The Micacocidin Production-Related RSc1806 Deletion Alters the Quorum Sensing-Dependent Gene Regulation of Ralstonia pseudosolanacearum Strain OE1-1.

The soil-borne phytopathogenic gram-negative bacterium Ralstonia solanacearum species complex (RSSC) produces staphyloferrin B and micacocidin as siderophores that scavenge for trivalent iron (Fe3+) in the environment, depending on the intracellular divalent iron (Fe2+) concentration. The staphyloferrin B-deficient mutant reportedly retains its virulence, but the relationship between micacocidin and virulence remains unconfirmed. To elucidate the effect of micacocidin on RSSC virulence, we generated the micacocidin productivity-deficient mutant (ΔRSc1806) that lacks RSc1806, which encodes a putative polyketide synthase/non-ribosomal peptide synthetase, using the RSSC phylotype I Ralstonia pseudosolanacearum strain OE1-1. When incubated in the condition without Fe2+, ΔRSc1806 showed significantly lower Fe3+-scavenging activity, compared with OE1-1. Until 8 days after inoculation on tomato plants, ΔRSc1806 was not virulent, similar to the mutant (ΔphcA) missing phcA, which encodes the LysR-type transcriptional regulator PhcA that regulates the expression of the genes responsible for quorum sensing (QS)-dependent phenotypes including virulence. The transcriptome analysis revealed that RSc1806 deletion significantly altered the expression of more than 80% of the PhcA-regulated genes in the mutant grown in medium with or without Fe2+. Among the PhcA-regulated genes, the transcript levels of the genes whose expression was affected by the deletion of RSc1806 were strongly and positively correlated between the ΔRSc1806 and the phcA-deletion mutant. Furthermore, the deletion of RSc1806 significantly modified QS-dependent phenotypes, similar to the effects of the deletion of phcA. Collectively, our findings suggest that the deletion of micacocidin production-related RSc1806 alters the regulation of PhcA-regulated genes responsible for QS-dependent phenotypes including virulence as well as Fe3+-scavenging activity. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Disproof of the Structures and Biosynthesis of Ergoynes, Gs-Polyyne-l-Ergothioneine Cycloadducts from Gynuella sunshinyii YC6258.

Some bacteria produce "bacterial polyynes" bearing a conjugated C≡C bond that starts with a terminal alkyne. Ergoynes A and B have been reported as sulfur-containing metabolites from Gynuella sunshinyii YC6258. These compounds were thought to be formed by cycloaddition between a bacterial polyyne (named Gs-polyyne) and l-ergothioneine. The biosynthetic gene clusters (BGCs), which may contribute to their synthesis, were present in the YC6258 genome. The biosynthetic origin of Gs-polyyne is interesting considering its rare 2-isopentyl fatty acyl skeleton. Here, the structures and biosynthesis of Gs-polyyne and ergoynes were verified by analytical, chemical, and genetic techniques. In the YC6258 extract, which was prepared considering their instability, Gs-polyyne was detected as a major LC peak, and ergoynes were not detected. The NMR data of the isolated Gs-polyyne contradicted the proposed structure and identified it as the previously reported protegenin A. The expression of Gs-polyyne BGC in Escherichia coli BL21(DE3) also yielded protegenin A. The cyclization between protegenin A and l-ergothioneine did not proceed during sample preparation; a base, such as potassium carbonate, was required. Overall, Gs-polyyne was identified as protegenin A, while ergoynes were determined to be artifacts. This cyclization may provide a derivatization to stabilize polyynes or create new chemical space.

Cycloheximide in the nanomolar range inhibits seed germination of Orobanche minor.

From the 992 samples of culture extracts of microorganisms isolated from soil in Japan, we found that the extract of Streptomyces sp. no. 226 inhibited Orobanche minor seed germination without significantly affecting the seed germination of Trifolium pratense and the growth of Aspergillus oryzae and Escherichia coli. Using ESI-MS, 1H-NMR, and 13C-NMR, we identified the active compound as cycloheximide. Cycloheximide had half-maximum inhibitory concentrations of 2.6 ng/mL for the inhibition of seed germination of O. minor and 2.5 µg/mL for that of the conidial germination of A. oryzae. Since cycloheximide is known to inhibit translation by interacting with ribosomal protein L28 (RPL28) in yeast, we investigated whether RPL protein of O. minor plays a critical role in the inhibition of O. minor seed germination. Our data suggested that O. minor RPL27A was not sensitive to cycloheximide by comparing it to the strain expressing S. cerevisiae RPL28. These findings suggest the presence of an unidentified mechanism by which cycloheximide hinders O. minor seed germination.

Structures and Biosynthesis of Caryoynencins, Unstable Bacterial Polyynes from Pseudomonas protegens Recombinant Expressing the cayG Gene.

Bacteria in certain genera can produce "bacterial polyynes" that contain a conjugated C≡C bond starting from a terminal alkyne. Protegenin A is a derivative of octadecanoic acid that contains an ene-tetrayne moiety. It was discovered in Pseudomonas protegens Cab57 and exhibits strong antioomycete and moderate antifungal activity. By introducing cayG, a cytochrome P450 gene from Burkholderia caryophylli, into P. protegens Cab57, protegenin A was converted into more complex polyynes, caryoynencins A-E. A purification method that minimized the degradation and isomerization of caryoynencins was established. For the first time, as far as we know, the 1H and 13C{1H} NMR signals of caryoynencins were completely assigned by analyzing the NMR data of the isolated compounds and protegenin A enriched with [1-13C]- or [2-13C]-acetate. Through the structural analysis of caryoynencins D/E and bioconversion experiments, we observed that CayG constructs the allyl alcohol moiety of caryoynencins A-C through sequential hydroxylation, dehydration, and hydroxylation. The recombinant strain exhibited a stronger antioomycete activity compared to the wild-type strain. This paper proposes a stable purification and structural determination method for various bacterial polyynes, and P. protegens Cab57 holds promise as an engineering host for the production of biologically active polyynes.

Astaxanthin protects human ARPE-19 retinal pigment epithelium cells from blue light-induced phototoxicity by scavenging singlet oxygen.

Age-related macular degeneration (AMD) is one of an increasing number of diseases that causes irreversible impairment and loss of vision in the elderly. AMD occurs by oxidative stress-mediated apoptosis of retinal pigment epithelium cells. The onset of AMD may be positively correlated with the exposure to blue light. We screened food-derived carotenoids for cytoprotective action against blue light irradiation using human ARPE-19 retinal pigment epithelium cells. This study revealed that blue light irradiation triggered apoptosis and oxidative stress in all-trans-retinal (atRAL)-exposed ARPE-19 cells by generating singlet oxygen (1O2), leading to significant cell death. We found that astaxanthin, a potent anti-oxidative xanthophyll abundant in several marine organisms including microalgae, salmon, and shrimp, significantly suppresses blue light-induced apoptotic cell death of atRAL-exposed ARPE-19 cells by scavenging 1O2. Mechanistic studies using the blue-light irradiated cells also demonstrated that the cytoprotective effects of astaxanthin can be attributed to scavenging of 1O2 directly. Our results suggest the potential value of astaxanthin as a dietary strategy to prevent blue light-induced retinal degeneration including AMD.

KEY POLICY HIGHLIGHTSBlue light irradiation triggered apoptosis and oxidative stress in all-trans-retinal (atRAL)-exposed human ARPE-19 retinal pigment epithelium cells by generating singlet oxygen (¹O₂), leading to significant cell death.Astaxanthin, a potent anti-oxidative xanthophyll abundant in several marine organisms including microalgae, salmon, and shrimp, significantly suppresses blue light-induced cell death of atRAL-exposed ARPE-19 cells.Astaxanthin inhibited apoptosis and oxidative stress induced by blue light by directly scavenging ¹O₂.

The transcription regulator ChpA affects the global transcriptome including quorum sensing-dependent genes in Ralstonia pseudosolanacearum strain OE1-1.

The gram-negative plant-pathogenic ß-proteobacterium Ralstonia pseudosolanacearum strain OE1-1 produces methyl 3-hydroxymyristate as a quorum sensing (QS) signal through methyltransferase PhcB and senses the chemical via the sensor histidine kinase PhcS. This leads to activation of the LysR family transcription regulator PhcA, which regulates the genes (QS-dependent genes) responsible for QS-dependent phenotypes, including virulence. The transcription regulator ChpA, which possesses a response regulator receiver domain and also a hybrid sensor histidine kinase/response regulator phosphore-acceptor domain but lacks a DNA-binding domain, is reportedly involved in QS-dependent biofilm formation and virulence of R. pseudosolanacearum strain GMI1000. To explore the function of ChpA in QS of OE1-1, we generated a chpA-deletion mutant (ΔchpA) and revealed that the chpA deletion leads to significantly altered QS-dependent phenotypes. Furthermore, ΔchpA exhibited a loss in its infectivity in xylem vessels of tomato plant roots, losing virulence on tomato plants, similar to the phcA-deletion mutant (ΔphcA). Transcriptome analysis showed that the transcript levels of phcB, phcQ, phcR, and phcA in ΔchpA were comparable to those in OE1-1. However, the transcript levels of 89.9% and 88.9% of positively and negatively QS-dependent genes, respectively, were significantly altered in ΔchpA compared with OE1-1. Furthermore, the transcript levels of these genes in ΔchpA were positively correlated with those in ΔphcA. Together, our results suggest that ChpA is involved in the regulation of these QS-dependent genes, thereby contributing to the behaviour in host plant roots and virulence of OE1-1.

Quorum Sensing-Dependent Invasion of Ralstonia solanacearum into Fusarium oxysporum Chlamydospores.

Strains of the Ralstonia solanacearum species complex (RSSC), although known as the causative agent of bacterial wilt disease in plants, induce the chlamydospores of many fungal species and invade them through the spores. The lipopeptide ralstonins are the chlamydospore inducers produced by RSSC and are essential for this invasion. However, no mechanistic investigation of this interaction has been conducted. In this study, we report that quorum sensing (QS), which is a bacterial cell-cell communication, is important for RSSC to invade the fungus Fusarium oxysporum (Fo). ΔphcB, a deletion mutant of QS signal synthase, lost the ability to both produce ralstonins and invade Fo chlamydospores. The QS signal methyl 3-hydroxymyristate rescued these disabilities. In contrast, exogenous ralstonin A, while inducing Fo chlamydospores, failed to rescue the invasive ability. Gene-deletion and -complementation experiments revealed that the QS-dependent production of extracellular polysaccharide I (EPS I) is essential for this invasion. The RSSC cells adhered to Fo hyphae and formed biofilms there before inducing chlamydospores. This biofilm formation was not observed in the EPS I- or ralstonin-deficient mutant. Microscopic analysis showed that RSSC infection resulted in the death of Fo chlamydospores. Altogether, we report that the RSSC QS system is important for this lethal endoparasitism. Among the factors regulated by the QS system, ralstonins, EPS I, and biofilm are important parasitic factors. IMPORTANCE Ralstonia solanacearum species complex (RSSC) strains infect both plants and fungi. The phc quorum-sensing (QS) system of RSSC is important for parasitism on plants, because it allows them to invade and proliferate within the hosts by causing appropriate activation of the system at each infection step. In this study, we confirm that ralstonin A is important not only for Fusarium oxysporum (Fo) chlamydospore induction but also for RSSC biofilm formation on Fo hyphae. Extracellular polysaccharide I (EPS I) is also essential for biofilm formation, while the phc QS system controls these factors in terms of production. The present results advocate a new QS-dependent mechanism for the process by which a bacterium invades a fungus.

The phc Quorum-Sensing System in Ralstonia solanacearum Species Complex.

Ralstonia solanacearum species complex (RSSC) strains are devastating plant pathogens distributed worldwide. The primary cell density-dependent gene expression system in RSSC strains is phc quorum sensing (QS). It regulates the expression of about 30% of all genes, including those related to cellular activity, primary and secondary metabolism, pathogenicity, and more. The phc regulatory elements encoded by the phcBSRQ operon and phcA gene play vital roles. RSSC strains use methyl 3-hydroxymyristate (3-OH MAME) or methyl 3-hydroxypalmitate (3-OH PAME) as the QS signal. Each type of RSSC strain has specificity in generating and receiving its QS signal, but their signaling pathways might not differ significantly. In this review, I describe the genetic and biochemical factors involved in QS signal input and the regulatory network and summarize control of the phc QS system, new cell-cell communications, and QS-dependent interactions with soil fungi.

Determinants of Subtype-Selectivity of the Anthelmintic Paraherquamide A on Caenorhabditis elegans Nicotinic Acetylcholine Receptors.

The anthelmintic paraherquamide A acts selectively on the nematode L-type nicotinic acetylcholine receptors (nAChRs), but the mechanism of its selectivity is unknown. This study targeted the basis of paraherquamide A selectivity by determining an X-ray crystal structure of the acetylcholine binding protein (AChBP), a surrogate nAChR ligand-binding domain, complexed with the compound and by measuring its actions on wild-type and mutant Caenorhabditis elegans nematodes and functionally expressed C. elegans nAChRs. Paraherquamide A showed a higher efficacy for the levamisole-sensitive [L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8)] nAChR than the nicotine-sensitive [N-type (ACR-16)] nAChR, a result consistent with in vivo studies on wild-type worms and worms with mutations in subunits of these two classes of receptors. The X-ray crystal structure of the Ls-AChBP-paraherquamide A complex and site-directed amino acid mutation studies showed for the first time that loop C, loop E, and loop F of the orthosteric receptor binding site play critical roles in the observed L-type nAChR selective actions of paraherquamide A. SIGNIFICANCE STATEMENT: Paraherquamide A, an oxindole alkaloid, has been shown to act selectively on the L-type over N-type nAChRs in nematodes, but the mechanism of selectivity is unknown. We have co-crystallized paraherquamide A with the acetylcholine binding protein, a surrogate of nAChRs, and found that structural features of loop C, loop E, and loop F contribute to the L-type nAChR selectivity of the alkaloid. The results create a new platform for the design of anthelmintic drugs targeting cholinergic neurotransmission in parasitic nematodes.

ß-1,4-Cellobiohydrolase is involved in full expression of phcA, contributing to the feedback loop in quorum sensing of Ralstonia pseudosolanacearum strain OE1-1.

After infecting roots of tomato plants, the gram-negative bacterium Ralstonia pseudosolanacearum strain OE1-1 activates quorum sensing (QS) to induce production of plant cell wall-degrading enzymes, such as ß-1,4-endoglucanase (Egl) and ß-1,4-cellobiohydrolase (CbhA), via the LysR family transcriptional regulator PhcA and then invades xylem vessels to exhibit virulence. The phcA-deletion mutant (ΔphcA) exhibits neither the ability to infect xylem vessels nor virulence. Compared with strain OE1-1, the egl-deletion mutant (Δegl) exhibits lower cellulose degradation activity, lower infectivity in xylem vessels, and reduced virulence. In this study, we analysed functions of CbhA other than cell wall degradation activity that are involved in the virulence of strain OE1-1. The cbhA-deletion mutant (ΔcbhA) lacked the ability to infect xylem vessels and displayed loss of virulence, similar to ΔphcA, but exhibited less reduced cellulose degradation activity compared with Δegl. Transcriptome analysis revealed that the phcA expression levels in ΔcbhA were significantly lower than in OE1-1, with significantly altered expression of more than 50% of PhcA-regulated genes. Deletion of cbhA led to a significant change in QS-dependent phenotypes, similar to the effects of phcA deletion. Complementation of ΔcbhA with native cbhA or transformation of this mutant with phcA controlled by a constitutive promoter recovered its QS-dependent phenotypes. The expression level of phcA in ΔcbhA-inoculated tomato plants was significantly lower than in strain OE1-1-inoculated plants. Our results collectively suggest that CbhA is involved in the full expression of phcA, thereby contributing to the QS feedback loop and virulence of strain OE1-1.

Discovery of Cyclic Lipopeptides Ralstopeptins A and B from Ralstonia solanacearum Species Complex and Analysis of Biosynthetic Gene Evolution.

Ralstonia solanacearum species complex (RSSC) strains are plant pathogens that produce lipopeptides (ralstonins and ralstoamides) by the polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) enzyme hybrid. Recently, ralstonins were found to be key molecules in the parasitism of RSSC to other hosts, Aspergillus and Fusarium fungi. The PKS-NRPS genes of RSSC strains in the GenBank database suggest the production of additional lipopeptides, although it has not been confirmed to date. Here, we report the genome-driven and mass-spectrometry-guided discovery, isolation, and structural elucidation of ralstopeptins A and B from strain MAFF 211519. Ralstopeptins were found to be cyclic lipopeptides with two amino acid residues less than ralstonins. The partial deletion of the gene encoding PKS-NRPS obliterated the production of ralstopeptins in MAFF 211519. Bioinformatic analyses suggested possible evolutionary events of the biosynthetic genes of RSSC lipopeptides, where intragenomic recombination may have occurred within the PKS-NRPS genes, reducing the gene size. The chlamydospore-inducing activities of ralstopeptins A and B, ralstonins A and B, and ralstoamide A in the fungus Fusarium oxysporum indicated a structural preference for ralstonins. Altogether, we propose a model for the evolutionary processes that contribute to the chemical diversity of RSSC lipopeptides and its relation to the endoparasitism of RSSC in fungi.

The behavior of Ralstonia pseudosolanacearum strain OE1-1 and morphological changes of cells in tomato roots.

The soil-borne Gram-negative ß-proteobacterium Ralstonia solanacearum species complex (RSSC) infects tomato roots through the wounds where secondary roots emerge, infecting xylem vessels. Because it is difficult to observe the behavior of RSSC by a fluorescence-based microscopic approach at high magnification, we have little information on its behavior at the root apexes in tomato roots. To analyze the infection route of a strain of phylotype I of RSSC, R. pseudosolanacearum strain OE1-1, which invades tomato roots through the root apexes, we first developed an in vitro pathosystem using 4 day-old-tomato seedlings without secondary roots co-incubated with the strain OE1-1. The microscopic observation of toluidine blue-stained longitudinal semi-thin resin sections of tomato roots allowed to detect attachment of the strain OE1-1 to surfaces of the meristematic and elongation zones in tomato roots. We then observed colonization of OE1-1 in intercellular spaces between epidermis and cortex in the elongation zone, and a detached epidermis in the elongation zone. Furthermore, we observed cortical and endodermal cells without a nucleus and with the cell membrane pulling away from the cell wall. The strain OE1-1 next invaded cell wall-degenerated cortical cells and formed mushroom-shaped biofilms to progress through intercellular spaces of the cortex and endodermis, infecting pericycle cells and xylem vessels. The deletion of egl encoding ß-1,4-endoglucanase, which is one of quorum sensing (QS)-inducible plant cell wall-degrading enzymes (PCDWEs) secreted via the type II secretion system (T2SS) led to a reduced infectivity in cortical cells. Furthermore, the QS-deficient and T2SS-deficient mutants lost their infectivity in cortical cells and the following infection in xylem vessels. Taking together, infection of OE1-1, which attaches to surfaces of the meristematic and elongation zones, in cortical cells of the elongation zone in tomato roots, dependently on QS-inducible PCDWEs secreted via the T2SS, leads to its subsequent infection in xylem vessels.

Transport Form and Pathway from the Intestine to the Peripheral Tissues and the Intestinal Absorption and Metabolism Properties of Oleamide.

This study aimed to obtain information on the transport form and pathway from the intestine to the peripheral tissues and on the intestinal absorption and metabolism properties of oleamide (cis-9-octadecenamide). Oleamide was primarily transported via the portal vein. Density gradient centrifugation indicated that plasma oleamide was enriched in the fractions containing albumin in the portal and peripheral blood. Oleamide formed a complex with albumin in an endothermic reaction (apparent Kd = 4.4 µM). The CD36 inhibitor inhibited the oleamide uptake into the intestinal epithelial Caco-2 cells, and oleamide decreased the cell surface CD36 level. The fatty acid amide hydrolase (FAAH) inhibitor increased the transepithelial transport of oleamide across Caco-2 cells and the plasma oleamide concentration in mice intragastrically administered with oleamide. These results indicate that oleamide is transported primarily via the portal vein as a complex with albumin. Furthermore, we suggest that oleamide is taken up via CD36 in the small intestine and degraded intracellularly by FAAH.

Competitive chrodrimanin B interactions with rat brain GABAA receptors revealed by radioligand binding assays.

Meroterpenoid compounds chrodrimanins produced by Talaromyces sp. YO-2 have been shown to act as competitive antagonists of silkworm larval GABAA receptors using electrophysiology, yet no further evidence has been provided to support such an action. We have investigated the actions of chrodrimanin B on rat brain GABAA receptors by binding assays with non-competitive ligand of GABAA receptors [3H]EBOB and competitive ligands [3H]gabazine and [3H]muscimol. Chrodrimanin B did not significantly affect the binding of [3H]EBOB while reducing the binding of [3H]gabazine and [3H]muscimol to the rat membrane preparations. Chrodrimanin B increased the dissociation constant Kd of [3H]gabazine and [3H]muscimol without significantly affecting the maximum binding, pointing to competitive interactions of chrodrimanin B with rat GABAA receptors in support of our previous observation that the compound acts as a competitive antagonist on the silkworm larval GABA receptor.

Genome Mining Discovery of Protegenins A-D, Bacterial Polyynes Involved in the Antioomycete and Biocontrol Activities of Pseudomonas protegens.

Some bacteria uniquely produce "bacterial polyynes", which possess a conjugated C≡C bond starting with a terminal alkyne, and use them as chemical weapons against hosts and competitors. Pseudomonas protegens Cab57, a biocontrol agent against plant pathogens, has an orphan biosynthetic gene cluster for bacterial polyynes (named protegenins). In this study, the isolation, structure elucidation, and biological characterization of protegenins A-D are reported. The structures of protegenins A-D determined by spectroscopic and chemical techniques were octadecanoic acid derivatives possessing an ene-tetrayne, ene-triyne-ene, or ene-triyne moiety. The protegenins exhibited weak to strong antioomycete activity against Pythium ultimum OPU774. The deletion of proA, a protegenin biosynthetic gene, resulted in the reduction of the antioomycete activity of P. protegens. The Gac/Rsm system, a quorum sensing-like system of Pseudomonas bacteria, regulated the production of protegenins. The production profile of protegenins was dependent on the culturing conditions, suggesting a control mechanism for protegenin production selectivity. P. protegens suppressed the damping-off of cucumber seedlings caused by P. ultimum, and this protective effect was reduced in the proA-deletion mutant. Altogether, protegenins are a new class of bacterial polyynes which contribute to the antioomycete and plant-protective effects of P. protegens.

PhcQ mainly contributes to the regulation of quorum sensing-dependent genes, in which PhcR is partially involved, in Ralstonia pseudosolanacearum strain OE1-1.

The gram-negative plant-pathogenic ß-proteobacterium Ralstonia pseudosolanacearum strain OE1-1 produces methyl 3-hydroxymyristate as a quorum sensing (QS) signal via the methyltransferase PhcB and senses the chemical through the sensor histidine kinase PhcS. This leads to functionalization of the LysR family transcriptional regulator PhcA, regulating QS-dependent genes responsible for the QS-dependent phenotypes including virulence. The phc operon consists of phcB, phcS, phcR, and phcQ, with the latter two encoding regulator proteins with a receiver domain and a histidine kinase domain and with a receiver domain, respectively. To elucidate the function of PhcR and PhcQ in the regulation of QS-dependent genes, we generated phcR-deletion and phcQ-deletion mutants. Though the QS-dependent phenotypes of the phcR-deletion mutant were largely unchanged, deletion of phcQ led to a significant change in the QS-dependent phenotypes. Transcriptome analysis coupled with quantitative reverse transcription-PCR and RNA-sequencing revealed that phcB, phcK, and phcA in the phcR-deletion and phcQ-deletion mutants were expressed at similar levels as in strain OE1-1. Compared with strain OE1-1, expression of 22.9% and 26.4% of positively and negatively QS-dependent genes, respectively, was significantly altered in the phcR-deletion mutant. However, expression of 96.8% and 66.9% of positively and negatively QS-dependent genes, respectively, was significantly altered in the phcQ-deletion mutant. Furthermore, a strong positive correlation of expression of these QS-dependent genes was observed between the phcQ-deletion and phcA-deletion mutants. Our results indicate that PhcQ mainly contributes to the regulation of QS-dependent genes, in which PhcR is partially involved.

Sodium sulfite causes gastric mucosal cell death by inducing oxidative stress.

Sulfites are commonly used as a preservative and antioxidant additives in the food industry. Sulfites are absorbed by the gastrointestinal tract and distributed essentially to all body tissues. Although sulfites have been believed to be safe food additives, some studies have shown that they exhibit adverse effects in various tissues. In this study, we examined the cytotoxic effect of sodium sulfite (Na2SO3) against rat gastric mucosal cells (RGM1) and further investigated its underlying molecular mechanism. We demonstrated that exposure to Na2SO3 exerts significant cytotoxicity in RGM1 cells through induction of oxidative stress. Exposure of RGM1 cells to Na2SO3 caused a significant formation of protein carbonyls and 8-hydroxy-2'-deoxyguanosine, major oxidative stress markers, with a concomitant accumulation of carbonylated protein-related aggregates. Furthermore, we found that incubation of lysozyme with Na2SO3 evokes protein carbonylation and aggregation via the metal ion-catalyzed free radical formation derived from Na2SO3. Our results suggest that Na2SO3 might lead to gastric tissue injury via induction of oxidative stress by the formation of Na2SO3-related free radicals.

The Spiral Trial: A multicenter, randomized, controlled trial of Spiral thread sutures versus conventional thread sutures to prevent thinning of uterine scars following elective cesarean section.

BACKGROUND: The aim of this randomized controlled trial (RCT) is to investigate whether Spiral-thread sutures are superior to conventional sutures (0-Vicryl) for preventing uterine scar thinning following elective cesarean section. METHODS: This multicenter, parallel-group RCT will be conducted in four hospitals across three medical regions in Japan to assess 200 women (≥20 years old) with singleton pregnancies who are scheduled to undergo cesarean sections. Eligible women will be randomly assigned (1:1 ratio) to receive either the conventional uterine suture continuous absorption thread, which is most commonly used in Japan, or the Spiral thread. The primary endpoint is the degree of scar thinning, measured by transvaginal ultrasonography 6-7 months postoperatively, to evaluate the position of the uterus (anterior or posterior tilt) and myometrial wound thickness. The degree of thinning will be compared between the groups, and four measurements (mm) of the thinning area, including caudal distance, depth of the depression, remaining thickness of the myometrium on the serous side of the most depressed area, and width of the depression, will be recorded in the sagittal view on transvaginal ultrasound. Secondary endpoints will include total operative time, suture application time (from birth to the end of uterine suturing), operative blood loss, number of additional Z-sutures or continuous sutures required to stop bleeding, maternal abnormality frequency (surgical complications and postoperative infections), surgeon's years of experience, and clinical interpretation of individual subscale scores. DISCUSSION: This study shall provide important evidence on the optimal suture for preventing hysterotomy wound thinning after the first cesarean section. TRIAL REGISTRATION: National Institute of Public Health, Japan: jRCT1062200001 (May 7, 2020; https://rctportal.niph.go.jp/en/detail?trial_id=jRCT1062200001) and Okayama University Certified Review Board: CRB6180001 (April 9, 2020, version 3.0).

Disproof of the Proposed Structures of Bradyoxetin, a Putative Bradyrhizobium japonicum Signaling Molecule, and HMCP, a Putative Ralstonia solanacearum Quorum-Sensing Molecule.

First, we revisited the reported NMR data of bradyoxetin, a putative cell density factor of Bradyrhizobium japonicum, and found some inconsistencies in the proposed structure. To elucidate the correct structure, we synthesized model oxetane compounds and confirmed that the NMR data of the synthetic compounds did not match those of the reported bradyoxetin. After reinterpreting the reported NMR data, we concluded that bradyoxetin must be chloramphenicol. Next, some derivatives of 2-hydroxy-4-((methylamino)(phenyl)methyl)cyclopentanone (HMCP), which is a putative quorum-sensing molecule of Ralstonia solanacearum, were synthesized. The NMR spectra of the synthesized compounds were completely different from those of the reported natural products. Based on theoretical studies, including the estimation of 1H and 13C NMR chemical shifts using density functional theory calculations, we confirmed the correctness of the structure of the synthesized compound. These results strongly suggest that the proposed structure of HMCP could be incorrect.

Oleamide rescues tibialis anterior muscle atrophy of mice housed in small cages.

Skeletal muscle atrophy causes decreased physical activity and increased risk of metabolic diseases. We investigated the effects of oleamide (cis-9,10-octadecanamide) treatment on skeletal muscle health. The plasma concentration of endogenous oleamide was approximately 30 nm in male ddY mice under normal physiological conditions. When the stable isotope-labelled oleamide was orally administered to male ddY mice (50 mg/kg), the plasma concentration of exogenous oleamide reached approximately 170 nm after 1 h. Male ddY mice were housed in small cages (one-sixth of normal size) to enforce sedentary behaviour and orally administered oleamide (50 mg/kg per d) for 4 weeks. Housing in small cages decreased tibialis anterior (TA) muscle mass and the cross-sectional area of the myofibres in TA muscle. Dietary oleamide alleviated the decreases in TA muscle and resulted in plasma oleamide concentration of approximately 120 nm in mice housed in small cages. Housing in small cages had no influence on the phosphorylation levels of Akt serine/threonine kinase (Akt), mechanistic target of rapamycin (mTOR) and ribosomal protein S6 kinase (p70S6K) in TA muscle; nevertheless, oleamide increased the phosphorylation levels of the proteins. Housing in small cages increased the expression of microtubule-associated protein 1 light chain 3 (LC3)-II and sequestosome 1 (p62), but not LC3-I, in TA muscle, and oleamide reduced LC3-I, LC3-II and p62 expression levels. In C2C12 myotubes, oleamide increased myotube diameter at ≥100 nm. Furthermore, the mTOR inhibitor, Torin 1, suppressed oleamide-induced increases in myotube diameter and protein synthesis. These results indicate that dietary oleamide rescued TA muscle atrophy in mice housed in small cages, possibly by activating the phosphoinositide 3-kinase/Akt/mTOR signalling pathway and restoring autophagy flux.