Differential effects on membrane permeability and viability of human keratinocyte cells undergoing very low intensity megasonic fields

Among different therapeutic applications of Ultrasound (US), transient membrane sonoporation (SP) - a temporary, non-lethal porosity, mechanically induced in cell membranes through US exposure - represents a compelling opportunity towards an efficient and safe drug delivery. Nevertheless, progresses in this field have been limited by an insufficient understanding of the potential cytotoxic effects of US related to the failure of the cellular repair and to the possible activation of inflammatory pathway. In this framework we studied the in vitro effects of very low-intensity US on a human keratinocyte cell line, which represents an ideal model system of skin protective barrier cells which are the first to be involved during medical US treatments. Bioeffects linked to US application at 1 MHz varying the exposure parameters were investigated by fluorescence microscopy and fluorescence activated cell sorting. Our results indicate that keratinocytes undergoing low US doses can uptake drug model molecules with size and efficiency which depend on exposure parameters. According to sub-cavitation SP models, we have identified the range of doses triggering transient membrane SP, actually with negligible biological damage. By increasing US doses we observed a reduced cells viability and an inflammatory gene overexpression enlightening novel healthy relevant strategies

Loss of correlation between HIV viral load and CD4+ T-cell counts in HIV/HTLV-1 co-infection in treatment naive Mozambican patients

Seven hundred and four HIV-1/2-positive, antiretroviral therapy (ART) naïve patients were screened for HTLV-1 infection. Antibodies to HTLV-1 were found in 32/704 (4.5%) of the patients. Each co-infected individual was matched with two HIV mono-infected patients according to World Health Organization clinical stage, age +/-5 years and gender. Key clinical and laboratory characteristics were compared between the two groups. Mono-infected and co-infected patients displayed similar clinical characteristics. However, co-infected patients had higher absolute CD4+ T-cell counts (P = 0.001), higher percentage CD4+ T-cell counts (P < 0.001) and higher CD4/CD8 ratios (P < 0.001). Although HIV plasma RNA viral loads were inversely correlated with CD4+ T-cell-counts in mono-infected patients (P < 0.0001), a correlation was not found in co-infected individuals (P = 0.11). Patients with untreated HIV and HTLV-1 co-infection show a dissociation between immunological and HIV virological markers. Current recommendations for initiating ART and chemoprophylaxis against opportunistic infections in resource-poor settings rely on more readily available CD4+ T-cell counts without viral load parameters. These guidelines are not appropriate for co-infected individuals in whom high CD4+ T-cell counts persist despite high HIV viral load states. Thus, for co-infected patients, even in resource-poor settings, HIV viral loads are likely to contribute information crucial for the appropriate timing of ART introduction

Intracellular targeting and functional analysis of single-chain Fv fragments in mammalian cells

In the past decade, intracellular antibodies have proven to be a useful tool in obtaining the phenotypic knock-out of selected gene function in different animal and plant systems. This strategy is based on the ectopic expression of recombinant forms of antibodies targeted towards different intracellular compartments, exploiting specific targeting signals to confer the new intracellular location. The functional basis of this technology is closely linked to the ability of intracellular antibodies to interact with their target antigens in vivo. This interaction allows either a direct neutralising effect or the dislodgement of the target protein from its normal intracellular location and, by this mechanism, the inactivation of its function. By using this approach, the function of several antigens has been inhibited in the cytoplasm, the nucleus, and the secretory compartments. In this article, we shall describe all the steps required for expressing single-chain Fv fragments in different subcellular compartments of mammalian cells and their subsequent use in knock-out experiments, starting from a cloned single-chain Fv fragment. This will include the analysis of the solubility properties of the new scFv fragment in transfected mammalian cells, the intracellular distribution of the antigen-antibody complex, and the resulting phenotype

Further insights on predictors of environmental tobacco smoke exposure during the pediatric age

Background: The smoking ban in public places has reduced Environmental Tobacco Smoke (ETS) exposure for non-smokers, but despite this, domestic environments still remain places at high risk of exposure, and, today, about 40% of children worldwide are exposed to ETS at home. The aims of the study are to investigate the contribution of several factors on ETS exposure among a group of Italian children and to evaluate the changes in smoking precautions adopted at home when the smoker is the mother, the father, or both parents, respectively. Methods: A cross-sectional study was performed on a sample of 519 Italian schoolchildren. Information was collected via a questionnaire. Results: 41.4% of the participants lived with at least one smoker. Almost half of the children exposed to ETS lived with one or more smokers who do not observe any home smoking ban. Lower maternal or paternal educational levels significantly increase the risk of ETS exposure at home and the “worst case” is represented by both parents who smoke. Conclusions: More effective preventive interventions are needed to protect children from ETS exposure. Some interventions should be specifically dedicated to smokers with a low educational level and to mothers that smoke

Rapid detection of copy number variations and point mutations in BRCA1/2 genes using a single workflow by ion semiconductor sequencing pipeline

Molecular analysis of BRCA1 (MIM# 604370) and BRCA2 (MIM #600185) genes is essential for familial breast and ovarian cancer prevention and treatment. An efficient, rapid, cost-effective accurate strategy for the detection of pathogenic variants is crucial. Mutations detection of BRCA1/2 genes includes screening for single nucleotide variants (SNVs), small insertions or deletions (indels), and Copy Number Variations (CNVs). Sanger sequencing is unable to identify CNVs and therefore Multiplex Ligation Probe amplification (MLPA) or Multiplex Amplicon Quantification (MAQ) is used to complete the BRCA1/2 genes analysis. The rapid evolution of Next Generation Sequencing (NGS) technologies allows the search for point mutations and CNVs with a single platform and workflow. In this study we test the possibilities of NGS technology to simultaneously detect point mutations and CNVs in BRCA1/2 genes, using the OncomineTM BRCA Research Assay on Personal Genome Machine (PGM) Platform with Ion Reporter Software for sequencing data analysis (Thermo Fisher Scientific). Comparison between the NGS-CNVs, MLPA and MAQ results shows how the NGS approach is the most complete and fast method for the simultaneous detection of all BRCA mutations, avoiding the usual time consuming multistep approach in the routine diagnostic testing of hereditary breast and ovarian cancers

Dual action of a dinoflagellate-derived precursor of Pacific ciguatoxins (P-CTX-4B) on voltage-dependent K(+) and Na(+) channels of single myelinated axons

The effects of Pacific ciguatoxin-4B (P-CTX-4B, also named gambiertoxin), extracted from toxic Gambierdiscus dinoflagellates, were assessed on nodal K(+) and Na(+) currents of frog myelinated axons, using a conventional voltage-clamp technique. P-CTX-4B decreased, within a few minutes, both K(+) and Na(+) currents in a dose-dependent manner, without inducing any marked change in current kinetics. The toxin was more effective in blocking K(+) than Na(+) channels. P-CTX-4B shifted the voltage-dependence of Na(+) conductance by about 14 mV towards more negative membrane potentials. This effect was reversed by increasing Ca(2+) in the external solution. A negative shift of about 16 mV in the steady-state Na(+) inactivation-voltage curve was also observed in the presence of the toxin. Unmodified and P-CTX-4B-modified Na(+) currents were similarly affected by the local anaesthetic lidocaine. The decrease of the two currents by lidocaine was dependent on both the concentration and the membrane potential during pre-pulses. In conclusion, P-CTX-4B appears about four times more effective than P-CTX-1B to affect K(+) channels, whereas it is about 50 times less efficient to affect Na(+) channels of axonal membranes. These actions may be related to subtle differences between the two chemical structures of molecules

The influence of position in overlap joints of Mg and Al alloys on microstructure and hardness of laser welds

Structure and properties of laser beam welding zone of dissimilar materials, AZ31 magnesium alloy and A5754 Aluminum alloy, are investigated. The microstructure and quality of the Mg/Al weld were studied by metallography, microhardness and optical microscopy. Differences in physical and mechanical properties of both materials, magnesium and aluminum, affect weldability and resistance of this combination, and lead to the formation of intermetallic compounds in the welded metal

Association of blood pressure with anxiety and depression in a sample of primary care patients

Introduction According to international scientific literature, and as summarized in the guidelines of the International Society of Hypertension, lowering of blood pressure can prevent cardiovascular accidents. Some studies suggest that hypertension, anxiety, and depression might be inversely correlated. Objective To investigate whether blood pressure is associated with anxiety and depression. Methods Cross-sectional design. Male and female primary care patients were enrolled, aged 40–80. Criteria of exclusion adopted: use of antidepressants or antipsychotics; previous major cardiovascular event; psychosis or major depression; Type 1-DM; pregnancy and hereditary disease associated to obesity. Anxiety and depression symptoms were assessed using HADS. Waist circumference, hip circumference, blood pressure, HDL, triglycerides, blood sugar, hypertension, albumin concentrations and serum iron were also assessed. Results Of the 210 subjects, 84 were men (40%), mean age was 60.88 (SD ± 10.88). Hypertension was found to correlate significantly to anxiety (OR = 0.38; 95% CI = 0.17–0.84), older age (OR = 3.96; 95% CI = 1.88–8.32), cigarette smoking (OR = 0.35; 95%CI = 0.13–0.94), high Body Mass Index (OR = 2.50; 95% CI = 1.24–5.01), Waist-hip ratio (OR = 0.09; 95% CI = 0.02–0.46) and the Index of comorbidity (OR = 16.93; 95% CI = 3.71–77.29). Conclusions An inverse association was found between anxiety and hypertension, suggesting the need to clinically manage these two dimensions in a coordinated way. Other findings are well known and already included in prevention campaigns. Further research is needed, also to better understand and explain the causative pathways of this correlation