Identification of sesquiterpene aldehydes as volatile antifungal compounds in Phaeolepiota aurea culture filtrate.

The culture filtrate extract of golden bootleg (Phaeolepiota aurea) exhibited strong antifungal activity in a bioassay for volatile compounds. Purification of active compounds through silica gel column chromatography and HPLC isolated two compounds, 1 and 2, with molecular weights of 234 and 259, respectively, as confirmed by mass spectrometry. NMR analysis identified these compounds as new sesquiterpenoids with the α-bourbonene skeleton. Both compounds had an aldehyde group, while 2 possessed a cyanohydrin group. These compounds were named phaeolep aldehydes A (1) and B (2). Phaeolep aldehyde A inhibited hyphae elongation of Penicillium citrinum, Aspergillus niger, Cladosporium sphaerospermum, and Alternaria brassicicola at 10 ppm, but did not exhibit any antibacterial activity. In contrast, phaeolep aldehyde B exhibited potent inhibitory effects on the spore germination of Pe. citrinum, achieving 58.2% inhibition at 0.25 ppm, outperforming phaeolep aldehyde A, which exhibited 25.7% inhibition at 10 ppm.

Priming of Immune System in Tomato by Treatment with Low Concentration of L-Methionine.

Various metabolites, including phytohormones, phytoalexins, and amino acids, take part in the plant immune system. Herein, we analyzed the effects of L-methionine (Met), a sulfur-containing amino acid, on the plant immune system in tomato. Treatment with low concentrations of Met enhanced the resistance of tomato to a broad range of diseases caused by the hemi-biotrophic bacterial pathogen Pseudomonas syringae pv. tomato (Pst) and the necrotrophic fungal pathogen Botrytis cinerea (Bc), although it did not induce the production of any antimicrobial substances against these pathogens in tomato leaf tissues. Analyses of gene expression and phytohormone accumulation indicated that Met treatment alone did not activate the defense signals mediated by salicylic acid, jasmonic acid, and ethylene. However, the salicylic acid-responsive defense gene and the jasmonic acid-responsive gene were induced more rapidly in Met-treated plants after infection with Pst and Bc, respectively. These findings suggest that low concentrations of Met have a priming effect on the phytohormone-mediated immune system in tomato.

Isolation of 2,2'-azoxybisbenzyl alcohol from Agaricus subrutilescens and its inhibitory activity against bacterial biofilm formation.

Virulence pathways in pathogenic bacteria are regulated by quorum sensing mechanisms, particularly biofilm formation through autoinducer (AI) production and sensing. In this study, the culture filtrate extracted from an edible mushroom, Agaricus subrutilescens, was fractionated to isolate a compound that inhibits biofilm formation. Four gram-negative bacteria (Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, and Enterobacter cloacae) and two gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus) were used for the bioassay. The bioassay-guided chromatographic separations of the culture filtrate extract resulted in the isolation of the compound. Further, spectroscopic analyses revealed the identity of the compound as 2,2'-azoxybisbenzyl alcohol (ABA). The minimum inhibitory and sub-inhibitory concentrations of the compound were also determined. Azoxybisbenzyl alcohol was significantly effective in inhibiting biofilm formation in all tested bacteria, with half-maximal inhibitory concentrations of 3-11 µg/mL. Additionally, the bioactivity of ABA was confirmed through the bioassays for the inhibition of exopolysaccharide matrixes and AI activities.

Strobilurin X acts as an anticancer drug by inhibiting protein synthesis and suppressing mitochondrial respiratory chain activity.

PURPOSE: Strobilurins act as antifungal agents by inhibiting the mitochondrial respiratory chain. The cytotoxic activity of strobilurins, focusing on its anticancer activities, has been reported. However, the mechanisms involved in these activities remain unclear. METHODS: The cytotoxic effects of strobilurin X isolated from the mycelium of Mucidula. venosolamellata were examined in human cancer cell lines (A549 and HeLa) and normal fibroblasts (WI-38). RESULTS: Strobilurin X significantly decreased the viability of A549 and HeLa cells compared to that in the WI-38 cells after 48 h of exposure. The EC50 values for cytotoxicity in the A549, HeLa, and WI-38 cells were 3.4, 5.4, and 16.8 µg/mL, respectively. Strobilurin X inhibited the mitochondrial respiratory chain and enhanced the release of lactate in the A549 cells. The IC50 value of strobilurin X against the mitochondrial respiratory chain complex III activity was 139.8 ng/mL. The cytotoxicity induced by strobilurin X was not completely rescued after adding uridine, methyl pyruvate, or N-acetyl cysteine. Furthermore, pharmacological approaches demonstrated that strobilurin X failed to modulate the mitogen-activated protein kinase family and phosphoinositide 3-kinase-Akt pathways; alternatively, it suppressed protein synthesis independent of uridine. CONCLUSION: Strobilurin X induced cytotoxicity by blocking the mitochondrial respiratory chain and suppressing protein synthesis. These findings may aid in the development of novel anticancer drugs using strobilurins.

Inhibition of melanoma cell proliferation by strobilurins isolated from mushrooms and their synthetic analogues.

Strobilurins A and X, isolated from Mucidula venosolamellata culture extracts, demonstrated potent inhibition of human melanoma G-361 cell proliferation. Strobilurin X exhibited milder inhibitory effects on human fibroblast cells (NB1RGB) compared to strobilurin A. Additional strobilurin-related compounds were isolated from the other mushroom species. Oudemansins A and B displayed weaker activities on G-361 cells than strobilurins A and B, respectively, emphasizing the importance of a conjugated double-bond structure. Among isolated compounds, strobilurin G showed the lowest IC50 value for G-361 cells. Additional strobilurins bearing various substituents on the benzene ring were synthesized. Synthetic intermediates lacking the methyl ß-methoxyacrylate group and a strobilurin analogue bearing modified ß-methoxyacrylate moiety showed almost no inhibitory activity against G-361 cells. The introduction of long or bulky substituents at the 4' position of the benzene ring of strobilurins enhanced the activity and selectivity, suggesting differential recognition of the benzene ring by G-361 and NB1RGB cells.

Association of bone health by calcaneal quantitative ultrasound with quadriceps strength and function one year after unilateral total knee arthroplasty.

Introduction: Although total knee arthroplasty (TKA) effectively improves knee pain and functional decline due to knee osteoarthritis, hip fractures are more likely to occur in the first year after surgery. Bone mineral density (BMD) measured by dual-energy X-ray absorptiometry (DXA) decreases in the first year after TKA, but it is not clear whether BMD measured by quantitative ultrasound (QUS) also shows a decrease. This study aimed to evaluate the change in QUS parameters before and 1 year after TKA and to investigate the association of QUS parameters with quadriceps strength and function 1 year after TKA. Methods: This prospective cohort study included 30 patients scheduled for their unilateral TKA. We assessed BMD using QUS before and 1 year after surgery. Quadriceps strength was assessed using a hand-held dynamometer, and function was assessed using a self-reported questionnaire. A linear mixed model estimated the mean difference in QUS parameters before and 1 year after TKA. A general linear model was also used to examine the association of QUS parameters with quadriceps strength and self-reported function at 1 year after surgery. Results: We found no significant decrease in the QUS parameters on either the surgical or non-surgical side at 1 year postoperatively compared to preoperatively. The QUS parameters were not associated with quadriceps strength or self-reported function on either the surgical or non-surgical side at 1 year postoperatively. Conclusions: QUS alone may not be sufficient to detect changes in BMD from before surgery to 1 year after TKA. Clinicians should evaluate BMD preoperatively to identify patients at high risk for hip fractures and develop a program to prevent postoperative hip fractures. Level of evidence: Level 3, Cohort study.

Characterization of Disease Resistance Induced by a Pyrazolecarboxylic Acid Derivative in Arabidopsis thaliana.

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is induced through the salicylic acid (SA)-mediated signaling pathway. Here, we characterized 3-chloro-1-methyl-1H-pyrazole-5-carboxylic acid (CMPA) as an effective SAR inducer in Arabidopsis. The soil drench application of CMPA enhanced a broad range of disease resistance against the bacterial pathogen Pseudomonas syringae and fungal pathogens Colletotrichum higginsianum and Botrytis cinerea in Arabidopsis, whereas CMPA did not show antibacterial activity. Foliar spraying with CMPA induced the expression of SA-responsible genes such as PR1, PR2 and PR5. The effects of CMPA on resistance against the bacterial pathogen and the expression of PR genes were observed in the SA biosynthesis mutant, however, while they were not observed in the SA-receptor-deficient npr1 mutant. Thus, these findings indicate that CMPA induces SAR by triggering the downstream signaling of SA biosynthesis in the SA-mediated signaling pathway.

Pt17 nanocluster electrocatalysts: preparation and origin of high oxygen reduction reaction activity.

We recently found that [Pt17(CO)12(PPh3)8]z (Pt = platinum; CO = carbon monoxide; PPh3 = triphenylphosphine; z = 1+ or 2+) is a Pt nanocluster (Pt NC) that can be synthesized with atomic precision in air. The present study demonstrates that it is possible to prepare a Pt17-supported carbon black (CB) catalyst (Pt17/CB) with 2.1 times higher oxygen reduction reaction (ORR) activity than commercial Pt nanoparticles/CB by the adsorption of [Pt17(CO)12(PPh3)8]z onto CB and subsequent calcination of the catalyst. Density functional theory calculation strongly suggests that the high ORR activity of Pt17/CB originates from the surface Pt atoms that have an electronic structure appropriate for the progress of ORR. These results are expected to provide design guidelines for the fabrication of highly active ORR catalysts using Pt NCs with a diameter of about 1 nm and thereby enabling the use of reduced amounts of Pt in polymer electrolyte fuel cells.

Effects of gelation concentration on cyclic deformation behavior of κ-carrageenan hydrogels.

The aim of this study is to elucidate the relationship between the network structure of κ-carrageenan hydrogels and their mechanical properties. First κ-carrageenan hydrogels have been prepared at different gelation concentrations, and then the mechanical behavior during four cyclic deformations has been examined at the same κ-carrageenan concentration. Young's modulus is higher for the gel prepared at 5 gL-1 (C05) compared to that for the gel prepared at 30 gL-1 (C30). C30 shows almost linear relation between the stress and the strain like an ideal rubber, while a residual strain appears in each cyclic deformation for C05. The extent of the residual strain depends on the maximum strain and the deformation speed, indicating that C05 deforms plastically to some extent. The residual strain for C05 decreases gradually even after a cyclic deformation and disappears in the case of a small strain as if there were a memory of the structure. The effects of the gelation concentration on the mechanical properties have been explained based on the network structure specific to κ-carrageenan hydrogels. The higher modulus for C05 has been attributed to the higher helix content and the plastic deformation of C05 to the loosely-aggregated crosslinks.

Verbal Suggestion Modulates the Sense of Ownership and Heat Pain Threshold During the "Injured" Rubber Hand Illusion.

The appearance of the self-body influences the feeling that one's body belongs to oneself, that is, a sense of ownership (SoO) and pain perception. This can be identified by measuring the SoO and pain thresholds after performing the rubber hand illusion (RHI) with an injured rubber hand. The generation of SoO is thought to be caused by multisensory integration of bottom-up factors (vision, proprioceptive, and touch), and by top-down factors, such as the context effect. The appearance is one of the context effects which may become more effective when used simultaneously with other context effects (e.g., verbal suggestion). However, in the RHI, when appearance and other context effects are used simultaneously, the effect is unclear. In this study, we attempted to identify the influence of verbal suggestion on the SoO and heat pain threshold (HPT). As a preliminary step, in Experiment 1, the "normal" rubber hand and "penetrated nail" as injured rubber hand were used to clarify the context effect with appearance alone during RHI (synchronous/asynchronous), which was conducted within-subjects. In Experiment 2, we only used the "penetrated nail" rubber hand to clarify the context effect with verbal suggestion and appearance during RHI. We randomly classified participants into two suggestion groups ("fear" and "no-fear"). The RHI (synchronous/asynchronous) was conducted for each group. In each experiment, the effect of each condition was assessed by subjective measures of SoO, such as questionnaire, and objective measures of SoO, such as proprioceptive drift and electrodermal activity. Following RHI in each condition, HPT was measured. The main finding was that, in the synchronous condition, the "penetrated nail" appearance with "fear" verbal suggestion modulated questionnaire and HPT, but not electrodermal activity. We conclude that the context-included multisensory integration affected the subjective factors because it contains a higher cognitive process by verbal suggestion.

Preparation of tricationic tris(pyridylpalladium(II)) metallacyclophane as an anion receptor.

A tricationic tris(pyridylpalladium(II)) metallacyclophane was prepared from 3,5-dibromopyridine by a successive treatment with tetrakis(triphenylphosphine)palladium(0), diphosphine, and silver salt. Single-crystal X-ray diffraction analysis revealed that the metallacyclophane incorporated one of three counter anions into its hole-shaped cavity to form multidentate C-H⋯anion interactions. Solution-phase 1H NMR experiments in DMSO-d6 indicated that the metallacyclophane exhibited selective binding behavior toward nitrate, tetrafluoroborate, p-toluenesulfonate, perchlorate, and hydrogen sulfate ions, whereas the hexafluoroantimonate ion exhibited only weak interaction toward the metallacyclophane. This anion recognition behavior was further demonstrated by an extraction experiment of water-soluble sulfonate dyes.

[Determination of Caffeine, Theobromine, and Theophylline in Chocolate Using LC-MS].

A simultaneous determination method for caffeine, theobromine, and theophylline in chocolate was developed. Three compounds were ultrasonically extracted twice (15 min at 50℃) in an acetonitrile-water (1 : 1, v/v). The extract was purified using Oasis HLB SPE cartridge, and the purified processed by LC-MS. The method exhibited recoveries of 97.4-100.2%, RSDs of repeatability of 1.0-2.8%, and RSDs of within-laboratory reproducibility of 2.0-7.9%. This method was simpler and more selective than existing methods, and was practical for caffeine, theobromine, and theophylline analysis in chocolate.

Quantitative Ultrasound of the Heel in Women With Knee Osteoarthritis.

The prevalence of multiple falls is higher in older women, and knee pain is associated with multiple falls in women; thus, it is important to detect individuals at risk of fractures among women with knee osteoarthritis. We aimed to clarify the association between knee osteoarthritis and the quantitative ultrasound (QUS) parameter, and investigate the clinical relevance of the QUS parameter with osteoarthritis-related characteristics in women with knee osteoarthritis. This was a cross-sectional study. We recruited 101 women with knee osteoarthritis at a local orthopedic hospital and 102 healthy individuals from the community. Heel QUS measurements were performed using the QUS device, measuring the stiffness index. We also assessed knee pain, quadriceps strength, and physical activity in women with knee osteoarthritis. We fitted a multiple regression model to estimate the association between knee osteoarthritis and the stiffness index. We also fitted 3 multiple regression models to investigate the clinical relevance of the QUS parameter with osteoarthritis-related characteristics in women with knee osteoarthritis. Compared with the control group, individuals with grade 2 osteoarthritis did not demonstrate a significant difference in stiffness index (p = 0.68); however, those with grade 3 osteoarthritis (p < 0.001) and 4 (p < 0.001) showed a lower stiffness index than that of the control group. Additionally, although the QUS parameter did not show an association with knee pain score (p = 0.70) or quadriceps strength (p = 0.11), we found a significant association between the QUS parameter and physical activity (p = 0.003). Our results demonstrate that women with moderate or severe knee osteoarthritis showed lower QUS parameters compared to healthy women, and the QUS parameter was associated with physical activity in women with knee osteoarthritis.

Turn-On Fluorescent Probe Based on a Dansyl Triarginine Peptide for Ganglioside Imaging.

Gangliosides play pivotal biological roles in the animal cell membranes, and it is vital to develop fluorescent probes for imaging them. To date, various artificial receptors for ganglioside imaging have been developed; however, turn-on fluorescence imaging for gangliosides with high contrast has not been achieved. We developed a simple fluorescent probe on the basis of a dansyl triarginine peptide for turn-on ganglioside imaging on the liposome membrane. The probe bound to monosialyl gangliosides and other anionic lipids with association constants was 105 M-1, which enhanced from 6-fold to 7-fold the fluorescence intensity. Upon binding to monosialyl ganglioside-containing giant liposomes, the turn-on probe selectively enhanced the fluorescence intensity compared with the other anionic lipids. This simple peptide probe for turn-on fluorescence imaging of gangliosides would provide a novel molecular tool for chemical biology.

Overexpression of neural miRNAs miR-9/9* and miR-124 suppresses differentiation to Müller glia and promotes differentiation to neurons in mouse retina in vivo.

MicroRNAs (miRNAs) are known to regulate gene expression and modulate cellular differentiation. MicroRNA-9/9* (miR-9/9*) and microRNA-124 (miR-124) are highly expressed in the central nervous system. In vivo function of miR-9/9* and miR-124 has been investigated in detail, whereas there remain some discrepancies regarding neural development. To this end, we electroporated miR-9/9*, miR-124 or miR-9/9*/124 expression plasmids into neonatal retinal progenitor cells (RPCs) in vivo and analyzed the fate of electroporated cells. Both miR-9/9* and miR-124 reduced the number of SOX9- and GS-positive cells and increased that of TUBB3-positive cells in the postnatal day 14 retina. No major effects on the proliferation and apoptosis of the electroporated cells were detected at least postnatal day 3. These indicated that miR-9/9* and miR-124 influence the cell fate of glial cells, thereby inducing their differentiation into neurons. Moreover, we found this cell fate modulation was occurred in RPCs indicating high-level expression of miRNA, but not in the low level. Our results strongly suggest that high-level miRNA overexpression is essential for directing cell fate by miR-9/9* and miR-124 interference.

Quantification of amygdalin, prunasin, total cyanide and free cyanide in powdered loquat seeds.

Amygdalin, prunasin, total cyanide and free cyanide concentrations in 12 powdered loquat seeds were investigated. Loquat (Eriobotrya japonica) is a species of flowering plant in the family Rosaceae, and its fruit is quite popular in Japan. Amygdalin and prunasin were measured using LC-MS/MS. Total cyanide was measured by enzymatic treatment, steam distillation and colorimetric quantification using the pyridine-pyrazolone method. Free cyanide was measured without enzymatic treatment. The mean concentrations of amygdalin, prunasin, total cyanide and free cyanide in powdered loquat seeds were 5900, 760, 410 and 44 mg/kg, respectively. The range of each quantitative value was extensive. Seven out of twelve samples were at risk for exceeding the acute reference dose (ARfD) of cyanide.

Teriparatide relieves ovariectomy-induced hyperalgesia in rats, suggesting the involvement of functional regulation in primary sensory neurons by PTH-mediated signaling.

Clinical studies have reported that teriparatide (TPTD), a human parathyroid hormone analog, reduces back pain in osteoporotic patients. However, the mechanistic insights of this pharmacological action remain elusive. This study investigated the antinociceptive effect of TPTD mainly on primary sensory neurons in ovariectomized (OVX) rats. The plantar test showed thermal hyperalgesia in the OVX rats, which was significantly, but not fully, recovered immediately after the initial TPTD administration. The von Frey test also demonstrated reduced withdrawal threshold in the OVX rats. This was partially recovered by TPTD. Consistently, the number and size of spinal microglial cells were significantly increased in the OVX rats, while TPTD treatment significantly reduced the number but not size of these cells. RNA sequencing-based bioinformatics of the dorsal root ganglia (DRG) demonstrated that changes in neuro-protective and inflammatory genes were involved in the pharmacological effect of TPTD. Most neurons in the DRG expressed substantial levels of parathyroid hormone 1 receptor. TPTD treatment of the cultured DRG-derived neuronal cells reduced the cAMP level and augmented the intracellular calcium level as the concentration increased. These findings suggest that TPTD targets neuronal cells as well as bone cells to exert its pharmacological action.

Normal aging hyperactivates innate immunity and reduces the medical efficacy of minocycline in brain injury.

Symptoms of many neurodegenerative diseases appear later in human life. However, young animal models for penetrating traumatic brain injury (pTBI) have been used to study neurodegenerative diseases and evaluate the efficacy of neuroprotective medicines. Possibly because of this discordance, effective neuroprotective drugs have still not been developed. For patients suffering from pTBI, aging is known to be a significant prognostic factor of mortality. In this study, we aimed to establish a model of aged pTBI animals using Drosophila melanogaster. We successfully generated aged pTBI flies as a new pTBI model showing increased neurodegeneration and higher mortality. To elucidate the mechanism of increased vulnerability in aged pTBI animals, we analyzed the GenBank-deposited transcriptome data of young and aged flies, demonstrating the importance of innate immunity genes for higher mortality in aged pTBI models. We found that in the context of pTBI, normal aging strongly activated the expression of antimicrobial peptide genes and upregulated the nuclear factor-κB gene in the immune deficiency pathway, but not the Toll pathway. Moreover, we found that minocycline increased the survival of young pTBI flies, but not aged pTBI flies. These results suggested that immune system activation under neurodegenerative conditions was involved in normal aging, thereby inhibiting the medicinal efficacy of neuroprotective drugs effective for young flies in aged flies.