Frequent In-Migration and Highly Focal Transmission of Dengue Viruses among Children in Kamphaeng Phet,Thailand

Revealing the patterns and determinants of the spread of dengue virus (DENV) at local scales is central to understanding the epidemiology and evolution of this major human pathogen. We performed a phylogenetic analysis of the envelope (E) genes of DENV-1, -2, -3, and -4 isolates (involving 97, 23, 5, and 74 newly collected sequences, respectively) sampled from school-based cohort and village-based cluster studies in Kamphaeng Phet, Thailand, between 2004 and 2007. With these data, we sought to describe the spatial and temporal patterns of DENV spread within a rural population where a future vaccine efficacy trial is planned. Our analysis revealed considerable genetic diversity within the study population, with multiple lineages within each serotype circulating for various lengths of time during the study period. These results suggest that DENV is frequently introduced into both semi-urban and rural areas in Kamphaeng Phet from other populations. In contrast, the persistence of viral lineages across sampling years was observed less frequently. Analysis of phylogenetic clustering indicated that DENV transmission was highly spatially and temporally focal, and that it occurred in homes rather than at school. Overall, the strength of temporal clustering suggests that seasonal bottlenecks in local DENV populations facilitate the invasion and establishment of viruses from outside of the study area, in turn reducing the extent of lineage persistence.     

Antagonistic activity against pathogenic bacteria by human vaginal lactobacilli

This study attempted to isolate lactobacilli strains from healthy vaginal ecosystem to search for a new effective antibacterial probiotic strain. The strains were identified and characterized for their probiotic properties including bile salt and acid tolerance, growth at acidic pH, their ability to utilize protein, starch, and lipid, the production of hydrogen peroxide and bacteriocin as well as their antibiotic resistance patterns. The antibacterial activity of the culture supernatant of these strains were tested against a wide range of Gram-positive and Gram-negative pathogenic bacteria including Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae. Salmonella typhi, and Salmonella typhimurium. None of the strains inhibited the growth of Gram-negative bacteria. Contrastly, the culture supernatant of strain L 22, identified as Lactobacillus reuteri, significantly inhibited all of the clinical isolates of methicillin-resistant S. aureus (MRSA). The antibacterial effect of the selected strain L 22 was further investigated. In the presence of L 22, the bacterial growth was assessed in vitro by viable bacterial counting. The numbers of viable cells were significantly lower in L 22-containing broth than those in the control by 6h. This finding clearly demonstrates that strain L 22 can produce an anti-MRSA effect. The antibacterial ability of the strain L 22 was fundamentally attributed to their bacteriocin production which can cause both cell inhibition and cell death.     

Anti Propionibacterium acnes activity of rhodomyrtone, an effective compound from Rhodomyrtus tomentosa (Aiton) Hassk. leaves

Propionibacterium acnes have been recognized as one of the main causative agents in pathogenesis of acne. Twenty one isolates of P. acnes isolated from acne lesions were screened for lipase and protease activity which are reported to be associated in acne and inflammation. Interestingly, all P. acnes isolates demonstrated lipase activity. Similarly, 90% of test P. acnes produced protease enzyme. Antibacterial activity of the ethanol extract of Rhodomyrtus tomentosa (Aiton) Hassk. leaves and rhodomyrtone, its principle compound were tested against P. acnes using broth macrodilution method. The MIC(90) values of the ethanol extract and rhodomyrtone were 32 and 0.5 μg/mL, respectively. The numbers of the bacterial cells were reduced at least 99% after treatment with the ethanol extract and rhodomyrtone within 72 and 24 h, respectively. Cytotoxicity test of the extract and rhodomyrtone was performed on human normal fibroblast. The IC(50) values of the ethanol extract and rhodomyrtone were 476 and more than 200 μg/mL, approximately 15 and 400 folds higher than the MIC(90) values indicating that both substances were very low cytotoxic which could be applied as topical therapeutic anti-acne agents.     

Boesenbergia pandurata (Roxb.) Schltr., Eleutherine americana Merr. and Rhodomyrtus tomentosa (Aiton) Hassk. as antibiofilm producing and antiquorum sensing in Streptococcus pyogenes

Biofilm formation has been demonstrated as a potentially important mechanism contributing to antibiotic treatment failure on Streptococcus pyogenes. It could play a significant role in recurrent and chronic infections. Boesenbergia pandurata (Roxb.) Schltr., Eleutherine americana Merr. and Rhodomyrtus tomentosa (Aiton) Hassk. have been previously reported from our laboratory as effective agents against S. pyogenes. Therefore, in the present study, we observed the effect of these plants on biofilm formation. The bacterial biofilms were quantified by safranin staining and absorbance at 492 nm. The results clearly demonstrated that all subinhibitory concentrations [1/32-1/2 minimal inhibitory concentration (MIC)] of E. americana (7.81-125 microg mL(-1)) and R. tomentosa (0.24-7.81 microg mL(-1)) extracts significantly prevented biofilm formation while 1/2MIC (7.81 microg mL(-1)) of B. pandurata extract produced this effect. The issue of antiquorum sensing of this pathogenic bacterium has been further explored. A correlation between antiquorum-sensing and antibiofilm-producing activities was demonstrated. Strong inhibition on quorum sensing was displayed with the extract of R. tomentosa. Eleutherine americana extract showed partial inhibition, while B. pandurata did not show this activity. By contrast, an assay of microbial adhesion to hydrocarbon revealed no changes in the cell-surface hydrophobicity of the treated organisms. Active organisms with the ability to inhibit quorum sensing and biofilm formation are worth studying as they may provide complimentary medicine for biofilm-associated infections.     

GCN2 Protein Kinase Is Required to Activate Amino Acid Deprivation Responses in Mice Treated with the Anti-cancer Agent l-Asparaginase

Asparaginase depletes circulating asparagine and glutamine, activating amino acid deprivation responses (AADR) such as phosphorylation of eukaryotic initiation factor 2 (p-eIF2) leading to increased mRNA levels of asparagine synthetase and CCAAT/enhancer-binding protein β homologous protein (CHOP) and decreased mammalian target of rapamycin complex 1 (mTORC1) signaling. The objectives of this study were to assess the role of the eIF2 kinases and protein kinase R-like endoplasmic reticulum resident kinase (PERK) in controlling AADR to asparaginase and to compare the effects of asparaginase on mTORC1 to that of rapamycin. In experiment 1, asparaginase increased hepatic p-eIF2 in wild-type mice and mice with a liver-specific PERK deletion but not in GCN2 null mice nor in GCN2-PERK double null livers. In experiment 2, wild-type and GCN2 null mice were treated with asparaginase (3 IU per g of body weight), rapamycin (2 mg per kg of body weight), or both. In wild-type mice, asparaginase but not rapamycin increased p-eIF2, p-ERK1/2, p-Akt, and mRNA levels of asparagine synthetase and CHOP in liver. Asparaginase and rapamycin each inhibited mTORC1 signaling in liver and pancreas but maximally together. In GCN2 null livers, all responses to asparaginase were precluded except CHOP mRNA expression, which remained partially elevated. Interestingly, rapamycin blocked CHOP induction by asparaginase in both wild-type and GCN2 null livers. These results indicate that GCN2 is required for activation of AADR to asparaginase in liver. Rapamycin modifies the hepatic AADR to asparaginase by preventing CHOP induction while maximizing inhibition of mTORC1.     

The mode of antistaphylococcal action of Eleutherine americana

The anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) activity and the possible mechanism of action of a crude extract from red bulbs of Eleutherine americana Merr. were investigated. The crude ethanolic extract from E. americana produced minimum inhibitory concentrations (MICs) of 62.5–1000 and 250 μg mL⁻¹ against MRSA isolates and the reference strains, respectively. Treatment of S. aureus ATCC 27664 with a crude extract at 2MIC reduced the inoculum size by 5 log at 24 h compared with the control. The combined effect of the extract and 7.5% NaCl on the enterotoxin-producing ATCC strain resulted in no detection of organisms within 24 h compared with the control. The release of cell materials after extract treatment was determined by measuring OD_260 nm, the treatment resulted in cytoplasmic leakage. Determination of OD_620 nm showed that the extract did not cause gross cell wall damage. However, observation of S. aureus cells under an electron microscope after treatment with 2MIC and 4MIC of the crude extract revealed that the extract caused damage to membrane morphology. A knowledge of the mechanism of action of the E. americana extract may offer useful hints in the search for novel antibacterial substance.     

Control of nanobiointerfaces generated from well-defined biomimetic polymer brushes for protein and cell manipulations

To better understand protein/material and cell/material interactions at the submolecular level, well-defined polymer brushes consisting of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) on silicon wafers were prepared by atom transfer radical polymerization (ATRP). Silicon wafers were treated with 3-(2-bromoisobutyryl)propyl dimethylchlorosilane (BDCS) to form a monolayer that acts as initiators for ATRP. Silicon-supported BDCS monolayers were soaked in a methanol/water mixture solution containing Cu(I)Br, bipyridine, and a sacrificial initiator. After MPC was added to the solution, ATRP was carried out for 18 h. The molecular weight and thickness of the PMPC brush layer on the silicon surface increased with an increase in the polymerization time. The dense polymer brushes were obtained by the "grafting from" system. By selective decomposition of the BDCS monolayer by UV light-irradiation, the PMPC brush region and the sizes were well controlled, resulting in fabricating micropatterns of the PMPC brushes. When the thickness of the PMPC brush layer was greater than 5.5 +/- 1.0 nm (3 h polymerization), serum protein adsorption and fibroblast adhesion were effectively reduced, i.e., proteins and cells could recognize such thin polymer brushes on the surface. In addition, the density of the adherent cells on the patterned PMPC brush surface could be controlled by changing the size of the pattern.     

General Control Nonderepressible 2 (GCN2) Kinase Protects Oligodendrocytes and White Matter during Branched-chain Amino Acid Deficiency in Mice

Branched-chain amino acid (BCAA) catabolism is regulated by branched-chain α-keto acid dehydrogenase, an enzyme complex that is inhibited when phosphorylated by its kinase (BDK). Loss of BDK function in mice and humans causes BCAA deficiency and epilepsy with autistic features. In response to amino acid deficiency, phosphorylation of eukaryotic initiation factor 2α (eIF2∼P) by general control nonderepressible 2 (GCN2) activates the amino acid stress response. We hypothesized that GCN2 functions to protect the brain during chronic BCAA deficiency. To test this idea, we generated mice lacking both Gcn2 and Bdk (GBDK) and examined the development of progeny. GBDK mice appeared normal at birth, but they soon stopped growing, developed severe ataxia, tremor, and anorexia, and died by postnatal day 15. BCAA levels in brain were diminished in both Bdk^−/− and GBDK pups. Brains from Bdk^−/− pups exhibited robust eIF2∼P and amino acid stress response induction, whereas these responses were absent in GBDK mouse brains. Instead, myelin deficiency and diminished expression of myelin basic protein were noted in GBDK brains. Genetic markers of oligodendrocytes and astrocytes were also reduced in GBDK brains in association with apoptotic cell death in white matter regions of the brain. GBDK brains further demonstrated reduced Sod2 and Cat mRNA and increased Tnfα mRNA expression. The data are consistent with the idea that loss of GCN2 during BCAA deficiency compromises glial cell defenses to oxidative and inflammatory stress. We conclude that GCN2 protects the brain from developing a lethal leukodystrophy in response to amino acid deficiencies.     

alpha-Glucosidase inhibitory activity of cyanidin-3-galactoside and synergistic effect with acarbose

Cyanidin-3-galactoside, a natural anthocyanin, was investigated for its alpha-glucosidase inhibitory activity. The IC(50) value of cyanidin-3-galactoside was 0.50 +/- 0.05 mM against intestinal sucrase. A low dose of cyanidin-3-galactoside showed a synergistic inhibition on intestinal alpha-glucosidase (maltase and sucrase) when combined with acarbose. A kinetic analysis showed that cyanidin-3-galactoside gave a mixed type inhibition against intestinal sucrase. The results indicated that cyanidin-3-galactoside was an alpha-glucosidase inhibitor and could be used in combination with acarbose for treatment of diabetes.