Screening of lipases for regioselective hydrolysis of peracetylated β-monosaccharides

The monodeacetylation of peracetylated-β-d-galactose (1) and peracetylated N-acetyl-β-d-glucosamine (2) by different lipases is here described. Lipases from different sources in an immobilized form were evaluated to find those that offer the higher activity and regioselectivity in the reactions. In the hydrolysis of 1, the lipase from Aspergillus niger was the most active one, although it hydrolyzed the anomeric position. Using the lipase from Candida rugosa, 30% yield of the corresponding 6-OH isomer was achieved. On the other hand, in the hydrolysis of 2, the lipase from A. niger was the most active and regioselective catalyst, producing more than 75% of the 6-OH derivative product.     

Enfuvirtide administration in HIV-positive transgender patient with soft tissue augmentation: US evaluation

Enfuvirtide is a large protein that should be injected subcutaneously to ensure an appropriate absorption. Here we report the case of a transgender HIV-positive patient receiving enfuvirtide with an individualized background regimen of antiretroviral drugs, who had previously undergone liquid silicone oil injections. We performed US scan to detect silicone-free areas for following enfuvirtide injections. US can be useful in the correct management of those patients with liquid silicone oil soft tissue augmentation who require subcutaneously injected drugs.     

Diverse human aldolase C gene promoter regions are required to direct specific LacZ expression in the hippocampus and Purkinje cells of transgenic mice

Aldolase C is selectively expressed in the hippocampus and Purkinje cells in adult mammalian brain. The gene promoter regions governing cell-specific aldolase C expression are obscure. We show that aldolase C messenger expression in the hippocampus is restricted to CA3 neurons. The human distal promoter region (-200/-1200 bp) is essential for beta-galactosidase (beta-gal) expression in CA3 neurons and drives high stripe-like beta-gal expression in Purkinje cells. The 200 bp proximal promoter region is sufficient to drive low brain-specific and stripe-like beta-gal expression in Purkinje cells. Thus, the human aldolase C gene sequences studied drive endogenous-like expression in the brain.     

Validation of the effect of helox on thermal conductance in homeotherms using heated models

It is commonly stated that mixtures of 80% helium and 20% oxygen (helox) increase thermal conductance, and hence heat flux, from homeotherms by roughly a factor of two. However, because helox affects heat loss by conduction and convection differently, its effect on heat flux should vary according to the relative contributions of these two routes to the overall heat flux. We used heated models made of three sizes of copper tubing and covered with various grades of synthetic fur to measure heat flux under air and helox atmospheres. Thermal conductance in helox (C_helox; W•m⁻² •°C⁻¹) was highly correlated with thermal conductance in air (C_air) and could be predicted by C_helox=1.704+1.726 C_air. The relative increase in heat flux under a helox atmosphere compared with that in air (conductance ratio; C_helox/C_air) varied from a maximum of 2.5 when thermal conductance is infinitely small and heat flux is predominantly determined by conduction to a minimum of 1.85 when animals are naked and heat flux is dominated by convection. These values follow the relation: C_helox/C_air=2.503−0.081 C_air. Body size has no significant effect on the conductance ratio.     

Oxidative DNA Damage and Activation of c-Jun N-Terminal Kinase Pathway in Fibroblasts from Patients with Hereditary Spastic Paraplegia

1.Hereditary spastic paraplegia (HSP) is a genetically heterogeneous group of neurodegenerative disorders affecting 1 in 10,000 individuals. The present study was aimed to elucidate the role played by reactive oxygen species (ROS) in the pathogenesis of this disease. 2. To address this question we used 7-11 passaged fibroblasts from HSP patients to measure the extent of DNA damage induced by H2O2 treatment and to evaluate the JNK phosphorylation level after hydrogen peroxide and serum stimuli. 3. The present study demonstrates that HSP cells compared to controls are more sensitive to DNA damages induced by H2O2 treatment, and that JNK phosphorylation levels are increased in HSP fibroblasts compared to controls after hydrogen peroxide and serum stimuli. These results suggest a ROS-mediated pathogenetic mechanism for this disease.     

Unusual adult-onset Leigh syndrome presentation due to the mitochondrial m.9176T>C mutation

Leigh syndrome (LS) is an incurable, nearly always fatal, neurodegenerative, pediatric disorder that results from respiratory chain failure. The most common mitochondrial DNA (mtDNA) mutations that result in LS are m.8993T→C/G and m.9176T→C/G, which were previously found in several patients with early-onset Leigh syndrome. Here, we describe clinical and molecular features of a novel pedigree, where LS developed in two siblings. The proband was a young woman with an unusual adult-onset LS. She harbored a homoplasmic m.9176T→C mutation, based on analysis of a muscle biopsy. In contrast, the brother died at a young age. This novel case report and literature review highlights the variability of phenotypic expression of the m.9176T→C mutation.     

Malvidin,a red wine polyphenol,modulates mammalian myocardial and coronary performance and protects the heart against ischemia/reperfusion injury

A moderate red wine consumption and a colored fruit-rich diet protect the cardiovascular system, thanks to the presence of several polyphenols. Malvidin-3-0-glucoside (malvidin), an anthocyanidine belonging to polyphenols, is highly present in red grape skin and red wine. Its biological activity is poorly characterized, although a role in tumor cell inhibition has been found. To analyze whether and to which extent, like other food-derived polyphenols, malvidin affects the cardiovascular function, in this study, we have performed a quantitative analysis by high-performance liquid chromatography of polyphenolic content of red grape skins extract, showing that it contains a high malvidin amount (63.93±12.50 mg/g of fresh grape skin). By using the isolated and Langendorff perfused rat heart, we found that the increasing doses (1–1000 ng/ml) of the extract induced positive inotropic and negative lusitropic effects associated with coronary dilation. On the same cardiac preparations, we observed that malvidin (10^?10–10^?6 mol/L) elicited negative inotropism and lusitropism and coronary dilation. Analysis of mechanism of action revealed that malvidin-dependent cardiac effects require the activation of the phosphatidylinositol 3-kinase (PI3K)/nitric oxide (NO)/cGMP/PKG pathway and are associated with increased intracellular cGMP and the phosphorylation of endothelial NO synthase (eNOS), PI3K–AKT, ERK1/2, and GSK-3β. AKT and eNOS phosphorylation was confirmed in human umbilical vein endothelial cell. We also found that malvidin act as a postconditioning agent, being able to elicit cardioprotection against ischemia/reperfusion damages. Our results show the cardioactivity of polyphenols-rich red grape extracts and indicate malvidin as a new cardioprotective principle. This is of relevance not only for a better clarification of the beneficial cardiovascular effects of food-derived polyphenols but also for nutraceutical research.     

Assessment of EGFR Mutations in Circulating Tumor Cell Preparations from NSCLC Patients by Next Generation Sequencing: Toward a Real-Time Liquid Biopsy for Treatment

Introduction

Assessment of EGFR mutation in non-small cell lung cancer (NSCLC) patients is mandatory for optimization of pharmacologic treatment. In this respect, mutation analysis of circulating tumor cells (CTCs) may be desirable since they may provide real-time information on patient''s disease status.

Experimental Design

Blood samples were collected from 37 patients enrolled in the TRIGGER study, a prospective phase II multi-center trial of erlotinib treatment in advanced NSCLC patients with activating EGFR mutations in tumor tissue. 10 CTC preparations from breast cancer patients without EGFR mutations in their primary tumors and 12 blood samples from healthy subjects were analyzed as negative controls. CTC preparations, obtained by the Veridex CellSearch System, were subjected to ultra-deep next generation sequencing (NGS) on the Roche 454 GS junior platform.

Results

CTCs fulfilling all Veridex criteria were present in 41% of the patients examined, ranging in number between 1 and 29. In addition to validated CTCs, potential neoplastic elements were seen in 33 cases. These included cells not fulfilling all Veridex criteria (also known as “suspicious objects”) found in 5 (13%) of 37 cases, and isolated or clustered large naked nuclei with irregular shape observed in 33 (89%) cases. EGFR mutations were identified by NGS in CTC preparations of 31 (84%) patients, corresponding to those present in matching tumor tissue. Twenty-five (96%) of 26 deletions at exon 19 and 6 (55%) of 11 mutations at exon 21 were detectable (P = 0.005). In 4 (13%) cases, multiple EGFR mutations, suggesting CTC heterogeneity, were documented. No mutations were found in control samples.

Conclusions

We report for the first time that the CellSearch System coupled with NGS is a very sensitive and specific diagnostic tool for EGFR mutation analysis in CTC preparations with potential clinical impact.