Identification of marker compounds in fermented Benincasa hispida and validation of the method for its analysis.

Fermentation is a process that improves health functionality by inducing the production and increase of bioactive compounds. In this study, to standardize the fermentation process for Benincasa hispida, marker compounds that are increased or produced during fermentation were identified based on UPLC-QTOF-MS/MS. Analysis method verification and content analysis were conducted using HPLC-PDA. The marker compounds produced or increased in content were identified as 2-furoic acid, 2,3-dihydroxybenzoic acid, and rubinaphthin A by comparing their retention times, UV and MS spectra, and molecular formulas with those reported in previous studies. In addition, the increase in the content of the marker compounds by fermentation was confirmed, and the analytical method was validated by measuring its specificity, linearity, limit of detection and quantitation, precision, and accuracy. These results suggest that the developed fermentation process, marker compound identification, and verified analysis method can be applied to develop potential functional food ingredients from fermented B. hispida.

Deep learning-based optic disc classification is affected by optic-disc tilt.

We aimed to determine the effect of optic disc tilt on deep learning-based optic disc classification. A total of 2507 fundus photographs were acquired from 2236 eyes of 1809 subjects (mean age of 46 years; 53% men). Among all photographs, 1010 (40.3%) had tilted optic discs. Image annotation was performed to label pathologic changes of the optic disc (normal, glaucomatous optic disc changes, disc swelling, and disc pallor). Deep learning-based classification modeling was implemented to develop optic-disc appearance classification models with the photographs of all subjects and those with and without tilted optic discs. Regardless of deep learning algorithms, the classification models showed better overall performance when developed based on data from subjects with non-tilted discs (AUC, 0.988 ± 0.002, 0.991 ± 0.003, and 0.986 ± 0.003 for VGG16, VGG19, and DenseNet121, respectively) than when developed based on data with tilted discs (AUC, 0.924 ± 0.046, 0.928 ± 0.017, and 0.935 ± 0.008). In classification of each pathologic change, non-tilted disc models had better sensitivity and specificity than the tilted disc models. The optic disc appearance classification models developed based all-subject data demonstrated lower accuracy in patients with the appearance of tilted discs than in those with non-tilted discs. Our findings suggested the need to identify and adjust for the effect of optic disc tilt on the optic disc classification algorithm in future development.

How are moral foundations associated with empathic traits and moral identity?

We examined the relationship between moral foundations, empathic traits, and moral identity using an online survey via Mechanical Turk. In order to determine how moral foundations contribute to empathic traits and moral identity, we performed classical correlation analysis as well as Bayesian correlation analysis, Bayesian ANCOVA, and Bayesian regression analysis. Results showed that individualizing foundations (harm/care, fairness/reciprocity) and binding foundations (ingroup/loyalty, authority/respect, purity/sanctity) had various different relationships with empathic traits. In addition, the individualizing versus binding foundations showed somewhat reverse relationships with internalization and symbolization of moral identity. This suggests that moral foundations can contribute to further understanding of empathic traits and moral identity and how they relate to moral behavior in reality. We discuss the implications of these results for moral educators when starting to teach students about moral issues. Supplementary Information: The online version contains supplementary material available at 10.1007/s12144-021-02372-5.

Melanocortin-4 receptors activate sympathetic preganglionic neurons and elevate blood pressure via TRPV1.

Melanocortin-4 receptors (MC4Rs) expressed by the central nervous system are essential regulators of energy homeostasis, and Mc4r mutation is the most common cause of human monogenic obesity. Notably, patients with obesity carrying Mc4r mutations are protected against obesity-induced hypertension, and MC4R agonists elevate blood pressure (BP). Although increased sympathetic tone by MC4Rs is suggested to underlie this phenotype, the detailed mechanisms remain unclear. Here, we investigate how MC4Rs regulate the sympathetic preganglionic neurons and find that MC4Rs activate these neurons via the protein kinase A-dependent activation of the transient receptor potential vanilloid 1 (TRPV1) channel. Importantly, we demonstrate that the inhibition of TRPV1 prevents MC4R-induced elevation of BP but does not affect MC4R-induced anorexia. We further show that TRPV1 is responsible for MC4R-dependent activation of the sympathetic preganglionic neurons by high-fat diet. Together, our results provide insight into how MC4Rs regulate sympathetic function.

Environmental obesogens (bisphenols, phthalates and parabens) and their impacts on adipogenic transcription factors in the absence of dexamethasone in 3T3-L1 cells.

Endocrine-disrupting chemicals (EDCs) are exogenous compounds that are capable of blocking or mimicking the action of bioidentical hormones. Obesogenic EDCs, commonly called obesogens, play an important role in adipogenesis. This study was carried out to determine the effects of select obesogens and their alternatives on adipogenesis in 3T3-L1 cells under dexamethasone (DEX)-free conditions. Preadipocytes were treated with a cocktail of 3-isobutyl-1-methylxanthine (IBMX) and insulin to which an obesogen (viz., bisphenol A (BPA) or its analogs BPS and BPF; dioctyl terephthalate; tris (2-ethylhexyl) trimellitate; or various parabens) had been added. A mixture containing IBMX, insulin, and DEX, which constitute the typical hormonal cocktail required for adipocyte differentiation, was used as the control against which the other groups were measured. The obesogens and the PBA analogs all had evident adipogenic effects under DEX-free conditions, as was determined by estimating the lipid accumulation levels in the cells using Oil Red O staining. Furthermore, the expression of adipogenic transcription factors (CCAAT/enhancer-binding protein-alpha, peroxisome proliferator-activated receptor-gamma, and adipocyte protein 2) was induced by 20 µM of BPA, BPS, or BPF at both the mRNA and protein levels, as determined through reverse transcription-polymerase chain reaction and western blot assays. Taken together, the results reveal that adipocyte differentiation can be induced by obesogens and their alternatives in the absence of DEX.

Liver-Specific Deletion of Mouse CTCF Leads to Hepatic Steatosis via Augmented PPARγ Signaling.

BACKGROUND & AIMS: The liver is the major organ for metabolizing lipids, and malfunction of the liver leads to various diseases. Nonalcoholic fatty liver disease is rapidly becoming a major health concern worldwide and is characterized by abnormal retention of excess lipids in the liver. CCCTC-binding factor (CTCF) is a highly conserved zinc finger protein that regulates higher-order chromatin organization and is involved in various gene regulation processes. Here, we sought to determine the physiological role of CTCF in hepatic lipid metabolism. METHODS: We generated liver-specific, CTCF-ablated and/or CD36 whole-body knockout mice. Overexpression or knockdown of peroxisome proliferator-activated receptor (PPAR)γ in the liver was achieved using adenovirus. Mice were examined for development of hepatic steatosis and inflammation. RNA sequencing was performed to identify genes affected by CTCF depletion. Genome-wide occupancy of H3K27 acetylation, PPARγ, and CTCF were analyzed by chromatin immunoprecipitation sequencing. Genome-wide chromatin interactions were analyzed by in situ Hi-C. RESULTS: Liver-specific, CTCF-deficient mice developed hepatic steatosis and inflammation when fed a standard chow diet. Global analysis of the transcriptome and enhancer landscape revealed that CTCF-depleted liver showed enhanced accumulation of PPARγ in the nucleus, which leads to increased expression of its downstream target genes, including fat storage-related gene CD36, which is involved in the lipid metabolic process. Hepatic steatosis developed in liver-specific, CTCF-deficient mice was ameliorated by repression of PPARγ via pharmacologic blockade or adenovirus-mediated knockdown, but hardly rescued by additional knockout of CD36. CONCLUSIONS: Our data indicate that liver-specific deletion of CTCF leads to hepatosteatosis through augmented PPARγ DNA-binding activity, which up-regulates its downstream target genes associated with the lipid metabolic process.

Microglial MERTK eliminates phosphatidylserine-displaying inhibitory post-synapses.

Glia contribute to synapse elimination through phagocytosis in the central nervous system. Despite the important roles of this process in development and neurological disorders, the identity and regulation of the "eat-me" signal that initiates glia-mediated phagocytosis of synapses has remained incompletely understood. Here, we generated conditional knockout mice with neuronal-specific deletion of the flippase chaperone Cdc50a, to induce stable exposure of phosphatidylserine, a well-known "eat-me" signal for apoptotic cells, on the neuronal outer membrane. Surprisingly, acute Cdc50a deletion in mature neurons causes preferential phosphatidylserine exposure in neuronal somas and specific loss of inhibitory post-synapses without effects on other synapses, resulting in abnormal excitability and seizures. Ablation of microglia or the deletion of microglial phagocytic receptor Mertk prevents the loss of inhibitory post-synapses and the seizure phenotype, indicating that microglial phagocytosis is responsible for inhibitory post-synapse elimination. Moreover, we found that phosphatidylserine is used for microglia-mediated pruning of inhibitory post-synapses in normal brains, suggesting that phosphatidylserine serves as a general "eat-me" signal for inhibitory post-synapse elimination.

Method validation of 12 kinds of food dye in chewing gums and soft drinks, and evaluation of measurement uncertainty for soft drinks.

We developed and validated a method for the simultaneous quantitative analysis of food dyes in two food matrices, i.e., chewing gum and soft drinks. Furthermore, we evaluated the stability of food dyes in these matrices with respect to the pH and acid content. The optimized and validated method is based on high-performance liquid chromatography-photodiode array (HPLC-PDA) and liquid chromatography-tandem spectroscopy; the proposed method could identify and quantify 12 dyes in the two matrices. The recoveries of the food dyes identified by HPLC-PDA analysis ranged from 98.61% to 118.42%, with relative standard deviations of 0.06-4.90%. In addition, the expanded uncertainties of the measurements ranged from 0.57 to 3.12%. Finally, the food dyes were found to be stable in the matrices over 30 days. Thus, we believe that the proposed analytical method is suitable for the identification and quantification of food dyes in chewing gum and soft drink samples.

Physicochemical, nutritional and functional properties of Cucurbita moschata.

Cucurbita moschata is widely planted in most parts of the world, and is rich in carotenoids, vitamins, dietary fiber, minerals, and phenolic compounds. It also has important medicinal value. Some related research has proven that Cucurbita moschata has the potential ability to induce anti-obesity, anti-diabetic, antibacterial, and anticancer effects. At the same time, it has attracted more attention in the medical field. These nutrients and bioactive compounds in Cucurbita moschata have important effects on human health. In order to make better use of this crop, it still needs further study. Therefore, the purpose of this article is to summarize the physicochemical properties and nutritional components of Cucurbita moschata, and to provide a reference for further research on the benefits of on human health.

p53 expression confers sensitivity to 5-fluorouracil via distinct chromatin accessibility dynamics in human colorectal cancer.

One of the most commonly used drugs in chemotherapy, 5-fluorouracil (5-FU) has been shown to be effective in only 10-15% of patients with colon cancer. Thus, studies of the mechanisms affecting 5-FU sensitivity in these patients are necessary. The tumor suppressor protein p53 is a transcription factor that serves important roles in cell apoptosis by regulating the cell cycle. It has also been characterized as a key factor influencing drug sensitivity. Furthermore, accessible chromatin is a hallmark of active DNA regulatory elements and functions as a crucial epigenetic factor regulating cancer mechanisms. The present study assessed the genetic regulatory landscape in colon cancer by performing RNA sequencing and Assay for Transposase-Accessible Chromatin sequencing, and investigated the effects of 5-FU on chromatin accessibility and gene expression. Notably, while treatment with 5-FU mediated global increases in chromatin accessibility, chromatin organization in several genomic regions differed depending on the expression status of p53. Since the occupancy of p53 does not overlap with accessible chromatin regions, the 5-FU-mediated changes in chromatin accessibility were not regulated by direct binding of p53. In the p53-expressing condition, the 5-FU-mediated accessible chromatin region was primarily associated with genes encoding cell death pathways. Additionally, 5-FU was revealed to induce open chromatin conformation at regions containing binding motifs for AP-1 family transcription factors, which may drive expression of apoptosis pathway genes. In conclusion, expression of p53 may confer 5-FU sensitivity by regulating chromatin accessibility of distinct genes associated with cell apoptosis in a transcription-independent manner.

Antiobesity effect of Brassica juncea cultivated in Jeongseon with optimized sinigrin content using 3T3-L1 adipocytes.

In recent years, Brassica juncea has been selected as a special agricultural crop in Jeongseon, Gangwon-do, Korea, and is actively grown there. However, there have been no studies on B. juncea cultivated in Jeongseon (BJJ). Sinigrin, an index component of cruciferous vegetables, has been reported to have antiobesity effects. In this study, we developed a method for obtaining a BJJ extract with optimized sinigrin content, and investigate the antiobesity properties of the BJJ extract and sinigrin. The optimal extraction conditions for BJJ were found to be with 60% ethanol, at 70°C, for 3 hr. Lipid accumulation and ROS production were significantly suppressed in both the BJJ extract and sinigrin-treated groups. Furthermore, BJJ extract and sinigrin were effectively controlled the expression of proteins that regulate lipid accumulation, fatty acid oxidation, and energy metabolism. Thus, BJJ extract containing sinigrin may be used as a health functional food material. PRACTICAL APPLICATIONS: Brassica juncea has been reported to be rich in flavonoids, polyphenols, and glucosinolate, which are secondary vegetable metabolites. In this study, an extraction method to optimize the content of sinigrin in BJJ was established, and the antiobesity mechanism for the extract was confirmed. Lipid accumulation and ROS production were significantly suppressed in both the BJJ extract and sinigrin-treated groups in the study. It was confirmed that the expression of proteins that regulate lipid accumulation, lipid synthesis, fatty acid oxidation, heat generation, and energy metabolism was effectively controlled by the BJJ extract and sinigrin. Therefore, the ethanol extraction method of this study are considered to be useful for the preparation of extracts using cruciferous vegetables, and BJJ extract containing sinigrin have the potential to be used as a health functional food material for obesity.

Radical Scavenging-Linked Anti-Adipogenic Activity of Aster scaber Ethanolic Extract and Its Bioactive Compound.

Aster scaber is a wild vegetable cultivated in Korea and is known to contain phytochemicals with various biological activities. The potential antioxidant and anti-obesity effects of A. scaber and their mechanism are yet to be reported. We evaluated the total phenolic, flavonoid, and proanthocyanidin contents and oxygen radical absorbance capacity of A. scaber ethanolic extract (ASE), and analyzed the major phenolic compounds of ASE. Antioxidant activity was measured at the chemical level through 2,2-diphenyl-1-picrylhydrazyl (DPPH), reducing power assay, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), and fluorescence recovery after photobleaching (FRAP) assay. In addition, it was measured in vitro through inhibition of Reactive oxygen species (ROS) production in 3T3-L1 adipocyte, and inhibition of lipid accumulation was also evaluated. ASE reduced the expression of enzymes involved in the production of ROS and increased the expression of antioxidant enzymes that reduce increased ROS levels. They also reduced the expression of adipogenesis transcription factors that regulate adipocyte differentiation in relation to ROS production, inhibited the expression of lipogenesis-related genes related to fat accumulation through AMP-activated protein kinase (AMPK) activity, and increased expression of lipolysis-related genes. Thus, ASE containing CGA (chlorogenic acid) inhibits ROS production in 3T3-L1 adipocytes, owing to its strong antioxidant activity, and inhibits lipid accumulation caused by oxidative stress. The extract can be used as a potential functional food material for reducing oxidative stress and obesity.

The Double-Edged Sword of a Calling: The Mediating Role of Harmonious and Obsessive Passions in the Relationship between a Calling, Workaholism, and Work Engagement.

Even though research on perceiving a calling has been growing, our understanding of its double-edged sword effects and psychological mechanisms remain unclear, especially in terms of work engagement and workaholism. Based on the heavy working investment (HWI) and dualistic model of passion (DMP) theories, we established a dual-path structural model to examine the effects of callings on work engagement and workaholism through two types of passion: harmonious (HP) and obsessive (OP) passions. Our results showed that the association between perceiving a calling and work engagement was partially mediated by HP, while the association between perceiving a calling and workaholism was fully mediated by OP. This study contributes to the literature in that it reveals how perceiving a calling has different effects on work engagement and workaholism through the HWI theoretical lens, as well as the mediating roles of HP and OP, based on the DMP theory. Our findings can be practically applied in organizations and counseling.

Development and validation of the English version of the Moral Growth Mindset measure.

Background: Moral Growth Mindset (MGM) is a belief about whether one can become a morally better person through efforts. Prior research showed that MGM is positively associated with promotion of moral motivation among adolescents and young adults. We developed and tested the English version of the MGM measure in this study with data collected from college student participants. Methods: In Study 1, we tested the reliability and validity of the MGM measure with two-wave data ( N = 212, Age mean = 24.18 years, SD = 7.82 years). In Study 2, we retested the construct validity of the MGM measure once again and its association with other moral and positive psychological indicators to test its convergent and discriminant validity ( N = 275, Age mean = 22.02 years, SD = 6.34 years). Results: We found that the MGM measure was reliable and valid from Study 1. In Study 2, the results indicated that the MGM was well correlated with other moral and positive psychological indicators as expected. Conclusions: We developed and validated the English version of the MGM measure in the present study. The results from studies 1 and 2 supported the reliability and validity of the MGM measure. Given this, we found that the English version of the MGM measure can measure one's MGM as we intended.

Enhancement of Immune Activities of Mixtures with Sasa quelpaertensis Nakai and Ficus erecta var. sieboldii.

The objective of the present study was to develop a concoction of natural products that could dramatically improve immune function with minimal possible side effects. Sasa quelpaertensis Nakai and Ficus erecta var. sieboldii are plants that are native to Jeju Island, Korea and are known to be rich in physiologically active substances. We prepared a mixture of different proportions and extraction conditions using two natural plants and determined their optimum mixing ratio and extraction method by assessing immune function-related biomarkers in RAW264.7 macrophages. Optimal extract (HR02/04(8:2)-W) was selected from in vitro experiments and its immunity-enhancing efficacy was evaluated in mice. After oral administration of extract to BALB/c mice for 2 weeks, nitric oxide production in the peritoneal exudate cells, natural killer cell cytotoxicity, cytokine expression in splenocytes, and total cell number of immune tissues and phenotype analysis were evaluated. Our results demonstrated that HR02/04(8:2)-W significantly enhanced the immune system by increasing natural killer cell activity, cytokine expression, and total number of cells in immune tissues. In conclusion, our study validates the role of HR02/04(8:2)-W in enhancing immunity and its potential development as a functional food.

NGL-1/LRRC4C-Mutant Mice Display Hyperactivity and Anxiolytic-Like Behavior Associated With Widespread Suppression of Neuronal Activity.

Netrin-G ligand-1 (NGL-1), encoded by Lrrc4c, is a post-synaptic adhesion molecule implicated in various brain disorders, including bipolar disorder, autism spectrum disorder, and developmental delay. Although previous studies have explored the roles of NGL-1 in the regulation of synapse development and function, the importance of NGL-1 for specific behaviors and the nature of related neural circuits in mice remain unclear. Here, we report that mice lacking NGL-1 (Lrrc4c-/- ) show strong hyperactivity and anxiolytic-like behavior. They also display impaired spatial and working memory, but normal object-recognition memory and social interaction. c-Fos staining under baseline and anxiety-inducing conditions revealed suppressed baseline neuronal activity as well as limited neuronal activation in widespread brain regions, including the anterior cingulate cortex (ACC), motor cortex, endopiriform nucleus, bed nuclei of the stria terminalis, and dentate gyrus. Neurons in the ACC, motor cortex, and dentate gyrus exhibit distinct alterations in excitatory synaptic transmission and intrinsic neuronal excitability. These results suggest that NGL-1 is important for normal locomotor activity, anxiety-like behavior, and learning and memory, as well as synapse properties and excitability of neurons in widespread brain regions under baseline and anxiety-inducing conditions.

Peroxisomes support human herpesvirus 8 latency by stabilizing the viral oncogenic protein vFLIP via the MAVS-TRAF complex.

Human herpesvirus 8 (HHV-8) is causally related to human malignancies. HHV-8 latent viral FLICE-inhibitory protein (vFLIP) is a viral oncoprotein that is linked to pathogenesis, but how its expression is regulated is largely unknown. In an attempt to understand the role of the mitochondrial antiviral signaling (MAVS) adaptor in HHV-8 infection, we discovered that vFLIP expression was post-translationally up-regulated by the MAVS signaling complex on peroxisomes. Furthermore, we demonstrated that vFLIP could be targeted to the peroxisomes, where it was oncogenically active, in a PEX19-dependent manner. Targeted disruption of vFLIP and MAVS interaction resulted in a decrease in vFLIP expression and selectively promoted death of latently HHV-8-infected cells, providing therapeutic potential for treating HHV-8 diseases. Collectively, our experimental results suggest novel involvement of peroxisomes and MAVS in the stabilization of vFLIP and thereby in the establishment or maintenance of HHV-8 latency and associated pathogenesis.

Three-dimensional brain-like microenvironments facilitate the direct reprogramming of fibroblasts into therapeutic neurons.

Biophysical cues can improve the direct reprogramming of fibroblasts into neurons that can be used for therapeutic purposes. However, the effects of a three-dimensional (3D) environment on direct neuronal reprogramming remain unexplored. Here, we show that brain extracellular matrix (BEM) decellularized from human brain tissue facilitates the plasmid-transfection-based direct conversion of primary mouse embryonic fibroblasts into induced neuronal (iN) cells. We first show that two-dimensional (2D) surfaces modified with BEM significantly increase the generation efficiency of iN cells and enhance neuronal transdifferentiation and maturation. Moreover, in an animal model of ischaemic stroke, iN cells generated on the BEM substrates and transplanted into the brain led to significant improvements in locomotive behaviours. We also show that compared with the 2D BEM substrates, 3D BEM hydrogels recapitulating brain-like microenvironments further promote neuronal conversion and potentiate the functional recovery of the animals. Our findings suggest that 3D microenvironments can boost nonviral direct reprogramming for the generation of therapeutic neuronal cells.

Epigenome mapping highlights chromatin-mediated gene regulation in the protozoan parasite Trichomonas vaginalis.

Trichomonas vaginalis is an extracellular flagellated protozoan parasite that causes trichomoniasis, one of the most common non-viral sexually transmitted diseases. To survive and to maintain infection, T. vaginalis adapts to a hostile host environment by regulating gene expression. However, the mechanisms of transcriptional regulation are poorly understood for this parasite. Histone modification has a marked effect on chromatin structure and directs the recruitment of transcriptional machinery, thereby regulating essential cellular processes. In this study, we aimed to outline modes of chromatin-mediated gene regulation in T. vaginalis. Inhibition of histone deacetylase (HDAC) alters global transcriptional responses and induces hyperacetylation of histones and hypermethylation of H3K4. Analysis of the genome of T. vaginalis revealed that a number of enzymes regulate histone modification, suggesting that epigenetic mechanisms are important to controlling gene expression in this organism. Additionally, we describe the genome-wide localization of two histone H3 modifications (H3K4me3 and H3K27Ac), which we found to be positively associated with active gene expression in both steady and dynamic transcriptional states. These results provide the first direct evidence that histone modifications play an essential role in transcriptional regulation of T. vaginalis, and may help guide future epigenetic research into therapeutic intervention strategies against this parasite.

Dynamic Long-Range Chromatin Interaction Controls Expression of IL-21 in CD4+ T Cells.

IL-21, a pleiotropic cytokine strongly linked with autoimmunity and inflammation, regulates diverse immune responses. IL-21 can be potently induced in CD4(+) T cells by IL-6; however, very little is known about the mechanisms underlying the transcriptional regulation of the Il21 gene at the chromatin level. In this study, we demonstrated that a conserved noncoding sequence located 49 kb upstream of the Il21 gene contains an enhancer element that can upregulate Il21 gene expression in a STAT3- and NFAT-dependent manner. Additionally, we identified enhancer-blocking insulator elements in the Il21 locus, which constitutively bind CTCF and cohesin. In naive CD4(+) T cells, these upstream and downstream CTCF binding sites interact with each other to make a DNA loop; however, the Il21 promoter does not interact with any cis-elements in the Il21 locus. In contrast, stimulation of CD4(+) T cells with IL-6 leads to recruitment of STAT3 to the promoter and novel distal enhancer region. This induces dynamic changes in chromatin configuration, bringing the promoter and the regulatory elements in close spatial proximity. The long-range interaction between the promoter and distal enhancer region was dependent on IL-6/STAT3 signaling pathway but was disrupted in regulatory T cells, where IL-21 expression was repressed. Thus, our work uncovers a novel topological chromatin framework underlying proper transcriptional regulation of the Il21 gene.