Loop recognition and copper-mediated disulfide reduction underpin metal site assembly of CuA in human cytochrome oxidase.

Maturation of cytochrome oxidases is a complex process requiring assembly of several subunits and adequate uptake of the metal cofactors. Two orthologous Sco proteins (Sco1 and Sco2) are essential for the correct assembly of the dicopper CuA site in the human oxidase, but their function is not fully understood. Here, we report an in vitro biochemical study that shows that Sco1 is a metallochaperone that selectively transfers Cu(I) ions based on loop recognition, whereas Sco2 is a copper-dependent thiol reductase of the cysteine ligands in the oxidase. Copper binding to Sco2 is essential to elicit its redox function and as a guardian of the reduced state of its own cysteine residues in the oxidizing environment of the mitochondrial intermembrane space (IMS). These results provide a detailed molecular mechanism for CuA assembly, suggesting that copper and redox homeostasis are intimately linked in the mitochondrion.

N-terminal domains mediate [2Fe-2S] cluster transfer from glutaredoxin-3 to anamorsin.

In eukaryotes, cytosolic monothiol glutaredoxins are proteins implicated in intracellular iron trafficking and sensing via their bound [2Fe-2S] clusters. We define a new role of human cytosolic monothiol glutaredoxin-3 (GRX3) in transferring its [2Fe-2S] clusters to human anamorsin, a physical and functional protein partner of GRX3 in the cytosol, whose [2Fe-2S] cluster-bound form is involved in the biogenesis of cytosolic and nuclear Fe-S proteins. Specific protein recognition between the N-terminal domains of the two proteins is the mandatory requisite to promote the [2Fe-2S] cluster transfer from GRX3 to anamorsin.

Determinants of the use of dietary supplements among secondary and high school students

Background: All over the world, including Poland, the sale of dietary supplements is increasing. More and more often, people including children and youths, use dietary supplements on their own initiative and without any medical indications or knowledge in this field. Objectives: Analysis of the conditions of using the dietary supplements with vitamins and minerals among secondary school and high school students in Poland. Material and methods: The study included 396 students aged 13-18 years (249 girls and 147 boys). Authors' questionnaire was used to evaluate the intake of dietary supplements. The use of cluster analysis allowed to distinguish groups of students with similar socio-demographic characteristics and the frequency of use of dietary supplements. Results: In the studied population of students three clusters were created that significantly differed in socio-demographic characteristics. In cluster 1 and 2, were mostly students who used dietary supplements (respectively, 56% of respondents and 100%). In cluster 1 there were mostly students coming from rural areas and small city, with a worse financial situation, mainly boys (56%), while cluster 2 was dominated by girls (81%) living in a big city, coming from families with a good financial situation and who were more likely to be underweight (28.8%). In cluster 3 there were mostly older students (62%), not taking dietary supplements. In comparison to cluster 2, they had lower frequency of breakfast consumption (55% vs. 69%), but higher frequency of the consumption of soft drinks, fast-food, coffee as well as salt use at the table. Conclusions: The results show that the use of dietary supplements in adolescence is a common phenomenon and slightly conditioned by eating behaviors. This unfavorable habit of common dietary supplements intake observed among students indicates the need for education on the benefits and risks of the supplements usage.

Family influences on breakfast frequency and quality among primary school pupils in Warsaw and its surrounding areas.

BACKGROUND: Regular consumption of a well balanced breakfast is a pre-requisite for normal growth and child development, along with the acquisition of proper eating habits. The family environment is crucial place where children learn such patterns of behaviour that form the basis for their development. OBJECTIVES: To determine how family factors affect the regular eating of breakfast and types of foodstuffs consumed in primary school pupils, including food purchases made from vending machines and school tuck shops. MATERIALS AND METHODS: Subjects were 836 pupils (435 girls and 401 boys, aged 6 - 13) from Warsaw and the surrounding areas. Appropriate socio-demographic data and relevant eating habits were obtained from direct interviewing of the subjects by means of a custom designed questionnaire. Statistical analyses were performed by the Kohonen type cluster analysis model and Chi-square test (Chi(2)); (p≤0.05). RESULTS: Three clusters of pupils were identified by their differing socio-demographics and eating habits (eg. rates of breakfast consumption, buying from vending machines or school tuck shops). The first and third clusters were mainly pupils from two-parent families with parents proportionally spending similar times at work, where respective breakfast (87% and 91%) and second breakfast (77% and 72%) consumption rates were also similar together with food shopping rates during school time (respectively 69% and 63%). Pupils with single-parents, multi-generation families or if both parents were profession- ally active, predominated in the second cluster. These ate breakfast (73%) and second breakfast (67%) more rarely, but more frequently shopped for food at school (84%). A small number of pupils had a packed second breakfast from home, rarely ate sandwiches, fruit and/or vegetables and dairy products but ate more sweets, sweet rolls and savoury snacks. However, a large number of subjects bought sandwiches, fresh fruit and/or vegetables and fast-food at school. CONCLUSIONS: Family factors were found to affect eating habits in children and adolescents regarding how often breakfast was eaten and the type of foodstuffs consumed. High consumptions of unhealthy food items for second breakfast were also observed. Single-parent pupils, those in multi-generation families or if both parents are employed rarely brought second breakfasts from home but frequently bought food from vending machines and school tuck shops. The results of the presented findings are significant towards planning an appropriate educational campaigns and health programmes targeted at children, adolescents and their families.

Comparing diabetic with non-diabetic overweight subjects through assessing dietary intakes and key parameters of blood biochemistry and haematology.

INTRODUCTION: An important way of preventing type 2 diabetes is by adopting a proper diet by which means appropriate control over blood glycaemia and lipids can be achieved. OBJECTIVES: To assess selected biochemical and haematological markers in overweight subjects or those suffering from type 2 diabetes in relation to their estimated dietary intake. MATERIAL AND METHODS: The study was conducted in 2012 on n = 86 overweight or obese subjects living in Warsaw or its environs, of whom n = 43 had type 2 diabetes. Dietary intakes were compared between non-diabetics (control group) and diabetics (test group) by 3 day records, whilst the relevant blood biochemistry and haematology results were obtained from medical records; with patient consent. RESULTS: Diabetic subjects had significantly higher serum glucose and CRP levels than controls, respectively; 190 vs 98 mg/ dl and 1.4 vs 1.1 mg/dl. Lipid profiles were however more significantly abnormal in controls, compared to diabetics with respectively; total cholesterol 220 vs 194 mg/dl, LDL-cholesterol 131 vs 107 mg/dl and triglycerides 206 vs 157 mg/dl. There were no significant differences in HDL-cholesterol; respectively 55 vs 51 mg/dl. In the diabetics, calorific intakes from carbohydrates, especially sugars, were significantly lower than controls i.e. 9% vs 13%. The proportional share of calories derived from dietary fats did not differ between groups, nevertheless a positive correlation was observed between dietary fat content with blood cholesterol concentrations in diabetics. CONCLUSIONS: Disorders of carbohydrate metabolism were confirmed in both overweight and diabetic (type 2) subjects. In addition, both groups demonstrated untoward lipid profiles that correlated with their improper nutrition.

Determinants of the level of circulating-tumor HPV16 DNA in patients with HPV-associated oropharyngeal cancer at the time of diagnosis.

Circulating tumor HPV DNA (ctHPV16) assessed in liquid biopsy may be used as a marker of cancer in patients with HPV-associated oropharyngeal cancer (HPV + OPC). Factors influencing the initial ctHPV16 quantity are not well recognized. In this study we aimed to establish what factors are related to the level of ctHPV16 at the time of diagnosis. 51 patients (37 men and 14 women, median age of 57 years old) with HPV + OPC prior to definitive treatment were included. ctHPV16 was measured by qPCR. Tumor and nodal staging were assessed according to AJCC8. Blood derived factors included squamous cell carcinoma antigen (SCC-Ag), serum soluble fragment of cytokeratin 19 (CYFRA 21-1), C-reactive protein (CRP), albumin level (Alb), neutrophils (Neut), thrombocytes (Plt) and lymphocyte (Lym) count, Neut/Lym ratio were assessed. The volumes of the primary tumor (TV) and involved lymph nodes (NV) were calculated using MRI, CT or PET-CT scans. Data were analysed using parametric and nonparametric methods. Variables for multivariable linear regression analysis were chosen based on the results from univariable analysis (correlation, univariable regression and difference). There were 9 (18%), 10 (19%) and 32 (63%) patients who had TV and NV assessed in MRI, CT or PET respectively. Primary tumor neither as T-stage nor TV was related to ctHPV16 level. Significant differences in the ctHPV16 between patients with high vs low pain (P = 0.038), NV (P = 0.023), TV + NV (P = 0.018), CYFRA 21-1 (P = 0.002), CRP (P = 0.019), and N1 vs N3 (P = 0.044) were observed. ctHPV16 was significantly associated with CYFRA 21-1 (P = 0.017), N stage (P = 0.005), NV (P = 0.009), TV + NV (P = 0.002), CRP (P = 0.019), and pain (P = 0.038). In univariable linear regression analysis the same variables predicted ctHPV16 level. In multivariable analyses, CYFRA 21-1 and CRP (both as categorical variables) were predictors of ctHPV16 level even above NV. ctHPV16 at presentation is driven by tumor volume measured mostly by N. CYFRA 21-1 and CRP are additional factors related to ctHPV16 prior to the treatment.

Successful Treatment of Adenoid Cystic Carcinoma with the Application of a High-Dose Stereotactic Body Radiotherapy Boost.

BACKGROUND: Adenoid cystic carcinoma (ACC) should be treated with a surgical procedure. Unfortunately, in some cases, such procedures are impossible to perform. In that event, radiotherapy can be used as a form of radical treatment, although ACC is established as a radio- and chemoresistant tumour. Therefore, unconventional fractionated radiotherapy needs to be considered. CASE PRESENTATION: Here, we present a case study of a patient with an unresectable tumour of the choanae and nasopharynx treated with a stereotactic radiotherapy boost in combination with conventional radiotherapy. We achieved complete clinical regression after application of a 1 × 18 Gy boost followed by conventional radiotherapy at 50 Gy in 25 fractions. The early and late tolerance of this treatment were positive. During the 2-year follow-up, local and distant recurrence were not detected. CONCLUSIONS: This case represents an individualized, modern and safe approach to unresectable ACC. This is one of the first cases to show the use of a combination of stereotactic and conventional radiotherapy in radical, conservative cancer treatment.

Cell type-specific differences in redox regulation and proliferation after low UVA doses.

Ultraviolet A (UVA) radiation is harmful for living organisms but in low doses may stimulate cell proliferation. Our aim was to examine the relationships between exposure to different low UVA doses, cellular proliferation, and changes in cellular reactive oxygen species levels. In human colon cancer (HCT116) and melanoma (Me45) cells exposed to UVA doses comparable to environmental, the highest doses (30-50 kJ/m2) reduced clonogenic potential but some lower doses (1 and 10 kJ/m2) induced proliferation. This effect was cell type and dose specific. In both cell lines the levels of reactive oxygen species and nitric oxide fluctuated with dynamics which were influenced differently by UVA; in Me45 cells decreased proliferation accompanied the changes in the dynamics of H2O2 while in HCT116 cells those of superoxide. Genes coding for proteins engaged in redox systems were expressed differently in each cell line; transcripts for thioredoxin, peroxiredoxin and glutathione peroxidase showed higher expression in HCT116 cells whereas those for glutathione transferases and copper chaperone were more abundant in Me45 cells. We conclude that these two cell types utilize different pathways for regulating their redox status. Many mechanisms engaged in maintaining cellular redox balance have been described. Here we show that the different cellular responses to a stimulus such as a specific dose of UVA may be consequences of the use of different redox control pathways. Assays of superoxide and hydrogen peroxide level changes after exposure to UVA may clarify mechanisms of cellular redox regulation and help in understanding responses to stressing factors.

Correction: Profiles of a broad spectrum of epigenetic DNA modifications in normal and malignant human cell lines: Proliferation rate is not the major factor responsible for the 5-hydroxymethyl-2'-deoxycytidine level in cultured cancerous cell lines.

[This corrects the article DOI: 10.1371/journal.pone.0188856.].

Profiles of a broad spectrum of epigenetic DNA modifications in normal and malignant human cell lines: Proliferation rate is not the major factor responsible for the 5-hydroxymethyl-2'-deoxycytidine level in cultured cancerous cell lines.

Active demethylation of 5-methylcytosine moiety in DNA occurs by its sequential oxidation to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine, catalysed by enzymes of the Ten-Eleven Translocation family proteins (TETs 1, 2 and 3). Here we analyzed for the first time all the intermediate products of DNA demethylation pathway in the form of deoxynucleosides (5-methyl-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine and 5-carboxy-2'-deoxycytidine as well as 5-(hydroxymethyl)-2'-deoxyuridine) using automated isotope-dilution online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. DNA was isolated from human malignant cell lines of colon adenocarcinoma (HCT 116), melanoma (Me45), myelogenous leukemia bone marrow blasts (K562), EBV-positive Burkitt's lymphoma lymphoblasts (Raji), EBV-negative Burkitt's lymphoma lymphoblasts (male-CA46 and female-ST486), as well as normal neonatal dermal fibroblasts (NHDF-Neo). The expression levels of TET1, TET2, TET3, SMUG1, and TDG genes were also assayed by RT-qPCR. Our results show a global erasure of 5-hydroxymethyl-2'-deoxycytidine and 5-carboxy-2'-deoxycytidine in DNA of cultured cells compared with DNA from primary malignant tissue. Moreover, malignant cells in culture have a quite different DNA epigenetic profile than cultured normal cells, and different types of malignant cells display different and characteristic profiles of DNA epigenetic marks. Similar analyses of a broader spectrum of epigenetic modifications, not restricted to 5-methyl-2'-deoxycytidine, could lead to better understanding of the mechanism(s) responsible for emergence of different types of cancer cells.

Human pulmonary artery endothelial cells in the model of mucopolysaccharidosis VI present a prohypertensive phenotype.

BACKGROUND: Mucopolysaccharidosis type VI (MPS VI) is an autosomal recessive lysosomal disorder caused by a deficient activity of N-acetylgalactosamine-4-sulfatase (ARSB). Pulmonary hypertension (PH) occurs in MPS VI patients and is a marker of bad prognosis. Malfunction of endothelium, which regulates vascular tonus and stimulates angiogenesis, can contribute to the occurrence of PH in MPS VI. AIM: The aim of the study was to establish a human MPS VI cellular model of pulmonary artery endothelial cells (HPAECs) and evaluate how it affects factors that may trigger PH such as proliferation, apoptosis, expression of endothelial nitric oxide synthase (eNOS), natriuretic peptide type C (NPPC), and vascular endothelial growth factor A (VEGFA). RESULTS: Increasing concentrations of dermatan sulfate (DS) reduce the viability of the cells in both ARSB deficiency and controls, but hardly influence apoptosis. The expression of eNOS in HPAECs is reduced up to two thirds in the presence of DS. NPPC shows a biphasic expression reaction with an increase at 50 µg/mL DS and reduction at 0 and 100 µg/mL DS. The expression of VEGFA decreases with increasing DS concentrations and absence of elastin, and increases with increasing DS in the presence of elastin. CONCLUSION: Our data suggest that MPS VI endothelium presents a prohypertensive phenotype due to the reduction of endothelium's proliferation ability and expression of vasorelaxing factors.

Induction of bystander effects by UVA, UVB, and UVC radiation in human fibroblasts and the implication of reactive oxygen species.

Radiation-induced bystander effects are various types of responses displayed by nonirradiated cells induced by signals transmitted from neighboring irradiated cells. This phenomenon has been well studied after ionizing radiation, but data on bystander effects after UV radiation are limited and so far have been reported mainly after UVA and UVB radiation. The studies described here were aimed at comparing the responses of human dermal fibroblasts exposed directly to UV (A, B, or C wavelength range) and searching for bystander effects induced in unexposed cells using a transwell co-incubation system. Cell survival and apoptosis were used as a measure of radiation effects. Additionally, induction of senescence in UV-exposed and bystander cells was evaluated. Reactive oxygen species (ROS), superoxide radical anions, and nitric oxide inside the cells and secretion of interleukins 6 and 8 (IL-6 and IL-8) into the medium were assayed and evaluated as potential mediators of bystander effects. All three regions of ultraviolet radiation induced bystander effects in unexposed cells, as shown by a diminution of survival and an increase in apoptosis, but the pattern of response to direct exposure and the bystander effects differed depending on the UV spectrum. Although UVA and UVB were more effective than UVC in generation of apoptosis in bystander cells, UVC induced senescence both in irradiated cells and in neighbors. The level of cellular ROS increased significantly shortly after UVA and UVB exposure, suggesting that the bystander effects may be mediated by ROS generated in cells by UV radiation. Interestingly, UVC was more effective at generation of ROS in bystanders than in directly exposed cells and induced a high yield of superoxide in exposed and bystander cells, which, however, was only weakly associated with impairment of mitochondrial membrane potential. Increasing concentration of IL-6 but not IL-8 after exposure to each of the three bands of UV points to its role as a mediator in the bystander effect. Nitric oxide appeared to play a minor role as a mediator of bystander effects in our experiments. The results demonstrating an increase in intracellular oxidation, not only in directly UV-exposed but also in neighboring cells, and generation of proinflammatory cytokines, processes entailing cell damage (decreased viability, apoptosis, senescence), suggest that all bands of UV radiation carry a potential hazard for human health, not only due to direct mechanisms, but also due to bystander effects.

Characteristic of c-Kit+ progenitor cells in explanted human hearts.

According to literature data, self-renewing, multipotent, and clonogenic cardiac c-Kit(+) progenitor cells occur within human myocardium. The aim of this study was to isolate and characterize c-Kit(+) progenitor cells from explanted human hearts. Experimental material was obtained from 19 adult and 7 pediatric patients. Successful isolation and culture was achieved for 95 samples (84.1%) derived from five different regions of the heart: right and left ventricles, atrium, intraventricular septum, and apex. The average percentage of c-Kit(+) cells, as assessed by FACS, ranged between 0.7 and 0.9%. In contrast to published data we do not observed statistically significant differences in the number of c-Kit(+) cells between disease-specific groups, parts of the heart or sexes. Nevertheless, c-Kit(+) cells were present in significant numbers (11-24%) in samples derived from three explanted pediatric hearts. c-Kit(+) cells were also positive for CD105 and a majority of them was positive for CD31 and CD34 (83.7 ± 8.6 and 75.7 ± 11.4%, respectively). Immunohistochemical analysis of the heart tissue revealed that most cells possessing the c-Kit antigen were also positive for tryptase, a specific mast cell marker. However, flow cytometry analysis has shown cultured c-Kit(+) cells to be negative for hematopoietic marker CD45 and mast cell marker CD33. Isolated c-Kit(+) cells display mesenchymal stem cell features and are thought to differentiate into endothelial cells.

An intrinsically disordered domain has a dual function coupled to compartment-dependent redox control.

The functional role of unstructured protein domains is an emerging field in the frame of intrinsically disordered proteins. The involvement of intrinsically disordered domains (IDDs) in protein targeting and biogenesis processes in mitochondria is so far not known. Here, we have characterized the structural/dynamic and functional properties of an IDD of the sulfhydryl oxidase ALR (augmenter of liver regeneration) located in the intermembrane space of mitochondria. At variance to the unfolded-to-folded structural transition of several intrinsically disordered proteins, neither substrate recognition events nor redox switch of its shuttle cysteine pair is linked to any such structural change. However, this unstructured domain performs a dual function in two cellular compartments: it acts (i) as a mitochondrial targeting signal in the cytosol and (ii) as a crucial recognition site in the disulfide relay system of intermembrane space. This domain provides an exciting new paradigm for IDDs ensuring two distinct functions that are linked to intracellular organelle targeting.

Targeting and maturation of Erv1/ALR in the mitochondrial intermembrane space.

The interaction of Mia40 with Erv1/ALR is central to the oxidative protein folding in the intermembrane space of mitochondria (IMS) as Erv1/ALR oxidizes reduced Mia40 to restore its functional state. Here we address the role of Mia40 in the import and maturation of Erv1/ALR. The C-terminal FAD-binding domain of Erv1/ALR has an essential role in the import process by creating a transient intermolecular disulfide bond with Mia40. The action of Mia40 is selective for the formation of both intra and intersubunit structural disulfide bonds of Erv1/ALR, but the complete maturation process requires additional binding of FAD. Both of these events must follow a specific sequential order to allow Erv1/ALR to reach the fully functional state, illustrating a new paradigm for protein maturation in the IMS.

Copper binding to chicken and human prion protein amylodogenic regions: differences and similarities revealed by Ni2+ as a diamagnetic probe.

Both human (h) and chicken (Ch) prion proteins (PrP) bind copper ions within the so called "tandem repeat" N-terminal region. Outside this region, hPrP possesses two additional copper binding sites, localized at His-96 and His-111 in the so called "amylodogenic" or neurotoxic region (residues 91-126). Also ChPrP possesses a similar region (ChPrP(105-140)) containing two His (His-110 and His-124) and an identical hydrophobic tail of 15 amino acids rich in Ala and Gly. The copper binding abilities within such region of ChPrP were investigated by NMR, CD and potentiometry using Ni(2+) as diamagnetic probe. The formation of diamagnetic metal complexes allowed to monitor the chemical shift and signal intensity variations and to determine the structural and kinetic features of the His-110 and His-124 metal binding sites. Finally a comparison between the hPrP and ChPrP metal binding abilities was performed. We found that the two prion proteins exhibited different copper and nickel preferences with the favoured metal binding sites localized at opposite His: His-110 for ChPrP, and His-111 for hPrP.

Copper(II) coordination outside the tandem repeat region of an unstructured domain of chicken prion protein.

Combined potentiometric, calorimetric and spectroscopic methods were used to investigate the Cu(2+) binding ability and coordination behaviour of some peptide fragments related to the neurotoxic region of chicken Prion Protein. The systems studied were the following protein fragments: chPrP(106-114), chPrP(119-126), chPrP(108-127), chPrP(105-127) and chPrP(105-133).The complex formation always starts around pH 4 with the coordination of an imidazole nitrogen, followed by the deprotonation and binding of amide nitrogens from the peptidic backbone. At neutral pH, the {N(im), 3N(-)} binding mode is the preferred one. The amide nitrogens participating in the binding to the Cu(2+) ion derive from residues from the N-terminus side, with the formation of a six-membered chelate ring with the imidazolic side chain.Comparison of thermodynamic data for the two histydyl binding domains (around His-110 and His-124), clearly indicates that the closest to the hexarepeat domain (His-110) has the highest ability to bind Cu(2+) ions, although both of them have the same coordination mode. Conversely, in the case of the human neurotoxic peptide region, between the two binding sites, located at His-96 and His-111, the farthest from the tandem repeat region is the strongest one. Finally, thermodynamic data show that chicken peptide is a distinctly better ligand for coordination of copper ions with respect to the human fragment.