Polyvinyl alcohol/chitosan wound dressings loaded with antiseptics.

Wound care remains a challenge in healthcare. This work aimed to develop a new polyvinyl alcohol (PVA)/chitosan (Ch) based wound dressing able to ensure protection, hydration and a controlled release of antiseptics, as alternative to actual treatments. Two distinct formulations (1:1 and 3:1, w/w) were prepared, sterilized by autoclaving and characterized concerning surface morphology, degradation over the time, mechanical properties and hydrophilicity. Both dressings revealed adequate properties for the intended purpose. The dressings were loaded with chlorhexidine (CHX) and polyhexanide (PHMB) and the drug release profiles were determined using Franz diffusion cells. The release of PHMB was more sustained than CHX, lasting for 2 days. As the amounts of drugs released by PVA/Ch 1:1 were greater, the biological tests were done only with this formulation. The drug loaded dressings revealed antibacterial activity against S. aureus and S. epidermidis, but only the ones loaded with PHMB showed adequate properties in terms of cytotoxicity and irritability. The application of this elastic dressing in the treatment of wounds in a dog led to faster recovery than conventional treatment, suggesting that the material can be a promising alternative in wound care.

The legacy of Columbus in American horse populations assessed by microsatellite markers.

Criollo horse populations descend from horses brought from the Iberian Peninsula over the period of colonization (15th to 17th century). They are spread throughout the Americas and have potentially undergone genetic hybridization with other breeds in the recent past. In this study, 25 autosomal microsatellites were genotyped in 50 horse breeds representing Criollo populations from 12 American countries (27 breeds), breeds from the Iberian Peninsula (19), one breed each from France and Morocco and two cosmopolitan horse breeds (Thoroughbred and Arabian). The genetic relationships among breeds identified five clusters: Celtic; Iberian; North American with Thoroughbred influence; most Colombian breeds; and nearly all other Criollo breeds. The group of "all other Criollo breeds" had the closest genetic relationship with breeds originating from the Iberian Peninsula, specifically with the Celtic group. For the whole set of Criollo breeds analysed, the estimated genetic contribution from other breeds was approximately 50%, 30% and 20% for the Celtic, Iberian and Arab-Thoroughbred groups, respectively. The spatial distribution of genetic diversity indicates that hotspots of genetic diversity are observed in populations from Colombia, Ecuador, Brazil, Paraguay and western United States, possibly indicating points of arrival and dispersion of Criollo horses in the American continent. These results indicate that Criollo breeds share a common ancestry, but that each breed has its own identity.

Fertility Assessment in Sorraia Stallions by Sperm-Fish and Fkbp6 Genotyping.

The Sorraia, a critically endangered indigenous Iberian horse breed, is characterized by low genetic variability, high rate of inbreeding, bad sperm quality and subfertility. Here, we studied 11 phenotypically normal but subfertile Sorraia stallions by karyotyping, sex chromosome sperm-FISH and molecular analysis of FKBP6 - a susceptibility locus for impaired acrosome reaction (IAR). The stallions had normal sperm concentration (>300 million cells/ml), but the numbers of progressively motile sperm (21%) and morphologically normal sperm (28%) were invariably low. All stallions had a normal 64,XY karyotype. The majority of sperm (89%) had normal haploid sex chromosome content, although 11% of sperm carried various sex chromosome aneuploidies. No correlation was found between the percentage of sperm sex chromosome abnormalities and inbreeding, sperm morphology or stallion age. Direct sequencing of FKBP6 exon 4 for SNPs g.11040315G>A and g.11040379C>A revealed that none of the stallions had the susceptibility genotype (A/A-A/A) for IAR. Instead, all animals had a G/G-A/A genotype - a testimony of low genetic variability. The findings ruled out chromosomal abnormalities and genetic predisposition for IAR as contributing factors for subfertility. However, low fertility of the Sorraia stallions could be partly attributed to relatively higher rate of sex chromosome aneuploidies in the sperm.

Derepression of a baker's yeast strain for maltose utilization is associated with severe deregulation of HXT gene expression.

AIMS: We undertook to improve an industrial Saccharomyces cerevisiae strain by derepressing it for maltose utilization in the presence of high glucose concentrations. METHODS AND RESULTS: A mutant was obtained from an industrial S. cerevisiae strain following random UV mutagenesis and selection on maltose/5-thioglucose medium. The mutant acquired the ability to utilize glucose simultaneously with maltose and possibly also sucrose and galactose. Aerobic sugar metabolism was still largely fermentative, but an enhanced respirative metabolism resulted in a 31% higher biomass yield on glucose. Kinetic characterization of glucose transport in the mutant revealed the predominance of the high-affinity component. Northern blot analysis showed that the mutant strain expresses only the HXT6/7 gene irrespective of the glucose concentration in the medium, indicating a severe deregulation in the induction/repression pathways modulating HXT gene expression. Interestingly, maltose-grown cells of the mutant display inverse diauxy in a glucose/maltose mixture, preferring maltose to glucose. CONCLUSION: In the mutant here reported, the glucose transport step seems to be uncoupled from downstream regulation, because it seems to be unable to sense abundant glucose, via both repression and induction pathways. SIGNIFICANCE AND IMPACT OF THE STUDY: We report here the isolation of a S. cerevisiae mutant with a novel derepressed phenotype, potentially interesting for the industrial fermentation of mixed sugar substrates.

Molecular structure in peripheral dog breeds: Portuguese native breeds as a case study.

Genetic variability in purebred dogs is known to be highly structured, with differences among breeds accounting for approximately 30% of the genetic variation. However, analysis of the genetic structure in non-cosmopolitan breeds and local populations is still limited. Nine Portuguese native dog breeds, and other peripheral dog populations (five) with regional affinities, were characterized using 16 microsatellites and 225 amplified fragment length polymorphism (AFLP) markers, and the pattern of genetic differentiation was investigated. Although the level of breed differentiation detected is below that of other dog breeds, there is in most cases a correlation between breed affiliation and molecular structure. AFLP markers and Bayesian clustering methods allowed an average of 73.1% of individuals to be correctly assigned to source populations, providing robust genotypic assessment of breed affiliation. A geographical genetic structure was also detected, which suggests a limited influence of African dogs on the Iberian breeds. The sampling effect on the estimation of population structure was evaluated and there was a 2.2% decrease in genetic differentiation among breeds when working animals were included. Genetic diversity of stray dogs was also assessed and there is no evidence that they pose a threat to the preservation of the gene pool of native dog breeds.

Genetic variation in BoLA microsatellite loci in Portuguese cattle breeds.

Major histocompatibility complex (MHC) typing based on microsatellites can be a valuable approach to understanding the selective processes occurring at linked or physically close MHC genes and can provide important information on variability and relationships of populations. Using microsatellites within or in close proximity with bovine lymphocyte antigen (BoLA) genes, we investigated the polymorphisms in the bovine MHC, known as the BoLA, in eight Portuguese cattle breeds. Additional data from non-BoLA microsatellite loci were also used to compare the variability between these regions. Diversity was higher in BoLA than in non-BoLA microsatellites, as could be observed by the number of alleles, allelic richness and observed heterozygosity. Brava de Lide, a breed selected for aggressiveness and nobility, presented the lowest values of observed heterozygosity and allelic richness in both markers. Results from neutrality tests showed few statistically significant differences between the observed Hardy-Weinberg homozygosity (F) and the expected homozygosity (F(E)), indicating the apparent neutrality of the BoLA microsatellites within the analysed breeds. Nevertheless, we detected a trend of lower values of observed homozygosity compared with the expected one. We also detected some differences in the levels of allelic variability among the four BoLA microsatellites. Our data showed a higher number of alleles at the BoLA-DRB3 locus than at the BoLA-DRBP1 locus. These differences could be related to their physical position in the chromosome and may reflect functional requirements for diversity.

Genetic diversity and relationships of Portuguese and other horse breeds based on protein and microsatellite loci variation.

There are three native Portuguese horse breeds: Lusitano, Sorraia and Garrano. This study compares diversity patterns of 17 protein and 12 microsatellite markers in these three as well as 30 other breeds to infer relationships among the breeds and to compare levels of polymorphism of these breeds for use in conservation efforts. The Garrano and the Lusitano showed a high level of genetic diversity, similar to that observed for most of the other analysed breeds, while the Sorraia and Friesian breeds showed low levels of variation for both genetic marker types. The combined protein and microsatellite data produced a tree that fit historical records well and with greater confidence levels than those for either data set alone. The combined genetic diversity and relationship information provides important baseline data for future breed conservation efforts, especially for a critically endangered breed such as the Sorraia.

A lost Sorraia maternal lineage found in the Lusitano horse breed.

A common female founder individual of the Portuguese horse breeds Sorraia and Lusitano was found while conducting research on the variation of the Lusitano mitochondrial DNA lineages in relation to studbook information. We obtained 416-bp control region sequences from 16 descendents of a female Sorraia founder (Pomba) still represented in the living population of the Lusitano, according to the most recent edition of this breed's studbook. The same haplotype was found for all analysed samples and belongs to the haplogroup described by several authors as having predominantly Iberian, South American and North African haplotypes bringing new insights on the relationship between the Sorraia and the other Iberian breeds. This work illustrates how weak the boundary of breed establishment can be, especially at the same geographical region. Using the same founders in different breeds is surely one of the explanations to frequently shared haplotypes among recent breeds, resulting in a lack of consistency between mtDNA sequences and breeds and/or geographical regions.

Major histocompatibility complex locus DRA polymorphism in the endangered Sorraia horse and related breeds.

The major histocompatibility complex (MHC) genes play well-defined roles in eliciting immune responses and combating infectious diseases. This genetic system is among the most polymorphic. The extent of genetic variation within a population has been directly correlated with fitness for many traits. The MHC class II locus DRA polymorphism was analysed in the endangered Sorraia horse, two other Portuguese and four New World horse breeds considered to be historically close to the Sorraia. Comparison of the Sorraia with other breeds demonstrated less MHC variation among Sorraia horses. If DRA polymorphism provides greater disease resistance, selective breeding to increase MHC polymorphism may increase fitness of this population.

Variation in the mitochondrial control region sequence between the two maternal lines of the Sorraia horse breed

The mitochondrial control region sequence was compared between the two surviving matrilineal lineages of the Sorraia horse. Two haplotypes were found, showing three differences between them. The reduced number of surviving maternal lineages emphasizes the importance of establishing a conservation plan for this endangered breed

Microsatellites in Portuguese autochthonous horse breeds: usefulness for parentage testing

The use of DNA technology for parentage testing is increasing every day. Most laboratories have improved their exclusion probabilities (PE) by the addition of DNA microsatellite loci to standard blood-typing results. The efficacy of each locus depends on the number of alleles detected and their frequencies in the breeds tested. Here we analyzed the usefulness of six microsatellites for routine parentage testing in three Portuguese autochthonous horse breeds: Lusitano, Sorraia and Garrano. The DNA loci analyzed - ASB2, HMS3, HMS7, HTG4, HTG10, and VHL20 - were chosen based upon the polymorphism detected in other breeds with Iberian horse influence. The estimated probability of exclusion of wrongly named parents (PE) was high, with values ranging from 88.5 per cent to 99.6 per cent

First epidemiological data on pathogenic leptospires isolated on the Azorean islands.

Insectivores (Erinaceus europaeus) and rodents (Rattus rattus, R. norvegicus and Mus musculus) from different islands of the Azores Archipelago were found to carry three distinct Leptospira interrogans s.l. serovars (copenhageni, icterohaemorrhagiae and ballum) which have never been previously investigated there. The house mouse and the black rat were the major Leptospira reservoirs showing isolation rates ranging from 0% for both species (in Graciosa) to 88% and 33%, respectively (in Sãao Miguel). This study also showed that the majority of the animals with positive kidney cultures exhibited specific agglutinins against the isolated strains of Leptospira. The observed isolation rates in the different islands, with a very interesting island variation in prevalence, suggest that small mammals, serving as sylvatic reservoirs of pathogenic leptospires, may represent an important risk to the health of humans and livestock, particularly in the islands of Terceira and Sãao Miguel.