Lactobacillus fermentum 5.2, L. plantarum 6.2, and L. plantarum 7.1) against Gardnerella vaginalis. Our results show that the lactobacilli have potential use as probiotics, since they have high hydrophobicity and autoaggregation properties and effectively adhere to vaginal cells. Metabolites secreted into the culture medium and whole cells of the strains under study are capable of interfering with the growth of G. vaginalis to different degrees. The elucidation of the antagonistic mechanisms as well as their effect on human cells may be useful in the development of a product containing such microorganisms or products secreted by them.Study of the probiotic potential of microorganisms isolated from fermented foods has been increasing, especially studies related to lactobacilli. In intestinal models, lactobacilli have demonstrated beneficial properties, such as anti-inflammatory activity and increased antibody production, but the molecular mechanisms involving probiotic and antagonistic action as well as their effect on human vaginal cells have not yet been fully elucidated. The aim of this study was to evaluate the functional and antagonistic properties of three strains of lactobacilli isolated from cocoa fermentation ( Probiotics are microorganisms capable of conferring health benefits to the host after correct administration. Lactic acid bacteria (LAB) are an integral part of the intestinal and genital microbiota of humans and other vertebrates .Probiotic can act in different ways: (1) competitively occupying receptors on mucosal epithelial cells ; (2) inhMost of the probiotics available on the market have human origin, due to the concept that (it was expected) their action would be accentuated in organisms of the same species from which the strain was primarily isolated. However, new studies have shown that microorganisms of extraintestinal origin, isolated from plants and fermented foods, exhibit promising effects in the treatment and prevention of numerous diseases –11.Cocoa is the main agricultural product in southern Bahia, and Brazil is one of the world's largest producers, along with Ghana and Côte d'Ivoire in Africa . The ferInterest in searching for new strains with probiotic potential has risen in the industrial and scientific sectors mainly due to the market demand for functional foods and therapeutics with lesser side effects and because of the numerous benefits attributed to these microorganisms , 15. ThePreliminary studies of our group showed that LAB isolated from this fermentative process were able to reduce intestinal inflammation induced in an experimental model of colitis in rats, decreasing the concentration of proinflammatory cytokines in serum, increasing level of IgA, and restoring tissue structure of the mucosa , 17. Lactobacillus counts and increased pathogenic microorganisms such as Gardnerella vaginalis /[(Ax + Ay)/2], where x and y indicate the absorbance of strains in the control tubes and (x + y) indicates the absorbance of the mixtures.For the coaggregation assay, aA0 − A2)/A0) × 100, where A0 indicates the absorbance at time 0 h and A2 indicates the absorbance after 2 h.To determine the degree of hydrophobicity, we used microbial adhesion to hydrocarbons (MATH), adapted from Rodríguez et al. , using x6 cells mL−1. Lactobacilli were grown in MRS broth for 18 h. After centrifugation , pellets were recovered, washed twice with 0.9% saline, and adjusted to 1 × 108 CFU mL−1 in RPMI supplemented with 10% FBS. Lactobacilli cell suspension was added to wells containing HMVII cells  and incubated at 37°C in a 5% CO2 atmosphere. Medium was added to the wells containing HMVII cells as a negative control. After 2 h of interaction, the cell monolayer was washed three times with 0.9% saline and treated with 0.25% trypsin-EDTA for 5 min. The determination of adhered lactobacilli was performed by serial dilution followed by plating on MRS agar. Plates were incubated for 48 h at 37°C and the colony forming units (CFU mL−1) were counted. The percentage of adhered lactobacilli was calculated by the following formula: adhesion (%) =  × 100.For the adhesion test, we used a methodology adapted from Santos et al. . Vaginal6 cells mL−1) were grown on glass coverslips with each one of the three strains of Lactobacillus tested in this study (1 × 108 UFC mL−1) and incubated for 2 h at 37°C in a 5% CO2 atmosphere. HMVII cells alone were used as control. Coverslips were washed three times with 0.9% saline and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.2. Next, dehydration was performed in series of increasing acetone concentrations . The samples were subjected to critical point drying and metallized with an approximately 20 nm thick gold layer to be observed in the scanning electron microscope Quanta 250 (FEI Company).In addition, scanning electron microscopy (SEM) was performed to visualize lactobacilli adhered to the vaginal cells after interaction. HMVII cells ; then wells were perforated in the agar in which culture supernatants of the different lactobacilli were added. Plates were incubated for 24 h at 37°C in a 5% CO2 atmosphere. After incubation, the presence or absence of inhibition halos around the wells was observed.First, an evaluation of the antimicrobial activity was made through the agar diffusion technique. The strain of−1 of the culture supernatants of the lactobacilli in Mueller-Hinton broth (MH) containing 5 × 105 CFU mL−1 of G. vaginalis. The same procedure was done with the following controls: lyophilized culture medium without lactobacilli (MRS control); MH without inoculum (control of sterility of the medium); MH containing 5 × 105 CFU mL−1 of G. vaginalis (positive control); and MH containing 5 × 105 CFU mL−1 of G. vaginalis and 12.5 μg mL−1 of chloramphenicol (negative control). The microtiter plates were incubated for 24 h at 37°C in a 5% CO2 atmosphere with inhibition being observed by the absence of turbidity in the wells. To confirm whether the supernatants had a bactericidal or bacteriostatic effect, the contents of the wells were plated onto blood agar and then incubated at 37°C in a 5% CO2 atmosphere for 24 h [Microdilution technique was performed to determine minimum inhibitory concentration (MIC) in 96-well microtiter plates according to the recommendations of the Clinical and Laboratory Standards Institute, CLSI . Serial  G. vaginalis, we used the bacterial coculture technique described by Coudeyras et al. [8 CFU mL−1 was made for each microorganism. The strain of G. vaginalis was cultivated alone (control) or with each strain of the three lines of Lactobacillus, in a ratio of 1 : 1 at 37°C in a 5% CO2 atmosphere for 24 h. Aliquots were removed after 4, 8, and 24 h, serially diluted, and plated onto blood agar plates to determine the microbial count of G. vaginalis after interaction. Plates were also incubated at 37°C for 24 h in a 5% CO2 atmosphere.To evaluate the influence of lactobacilli on the growth ofs et al. . The ass Lactobacillus strains to antimicrobials was determined by the modified agar diffusion method of CLSI [μg), amoxicillin (10 μg), ampicillin (10 μg), cefalotin (30 μg), ciprofloxacin (5 μg), clindamycin (2 μg), chloramphenicol (30 μg), erythromycin (10 μg), gentamicin (10 μg), nitrofurantoin (300 μg), norfloxacin (10 μg), penicillin G (10 μg), tetracycline (30 μg), and vancomycin (30 μg).Susceptibility of of CLSI . Lactoba of CLSI . The antp < 0.05.All experiments were performed in triplicate. Quantitative data are presented by mean and standard deviations that were analyzed using GraphPad Prism 5.01. Statistical differences between mean values were determined using ANOVA and Tukey's posttest with  L. plantarum isolated from cocoa fermentation showed autoaggregation values of 18.08 and 20.94% [ L. fermentum strains isolated from fermented Chinese products presented autoaggregation ranging from 0.86 to 65.15% [All three strains of lactobacilli tested in this study showed percentage of autoaggregation around 30% after 5 h of incubation . Autoaggo 65.15%  L. fermentum 5.2 and L. plantarum 6.2 showed moderate hydrophobicity  while L. plantarum 7.1 was highly hydrophobic (71.20%). These values of hydrophobicity are much higher than those found for other lactobacilli isolated from cocoa fermentation. Ramos et al. [ L. fermentum and three strains of L. plantarum, obtained hydrophobicity values ranging from zero to 1.4%. Santos and coauthors [ L. fermentum and 6 strains of L. plantarum, obtained values that varied from 3.5 to 16.9%, with the highest value attributed to a strain of L. plantarum.Hydrophobicity, also known as microbial adhesion to hydrocarbons (MATH), together with autoaggregation, is considered an important bacterial surface feature and can be classified into 3 categories: low (MATH < 33%), medium (33% < 66%), or high (MATH > 66%) . In thiss et al. , testingoauthors , analyzi Lactobacillus to HMVII epithelial vaginal cells, the strains L. fermentum 5.2 and L. plantarum 6.2 showed similar or almost equal percentage , whereas L. plantarum 7.1 was significantly more adhesive (55.75%). It was possible to confirm this result by scanning electron microscopy images. L. plantarum 7.1 presented more bacteria adhered to HMVII cells when compared to the other two strains , as well as adhesion to host cells, are important criteria for the selection of probiotic bacteria strains , 21, 28. L. plantarum strains used in this study (but not L. fermentum 5.2) showed antimicrobial activity against G. vaginalis, notable by the presence of inhibition halos around the wells. The supernatant halos of L. plantarum 6.2 and L. plantarum 7.1 were 12 and 11 mm, respectively  was determined, inhibition ofrnatants . L. plantarum supernatants alone may be related to their acidity, since the supernatants of L. plantarum 6.2 and L. plantarum 7.1 had pH of 3.81 and 3.77, respectively, while the pH of the supernatant of L. fermentum 5.2 was 4.78  isolated from poultry litter was 3.83 and 3.88, respectively.Antibacterial activity ofwas 4.78 . The cul of 3.81 . On the  of 4.16 . Poppi e of 4.16  showed t Escherichia coli, Staphylococcus aureus, Proteus vulgaris, Klebsiella pneumoniae, and Gardnerella vaginalis [ Salmonella typhimurium and Shigella dysenteriae when the pH was adjusted to values above 7.0. Antagonistic effects related to acid production (mainly lactic acid) have already been demonstrated for lactobacilli isolated from several sources. In these studies, exposure to high temperature or protease treatments did not significantly alter the antimicrobial activity of culture supernatants [Studies by other authors using the agar diffusion technique have shown that lactobacilli culture supernatants isolated from the vaginal microenvironment displayed inhibitory activity againstaginalis –40. Afteaginalis . The samaginalis , where mrnatants , 40. L. fermentum strain isolated from cocoa fermentation was able to inhibit the growth of S. aureus with an MIC of 20 mg mL−1. This effect, as found in our study for L. plantarum strains, was bactericidal and was confirmed by plating of treated culture. Similarly, a culture supernatant of a L. paracasei strain isolated from fermented milk was also shown to inhibit bacterial growth of pathogens, especially E. coli, with an MIC of 15.6 mg mL−1 [ Serratia marcescens, values around 0.16 mg mL−1 of the culture supernatants from strains belonging to the species L. acidophilus and L. plantarum were required [In a study conducted by Melo et al. , the cul mg mL−1 . Howeverrequired . G. vaginalis, greater than 40% ( G. vaginalis bacteria by Lactobacillus strains is a well-known and desired effect of strains for potential vaginal probiotic application. In fact, other authors found that vaginal isolates of L. acidophilus, L. gasseri, and L. jensenii showed high coaggregation activity against C. albicans, E. coli, and G. vaginalis [ L. salivarius and L. gasseri with G. vaginalis. In addition, strains of L. fermentum and L. plantarum isolated from cocoa fermentation efficiently coaggregated with E. coli, Shigella flexneri, Salmonella enterica, L. monocytogenes, and S. aureus [The three strains of lactobacilli tested in our study showed high coaggregation values after incubation withthan 40% . Reductiaginalis  demonstr. aureus , 30. Coa. aureus . Lactobacillus strains were able to reduce by one log unit the microbial counts of G. vaginalis after 24 hours of incubation when compared to G. vaginalis growing alone ( L. fermentum 5.2 was able to maintain inhibitory activity against G. vaginalis during all the time period evaluated. It has been previously found that L. acidophilus, L. jensenii, L. gasseri, and L. crispatus isolated from the vaginal microbiota of healthy women showed inhibitory activity, demonstrated by the coculture technique, against G. vaginalis and Prevotella bivia with stable inhibition from the first hour [ L. rhamnosus strain, demonstrated inhibition of G. vaginalis, P. bivia, and C. albicans after 8 hours, with significant inhibition of G. vaginalis after 24 hours. Other pathogens that are also capable of causing bacterial vaginosis, such as E. coli and S. aureus, also have their growth affected when cocultivated with strains of L. plantarum and L. fermentum: after 24 hours, a decrease of up to three logs was observed when compared to controls [The coculture technique is able to assess the influence of one microorganism on the growth of another when both are incubated together. We observed that allng alone . Only L.rst hour . These rrst hour  who, usicontrols .8 lactobacilli per mL was used in the coaggregation and coculture assays. Results found were satisfactory and promising, since such concentration was able to inhibit the growth of G. vaginalis after interaction. Commercial formulations and in vivo studies show that a concentration ranging from 108 to 109 CFU is required to achieve the same result [In the present study, a concentration of 10e result –49. Lactobacillus and does not present a risk of being transferred through horizontal genetic transfer to the bacteria of the native intestinal microbiota [Susceptibility of lactobacilli isolated from cocoa fermentation to different antimicrobials is shown in crobiota , 51, 52. G. vaginalis by lactobacilli and their metabolites, as well as evaluating the immunomodulatory capacity of lactobacilli isolated from cocoa fermentation.Lactobacilli used in this study may protect the vaginal environment through multiple mechanisms, including adhesion to the epithelium, coaggregation with potential pathogens, and production of antagonistic molecules. They are promissory strains for the development of prophylactic agents. These results may serve as a basis for further studies aimed at investigating molecular mechanisms related to the inhibition of


To describe the prevalence of alcohol dependence and to explore the relationship between alcohol dependence and newly detected hypertension in China.A multistage stratified cluster sampling method was used to obtain samples from February to June 2013. The Michigan Alcoholism Screening Test was used to estimate alcohol dependence level. A standard questionnaire measured other independent variables. Enumeration data were analyzed using chi-square; quantitative data were analyzed using t-tests. Spearman correlation analysis and multivariate logistic regression analysis were performed to identify the relationship between alcohol dependence and hypertension.P < 0.01). Alcohol dependence was positively correlated with systolic blood pressure  and diastolic blood pressure  and was an independent risk factor for hypertension after adjusting for confounders .The alcohol dependence rate was 11.56%; 22.02% of males (3854/17501) and 1.74% of females (324/18656) were classified as alcohol dependent. The newly detected hypertension rate was 9.46% (3422/36157). Significant associations were found between alcohol dependence levels and blood pressure (Alcohol dependence was high and associated with hypertension. Health education and precautions against alcoholism should be implemented in Xuzhou city. Alcohol dependence involves a range of behavioral, cognitive and physiological symptoms, of which the main characteristic is “preoccupation with alcohol overriding other interests, persistent drinking despite obvious harm and withdrawal symptoms on stopping” . AlcoholAlcohol consumption has a causal impact on a number of diseases, including several types of cancer, ischemic heart disease, ischemic and hemorrhagic stroke and liver cirrhosis . QuintanThis population-based, cross-sectional survey was conducted in Xuzhou, Jiangsu, China, from February 2013 to June 2013. The Probability proportional to size was used to select the sample from all 11 regions in Xuzhou city. First, each region selected five subdistricts/townships in urban/rural areas. Second, each subdistrict/township selected five communities/villages. Final, each household selected one person who had lived in their current residence for at least 5 years and was at least 18 years old with a Kish selection table. Participants who met one of the following criteria were excluded: (1) all patients with a previous diagnosis of hypertension were excluded because behavior control might be a confounder; (2) members of the floating population or temporary residents; (3) individuals with a disability or other severe diseases . Participants were interviewed face-to-face by trained investigators on the day of their regular medical appointments. A total of 39,454 people were selected and 36,157 participated in the survey. The overall response rate was 91.6%.Each participant was measured three times blood pressure in a sitting position after 5 min of rest by trained and certified observers according to the recommended procedure by the American Heart Association . The par2), normal weight (18.5 kg/m2 to 23.9 kg/m2), overweight (24.0 kg/m2 to 27.9 kg/m2) and obese (≥28.0 kg/m2) [Age, sex, body mass index (BMI), marital status, average monthly income, level of education, cigarette smoking, physical activity and family history of hypertension were recorded using a standard self-designed questionnaire. Body height, weight and blood pressure were measured using standard equipment and methods, and BMI was defined as underweight < 18.5 kg/m.5 kg/m2,0 kg/m2) . Alcohol0 kg/m2)  was used0 kg/m2) . Higher 0 kg/m2) . The sen0 kg/m2) .All the investigators and staff members were carefully trained using standardized protocols for the questionnaires, blood pressure measurements and anthropometric measurements.All statistical analyses were conducted using SPSS 18.0 . Data were entered twice independently using EpiData 3.1 and checked for errors and inconsistencies to enhance accuracy. Enumeration data were compared using chi-square and quantitative data were compared using t-tests. Spearman’s rank correlation was used to estimate the relationship between alcohol dependence and newly detected hypertension. Logistic regression analysis was performed to measure the probability that alcohol dependence predicts hypertension with adjusted risk factors including age, sex, BMI, marital status, average monthly income, level of education, cigarette smoking and physical activity. All confidence intervals (CIs), estimations and tests used a two-sided error rate of 5%. Continuous variables were described using mean ± standard error or median and quartiles.P > 0.05). The characteristics of the participants are shown in Table There were 39,454 participants and 3297 individuals declined to take part. The mean age of participants was 45.5 ± 16.6 years, and 48.40% were male. The mean age of non-respondents was 45.1 ± 16.8 years, and 49.1% were male. 6178 participants (18.8%) who had the history of hypertension were excluded. There were no significant differences between participants and non-respondents . A total of 4178 participants (11.56%) met the criteria of alcohol dependence. There were significant differences in alcohol dependence for sex, age, areas, marital status, average monthly income, education level, physical activity level and body type  . Spearman’s rank correlation analysis also showed that alcohol dependence was positively correlated with systolic (r = 0.071) and diastolic (r = 0.077) blood pressure . Individuals who consumed alcohol had significantly increased risk of hypertension compared with non-drinking , after adjusting for age, sex, BMI, marital status, average monthly income, level of education, family history of hypertension, cigarette smoking, physical activity and area.A total of 4999 participants were classified as alcohol drinking. The rate of alcohol drinking was 13.83% (4999/36157). The rate of alcohol drinking of men was higher than that of women(χP < 0.01).Of the 36,157 participants, 3422 had hypertension and the prevalence was 9.46%. Participants’ systolic blood pressure and diastolic blood pressure were 120.3 ± 11.6 mmHg and 77.0 ± 7.7 mmHg, respectively. The Table P < 0.01; for light alcohol dependence, OR: 1.35, 95% CI: 1.11–1.64, P < 0.01; for medium alcohol dependence, OR: 1.83, 95% CI: 1.40–2.41, P < 0.01. These results are shown in Table Multiple logistic regression models indicated that alcohol dependence was an independent risk factor for hypertension after adjusting for confounders including age, sex, BMI, marital status, average monthly income, level of education, family history of hypertension, cigarette smoking, physical activity and area; the total OR for alcohol dependence was 1.37, 95% CI: 1.19–1.71. For low alcohol dependence, OR: 1.44, 95% CI: 1.14–1.81, This study produced three main findings. First, alcohol dependence was associated with increased odds of having hypertension. Second, the association between alcohol dependence and hypertension increased with greater levels of alcohol dependence. Third, the alcohol drinking rate and the alcohol dependence rate were 13.83% and 11.56%, respectively.As the results indicate, the alcohol drinking rate of participants living in Xuzhou was lower than the average drinking rate in China  (21.10%)r = 0.513, P = 0.023; r = 0.571, P = 0.005, respectively) [Numerous epidemiologic studies have been conducted to clarify the association between alcohol consumption and hypertension. However, the resultant findings are not consistent. Some studies have shown that alcohol consumption increases the risk of hypertension. For example, Li et al. reported that non-drinking Chinese adults had a lower prevalence of hypertension than current drinking . Wang anctively) , which ictively)  demonstrctively) . In addictively) , 34. Forctively) . Howeverctively) . Moreovectively) , 34.P < 0.001) and a trend toward decreased risk of hypertension with alcohol consumption 11 to 20 g/d , whereas a significantly increased risk of hypertension was associated with heavy alcohol consumption of 21 to 30 g/d  and 31 to 40 g/d  [A meta-analyses of 16 prospective studies with 227,656 subjects  demonstrated that alcohol use is consistently associated with increased hypertension risk. This meta-analysis showed that, compared with non-drinking, heavy alcohol consumption in men is associated with increased risk of hypertension, and low and moderate alcohol consumption is associated with a trend toward increased risk of hypertension. Among women, the meta-analysis indicated protective effects at < 10 g/d  . These f+ and Na+ in vascular smooth muscle cells and causes vasoconstriction [The multiple logistic regression models showed that alcohol dependence was an independent risk factor for hypertension even after adjusting for confounders, and the odds ratios gradually increased with greater alcohol dependence. However, the biological mechanism underlying the development of hypertension in association with alcohol use remains unclear. Several hypotheses have been suggested to explain this process: (1) alcohol influences the function of the vascular endothelium by activating the renin–angiotensin–aldosterone system and inhibiting vasodilation ; (2) thetriction ; and (6)triction , 42. HowAlthough the confounding factor that behavior control might influence living habits was adjusted by excluding patients who had already been diagnosed before our study, some limitations should be noted. First, this was a cross-sectional study; therefore, we could establish only an association between variables, not the presence of causation. Further randomized controlled or cohort studies are required. Second, information about alcohol dependence was self-reported by participants, not objectively measured. Therefore, the possibility of recall bias cannot be completely rejected. Third, dietary habits were not considered in this study; drinking generally consume a salty and unbalanced diet , which mIn summary, our study showed that the prevalence of alcohol dependence is a higher in the Chinese city of Xuzhou City, and alcohol dependence was associated with increased hypertension risk. Thus, one of the priorities should be given to strengthening health education and precautionary and family health measures for excessive drinking. Further well-designed studies are needed to strengthen our conclusions.


Inhaled steroid resistance is an obstacle to asthma control in asthmatic smokers. The reasons of this phenomenon are not yet entirely understood. Interaction of drug particles with environmental tobacco smoke (ETS) could change the aerodynamic profile of the drug through the particle coagulation phenomenon. Aim of the present study was to examine whether steroid particles interact with smoke when delivered in the presence of ETS.Beclomethasone-hydrofluoralkane (BDP-HFA) pMDI particle profile was studied after a single actuation delivered in ambient air or in the presence of ETS in an experimental chamber using a light scattering Optical Particle Counter capable of measuring the concentrations of particle sized 0.3–1.0, 1.1–2.0, 2.1–3.0, 3.1–4.0, 4.1–5.0, and > 5.1 μm in diameter with a sampling time of one second. The number of drug particles delivered after a single actuation was measured as the difference between total particle number after drug delivery and background particle number. Two groups of experiments were carried out at different ambient background particle concentrations. Two-tail Student's t-test was used for statistical analysis.When delivered in ambient air, over 90% of BDP-HFA particles were found in the 0.3–1.0 μm size class, while particles sized 1.1–2.0 μm and 2.1–3.0 represented less than 6.6% and 2.8% of total particles, respectively. However, when delivered in the presence of ETS, drug particle profile was modified, with an impressive decrease of 0.3–1.0 μm particles, the most represented particles resulting those sized 1.1–2.0 μm , and 2.1–3.0 μm particles accounting up to 31% of total particles.Our data suggest that particle interaction between inhaled BDP-HFA pMDI and ETS takes place in the first few seconds after drug delivery, with a decrease in smaller particles and a concurrent increase of larger particles. The resulting changes in aerosol particle profile might modify regional drug deposition with potential detriment to drug efficacy, and represent a new element of steroid resistance in smokers. Although the present study does not provide any functional or clinical assessment, it might be useful to advise smokers and non smokers with obstructive lung disease such as asthma or COPD, to avoid to act inhaled drugs in the presence of ETS in order to obtain the best therapeutic effect. Suspended particles in aerosol phase are subject to dynamic changes, mainly due to the process of coagulation, whereby particles collide with one another, and adhere to form larger particles, due mainly to brownian motion ,2. ColliETS is one of the most common cause of indoor pollution , and is Although inhaled steroids are the cornerstone of asthma therapy, their efficacy is dramatically reduced in asthmatic smokers, compromising asthma control, a phenomenon called "steroid resistance" -24. So fAccording to pharmaceutical guidelines, inhaled steroids are studied in ambient air , and no The aim of the present report was to investigate if particles size distribution of beclomethasone-hydrofluoralkane pressurized metered dose inhaler (BDP-HFA pMDI) is modified when the pMDI aerosol is delivered in the presence of ETS.3 acrylic chamber along with temperature and relative humidity control devices, and with the inner surface coated with antistatic paint to avoid losses of particles due to electrostatic charges. A scheme of the experimental setting is shown in Figure A series of experiments was carried out by measuring BDP-HFA pMDI  particle size distribution in ambient air (background room air) or in the presence of ETS (background room air additioned with tobacco smoke produced by consuming a smouldering cigarette for 0.5 cm from the tip). The optical particle counter based on real time laser diffraction  was used, capable of measuring the concentration of particles in the range size of 0.3–1.0, 1.1–2.0, 2.1–3.0, 3.1–4.0, 4.1–5.0, and > 5.1 μm with a sampling time of 1 second . The anaTwo different groups of experiments were carried out at different times using the same laboratory setting, but with a different serial number of the same particle counter model, and at different ambient background particle concentrations. Background particle concentrations after ETS injection were similar in the different experiments. The first group of experiments was done in triplicate (three ambient and three ETS tests), while the second one was done in quadruplicate. Statistical data analysis was performed with two-tail Student's t-test.An explanatory picture of real time measurement before and after the drug delivery in ambient air is shown in Figure Table Background particle concentrations were stable during the tests, with a mean ± SD particle concentration (pooled data) of 1545 ± 163, 51 ± 35, 58 ± 42, 30 ± 34, 13 ± 14 and 35 ± 31 p/l for the same size classes, respectively.Figure When the same set of experiments was carried out with BDP-HFA pMDI delivered in the presence of ETS, the distribution of the particle diameter changed, with a significant decrease of particle number for particle sized 0.3–1.0 μm, as compared to background particle concentrations  of 624511 ± 3218, 14542 ± 854, 4239 ± 478, 112 ± 43, 27 ± 24, and 34 ± 28 p/l for particle diameters of 0.3–1.0, 1.1–2.0, 2.1–3.0, 3.1–4.0, 4.1–5.0, and > 5.1 μm, respectively. The exceedingly high prevalence of submicrometric particles with a very low number of coarse particles is consistent with the particle composition of ETS aerosol assessed by previous studies .As shown in Figure A similar set of experiments (test group 2), was carried at a different time, with the same model of a particle counter of a different serial number, and with different background air conditions , while a mean ± SD increase of 1609 ± 46, 139 ± 9, 11 ± 4, 2 ± 2, 1 ± 2 p/l was observed for particle sized 1.1–2.0, 2.1–3.0, 3.1–4.0, 4.1–5.0, and > 5.1 μm, respectively  of 584934 ± 2764, 2422 ± 214, 158 ± 58, 25 ± 24, 9 ± 13, and 10 ± 13 p/l, for particle diameters of 0.3–1.0, 1.1–2.0, 2.1–3.0, 3.1–4.0, 4.1–5.0, and > 5.1 μm, respectively.As shown in Figure Our results indicate that delivery in the presence of ETS can affect pMDI steroid particle size distribution with a shift towards larger particle size taking place in a few seconds. In the two groups of experiments with different ambient backgrounds the particle frequency in ETS showed the disappearance of particles sized 0.3–1.0 μm in diameter, with an increase mainly in 1.1–2.0 and 2.1–3.0 μm particles.We choose extrafine HFA formulation BDP because it is a widely used inhaled steroid and because it is the only inhaled corticosteroid included in the list of essential drugs of the World Health Organization . BronchiThe growth in particle size might change drug deposition in the small airways, which are involved in inflammation processes and bronchoconstriction both in asthma and in COPD, even though such a principle may not be working for other drugs. In fact, Usmani and co-workers reported that larger particles were more efficacious than smaller particles for the beta-agonist albuterol . InteracAlthough active and passive smoking are regarded as important risk factors for asthma exacerbations, and in spite of the fact that the issue of poor asthma control in current smokers is drawing attention also in the primary care setting ,33, no sThe results obtained with the real time laser-operated analyser were consistent and repeatable. However this technique has some limitations, such as the possibility of interferences due to coincidence losses in particle count at high particle concentrations: therefore, data for particles sized 0.3–1.0 μm in diameter were corrected according to a proper equation. In addition, our data showed a concurrent increase in the number of particles with larger size class, a result difficult to explain by a technical artifact. Another limitation of the optical particle counter technology is that it measures only particle number and size, without characterizing the kind of particles measured and determining their mass. The results are therefore only suggestive of particle coagulation. Further studies with different methodologies such as Andersen cascade impactor and Scanning Electron Microscopy technology are needed to provide such an information and confirm the presence of the phenomenon . As for BDP-HFA pMDI particle profile showed small differences in the two different tests groups carried out at different times in ambient air: particle frequency was 90.2%, 6.7%, and 2.8% in test group 1, and 96.2%, 3.5%, and 0.4% in test group 2, for particles sized 0.3–1.0, 1.1–2.0, and 2.1–3.0 μm in diameter, respectively (see Figure vs ETS profile, accounting for over 97% of total particles, suggesting only minor changes in overall regional deposition of the drug [It should be underlined that, although in the presence of ETS the aerodynamic profile of BDP-HFA pMDI particles was deeply altered, the "load" of particles ranging between 0.3 and 3.0 μm in diameter, altogether, did not change when comparing ambient air the drug -31.Although much higher than the ambient air counterpart, the mean levels of background submicrometric particle concentrations used in the experiments with ETS represent real world indoor concentrations which can be currently encountered in ETS polluted places, reaching levels of several hundred thousand particles per liter ,9,10,27.5 to ~6 × 104/cm3 in total ETS particles in the first 15 minutes since generation [The possibility of spontaneous "self coagulation" of ETS deserve a comment. A particle size increase between 20 and 50% along with a concurrent reduction in total particle number were reported by Morawska and co-workers during the first 30 to 60 minutes after of ETS production [Another issue to be discussed is the rapidity of particle interaction. In a recent study, Seipenbush and co-workers were able to demonstrate that the injection of 0.2 μm sebacate droplets into a background aerosol of platinum nanoparticles induced a heterogeneous coagulation process measurable in the first 4 minutes of interaction . The autIn summary, our data showed that the particle profile of the inhaled steroid BDP-HFA pMDI is altered when the drug is delivered in the presence of ETS. Although further studies are needed to confirm these findings, and although the present study does not provide any functional or clinical assessment, the results are relevant to the phenomenon of steroid resistance .Smoking has been shown to impair the clinical response to systemic steroids through biochemical mechanisms affecting steroid pharmacodynamics at the cellular level ,42. Our The authors declare that they have no competing interests.GI and AR conceived the study, carried out the particle interaction studies, and performed the statistical analysis. CDM, RM, GN and RB participated in the design of the study, and helped to draft the manuscript. All authors read and approved the final manuscript.


Enterococcus faecalis S37, a strain recently isolated from the chicken feces. Enterocin S37 has a molecular weight comprised between 4 and 5 kDa. It remained active after 1 h at 80oC and at pH values ranging from 4.0 to 9.0. Furthermore, cell-free supernatant of Enterococcus faecalis S37 and purified enterocin S37 were active against Gram-positive bacteria including Listeria monocytogenes EGDe, L. innocua F, Enterococcus faecalis JH2-2, and Lactobacillus brevis F145. The purification of enterocin S37 was performed by ammonium sulfate precipitation followed up by hydrophobic-interaction chromatography procedures. Treatment of enterocin S37 with proteinase K, α-chymotrypsin, and papain confirmed its proteinaceous nature, while its treatment with lysozyme and lipase resulted in no alteration of activity. Enterocin S37 is hydrophobic, anti-Listeria and likely acting by depletion of intracellular K+ ions upon action on KATP channels. This study contributed to gain more insights into the mode of action of enterocins.The aim of this research was to purify and characterize the mode of action of enterocin S37, a bacteriocin produced by  Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria that deserve considerable interest for their use as natural and nontoxic food preservatives as well as for their potential use in human and veterinary applications, and in the animal production field . In 1976β-methyl lanthionine (hence the name lantibiotics) and dehydrated residues. Nisin is the most characterized of the class I bacteriocins. Class II is defined as small heat-stable nonlanthionine containing membrane active peptides characterized by the presence of a Gly-Gly processing site in the bacteriocins precursor, the presence of amphiphilic helices with varying amounts of hydrophobicity and moderate to high-heat stability. These were further subdivided into three subgroups named subclass IIa, which are Listeria active peptides and YGNGV box in the N-terminal [Klaenhammer  suggesteterminal ; subclasterminal  and subcterminal . Class Iterminal .Enterococcus strains from various ecosystems were characterized as antagonists of broad range of foodborne pathogens [Staphylococcus and Streptococcus and Gram-negative bacteria like Campylobacter or Salmonella, strains of medical interest [E. faecalis from chicken drop [Many athogens , 13. Theathogens . Most ofathogens , 4 and aathogens . As repointerest , 4, 16. ken drop  which apE. faecalis S37 was isolated from poultry feces collected in a French farm located in Ancenis (West of France) [E. faecalis S37 was identified by sequencing whole 16S rRNA gene [E. faecalis S37 showed in first instance an antagonism directed against strains belonging to Listeria and Campylobacter species [E. faecalis S37, E. faecalis JH2-2, Lactobacillus brevis F1.114, Lb. bulgaricus 340, Lb. brevis F145, Pseudomonas sp. 131, Escherichia coli CIP 76.24, Salmonella montevideo, Sal. enterica serotype enteritidis CIP 81.3, Staphylococcus epidermidis CIP 68.21, Bacillus cereus CIP 78.3,and Leuconostoc mesenteroides subsp. mesenteroides DSM 20240 were grown on Brain Heart Infusion broth (BHI) medium , whilst L. monocytogenes EGDe and L. innocua F were grown on Elliker medium (AES). Campylobacter jejuni NCTC 11168 was grown in Brucella medium (AES).The antibacterial activity of enterocin S37 was assessed against the Gram-negative and Gram-positive bacteria listed in μL from overnight culture of targeted strains was added to 20 ml of Elliker or BHI soft agar medium already containing 0.8% (w/v) of agar . Wells were performed in solid agar and 50 μl of each sample tested was poured into the wells. The Petri dishes were left at room temperature, in sterile conditions, for 1 h before incubation for 18 h at adequate temperature according to the target strain to be tested. After this period of incubation, the antibacterial activity was detected by observing the inhibition zones around the well containing the CFS. It should be noted that supernatant was obtained by centrifuging at 15000 g during 30 min at 4°C, an overnight culture of E. faecalis grown at 37°C for 18 to 24 h, on de Man Rogosa Sharpe (MRS) medium [μm membrane .The antibacterial activity was measured by the well known agar diffusion test . For thi) medium  (AES). T2HPO4, 25 mM KH2PO4, pH 7) filtered through 0.2 μm membrane (Sartorius). These samples were tested by agar diffusion test on L. monocytogenes EGDe strain, as described previously. One arbitrary unit was defined as the reciprocal of the lowest dilution that did not show growth of the target strain.Arbitrary Unit (AU) was calculated as described by Batdorj et al. . Serial 2HPO4, 25 mM KH2PO4, pH 7); the active one was applied onto RP-HPLC column  previously equilibrated with solvent A . Elution was performed by using a gradient from 0% solvent B  to 100% solvent B in 45 min. The flow rate was adjusted to 3 ml.min−1 and the absorbance was recorded at 220 and 280 nm. Fractions of 10 ml were collected and tested for their antibacterial activity. The acetonitrile was removed from the active fractions by Speed-Vac concentrator  and pH was adjusted to 6.8 using phosphate buffer . The active fractions were pooled and the purification of the bacteriocin was continued by RP-HPLC on a Waters Alliance apparatus with Millennium software . Concentrated bacteriocin (80 μl) was injected into an analytical C18 column  equilibrated in 0% solvent C (0.045% TFA in H2O) and 50% solvent D . The elution was performed with a linear gradient from 30% to 100% solvent D in 30 min. The flow rate was adjusted to 0.8 ml min−1. The eluted peaks were detected by spectrophotometry measuring the absorbance between 210 and 300 nm with a photo diode array detector  and collected manually. The fractions were then concentrated in a Speed-Vac concentrator.The CFS neutralized and filtered (400 ml) was precipitated with ammonium sulfate to 80% saturated solution. Three distinct fractions were recovered and were resuspended in 40 ml of phosphate buffer (25 mM Kα-chymotrypsin (1 mg · ml−1), papain (1 mg · ml−1), proteinase K (0.5 mg/ml−1), lysozyme (2 mg · ml−1), and lipase (1 mg · ml−1)  on the antibacterial activity of enterocin S37 was checked by the agar diffusion assay as described by Batdorj et al. [μl of supernatant at 80°C for 15 min, 80°C for 1 h and 90°C for 15 min. After cooling in ice, each sample was tested for its antagonism by agar well diffusion assay [The effect of j et al. . CFS wasj et al. . The staon assay .L. monocytogenes EGDe and incubated overnight at  37°C.In order to determine the apparent molecular mass of enterocin S37, the SDS-PAGE gel was cut into two parts. The part of the gel containing the molecular markers and the samples were stained by silver nitrate coloration method , and theL. monocytogenes EGDe at 30°C in tryptic soy broth (Biokar diagnostics) supplemented with 0.6% (w/v) yeast extract  and called TSYE broth (TSYEB). Enterocin S37 was added at 2- 4- 6- 8- and 10-fold the minimal inhibitory concentration (MIC). The MIC was determined for L. monocytogenes EGDe according to Naghmouchi et al. [Resistant variants to enterocin S37 were developed by stepwise culture of strain of i et al.  using miL. monocytogenes EGDe and its bacteriocin-resistant variant were grown at 30°C for 24 h in TSYEB in the presence of enterocin S37 at 5 μg · ml−1and K+ channel modulators such as pinacidil (Pi) at 10 μg · ml−1, cromakalim (Cro) at 10 μg · ml−1 and glipizide (Gli) at 10 μg · ml−1 Combination of enterocin S37 with each of K+ channel modulator was also realized in similar conditions. All these K+ channel modulators were obtained from Sigma-Aldrich (Germany). Growth was monitored by measuring the optical density at 595 nm (OD595) of 250 μl of freshly inoculated medium placed into microplate wells [te wells .+ channel modulator was calculated as described by Naghmouchi et al. [595(x)/OD595(i)) . The value (x) was the culture containing inhibitor and (i) was the uninhibited control culture. Data were expressed as the percent change in IA obtained with the enterocin S37/K+ channel modulator.The inhibitory activity (IA) of enterocin S37 in presence or absence of each Ki et al. . ClearlyE. faecalis S37 displayed activities against E. faecalis JH2-2, Lb. brevis F1.114, Lb. brevis F145, L. monocytogenes EGDe, and L. innocua F. However, none antibacterial activity was detected against the remaining strains listed in Campylobacter activity previously observed [Enterococcus genus is interesting in order to generate novel knowledge on immunity proteins and cross-resistance within this genus.CFS of E. faecalis S37 displayed activity against the target strains E. faecalis JH2-2, Lb. brevis F1.114, Lb. brevis F145, L. monocytogenes EGDe and L. innocua F even after treatments with lipase (1 mg · ml−1) and lysozyme (2 mg · ml−1). The antibacterial activity observed upon lipase and lysozyme treatments was similar to that observed with the untreated supernatant used as positive controls. However, treatment with proteolytic enzymes, such as α-chymotrypsin, papain, and proteinase K resulted in the loss of the bacteriocin activity ; the experiment was not conclusive because of toxicity of this compound, even at low concentrations, towards the target strains.CFS of activity . The staactivity . Attempt2O, and 0.05% TFA. The purity of each active fraction collected after each step was checked on SDS-PAGE ; subsequently, the active fraction was passed through two successive RP-HPLC columns. Active fractions  were eluted at 60% of elution buffer containing 80% acetonitrile, 20% HSDS-PAGE .L. monocytogenes EGDe with 1% volume transfer (v/v). The other proteins bands revealed by electrophoresis were devoid of antibacterial activity. The antimicrobial activity of each sample, obtained during purification process, was also checked by agar diffusion test on Petri dish  and inhibitor (glipizide) affected differently the growth of L. monocytogenes EGDe and its bacteriocin-resistant variant to enterocin S37 obtained in this research. As shown in μg · ml−1) and pinacidil (10 μg · ml−1). Effect of the combinations of enterocin S37 with cromakalim (10 μg · ml−1) and glipizide (10 μg · ml−1) was less important on L. monocytogenes EGDe bacteriocin-resistant variant.Both K8 cfu/g of feces [Enterococci are known to be widespread in nature. For most enterococcal species, the predominant habitat is the gastrointestinal tract of animals and humans where they can be found in numbers as high as 10of feces . Enterocof feces . Additioof feces .E. faecium and E. faecalis for their capabilities to produce enterocins and they concluded that 51% of isolates were producers of enterocins and 46% of them were able to produce more than one enterocin. The strains producing enterocins were carrying at least one structural gene coding for the most known enterocins  [E. faecalis as sources of enterocins [E. faecalis recently isolated from chicken feces [L. monocytogenes EGDe and casually against Campylobacter jejuni NCTC 11168  has a molecular weight < 10 kDa, (iii) was sensitive to proteases, and (iv) was stable at different pH and high temperatures.Many enterococci were reported to produce bacteriocins called enterocins, which are various, having great diversity in their structure, and active against numerous microorganisms, especially foodborne pathogens and against microorganisms of environmental and medical interests , 14, 23.nd cylL) . Other sterocins , 25. Takterocins . We haveen feces . EnterocTC 11168 . However−1 to 51,200 AU · ml−1) during the purification process  and one K+ channel blocker (glipizide). Cromakalim was shown to act on ATP-sensitive potassium channels and caused membrane hyper polarization, pulls their membrane potential away from the threshold [+ channels, reducing K+ conductance and causing depolarization of the membrane, which leads to Ca++ ions influx through voltage-sensitive Ca++ channels, causing rising of the intracellular concentrations of Ca++ ions [L. monocytogenes strains was studied and resulted to be overall weak. Thus, the inhibition of wildtype (sensitive phenotype) to cromakalim and glipizide was estimated to 14.29% ± 1.38 and 17.14% ± 4.08 while the inhibition of the mutant strain (resistant phenotype) was evaluated to 4.46% ± 3.53 and 8.93% ± 1.06. Pinacidil showed high-inhibitory activity on the wildtype (86.79% ± 1.78) and interestingly not on the mutant strain (19.64% ± 4.02).For the first time, the effect of Khreshold . In the hreshold . Pinacidhreshold . Finallya++ ions , 32. Fur+ channel modulators suggested an implication of enterocin S37 in a depletion of intracellular K+ level, by an action on KATP channels rather than Kv channels. These data are in good agreement with those obtained formerly with nisin A, whose antibacterial activity was connected to KATP channels with concomitant loss of all intracellular K+ and ATP [+ and affecting sensitive cells growth. The resistant variant was able to grow but was slightly affected by the combination of enterocin S37/K+ modulators, except for pinacidil, where the inhibitory effect has reached 49.79% ± 3.36, instead of 23.84% ± 7.73 and 24.11% ± 7.07 when cromakalim and glipizide were added, respectively (Combinations of enterocin S37 and different K and ATP . As for  and ATP , 34, entectively .L. monocytogenes EGDe. However, the combination of enterocin S37/K+ channel modulators impacted strongly the growth of resistant variant. Otherwise, the K+ channel modulators enhanced the inhibitory activity of enterocin S37 on sensitive strain of L. monocytogenes EGDe, with values reaching, 92.86% ± 5.90 and 94.29% ± 4.55 when enterocin S37 was combined with glipizide and pinacidil, respectively. Combination of cromakalim and enterocin S37 resulted in 84.29% ±  2.5 inhibition of the growth of sensitive strain; this data is equal to that obtained with enterocin S37 alone 84.21% ± 5.87 (It was also well established that many bacteriocins, including enterocins, interact with membrane lipids leading to the formation of pores and subsequently to the loss of intracellular components, resulting thereof in the cell death –39. The % ± 5.87 . However% ± 5.87 .The mode of action of enterocin S37 could be triggered by potential interaction with cell membrane. The determination of aminoacid sequence of enterocin S37 is under consideration.


The results obtained in the present study reveal that the residues of the three herbicides under investigation were predominantly confined to the upper soil layer (0–6 cm). Comparatively, low mobility of these herbicides in soils could be due to strong adsorption of these chemical to soil colloids.A laboratory experiment was conducted by using three herbicides, two from dinitroaniline group and one from thiocarbamate group to know their degree of downward movement (leachability) through soil columns and their contribution in ground water contamination. Soil columns were loaded with Pendimethalin, Benthiocarb and Oryzalin at doses of 10.0, 10.0 and 7.7 kg/ha, respectively. After 30 days soil samples were analyzed from each segments  for Benthiocarb and Pendimethalin by GLC equipped with Ni OCOrganic CarbonCECCation Exchange CapacityGLCGas Liquid ChromatographGCGas ChromatographGC-MSGas Chromatography-Mass SpectrometryHPLCHigh Performance Liquid ChromatographLC-MSLiquid Chromatography-Mass SpectrometryESElectro Sprayet al., et al., The use of pesticides in crop production is inevitable to avoid the losses due to various pests infesting it at different stages of the growth. The extent to which pesticides are susceptible to transport through and from soil, and contribute to nonpoint source pollution, is dependant on the processes of biodegradation and sorption which determine the longevity and mobility of the pesticide in the soil, respectively. The movement of pesticides through soil is an important process that determines their fate in both soil and aquatic environments. Pendimethalin , Benthiocarb  and Oryzalin [S-4-chlorobenzyl diethyl thiocarbamate] –3 are usSoil sample collected at 0–15 cm depth from farmer's plot at Maricha village, Nadia, West Bengal, India were used after air drying and sieving through 2 mm sieve. A 36 cm long methacrylate column with an inner diameter of 6 cm were cut into six sections (6 cm long each) and sealed with a water-proof silicone adhesive tape. The bottom ring was filled with glass wool. A muslin cloth was fixed with rubber ring on the outer side of columns to avoid contamination of leachate with soil particles. Five rings were hand-packed with sandy loam soil (pH 7.2) collected from 0–15 cm depth after air drying and sieving through 2 mm sieve.+)/µg.Before applying the herbicides, soil columns were wetted to their apparent water holding capacity by applying 0.01 M calcium chloride to the top of the column. After this initial equilibrium, columns were loaded with Pendimethalin, Benthiocarb and Oryzalin at the doses of 10.0, 10.0 and 7.7 kg/ha, respectively. After 24 h of application, the columns were irrigated daily using 0.01 M calcium chloride solution. After 30 days soil samples were analyzed from each segment. The soil of the experimental site was silty clay loam in texture, tending towards neutral in reaction with pH 7.25, OC 0.42% and poses relatively low N status and high P, K status. The soil consists 12% sand, 65% silt and 23% clay having CEC at 25 cmol . Leachates collected daily were analyzed by partitioning the residues into dichloromethane phase thrice (100+50+50 mL).The combined organic phase was evaporated to near dryness under reduced pressure at 40 °C  coupled with Chemito 5000 data processor for Benthiocarb and Pendimethalin. The HP-5 capillary column (30 m × 0.32 mm i.d.) of 0.25 µm film thickness was used. The temperatures were: oven 210 °C, injector 225 °C and detector 350 °C. Flow rate of carrier gas (nitrogen) was 1.8 mL/min (split). The retention time, limit of detection (LOD) and limit of quantification (LOQ) were 9.13 min, 0.01 µg/g and 0.04 µg/g for Benthiocarb  with Nithiocarb  and 13.3thiocarb . The ana18), mobile phase , wave length (285 nm) and flow rate at 1 mL/min. The retention time, limit of detection (LOD) and limit of quantification (LOQ) were 6.880 min, 0.01 µg/g and 0.05 µg/g for Oryzalin  with model JASCO UV 1575 UV-VIS detector equipped with Chemito 5000 data processor applying the analytical conditions: column  with the above mentioned herbicides. The average recoveries of Pendimethalin were above 89 and 94 percent in soil and leachates, respectively. The corresponding recovery values for Benthiocarb and Oryzalin were above 87, 93 and 91, 96%, respectively .The residue content was calculated by using the formula:Where,1 = Area of Oryzalin from sample, in chromatogramA									2 = Area of Oryzalin from standard, in chromatogramA									1 = Total volume of sample (in mL)V									C = Concentration of analytical standard in ppm (µg/mL) × µl injected									W = Weight of the sample (in gm)									2 = Injected volume of sample (in µl)V									f = Recovery factorR									Linearity was evaluated by linear regression analysis.oe−KI) was confirmed by testing the statistical significance of correlation coefficient. The half-life values were calculated from dissipation constant calculated from regression analysis.The residue data were subjected to regression analysis and the fit of the data to first order kinetics . Comparatively, low mobility of these herbicides in soils could be due to strong adsorption of these chemical to soil colloids. The mobility of Oryzalin was greater in the soil column than Pendimethalin and Benthiocarb due to different soil physio-chemical properties. Comparatively low mobility of these herbicides in soil could be attributed to its strong adsorption by an alluvial soil. Due to low mobility, the chance of leaching of benthiocarb is less than other two herbicides. Therefore, the power of ground water contamination is negligible for Benthiocarb than dinitroaniline group of herbicide.


This study was conducted to assess the dose proportionality, safety, and tolerability of fentanyl buccal tablet (FBT) in Japanese volunteers.max), time to Cmax (tmax), area under the serum fentanyl concentration-time curve (AUC) from time 0 to infinity (AUC0–∞), and AUC from 0 to the last quantifiable concentration (AUC0–last) were summarized using descriptive statistics. Dose proportionality was claimed if the ln-ln plots of Cmax, AUC0–∞, and AUC0–last vs. dose were linear and the 90% confidence intervals (CI) of the slopes were within 0.8927 and 1.1073. The safety population comprised volunteers who received ≥1 FBT.Healthy, opioid-naive Japanese adults received single-dose FBT 100, 200, 400, and 800 µg in a randomized, open-label, crossover fashion. Naltrexone was given to minimize the opioid effects of fentanyl. Peak serum fentanyl concentration . However, the increase in systemic exposure with dose appeared linear, and a post hoc analysis of partial AUCs from time 0 to 8, 12, 18, and 24 hours supported dose proportionality. Median tmax of 90 minutes (range 30–180 minutes) was independent of dose. Adverse events (AEs) were mild or moderate. The most frequent AEs were nausea (N = 9), dizziness (N = 8), headache (N = 6), somnolence (N = 6), dyspepsia (N = 5), and vomiting (N = 3). No application-site or serious AEs were reported.Twenty-five volunteers were enrolled, 23 were included in the safety population (mean age 35.3 years), and 19 completed the study. The assessment of dose proportionality did not meet the statistical criteria (slope [90% CI]: 0.9118  for CSystemic exposure to FBT was approximately dose proportional across the range 100 µg to 800 µg in healthy Japanese adults. Adverse events were mild or moderate. FENTORA®, Cephalon, Inc., Frazer, PA, USA) is approved for marketing in the United States for the management of breakthrough pain in patients with cancer who are already receiving and who are tolerant to around-the-clock opioid therapy for their underlying persistent cancer pain , 1 + [ln(1.25)/ln(high/low)], where high = 800 μg (highest dose) and low = 100 μg (lowest dose). It resulted in . Dose proportionality was concluded if the plot of pharmacokinetic parameter vs. dose indicated linearity  and the 90% CI for the slope fell within the range 0.8927 to 1.1073.The sample size of volunteers was not based on a statistical power calculation but reflects the typical number of volunteers enrolled in pharmacokinetic studies. Descriptive statistics were used to summarize the pharmacokinetic parameters for FBT by dose. The slopes were obtained for natural log (ln)-transformed pharmacokinetic parameters vs. ln(dose). The primary methodology used to assess dose proportionality from 100 µg to 800 µg was a comparison of the 90% confidence interval (CI) of the slopes with a criterion CI, based on a power model . A mixed0–last, AUC0–∞, and Cmax. Treatment order, treatment, and period were fixed effects, and volunteer nested within treatment order was a random effect. The statistical analyses were performed with SAS PROC MIXED software.Analyses of variance were performed for pairwise comparisons on the dose-normalized and ln-transformed AUC0–∞ and AUC0–last was applied to the partial AUC values for AUC0–8, AUC0–12, AUC0–18, and AUC0–24.In the post hoc analysis, the same statistical analysis model for dose proportionality used on AUCThe pharmacokinetic population comprised volunteers who completed ≥2 periods of the study. The safety analysis population in this study comprised all volunteers who received ≥1 dose of FBT.Twenty-five volunteers were enrolled in the study; 23  aged 20 to 51 years  were incThe mean serum fentanyl concentration-time curves for FBT 100, 200, 400, and 800 µg are illustrated in max occurred at a median of 90 minutes (range 30–180 minutes) for all doses. Mean Cmax, AUC0–∞, and AUC0–last increased with increasing doses of FBT . The slopes (90% CIs) were 0.9118  for Cmax, 1.0756  for AUC0–∞, and 1.0992  for AUC0–last. Dose proportionality was not observed by analysis of variance performed for pairwise comparisons on ln-transformed and dose-normalized AUC0–last, AUC0–∞, and Cmax.Statistical analysis showed that the slopes of the plots for ln-transformed C0–∞, AUC0–last, and t½ values were probably underestimated. For this reason, a post hoc analysis was performed on partial AUCs.The statistical analysis may have failed to show dose proportionality because, since serum fentanyl levels fell below the limit of quantitation after 18 hours after the administration of FBT 100 µg and 200 µg in some volunteers, AUC0–8 , AUC0–12 , AUC0–18 , and AUC0–24 . An analysis of variance was also performed for pairwise comparisons on ln-transformed and dose-normalized AUC0–8, AUC0–12, AUC0–18, and AUC0–24. Dose proportionality was found for AUC0–18 and AUC0–24, but not for AUC0–8 or AUC0–12 over the 100 to 800 µg dose range.In the post hoc analysis of partial AUCs, the criteria for dose proportionality were met. The slopes of ln-transformed AUC vs. ln-transformed dose were close to 1 and their 90% CIs fell within the predefined range required for dose proportionality: AUCThe mean buccal dwell times for FBT were 48.95, 59.24, 59.27, and 68.14 minutes for the 100, 200, 400, and 800 µg doses, respectively.max of 90 minutes) or 4 hours after administration of any of the doses.As stated previously, naltrexone was administered to these healthy volunteers to minimize the opioid effects of fentanyl. In the safety analysis set, 18 volunteers had ≥1 AE, and all AEs were mild or moderate. No volunteers in the safety analysis set had serious AEs or discontinuations resulting from AEs. The most frequent AEs are summarized in max, AUC0–last, and AUC0–∞ across the dose range of FBT 100 to 800 µg in healthy Japanese volunteers. These parameters appeared to be approximately dose proportional, though they did not meet the statistical criteria for dose proportionality. Post hoc analysis of partial areas from time zero to serum sampling times at 8, 12, 18, and 24 hours  showed that all of these truncated areas were proportional to dose. The mean buccal dwell times for FBT ranged from 49 to 68 minutes; however, no association has been found between variations in dwell time and FBT pharmacokinetic parameters and early exposure as measured by Cmax, tmax, and AUC0–tmax′[The primary objective of this study was to evaluate the dose proportionality of fentanyl CUC0–tmax′.Safety and tolerability findings would not be expected to reflect those in patients because the healthy volunteers in this study were administered oral naltrexone to minimize the opioid effects of fentanyl. All AEs were mild or moderate. The most common AEs were nausea, dizziness, headache, somnolence, dyspepsia, and vomiting, a finding similar to that previously reported in healthy volunteers administered FBT and naltrexone ,8–10.A number of elements intrinsic to open-label studies in healthy individuals limit the interpretation of results, such as differences in drug metabolism in healthy persons compared with patients who have medical conditions, such as hepatic or renal impairment. Unlike healthy individuals, patients with chronic medical conditions may be taking multiple pharmacologic therapies that could alter drug metabolism.In conclusion, although the primary results from this study did not meet the statistical criteria for dose proportionality over the FBT dose range of 100 to 800 µg, a post hoc analysis examining AUCs for 0–8, 0–12, 0–18, and 0–24 hours supported dose proportionality.


Among 777 endometrial cancer cases and 1550 controls from Italy and Switzerland, odds ratio was 1.7  for diabetes, and 5.1 for obese diabetic women as compared with non-obese non-diabetic ones. Diabetes shows a supramultiplicative effect with body mass index, but not with physical activity or hypertension. An increased risk of endometrial cancer in diabetic women has often been reported ; physical activity at age 30–39 years was considered.For both cases and controls, data were collected by trained interviewers during their hospital stay using similar structured questionnaires covering education, socio-economic factors, lifestyle habits, anthropometric measures and physical activity at various ages, a validated food-frequency section, menstrual and reproductive history, and use of oral contraceptive (OC) and hormone replacement therapy (HRT). Details were also collected on personal medical history, including diabetes and hypertension. Body mass index at diagnosis and at age 30–39 years were computed as weight/heightχ2 distribution with one degree of freedom.Odds ratios (OR) according to history of diabetes and corresponding 95% confidence intervals (CIs), were estimated using multiple logistic regression models. The models included terms for age, year of interview, study centre, years of education, parity, menopausal status, OC and HRT use, plus BMI, physical activity and history of hypertension when appropriate. To test for heterogeneity, we compared the differences between the −2 log likelihood of the models with and without interaction term with the vs 4.6%), with an OR of 1.7 (95% CI: 1.2–2.5) after detailed adjustment for BMI at diagnosis. The OR for diabetes was 2.0 (95% CI: 1.4–2.9) in the absence of adjustment for BMI, and 1.9 (95% CI: 1.3–2.7) when a term for BMI at age 30–39 was included in the model. The OR was 2.4 (95% CI: 1.9–3.1) for women with a BMI at diagnosis ⩾30 kg m−2, 1.6 (95% CI: 1.3–2.0) for women with a BMI at age 30–39 ⩾25 kg m−2, 1.4 (95% CI: 1.1–1.8) for women with a low level of physical activity at age 30–39, and 1.2 (95% CI: 1.0–1.5) for women with a history of hypertension.−2), the OR was 1.4 for non-obese women with diabetes, but rose to 5.1 for obese diabetic women. Corresponding ORs for women with diabetes were 1.8 with BMI at age 30–39 years <25 kgm−2, and 3.4 with BMI at age 30–39 years ⩾25 kg m−2. However, the interaction between diabetes and BMI at diagnosis or at 30–39 years was not statistically significant. As compared with non-diabetic women with moderate or high physical activity, the OR was similar in diabetic women reporting moderate/high (OR=1.9) or low physical activity (OR=2.0). Similarly, the ORs were comparable in diabetic women without (OR=1.8) and with (OR=2.1) a history of hypertension, as compared with non-diabetic women with no hypertension.The relation between diabetes and endometrial cancer risk was also examined in strata of age, menopausal status, parity, OC and HRT use, but no meaningful differences in risk emerged.This large case–control study on endometrial cancer confirms and provides further quantitative evidence that diabetes is independently related to endometrial cancer risk. The association was partly, but not totally, explained by overweight. As in other epidemiological studies  (This is a hospital-based case–control study with some of the related weaknesses and strengths. Diabetics may be more frequently admitted to hospital, although we excluded from the comparison group all women hospitalised for chronic and metabolic conditions. Furthermore, the 4.6% prevalence of diabetes in middle age and elderly women in our comparison group is similar to that of population-based surveys (


T and GBL13, were isolated as one of intestinal lactobacilli from the faecal specimens from different thoroughbreds of the same farm where they were born in Hokkaido, Japan. They were Gram-positive, facultatively anaerobic, catalase-negative, non-spore-forming and non-motile rods. KBL13T and GBL13 homofermentatively metabolize glucose, and produce lactate as the sole final product from glucose. The 16S rRNA gene sequence, DNA–DNA hybridization, DNA G+C content and biochemical characterization indicated that these two strains, KBL13T and GBL13, belong to the same species. In the representative strain, KBL13T, the DNA G+C content was 34.3 mol%. Lactobacillus salivarius JCM 1231T  is the type strain most closely related to the strain KBL13T as shown in the phylogenetic tree, and the 16S rRNA gene sequence identity showed 96.0 % (1425/1484 bp). Comparative 16S rRNA gene sequence analysis of this strain indicated that the two isolated strains belong to the genus Lactobacillus and that they formed a branch distinct from their closest relatives, L. salivarius, Lactobacillus aviarius, Lactobacillus saerimneri and Lactobacillus acidipiscis. DNA–DNA reassociation experiments with L. salivarius and L. aviarius confirmed that KBL13T represents a novel species, for which the name Lactobacillus hayakitensis sp. nov. is proposed. The type strain is KBL13T (=JCM 14209T=DSM 18933T).Two strains, KBL13 Lactobacillus flora of thoroughbred gastrointestinal contents by using the culturing method  at 4 °C to our laboratory within 24 h. The initial processing and subsequent weighing and dilution of the specimens were carried out under anaerobic conditions. Each dilution was then spread on to BL agar plates  and incubated anaerobically at 37 °C for 2 days. All further cultivation was performed at 37 °C in ABCM broth . The 16S rRNA gene sequences of the isolates were determined as described previously . The closest known relatives of the isolates were determined by performing database searches, and the sequences of closely related species were retrieved from the DDBJ database. Multiple alignments of the sequences were carried out with the clustal_x program, version 1.18 , the physiological characteristics of KBL13T and GBL13 were compared with those of L. salivarius JCM 1231T  and JCM 1150 as shown in Table 1L. salivarius JCM 1231T and JCM 1150, L. aviarius subsp. aviarius JCM 5666T and L. aviarius subsp. araffinosus JCM 5667T used in the study were obtained from the Japan Collection of Microorganisms. A high similarity of 96.0 % (1425/1484 bp) was observed in the 16S rRNA gene sequences of KBL13T and L. salivarius JCM 1231T. Identical tree topologies were obtained by using the maximum-likelihood and maximum-parsimony methods .In a neighbour-joining dendrogram created based on the sequence of KBL13T and GBL13 were 34.3 and 34.8 mol%, respectively. The DNA G+C content of their closest relatives, L. salivarius JCM 1231T and L. aviarius subsp. araffinosus JCM 5667T was 34.7 and 41.3 mol%, respectively. The DNA G+C content of KBL13T was found to be within the range of 32.0–55.0 mol%, which is the range reported for Lactobacillus species . Other biochemical tests, such as those on motility, growth at a fixed temperature and gas production from glucose, were performed by using the methods described by T and GBL13 were found to be the same species, KBL13T was used as a representative strain in the experiments described below.The sugar fermentation patterns were determined using the API 50CH system (bioMérieux) according to the manufacturer's instructions. The results were recorded after 48 h at 37 °C. The isomer of lactic acid produced from glucose was determined by using an F-kit .T is not able to grow in 4.5 % NaCl and at 15 °C, but grows in 3.0 % NaCl and at 45 °C. Cells are catalase-negative. Glucose is metabolized homofermentatively and lactate is the sole final product. Strain KBL13T produces l(+)-lactic acid. Acid is produced from glucose, fructose, mannose, mannitol, N-acetyl-d-glucosamine, arbutin, aesculin, salicin, cellobiose, maltose, sucrose and gentiobiose. Amygdalin and raffinose are weakly fermented. In this species, some strains cannot ferment N-acetyl-d-glucosamine, arbutin and raffinose. The DNA G+C content of the type strain is 34.3 %, and the cell wall composition of the strain exhibits the Lys–Asp peptydoglycan type. Cells are Gram-positive, 3.0–5.0 μm long and 1.0–1.5 μm wide, non-motile and non-spore-forming rods. They occur singly or in pairs. Colonies are small (1.5 mm), circular to slightly irregular, convex, with a smooth to rough surface, and white when grown on MRS agar. The optimum growth temperature is 37 °C. Strain KBL13T (=JCM 14209T=DSM 18933T), was isolated from the faeces of a thoroughbred.The type strain, KBL13


R. felis has been a matter of debate, mostly due to the contamination of an initial R. felis culture by R. typhi. However, the first stable culture of R. felis allowed its precise phenotypic and genotypic characterization, and demonstrated that this species belonged to the spotted fever group rickettsiae. Later, its genome sequence revealed the presence of two forms of the same plasmid, physically confirmed by biological data. In a recent article, Gillespie et al. :e266.) used a bioinformatic approach to refute the presence of the second plasmid form, and proposed the creation of a specific phylogenetic group for R. felis.Since its first detection, characterization of R. felis strain URRWXCal2T, but observed that the plasmid content of this species, from none to 2 plasmid forms, may depend on the culture passage history of the studied strain. We also demonstrated that R. felis does not cultivate in Vero cells at 37°C but generates plaques at 30°C. Finally, using a phylogenetic study based on 667 concatenated core genes, we demonstrated the position of R. felis within the spotted fever group.In the present report, we, and five independent international laboratories confirmed unambiguously by PCR the presence of two plasmid forms in R. felis, which unambiguously belongs to the spotted fever group rickettsiae, may contain up to two plasmid forms but this plasmid content is unstable.We demonstrated that  Rickettsia felis (R. felis) was first detected in 1990 in American Ctenocephalides felis fleas using electron microscopy, and named the ELB agent after the Elward laboratory  where the flea colony was raised R. felis, several characteristics of R. typhiR. felis culture with R. typhiR. typhi and R. felisR. felis was cultivated from cat fleas at low temperature (28°C), and was established for the first time 2T in two official collections: the American Type Culture Collection (ATCC VR-1525), and the Collection de Souches de l'Unité des Rickettsies (CSUR R121). Subsequently, another two teams were able to successfully grow R. felis, also at temperatures ≤32°C 2T demonstrated that its antibiotic susceptibility R. felis strain URRWXCal2Ti. e., a large, , and a small  forms. The pRF and PRFδ plasmids had identical sequences with the exception of an additional 24 ORFs in pRF. Then, we confirmed these results by PCR assays specifically targeting each plasmid Ctenocephalides felis fleas from various locations. However, in a recent bioinformatic analysis of the R. felis genome sequence, Gillespie et al. questioned our results and proposed that the pRFδ plasmid was an artefact from genome assembly et al. who failed to detect the pRFδ plasmid in R. felis strain LSU C. felis fleas at Louisiana State University R. felis in a fourth phylogenetic lineage within the Rickettsia genus et al. were not appropriate. Therefore, we asked five independent laboratories worldwide to check the presence of two plasmids in R. felis. We also evaluated the presence of both plasmids in various specimens and in cloned R. felis. Finally, we conducted a phylogenetic study based on 667 concatenated Rickettsia core protein-encoding genes, and tested new specimens.As our previous work had been carefully performed and experimentally confirmed, we believe that the conclusions of Gillespie R. felis cultivated from the initial isolate R. felis strain URRWXCal2T. However, in order to obtain indisputable results, we proposed five independent laboratories worldwide to perform these PCR assays. To avoid any interpretation bias, we provided these laboratories with anonymized kits. All five laboratories obtained similar PCR results  but only the large plasmid form in the remaining five clones. In addition, only the small plasmid form was detected from the positive control DNA extracted from R. felis with a 50 cell culture passage history. These data demonstrated that the plasmid content of R. felis may vary according to culture history, with a small plasmid form being unstable. R. felis may exhibit from two  to one plasmid form (R. felis with a 50 cell culture passages). Such a phenomenon was recently reported by Baldridge et al. who demonstrated that R. peacockii lost its plasmids during serial passage in cell culture Using all four primer pairs, we obtained PCR products of the expected sizes from  results , Table 1 at 37°C . Of 20 cet al. speculated, based on an in silico analysis, that the small plasmid form detected in R. felis was an artefact of genome assembly R. felis, but found them both by pulsed field gel electrophoresis , R. africae (as ESF agent), and Legionella pneumophila (as Tatlock agent) R. prowazekii  was found in ticks in Africa and Mexico R. conorii was reported to infect mites and lice R. bellii was found in insects (unpublished data), although both species were believed to be strictly associated with ticks. Therefore, a simplification of the relationship between ecological niche, pathogenicity and phylotype is not possible, and these findings forced rickettsiologists to define more carefully rickettsiae as either pathogenic or of unknown pathogenicity. Moreover, by deliberately omitting SFG rickettsiae of unknown pathogenicity (such as the SFG species R. montanensis) the authors proposed in their The hypothesis that et al.R. feliset al. who could detect only one plasmid R. felis was an artefact of our genome assembly. It is clear in other bacterial genera that the plasmid content may vary from one strain to another and that plasmids may not be stably maintained in culture R. felis may have one, two or no plasmid, depending on the strain or the culture passage history. This plasmidic instability, which we also identified in R. africae (unpublished data), was also described recently in R. peacockiiR. prowazekii or R. conorii.We were surprised that the only hypothesis produced by Gillespie R. felis, the choice of genes for infering phylogenies should be extremely careful. For example, Woese clearly demonstrated discrepancies between the phylogenies obtained using the ribosomal operon and amino-acyl –tRNA synthetases et al.R. felis, between SFG and TG rickettsiae, contradicting biological data and their own work R. felis may be subject to gene recombination with R. typhi as these two rickettsiae can meet in the same host flea. However, the current position of R. felis and its clustering with R. australis and R. akari is exactly the same as that defined in as early as 1999 Rickettsia species based on the concatenation of their core gene set and demonstrated unambiguously that R. felis belongs to the spotted fever group. Finally, classification of rickettsiae based on current knowledge of host specificity is not reliable.Regarding the phylogenetic position of R. felis does not grow at 37°C. This result confirms the fact that authors who initially reported a culture of this species at 37°C R. felisR. typhi, which they later acknowledged R. felis were similar to those of R. typhiWe also demonstrated for the first time using a cloning method in Vero cells that R. felis. We clearly demonstrated that R. felis is a SFG rickettsia, that it does not grow at 37°C and that it has, without any possible doubt, two plasmids. Finally, we think that it would have been fair, since our R. felis strain is available, to check the presence of plasmids prior concluding that our data resulted from an error.We believe that it was important to clarify the status of R. felis strain URRWXCal2T kept frozen at -80°C since initial isolation was cultivated in XTC2 cells as previously described R. felis using the QIAmp Tissue kit . The pRFδ plasmid was detected using the primers pRFa and pRFd , 2.5 µL High Fidelity PCR Buffer 10X, 0.5 µL of a 10mM dNTP mixture, 1 µL of 50 mM MgSO4, 0.5 µL of each primer (10mM), and 17.9 µL sterile water. Amplification conditions included an initial denaturation at 94°C for 2 min followed by 25 cycles comprised of 94°C for 30sec., 55°C for 30 sec., and 68°C for 2 min. PCR products were resolved in 1% agarose gels with ethidium bromide.R. felis strain URRWXCal2T (DNA1), R. conorii strain Malish 7 (DNA2) and R. africae strain ESF-5 (DNA3) (5′-CCTATGGCTATTATGCTTGC-3′)–AR (5′-ATTGCAAAAAGTACAGTGAACA-3′) specific for the citrate synthase (gltA)-encoding gene. Each primer pair was tested on each of the three DNA specimens , 2.5 µL Buffer, 2.5 µL dNTP, 1 µL MgCl2, 0.5 µL of each primer, and 13.0 µL sterile water. Amplification conditions included an initial denaturation at 94°C for 15 min followed by 39 cycles comprised of 94°C for 1 min., 54°C for 30 sec., and 72°C for 2 min. The reaction was completed by a final elongation step at 72°C for 5 min.We prepared a kit that contained DNA from 5 (DNA3)  extracte5 (DNA3) . The kit5 (DNA3) , and AF pecimens . For eacR. felis-positive (strain LSU) C. felis fleas Archaeopsylla erinacei fleas from Algeria previously found to contain R. felis strain Rf2125 K. Macaluso provided the expert laboratory L1 with DNA from five 4 plaque forming units of R. felis strain URRWXCal2T , we inoculated Vero cells at 30°C and 37°C and performed a plaque assay as previously described R. felis clones grown from single R. felis cells .Using tenfold dilutions of a suspension containing 10is cells . DNA extR. felis among Rickettsia species, we based our analysis on the 704 core protein-coding genes identified by Blanc et al. by comparison of 7 rickettsial genomes R. slovaca and R. akari genomes To estimate the phylogenetic position of


In this work, the anti-inflammatory activity of two COS mixtures is characterized in vivo , following the carrageenan-induced paw edema method. This is a widely accepted animal model of acute inflammation to evaluate the anti-inflammatory effect of drugs. Our data suggest that COS possess anti-inflammatory activity, which is dependent on dose and, at higher doses, also on the molecular weight. A single dose of 500 mg/kg b.w. weight may be suitable to treat acute inflammation cases; however, further studies are needed to ascertain the effect upon longer inflammation periods as well as studies upon the bioavailability of these compounds.All the reports to date on the anti-inflammatory activity of chitooligosaccharides (COS) are mostly based on  N-acetyl-d-glucosamine and deacetylated glucosamine units [in vitro tests [in vivo upon inflammation induced by carrageenan.Chitooligosaccharides (COS) are partially hydrolyzed products of chitosan, a biopolymer composed of β-1–4)-linked –4-linkedro tests –4. It haro tests ; furtherro tests . This pr50 value in mice, for both COS, was found to be above 1,000 mg/kg b.w.Both COS mixtures administered orally at doses between 50–1,000 mg/kg b.w., didn’t generate any significant change in the autonomic or behavioural responses during the observation period. Therefore, the oral LDEdema induced by phlogistic agents is a widely accepted model for the evaluation of anti-inflammatory effect of drugs . Carragep < 0.05) decreased paw edema at 2, 3 and 6 h after carrageenan injection compared to saline, with inhibition levels of 67.92%, 71.61% and 78.79%, respectively (p < 0.05) decreased the paw volume at 3 and 6 h after carrageenan administration compared to vehicle control (p < 0.05) stronger effect −73.42% and 78.13%, than COS5–63.20% and 71.88% at 3 and 6 h, respectively.To demonstrate the validity of the carrageenan-induced paw edema test, mice were administered INN orally as a positive control at a dosage of 10 mg/kg b.w. 1 h before carrageenan injection. As expected, INN significantly —and 5.3 kDa (COS5)—and possessing a degree of deacetylation in the 80–85% range, were purchased from Nicechem . Both compounds were derived from crab shells. All chemicals used in this work were purchased from Sigma-Brazil.ad libitum. Groups of six mice were used and the studies were carried out in accordance with current guidelines for the veterinary care of laboratory animals [Balb/c male mice (6 weeks), weighing between 27 and 32 g, were used in the experiments. The animals were purchased from Centro Multidisciplinar para Investigação Biológica na Área da Ciência em Animais de Laboratório–CEMIB at the University of Campinas (UNICAMP), and were kept in polyethylene boxes (n = 6), in a controlled environment—constant temperature (24 ± 2 °C) with a 12 h light-dark cycle and relative humidity of 40–70%. They were kept without food for 24 h before the experiment and water was  animals , and werMice were divided into seven groups, each containing six animals. COS mixtures were administered orally, in varying doses  to these animals, using saline solution as a vehicle. A group of animals treated with the vehicle served as control. They were continuously observed for 4 h to detect changes in their autonomic or behavioural responses viz. alertness, spontaneous activity, irritability, pinna reflex, corneal reflex, urination, salivation and piloerection . Any morsteroid hormones). A group of animals treated with the vehicle served as negative control. Edema was induced by injecting 0.02 ml of 2.5% carrageenan in sterile saline into the plantar surface of the right hind paw. The difference of volumes between the basal and sequential measurements in the right hind paw was calculated as the edema formation. The paw volume was measured in an Ugo Basile plethysmometer , at 0.5, 1, 2, 3, 4, 6, 8, 24 and 48 h.Anti-inflammatory activity was studied by carrageenan-induced paw edema method . Mice wen = 6). Statistical significance was determined by analysis of variance and subsequent Duncan’s multiple range test (p < 0.05). The analysis was performed using Statistical Package for Social Sciences – SPSS statistical software .The results are expressed as mean ± SEM  was comparable to that of indomethacin, but during a shorter period. A single dose of 500 mg/kg b.w. may indeed be suitable to treat acute inflammation cases; however, further studies are needed to ascertain the effect upon longer inflammation periods as well as studies upon the bioavailability of these compounds.


Crithidia fasciculata (CfGspS) obeys a rapid equilibrium random ter-ter model with kinetic constants KGSH = 609 μm, KSpd = 157 μm and KATP = 215 μm. Phosphonate and phosphinate analogues of glutathionylspermidine, previously shown to be potent inhibitors of GspS from Escherichia coli, are equally potent against CfGspS. The tetrahedral phosphonate acts as a simple ground state analogue of glutathione (GSH) (Ki ∼ 156 μm), whereas the phosphinate behaves as a stable mimic of the postulated unstable tetrahedral intermediate. Kinetic studies showed that the phosphinate behaves as a slow-binding bisubstrate inhibitor [competitive with respect to GSH and spermidine (Spd)] with rate constants k3 (on rate) = 6.98 × 104 m−1·s−1 and k4 (off rate) = 1.3 × 10−3 s−1, providing a dissociation constant Ki = 18.6 nm. The phosphinate analogue also inhibited recombinant trypanothione synthetase (EC 6.3.1.9) from C. fasciculata, Leishmania major, Trypanosoma cruzi and Trypanosoma brucei with Kiapp values 20–40-fold greater than that of CfGspS. This phosphinate analogue remains the most potent enzyme inhibitor identified to date, and represents a good starting point for drug discovery for trypanosomiasis and leishmaniasis.Glutathionylspermidine is an intermediate formed in the biosynthesis of trypanothione, an essential metabolite in defence against chemical and oxidative stress in the Kinetoplastida. The kinetic mechanism for glutathionylspermidine synthetase (EC 6.3.1.8) from  N1,N8-bis(glutathionyl)spermidine] and trypanothione reductase, which replaces not only glutathione/glutathione reductase but also thioredoxin/thioredoxin reductase in mammalian cells , in a two-step reaction with glutathionylspermidine as an intermediate –20. Both.5.1.78) . Previou.5.1.78) ,27. Howeynthesis . AlthougJ748279)  and putaShigella ,30. The g growth , and a s(CfGspS) . Glutath(CfGspS) . A previnhibitor .CfGspS /Km]). The y-intercept of k4 of 1.3 × 10−3·s−1. Thus, the overall dissociation half-life for the complex is 0.14 h . For an inhibitor of this type, the dissociation constant (Ki) is then obtained from the ratio of k4/k3, yielding a Ki of 18.6 nm. To confirm the Ki value, v0 and vs obtained at different concentrations of inhibitor were fitted to the equation vs = v0/(1 + [I]/Kiapp) by nonlinear regression, yielding a Kiapp value of 31.1 ± 2.1 nm, and true Ki value was calculated to be 19.0 ± 1.3 nm, using the relationship Ki = Kiapp/(1 + [S]/Km). Thus both methods of determining Ki are in excellent agreement.In contrast to the simple, linear inhibition shown by the phosphonate, time-dependent inhibition was observed for the phosphinate mimic . In reacntration . These r  ≥ 10 [E]), and the reaction was then initiated with substrate. Under these conditions, a slow release of inhibitor is observed until a steady state is reached. Provided that there is no significant enzyme inactivation, substrate depletion or product inhibition, this steady state should be identical to the steady state established when initiating with enzyme  > [E] , it proved impossible to measure enzyme activity upon 100-fold dilution into the assay mixture. Instead, high concentrations of inhibitor (200 μm), enzyme (20 μm) and ATP (400 μm) were preincubated on ice for 60 min and then applied to a desalting column to remove all free inhibitor. The following reactions were then analysed: (a) enzyme-only control , whereas for an uncompetitive inhibitor, kobs increases in a hyperbolic fashion with increasing concentrations of substrate. As kobs increases with increasing concentrations of ATP  was determined in the absence of GSH or Spd plus or minus 1 μm phosphinate over 30 min. After correction for the background rate due to auto-oxidation of NADPH and hydrolysis of ATP in the coupling system, the net rates of endogenous ATPase activity (∼ 0.01% of kcat) in the presence and absence of inhibitor are 1.4 (± 0.9) × 10−3 and 3.0 (± 1.5) × 10−3·s−1, respectively (mean of three determinations). This shows that the inhibitor is not turned over by the enzyme. However, this method is insufficiently sensitive to detect a single phosphorylation event.To determine whether the phosphinate is turned over by CfGspS is potently inhibited by the phosphinate inhibitor, it remained to be determined whether the homologous enzyme, TryS, could also be inhibited in a similar manner. Owing to the various pH optima, Km values for substrates and GSH substrate inhibition profiles of the various TryS enzymes to be compared , a uniform assay was used for IC50 determination, i.e. 2 mm Spd, 0.2 mm GSH, 2 mm ATP, 100 mm (K+) Hepes (pH 7.2). This allows for direct comparison of the data collected for all the enzymes under conditions that approximate to the physiological metabolite levels found in these organisms .TryS enzymes from eviously –19. In am (K+) Hepes (pH 7.3), 0.2 mm NADH, 1 mm phosphoenolpyruvate, 5 mm dithiothreitol or Tris(2-carboxyethyl)phosphine hydrochloride, 0.5 mm EDTA, 10 mm MgSO4, 2 U·mL−1l-lactate dehydrogenase, and 2 U·mL−1 pyruvate kinase , with varying amounts of ATP, GSH and Spd in a total volume of 1 mL. Rates are expressed in moles of substrate utilized per second per mole of enzyme. To determine the kinetic mechanism, data were collected for GspS at a range of substrate concentrations. A complete matrix of rates as a function of substrate concentration  was gathered, so that for any given concentration of any one substrate the rates were measured over the entire range of the remaining two substrates. When fixed concentrations of each of these substrates were used, the final concentrations for ATP, GSH and Spd were 0.5, 1 and 1 mm respectively, unless otherwise stated. The assay was initiated by adding GspS (300 nm) and, after a lag of 10 s, the linear decrease in absorbance was monitored for up to 1 min. Data were then globally fitted by nonlinear regression to all possible models for rapid equilibrium ter-reactant systems . The Kiapp values of the inhibitor against each enzyme were determined using the following tight-binding inhibition equation [E] was fixed at 100 nm: In this equation, equation  Eqn 8),,s is a s


We analysed two case–control studies of women from Italy and Switzerland, including 195 cases of oral and pharyngeal cancers and 1113 controls. The multivariate odds ratio was 4.6 for heavy smokers and 2.7 for high alcohol intake. Vegetables, fruit, β-carotene and wholegrain foods were inversely, butter and retinol directly, related to risk. © 2000 Cancer Research Campaign


Objective: The purpose of this study was to evaluate the prevalence of             Chlamydia trachomatis in our rural obstetric population and assess the appropriateness             of selective vs. universal prenatal screening.					Methods: Between April 1, 1991 and May 1, 1993, 1,587 patients were             screened at their first prenatal visit using a C. trachomatis antigen test. Patients who were             unmarried, younger than 20 years of age, or had a history of a previous sexually transmitted             disease (STD) were classified as being at high risk for C. trachomatis. All others were             considered low risk for C. trachomatis.							Results: The overall prevalence of C. trachomatis was 2.0%.             There was a significant difference (P < 0.001) in the 1,128 patients             considered low risk  compared to the 459 patients             with one or more identifiable risk factors .					Conclusions: Routine prenatal screening for C. trachomatis in our             population is not appropriate for low-risk patients.


Supplementary data are given on the following: (1) the full taxonomic list of invertebrates ( Next in order of abundance were saprophages, 36.5% (31.7–41.3%); pests of oilseed rape, 13.7% (10.9–16.6%); predators, 5.7% (4.4–7.0%); pollinators , 3.2% (2.0–4.3%); and parasitoids, 1.5% (1.2–1.8%) (Annex 1 in Ref 1.1log-transformed) area of a field and the number of invertebrates sampled there . On average, 6.2% more invertebrates were sampled in the large fields  than in the small ones : 387 (95% C.I. = 334–440) vs. 363 (237–488), respectively. More pronounced differences emerged when the landscape context, i.e. the type of field surroundings, was taken into account. In an open landscape, 12% fewer invertebrates were sampled in the small fields than in the large ones , presumably because of the small sample size and the confounding influence of edge habitat variability (see below).There was a weak but insignificant relationship between the . Specifically, we found that the number of all invertebrates sampled was positively correlated with the area of dirt roads . Further, both the number and percentage (%) of the herbivorous insects were positively correlated with the length of linear woody features surrounding the fields  and wooded area ; and the numbers of all invertebrates and Diptera  were correlated with the length of dirt roads . Interestingly, we found that the number of the pooled four species of true weevils of the tribe Ceutorhynchus spp.  was positively correlated with the area of permanent vegetation , and with the total wooded area . Similarly, the number of Coleoptera  was positively correlated with the area of forests .Analysis of the eight edge habitat variables measured within a 100 m radius of the invertebrate sampling points  in the 3r = −0.462, P = 0.009).The only negative statistically significant relationship was between the percentage (%) of herbivorous insects and the area of woods .The concentrations of Na, Ca, Mg, Cu, Zn, Fe, As, Co and Pb varied significantly between two oilseed rape pest taxa, 22.1−1; foliar fertiliser  and magnesium sulphate (3 kg ha−1). Also applied were herbicides ; insecticides between March and May  targeting herbivorous insects, mostly stem weevils Ceutorhynchus spp. and pollen beetles Meligethes spp.; and fungicides .The 35 winter oilseed rape fields were managed using conventional amounts of agrochemicals, including pesticides and fertilisers. The landowner  supplied management data on agricultural practices in a few large fields for the study year; both the timing and use of agrochemicals were similar in all the other fields. The fields were sown (winter oilseed rape cultivar: PRW 31 F-1) in the second half of September 2014. Each of three mineral fertilisers  was used in a dose of 300 kg ha2.2We used reference materials for each of the AAS measurements. These were blind tests, i.e. they contained the same chemical composition as a particular sample, but were devoid of the analysed biological material. The analytical procedure for preparing these samples was the same as in the case of our ones. We used standardised samples obtained from SGAB Analytica, Luleå Technical University, Luleå, Sweden and Fürst Medical Laboratory, Billingstad, Oslo, Norway, Certified Values and Uncertainty NCS ZC, i.e. standards of a particular quality for each kind of tissue and chemical element. The analytical measurement process was validated using reference materials, i.e. CVU  provided by SGAB Analytica, Luleå Technical University, Luleå, Sweden and Fürst Medical Laboratory, Billingstad, Oslo, Norway, Certified Values and Uncertainty NCS ZC. Reference values amounted to 0.25 ± 0.05–32.7 ± 1.8 for the target chemical elements. The average (±SD) values determined for the target elements (20 measurements in the invertebrate and plant samples) were 0.19 ± 0.05–34.8 ± 1.07. The precision of the method, understood as the degree of conformity between the results of multiple analyses performed on the same sample, was 5% .2.3P-values adjusted to multiple comparisons) would be justified, thus permitting a robust biological interpretation of our observations.Pre-analysis of our habitat variables quantified for the individual oilseed rape fields showed strong collinear associations (tested by Spearman's and Pearson's correlation coefficients) between some of these variables. Principal Component Analysis (PCA) was applied to reduce collinearity among them see . But becThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.