Characterisation of a Leishmania mexicana knockout lacking guanosine diphosphate-mannose pyrophosphorylase

In eukaryotes, the enzyme GDP-mannose pyrophosphorylase (GDP-MP) is essential for the formation of GDP-mannose, the donor of activated mannose for all glycosylation reactions. Unlike other eukaryotes, where deletion of GDP-mannose pyrophosphorylase is lethal, deletion of this gene in Leishmania mexicana has no effect on viability, but leads to the generation of avirulent parasites. In this study, we show that the null mutants have a perturbed morphology and cytokinesis, retarded growth and increased adherence to the substratum where they form large colonies. The null mutants attach avidly to mouse macrophages, but unlike the wild type organisms, they do not bind to the complement receptor 3 and are slow to induce phagocytosis. Once internalised, they localise to the phagolysosome, but in contrast to wild type organisms which transform into the intracellular amastigote and establish in the macrophage, they are cleared by 24 h in culture and by 5 h in vivo. The null mutants are hypersensitive to human but not mouse complement and to temperature and acidic pH. Surprisingly, in view of the lack of several known host-protective antigens, injection of the mutant parasites into BALB/c mice confers significant and long lasting protection against infection, suggesting that these temperature sensitive mutants are an attractive candidate for a live attenuated vaccine.     

FISH glossary: an overview of the fluorescence in situ hybridization technique

The introduction of FISH (fluorescence in situ hybridization) marked the beginning of a new era for the study of chromosome structure and function. As a combined molecular and cytological approach, the major advantage of this visually appealing technique resides in its unique ability to provide an intermediate degree of resolution between DNA analysis and chromosomal investigations while retaining information at the single-cell level. Used to support large-scale mapping and sequencing efforts related to the human genome project, FISH accuracy and versatility were subsequently capitalized on in biological and medical research, providing a wealth of diverse applications and FISH-based diagnostic assays. The diversification of the original FISH protocol into the impressive number of procedures available these days has been promoted throughout the years by a number of interconnected factors: the improvement in sensitivity, specificity and resolution, together with the advances in the fields of fluorescence microscopy and digital imaging, and the growing availability of genomic and bioinformatic resources. By assembling in a glossary format many of the "acronymed" FISH applications published so far, this review intends to celebrate the ability of FISH to re-invent itself and thus remain at the forefront of biomedical research.     

Copper acquisition by the SenC protein regulates aerobic respiration in Pseudomonas aeruginosa PAO1

Actinophage TG1 forms stable lysogens by integrating at a unique site on chromosomes of Streptomyces strains. The phage ( attP _TG1 ) and bacterial ( attB _TG1 ) attachment sites for TG1 were deduced from comparative genomic studies on the TG1-lysogen and nonlysogen of Streptomyces avermitilis . The attB _TG1 was located within the 46-bp region in the dapC gene (SAV4517) encoding the putative N -succinyldiaminopimelate aminotransferase. TG1-lysogens of S. avermitilis , however, did not demand either lysine or diaminopimelate for growth, indicating that the dapC annotation of S. avermitilis requires reconsideration. A bioinformatic survey of DNA databases using the fasta program for the attB _TG1 sequence extracted possible integration sites from varied streptomycete genomes, including Streptomyces coelicolor A3(2) and Streptomyces griseus . The gene encoding the putative TG1 integrase ( int _TG1 ) was located adjacent to the attP _TG1 site. TG1 integrase deduced from the int _TG1 gene was a protein of 619 amino acids having a high sequence similarity to φC31 integrase, especially at the N-terminal catalytic region. By contrast, sequence similarities at the C-terminal regions crucial for the recognition of attachment sites were moderate or low. The site-specific recombination systems based on TG1 integrase were shown to work efficiently not only in Streptomyces strains but also in heterologous Escherichia coli .     

Multiple origins of the western European house mouse in the Aeolian Archipelago: clues from mtDNA and chromosomes

The expansion of Mus musculus domesticus from its origin has been studied in detail. The colonization routes and times depended on its commensal habits which favoured a rapid and recent dispersal, making it difficult to unravel the expansion pattern. The situation is still obscure in the central Mediterranean area. Mitochondrial D-loop was sequenced for 65 mice from the Aeolian Archipelago and the sixteen haplotypes identified were compared with the 528 available mouse haplotypes. The central Mediterranean phylogeography, the demographic history of the Aeolian mice and the relationships between mtDNA and karyotypes was investigate. Five lineages are present, belonging to five of the haplogroups previously described for the Mediterranean basin, and most individuals fall within the European haplogroups. The Archipelago was subjected to multiple colonizations and chromosomal and molecular data agree in indicating Sicily and Italy as possible sources of colonization in recent times. Nevertheless, the signatures of earlier colonizations might have been lost through extinction and admixing of mice due to human movements. Drastic events during the entire colonization process have led to the present-day random distribution of haplotypes. Furthermore, Salina emerges as an ancestral condition and no relation between karyotype composition and haplotype variability was highlighted.     

Star Trk(B): The astrocyte path to neurodegeneration

Comment on: Colombo E, et al. J Exp Med 2012; 209:521-35.     

Recovering the osteoblastic differentiation potential of mesenchymal stem cells derived from diabetic rats by photobiomodulation therapy

Autologous cell-based therapy for bone regeneration might be impaired by diabetes mellitus (DM) due to the negative effects on mesenchymal stem cells (MSCs) differentiation. Strategies to recover their osteogenic potential could optimize the results. We aimed to evaluate the effect of photobiomodulation (PBM) therapy on osteoblast differentiation of rats with induced DM. Bone marrow MSCs of healthy and diabetic rats were isolated and differentiated into osteoblasts (OB and dOB, respectively). dOB were treated with PBM therapy every 72 hour (660 nm; 0.14 J; 20 mW; 0.714 W/cm², and 5 J/cm²). Cell morphology, viability, gene and protein expression of osteoblastic markers, alkaline phosphatase (ALP) activity, and the mineralized matrix production of dOB-PBM were compared to dOB. PBM therapy improved viability of dOB, increased the gene and protein expression of bone markers, the ALP activity and the mineralized matrix production. PBM therapy represents an innovative therapeutic approach to optimize the treatment of bone defects in diabetic patients.