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.

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.

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.

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.

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.

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.

[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.

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.

Continuous, intrinsic magnetic depletion of erythrocytes from whole blood with a quadrupole magnet and annular flow channel; pilot scale study.

The ability to separate RBCs from the other components of whole blood has a number of useful clinical and research applications ranging from removing RBCs from typical clinical blood draw, bone marrow transplants to transfusions of these RBCs to patients after significant blood loss. Viewed from a mechanistic/process perspective, there are three routine methodologies to remove RBCs: 1) RBCs lysis, 2) separation of the RBCs from the nucleated cells (i.e., stem cells) based on density differences typically facilitated through centrifugation or sedimentation agents, and 3) antibody based separation in which a targeted RBC is bound with an affinity ligand that facilitates its removal. More recently, several microfluidic based techniques have also been reported. In this report, we describe the performance of continuous RBC separation achieved by the deflection of intrinsically magnetic, deoxygenated RBCs as they flow through a magnetic energy gradient created by quadrupole magnet. This quadrupole magnetic, with aperture of 9.65 mm, has a maximum field of B0 = 1.36 T at the pole tips and a constant field gradient of B0 /r0 = 286 T/m. The annular flow channel, contained within this quadrupole magnet, is 203 mm long, has an inner radius of 3.98 mm, and an inner, outer radius of 4.36 mm, which corresponds to an annulus radius of 380 micrometer. At the entrance and exit to this annular channel, a manifold was designed which allows a cell suspension and sheath fluid to be injected, and a RBC enriched exit flow (containing the magnetically deflected RBCs) and a RBC depleted exit flow to be collected. Guided by theoretical models previously published, a limited number of operating parameters; total flow rate, flow rate ratios of flows in and flow out, and ratios of RBC to polystyrene control beads was tested. The overall performance of this system is consistent with our previously presented, theoretical models and our intuition. As expected, the normalized recovery of RBCs in the RBC exit fraction ranged from approximately 95% down to 60%, as the total flow rate through the system increased from 0.1 to 0.6 ml/min. At the cell concentrations studied, this corresponds to a flow rate of 1.5 × 106 -9 × 106 cells/min. While the throughput of these pilot scale studies are slow for practical applications, the general agreement with theory, and the small cross-sectional area in which the actual separation is achieved, 77 mm2 (annulus radius times the length), and corresponding volume of approximately 2 mls, suggests the potential to scale-up a system for practical applications exists and is actively being pursued.