Multilocus phylogeography of the common midwife toad, Alytes obstetricans (Anura, Alytidae): Contrasting patterns of lineage diversification and genetic structure in the Iberian refugium.

Recent investigations on the evolutionary history of the common midwife toad (Alytes obstetricans) revealed high levels of geographically structured genetic diversity but also a situation where delineation of major historical lineages and resolution of their relationships are much more complex than previously thought. We studied sequence variation in one mitochondrial and four nuclear genes throughout the entire distribution range of all recognized A. obstetricans subspecies to infer the evolutionary processes that shaped current patterns of genetic diversity and population subdivision. We found six divergent, geographically structured mtDNA haplogroups diagnosing population lineages, and varying levels of admixture in nuclear markers. Given the timeframe inferred for the splits between major lineages, the climatic and environmental changes that occurred during the Pleistocene seem to have shaped the diversification history of A. obstetricans. Survival of populations in allopatric refugia through the Ice Ages supports the generality of the "refugia-within-refugia" scenario for the Iberian Peninsula. However, lineages corresponding to subspecies A. o. almogavarii, A. o. pertinax, A. o. obstetricans, and A. o. boscai responded differently to Pleistocene climatic oscillations after diverging from a common ancestor. Alytes o. obstetricans expanded northward from a northern Iberian refugium through the western Pyrenees, leaving a signal of contrasting patterns of genetic diversity, with a single mtDNA haplotype north of the Pyrenees from SW France to Germany. Both A. o. pertinax and A. o. boscai are widespread and genetically diverse in Iberia, the latter comprising two divergent lineages with a long independent history. Finally, A. o. almogavarii is mostly restricted to the north-eastern corner of Iberia north of the Ebro river, with additional populations in a small region in south-eastern France. This taxon exhibits unparalleled levels of genetic diversity and little haplotype sharing with other lineages, suggesting a process of incipient speciation.

Development and characterization of microsatellite markers for Brazilian four-eyed frogs (genus Pleurodema) endemic to the Caatinga biome.

We used pyrosequencing to develop microsatellite markers for the Brazilian four-eyed frog Pleurodema diplolister and tested the microsatellite markers for cross-amplification in its sister Pleurodema alium, which are both endemic species of the Caatinga biome in northeastern Brazil. We used multiplex sets to amplify and genotype 30 individuals of P. diplolister from three different populations and 10 individuals of P. alium from a single population. We successfully amplified 24 loci for P. diplolister, 13 of which we were able to amplify in P. alium. All loci were polymorphic. Significant deviations from the Hardy-Weinberg equilibrium and the presence of null alleles were only consistently detected at one locus (Pleu9). These markers will enable the study of geographic genetic diversity and evolutionary processes in these two Caatinga endemics, and the inclusion of genetic data for conservation planning of the Caatinga biome.

Development of microsatellite markers for the Neotropical endemic Brazilian Guanabara frog, Euparkerella brasiliensis, through 454 shotgun pyrosequencing.

The new-generation 454 GS-FLX Titanium pyrosequencing was used to isolate microsatellite markers for the Brazilian Guanabara frog, Euparkerella brasiliensis, an Atlantic forest endemic species. Three multiplex polymerase chain reaction sets were optimized for genotyping of 11 polymorphic (di- and tetranucleotide) microsatellite markers. Genetic diversity was assessed in 21 individuals from a population (Reserva Ecológica de Guapiaçu, REGUA) located in the central region of the Rio de Janeiro State, in Brazil. The mean number of alleles per locus ranged from 3 to 12. Observed and expected heterozygosities ranged from 0.095 to 0.905 and from 0.094 to 0.904, respectively. After using the Bonferroni correction for multiple tests, there was no evidence of linkage disequilibrium between pairs of loci but deviations for Hardy-Weinberg equilibrium were found in 4 loci. We found no evidence for allele dropouts or stuttering, but we detected the presence of null alleles at loci Eb10 and Eb36. These markers will be useful for analyses of fine-scale population structure and determination of relative effects of habitat loss and fragmentation on population genetic variability within species.

Characterization of polymorphic microsatellite markers for the Neotropical leaf-frog Phyllomedusa burmeisteri and cross-species amplification.

Twelve polymorphic tetranucleotide microsatellite loci were isolated and characterized for the leaf-frog Phyllomedusa burmeisteri, an endemic species of the Brazilian Atlantic forest. These loci were screened in 25 individuals from two populations of the Minas Gerais State (Carangola and Juiz de Fora). The number of alleles per locus ranged from 3 to 16 (mean = 8). Observed and expected heterozygosities ranged from 0.25 to 0.92 and 0.56 to 0.92, respectively. Evidence for both the presence of null alleles and Hardy- Weinberg equilibrium deviations were found in loci Phybu4, Phybu17, and Phybu21. Genotypic disequilibrium for each pair of loci across populations was not significant. Cross-species amplification was successful for 11 of the 12 developed loci for the sister-species, P. bahiana. These microsatellites will be important for future fine-scale population structure analyses.

Novel C1q assay reveals a clinically relevant subset of human leukocyte antigen antibodies independent of immunoglobulin G strength on single antigen beads.

It has been known for 40 years that cytotoxic human leukocyte antigen (HLA) antibodies are associated with graft rejection. However, the complement-dependent cytotoxicity assay (CDC) used to define these clinically deleterious antibodies suffers from a lack of sensitivity and specificity. Recently, methods exploiting immunoglobulin G (IgG) antibody binding to HLA single antigen beads (SAB) have overcome sensitivity and specificity drawbacks but introduced a new dilemma: which of the much broader set of antibodies defined by these methods are clinically relevant. To address this, we developed a complement-fixing C1q assay on the HLA SAB that combines sensitivity, specificity, and functional potential into one assay. We compared the CDC, IgG, and C1q assays on 96 sera having 2,118 defined antibodies and determined that CDC detects only 19% of complement-fixing antibodies detected by C1q, whereas C1q detects only 47% of antibodies detected by IgG. In the same patient, there is no predictability by IgG mean fluorescence intensity (MFI) as to which of the antibodies will bind C1q because fixation is independent of MFI values. In 3 clinical studies, C1q(+) antibodies appear to be more highly correlated than those detected by IgG alone for antibody-mediated rejection in hearts as well as for kidney transplant glomerulopathy and graft failure.

Pyoderma gangrenosum: an Indian perspective.

BACKGROUND: Pyoderma gangrenosum (PG) is an uncommon, idiopathic, neutrophilic skin disease sometimes associated with systemic diseases. To our knowledge, there have been no case series of PG reported from India. AIM: To study the clinical features, associated systemic diseases and treatment methods in our case series. METHODS: In total, 18 cases of PG diagnosed at our institution in Mangalore were evaluated in a prospective study. RESULTS: Ulcerative PG was the most common variant, seen in all except one case. Paediatric patients constituted less than a quarter (27.77%) of the patients. Systemic diseases were associated with the disease in over half of the patients (55.55%). The pathergy test was positive in seven patients (38.88%), and all but one had associated systemic disease. Corticosteroids were mainly used for management. CONCLUSIONS: PG is a rare disease in India, but paediatric PG cases were relatively common. Ulcerative PG was the commonest type, and over half of the patients had associated systemic diseases. There were a significant number of patients with associated systemic disease who had a positive pathergy test.

Rhinoentomophthoromycosis with mutilation.

Rhinoentomophthoromycosis is a rare subcutaneous zygomycosis. We report a case of rhinoentomophthoromycosis with mutilation, which is an even more unusual phenomenon, and discuss the clinical features and management of this rare dermatosis.

Genetic exchange across a hybrid zone within the Iberian endemic golden-striped salamander, Chioglossa lusitanica.

The study of hybrid zones resulting from Pleistocene vicariance is central in examining the potential of genetically diverged evolutionary units either to introgress and merge or to proceed with further isolation. The hybrid zone between two mitochondrial lineages of Chioglossa lusitanica is located near the Mondego River in Central Portugal. We used mitochondrial and nuclear diagnostic markers to conduct a formal statistical analysis of the Chioglossa hybrid zone in the context of tension zone theory. Key results are: (i) cline centres are not coincident for all markers, with average widths of ca. 2-15 km; (ii) heterozygote deficit was not observed across loci near the transect centre; (iii) associations of parental allele combinations ('linkage disequilibrium'R) were not detected either across loci or across the transect. These observations suggest that the Chioglossa hybrid zone is not a tension zone with strong selection against hybrids but instead one shaped mostly by neutral mixing. The patterns uncovered suggest a complex history of populations over a small scale that may be common in southern Pleistocene refugia.

[Limb amputations carried out in hospitals of the national health service in the years from 1990 to 1993]. / Amputações de membros efectuadas nos hospitais do Serviço Nacional de Saúde nos anos de 1990 a 1993.

This study was to assess the number of limbs amputations in Portugal State Hospitals over a four years period (1990 to 1993), analysing their incidence according to etiology, sex, average age, duration of hospitalization, level of amputation and mortality rate. The authors emphasise the levels of amputation which are mot favourable for an effective rehabilitation.

Osseous metastases from renal cell carcinoma: embolization and surgery for restoration of function. Work in progress.

Five patients underwent preoperative embolization of osseous metastases from renal cell carcinoma. The group consisted of four men and one woman who ranged in age from 46 to 79 years. The lesions were located in the pubic ramus and acetabulum, proximal femur, femoral midshaft, proximal humerus, and proximal tibia. All embolizations were performed within 24 hours of surgery. The internal fixation and tumor curettage was accomplished with estimated perioperative blood loss ranging from 10 ml to 1,250 ml. All patients had significant restoration of function following surgery. We suggest that preoperative embolization is an important and efficacious adjunct in the management of hypervascular renal cell osseous metastases.

The diagnostic dilemma of the posterior mediastinal thymus: CT manifestations.

Extension of the normal thymus into the posterior mediastinum is rare. The CT appearance of this anomaly in an infant is presented. A mass of soft-tissue density extended from the anterior mediastinum to the posterior chest wall. The absence of any tissue-cleavage plane in the lesion and a smooth continuous lateral margin are signs of posterior extension of the thymus, and they help to distinguish this from a normal anterior thymus being present with a posterior tumor.

Femoral artery spasm in children: catheter size is the principal cause.

The incidence of femoral artery spasm and subsequent thrombosis complicating angiography is greater in children than adults. This study was designed to assess femoral artery spasm and to determine its causes in children. In 100 unselected, consecutive infants and children undergoing left heart angiocardiography by the Seldinger technique, angiography of the femoral artery was performed to include the puncture site after completion of angiocardiography. Arterial spasm was quantified and, with mathematical analysis, related to various clinical and angiographic factors. Some degree of arterial spasm was noted in 62% of all cases; it was more frequent and severe in neonates. Although it was more intense in the infant with a small femoral artery, analysis of the data indicates that patient age, weight, length of catheterization, size of femoral artery, and catheter size have only minimal relative importance in the occurrence and severity of arterial spasm. The most important factor in spasm is the relative size of catheter to artery.

Seldinger sinography.

It is very important to have a thorough knowledge of the anatomy of cutaneous sinuses and fistulae prior to surgical intervention. This paper describes a modification of the Seldinger technique for the demonstration of these sinuses and fistulae, especially those extending into bone.