Impacts of Subchronic and Mild Social Defeat Stress on Plasma Putrefactive Metabolites and Cardiovascular Structure in Male Mice.

Psychosocial stress precipitates mental illnesses, such as depression, and increases the risk of other health problems, including cardiovascular diseases. In this study, we observed the effects of psychosocial stress on the histopathological features of systemic organs and tissues in a mouse psychosocial stress model, namely the subchronic and mild social defeat stress (sCSDS) model. There were several pathological findings in the tissues of both sCSDS and control mice. Mild fibrosis of the heart was observed in sCSDS mice but not in control mice. Extramedullary hematopoiesis in the spleen and hemorrhage in the lungs were observed in both the control and sCSDS mice. Focal necrosis of the liver was seen only in control mice. Furthermore, putrefactive substances in the blood plasma were analyzed because these metabolites originating from intestinal fermentation might be linked to heart fibrosis. Among them, plasma p-cresyl glucuronide and p-cresyl sulfate concentrations significantly increased owing to subchronic social defeat stress, which might influence cardiac fibrosis in sCSDS mice. In conclusion, several pathological features such as increased cardiac fibrosis and elevated plasma putrefactive substances were found in sCSDS mice. Thus, sCSDS mice are a potential model for elucidating the pathophysiology of psychosocial stress and heart failure.

Comparative environmental RNA and DNA metabarcoding analysis of river algae and arthropods for ecological surveys and water quality assessment.

Environmental DNA (eDNA) metabarcoding is widely used for species analysis, while the use of environmental RNA (eRNA) metabarcoding is more limited. We conducted comparative eDNA/eRNA metabarcoding of the algae and arthropods (aquatic insects) in water samples from Naka River, Japan, to evaluate their potential for biological monitoring and water quality assessment. Both methods detected various algae and arthropod species; however, their compositions were remarkably different from those in traditional field surveys (TFSs), indicating low sensitivity. For algae, the species composition derived from eDNA and eRNA metabarcoding was equivalent. While TFSs focus on attached algae, metabarcoding analysis theoretically detects both planktonic and attached algae. A recently expanded genomic database for aquatic insects significantly contributed to the sensitivity and positive predictivity for arthropods. While the sensitivity of eRNA was lower than that of eDNA, the positive predictivity of eRNA was higher. The eRNA of terrestrial arthropods indicated extremely high or low read numbers when compared with eDNA, suggesting that eRNA could be an effective indicator of false positives. Arthropod and algae eDNA/eRNA metabarcoding analysis enabled water quality estimates from TFSs. The eRNA of algae and arthropods could thus be used to evaluate biodiversity and water quality and provide insights from ecological surveys.

RAID: Regression Analysis-Based Inductive DNA Microarray for Precise Read-Across.

Chemical structure-based read-across represents a promising method for chemical toxicity evaluation without the need for animal testing; however, a chemical structure is not necessarily related to toxicity. Therefore, in vitro studies were often used for read-across reliability refinement; however, their external validity has been hindered by the gap between in vitro and in vivo conditions. Thus, we developed a virtual DNA microarray, regression analysis-based inductive DNA microarray (RAID), which quantitatively predicts in vivo gene expression profiles based on the chemical structure and/or in vitro transcriptome data. For each gene, elastic-net models were constructed using chemical descriptors and in vitro transcriptome data to predict in vivo data from in vitro data (in vitro to in vivo extrapolation; IVIVE). In feature selection, useful genes for assessing the quantitative structure-activity relationship (QSAR) and IVIVE were identified. Predicted transcriptome data derived from the RAID system reflected the in vivo gene expression profiles of characteristic hepatotoxic substances. Moreover, gene ontology and pathway analysis indicated that nuclear receptor-mediated xenobiotic response and metabolic activation are related to these gene expressions. The identified IVIVE-related genes were associated with fatty acid, xenobiotic, and drug metabolisms, indicating that in vitro studies were effective in evaluating these key events. Furthermore, validation studies revealed that chemical substances associated with these key events could be detected as hepatotoxic biosimilar substances. These results indicated that the RAID system could represent an alternative screening test for a repeated-dose toxicity test and toxicogenomics analyses. Our technology provides a critical solution for IVIVE-based read-across by considering the mode of action and chemical structures.

In silico systems for predicting chemical-induced side effects using known and potential chemical protein interactions, enabling mechanism estimation.

In silico models for predicting chemical-induced side effects have become increasingly important for the development of pharmaceuticals and functional food products. However, existing predictive models have difficulty in estimating the mechanisms of side effects in terms of molecular targets or they do not cover the wide range of pharmacological targets. In the present study, we constructed novel in silico models to predict chemical-induced side effects and estimate the underlying mechanisms with high general versatility by integrating the comprehensive prediction of potential chemical-protein interactions (CPIs) with machine learning. First, the potential CPIs were comprehensively estimated by chemometrics based on the known CPI data (1,179,848 interactions involving 3,905 proteins and 824,143 chemicals). Second, the predictive models for 61 side effects in the cardiovascular system (CVS), gastrointestinal system (GIS), and central nervous system (CNS) were constructed by sparsity-induced classifiers based on the known and potential CPI data. The cross validation experiments showed that the proposed CPI-based models had a higher or comparable performance than the traditional chemical structure-based models. Moreover, our enrichment analysis indicated that the highly weighted proteins derived from predictive models could be involved in the corresponding functions of the side effects. For example, in CVS, the carcinogenesis-related pathways (e.g., prostate cancer, PI3K-Akt signal pathway), which were recently reported to be involved in cardiovascular side effects, were enriched. Therefore, our predictive models are biologically valid and would be useful for predicting side effects and novel potential underlying mechanisms of chemical-induced side effects.

Safety Pharmacological Evaluation of the Coffee Component, Caffeoylquinic Acid, and Its Metabolites, Using Ex Vivo and In Vitro Profiling Assays.

Although coffee components have gained interest for use as pharmaceuticals, little is known about their safety pharmacological effects. Hence, we aimed to evaluate the safety pharmacological effects of a chlorogenic acid (CGA)-related compound contained in coffee, 5-O-caffeoylquinic acid (5-CQA), and its metabolites, 5-O-feruloylquinic acid (5-FQA), caffeic acid (CA), and ferulic acid (FA). Langendorff perfused heart assay, electrophysiological assay of acute rat hippocampal slices, and in vitro Magnus assay of gastrointestinal tracts were conducted at 1-100 µM. Moreover, in vitro profiling assays against 38 major targets were conducted. In the Langendorff assay, no significant adverse effects were observed. In the electrophysiological assay, although epileptiform discharge rates were increased at 10 µM CA with 4-aminopyridine, and area under the curve (AUC) and number of population spike were increased at 10 µM FA with bicuculline, dose dependency was not confirmed, and no significant changes were observed at 1 µM and by CGAs alone. In the Magnus assay, a slight increase in contraction activity was observed at >1 µM FA in the stomach fundi and 100 µM 5-CQA in the ileum, suggesting enterokinesis promotion. No significant interactions were observed in the in vitro profiling assays. Therefore, CGAs could have a fundamental function as safe pharmaceuticals.

Three-dimensional cultured tissue constructs that imitate human living tissue organization for analysis of tumor cell invasion.

Preventing cancer metastasis requires a thorough understanding of cancer cell invasion. These phenomena occur in human 3-D living tissues. To this end, we developed a human cell-based three-dimensional (3-D) cultured tissue constructs that imitate in vivo human tissue organization. We investigated whether our 3-D cell culture system can be used to analyze the invasion of human oral squamous cell carcinoma (OSCC) cells. The 3-D tissue structure consisted of five layers of normal human dermal fibroblasts along with human dermal lymphatic endothelial cell tubes and was generated by the cell accumulation technique and layer-by-layer assembly using fibronectin and gelatin. OSCC cells with different lymph metastatic capacity were inoculated on the 3-D tissues and their invasion through the 3-D tissue structure was observed. Conventional methods of analyzing cell migration and invasion, that is, 2-D culture-based transwell and Matrigel assays were also used for comparison. The results using the 3-D cultured tissue constructs were comparable to those obtained using conventional assays; moreover, use of the 3-D system enabled visualization of differential invasion capacities of cancer cells. These results indicate that our 3-D cultured tissue constructs can be a useful tool for analysis of cancer cell invasion in a setting that reflects the in vivo tissue organization. © 2018 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 292-300, 2019.

Development of vascularized iPSC derived 3D-cardiomyocyte tissues by filtration Layer-by-Layer technique and their application for pharmaceutical assays.

In vitro development of three-dimensional (3D) human cardiomyocyte (CM) tissues derived from human induced pluripotent stem cells (iPSCs) has long been desired in tissue regeneration and pharmaceutical assays. In particular, in vitro construction of 3D-iPSC-CM tissues with blood capillary networks have attracted much attention because blood capillaries are crucial for nutrient and oxygen supplies for CMs. Blood capillaries in 3D-iPSC-CM tissues will also be important for in vitro toxicity assay of prodrugs because of the signaling interaction between cardiomyocytes and endothelial cells. Here, we report construction of vascularized 3D-iPSC-CM tissues by a newly-discovered filtration-Layer-by-Layer (LbL) technique for cells, instead of our previous centrifugation-LbL technique. The filtration-LbL allowed us to fabricate nanometer-sized extracellular matrices (ECM), fibronectin and gelatin (FN-G), films onto iPSC-CM surfaces without any damage and with high yield, although centrifugation-LbL induced physical stress and a lower yield. The fabricated FN-G nanofilms interacted with integrin molecules on the cell membrane to construct 3D-tissues. We found that the introduction of normal human cardiac fibroblasts (NHCFs) into the iPSC-CM tissues modulated organization and synchronous beating depending on NHCF ratios. Moreover, co-culture with normal human cardiac microvascular endothelial cells (NHCMECs) successfully provided blood capillary-like networks in 3D-iPSC-CM tissues, depending on NHCF ratios. The vascularized 3D-iPSC-CM tissues indicated significantly different toxicity responses as compared to 2D-iPSC-CM cells by addition of doxorubicin as a model of a toxic drug. The constructed vascularized 3D-iPSC-CM tissues would be a promising tool for tissue regeneration and drug development. STATEMENT OF SIGNIFICANCE: In vitro fabrication of vascularized three-dimensional (3D) human cardiomyocyte (CM) tissues derived from human induced pluripotent stem cells (iPSCs) has attracted much attention owing to their requirement of much amount of nutrition and oxygen, but not yet published. In this manuscript, we report construction of vascularized 3D-iPSC-CM tissues by a newly-discovered filtration-Layer-by-Layer (LbL) technique. The filtration-LbL fabricates nanometer-sized fibronectin and gelatin (FN-G) films onto iPSC-CM surfaces. The FN-G nanofilms induce cell-cell interactions via integrin molecules on cell surfaces, leading to construction of 3D-tissues. The constructed vascularized 3D-iPSC-CM tissues would be a promising tool for tissue regeneration and drug development. We believe that this manuscript has a strong impact and offers important suggestions to researchers concerned with biomaterials and tissue engineering.