M2-polarized macrophages. P2X7 is temporarily expressed in M1 macrophages. Metabotropic P2Y receptors showed a distinct expression profile in M1 and M2-polarized macrophages: Gαq coupled P2Y1 and P2Y6 are exclusively upregulated in M2, whereas Gαi P2Y13 and P2Y14 are overexpressed in M1. This consequently leads to functional differences between M1 and M2 in response to adenosine di-phosphate stimulation (ADP): In contrast to M1, M2 showed increased cytoplasmatic calcium after ADP stimulation. In the present study we show that bone marrow-derived macrophages express a unique repertoire of purinergic receptors. We show for the first time that the repertoire of purinergic receptors is highly flexible and quickly adapts upon pro- and anti-inflammatory macrophage differentiation with functional consequences to nucleotide stimulation.

Proteomic Signature of Dysfunctional Circulating Endothelial Colony-Forming Cells of Young Adults.

Background A subpopulation of endothelial progenitor cells called endothelial colony-forming cells (ECFCs) may offer a platform for cellular assessment in clinical studies because of their remarkable angiogenic and expansion potentials in vitro. Despite endothelial cell function being influenced by cardiovascular risk factors, no studies have yet provided a comprehensive proteomic profile to distinguish functional (ie, more angiogenic and expansive cells) versus dysfunctional circulating ECFCs of young adults. The aim of this study was to provide a detailed proteomic comparison between functional and dysfunctional ECFCs. Methods and Results Peripheral blood ECFCs were isolated from 11 subjects (45% men, aged 27±5 years) using Ficoll density gradient centrifugation. ECFCs expressed endothelial and progenitor surface markers and displayed cobblestone-patterned morphology with clonal and angiogenic capacities in vitro. ECFCs were deemed dysfunctional if <1 closed tube formed during the in vitro tube formation assay and proliferation rate was <20%. Hierarchical functional clustering revealed distinct ECFC proteomic signatures between functional and dysfunctional ECFCs with changes in cellular mechanisms involved in exocytosis, vesicle transport, extracellular matrix organization, cell metabolism, and apoptosis. Targeted antiangiogenic proteins in dysfunctional ECFCs included SPARC (secreted protein acidic and rich in cysteine), CD36 (cluster of differentiation 36), LUM (lumican), and PTX3 (pentraxin-related protein PYX3). Conclusions Circulating ECFCs with impaired angiogenesis and expansion capacities have a distinct proteomic profile and significant phenotype changes compared with highly angiogenic endothelial cells. Impaired angiogenesis in dysfunctional ECFCs may underlie the link between endothelial dysfunction and cardiovascular disease risks in young adults.

Sex-dependent dynamics of metabolism in primary mouse hepatocytes.

The liver is one of the most sexually dimorphic organs. The hepatic metabolic pathways that are subject to sexual dimorphism include xenobiotic, amino acid and lipid metabolism. Non-alcoholic fatty liver disease and hepatocellular carcinoma are among diseases with sex-dependent prevalence, progression and outcome. Although male and female livers differ in their abilities to metabolize foreign compounds, including drugs, sex-dependent treatment and pharmacological dynamics are rarely applied in all relevant cases. Therefore, it is important to consider hepatic sexual dimorphism when developing new treatment strategies and to understand the underlying mechanisms in model systems. We isolated primary hepatocytes from male and female C57BL6/N mice and examined the sex-dependent transcriptome, proteome and extracellular metabolome parameters in the course of culturing them for 96 h. The sex-specific gene expression of the general xenobiotic pathway altered and the female-specific expression of Cyp2b13 and Cyp2b9 was significantly reduced during culture. Sex-dependent differences of several signaling pathways increased, including genes related to serotonin and melatonin degradation. Furthermore, the ratios of male and female gene expression were inversed for other pathways, such as amino acid degradation, beta-oxidation, androgen signaling and hepatic steatosis. Because the primary hepatocytes were cultivated without the influence of known regulators of sexual dimorphism, these results suggest currently unknown modulatory mechanisms of sexual dimorphism in vitro. The large sex-dependent differences in the regulation and dynamics of drug metabolism observed during cultivation can have an immense influence on the evaluation of pharmacodynamic processes when conducting initial preclinical trials to investigate potential new drugs.

The potential roles of m6A modification in regulating the inflammatory response in microglia.

BACKGROUND: Microglia are key regulators of the inflammatory response in the brain. Adenosine in RNAs can be converted to m6A (N6-methyladenosine), which regulates RNA metabolism and functions as a key epitranscriptomic modification. The m6A modification pattern and m6A-related signatures under pro-inflammatory and anti-inflammatory conditions of microglia remain unclear. METHODS: Primary rat microglia were differentiated into pro-inflammatory M1-like (M1-L), anti-inflammatory M2-like (M2-L), and resting, unstimulated (M0-L) phenotypes. m6A mRNA and lncRNA epitranscriptomic microarray analyses were performed, and pathway analysis was conducted to understand the functional implications of m6A methylation in mRNAs and lncRNAs. The m6A methylation level and gene expression of mRNAs and lncRNAs were subsequently verified by m6A Me-RIP and qRT-PCR. RESULTS: A total of 1588 mRNAs and 340 lncRNAs, 315 mRNAs and 38 lncRNAs, and 521 mRNAs and 244 lncRNAs were differentially m6A methylated between M1-L and M0-L (M1-L/M0-L), M2-L and M0-L (M2-L/M0-L), M2-L and M1-L (M2-L/M1-L), respectively. Furthermore, 4902 mRNAs, 4676 mRNAs, and 5095 mRNAs were identified distinctively expressed in M1-L/M0-L, M2-L/M0-L, and M2-L/M1-L, respectively. Pathway analysis of differentially m6A methylated mRNAs and lncRNAs in M1-L/M0-L identified immune system, signal transduction, and protein degradation processes. In contrast, the distinct m6A methylated mRNAs in M2-L/M0-L were involved in genetic information processing, metabolism, cellular processes, and neurodegenerative disease-related pathways. We validated m6A methylation and the expression levels of five mRNAs and five lncRNAs, which were involved in upregulated pathways in M1-L/M0-L, and five mRNAs involved in upregulated pathways in M2-L/M0-L. CONCLUSIONS: These findings identify a distinct m6A epitranscriptome in microglia, and which may serve as novel and useful regulator during pro-inflammatory and anti-inflammatory response of microglia.

Head and Neck Cancer Cell Death due to Mitochondrial Damage Induced by Reactive Oxygen Species from Nonthermal Plasma-Activated Media: Based on Transcriptomic Analysis.

Mitochondrial targeted therapy is a next-generation therapeutic approach for cancer that is refractory to conventional treatments. Mitochondrial damage caused by the excessive accumulation of reactive oxygen species (ROS) is a principle of mitochondrial targeted therapy. ROS in nonthermal plasma-activated media (NTPAM) are known to mediate anticancer effects in various cancers including head and neck cancer (HNC). However, the signaling mechanism of HNC cell death via NTPAM-induced ROS has not been fully elucidated. This study evaluated the anticancer effects of NTPAM in HNC and investigated the mechanism using transcriptomic analysis. The viability of HNC cells decreased after NTPAM treatment due to enhanced apoptosis. A human fibroblast cell line and three HNC cell lines were profiled by RNA sequencing. In total, 1 610 differentially expressed genes were identified. Pathway analysis showed that activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) were upstream regulators. Mitochondrial damage was induced by NTPAM, which was associated with enhancements of mitochondrial ROS (mtROS) and ATF4/CHOP regulation. These results suggest that NTPAM induces HNC cell death through the upregulation of ATF4/CHOP activity by damaging mitochondria via excessive mtROS accumulation, similar to mitochondrial targeted therapy.

Experimental long-term diabetes mellitus alters the transcriptome and biomechanical properties of the rat urinary bladder.

Diabetes mellitus (DM) is the leading cause of chronic kidney disease and diabetic nephropathy is widely studied. In contrast, the pathobiology of diabetic urinary bladder disease is less understood despite dysfunctional voiding being common in DM. We hypothesised that diabetic cystopathy has a characteristic molecular signature. We therefore studied bladders of hyperglycaemic and polyuric rats with streptozotocin (STZ)-induced DM. Sixteen weeks after induction of DM, as assessed by RNA arrays, wide-ranging changes of gene expression occurred in DM bladders over and above those induced in bladders of non-hyperglycaemic rats with sucrose-induced polyuria. The altered transcripts included those coding for extracellular matrix regulators and neural molecules. Changes in key genes deregulated in DM rat bladders were also detected in db/db mouse bladders. In DM rat bladders there was reduced birefringent collagen between detrusor muscle bundles, and atomic force microscopy showed a significant reduction in tissue stiffness; neither change was found in bladders of sucrose-treated rats. Thus, altered extracellular matrix with reduced tissue rigidity may contribute to voiding dysfunction in people with long-term DM. These results serve as an informative stepping stone towards understanding the complex pathobiology of diabetic cystopathy.