The association between body mass index (BMI) at the time of breast cancer diagnosis and the prognosis of breast cancer patients remains controversial. Furthermore, the association between BMI and prognosis with respect to different breast cancer subtypes is not clearly defined.We analyzed data from 41,021 invasive breast cancer patients between January 1988 and February 2008 from the Korean Breast Cancer Registry (KBCR) database. Overall survival (OS) and breast cancer-specific survival (BCSS) were analyzed using the Kaplan-Meier method and Cox’s proportional hazard regression model among all patients and specific breast cancer subtypes with respect to BMI categories.A U-shaped association between BMI and mortality was observed in the total cohort. Underweight and obese individuals exhibited worse OS  and BCSS  than normal-weight individuals. In the estrogen receptor (ER) and/or progesterone receptor (PR)+/human epidermal growth factor receptor 2 (HER2) - subgroup, obese individuals exhibited worse OS (1.48 [1.18 to 1.85]) and BCSS (1.31 [1.13 to 1.52]) than normal-weight individuals. Conversely, in the ER and PR-/HER2+ subgroup, underweight individuals exhibited worse OS (1.68 [1.12 to 2.47]) and BCSS (1.79 [1.11 to 2.90]) than normal-weight individuals.We observed a U-shaped relationship between BMI at diagnosis and poor OS and BCSS among all breast cancer patients. However, obesity in the ER and/or PR+/HER2- subgroup and underweight in the ER and PR-/HER2+ subgroup were poor prognostic factors. Therefore, BMI at diagnosis and breast cancer subtype should be considered simultaneously in various treatment decision processes and surveillance schedules. The association between body mass index (BMI) at the time of breast cancer diagnosis and the prognosis of breast cancer patients remains controversial despite many studies, including single institution, multi-center, and population-based studies, meta-analyses, and randomized controlled trials –24. In mPrevious studies have not adequately demonstrated an association between BMI at the time of breast cancer diagnosis and prognosis in breast cancer patients with respect to breast cancer subtypes. Recent advances in our understanding of breast cancer biology based on molecular techniques allow us to divide breast cancer into at least four subtypes , 26. TheMoreover, there are certain differences between Asian and Western regions with respect to the prevalence of obesity. Although the prevalence of obesity is lower in Asians, the health risks associated with obesity occur at a lower BMI in Asian populations –29. TherThe aim of this study was to investigate the prognostic significance of BMI at the time of breast cancer diagnosis in all breast cancer patients and in each breast cancer subtype by analyzing overall survival (OS) and breast cancer-specific survival (BCSS) using population-based data from the Korean Breast Cancer Registry (KBCR) database.The KBCR database is a web-based, prospectively maintained nationwide database managed by the Korean Breast Cancer Society (KBCS). One hundred and two institutions have voluntarily participated in this registry since 1997. Before inserting personal information along with various datasets, written informed consent should be mandatory from the patient. From the initial conception of KBCR database, principal investigators from every single institution have agreed on the principles and process of utilizing this database for research purposes. After 2000, an online registration program was implemented, and the database has been actively utilized for various research studies on breast cancer in Korea , 30. EssAccording to the guidelines of utilizing KBCR database, this study was approved by the institutional Review Board (IRB) of St. Vincent’s Hospital, College of Medicine, The Catholic University, where the first author of this article is affiliated (VC14RISI0234).In this study, we selected and assessed invasive breast cancer patients who underwent curative surgery between January 1988 and February 2008. To achieve a more accurate analysis, we excluded patients treated with neoadjuvant therapy and patients for whom essential registry data  and ER/PR status were not available. Patients with distant metastasis at the time of diagnosis were excluded, because distant metastasis is the worst prognostic factor compared with other prognostic factors  and serves as confounding factor for survival analysis.The data on the remaining 41,021 patients were included in the final analysis.All patients were categorized into five subgroups according to the expression of ER, PR and HER2 as follows: (a) ER and/or PR+/HER2-; (b) ER and/or PR+/HER2+; (c) ER and PR-/HER2+; (d) ER and PR-/HER2-; and (e) unknown. All patients for whom ER/PR expression but not HER2 expression information was available were categorized into the unknown group.Positive staining for ER or PR was defined as the positive staining of ≥10 % nuclei in ten high-power fields, and HER2 positivity was defined as 3+ immunohistochemical (IHC) staining or HER2 gene amplification by fluorescence in situ hybridization (FISH). Cases of 2+ HER2 by IHC without a FISH result were treated as HER2-negative.Patient survival data, including the date and cause of death, were obtained from the Korean Central Cancer Registry, Ministry of Health and Welfare, Korea.2), underweight BMI (<18.5 kg/m2), overweight BMI (25.0 – 29.9 kg/m2) and obese BMI (≥30 kg/m2) according to the guidelines of the World Health Organization (WHO) [BMI was calculated by dividing weight (kg) by height (m) squared. The BMI at diagnosis was categorized as normal BMI (18.5 - 24.9 kg/mon (WHO) .p value of less than 0.05 was considered significant. All statistical analyses were performed using the SAS software for Windows .The patient characteristics were compared with respect to BMI category  using the chi-square test. The chi-square test and analysis of variance (ANOVA) were used to determine differences in the clinicopathological features between groups. With respect to survival analyses, we explored OS and BCSS using data from the KBCR database. OS was defined as the time from the initial diagnosis of primary breast cancer to death from any cause. BCSS was defined as survival until death from breast cancer. Survival curves were estimated using the Kaplan-Meier method. Log-rank tests were performed for the comparison of survival curves. Multivariate analyses were conducted using Cox’s proportional-hazard regression models to study the effect of BMI at diagnosis on OS and BCSS. The parameters included in the multivariate analysis model were as follows: patient age; tumor size; histologic grade; lymph node status; operation method; adjuvant treatment; ER/PR status and HER2 expression. A p < 0.001). Obese patients had larger tumors (p < 0.001), high frequencies of axillary lymph node metastasis (p < 0.001), histologically high-grade lesions (p = 0.003), and negative ER and PR (p = 0.002) and HER2 expression (p = 0.004) compared with underweight patients.For the 41,021 patients included in our analysis, the mean age at breast cancer diagnosis was 48 years . The baseline characteristics are presented in Table p < 0.001). The ER and/or PR+/HER2- and ER and PR-/HER2- subtypes were more prevalent in the obese BMI category. However, the ER and/or PR+/HER2+ subtype was more prevalent in the underweight BMI category.Breast cancer subgroups categorized according to the expression of ER, PR and HER2 exhibited a significant association with BMI categories , as did those in the obese BMI category . Patients in the underweight BMI category  and the obese BMI category  exhibited significantly worse BCSS compared with those in the normal BMI category.After adjusting for poor prognostic factors, such as tumor size, axillary lymph node metastasis, histologic grade, ER, PR and HER2 expression, a U-shaped association between BMI and mortality was observed in the total cohort (Table p = 0.0006) and BCSS  compared with those in the normal BMI category (Table p = 0.1269) and BCSS (p = 0.2684) rate between patients in the normal BMI category and the underweight BMI category. Conversely, in the ER and PR-/HER2+ subgroup, patients in the underweight BMI category exhibited significantly worse OS  and BCSS  compared with those in the normal BMI category. However, obese BMI was not associated with decreased OS (p = 0.4247) or BCSS (p = 0.5683) in the ER and PR-/HER2+ subgroup. In the ER and/or PR+/HER2+ and ER and PR-/HER2- subgroups, BMI categories did not exhibit a significant association with OS and BCSS. In the unknown subgroup, patients in the obese BMI category exhibited significantly worse OS  but not worse BCSS  compared with those in the normal BMI category.In the ER and/or PR+/HER2- subgroup, patients in the obese BMI category exhibited significantly worse OS , permitting a detailed examination across multiple BMI categories during the long follow-up period. Furthermore, our study is the first to investigate the prognostic significance of BMI in four different breast cancer subtypes. However, our study was limited by the information available in the KBCR database. First, registered patients in the KBCR database were heterogeneous with respect to breast cancer stage, IHC staining results, and presence of comorbidities. Additionally, the essential registry data (BMI and HR expression) were only available for 62.84 % of invasive breast cancer patients in the KBCR database. Therefore, the possibility of selection bias remains. Second, although total sample size is larger compared with previous studies, the sample size of underweight and obese BMI subjects is small to draw a conclusion on the independent effect of BMI. Finally, the ethnic homogeneity of the KBCR database may limit the generalizability of our finding to other racial and ethnic groups.2). Among breast cancer patients with ER and/or PR+/HER2- tumors, obese individuals exhibit significantly poorer OS and BCSS, whereas among those with ER and PR-/HER2+ tumors, underweight patients exhibit significantly poorer OS and BCSS compared with breast cancer patients with normal BMIs. Although obesity and underweight BMI at diagnosis are poor prognostic factors in pooled breast cancer samples, BMI at diagnosis exhibited a different impact on breast cancer prognosis in specific breast cancer subtypes. Therefore, BMI at diagnosis and breast cancer subtype should be considered simultaneously in various treatment decision processes and surveillance schedules.In conclusion, our results indicated a U-shaped relationship between BMI at diagnosis and poor OS and BCSS among all breast cancer patients, with the lowest risk observed among breast cancer patients with normal BMIs (18.5 - 24.9 kg/m


To estimate the burden of road traffic injuries and deaths for all road users and among different road user groups in Africa.We searched MEDLINE, EMBASE, Global Health, Google Scholar, websites of African road safety agencies and organizations for registry- and population-based studies and reports on road traffic injury and death estimates in Africa, published between 1980 and 2015. Available data for all road users and by road user group were extracted and analysed. We conducted a random-effects meta-analysis and estimated pooled rates of road traffic injuries and deaths.We identified 39 studies from 15 African countries. The estimated pooled rate for road traffic injury was 65.2 per 100 000 population  and the death rate was 16.6 per 100 000 population (95% CI: 15.2–18.0). Road traffic injury rates increased from 40.7 per 100 000 population in the 1990s to 92.9 per 100 000 population between 2010 and 2015, while death rates decreased from 19.9 per 100 000 population in the 1990s to 9.3 per 100 000 population between 2010 and 2015. The highest road traffic death rate was among motorized four-wheeler occupants at 5.9 per 100 000 population (95% CI: 4.4–7.4), closely followed by pedestrians at 3.4 per 100 000 population (95% CI: 2.5–4.2).The burden of road traffic injury and death is high in Africa. Since registry-based reports underestimate the burden, a systematic collation of road traffic injury and death data is needed to determine the true burden. Global status report on road safety, the WHO African Region had the highest rate of fatalities from road traffic injuries worldwide at 26.6 per 100 000 population for the year 2013.,,,,,,In Africa, the number of road traffic injuries and deaths have been increasing over the last three decades. Global status report on road safety presented the first modelled regional estimate of a road traffic death rate, which was used to statistically address the underreporting of road traffic deaths by countries with an unreliable death registration system.The 2009This study aimed to review existing literature on published studies, registry-based reports and unpublished articles on the burden of road traffic injuries and deaths in the African continent to generate a continent-wide estimate of road traffic injuries and deaths for all road users and by road user type .http://www.who.int/bulletin/volumes/94/7/15-163121). There was no language restriction.We searched MEDLINE, EMBASE, Global Health, Google Scholar, websites of road safety agencies and relevant organizations within Africa for articles published between 1980 and 2015 . The seaBox 11. africa/ or exp africa, northern/ or exp algeria/ or exp egypt/ or exp libya/ or exp morocco/ or exp tunisia/ or exp “africa south of the sahara”/ or exp africa, central/ or exp cameroon/ or exp central african republic/ or exp chad/ or exp congo/ or exp “democratic republic of the congo”/ or exp equatorial guinea/ or exp gabon/ or exp africa, eastern/ or exp burundi/ or exp djibouti/ or exp eritrea/ or exp ethiopia/ or exp kenya/ or exp rwanda/ or exp somalia/ or exp sudan/ or exp tanzania/ or exp uganda/ or exp africa, southern/ or exp angola/ or exp botswana/ or exp lesotho/ or exp malawi/ or exp mozambique/ or exp namibia/ or exp south africa/ or exp swaziland/ or exp zambia/ or exp zimbabwe/ or exp africa, western/ or exp benin/ or exp burkina faso/ or exp cape verde/ or exp cote d'ivoire/ or exp gambia/ or exp ghana/ or exp guinea/ or exp guinea-bissau/ or exp liberia/ or exp mali/ or exp mauritania/ or exp niger/ or exp nigeria/ or exp senegal/ or exp sierra leone/ or exp togo/2. exp vital statistics/ or exp incidence 3. .tw.4. (disease adj3 burden).tw.5. exp “cost of illness”/6. exp quality-adjusted life years/7. QALY.tw.8. Disability adjusted life years.mp.9. .tw.10. exp risk factors/11. 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 1012. road traffic accident*.mp. 13. RTAs.mp. 14. road traffic injur*.mp. 15. traffic crash*.mp. 16. exp Accidents, Traffic/17. exp air bags/ or exp child restraint systems/ or exp seat belts/18. exp motor vehicles/ or exp automobiles/ or exp motorcycles/19. 12 or 13 or 14 or 15 or 16 or 17 or 1820. 1 and 11 and 1921. limit 20 to (yr = ”1980 –CurrentWe included a study in the review if it met the following criteria: (i) conducted between 1980 and 2015 and that the study was done in an African country; (ii) clearly referred to road traffic crashes, injuries or deaths; (iii) referred data came from a population- or registry-based data system; (iv) registry-based hospital data with the underlying cause of death data coded in the International Classification of Disease and Related Health Problems, 10th revision (ICD-10), with codes V01–V89; (v) directly attempted to estimate the number or rate of road traffic crashes, injuries or deaths in a particular African country or the region as a whole; or (vi) provided any other information  that may further help to understand the burden and determinants of road traffic crashes and policy response in the African region.We excluded studies if they: (i) referred to deaths by other means of transportation including water, air and other unspecified transport means; (ii) were mainly reviews, viewpoints and commentaries; (iii) did not have a clearly defined study design, data capture and analysis method; and (iv) had not clearly defined and consistently applied a case definition of a road traffic crash, injury or fatality.For this study, a crash is defined as a road traffic collision that resulted in an injury or fatality. Injury refers to non-fatal cases from a road traffic crash.For each full text accessed, we checked if the study method had flaws in the design and execution. For the registry-based studies, we examined the study design, completeness, the appropriateness of statistical and analytical methods employed and if the limitations were explicitly stated. For each study, we assessed if the reported sample size or study population was appropriate to provide a representative estimate and if the heterogeneities within and between population groups undermine the pooled estimates. Studies not meeting this quality assessment were excluded.Available data from all selected studies were extracted twice, compiled and stored in a spreadsheet. For each study, data on the country, study period, study design, sample size, mean age and case definitions were extracted . ReporteAll extracted data on road traffic crashes, injuries and deaths were converted to rates per 100 000 population. Studies were subdivided into population- and registry-based studies and analysed separately for all road users and by road user category. A random effects meta-analysis was conducted on extracted road traffic crash, injury and death rates. To give a better understanding of the data distribution and comparisons with the pooled estimates and the confidence intervals, we further presented the range, median and data points within each data set. All statistical analyses were done in Excel 2010  and Stata version 13.1 .,––––The review identified 39 studies reporting on 15 African countries . Six wer,From all registry-based studies, Nigeria recorded the highest and lowest total crash rate at 716.57 per 100 000 population and 2.9 per 100 000 population, in 1990 and 2011, respectively.,,,,,,From the available population-based studies, Nigeria reported the highest number of road traffic injury and death rates at 4120 per 100 000 population and 160 per 100 000 population, respectively. The road traffic injury rate is the highest recorded in any single study in Africa. Algeria and Ghana also reported high road traffic injury rates at 700 and 938 per 100 000 population, respectively.From 1990 to 2015, road traffic injury rates increased from 40.7 to 92.9 per 100 000 population . In cont,,,From individual studies, road traffic death rates among pedestrians ranged from 0.26 per 100 000 population in Nigeria in 2007 to 13 per 100 000 population in South Africa in 2003.,,Our study reflects the difficulties that many experts have noted in describing the extent of road traffic crashes, injuries and deaths in Africa, for which modelling based on scarce and variable information, may not necessarily provide a reliable estimate.,51 as noted in Ghana, South Africa and Zambia., ,,The subgroup analysis showed that injury rates increased and death rates decreased between 1990 and 2015. A high road traffic injury number may reflect the effect of economic growth on the burden of road traffic injury in the region, which may be associated with increased travel and exposure to a hazardous traffic environment.,Our findings further revealed that the highest rates of casualties are among motorized four-wheeler occupants and pedestrians. A WHO report shows that 43% and 38% of road traffic deaths in the African Region occurred among motorized four-wheeler occupants and pedestrians, respectively.,,Meanwhile, a major challenge for the response to road crashes in Africa is the lack of reliable information and data that can inform an evidence-based public health response.Our study has the following limitations. Population-based studies on road traffic crashes in Africa, which would have been more reliable than registry-based studies, were not available. Population-based studies may have given insights on the extent of road users’ exposure to traffic risk, mode and frequency of road travel, distance travelled, number of road commuters and the conditions of the road. In the absence of such information, we have not based our estimates on an appropriate travel exposure denominator, thus limiting an understanding of the reasons behind the reported road traffic crash, injury and death rates and trends.The available registry-based studies varied in their quality. They reported questionable values and trends and provided uncertain estimates. Lack of appropriate case definition for road traffic fatalities and incomplete breakdown of road traffic crash estimates by road user type were major limitations. Additionally, the non-fatal injury figures reported by the different studies varied with respect to severity and outcome. These variations could have affected our meta-analyses.While we applied the UN population data for Africa to estimate rates where relevant national reference population data were unavailable, there were no comparable data to use for subnational studies. In addition, the data employed for this analysis were generated only from 15 countries, which is relatively small to accurately reflect the overall situation in the region. Hence, our estimates should be interpreted against these limitations.In conclusion, our study suggests that the burden of road traffic injuries in Africa is high and there is an underestimation of road traffic fatalities. Improved road traffic injury surveillance across African countries may be useful in identifying relevant data gaps and developing contextually feasible prevention strategies in these settings.


Differences in the content of dietary fiber and its fractions were observed in the examined products. The highest total dietary fiber content was detected in boiled buckwheat groats, while the lowest - in boiled barley groats. The scientific achievements of this research could help consumers to choose those cereal products available on the market, such as barley and buckwheat groats, which are a rich source of antioxidative compounds and dietary fiber.In recent years, there has been an ever-increasing interest in the research of polyphenols obtained from dietary sources, and their antioxidative properties. The purpose of this study was to determine the effect of boiling buckwheat and barley groats on the antioxidant properties and dietary fiber composition. Antioxidative properties were investigated using methyl linoleate model system, by assessing the DPPH  radical scavenging activity and metal chelating activity. The results were compared with butylated hydroxytoluene (BHT). Raw barley and buckwheat groats extracts showed higher DPPH scavenging ability compared to boiled barley and buckwheat groats extracts. Raw barley groats extract exhibited higher antioxidant activity than boiled groats extract in the methyl linoleate emulsion. Higher chelating ability in relation to Fe (II) ions was observed for boiled groats extracts as compared to raw groats extracts. BHT showed small antiradical activity and metal chelating activity, while showing higher antioxidative activity in emulsion system. The analysis of groats extracts using HPLC method showed the presence of rutin, catechin, quercetin, gallic,  One of the possible modifications of dietary composition, aiming at improving its health promoting properties, is to increase the content of natural non-nutritive food substances with beneficial biological properties. In this group, apart from antioxidant vitamins, carotenoids, minerals and dietary fiber, we can classify low-molecular-weight secondary metabolites of plants , 2.Cereals and pseudo cereals are an important source of macronutrients and bioactive substances with antioxidant activity . GrowingThe technological production process of buckwheat and barley groats includes such stages as cleaning and thermal conditioning (roasting) of grains, size sorting, dehulling, sorting after dehulling and sorting of groats connected with the separation of waste and by-products . The mosFagopyrum esculentum Moench), Kora cultivar were obtain from Breading Station Palikije (Poland) and were used for production of buckwheat groats. Raw buckwheat groats were roasted and dehulled in industrial environment. Barley and buckwheat groats were used both as raw as well as boiled material. The groats were boiled with water/groats ratio of 2:1 (v/v), for 30 min until water has been completely absorbed. After boiling the groats were lyophilized and then comminuted in a Cyclotec mill.Grains of barley Antek cultivar were obtained from Breading Station Danko (Poland) and were used for production of barley groats. Buckwheat grains ; acetone, methanol, ethanol, sodium carbonate POCH (Poland), methyl linoleate Nu-Chek Prep. (USA); BHT Merck (Germany), thermostable α-amylase from Novozymes (Denmark). All other chemicals were of analytical grade.The following chemicals were used: 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent (FCR), (+) catechin, 3-(2-pyridyl)-5,6-bis (4-phenyl-sulfonic acid)-1,2,4-triazine (Ferrozine), Tween 20, α-amylase, pepsin, pancreatin, phenolic acids: et al. [Phenolic compounds were extracted from ground samples according to Amarowicz et al.  with 80 et al. . Aliquotet al. [p-hydroxybenzoic acid, and catechin at 320 nm for p-coumaric, o-coumaric, sinapic, ferulic acids, and at 360 nm for rutin and quercetin.The content of flavonoids and phenolic acids was estimated using the method described by Drożdżyńska et al. . Fast liet al. [The ability to inhibit autoxidation of methyl linoleate was determined following Lingnert et al. . The metet al. [The capacity of the prepared extract to scavenge the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) was monitored according to the method of Sanchez-Moreno et al. . DPPH , soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) was estimated according to Asp et al. . Dietaryet al. , as modiet al. .P < 0.05). Spearman’s rank correlation was used to determine the correlation coefficient between the content of phenolic compounds and the content of fiber and its fractions in groats, and their antioxidative properties.All data were subjected to analysis of variance (ANOVA) using the general linear models of statistical analysis system software (version 8). The significant differences among multiple groups were determined by Tukey’s test . Boiling of barley and buckwheat groats also affected the content of flavonoids and phenolic acids (Table p-coumaric acid (0.91 mg/kg DM extract) in comparison to raw buckwheat (7.26 mg/kg DM extract). Heat treatment of barley groats caused a decrease in the content of sinapic acid (19 %) in comparison to raw barley groats and a slight increase in ferulic acid content (3 %). Boiling of buckwheat groats did not induce any change in the content of rutin. Higher content of polyphenols in boiled buckwheat groats was possibly due to their partial release from the bound form with proteins as a result of boiling. Moreover, phenolics may also be associated with other plant components such as carbohydrates. Technological processing can modify the polyphenol content of foods in several ways [et al. [et al. [et al. [The total phenolic content in buckwheat groats was higher than in the barley groats Table . Boilingds Table . Boiled ral ways . The infral ways . Kreft epic acid  % in com [et al.  showed t [et al. . On the  [et al.  and an i [et al. . Zielińs [et al.  observedThe free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity for different kinds of groats extract are shown in Fig. Figure The ability of the extracts to chelate iron ions was presented in Fig. et al. [The antioxidant activity of extracts is not always dependent on the level of phenolic compounds, but can be different depending on used methods . Therefoet al.  showed tet al. , 29. Theet al. .The highest content of total dietary fiber (TDF) was shown in boiled buckwheat groats (16.45 %), while the lowest in boiled barley groats (7.99 %) Table . The higr = 0.80).The groats showed varied content of dietary fiber fractions. The highest lignin level was found in boiled buckwheat groats (6.24 %), while the lowest in boiled barley groats (0.12 %). The highest content of cellulose was observed for raw barley groats (4.00 %), while the lowest for raw buckwheat groats and boiled barley groats, 0.67 and 0.78 %, respectively. A significantly higher content of hemicelluloses was found in raw buckwheat groats (6.94 %) in comparison with other groats. It was shown that the content of IDF, neutral detergent fiber (NDF) and lignin in groats was correlated with antioxidant activities  ratio does not affect the nutritional properties of buckwheat groats in any negative way. It should be emphasized that this is the recommended way of cooking buckwheat groats. Extracts of boiled buckwheat groats showed a significantly higher content of polyphenols and total dietary fiber than raw buckwheat groats. Both boiled groats extracts formed complexes with iron ions to a higher degree than raw groats extracts. Buckwheat and barley have a high potential as materials for functional food development and production. They would be an ideal possible base for the design of novel foods.


Escherichia coli (EAEC) prototype strain 042 whose entire characterization was essentially obtained from studies performed with the purified toxin. Here we show that Pet is not exclusive to EAEC. Atypical enteropathogenic Escherichia coli (aEPEC) strains, isolated from diarrhea cases, express Pet and its detection in supernatants of infected HEp-2 cells coincides with the appearance of cell damage, which, in turn, were similar to those described with purified Pet. Pet secretion and the cytotoxic effects are time and culture medium dependent. In presence of DMEM supplemented with tryptone cell rounding and detachment were observed after just 5 h of incubation with the bacteria. In the absence of tryptone, the cytotoxic effects were detected only after 24 h of infection. We also show that, in addition to the prototype EAEC, other pet+ EAEC strains, also isolated from diarrhea cases, induce cellular damage in the same degree as the aEPEC. The cytotoxic effects of EAEC and aEPEC strains were significantly reduced in the presence of a serine protease inhibitor or anti-Pet IgG serum. Our results show a common aspect between the aEPEC and EAEC and provide the first evidence pointing to a role of Pet in aEPEC pathogenesis.Plasmid encoded toxin (Pet) is a serine protease originally described in enteroaggregative  Most bacterial enterotoxins, with a few exceptions, are proteases known to catalyze the hydrolytic cleavage of proteins and peptides in order to improve nutrient uptake by the bacteria . As a co Escherichia coli (EAEC), is certainly the best studied member of the SPATE family [α-fodrin activity, leading to a loss of actin stress fibers, cytoskeleton contraction and, finally, to cell rounding and detachment [The plasmid encoded toxin (Pet), so far described only in enteroaggregativeE family –7. This E family , where itachment . In additachment .EAEC, defined by its aggregating pattern of adherence to epithelial cells , has bee Escherichia coli (aEPEC) have been associated with both acute childhood diarrhea [ E. coli (DEC) pathotypes [Atypical enteropathogenicdiarrhea –16 and pdiarrhea , 18. Howthotypes –21. As athotypes . E. coli (DEC) were found in aEPEC strains through the amplification of small fragments of the gene [The presence of genes encoding virulence factors in plasmids, PAIs, transposons, or bacteriophages has allowed genetic recombination that may contribute to pathogenesis. Recently, SPATE-encoding genes from diarrheagenicthe gene .The expression of enterotoxins and cytotoxins has been shown to be determinant in the pathogenic processes caused by several enteric bacteria. However the global mechanism of diarrhea caused by aEPEC, which includes ion loss to the intestinal lumen and the secretion of enterotoxins, is poorly understood. For these reasons, we investigated the expression of Pet in aEPEC strains isolated from diarrhea cases . In thisOur results point to the horizontal transfer of genes encoding the autotransporter protein, which can be an important factor in the emergence of highly virulent strains. We also show that EAEC strains, also obtained from diarrhea cases , induce  pet gene, isolated from cases of acute diarrhea [ pet gene was included as negative control [For this study we selected four aEPEC  and thrediarrhea , 23, 27.μg/mL) [ E. coli HB101 strain was used as a negative control [The EAEC 042  and Pet μg/mL) , 9 were  control .μL bacterial culture was seeded in 3 mL DMEM  or DMEM containing tryptone 1% (DMEM-tryptone) and cultured at 37°C with shaking at 200 rpm until the exponential phase.Bacteria were cultivated in Luria-Bertani (LB)  agar or LB broth before each experiment. For the cytotoxicity assays, 100 2, at 37°C. The subcultures were harvested with 10 mM EDTA and 0.25% trypsin in PBS, pH 7.4. For experimental use, the trypsinized cells were seeded, at 2 × 105 cells/well, in 24-well culture plates containing 13 mm round cover glasses. The cells were then submitted to cytotoxicity assays.HEp-2 cells (ATCC N° CCL-23) were grown in DMEM  supplemented with 10% fetal bovine serum (FBS)  in a humidified atmosphere with 5% COμg of protein were loaded in each well in a 12% SDS-polyacrylamide gel [The expression of Pet was verified by immunoblotting performed with the IgG enriched fraction of a rabbit polyclonal anti-Pet serum (anti-Pet IgG) . Brieflymide gel , 32. Aftμg/mL of anti-Pet IgG for 1 h at room temperature. The membrane was then washed and incubated for 1 h with anti-rabbit IgG diluted 1 : 5.000 and analyzed by either Diaminobenzidine (DAB)  or ECL chemiluminescence system (Amersham Biosciences), according to the manufacturer's instructions.After the transfer and blocking stages, the membranes were probed with 200 Cell cultures were infected, in the presence of 10% FBS, at an MOI of 10 with the different bacterial strains cultivated in DMEM or DMEM-tryptone, and then incubated for 5 or 24 h, respectively, at 37°C.μg/mL, and the prototype EAEC 042 were used as positive controls, while strain aEPEC BA 3170 (pet−) and E. coli HB101 served as negative controls.To evaluate the cytotoxic effects, the cells were washed twice with PBS, fixed with 70% methanol, and stained with 10% Giemsa . The parameters used for the cytotoxicity evaluation were based on the criteria defined by Saidi and Sears  and NavaIn order to confirm the expression of Pet in this system, the culture medium was removed and precipitated with trichloroacetic acid (TCA). The precipitate was then submitted to SDS/PAGE and immunodetection of the toxin was performed with anti-Pet IgG, developed with DAB.μL of each culture was preincubated with anti-Pet IgG at the concentrations of 125 μg/mL and 250 μg/mL for 30 min at 37°C. Alternatively, the same cultures were preincubated with PMSF at the concentrations of 0.3 mM, 0.6 mM, and 1.25 mM for 15 min at 37°C. The preincubated aliquots were then added to the cells in fresh medium and incubated for 5 h at 37°C prior to standard fixation and staining. The positive controls were the prototype EAEC 042 and Pet (200 μg/mL) preincubated with the anti-Pet IgG or PMSF. Strains cultivated with the culture medium only were also used as controls. All experiments were performed at least three times, each time in triplicate.The neutralization test was performed using anti-Pet IgG  or the sP < 0.05.All data were analyzed using GraphPad Prism 5.0 . For use in nonparametric analyses, the Friedman with Dunn's multiple comparison test was performed. In parametric analyses, the 1-way ANOVA with Tukey's multiple comparison test was carried out. Shapiro-Wilk normality test was used for all data. Results were considered statistically significant when  pet gene has been identified in aEPEC strains [ pet+ aEPEC strains. Culture supernatants were precipitated with TCA and subjected to immunoblotting with anti-Pet IgG [ E. coli strain HB101, used as a negative control , the cytotoxic effects induced by these strains could be compared after any given incubation time. We show here that aEPEC clearly induces cell rounding and detachment in the same degree as EAEC, or even more.μg/mL and 250 μg/mL, as compared to the cultures infected with untreated bacteria , could not deliver Pet to epithelial cells, as opposed to the observation with EAEC 042, suggesting that adhesion of EAEC to the cell is important for proper toxin delivery . cah, ehaA, ehaC, and espI [However, the aEPEC strains studied here were classified, based on adhesion assays, as nonadherent to epithelial cells . Althougand espI . Furtherand espI . Therefoand espI , 38. Forand espI .To establish a correlation between the cellular damage observed in this work and toxin Pet, we preincubated the bacteria with either the serine protease inhibitor PMSF or the rabbit anti-Pet IgG enriched fraction. Both treatments considerably reduced or even completely prevented the damage induced to the cells. These results show that the cytotoxic effect induced by both DEC categories is due to toxin Pet. pet gene in aEPEC and its high heterogeneity [ cah, ehaA, ehaC, and espI could be detected, but not AAF/II encoding genes (unpublished data) or other SPATE genes [The presence of genes encoding virulence factors in mobile units, such as plasmids, favors the horizontal transmission of these factors between bacteria and could explain the presence of theogeneity , 21, 39.ogeneity . Such coTE genes . In addi Escherichia coli (tEPEC) express different serine proteases [ E. coli secreted protein C (EspC) [α-fodrin and alters the actin fibers of the cytoskeleton, inducing similar damage [ pet+ studied here lack the espC gene sequence as well as other SPATE encoding genes [An additional interesting aspect that relates the SPATEs and the DEC pathotypes is that EAEC and typical enteropathogenicroteases , 40. EAEC (EspC) . EspC waC (EspC) . Like Per damage . Howeverr damage . Interesng genes . espC gene and whose single pathogenic mechanism described so far is the attaching and effacing lesion, Pet might play an important role in cytotoxicity.To better understand the processes involving SPATEs, which present such diverse activities, they should be characterized in the context of each pathogen that expresses them. In the case of aEPEC, which does not present theBoth EAEC and aEPEC are known to present a high heterogeneity of virulence factors and have been isolated from both diarrheic and nondiarrheic patients. Afset et al.  and Nguy pet gene was previously identified in aEPEC through the amplification of a fragment of 302 bp [ pet gene, the importance of bacterial adherence for toxin delivery, and the contribution of Pet to aEPEC pathogenicity are the goals of our next studies.Though thef 302 bp , we showf 302 bp , 27. Det


Aspergillus awamori (AA), fructooligosaccharide (FOS), and combined Aspergillus awamori and fructooligosaccharide (AA + FOS) on growth, digestibility, blood parameters, and expression of some growth-related genes. A total of 60 broiler chicks at the age of 15 d were divided into a control group (n = 15) and 3 treatment groups. The control group was fed a basal diet, and the treatment groups were fed basal diets supplemented with 0.05% AA, 0.05% FOS, and combined of 0.05% AA and 0.05% FOS. Results from measurement of growth performance and digestibility revealed a significant increase in the body weight gain with improved feed conversion rate in the experimental groups. Interestingly, dry matter digestibility (DMD) and crude protein utilization (CPU) were improved. In addition, plasma total cholesterol and low density lipoprotein-cholesterol (LDL-C) were decreased, while plasma high density lipoprotein-cholesterol (HDL-C) was increased by feeding AA, FOS, and AA + FOS. Expressions of growth hormone secretagogue receptor (GHSR), insulin-like growth factor 1 (IGF-1), and insulin-like growth factor 1 receptor (IGF1R) were increased in experimental groups. In conclusion, the supplementation of either Aspergillus awamori or fructooligosaccharide or both improves digestibility and growth performance probably by promoting skeletal muscle protein metabolism.This study was conducted to show the effect of  Aspergillus species  are used as probiotics [ Aspergillus species are extensively used for industrial enzyme production (mainly a-amylases and glucoamylases) due to their superior ability to secrete enzymes [ Aspergillus awamori is a fungus used for food processing in Japan [ A. awamori are given generally recognized as safe (GRAS) status from Food and Drug Administration (FDA) [ Aspergillus probiotic include the reduction of gut pH, production of some digestive enzymes and vitamins, production of antibacterial substances , reduction of cholesterol level in the blood, stimulation of immune system, suppression of bacterial infections, removal of carcinogens, improvement of calcium absorption, and reduction of faecal enzyme activity as well as reconstruction of normal intestinal microflora disorders caused by diarrhoeas, antibiotic therapy, and radiotherapy [Probiotics are live cultures of organisms supplemented in animal diets that can beneficially affect the host animal by improving the microbial balance in the gut . Recentlobiotics –4. Asper enzymes . Saleh ein Japan . The proon (FDA) . The phyotherapy . Fructooligosaccharides can be used as alternative of antibiotics to enhance the growth and production efficiency of broilers [β-linkages between fructose monomers cannot be hydrolyzed by enzymes of endogenous origin [ Fructooligosaccharides have been shown to enhance the growth of Bifidobacterium and Lactobacillus but inhibit Escherichia coli and Salmonella in the large intestine [Prebiotics have been described as nondigestible food substances that selectively stimulate the growth of favorable species of bacteria in the gut, thereby benefitting the host . These sbroilers  and theys origin . The phyntestine , 14.The combination of prebiotic and probiotic is referred as synbiotic . It has GHSR), insulin-like growth factor 1 (IGF1), and insulin-like growth factor 1 receptor (IGF1R) are important genes of this axis which have a significant role in muscles development and growth. The IGF1 gene stimulates glucose uptake, amino acid uptake, and protein synthesis and inhibits protein degradation by satellite cell-derived myotubes [ IGF1 gene in the muscle tissue leads to enhanced muscle growth in chicks [The growth and development of chickens are primarily regulated by genes of the somatotropic axis. Ghrelin receptor, or growth hormone secretagogue receptor . The control group was fed a basal diet, and the experimental treatment groups were fed the basal diets supplemented with 0.05% Aspergillus awamori (AA) as probiotic, 0.05% fructooligosaccharide (FOS) as prebiotic, and combined 0.05% A. awamori and 0.05% fructooligosaccharide (AA + FOS) as synbiotic. The birds were given the experimental diets from 15 to 37 days of age. The experiment was conducted in a normal room with 14 h light: 10 h dark cycle. Room temperature was maintained at 23–25°C with relative humidity from 50 to 70% throughout the experiment. All experiments were performed in accordance with institutional guidelines concerning animal use.Sixty, one-day-old unsexed Lohmann broilers were housed in an electrically heated battery brooder and provided with water and commercial starter diet  and metabolizable energy (ME) 13.39 MJ/kg) until 12 days of age. The chicks were housed in individual cages and fed the basal diet from 12 to 15 days of age. The composition of the basal diet  is shown in Body weight was recorded every 3 days and feed intake was recorded daily during the experimental period. At the end of the experimental period, the birds were weighted and slaughtered then dissected to measure the weights of breast muscle, liver, and abdominal fat. Blood samples were collected in heparinized test tubes and quickly centrifuged  to separate the plasma. Plasma and meat samples were stored at −20°C and −8°C, respectively, until further analysis.Utilization coefficients of nutrients were calculated for dry matter (DM), crude protein (CP), crude fiber (CF), and ether extracts (EE) by analysing the diets and collecting faces compassion for the last three days of the experiment; excreta were collected and weighted from each bird. Then the samples were dried by the drying oven and grinded. The crude protein, crude fiber, and ether extracts were analysed. The calculations were as follows: nitrogen retention (%) = /total nitrogen intake × 100; the same methods were used for all coefficients of nutrients.Total cholesterol level, triglyceride, HDL-C, and LDL-C were measured calorimetrically using commercial kits  according to the procedure outlined by the manufacturer.μg per sample) was reverse transcribed into cDNA using Revert Aid H minus Reverse Transcriptase and as described by manufacturer . The IGF1 forward primer 5′AGCTGTTCGAATGATGGTGTTT3′ and reverse primer 5′GCCCCAGCATTCTCTTTCCTT3′, IGF1R forward primer 5′TCCAACACAACACTGAAGAATC3′ and reverse primer 5′ACCATATTCCAGCTATTGGAGC3′, GHSR forward primer 5′GTCGCCTGCGTCCTCCTCTT3′ and reverse primer 5′ACGGGCAGGAAAAAGAAGATG3′, GAPDH forward primer 5′GTGAAAGTCGGAGTCAACGG3′ and reverse primer 5′ACAGTGCCCTTGAAGTGTCC3′ were used to amplify partial clones of IGF1, IGF1R, GHSR, and the house-keeping gene GAPDH, respectively. The PCR was carried out in a reaction volume of 25 μL, containing 1.0 μL cDNA template (approximately 50 ng), 0.5 μL (0.20 mM) dNTP, 2.5 μL buffer, 1.5 μL (2.5 mM) MgCl2, 1.0 μL 10 μmoL/L forward primer, 1.0 μL 10 μmoL/L reverse primer, 0.5 μL Taq DNA polymerase , and 17.0 μL nuclease free water. Thermal cycling parameters were as follows: initial denaturation at 94°C for 5 min, 35 cycles of amplification , and final extension at 72°C for 5 min.Each muscle sample was homogenized and a total RNA was extracted using total RNA purification kit following the manufacturer protocol . The extracted total RNA . Tukey's multiple comparison test was used to identify which treatments conditions were significantly different from each other at a significance level of Data presented in Dry matter digestibility (DMD), crude protein utilization (CPU), and crude fiber utilization (CFU) were all improved by feeding AA and FOS, while extract utilization (EEU) was only improved by feeding combination of AA and FOS . GHSR, IGF1, and IGF1R genes in muscles after treatment in comparison to control group and to the housekeeping gene, GAPDH, are presented in Figures  GHSR and IGF1R were increased by feeding AA and FOS and highly significant by the combination of AA and FOS. However, the increased level of IGF1 mRNA was nearly similar after feeding the three experimental diets.Changes in transcription levels of Aspergillus awamori (AA) and fructooligosaccharide (FOS) and its mechanism in broiler chickens. The combined feeding AA and FOS synergistically promoted the broiler performance. The improvement in weight gain and feed efficiency due to the combination of AA and FOS may be partially due to the increase in metabolic energy of the feed [ fructooligosaccharide does not selectively enrich for beneficial bacterial populations. Instead, it is thought to act by binding and removing pathogens from the intestinal tract and stimulation of the immune system [The main aim of the present study was to show how the growth performance, digestibility, and protein metabolism can be improved by feedingthe feed  or due tthe feed . The frue system . Aspergillus [ IGF1 gene exerts anabolic actions on skeletal muscle tissue. These actions include stimulation of amino acids uptake and incorporation into protein, uridine and thymidine synthesis into nucleic acid glucose uptake, cell proliferation, and suppression of protein degradations [ IGFI and higher IGFI mRNA levels as compared to chickens with low growth rate [In this study we have found that body weight and breast muscle weight were increased by AA and FOS. Similarly, Yamamoto et al. have noticed a significant increase in carcass weight and breast muscle weight of broilers after feeding on diets containing 0.05 and 1% of AA . This seergillus . Guobin ergillus . Also, Iergillus . The IGFadations . All IGFwth rate . This suwth rate . In addiwth rate . Fermacto, which is a commercial fermentation product at levels of 0.15 and 0.3%, have a significant lower abdominal fat [ Aspergillus oryzae at 0.1% in diet significantly lowered serum cholesterol and triglyceride in broiler chickens [ Aspergillus could be related to an inhibitor of 3-hydroxyl-3-methylglutaryl-coenzyme (HMG-CoA) reductase [Abdominal fat weight was decreased by feeding AA and FOS. This agrees with Navidshad et al. who found that broilers fed on diets containinginal fat . We suggchickens . The meceductase , 34. It eductase  and prebeductase . Aspergillus awamori [ Aspergillus awamori possesses the ability to digest raw starches [ Bifidobacterium and Lactobacillus, which had the action of precipitation and assimilation with bile salt [GHSR) is a candidate gene for food intake and food conversion rate and so its absence decreases the food conversion rate and subsequently fat content and body weight [ GHSR after supplementation of AA and FOS. This elevation is therefore likely to be responsible for, or associated with, improved digestibility and food conversion rate. Further investigations are needed to validate this possibility.Utilization coefficients of nutrients dry matter (DM), crude protein (CPU), crude fiber (CFU), and ether extracts (EEU) were all improved by feeding AA and FOS. The broilers do not produce some enzymes such as cellulase and xylanase which are required for the digestion of soluble nonstarch polysaccharides. These enzymes can be produced by awamori , 3, 37 a awamori . Furtherstarches . In thisstarches . Some instarches . Additioile salt  thus incile salt , 42 and y weight , 21. In  Aspergillus awamori and fructooligosaccharide to the broiler diets probably by promoting skeletal muscle protein metabolism.We concluded that growth performance and digestibility can be improved by supplementation of both


Using field and experimental observations on Baltic copepods and bloom-forming diazotrophic filamentous cyanobacteria, we show that cyanobacteria may in fact support zooplankton production during summer. To highlight this side of zooplankton-cyanobacteria interactions, we conducted: (1) a field survey investigating linkages between cyanobacteria, reproduction and growth indices in the copepod Acartia tonsa; (2) an experiment testing relationships between ingestion of the cyanobacterium Nodularia spumigena  and organismal responses  in the copepod A. bifilosa; and (3) an analysis of long term (1999–2009) data testing relationships between cyanobacteria and growth indices in nauplii of the copepods, Acartia spp. and Eurytemora affinis, in a coastal area of the northern Baltic proper. In the field survey, N. spumigena had positive effects on copepod egg production and egg viability, effectively increasing their viable egg production. By contrast, Aphanizomenon sp. showed a negative relationship with egg viability yet no significant effect on the viable egg production. In the experiment, ingestion of N. spumigena mixed with green algae Brachiomonas submarina had significant positive effects on copepod oxidative balance, egg viability and development of early nauplial stages, whereas egg production was negatively affected. Finally, the long term data analysis identified cyanobacteria as a significant positive predictor for the nauplial growth in Acartia spp. and E. affinis. Taken together, these results suggest that bloom forming diazotrophic cyanobacteria contribute to feeding and reproduction of zooplankton during summer and create a favorable growth environment for the copepod nauplii.It is commonly accepted that summer cyanobacterial blooms cannot be efficiently utilized by grazers due to low nutritional quality and production of toxins; however the evidence for such effects  One can speculate that when preferred prey is scarce, zooplankton may increase feeding on the abundant cyanobacteria, despite their inadequate biochemical composition and toxicity. We know, for example, from terrestrial ecology that rainforest-dwelling parrots consume toxic foods during dry seasons when other, non-toxic, food is limited Toxic blooms of filamentous cyanobacteria are proliferating worldwide due to the climate change and eutrophication Aphanizomenon sp., Nodularia spumigena and Dolichospermum spp. Pseudocalanus acuspes in the southern Baltic Sea indicates that cyanobacteria contribute substantially to its diet during summer in situ even when alternative food is present In the Baltic Sea, summer blooms of diazotrophic filamentous cyanobacteria are dominated by Similarly, effects of filamentous cyanobacteria on survival and development are often contradictory, with both negative Acartia tonsa to phytoplankton community structure, with specific focus on the effects of the bloom-forming cyanobacteria. Then, we conducted a follow-up laboratory experiment, where ingestion of the cyanobacterium was measured in A. bifilosa using molecular diet analysis and related to the copepod oxidative status, and recruitment. Finally, we analyzed long-term data on growth indices in nauplii of Acartia spp. and Eurytemora affinis in relation to cyanobacteria bloom intensity.Here, we explored linkages between Baltic Sea diazotrophic filamentous cyanobacteria and fitness-related responses in copepods: reproductive output, juvenile development and growth, and oxidative status. To evaluate these connections, we conducted a set of interrelated studies in the northern Baltic proper. First, in a field survey, we used a correlative approach to relate reproduction (egg production and their viability) of the copepod The sampling was conducted within Swedish National Marine Monitoring in the Baltic Sea and no specific permissions were required for the sampling locations of this study. Also, we did not require ethical approval to conduct this research as no animals considered in any animal welfare regulations and no endangered or protected species were involved in either field or experimental studies.Sampling was conducted biweekly during cyanobacterial bloom  at three stations in the north-west Baltic proper ; each species of filamentous cyanobacteria was treated as a separate category regardless of size.Sea surface temperature (SST) was measured on each sampling occasion. Integrated phytoplankton samples were collected by a hose (inner diameter 19 mm) at stns H2 and H4 (0–14 m) and stn B1 (0–20 m), and preserved with acidic Lugol's solution. Using an inverted microscope (Wild M40), cells counts (>2 µm) and biovolume analysis were conducted according to HELCOM monitoring guidelines A. tonsa females were sorted upon arrival to the laboratory; the choice of species was based on relative abundance of copepod species in the samples and availability of adult females. They were incubated individually for 24 h in darkness at ambient (±2°C) temperature, in microplate wells  filled with 5 mL of 6-µm filtered seawater. The mortality of the females during the incubation was <5%.Copepods were sampled by vertical hauls (0–10 m) using a WP2 net  and brought to the laboratory in 20 L insulated containers. Using a wide-mouth pipette, adult −1 day−1) was recorded by counting number of eggs in each well under a stereomicroscope . No crumbled or empty egg shells were found, indicating absence of egg cannibalism. Live females were individually placed in Eppendorf tubes, frozen at −80°C and stored for a few weeks before RNA analysis. For egg viability  analysis, all eggs from each well were transferred to a depression slide and stained with TO-PRO-1 iodide (Molecular Probes) −1 day−1) was calculated by multiplying EV% with EPR.The egg production rate  and the cyanobacterium Nodularia spumigena  were grown under irradiance of 13.7 µmol photons m−2 s−1 for 16 h a day, and at 18°C, in f/2 medium (without silica) and Z8 (without nitrogen), respectively In August 2010, copepods were collected, with a 150 µm plankton net in the Storfjärden Bay, Western Finland  was used as a sole food, and in the treatments with cyanobacteria, B. submarina (971±208 µg C L−1) were mixed with N. spumigena (102±18 µg C L−1); this mixture approximates average N. spumigena contribution to the summer phytoplankton community in the northern Baltic proper  were incubated in two alternative feeding regimes, with or without c proper . The botN. spumigena in the copepod gut content and to characterize oxidative status of the copepods, adult animals recovered from the incubation bottles were placed into microcentrifuge tubes with 0.7 mL phosphate buffer saline (PBS) and 100 µm glass beads and homogenized for 4 minutes using FastPrep with cooling. The tubes were thereafter centrifuged at 4°C for 5 min with 10 000×g.To determine amount of N. spumigena using qPCR. The Nodularia-specific primers were used to amplify ∼200 bp fragment of N. spumigena 16S rDNA in the guts of copepods −4 ng), DNA extracted from a culture of N. spumigena was used 2>0.99), with amplification efficiency of 95–100%. As a reference sample, the feeding media containing N. spumigena but no copepods following the same procedure as for the test samples was used. A standard curve and no template controls (water) in triplicates were included in all runs. To check for non-specific products, DNA melt-curve analysis was performed after each qPCR experiment.For DNA extraction, 50 µL of the homogenate were used by mixing with 50 µL 20% Chelex To measure the intracellular soluble antioxidant capacity, the homogenized copepod samples were analyzed using oxygen radical absorption capacity  assay ik is assigned stage value , in number of copepods at that stage, and NS – total number of individuals To estimate copepod reproductive output, the female EPR, the EV% and early nauplial development  were used. To determine EPR, all eggs were counted and related to the number of live females in the corresponding bottle. For EV% measurements, ∼50 eggs per bottle were analyzed with TO-PRO-1 iodide staining www.2.ecology.su.se) were used to study effects of various phytoplankton groups and physical factors on copepod growth and recruitment, with particular focus on the effects of cyanobacteria. As a proxy for copepod growth during early life stages, we used RNA:DNA ratio in the nauplii  of Acartia spp.  and Eurytemora affinis. In the study areas, A. bifilosa dominate in summer Acartia communities, contributing 62 to 94% on the long-term basis . Specifically, we considered that biotic  and abiotic  variables integrate local environmental variability, and could define an adequate environmental framework for copepod growth.Zooplankton collected in the northern Baltic proper within SNMMP and SYVAB's marine monitoring program in Himmerfjärden Bay . Phytoplankton samples were collected as integrated hose samples, preserved and analyzed in the same way as in the summer field survey Acartia spp. and E. affinis. The younger nauplial stages (NI–NII) were considered non-feeding later preserved samples, rinsed and transferred to Eppendorf tubes (5–7 ind. sample−1); the two copepod genera were treated separately. Nucleic acids were quantified with fluorometric high-range RiboGreen  using the same instrumentation as for RNA analysis in the summer field survey RNA and DNA contents were measured in nauplii (NIII–NVI) of www.smhi.se (SHARK database) and www.2.ecology.su.se/dbhfj/index.htm, and data on copepod growth- and reproduction-related variables are provided as Supporting Information  with normal error structure and either identity or log-link function were applied using STATISTICA 10 . For all GLMs, the response variables and biovolume data were Box-Cox transformed to approach normal distribution. Akaike Information Criterion (AIC) was used to optimize the number and combination of predictive variables included. To validate the models, the Wald statistic was used to check the significance of the regression coefficient for each parameter, a likelihood ratio test was used to evaluate the statistical significance of including or not including each parameter and model goodness of fit was checked using deviance and Pearson χAphanizomenon sp., N. spumigena and Dolichospermum spp.], autotrophs in each size fraction , total phytoplankton biovolume, salinity and SST]. Copepod PL was also included as independent variable for RNA and EPR GLMs were used to examine relationships between the dependent variables  and the explanatory variables (biovolumes of filamentous cyanobacteria species [N. spumigena observed in the summer field survey and recent reports on high levels of antioxidants in cyanobacteria N. spumigena. The amount of Nodularia in copepod guts determined by qPCR was used as a measure of grazing.Based on the positive effects of Acartia spp. and E. affinis) and station (B1 and H4), because of the significant differences in RNA:DNA ratio between the species and differences in phytoplankton community structure between the stations. Only those phytoplankton groups that contributed>5% to the total phytoplankton biovolume were considered (Mesodinium rubrum (autotrophic ciliate), (5) nitrogen fixing filamentous Cyanobacteria, (6) Cryptophyceae, (7) large (>10 µm) Prymnesiophyceae, and (8) Prasinophyceae. We also included biovolumes of total phytoplankton and phytoplankton without cyanobacteria. The monthly NAO index values were taken from the Climate Prediction Center, Washington, DC (ftp://ftp.cpc.ncep.noaa.gov/wd52dg/data/indices/nao_index.tim). A high, positive winter NAO index  indicates mild and rainy winters, while low, negative values occur during cold winters over Europe. A warmer winter is generally followed by a warmer spring  with early onset of the growth season.GLMs were used to examine relationships between the nauplial RNA:DNA ratio and biovolume of each phytoplankton group and abiotic variables that have been reported to affect zooplankton in the Baltic Sea . Wilcoxon signed rank test was used to determine the significance of differences in nauplial RNA:DNA ratio between the stations (B1 and H4) and between the months (July and August) within the stations, whereas paired t-test was used to evaluate these differences in phytoplankton. The models were constructed for each taxa  and highest contribution of filamentous cyanobacteria (29%) observed at stns H4 and H2, respectively , and a significantly positive correlation between the cyanobacteria and the smallest phytoplankton size class , whereas no correlation was found for the intermediate size class . Among the cyanobacteria, Aphanizomenon sp. had the highest significant negative correlation with phytoplankton>15 µm  and N. spumigena had a moderate positive correlation with phytoplankton 2–5 µm .Phytoplankton communities differed among the stations, with the highest total phytoplankton  but not the RNA content  and the effect was negative. No statistically significant model for EPR was found for stn H2, which had significantly lower EPR compared to stns B1 and H4 ; none of the controls were positive . Moreover, the nauplial RNA:DNA ratio was also significantly higher at stn H4 than at stn B1 . Therefore, to describe responses of nauplial RNA:DNA ratio  were significantly predictive. For E. affinis, only models incorporating total phytoplankton, filamentous cyanobacteria, diatoms and SST were significant  was significantly advanced if their mothers were feeding on the cyanobacterium, thus effectively increasing recruitment  direct increase of macro- and micronutrient intake by copepods that use cyanobacteria as an additional food source; (2) supplementing copepod's diet with phytochemicals, such as polyphenols, vitamins and various antioxidants, that may enhance physiological responses pumigena , and thuN. spumigena increased the antioxidant defenses and improved oxidative status as indicated by the ORAC:TBARS ratio , which was also a positive predictor for egg production; whereas these were not available for the copepods in the experiment. There might also be differences between the test species, A. tonsa (field survey) and A. bifilosa (experiment), in their sensitivity to the cyanobacterium effects and dependency on other food sources and environmental factors. In particular, A. bifilosa might have been avoiding to ingest larger filaments in the experiment due to food selection against this cyanobacterium Brachiomonas with or without Nodularia) was provided at the same amount. As a result, lower food intake when feeding in mixtures containing cyanobacteria would translate into lower egg production, which was observed in this copepod species.Although the net effects of cyanobacteria on copepod reproduction  appear either neutral or positive, there is some variation among the species and studies. For example, in the summer field survey, effects of positive , whereasoduction . This diN. spumigena colonies do not form bundles, are not as rigid Aphanizomenon sp., which was found to negatively affect EPR  in the northern Baltic proper. Variables measured: EPR (egg production rate), VEPR (viable egg production rate), RNA , and PL (prosome length).(XLSX)Click here for additional data file.Table S2Acartia bifilosa: Data on feeding, reproduction and nauplii development obtained in the laboratory experiment conducted in August 2010, in the Storfjärden Bay, Western Finland. Variables measured: EPR (egg production rate), VEPR (viable egg production rate), RNA , and PL (prosome length).(XLSX)Click here for additional data file.Table S3Acartia sp. and Eurytemora affinis: Long-term data  on RNA:DNA ratio in nauplii at stns B1 and H4 in the northern Baltic proper. The mean values are based on 3–4 replicate samples analyzed per year (July-August).(XLSX)Click here for additional data file.Table S4Growth and reproduction indices observed in the (A) field survey, (B) experimental study, and (C) long-term dataset. Data are presented as means with their standard deviations; abbreviations as in (DOC)Click here for additional data file.Table S5Pearson r for correlations among phytoplankton groups at stn B1 and stn H4. Significant correlations are in bold face (p<0.05). Abbreviations: CHLO – Chlorophyceae, CHRY – Chrysophyceae, CRYP – Cryptophyceae, CYAN – Cyanophyceae, DIAT – Diatomea, DINO – Dinophyceae, EUGL – Euglenophyceae, MESO – Mesodinium rubrum (Myrionecta rubrum), PRAS – Prasinophyceae, PRYM – Prymnesiophyceae, UNID – unidentified flagellates, and HDIN – heterotrophic dinoflagellates.(DOC)Click here for additional data file.Table S6Summary of the observed effects linking filamentous cyanobacteria to reproductive output, growth indices and oxidative status in copepods. Abbreviations as in (DOC)Click here for additional data file.Protocol S1Oxidative stress biomarkers.(DOCX)Click here for additional data file.


Escherichia coli and also to find out the enzymatic mechanisms of β-lactam antibiotics resistance.Multi-drug resistance (MDR) in Gram-negative organisms is an alarming problem in the world. MDR and extensively-drug resistance (XDR) is in increasing trend due to the production of different types of beta (β)-lactamases. Thus the aim of this study was to document the incidence of MDR and XDR in clinical isolates of Escherichia coli (E. coli) identified by standard laboratory methods were studied. Antibiotic susceptibility profile was performed for all the isolates and the suspected isolates were phenotypically tested for the production of extended spectrum β-lactamase (ESBL), metallo β-lactamase (MBL) and AmpC β-lactamase (AmpC) by recommended methods.Two hundred clinical isolates of Around three-fourth (78%) of the total isolates were multi-drug resistant. ESBL, MBL and AmpC production was found in 24%, 15% and 9% of isolates respectively. Amikacin, chloramphenicol and colistin were found to be the most effective antibiotics.High percentage of MDR was observed. β-lactamase mediated resistance was also high. Thus, regular surveillance of drug resistance due to β-lactamases production and infection control policy are of utmost importance to minimize the spread of resistant strains. Resistance to broad spectrum β-lactams, mediated by extended spectrum β-lactamases (ESBL), metallo β-lactamases (MBL) and AmpC β-lactamases (AmpC) enzymes is an increasing problem worldwide . PresencKlebsiella pneumoniae (K. pneumoniae), Klebsiella oxytoca, Escherichia coli (E. coli), Proteus mirabilis, Enterobacter cloacae, Morganella morganii, Serratia marcescens, Shigella dysenteriae, Pseudomonas aeruginosa, Burkholderia cepacia, Capnocytophaga ochracea, Citrobacter species, and Salmonella species [ESBLs were first identified in 1983. Since the time, they have been found worldwide in a number of organisms, including  species -10.E. coli and K. pneumoniae, is now a critical concern for the development of therapies against bacterial infection. The major ESBL producer was K. pneumoniae before 2000, but now E. coli has become an important ESBL carrier in Western countries [The emergence of ESBL producing bacteria, particularly ountries -13. Sincountries . Therefoountries . The MBLountries .E. coli and Klebsiella spp. is an emerging problem [AmpC-mediated β-lactam resistance in  problem . High le problem ,19.E. coli and to determine whether any alternative regimens are available to treat them.The production of β-lactamases is the single most prevalent mechanism responsible for resistance to β-lactams among clinical isolates belonging to the family of Entrobacteriaceae . Over thE. coli , identified by standard microbiological technique [A total of 200 random, non-redundant and non-repetitive community acquired clinical isolates of echnique  in the bE. coli 25922 was used as a control organism for antibiotic sensitivity testing.Antibiograms of the isolates were determined by modified Kirby-Bauer disk diffusion method on Mueller-Hinton agar standard media using commercially prepared disks  in compliance with Clinical and Laboratory Standards Institute (CLSI) guidelines . AntimicMDR, XDR and PDR isolates were identified according to the guidelines recommended by joint initiative of the European Centre for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC) . AccordiE. coli were examined for their susceptibility to 3rd generation cephalosporins by using ceftazidime (30 μg) and cefotaxime (30 μg) disks. The isolates showing diameter of ≤ 22 mm zone of inhibition for ceftazidime and/or ≤ 27 mm for cefotaxime were considered as ESBLs suspects as per National Committee for Clinical Laboratory Standards (NCCLS) guidelines [et al. [Isolates of idelines . All sus [et al. .Isolates that were found resistant to imipenem, meropenem or third generation cephalosporins (ceftazidime) in Kirby Bauer disk diffusion method were presumptively considered MBL producers and were confirmed by the imipenem disk with EDTA methods. Briefly, the test inoculums (comparable to 0.5 McFarland standards) were prepared and transferred on to Mueller Hinton agar plates. Two imipenem (10ug) disks were placed on the surface of agar plate and 10 μl EDTA solutions was added to one of them to obtain a desired concentration of 750 μg. Plates were incubated for 16 to 18 hours at 35°C. An increase in zone size of more than or equal to 7 mm for imipenem-EDTA disk compared to imipenem disk alone indicated MBL producer strain as described by Yong et al. .E. coli isolates resistant to cefoxitin in Kirby-Bauer disk diffusion method were confirmed for AmpC β-lactamase production by modified Hodge test. In the test, a cefoxitin susceptible E. coli indicator strain (ATCC 25922) was plated on Muller Hinton agar medium and the cefoxitin disk was placed. Test organism was streaked toward the cefoxitin disk. If the test organism expressed AmpC, it hydrolyzed the cefoxitin and showed growth along the intersection of the streak and the zone of inhibition from the cefoxitin disk [All tin disk .The samples used in this study were from routine clinical specimens; however, verbal consent was taken from the patients. The ethical approval was obtained from Institutional Review Committee of Chitwan Medical College Teaching Hospital (CMCTH), Bharatpur, Nepal to conduct the study.E. coli isolates tested exhibited resistance to amoxicillin-clavulanic acid and 77 % of them remained resistant to ciprofloxacin whereas all the isolates were susceptible to colistin and few isolates (7%) were resistant to imipenem  isolates were MDR and 14 (7%) isolates were XDR whereas no PDR isolate was identified , 30 (15%) and 18 (9%) isolates respectively  were producers of both ESBL and MBL, 8 (4%) isolates synthesized both ESBL and AmpC whereas 6 3%) isolates produced both MBL and Amp C. All the three types of β-lactamases  were detected in 4 (2%) isolates  whereas no isolates were resistant to cytoplasmic membrane damaging agent Table .Table 4Very minor numbers of isolates (4%) were resistant to only one antibiotic whereas majorities 85%) of isolates were resistant to at least three antibiotics  and amikacin as the most effective antibiotics has also been defined by other authors and it can be concluded that amikacin is a promising regimen for the empirical therapy [In current study we investigated 200 clinical isolates of floxacin . Howeverfloxacin . Among t therapy -31. ChloE. coli was also observed by Ibrahim et al. (92.2%) from Sudan [E. coli in Nepal.In present study, of total isolates tested, 156 (78%) isolates were MDR. Similarly, higher rate of MDR was also reported by Sharma et al. (90.8%)  whereas om Sudan . As it wE. coli isolates showed relatively high resistance rates to ciprofloxacin (91%). This has been hypothesized to be related to the inappropriate use (over-use and miss-use) of fluoroquinolones for humans [All MDR isolates in this study were resistant to amoxicillin-clavulanic acid. Resistance to fluoroquinolones varies geographically and is an emerging problem in both developed and developing countries ,35. In tr humans . Also, pr humans . Majoritr humans . All XDRE. coli have been described in hospitals to cause various outbreaks, but their presence in community has also been documented [E. coli strains were detected. Higher prevalence of ESBL producing E. coli has also been reported by other authors [ESBL-producing cumented -40. The  authors ,41,42 bu authors . This lo authors . This hiSusceptibility pattern of ESBL-producing isolates showed that these strains are not only resistant to β-lactams but also to other classes of antibacterials including gentamicin, cotrimoxazole and fluoroquinolones. In this study, we found that the β-lactamases producing isolates were resistant to most of the antibiotics tested but amikacin, imipenem, chloramphenicol and colistin showed promising efficacy against ESBL and AmpC producing isolates, which is in accordance with the other report from Nepal . The carMulti type β-lactamases producing isolates were also reported in our study. Among total tested isolates, 10 (5%) isolates were producers of both ESBL and MBL. ESBL together with AmpC production was seen in 8 (4%) isolates, whereas, 6 (3%) isolates produced both MBL and AmpC and 4 (2%) isolates did all, ESBL, MBL and AmpC. Similar result of ESBL with AmpC production (5.4%) was also documented by Kaur et al. from India .Enterobacteriaceae and currently disseminated worldwide [Over the past 20 years there has been increased resistance to β-lactam antibiotics due to production of ESBL mediated by plasmids. This type of resistance is now observed in all species of orldwide . In thisorldwide .In the present study, we found that all the isolates were resistant to at least one agent and 170 (85%) isolates were resistant to at least 3 agents while 22 (11%) isolates were resistant to two agents and 8 (4%) isolates were resistant to only one antimicrobial agent.E. coli and high rate of ESBL, MBL and AmpC β-lactamase production in our set up. A strict hospital infection control policies and a prudent use of antimicrobial regimens are to be adopted by the concerned people to minimize the development of resistant strains. It is also essential to have a regular and routine monitoring of ESBL, MBL and AmpC β-lactamase producing clinical isolates in clinical laboratories. Regular nation wise surveillance of multidrug resistance seems necessary step to combat the severity of infections caused by MDR pathogens. Further studies at molecular level may be beneficial to rule out the cause of MDR pattern.This study concluded that, there is high prevalence of community acquired MDR


Aim: The aim of this study was to compare the prevalence and severity of depression in chronic hepatitis B (CHB) patients, chronic hepatitis C (CHC) patients, and healthy participants.Methods: Two hundred and fifty-three persons participated in this cross-sectional study between 2011 and 2012 in Imam Khomeini Hospital. The prevalence and severity of depression were assessed using the Hamilton Scale.Results: There was significantly higher prevalence of depression in CHC patients (35.9%) than in healthy participants (11.3%) and CHB patients (19.8%) (both P < 0.01). However, CHB and CHC patients did not differ significantly in their depression prevalence after excluding inactive hepatitis B surface antigen (HBsAg) carriers . Inactive HBsAg carriers differed significantly from CHB patients—excluding inactive HBsAg carriers—in depression prevalence . No statistically significant difference was found in depression severity between all groups (P > 0.05).Conclusion: Depression screening in chronic hepatitis B and chronic hepatitis C patients may be beneficial in disease management. The incidence of depression is increased in patients with major chronic medical disorders, such as conditions involving immune and inflammatory mechanisms . HepatitPrevious studies showed higher rates of depression in CHC patients , 8–13 anThe study protocol was approved by the Institutional Review Board at Tehran University of Medical Sciences. This cross-sectional study was conducted between November 2011 and December 2012. The setting for participants’ recruitment was the hepatitis clinic of Imam Khomeini Hospital, which is a tertiary care medical centre in Tehran. Convenience sampling strategy was used. The investigators explained the design and purpose of the study to participants who met eligibility criteria before obtaining informed consent.The inclusion criterion for patients was HBV or HCV infection diagnosed at least 6 months previously, using polymerase chain reaction (PCR). Patients with clinical symptoms or signs of cirrhosis or portal hypertension were excluded. Further exclusion criteria were antiviral therapy, any chronic physical or psychiatric disease or malignancy, past history of chronic physical or psychiatric diseases or malignancy, and co-infection with human immunodeficiency virus (HIV), HCV, or HBV. Finally, inactive hepatitis B surface antigen (HBsAg) carriers were defined as CHB patients without liver injuries, identified with liver enzyme tests or liver biopsy.Healthy participants were hospital staff members free from known physical or psychiatric diseases, and who were not using any drugs or medication.The outcomes were the rate and severity of depression for each group. All participants were interviewed by one trained psychologist using a 17-item Hamilton Depression Rating Scale (HAMD-17). A minimum of 20 minutes was allocated to each clinical interview. HAMD-17 includes 17 items , aiming to identify the presence and severity of depression. A validity and reliability study was performed and the cut-off score was accepted as 10. Categoric scores for depression severity were determined as: no depression, score <10; mild depression, score 10–13; moderate depression, score 14–17; severe depression, score >17).Demographic and clinical characteristics of all participants were recorded using a checklist. In addition, CHC patients' clinical characteristics, including serum level alanine aminotransferase enzyme (ALT), viral load, fibrosis stage of liver, genotype of virus, and past history of intravenous (IV) drug abuse were recorded. Viral load and genotype of virus, measured with PCR test, and fibrosis stage of liver were identified with liver biopsy. If there was a history of IV drug abuse, participants were considered to be drug-free if they had not injected in the previous 4 months and recorded with a data collection form.2 test and Kruskal-Wallis test, respectively. Inactive HBsAg carriers were also compared with CHB patients, CHC patients, and healthy participants using the Pearson χ2 tests. Either the Pearson χ2 test or Fisher’s exact test were used to compare depression prevalence in different subgroups of CHC patients. All P-values were two-tailed and a P < 0.05 was considered significant. SPSS 16.0 was used for statistical analyses.In this study, the three groups were compared for proportion and severity of depression, using the Pearson χEighty-one CHB patients, 92 CHC patients, and 80 healthy participants met the eligibility criteria and were analysed. Participants’ clinical and demographic characteristics are summarized by group in P = 0.003). In addition, CHC patients showed a higher rate of depression than healthy participants (P = 0.000) or CHB patients (P = 0.019). However, CHB patients did not differ significantly from CHC patients in terms of prevalence of depression after excluding inactive HBsAg carriers . There was no statistically significant difference between inactive HBsAg carriers and healthy participants . Inactive HBsAg carriers and CHB patients—excluding inactive HBsAg carriers—differed significantly in their prevalence of depression .Nine (11.3%) healthy participants and 49 (28.3%) patients with hepatitis, including 16 (19.8%) CHB patients and 33 (35.9%) CHC patients were depressed. There was a significantly higher rate of depression in patients with hepatitis than in healthy participants (P = 0.120).P = 0.006) and alcohol drinkers (P = 0.012), but this prevalence was not associated with viral load, HCV genotype, fibrosis stage, or serum ALT level   .Table 3et al. demonstrated that a higher prevalence of major depressive disorder (MDD) could be observed among CHC patients than in CHB patients or controls and the risk of MDD was not statistically different between the CHB group and controls  [et al. showed that both CHC and CHB groups had a higher frequency of some degree of depression (72.6% and 58.6%) than healthy subjects (37.8%), and CHC patients had more depressive features than CHB [et al. reported no significant difference in the rate of psychiatric diagnosis (mainly depression) between the CHB and CHC patient groups [vs. 11.3%), and that CHC patients are more vulnerable to depression than those with CHB (35.9% vs. 19.8%). Depressed CHC patients, CHB patients, and healthy participants tend towards lower depression severity scores, but the differences did not reach statistical significance.Previous studies comparing the prevalence of depression in CHC, CHB patients and healthy participants reported varying results. Carta  = 0.58) . Qureshit groups . This stet al. found that inactive adult HBsAg carriers exhibited significantly greater depression than healthy controls [et al. demonstrated that, in respect of the extent of depression, children with HBV infection were no different from those who were HBsAg carriers, nor from healthy controls [vs. 11.3%).Atesci controls ; howevercontrols . In the In addition, the present study analysed the risk factors associated with depression among CHC patients. Smoking and alcohol consumption have increased the rate of depression in CHC patients, while viral load, liver fibrosis stage, HCV genotype, serum ALT level, past history of IV drug abuse, marital status, and gender have not had any influence. Hence infection with HBV or HCV may not be the only cause of higher rates of depression in these patients. The chronic nature of the disease, stigmatization, or patients' perceptions of adverse outcomes of disease may be the additional causes. Depression screening in CHB and CHC patients, especially smokers or alcohol consumers, may be beneficial in disease management.This study has some limitations, including non-probability sampling and the absence of group matching. It still has some strength: during this study a single interviewer—who was a trained psychologist instead of a trained general practitioner—completed all of the questionnaires; in addition, the questionnaire measures the severity of depression, as well as assessing its presence.In conclusion, our findings suggest that depression screening in chronic hepatitis B and chronic hepatitis C patients may be beneficial in disease management. However, it is necessary to conduct better-developed studies with adequate sample size to show the effects of depression screening on chronic hepatitis management in this population.Conflict of interest statement: none declared.