Antiherpes simplex virus type 2 activity of the antimicrobial peptide subtilosin

In the present study we evaluated the antiviral activity of subtilosin, a cyclical peptide isolated from Bacillus amyloliquefaciens, against herpes simplex virus type 2 (HSV-2) in cell cultures and we investigated subtilosin mode of action. We determined, using a virus yield inhibition assay, that non cytotoxic concentrations of subtilosin inhibit HSV-2 replication in Vero cell cultures. Subtilosin strongly inhibited extracellular and total virus production even when it was added at 8 h post-infection indicating that not only virus release but also viral particle formation is impeded by the antiviral peptide. Although viral glycoprotein gD level of expression is not affected by the bacteriocin, an altered pattern of gD intracellular localization was detected by immunofluorescence assay in subtilosin treated culture. On the other hand, at high concentrations subtilosin displays virucidal action.Fil: Quintana, Verónica Mara. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; ArgentinaFil: Torres, Nicolás. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Wachsman, Mónica B.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; ArgentinaFil: Sinko, Patrick J.. State University of New Jersey; Estados UnidosFil: Castilla, Viviana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; ArgentinaFil: Chikindas, Michael. State University of New Jersey; Estados Unido

Mode of action of lactocin 160, a bacteriocin from vaginal Lactobacillus rhamnosus.

OBJECTIVES: To determine the mechanism of antimicrobial action of lactocin 160, a bacteriocin produced by the healthy vaginal strain of Lactobacillus rhamnosus, using an established model, with Micrococcus luteus ATCC 10420 as a test organism. METHODS: Sensitivity of M. luteus to lactocin 160 was determined by the diffusion assay. Loss of cellular ATP in the lactocin-treated cells was elucidated using a commercially available ATP determination kit (luciferin-luciferase bioluminescence assay). Luminescence intensity as a reflection of ATP quantity was determined using a luminometer. Dissipation of membrane potential (Deltapsi) was studied using fluorophore DiSC3(5) with the fluorescence spectrum sensitive to changes in Deltapsi. RESULTS: Lactocin 160 inhibited growth of M. luteus ATCC 10420 at a concentration of 5 microg/ml. There were no significant changes in the intracellular ATP level of M. luteus upon the addition of 20 microg/ml of lactocin 160. However, the extracellular ATP level increased significantly. This means that the treatment of cells with lactocin 160 resulted in an efflux of ATP from inside the cells. Therefore, a partially purified lactocin 160 preparation (16 microg /ml of the bacteriocin in the sample) killed sensitive cells and dissipated 3.12 +/- 0.36% of Deltapsi. CONCLUSION: Lactocin 160 has a mode of action typical for bacteriocins. It disturbs the cellular membrane (Deltapsi dissipation) and induces ATP efflux, most likely because of the pore formation, which is a common mechanism of action for many bacteriocins

Safety Study of an Antimicrobial Peptide Lactocin 160, Produced by the Vaginal Lactobacillus rhamnosus

Objective. To evaluate the safety of the antimicrobial peptide, lactocin 160. Methods. Lactocin 160, a product of vaginal probiotic Lactobacillus rhamnosus 160 was evaluated for toxicity and irritation. An in vitro human organotypic vaginal-ectocervical tissue model (EpiVaginal) was employed for the safety testing by determining the exposure time to reduce tissue viability to 50% (ET-50). Hemolytic activity of lactocin160 was tested using 8% of human erythrocyte suspension. Susceptibility of lactobacilli to lactocin160 was also studied. Rabbit vaginal irritation (RVI) model was used for an in vivo safety evaluation. Results. The ET-50 value was 17.5 hours for lactocin 160 (4.9 hours for nonoxynol 9, N9). Hemolytic activity of lactocin 160 was 8.2% (N9 caused total hemolysis). Lactobacilli resisted to high concentrations of peptide preparation. The RVI model revealed slight vaginal irritation. An average irritation index grade was evaluated as “none.” Conclusions. Lactocin 160 showed minimal irritation and has a good potential for intravaginal application

MUTAGENICITY OF KOJIC ACID PRODUCED FROM WILD TYPE Aspergillus oryzae

Kojic acid (KA) is a multipurpose natural compound, commonly used in the food and cosmetics industry. It is produced by different types of molds especially by the species Aspergillus oryzae. In this study, we test the mutagenicity of local produced kojic acid PKA produced by the wild-type strain of A. oryzae as well as the standard commercially produced kojic acid SKA and ascorbic acid SAA for comparison to stop food manufacturers doubts about using KA. AMES test with Salmonella enterica ATCC 29629 strain TA1535 and S9 liver enzyme for metabolic activation of the tested compounds were utilized in this study by direct and indirect methods were used in the test. The study results showed that the tested PKA kojic acid had cannot induce reverse mutation in the strain ATCC 29629TA1535 used in the test in contrast with the positive control in direct and indirect methods, even where the tested acids were treated with S9 liver enzymes with or without pre-incubation for three hours at 37 °C hadn’t given positive results on TA1535. The used concentration of 1% and 10% S9 liver enzymes hadn’t metabolically activated the three acids. 6 mg/plate of KA inhibited the growth of TA1535. SAA gave the same negative results as PKA and SKA. In conclusion, the tested PKA produced by wild-type A. oryzae was not has mutagenic effect on bacterial strain TA1535   and gave the same effect as the commonly used as food additive SAA and SKA even when treated with S9 liver enzymes

Spermicidal Activity of the Safe Natural Antimicrobial Peptide Subtilosin

Bacterial vaginosis (BV), a condition affecting millions of women each year, is primarily caused by the gram-variable organism Gardnerella vaginalis. A number of organisms associated with BV cases have been reported to develop multidrug resistance, leading to the need for alternative therapies. Previously, we reported the antimicrobial peptide subtilosin has proven antimicrobial activity against G. vaginalis, but not against the tested healthy vaginal microbiota of lactobacilli. After conducting tissue sensitivity assays using an ectocervical tissue model, we determined that human cells remained viable after prolonged exposures to partially-purified subtilosin, indicating the compound is safe for human use. Subtilosin was shown to eliminate the motility and forward progression of human spermatozoa in a dose-dependent manner, and can therefore be considered a general spermicidal agent. These results suggest subtilosin would be a valuable component in topical personal care products aimed at contraception and BV prophylaxis and treatment

Quorum sensing and its inhibition mechanisms

The article is a brief literature review. This article provides an overview of the Quorum Sensing system in bacterial communities, highlighting the peculiarities of the system for gram-positive and gramnegative microorganisms. Basic information about the three existing Quorum Sensing systems is presented. Information is also given about different types of autoinducers, which are signaling molecules that trigger a cascade of behavioral reactions. The importance of the Quorum Sensing system as one of the fundamental mechanisms in the formation and regulation of bacterial biofilms is described, emphasizing the significance of biofilm microorganisms for modern clinical medicine and their impact on aggravating the issue of antibiotic resistance. The main mechanisms of inhibiting bacterial quorum, including by other microorganisms, are presented. The work discusses enzymatic and non-enzymatic methods of inhibiting the Quorum Sensing system, points of application and mechanisms of action. Some microorganisms with confirmed enzymatic activity by Quorum Quenching are indicated. Also presented are registered cases of suppression of other bacteria by microorganisms through the Quorum Sensing inhibitors system

The Role of Phonological Awareness and Phonetic Radical Awareness in Acquiring Chinese Literacy Skills in Learners of Chinese as a Second Language

There is much research into the roles of phonological awareness and phonetic radical awareness in the development of Chinese character reading and writing skills in native-speaking children, but there is comparatively little work on the relationship between such metalinguistic skills and character literacy skills in adult learners of Chinese a second language (CSL). In this study, we explored this issue with 83 Arabic and English CSL learners who had studied Chinese in their home country. Their knowledge of phonological awareness, phonetic radical awareness, and Chinese character reading and writing was measured. There were two main findings. Firstly, the learners’ phonological awareness, but not their phonetic radical awareness, predicted the acquisition of character reading and writing skills directly or indirectly. Secondly, phonetic radical awareness did not mediate the effect of phonological awareness on character reading and writing skills. The results point to the different roles that phonological awareness and phonetic radical awareness play in the development of character literacy skills, and the still unclear relationship between phonological awareness and phonetic radical awareness. These findings are important for understanding the contribution of phonological awareness and phonetic radical awareness to the acquisition of character literacy skills for CSL learners