The crucial role of particle surface reactivity in respirable quartz-induced reactive oxygen/nitrogen species formation and APE/Ref-1 induction in rat lung

Persistent inflammation and associated excessive oxidative stress have been crucially implicated in quartz-induced pulmonary diseases, including fibrosis and cancer. We have investigated the significance of the particle surface reactivity of respirable quartz dust in relation to the in vivo generation of reactive oxygen and nitrogen species (ROS/RNS) and the associated induction of oxidative stress responses in the lung. Therefore, rats were intratracheally instilled with 2 mg quartz (DQ12) or quartz whose surface was modified by either polyvinylpyridine-N-oxide (PVNO) or aluminium lactate (AL). Seven days after instillation, the bronchoalveolar lavage fluid (BALF) was analysed for markers of inflammation (total/differential cell counts), levels of pulmonary oxidants (H₂O₂, nitrite), antioxidant status (trolox equivalent antioxidant capacity), as well as for markers of lung tissue damage, e.g. total protein, lactate dehydrogenase and alkaline phosphatase. Lung homogenates as well as sections were investigated regarding the induction of the oxidative DNA-lesion/oxidative stress marker 8-hydroxy-2''-deoxyguanosine (8-OHdG) using HPLC/ECD analysis and immunohistochemistry, respectively. Homogenates and sections were also investigated for the expression of the bifunctional apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) by Western blotting and immunohistochemistry. Significantly increased levels of H₂O₂ and nitrite were observed in rats treated with non-coated quartz, when compared to rats that were treated with either saline or the surface-modified quartz preparations. In the BALF, there was a strong correlation between the number of macrophages and ROS, as well as total cells and RNS. Although enhanced oxidant generation in non-coated DQ12-treated rats was paralleled with an increased total antioxidant capacity in the BALF, these animals also showed significantly enhanced lung tissue damage. Remarkably however, elevated ROS levels were not associated with an increase in 8-OHdG, whereas the lung tissue expression of APE/Ref-1 protein was clearly up-regulated. The present data provide further in vivo evidence for the crucial role of particle surface properties in quartz dust-induced ROS/RNS generation by recruited inflammatory phagocytes. Our results also demonstrate that quartz dust can fail to show steady-state enhanced oxidative DNA damage in the respiratory tract, in conditions were it elicits a marked and persistent inflammation with associated generation of ROS/RNS, and indicate that this may relate to compensatory induction of APE/Ref-1 mediated base excision repair.     

Interaction of ouabain with the Na(+) pump in intact epithelial cells

To determine the specificity and efficacy of [(3)H]ouabain binding as a quantitative measure of the Na(+) pump (Na(+), K(+)-ATPase) and as a marker for the localization of pumps involved in transepithelial Na(+)-transport, we analyzed the interaction of [(3)H]ouabain with its receptor in pig kidney epithelial (LLC-PK(1)) cells. When these epithelial cells are depleted of Na(+) and exposed to 2 muM [(3)H]ouabain in a Na(+)-free medium, binding is reduced by 90 percent. When depleted of K(+) and incubated in a K(+)- free medium, the ouabain binding rate is increase compared with that measured at 5 mM. This increase is only demonstable when Na(+) is present. The increased rate could be attributed to the predominance of the Na(+)-stimulated phosphorylated form of the pump, as K(+) is not readily available to stimulate dephosphorylation. However, some binding in the K(+)-free medium is attributable to pump turnover (and therefore, recycling of K(+)), because analysis of K(+)-washout kinetics demonstrated that addition of 2 muM ouabain to K(+)-depleted cells increased the rate of K(+) loss. These results indicate that in intact epithelial cells, unlike isolated membrane preparations, the most favorable condition for supporting ouabain binding occurs when the Na(+), K(+)-ATPase is operating in the Na(+)-pump mode or is phosphorylated in the presence of Na(+). When LLC-PK(1) cells were exposed to ouabain at 4 degrees C, binding was reduced by 97 percent. Upon rewarming, the rate of binding was greater than that obtained on cells kept at a constant 37 degrees C. However, even at this accelerated rate, the time to reach equilibrium was beyond what is required for cells, swollen by exposure to cold, to recover normal volume. Thus, results from studies that have attempted to use ouabain to eliminate the contribution of the conventional Na(+) pump to volume recovery must be reevaluated if the exposure to ouabain was done in the cold or under conditions in which the Na(+) pump is not operating.     

Effect of nitrogen source and concentration to produce proteins in mass cultures of the microalgae <i>Chaetoceros muelleri</i>

Proteins are one of the major metabolites in biomass from microalgae that constitute the diet of marine organisms grown in aquaculture, and are essential for their growth. The quantity of this component is influenced by nutrients, temperature and light intensity, among others. We examined the growth, biomass production and protein of Chaetoceros muelleri with two sources of nitrogen (nitrate and urea) at three concentrations, using the medium f/2 (0.88 mol/L) (nitrates) as control. The treatments were the medium 2f (3.53 mol/L) and 4f (7.05 mol/L) with NO_3^-, and the medium f/2 (0.88 mol/L), 2f (3.53mol/L) and 4f (7.05 mol/L) with urea. In general, the productive parameters were greater using urea than nitrate in the media. Higher cell concentrations (2.83 x 10⁶ cell/mL), average and cumulative growth rates (1.50 div/day and 6.01 divisions), dry weight (0.0044 g/L), and proportion of proteins (23.74%) were found when urea was used as the N source. However, most of the bands on the electrophoretic profile were present in the mediums with NO_3^- (~6.5 to 90 kDa).     

HIBCH mutations can cause Leigh-like disease with combined deficiency of multiple mitochondrial respiratory chain enzymes and pyruvate dehydrogenase

Background

Deficiency of 3-hydroxy-isobutyryl-CoA hydrolase (HIBCH) caused by HIBCH mutations is a rare cerebral organic aciduria caused by disturbance of valine catabolism. Multiple mitochondrial respiratory chain (RC) enzyme deficiencies can arise from a number of mechanisms, including defective maintenance or expression of mitochondrial DNA. Impaired biosynthesis of iron-sulphur clusters and lipoic acid can lead to pyruvate dehydrogenase complex (PDHc) deficiency in addition to multiple RC deficiencies, known as the multiple mitochondrial dysfunctions syndrome.

Methods

Two brothers born to distantly related Pakistani parents presenting in early infancy with a progressive neurodegenerative disorder, associated with basal ganglia changes on brain magnetic resonance imaging, were investigated for suspected Leigh-like mitochondrial disease. The index case had deficiencies of multiple RC enzymes and PDHc in skeletal muscle and fibroblasts respectively, but these were normal in his younger brother. The observation of persistently elevated hydroxy-C4-carnitine levels in the younger brother led to suspicion of HIBCH deficiency, which was investigated by biochemical assay in cultured skin fibroblasts and molecular genetic analysis.

Results

Specific spectrophotometric enzyme assay revealed HIBCH activity to be below detectable limits in cultured skin fibroblasts from both brothers. Direct Sanger sequence analysis demonstrated a novel homozygous pathogenic missense mutation c.950G <A; p.Gly317Glu in the HIBCH gene, which segregated with infantile-onset neurodegeneration within the family.

Conclusions

HIBCH deficiency, a disorder of valine catabolism, is a novel cause of the multiple mitochondrial dysfunctions syndrome, and should be considered in the differential diagnosis of patients presenting with multiple RC deficiencies and/or pyruvate dehydrogenase deficiency.

     

Selective use of abciximab in coronary stenting: overall outcomes can still be equivalent to those in the EPISTENT treatment group

BACKGROUND: The EPISTENT trial reported improved early outcomes with routine use of abciximab after coronary stenting. Increasing use of stents means that routine abciximab adds significantly to costs of percutaneous coronary intervention (PCI). This paper reports the results of a protocol encouraging restriction of abciximab therapy to high-risk patients only. METHODS: Data were collected prospectively over a 34-month period for patients undergoing PCI with stenting. In addition to those who fulfilled criteria for inclusion in the EPISTENT trial, patients treated in the setting of acute myocardial infarction (AMI) were studied. Demographic data, procedural details and early clinical outcomes were recorded. RESULTS: Of 808 patients studied, 601 fulfilled EPISTENT inclusion criteria and comprised 367 patients (45%) treated for stable angina and 234 (30%) treated for unstable or post-infarct angina. The additional 207 patients (25%) were treated during AMI. The 808 patients received a total of 981 stents. Abciximab was given in only 88 cases (10.9%). Major adverse clinical events occurred in 39 patients (4.8%). CONCLUSION: Selective use of abciximab for patients undergoing coronary stenting can be associated with outcomes equivalent to those reported for routine use, but with significant cost savings.     

(Cost)-effectiveness of a multi-component intervention for adults with epilepsy: study protocol of a Dutch randomized controlled trial (ZMILE study)

Background

In patients with epilepsy, poor adherence to anti-epileptic drugs has been shown to be the most important cause of poorly controlled epilepsy. Furthermore, it has been noted that the quality of life among patients with epilepsy can be improved by counseling and treatments aimed at increasing their self-efficacy and concordance, thus stimulating self-management skills. However, there is a need for evidence on the effectiveness of such programs, especially within epilepsy care. Therefore, we have developed a multi-component intervention (MCI) which combines a self-management/education program with e-Health interventions. Accordingly, the overall objective of this study is to assess the (cost)-effectiveness and feasibility of the MCI, aiming to improve self-efficacy and concordance in patients with epilepsy.

Methods

A RCT in two parallel groups will be conducted to compare the MCI with a control condition in epilepsy patients. One hundred eligible epilepsy patients will be recruited and allocated to either the intervention or control group. The intervention group will receive the MCI consisting of a self-management/education program of six meetings, including e-Health interventions, and will be followed for 12 months. The control group will receive care as usual and will be followed for 6 months, after which patients will be offered the possibility of participating in the MCI. The study will consist of three parts: 1) a clinical effectiveness study, 2) a cost-effectiveness study, and 3) process evaluation. The primary outcome will be self-efficacy. Secondary outcomes include adherence, side effects, change in seizure severity & frequency, improved quality of life, proactive coping, and societal costs. Outcome assessments will be done using questionnaires at baseline and after 3, 6, 9, and 12 months (last two applicable only for intervention group).

Discussion

In times of budget constraints, MCI could be a valuable addition to the current healthcare provision for epilepsy, as it is expected that higher concordance and self-efficacy will result in reduced use of healthcare resources and an increased QOL. Accordingly, this study is aimed helping patients to be their own provider of health care, shifting epilepsy management from professionals to self-care by patients equipped with appropriate skills and tools.

Trial registration number

NTR4484.

     

Phylogeny of a new supertribe Cephenniitae with generic review of Eutheiini and description of a new tribe Marcepaniini (Coleoptera: Staphylinidae: Scydmaeninae)

Genera of Eutheiini are reviewed and Eutheimorphus is removed from this tribe of ant‐like stone beetles (Scydmaeninae) and transferred to Cephenniini. A monogeneric Marcepaniini trib.n. is described to accommodate Marcepania gen.n. from Malaysia, with five species: M. semengohensis sp.n. (the type species of Marcepania), M. tuberculata sp.n. , M. seramaensis sp.n. , M. minutissima sp.n. and M. elongata sp.n. A phylogenetic analysis of all genera of Cephenniini, Eutheiini and Marcepaniini based on adult morphological characters resulted in recovering a well‐supported monophyletic clade Eutheiini + (Marcepaniini + Cephenniini) and these tribes are included in Cephenniitae stat.n. (Eutheiini and Cephenniini are therefore removed from Scydmaenitae). Only a weak support for monophyly of Eutheiini was found, but morphological characters allow for maintaining this presumably relic group as a separate tribe. Previously proposed monophyletic groups within Cephenniini were recovered as such, but after inclusion of Eutheimorphus, a sister taxon to the ‘Cephennomicrus group’, the latter lineage gained weak statistical support. The evolutionary history of Cephenniitae is discussed, with focus on known northern hemisphere fossils classified in Scydmaenitae and Hapsomelitae, but possibly closely allied to Cephenniitae. Establishing the supertribe Cephenniitae is the first step toward a profound reclassification of Scydmaeninae on a robust phylogenetic basis. This published work has been registered in ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:B0E1B12D-9587-4C4F-A908-A12A0C424A8C .     

Intradiscal application of rhBMP-7 does not induce regeneration in a canine model of spontaneous intervertebral disc degeneration

IntroductionStrategies for biological repair and regeneration of the intervertebral disc (IVD) by cell and tissue engineering are promising, but few have made it into a clinical setting. Recombinant human bone morphogenetic protein 7 (rhBMP-7) has been shown to stimulate matrix production by IVD cells in vitro and in vivo in animal models of induced IVD degeneration. The aim of this study was to determine the most effective dose of an intradiscal injection of rhBMP-7 in a spontaneous canine IVD degeneration model for translation into clinical application for patients with low back pain.MethodsCanine nucleus pulposus cells (NPCs) were cultured with rhBMP-7 to assess the anabolic effect of rhBMP-7 in vitro, and samples were evaluated for glycosaminoglycan (GAG) and DNA content, histology, and matrix-related gene expression. Three different dosages of rhBMP-7 (2.5 μg, 25 μg, and 250 μg) were injected in vivo into early degenerated IVDs of canines, which were followed up for six months by magnetic resonance imaging (T2-weighted images, T1rho and T2 maps). Post-mortem, the effects of rhBMP-7 were determined by radiography, computed tomography, and macroscopy, and by histological, biochemical (GAG, DNA, and collagen), and biomolecular analyses of IVD tissue.ResultsIn vitro, rhBMP-7 stimulated matrix production of canine NPCs as GAG deposition was enhanced, DNA content was maintained, and gene expression levels of ACAN and COL2A1 were significantly upregulated. Despite the wide dose range of rhBMP-7 (2.5 to 250 μg) administered in vivo, no regenerative effects were observed at the IVD level. Instead, extensive extradiscal bone formation was noticed after intradiscal injection of 25 μg and 250 μg of rhBMP-7.ConclusionsAn intradiscal bolus injection of 2.5 μg, 25 μg, and 250 μg rhBMP-7 showed no regenerative effects in a spontaneous canine IVD degeneration model. In contrast, intradiscal injection of 250 μg rhBMP-7, and to a lesser extent 25 μg rhBMP-7, resulted in extensive extradiscal bone formation, indicating that a bolus injection of rhBMP-7 alone cannot be used for treatment of IVD degeneration in human or canine patients.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0625-2) contains supplementary material, which is available to authorized users.