Beneficial Effects of Fermentation of Red Chili Pepper Using Lactococcus lactis subs. Cremoris RPG-HL-0136 in High-Fat Diet-Induced Obese Mice.

Red chili pepper is a beneficial natural spicy food that has antiobesity and antitype II diabetes effects, but it is not conducive to in-depth research as a dietary strategy to treat obesity. This study aims to investigate the beneficial effects of red chili pepper, fermented with a novel Lactococcus lactis subs. cremoris RPG-HL-0136. LC-MS/MS analysis is conducted to detect the content of capsaicin and dihydrocapsaicin, and no significant difference is observed between the nonfermented red chili pepper (NFP) (W/W) and the prepared L. lactis subs. cremoris RPG-HL-0136-fermented chili mixture (LFP). After establishing a high-fat diet-induced obese type II diabetic mouse model, the effects on weight gain, weight loss of liver and testicular fat, total cholesterol, triglyceride, fasting glucose, insulin, and homeostatic model assessment for insulin resistance in LFP were evaluated to be better than those in NFP following 10 weeks of interventions. All animal experiments were approved by the Institutional Animal Care and Use Committee of Xinxiang medical university. NFP and LFP could increase the expression of transient receptor potential vanilloid subfamily 1, peroxisome proliferator-activated receptor-alpha and caspase-2 in the high-fat mice. Compared with unfermented red chili pepper, the fermented red chili pepper complex significantly reduced LPS, tumor necrosis factor-alpha, and interleukin-6 in serum (P < .05). Intake of LFP significantly increased the expression of claudin-1 and occludin in the colon of the high-fat mice (P < .05), and there was no damage to the stomach and colon. This study provides scientific evidence that red chili pepper, fermented with L. lactis subs. cremoris RPG-HL-0136, may be beneficial for future treatment of obesity and accompanying diabetes. (IACUC.No.XYLL-20200019).

Selenophosphate synthetase 1 deficiency exacerbates osteoarthritis by dysregulating redox homeostasis.

Aging and mechanical overload are prominent risk factors for osteoarthritis (OA), which lead to an imbalance in redox homeostasis. The resulting state of oxidative stress drives the pathological transition of chondrocytes during OA development. However, the specific molecular pathways involved in disrupting chondrocyte redox homeostasis remain unclear. Here, we show that selenophosphate synthetase 1 (SEPHS1) expression is downregulated in human and mouse OA cartilage. SEPHS1 downregulation impairs the cellular capacity to synthesize a class of selenoproteins with oxidoreductase functions in chondrocytes, thereby elevating the level of reactive oxygen species (ROS) and facilitating chondrocyte senescence. Cartilage-specific Sephs1 knockout in adult mice causes aging-associated OA, and augments post-traumatic OA, which is rescued by supplementation of N-acetylcysteine (NAC). Selenium-deficient feeding and Sephs1 knockout have synergistic effects in exacerbating OA pathogenesis in mice. Therefore, we propose that SEPHS1 is an essential regulator of selenium metabolism and redox homeostasis, and its dysregulation governs the progression of OA.

Selenoprotein H is an essential regulator of redox homeostasis that cooperates with p53 in development and tumorigenesis.

Selenium, an essential micronutrient known for its cancer prevention properties, is incorporated into a class of selenocysteine-containing proteins (selenoproteins). Selenoprotein H (SepH) is a recently identified nucleolar oxidoreductase whose function is not well understood. Here we report that seph is an essential gene regulating organ development in zebrafish. Metabolite profiling by targeted LC-MS/MS demonstrated that SepH deficiency impairs redox balance by reducing the levels of ascorbate and methionine, while increasing methionine sulfoxide. Transcriptome analysis revealed that SepH deficiency induces an inflammatory response and activates the p53 pathway. Consequently, loss of seph renders larvae susceptible to oxidative stress and DNA damage. Finally, we demonstrate that seph interacts with p53 deficiency in adulthood to accelerate gastrointestinal tumor development. Overall, our findings establish that seph regulates redox homeostasis and suppresses DNA damage. We hypothesize that SepH deficiency may contribute to the increased cancer risk observed in cohorts with low selenium levels.

Essential Experimental Methods for Identifying Bonghan Systems as a Basis for Korean Medicine: Focusing on Visual Materials from Original Papers and Modern Outcomes.

In the 1960s, through studies on Korean Medicine, Bonghan Kim proposed the Bonghan systems (BS) as the anatomical reality of the acupuncture meridians based on various experimental data. Since 2002, several groups, mainly led by a team at Seoul National University, who renamed the BS as the primo vascular system (PVS), have published around 70 papers showing biological structures corresponding to the BS. However, it is still difficult for other researchers to find them, especially under the skin, which Bonghan Kim first reported as acupuncture points, due to similar-looking biological tissues, for example, the lymphatic vessels, and such artifacts as blood clots or fascia debris. To solve these drawbacks, we examined the main methods for identifying the BS by comparing the original papers with the modern outcomes in terms of the common physical/chemical characteristics of the BS. In addition, effective methods of staining and microscopic observations discovered by modern teams are synthetically explained using visual materials such as diagrams and photos. Through the essentially organized methods in this review paper, we suggest that one can find the BS under the skin as putative acupuncture points by tracing the intraexternal BS, from which a new Korean Medicine will be born.

Selenium utilization in thioredoxin and catalytic advantage provided by selenocysteine.

Thioredoxin (Trx) is a major thiol-disulfide reductase that plays a role in many biological processes, including DNA replication and redox signaling. Although selenocysteine (Sec)-containing Trxs have been identified in certain bacteria, their enzymatic properties have not been characterized. In this study, we expressed a selenoprotein Trx from Treponema denticola, an oral spirochete, in Escherichia coli and characterized this selenoenzyme and its natural cysteine (Cys) homologue using E. coli Trx1 as a positive control. (75)Se metabolic labeling and mutation analyses showed that the SECIS (Sec insertion sequence) of T. denticola selenoprotein Trx is functional in the E. coli Sec insertion system with specific selenium incorporation into the Sec residue. The selenoprotein Trx exhibited approximately 10-fold higher catalytic activity than the Sec-to-Cys version and natural Cys homologue and E. coli Trx1, suggesting that Sec confers higher catalytic activity on this thiol-disulfide reductase. Kinetic analysis also showed that the selenoprotein Trx had a 30-fold higher Km than Cys-containing homologues, suggesting that this selenoenzyme is adapted to work efficiently with high concentrations of substrate. Collectively, the results of this study support the hypothesis that selenium utilization in oxidoreductase systems is primarily due to the catalytic advantage provided by the rare amino acid, Sec.

Endogenous hydrogen sulfide production is essential for dietary restriction benefits.

Dietary restriction (DR) without malnutrition encompasses numerous regimens with overlapping benefits including longevity and stress resistance, but unifying nutritional and molecular mechanisms remain elusive. In a mouse model of DR-mediated stress resistance, we found that sulfur amino acid (SAA) restriction increased expression of the transsulfuration pathway (TSP) enzyme cystathionine γ-lyase (CGL), resulting in increased hydrogen sulfide (H2S) production and protection from hepatic ischemia reperfusion injury. SAA supplementation, mTORC1 activation, or chemical/genetic CGL inhibition reduced H2S production and blocked DR-mediated stress resistance. In vitro, the mitochondrial protein SQR was required for H2S-mediated protection during nutrient/oxygen deprivation. Finally, TSP-dependent H2S production was observed in yeast, worm, fruit fly, and rodent models of DR-mediated longevity. Together, these data are consistent with evolutionary conservation of TSP-mediated H2S as a mediator of DR benefits with broad implications for clinical translation. PAPERFLICK:

Primo vascular system in human umbilical cord and placenta.

The primo vascular system (PVS) has been observed in various animals such as mice, rats, rabbits, dogs, swine, and cow, but not in humans. In this work, we report on the observation of a human PVS on both the epithelial fascia and inside the blood vessels of the umbilical cord (UC). The main morphological characteristics of the primo vessels (PVs) and primo nodes (PNs) from the human UC were in agreement with those of the PVS in various animal organs, including the thicknesses and the transparency of the PVs, the sizes of the PNs, the broken-line arrangement of the rod-shaped nuclei, the sparse distribution of nuclei, and the presence of hollow lumens in the central inner parts of the PNs. It was rather surprising that the human PV was not thicker than the PVs from small animals. The difference between the PVS and blood/lymph vessels was confirmed using immunofluorescence staining of von Willebrand factor, CD31, LYVE-1, and D2-40. The positive expression of the PVS to proliferating cell nuclear antigen, a cell-proliferation marker, was consistent with the recent finding of very small embryonic-like stem cells in the PVS of mice.

Characterization of mammalian selenoprotein o: a redox-active mitochondrial protein.

Selenoproteins exhibit diverse biological functions, most of which are associated with redox control. However, the functions of approximately half of mammalian selenoproteins are not known. One such protein is Selenoprotein O (SelO), the largest mammalian selenoprotein with orthologs found in a wide range of organisms, including bacteria and yeast. Here, we report characterization of mammalian SelO. Expression of this protein could be verified in HEK 293T cells by metabolic labeling of cells with 75Se, and it was abolished when selenocysteine was replaced with serine. A CxxU motif was identified in the C-terminal region of SelO. This protein was reversibly oxidized in a time- and concentration-dependent manner in HEK 293T cells when cells were treated with hydrogen peroxide. This treatment led to the formation of a transient 88 kDa SelO-containing complex. The formation of this complex was enhanced by replacing the CxxU motif with SxxC, but abolished when it was replaced with SxxS, suggesting a redox interaction of SelO with another protein through its Sec residue. SelO was localized to mitochondria and expressed across mouse tissues. Its expression was little affected by selenium deficiency, suggesting it has a high priority for selenium supply. Taken together, these results show that SelO is a redox-active mitochondrial selenoprotein.

Evidence for the Primo Vascular System above the Epicardia of Rat Hearts.

We for the first time reported evidence for the existence of a novel network, a PVS, abovethe epicardium of the rat heart. (1) We were consecutively able to visualize the PVs and the PNs above the epicardial spaces of five rats' hearts by using Cr-Hx spraying or injection. (2) Hematoxylin and eosin (H&E) and toluidine blue staining of the PVs and the PNs showed that they consisted of a basophilic matrix; specifically the PNs contained several mast cells, some of which were degranulating into pericardial space. Also, 4', 6-diamidino-2 phenylindole (DAPI) images of the PVs and the PNs showed that they contained various kinds of cells. (3) Transmission electron microscopic (TEM) longitudinal image of the PVs showed that the sinuses contained many granules with high-electron-density cores in parallel with putative endothelial cells. (4) TEM images of the PNs demonstrated that they consisted of lumen-containing cells surrounded by fibers and that they had mast cells that were degranulating toward the epicardium of the rat heart. The above data suggest that mast-cells-containing novel network exists above the epicardium of the rat heart.

Toward a theory of the primo vascular system: a hypothetical circulatory system at the subcellular level.

This paper suggests a theoretical framework for the primo vascular system (PVS), a hypothetical circulatory system, in which extracellular DNA microvesicles interact to form and break down cell structures. Since Bonghan Kim reported the existence of Bonghan ducts and the SNU research team reinvestigated and named it the PVS, there has been series of studies trying to examine its structure and functions. In this paper, we hypothesize that the PVS is the network system in which extracellular DNA microvesicles circulate and interact at the subcellular level, forming and breaking down cell structures. This idea integrates A. Béchamp's idea of microzymas and Bonghan Kim's idea of sanals. A proof of this idea may complement modern medical theory, perhaps providing an essential clue for an alternative solution dealing with modern healthcare problem.

Composition of the extracellular matrix of lymphatic novel threadlike structures: is it keratin?

Background. The lumen of novel threadlike structures (NTSs) is enclosed by a single layer of endothelial cells surrounded by extracellular matrix (ECM). We hypothesized that collagen may be a component of the ECM associated with lymphatic NTSs. Methods. Six female New Zealand white rabbits were anesthetized, and the NTS structures within lymphatic vessels were identified by contrast-enhanced stereomicroscopy or alcian blue staining. Isolated NTS specimens were stained with acridine orange, YOYO-1, and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). The structural and molecular composition of the ECM was investigated using transmission electron microscopy (TEM), electrospray ionization-mass spectrometry, and proteomic analysis. Results. The lymph vessel wall was stained red by DiI, and rod-shaped nuclei were stained green by YOYO-1. The area surrounding the NTS was also stained red and contained green rod-shaped nuclei. TEM images showed that the NTS consisted of many ECM fibers and the ECM fibers appeared to be ~100 nm in diameter and had narrowly spaced striated bands. Proteomic analysis of the lymphatic NTS-associated ECM identified 4 proteins: keratin 10, cytokeratin 3, cytokeratin 12, and soluble adenylyl cyclase. Conclusion. The TEM study suggested that the lymphatic NTS-associated ECM did not contain collagen. This was confirmed by proteomic analysis, which showed that keratin was the major component of the ECM.

Novel threadlike structures may be present on the large animal organ surface: evidence in Swine model.

Background. The types of embryonic development probably provoke different paths of novel threadlike structure (NTS) development. The authors hypothesized that NTS may be easily observed on the surface of swine intestines by using trypan blue staining method and visualization under an optical microscope. Methods. General anesthesia was administered to 2 Yorkshire pigs. The abdominal walls of the pigs were carefully dissected along the medial alba. NTSs were identified on organ surfaces under a stereoscopic microscope after trypan blue staining. Isolated NTS specimens obtained from the large intestine were subjected to 4',6-diamidino-2-phenylindole (DAPI) staining and observed using the polarized light microscopy to confirm whether the obtained structure fits the definition of NTS. Results. We found elastic, semitransparent threadlike structures (forming a network structure) that had a milky-white color in situ and in vivo in swine large intestines. The samples showed distinct extinction of polarized light at every 90 degrees, and nucleus was shown to be rod shaped by DAPI staining, indicating that they meet the criteria of NTS. Conclusion. We used a swine model to demonstrate that NTS may be present on large animal organ surfaces. Our results may permit similar studies by using human specimens.

Visualization of the network of primo vessels and primo nodes above the pia mater of the brain and spine of rats by using Alcian blue.

By spraying and injecting Alcian blue into the lateral ventricle, we were able to visualize the network of the nerve primo vascular system above the pia mater of the brain and spine of rats. Staining these novel structures above the pia mater with 4',6-diamidino-2-phenylindole demonstrated that they coexisted in cellular and extracellular DNA forms. The cellular primo node consisted of many cells surrounded by rod-shaped nuclei while the extracellular primo node had a different morphology from that of a general cell in terms of DNA signals, showing granular DNA in a threadlike network of extracellular DNA. Also, differently from F-actin in general cells, the F-actin in the primo vessel was short and rod-shaped. Light and transmission electron microscopic images of the PN showed that the nerve primo vascular system above the pia mater of the brain and spine was a novel dynamic network, suggesting the coexistence of DNA and extracellular DNA. Based on these data, we suggest that a novel dynamic system with a certain function exists above the pia mater of the central nerve system. We also discuss the potential of this novel network system in the brain and spine as related to acupuncture meridians and neural regeneration.

Protocol for the observation of the primo vascular system in the lymph vessels of rabbits.

Molecular-level understanding of the structure and the functions of the lymphatic system has greatly enhanced the importance of this second circulation system, especially in connection with cancer metastasis and inflammation. Recently, a third circulatory system, the primo vascular system (PVS) was found in various parts of an animal's body, especially as threadlike structures floating in the lymphatic flow in lymph vessels. Although the medical significance of this emerging system will require much work in the future, at present, several important suggestions in connection with immune cells, stem cells, and cancer metastasis have already appeared. Experiments to observe the PVS in the lymph vessels near the caudal vena cava of rabbits and rats have been performed by several independent teams, but reproduction requires considerable skill and technical know-how. In this article, we provide a detailed protocol to detect the PVS inside the lymph vessels of a rabbit. Detection and isolation are the first steps in unraveling the physiological functions of the PVS, which awaits intensive research.

Growth of microgranules into cell-like structures in fertilized chicken eggs: hypothesis for a mitosis-free alternative pathway.

According to Bonghan Kim's theory of anatomical reality for acupuncture meridians, DNA microgranules known as Sanals are key functional components in the primo vascular system (formerly the Bonghan system). To investigate this issue, we developed a new system, an incubator bound to a phase-contrast microscope, in which we cultivated and then observed for 10 hours microgranules taken from 3-day-old chick embryos and from blastoderms of fertilized chicken eggs. With this system, we found that, over time, the microgranules grew in circular patterns to become cell-like structures. In the embryo specimens, we found two distinctive microgranule growths, which developed into cell-like structures over 10 hours. In the first case, a microgranule of about 1.0 µm in size developed into a 3.3-µm-sized cell-like structure, with a pattern of concentric circles. The growth rate of the diameter of the first microgranule was, on average, 0.23 µm/hour. In the second case, a 2.5-µm-sized microgranule developed into a 5.4-µm-sized cell-like structure, which also exhibited a pattern of concentric circles. The average growth rate of the diameter of the second microgranule was 0.31 µm/hour. In the blastoderm specimens from the fertilized chicken egg, we also found three distinctive concentric growths. Interestingly, one of the three blastoderm microgranules grew very quickly, from about 2.5 µm in size to about 5.5 µm in size during 5 minutes of incubation. This was followed by steady growth to about 7.0 µm in size during the next 10 hours of incubation. In the final step of our investigation, we confirmed that the cell-like structures that had grown from the microgranules stained by acridine orange had DNA signals. We believe that the data obtained with our experimental method provide a clue that a mitosis-free alternative pathway for cell formation may, indeed, exist. We also suggest that this new function of microgranules (Sanals) might be related with the acupuncture meridian called the primo vascular system.

Inhibition of pentylenetetrazole-induced seizure in mice by using a 4 Hz magnetic field: a comparative study with a 60 Hz magnetic field.

We investigated the comparative effects of 4 and 60 Hz magnetic fields on pentylenetetrazole (PTZ)-induced seizure in mice. For this study, we measured the latent time to seizure, seizure duration, and lethality induced by PTZ in mice exposed to 4 and 60 Hz magnetic fields (MF) for 30 min. Compared to sham-exposed controls, the latent time to tail twitching and seizure in the 4 Hz MF group was significantly decreased while the latent time to seizure in the 60 Hz MF group was significantly increased. The seizure duration in the 4 Hz MF group was significantly decreased while that in the 60 Hz MF group was significantly increased. More importantly, while the mice exposed to a 60 Hz MF experienced significantly increased lethality after seizure convulsion, those exposed to a 4 Hz MF showed no lethality, with a shortening of the duration of seizure. This beneficial effect of a 4 Hz MF on seizure has the same implication as the anti-oxidative effects of a 4 Hz MF observed in our previous work. The results of our current and previous works indicate that a 4 Hz MF may be used as a therapeutic physical agent for the treatment of oxidative stress-induced diseases, including seizure, with or without chemical drugs.

Expression of keratin 10 in rat organ surface primo-vascular tissues.

The primo-vascular system is described as the anatomical structure corresponding to acupuncture meridians and has been identified in several tissues in the body, but its detailed anatomy and physiology are not well understood. Recently, the presence of keratin 10 (Krt10) in primo-vascular tissue was reported, but this finding has not yet been confirmed. In this study, we compared Krt10 expression in primo-vascular tissues located on the surface of rat abdominal organs with Krt10 expression on blood and lymphatic vessels. Krt10 protein (approximately 56.5 kDa) was evaluated by western blot analysis and immunohistochemistry. Krt10 (IR) in the primo-node was visualized as patchy spots around each cell or as a follicle-like structure containing a group of cells. Krt10 IR was also identified in vascular and lymphatic tissues, but its distribution was diffuse over the extracellular matrix of the vessels. Thus Krt10 protein was expressed in all three tissues tested, but the expression pattern of Krt10 in primo-vascular tissue differed from those of blood and lymphatic vascular tissues, suggesting that structural and the regulatory roles of Krt10 in primo-vascular system are different from those in blood and lymphatic vessels.

Analyses of fruit flies that do not express selenoproteins or express the mouse selenoprotein, methionine sulfoxide reductase B1, reveal a role of selenoproteins in stress resistance.

Selenoproteins are essential in vertebrates because of their crucial role in cellular redox homeostasis, but some invertebrates that lack selenoproteins have recently been identified. Genetic disruption of selenoprotein biosynthesis had no effect on lifespan and oxidative stress resistance of Drosophila melanogaster. In the current study, fruit flies with knock-out of the selenocysteine-specific elongation factor were metabolically labeled with (75)Se; they did not incorporate selenium into proteins and had the same lifespan on a chemically defined diet with or without selenium supplementation. These flies were, however, more susceptible to starvation than controls, and this effect could be ascribed to the function of selenoprotein K. We further expressed mouse methionine sulfoxide reductase B1 (MsrB1), a selenoenzyme that catalyzes the reduction of oxidized methionine residues and has protein repair function, in the whole body or the nervous system of fruit flies. This exogenous selenoprotein could only be expressed when the Drosophila selenocysteine insertion sequence element was used, whereas the corresponding mouse element did not support selenoprotein synthesis. Ectopic expression of MsrB1 in the nervous system led to an increase in the resistance against oxidative stress and starvation, but did not affect lifespan and reproduction, whereas ubiquitous MsrB1 expression had no effect. Dietary selenium did not influence lifespan of MsrB1-expressing flies. Thus, in contrast to vertebrates, fruit flies preserve only three selenoproteins, which are not essential and play a role only under certain stress conditions, thereby limiting the use of the micronutrient selenium by these organisms.

Dietary selenium affects host selenoproteome expression by influencing the gut microbiota.

Colonization of the gastrointestinal tract and composition of the microbiota may be influenced by components of the diet, including trace elements. To understand how selenium regulates the intestinal microflora, we used high-throughput sequencing to examine the composition of gut microbiota of mice maintained on selenium-deficient, selenium-sufficient, and selenium-enriched diets. The microbiota diversity increased as a result of selenium in the diet. Specific phylotypes showed differential effects of selenium, even within a genus, implying that selenium had unique effects across microbial taxa. Conventionalized germ-free mice subjected to selenium diets gave similar results and showed an increased diversity of the bacterial population in animals fed with higher levels of selenium. Germ-free mice fed selenium diets modified their selenoproteome expression similar to control mice but showed higher levels and activity of glutathione peroxidase 1 and methionine-R-sulfoxide reductase 1 in the liver, suggesting partial sequestration of selenium by the gut microorganisms, limiting its availability for the host. These changes in the selenium status were independent of the levels of other trace elements. The data show that dietary selenium affects both composition of the intestinal microflora and colonization of the gastrointestinal tract, which, in turn, influence the host selenium status and selenoproteome expression.