Streamlined ion torrent PGM-based diagnostics: BRCA1 and BRCA2 genes as a model

To meet challenges in terms of throughput and turnaround time, many diagnostic laboratories are shifting from Sanger sequencing to higher throughput next-generation sequencing (NGS) platforms. Bearing in mind that the performance and quality criteria expected from NGS in diagnostic or research settings are strikingly different, we have developed an Ion Torrent''s PGM-based routine diagnostic procedure for BRCA1/2 sequencing. The procedure was first tested on a training set of 62 control samples, and then blindly validated on 77 samples in parallel with our routine technique. The training set was composed of difficult cases, for example, insertions and/or deletions of various sizes, large-scale rearrangements and, obviously, mutations occurring in homopolymer regions. We also compared two bioinformatic solutions in this diagnostic context, an in-house academic pipeline and the commercially available NextGene software (Softgenetics). NextGene analysis provided higher sensitivity, as four previously undetected single-nucleotide variations were found. Regarding specificity, an average of 1.5 confirmatory Sanger sequencings per patient was needed for complete BRCA1/2 screening. Large-scale rearrangements were identified by two distinct analyses, that is, bioinformatics and fragment analysis with electrophoresis profile comparison. Turnaround time was enhanced, as a series of 30 patients were sequenced by one technician, making the results available for the clinician in 10 working days following blood sampling. BRCA1/2 genes are a good model, representative of the difficulties commonly encountered in diagnostic settings, which is why we believe our findings are of interest for the whole community, and the pipeline described can be adapted by any user of PGM for diagnostic purposes.     

Review of genitourinary tuberculosis with focus on end-stage renal disease

Tuberculosis (TB) is a current public health problem, remaining the most common worldwide cause of mortality from infectious disease. Recent studies indicate that genitourinary TB is the third most common form of extra-pulmonary disease. The diagnosis of renal TB can be hypothesized in a non-specific bacterial cystitis associated with a therapeutic failure or a urinalysis with a persistent leukocyturia in the absence of bacteriuria. We report on the case of a 33-year-old man who presented on admission end stage renal disease (ESRD) secondary to renal TB and a past history of pulmonary TB with important radiologic findings. The diagnosis was based on clinical findings despite all cultures being negative. Empiric treatment with tuberculostatic drugs was started and the patient became stable. He was discharged with no symptom, but without renal function recovery. He is on maintenance hemodialysis three times a week. TB is an important cause of kidney disease and can lead to irreversible renal function loss.     

Copper ability to induce premature senescence in human fibroblasts

Human diploid fibroblasts (HDFs) exposed to subcytotoxic concentrations of oxidative or stressful agents, such as hydrogen peroxide, tert-butylhydroperoxide, or ethanol, undergo stress-induced premature senescence (SIPS). This condition is characterized by the appearance of replicative senescence biomarkers such as irreversible growth arrest, increase in senescence-associated β-galactosidase (SA β-gal) activity, altered cell morphology, and overexpression of several senescence-associated genes. Copper is an essential trace element known to accumulate with ageing and to be involved in the pathogenesis of some age-related disorders. Past studies using either yeast or human cellular models of ageing provided evidence in favor of the role of intracellular copper as a longevity modulator. In the present study, copper ability to cause the appearance of senescent features in HDFs was assessed. WI-38 fibroblasts exposed to a subcytotoxic concentration of copper sulfate presented inhibition of cell proliferation, cell enlargement, increased SA β-gal activity, and mRNA overexpression of several senescence-associated genes such as p21, apolipoprotein J (ApoJ), fibronectin, transforming growth factor β-1 (TGF β1), insulin growth factor binding protein 3, and heme oxygenase 1. Western blotting results confirmed enhanced intracellular p21, ApoJ, and TGF β1 in copper-treated cells. Thus, similar to other SIPS-inducing agents, HDF exposure to subcytotoxic concentration of copper results in premature senescence. Further studies will unravel molecular mechanisms and the biological meaning of copper-associated senescence and lead to a better understanding of copper-related disorder establishment and progression.

Electronic supplementary material

The online version of this article (doi:10.1007/s11357-011-9276-7) contains supplementary material, which is available to authorized users.     

The effect of ballroom dance on balance and functional autonomy among the isolated elderly

The aim of the present study was to analyze the influence of a ballroom dancing program on the functional autonomy and physical balance of institutionalized elderly individuals. The study enrolled 75 sedentary elderly subjects from long-term institutions who were randomly divided into a ballroom dance program group (EG; n=39) and a control group (CG; n=36). The protocol of the Latin American Group for Maturity (GDLAM) was used to evaluate functional autonomy. Physical balance was analyzed using a stabilometer and posture meter platforms. The level of significance in statistical tests was set at p<0.05. Regarding the physical balance evaluation, only the members of the EG achieved a significant reduction in weight (Δ=-0.98 kg) following the experiment, both in the intragroup (p=0.002) and in the intergroup analysis (p=0.012). In the evaluation of functional autonomy, only the EG showed a significant reduction in the execution time of all the tests and in the GDLAM index: GI (Δ=-6.99), both in the intragroup (p<0.001) and in the intergroup analysis (p=0.011). Thus, it can be inferred that sedentary elderly individuals who are residents of long-term institutions can improve their functional autonomy and balance with a ballroom dance program.     

Angiogenesis and lymphangiogenesis in mucoepidermoid carcinoma of minor salivary glands

J Oral Pathol Med (2012) 41: 603-609 Background: Mucoepidermoid carcinoma is the most common malignant tumor of salivary glands. This tumor is characterized by a great variability in clinical behavior, and little is known about the pathological mechanisms involved in its variance. Angiogenesis is an important step in tumor progression and is believed to be an essential event for metastatic dissemination. Methods: We aimed to investigate angiogenesis and lymphangiogenesis in mucoepidermoid carcinoma measuring the density of neoformed and lymphatic vessels using CD105 and D2-40 antibodies, respectively, and by immunohistochemical evaluation of VEGF-A and VEGF-C proteins. It was also investigated the expression of D2-40 in neoplastic cells. Results: We studied 26 cases of mucoepidermoid carcinoma, which showed great angiogenic activity measured by neoformed vessel density. However, a low density of lymphatics was observed. VEGF-A, VEGF-C, and D2-40 were commonly detected in mucoepidermoid carcinoma, but only VEGF-A expression correlated with neoformed vessel density. Recurrence and nodal metastasis were associated with low VEGF-A expression and low neoformed vessel density, indicating that impaired angiogenesis could lead to an aggressive phenotype. Conclusions: Angiogenesis seems important in the modulation of mucoepidermoid carcinoma pathogenesis; however, none of the parameters analyzed could predict tumor behavior.