In Vitro Effect of Copper (I) Complex [Cu(NN1)2](ClO4) on Vibrio harveyi BB170 Biofilm Formation.

Biofilm formation in pathogenic bacteria is an important factor of resistance to antimicrobial treatments, allowing them to survive for a long time in their hosts. In the search for new antibiofilm agents, in this work we report the activity of a copper (I) complex, [Cu(NN1)2]ClO4, synthesized with Cu (I) and NN1, an imine ligand 6-((quinolin-2-ylmethylene)amino)-2H-chromen-2-one, a derivate of natural compound coumarin. The antibacterial and antibiofilm capacity was evaluated in Vibrio harveyi BB170 used as model bacteria. Antibacterial activity was measured in vitro by minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and half-maximal inhibitory concentration (IC50) determination. Antibiofilm capacity of copper (I) complex was analyzed by different concentrations of IC50 values. The results showed that the sub-IC50 concentration, 12.6 µg/mL of the copper (I) complex, was able to reduce biofilm formation by more than 75%, and bacterial viability was reduced by 50%. Inverted and confocal laser scanning microscopy showed that the [Cu(NN1)2]ClO4 complex affected the biofilm structure. Therefore, the copper (I) complex is effective as an antibiofilm compound in V. harveyi BB170.

Efectos clínicos de Lactobacillus reuteri en el tratamiento de la gingivitis: Ensayo clínico aleatorizado controlado / Clinical effect of Lactobacillus reuteri in the treatment of gingivitis: a randomized controlled clinical trial

RESUMEN: Objetivo: Evaluar la eficacia de L. reuteri como adjunto en el tratamiento de la gingivitis. Material y Métodos: Se realizó un ensayo clínico aleatorizado placebo controlado en sujetos con gingivitis durante 3 meses. El grupo test recibió una tableta por día de la cepa probiótica Lactobacillus reuteri (dosis 2x10(8) UFC por día), el grupo control recibió las mismas tabletas pero sin bacterias vivas. La variable de resultado principal fue el índice gingival (IG), y las variables de resultado secundarias fueron el índice de placa (IP) y el índice de sangrado al sondaje (IS). Se realizó comparación intra e inter-grupos en el basal y al finalizar la intervención (3 meses). Resultados: Fueron incluidos en el análisis un total de 30 sujetos (15 test, 15 control). No hubo diferencias estadísticamente significativas entre los grupos en el basal (p> 0.05). Después de 3 meses de intervención se produjo en ambos grupos una disminución estadísticamente significativa en el índice gingival, índice de sangrado al sondaje e índice de placa (p< 0.05). Se detectó una significativa reducción en el número de sitios con IG 2 solo en el grupo test (p< 0.05). Conclusiones: El uso de tabletas de probiótico con L. reuteri como adjunto en el tratamiento de la gingivitis, produce una significativa reducción en el número de sitios que presentan inflamación más severa.

ABSTRACT: Aim: To evaluate the efficacy of L. reuteri as adjunct in the treatment of the gingivitis. Materials and Methods: A placebo-controlled clinical trial was conducted in gingivitis subjects for 3 months. Test treatment consisted of the administration of one tablet per day containing the probiotic strain Lactobacillus reuteri (doses 2x10(8) UFC per day), the control group received the same tablets but without live bacteria. The main outcome variable was the change in gingival index (GI), and the secondary outcome variables were the plaque index (PII) and the bleeding on probing (BoP). Outcome variables were compared between and within groups at baseline and at the end of intervention (3 months). Results: A total of 30 subjects (15 test, 15 control) were included in the analysis. No statistically significant differences were found between the groups at baseline (p> 0.05). Both treatment groups experienced a statistically significant improvement in the GI, PII and BoP (p < 0.05). There was a significant reduction in the number of sites with GI 2 only in the test group (p< 0.05). Conclusions: The use of probiotic tablets containing L. reuteri produces a significant reduction in the number of sites with severe inflammation.

Neisseria gonorrhoeae Modulates Immunity by Polarizing Human Macrophages to a M2 Profile.

Current data suggest that Neisseria gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and antigen-presenting cells. The present report is focused on gonococcus evasion mechanism on macrophages (MФ) and its impact in the subsequent immune response. In response to various signals MФ may undergo classical-M1 (M1-MФ) or alternative-M2 (M2-MФ) activation. Until now there are no reports of the gonococcus effects on human MФ polarization. We assessed the phagocytic ability of monocyte-derived MФ (MDM) upon gonococcal infection by immunofluorescence and gentamicin protection experiments. Then, we evaluated cytokine profile and M1/M2 specific-surface markers on MФ challenged with N. gonorrhoeae and their proliferative effect on T cells. Our findings lead us to suggest N. gonorrhoeae stimulates a M2-MФ phenotype in which some of the M2b and none of the M1-MФ-associated markers are induced. Interestingly, N. gonorrhoeae exposure leads to upregulation of a Programmed Death Ligand 1 (PD-L1), widely known as an immunosuppressive molecule. Moreover, functional results showed that N. gonorrhoeae-treated MФ are unable to induce proliferation of human T-cells, suggesting a more likely regulatory phenotype. Taken together, our data show that N. gonorroheae interferes with MФ polarization. This study has important implications for understanding the mechanisms of clearance versus long-term persistence of N. gonorroheae infection and might be applicable for the development of new therapeutic strategies.

Novel missense mutation of the FAM83H gene causes retention of amelogenin and a mild clinical phenotype of hypocalcified enamel.

OBJECTIVE: Amelogenesis imperfecta (AI) is a group of clinically and genetically heterogeneous inherited conditions, causing alterations in the structure of enamel and chemical composition of enamel matrix during development. The objective of this study was to compare the clinical, radiographic, histological and immunohistochemical phenotypes of subjects affected with hypocalcified AI from three Chilean families and identify causal mutations in the FAM83H gene. DESIGN: The diagnosis was made using clinical, radiographic, histological and genealogical data from the patients, who were evaluated according to the classification criteria by Witkop. PCR and Sanger sequencing of the complete coding sequence and surrounding intron regions of the FAM83H gene were conducted. The structural study of the affected teeth was performed with light microscopy, scanning electron microscopy and immunohistochemistry. RESULTS: The probands of the three families were diagnosed with hypocalcified AI, but in only one of them the missense variant p.Gly557Cys was identified. This variant was not present in the SNP database or in 100 healthy controls and segregated with the disease in the affected family. Using light microscopy, a normal prismatic structure was observed in all three cases. However, the ultrastructure was found to be affected in two of the cases, showing persistence of organic matter including amelogenins. CONCLUSIONS: These results suggest that FAM83H missense mutation reported in one of the families analyzed in this study might cause a phenotype of hypocalcified enamel more attenuated with retention of amelogenin.

DNA from oral bacteria by sodium hydroxide-paper method suitable for polymerase chain reaction.

In the oral cavity, we can find a complex mixture of microorganisms, commensals, and pathogens. The studies of normal oral microbiota, as well as the studies of much oral pathology (e.g., caries, periodontitis), involve the isolation and cultivation of these microorganisms and their molecular analysis. The aim of this study was to validate a quick, easy, efficient, and inexpensive DNA extraction method for the recovery of genomic DNA from gram-positive and gram-negative oral bacteria to be used in polymerase chain reaction amplification. This method worked great with all samples analyzed, providing an approach to extract DNA for different microorganisms.

An artifact in studies of gene regulation using ß-galactosidase reporter gene assays.

Reporter gene assays are important tools for evaluating gene expression. A frequently used assay measures the activity of ß-galactosidase (ß-gal) expressed from lacZ in plasmid or genomic constructions. Such constructions are often used to interrogate the ability of DNA (query DNA), potentially encoding a transcription factor, to regulate in trans the expression of a promoter fused to the reporter lacZ. Query DNA is frequently inserted into a second plasmid within the α-subunit of ß-gal, interrupting its function. However, this plasmid can induce up-expression of ß-gal even when void of query DNA, leading to confusion between artifact and authentic regulation.

Bioinformatic prediction and experimental verification of Fur-regulated genes in the extreme acidophile Acidithiobacillus ferrooxidans.

The gamma-proteobacterium Acidithiobacillus ferrooxidans lives in extremely acidic conditions (pH 2) and, unlike most organisms, is confronted with an abundant supply of soluble iron. It is also unusual in that it oxidizes iron as an energy source. Consequently, it faces the challenging dual problems of (i) maintaining intracellular iron homeostasis when confronted with extremely high environmental loads of iron and (ii) of regulating the use of iron both as an energy source and as a metabolic micronutrient. A combined bioinformatic and experimental approach was undertaken to identify Fur regulatory sites in the genome of A. ferrooxidans and to gain insight into the constitution of its Fur regulon. Fur regulatory targets associated with a variety of cellular functions including metal trafficking (e.g. feoPABC, tdr, tonBexbBD, copB, cdf), utilization (e.g. fdx, nif), transcriptional regulation (e.g. phoB, irr, iscR) and redox balance (grx, trx, gst) were identified. Selected predicted Fur regulatory sites were confirmed by FURTA, EMSA and in vitro transcription analyses. This study provides the first model for a Fur-binding site consensus sequence in an acidophilic iron-oxidizing microorganism and lays the foundation for future studies aimed at deepening our understanding of the regulatory networks that control iron uptake, homeostasis and oxidation in extreme acidophiles.

In vivo formation of glutamyl-tRNA(Gln) in Escherichia coli by heterologous glutamyl-tRNA synthetases.

Two types of glutamyl-tRNA synthetase exist: the discriminating enzyme (D-GluRS) forms only Glu-tRNA(Glu), while the non-discriminating one (ND-GluRS) also synthesizes Glu-tRNA(Gln), a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). Testing the capacity to complement a thermosensitive E. coli gltX mutant and to suppress an E. coli trpA49 missense mutant we examined the properties of heterologous gltX genes. We demonstrate that while Acidithiobacillus ferrooxidans GluRS1 and Bacillus subtilis Q373R GluRS form Glu-tRNA(Glu), A. ferrooxidans and Helicobacter pylori GluRS2 form Glu-tRNA(Gln) in E. coli in vivo.