Is quantitative real time polymerase chain reaction MCAM transcript assay really suitable for prognostic and predictive management of melanoma patients?

Background: Recent advances in next generation sequencing (NGS) technology have enabled comprehensive and accurate screening of the entire genomic region of BRCA1/2 genes and, to date, many studies report the effectiveness of these technologies. Here we show that Gene Scan (GS) labeling Quality Control (QC), performed before massive parallel pyrosequencing, coupled with Multiple Amplicon Quantification software (MAQ-S) analysis is a rapid and powerful tool in the detection of deleterious BRCA mutations carried by different patients. Methods: GS labeling QC assay was performed according to the manufacturers' instructions and MAQ-S software was employed for analysis results. Results: GS labeling QC was able to detect 14 different BRCA frameshift mutations in our patients. In addition, two novel BRCA mutations (c.1893_1894in5TTAAGCCCACAAAT in BRCA1 gene and c.9413_9414insT in BRCA2 gene) were identified. Conclusion: We prove that a simple QC step may represent a valid and useful tool for a rapid detection of frameshift mutations in BRCA genes. For this reason, we recommend using this approach before massive parallel sequencing. (C) 2014 Elsevier B.V. All rights reserved

Gene symbol: CYP21A2. Disease: Non-classic 21-Hydroxylase deficiency.

No Abstrac

Surfactant and Varespladib Co-Administration in Stimulated Rat Alveolar Macrophages Culture

Antibiotic resistance emerged shortly after the introduction of antibiotics into the field of medicine, bringing about a challenging concern. Resistance to antibiotics is encoded by antibiotic resistance genes, among other mechanisms. Antibiotic resistance genes are highly regulated genes that are expressed when antibiotics are introduced. The current research focuses on one resistance gene ytbD, encoded by Bacillus subtilis. This research will give an insight into ytbD regulation by observing the spatial and temporal expression of a luciferase reporter gene fused to the ytbD promoters. The expression pattern is observed under the control of different lengths of ytbD promoters when ribosome-targeting, including chloramphenicol, and nonribosom-targeting antibiotics are introduced. To build the different lengths of the promoters, we designed primers that will include or exclude predicted regulatory sequences during the engineering of the reporter strains. In doing so, we are trying to test if these upstream sequences play a role in the regulation of ytbD and whether antibiotics will affect the regulation pattern

Ex Vivo Effect of Varespladib on Secretory Phospholipase A2 Alveolar Activity in Infants with ARDS

Background: Secretory phospholipase A2 (sPLA2) plays a pivotal role in acute respiratory distress syndrome (ARDS). This enzyme seems an interesting target to reduce surfactant catabolism and lung tissue inflammation. Varespladib is a specifically designed indolic sPLA2 inhibitor, which has shown promising results in animals and adults. No specific data in pediatric ARDS patients are yet available. Methods: We studied varespladib in broncho-alveolar lavage (BAL) fluids obtained ex vivo from pediatric ARDS patients. Clinical data and worst gas exchange values during the ARDS course were recorded. Samples were treated with saline or 10-40-100 mu M varespladib and incubated at 37 degrees C. Total sPLA2 activity was measured by non-radioactive method. BAL samples were subjected to western blotting to identify the main sPLA isotypes with different sensitivity to varespladib. Results was corrected for lavage dilution using the serum-to-BAL urea ratio and for varespladib absorbance. Results: Varespladib reduces sPLA2 activity (p<0.0001) at 10,40 and 100 mu M; both sPLA2 activity reduction and its ratio to total proteins significantly raise with increasing varespladib concentrations (p<0.001). IC50 was 80 mu M. Western blotting revealed the presence of sPLA2-IIA and -IB isotypes in BAL samples. Significant correlations exist between the sPLA2 activity reduction/proteins ratio and PaO2 (rho=0.63; p<0.001), PaO2/FiO(2) (rho=0.7; p<0.001), oxygenation (rho=-0.6; p<0.001) and ventilation (rho=-0.4; p=0.038) indexes. Conclusions: Varespladib significantly inhibits sPLA2 in BAL of infants affected by post-neonatal ARDS. Inhibition seems to be inversely related to the severity of gas exchange impairment

Western blot assay of BAL samples for the two main sPLA2 isotypes that respond differently to varespladib inhibition (sPLA2-IB and sPLA2-IIA).

<p>Illustrative results of four ARDS patients (and human recombinant proteins, as controls) are shown. All patients presents sPLA2-IIA, while some also shown the presence of the isotype –IB. sPLA2: secretory phospholipase A2.</p

sPLA2 activity in the four BAL aliquots treated either with normal saline (basal), or varespladib at 10, at 40 and at 100 µM.

<p>Overall difference in median sPLA2 activity is significant at the Friedman <i>Q</i>-test (see text for details). <i>p</i>-values describe <i>post-hoc</i> comparison: <i>p</i> = 0.013 between basal and varespladib10; <i>p</i> = 0.007 between basal and varespladib40; <i>p</i> = 0.005 between varespladib100 and each other measurement. sPLA2: secretory phospholipase A2.</p

Surfactant and Varespladib Co-Administration in Stimulated Rat Alveolar Macrophages Culture

Acute lung injury is a life-threating condition characterized by surfactant dysfunction and raised secretory phospholipase A2 (sPLA(2)) activity. Varespladib is a sPLA(2) inhibitor shown to be effective in animal models of acute lung injury. We aimed at investigating the effect of co-administration of surfactant and varespladib on sPLA(2) activity. Alveolar macrophages were cultured and stimulated with lipopolysaccharide and then treated with either varespladib, surfactant, varespladib followed by surfactant or nothing. sPLA(2) activity, free fatty acids, tumour necrosis factor-alpha (TNF-alpha) and protein concentrations were measured in culture supernatants. Treatment with varespladib (p=0.019) and varespladib + surfactant (p=0.013), reduced the enzyme activity by approximately 15\% from the basal level measured in the untreated cultures. Surfactant, varespladib and varespladib + surfactant, respectively decreased free fatty acids by -45\% (p=0.045), 62\% (p=0.009) and -48\% (p=0.015), from the baseline concentration of the untreated cultures. Varespladib and poractant-alpha co-administration reduces sPLA(2) activity and free fatty acids release in cultured rat alveolar macrophages, although a clear drug synergy was not evident. Since co-administration may be useful to reduce inflammation and surfactant inactivation in acute lung injury, further in vivo studies are warranted to verify its clinical usefulness

Negative correlation between the reduction of sPLA2 total activity and protein content.

<p>Grey lines represent linear regression line and its 95% confidence interval (rho = 0.906; <i>p</i><0.001; R² = 0.73).</p

Clinical data.

<p>Data are expressed as median (i.q. range) or number (%). GA: gestational age at the birth; OI: oxygenation index; VI: ventilatory index; RSV: respiratory syncitial virus; BPD: broncho-pulmonary dysplasia.</p