Disturbances of the circadian rhythm of cortisol secretion are associated with depression, coronary calcification, and higher all-cause and cardiovascular mortality.The primary aim of this study was to test the associations between midnight salivary cortisol (MSC), depression and HbA1c, and control for behavioural, environmental and intra individual factors with possible impact on cortisol secretion, like smoking, physical inactivity, season, medication, diabetes duration, severe hypoglycemia episodes, age and gender in patients with type 1 diabetes. Secondary aims were to present MSC levels for a reference group of non-depressed type 1 diabetes patients with a healthy life style , and to explore seasonal variations.A cross-sectional population based study of 196 patients (54% men and 46% women) aged 18–59 years that participated in a randomized controlled trial targeting depression in type 1 diabetes. Depression was assessed by the Hospital Anxiety and Depression Scale-depression subscale. MSC, HbA1c, serum-lipids, blood pressure, waist circumference and data from medical records and the Swedish National Diabetes Registry were collected.Thirty four patients (17%) had MSC ≥9.3 nmol/L, which was associated with smoking (AOR 5.5), spring season (AOR 4.3), physical inactivity (AOR 3.9), self-reported depression (AOR 3.1), and older age (per year) (AOR 1.08). HbA1c >70 mmol/mol (>8.6%) (AOR 4.2) and MSC ≥9.3 nmol/L (AOR 4.4) were independently linked to self-reported depression. Season was strongly associated with MSC levels and no other variables studied showed seasonal variations. In a reference group of 137 non-depressed patients with a healthy life style  the median MSC level was 4.6 nmol/L (range 1.9–23.0).In this study of patients with type 1 diabetes high MSC was linked to smoking, physical inactivity, depression, season and older age. Thus a high cortisol value identified three major targets for treatment in type 1 diabetes. Depression is common in persons with diabetes ,2, affecThe main hypothesis of this study was that a disturbed circadian rhythm manifested by high midnight cortisol is associated with depression and with impaired glycemic control in patients with type 1 diabetes. The primary aim of this study was to test the associations between midnight salivary cortisol (MSC), depression and HbA1c, and control for behavioural, environmental and intra individual factors with possible impact on cortisol secretion like smoking, physical inactivity, season, medication, diabetes duration, severe hypoglycemia episodes, age and gender in patients with type 1 diabetes. Secondary aims were to present MSC levels for a reference group of non-depressed type 1 diabetes patients with a healthy life style , and to explore seasonal variations.This study presents cross sectional baseline data from the randomized control trial (RCT) “Psychological variables and hyperglycemia in diabetes mellitus”  which targets psychological symptoms in patients with diabetes and inadequate glycemic control in a population based cohort of patients with type 1 diabetes. A first baseline study showed that depression, obesity and smoking were independently associated with high HbA1c ,35 will To explore variables associated with high MSC we consecutively recruited 196 persons. Patients attended the only specialist diabetes outpatient clinic in a county with a population of 125,000 in South Sweden during 2009 . Inclusi= 0.33), mean HbA1c (p = 0.34), abdominal obesity (p = 0.38), physical inactivity (p = 0.68), clinical psychiatric diagnosis (p = 0.71), hypertension (p = 0.80), hyperlipidemia (p = 0.80), diabetes duration (p = 0.89), or self-reported depression (p >0.99).There were 62 patients who chose not to deliver MSC samples and 23 who failed to deliver proper samples. These 85 patients did not differ from the 196 included patients regarding smoking (p = 0.13), age (p = 0.15), gender (p = 0.30), use of antidepressants  and blood pressure were measured.Data were also collected from the Swedish National Diabetes Register (S-NDR), and from computerized medical records from the Departments of Internal Medicine, Ophthalmology, and Psychiatry (only drug prescription data), and from Primary Care clinics.The study was approved by the Regional Ethical Review Board of Linköping University . All patients provided written informed consent.Each patient collected one MSC sample between 23.30 and 00.30 hours, using the Salivette sampling method  ,22,29-33th percentile was 8.9 nmol/L [In a healthy population without diabetes, late night cortisol ranged from 1.4 to 16.7 nmol/L, and the 959 nmol/L . Mean MSMSC samples were collected between 29/03/2009 and 18/01/2010, which was divided into three periods. The first period 29/03/2009 until 31/05/2009 was defined as spring; the second period 01/06/2009 until 31/08/2009 was defined as summer; and the third period 09/01/2009 until 18/01/2010 was defined as autumn/winter.Self-reported depression was assessed by Hospital Anxiety and Depression Scale-depression subscale (HADS-D) consisting of 7 statements with 4 response alternatives from 0 to 3, using the recommended ≥8 points as cut off level ,34,36. PClinical psychiatric diagnoses were established clinically prior to recruitment, were dichotomized as having or not having a psychiatric diagnosis, and used mainly for validation of the HADS-D.Smokers were defined as patients having smoked any amount of tobacco during the last year.Physical inactivity was defined as moderate activities, such as 30 minutes of walking, less than once a week.3), which was defined as high HbA1c  [Venous HbA1c was analysed with high pressure liquid chromatography, HPLC - variant II, Turbo analyzer  . HbA1c wmol/mol) .Serum-lipids were analysed with the enzymatic colour test . Hyperlipidemia was defined as S-Cholesterol >4.5 mmol/L and/or S-Low density lipoprotein cholesterol >2.5 mmol/L ; or use of lipid lowering drugs independent of lipid blood levels. HbA1c and lipids were analysed at the department of Clinical Chemistry, Växjö Central Hospital.Blood pressure was measured in the sitting position. Hypertension was defined as systolic blood pressure >130 mm Hg and/or diastolic blood pressure >80 mm Hg ; or use of antihypertensive drugs independent of blood pressure levels.WC was measured between the lowest rib margin and iliac crest by a nurse. Abdominal obesity was defined as WC ≥1.02 m. for men, and as WC ≥0.88 m. for women .A severe hypoglycemic episode was defined as needing help from another person due to hypoglycemia, and episodes occurring during the last 6 months were registered .t-test was used for analyses of mean differences. Non-parametric distribution was presented as median values (quartile (q)1, q3; range), and analyses were performed with Kruskal-Wallis test or Mann-Whitney U test. MSC was dichotomized at 9.3 nmol/L [P ≤0.05 was considered statistically significant.SPSS® version 18  was used for statistical analyses. Fisher’s exact test (two-tailed) was used to analyse differences of prevalence. Continuous variables, normally distributed, were presented as mean ± SD, and Student’s 3 nmol/L . Crude oIn this study of 196 patients with type 1 diabetes, 54% men and 46% women with mean age 41.3 (range 18–59) years and mean diabetes duration 21.1 (range 1–55) years, we analyzed variables associated with high MSC levels and self-reported depression. Baseline characteristics and gender differences are presented in Table 1, q3, range: 3.1, 7.5; 1.9–47.0) nmol/L  and the prevalence rates of high MSC ≥9.3 nmol/L) (p = 0.013) were highest in the spring samples and lowest in the autumn/winter samples (Table  nmol/L (Thirty four patients (17%) had MSC ≥9.3 nmol/L, which was associated with smoking (AOR 5.5), spring AOR 4.3), physical inactivity (AOR 3.9), self-reported depression (AOR 3.1), and older age (per year) (AOR 1.08)  nmol/L was 4.6  for 137 (55% men) non-depressed, non-smoking and physically active patients with median (range) age 43 (20–59) years. In these 137 patients MSC ≥9.3 nmol/L was associated with season, spring (AOR 7.9 (1.6–37.8), p = 0.010), summer (AOR 1.9 (0.2–14.5), p = 0.54), autumn/winter (AOR 1) (reference); but not with age (per year) (1.05 (0.99–1.11), p = 0.084), or gender (p = 0.59). In spring median MSC  nmol/L was (n = 50) 6.6 , in summer (n = 36) 3.5 ; and in autumn/winter (n = 51) 3.5 , p <0.001. Median age (range) years in spring was 46 (20–59); in summer 39 (20–59); in autumn/winter 44 (22–59), p = 0.022. Median age did not differ between patients recruited in spring and autumn/winter (p = 0.67).Median MSC (qMSC ≥9.3 nmol/L (AOR 4.4), HbA1c >70 mmol/L (>8.6%) (AOR 4.2) and antidepressants (AOR 4.9) were independently associated with self-reported depression Table .The associations (COR (CI), p) were significant between self-reported depression and “clinical psychiatric diagnosis and use of antidepressants” (9.0 (2.5–32.1), 0.001 (n = 13)), and between self-reported depression and “clinical psychiatric diagnosis without use of antidepressants” (5.7 (1.5–21.3), 0.009 (n = 14)), with “no clinical psychiatric diagnosis/antidepressants” as reference (n = 169).In this population based study of 196 patients with type 1 diabetes, smoking, physical inactivity, depression and age, were associated with high MSC (≥9.3 nmol/L), whereas HbA1c was not. High MSC and high HbA1c (>70 mmol/L (>8.6%)) were independently associated with depression. A seasonal variation was found with the highest prevalence of high MSC levels in spring and the lowest in autumn/winter. The main links between these variables are illustrated in Figure Strengths of our study are first that we systematically investigated factors that could confound our results such as use of antidepressants or inhaled steroids, and seasonal changes in cortisol secretion. Secondly, we determined factors not associated with high MSC, i.e. HbA1c, antidepressants and diabetes duration. Third, the population of patients with type 1 diabetes was large and well defined. Pregnant women, patients with severe somatic or psychiatric disorders including substance abuse, and patients using systemic corticosteroid treatment, were excluded, all factors that knowingly affect cortisol levels. Fourth, we thoroughly examined and found that the eligible 85 patients who did not deliver salivary cortisol samples did not differ from the 196 included patients. This suggests that our results could be generalized to a larger population of patients with type 1 diabetes.Limitations to our study are first that self-reported depression was not confirmed by a diagnostic interview. Yet, clinical psychiatric diagnoses, both for those using and not using antidepressants, were clearly associated with self-reported depression. Secondly, HbA1c and MSC were only measured once, but a demand for repeated measurements would probably have resulted in a lower participation rate due to the inconvenience with both venous and midnight sampling. Third, there was no data from the middle of January until the end of March which makes it impossible to exclude seasonality in depressive symptoms, though we did not find any. Fourth, to confirm the seasonality of MSC levels there is a need for repeated measurements throughout the year.A normal circadian rhythm of cortisol is characterized by maximum levels in the morning and minimum levels at midnight . In thisWe found the highest midnight cortisol levels in spring and the lowest in autumn/winter which is a new finding. First, we have not found any previous study where seasonal variations of midnight salivary cortisol were analysed; measurements have been performed during daytime and late evening. Second, according to a review of circannual hormonal changes, basal levels of circulating glucocorticoids seem to be lower during the spring and summer and peak during the autumn and winter . HoweverHypercortisolemia is a known cause of hyperglycemia , but we The absence of associations between depression and gender, physical inactivity and smoking differ from previous research ,23,24. FWe chose a 30 minutes (60 minutes for brushing the teeth) restriction period of eating etc. before MSC sampling. A variety of restriction periods before salivary cortisol sampling are found in the literature: 15 minutes ,22, 30 mThe ECLIA method used to analyze MSC in our study is well validated -32, but Salivary cortisol will probably be used more in future clinical practice and research as it can be sampled at home, is noninvasive, painless and stress free ,32. HighHigh levels of MSC linked to depression, smoking and physical inactivity highlights three main targets in diabetes care, as a disturbance of the circadian rhythm of cortisol is associated with coronary calcification, all-cause and cardiovascular mortality ,14.The additional link between depression and high HbA1c emphasizes the severity of depression in patients with type 1 diabetes. Routine systematic depression evaluation at diabetes control visits is suggested. A high cortisol level may help to emphasize to medical professionals and patients alike the necessity of taking action against depression, smoking and physical inactivity.1: The first quartile; q3: The third quartile; RCT: Randomized controlled trial; SAD: Seasonal affective disorder; S-NDR: Swedish national diabetes registry; WC: Waist circumference.AOR: Adjusted odds ratio; CI: Confidence interval; COR: Crude odds ratio; DCCT: Diabetes control and complication trial; ECLIA: Electrochemiluminescence immunoassay; HADS-D: Hospital anxiety and depression scale-depression subscale; HPA axis: Hypothalamic-pituitary-adrenal axis; IFCC: International federation of clinical chemistry; MSC: Midnight salivary cortisol; qThe authors declare that they have no conflicts of interest that could be perceived as prejudicing the impartiality of the research reported.EOM, MT, MH, ML-O and HOT participated as investigators and reviewed and edited the manuscript. EOM, ML-O, MT and MH contributed to the study design and implementation. EOM, HOT and ML-O contributed to the analysis and wrote the statistical methods. EOM wrote the manuscript, and is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6823/14/75/prepub


Amaranthus hybridus, Amaranthus sp., Cucurbita maxima, Ipomoea batatas, Solanum villosum, Solanum scabrum, and Vigna unguiculata were explored for their capabilities to accumulate selenium when grown on selenium enriched soil and for use as a potential source of selenium enriched functional foods. Their selenium contents were determined by spectrophotometry using the complex of 3,3′-diaminobenzidine hydrochloride (DABH) as a chromogen. The mean concentrations in the leaves were found to range from 7.90 ± 0.40 to 1.95 ± 0.12 μg/g dry weight (DW), with C. maxima accumulating the most selenium. In stems, the accumulated selenium content ranged from 1.12 ± 0.10 μg/g in Amaranthus sp. to 5.35 ± 0.78 μg/g DW in C. maxima and was hence significantly different (P < 0.01). The cancer cell line MDA-MB-231 was used in cytotoxicity assays to determine the anticancer potential of these extracts. With exception of S. scabrum and S. villosum, no cytotoxicity was detected for the selenium enriched vegetable extracts up to 100 μg/mL concentration. Hence, following careful evaluation the studied vegetables may be considered as selenium enriched functional foods.Selenium deficiency in humans has been associated with various diseases, the risks of which can be reduced through dietary supplementation. Selenium accumulating plants may provide a beneficial nutrient for avoiding such illnesses. Thus, leafy vegetables such as Selenium is a semimetallic, biologically active trace element that forms bonds with carbon whose strength is in the range of common covalent bonds. Commonly occurring in selenoamino acids and selenoproteins , 2, seleμg/day) and toxicity (greater than 400 μg/day) [μg Se/day intake is commonly accepted as the reference labelling value (RLV) [Whereas selenium is essential as trace element, it can cause acute or chronic toxicity at higher serum concentrations –17. Its  μg/day) , requiri μg/day) , 19. The μg/day) . These rue (RLV) . Allium sativum (garlic) [ A. cepa (onions) [ A. fistulosum (green onion) [ Brassica juncea (mustard) [ Brassica oleracea (wild cabbage and related cultivars) [ Astragalus bisulcatus (milk-vetch) [ A. racemosus  [ Cucurbita pepo (summer squash) [ Stanleya pinnata (desert princesplume) [ Amaranthus hybridus , Amaranthus sp. (unidentified), Cucurbita maxima (pumpkin), Ipomoea batatas , Solanum scabrum , S. villosum , and Vigna unguiculata (cowpeas)  , A. cepa(onions) , A. fistn onion) , Brassicmustard) , 30, Braltivars) , Astragak-vetch) , 33, A.  squash) , 35, andesplume) , 30. In cowpeas)  were inv2SeO4 solution was applied to each 100 m2 portion of the nursery. During the growth period, watering during the morning and evening every day and regular weeding were carried out.The selected leafy vegetable species were grown on selenium enriched soil (except for control purpose) at the nursery site of the Botany Department, University of Dar es Salaam, following standard procedures . To enri Amaranthus spp., V. unguiculata, and C. maxima while S. villosum, S. scabrum, and I. batatas were harvested at the age of six weeks based on differences in growth rate. The late harvest was done three weeks after the early harvest of each vegetable species. Samples were rinsed with tap water, followed by distilled water, and were then separated into stems and leaves. The samples were chopped into pieces, packed in separate plastic bags and transferred to the laboratory for oven drying to constant weight at a temperature between 50°C and 60°C, and ground to powder following an established procedure [ Amaranthus spp., seeds were also harvested. The seeds were first left to mature and dry and then harvested and further air-dried before being ground to powder for homogenization. Powdered seeds, leaves, and stem samples were stored in a freezer (at −4°C) prior to analysis.Vegetable samples were harvested at early and late stages of their growth. Early sampling was done at the age of three weeks after seed germination forrocedure . For AmaSoil samples were collected in three stages that involved random sampling using shovel. The first collection was done immediately after nursery preparation before sowing seeds and application of selenium. This was necessary in order to determine any remaining traces of selenium left in the soil as 10 g Se/ha was applied to the area three years earlier before this study. Following application of selenium, on the next day, random sampling to collect a second set of soil samples was done. The soil samples were then air-dried, packed in plastic bags, and stored in the freezer (at −4°C) until the analysis. After all vegetable samples and seeds were harvested; the third set of soil sample was collected to determine the amount of selenium left in the soil.3; 70%), hydrogen peroxide , toluene, concentrated ammonia (NH3) solution, 0.5 mol L−1 sulphuric acid, and 3,3′-diaminobenzidine hydrochloride (DABH) solution that was freshly prepared by dissolving 0.125 g 3,3′-DABH in 25 mL acetone and then stored in amber-coloured bottle. A solution of sodium selenate (Na2SeO4) was used both for soil application and as a standard. Deionized water was used for preparing reagent solutions and samples throughout.Analytical grade reagents and HPLC grade solvents were used as received from commercial suppliers, without further purification: nitric acid  was applied. A Shimadzu-240 UV spectrophotometer equipped with 1 cm high quartz and silica cuvettes was used for reading the absorbance of the Se-3,3′-DABH complex (piazselenol). All laboratory glassware was kept overnight in 10% nitric acid solution to remove contaminants and was rinsed with deionized water and dried in a dust-free oven ahead of use. Whatman cellulose filter papers were used for filtration of the digested samples in order to remove the remained silica.μg/mL stock solution was prepared by dissolving 0.219 g Na2SeO4 in 100 mL deionized water; appropriate amounts were diluted with deionized water (pH adjusted to 1.8 ± 0.1 using 0.5 M H2SO4) to 0.1 μg/mL, 0.2 μg/mL, 0.3 μg/mL, 0.5 μg/mL, 0.7 μg/mL, 0.9 μg/mL, and 1.1 μg/mL concentrations, which were used for construction of a calibration curve. The absorbance of selenium plotted against concentration gave a linear correlation, which was then used to determine the unknown selenium concentration of the samples.For determination of the selenium content of extracts the method established by Katamto and Al-Zehouri was adopted . A 920 μ3) and 4 mL hydrogen peroxide (30% H2O2) in 100 mL conical flasks. The mixture was subjected to heat at 70 ± 5°C for 35 minutes using a microwave oven. The solution was filtered and diluted to 100 mL with deionized water. The selenium concentration was determined by spectrophotometric analysis using 3,3′-diaminobenzidine hydrochloride (DABH) as chromogen [3). The colored complex was extracted with 10 mL toluene and its absorbance was measured at 420 nm. Standard deviation (SD) was calculated for triplicate measurements and the concentration was calculated using the regression equation of the concentration versus UV absorbance calibration (vide supra).A 1 g portion of each of the powdered dry leaves, stems, seed, and soil sample was digested with 12 mL concentrated nitric acid  to ensure exponential growth for the duration of the assay. After a 24 h preincubation growth, the medium was replaced with experimental medium containing the appropriate extract concentrations (100–0.1 μg/mL) or vehicle controls (0.1% or 1.0% v/v DMSO). After 72 h of incubation, cell viability was measured using Alamar Blue reagent  according to the manufacturer's instructions. Absorbance was measured at 570 nm with 600 nm as a reference wavelength. Results were expressed as mean ± standard error for six replicates as a percentage of vehicle control (taken as 100%). Extract that showed <80% viability was considered as a positive hit for cytotoxicity. Such extracts were further subjected to new cell library testing under serial dilutions to obtain LD50. Experiments were performed independently at least six times followed by statistical analyses.Cytotoxic activity of the aqueous vegetable extracts was evaluated against the MDA-MB-231 human cancer cell line as previously reported by Irungu et al. . MDA-MB-t-test whereby P < 0.05 was considered to be statistically significant.Comparison of the concentrations of Se accumulated in different leafy vegetable species was achieved using Kruskal-Wallis Analysis of Variance (ANOVA) and Dunn's Multiple Comparison test. Statistical analyses for cytotoxicity assay were performed using a two-tailed Student's 2SeO4 solution  of 0.9895, as summarized in The selenium concentration of the vegetable extracts was determined by UV spectrophotometric analyses with detection of the chromogen 3,3′-diaminobenzidine hydrochloride- (DABH-) selenium complex at = 420 nm . For val C. maxima, 7.90 ± 0.40 μg/g and 6.49 ± 0.26 μg/g dry weight (DW) for late and early harvest, respectively, while lowest concentration was found in Amaranthus sp., 2.21 ± 0.09 μg/g and 1.95 ± 0.12 μg/g DW, for late and early harvest, respectively.All seven investigated leafy vegetable species were found to accumulate selenium; that is, they possessed higher concentrations than the controls , albeit  Amaranthus and Solanum, are usually consumed together with their leaves. Determination of the selenium content of the stems indicated selenium accumulation, with a similar trend in between the species to that observed for the leaves, yet in lesser extent ( Amaranthus sp. accumulated the lowest amount of selenium (1.12 ± 0.10 μg/g DW) whereas V. unguiculata the highest (3.39 ± 0.41 μg/g DW); among the samples harvested at a late time point, the lowest concentration was observed for Amaranthus sp. (1.83 ± 0.05 μg/g DW) and the highest for C. maxima (5.35 ± 0.78 μg/g DW)  .Variance analyses indicated that the increased selenium concentration of the vegetables that were grown on selenium enriched soil was statistically significant, when compared to the control samples. The differences between late and early harvests of leaves and stems were, however, not significant . Thus, thttp://dx.doi.org/10.1155/2015/549676) indicated that the difference in selenium uptake for leaves was significant only in between C. maxima and Amaranthus sp. (P < 0.01) but not the other plants. For the stem samples, the difference in selenium uptake was statistically significant between V. unguiculata and Amaranthus sp. for early harvested samples (P < 0.05) and between C. maxima and Amaranthus sp. (P < 0.01) for late harvested stems. Although the differences of the selenium uptake of various plants were not statistically significant, they may still have a biological impact considering the amount of plants consumed by a human.Statistical analyses  indicated that the difference in selenium uptake of leaves was significant only in between C. maxima and Amaranthus sp (p<0.01), but not the other plants. For the stem samples, the difference in selenium uptake was statistically significant between V. unguiculata and Amaranthus sp for early harvested samples (p<0.05) and between C. maxima and Amaranthus sp (p<0.01) for late harvested stems.


Staphylococcus aureus is a notorious pathogen with a propensity to cause chronic, non-healing wounds. Bacterial persisters have been implicated in the recalcitrance of S. aureus infections, and this motivated us to examine the persistence of S. aureus to ciprofloxacin, a quinolone antibiotic. Upon treatment of exponential phase S. aureus with ciprofloxacin, we observed that survival was a non-monotonic function of ciprofloxacin concentration. Maximal killing occurred at 1 µg/mL ciprofloxacin, which corresponded to survival that was up to ~40-fold lower than that obtained with concentrations ≥ 5 µg/mL. Investigation of this phenomenon revealed that the non-monotonic response was associated with prophage induction, which facilitated killing of S. aureus persisters. Elimination of prophage induction with tetracycline was found to prevent cell lysis and persister killing. We anticipate that these findings may be useful for the design of quinolone treatments. Staphylococcus aureus is a dangerous, Gram-positive pathogen that is frequently associated with nosocomial infections [S. aureus infections can be difficult to cure because many subspecies are resistant to one or more antibiotics , multidrug-resistant (MDR) S. aureus) [S. aureus contain high levels of persisters [S. aureus has several [fections ,2,3. S.  aureus)  and bothrsisters ,6,7,8,9.rsisters ,11,12,13rsisters . Such do several ,17,18,19 several ,21,22.S. aureus NCTC 8325 to ciprofloxacin. Interestingly, we observed that survival of S. aureus was at a minimum at 1 µg/mL ciprofloxacin and increasing the concentration further led to increased survival. At ≥ 5 µg/mL ciprofloxacin, biphasic killing kinetics were observed, and the abundance of survivors reached a plateau with regard to ciprofloxacin concentration, confirming that those cells were persisters. On the basis of previous studies that investigated the impact of quinolones on transcription, translation, and prophage induction [S. aureus infections.Here, we investigated the persistence of nduction ,25,26,27S. aureus to ciprofloxacin, we measured survival of cultures treated with different concentrations as a function of time . Inspection of the survival curves for 5, 10, and 50 µg/mL depicted classic biphasic killing kinetics  to measure cell lysis, we observed that lysis occurred with ciprofloxacin concentrations of 0.25, 0.5, and 1 µg/mL as indicated by significant reductions in culture density after 2 h of treatment  , corresponded to the treatments that exhibited lysis, whereas bacteriophage release from the 5 and 10 µg/mL treatments were quantitatively similar and not significantly different from the untreated control  a. Altern control b.Collectively, these data suggested that prophage induction and cell lysis were responsible for the reduction in survival at 1 µg/mL ciprofloxacin compared to the higher concentrations tested. Further, these data provide support for a mechanism where the non-monotonic survival curve is the result of persister killing that depends on prophage induction.p-values < 0.05)  b. FurtheIn addition, we performed experiments in which tetracycline was added 1 h after treatment with ciprofloxacin , which aS. aureus [recA-TIR-gfpmut2) at both 1 and 50 µg/mL ciprofloxacin; PrecA is part of the S. aureus SOS response that is induced by ciprofloxacin [recA reporter was highest under treatment with 1 µg/mL ciprofloxacin and much reduced in cultures treated with 50 µg/mL , Pearl and colleagues found that the physiology of persisters to ampicillin protected them from endogenous prophage induction but did not render them immune to cell death caused by super-infection [Whether prophage that were endogenous to persisters were responsible for the killing or whether it was associated with super-infection of persisters with bacteriophage released from dead cells was not determined here. In a previous study, using a high persistent mutant of nfection . The extnfection ,41) and nfection ,43,44) cStrains used in this study are listed in E. coli SA08B prepared using the Mix and Go E. coli Transformation Kit  and transformants were plated immediately on LB agar supplemented with 100 µg/mL ampicillin and incubated overnight at 37 °C. Successful ligation was confirmed by colony PCR and DNA sequencing . S. aureus genomic DNA used as the PCR template was extracted using a DNeasy Blood and Tissue Kit  following the manufacturer’s instructions. As S. aureus is a Gram-positive organism, the lysis step was modified such that the cells were incubated in 500 µg/mL lysostaphin, 20 mM Tris-HCl, 2 mM EDTA (pH 8.0), and 1.2% Triton X-100 at 37 °C for 1 h; Proteinase K was added and the sample was incubated at 56 °C for an additional 2 h. Plasmids were extracted using a QIAprep Spin MiniPrep Kit (Qiagen).Generalized cloning procedures are as follows: DNA inserts were PCR amplified from plasmid or chromosomal DNA with Phusion DNA Polymerase . Inserts and vectors were digested with restriction enzymes (New England Biolabs) according to the manufacturer’s protocols, and the vectors were treated with Antarctic Phosphatase (New England Biolabs) to circumvent self-ligation before the inserts and vectors were ligated together using Quick Ligase (New England Biolabs). Ligation products were transformed into chemically competent E. coli-staphylococcal shuttle vector with a cadmium-inducible cassette Pcad-cadC [recA reporter plasmid, pES10. The primers used for constructing pES10 are listed in gfpmut2, derived from pUA66 [cad-cadC (generating pES1); primers used to amplify gfpmut2 were designed so that a gene10 ribosomal binding site (RBS) was inserted upstream of gfpmut2. In order to generate a versatile backbone, we then synthesized a multiple cloning site by the extension of two overlapping oligonucleotides using the Klenow fragment , and we replaced Pcad-cadC of pES1 with this MCS (using the SphI and KpnI restriction sites) (generating pES4). In work unrelated to the data presented above, replacement of the cadmium cassette of pES1 with different promoters failed to generate a high fluorescence signal (data not shown). Low fluorescence of staphylococcal reporters has been reported to be highly sensitive to the RBS region when compared within the same backbone, with the RBS regions ranked as gene10 < sarA < sod < hld with respect to fluorescence output [E. coli [sod RBS region in the staphylococcal pCN vectors [gene10 RBS region was replaced with the TIR sequence (TIR: 5′-TGATTAACTTTATAAGGAGGAAAAACATATG-3′), generating pES5. Finally, PrecA, consisting of 505 bp upstream of the start codon of recA, was amplified from S. aureus genomic DNA and ligated at SbfI and KpnI sites of pES5 to generate pES10.pCN51 is a high copy cad-cadC , and it om pUA66 , into pCe output . In a la[E. coli  was founE. coli SA08B , which was designed with a methylation pattern to allow direct transfer of DNA from E. coli to wild-type S. aureus strains. However, we were unsuccessful in directly transforming intact plasmids from E. coli SA08B to S. aureus NCTC 8325, and therefore, we used S. aureus RN4220 as a cloning intermediate. Sequence-verified plasmids from E. coli SA08B were transformed into electrocompetent S. aureus RN4220 cells following electroporation protocols described previously [S. aureus 80α lysogen RN10395 was used to facilitate plasmid transfer from RN4220 to NCTC 8325, as phage 80α is a helper phage that enhances competence for transformation of the strain bearing it [2 and 25 µL of the infective lysate and incubated for 15–20 min at room temperature. Then, 100 µL of 1 M sodium citrate was added, and the culture was incubated for 0.5–1 h at 37 °C. The culture was added to 3 mL of melted top agar, mixed gently, and poured immediately on a phage agar plate containing 10 µg/mL ERM. When solidified, plates were incubated at 37 °C overnight. Single colonies were inoculated into 3 mL LB containing 10 µg/mL ERM, grown overnight, mixed with equal volumes of LB-glycerol and stored as a −80 °C stock. Function of the plasmids in each clone was confirmed by fluorescence enhancement following CIP treatment compared with strains harboring the promoterless control vector, pES5.Vectors were maintained in the cloning strain eviously , and traaring it . Transduaring it ,45. In oS. aureus overnight culture grown in LB at 37 °C was diluted to ~105 CFU/mL and added to the antibiotic dilution series at a 1:1 ratio. The 96-well plate was then incubated statically at 37 °C overnight, and the MIC was defined as the lowest antibiotic concentration inhibiting visible growth. Interestingly, in a number of these plates, biofilm formation was observed in the well just below the MIC. All lower concentrations had turbid wells with no biofilm colonies when the liquid was removed; however, just below the MIC, the bulk fluid lacked turbidity, but defined biofilm colonies were visible on the bottom of the well. When observed, this was still considered growth, and the MIC was classified as one concentration above that well.Quantifications of MICs were conducted in LB in 96-well plates using a procedure described previously . BrieflyS. aureus NCTC 8325 from a −80 °C stock were used to inoculate 3 mL of LB, and the culture was grown for 16 h at 37 °C and 250 rpm. Overnight cultures were diluted to an OD600 = 0.01 in 50 mL of LB in 500 mL baffled flasks and grown at 37 °C and 250 rpm. At OD600 ~0.4–0.6, 10 mL aliquots were transferred to 250 mL baffled flasks. When more than 50 mL of culture was required, identical 50 mL cultures were pooled prior to distribution of 10 mL aliquots. Where applicable, tetracycline was added to cultures at a final concentration of 10 µg/mL immediately prior to or 1 h following treatment with ciprofloxacin. Ciprofloxacin was added to cultures at final concentrations ranging from 0–50 µg/mL. At designated time points, samples were removed for persister enumeration and OD600 measurements. For cell counts, samples were removed (200 µL to 1 mL depending on the time point), and the volume was brought up to 1 mL with phosphate-buffered saline (PBS) as needed. To remove the antibiotic, cells were collected by centrifugation , 900 µL of the supernatant was removed, 900 µL of PBS was added, the cell pellet was resuspended, and these steps were repeated a second time. Samples were then serially diluted in PBS, and 10 µL spots were plated on LB agar. To improve the limit of detection, 100 µL spots were plated in some cases. Plates were incubated at 37 °C for ~20 h, and persisters were enumerated by counting CFUs. The limit of detection (1 colony in the zero dilution of a 100 µL spot) was 1.7 log10(CFU/mL). To measure cell density, samples were removed from flasks, and OD600 was measured with a Synergy H1 Hybrid Multi-Mode microplate reader .S. aureus NCTC 8325 cultures were filtered with 0.22 µm syringe filters, and the effluent were stored at −20 °C until subsequent analysis. Since different concentrations of ciprofloxacin in the different sample lysates could have impacted PFU measurements, all undiluted lysates were adjusted to contain the same ciprofloxacin concentration, which was 10 µg/mL. Control experiments with samples whose ciprofloxacin concentrations were not adjusted in this manner demonstrated that this step did not alter PFU measurements , after which phage plaques were enumerated.Phage buffer, top agar, and bottom agar were prepared as previously described . To obtaurements . 10-foldS. aureus NCTC 8325 containing pES10 with the addition of 10 µg/mL ERM for plasmid retention. At designated time points, samples were removed, the cells were collected by centrifugation , the media was removed, and the remaining cell pellet was resuspended in 320 µL of PBS. The OD600 of each sample was adjusted to 0.2 in PBS in a total volume of 300μL and GFP fluorescence was measured in black, clear bottom 96-well plates using 485/515 nm excitation/emission on a Synergy H1 Hybrid Multi-Mode microplate reader (BioTek). Fluorescence values of equivalent volumes of PBS were used as blanks.The same protocol used for the antibiotic treatment assays described above was used for fluorescence assays of t-tests with unequal variances and the null hypothesis that the means of the two samples were equivalent. p-Values less than 0.05 were considered significant.The statistical significances reported were determined using two-tailed Student’s S. aureus strains harbor prophage [S. aureus infections. At ciprofloxacin concentrations around the OBC, survival of S. aureus is severely compromised (S. aureus but significantly limit induction of prophage.Since the majority of prophage  and non-prophage ,31, we ppromised , but bacpromised . Given t


The experimental design consisted of the factorial combination of treatment  at three doses  and two time-course applications (at the second and fourth week after transplantation) and the effects were recorded at flowering and maturity. Both biostimulants contained different amounts of indoleacetic acid and isopentenyladenosine; the AH spectra exhibited amino acid functional groups in the peptidic structure, while the RG spectra showed the presence of polyphenols, such as resveratrol. These results revealed that at flowering, RG and AH increased the weights of fresh leaves and fruits and the number of green fruits, whereas at maturity, the biostimulants most affected the fresh weight and number of red fruits. At flowering, the leaves of the treated plants contained high amounts of epicatechin, ascorbic acid, quercetin, and dihydrocapsaicin. At maturity, the leaves of the treated plants exhibited elevated amounts of fructose, glucose, chlorogenic, and ferulic acids. Moreover, green fruits exhibited a high content of chlorogenic acid, p-hydroxybenzoic acid, p-coumaric acid and antioxidant activity, while both AH- and RG-treated red fruits were highly endowed in capsaicin. The 1H high-resolution magic-angle spinning (HRMAS)-nuclear magnetic resonance (NMR) spectra of red fruits revealed that both products induced a high amount of NADP+, whereas RG also increased glucose, fumarate, ascorbate, thymidine and high molecular weight species. Our results suggested that AH and RG promoted plant growth and the production of secondary metabolites, such as phenols.Two biostimulants, one derived from alfalfa plants (AH) and the other obtained from red grape (RG), were chemically characterized using enzyme linked immuno-sorbent assays, Fourier transform infrared (FT-IR) and Raman spectroscopies. Two doses (50 and 100 mL L Substances and materials, with the exception of nutrients and pesticides, which, when applied to plants, seeds or growing substrates in specific formulations, have the capacity to modify physiological processes of plants in a way that provides potential benefits to growth, development and/or stress response” . Pepper was selected because it is a representative species of the Mediterranean region and it has a short developmental period. In addition, it also has a long history as a source of healthy and biologically active compounds, including flavonoids, phenols, carotenoids, capsaicinoids, vitamins, and anticancer properties  plants , and the second was obtained by cool extraction  were used. One was produced by fully controlled enzymatic hydrolysis using alfalfa .Infrared spectral acquisition was performed on solid samples using a Nicolet 5700 FT-IR equipped with a diamond attenuated total reflectance (ATR) accessory and a DTGS  detector. The total number of scans averaged for each spectrum was 64 with a resolution of 4 cmRaman spectra of samples were registered in solid state with a Renishaw Raman RM2000 instrument, which was equipped with an electrically cooled CCD camera and an excitation line using a He/Ne laser at 632.8 nm. Each spectrum was registered using a 10 s measurement time.Capsicum chinense L. cv. Fuoco della Prateria) were homogenously selected per growth and one seedling was transplanted per pot. The plants were grown until maturity in a tunnel maintained at 25/15°C day/night, receiving natural light. Treatment was applied by spraying the diluted products on the surface of the leaves at two dosages: 50 and 100 mL L−1 for RG, and 25 and 50 mL L−1 for AH by spraying each one with 4.5 mL of RG or AH on the leaves. The plants were treated at the second and fourth week after transplantation, and two collection periods were used: at flowering (time 1) and maturity (time 2) . All treatments were irrigated with half-strength Hoagland's nutrient solution , a cool extract from red grape (RG), and an alfalfa hydrolyzed (AH), with two application doses, which were supplied at two randomized blocks of pots, consisting of ten replications for a total of 100 pots. The pots were filled with 2 L perlite/vermiculite (1/1 w/w) mixture per pot. At the start of the trial, 7 day-old pepper seedlings  were determined according to Arnaldos et al.  with galAscorbic acid was extracted from five grams of tissue, homogenized until uniform consistency in a meta-phosphoric acid and acetic acid solution. The concentration of ascorbic acid was determined according to the ISO 6557 method.2O (50:50) at a flow rate of 1.0 mL min−1 at 25°C; the detection was performed at 278 nm; the separation was obtained in isocratic elution for 10 min and then a linear gradient of 50–90% methanol (Sigma) for 10 min. The capsaicin and dihydrocapsaicin concentration in the extracts was obtained using a stock solution of standard capsaicin (2 mg L−1)  and dihydrocapsaicin (2 mg L−1) (Fluka) in methanol (Sigma), transferred into a vial and kept cool at 4°C prior to use.To determine the levels of capsaicin and dihydrocapsaicin, two grams of tissue was extracted by treatment with 20 mL of acetone and homogenization with an Ultra-Turrax T 25  for 30 s at 17,500 rpm. The extracts were filtered through a Whatman No. 42 filter paper followed by a 0.45-μm nylon membrane prior to high-performance liquid chromatography (HPLC) analysis. A total of 10 extracts were analyzed. The HPLC system consisted of a model  equipped with a DAD detector  and autosampler . A ODS-2  column was eluted with methanol/H−1 dry weight.β-Carotene and lycopene were extracted and quantified using ultraviolet-visible (UV Vis) spectrophotometric assays as previously described by Rodríguez-Amaya . These r2O phosphate buffer (pH 7.2) with 0.5% TSP, i.e., 3-(trimethylsilyl)-propionic- 2,2,3,3-d4 acid sodium salt, were then added. The HRMAS-NMR spectra were recorded at 298 K using a Bruker AVANCE spectrometer operating at a 1H frequency of 400.13 MHz, equipped with a 4 mm HRMAS dual channel probe head and spinning samples at 7 kHz. 1H NMR spectra were referenced to the methyl group signal at δ 0.00 ppm of TSP, while the 13CNMR spectra were referenced to the TSP δ 0.00 ppm. The 1H-HRMAS-NMR spectra were acquired using a water suppression pulse sequence, noesypr1D (Bruker library), using 32 K data points over a 4807 Hz spectral width and addition of 256 transients. A recycle delay of 3 s and a delay for the efficient NOE effect equal to 150 ms were used. The 90° pulse length was 5.3 μs, and saturation of the water residual signal was achieved by irradiating during recycle delay at δ equal to 4.70 ppm. Each spectrum was FT transformed with 64 K data points and manually phased and base-lined, and a line broadening factor equal to 0.3 Hz was applied to the FID prior FT. 13C-HRMAS-NMR spectra were acquired using the power-gated decoupling sequence, zgpg30 (Bruker library), with a 30° flip angle pulse of 5.0 μs. The experiments were performed using 64 K data points over a 22,123 Hz (220 ppm) spectral width by the addition of 64 K transients with a recycle delay of 3 s. Each spectrum was FT transformed with 128 K data points and manually phased and base-lined, and a line broadening factor of 0.5 Hz was applied to the FID. The 1H-1H TOCSY experiment was acquired in the TPPI phase sensitive mode, with a 4807 Hz spectral width in both dimensions, 100 ms of spin-lock time of 4500 Hz, 2 K data points in f2, and 1 K increments in f1, each with 32 scans. The 1H-13C HSQC spectra were acquired in the TPPI phase-sensitive mode, with a 4807 Hz spectral width in f2 dimension and a 15,083 Hz spectral width in f1. 2 K data points in f2 and 1 K increments in f1, each with 32 scans, were used. All of the 1H NMR spectra were baseline corrected, and aligned using XWINNMR 3.5 software . Each spectrum was divided into intervals equal to 0.06 ppm (buckets) in a range from 0.06 to 9.00 ppm, with the exclusion of the water region from 4.74 to 4.86 ppm, using AMIX 3.5 software . All integrated buckets were scaled by using the ACD bucketing method within ACDlab 8.0 software to a signal intensity of the peak at 3.81 ppm such that the NMR spectra were bucketed in 149 variables. The area under each bin was integrated and normalized with respect to the sum of all integrals, which was set equal to 100. The NMR data were analyzed using the means of the partial least squares projections to latent structures–discriminant analysis (PLS-DA) as previously described in Ritota et al.  were extracted and the relative scores were determined. Only PCs with an eigenvalue >1 were considered for discussion. Differences between groups of means were obtained using the Student-Newman-Keuls test at P ≤ 0.05. All statistics were made by SPSS software version 19 , concentration , time from treatment (flowering and maturity), and maturation stage of fruits (red and green peppers). To identify the structure of the interdependences of the main parameters of the leaves and fruits, joint principal components analysis (PCA) was performed on the following variables: the weight of fresh leaves and fruits, glucose, fructose, total phenolic acids, chlorogenic, caffeic, ferulic, P ≤ 0.05), TOC (18.8 and 1.23 %) (P ≤ 0.05), total phenols (2576 and 970 mg L−1) (P ≤ 0.05), and IAA (18.46 and 2.92 nmol mg−1 C) (P ≤ 0.05). Smaller differences were found in the amount of total sugars (P ≤ 0.05) and IPA, which were slightly higher in RG  compared to AH .The chemical characteristics of the two biostimulants are listed in Table −1 spectral region because in this area, the characteristic groups are absorbed and the “finger fingerprint” region is included. Consequently, in this region, any structural differences can be detected. Both spectra are characterized by a number of complementary bands of varying intensities. The bending vibration of the NH2 group appears as a shoulder at 1630 cm−1 in the FT-IR spectrum. Similarly, the NH2 bending vibration in the Raman spectrum is very weak and is located at 1607 cm−1. In the IR spectrum, the strong band at 1576 cm−1, which is absent in the Raman spectrum, is assigned to the asymmetric stretching vibration of COO− in the Asp, Glu and NH2 bending vibration in Gln and Lys, respectively. A band at 1515 cm−1 is only observed in the IR spectrum and may be assigned to the stretching vibration of the aromatic ring and NH bending motion in Try and Trp. The band at 1455 cm−1 in the IR spectrum is assigned to the asymmetric bending of CH3. The corresponding band in the Raman spectrum appears at 1450 cm−1. The strong band at 1398 cm−1 and a shoulder at 1349 cm−1 in the IR spectrum are attributed to the symmetric stretching vibration of COO− and symmetric bending of CH3, respectively. The corresponding bands in the Raman spectrum appear at 1409 and 1350 cm−1. A medium band in the IR spectrum at 1078 cm−1 can also be attributed to the ρ (NH2) and C-O stretching vibration. The bands at 1203, 1110–1100, 1068–1012 cm−1 correspond to the stretching vibration of C–O instead the bands in the range 996–665 cm−1, which are assigned to aromatic CH out-of-plane bending vibrations.The FT-IR and Raman spectra of AH are shown in Figure −1, in the FT-IR spectrum, is assigned to the stretching vibration of carbonyl (C=O) in the aldehyde group  absorbs , indicating the existence of both aliphatic and aromatic C=C double bonds as in the case of the polyphenols caffeic and isoferulic acids  and δ (CH3), although some contribution from ring stretching and δ (OH) is also possible in these bands. The medium band appearing at 1267 cm−1 is attributed to the ν (C–O), characteristic of phenolic compounds as also detected in the FT-IR spectrum. The very intense band at 1154 cm−1 is assigned to δ (CH) in aromatic compounds, but this band is also characteristic of polyenes, such as carotenoids and in the polyphenol resveratrol  of the aromatic moieties and ν (C-C) of the aliphatic parts. The bands observed at 900–600 cm−1 can be attributed to skeletal vibrations, i.e., vibrations involving δ (CCC) motions. Finally, the strong band appearing at 370 cm−1 is attributed to bending vibrations in polyphenols δ (CCO) and also to δ (CC=C) bending of the vinylidene group in resveratrol , concentration (P ≤ 0.001) and time from treatment (P ≤ 0.001), whereas the post-hoc test showed significant differences between the treated and control plants , total fruits (2.2-fold UNT) and number of fruits (2.4-fold UNT) (Table −1) and UNT were observed for RG at a rate of 50 mL L−1  compared to 25 mL L−1 AH (29.9 g) and 100 mL L−1 RG (23.1 g) and UNT (16.4 g) , concentration (P ≤ 0.001), time from treatment (P ≤ 0.001), and maturation of fruits (P ≤ 0.001). Significant differences between treated and untreated plants were obtained using the post-hoc test.The chemical composition of leaves and fruits was significantly affected by treatment (p-hydroxybenzoic acid (0.85), fresh weight (0.76) and p-coumaric acid (0.75). The total phenolic acids (0.60) and ß-carotene (0.60) were potentially correlated with factor 3 (11% of the variance). The data were plotted according to PC1 and PC2  (P ≤ 0.05), ascorbic acid (1928 and 608 mg kg−1) (P ≤ 0.05), quercetin (0.49 μg g−1 vs. nd) and dihydrocapsaicin (1.47 mg Fe 2+ kg−1 vs. nd) compared to time 2 (maturity) (Table −1) (P ≤ 0.05), glucose (611 and 349 mg g−1) (P ≤ 0.05), and chlorogenic (55 μg g−1 vs. nd) and ferulic (7.52 and 2.32 μg g−1) (P ≤ 0.05) acids were found at time 2 with respect to time 1 (Table −1) (P ≤ 0.05) and antioxidant activity (3942 and 3668 mg Fe2+ kg−1) (P ≤ 0.05) than cluster b (Table p-hydroxybenzoic acid (0.57 and 1.22 μg g−1) (P ≤ 0.05) and ß-carotene (1.85 and 2.27 μg g−1) (P ≤ 0.05) (Table −1) (P ≤ 0.05), quercetin (0.42 vs. nd), dihydrocapsaicin (0.84 vs. nd), ascorbic acid (2026 and 3942 μg g−1) (P ≤ 0.05), and lower content in glucose (308 and 579 mg g−1) (P ≤ 0.05), fructose (157 and 337 mg g−1) (P ≤ 0.05), chlorogenic acid (nd vs. 66), and ferulic acid (1.27 and 11.7 μg g−1) (P ≤ 0.05) than cluster c (Table p-coumaric acid (0.59 and 3.21 μg g−1) (P ≤ 0.05) and caffeic acid (1.54 and 5.76 μg g−1) (P ≤ 0.05) in the former compared to the latter , quercetin (0.96), ascorbic acid (0.93), and epicatechic acid (0.93), and chlorogenic acid (−0.97), fructose (−0.97), glucose (−0.90), and ferulic acid (−0.86). Factor 2 accounted for 20% and was correlated with  Figures , which ip-hydroxybenzoic acid (0.70), and epicatechic acid (0.67), and it was negatively correlated with fructose (−0.89), glucose (−0.85), and ß-carotene (−0.72). Factor 2 accounted for 32% and was positively correlated with quercetin (0.91), antioxidant activity (0.87), and lycopene (0.82), and it was negatively with ascorbic acid (−0.82). Caffeic acid (0.81) and dihydrocapsaicin (0.67) were potentially correlated with factor 3 (15% of the variance). The data were plotted according to PC1 and PC2  (P ≤ 0.05) and total phenolic acids (4529 and 1401 mg GAE kg−1) (P ≤ 0.05) than the red fruits, whereas the red fruits were highly endowed in fructose (1903 and 508 mg g−1) (P ≤ 0.05) and glucose (1815 and 793 mg g−1) (P ≤ 0.05) with respect to the green fruits (Tables −1), whereas both quercetin and antioxidant activity were higher at time 2 than time 1  (P ≤ 0.05)  (P ≤ 0.05), p-hydroxybenzoic acid (1.34 vs. 1.05 and 1.04 μg g−1) (P ≤ 0.05), p-coumaric acid (0.94 vs. 0.32 and 0.37 μg g−1) (P ≤ 0.05) and antioxidant activity (14819 vs. 13132 and 12071 mg Fe2+ kg−1) (P ≤ 0.05)  (P ≤ 0.05) , total phenolic acids (0.86),  Figures , which i, total p1H-HRMAS-NMR spectrum of AH-treated red fruits is shown in Figure 1H and 13C chemical shifts based on 1D- and 2D TOCSY experiments are supported by chemical shifts previously reported in the literature  due to malate based on the cross-peaks in the TOCSY spectrum with peaks at δ = 2.39 and 4.31 ppm, β-CH and α-CH, respectively , phenylalanine, tryptophan, and tyrosine were identified, as previously described in detail by Ritota et al.  and/or deoxythymidine triphosphate (dTTP) and HMW species, while the untreated pepper was characterized by a higher amount in glutamate and/or glutamine and/or 2-oxoglutarate.The PLS-DA classification model (data not shown) was obtained to discriminate the effect of both biostimulants on quantities of the corresponding metabolite in pepper. For example, untreated pepper contained higher amounts of methylnicotinamide, cytidine, trigonelline, and imidazole derivatives compared to pepper treated with AH. Thus, the treated pepper showed the highest amount of NADPApplication of AH and RG biostimulants in pepper plant cultivation has demonstrated strong positive effects on growth, development and fruit quality. The beneficial effects observed indicate the presence of more than one group of plant growth-promoting substances/hormones. Interestingly, IAA and IPA were found in both biostimulants at different amounts, as shown in Table After the first application of AH and RG, the biostimulant effect was visible early in terms of quantitative characteristics, such as a sharp increase in the weight and number of fresh leaves and fruits . This was a typical short-time effect that has been previously observed in treated plants with HSs or other biostimulants  may modulate carbon and nitrogen metabolism by increasing the activity of enzymes involved in glycolysis, the Krebs cycle and nitrate assimilation  are present in commercial cultivars of chili peppers at different maturation stages . However, red fruits at time 1 and 2 and green fruits at time 2 also exhibited considerable antioxidant activity (1.25–1.46-fold UNT). Importantly, lycopene was found only in fruits at maturity and its amount showed low to high increases in the treated fruits. Capsaicin is an active component, which accounts for the pharmaceutical properties of peppers. It is well established that the level of capsaicin in a given variety can vary depending on the light intensity and temperature at which the plant is grown, the age of the fruit, and the position of the fruit on the plant. In our study, the amount of capsaicin sharply increased l et al. , who fouMost frequently, biostimulants as well as humic substances may modulate nitrogen metabolism (Schiavon et al., On the basis of our data, the two biostimulants demonstrated ameliorating properties on the growth of pepper plants from flowering to fruit maturity. With this perspective, long-term experiments should collect information on the nutritive and health-promoting compounds of a biostimulated crop.The use of biostimulants should also be increased to reduce the excessive inputs of mineral fertilization, which is a source of pollution and a risk for environmental fitness. Moreover, from a management perspective, cultivation wastes might turn into raw materials, thus permitting a reduction in disposal costs.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The identification of acid mucopolysaccharide by the liquid chromatography/tandem mass spectrometry method (LC-MS/MS) of the predominant disaccharide units of glycosaminoglycans (GAGs)  after methanolysis is validated and applicable for mucopolysaccharidosis (MPS) type determination.2H6] CS, [2H6] DS and [2H6] HS, were prepared in-house by deuteriomethanolysis of CS, DS and HS.A total of 76 urine samples were collected and analyzed, from nine MPS I patients, 13 MPS II patients, seven MPS III patients, eight MPS VI patients, and 39 normal controls. Urinary GAG was first precipitated by the Alcian blue method followed by a treatment of 3 N HCl methanol. The protonated species of the methylated disaccharide products were detected by using a multiple reaction monitoring experiment. Internal standards, [m/z 426.1 → 236.2 for DS (m/z 432 → 239 for dimers derived from [2H6] CS and [2H6] DS) and m/z 384.2 → 161.9 for HS (m/z 390.4 → 162.5 for the [2H6] HS dimer). The quantities of DS and HS were determined, which varied from one MPS type to the other. The results can be used to evaluate the severity of MPS subgroups, as well as urinary GAG amelioration at follow-up after enzyme replacement therapy (ERT).One particular disaccharide for each GAG was selected, in which the parent ion and its daughter ion after collision were The modified LC–MS/MS method for MPS type determination is specific, sensitive, validated, accurate, and applicable for simultaneous quantifications of urinary DS and HS. This method can help to make correct diagnosis of MPS patients and evaluate the effectiveness of ERT. The mucopolysaccharidoses (MPS), known as a group of lysosomal storage disorders, are caused by the deficiency of specific enzymes that catalyze the stepwise degradation of glycosaminoglycans . There are at least 11 known enzymes involved in the catabolism of dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), chondroitin sulfate (CS), and hyaluronic acid (HA) . Any enzAn accurate diagnosis is achieved generally by three sequential tests: the quantification of urinary GAG, two-dimensional electrophoresis (2-D EP) qualitative analysis, and leukocyte enzyme activity assay –12. UrinThe polysaccharide chains are elongated by the sequential addition of alternating acidic and amino sugars, donated by their UDP-derivatives. Amino sugars are essential components of GAGs, glycoproteins, and glycolipids. Members of the GAG family vary in the type of hexosamine, hexose or hexuronic acid unit they contain  . The spe+) mode. The quadrupoles, Q1 and Q3, were tuned with unit resolution, and the MS conditions were optimized for maximum signal intensity. The LC-MS/MS analysis was performed on a 4000 QTRAP LC-MS/MS system  equipped with a TurboIonSpray , and Agilent 1260 Infinity HPLC pump and autosampler . The experimental parameters were set and are clearly described in the following section. Data were acquired and processed using Analyst 1.5.2™ software (AB Sciex). Calibrations of GAG standards and internal standards were performed with every batch of samples.The LC-MS/MS method for GAG disaccharide analysis was modified according to the literature reported by Auray-Blais et al. . For GAGA total of 76 urine samples were collected and analyzed from nine MPS I patients, 13 MPS II patients, seven MPS III patients, eight MPS VI patients, and 39 normal controls. MPS I phenotypes comprised five MPS I-Hurler/ Scheie (H/S) and four MPS I-S patients according to their progressive central nervous system and the severity of somatic manifestations. They have received enzyme replace therapy (ERT) in a varied duration, from one month to 8.8 years, and the ages of ERT initiation were from 0.7 to 34.9 years old. In those, two patients expired due to respiratory infection and complications. For MPS II phenotypes, eight severe and five mild forms were inclusive in this study. All 13 patients have received ERT and the duration of receiving ERT was from 1.3 to 7.3 years. The ages of ERT initiation were varied, from 4.5 to 18.3 years old. The seven MPS III patients were identified as one IIIA, three IIIB, and three IIIC and all patients were confirmed by leukocyte enzymatic assays. No MPS VI phenotypes were further classified. Soft tissue and skeletal disease presented in MPS VI patients, but without central nervous system (CNS) involvement. These patients have received ERT from 3 to 10.3 years, and the ages of ERT initiation were from 4.4 to 21.2 years old. Normal controls (n = 39) were chosen from out-patient department, and informed consent was obtained from all normal population and MPS patients. The study was approved by the ethics committee of Mackay Memorial Hospital, Taipei, Taiwan.N-acetylhexosamine dimers  DS, [2H6] CS, and [2H6] HS was ready for LC-MS/MS analysis. The GAG internal standard mixture was stable in 4°C for seven days.The internal standards including  CS and [2H6] DS dimers , as well as the molecular ion transition m/z 390.2 → 168 for the [2H6] HS dimer (retention time = 3.39 ± 0.06 minutes). Quantifications were achieved using peak areas that were processed using Analyst 1.5.2™ software (AB Sciex).One particular disaccharide for each GAG was selected, in which the parent ion and its daughter ion after collision were The within-run and between-run precisions were determined by an assay of total 18 samples in low, medium, and high concentrations of DS and HS using the method described earlier. All 18 samples were run in triplicate for within-run analysis, and a duplicate analysis of each sample (n = 18) was performed on three different days sequentially. The average within-run and between-run CV was 9.7% (low conc.), 6.4% (medium conc.), and 5.9% (high conc.), as well as 11.8%, 7.6%, and 9.3%, respectively. The analytical recovery was also investigated by spiked standards with known concentrations of DS and HS (20 μg/mL for each standard) into pooled normal urine samples as in the precision studies. The recoveries of this LC-MS/MS assay were 94.3% for DS (ranging from 91.9% to 97.6%), and 95.1% for HS (ranging from 92.3% to 98.4%). The linearity study of this method was carried out with concentrations ranging from 12.5 to 200 μg/mL for DS, and 3.125 to 50 μg/mL for HS. Each concentration was performed in triplicate. Linearity of DS and HS was calculated individually and the correlation coefficients (r) were 0.9914 for DS and 0.9935 for HS, respectively.In this study, a total of 76 urine samples were collected and analyzed. As shown in Figure The mass spectrums of the normal control and each MPS type are illustrated in Figure p < 0.005 for DS; p < 0.001 for HS), and the concentrations were 276.9 ± 301.0 and 7.3 ± 8.7 μg/mL, respectively. When comparing the quantities of DS and HS in MPS I and II patients, the urinary HS in the severe form of MPS II was significantly greater than that of MPS I and the mild form of MPS II , and the DS was slightly lower than that found in MPS I and the mild form of MPS II patients  . A very small amount of DS (0.73 μg/mL) was found in MPS III patients. The predominantly affected GAG in MPS VI patients was DS, which was seen to be moderately elevated, approximately 105.5 μg/mL on average, and a relatively little amount of HS (1.26 μg/mL) was observed. No significant difference was found when comparing DS values between the severe form of MPS II and MPS VI. This was a reliable indicator to discriminate MPS VI from MPS I and II (p < 0.01 and p < 0.05). The quantifications of DS and HS in different MPS types are compared and shown in Figures The average concentrations (in μg/mL) of urinary DS and HS found in different MPS types and the normal controls were determined. The majority of urinary GAG found in normal control was CS (71.9 ± 46.6 μg/mL) with very little DS and HS . In MPS I patients, DS and HS were simultaneously detected and were shown to be significantly elevated when comparing with normal control  and particular disaccharide units of the affected GAGs were measured for three successive years. The average reductions of urinary GAGs after ERT in MPS I (n = 9), II (n = 13), and VI (n = 9) patients were significant. In the first year, the reductions were 62.0%, 71.5%, and 61.9%, respectively. In the second year, they were 72.5%, 85.5% and 77.9%. In the third year, they were 71.9%, 88.1% and 87.6%  is another merit of our method modification. Combined with the addition of internal standards, the eluted positions of CS, DS, or HS can be precisely identified. In addition, the relative concentrations of CS, DS, and HS in the urine sample can be obtained by integrating the peak areas between the individual GAG and that of internal standard with known concentration. The internal standard can also be used as a quality assurance in LC-MS/MS analysis.m/z 426.1 → 236.2 for DS and m/z 384.2 → 161.9 for HS.Based on the results detected by LC-MS/MS method, the quantities of affected GAGs are distinctive for MPS type determinations. The results are fully matched with those examined by 2-D EP method, and correspond well with the leukocyte enzymatic assay. The LC-MS/MS method used for MPS type determination has many advantages, including high sensitivity, high specificity, high throughput, high reliability, and analytic automation. The highly sensitive of LC-MS/MS method was achieved and the lower limit of quantitation (LLOQ) was less than 0.06 μg and 0.065 μg with 100 μL urine for DS and HS, respectively. The high specificity of MRM detection allowed specific detection of DS and HS, in which the parent ion and its daughter ion after collision were vs. 22.9 μg/mL). The results also showed that the concentrations of DS were varied among MPS I, MPS II, and MPS VI. The quantities of DS detected from MPS VI patients was significantly lower than those with MPS I and the mild form MPS II patients; but was equivalent to those of severe form MPS II patients. As very little or no HS was seen in the samples from MPS VI patients, and this can be used as a marker.The LC-MS/MS method can also be used for MPS subgroup classifications, particularly for MPS II. In this application, the concentration of HS is the key factor used to distinguish the mild form of MPS II from the severe form. The results obtained in this study showed that the concentrations of HS detected from the samples of the severe form MPS II patients were significantly higher than those of the mild form MPS II patients. The increase was over 35 times greater (824.0 Disease-specific treatments to replace specific enzyme activity include ERT for the non-neurologic manifestations of MPS patients –31, and The LC-MS/MS method used for MPS type determination is specific, sensitive, validated, reproducible, accurate and applicable for simultaneous quantification of urinary DS and HS. By using this method, MPS can be properly diagnosed. As well, this method can be used to evaluate the effectiveness of follow-up therapy.


Concentrations of La, Ce, Eu, Gd, and Ca were significantly lower in nondrinkers’ semen than in semen from drinkers. Significant differences were detected between La, Ce, Eu, Gd, and Ca concentrations in semen from nondrinkers and moderate drinkers. Concentrations of La, Ce, and Gd in semen of short-term smokers were significantly lower than those in extremely long-term smokers. Significant differences were also detected between La concentration in semen from a group of short-term smokers and that of a group of long-term smokers. Positive correlations were found between La, Ce, Eu, Gd, and Ca concentrations in semen. La, Ce, Gd, and Ca concentrations in semen were positively associated with progressive motility and percentage of normal spermatozoa. Positive correlations were found between Ca and sperm concentration. Concentrations of La, Ce, and Gd were negatively associated with sperm concentration, whilst Ca concentration was negatively associated with volume of ejaculate. At the examined level, La, Ce, Eu, and Gd did not affect sperm quality, whereas alcohol consumption and smoking might have increased the level of rare earth elements in semen.The aim of the present study was to measure lanthanum (La), cerium (Ce), europium (Eu), and gadolinium (Gd) concentrations in human semen and correlate the results with sperm quality. The median semen content of La was 19.5 µg kg The elements lanthanum (La), cerium (Ce), europium (Eu), and gadolinium (Gd) belong to this group. During the past few years, the widespread application of rare earth elements has been extensively reported in various fields including agriculture, animal husbandry, electronics, energy, fuel additives, and modern biomedicine. Rare earth ions initially enter organs; after transforming into metastable hydrogen oxide particles, they selectively accumulate in the liver and other reticular structures. This leads to enrichment of REEs in these tissues  undergoing routine infertility evaluation at an infertility clinic: “NZOZ Medical Center Co. Prof. dr. hab. med. Wiesław Szymański, Dr. med. Marek Szymański” and “Almed - Genito-Urinary Medicine Clinic - dr G. Ludwikowski.” The inclusion criteria were male partners of infertile couples and attending fertility clinic for fertility evaluation  and also served as the control group. Group II consisted of males identified as having semen anomalies as the only reason for their infertility with abnormal volume of semen, abnormal concentration, morphology, or motility of spermatozoa; males with more than one abnormal semen variables; and males with no spermatozoa in the ejaculate (N = 85).Participants were divided into two groups based on their ejaculate parameters. Group I consisted of healthy male partners of infertile couples with normal ejaculate [normozoospermia; N = 80; group II N = 82). The method used for determination of La, Ce, Eu, Gd, and Ca concentrations in semen samples is described elsewhere  and heated at 100 °C (1 h). After self-cooling, 6 ml of bidistilled water was added, and a solution was mixed up. La, Ce, Eu, Gd, and Ca concentrations in semen samples were determined using inductively coupled plasma–mass spectrometry . Recoveries were assessed trough sample dilution. Five-fold diluted samples showed differences in concentrations compared with the original sample, which was <20 %. The limit of detection for La and Ce was 0.3 µg kg−1, for Eu and Gd was 0.3 ng kg−1, and for Ca was 0.33 µg kg−1. limit of quantification for La and Ce was 0.9 µg kg−1, for Eu and Gd was 0.9 ng kg−1, and for Ca was 0.33 µg kg−1. Precision of the measurements for La, Ce, Eu, Gd, and Ca was 9, 7, 12, 10, and 8 %, respectively. In the determination of Eu, 35 % of samples were lower than the method detection limit (MDL). For Gd, Ce, La, and Ca no results were lower than the MDL.Seminal La, Ce, Eu, Gd, and Ca were measured in 162 of 168 samples , and group II . Ethical approval was obtained for this study from the Local Committee of Bioethics of Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz (KB/538/2007).The medians were calculated and analyzed using computer program Statistica StatSoft, version 10.0 . The median values of La, Ce, Eu, Gd, and Ca semen concentrations were compared using U-Mann–Whitney and Kruskal–Wallis tests. The relationships between La, Ce, Eu, Gd, and Ca semen concentrations and semen-quality parameters were calculated using Spearman’s rank correlation coefficients, and −1 dry weight (dw) (median 19.5). The semen content of Ce was 41.9 µg kg−1 dw (median 4.52–167). Concentration of Ce was the lowest of the measured REEs and was 0.68 µg kg−1 dw (median 0.06–1.95). The range of Gd content in semen was from 0.38 to 12.0 µg kg−1 dw (median 3.19). In the present study, the range of seminal Ca content was from 484 to 17,191 mg kg−1 dw (median 4063). Our study also showed that semen of men from group I had greater La, Ce, and Gd concentrations than semen of men from group II, but the differences were not significant. Median concentration of Eu in semen of group I and II males was similar. The concentration of Ca in semen of group I males was lower than in semen from group II men [the difference was not significant  and group II , concentrations of La, Ce, Eu, Gd, and Ca (Eu and Ca only in group II) was significantly lower in nondrinkers’ semen than in semen from drinkers Table . A signiIn the group of all men (group I + II) and group II , concentrations of La, Ce, and Gd in semen of short-term smokers were significantly lower than those in semen of extremely long-term smokers Table . A signiWe found a significant positive correlation between La, Ce, Eu, Gd, and Ca concentrations in semen. In the group of all males, concentrations of La, Ce, and Gd in semen were positively associated with progressive motility and percentage of normal spermatozoa. In group I , La, Gd and Ca concentrations were also positively associated with progressive motility and percentage of normal spermatozoa, whilst the concentration of Ce was negatively correlated with the percentage of spermatozoa with progressive motility. We also found a significant positive correlation between Ce concentration in semen and percentage of normal spermatozoa and between Ca and sperm concentration. In group II , concentrations of La, Ce, and Gd in semen were negatively associated with sperm concentration, whereas Ca concentration was negatively associated with volume of ejaculate Table . Our stu−1 dw, respectively. There are no data concerning this subject in the literature. However, we found some information about concentrations of this element in blood, serum, urine, teeth, and hair. Concentrations of La and Ce in blood plasma of adult males and females living in northern Germany ranged from “not traceable” to 20 ng l−1 for La and to 30 ng l−1 for Ce  were significantly greater than data from a study by Morita et al. , 3.1 ± 2.15 (Ce), 0.21 ± 0.08 (Eu), whereas Gd in urine and La in serum were at concentrations <1 µg l−1. Peng et al. (−1) in young children living nearest to the REE-mining area. The next greatest (472 ng g−1) was in those living nearer to the REEs mining area, and the lowest content (97.4 ng g−1) was in those living in the control area. The REE content in the mothers’ hair was 1510 ng g−1 in the high-exposure area, 242 ng g−1 in the low-exposure area, and 59.2 ng g−1 in the control area. Brown et al. (−1 respectively). The range of Ce concentration was greater in teeth in Ugandan children (0.04–0.36 mg kg−1) compared with teeth in children from the UK (0.005–0.09 mg kg−1). The precise mechanism by which these elements are transferred from the circulatory blood into semen is unclear. In the present study, the median value of seminal Ca content was 4063 mg kg−1 dw. Surprisingly, communications in this specific field are also rather limited. Seminal concentration of Ca was reported by Sørensen et al. . Our results do not support this finding; however, it must be emphasized that we measured total concentrations of La, Ce, and Gd in semen and not the effect on spermatozoa of ionized form of this elements. Studies on the relationship between Ca and semen parameters are contradictory. Our study showed that increased seminal Ca levels were associated with increased progressive motility, sperm concentration, and percentage of normal sperm cells. These findings are in accordance with Bassey et al. (Our study shows positive correlations between La, Ce, Eu, Gd, and Ca concentrations in semen. Lanthanides act as Ca analogues in biological systems, and trivalent lanthanide ions can replace Ca at metal-binding sites (Wang et al. , Saling  reportedl et al. , both Cey et al.  who alsoy et al.  showed ty et al.  and Wongy et al.  found noy et al. . All of For the first time, the contents of REEs in human semen were determined and described. Similarly, REE relationships with semen quality, concentration of seminal Ca, and lifestyle habits were reported for the first time. Summarizing our results, although we could not unequivocally prove the influence of La, Ce, Eu, and Gd concentrations on sperm quality as a whole, we found both positive and negative association of La, Ce, Eu and Gd concentrations with semen-quality parameters, i.e., volume of ejaculate, sperm concentration, motility, and morphology. In addition, our findings suggest that alcohol consumption and smoking might increase levels of REEs in semen. However, it is unclear how these elements are transferred into the semen, and a review of the literature reveals that there is little information about the possible impact of REEs on human health. Therefore, further research on La, Ce, Eu, and Gd and the toxicological, human, and public health consequences is urgently required.


P < 0.05) in the reactive substances to thiobarbituric acid levels, nitrite content, and superoxide dismutase activity and high rates of DNA damage in leukocytes were observed when compared with healthy women group and baseline levels. Similarly, after the second cycle, the same parameters were increased (P < 0.05) when compared with baseline levels. Increase in catalase activity was detected only after the fourth cycle and reduced glutathione levels and glutathione peroxidase activity were decreased in all cycles when compared with healthy women, as well as after the second and fourth chemotherapy cycles compared to baseline (P < 0.05). Patients with breast cancer presented an indicative of oxidative stress before, during, and after chemotherapy, as well as increased genotoxic damage in all stages of treatment, demonstrating the clinical applicability of this investigation.The aim of this study was to evaluate the oxidative parameters of erythrocytes and genotoxicity in leukocytes of patients with breast cancer. Oxidative parameters were detected by spectrophotometry and genotoxic damage by single cell gel electrophoresis. Twenty-eight women with breast cancer were monitored before chemotherapy and after the second and fourth cycles of therapy with cyclophosphamide and doxorubicin. After the fourth cycle, increases ( The etiology of breast cancer possesses a multifactorial origin , 2, showMost chemotherapeutic agents are not specific against neoplastic cells, also affecting normal cells , which rReactive oxygen species (ROS) represent important factor in carcinogenesis and may play a role in initiation and progression of tumors. Free radicals stimulate oxidative DNA damage, contributing to mutagenesis, which is essential for the process of tumor initiation –13. Unre2 and cyclophosphamide 600 mg/m2) and 28 patients not exposed to the chemotherapy. The patients were exposed to four 21-day cycles with intravenous AC. The unexposed group consisted of individuals who had not been exposed to genotoxic agents  and who were free of any malignant neoplasm or clinical, biochemical, hematological, hepatic, cardiovascular, renal, or endocrine manifestations. Blood samples were collected with EDTA or heparin by venipuncture using vacutainers, maintained at 4°C during transport to the laboratory, and immediately processed. Three collections of the peripheral blood were carried out during four cycles of chemotherapy: the first collection was performed before the beginning of treatment (C0), 21 days after the second cycle of chemotherapy (C2), and 21 days after the fourth cycle (C4).The subjects were patients diagnosed with ductal breast cancer under treatment at the Department of Oncology, São Marcos Hospital, Teresina, Piauí, Brazil, from August 2012 to February 2013. This clinical study was approved by the Research Ethics Committee of University Center UNINOVAFAPI (registration number 0406.0.043.00011). This study involved a total of 56 individuals including 28 patients exposed to chemotherapy by the AC protocol  and the demographic, medical and lifestyle were summarized in  status, were obtained and analyzed from medical records. The descriptive statistics for such variables are listed in Details about clinical features, such as cancer site, clinical stage and HER-2/neu, ER (estrogen receptor), and PR (progesterone receptor)single cell gel electrophoresis (SCGE)) assay was performed as described by Singh et al. [μL) were embedded in 95 μL of 0.75% low-melting point agarose, which was immediately added to the surface of a precoated (1.5% agarose) microscope slide. When the agarose had solidified, the slides were placed in lysis buffer  containing freshly added 1% (v/v) Triton X-100 and 10% (v/v) dimethyl sulfoxide (DMSO) for a minimum of 1 day and a maximum of 7 days. After treatment with lysis buffer, the slides were incubated in freshly made alkaline buffer  for 20 min and the DNA was electrophoresed for 20 min at 25 V (0.90 V/cm) and 300 mA after which the buffer was neutralized with 0.4 M Tris (pH 7.5) and dried overnight. Gels were rehydrated for 5 min in distilled water and then stained for 15 min (37°C) with a solution containing the following sequence: 34 mL of Solution B  and 66 mL of Solution A (5% sodium carbonate). The staining was stopped with 1% acetic acid and the gels were air dried. Analyses (100 cells/patient) were carried out by light microscopy (Olympus CX40) at 100x magnification with immersion oil. Images of cells (50 cells/slide in two replicates) were evaluated for the following: (i) damage index (DI), in which each cell was classified into classes  according to tail size and cell shape [The alkaline comet  was added in white tube. In another test tube 500 μL of Griess reagent and 500 μL of the homogenate at 10% of the erythrocytes (sodium phosphate buffer 50 mM pH 7.4) (Test) were added. The spectrophotometric measurement was performed at 560 nm. Results were expressed in μM/mg protein.The determination of nitrite content was based on the Griess reaction  in whichμL of homogenate, 1 mL of trichloroacetic acid 10%, and 1 mL of thiobarbituric acid 0.67% were mixed and stirred. Subsequently, this mixture was maintained in a bath of boiling water for 15 min and freshened under running water. After cooling, 2 mL of n-butanol was added and centrifuged at 1.200 rpm/5 min and the butanol phase was read spectrophotometrically at 535 nm. Results were expressed as nmol/mL.Blood samples were centrifuged at 3000 rpm at 4°C during 5 minutes. Plasma was removed and a pellet of erythrocytes was washed with a cold solution of NaCl 0.9% and centrifuged. An erythrocytes' homogenate 10% diluted in phosphate buffer sodium 50 mM and pH 7.4 was stored at −20°C. Lipid peroxidation was measured by TBARS levels, a method previously described by Draper and Hadley (1990) , 28. 250μL of distilled water and 80 μL of trichloroacetic acid 50%. After centrifugation at 3.000 rpm for 15 min, 400 μL from the supernatant was collected and added to 800 μL of Tris-HCl 0.4 M, pH 8.9, and 20 μL of DTNB 0.01 M. One minute later, spectrophotometric measurement was performed at 412 nm. Concentration of GSH was expressed in mg/g of hemoglobin.Determination of GSH was based on the Ellman reaction  with some modifications described by Sedlak and Lindsay . Four hu2O2), oxidizing reduced glutathione (GSH) to form oxidized glutathione (GSSG). GSSG is then reduced by glutathione reductase (GR) and β-nicotinamide adenine dinucleotide phosphate (NADPH) forming NADP+ (resulting in decreased absorbance at 340 nm) and recycling the GSH. Because GPx is limiting, the decrease in absorbance at 340 nm is directly proportional to the GPx concentration. 1 unit of GPx-1 = the amount of enzyme necessary to catalyze the oxidation (by H2O2) of 1.0 μmole GSH to GSSG, per minute at 25°C, pH 7.0. Results were expressed in U/g of hemoglobin.The glutathione peroxidase activity coupled assay was determined by Paglia and Valentine . GPx cat2O2 (18 mL), Tris HCl 1 M, EDTA pH 8.0 5 mM (1.0 mL), and H2O (0.8 mL). The reading was carried out in a quartz cuvette at 230 nm with 980 μL of reaction medium plus 20 μL erythrocytes' homogenate prepared in sodium phosphate buffer 50 mM, pH 7.4 [Erythrocytes' homogenate in pH 7.4 was centrifuged  and the supernatant was used to quantify catalase activity. The reaction medium was prepared with H, pH 7.4 .c reduction rate was determined by superoxide radicals using the xanthine-xanthine oxidase system as a source of superoxide anion (O2−) [2− in the presence of cytochrome c.Erythrocytes homogenate prepared in sodium phosphate buffer 50 mM, pH 7.4, was centrifuged  and supernatants were used for testing superoxide dismutase (SOD) activity. Cytochrome n (O2−) . ResultsP < 0.05) using the Graphpad program  and SPSS . Correlations among data obtained were calculated using Spearman's correlation coefficient.In order to determine statistical differences, data expressed as mean ± standard error of the mean (S.E.M.) were compared by one-way analysis of variance (ANOVA) followed by the Newman-Keuls test  level, nitrite content, GSH concentration, and GPx, CAT, and SOD activities.P < 0.05) increased, as demonstrated by basal TBARS (1.42 ± 0.45 nM/mg of protein) and nitrite (1.16 ± 0.62 μM/mg protein) contents in erythrocytes of patients with breast cancer when compared with the control group  (P < 0.05). When these same patients were submitted to chemotherapeutics (combination of cyclophosphamide and doxorubicin), such increases in both TBARS (4.76 ± 0.68 and 11.98 ± 0.65 nM/mg of protein) and nitrite ion levels (1.81 ± 0.02 and 3.49 ± 0.07 μM/mg of protein) were also detected in C2 and C4, respectively . With AC chemotherapy, there was decrease in GSH levels in C2 (46.6%) and C4 groups (50.9%) (P < 0.05). Similarly, baseline levels (C0) also presented diminution of 21.4% and 27.7% in C2 and C4 groups, respectively  in comparison with control group (36.13 ± 7.65 U/g protein) (ectively .P < 0.05).GPx activity  showed rμM/mg) exhibited a significant increase (44.3%) when compared to the control group (15.82 ± 1.21 μM/mg), C0, and C2  (μM/mg) when compared with control group. After chemotherapy (2.63 ± 0.65 μM/mg), its activity increased about 93.4 and 54.7% in relation to the control group (1.36 ± 0.62 μM/mg) and baseline (1.70 ± 0.43 μM/mg), respectively.In relation to the catalase levels, only treated patients  . Similar, resp.)  also incDNA in the tail was organized into five classes: (i) class 0: undamaged, with no tail; (ii) class 1: with tail shorter than the diameter of the head (nucleus); (iii) class 2: with tail length between one and two times the diameter of the head; (iv) class 3: with tail longer than two times the diameter of the head; and (v) class 4: comets with no heads  Figure .P < 0.05) in the classes of DNA damage in lymphocytes of patients with breast cancer (C0) in the control group. This condition is increased (P < 0.05) in C2 and C4  compared to the control group (44.43 ± 1.67). After AC chemotherapy, there was an increase of 66.25 and 105.2% of damage index in C2 (164.4 ± 6.36) and C4 groups (202.9 ± 5.34) in comparison with C0 group (98.89 ± 5.56). In a similar way, an increase of 23.2% after the fourth cycle was noted when compared to the C2 group . status (C0). Similarly, in C4 patients, an increase of 15.8% was observed in comparison with the frequency of C2.An increase of 171.1% in frequency of DNA damage before chemotherapy (C0) (61.00 ± 2.01) was observed when compared to the CG (22.50 ± 0.94) . AC chemP > 0.05). However, a significant positive correlation (correlation factor = 0.389 and P = 0.041) was observed between race and nitrite levels after chemotherapy (Q4) and a negative correlation was observed with nitrite levels  in the diagnosis and activity of superoxide dismutase  after chemotherapy (Q4) to marital status. There was also a negative correlation  between the practice of physical exercises and malondialdehyde levels during chemotherapy. No correlation was observed between ER and PR receptors with oxidative stress, except between HER2 and glutathione peroxidase in Q2 group with 0.412 correlation factor P = 0.29.There was no correlation between sperm risk factors, age, smoking, and family history with the disease, as well as the levels of oxidative stress assessed by measurements of enzymes catalase, superoxide dismutase and glutathione peroxidase and malondialdehyde levels, nitrite, and reduced glutathione  and nitrite levels (Q4), as well as between frequency of damage (QD) and nitrite in group Q4 and Q2 catalase group.Regarding the genotoxicity and oxidative stress, positive correlations were observed for the contents of DNA damage assessed at diagnosis (QD) compared to those obtained during (Q2) and after (Q4) chemotherapy, with 0.663 correlation factor 0.537 and  in situ (DCIS) is the most common noninvasive or preinvasive type with chances of a recurrence under 30% within 5–10 years after initial diagnosis. On the other hand, invasive ductal carcinoma (IDC), known as infiltrating ductal carcinoma, is the most common type of invasive breast cancer, representing around 80% of cases. About two-thirds of women are 55 or older when they are diagnosed with an IDC [Breast cancer is the second most common cancer in women over the age of 50. It is often first detected as an abnormality on a mammogram before the patient or health care provider feels it. Early cases may be asymptomatic, and pain and discomfort are typically not present. Breast cancer can begin in different areas of the breast, such as ducts and lobules. Ductal carcinomah an IDC .Many studies have reported that reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved in the etiology and progression of various cancers –38. ThesAn alternative method of analyzing oxidative stress is achieved by quantification of lipid peroxidation. The lipid radical is unstable and degrades very rapidly into secondary products. Most of them are electrophilic aldehydes, such as TBARS, which is the main marker of oxidative injury in the unsaturated lipids in cell membranes, leading to oxidation of fatty acids (LH) and formation of the lipid radical (L•) . Therefo2− generation and revealed an increase according to the treatment when compared to the baseline. Previous findings found analogous results, exhibiting increases in lipid oxidation activated by NO2− levels or nitric oxide [The evaluation of nitrite concentration has been used as an index of endogenous NO production in biological systems in distinct pathological processes beyond its physiological properties such as vasodilation, neurotransmission, and immune response , 18. It ic oxide , 37. Nitic oxide . Prior aic oxide .The extent of oxidative damage depends not only on ROS levels, but also on mechanisms of cellular antioxidant defenses. Low level of GSH, a molecule of critical importance in maintaining the stability of erythrocytes membranes, is related to cellular defense against xenobiotics and harmful compounds such as free radicals and hydroperoxides . This drMetabolites generated by CMF  induced lipid peroxidation by inactivation of GSH levels and SOD, CAT, GPx, and GST activities in erythrocytes of patients with breast cancer, thereby rendering the system inefficient in management of the free radical attack. Acrolein and phosphoramide mustard are the metabolites of cyclophosphamide that are among the causative agents, which reduce the activity of SOD, CAT, GPX, glutathione-S-transferase, and glucose-6-phosphate dehydrogenase in erythrocytes of CMF treated breast cancer patients . In the 2) and cyclophosphamide (600 mg/m2) also showed reduced CAT, SOD, GSH, and GPx activity and increased TBARS levels [In relation to the CAT levels, our findings did not show differences before and during chemotherapy, presenting only increasing activity after treatment. On the other hand, while some studies found increases only after chemotherapy , 49, othS levels . Some ofS levels .10 Da of DNA) [In this study, the genotoxic profile assay of the patients with breast cancer under treatment with AC was also investigated. This evaluation was carried out by alkaline comet assay, a well-established, simple, versatile, rapid, visual, and sensitive tool used to assess DNA damage and repair quantitatively as well as qualitatively in individual cell populations . Some ot of DNA) .The pathological condition significantly raised the damage indices and frequencies in lymphocytes when compared with the normal control group, confirming previous investigations performed by Sánchez-Suárez et al.  and Agno in vivo clastogenic activity [In fact, antineoplastic agents are currently used in clinical studies which induce breaks in mammalian DNA strands as seen with topoisomerase I (camptothecin) and topoisomerase II (etoposide) inhibitors and 5-FU . This, factivity , 60. Moractivity , 45, 61.activity .Patients with breast cancer under chemotherapy presented antioxidant status indicative of oxidative stress before, during, and after chemotherapy, as well as increasing genotoxic damage in all stages of the treatment. These results highlight the importance of monitoring patients in chemotherapy, especially using cytogenetic and molecular markers in order to provide new prognostic findings to the treatment as a strategy to reduce recurrences and to improve quality of life.