Novel mechanism of bacteriocin secretion and immunity carried out by lactococcal multidrug resistance proteins

A natural isolate of Lactococcus lactis was shown to produce two narrow spectrum class II bacteriocins, designated LsbA and LsbB. The cognate genes are located on a 5.6-kb plasmid within a gene cluster specifying LmrB, an ATP-binding cassette-type multidrug resistance transporter protein. LsbA is a hydrophobic peptide that is initially synthesized with an N-terminal extension. The housekeeping surface proteinase HtrA was shown to be responsible for the cleavage of precursor peptide to yield the active bacteriocin. LsbB is a relatively hydrophilic protein synthesized without an N-terminal leader sequence or signal peptide. The secretion of both polypeptides was shown to be mediated by LmrB. An L. lactis strain lacking plasmid-encoded LmrB and the chromosomally encoded LmrA is unable to secrete either of the two bacteriocins. Complementation of the strain with an active LmrB protein resulted in restored export of the two polypeptides across the cytoplasmic membrane. When expressed in an L. lactis strain that is sensitive to LsbA and LsbB, LmrB was shown to confer resistance toward both bacteriocins. It does so, most likely, by removing the two polypeptides from the cytoplasmic membrane. This is the first report in which a multidrug transporter protein is shown to be involved in both secretion and immunity of antimicrobial peptides.

Entropy dynamics for a propeller-shaped quantum Brownian molecular rotator

We investigate and analyze the time dependence of the so-called differential entropy as a measure of the dynamical stability of a one-dimensional, propeller-shaped quantum Brownian molecular rotator. The larger the entropy change, the more profound the instability (lower control) of the rotator. The quantum molecular rotator is modelled by the quantum Caldeira-Leggett master equation while assuming the external harmonic field for the rotator. Rotational stability is found relatively high for the constructed Gaussian states, especially for molecules with a larger number of blades.Publishe

Hsp90 governs dispersion and drug resistance of fungal biofilms

Fungal biofilms are a major cause of human mortality and are recalcitrant to most treatments due to intrinsic drug resistance. These complex communities of multiple cell types form on indwelling medical devices and their eradication often requires surgical removal of infected devices. Here we implicate the molecular chaperone Hsp90 as a key regulator of biofilm dispersion and drug resistance. We previously established that in the leading human fungal pathogen, Candida albicans, Hsp90 enables the emergence and maintenance of drug resistance in planktonic conditions by stabilizing the protein phosphatase calcineurin and MAPK Mkc1. Hsp90 also regulates temperature-dependent C. albicans morphogenesis through repression of cAMP-PKA signalling. Here we demonstrate that genetic depletion of Hsp90 reduced C. albicans biofilm growth and maturation in vitro and impaired dispersal of biofilm cells. Further, compromising Hsp90 function in vitro abrogated resistance of C. albicans biofilms to the most widely deployed class of antifungal drugs, the azoles. Depletion of Hsp90 led to reduction of calcineurin and Mkc1 in planktonic but not biofilm conditions, suggesting that Hsp90 regulates drug resistance through different mechanisms in these distinct cellular states. Reduction of Hsp90 levels led to a marked decrease in matrix glucan levels, providing a compelling mechanism through which Hsp90 might regulate biofilm azole resistance. Impairment of Hsp90 function genetically or pharmacologically transformed fluconazole from ineffectual to highly effective in eradicating biofilms in a rat venous catheter infection model. Finally, inhibition of Hsp90 reduced resistance of biofilms of the most lethal mould, Aspergillus fumigatus, to the newest class of antifungals to reach the clinic, the echinocandins. Thus, we establish a novel mechanism regulating biofilm drug resistance and dispersion and that targeting Hsp90 provides a much-needed strategy for improving clinical outcome in the treatment of biofilm infections

Molecular characterization of old local grapevine varieties from South East European countries

South East European (SEE) viticulture partially relies on native grapevine varieties, previously scarcely described. In order to characterize old local grapevine varieties and assess the level of synonymy and genetic diversity from SEE countries, we described and genotyped 122 accessions from Albania, Federation of Bosnia and Herzegovina (B&H), Croatia, Macedonia, Moldova, Montenegro, Republika Srpska (Bosnia and Herzegovina) and Romania on nine most commonly used microsatellite loci. As a result of the study a total of 86 different genotypes were identified. All loci were very polymorphic and a total of 96 alleles were detected, ranging from 8 to 14 alleles per locus, with an average allele number of 10.67. Overall observed heterozygosity was 0.759 and slightly lower than expected (0.789) while gene diversity per locus varied between 0.600 (VVMD27) and 0.906 (VVMD28). Eleven cases of synonymy and three of homonymy have been recorded for samples harvested from different countries. Cultivars with identical genotypes were mostly detected between neighboring countries. No clear differentiation between countries was detected although several specific alleles were detected. The integration of the obtained genetic data with ampelographic ones is very important for accurate identification of the SEE cultivars and provides a significant tool in cultivar preservation and utilization.

Microdevices for extensional rheometry of low viscosity elastic liquids : a review

Extensional flows and the underlying stability/instability mechanisms are of extreme relevance to the efficient operation of inkjet printing, coating processes and drug delivery systems, as well as for the generation of micro droplets. The development of an extensional rheometer to characterize the extensional properties of low viscosity fluids has therefore stimulated great interest of researchers, particularly in the last decade. Microfluidics has proven to be an extraordinary working platform and different configurations of potential extensional microrheometers have been proposed. In this review, we present an overview of several successful designs, together with a critical assessment of their capabilities and limitations

Determination of the target nucleosides for members of two families of 16S rRNA methyltransferases that confer resistance to partially overlapping groups of aminoglycoside antibiotics

The 16S ribosomal RNA methyltransferase enzymes that modify nucleosides in the drug binding site to provide self-resistance in aminoglycoside-producing micro-organisms have been proposed to comprise two distinct groups of S-adenosyl-l-methionine (SAM)-dependent RNA enzymes, namely the Kgm and Kam families. Here, the nucleoside methylation sites for three Kgm family methyltransferases, Sgm from Micromonospora zionensis, GrmA from Micromonospora echinospora and Krm from Frankia sp. Ccl3, were experimentally determined as G1405 by MALDI-ToF mass spectrometry. These results significantly extend the list of securely characterized G1405 modifying enzymes and experimentally validate their grouping into a single enzyme family. Heterologous expression of the KamB methyltransferase from Streptoalloteichus tenebrarius experimentally confirmed the requirement for an additional 60 amino acids on the deduced KamB N-terminus to produce an active methyltransferase acting at A1408, as previously suggested by an in silico analysis. Finally, the modifications at G1405 and A1408, were shown to confer partially overlapping but distinct resistance profiles in Escherichia coli. Collectively, these data provide a more secure and systematic basis for classification of new aminoglycoside resistance methyltransferases from producers and pathogenic bacteria on the basis of their sequences and resistance profiles

Genetics of Mechanosensation in the Heart

Mechanosensation (the ultimate conversion of a mechanical stimulus into a biochemical signal) as well as mechanotransduction (transmission of mechanically induced signals) belong to the most fundamental processes in biology. These effects, because of their dynamic nature, are particularly important for the cardiovascular system. Therefore, it is not surprising that defects in cardiac mechanosensation, are associated with various types of cardiomyopathy and heart failure. However, our current knowledge regarding the genetic basis of impaired mechanosensation in the cardiovascular system is beginning to shed light on this subject and is at the centre of this brief review

Biofilms of non-Candida albicans Candida species : quantification, structure and matrix composition

Most cases of candidiasis have been attributed to C. albicans, but recently, non- Candida albicans Candida (NCAC) species have been identified as common pathogens. The ability of Candida species to form biofilms has important clinical repercussions due to their increased resistance to antifungal therapy and the ability of yeast cells within the biofilms to withstand host immune defenses. Given this clinical importance of the biofilm growth form, the aim of this study was to characterize biofilms produced by three NCAC species, namely C. parapsilosis, C. tropicalis and C. glabrata. The biofilm forming ability of clinical isolates of C. parapsilosis, C. tropicalis and C. glabrata recovered from different sources, was evaluated by crystal violet staining. The structure and morphological characteristics of the biofilms were also assessed by scanning electron microscopy and the biofilm matrix composition analyzed for protein and carbohydrate content. All NCAC species were able to form biofilms although these were less extensive for C. glabrata compared with C. parapsilosis and C. tropicalis. It was evident that C. parapsilosis biofilm production was highly strain dependent, a feature not evident with C. glabrata and C. tropicalis. Scanning electron microscopy revealed structural differences for biofilms with respect to cell morphology and spatial arrangement. Candida parapsilosis biofilm matrices had large amounts of carbohydrate with less protein. Conversely, matrices extracted from C. tropicalis biofilms had low amounts of carbohydrate and protein. Interestingly, C. glabrata biofilm matrix was high in both protein and carbohydrate content. The present work demonstrates that biofilm forming ability, structure and matrix composition are highly species dependent with additional strain variability occurring with C. parapsilosis.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/28341/2006, PDTC/BIO/61112/200

Development of a High-Throughput Candida albicans Biofilm Chip

We have developed a high-density microarray platform consisting of nano-biofilms of Candida albicans. A robotic microarrayer was used to print yeast cells of C. albicans encapsulated in a collagen matrix at a volume as low as 50 nL onto surface-modified microscope slides. Upon incubation, the cells grow into fully formed “nano-biofilms”. The morphological and architectural complexity of these biofilms were evaluated by scanning electron and confocal scanning laser microscopy. The extent of biofilm formation was determined using a microarray scanner from changes in fluorescence intensities due to FUN 1 metabolic processing. This staining technique was also adapted for antifungal susceptibility testing, which demonstrated that, similar to regular biofilms, cells within the on-chip biofilms displayed elevated levels of resistance against antifungal agents (fluconazole and amphotericin B). Thus, results from structural analyses and antifungal susceptibility testing indicated that despite miniaturization, these biofilms display the typical phenotypic properties associated with the biofilm mode of growth. In its final format, the C. albicans biofilm chip (CaBChip) is composed of 768 equivalent and spatially distinct nano-biofilms on a single slide; multiple chips can be printed and processed simultaneously. Compared to current methods for the formation of microbial biofilms, namely the 96-well microtiter plate model, this fungal biofilm chip has advantages in terms of miniaturization and automation, which combine to cut reagent use and analysis time, minimize labor intensive steps, and dramatically reduce assay costs. Such a chip should accelerate the antifungal drug discovery process by enabling rapid, convenient and inexpensive screening of hundreds-to-thousands of compounds simultaneously