Overexpression of superoxide dismutase 1 protects against beta-amyloid peptide toxicity: effect of estrogen and copper chelators

Beta-amyloid peptides (Abeta) are major constituents of senile plaques in Alzheimer's disease (AD) brain and contribute to neurodegeneration, operating through activation of apoptotic pathways. It has been proposed that Abeta induces death by oxidative stress, possibly through the generation of peroxynitrite from superoxide and nitric oxide. Estrogen is thought to play a protective role against neurodegeneration through a variety of mechanisms including scavenging of reactive oxygen species (ROS). In this study, we have challenged with Abeta, either in the presence or in the absence of 17beta-estradiol, differentiated human neuroblastoma SH-SY5Y cells (named line SH) and the same line overexpressing anti-oxidant enzyme superoxide dismutase 1 (SOD1; named line WT). We have observed that: (1) WT cells are less susceptible than SH cells to Abeta insult; (2) caspase-3, but not caspase-1, is involved in Abeta-induced apoptosis in this system; (3) estrogen protects both lines, without significantly affecting SOD activity; and (4) copper chelators prevent Abeta-induced toxicity. Our results further support the notion that anti-oxidant therapy might be beneficial in the treatment of AD by preventing activation of selected apoptotic pathways.     

A signal, from human mtDNA, of postglacial recolonization in Europe

Mitochondrial HVS-I sequences from 10,365 subjects belonging to 56 populations/geographical regions of western Eurasia and northern Africa were first surveyed for the presence of the T-->C transition at nucleotide position 16298, a mutation which has previously been shown to characterize haplogroup V mtDNAs. All mtDNAs with this mutation were then screened for a number of diagnostic RFLP sites, revealing two major subsets of mtDNAs. One is haplogroup V proper, and the other has been termed "pre*V," since it predates V phylogenetically. The rather uncommon pre*V tends to be scattered throughout Europe (and northwestern Africa), whereas V attains two peaks of frequency: one situated in southwestern Europe and one in the Saami of northern Scandinavia. Geographical distributions and ages support the scenario that pre*V originated in Europe before the Last Glacial Maximum (LGM), whereas the more recently derived haplogroup V arose in a southwestern European refugium soon after the LGM. The arrival of V in eastern/central Europe, however, occurred much later, possibly with (post-)Neolithic contacts. The distribution of haplogroup V mtDNAs in modern European populations would thus, at least in part, reflect the pattern of postglacial human recolonization from that refugium, affecting even the Saami. Overall, the present study shows that the dissection of mtDNA variation into small and well-defined evolutionary units is an essential step in the identification of spatial frequency patterns. Mass screening of a few markers identified using complete mtDNA sequences promises to be an efficient strategy for inferring features of human prehistory.     

Pharmacological evidence for system-dependent involvement of protein kinase C isoenzymes in phorbol ester-suppressed gap junctional communication

Several phorbol esters are potent activators of protein kinase C. They down-regulate gap junctional intercellular communication and induce phosphorylation of connexin43, but the sensitivity and extent of responses vary much between systems. We asked whether the total protein kinase C enzyme activity or the protein kinase C isoenzyme constitution was of importance for such variations. Some fibroblastic culture systems were compared. It was concluded that the total protein kinase C enzyme activity did not determine the sensitivity to phorbol esters. Furthermore, the use of isotype-specific inhibitors of protein kinase C indicated that protein kinase C alpha, delta, and epsilon may be involved to different extents in different fibroblastic systems in the response to phorbol esters.     

Molecular Dissection of the Basal Clades in the Human Y Chromosome Phylogenetic Tree

One hundred and forty-six previously detected mutations were more precisely positioned in the human Y chromosome phylogeny by the analysis of 51 representative Y chromosome haplogroups and the use of 59 mutations from literature. Twenty-two new mutations were also described and incorporated in the revised phylogeny. This analysis made it possible to identify new haplogroups and to resolve a deep trifurcation within haplogroup B2. Our data provide a highly resolved branching in the African-specific portion of the Y tree and support the hypothesis of an origin in the north-western quadrant of the African continent for the human MSY diversity.     

In vitro genotoxic effects of titanium dioxide nanoparticles (n‐TiO2) in human sperm cells

Titanium dioxide nanoparticles (TiO₂‐NPs) are one of the most widely engineered nanoparticles used. The study has been focused on TiO ₂‐NPs genotoxic effects on human spermatozoa in vitro. TiO ₂‐NPs are able to cross the blood–testis barrier induced inflammation, cytotoxicity, and gene expression changes that lead to impairment of the male reproductive system. This study presents new data about DNA damage in human sperms exposed in vitro to two n‐TiO ₂ concentrations (1 µg/L and 10 µg/L) for different times and the putative role of reactive oxygen species (ROS) as mediators of n‐TiO ₂ genotoxicity. Primary n‐TiO ₂ characterization was performed by transmission electron microscopy. The dispersed state of the n‐TiO ₂ in media was spectrophotometrically determined at 0, 24, 48, and 72 hr from the initial exposure. The genotoxicity has been highlighted by different experimental approaches (comet assay, terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL] test, DCF assay, random amplification of polymorphic DNA polymerase chain reaction [RAPD‐PCR]). The comet assay showed a statistically significant loss of sperm DNA integrity after 30 min of exposure. Increased threshold of sperm DNA fragmentation was highlighted after 30 min of exposure by the TUNEL Test. Also, the RAPD‐PCR analysis showed a variation in the polymorphic profiles of the sperm DNA exposed to n‐TiO ₂. The evidence from the DCF assay showed a statistically significant increase in intracellular ROS linked to n‐TiO ₂ exposure. This research provides the evaluation of n‐TiO ₂ potential genotoxicity on human sperm that probably occurs through the production of intracellular ROS.     

1064‐nm‐resonant gold nanorods for photoacoustic theranostics within permissible exposure limits

Therapeutic and diagnostic methods based on photomechanical effects are attracting much current attention in contexts as oncology, cardiology and vascular surgery, for such applications as photoacoustic imaging or microsurgery. Their underlying mechanism is the generation of ultrasound or cavitation from the interaction of short optical pulses with endogenous dyes or targeted contrast agents. Among the latter, gold nanorods are outstanding candidates, but their use has mainly been reported for photoacoustic imaging and photothermal treatments. Conversely, much less is still known about their value as a precision tool for photomechanical manipulations, such as to impart local damage with high spatial resolution through the expansion and collapse of microbubbles. Here, we address the feasibility of gold nanorods exhibiting a distribution of surface plasmon resonances between about 900 to above 1100 nm as a contrast agent for photoacoustic theranostics. After testing their cytotoxicity and cellular uptake, we discuss their photostability and use to mediate cavitation and the photomechanical destruction of targeted cells. We find that the choice of a plasmonic band peaking around 1064 nm is key to enhance the translational potential of this approach. With respect to the standard alternative of 800 nm, at 1064 nm, relevant regulations on optical exposure are less restrictive and the photonic technology is more mature.      

Strain‐Based In Situ Study of Anion and Cation Insertion into Porous Carbon Electrodes with Different Pore Sizes

The expansion of porous carbon electrodes in a room temperature ionic liquid (RTIL) is studied using in situ atomic force microscopy (AFM). The effect of carbon surface area and pore size/pore size distribution on the observed strain profile and ion kinetics is examined. Additionally, the influence of the potential scan rate on the strain response is investigated. By analyzing the strain data at various potential scan rates, information on ion kinetics in the different carbon materials is obtained. Molecular dynamics (MD) simulations are performed to compare with and provide molecular insights into the experimental results; this is the first MD work investigating the pressure exerted on porous electrodes under applied potential in a RTIL electrolyte. Using MD, the pressure exerted on the pore wall is calculated as a function of potential/charge for both a micropore (1.2 nm) and a mesopore (7.0 nm). The shape of the calculated pressure profile matches closely with the strain profiles observed experimentally.     

Genomic Analysis of Sleeping Beauty Transposon Integration in Human Somatic Cells

The Sleeping Beauty (SB) transposon is a non-viral integrating vector system with proven efficacy for gene transfer and functional genomics. However, integration efficiency is negatively affected by the length of the transposon. To optimize the SB transposon machinery, the inverted repeats and the transposase gene underwent several modifications, resulting in the generation of the hyperactive SB100X transposase and of the high-capacity “sandwich” (SA) transposon. In this study, we report a side-by-side comparison of the SA and the widely used T2 arrangement of transposon vectors carrying increasing DNA cargoes, up to 18 kb. Clonal analysis of SA integrants in human epithelial cells and in immortalized keratinocytes demonstrates stability and integrity of the transposon independently from the cargo size and copy number-dependent expression of the cargo cassette. A genome-wide analysis of unambiguously mapped SA integrations in keratinocytes showed an almost random distribution, with an overrepresentation in repetitive elements (satellite, LINE and small RNAs) compared to a library representing insertions of the first-generation transposon vector and to gammaretroviral and lentiviral libraries. The SA transposon/SB100X integrating system therefore shows important features as a system for delivering large gene constructs for gene therapy applications.