Anti-viral effect of usenamine a using SARS-CoV-2 pseudo-typed viruses.

The escalating pandemic brought about by the novel SARS-CoV-2 virus is threatening global health, and thus, it is necessary to develop effective antiviral drugs. Usenamine A is a dibenzo-furan derivative separated from lichen Usnea diffracta showing broad-spectrum activity against different viruses. We evaluate that usenamine A has antiviral effects against novel SARS-CoV-2 Delta variant pseudotyped viruses (PVs) in A549 cells. In addition, usenamine A significantly suppresses SARS-CoV-2 PV-induced mitochondrial depolarization, elevated reactive oxygen species (ROS) levels, apoptosis, and inflammation. Usenamine A also causes the SARS-CoV-2 spike protein to become less stable. Thus, usenamine A shows potential as an antiviral drug that can provide protection against COVID-19.

Development of a screening platform to discover natural products active against SARS-CoV-2 infection using lung organoid models.

BACKGROUND: Natural products can serve as one of the alternatives, exhibiting high potential for the treatment and prevention of COVID-19, caused by SARS-CoV-2. Herein, we report a screening platform to test the antiviral efficacy of a natural product library against SARS-CoV-2 and verify their activity using lung organoids. METHODS: Since SARS-CoV-2 is classified as a risk group 3 pathogen, the drug screening assay must be performed in a biosafety level 3 (BSL-3) laboratory. To circumvent this limitation, pseudotyped viruses (PVs) have been developed as replacements for the live SARS-CoV-2. We developed PVs containing spikes from Delta and Omicron variants of SARS-CoV-2 and improved the infection in an angiotensin-converting enzyme 2 (ACE2)-dependent manner. Human induced pluripotent stem cells (hiPSCs) derived lung organoids were generated to test the SARS-CoV-2 therapeutic efficacy of natural products. RESULTS: Flavonoids from our natural product library had strong antiviral activity against the Delta- or Omicron-spike-containing PVs without affecting cell viability. We aimed to develop strategies to discover the dual function of either inhibiting infection at the beginning of the infection cycle or reducing spike stability following SARS-CoV-2 infection. When lung cells are already infected with the virus, the active flavonoids induced the degradation of the spike protein and exerted anti-inflammatory effects. Further experiments confirmed that the active flavonoids had strong antiviral activity in lung organoid models. CONCLUSION: This screening platform will open new paths by providing a promising standard system for discovering novel drug leads against SARS-CoV-2 and help develop promising candidates for clinical investigation as potential therapeutics for COVID-19.

Identification of cell-biologic mechanisms of coronary artery spasm and its ex vivo diagnosis using peripheral blood-derived iPSCs.

BACKGROUND: Although vasospastic angina (VSA) is known to be caused by coronary artery spasm, no study has fully elucidated the exact underlying mechanism. Moreover, in order to confirm VSA, patients should undergo invasive coronary angiography with spasm provocation test. Herein, we investigated the pathophysiology of VSA using peripheral blood-derived induced pluripotent stem cells (iPSCs) and developed an ex vivo diagnostic method for VSA. METHODS AND RESULTS: With 10 mL of peripheral blood from patients with VSA, we generated iPSCs and differentiated these iPSCs into target cells. As compared with vascular smooth muscle cells (VSMCs) differentiated from iPSCs of normal subjects with negative provocation test, VSA patient-specific iPSCs-derived VSMCs showed very strong contraction in response to stimulants. Moreover, VSA patient-specific VSMCs exhibited a significant increase in stimulation-induced intracellular calcium efflux (Changes in the relative fluorescence unit [ΔF/F]; Control group vs. VSA group, 2.89 ± 0.34 vs. 10.32 ± 0.51, p < 0.01), and exclusively induced a secondary or tertiary peak of calcium efflux, suggesting that those findings could be diagnostic cut-off values for VSA. The observed hyperreactivity of VSA patient-specific VSMCs were caused by the upregulation of sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) due to its enhanced small ubiquitin-related modifier (SUMO)ylation. This increased activity of SERCA2a was reversed by treatment with ginkgolic acid, an inhibitor of SUMOylated E1 molecules (pi/µg protein; VSA group vs. VSA + ginkgolic acid, 52.36 ± 0.71 vs. 31.93 ± 1.13, p < 0.01). CONCLUSIONS: Our findings showed that abnormal calcium handling in sarco/endoplasmic reticulum could be induced by the enhanced SERCA2a activity in patients with VSA, leading to spasm. Such novel mechanisms of coronary artery spasm could be useful for drug development and diagnosis of VSA.

Tryptophan-dependent and -independent secretions of tryptophanyl- tRNA synthetase mediate innate inflammatory responses.

While cytoplasmic tryptophanyl-tRNA synthetase (WARS1) ligates tryptophan (Trp) to its cognate tRNAs for protein synthesis, it also plays a role as an innate immune activator in extracellular space. However, its secretion mechanism remains elusive. Here, we report that in response to stimuli, WARS1 can be secreted via two distinct pathways: via Trp-dependent secretion of naked protein and via Trp-independent plasma-membrane-derived vesicles (PMVs). In the direct pathway, Trp binding to WARS1 induces a "closed" conformation, generating a hydrophobic surface and basic pocket. The Trp-bound WARS1 then binds stable phosphatidylinositol (4,5)-biphosphate and inner plasma membrane leaflet, passing across the membrane. In the PMV-mediated secretion, WARS1 recruits calpain 2, which is activated by calcium. WARS1 released from PMVs induces inflammatory responses in vivo. These results provide insights into the secretion mechanisms of WARS1 and improve our understanding of how WARS1 is involved in the control of local and systemic inflammation upon infection.

Polysaccharides of Weissella cibaria Act as a Prebiotic to Enhance the Probiotic Potential of Lactobacillus rhamnosus.

This work aimed to investigate the effect of EPS (extracellular polysaccharide) of Weissella cibaria as a prebiotic to promote the growth and antibacterial properties of Lactobacillus rhamnosus. The morphological, growth behavior, and antibacterial properties of L. rhamnosus were determined in MRSB (de Man Rogosa Sharpe broth) supplemented with different concentrations of EPS (0.1-2%). The results revealed that the incorporation of the EPS (2%) in MRSA improved the bacterial growth in terms of colony-forming unit (CFU, 0.7 × 105 CFU/mL) compared to L. rhamnosus grown in bare MRSA. The SEM observation revealed that EPS incorporation in the MRSB culture media does not affect the morphological properties of L. rhamnosus. Moreover, it was confirmed that the extract of probiotics cultured in MRSA supplemented with EPS (2%) was exhibited strong antibacterial and antibiofilm activity against targeted pathogens. This L. rhamnosus extract was found to be biocompatible evidanced by erythrocyte hemolysis assay. These results confirmed that EPS regulates the growth of probiotics, resists pathogen infection, and biocompatibility.

Review of the Current State of Freely Accessible Web Tools for the Analysis of 16S rRNA Sequencing of the Gut Microbiome.

Owing to the emergence and improvement of high-throughput technology and the associated reduction in costs, next-generation sequencing (NGS) technology has made large-scale sampling and sequencing possible. With the large volume of data produced, the processing and downstream analysis of data are important for ensuring meaningful results and interpretation. Problems in data analysis may be encountered if researchers have little experience in using programming languages, especially if they are clinicians and beginners in the field. A strategy for solving this problem involves ensuring easy access to commercial software and tools. Here, we observed the current status of free web-based tools for microbiome analysis that can help users analyze and handle microbiome data effortlessly. We limited our search to freely available web-based tools and identified MicrobiomeAnalyst, Mian, gcMeta, VAMPS, and Microbiome Toolbox. We also highlighted the various analyses that each web tool offers, how users can analyze their data using each web tool, and noted some of their limitations. From the abovementioned list, gcMeta, VAMPS, and Microbiome Toolbox had several issues that made the analysis more difficult. Over time, as more data are generated and accessed, more users will analyze microbiome data. Thus, the availability of free and easily accessible web tools can enable the easy use and analysis of microbiome data, especially for those users with less experience in using command-line interfaces.

Current status of foodservice nutrition management and effects of welfare facility support for the elderly in Cheongju City.

BACKGROUND/OBJECTIVES: South Korea is representative of countries experiencing rapid societal aging. This study aimed to understand the current status of foodservice nutrition management provided to welfare facilities for the elderly and to understand improvements after support from "the Center for Social Welfare Foodservice Management (CSWFM)" in Cheongju City. SUBJECTS/METHODS: The status of foodservice nutrition management was assessed by dietitians and hired by the CSWFM, who visited 40 welfare facilities (registered members of the CSWFM) for the elderly in Cheongju City. After visiting each facility three times from July to December 2019, the results of inspections on four areas, that is, 'menu', 'meal provision', 'cooking', and 'distribution' management for 2nd and 3rd visits (support visits) were compared with results obtained at initial visits. RESULTS: Before support as determined during 1st visits, compliance rates with 'menu', 'meal provision', 'cooking', and 'distribution' requirements were 72.1%, 75.5%, 58.3%, and 77.5%, respectively. The mean compliance rate for all 15 items on the questionnaire used was 70.8%. Items with low compliance rates were 'Is the soup provided by foodservice at the recommended salinity?' (compliance rate 37.5%) and 'Is the foodservice cooking conducted by referring to a recipe?' (42.5%). At the two support visits, mean compliance rates increased significantly (P < 0.01, P < 0.001), mean total score had significantly increased from 71.80 to 90.26 (P < 0.001), and mean soup salinity decreased significantly from 0.82% at 1st visits to 0.68% (P < 0.001) and 0.56% (P < 0.001) at the 1st and second follow-up visits. CONCLUSIONS: These results show that the status of nutrition management at welfare facilities for the elderly was much improved by CSWFM involvement, and indicate the CSWFM should continuously provide nutrition management support to facilities and that finances and opportunities for more welfare facilities for the elderly be expanded.

Impact of industrial effluents on the environment and human health and their remediation using MOFs-based hybrid membrane filtration techniques.

The hazardous risk posed by industrial effluent discharge into the ecosystem has raised a plethora of environmental issues, public health, and safety concerns. The effluents from industries such as tanning, leather, petrochemicals, pharmaceuticals, and textiles are create significant stress on the aquatic ecosystem, which induces significant toxicity, involved in endocrine disruptions, and inhibits reproductive functions. Therefore, this review presented an overall abridgment of the effects of these effluents and their ability to synergize with modern pollutants such as pharmaceuticals, cosmetic chemicals, nanoparticles, and heavy metals. We further emphasize the metal organic framework (MOF) based membrane filtration approach for remediation of industrial effluents in comparison to the traditional remediation process. The MOF based-hybrid membrane filters provide higher reusability, better adsorption, and superior removal rates through the implication of nanotechnology, while the traditional remediation process offers poorer filtration rates and stability.

Enhanced Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood and Using Their Mesoderm Differentiation Ability to Regenerate Infarcted Myocardium.

Тhe most pressing issue in generating induced pluripotent stem cells (iPSCs) in clinical practice is the cell source. Compared to human dermal fibroblasts (HDFs), which have been widely used, human peripheral blood could be a more easily obtainable alternative. However, iPSCs generated from fresh peripheral blood require inconvenient specific methods including isolation. Recently, we succeeded in isolating and culturing human heart-derived circulating cells called circulating multipotent stem (CiMS) cells. Here, we investigated the generation efficiency of CiMS-derived iPSCs (CiMS-iPSCs) and tested their differentiation potential into mesodermal lineages and cardiovascular cells. We isolated and cultured CiMS cells from peripheral mononuclear cells with a high efficiency. Moreover, our method succeeded in reprogramming the CiMS cells and generating iPSCs with higher efficiency compared to when HDFs were used. Compared to HDF-iPSCs or human embryonic stem cells (hESCs), CiMS-iPSCs showed high differentiation potential into mesodermal lineage cells and subsequently into endothelial cells, vascular smooth muscle cells, and cardiomyocytes. Further, we checked the epigenetic status of each cell type. While methylation of the CpG site of the brachyury T promoter did not differ between cell types, the histone H3 lysine 4 trimethylation level in the brachyury T promoter region was enhanced in CiMS-iPSCs, compared to that in other cell types. In contrast, histone H3 lysine 9 acetylation was downregulated during the differentiation process of the CiMS-iPSCs. In the myocardial infarction model, the CiMS-iPSCs group showed more therapeutic potential in regenerating the myocardium than other cell types. Our study showed a new method to isolate human heart-derived stem cells from human peripheral blood and to generate iPSCs efficiently. Due to epigenetic memory, these CiMS-iPSCs easily differentiated into cardiovascular lineage cells, resulting in improved efficiency in vivo. These results suggest that our new method using CiMS cells has therapeutic potential in regenerative medicine using cell therapy.

Mitochondrial dysfunction associated with autophagy and mitophagy in cerebrospinal fluid cells of patients with delayed cerebral ischemia following subarachnoid hemorrhage.

Decreased mitochondrial membrane potential in cerebrospinal fluid (CSF) was observed in patients with subarachnoid hemorrhage (SAH) accompanied by delayed cerebral ischemia (DCI). However, whether abnormal mechanisms of mitochondria are associated with the development of DCI has not been reported yet. Under cerebral ischemia, mitochondria can transfer into the extracellular space. Mitochondrial dysfunction can aggravate neurologic complications. The objective of this study was to evaluate whether mitochondrial dysfunction might be associated with autophagy and mitophagy in CSF cells to provide possible insight into DCI pathogenesis. CSF samples were collected from 56 SAH patients (DCI, n = 21; and non-DCI, n = 35). We analyzed CSF cells using autophagy and mitophagy markers (DAPK1, BNIP3L, BAX, PINK1, ULK1, and NDP52) via qRT-PCR and western blotting of proteins (BECN1, LC3, and p62). Confocal microscopy and immunogold staining were performed to demonstrate the differentially expression of markers within dysfunctional mitochondria. Significant induction of autophagic flux with accumulation of autophagic vacuoles, increased expression of BECN1, LC3-II, and p62 degradation were observed during DCI. Compared to non-DCI patients, DCI patients showed significantly increased mRNA expression levels (2-ΔCt) of DAPK1, BNIP3L, and PINK1, but not BAX, ULK1, or NDP52. Multivariable logistic regression analysis revealed that Hunt and Hess grade ≥ IV (p = 0.023), DAPK1 (p = 0.003), and BNIP3L (p = 0.039) were related to DCI. Increased mitochondrial dysfunction associated with autophagy and mitophagy could play an important role in DCI pathogenesis.

Antiviral Activity of Chrysin against Influenza Virus Replication via Inhibition of Autophagy.

Influenza viruses cause respiratory infections in humans and animals, which have high morbidity and mortality rates. Although several drugs that inhibit viral neuraminidase are used to treat influenza infections, the emergence of resistant viruses necessitates the urgent development of new antiviral drugs. Chrysin (5,7-dihydroxyflavone) is a natural flavonoid that exhibits antiviral activity against enterovirus 71 (EV71) by inhibiting viral 3C protease activity. In this study, we evaluated the antiviral activity of chrysin against influenza A/Puerto Rico/8/34 (A/PR/8). Chrysin significantly inhibited A/PR/8-mediated cell death and the replication of A/PR/8 at concentrations up to 2 µM. Viral hemagglutinin expression was also markedly decreased by the chrysin treatment in A/PR/8-infected cells. Through the time course experiment and time-of-addition assay, we found that chrysin inhibited viral infection at the early stages of the replication cycle. Additionally, the nucleoprotein expression of A/PR/8 in A549 cells was reduced upon treatment with chrysin. Regarding the mechanism of action, we found that chrysin inhibited autophagy activation by increasing the phosphorylation of mammalian target of rapamycin (mTOR). We also confirmed a decrease in LC3B expression and LC3-positive puncta levels in A/PR/8-infected cells. These results suggest that chrysin exhibits antiviral activity by activating mTOR and inhibiting autophagy to inhibit the replication of A/PR/8 in the early stages of infection.

Anticancer Effects of Propionic Acid Inducing Cell Death in Cervical Cancer Cells.

Recent studies found that short-chain fatty acids (SCFAs), which are produced through bacterial fermentation in the gastrointestinal tract, have oncoprotective effects against cervical cancer. The most common SCFAs that are well known include acetic acid, butyric acid, and propionic acid, among which propionic acid (PA) has been reported to induce apoptosis in HeLa cells. However, the mechanism in which SCFAs suppress HeLa cell viability remain poorly understood. Our study aims to provide a more detailed look into the mechanism of PA in HeLa cells. Flow cytometry analysis revealed that PA induces reactive oxygen species (ROS), leading to the dysfunction of the mitochondrial membrane. Moreover, PA inhibits NF-κB and AKT/mTOR signaling pathways and induces LC3B protein levels, resulting in autophagy. PA also increased the sub-G1 cell population that is characteristic of cell death. Therefore, the results of this study propose that PA inhibits HeLa cell viability through a mechanism mediated by the induction of autophagy. The study also suggests a new approach for cervical cancer therapeutics.

Ciliogenesis is Not Directly Regulated by LRRK2 Kinase Activity in Neurons.

Mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of familial Parkinson's disease (PD). The increase in LRRK2 kinase activity observed in the pathogenic G2019S mutation is important for PD development. Several studies have reported that increased LRRK2 kinase activity and treatment with LRRK2 kinase inhibitors decreased and increased ciliogenesis, respectively, in mouse embryonic fibroblasts (MEFs) and retinal pigment epithelium (RPE) cells. In contrast, treatment of SH-SY5Y dopaminergic neuronal cells with PD-causing chemicals increased ciliogenesis. Because these reports were somewhat contradictory, we tested the effect of LRRK2 kinase activity on ciliogenesis in neurons. In SH-SY5Y cells, LRRK2 inhibitor treatment slightly increased ciliogenesis, but serum starvation showed no increase. In rat primary neurons, LRRK2 inhibitor treatment repeatedly showed no significant change. Little difference was observed between primary cortical neurons prepared from wild-type (WT) and G2019S+/- mice. However, a significant increase in ciliogenesis was observed in G2019S+/- compared to WT human fibroblasts, and this pattern was maintained in neural stem cells (NSCs) differentiated from the induced pluripotent stem cells (iPSCs) prepared from the same WT/G2019S fibroblast pair. NSCs differentiated from G2019S and its gene-corrected WT counterpart iPSCs were also used to test ciliogenesis in an isogenic background. The results showed no significant difference between WT and G2019S regardless of kinase inhibitor treatment and B27-deprivation-mimicking serum starvation. These results suggest that LRRK2 kinase activity may be not a direct regulator of ciliogenesis and ciliogenesis varies depending upon the cell type or genetic background.

Anticancer Activity of Lesbicoumestan in Jurkat Cells via Inhibition of Oxidative Stress-Mediated Apoptosis and MALT1 Protease.

This study explores the potential anticancer effects of lesbicoumestan from Lespedeza bicolor against human leukemia cancer cells. Flow cytometry and fluorescence microscopy were used to investigate antiproliferative effects. The degradation of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) was evaluated using immunoprecipitation, Western blotting, and confocal microscopy. Apoptosis was investigated using three-dimensional (3D) Jurkat cell resistance models. Lesbicoumestan induced potent mitochondrial depolarization on the Jurkat cells via upregulated expression levels of mitochondrial reactive oxygen species. Furthermore, the underlying apoptotic mechanisms of lesbicoumestan through the MALT1/NF-κB pathway were comprehensively elucidated. The analysis showed that lesbicoumestan significantly induced MALT1 degradation, which led to the inhibition of the NF-κB pathway. In addition, molecular docking results illustrate how lesbicoumestan could effectively bind with MALT1 protease at the latter's active pocket. Similar to traditional 2D cultures, apoptosis was markedly induced upon lesbicoumestan treatment in 3D Jurkat cell resistance models. Our data support the hypothesis that lesbicoumestan is a novel inhibitor of MALT1, as it exhibited potent antiapoptotic effects in Jurkat cells.

Quantitative Proteomic Analysis of Primitive Neural Stem Cells from LRRK2 G2019S-Associated Parkinson's Disease Patient-Derived iPSCs.

Parkinson's disease (PD) is a common neurodegenerative disease, causing movement defects. The incidence of PD is constantly increasing and this disease is still incurable. Thus, understanding PD pathophysiology would be pivotal for the development of PD therapy, and various PD models have thus been already developed. Through recent advances in reprogramming techniques, a primitive neural stem cell (pNSC) derived from PD patient induced pluripotent stem cells (iPSCs) could be potentially used as a reproducible and reliable experimental system to analyze the effect of the leucine-rich repeat kinase 2 G2019S mutation (LK2GS) in neural cells. Here, we investigated the advantages of such a model system through quantitative proteomic analysis of pNSCs from normal control iPSCs and familial PD patient iPSCs harboring LK2GS. We confirmed that the expression of molecules known to be involved in PD pathogenesis, such as oxidative stress-, cell adhesion-, and cytoskeleton-related proteins, were altered in the LK2GS pNSC. In addition, we showed that down-regulation of Ku80, which was found in the proteomic analysis with LK2GS pNSCs, resulted in apoptosis induced by DNA damage response. Taken together, we suggest that pNSCs from PD iPSCs could provide a reliable and useful model system to study PD. Moreover, the highly expandable pNSC is suitable for multi-omics approaches to understand PD pathologies and discover therapeutic targets for PD.

Neuroprotective Effects of Cryptotanshinone in a Direct Reprogramming Model of Parkinson's Disease.

Parkinson's disease (PD) is a well-known age-related neurodegenerative disease. Considering the vital importance of disease modeling based on reprogramming technology, we adopted direct reprogramming to human-induced neuronal progenitor cells (hiNPCs) for in vitro assessment of potential therapeutics. In this study, we investigated the neuroprotective effects of cryptotanshinone (CTN), which has been reported to have antioxidant properties, through PD patient-derived hiNPCs (PD-iNPCs) model with induced oxidative stress and cell death by the proteasome inhibitor MG132. A cytotoxicity assay showed that CTN possesses anti-apoptotic properties in PD-hiNPCs. CTN treatment significantly reduced cellular apoptosis through mitochondrial restoration, such as the reduction in mitochondrial reactive oxygen species and increments of mitochondrial membrane potential. These effects of CTN are mediated via the nuclear factor erythroid 2-related factor 2 (NRF2) pathway in PD-hiNPCs. Consequently, CTN could be a potential antioxidant reagent for preventing disease-related pathological phenotypes of PD.

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells.

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Iroquois Homeobox Protein 2 Identified as a Potential Biomarker for Parkinson's Disease.

The diagnosis of Parkinson's disease (PD) is initiated after the occurrence of motor symptoms, such as resting tremors, rigidity, and bradykinesia. According to previous reports, non-motor symptoms, notably gastrointestinal dysfunction, could potentially be early biomarkers in PD patients as such symptoms occur earlier than motor symptoms. However, connecting PD to the intestine is methodologically challenging. Thus, we generated in vitro human intestinal organoids from PD patients and ex vivo mouse small intestinal organoids from aged transgenic mice. Both intestinal organoids (IOs) contained the human LRRK2 G2019S mutation, which is the most frequent genetic cause of familial and sporadic PD. By conducting comprehensive genomic comparisons with these two types of IOs, we determined that a particular gene, namely, Iroquois homeobox protein 2 (IRX2), showed PD-related expression patterns not only in human pluripotent stem cell (PSC)-derived neuroectodermal spheres but also in human PSC-derived neuronal cells containing dopaminergic neurons. We expected that our approach of using various cell types presented a novel technical method for studying the effects of multi-organs in PD pathophysiology as well as for the development of diagnostic markers for PD.

NFATc1+CD31+CD45- circulating multipotent stem cells derived from human endocardium and their therapeutic potential.

Many studies have shown the existence of cardiac stem cells in the myocardium and epicardial progenitor cells in the epicardium. However, the characteristics of stem cells in the endocardium has not been fully elucidated. In this study, we investigated the origin of newly identified cells in the blood and their therapeutic potential. The new population of cells, identified from human peripheral blood, was quite different from previously reported stem cells. These newly identified cells, which we named Circulating Multipotent Stem (CiMS) cells, were multipotent, and therefore differentiated into multiple lineages in vitro and in vivo. In order to determine the origin of these cells, we collected peripheral blood from a group of patients who underwent bone marrow, liver, heart, or kidney transplantation. We identified the endocardium as the origin of these cells because the Short Tandem Repeat profile of CiMS cells from the recipient had changed from the recipient's profile to the donor's profile after heart transplantation. CiMS cells significantly increased after stimuli to the endocardium, such as catheter ablation for arrhythmia or acute myocardial infarction. CiMS cells circulate in human peripheral blood and are easily obtainable, suggesting that these cells could be a promising tool for cell therapy.

CAGE-miR-140-5p-Wnt1 Axis Regulates Autophagic Flux, Tumorigenic Potential of Mouse Colon Cancer Cells and Cellular Interactions Mediated by Exosomes.

Although the cancer/testis antigen CAGE has been implicated in tumorigenesis, the molecular mechanisms of CAGE-promoted tumorigenesis remain largely unknown. CT26Flag-CAGE cells, CT26 (mouse colon cancer cells) cells stably expressing CAGE, were established to investigate CAGE-promoted tumorigenesis. Down-regulation of CAGE led to decreased autophagic flux in CT26Flag-CAGE cells. CAGE interacted with Beclin1, a mediator of autophagy. The CT26Flag-CAGE cells showed enhanced autophagosome formation and displayed greater tumor spheroid-forming potential than CT26 cells. MicroRNA array analysis revealed that CAGE decreased the expression of various microRNAs, including miR-140-5p, in CT26 cells. CAGE was shown to bind to the promoter sequences of miR-140-5p. MiR-140-5p inhibition increased the tumorigenic potential of and autophagic flux in CT26 cells. A miR-140-5p mimic exerted negative effects on the tumorigenic potential of CT26Flag-CAGE cells and autophagic flux in CT26Flag-CAGE cells. MiR-140-5p was predicted to bind to the 3'-UTR of Wnt1. CT26Flag-CAGE cells showed higher expression of Wnt1 than CT26 cells. Down-regulation of Wnt1 decreased autophagic flux. Luciferase activity assays showed the direct regulation of wnt1 by miR-140-5p. Tumor tissue derived from the CT26Flag-CAGE cells revealed higher expressions of factors associated with activated mast cells and tumor-associated macrophages than tumor tissue derived from CT26 cells. Culture medium from the CT26Flag-CAGE cells increased autophagic flux in CT26 cells, mast cells and macrophages. Culture medium from the CT26Flag-CAGE cells increased CD163 and autophagic flux in CT26 cells, mast cells, and macrophages in a Wnt1-dependent manner. Exosomes from CT26Flag-CAGE cells increased autophagc flux in CT26 cells, mast cells, and macrophages. Exosomes from CT26Flag-CAGE cells increased the tumorigenic potential of CT26 cells. Wnt1 was shown to be present within the exosomes. Recombinant Wnt1 protein increased autophagic flux in CT26, mast cells, and macrophages. Recombinant wnt1 protein mediated interactions between the CT26 cells, mast cells, and macrophages. Our results showed novel roles for the CAGE-miR-140-5p-Wnt1 axis in autophagic flux and cellular interactions mediated by exosomes.