Peroxynitrite Mediates Nitric Oxide–Induced Blood–Brain Barrier Damage

Using the in vitro blood-brain barrier (BBB) model ECV304/C6, which consists of cocultures of human umbilical vein endothelial-like cells (ECV304) and rat glioma cells (C6), the role of peroxynitrite (OONO-) in nitric oxide (NO*)-mediated BBB disruption was evaluated. Endothelial cell cultures were exposed to NO* gas, in the presence or absence of the OONO- blocker FeTPPS. Separate exposure to NO* and OONO- resulted in endothelial cell cytotoxicity and a decline in barrier integrity. Unfortunately, FeTPPS induced significant detrimental effects on model BBB integrity at a concentration of 300 microM and above. At 250 microM (the highest concentration usable), FeTPPS displayed a trend toward prevention of NO* elicited perturbation of barrier integrity. Dichlorofluorescein diacetate is oxidized to fluorescent dichlorofluorescein by OONO- but only marginally by NO* or O2*-. We observed large and rapid increases in fluorescence in ECV304 preloaded cells following NO* exposure, which were blocked by FeTPPS. Furthermore, using an antinitrotyrosine antibody we detected the nitration of endothelial cell proteins following NO* exposure and conclude that NO*-mediated BBB dysfunction is predominantly elicited by OONO- and not NO*. Proposed mechanisms of NO*-mediated OONO- elicited barrier dysfunction and damage are discussed.     

Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha

Introduction

A surprising feature of the inflammatory infiltrate in rheumatoid arthritis is the accumulation of neutrophils within synovial fluid and at the pannus cartilage boundary. Recent findings suggest that a distinct subset of IL-17-secreting T-helper cells (T_H17 cells) plays a key role in connecting the adaptive and innate arms of the immune response and in regulating neutrophil homeostasis. We therefore tested the hypothesis that synovial fibroblasts bridge the biological responses that connect T_H17 cells to neutrophils by producing neutrophil survival factors following their activation with IL-17.

Methods

IL-17-expressing cells in the rheumatoid synovium, and IL-17-expressing cells in the peripheral blood, and synovial fluid were examined by confocal microscopy and flow cytometry, respectively. Peripheral blood neutrophils were cocultured either with rheumatoid arthritis synovial fibroblasts (RASF) or with conditioned medium from RASF that had been pre-exposed to recombinant human IL-17, TNFα or a combination of the two cytokines. Neutrophils were harvested and stained with the vital mitochondrial dye 3,3'-dihexyloxacarbocyanine iodide before being enumerated by flow cytometry.

Results

T_H17-expressing CD4⁺ cells were found to accumulate within rheumatoid synovial tissue and in rheumatoid arthritis synovial fluid. RASF treated with IL-17 and TNFα (RASF_IL-17/TNF) effectively doubled the functional lifespan of neutrophils in coculture. This was entirely due to soluble factors secreted from the fibroblasts. Specific depletion of granulocyte–macrophage colony-stimulating factor from RASF_IL-17/TNF-conditioned medium demonstrated that this cytokine accounted for approximately one-half of the neutrophil survival activity. Inhibition of phosphatidylinositol-3-kinase and NF-κB pathways showed a requirement for both signalling pathways in RASF_IL-17/TNF-mediated neutrophil rescue.

Conclusion

The increased number of neutrophils with an extended lifespan found in the rheumatoid synovial microenvironment is partly accounted for by IL-17 and TNFα activation of synovial fibroblasts. T_H17-expressing T cells within the rheumatoid synovium are likely to contribute significantly to this effect.     

Mutational changes in S-cone opsin genes common to both nocturnal and cathemeral Aotus monkeys

Aotus is a platyrrhine primate that has been classically considered to be nocturnal. Earlier research revealed that this animal lacks a color vision capacity because, unlike all other platyrrhine monkeys, Aotus has a defect in the opsin gene that is required to produce short-wavelength sensitive (S) cone photopigment. Consequently, Aotus retains only a single type of cone photopigment. Other mammals have since been found to show similar losses and it has often been speculated that such change is in some fashion tied to nocturnality. Although most species of Aotus are indeed nocturnal, recent observations show that Aotus azarai, an owl monkey species native to portions of Argentina and Paraguay, displays a cathemeral activity pattern being active during daylight hours as frequently as during nighttime hours. We have sequenced portions of the S-cone opsin gene in A. azarai and Aotus nancymaae, the latter a typically nocturnal species. The S-cone opsin genes in both species contain the same fatal defects earlier detected for Aotus trivirgatus. On the basis of the phylogenetic relationships of these three species these results imply that Aotus must have lost a capacity for color vision early in its history and they also suggest that the absence of color vision is not compulsively linked to a nocturnal lifestyle.     

Meibum lipid composition in Asians with dry eye disease

Background

Previous lipidomic analyses of the human meibum had largely focused on individuals from non-Asian populations, despite the higher prevalence of dysfunctional tear syndrome (DTS) observed across Asia. Information pertaining to the alterations in lipid profiles in relation to DTS onset and progression is also lacking and warrants comprehensive experimental analysis.

Methodologies/Principal Findings

We examined the meibum lipidome of 27 DTS patients and 10 control subjects for a total of 256 lipid species from 12 major lipid classes, including cholesteryl ester (CE), wax ester (WE), triacylglyceride (TAG), (O-acyl)-ω-hydroxy fatty acid (OAHFA), glycerophospholipids (phosphatidylcholine, PC; phosphatidylethanolamine, PE; phosphatidylinositol, PI; phosphatidylglycerol, PG) and sphingolipids (sphingomyelin, SM; ceramide, Cer; glucosylceramide, GluCer; dihexosylceramide, DihexCer). Neutral lipids were analysed using high-performance liquid-chromatography coupled with mass spectrometry (HPLC/MS) and tandem mass spectrometry (MS/MS) was used for the qualitative and quantitative analysis of polar lipid species. DTS patients were classified into three severity groups (i.e. mild, moderate and severe) based on the ocular surface disease index (OSDI). A significantly lower level of TAG (p<0.05) was observed in patients under the moderate category compared to the mild category. Notably, a number of OAHFA species displayed consistently decreasing levels that correlate with increasing disease severity. An attempt was also made to investigate the changes in meibum lipid profiles of DTS patients compared to normal individuals classified based on OSDI score. Several unsaturated TAG and PC species were found at significantly higher levels (p<0.05) in patients than controls.

Conclusion

The current study presents, for the first time, a comprehensive lipidome of meibum from individuals of an Asian ethnicity, which can potentially offer new insights into the higher prevalence of DTS observed amongst Asian populations. This study also represents an attempt towards identification of lipid species in meibum which could serve as marker for DTS.     

Shotgun proteomics of cyanobacteria--applications of experimental and data-mining techniques.

Cyanobacteria are photosynthetic bacteria notable for their ability to produce hydrogen and a variety of interesting secondary metabolites. As a result of the growing number of completed cyanobacterial genome projects, the development of post-genomics analysis for this important group has been accelerating. DNA microarrays and classical two-dimensional gel electrophoresis (2DE) were the first technologies applied in such analyses. In many other systems, 'shotgun' proteomics employing multi-dimensional liquid chromatography and tandem mass spectrometry has proven to be a powerful tool. However, this approach has been relatively under-utilized in cyanobacteria. This study assesses progress in cyanobacterial shotgun proteomics to date, and adds a new perspective by developing a protocol for the shotgun proteomic analysis of the filamentous cyanobacterium Anabaena variabilis ATCC 29413, a model for N(2) fixation. Using approaches for enhanced protein extraction, 646 proteins were identified, which is more than double the previous results obtained using 2DE. Notably, the improved extraction method and shotgun approach resulted in a significantly higher representation of basic and hydrophobic proteins. The use of protein bioinformatics tools to further mine these shotgun data is illustrated through the application of PSORTb for localization, the grand average hydropathy (GRAVY) index for hydrophobicity, LipoP for lipoproteins and the exponentially modified protein abundance index (emPAI) for abundance. The results are compared with the most well-studied cyanobacterium, Synechocystis sp. PCC 6803. Some general issues in shotgun proteome identification and quantification are then addressed.     

Experiences of Domestic Violence and Mental Disorders: A Systematic Review and Meta-Analysis

Background

Little is known about the extent to which being a victim of domestic violence is associated with different mental disorders in men and women. We aimed to estimate the prevalence and odds of being a victim of domestic violence by diagnostic category and sex.

Methods

Study design: Systematic review and meta-analysis. Data Sources: Eighteen biomedical and social sciences databases (including MEDLINE, EMBASE, PsycINFO); journal hand searches; scrutiny of references and citation tracking of included articles; expert recommendations, and an update of a systematic review on victimisation and mental disorder. Inclusion criteria: observational and intervention studies reporting prevalence or odds of being a victim of domestic violence in men and women (aged ≥16 years), using validated diagnostic measures of mental disorder. Procedure: Data were extracted and study quality independently appraised by two reviewers. Analysis: Random effects meta-analyses were used to pool estimates of prevalence and odds.

Results

Forty-one studies were included. There is a higher risk of experiencing adult lifetime partner violence among women with depressive disorders (OR 2.77 (95% CI 1.96–3.92), anxiety disorders (OR 4.08 (95% CI 2.39–6.97), and PTSD (OR 7.34 95% CI 4.50–11.98), compared to women without mental disorders. Insufficient data were available to calculate pooled odds for other mental disorders, family violence (i.e. violence perpetrated by a non-partner), or violence experienced by men. Individual studies reported increased odds for women and men for all diagnostic categories, including psychoses, with a higher prevalence reported for women. Few longitudinal studies were found so the direction of causality could not be investigated.

Conclusions

There is a high prevalence and increased likelihood of being a victim of domestic violence in men and women across all diagnostic categories, compared to people without disorders. Longitudinal studies are needed to identify pathways to being a victim of domestic violence to optimise healthcare responses.     

Defence Cheats Can Degrade Protection of Chemically Defended Prey

Many species defend themselves against enemies using repellent chemicals. An important but unanswered question is why investment in chemical defence is often variable within prey populations. One explanation is that some prey benefit by cheating, paying no costs of defence, but gaining a reduced attack rate because of the presence of defended conspecifics. Two important assumptions about predator behaviour must be met to explain cheating as a stable strategy: first, predators increase attack rates as cheats increase in frequency; second, defended prey survive attacks better than non‐defended conspecifics. We lack data from wild predators that evaluate these hypotheses. Here, we examine how changes in the frequency of non‐defended ‘cheats’ affect predation by wild birds on a group of otherwise defended prey. We presented mealworm larvae that were either edible (‘cheats’) or unpalatable (bitter tasting), and varied the proportion of cheats from 0 to 1 by increments of 0.25. We found strong frequency‐dependent effects on the birds' foraging behaviour, with the proportion of prey attacked increasing nonlinearly with the frequency of cheats. We did not, however, observe that birds taste‐rejected defended prey at the site of capture. One explanation is that wild birds may not assess prey palatability at the site of capture, but do this elsewhere. If so, defended and undefended prey may pay high costs of initial attack and relocation away from ecologically favourable locations. Alternatively, defended prey may not be taste‐rejected because with acute time constraints, wild birds do not have time to make fine‐grained decisions during feeding. We discuss the data in relation to the evolutionary ecology of prey defences.     

Perceived Obstacles of Colorectal Cancer Screening and Their Associated Factors among 10,078 Chinese Participants

Purpose

to evaluate the proportion of self-referred screening participants having various psychological barriers and the factors associated with these barriers.

Methods

A territory-wide bowel cancer screening centre sent an invitation via the media to all Hong Kong residents aged 50–70 years who were asymptomatic of CRC to join a free screening programme. Upon attendance they were requested to complete self-administered surveys on their perceived barriers of screening. Binary logistic regression analyses were used to evaluate the factors associated with these barriers.

Results

From 10,078 consecutive screening participants (mean age 57.5 years; female 56.4%) completed the surveys between May 2008 to September 2012. There were high proportions who agreed or strongly agreed with the following barriers: financial difficulty (86.0%), limited service accessibility (58.2%), screening-induced bodily discomfort (55.2%), physical harm (44.4%), embarrassment (40.1%), apprehension (38.8%) and time constraints (13.9%). From regression models, older participants (aged ≥56) were less likely to have these barriers (Adjusted odds ratio [AOR] ranged from 0.738 to 0.952) but they encountered more difficulties to access to screening services (AOR ranged from 1.141 to 1.371). Female subjects were more likely to encounter most of these barriers (AOR ranged from 1.188 to 2.179). Participants who were uncertain of the necessity of CRC screening for people aged ≥50 were more likely to report these barriers (AOR ranged from 1.151 to 1.671).

Conclusion

The proportions of perceptual barriers of CRC screening were high among these participants. Those with these associated factors should receive more thorough explanation of the screening test procedures.     

Use of HbA1c in the Identification of Patients with Hyperglycaemia Caused by a Glucokinase Mutation: Observational Case Control Studies

Aims

HaemoglobinA1c (HbA1c) is recommended for diabetes diagnosis but fasting plasma glucose (FPG) has been useful for identifying patients with glucokinase (GCK) mutations which cause lifelong persistent fasting hyperglycaemia. We aimed to derive age-related HbA1c reference ranges for these patients to determine how well HbA1c can discriminate patients with a GCK mutation from unaffected family members and young-onset type 1 (T1D) and type 2 diabetes (T2D) and to investigate the proportion of GCK mutation carriers diagnosed with diabetes using HbA1c and/or FPG diagnostic criteria.

Methods

Individuals with inactivating GCK mutations (n = 129), familial controls (n = 100), T1D (n = 278) and T2D (n = 319) aged ≥18years were recruited. Receiver Operating Characteristic (ROC) analysis determined effectiveness of HbA1c and FPG to discriminate between groups.

Results

HbA1c reference ranges in subjects with GCK mutations were: 38–56 mmol/mol (5.6–7.3%) if aged ≤40years; 41–60 mmol/mol (5.9–7.6%) if >40years. All patients (123/123) with a GCK mutation were above the lower limit of the HbA1c age-appropriate reference ranges. 69% (31/99) of controls were below these lower limits. HbA1c was also effective in discriminating those with a GCK mutation from those with T1D/T2D. Using the upper limit of the age-appropriate reference ranges to discriminate those with a mutation from those with T1D/T2D correctly identified 97% of subjects with a mutation. The majority (438/597 (73%)) with other types of young-onset diabetes had an HbA1c above the upper limit of the age-appropriate GCK reference range. HbA1c ≥48 mmol/mol classified more people with GCK mutations as having diabetes than FPG ≥7 mmol/l (68% vs. 48%, p = 0.0009).

Conclusions

Current HbA1c diagnostic criteria increase diabetes diagnosis in patients with a GCK mutation. We have derived age-related HbA1c reference ranges that can be used for discriminating hyperglycaemia likely to be caused by a GCK mutation and aid identification of probands and family members for genetic testing.