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.     

Bacterial species and evolution: Theoretical and practical perspectives

A discussion of the species problem in modern evolutionary biology serves as the point of departure for an exploration of how the basic science aspects of this problem relate to efforts to map bacterial diversity for practical pursuits—for prospecting among the bacteria for useful genes and gene-products. Out of a confusing array of species concepts, the Cohesion Species Concept seems the most appropriate and useful for analyzing bacterial diversity. Techniques of allozyme analysis and DNA fingerprinting can be used to put this concept into practice to map bacterial genetic diversity, though the concept requires minor modification to encompass cases of complete asexuality. Examples from studies of phenetically definedBacillus species provide very partial maps of genetic population structure. A major conclusion is that such maps frequently reveal deep genetic subdivision within the phenetically defined specles; divisions that in some cases are clearly distinct genetic species. Knowledge of such subdivisions is bound to make prospecting within bacterial diversity more effective. Under the general concept of genetic cohesion a hypothetical framework for thinking about the full range of species conditions that might exist among bacteria is developed and the consequences of each such model for species delineation, and species identification are discussed. Modes of bacterial evolution, and a theory of bacterial speciation with and without genetic recombination, are examined. The essay concludes with thoughts about prospects for very extensive mapping of bacterial diversity in the service of future efforts to find useful products. In this context, evolutionary biology becomes the handmaiden of important industrial activities. A few examples of past success in commercializing bacterial gene-products from species ofBacillus and a few other bacteria are reviewed.     

A mRNA landscape of bovine embryos after standard and MAPK-inhibited culture conditions: a comparative analysis

Background

Genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass (ICM) samples using microarray analysis. As MAPK inhibition can increase the NANOG/GATA6 ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a MAPK inhibitor and changes in gene expression in the inner cell mass were analysed.

Results

Between morula and blastocyst 3,774 genes were differentially expressed and the largest differences were found in blastocyst up-regulated genes. Gene ontology (GO) analysis shows lipid metabolic process as the term most enriched with genes expressed at higher levels in blastocysts. Genes with higher expression levels in morulae were enriched in the RNA processing GO term. Of the 497 differentially expressed genes comparing ICM and TE, the expression of NANOG, SOX2 and POU5F1 was increased in the ICM confirming their evolutionary preserved role in pluripotency. Several genes implicated to be involved in differentiation or fate determination were also expressed at higher levels in the ICM. Genes expressed at higher levels in the ICM were enriched in the RNA splicing and regulation of gene expression GO term. Although NANOG expression was elevated upon MAPK inhibition, SOX2 and POU5F1 expression showed little increase. Expression of other genes in the MAPK pathway including DUSP4 and SPRY4, or influenced by MAPK inhibition such as IFNT, was down-regulated.

Conclusion

The data obtained from the microarray studies provide further insight in gene expression during bovine embryonic development. They show an expression profile in pluripotent cells that indicates a pluripotent, epiblast-like state. The inability to culture ICM cells as stem cells in the presence of an inhibitor of MAPK activity together with the reported data indicates that MAPK inhibition alone is not sufficient to maintain a pluripotent character in bovine cells.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1448-x) contains supplementary material, which is available to authorized users.     

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).     

Stimulation of Cloudman melanoma tyrosinase activity occurs predominantly in G2 phase of the cell cycle

A widely accepted notion is that an increasing cellular cyclic AMP (cAMP) concentration is prerequisite for increasing tyrosinase activity and melanin synthesis and for regulating proliferation of pigment cells. alpha-Melanocyte stimulating hormone (alpha-MSH) increases cAMP and tyrosinase activity in Cloudman melanoma cells. Prostaglandins (PGs) E1 and E2 increase melanoma cell tyrosinase activity and inhibit proliferation. Both PGs, but not alpha-MSH, block the progression of Cloudman melanoma cells from G2 phase of the cell cycle into M or G1. Only PGE1 and not PGE2 causes an elevation of cellular cAMP concentrations. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine (DDA) at 5 x 10(-4) M effectively blocks the increased cAMP synthesis by cells treated with 10 micrograms/ml PGE1. The addition of DDA, however, enhances the melanogenic response of melanoma cells to 10 micrograms/ml PGE1 or PGE2, 10(-7) M alpha-MSH, 10(-4) M isobutylmethylxanthine, 10(-4) M dibutyryl cyclic AMP. DDA also augments the effects of PGE1 or PGE2 on the melanoma cell cycle. Moreover, when DDA is added concomitantly with alpha-MSH, more cells are recruited into G2 than observed in untreated controls. Neither alpha-MSH nor DDA alone has any effect on the cell cycle. These findings undermine the role of cAMP in the melanogenic process and suggest that blocking melanoma cells in G2 may be required for the remarkable stimulation of tyrosinase activity observed with PGE1 or PGE2 alone or in combination with DDA. The observed block in G2 may be essential for the synthesis of sufficient mRNA, which is required for stimulation of tyrosinase activity.     

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.