Even free radicals should follow some rules: a guide to free radical research terminology and methodology.

Free radicals and oxidants are now implicated in physiological responses and in several diseases. Given the wide range of expertise of free radical researchers, application of the greater understanding of chemistry has not been uniformly applied to biological studies. We suggest that some widely used methodologies and terminologies hamper progress and need to be addressed. We make the case for abandonment and judicious use of several methods and terms and suggest practical and viable alternatives. These changes are suggested in four areas: use of fluorescent dyes to identify and quantify reactive species, methods for measurement of lipid peroxidation in complex biological systems, claims of antioxidants as radical scavengers, and use of the terms for reactive species

Interactions among mitochondrial proteins altered in glioblastoma

Mitochondrial dysfunction is putatively central to glioblastoma (GBM) pathophysiology but there has been no systematic analysis in GBM of the proteins which are integral to mitochondrial function. Alterations in proteins in mitochondrial enriched fractions from patients with GBM were defined with label-free liquid chromatography mass spectrometry. 256 mitochondrially-associated proteins were identified in mitochondrial enriched fractions and 117 of these mitochondrial proteins were markedly (fold-change ≥2) and significantly altered in GBM (p ≤ 0.05). Proteins associated with oxidative damage (including catalase, superoxide dismutase 2, peroxiredoxin 1 and peroxiredoxin 4) were increased in GBM. Protein–protein interaction analysis highlighted a reduction in multiple proteins coupled to energy metabolism (in particular respiratory chain proteins, including 23 complex-I proteins). Qualitative ultrastructural analysis in GBM with electron microscopy showed a notably higher prevalence of mitochondria with cristolysis in GBM. This study highlights the complex mitochondrial proteomic adjustments which occur in GBM pathophysiology

Benefits and risks of the hormetic effects of dietary isothiocyanates on cancer prevention

The isothiocyanate (ITC) sulforaphane (SFN) was shown at low levels (1-5 µM) to promote cell proliferation to 120-143% of the controls in a number of human cell lines, whilst at high levels (10-40 µM) it inhibited such cell proliferation. Similar dose responses were observed for cell migration, i.e. SFN at 2.5 µM increased cell migration in bladder cancer T24 cells to 128% whilst high levels inhibited cell migration. This hormetic action was also found in an angiogenesis assay where SFN at 2.5 µM promoted endothelial tube formation (118% of the control), whereas at 10-20 µM it caused significant inhibition. The precise mechanism by which SFN influences promotion of cell growth and migration is not known, but probably involves activation of autophagy since an autophagy inhibitor, 3-methyladenine, abolished the effect of SFN on cell migration. Moreover, low doses of SFN offered a protective effect against free-radical mediated cell death, an effect that was enhanced by co-treatment with selenium. These results suggest that SFN may either prevent or promote tumour cell growth depending on the dose and the nature of the target cells. In normal cells, the promotion of cell growth may be of benefit, but in transformed or cancer cells it may be an undesirable risk factor. In summary, ITCs have a biphasic effect on cell growth and migration. The benefits and risks of ITCs are not only determined by the doses, but are affected by interactions with Se and the measured endpoint

Alarmins MRP8 and MRP14 Induce Stress Tolerance in Phagocytes under Sterile Inflammatory Conditions

Hyporesponsiveness by phagocytes is a well-known phenomenon in sepsis that is frequently induced by low-dose endotoxin stimulation of Toll-like receptor 4 (TLR4) but can also be found under sterile inflammatory conditions. We now demonstrate that the endogenous alarmins MRP8 and MRP14 induce phagocyte hyporesponsiveness via chromatin modifications in a TLR4-dependent manner that results in enhanced survival to septic shock in mice. During sterile inflammation, polytrauma and burn trauma patients initially present with high serum concentrations of myeloid-related proteins (MRPs). Human neonatal phagocytes are primed for hyporesponsiveness by increased peripartal MRP concentrations, which was confirmed in murine neonatal endotoxinemia in wild-type and MRP14(-/-) mice. Our data therefore indicate that alarmin-triggered phagocyte tolerance represents a regulatory mechanism for the susceptibility of neonates during systemic infections and sterile inflammation

Molecular mechanisms of drug resistance in natural Leishmania populations vary with genetic background

The evolution of drug-resistance in pathogens is a major global health threat. Elucidating the molecular basis of pathogen drug-resistance has been the focus of many studies but rarely is it known whether a drug-resistance mechanism identified is universal for the studied pathogen; it has seldom been clarified whether drug-resistance mechanisms vary with the pathogen's genotype. Nevertheless this is of critical importance in gaining an understanding of the complexity of this global threat and in underpinning epidemiological surveillance of pathogen drug resistance in the field. This study aimed to assess the molecular and phenotypic heterogeneity that emerges in natural parasite populations under drug treatment pressure. We studied lines of the protozoan parasite Leishmania (L.) donovani with differential susceptibility to antimonial drugs; the lines being derived from clinical isolates belonging to two distinct genetic populations that circulate in the leishmaniasis endemic region of Nepal. Parasite pathways known to be affected by antimonial drugs were characterised on five experimental levels in the lines of the two populations. Characterisation of DNA sequence, gene expression, protein expression and thiol levels revealed a number of molecular features that mark antimonial-resistant parasites in only one of the two populations studied. A final series of in vitro stress phenotyping experiments confirmed this heterogeneity amongst drug-resistant parasites from the two populations. These data provide evidence that the molecular changes associated with antimonial-resistance in natural Leishmania populations depend on the genetic background of the Leishmania population, which has resulted in a divergent set of resistance markers in the Leishmania populations. This heterogeneity of parasite adaptations provides severe challenges for the control of drug resistance in the field and the design of molecular surveillance tools for widespread applicability

Enhancing the immunogenicity of tumour lysate-loaded dendritic cell vaccines by conjugation to virus-like particles

BACKGROUND: Tumour cell lysates are an excellent source of many defined and undefined tumour antigens and have been used clinically in immunotherapeutic regimes but with limited success. METHODS: We conjugated Mel888 melanoma lysates to rabbit haemorrhagic disease virus virus-like particles (VLP), which can act as vehicles to deliver multiple tumour epitopes to dendritic cells (DC) to effectively activate antitumour responses. RESULTS: Virus-like particles did not stimulate the phenotypic maturation of DC although, the conjugation of lysates to VLP (VLP-lysate) did overcome lysate-induced suppression of DC activation. Lysate-conjugated VLP enhanced delivery of antigenic proteins to DC, while the co-delivery of VLP-lysates with OK432 resulted in cross-priming of naïve T cells, with expansion of a MART1(+) population of CD8(+) T cells and generation of a specific cytotoxic response against Mel888 tumour cell targets. The responses generated with VLP-lysate and OK432 were superior to those stimulated by unconjugated lysate with OK432. CONCLUSION: Collectively, these results show that the combination of VLP-lysate with OK432 delivered to DC overcomes the suppressive effects of lysates, and enables priming of naïve T cells with superior ability to specifically kill their target tumour cells

Perinatal Tobacco Smoke Exposure Increases Vascular Oxidative Stress and Mitochondrial Damage in Non-Human Primates

Epidemiological studies suggest that events occurring during fetal and early childhood development influence disease susceptibility. Similarly, molecular studies in mice have shown that in utero exposure to cardiovascular disease (CVD) risk factors such as environmental tobacco smoke (ETS) increased adult atherogenic susceptibility and mitochondrial damage; however, the molecular effects of similar exposures in primates are not yet known. To determine whether perinatal ETS exposure increased mitochondrial damage, dysfunction and oxidant stress in primates, archived tissues from the non-human primate model Macaca mulatta (M. mulatta) were utilized. M. mulatta were exposed to low levels of ETS (1 mg/m3 total suspended particulates) from gestation (day 40) to early childhood (1 year), and aortic tissues were assessed for oxidized proteins (protein carbonyls), antioxidant activity (SOD), mitochondrial function (cytochrome oxidase), and mitochondrial damage (mitochondrial DNA damage). Results revealed that perinatal ETS exposure resulted in significantly increased oxidative stress, mitochondrial dysfunction and damage which were accompanied by significantly decreased mitochondrial antioxidant capacity and mitochondrial copy number in vascular tissue. Increased mitochondrial damage was also detected in buffy coat tissues in exposed M. mulatta. These studies suggest that perinatal tobacco smoke exposure increases vascular oxidative stress and mitochondrial damage in primates, potentially increasing adult disease susceptibility

Understanding the importance of selenium and selenoproteins in muscle function

Selenium is an essential trace element. In cattle, selenium deficiency causes dysfunction of various organs, including skeletal and cardiac muscles. In humans as well, lack of selenium is associated with many disorders, but despite accumulation of clinical reports, muscle diseases are not generally considered on the list. The goal of this review is to establish the connection between clinical observations and the most recent advances obtained in selenium biology. Recent results about a possible role of selenium-containing proteins in muscle formation and repair have been collected. Selenoprotein N is the first selenoprotein linked to genetic disorders consisting of different forms of congenital muscular dystrophies. Understanding the muscle disorders associated with selenium deficiency or selenoprotein N dysfunction is an essential step in defining the causes of the disease and obtaining a better comprehension of the mechanisms involved in muscle formation and maintenance

Downregulation of Integrin β4 Decreases the Ability of Airway Epithelial Cells to Present Antigens

Airway epithelial cells have been demonstrated to be accessory antigen presentation cells (APC) capable of activating T cells and may play an important role in the development of allergic airway inflammation of asthma. In asthmatic airways, loss of expression of the adhesion molecule integrin β4 (ITGB4) and an increase in Th2 inflammation bias has been observed in our previous study. Given that ITGB4 is engaged in multiple signaling pathways, we studied whether disruption of ITGB4-mediated cell adhesion may contribute to the adaptive immune response of epithelial cells, including their ability to present antigens, induce the activate and differentiate of T cells. We silenced ITGB4 expression in bronchial epithelial cells with an effective siRNA vector and studied the effects of ITGB4 silencing on the antigen presentation ability of airway epithelial cells. T cell proliferation and cytokine production was investigated after co-culturing with ITGB4-silenced epithelial cells. Surface expression of B7 homologs and the major histocompatibility complex (MHC) class II was also detected after ITGB4 was silenced. Our results demonstrated that silencing of ITGB4 resulted in impaired antigen presentation processes and suppressed T cell proliferation. Meanwhile, decrease in Th1 cytokine production and increase in Th17 cytokine production was induced after co-culturing with ITGB4-silenced epithelial cells. Moreover, HLA-DR was decreased and the B7 homologs expression was different after ITGB4 silencing. Overall, this study suggested that downregulation of ITGB4 expression in airway epithelial cells could impair the antigen presentation ability of these cells, which further regulate airway inflammation reaction in allergic asthma