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The beginning of the 21st century has witnessed a growth in our understanding of the importance of planning urban landscapes in the context of urban population growth and unpredictable climatic conditions. In the search for responses to the challenges set by the development of contemporary urban landscapes, researchers have offered solutions based on the concept of sustainable and resilient cities, whose spatial development would be based on an interdisciplinary approach to strategy development: biodiversity, urban ecological networks and connectivity, multifunctionality and modularity. Although the concepts of a green infrastructure, in their spatial and functional dimensions, allow the application of such strategies, there are still problems when it comes to implementation and measuring the results achieved. At the same time, there is a growing discussion of the important role played by urban forestry in the context of the collaborative planning of urban landscapes and the application of the ideas of a green infrastructure. The key question is: what are the modalities of application of the concept of green infrastructure in the process of planning the development of the modern city and how can the resulting benefits be evaluated? With the modalities of application of the concept of green infrastructure in mind, we discuss its multi-scale and multifunctional dimensions as applied in the case of Serbia. The realisation of the green infrastructure concept is presented through the example of the Urban Forest Management Plan for the City of Belgrade - Mladenovac Municipality. The results of using the spatial-ecological approach in creating the plan and establishing connectivity as a new aim in forest management planning show that the implementation of the green infrastructure concept, and the achieved multifunctional ecosystem values, can be presented on the basis of the parameters of landscape metrics. In light of the new urban world, future research should focus on the application of the landscape ecological approach of the green infrastructure concept in collaborative planning at the urban landscape scale, which allows the creation of ecosystem services and benefits to human well-being.

Hepatitis C virus infection is the most common chronic blood-borne infection and one of the most important causes of chronic liver disease. Knowing the predictors associated with pegylated interferon/ribavirin (PEG-IFN/RBV) combination therapy response is important for evidence-based treatment recommendations. The goal of this study was to identify host and viral factors of response to PEG-IFN/RBV treatment in chronic hepatitis C genotype 1b patients. We have examined the relationship between gender, age, level of alanine aminotransferase (ALT), viral load and liver fibrosis progression on therapy response. ALT level and viral load were evaluated before starting treatment with combination therapy. The elevated levels of ALT and route of HCV transmission were found to be significantly associated with the response to therapy in HCV-infected patients. Our findings may be useful for estimating a patient’s likelihood of achieving sustained viral response. [Projekat Ministarstva nauke Republike Srbije, br. OI 173049 i br. TR 37021]

The objectives were to examine if there is a causal relationship between osteosarcopenic adiposity (OSA) syndrome (coexistence of osteopenia/osteoporosis, sarcopenia, and excess adiposity) and cardiometabolic disorders or if these disorders initiate the development of OSA and its worsening. The search was conducted in PubMed, Scopus, and Web of Science to include articles up to the end of 2023. Of n=539 articles retrieved, n=15 met the eligibility criteria. Only studies conducted in adults and with all three body composition compartments (bone, muscle/lean, adipose) measured were considered. The results revealed that several cardiometabolic disorders, namely, hypertension, dyslipidemia (elevated total and LDL-cholesterol, lower HDL-cholesterol), insulin resistance, hyperglycemia, lower serum vitamin D, and some inflammatory markers were accompanied by OSA. In most cases, the OSA phenotype was associated with worse outcomes than cases with healthy or less impaired body composition. Our initial questions about the reciprocal cause-and-effect relationships could be surmised with more certainty for the OSA and some cardiovascular risks (hypertension, dyslipidemia) and some metabolic abnormalities (several inflammatory markers). The results of this review underscore the importance of body composition in health and from a clinical perspective, all three body composition compartments should be measured by standardized technologies using regulated diagnostic criteria to identify OSA. Randomized trials and prospective studies in diverse groups of older and younger individuals are necessary to determine if the relationships between OSA and clinical endpoints are causal and reversible through intervention and to uncover the mechanisms., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

As the existing data on the correlation of adiposity with adverse outcomes of carotid endarterectomy (CEA) are inconsistent, the aim of the present study is to examine the correlation of an increased body mass index with 30-day complications after carotid endarterectomy. The cohort study comprises 1586 CEAs, performed at the Clinic for Vascular Surgery in Belgrade, from 2012-2017. Out of them, 550 CEAs were performed in patients with normal body mass index (18.5-24.9), 750 in overweight (25.0-29.9), and 286 in obese (≥30) patients. The association of overweight and obesity with early outcomes of carotid endarterectomy was assessed using univariate and multivariate logistic regression analysis. Overweight patients, in whom CEAs were performed, were significantly more frequently males, compared to normal weight patients-Odds Ratio (OR) 1.51 (95% confidence interval- 1.19-1.89). Moreover, overweight patients significantly more frequently had non-insulin-dependent diabetes mellitus-OR 1.44 (1.09-1.90), and more frequently used ACEI in hospital discharge therapy-OR 1.41 (1.07-1.84) than normal weight patients. Additionally, the CEAs in them were less frequently followed by bleedings-OR 0.37 (0.16-0.83). Compared to normal weight patients, obese patients were significantly younger-OR 0.98 (0.96-0.99), and with insulin-dependent and non-insulin-dependent diabetes mellitus-OR 1.83 (1.09-3.06) and OR 2.13 (1.50-3.01) respectively. They also more frequently had increased triglyceride levels-OR 1.36 (1.01-1.83), and more frequently used oral anticoagulants in therapy before the surgery-OR 2.16 (1.11-4.19). According to the results obtained, overweight and obesity were not associated with an increased death rate, transient ischemic attack (TIA), stroke, myocardial infarction, or with minor complications, and the need for reoperation after carotid endarterectomy. The only exception was bleeding, which was significantly less frequent after CEA in overweight compared to normal weight patients., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Vukašinović et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Precautionary allergen labels (PAL) should be used to indicate the possibility of allergen presence in the food. This study aimed to determine the prevalence and types of precautionary labeling statements on different pre-packaged food products in retail stores in Belgrade, Serbia, as well as to assess consumers' attitudes and behavior towards PAL statements., Methods: This was a descriptive study. The following characteristics of 1404 pre-packaged foods were analyzed: prevalence of PAL, listed food allergens on PAL, and the types of the advisory terminology. In the group of 275 participants (94 with food allergies, and 181 persons who purchasing food for a household member with food allergy) reading practice of PAL, purchasing practice based on PAL, and the opinion about PAL statements credibility were evaluated., Results: Overall, 33.9% of products had precautionary statements for one or more allergens. "Tree nuts" were the most common allergens listed in the PAL. The most common type of PAL was "May contain traces of x [allergen]" (52.7%). The PAL was always read by half of the participants. Less than half (43.3%) of the participants incorrectly believed that PAL is regulated by national law. A quarter of participants thought that the PAL statements are trustworthy., Conclusion: PAL statements frequently are not user-friendly and are not providing sufficient protection for food allergic patients. To gain buyers' confidence, protect health and provide security, the necessity for the strategies that would regulate PAL by the law exists., Competing Interests: Conflict of interest The authors declare that there is no conflict of interest., (Copyright © 2022 Davidović et al. Published by Tehran University of Medical Sciences.)

Background: Type 2 diabetes mellitus (T2DM) is commonly associated with hyperglycemia, dyslipidemia, oxidative stress and inflammation which are well known cardiovascular risk factors. Pomegranate peel polyphenols have a proven hypolipemic, antioxidant and anti-inflammatory activity. However, there is a lack of clinical studies that would confirm its antioxidant and anti-inflammatory effects in diabetic patients. The potential of pomegranate peel extract (PoPEx) to counteract inflammation and oxidative stress in T2DM patients was investigated. For this purpose, a randomized, double-blind placebo-controlled study involving adult T2DM patients treated with PoPEx or placebo for eight-weeks was conducted., Methods: Patients were randomly divided into two groups: the first group (n = 30) received capsules containing PoPEx 250 mg twice daily, while the placebo group (n = 30) received placebo capsules twice daily. Plasma concentration of inflammatory factors (interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and high sensitivity C reactive protein (hsCRP)), oxidative stress biomarkers (thiobarbituric acid reactive substances (TBARS), nitrites (NO2-), superoxide anion radical (O2-), hydrogen peroxide (H2O2), total antioxidant capacity (TAC)), homocysteine and lipid profile were analyzed., Results: The PoPEx treatment showed a significant reduction of inflammatory factors (IL-6, TNF-α, hsCRP), oxidative stress biomarkers (TBARS, NO2-, O2-) and homocysteine, while the TAC was increased. Moreover, a significant improvement in lipid profile was observed in the PoPEx group. Additional analysis showed a significant inverse correlation between the decrements of all measured inflammatory markers and TAC in the PoPEx group., Conclusions: The study demonstrated that eight-week-long PoPEx administration had favorable effects on inflammatory status and oxidative stress biomarkers in diabetic patients., Competing Interests: The authors declare no conflict of interest. Vladimir Jakovljević and Dragan M Djuric are serving as Guest editors of this journal. We declare that Vladimir Jakovljević and Dragan M Djuric had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Peter A. McCullough., (© 2022 The Author(s). Published by IMR Press.)

Nutrition and health claims (NHCs) are a powerful tool that influence consumers' final decision on the choice of food products. The purposes of this repeated cross-sectional study were to (i) assess the prevalence of pre-packaged food products containing nutrition and health claims among different food categories, (ii) to determine the type of NHCs labelled on the examined food products, and (iii) to evaluate the trend in the use of NHCs in comparison to the 2012 survey. The survey was conducted immediately before the full enforcement of the new national legislation on NHCs in 2020. It comprised 3141 pre-packaged food products from 10 product categories. In total, 21.2% of food products contained any claim (19.4% contained any nutrition claim; 8.2% contained any health claim). In comparison to the 2012 survey, we observed a rising trend in the presence of NHCs; the use of nutrition claims on food products increased three times and the use of health claims increased 1.3 times in the 2020 survey. Bearing in mind that NHCs are a powerful tool guiding consumers' food purchase decisions, NHCs should be supported by precise legislation and strict surveillance by the public health authorities.

Visual interpretation of cervical biopsies is subjective and variable, generally showing fair to moderate inter-reader agreement in distinguishing high from low grade cervical intraepithelial neoplasia (CIN). We investigated the performance of two objective p16 quantitative tests in comparison with visual assessment: (i) p16-mRNA assay and (ii) digital analysis of sections stained for p16 protein. The primary analysis considered 232 high-risk human papilloma virus positive (HPV+) samples from diagnostic cervical specimens. A p16 RT-qPCR (p16-mRNA assay) was run on mRNA extracted from formalin-fixed paraffin-embedded sections. Two p16 immunohistochemistry (IHC) readings, a visual read by a histopathologist (Visual IHC) and a digital read of a high-resolution scan (Digital IHC), were done on adjacent sections. The worst reviewed CIN grade (agreed by at least two histopathologists) from up to two biopsies and a loop excision was taken, with CIN2/3 as the primary endpoint. Visual IHC attained a specificity of 70% (95%CI 61-77) for 85% (95%CI 77-91%) sensitivity. The four-point Visual IHC staining area under the curve (AUC) was 0.77 (95%CI 0.71-0.82), compared with 0.71 (95%CI 0.64-0.77) for p16-mRNA and 0.67 (95%CI 0.60-0.74) for Digital IHC. Spearman rank-order correlations were: visual to p16-mRNA 0.41, visual to digital 0.49 and p16-mRNA to digital: 0.22. The addition of p16-mRNA assay to visual reading of p16 IHC improved the AUC from 0.77 to 0.84 (p = 0.0049). p16-mRNA testing may be complementary to visual IHC p16 staining for a more accurate diagnosis of CIN, or perhaps a substitute in locations with a lack of skilled pathologists., (© 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Clinically aggressive disease behavior is difficult to predict in men with low to intermediate clinical risk prostate cancer and methylation biomarkers may be a valuable adjunct for assessing the management of these patients. We set to evaluate the utility of DNA methylation to identify high risk disease in men currently considered as low or intermediate risk. DNA was extracted from formalin-fixed paraffin-embedded transurethral prostate resection tissues collected during the years 1990-96 in a watchful-waiting cohort of men in the UK. The primary end point was death of prostate cancer, assessed by reviewing cancer registry records from 2009. Methylation was quantified by pyrosequencing assays for six genes (HSPB1, CCND2, TIG1, DPYS, PITX2, and MAL) with established biomarker value in prostate cancer. A novel prognostic methylation score was developed by multivariate Cox modelling using the six methylation biomarkers in 385 men with low-and-intermediate clinical risk variables and its prognostic value compared to two previously defined clinically-derived risk scores. Methylation score was the most significant variable in univariate and bivariate analysis in men with low-to-intermediate CAPRA risk score. When combined with CAPRA score the hazard ratio was 2.02; 95% confidence interval, 1.40-2.92. For a methylation score sensitivity of 83% the specificity was 44%, while the maximum achieved sensitivity by CAPRA was 68% at a specificity of 44%. The derived methylation score is a strong predictor of aggressive prostate cancer that could have an important role in directing the management of patients with low-to-intermediate risk disease. The estimated areas under the curve (AUC) at 10 years of follow-up were 0.62 (95% CI: 0.51, 0.70) and 0.74 (95% CI: 0.65, 0.82) for CAPRA, and combined (CAPRA + methylation) risk score (CRS) respectively.

High-risk human papillomavirus (hrHPV) DNA tests have excellent sensitivity for detection of cervical intraepithelial neoplasia 2 or higher (CIN2+). A drawback of hrHPV screening, however, is modest specificity. Therefore, hrHPV-positive women might need triage to reduce adverse events and costs associated with unnecessary colposcopy. We compared the performance of HPV16/18 genotyping with a predefined DNA methylation triage test (S5) based on target regions of the human gene EPB41L3, and viral late gene regions of HPV16, HPV18, HPV31 and HPV33. Assays were run using exfoliated cervical specimens from 710 women attending routine screening, of whom 38 were diagnosed with CIN2+ within a year after triage to colposcopy based on cytology and 341 were hrHPV positive. Sensitivity and specificity of the investigated triage methods were compared by McNemar's test. At the predefined cutoff, S5 showed better sensitivity than HPV16/18 genotyping (74% vs 54%, P = 0.04) in identifying CIN2+ in hrHPV-positive women, and similar specificity (65% vs 71%, P = 0.07). When the S5 cutoff was altered to allow equal sensitivity to that of genotyping, a significantly higher specificity of 91% was reached (P < 0.0001). Thus, a DNA methylation test for the triage of hrHPV-positive women on original screening specimens might be a valid approach with better performance than genotyping., (© 2016 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Background: The aim of this study was to determine sex differences in the prevalence of cardiovascular health (CVH) metrics according to the CVH status., Methods: The cross-sectional, population-based study involved 2250 women and 1920 men aged ≥18 years that participated in the 2010 National Health Survey in the Republic of Srpska, Bosnia and Herzegovina. Prevalence of CVH behaviours (smoking, body mass index, physical activity, diet), CVH factors (cholesterol, fasting blood glucose and blood pressure, plus smoking) and ideal CVH were estimated according to the American Heart Association criteria for ideal, intermediate and poor levels. Association between sex and ideal CVH categories was analyzed with multivariable logistic regression analysis across three age stratums., Results: A higher prevalence of ideal CVH metrics was seen in women for smoking status, body mass index, healthy diet score and blood pressure, and in men for physical activity and total cholesterol. Women from all age groups had better CVH behaviours (odds ratio [OR] = 1.40 for the youngest; OR = 2.05 for middle-aged; and OR = 2.03 for older-aged women), while only women from the youngest age group had better CVH factors (OR = 5.09). In line with this, ideal overall CVH prevailed in younger and middle-aged women in comparison to men of the same ages (OR = 3.01 and OR = 2.25, respectively), while disappeared in older ones., Conclusions: Significant differences in the prevalence of CVH metrics between men and women in the Republic of Srpska should be considered in cardiovascular disease prevention., (© The Author 2015. Published by Oxford University Press on behalf of the European Public Health Association. All rights reserved.)

Methylation of human papillomavirus (HPV) and host genes may predict cervical cancer risk. We examined the methylation status of selected sites in HPV16 and human genes in DNA extracted from exfoliated cervical cell samples of 244 women harboring HPV16-positive cancer or cervical intraepithelial neoplasia (CIN) or negative for intraepithelial lesions or malignancy (NILM). We quantified the methylation of CpG sites in the HPV16 L1 gene (CpG 6367 and 6389) and in the human genes EPB41L3 (CpG 438, 427, 425) and LMX1 (CpG 260, 262, 266, 274) following bisulfite treatment and pyrosequencing. Receiver operating characteristic (ROC) curves were used to analyze the diagnostic utility of methylation level for the different sites and for a joint predictor score. Methylation in all sites significantly increased with lesion severity (p < 0.0001). Area under the curve (AUC) was highest among the CIN2/3 vs. cancer ranging from 0.786 to 0.853 among the different sites. Site-specific methylation levels strongly discriminated CIN2/3 from NILM/CIN1 and cancer from CIN2/3 (range of odds ratios [OR]: 3.69-12.76, range of lower 95% confidence bounds: 1.03-4.01). When methylation levels were mutually adjusted for each other EPB41L3 was the only independent predictor of CIN2/3 vs. NILM/CIN1 contrasts (OR = 9.94, 95%CI: 2.46-40.27). High methylation levels of viral and host genes are common among precancerous and cancer lesions and can serve as independent risk biomarkers. Methylation of host genes LMX1 and EPB41L3 and of the viral HPV16 L1 sites has the potential to distinguish among precancerous lesions and to distinguish the latter from invasive disease., (© 2014 UICC.)

Introduction: The pandemic of obesity in adolescents is one of the challenges of public health., Aim: The aim of this study was to examine the association of overweight with demographic, socioeconomic and lifestyle factors among Serbian adolescents., Method: A cross-sectional study of 2139 adolescents aged 10 to 19 years was carried out. Data used in this study were from the 2006 Health Survey. In accordance with the international sex- and age-specific Body Mass Index cut-off points, all participants were classified as being normal weight or overweight, including obese. The association between the risk factors and overweight were examined using a multivariate logistic regression model., Results: The study showed that 28.9% of boys and 17.0% of girls were overweight, while 14.5% of boys and 8.1% of girls were obese. Boys were more likely to be overweight/obese, compared with girls. Being younger (p< 0.01 for 14 to 15 years) and (p< 0.01, for 16 to 19 years), engaging in physical activities that last less than 7 hours a week, in such a manner that they breathe quickly and become sweaty, (p< 0.01) and skipping breakfast (p< 0.05) were risk factors significantly associated with overweight among adolescents. No significant association was found with wealth index., Conclusion: These findings should be an integral part of further preventive interventions, especially oriented towards younger adolescents, who are physically inactive, have a habit of skipping breakfast and are boys.

Testing for high-risk (hr) types of human papillomavirus (HPV) is highly sensitive as a screening test of high-grade cervical intraepithelial neoplastic (CIN2/3) disease, the precursor of cervical cancer. However, it has a relatively low specificity. Our objective was to develop a prediction rule with a higher specificity, using combinations of human and HPV DNA methylation. Exfoliated cervical specimens from colposcopy-referral cohorts in London were analyzed for DNA methylation levels by pyrosequencing in the L1 and L2 regions of HPV16, HPV18, HPV31 and human genes EPB41L3, DPYS and MAL. Samples from 1,493 hrHPV-positive women were assessed and of these 556 were found to have CIN2/3 at biopsy; 556 tested positive for HPV16 (323 CIN2/3), 201 for HPV18 (73 CIN2/3) and 202 for HPV31 (98 CIN2/3). The prediction rule included EPB41L3 and HPV and had area under curve 0.80 (95% CI 0.78-0.82). For 90% sensitivity, specificity was 36% (33-40) and positive predictive value (PPV) was 46% (43-48). By HPV type, 90% sensitivity corresponded to the following specificities and PPV, respectively: HPV16, 38% (32-45) and 67% (63-71); HPV18, 53% (45-62) and 52% (45-59); HPV31, 39% (31-49) and 58% (51-65); HPV16, 18 or 31, 44% (40-49) and 62% (59-65) and other hrHPV 17% (14-21) and 21% (18-24). We conclude that a methylation assay in hrHPV-positive women might improve PPV with minimal sensitivity loss., (© 2014 The Authors. UICC.)

Background: Prostate cancer has a variable clinical behaviour with frequently unpredictable outcome. DNA methylation plays an important role in determining the biology of cancer but prognostic information is scanty. We assessed the potential of gene-specific DNA methylation changes to predict death from prostate cancer in a cohort of untreated men in the UK., Methods: This was a population-based study in which cases were identified from six cancer registries in Great Britain. DNA was extracted from formalin-fixed paraffin wax-embedded transurethral prostate resection tissues collected during 1990-96 from men with clinically-localised cancer who chose not to be treated for at least 6 months following diagnosis. The primary end point was death from prostate cancer. Outcomes were determined through medical records and cancer registry records. Pyrosequencing was used to quantify methylation in 13 candidate genes with established or suggested roles in cancer. Univariate and multivariate Cox models were used to identify possible predictors for prostate cancer-related death., Results: Of 367 men, 99 died from prostate cancer during a median of 9.5 years follow-up (max = 20). Univariately, 12 genes were significantly associated with prostate cancer mortality, hazard ratios ranged between 1.09 and 1.28 per decile increase in methylation. Stepwise Cox regression modelling suggested that the methylation of genes HSPB1, CCND2 and DPYS contributed objective prognostic information to Gleason score and PSA with respect to cancer-related death during follow-up (p = 0.006)., Conclusion: Methylation of 13 genes was analysed in 367 men with localised prostate cancer who were conservatively treated and stratified with respect to death from prostate cancer and those who survived or died of other causes. Of the 13 genes analysed, differential methylation of HSPB1, CCND2 and DPYS provided independent prognostic information. Assessment of gene-methylation may provide independent objective information that can be used to segregate prostate cancers at diagnosis into predicted behavioural groups.

Genomic changes affecting tumour suppressor genes are fundamental to cancer. We applied SNP array analysis to a panel of testicular germ cell tumours to search for novel tumour suppressor genes and identified a frequent small deletion on 6q25.3 affecting just one gene, ZDHHC14. The expression of ZDHHC14, a putative protein palmitoyltransferase with unknown cellular function, was decreased at both RNA and protein levels in testicular germ cell tumours. ZDHHC14 expression was also significantly decreased in a panel of prostate cancer samples and cell lines. In addition to our findings of genetic and protein expression changes in clinical samples, inducible overexpression of ZDHHC14 led to reduced cell viability and increased apoptosis through the classic caspase-dependent apoptotic pathway and heterozygous knockout of ZDHHC14 increased [CORRECTED] cell colony formation ability. Finally, we confirmed our in vitro findings of the tumour suppressor role of ZDHHC14 in a mouse xenograft model, showing that overexpression of ZDHHC14 inhibits tumourigenesis. Thus, we have identified a novel tumour suppressor gene that is commonly down-regulated in testicular germ cell tumours and prostate cancer, as well as given insight into the cellular functional role of ZDHHC14, a potential protein palmitoyltransferase that may play a key protective role in cancer., (© 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

Background: High risk human papillomavirus (HR-HPV) infection is common and only a small minority of infections become persistent and lead to cervical cancers. Women positive for HR-HPV usually require a second test to avoid unnecessary colposcopies and over treatment. Elevated DNA methylation of HR-HPV L1 and L2 genes in high grade disease has emerged as a promising molecular triage tool., Objectives: Our aim was to accurately measure methylation levels at selected CpG positions in the HPV18, HPV31 and HPV33 genomes. We focused on the L2, L1, URR and E6 regions because these were previously shown to be interesting areas for study., Study Design: Pyrosequencing was used to measure methylation in 208 HPV18, 207 HPV31, and 126 HPV33 positive women selected from a London colposcopy referral population., Results: After adjustment for multiple testing, at FDR 5%, elevated methylation was significantly associated with cervical intraepithelial neoplasia grades 2 or worse (CIN2+) in all investigated CpGs in HPV18 L2 and L1. Two of 6 L2 and 12 of 15 L1 sites in HPV31 and 6 of 8 L2 and 3 of 13 L1 sites in HPV33 showed significantly elevated methylation in CIN2+. Methylation of CpG sites in the URR and E6 region of the HPV types was low and most differences were not significant., Conclusion: Elevated methylation of CpG sites in the L1 and L2 regions of HPV18, HPV31 and HPV33 is associated with CIN2+ and a panel test may be useful for triage of women with HR-HPV infections., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: Testing for high risk human papillomavirus (HR-HPV) is increasing; however due to limitations in specificity there remains a need for better triage tests. Research efforts have focused recently on methylation of human genes which show promise as diagnostic classifiers., Methods: Methylation of 26 genes: APC, CADM1, CCND2, CDH13, CDKN2A, CTNNB1, DAPK1, DPYS, EDNRB, EPB41L3, ESR1, GSTP1, HIN1, JAM3, LMX1, MAL, MDR1, PAX1, PTGS2, RARB, RASSF1, SLIT2, SOX1, SPARC, TERT and TWIST1 was measured by pyrosequencing in cytology specimens from a pilot set of women with normal or cervical intraepithelial neoplasia grade 3 (CIN3) histology. Six genes were selected for testing in Predictors 1, a colposcopy referral study comprising 799 women. The three genes EPB41L3, DPYS and MAL were further tested in a second colposcopy referral study, Predictors 2, comprising 884 women., Results: The six genes selected from the pilot: EPB41L3, EDNRB, LMX1, DPYS, MAL and CADM1 showed significantly elevated methylation in CIN2 and CIN3 (CIN2/3) versus ≤CIN1 in Predictors 1 (p<0.01). Highest methylation was observed in cancer tissues. EPB41L3 methylation was the best single classifier of CIN2/3 in both HR-HPV positive (p<0.0001) and negative samples (p=0.02). Logistic regression modeling showed that other genes did not add significantly to EPB41L3 and in Predictors 2, its classifier value was validated with AUC 0.69 (95% CI 0.65-0.73)., Conclusion: Several methylated genes show promise for detecting CIN2/3 of which EPB41L3 seems the best. Methylated human gene biomarkers used in combination may be clinically useful for triage of women with HR-HPV infections., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Aim: We investigated if methylation of candidate genes can be useful for predicting prostate cancer (PCa) specific death., Patients & Methods: Methylation of PITX2, WNT5a, SPARC, EPB41L3 and TPM4 was investigated in a 1:2 case-control cohort comprising 45 men with cancer of Gleason score ≤ 7 who died (cases), and 90 men who were alive or died of other causes with survival time longer than the cases (controls). A univariate conditional logistic regression model was fitted by maximizing the likelihood of DNA methylation of each gene versus the primary end point., Results: A 10% increase in methylation of PITX2 was associated with PCa related death with OR 1.56 (95% CI: 1.17-2.08; p = 0.005)., Conclusion: Our study strengthens prior findings that PITX2 methylation is useful as a biomarker of poor outcome of PCa and in addition we also suggest that it may be particularly useful in men with low Gleason score.

DNA methylation changes in human papillomavirus type 16 (HPV16) DNA are common and might be important for identifying women at increased risk of cervical cancer. Using recently published data from Costa Rica we developed a classification score to differentiate women with cervical intraepithelial neoplasia grade 2 or 3 (CIN2/3) from those with no evident high-grade lesions. Here, we aim to investigate the performance of the score using data from the UK. Exfoliated cervical cells at baseline and 6-months follow-up were analyzed in 84 women selected from a randomized clinical trial of women undergoing surveillance for low-grade cytology. Selection of women for the methylation study was based on detectable HPV16 in the baseline sample. Purified DNA was bisulfite converted, amplified and pyrosequenced at selected CpG sites in the viral genome (URR, E6, L1 and L2), with blinding of laboratory personnel to the clinical data. The primary measure was a predefined score combining the mean methylation in L1 and any methylation in L2. At the second follow-up visit, 73/84 (87%) women were HPV16 positive and of these 25 had a histopathological diagnosis of CIN2/3. The score was significantly associated with CIN2/3 (area under curve = 0.74, p = 0.002). For a cutoff with 92% sensitivity, colposcopy could have been avoided in 40% (95% CI 27-54%) of HPV16 positive women without CIN2/3; positive predictive value was 44% (32-58%) and negative predictive value was 90% (71-97%). We conclude that quantitative DNA methylation assays could help to improve triage among HPV16 positive women., (Copyright © 2013 UICC.)

Eating disorders are considered chronic diseases of civilization. The most studied and well known are anorexia and bulimia nervosa. Anorexia is considered one of the most common psychiatric problems of girls in puberty and adolescence. Due to high mortality and morbidity as well as the increasing expansion of these diseases, it is clear why the amount of research on these diseases is growing worldwide. Eating disorders lead to numerous medical complications, mostly due to late diagnosis. The main characteristic of these diseases is changed behavior in the nutrition, either as an intentional restriction of food, i.e. extreme dieting, or overeating, i.e. binge eating. Extreme dieting, skipping meals, self-induced vomiting, excessive exercise, and misuse of laxatives and diuretics for the purpose of maintaining or reducing body weight are characteristic forms of compensatory behavior of patients with eating disorder. The most appropriate course of treatment is determined by evaluating the patient's health condition, associated with behavior and eating habits, the experience of one's own body, character traits of personality, and consequently the development and functioning of the individual. The final treatment plan is individual. Eating disorders are a growing medical problem even in this part of the world. Prevention should be planned in cooperation with different sectors so as to stop the epidemic of these diseases.

The environmental impacts of ski resorts in the Balkan region are great and can lead to landscape degradation and loss of land functionality. In this study, we present an example of the negative effects of human activities at the Stara Planina ski resort in southeastern Serbia. The objective of this study is detailed analysis of the characteristics of environmental impacts at the Stara Planina. The management of the ski area and ski slope development caused severe degradation of topsoil and native vegetation. The morphological characteristics of the area, lithological properties of the exposed material and climate conditions resulted in various geomorphic impacts, including rills, deep gullies, solifluctions and debris from rock weathering. Significant changes in land usage altered hydrological conditions, resulting in more frequent torrential floods in the downstream sections of the Zubska River and increased the sediment yield. Environmental impacts were analyzed in the immediate and wider zones of the ski resort in accordance with the specific topography and visual exposure. The restoration and erosion control measures have stopped degradation processes and helped to rehabilitate the appearance and functions of the landscape. The results show the importance of considering lithological (the type and characteristics of minerals present) and hydrological (precipitation, water storage capacity of soil, runoff) factors under the conditions of significant changes in land usage. The results of this investigation can contribute to the improvement of planning processes and the implementation of development projects in ski areas.

Background: The need for new prognostic factors in breast cancer is ever increasing as breast cancer management evolves. Aberrant DNA methylation plays a pivotal role in cancer development and progression; DNA methylation-based biomarkers may provide independent prognostic information. We used pyrosequencing to investigate the prognostic potential of quantitative DNA methylation of a large set of candidate genes in a Korean single-institution series of operable breast cancer., Methods: Absolute DNA methylation in 20 candidate genes from an initial set of 30 genes was measured by pyrosequencing of bisulfite converted DNA in 121 fresh frozen breast cancer cases. Survival analyses used continuous and categorized (quintile-based) gene methylation data with time to recurrence (TTR) as an endpoint. Prognostic abilities of gene-only and risk-score models were explored., Results: Median follow-up was 5.1 years; 25 recurrences (21%) were observed. Nodal status, methylation of TWIST1, SLIT2 (both as continuous and categorized variables) and APC, HLA-A, NKX2-5, SERPINB5, SFN (as categorized variables) were significantly prognostic; grade showed a prognostic trend. A multivariate model containing nodal status, grade and TWIST1 was a best fit (p< 0.001) in stepwise regression; risk-score based on this model separated patients into 3 distinct risk-groups (p< 0.001). A gene-only model based on TWIST1 and SFN also classified patients into distinct risk-groups (p=0.009)., Conclusions: This study shows that accurate quantitative measurement of DNA methylation by pyrosequencing identifies a small set of genes with independent prognostic potential in breast cancer. These genes complement the current clinico-pathological prognostic factors and appear to be potential biomarkers that warrant further validation.

Introduction: The association of obesity with renal function disorders has been demonstrated by some studies., Objective: The aim of this study was to assess the prevalence of renal insufficiency (RI), measured as glomerular filtration rate (GFR) < 60 ml/min and impact of weight loss on kidney function in a cohort of 109 obese patients (body mass index - BMI > 25 kg/m2), without previous kidney disease, and who underwent the dietician's treatment., Methods: According to body mass index (BMI), the patients were classified as overweight (25-29.9 kg/m2, 25 patients), obesity grade 1 (30-34.9 kg/m2, 37 patients), obesity grade II (35 to 39.9 kg/m2, 23 patients), and obesity grade III (> 40 kg/m2, 24 patients). Data on hypertension, cardiovascular diseases, diabetes, family illness history on diabetes, obesity and hypertension, smoking and medications, lipid profile, serum creatinine (sCr) measured before and after diet, and urine examination were collected from the patients' records. GFR was estimated using MDRD formula (modification of diet in renal disease)., Results: The patients groups were similar in age and co-morbidities, lipids, and sCr values. In comparison to other obese patients, blood pressure was the highest in obesity grade III patients (p = 0.0001). Mean GFR rate before diet was satisfactory in all studied groups. RI was present in 12.8% patients. After diet nine patients still had GFR < 60 ml/min, while lipids decreased in all groups. Patients with the highest decrease of BMI also showed best improvement in GFR. The risk for the development of decreased GFR was higher in elderly patients (estimated rate: -0.434, p < 0.0001)., Conclusion: Obesity is a potentially reversible risk factor for the development of decreased GFR. The relationship between obesity and decreased GFR may be mediated by the presence of known cardiovascular risk factors. In order to clarify the obesity influence on renal functioning, further studies are needed.

Background/aim: Iron is a vital constituent of hemoglobin, myoglobin, and some mitochondrial enzymes; therefore, body iron deficiency may result in reduced aerobic capacity. The aim of this study was to evaluate the effects of daily oral iron supplementation on body iron status, and the maximal oxygen uptake (VO2max) in female athletes with latent iron deficiency, as well as with iron-deficiency anemia., Methods: A total of 37 female volleyball players were included in the study. Seventeen female athletes had latent iron deficiency, and 20 ones iron deficiency anemia. Both groups were divided into the experimental and the control group. The experimental groups received a daily oral iron supplement (200 mg ferrous sulfate), for a two-month training course. Iron status was determined by serum parameters as follows: red blood cells count, hemoglobin concentration, serum iron and ferritin levels, an unsaturated iron binding capacity, total iron binding capacity and transferrin saturation. VO2max was determined by an indirect test., Results: Statistical difference between the latent iron deficient group versus the iron deficient anemic group was found regarding VO2max (p < 0.001). There were correlations between hemoglobin concentration and VO2max in the latent iron deficient group, as well as in the iron deficient anaemic group (p < 0.05). After two months, there was a significant increase in VO2max in all groups (from 7.0% to 18.2%). Values of VO2max at the end of training period were significantly different (45.98 +/- 1.76 vs 42.40 +/- 1.22 mL/kg/min; p < 0.001) between the experimental and the control group only in female athletes with iron deficiency anemia. After the supplementation, markers of iron status were significantly higher in supplemented groups than in the controls., Conclusion: VO2max was significantly lower in the iron deficient anemic group versus the latent iron deficient group. Iron supplementation during a two-month training period significantly improved body iron status in the iron deficient female athletes with or without anemia, and significantly increased VO2max only in the subjects with iron deficiency anemia.

Aberrant DNA methylation plays a pivotal role in carcinogenesis and its mapping is likely to provide biomarkers for improved diagnostic and risk assessment in prostate cancer (PCa). We quantified and compared absolute methylation levels among 28 candidate genes in 48 PCa and 29 benign prostate hyperplasia (BPH) samples using the pyrosequencing (PSQ) method to identify genes with diagnostic and prognostic potential. RARB, HIN1, BCL2, GSTP1, CCND2, EGFR5, APC, RASSF1A, MDR1, NKX2-5, CDH13, DPYS, PTGS2, EDNRB, MAL, PDLIM4, HLAa, ESR1 and TIG1 were highly methylated in PCa compared to BPH (p < 0.001), while SERPINB5, CDH1, TWIST1, DAPK1, THRB, MCAM, SLIT2, CDKN2a and SFN were not. RARB methylation above 21% completely distinguished PCa Separation based on methylation level of SFN, SLIT2 and SERPINB5 distinguished low and high Gleason score cancers, e.g. SFN and SERPINB5 together correctly classified 81% and 77% of high and low Gleason score cancers respectively. Several genes including CDH1 previously reported as methylation markers in PCa were not confirmed in our study. Increasing age was positively associated with gene methylation (p < 0.0001).Accurate quantitative measurement of gene methylation in PCa appears promising and further validation of genes like RARB, HIN1, BCL2, APC and GSTP1 is warranted for diagnostic potential and SFN, SLIT2 and SERPINB5 for prognostic potential.

Introduction: Eating disorders indicate unhealthy habits in nutrition and/or behaviour in the feeding and maintaining of body weight. The main characteristic of these diseases is changed behaviour in nutrition, either as an intentional restriction of food, namely extreme dieting or overeating, i.e. binge eating. Extreme dieting, skipping meals, self-induced vomiting, excessive exercise, and misuse of laxatives and diuretics for the purpose of maintaining or reducing body weight are the forms of compensatory behaviour., Objective: The purpose of the present research was to determine the presence of different inappropriate compensatory behaviours among eating disordered patients., Methods: The experimental group included 35 female eating disordered patients of 23.02 +/- 3.46 years on average, with anorexia or bulimia nervosa. The control group consisted of 70 girls aged 23.1 +/- 3.0 years on average. Each participant completed a "24-hour Recall Questionnaire" and the "Eating Disorder Diagnostic Scale"., Results: A high statistically significant difference existed in the presence of all compensatory behaviours in the experimental and control group, regarding vomiting (chi2 = 40.6; p < 0.001), misuse of laxatives and diuretics (chi2 = 33.7; p < 0.001), extreme dieting (chi2 = 23.4; p < 0.001) and excessive exercising (chi2 = 27.1; p < 0.001)., Conclusion: Eating disordered patients showed a significantly higher incidence of all evaluated forms of compensatory behaviour in comparison with the control group. This report confirms the presence of specific symptomatology of anorexia and bulimia patients.

Richard Morton was a distinguished physician of the 17th century. He was born in Suffolk, England, on July 30th 1637. Morton published three works but his landmark paper was "Phthisiologia, seu exercitationes de phthisi, tribus libris comprehensae" published in 1689, dedicated to William III. The book established his reputation at home and abroad lasting for over a century. Pulmonary tuberculosis was very frequent in the 17th century in England. He was the first physician ever to state that tubercles were always present in its pulmonary form. When we add to these momentous observations and their rational explanation the facts that he was the first physician to state categorically that tubercles are always present in phthisis, we must agree that Morton richly deserves his honoured place in the long list of those who have contributed to the solution of the problem of tuberculosis. Morton first described and gave conclusions of numerous today well known and already examined illnesses. In 1694 he gave first notes about the psychiatric illness which we today call "anorexia nervosa", calling it "nervous consumption". His chapters on treatment are long and contain a sound basis of common sense as indicated by his instructions on general management. He stresses the need for an adequate diet, an environment free from fog and smoke, and the desirability of ensuring a moderate amount of exercise. All Morton's therapeutic dicta are in their humanity and thoughtful care in striking contrast to the regimen of copious bleeding and semi-starvation inflicted by the later generation of physicians. Confirmation of his achievements and his teaching can be found in today's medical practice.

Keratoacanthoma (KA) is difficult to histologically distinguish from squamous cell carcinoma (SCC). Therefore, although KA is a benign self-resolving skin lesion, KA is commonly treated as SCC. Biomarkers to distinguish KA and SCC would thus be desirable. In search for specific markers, paraffin-embedded tissue samples from 25 SCC and 64 KA were arranged in a tissue microarray (TMA) and stained for immunologic cell-markers CD3, CD20 and CD68 as well as for proteins considered of relevance in tumorgenesis, namely NF kappaB/p65, I kappaB-alpha, STAT3, p53, TRAP-1, pRB, phosphorylated pRb, Cyld, p21, p16(INK4), Survivin, Bcl-xL, Caspase 3, Bak, FLK-1/VEGF-r2 and Ki-67. In addition, the tumors were tested for presence of human papillomavirus by PCR. We detected that the two lesions differed significantly in expression of Bcl-xL which was present in 84% of the SCC compared with only 15% in the KA (p < 0.001). The lower expression of the antiapoptotic protein Bcl-xL in KA is consistent with a possible role of apoptosis in the regression of KA., ((c) 2008 Wiley-Liss, Inc.)

Background/aim: Exertional heat stress is common problem in military services. The aim was to exemine changes in serum concentrations of some enzymes in soldiers during exertional heat stress test (EHST) as well as the effects of 10-days passive or active acclimatization in climatic chamber., Methods: Forty male soldiers with high aerobic capacity, performed EHST either in cool (20 degrees C, 16 degrees C Wet bulb globe temperature--WBGT), or hot (40 degrees C, 25 degrees C WBGT) environment, unacclimatized, or after 10 days of passive or active acclimation. Physiological strain was measured by tympanic temperatures (Tty) and heart rates (HR). Concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatine-kinase (CK) were measured in blood samples collected before and immediately after EHST., Results: Exertional heat stress test in hot conditions induced physiological heat stress (increase in Tty and HR), with significant increase in concentrations of all enzymes in unacclimatized group: ALT (42.5 +/- 4.2 before vs 48.1 +/- 3.75 U/L after EHST, p < 0.01), AST (24.9 +/- 5.1 vs 33.4 +/- 4.48 U/L, p < 0.01), LDH (160.6 +/- 20.2 vs 195.7 +/- 22.6 U/L, p < 0.001) and CK (215.5 +/- 91.2 vs 279.1 +/- 117.5 U/L, p < 0.05). In acclimatized soldiers there were no significant changes in concentrations of ALT and AST, while concentration of CK was significantly higher. Concentrations of LDH were significantly higher in all investigated groups, regardless of temperature conditions., Conclusion: In trained soldiers, 10-days passive or active acclimatization in climatic chamber can prevent increase in serum concentrations of ALT and AST, induced by exertional heat stress. Increase of serum concentrations of CK and LDH was induced by physical strain itself, with no additional effect of heat stress.

The interaction between UV-B irradiation and cutaneous human papillomaviruses (HPV) has been suggested to be of relevance for the development of non-melanoma skin cancers. We investigated the activity within the upstream regulatory region (URR) of the HPV types 8, 38, 92, 93 and 96, as well as their responsiveness to UV-B irradiation and cellular differentiation. Promoter activities were higher in HaCaT than in SiHa cells, corresponding to the HPV tissue tropism. Transcriptional start sites were mapped at P(92) (HPV-38), P(45) (HPV-92), P(7439) (HPV-93) and P(256) (HPV-96). Transcription from HPV-8, 93 and 96 URR was up-regulated by cellular differentiation, linking the activity of these HPVs to the cellular state. UV-B irradiation activated HPV-8 but inhibited HPV-38 and HPV-93 whereas HPV-92 and 96 were not affected. As there are variable UV-B responses among the HPV types, further studies of interactions between UV-B and HPV need to consider the HPV type.

Recent studies have suggested an association between human papillomaviruses (HPVs), particularly species 2 members of the genus Betapapillomavirus, and squamous cell carcinoma (SCC) of the skin. As most of these viruses are uncharacterized, molecular characterization and epidemiology are needed to advance our understanding of their significance in carcinogenesis. This study determined the complete genomes of four betapapillomaviruses of species 2 from skin lesions designated HPV-107, -110 and -111 and FA75[KI88-03], an isolate of an unpublished HPV type, and analysed their prevalence and viral loads in biopsies from SCC, actinic keratosis (AK), basal cell carcinoma, seborrhoeic keratosis and the healthy skin of 263 immunocompetent patients by HPV type-specific real-time PCR assays. Seventeen patients (6.5 %) harboured at least one of the four HPV types in their lesion, whereas seven patients (2.7 %) harboured one or more of the HPV types in healthy skin. Overall, the four viruses were more common in AK than in healthy skin (odds ratio 5.0, 95 % confidence interval 1.4-17.5), but the prevalence and viral loads were low. This characterization of HPV-107, -110 and -111 and FA75[KI88-03] expands the heterogeneity of members of species 2 of the genus Betapapillomavirus. However, as these types were found in only a few samples and in low amounts, a possible role in carcinogenesis remains elusive.