Advantage of high-resolution melting curve analysis over conformation-sensitive gel electrophoresis for mutational screening of BRCA1 and BRCA2 genes

BackgroundMutation screening of BRCA1 and BRCA2 (BRCAs) genes is a time-consuming and costly procedure that demands faster and cheaper alternative methods for routine diagnostics. The present study is aimed at comparing the results obtained with screening mutations methods, conformation sensitive gel electrophoresis (CSGE) and high-resolution melting analysis (HRMA), for BRCAs attending to their specificity, sensitivity, reliability and cost-efficiency.MethodsWe included 52 DNA samples of index patients from high-risk families. The mutational screening was performed by CSGE according to the Ganguly (1993) method and HRMA according to a modified De Leeneer (2008) method. The assays were performed in 384 well plates in the LightCycler 480 (Roche). All PCR products showing altered patterns were confirmed by sequencing.ResultsThe results obtained with the mutational study of BRCAs genes showed that HRMA exhibited higher sensitivity than CSGE as it was able to detect a wide mutational spectra of genetic variants in a larger number of samples. Aditionally, the combination of HRMA with hybiridization probes in a second step of the assay allows the specific confirmation of mutations. Furthermore, HRMA use less time, allowing the reduction of analysis time.ConclusionsHRMA offers clear advantages over CSGE for the mutation screening of BRCAs genes as it has greater sensitivity and higher efficiency and it is less time-consuming.     

Relationship between outer membrane alterations and susceptibility to antimicrobial agents in isogenic strains of Klebsiella pneumoniae

The activities of beta-lactams, chloramphenicol, tetracycline, fluoroquinolones and aminoglycosides against Klebsiella pneumoniae C3 (O1:K66, producing porins OmpK35 and OmpK36) and a set of isogenic mutants derived from it lacking the O antigen of lipopolysaccharide (LPS), capsular K antigen, or one or both porins were determined. MICs remained within one dilution step in mutants deficient in antigen O, in capsule or in one of the two porins. No increases in the MICs of aminoglycosides, fluoroquinolones, tetracycline and chloramphenicol were observed for strains deficient in the two porins, but the MICs of ampicillin, cephalothin, cefoxitin, cefotaxime and ceftazidime for this type of mutant increased four- to >256-fold. The highest MICs of beta-lactams were obtained in a porin-deficient mutant expressing increased beta-lactamase activity. It is concluded that isolated outer membrane alterations in K. pneumoniae are not decisive factors in increasing resistance to antimicrobial agents, but porin loss co-operates with beta-actamase production to increase resistance to beta-lactams.     

Analysis of genes encoding penicillin-binding proteins in clinical isolates of Acinetobacter baumannii

There is limited information on the role of penicillin-binding proteins (PBPs) in the resistance of Acinetobacter baumannii to β-lactams. This study presents an analysis of the allelic variations of PBP genes in A. baumannii isolates. Twenty-six A. baumannii clinical isolates (susceptible or resistant to carbapenems) from three teaching hospitals in Spain were included. The antimicrobial susceptibility profile, clonal pattern, and genomic species identification were also evaluated. Based on the six complete genomes of A. baumannii, the PBP genes were identified, and primers were designed for each gene. The nucleotide sequences of the genes identified that encode PBPs and the corresponding amino acid sequences were compared with those of ATCC 17978. Seven PBP genes and one monofunctional transglycosylase (MGT) gene were identified in the six genomes, encoding (i) four high-molecular-mass proteins (two of class A, PBP1a [ponA] and PBP1b [mrcB], and two of class B, PBP2 [pbpA or mrdA] and PBP3 [ftsI]), (ii) three low-molecular-mass proteins (two of type 5, PBP5/6 [dacC] and PBP6b [dacD], and one of type 7 (PBP7/8 [pbpG]), and (iii) a monofunctional enzyme (MtgA [mtgA]). Hot spot mutation regions were observed, although most of the allelic changes found translated into silent mutations. The amino acid consensus sequences corresponding to the PBP genes in the genomes and the clinical isolates were highly conserved. The changes found in amino acid sequences were associated with concrete clonal patterns but were not directly related to susceptibility or resistance to β-lactams. An insertion sequence disrupting the gene encoding PBP6b was identified in an endemic carbapenem-resistant clone in one of the participant hospitals.     

Glycosylphosphatidylinositol-specific phospholipase D in blood serum: is the liver the only source of the enzyme?

In cases of systemic inflammatory response syndrome, sepsis, and septic shock, the activity of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) in serum amounts to 20 to 25% of the activity found in a healthy control group. The activity of serum GPI-PLD is positively correlated with inflammatory markers and counts of monocytes and stab cells (bands) and negatively correlated with polymorphonuclear neutrophils and lymphocytes in severe diseases. This indicates a yet unknown involvement of the inflammatory system in GPI-PLD liberation and suggests that the liver is not the only source of the plasma enzyme. Plasma was shown to contain an effective inhibitor of GPI-PLD which is soluble in organic solvents. Its concentration in capillary plasma is 20-fold higher than in venous plasma. To find possible other sources of plasma GPI-PLD besides the liver, the GPI-degrading activity was measured in different organs of the rat. Product formation was analysed using [125I]TID-labeled GPI-AP.     

Immunohistochemical,genetic and epigenetic profiles of hereditary and triple negative breast cancers. Relevance in personalized medicine

This study aims to identify the profile of immunohistochemical (IHC) parameters, copy number aberrations (CNAs) and epigenetic alterations [promoter methylation (PM) and miR expression] related to hereditary (H) and triple negative (TN) breast cancer (BC). This profile could be of relevance for guiding tumor response to treatment with targeting therapy. The study comprises 278 formalin fixed paraffin-embedded BCs divided into two groups: H group, including 88 hereditary BC (HBC) and 190 non hereditary (NHBC), and TN group, containing 79 TNBC and 187 non TNBC (NTNBC). We assessed IHC parameters (Ki67, ER, PR, HER2, CK5/6, CK18 and Cadherin-E), CNA of 20 BC related genes, and PM of 24 tumor suppressor genes employing MLPA/MS-MLPA (MRC Holland, Amsterdam). MiR-4417, miR-423-3p, miR-590-5p and miR-187-3p expression was assessed by quantitative RT-PCR (Applied Biosystems). Binary logistic regression was applied to select the parameters that better differentiate the HBC or TN groups. For HBC we found that, ER expression, ERBB2 CNA and PM in RASSF1 and TIMP3 were associated with NHBC whereas; MYC and AURKA CNA were linked to HBC. For TNBC, we found that CDC6 CNA, GSTP1 and RASSF1 PM and miR-423-3p hyperexpression were characteristic of NTNBC, while MYC aberrations, BRCA1 hypermethylation and miR-590-5p and miR-4417 hyperexpression were more indicative of TNBC. The selected markers allow establishing BC subtypes, which are characterized by showing similar etiopathogenetic mechanisms, some of them being molecular targets for known drugs or possible molecular targets. These results could be the basis to implement a personalized therapy.     

Combined therapy for migraine prevention? Clinical experience with a beta-blocker plus sodium valproate in 52 resistant migraine patients

The aim was to explore whether combining a beta-blocker and sodium valproate could lead to an advantage in efficacy in patients with migraine previously resistant to the two medications in monotherapy. Fifty-two patients (43 women) with a history of episodic migraine with or without aura, and previously unresponsive to beta-blockers and sodium valproate in monotherapy, were treated with a combination of propranolol or nadolol and sodium valproate in an open-label fashion. Eight patients (15%) discontinued due to adverse events. Fifteen (29%) did not respond. The remaining 29 cases (56%) showed response (> 50% reduction in migraine days). The response was excellent in nine (17%). From this open trial, combination therapy with a beta-blocker and sodium valproate appears to be a good migraine preventative in some previously resistant migraine cases. Controlled trials are now necessary to determine the true advantage in efficacy of this combination in difficult to treat migraineurs.