Potential probiotic of Lactobacillus strains isolated from the intestinal tracts of pigs and feces of dogs with antibacterial activity against multidrug-resistant pathogenic bacteria.

The occurrence of multidrug-resistant pathogenic bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Acinetobacter baumannii (MDRAB), extended-spectrum ß-lactamase (ESBL) Escherichia coli, and Pseudomonas aeruginosa, has become a serious problem in animals and public. The objective of this study was to identify and isolate lactic acid bacterial (LAB) strains from the intestinal tracts of pigs and feces of dogs and then characterize them as potential probiotics with antimicrobial activity against multidrug-resistant pathogenic bacteria. In a preliminary isolation screening, 45 of 1167 isolated LAB strains were found to have anti-S. aureus ATCC 27,735 activity. Using 16S rDNA and 16S-23S rDNA intergenic spacer region (ISR) sequences, five of these isolates were further identified as Lactobacillus animalis 30a-2, Lactobacillus reuteri 4-12E, Weissella cibaria C34, Lactococcus lactis 5-12H, and Lactococcus lactis 6-3H. Antimicrobial substance assays suggest that the L. lactis 5-12H, L. lactis 6-3H, L. animalis 30a-2, L. reuteri 4-12E, and W. cibaria C34 strains might produce bacteriocins and hydrogen peroxide (H2O2) as antimicrobial substances. The L. animalis 30a-2 and W. cibaria C34 strains were further characterized for probiotic properties and shown to have high acid and bile salt tolerance. Additionally, they have broad antimicrobial spectra, and can significantly repress the growth of all of the tested strains of MRSA isolates, some MDRAB, ESBL E. coli, and P. aeruginosa isolates, along with food-borne pathogenic bacteria such as Bacillus cereus ATCC 11778, Listeria monocytogens ATCC 19111, Salmonella spp., Shigella spp., and Yersinia enterocolitica BCRC 12986. This is the first report of H2O2-producing L. animalis 30a-2 and W. cibaria C34 isolated from the intestinal tracts of pigs and feces of dogs that have good antimicrobial activity against multidrug-resistant and food-borne pathogenic bacteria and have excellent probiotic properties.

CaNRT2.1 Is Required for Nitrate but Not Nitrite Uptake in Chili Pepper Pathogen Colletotrichum acutatum.

Chili peppers are an important food additive used in spicy cuisines worldwide. However, the yield and quality of chilis are threatened by anthracnose disease caused by Colletotrichum acutatum. Despite the impact of C. acutatum on chili production, the genes involved in fungal development and pathogenicity in this species have not been well characterized. In this study, through T-DNA insertional mutagenesis, we identified a mutant strain termed B7, which is defective for the growth of C. acutatum on a minimal nutrient medium. Our bioinformatics analysis revealed that a large fragment DNA (19.8 kb) is deleted from the B7 genome, thus resulting in the deletion of three genes, including CaGpiP1 encoding a glycosylphosphatidyl-inisotol (GPI)-anchored protein, CaNRT2.1 encoding a membrane-bound nitrate/nitrite transporter, and CaRQH1 encoding a RecQ helicase protein. In addition, T-DNA is inserted upstream of the CaHP1 gene encoding a hypothetical protein. Functional characterization of CaGpiP1, CaNRT2.1, and CaHP1 by targeted gene disruption and bioassays indicated that CaNRT2.1 is responsible for the growth-defective phenotype of B7. Both B7 and CaNRT2.1 mutant strains cannot utilize nitrate as nitrogen sources, thus restraining the fungal growth on a minimal nutrient medium. In addition to CaNRT2.1, our results showed that CaGpiP1 is a cell wall-associated GPI-anchored protein. However, after investigating the functions of CaGpiP1 and CaHP1 in fungal pathogenicity, growth, development and stress tolerance, we were unable to uncover the roles of these two genes in C. acutatum. Collectively, in this study, our results identify the growth-defective strain B7 via T-DNA insertion and reveal the critical role of CaNRT2.1 in nitrate transportation for the fungal growth of C. acutatum.

Inhibition of LPS-Induced Oxidative Damages and Potential Anti-Inflammatory Effects of Phyllanthus emblica Extract via Down-Regulating NF-κB, COX-2, and iNOS in RAW 264.7 Cells.

Phyllanthus emblica is an edible nutraceutical and functional food in the Asia area with medicinal and nutritive importance. The fruit extract of P. emblica is currently considered to be one of the effective functional foods for flesh maintenance and disease treatments because of its antioxidative and immunomodulatory properties. We examined the antioxidant abilities of the fruit extract powder by carrying out 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, iron reducing power, and metal chelating activity analysis and showed excellent antioxidative results. In 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the result showed that the samples had no cytotoxic effect on RAW 264.7 cells even at a high concentration of 2 mg/mL. To investigate its immunomodulatory function, our estimation was to treat it with lipopolysaccharide (LPS) in RAW 264.7 cells to present anti-inflammatory capacities. The extract decreased reactive oxygen species (ROS) production levels in a dose-dependent manner measured by flow cytometry. We also examined various inflammatory mRNAs and proteins, including nuclear factor-κB (NF-κB), inducible nitric oxide synthases (iNOS), and cyclooxygenase-2 (COX-2). In quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting assay, all three targets were decreased by the extract, also in a dose-dependent manner. In conclusion, P. emblica fruit extract powder not only lessened antioxidative stress damages, but also inhibited inflammatory reactions.

A positive feedback loop between HEAT SHOCK PROTEIN101 and HEAT STRESS-ASSOCIATED 32-KD PROTEIN modulates long-term acquired thermotolerance illustrating diverse heat stress responses in rice varieties.

Heat stress is an important factor that has a negative impact on rice (Oryza sativa) production. To alleviate this problem, it is necessary to extensively understand the genetic basis of heat tolerance and adaptability to heat stress in rice. Here, we report the molecular mechanism underlying heat acclimation memory that confers long-term acquired thermotolerance (LAT) in this monocot plant. Our results showed that a positive feedback loop formed by two heat-inducible genes, HEAT SHOCK PROTEIN101 (HSP101) and HEAT STRESS-ASSOCIATED 32-KD PROTEIN (HSA32), at the posttranscriptional level prolongs the effect of heat acclimation in rice seedlings. The interplay between HSP101 and HSA32 also affects basal thermotolerance of rice seeds. These findings are similar to those reported for the dicot plant Arabidopsis (Arabidopsis thaliana), suggesting a conserved function in plant heat stress response. Comparison between two rice cultivars, japonica Nipponbare and indica N22 showed opposite performance in basal thermotolerance and LAT assays. 'N22' seedlings have a higher basal thermotolerance level than cv Nipponbare and vice versa at the LAT level, indicating that these two types of thermotolerance can be decoupled. The HSP101 and HSA32 protein levels were substantially higher in cv Nipponbare than in cv N22 after a long recovery following heat acclimation treatment, at least partly explaining the difference in the LAT phenotype. Our results point out the complexity of thermotolerance diversity in rice cultivars, which may need to be taken into consideration when breeding for heat tolerance for different climate scenarios.

Identifying 8-hydroxynaringenin as a suicide substrate of mushroom tyrosinase.

A biotransformed metabolite of naringenin was isolated from the fermentation broth of Aspergillus oryzae, fed with naringenin, and identified as 8-hydroxynaringenin based on the mass and (1)H- and (13)C-NMR spectral data. The compound showed characteristics of both an irreversible inhibitor and a substrate of mushroom tyrosinase in preincubation and HPLC analysis. These results demonstrate that 8-hydroxynaringenin belongs to a suicide substrate of mushroom tyrosinase. The partition ratio between the compound's molecules in the formation of product and in the inactivation of the enzyme was determined to be 283 +/- 21. The present study's results, together with our previous findings, which proved that both 8-hydroxydaidzein and 8-hydroxygenistein are suicide substrates of mushroom tyrosinase, show that 7,8,4'-trihydroxyl functional groups on flavonoids' skeletons play important roles in producing suicide substrate properties toward mushroom tyrosinase.

Auditory cortical evoked potentials in tinnitus patients with normal audiological presentation.

BACKGROUND/PURPOSE: It is widely assumed that damage to the peripheral hearing system is an essential prerequisite for the occurrence of tinnitus. However, previous studies have failed to target tinnitus patients with normal hearing. This study aims to investigate if tinnitus patients with normal audiological presentation demonstrate increased intensity dependence at the selected frequencies. METHODS: This study applied auditory cortical evoked potential test to investigate nine tinnitus patients with normal audiological presentation and nine age- and sex-matched healthy subjects without tinnitus. Auditory cortical evoked potentials (N1-P2) were elicited from stimuli at four frequencies (4000, 2000, 1000 and 500 Hz) with five intensities (50, 56, 62, 68 and 74 dB nHL). Intensity dependences by latency of N1 and amplitude of N1-P2 were surveyed at midline electrodes. RESULTS: The results showed that the intensity dependence by latency of N1 to the pooled frequencies at three midline electrodes, e.g. Fz, Cz and Pz, revealed non-significant difference. However, significant differences existed in the intensity dependence of amplitude N1-P2 to the pooled frequencies at the Fz and Cz positions. These differences suggested that tinnitus patients tended to respond less to increased sound intensity and were inclined to weaker intensity dependence. CONCLUSION: Increased intensity dependence of N1-P2 component at the selected frequencies cannot be demonstrated in tinnitus patients with normal hearing. Restated, the edge frequency phenomenon fails to present in tinnitus patients with normal hearing, a different characteristic from tinnitus patients with hearing loss.

Characterization of plasma proteome in biliary atresia.

BACKGROUND: Biliary atresia (BA) is a disorder during infancy with unknown etiology in which progression frequently leads to liver cirrhosis. Plasma proteome is characterized in this study. METHODS: Twelve paired plasma samples from 6 children with BA who received surgical correction at early stage and then liver transplantation at late stage of liver cirrhosis were studied. Plasma samples from 2 subjects without liver disorder were used as normal reference for 2-dimensional gel electrophoresis and for identification of protein spots by mass spectrometric analysis. Plasma samples from another 3 normal subjects (with a total of 5) were used for nephelometric quantification of immunoglobulin kappa light chain in comparison with patients' samples. RESULTS: Among the protein spots detected, ranging from 6 to 200 kDa mass with pIs of 3-10, significant up-regulation of immunoglobulin kappa light chain was found at the late stage of BA, which was subsequently confirmed by nephelometric analysis. Conversely, significant decrease of apolipoprotein (Apo) A-I and C-II, haptoglobin alpha2 and beta chain, and transthyretin were detected during the progression of BA. CONCLUSIONS: Increased immunoglobulin kappa light chain detected in late-stage BA characterizes adverse immune modulation in this disorder. Decreased apolipoproteins, haptoglobin and transthyretin levels might be potential markers of progressive liver injury, fibrosis and defective lipid metabolism in BA.