Novel cyanine dyes and homodimeric styryl dyes as fluorescent probes for assessment of lactic acid bacteria cell viability.

Innovations in labeling techniques and in the design and synthesis of dye structures are closely related to the development of service equipment such as light sources and detection methods. Novel styryl homodimers and monomethine cyanine dyes were synthesized and their staining abilities for discrimination between live and dead lactic acid bacterial cells were investigated. The dyes were combined in pairs based on their excitation and emission maxima and the capacity to penetrate through cell membranes of viable bacterial cells. The absorption maxima in the same region and the large Stocks shifts of the styryl derivatives allowed viability analysis to be done with epifluorescent microscope with a very basic configuration - one light source about 480nm and one filter for the fluorescent emissions. A staining protocol was developed and applied for live/dead analysis of Bulgarian yoghurt starters. The live cells quantification by the fluorescence dyes coincided well with the results of the much more time-consuming tests by plate counting. Thus, the proposed dye combinations are appropriate for rapid viability estimation in small laboratories that may have conventional equipment.

Antifungal activity and identification of Lactobacilli, isolated from traditional dairy product "katak".

Filamentous moulds are the main spoilage microorganisms, responsible for significant economic losses and several healthy risks in human food chain. The lactic acid bacteria (LAB), especially lactobacilli could be a natural antagonist of these dangerous organisms. In Bulgaria, a very limited data exists on the antifungal activity of LAB microbiota of fermented dairy products. In the present study, four active strains were isolated from traditional fermented curd/yogurt-like product "katak", produced in Bulgaria from centuries. The new isolates KR3, KR4, KR51 and KR53 were identified by API 50 CH biochemical test and different molecular methods (species-specific PCR, RAPD-PCR and 16S rDNA sequence analysis) as Lactobacillus brevis. According to our knowledge, this is the first data on the molecular characterization of the Lactobacillus microbiota of "katak". A broad spectrum of antifungal activity of the four L. brevis KR strains against test-cultures representatives of carcinogenic, toxigenic, deteriorative and allergenic fungi from the genera Aspergillus, Fusarium, Penicillium and Trichoderma was estimated. Strains L. brevis KR3, KR4 and KR51 completely suppress the growth of Penicillium claviforme, Aspergillus awamori and Aspergillus niger. With regard to Aspergillus flavus and Trichoderma viride, a lower and strain-specific inhibitory activity was observed. The antifungal activity of our new L. brevis isolates seems to be a promising advantage of these four strains, suggesting their potential applications in different food technologies as bio-preservative agents against moulds.

Tyrosine kinase inhibitor tyrphostin AG490 retards chronic joint inflammation in mice.

Tyrphostin AG490 is a Janus kinase (JAK) 2 inhibitor that is clinically used as an anticancer agent and is also effective in various models of inflammatory and autoimmune diseases. In this study, we examined the effects of tyrphostin AG490 on the development of collagenase-induced osteoarthritis (CIOA). Our results showed that tyrphostin-ameliorated cartilage and bone destructions. This effect was associated with decreased expression of signal transducers and activators of transcription 3 (STAT3), phosphorylated JAK2, Dickkopf homolog 1, and receptor activator of nuclear factor κB ligand (RANKL) in the joints of arthritic mice. Tyrphostin AG490 suppressed STAT3 phosphorylation and the expression of tumor necrosis factor-related apoptosis-inducing ligand and RANKL by synovial fluid cells. The drug inhibited RANKL-induced osteoclast differentiation in vitro. Molecules, such as tyrphostin AG490 that limit bone erosion and influence osteoclast generation, might have therapeutic utility in joint degenerative disorders.