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In Press, journal pre-proof : articles in press are accepted, peer reviewed articles that are not yet assigned to volumes/issues, but are citable using DOI.; International audience; Methionine (Met) can be oxidized to methionine sulfoxide (MetO), which exist as R- and S-diastereomers. Present in all three domains of life, methionine sulfoxide reductases (MSR) are the enzymes that reduce MetO back to Met. Most characterized among them are MSRA and MSRB, which are strictly stereospecific for the S- and R-diastereomers of MetO, respectively. While the majority of MSRs use a catalytic Cys to reduce their substrates, some employ selenocysteine. This is the case of mammalian MSRB1, which was initially discovered as selenoprotein SELR or SELX and later was found to exhibit an MSRB activity. Genomic analyses demonstrated its occurrence in most animal lineages, and biochemical and structural analyses uncovered its catalytic mechanism. The use of transgenic mice and mammalian cell culture revealed its physiological importance in the protection against oxidative stress, maintenance of neuronal cells, cognition, cancer cell proliferation, and the immune response. Coincident with the discovery of Met oxidizing MICAL enzymes, recent findings of MSRB1 regulating the innate immunity response through reversible stereospecific Met-R-oxidation of cytoskeletal actin opened up new avenues for biological importance of MSRB1 and its role in disease. In this review, we discuss the current state of research on MSRB1, compare it with other animal Msrs, and offer a perspective on further understanding of biological functions of this selenoprotein.

Accumulating reports have suggested that an inability to clear dead cells is a cause of inflammation and cancer and that the fascia is associated with cancer metastasis. Novel corpuscle-connected filiform structures (CCFSs) entangled on the fascia of the internal organs of rats were selectively visualized with high repeatability (93%) under vital staining with Janus Green B (JGB). Serial sections of CCFSs stained with haematoxylin and eosin and with Mattson Trichrome were microscopically examined. In parallel, the immunohistochemistry with CD31, Lyve 1, scanning electron microscopy, and transmission electron microscopy (TEM) were also used to investigate the novel properties of the CCFSs. All of the CCFSs enclosed JGB-stained granules, which was verified by the impromptu coupling of stereo and light microscopes and fluorescence-activated cell sorting. The functions of the CCFSs were discovered under fluorescence microscopy and confocal laser scanning microscopy to be implicated in cell death: fragmented DNAs and f-actins with extracellular DNAs. The dead cells in the CCFSs were verified by using terminal deoxynucleotidyl transferase dUTP nick end labelling and TEM. Moreover, the CCFS had a relatively-high concentration of calcium, a main element for cell death in tissue, which was measured in 2-dimensionally images using time-of-flight secondary ion mass spectrometry. Taken these results, we report for the first time that novel reticular CCFSs are widely entangled on the fascia of the internal organs of rats with the implication for cell death.

Classically activated pro-inflammatory macrophages are generated from naive macrophages by pro-inflammatory cues that dynamically reprogram their fuel metabolism toward glycolysis. This increases their intracellular reactive oxygen species (ROS) levels, which then activate the transcription and release of pro-inflammatory mediators. Our study on mice that lack methionine sulfoxide reductase (Msr)-B1 shows that the resulting partial loss of protein methionine reduction in pro-inflammatory macrophages creates a unique metabolic signature characterized by altered fuel utilization, including glucose and pyruvate. This change also associates with hyper-inflammation that is at least partly due to sustained oxidation of an exposed methionine residue (M44) on glyceraldehyde 3-phosphate dehydrogenase (GAPDH), thereby inducing GAPDH aggregation, inflammasome activation, and subsequent increased interleukin (IL)-1β secretion. Since MsrB1-knockout mice exhibit increased susceptibility to lipopolysaccharide (LPS)-induced sepsis, the MsrB1-GAPDH axis may be a key molecular mechanism by which protein redox homeostasis controls the metabolic profile of macrophages and thereby regulates their functions.

Selenium (Se) is an essential micronutrient for humans and animals and can have beneficial effects on plants. A low concentration of Se can improve plant growth; however, high concentrations can be...

The structure of human MICAL3 provides information about its mechanism., MICAL is an oxidoreductase that participates in cytoskeleton reorganization via actin disassembly in the presence of NADPH. Although three MICALs (MICAL1, MICAL2 and MICAL3) have been identified in mammals, only the structure of mouse MICAL1 has been reported. Here, the first crystal structure of human MICAL3, which contains the flavin-containing monooxygenase (FMO) and calponin-homology (CH) domains, is reported. MICAL3 has an FAD/NADP-binding Rossmann-fold domain for mono­oxygenase activity like MICAL1. The FMO and CH domains of both MICAL3 and MICAL1 are highly similar in structure, but superimposition of the two structures shows a different relative position of the CH domain in the asymmetric unit. Based on kinetic analyses, the catalytic efficiency of MICAL3 dramatically increased on adding F-actin only when the CH domain was available. However, this did not occur when two residues, Glu213 and Arg530, were mutated in the FMO and CH domains, respectively. Overall, MICAL3 is structurally highly similar to MICAL1, which suggests that they may adopt the same catalytic mechanism, but the difference in the relative position of the CH domain produces a difference in F-actin substrate specificity.

International audience; Methionine oxidation is involved in regulating the protein activity and often leads to protein malfunction. However, tools for quantitative analyses of protein-specific methionine oxidation are currently unavailable. In this work, we developed a biological sensor that quantifies oxidized methionine in the form of methionine-R-sulfoxide in target proteins. The biosensor "tpMetROG" consists of methionine sulfoxide reductase B (MsrB), circularly permuted yellow fluorescent protein (cpYFP), thioredoxin, and protein G. Protein G binds to the constant region of antibodies against target proteins, specifically capturing them. Then, MsrB reduces the oxidized methionine in these proteins, leading to cpYFP fluorescence changes. We assessed this biosensor for quantitative analysis of methionine-R-sulfoxide in various proteins, such as calmodulin, IDLO, LegP, Sacde, and actin. We further developed an immunosorbent assay using the biosensor to quantify methionine oxidation in specific proteins such as calmodulin in animal tissues. The biosensor-linked immunosorbent assay proves to be an indispensable tool for detecting methionine oxidation in a protein-specific manner. This is a versatile tool for studying the redox biology of methionine oxidation in proteins.

Lactococcus lactis subsp. cremoris is a lactic acid bacterium commonly used in the cheese manufacturing industry. It is known to produce antibacterial peptides and has recently received attention for its role as a probiotic strain. Here, we report the isolation of a new strain, Lactococcus lactis subsp. cremoris RPG-HL-0136 (RPG0136) from dried compost, which exhibits strong antibacterial activity. When RPG0136 was fed to mice, it increased the intestinal population of two beneficial bacteria, Lactobacillus and Bifidobacterium, whereas it decreased the intestinal population of two harmful bacteria, Bacteroides and Enterobacter. In addition, it increased the concentration of short-chain fatty acids, including acetic acid, propionic acid, and butyric acid, with a simultaneous decrease in pH, and accelerated the catabolic degradation of proteins, lipids, and starch. Lastly, RPG0136 increased the plasma IgG and intestinal mucosal SIgA concentrations and upregulated Reg3r, MUC1, and MUC2 expression to improve the intestinal mucosal immune function. The results of this study suggest that RPG0136 is a potential probiotic strain that supports the growth of a beneficial microbiome by promoting the synthesis of organic acids and enhancing intestinal immune function.

Wheat is one of the most important grains. Its consumption is increasing globally. Many countries are making efforts to increase the extent of wheat harvest. It is known that plant growth promoting rhizobacteria (PGPRs) have beneficial effects on various plants. Two PGPRs including Paenibacillus pabuli strain P7S (PP7S) and Pseudomonas nitroreducens strain IHB (PnIHB) were employed to investigate effects of PGPRs on early growth of three wheat cultivars (Koso, Seakumkang, and Jokyung). While PP7S had adverse effects on Seakumkang and Jokyung, PP7S had positive effects on Koso except root length compared to control group having no treatment of PP7S. However, all treatments with PnIHB had adverse effects on germination rate, root/shoot lengths, vigor index, and dry root/shoot weights of all three wheat cultivars. These positive effects with PP7S on Koso might be related to the earlier emergence of wheat seed above soil which is known to be an indicator of increased yield. Results of the present study suggest that if proper PGPR strains are selected, they could have positive effects on early growth rate of a wheat cultivar.

Microglia are the macrophages that reside in the brain. Activated microglia induces further activation of astrocytes and neuronal cells for mounting an immune response. However, activated microglia release neurotoxic mediators causing neuroinflammation, which is associated with chronic etiology of neurodegenerative diseases. We investigated the effect of ethanol extract of Physalis alkekengi L. var. francheti fruit (PAFE) on neuronal cell death mediated by activated microglia. PAFE decreased NO production and IL-6 secretion in LPS-stimulated BV-2 and primary microglial cells without reducing cell viability. Consistently, treatment with PAFE decreased iNOS and COX-2 expression and ERK phosphorylation in LPS-stimulated BV-2 cells. Finally, apoptosis of N2a cells grown in conditioned media prepared from LPS-stimulated BV-2 cells containing PAFE was inhibited via downregulation of the Bax/Bcl-2 ratio. Taken together, PAFE alleviates neuronal cell death by reducing neurotoxic mediators such as NO and IL-6 from activated microglia. Therefore, it could be a potential candidate to treat neurodegenerative diseases caused by chronic neuroinflammation.

International audience; Aberrant production of reactive oxygen species (ROS) leads to tissue damage accumulation, which is associated with a myriad of human pathologies. Although several sensors have been developed for ROS quantification, their applications for ROS-related human physiologies and pathologies still remain problematic due to the unstable nature of ROS. Herein, we developed Trx1-cpYFP-fRMsr (TYfR), a genetically-encoded fluorescent biosensor with the remarkable specificity and sensitivity toward fMetRO (free Methionine-R-sulfoxide), allowing for dynamic quantification of physiological levels of fMetRO, a novel indicator of ROS and methionine redox status in vitro and in vivo. Moreover, using the sensor, we observed a significant fMetRO enrichment in serum from patients with acute coronary syndrome, one of the most severe cardiovascular diseases, which becomes more evident following percutaneous coronary intervention. Collectively, this study proposes that fMetRO is a novel biomarker of tissue damage accumulation in ROS-associated human pathologies, and that TYfR is a promising tool for quantifying fMetRO with potentials in versatile applications.

Wheat leaf rust caused by Puccinia triticina is a destructive fungal disease causing considerable grain yield loss. In this study, we developed a novel assay to test the rust resistance of detached wheat leaves on defined media with retarded senescence. We observed that salicylic and jasmonic acid confer leaf rust resistance to a susceptible Keumkang wheat (Triticum aestivium L.). Transcription analysis revealed that atchi8 was highly expressed with an increased chitinase activity in the salicylic acid-treated leaves, while expression of PR-9, atpodL, and PR-5 increased in the jasmonic acid-treated leaves. Additionally, the metabolic profile suggested that the phenylalanine pathway might link flavonoid production to leaf rust resistance in the salicylic acid-treated leaves, while the alanine, aspartate, and glutamate metabolism might control the production of other amino acids to enhance pathogen stress response in the jasmonic acid-treated leaves. Finally, all identified genes and metabolites could be potential targets for screening chemical compounds for leaf rust resistance. Future studies on the underlying mechanisms of leaf rust resistance obtained by exogenous treatment of salicylic and jasmonic acids remain necessary.

Wheat yield is decreasing due to climate change, and a method to prevent decreasing yield during drought stress is desirable. In this study, wheat cultivars (Koso and Jokyung) were treated with 15% polyethylene glycol-6000 (PEG) and PEG + Si solution (6.5, 8.7, 13.1 and 26.1 mM). The effect of Si treatment on the alleviation of drought stress was measured using the germination test, shoot relative water content (RWC), seedling stage observation, and quantitative real time polymerase chain reaction (qRT-PCR). The results of root/shoot length ratio and shoot length ratio showed that Si treatment induced the alleviation of drought stress in Jokyung cultivar. The result of qRT-PCR showed the alleviation of drought stress in Koso cultivar. In addition, the results with shoot RWC and seedling stage observation showed that the alleviation effects of Si treatment was observed with both Koso and Jokyung cultivar at the high concentration of Si (26.1 mM). All these results suggest that Si treatment at a high concentration could be employed to alleviate drought stress in wheat.

Aegilops tauschii (Ae. tauschii) is a diploid (2n = 2x = 14) wild grass species, which has been reported as the progenitor of hexaploid wheat (Triticum aestivum) with D-genome. In this study, 68 Ae. tauschii accessions with diverse geographical backgrounds were investigated for their resistance to infection by the leaf rust fungi Puccinia triticina. Two Ae. tauschii accessions that exhibited hyper-resistance to leaf rust at both seedling and adult stages were identified. Utilizing two susceptible Ae. tauschii ecotypes and keumkang, a common Korean wheat cultivar known to be susceptible to leaf rust, as the negative control, further investigations were conducted for understanding the mechanism underlying immunity to leaf rust disease of these two resistant accessions. Resistant accessions displayed the increased β-1,3-glucanase activity to prevent fungal penetration and the better peroxidase activity to cope with leaf rust-induced oxidative stress. Moreover, transcriptional analyses reveal the important role of the LRR receptor-like serine/threonine-protein kinase FLS2 (lrr) to the disease resistance of the two ecotypes. Ae. tauschii is a remarkable genetic source, especially for abiotic and biotic stress resistance genes, as the plant is known for its wide-ranging geographical habitat and adaptability to different environments. This, combined with the fact that Ae. tauschii and wheat share a close evolutionary relationship, is indicative of the immense benefit of using Ae. tauschii as a material for improving the quality of synthetic wheat. Our aim was to identify and evaluate the strongest Ae. tauschii contenders for breeding leaf rust-resistant synthetic wheat.

Sphingolipids have been implicated in the etiology of chronic metabolic diseases. Here, we investigated whether sphingolipid biosynthesis is associated with the development of adipose tissues and metabolic diseases. SPTLC2, a subunit of serine palmitoyltransferase, was transcriptionally upregulated in the adipose tissues of obese mice and in differentiating adipocytes. Adipocyte-specific SPTLC2-deficient (aSPTLC2 KO) mice had markedly reduced adipose tissue mass. Fatty acids that were destined for the adipose tissue were instead shunted to liver and caused hepatosteatosis. This impaired fat distribution caused systemic insulin resistance and hyperglycemia, indicating severe lipodystrophy. Mechanistically, sphingosine 1-phosphate (S1P) was reduced in the adipose tissues of aSPTLC2 KO mice, and this inhibited adipocyte proliferation and differentiation via the downregulation of S1P receptor 1 and decreased activity of the peroxisome proliferator–activator receptor γ. In addition, downregulation of SREBP (sterol regulatory element–binding protein)-1c prevented adipogenesis of aSPTLC2 KO adipocytes. Collectively, our observations suggest that the tight regulation of de novo sphingolipid biosynthesis and S1P signaling plays an important role in adipogenesis and hepatosteatosis.

Endogenously produced hydrogen sulfide was proposed to be an underlying mechanism of lifespan extension via methionine restriction. However, hydrogen sulfide regulation and its beneficial effects via methionine restriction remain elusive. Here, we identified the genes required to increase hydrogen sulfide production under methionine restriction condition using genome-wide high-throughput screening in yeast strains with single-gene deletions. Sulfate assimilation-related genes, such as MET1, MET3, MET5, and MET10, were found to be particularly crucial for hydrogen sulfide production. Interestingly, methionine restriction failed to increase hydrogen sulfide production in mutant strains; however, it successfully extended chronological lifespan and reduced reactive oxygen species levels. Altogether, our observations suggested that increased hydrogen sulfide production via methionine restriction is not the mechanism underlying extended yeast lifespan, even though increased hydrogen sulfide production occurred simultaneously with yeast lifespan extension under methionine restriction condition.

Dietary restriction (DR) without malnutrition is associated with longevity in various organisms. However, it has also been shown that reduced calorie intake is often ineffective in extending life span. Selecting optimal dietary regimens for DR studies is complicated, as the same regimen may lead to different outcomes depending on genotype and environmental factors. Recent studies suggested that interventions such as moderate protein restriction with or without adequate nutrition (e.g., particular amino acids or carbohydrates) may have additional beneficial effects mediated by certain metabolic and hormonal factors implicated in the biology of aging, regardless of total calorie intake. In particular, it was shown that restriction of a single amino acid, methionine, can mimic the effects of DR and extend life span in various model organisms. We discuss the beneficial effects of a methionine-restricted diet, the molecular pathways involved, and the use of this regimen in longevity interventions.

Selenium is a vital trace element present as selenocysteine (Sec) in proteins that are, thus, known as selenoproteins. Humans have 25 selenoproteins, most of which are functionally characterized as oxidoreductases, where the Sec residue plays a catalytic role in redox regulation and antioxidant activity. Glutathione peroxidase plays a pivotal role in scavenging and inactivating hydrogen and lipid peroxides, whereas thioredoxin reductase reduces oxidized thioredoxins as well as non-disulfide substrates, such as lipid hydroperoxides and hydrogen peroxide. Selenoprotein R protects the cell against oxidative damage by reducing methionine-R-sulfoxide back to methionine. Selenoprotein O regulates redox homeostasis with catalytic activity of protein AMPylation. Moreover, endoplasmic reticulum (ER) membrane selenoproteins (SelI, K, N, S, and Sel15) are involved in ER membrane stress regulation. Selenoproteins containing the CXXU motif (SelH, M, T, V, and W) are putative oxidoreductases that participate in various cellular processes depending on redox regulation. Herein, we review the recent studies on the role of selenoproteins in redox regulation and their physiological functions in humans, as well as their role in various diseases.

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. 75Se 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.

Summary 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 (H 2 S) production and protection from hepatic ischemia reperfusion injury. SAA supplementation, mTORC1 activation, or chemical/genetic CGL inhibition reduced H 2 S production and blocked DR-mediated stress resistance. In vitro, the mitochondrial protein SQR was required for H 2 S-mediated protection during nutrient/oxygen deprivation. Finally, TSP-dependent H 2 S 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 H 2 S as a mediator of DR benefits with broad implications for clinical translation. PaperFlick

Thermotolerance in plants is a topic of concern given the current trends in global warming. Here, we aimed to develop a rapid and reproducible screening method for selection of heat stress-tolerant wheat varieties to expedite the breeding process. We tested the robustness of the screen in three Korean wheat cultivars, “BackJung”, “KeumKang”, and “ChoKyeong”. We showed that 4-day-old seedlings of “KeumKang” had the highest survival rates after a 45 °C treatment for 20 h. Moreover, the ability to retain chlorophyll and antioxidant activity was also highest in “KeumKang”. The increase in malondialdehyde content in “ChoKyeong” indicated that this cultivar showed the greatest damage after heat stress. Collectively, our results showed that “KeumKang” is the most heat-tolerant cultivar of the three examined. In conclusion, the most reliable and rapid screening method in our investigation was survival rate examined at lethal temperature.

Accumulation of oxidized amino acids, including methionine, has been implicated in aging. The ability to reduce one of the products of methionine oxidation, free methionine-R-sulfoxide (Met-R-SO), is widespread in microorganisms, but during evolution this function, conferred by the enzyme fRMsr, was lost in metazoa. We examined whether restoration of the fRMsr function in an animal can alleviate the consequences of methionine oxidation. Ectopic expression of yeast fRMsr supported the ability of Drosophila to catalyze free Met-R-SO reduction without affecting fecundity, food consumption, and response to starvation. fRMsr expression also increased resistance to oxidative stress. Moreover, it extended lifespan of flies in a methionine-dependent manner. Thus, expression of an oxidoreductase lost during evolution can enhance metabolic and redox functions and lead to an increase in lifespan in an animal model. More broadly, our study exposes the potential of a combination of genetic and nutritional strategies in lifespan control.

It has been reported that water at the interface of a hydrophilic thin film forms an exclusion zone, which has a higher density than ordinary water. A similar phenomenon was observed for a hydrated hydrophilic ceramic powder, and water turns into a three-dimensional cell-like structure composed of high density water and low density water. This structured water appears to have a stimulative effect on plant growth. This report outlines our study of antioxidant properties of this structured water and its effect on cell bioactivities. Culturing media which were prepared utilizing this antioxidant structured water promoted the viability of RAW 264.7 macrophage cells by up to three times. The same tendency was observed for other cells including IEC-6, C2C12, and 3T3-L1. Also, the cytokine expression of the splenocytes taken from a mouse spleen increased in the same manner. The water also appears to suppress the viability of cancer cell, MCF-7. These results strongly suggest that the structured water helps the activities of normal cells while suppressing those of malignant cells.

Transdermal drug delivery is the way to transport drug carriers, such as nanoparticles, across the skin barrier to the dermal and/or subcutaneous layer. In order to control the transdermal drug delivery process, based on the heterogeneous and nonlinear structures of the skin tissues, we developed a novel electromechanical method combining in vivo local skin impedance probing, subcutaneous micro-injection of colloidal nanoparticles, and transcutaneous electrical stimulation. Experiments on the nude mice using in vivo fluorescence imaging exhibited significantly different apparent diffusion patterns of the nanoparticles depending on the skin impedance: Anisotropic and isotropic patterns were observed upon injection into low and high impedance points, respectively. This result implies that the physical complexity in living tissues may cause anisotropic diffusion of drug carriers, and can be used as a parameter for controlling drug delivery process. This method also can be combined with microneedle-based drug release systems, micro-fabricated needle-electrodes, and/or advanced in vivo targeting/imaging technologies using nanoparticles.

International audience; The human genome contains 25 genes coding for selenocysteine-containing proteins (selenoproteins). These proteins are involved in a variety of functions, most notably redox homeostasis. Selenoprotein enzymes with known functions are designated according to these functions: TXNRD1, TXNRD2, and TXNRD3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4, and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine sulfoxide reductase B1), and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein-encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15-kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP), SELENOT (selenoprotein T, SELT), SELENOV (selenoprotein V, SELV), and SELENOW (selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing, and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates.

After incubation with Janus Green B in the peritoneal cavities of rats, lymph-vessel-like structures (LSs) were noticed under a stereomicroscope to run parallel to each other with rectangular branches on the inner abdominal wall; rarely were these LSs seen to connect with peritoneal LSs. These LSs were identified by using fluorescence microscopy (FM) at a magnification of 1,000X and confocal laser scanning microscopy (CLSM) to be lymph-vessel-like bundle structures (LBSs). Serial cross-sections of these LBSs were microscopically examined by using hematoxylin and eosin staining, Mattson trichrome staining, terminal deoxynucleotidyl transferase dUTP nick end labeling, and immunohistochemistry (IHC) with Lyve 1 and CD 31. The histology data from these LBSs revealed such novel characteristics as parallel clusters of live cells wrapped by collagen fibers of the fascia and an IHC different from those of lymphatic and blood vessels, being the novel bundle structures (NBSs). Under FM and CLSM with optical sections, some of the NBSs were observed to be able to swell like tiny balloons, implying that the bundle structures were hollow. Moreover, transmission electron microscopy images of two different cross-sections of an NBS showed it to be composed of four parallel tubules involving three kinds of sinuses with neither axons nor Schwann cells in the outermost wall, thus being a novel tubular-bundle structure (NTBS). The results of this research make evident with high repeatability (10/11) that beyond the orthodoxy of a single-tube system of blood and lymph vessels, a system of NTBSs is widely entangled in the fascia of the inner abdominal wall of a rat. Thus, the author suggests that NTBS-related functions and the entire NTBS network should be explored.

Bread wheat (Triticum aestivum L.) is a popular cereal crop worldwide, but its future use is threatened by its limited genetic diversity because of the evolutionary bottleneck limiting its ability to combat abiotic and biotic stresses. However, synthetic hexaploid wheat (SHW) is known for its genetic diversity resulting from of the artificial crossing used to transfer elite genes from donors. SHW is therefore a potential source for genetic variations to combat stress. We studied two SHW lines from CYMMIT (cSHW339464 and cSHW339465) and a Korean bread wheat (cv. KeumKang) to determine their ability to tolerate heat stress and leaf rust infection. Our results showed that cSHW339464 could tolerate heat stress because of its maintained-green phenotype, high accumulation of anthocyanin, antioxidant activity (DPPH), proline content, and the response of heat-shock proteins after being challenged by heat stress. On the other hand, cSHW339465 is resistant to leaf rust and can inhibit the growth of pathogens on the leaf surface, owing to the induction of genes encoding β-1,3-glucanase and peroxidase and subsequent enzyme activities. In conclusion, these two SHW lines could prove good candidates contributing to the improvement of current wheat resources.

Redox control of protein function involves oxidation and reduction of amino acid residues, but the mechanisms and regulators involved are insufficiently understood. Here, we report that in conjunction with Mical proteins, methionine-R-sulfoxide reductase B1 (MsrB1) regulates mammalian actin assembly via stereoselective methionine oxidation and reduction in a reversible, site-specific manner. Two methionine residues in actin are specifically converted to methionine-R-sulfoxide by Mical1 and Mical2 and reduced back to methionine by selenoprotein MsrB1, supporting actin disassembly and assembly, respectively. Macrophages utilize this redox control during cellular activation by stimulating MsrB1 expression and activity as a part of innate immunity. We identified the regulatory role of MsrB1 as a Mical antagonist in orchestrating actin dynamics and macrophage function. More generally, our study shows that proteins can be regulated by reversible site-specific methionine-R-sulfoxidation.

We found evidence that spontaneous self-assembly of DNA molecules from yolk granules occurred during the very early stage of egg fertilization. In order to find solid evidence for self-assembly of DNA molecules, we collected many available data in different stages of fertilized eggs, making a data table. At first by using acridine orange vital staining to demonstrate DNA, we noticed that some yolk granules emitted DNA signals that gradually increased with increasing incubation time from very small sizes to much larger nucleus-like structures. For convincing evidence, we also used another vital dye, Hoechst 33258 DNA-specific dye, to trace the changes in the yolk granules. The patterns of the DNA signals from yolk granules stained with Hoechst 33258 were the same as those from the yolk granules stained with acridine orange. A partial phase contrast microscopic image of the changes in the yolk granules showed some liquid-like material around the granules before the formation of the nucleus-like structures. Concomitant use of fluorescence and partial phase contrast microscopy suggested that these liquid-like materials may have been released from yolk granules in which spontaneous self-assembly of DNA molecules had occurred. Finally, in order to verify whether the DNA signals came from real DNA molecules or not, by using deoxyribonuclease I (DNAse), we confirmed that the nucleus-like structures were really assembled DNA molecules. Thus, in this article, we report evidence for the self-assembly of DNA molecules toward cell-like structures and discuss our findings, comparing them with those in the works of other pioneers, especially Antoine Bechamp, Olga Lepeshinskaya and Bong Han Kim, who insisted on the existence of a mitosis-free alternative pathway for generating new cells.

Novel threadlike structures (NTSs) on the surfaces of mammalian abdominal organs have recently attracted interests regarding their ability to transport fluid, enable cell migration, and possibly facilitate cancer metastasis. Nevertheless, histological studies of NTSs have been sporadic and often have inconsistent interpretations of the NTS internal structure. In this article, we provide a synthetic and consistent view of the NTS internal structure: the NTS is a loose bundle of fibrous stroma that forms interstitial channels and microsinusoids infiltrated with inflammatory cells. The fibroblasts are embedded in the stroma and mostly aligned along the major axis of the NTS. The sinusoids, which are in inconsecutive cross sections, have boundaries more or less delineated by extracellular fibers, partly surrounded by endothelial-like cells, or both. We compare these morphological features to other well-known connective tissues (i.e., trabecular meshwork and lymphatic capillary) and discuss the biomechanical and biological functions of NTSs based on their structural characteristics.

With a combination of cultivation and phase-contrast and fluorescence microscopic observation, we first found that fusion of extracellular vesicles with or without membranes occurred in fertilized chicken eggs. In order to find solid evidence for fusion, we collected many fusion data from various tissues; primo vessels and pancreases of mice and pancreases and omentums of rats. Especially, by using acridine orange vital staining to demonstrate DNA and phase-contrast and fluorescence microscopy for long real-time observation, we found that many of the extracellular vesicles involved in the fusion process contained DNAs. The fusions fall into two main patterns: pattern A characterizes a fusion of less agitated extracellular vesicles without membranes. Pattern B is a fusion of vigorously vibrating extracellular vesicles in a certain membrane. Considering all data, tables, pictures and movies, we were able to show fusions of DNA extracellular vesicles without or with membranes in several tissues of three species. Interestingly, some of the fused structures share the same morphology as normal cell's in terms of overall shape, size and DNA signals in the center. Thus, in this article we first report the evidence for the fusion of extracellular vesicles with/without DNA toward a specific structure and discuss our findings by comparing with those of other pioneer's works in search for a mitosis-free alternative pathway for generating new cells.

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.

© 2016, National Academy of Sciences. All rights reserved. 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.

Reactive oxygen species (ROS) oxidize methionine to a mixture of methionine-S-sulfoxide and methionine-R-sulfoxide. Methionine is also oxidized by various flavin-containing monooxygenases in a partially or fully stereospecific manner. Methionine sulfoxide can be reduced by methionine sulfoxide reductase (Msr) proteins in four different families. Both free and protein-based forms of methionine-S-sulfoxide are reduced by MsrA, whereas protein-based methionine-R-sulfoxide is reduced by MsrB. Among the three known mammalian MsrBs, only MsrB1 is a selenoprotein, containing a selenocysteine residue in place of the catalytic cysteine. Free methionine-R-sulfoxide reductase (fRMsr) reduces methionine-R-sulfoxide in the free, but not protein-based state, and is found only in unicellular organisms, whereas MsrA and MsrB are present in organisms in all three kingdoms of life. MsrP reduces both methionine-S-sulfoxide and methionine-R-sulfoxide, particularly in membrane-embedded proteins, with assistance from MsrQ, which is involved in electron transport in the mitochondrial respiratory chain. MsrP cannot utilize thioredoxin as a reducing agent. Msrs are oxidoreductases that protect against the effects of oxidative stress by increasing oxidative stress resistance and repairing damaged proteins via cyclic methionine oxidation/reduction. In addition to its two main functions, MsrB1 reversibly regulates actin assembly in conjunction with Mical, making methionine oxidation similar to other reversible posttranslational modifications. This finding highlights a new era in the understanding of Msr function and should facilitate further studies of the physiological role of Msrs.

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.