Purpose. Hesperetin and naringenin are naturally common flavonoids reported to have antioxidative effects. This study was performed to investigate whether either hesperetin or naringenin has a protective effect against apoptosis on retinal ischemia/reperfusion (I/R) injury. Methods. Retinal I/R was induced by increasing the intraocular pressure to 150 mmHg for 60 minutes. Thirty-three male Wistar albino rats were randomised into 5 groups named control, I/R + sham, I/R + solvent (DMSO), I/R + hesperetin, and I/R + naringenin. Animals were given either hesperetin, naringenin, or the solvent intraperitoneally immediately following reperfusion. Thickness of retinal layers and retinal cell apoptosis were detected by histological analysis, tunel assay, and immunohistochemistry assay. Results. Hesperetin and naringenin attenuated the I/R-induced apoptosis of retinal cells in the inner and outer nuclear cells of the rat retina. Retinal layer thickness of the naringenin treatment group was significantly thicker than that of the hesperetin, sham, and solvent groups (P < 0.05). Conclusions. Hesperetin and naringenin can prevent harmful effects induced by I/R injury in the rat retina by inhibiting apoptosis of retinal cells, which suggests that those flavanones have a therapeutic potential for the protection of ocular ischemic diseases. Disruption of the retinal blood supply, called “retinal ischemia,” decreases the delivery of oxygen and essential nutrients to the retina and results in cell death. Restoration of this disrupted retinal circulation is called “reperfusion” and aggravates ischemic injury by several biochemical processes that include oxidative stress, calcium overload caused by glutamate excitotoxicity, inflammation, cellular necrosis, and apoptosis . Many ocFlavonoids are polyphenolic compounds with various pharmacological properties and act as scavengers of free radicals by OH groups in their molecular structure , 8. AccoFor this study's purposes, it is important to note that the beneficial effect of hesperetin and naringenin on I/R-induced myocardial, cerebral, renal, and pancreatic injury has been demonstrated –21. HoweThirty-three healthy male Wistar albino rats weighing 250–300 g were obtained from Saki Yenilli Animal Laboratory, Ankara, Turkey. The animals were housed for a 12-h light/12-h dark cycle. Animal experiments were conducted in accordance with the Statement on the Use of Animals of the Association for Research in Vision and Ophthalmology. The institutional review board of the University of Canakkale Onsekiz Mart, Turkey, approved the research.Rats were anesthetised with intramuscular ketamine chloride  and xylazine . Corneal analgesia was achieved by using a topical application of 0.5% proparacaine hydrochloride . To preserve body temperature of the rats, they were placed in containers to cover their body. The anterior chamber of the right eye was cannulated with a 30-gauge infusion needle connected to a saline bottle. Retinal acute ischemia was induced by elevating the bottle to a height of 2 m to maintain an intraocular pressure of 150 mmHg for 60 minutes. Retinal ischemia was confirmed by the loss of red reflex and its return just after reperfusion. After a 60-minute period of ischemia, the IOP was returned to its normal values by removing the infusion cannula. Ofloxacin (0.3%) was applied topically to the eye to prevent infection. The rats in the control group were administered by cannulating a 30-gauge needle into the anterior chamber without elevating IOP.n = 6), I/R + sham (n = 6), I/R + solvent (DMSO) (n = 7), I/R + hesperetin (n = 7), and I/R + naringenin (n = 7). Animals were given either drug (hesperetin or naringenin) or DMSO intraperitoneally immediately following reperfusion.Hesperetin  and naringenin  were freshly prepared by dissolving the powder in dimethyl sulfoxide (DMSO) for intraperitoneal administration. The rats were randomly assigned to five groups: control  and stained with haematoxylin and eosin (H&E). Retinal thickness was quantified in three separate positions: central (100–150 μm from the optic nerve), peripheral (100–150 μm from the ora serrata), and midperipheral . Two representative sections were selected from the same three positions randomly for each eye, from which measurements were taken and their values averaged [The rats were anesthetised by intraperitoneal administration of 80 mg/kg ketamine and 4 mg/kg xylazine, 48 hours after retinal I/R injury. Eyes tissue samples were obtained for histopathological and immunochemistry investigation. Eyes samples fixed with 10% formaldehyde solution for 48 hours, embedded in paraffin. Sections 5-Leica, RM45 and stμm-thick sections were obtained, and the slides were air-dried and the tissue deparaffinised. Slides were washed in 0.01 mol/L phosphate-buffered saline (PBS). After washes with PBS, an antigen retrieval solution  was applied for 10 minutes at 100°C in a microwave oven, and endogenous peroxidase was eliminated by incubation in 3% H2O2 in pH 7.4 in PBS (0.01 M) for 10 minutes. After washing, specimens were treated with a blocking serum  at room temperature for 10 minutes. The sections were incubated with rabbit polyclonal anti-Caspase 3  and reacted with tissue specimens at room temperature for 90 minutes. Sections were then washed three times with PBS and incubated with biotinylated secondary antibody . Streptavidin peroxidase  was given at room temperature for 20 minutes. 3,3′-Diaminobenzidine (DAB) was used as a chromogen, and sections were counterstained with haematoxylin.The eye samples were fixed in 10% formaldehyde solution and embedded in paraffin. Immunohistochemical reactions were performed according to the avidin biotin-peroxidase complex technique described by Hsu et al. . Five μmApoptotic cells were visualized by using a terminal deoxynucleotidyl transferase dUTP nick-end labeling (tunel) assay kit . In brief, paraffin sections were deparaffinised in the xylene for 3 × 5 minutes, washing with absolute ethanol (2 × 5 minutes), washing once with 95% ethanol and once with 70% ethanol, and after that washing by applying 20 mg/mL proteinase K (20 min.). Endogenous peroxidase activity was inhibited by incubation with 3% hydrogen peroxide. Sections were incubated with an equilibration buffer for 10 to 30 minutes and then with TdT-enzyme, in a humidified atmosphere at 37°C, for 60 minutes. They were subsequently put into prewarmed working strength stop/wash buffer at room temperature for 10 minutes and incubated with a blocking buffer for 30 minutes. Each step was separated by thorough washes in tris-buffered saline (TBS). Labeling was revealed using DAB, counter staining was performed using haematoxylin, and sections were dehydrated, cleared, and mounted .P < 0.05.The data were expressed as mean ± standard deviation (SE). Differences among the groups were evaluated using one-way analysis of variance (ANOVA). The Bartlett test was used to determine whether the data were heterogeneous or homogeneous. The Bonferroni multiple comparison procedure was then applied to identify differences between means. Differences were considered significant at P < 0.05, P < 0.05, P < 0.05, Excepting the naringenin treatment group, 48 hours after retinal I/R injury the overall retinal thickness and other layers of the retina were significantly decreased compared to the control group . We performed immunohistochemistry to detect activated caspase-3. The number of caspase-3 positive cells was significantly less in the retina of rats treated with hesperetin or naringenin than in the retina of the sham and solvent groups, which had retinal damage induced by I/R  on the retinal damage caused by I/R in a rat retinal model by studying retinal morphology and immunohistochemistry. The retinal thickness and the quantity of apoptotic cells were compared between I/R groups and the control group. It was found that naringenin and hesperetin markedly reduced retinal cell injury following I/R in comparison to the sham and solvent groups. In addition, we also found that the treatment of naringenin inhibited the apoptosis of the retinal cells and reduced the thinning of the retinal thickness more effectively when compared to hesperetin.α-tocopherol, particularly for urgent antioxidative treatment needs [2+) vasodilatation after I/R, as well as protect vascular function [Flavonoids and diets rich with flavonoids have been reported to maintain treatment for several diseases with their powerful antioxidant activity, which also modulates the enzymatic activity , 26. Antnt needs . Flavonont needs  showed tfunction . Our finThe neuroprotective effect of flavanones on cerebral I/R injury was shown in recent studies , 31–33. This study shows that retinal cell injury seems to occur 2 days after I/R, as demonstrated with the sham group . It is cAn important limitation in this study is that only one dose of both hesperetin and naringenin was studied. Therefore, further studies are needed to evaluate the protective effect of these drugs in different doses. In addition, the current study did not examine the mechanism of the neuroprotective effects of hesperetin and naringenin.In conclusion, this study is important as it is the first to clearly show the protective role of three-hydroxyl-inclusive flavanones (hesperetin and naringenin) during retinal apoptosis and morphology with the retinal I/R injury in rats. Further investigations are necessary to evaluate the therapeutic potential and mechanism of hesperetin and naringenin for the prevention of ocular ischemic diseases.


Hyperphosphatemia, serum phosphorus ≥ 4.4 mg/dL, is associated with increased risk for chronic kidney disease and cardiovascular disease. Previous studies have shown a weak association between dietary phosphorus intake and serum phosphorus concentrations. While much less common in the general population, hypophosphatemia (< 2.5 mg/dL) may be associated with metabolic syndrome and obesity.Using three cycles from the National Health and Nutrition Examination Survey (NHANES) (2005–2010), this study evaluated independent risk factors for hyperphosphatemia and hypophosphatemia.Risk factors for hyperphosphatemia included higher adjusted calcium , increasing cholesterol , female gender  and low hemoglobin . Advanced age was protective . Models that included fasting serum glucose found lower body mass index (BMI) to be protective  and adjusting for serum vitamin D and parathyroid hormone removed the association with low hemoglobin and BMI. Risk factors for hypophosphatemia included the following protective factors: higher albumin , higher BUN , corrected calcium  and female gender . In men, higher fasting glucose levels increased risk .This study is the first to show an association between low hemoglobin levels and increased risk for hyperphosphatemia among individuals without chronic kidney disease. We did not find any association between diabetes mellitus, increasing BMI or fasting glucose levels and hypophosphatemia. Determinants of serum phosphorus concentrations include dietary intake, gastrointestinal absorption of phosphorus, urinary excretion and shifts in phosphorus between the intracellular and extracellular spaces . PreviouA recent study evaluating risk factors for hyperphosphatemia found an association between elevated phosphate levels and poverty, although it is not clear which intermediary factors associated with poverty would simultaneously increase phosphate levels . It is pIn patients with ESRD and kidney transplant recipients, a previous study  found a Other known risk factors for hyperphosphatemia include female gender although the reasons for this association are unclear . One stuIn contrast with hyperphosphatemia, hypophosphatemia is much less common in the general, non-hospitalized population. Low serum phosphate levels are associated with reduced cardiac output and also with risk for arrhythmia . HoweverThe aim of the present study was to evaluate potential risk factors for hyperphosphatemia and hypophosphatemia in a large population based survey of patients without kidney disease, the National Health and Nutrition Examination Surveys (NHANES) from 2005–10, including anemia as well as investigate the possible relationship between exposure to environmental phenols, specifically and mediating the association between poverty and hyperphosphatemia.The NHANES is a nationally representative, cross-sectional survey, which was designed by the National Center of Health Statistics (NCHS) to be a representative sample of the non-institutionalized US population and used to assess the health and nutritional status of Americans. A total of 31,034 adults and children participated in NHANES 2005–2010 . These NHANES cycles were approved by the Institutional Review Board of the National Center for Health Statistics. All NHANES participants who were ≥18 years of age provided informed consent.Participants <18 years of age were excluded from this study . Participants who stated that they had been told that they had weak/failing kidneys or had received dialysis in the past 12 months (n = 534) were also excluded from the study as were those with an estimated glomerular filtration rate <60 (n=486) and a serum creatinine > 1.3 mg/dL (n = 486). Vitamin D and parathyroid measurements were only available from the 2005–6 cycle (n = 5061) and ferritin and transferrin receptor saturation were only available on women for 2005–2010 for a smaller sample size . For the environmental phenols and pesticides, only one third of the sample was used per cycle  and special sampling weights provided by NHANES were used for each cycle. For the fasting glucose measurements, only those participants who were examined in the morning sample were included in sample   versus subsequent cycles .In NHANES 2005–6, serum creatinine, blood urea nitrogen (BUN), calcium, total cholesterol, phosphorus, uric acid and albumin were measured using the Beckman Synchron LX20 method  . SimilarThe environmental phenols (bisphenol A (BPA), benzopheonone-3 (BP-3) and one cholorphenols triclosan were measured in all years using solid phase extraction coupled on-line to high performance liquid chromatography -34.Two 24 -hour dietary recalls were used to assess the intake of specific micronutrients including phosphorus. In order to ensure accuracy, the dietary recall interview included a set of 3 dimensional measuring guides and the interview asked if the dietary intake was usual or unusual for that person and then the material is provided as an attachment as described in the NHANES documentation . Other m)-1.154 × age-0.203 × 0.742  × 1.212 (if black). Furthermore, the CKD-Epi formula was also used to estimate GFR where eGFR=144 × min a × max -1.209 × 0.993Age [1.018 if Female] × [1.159 if Black]  . Low hemDemographic information was also collected by NHANES including information on the participants’ gender, age, race/ethnicity with race/ethnicity defined as non-Hispanic white, non-Hispanic black, Mexican-American, other Hispanic and other race. Body mass index was calculated based on weight and height measurements with weight (kilograms) divided by height (meters squared). Diabetes mellitus was defined as a self-reported physician diagnosis of diabetes at the time of the interview. Poverty to income ratio was used as the main variable to assess poverty status, which is an index calculated by dividing family income by a poverty threshold specific to family size. Living in poverty was defined as having a poverty-to income ratio < 1. Obesity was defined as having a body mass index (BMI) ≥ 30 kg/m . DiabeteSteroid use was categorized as yes if any participant reported the use of prednisone, prednisolone, methylprednisolone, or methylprednisolone acetate within the last 30 days. This data was extracted from the medication questionnaire.Serum phosphate levels were dichotomized by presence (≥ 4.4 mg/dL) or absence (< 4.4 mg/dL & ≥ 2.5 mg/dL) of hyperphosphatemia or presence (< 2.5 mg/dL) or absence (≥ 2.5 mg/dL & <4.4 mg/dL) of hypophosphatemia. Additionally hyperphosphatemia was also evaluated in relationship to lower levels of serum phosphate (< 3.5 mg/dL). Bivariate analyses were conducted for risk factors for hyperphosphatemia or hypophosphatemia and included an evaluation of biochemical, sociodemographic, pesticide and phenol exposure and health history and dietary variables. Variables that were significant at p < 0.05 were included in multivariate logistic regression models analyzing independent risk factors for hyperphosphatemia or hypophosphatemia. All analyses were conducted using Stata 12.0.Sample weights were used as provided by NHANES in the analysis to combine weights from all three cycles following the NHANES analytic guidelines . As descThere were 19,380 persons ≥18 years of age from the three cycles of the NHANES (2005–2010) that were included in the analysis after exclusions based on age, laboratory parameters or self-reported conditions, although a smaller number was available in multivariate analyses based on missing data for certain parameters. A smaller number from the 2005–6 cycle included data on vitamin D and parathyroid hormone , as well as those that had morning blood draw for fasting glucose  and were assessed for environmental phenol exposures . One hundred and forty-four reported current use of oral steroids > 30 days, which represented a weighted percent of 0.50% (95% CI 0.41-0.61). In the total population evaluated, 15.29% (95% CI 14.23-16.41) had hyperphosphatemia (phosphorus ≥ 4.4 mg/dL) and 0.59% (95% CI 0.43-0.82) had hypophosphatemia (phosphorus ≤ 2.5 mg/dL).In bivariate analysis, risk factors for hyperphosphatemia included higher levels of albumin p < 0.01), higher vitamin D (p = 0.01), lower parathyroid hormone p = 0.02), higher corrected calcium (< 0.01), lower fasting glucose (< 0.01) and lower hemoglobin levels (p = 0.03) , benzopheone-3 (BP-3) and tricolsan and hyperphosphatemia. We also evaluated risk factors for high serum phosphate ≥ 4.4 in comparison to those with low serum phosphate < 3.5 finding similar risk factors to those described above with the exception of a higher BUN and a lower uric acid and serum creatinine in those with hyperphosphatemia in comparison with those with a serum phosphate < 3.5. In multivariate logistic regression analysis , increasing calcium , higher cholesterol , female gender  and low hemoglobin  were associated with increased risk for hyperphosphatemia , lower BUN (p=0.02), higher corrected calcium level (p < 0.01) and higher uric acid (p = 0.02). Lower ferritin was protective (p < 0.01), as was being female (p = 0.01) and having a lower BMI (p < 0.01). Being obese increased risk (p = 0.06) Table . In multIn these cycles of NHANES, there was relatively high percentage of individuals without chronic kidney disease who had hyperphosphatemia (15.29%) in comparison with previous analyses of earlier cycles (NHANES III – 1988–1994) which found 14% in the US population without excluding those with CKD and using a lower threshold ≥ 4.0 mg/dL . The higThis is the first study to evaluate risk factors for hyperphosphatemia and hypophosphatemia in a large population of US adults without chronic kidney disease. While these results suggest some known factors associated with hyperphosphatemia including an association with increasing calcium levels and female gender and a reduced risk with age, we also found increased risk with anemia, as indicated by low hemoglobin levels, as well as an association with lower BMI. Previous population based studies have found an association between kidney function and anemia in adults with a eGFR < 60 mL/minute but not in adults with normal kidney function . As suchAdditionally, low vitamin D levels decrease gut phosphorus absorption, as vitamin D acts on intestinal epi-thelial cells to increase intestinal absorption of calcium and phosphorus, and hence may be associated with hyperphosphatemia . AdditioIn this adjusted model for hyperphosphatemia, the association with low hemoglobin was no longer statistically significant, although it is possible that the smaller model (only one third the size of the larger model) was not sufficiently powered. When the association between low hemoglobin and hyperphosphatemia was assessed stratifying on vitamin D level > 30 ng/ml versus vitamin D level ≤ 30 ng/ml, the odds ratios were not significantly different in between models (OR 1.10 versus OR 1.29) or in comparison with the adjusted model OR 1.33, suggesting that neither confounding nor interaction plays an important role in defining the inter-relationships between serum phosphorus levels, hemoglobin and vitamin D.Ideally, future cycles of NHANES will include vitamin D and parathyroid hormone levels tested in all participants, as this was one of the limitations of our study, that we were unable to test these associations using one cycle of NHANES.It is not clear what mechanism is responsible for the increased serum phosphorus levels associated with anemia. Studies of hyperphosphatemia have been almost exclusively in patients with kidney disease, however, given the associations between hyperphosphatemia, cardiovascular disease and increased mortality , it is iWe found a low percentage of overall hypophosphatemia in these cycles of NHANES (0.59%). There is limited data on the prevalence of hypophosphatemia in the general population, although approximately 2-20% of hospitalized patients may have hypophosphatemia ,51, withIn contrast to the studies by Haglin  and HaglFuture studies need to be longitudinal in nature so as to better understand the mechanisms involved in explaining the relationship between hypophosphatemia and either obesity or serum glucose levels based on gender. Our current understanding is associative in nature and does not evaluate the temporality of relationships.The author has no competing interests.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/14/178/prepub


Quercus aquifolioides) grown at its upper elevational limit compared to lower elevations, to test whether the elevational limit of alpine shrubs (<3 m in height) are determined by carbon limitation or growth limitation. We studied the seasonal variations in non-structural carbohydrate (NSC) and its pool size in Q. aquifolioides grown at 3000 m, 3500 m, and at its elevational limit of 3950 m above sea level (a.s.l.) on Zheduo Mt., SW China. The tissue NSC concentrations along the elevational gradient varied significantly with season, reflecting the season-dependent carbon balance. The NSC levels in tissues were lowest at the beginning of the growing season, indicating that plants used the winter reserve storage for re-growth in the early spring. During the growing season, plants grown at the elevational limit did not show lower NSC concentrations compared to plants at lower elevations, but during the winter season, storage tissues, especially roots, had significantly lower NSC concentrations in plants at the elevational limit compared to lower elevations. The present results suggest the significance of winter reserve in storage tissues, which may determine the winter survival and early-spring re-growth of Q. aquifolioides shrubs at high elevation, leading to the formation of the uppermost distribution limit. This result is consistent with a recent hypothesis for the alpine treeline formation.Many studies have tried to explain the physiological mechanisms of the alpine treeline phenomenon, but the debate on the alpine treeline formation remains controversial due to opposite results from different studies. The present study explored the carbon-physiology of an alpine shrub species ( Sergyemla, the Tibet-Qinghai Plateau et al. (2010) also reported that the growth of Scots pine (Pinus sylvestris L.) in a treeline population in eastern Finnish Lapland, suffered from resources limitation under the current climatic conditions et al. (2002) et al.  Scientists have still not convincingly fully explained the physiological mechanism driving the alpine treeline formation on a global scale. Two dominant hypotheses, the carbon limitation, known also as source limitation, and the growth limitation , have received increasing attention in the debate about the alpine treeline phenomenon. The carbon limitation proposes that the alpine treeline trees may suffer from insufficient carbon supplies as a result of a shortage of photo-assimilates caused by low temperatures and short growing seasons Among all reserves, non-structural carbohydrates (NSC) are the most important food reserve compounds in trees Mobile carbon reserves in trees are known to exhibit seasonal variations Annual patterns of carbohydrate storage and mobilization varied with species, genotypes, and tissues Quercus aquifolioides Rehd. et Wils are also distributed to a certain elevational limit and formed an abrupt transition in life-form dominance. Q. aquifolioides, an endemic sclerophyllous evergreen broadleaved species, is mainly concentrated in the Hengduan Mountains , southwestern China Q. aquifolioides shrubs at the uppermost distribution, compared to plants at lower elevations, have higher levels of mobile carbohydrates during the growing season but lower concentrations during winter, as expected by a recently theory of winter C-shortage in treeline trees proposed by Li et al.(2008) Q. aquifolioides plants grown at 3000 m, 3500 m, and 3950 m a.s.l. (20 m below the uppermost limit of 3970 m a.s.l.) along an elevational gradient on Zheduo Mt., southwestern China.Previous studies have focused on trees rather than shrubs, although just like the alpine treeline, the alpine shrubs (<3 m in height) such as The present study was carried out within the study area of the Gong-Ga Mountain Ecosystem Research Station, Institute of Mountain Hazards and Environment. Neither the research species nor the area is legally protected. No specific permits were required for the described study.2O) of 6.54±0.50 and similar fertility on the study slope −1 soil, and the total available soil N, P, and K are 0.13±0.01, 0.01±0.008, and 0.13±0.04 mg g−1 soil, respectively . The soil organic matter is 30.51±2.20 mg g−1 soil. There are no statistically significant differences in soil nutrient elements among the sites at the 3 elevations .The study was carried out on a southeast-facing slope of Mt. Zeduo  located on the eastern edge of the Qinghai-Tibet Plateau, southwestern China. The climate belongs to the Tibetan Plateau Climate Zone characterized by abundant sunshine, cold winters, and cool summers Q. aquifolioides (oak) stand with an age of 30–35 years old on the sunny slope of Mt. Zeduo is naturally generated since the 1970s, and formed a pure shrub-stand consisting of multi-stem oak clumps (5–10 stems per clump) ranging from 2850 to 3970 m a.s.l. (the uppermost limit). A few individuals of Picea balfouriana, Lyonia ovalifolia, Betula platyphylla, Rosa moyesii, and Sabina procumbens coexisted in the stand. The understory layer is poorly developed due to the hot and dry conditions. Within the oak shrub stand, we established a 200 meters-long and 20 meters-wide sample strip at each elevation of 3000 m, 3500 m, and 3950 m a.s.l. (20 meters below the uppermost distribution) in April 2008. Characteristics of the study sites/stands are summarized in Quercus aquifolioides tissues  were sampled 7 (current-year tissues) and 11 times (≥1-yr-old tissues) at approximately monthly intervals from April 2008 through October 2009, respectively. The pheonological stage of the oak plants along the elevational gradient was taken into account to determine the sampling time: i.e. (1) April 15–20, 2008/2009 (before bud burst); (2) May 5–10, 2008/2009 (after bud burst); (3) mid-June, mid-July, and mid-August, 2008/2009 (during the growing season); (4) September 15–17, 2008/2009 (at the end of the growing season); (5) October 11–16, 2008/2009, and January 14–19, 2009 (during winter dormancy). If a sampling date was a clear day, samples were then taken around noon to minimize the diurnal NSC fluctuations between samples For each sampling date, six healthy and undamaged oak clumps (>30 m apart from each other) within each study strip were randomly selected. Roots, stem, branches and leaves were sampled. Roots samples were collected by carefully excavating roots originating from the selected individuals. Root samples were divided into four categories: fine roots (diameter <2.5 mm), medium roots (diameter = 2.5–5 mm), coarse roots (diameter >5 mm), and taproots. Roots samples were washed to remove other materials such as soil particles and dead roots. Each stem sample was a mixture of 3 stem discs taken from the lower, middle and upper one-third stem section of each sample clump. Current-year and previous-year leaves, current-year, 1-year-old, and previous-year (>2 years old) branches were collected from the downslope crown side of each selected clump. Samples for branches, stem and roots included bark, and were cut in small pieces. All samples were immediately stored in a cool box and killed in a microwave oven (40 s at 600 W) and dried to constant mass at 65°C.dry mass in an unit of fresh biomass oven-dried at 70°C to a constant weight. At the community level, dry biomass of each biomass component was calculated by multiplying mean clump biomass by clump density which was determined in six plots of 20 m×20 m at each elevation.In September 2009, six average clumps similar to those used for carbohydrate analyses within each study strip were selected for estimating the biomass of individual clump. Aboveground biomass cut from the ground surface level was separated into different categories corresponding to the tissue category for NSC analysis. Root biomass was investigated by digging out the entire root system, which was also separated into fine roots, medium roots, coarse roots, and taproots, corresponding to those used for NSC analysis. Fresh biomass for each component was measured and recorded. At the individual clump level, dry mass of each biomass component was calculated using the fresh biomass and a content of Samples were analyzed using the anthrone method to measure starch and soluble sugars 4 was added and the starch was hydrolyzed for 15 min. 4 ml distilled water was added and the mixture centrifuged at 5000 g for 10 min. The pellets were extracted one more time with 2 ml 4.6 M HClO4. The combined supernatants were adjusted to 20 ml. Starch concentration was measured spectrophotometrically at 625 nm using anthrone reagent, and calculated by multiplying the glucose concentration by the conversion factor of 0.9 Starch was extracted from the ethanol-insoluble pellet after removing the ethanol by evaporation. Starch in the residue was released by boiling in 2 ml distilled water for 15 min in a boiling water bath. After cooling to room temperature, 2 ml 9.2 M HClONonstructural carbohydrate is defined as the sum of the starch plus the total soluble sugars within each tissue sample for each sample date. The NSC pool size was calculated by multiplying tissue biomass by their corresponding mean NSC concentrations in each sample.n = 6). Normality of distribution and homogeneity of data  were checked (Kolmogorov-Smirnov-Test) before any further statistical analysis. Two-way repeated ANOVAs were performed with elevation and sampling date as factors to determine the effects of elevation and sampling date on concentrations of NSC and its components for each tissue type. Differences in mean concentrations of NSC and its components, biomass, and NSC-pools for each tissue type were compared by using the least significant ranges test at 5% level. The SPSS (version 17.0) statistical software  was used to perform the analyses.All statistical tests treated individual shrub clump as replicates  (P = 0.011) and for soluble sugars in 1-year-old branches (P = 0.033)  , Fig. 1.= 0.033) . In all = 0.033) , indicatP<0.001 for both NSC and sugars), whereas the mean starch content was 4.65% during the growing season and 3.46% during the dormant season (P<0.001) (data not shown).Within each tissue type and elevation where plants grown, concentrations of NSC and soluble sugars in tissues tended to be higher during the dormant season than those during the growing season  and 2, bP<0.001), previous-year leaves (P<0.001), current-year branches (P<0.001), 1-year-old branches (P<0.001), and previous-year branches (P<0.001) (P = 0.036), stem (P = 0.05), taproots (P = 0.002), coarse roots (P<0.001), medium roots (P<0.001), and fine roots (P = 0.038) (P = 0.04), current-year branches (P = 0.033), 1-year-old branches (P = 0.002), previous-year branches (P = 0.005), coarse roots (P<0.001), and medium roots (P = 0.023) (The concentrations of soluble sugars significantly increased with increasing elevation in current-year leaves (P<0.001) , Fig. 3.= 0.038) . The tis= 0.023) .The elevational effects on starch and NSC concentrations were more marked during the winter season (October–April) rather than during the growing season (May–September) . During The pooled data across sampling dates indicated that tissue NSC concentrations tended to increase with increasing elevation in leaves and branches, but to decrease in stem wood and roots . Starch The ratio of soluble sugars to starch within each tissue showed similar patterns of increased trend with increasing elevation for both seasons (except for the current year leaves at 3500 m during the growing season) . Plants The biomass at tissue level showed a decreasing trend with increasing elevation, except the fine roots biomass increased with increasing elevation . CorrespAt the tissue level, the NSC pool size tended to decrease with increasing elevation, except the NSC pool size in fine roots increased significantly with increasing elevation . CorrespThe present study showed that tissue mobile carbohydrate concentrations varied with time , and eleet al.  et al. (2009) found that two coniferous species  grown at the timberline (4300–4400 m a.s.l.) had a greater relative abundance of NSC compared to the same species grown at lower elevations in southeastern Tibetan Plateau in the growing season et al. (2008) found that the treeline coniferous trees  suffered from a ‘carbon limitation’ during and after dormancy in winter and in the early spring, but not during the growing season Levels of NSC concentrations within each tissue type did not show any differences in plants grown at the upper elevational limit compared to those at lower elevations during the growing season . Similaret al. (2006) showed that not leaves and branches but roots of four woody species tended to decrease the mobile carbohydrates concentrations with increasing elevation up to their elevational limit at the end of growing season  found that roots of Abies georgei trees grown at the alpine treeline (4330 m a.s.l.) had significantly lower NSC concentration (P<0.001) than those at 3480 m a.s.l. on Sergyemla Mt., SW China, during the growing season Pooled data in the present study found that leaves and branches showed an increasing trend but stem and roots exhibited a decreasing trend of tissue NSC concentrations with increasing elevation . In consQ. aquifoliodes is green and allows bark photosynthesis to produce glucose. For example, light-saturated photosynthetic rate of current-year braches in Quercus robur was found to reach 1.89 µmol m−2 s−1 in winter and 2.73 µmol m−2 s−1 in summer Except for the season-dependent elevational effects, the present study also found that the elevational effects on levels of NSC and its components were tissue-dependent  and 3. SQ. aquifoliodes trees need more nonstructural carbohydrate during winter for their higher maintenance respiration and higher basal respiration rate of sapwood Abies lasiocarpa, Bansal and Germino (2008) observed greater NSC concentrations in the aboveground tree tissues at the upper limit when respiration decreased et al. (1980) in situ rather than translocating them to stem and roots during winter. These may be the reasons for why decreased NSC concentrations with increasing elevation were found in stem and roots of evergreen plants in the present study and elsewhere Both source and sink tissue influence the carbon balance at a tree level The total NSC pool size at individual level was considered to better answer the question of whether the alpine treeline trees physiologically suffer from a carbon limitation, because the treeline trees may compensate for decreases in plant size by accumulating more NSC in plant tissues et al. (2008) proposed that trees growing at the elevational or latitudinal climate limit rely not only on the total NSC concentration, but also require a sufficiently high sugar-starch ratio to overwinter successfully et al. (2008) suggested that trees at their upper elevational limit need a minimum sugar-starch ratio of about three to overwinter et al.'s suggestion et al. (2007) found that the sugar-starch ratio in zoysiagrass (Zoysia spp.) was significantly positively correlated with cold hardiness et al. (2003) also found that the sugar-starch ratio in leaves of Arabidopsis thaliana grown at 23°C was much lower (<1.5) than in leaves of plants grown at 5°C (up to 2.5) The ratio of sugars to starch may help to explain the alpine treeline phenomenon Q. aquifoliodes plants varied significantly with season  hypothesis Quercus petraea trees is dependent on storage compounds et al. (2001) demonstrated that both source tissues and sink tissues of mature evergreen Quercus ilex participated in supplying carbon for re-growth in the early spring et al. (2009) suggested that evergreen trees relied on mobile carbon reserves to a lesser extent than deciduous trees during budbreak Q. aquifolioides at its elevational limit. It is, therefore, most possible that significantly lower winter NSC storage in sink tissues at the elevational limit, compared to lower elevations , resulting in the formation of the alpine treeline Levels of mobile carbohydrates concentrations in h season , Table 2h season , which ih season  and 2. T


Clostridium difficile infection (CDI) is an important cause of morbidity and mortality internationally, yet there are important regional differences in the epidemiology and microbiology of disease. Most reports have come from North America and Europe, with limited information from other regions, including the Middle East. Given the changes in the epidemiology of CDI in developed countries, particularly associated with the dissemination of hypervirulent epidemic clones, an understanding of the epidemiology and microbiology of CDI in diverse regions is warranted. This study involved collection of stool samples from individuals with diarrhea at the Isfahan University of Medical Sciences Teaching Hospital, Isfahan, Iran, between October 2010 and March 2011. Selective enrichment culture for C. difficile was performed and isolates were characterised using ribotyping, PCR for the detection of tcdA, tcdB and cdtB genes, and tcdC sequence analysis.Clostridium difficile was isolated from 19/89 (21%) stool samples of 17/86 (20%) patients. 13/17 (77%) cases of CDI were hospital-associated. Patients with CDI were significantly older (43 ± 28y) than those with non-CDI diarrhea (P = 0.018). All isolates were toxigenic, and possessed genes encoding for toxins A and B. Six (32%) of 19 isolates also possessed cdtB. Twelve ribotypes were identified. Ribotype 078/toxinotype V was most common, accounting for 4 (21%) of isolates. A single isolate of a different toxinotype V ribotype was identified, as was a toxinotype XXIV isolate. The remaining isolates consisted of 9 different toxinotype 0 ribotypes.CDI is an important cause of diarrhea in patients in this hospital. The diversity of ribotypes was striking, and the number of different types suggests the presence of a broad range of strains in the community, the hospital or both. The predominance of toxinotype V strains, which have been associated with community-associated disease and food animals, was unexpected and possible sources of this type require further investigation. Clostridium difficile is a leading cause of hospital-associated and antimicrobial-associated diarhea, and is of significant concern because of the increasing morbidity, mortality and relapse rates [Clostridium difficile infection (CDI) has been reported throughout much of the world, but most data come from developed countries in North America and Europe. Limited information is available regarding the role of C. difficile in diarrheic hospital patients in people in Iran or other Middle Eastern countries, or about C. difficile strains that are involved. Therefore, the aim of present study was to determine the prevalence of C. difficile in diarrheic patients in hospital in Isfahan, Iran, and to characterize isolates.se rates , along wse rates . Some ofse rates  and to ase rates ,5. ClostThe study was conducted at the Isfahan University of Medical Sciences Teaching Hospital, Isfahan, Iran, the largest tertiary care medical centre in the region with 800 beds, three intensive care units and all major clinical specialties. Aliquots of stool specimens that had been collected from diarrheic patients between October 2010 and March 2011 for other diagnostic testing were studied. Both patients that were admitted to hospital with diarrhea and those who developed diarrhea during hospitalization were included. Diarrhea was defined as watery, loose or unformed stool passed at a frequency of three times or more per 24 hours. Demographic and basic medical information were collected from the medical record. Cases were classified as community associated (CA) if the onset of symptoms occurred prior to, or within 48 h of admission and if they had not been hospitalized in the preceding 3 months . Cases wC. difficile selective enrichment broth containing 40 g/l proteose peptone, 5.0 g/l disodium hydrogen phosphate, 0.1 g/l magnesium sulphate, 2.0 g/l sodium chloride, 6.0 g/l fructose and 1.0 g/l sodium taurocholate supplemented with cysteine hydrochloride, norfloxacin and moxalactam, and anaerobically incubated at 37 C for 5–7 days. Two ml of each culture were then added to an equal volume of absolute ethanol, mixed and left at room temperature for 1 h. Alcohol shocked cultures were then centrifuged (4000 rpm/10 min) and the pellet was streaked onto C. difficile moxalactam norfloxacin (CDMN) agar (Oxoid) and incubated anaerobically at 37 C for 24-48 h. Suspect colonies were subcultured and identified as C. difficile on the basis of characteristic colony morphology, odour, Gram stain morphology and L-proline aminopeptidase test . All isolates were screened for the presence of genes encoding toxin A (tcdA), toxinB (tcdB), the binding component of CDT (cdtB) and triose phosphate isomerase (tpi) as have been previously described [tcdC sequence analysis [http://www.webcitation.org/64HSI8B56) , the appropriate numerical designation (i.e. 078) was used. Otherwise, internal nomenclature was used.Selective enrichment culture was performed. Approximately 5 g of stool was inoculated into 25 ml of escribed . Isolate4000 rpm/ min and escribed  and tcdCanalysis . For ribP value of <0.05 was considered significant.Categorical comparisons were performed using Fisher’s exact test while t-test was used for continuous data. A Clostridium difficile was isolated from 19/89 (21%) of stool samples from 17/86 (20%) patients; 8/36 (22%) women and 9/50 (18%) men (P = 0.78). 13/17 (76%) cases were HA-CDI while the other 4 (24%) were CA-CDI. Patients with CDI were significantly older (43 ± 28y) than those with non-CDI diarrhea (P = 0.018). Five (29%) CDI patients were over the age of 65 compared to 6/69 (8.7%) without non-CDI diarrhea (P = 0.037). Fifteen (88%) of CDI patients had received antimicrobials prior to the onset of diarrhea, compared to 58 (84%) others (P = 1.0).Eighty-six diarrheic patients were enrolled. Two patients had separate episodes of diarrhea 62 and 33 days apart, and the same ribotype was isolated from each episode. Follow-up data were limited but this represents a minimal recurrence rate of 12%. Two (12%) patients with CDI died during hospitalization, however death was only attributed to CDI in 1 (5.9%) individual.tcdA and tcdB, six (32%) isolates also possessed cdtB. Four of these were ribotype 078 and toxinotype V, and contained a 39 bp deletion and C184T nonsense mutation in tcdC. One ribotype was a toxinotype V isolate that differed from ribotype 078 but had the same tcdC deletion and mutation. Patients carrying these five isolates were admitted over a period of approximately 2 months, with no more than one per week All 4 patients from which ribotype 078 was isolated were from rural areas, but had hospital-associated diarrhea. The mean age of patients with ribotype 078 was 29.2y compared to 49.1y for other ribotypes, but this was not statistically significant  was consistent with many studies of hospitalized individuals in other regions, albeit higher than two recent Iranian studies that reported isolation of toxigenic  samples ,14. Othe samples , 10.5% i samples , and 13. samples . HoweverAs is consistent with other reports, CDI was associated with older individuals. However, in this study, the CDI patients were younger (mean 43y) compared to most other reports ,18 and lC. difficile at the time of admission. Since there was a wide diversity in types and little evidence of temporal clustering  was consistent with major reported human types. The diversity of ribotypes within toxinotype 0 strains and the differences between common toxinotype 0 ribotypes in this versus other studies was interesting. The relatively high prevalence of ribotype 078 was consistent with two studies from Kuwait that identified ribotype 078 as one of the predominant types ,24. AddiC. difficile might be an important enteric pathogen in patients in Iranian hospitals. Efforts must be undertaken to diagnose CDI to allow for targeted therapy and to provide a better understanding of this important disease. While some aspects identified here are consistent with reports of CDI in other areas, further study of the epidemiology and microbiology of CDI in this region is required to explore some apparent differences, such as the younger age distribution and the predominance of ribotype 078. While ribotype 027 was not identified, further surveillance is required to determine whether this strain is indeed present in the country and to monitor for its emergence.This study indicates that The authors declare that they have no competing interests.MJ, JSW and KW designed the study. FK coordinated sample and medical record data collection. MJ performed the laboratory tests under the direction of JSW. All authors contributed to writing and reviewing the final manuscript. All authors read and approved the final manuscript.


Xiao-Xu-Ming decoction (XXMD) on neurovascular unit and to examine the role of PI3K (phosphatidylinositol 3-kinase)/Akt pathway in this protection. The cerebral ischemia was induced by 90 min of middle cerebral artery occlusion. Cerebral infarct area was measured by tetrazolium staining, and neurological function was observed at 24 h after reperfusion. DNA fragmentation assay, combined with immunofluorescence, was performed to evaluate apoptosis of neuron, astrocyte, and vascular endothelial cell which constitute neurovascular unit. The expression levels of proteins involved in PI3K/Akt pathway were detected by Western blot. The results showed that XXMD improved neurological function, decreased cerebral infarct area and neuronal damage, and attenuated cellular apoptosis in neurovascular unit, while these effects were abolished by inhibition of PI3K/Akt with LY294002. We also found that XXMD upregulated p-PDKl, p-Akt, and p-GSK3β expression levels, which were partly reversed by LY294002. In addition, the increases of p-PTEN and p-c-Raf expression levels on which LY294002 had no effect were also observed in response to XXMD treatment. The data indicated the protective effects of XXMD on neurovascular unit partly through the activation of PI3K/Akt pathway.In the present study, we used a focal cerebral ischemia and reperfusion rat model to investigate the protective effects of  Current biomedical research about stroke is focusing on treating neurovascular unit (NVU), and it is widely accepted that the key to effective therapy lies in restoring normal function of NVU.NVU is a functionally and structurally interdependent multicellular complex which consists of endothelial cells, basal lamina, pericytes, astrocytes, and neurons , and theβ (GSK3β) β (GSK3β) (GSK3β) . AlthougTo date, a large body of basic studies have indicated that numerous agents or options could reduce cerebral infarct area and improve neurological deficits with animal models of stroke –21 and tXiao-Xu-Ming decoction (XXMD), one classic TCM formula recorded by Sun Simiao of the Chinese ancient Tang Dynasty, has been widely used in the treatment of stroke. It has aroused extensively preclinical and clinical studies for its high efficacy [Traditional Chinese medicine (TCM) which is derived from natural plants and animals may act on multiple targets, and its flexible and comprehensive treatment strategies usually generate unexpected outcomes. These characteristics of TCM coincide with the view of NVU protection. Based on this fact, TCM has become the focus of medical research –27. Xiaoefficacy –32. Lineefficacy , 30. A refficacy . Most imefficacy , 32. Howβ , total GSK3β, GAPDH, and horseradish-peroxidase- (HRP-) linked anti-rabbit antibody purchased from Cell Signaling Technology  were used for Western blot analysis. Mouse monoclonal antibodies for NeuN and GFAP from Millipore , CD31 from Abcam , and Alexa Fluor 555 anti-mouse antibody from life technologies  were applied to immunofluorescence. LY294002 (PI3K inhibitor), the primary antibodies for phospho-PDK1 , total PDK1, phospho-PTEN , total PTEN, phospho-Akt , total Akt, phospho-c-Raf , total c-Raf, phospho-GSK3Herba Ephedrae, cassia twig, Radix Paeoniae Alba, Rhizome Chuanxiong, Radix Ginseng, Radix Stephaniae Tetrandrae, Radix Scutellariae, Semen Armeniacae Amarum, Radix Aconiti Praeparata, Radix Glycyrrhizae, Radix Ledebouriellae, and fresh Rhizoma Zingiberis Recens in a ratio of 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1.5 : 5. The crude drugs were purchased from Traditional Chinese Medicine Pharmacy of Zhongshan Hospital, Fudan University. XXMD was prepared as previously described with small modifications [The XXMD consists of twelve crude drugs including ications . After tOne hundred and six male Sprague-Dawley rats weighing 250–280 g  were housed in groups of four with free access to food and water and maintained in temperature (22 ± 2°C) and humidity-controlled (55 ± 5%) room with 12:12 h light-dark cycle. Prior to experimental manipulation, rats were handled daily for 3 days.drat = dhuman × 0.71/0.11 [We usually apply the water extract of XXMD in humans and the common human daily dosage of XXMD is 165 g/75 kg in body weight. According to the formula, .71/0.11 , the com.71/0.11 , we ther.71/0.11 . In the For drug treatment, the rats in XXMD-treated groups were orally administered with XXMD (60 g/kg/day) and others in the Sham, I/R groups were given the same volume of distilled water for 3 days before cerebral ischemic surgery. The drug administrations were performed twice a day at 8:00 and 18:00 and continued until animals were sacrificed. The above procedures were carried out by one investigator who was aware of the animal grouping but did not participate in the sequent investigations. All experimental protocols and animal handling procedures were approved by the Animal Care and Use Committee (ACUC) of Fudan University, and were consistent with the National Institutes of Health Guide for the Care and Use of laboratory Animals.Focal cerebral ischemia and reperfusion were performed as described previously . BrieflyIt should be noted that there are several technical factors in the MCAO rat model which may affect cerebral infarct area, such as physical differences in the employed monofilament suture, the depth of the insertion, and accidental premature reperfusion –38. In oμL of LY294002 solution or the small amount of vehicle (DMAO + ETOH) was injected intracerebroventricularly on the ischemic side at 30 min before operation. The experimental design for the present study was shown in  To investigate the function of PI3K after cerebral ischemia and reperfusion, several rats in XXMD-treated group were pretreated with LY294002, the PI3K special inhibitor, as described previously  with smaExamination of neurological deficits was performed at 24 h after reperfusion by one investigator who was unaware of the experiment design. The neurological deficits were scored on a four-point scale described by Hara et al.  with a mCerebral infarct area was measured by 2,3,5-triphenyl tetrazolium chloride  staining. Rats were killed at 24 h after reperfusion and perfused transcardially with normal saline. Brains were sectioned into six coronal slices from rostal to caudal and stained with 1% TTC staining at 37°C for 20 min away from light. Then the brain tissues were differentiated according to white-colored infarct area and red-purple noninfarct area. Cerebral infarct areas were calculated using microscope image-analysis software  according to the following formula: /contralateral hemisphere area) × 100%.μm on a rotary microtome. To assess brain injury, Nissl staining was performed with toluidine blue. Briefly, slices were dewaxed, dehydrated, and stained with 1% toluidine blue  at 50°C for 30 min. Then the slices were rinsed and cleared in graded ethanol and xylene and coverslipped under permount. The photomicrographs were captured with a light microscope. And the number of intact cells in penumbra of ischemic cortex was counted throughout five lesion regions randomly.At 24 h after reperfusion, the rats were deep anesthetized with 10% chloral hydrate  and perfused transcardially with 200 mL normal saline followed by 4% paraformaldehyde . The brains were removed and postfixed in the same fixative for 24 h at 4°C. After dehydration in graded ethanol and xylene, the brains were embedded in paraffin and sectioned into slices of 5 In order to detect the extent of DNA fragmentation of different cells in NVU, double staining of TUNEL and immunofluorescence labeling with different antibodies were performed and the positive cells were counted. In this study, we chose anti-NeuN, anti-GFAP, and anti-CD31 antibodies, the markers of neuron, astrocyte, and vascular endothelial cell, combined with TUNEL staining to evaluate apoptosis and its location, and to further clarify the roles of multiple cells in NVU after ischemia and reperfusion.μm. We performed immunofluorescence labeling with anti-NeuN, anti-GFAP, and anti-CD31 antibodies and TUNEL staining with an In Situ Cell Death Detecting Kit , respectively, according to the method described by Zhou et al. [At 24 h after reperfusion, rats were deep anesthetized and the brains were rapidly removed and cut into slices of 10 u et al.  and the  Western blots were used to evaluate expression levels of proteins involved in PI3K/Akt pathway at 24 h after reperfusion. Whole cell protein of ischemic penumbra was extracted from the fresh brain. Equal volumes and qualities of protein solutions were separated by electrophoresis of appropriate concentrations of polyacrylamide gels and transferred to the poly-vinylidnene fluoride membranes . Then the membranes were placed in 5% skim milk, prepared with Tris-buffered saline with 0.1% Tween-20 (TBST) to block nonspecific binding, and incubated with primary antibodies at 4°C overnight. In the following 2 h, the membranes were incubated with horse-radish peroxidase-conjugated secondary anti-rabbit antibody after washing with TBST for 10 min × 3. Eventually, the targeted antigens were detected by standard chemical luminescence methods  with Fluor Chem FC 2 gel imaging system . GAPDH protein was used as a loading control. Phosphorylation levels of the targeted proteins were analyzed by total levels of corresponding proteins. Western blots were duplicated with three independent sets. Band intensities were measured with Quantity One software .t-tests using SPSS 11.5 for Windows . All were considered statistically significant for P values < 0.05. When all the data have been collected, the animal grouping was released. Data are presented as the means ± standard error of the mean (SEM). Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test or unpaired Student's The occlusion for 90 min followed by reperfusion for 24 h led to an infarct area and marked neurological deficits in rats. The results indicated that XXMD treatment resulted in a significant reduction in infarct area compared to the I/R group . In addition, the rats subjected to cerebral ischemia and reperfusion showed noticeable neurological deficits which were markedly attenuated by XXMD treatment, while rats in the sham group performed normally after operation. To evaluate the contribution of PI3K/Akt pathway to cerebral infarct area and neurological deficits, we investigated the effects of inhibiting PI3K with the specific inhibitor LY294002 on cerebral infarct area and neurological function at 24 h after reperfusion. Larger infarct area was observed in LY294002-treated rats (39.01 ± 2.27%) compared with the rats in the XXMD60 group. Additionally, we found a smaller infarct area in the XXMD60 + vehicle group (24.25 ± 2.23%) compared with the I/R group Figures . LikewisNissl staining showed neuronal injury in penumbra of ischemic cortex at 24 h after reperfusion. The images showed that most cells with intercellular space enlarged were shrunk in the ischemic cortex and had deep color staining indicative of injury in the I/R group. However, these characteristic changes were not observed in the sham group and were improved by XXMD treatment. Interestingly, LY294002 blocked the effect of XXMD on neuronal injury. Furthermore, there were more intact cells in the ischemic cortex of the XXMD-treated rats in the absence of LY294002 compared to the I/R group .Double staining of TUNEL and immunofluorescence labeling with NeuN were performed to evaluate neuron apoptosis. As shown in To comprehensively determine apoptosis of different cells in NVU, we next performed double staining for TUNEL and immunofluorescence labeling with GFAP to evaluate astrocyte apoptosis. As represented in the ischemic cortex, the number of TUNEL and GFAP double stained cells was markedly increased in penumbra of ischemic cortex at 24 h after reperfusion. These changes were largely reversed in the XXMD60 group but worsened in the presence of LY294002. In all cases, double stained cells were hardly observed in the sham group. Percentages of apoptotic astrocyte in different groups were similar with the above findings .The data revealed that astrocyte, as the key component of NVU, was involved in apoptosis induced by stroke with reperfusion. However, astrocyte apoptosis was decreased by treatment with XXMD. In view of the important role of astrocyte, the results further indicated that there were more normal astrocytes which promoted neuronal survival by XXMD treatment.Intriguingly, the results of CD31 and TUNEL double staining in different groups were consistent with antiapoptotic effects of XXMD on neuron and astrocyte after ischemia and reperfusion. As reperfusion occurred, vascular endothelial cell, another considerable component of NVU, was subjected to severe damage unavoidably and showed the changes of apoptosis. However, XXMD treatment significantly reduced vascular endothelial cell apoptosis which was prevented by intracerebroventricular injection of LY294002 .β, whereas XXMD treatment maintained the phosphorylation levels of these proteins, which were reversed upon pretreatment with LY294002. Changes in total levels of PDK1, Akt, and GSK3β were not observed in different groups at 24 h after reperfusion. Additionally, our data indicated that p-PTEN levels were decreased after ischemia and reperfusion, and XXMD treatment attenuated the declines in p-PTEN after ischemia. However, LY294002 administration had no effect on the expression of p-PTEN in the XXMD-treated rats. Another surprising finding in this study was that XXMD treatment in the absence or presence of LY294002 effectively upregulated the expression of p-c-Raf, which was decreased after cerebral ischemia and reperfusion. Moreover, the data showed that total PTEN and c-Raf levels of different groups were not significantly different. Since the effects of the PI3K inhibitor indicated that XXMD may exert NVU protection via PI3K/Akt signaling pathway, we further examined whether XXMD regulated PI3K/Akt pathway following focal cerebral ischemia and reperfusion. The expression levels of related proteins in the penumbra of ischemic area were detected by Western blots. As shown in Figures Stroke, as one of the leading causes of human death and disability, has become a severe threat to human health. Although the researchers have conducted plenty of preclinical studies and obtained many striking outcomes, translating bench success to the bedside proof of efficacy and safety has been frustrating . There iIn recent years, several scientists have focused on TCM, a potential and promising therapeutic approach for cerebral ischemia and investigated its efficacy and mechanism. XXMD is beneficial for the patients with stroke. A spate of studies has indicated that XXMD is prepared from twelve medical herbs containing a number of active fractions –46. The In the present study, we further investigated the NVU protective effects of XXMD after ischemia and reperfusion. The results clearly demonstrated that PI3K/Akt pathway played the crucial roles in NVU protection of XXMD against focal cerebral ischemia and reperfusion injury in rats. Increased attention has recently been drawn to NVU protection which has quite significant implications for cerebral ischemia and reperfusion injury. Given to its important role, we focused on evaluating apoptosis of neuron, astrocyte, and vascular endothelial cell, respectively, which were important components of NVU. The results suggested that apoptosis was a common event after ischemia and reperfusion. Although apoptosis mainly located in neurons, other components of NVU were also involved in this event, such as astrocytes and vascular endothelial cells. These results further indicated that it was necessary to pay attention to NVU protection in treatment of stroke. XXMD had multiple targets to reduce apoptosis of different cells in NVU. Moreover, obvious improvements of neurological deficits and infarct area were also observed in XXMD-treated group at 24 h after reperfusion. β, and c-Raf were decreased in the penumbra of ischemic cortex at 24 h after reperfusion, and XXMD treatment improved phosphorylation levels of these proteins. The data also suggested that LY294002 reversed the effects of XXMD on phosphorylation levels of related proteins except for PTEN and c-Raf. These implicated that PI3K/Akt pathway contributed to neurovascular unit protection of XXMD after stroke with reperfusion  as the upstream of Akt may activate Akt by directly phosphorylating Thr308 and indirectly phosphorylating Ser473  and somePlenty of reports have shown that PTEN which prevents recruitment of Akt to the membrane for phosphorylation is a negative regulator of Akt , 66 and Akt, the serine-threonine kinase, is a key molecule in PI3K/Akt pathway and controls survival and apoptosis , 72 and β activity by phosphorylating it at Ser9 and further promotes neuronal survival [β phosphorylation of groups subjected to different administrations. The data showed a reduction in p-GSK3β at 24 h after reperfusion, but it was blocked by XXMD treatment. Additionally, PI3K inhibitor was involved in PI3K/Akt pathway and partly prohibited the expression of p-GSK3β in rats administrated with XXMD treatment. Activated Akt downregulates GSK3survival , 73, 74.c-Raf, a mitogen-activated protein kinase, is a crucial target of RAS/MARK pathway. Previous studies show that Akt mediates phosphorylation of c-Raf at Ser259 and inhibits Raf kinase activity , thus prAbove all, we highlighted NVU protection of XXMD and elucidated the role of PI3K/Akt pathway during this course in the current study. The findings indicated that XXMD reduced cerebral infarct area and improved neurological function after ischemia and reperfusion. More importantly, we found that XXMD could protect NVU from ischemia-induced apoptosis and may exert its effects, at least in part, via the PI3K/Akt signaling pathway. Whether other pathways or mechanisms were involved in the effects of XXMD on cerebral ischemia and reperfusion injury needs our further exploration.β, and c-Raf. Taken as a whole, these effects on PI3K/Akt pathway may help explain the NVU protection of XXMD. In light of these observations, our data revealed the underlying mechanisms of XXMD treatment for stroke and provided more support for clinical applications of XXMD.Focal cerebral ischemia and reperfusion injury were successfully induced by MCAO. Cellular apoptosis in NVU was increased in penumbra of ischemic cortex, and PI3K/Akt pathway dysfunction occurred after cerebral ischemia and reperfusion. The results indicated that treatment with XXMD reduced infarct area and cellular apoptosis and improved function outcomes after stroke. Moreover, we demonstrated that such protection may involve blunting the decreases in phosphorylation of PTEN, PDK1, Akt, GSK3


KRAS mutation in patients with metastatic CRC (mCRC) based on synchronous or metachronous status.The epidermal growth factor receptor (EGFR)/RAS/RAF/MEK/MAPK pathway is an important pathway in the carcinogenesis, invasion and metastasis of colorectal cancers (CRCs). We conducted a retrospective study to determine the prognostic values of EGFR expression and KRAS was performed. The clinicopathological features of these mCRC patients were retrospectively investigated according to EGFR expression and KRAS mutation status. Moreover, we analyzed the prognostic values of EGFR expression and KRAS mutation among these patients.From October 2002 to March 2012, 205 patients with mCRC were retrospectively analyzed; 98 were found to have metachronous mCRC while 107 were found to have synchronous mCRC. The EGFR expressions were determinate by IHC (immunohistochemistry) analysis and categorized 1+ (weak intensity), 2+ (moderate intensity), and 3+ (strong intensity). Genomic DNA was isolated from frozen primary CRC tissues and direct sequencing of KRAS mutation was investigated in 205 patients and mutations were noted in 88 of those patients (42.9%). In patients with metachronous mCRC, positive EGFR expression was significantly correlated with well-and moderately-differentiated tumors (P = 0.028), poorer disease-free survival (DFS) (P < 0.001), and overall survival (OS) (P < 0.001). Furthermore, positive EGFR expression was a significant independent prognostic factor of DFS  and OS  in metachronous mCRC patients. KRAS mutation status was not significantly related to DFS and OS of patients with metachronous mCRC; likewise, KRAS mutation status was not significantly different in the progression-free survival (PFS) and OS of patients with synchronous mCRC .Of the 205 patients with mCRC, EGFR expression was analyzed in 167 patients, and positive EGFR expression was noted in 140 of those patients (83.8%). KRAS mutation did not have prognostic value in patients with metachronous or synchronous mCRC.The present study demonstrated that EGFR expression has prognostic value only for patients with metachronous mCRC. However,  Colorectal cancer (CRC) is the third most common cancer and the third leading cause of cancer death in the United States where an estimated 142,820 newly diagnosed cases of CRC and an estimated 50,830 cancer deaths from CRC were reported in 2013 [KRAS mutation has been studied for the predictive value of tumor response to anti-EGFR treatment and also has been confirmed to be the highly predictive of resistance to anti-EGFR treatment [KRAS mutation in synchronous and metachronous mCRC remains controversial [EGFR is a 170-KDa transmembrane receptor with an intracellular tyrosine kinase domain. EGFR is a member of the ErbB receptor family. After EFGR is bounded by EGF, EFGR forms a functionally active dimer (homodimer or heterodimer) that causes phosphorylation of tyrosine kinases in the intracellular domain of EGFR. Subsequently, complex intracellular signals to the cytoplasm and then to the nucleus are triggered by this phosphorylation . Two majreatment -18, the oversial . TherefoThis retrospective study included 205 patients with histologically proven synchronous or metachronous mCRC who received surgical treatment from a single-institution between October 2002 and July 2012. The present study was approved by the Institutional Review Board of the Kaohsiung Medical University Hospital. Patients’ clinical outcomes and survival statuses were regularly followed up. Available variables included: age of diagnosis, sex, tumor location, histological type, TNM classification, vascular invasion, perineural invasion, and preoperative and postoperative serum level of CEA. The TNM classification was defined according to the criteria of the American Joint Commission on Cancer/International Union Against Cancer (AJCC/UICC) . All patFormalin-fixed and paraffin-embedded tissue blocks were cut into 3 μm sections and deparaffinized, rehydrated, and autoclaved at 121°C for 5 min in Target Retrieval solution , pH 6.0, to retrieve antigens. Endogenous peroxidase was blocked by 3% hydrogen peroxide for 5 min at room temperature. After washing with a Tris buffer solution, the sections were incubated with EGFR for 1 hour at room temperature. Then, DAKO REAL EnVision Detection System-HRP  was applied for 30 minutes at room temperature. Finally, sections were incubated in 3′, 3-diaminobenzidine for 5 minutes, followed by Mayer’s hematoxylin counterstaining. Dehydration was performed through two changes of 95% ethanol and two changes of 100% ethanol, and the samples were cleared in three changes of xylene and then mounted. Negative controls were obtained by replacing the primary antibody with non-immune serum. Immunoreactivity of EGFR was evaluated by two independent researchers who were blinded to patient outcome.Expression patterns of EGFR were determined in a semi-quantitative manner by light microscopy. Immunoreactivity for EGFR (membrane staining) was categorized in accordance with the presence of tumor cell staining and staining intensity. The intensity of EGFR immunoreactivity was scored with a 3-tier system as follow ,30: 1+  digestion and the phenol/chloroform extraction procedure according to the method outlined by Sambrook et al. . The desly study ,32.KRAS mutation was compared using a Chi-square test  and Student t-test (for continuous variables). The Cox proportional-hazards model was used for univariate and multivariate analyses to identify the independent prognostic factors for OS, DFS and PFS. OS, DFS, and PFS were calculated by the Kaplan-Meier method, and the differences in survival rates were analyzed by the log-rank test. A P value less than 0.05 was considered to be statistically significant.All data were statistically analyzed using the Statistical Package for the Social Sciences, version 19.0 . The correlation between clinicopathological features and EGFR expression or KRAS mutation status was evaluated in 205 patients, and mutation was noted in 88 of those patients (42.9%).Of the 205 patients with mCRC, 98 patients (47.8%) were metachronous and 107 patients (52.2%) were synchronous. The mean age of the 205 patients was 61.0 ± 12.8  years of age. There were 120 males and 85 females. The median follow-up time for the 205 patients was 30.2 ± 20.9  months. Immunohistochemical analyses for EGFR expression were performed in 174 patients and positive expression was noted in 140 of those patients (80.5%). P < 0.001) and DFS  rates of patients with positive EGFR expression were significantly poorer than those of patients with negative EGFR expression.The clinical and pathological data regarding the 98 patients with metachronous mCRC are summarized in Table KRAS mutation status was evaluated in 98 patients and mutation was noted in 44 of those patients (44.9%). There were no significant differences in mean ages, tumor location, histological type, AJCC/UICC cancer stage, retrieved lymph node number, vascular invasion, perineural invasion, pre-operative serum CEA level, and post-operative serum CEA level between patients with wild-type KRAS and those with mutated KRAS. OS  and DFS  were not significantly different between patients with wild-type KRAS and those with mutated KRAS.P = 0.523) and PFS  were not significantly different between patients with positive EGFR expression and those negative EGFR expression. The differences of clinical and pathological data regarding the patients with metachronous mCRC and the patients with synchronous mCRC are summarized in Additional file The clinical and pathological data regarding the 107 patients with synchronous mCRC are summarized in Table KRAS mutation status was evaluated in 107 patients, and mutation was noted in 44 of those patients (41.1%). There were no significant differences in mean ages, gender, tumor location, histological type, tumor depth, lymph node metastasis, retrieved lymph node number, vascular invasion, perineural invasion, pre-operative serum CEA level, and post-operative serum CEA level between patients with wild-type KRAS and those with mutated KRAS. Moreover, OS  and PFS  were also not significantly different.P = 0.028; HR, 3.090; 95% CI, 1.130–8.445) and DFS . However, KRAS mutation was not a significant prognostic factor for OS  and DFS . The Kaplan-Meier survival analysis also demonstrated that patients with positive EGFR expressions had worse OS (P = 0.003) and DFS (P < 0.001) , respectively. The 5-year OS rates of patients with positive EGFR expression and those with negative EGFR expression were 23% and 79%, respectively. The median DFS times of patients with positive EGFR expression and those with negative EGFR expression were 20.96 and 50.17 months , respectively. The 3-year DFS rates of patients with positive EGFR expression and those with negative EGFR expression were 16% and 51%, respectively.The univariate and multivariate analyses were performed to investigate independent prognostic factors for OS and DFS in patients with metachronous mCRC using the Cox proportional-hazards model Table . PositivKRAS and those with mutated KRAS in terms of OS (P = 0.461) and DFS (P = 0.783) , respectively. The median DFS times of patients with wild-type KRAS and those with mutated KRAS were 37.90 and 22.80 months , respectively. Furthermore, we analyzed the OS and DFS of patients with wild-type KRAS (N = 54), mutated KRAS codon 12 (N = 32), and mutated KRAS codon 13 (N = 12). No significant difference was noted in terms of OS (P = 0.656) and DFS (P = 0.977)  Figure C and 2D.P = 0.883) and PFS (P = 0.945) , respectively. The median PFS times of patients with positive EGFR expression and those with negative EGFR expression were 8.20 and 11.70 months , respectively. In addition, the Kaplan-Meier survival analysis demonstrated no significant difference between patients with wild-type KRAS and those with mutated KRAS in terms of OS (P = 0.544) and PFS (P = 0.555) , respectively. The median PFS times of patients with wild-type KRAS and those with mutated KRAS were 9.30 and 11.70 months , respectively. Furthermore, we analyzed the OS and DFS of patients with wild-type KRAS (N = 63), mutated KRAS codon 12 (N = 37), and mutated KRAS codon 13 (N = 7). No significant difference was noted in terms of OS (P = 0.656) and PFS (P = 0.977) , in concordance with the mutation rate of KRAS in CRC (35% to 42%) [P = 0.028), in accordance with the report of Andreyev et al. [Of the 205 patients analyzed in this study, 98 patients had metachronous and 107 had synchronous mCRC. Positive EGFR expression was found in 80.5% patients through immunohistochemical analyses. The positive rate of EGFR expression in CRC was reported to be 25% to 82% [ to 42%) . In our v et al. -8,33,34.P = 0.06). EFGR extent ≧25% was a significant factor for locoregional recurrence . Theodoropoulos et al. [P = 0.001), which implied that EGFR overexpression was associated with tumor invasion. Furthermore, they also demonstrated a trend between positive EGFR expression and poorer OS. Deng et al. [P = 0.046); however, the association was not noted in stage IV patients, which is in agreement with our present study. The association between EFGR expression and worse survival has also been noted in other malignancies, such as gastric cancer [For the prognostic value of EGFR for patients with metachronous mCRC, we have demonstrated EGFR as an independent negative prognostic factor for OS and DFS by multivariate Cox proportional-hazards model. The Kaplan-Meier survival analysis also showed that patients with positive EGFR expression had worse OS and DFS. Galizia et al.  have shos et al.  reportedg et al.  reportedc cancer ,37, esopc cancer , and brec cancer . In contc cancer  reportedKRAS mutation status was found not to be a significant independent prognostic factor of OS, DFS, and PFS for patients with metachronous mCRC and synchronous mCRC. Roth et al. [KRAS mutation from 1299 patients with stages II and III colon cancer. No significant association between survival  and KRAS mutation status was demonstrated. Moreover, no difference was noted between survival  and type of KRAS mutation stratified by condon 12 and 13 in patients with stages II and III colon cancer, which is in agreement with our analyses of patients with metachronous mCRC. Rose et al. [KRAS mutation status in 110 patients with metachronous and synchronous mCRC. The OS of patients with metachronous and those with synchronous mCRC was not influenced by KRAS mutation status , which is also consistent with our present study. Three studies [KRAS mutation status in CRC patients also showed that there was no prognostic value of KRAS mutation status for OS and PFS. Recently, a systemic review and meta-analysis regarding the prognostic value of KRAS mutation status demonstrated that there was no association between KRAS mutation status and the prognosis of patients with CRC [KRAS were significantly associated with an increased risk of relapse and death and a significant association between failure-free survival and G12V of mutated KRAS in Duke’s C patients. Richman et al. [KRAS compared to patients with wild-type KRAS. Roth et al. indicated that the KRAS mutations were assessed by each referring center according to three types of methodologies, and meta-analysis results could be affected by variations between trials [KRAS mutations in CRC patients treated with cetuximab, it was concluded that mCRC patients with mutated KRAS could have worse PFS and OS when treated with cetuximab [Through the multivariate Cox proportional-hazards analyses and Kaplan-Meier survival analysis used in this study, h et al.  reportede et al.  assessed studies -23 from with CRC . In contwith CRC ,26 evalun et al.  reportedn trials . In a meetuximab .KRAS mutation simultaneously in patients with metachronous and synchronous mCRC. However, there are some limitations to the present study. First, the present study is a single-institution retrospective study. The study’s relatively small sample size is another limitation. Third, the KRAS mutation status was evaluated only as wild-type and mutant (codon 12 and codon 13) and we did not analyze other rare mutation types.To our best knowledge, this study is the first to evaluate the prognostic values of EGFR expression and KRAS mutation did not have prognostic value for patients with metachronous or synchronous mCRC.In conclusion, we have demonstrated that EGFR expression has prognostic value only for patients with metachronous mCRC, while having no such value for patients with synchronous mCRC. Our data indicate EGFR as an independent negative prognostic factor for OS and DFS in patients with metachronous mCRC. Analyzing EGFR expression may help identify high-risk patients requiring more aggressive therapeutic modalities in the setting of metachronous mCRC. However, EGFR: Epidermal growth factor receptor; CRC: Colorectal cancer; DFS: Disease-free survival; OS: Overall survival; PFS: Progression-free survival; CEA: Carcinoembryonic antigen; AJCC: American joint commission on cancer; IHC: Immunohistochemistry; PD: Poorly differentiated; MD: Moderately differentiated; WD: Well differentiated.The authors declare that they have no competing interest.CWH analyzed the data and wrote the manuscript. HLT, CMH, CJM, CYL, CHK and DCW made substantial contributions in data acquisition, statistical analyses, and data interpretation, and helped in manuscript preparation. YTC and CYC participated in making formalin-fixed and paraffin-embedded tissue blocks, immunohistochemical staining of EGFR, and interpretation of EGFR expression. JYW participated in study design and coordination. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/599/prepubBaseline characteristics of metachronous and synchronous metastatic colorectal cancer patients.Click here for file


To study mortality rates among patients with diabetes and concomitant atrial fibrillation (AF), prescribed different cardiovascular drugs in primary health care.Study population consisted of men (n = 1319) and women (n = 1094) aged ≥45 years from a database including 75 primary care centres in Sweden. Cox regression analysis, with hazard ratios (HRs), 95% confidence interval (95% CIs) and mortality (years to death) as outcome, and Laplace regression, with difference in time to first 10% mortality (with 95% CI), were performed. Independent variables were prescribed cardiovascular drugs. Regression models were adjusted for a propensity score calculated separately for each prescribed drug class .Overall mortality was lower in the whole sample for anticoagulants vs no treatment ; and among patients < 80 years for anticoagulants vs. antiplatelets ; while among individuals aged ≥80 years, antiplatelets  and anticoagulants  vs. no treatment were equally effective. Statins were associated with lower mortality among those <80 years . Laplace regression models in the whole sample, with years to first 10% of total mortality as outcome, were significant for: among patients < 80 years anticoagulants vs. no treatment 2.70 years (95% CI 0.04-5.37), anticoagulants vs. antiplatelets 2.31 years (95% CI 0.84-3.79), and those ≥80 antiplatelets vs. no treatment 1.78 years (95% CI 1.04-2.52).Our findings suggest that antiplatelets could exert a beneficial effect among those above 80 years. Diabetes is a chronic disease affecting around 4.5% of the Swedish population . DiabeteThe overall lifetime risk of developing AF from the age of 40 years has been estimated at 25%, irrespective of sex . HoweverIn contrast, women with AF have a higher excess mortality risk than in men with AF , and womThe risk of stroke is markedly increased in AF patients, and anticoagulants are preferred to antiplatelets due to their superiority in stroke prevention . HoweverIn terms of pharmacological treatment and prevention of macrovascular complications in patients with diabetes, multifactorial treatment has been found to be effective , in partThe primary aim of the present study was to compare the effects of antiplatelets and anticoagulants on mortality in men and women with diabetes and AF. A further aim was to understand the effects on mortality of cardiovascular drugs that are commonly prescribed to these patients.Extractor software  to extract individual electronic patient records (EPRs). National identification numbers were replaced with new unique serial numbers to ensure anonymity. The files were linked to a database constructed using the Total Population Register, the Inpatient Register and the Swedish Cause of Death Register [The study used individual-level patient data from 75 primary health care centers (PHCCs), 48 of which were located in Stockholm County. Individuals attending any of the participating PHCCs between 2001 and 2008 were included in the study. We used Register , which cRegister , were usEthical approvals were obtained from regional ethical boards at Karolinska Institutet and the Lund University.st 2001 and December 31st 2007 were included in this study. They were identified by the presence of the ICD-10  codes for AF (I48) and diabetes mellitus (E10-14) in the medical records. In total, 2,413 individuals, 1,319 men and 1,094 women, who were ≥45 years of age at the time of AF diagnosis were identified and included [All persons diagnosed with both diabetes and AF who attended the 75 PHCCs between January 1included .st 2007.Time to death in the period from registration of AF diagnoses until December 31Drugs prescribed to individuals in the study population during the assessment period were recorded by Anatomic Therapeutic Chemical (ATC) Classification. With regard to anti-thrombotic drugs (B01A), patients were divided into three groups: no treatment, anti-platelet treatment with no anticoagulant treatment (B01AC), including acetylsalicylic acid , anticoagulant treatment with no anti-platelet treatment (B01AA), and finally a group who had received both anticoagulant and anti-platelet treatment. Diuretic drugs (C03) were recorded as thiazides or related agents, and were also registered when in combination with other drugs , loop diuretics (C03C) or potassium-saving diuretics (C03D), including aldosterone antagonists (C03DA). Furthermore, the following cardiovascular agents were recorded: heart-active drugs (C01), beta blockers (C07), calcium receptor-blocking agents (C08), and RAS-blocking agents (C09). Lipid-lowering drugs (C10A), including statins (C10AA), were also recorded.Gender: Men and women.age groups 45–54, 55–64, 65–74, 75–84 and ≥85 years. Individuals younger than 45 years were excluded.Individuals were divided into the following Educational level was categorized as ≤9 years , 10–12 years  and >12 years (college and/or university studies).Marital status was classified as married, unmarried, divorced or widowed.We identified the following cardiovascular co-morbidities from the EPRs among individuals in the study population: hypertension (I10-15), coronary heart disease , heart failure , non-rheumatic valvular diseases (I34-38), cardiomyopathy (I42) and cerebrovascular diseases , including intracranial bleeding (I60-62).t-test, chi-square analysis and Fisher’s exact test.Differences in means and distributions between men and women were compared by Student’s th percentile was chosen, as the results are more reliable in Laplace regression if both exposed and unexposed individuals have a sufficient number of fatalities (events) during the follow-up. Laplace regression also uses bootstrap technique in the estimation of confidence intervals. Since different distributions and mathematical calculations are used to obtain results in Cox and Laplace regression, respectively, putting emphasis on findings significant with both methods reduces the risk of chance findings.Follow-up analyses were performed using Cox regression with hazard ratios (HRs) and 95% confidence interval (95% CI), using time to death as the outcome. Laplace regression was used to calculate the difference in years until any given percentage of the participants prescribed pharmacotherapies vs. those without these particular drugs die ,20. We cModels were tested for interaction between sex and each pharmacotherapy. Analyses were performed for individuals <80 and ≥80 years of age. 80 years has commonly been referred to as a cut-off age for stroke prophylaxis , as wellIn the regression models, prescription of antithrombotic drugs (anti-platelets or anticoagulants), digitalis, diuretics , beta blockers, RAS-blocking agents, calcium receptor-blocking agents and statins were used as independent factors in the analyses. For antithrombotic drugs we used three groups: no antithrombotic treatment, anti-platelets only and anticoagulants only. We excluded the group with both anti-platelets and anticoagulants, as it would be hard to interpret the effects seen. We excluded specific anti-arrhythmic agents, potassium-saving agents other than aldosterone antagonists and lipid-lowering agents other than statins due to their low prescription rates. We did not divide RAS-blocking agents into ACE inhibitors and ARB, or calcium receptor-blockers into those with vessel- and heart-specific effects. Adjustment was made for a propensity score comprising age group, cardiovascular co-morbidities , educational level, marital status, and all pharmacotherapies . A uniqup-value for two-sided tests of <0.01 was considered statistically significant. This cut-off was used due to the multiple comparisons between men and women. A p-value of <0.05 was considered statistically significant for variables in the Cox and Laplace regression analyses, using 95% confidence intervals.A p = 0.0012). Overall, men were significantly younger and had more education than did women, while significantly more women were widowed than were men. HF and valvular disease were more common among women, while CVDs were more common among men. CHD and hypertension were more common among women than men  are shown separately for men (n = 1319) and women (n = 1094) in Table 3 are shoTable Table Significant interactions were found regarding sex and digitalis (p = 0.002), as well as sex and loop diuretics (p = 0.003). For digitalis, in men HR was 1.13 (95% CI 0.76-1.68) and in women 0.54 (95% CI 0.35-0.82), and for loop diuretics, in men HR was 2.51 (95% CI 1.39-4.53) and in women 0.91 (95% CI 0.54-1.53).The main findings of this study was that anticoagulants were superior in reducing mortality among individuals with diabetes and AF below 80 years of age, while antiplatelet therapy seemed equally effective as anticoagulants among individuals 80 years and above. An unexpected finding was that mortality was reduced in women who were prescribed digitalis, but not in men. In addition, mortality was increased in men prescribed loop diuretics. Furthermore, we found that mortality was reduced in patients aged below 80 years prescribed statins, and also among those below 80 years prescribed beta blockers.One might have expected that all prescribed pharmacotherapies would decrease mortality among the population in this study. However, the benefits of a drug could be offset by side effects, as in the case of disopyramide, quinidine and sotalol, which are known to be associated with increased mortality . The stuAn interesting finding was the equally positive effects of antiplatelets and anticoagulants on mortality among individuals ≥80 years of age, making findings from a Cochrane review accountable also for patients with diabetes and AF . AlthougPrescription of digitalis was associated with decreased mortality only among women. It seems, according to a review, that beneficial effects of digitalis are obtained at low digoxin concentrations, while high serum levels were associated with increased mortality . PositivThe finding of an association between prescription of loop diuretics with an increased mortality among men could be explained by concomitant presence of CHF, as CHF in AF patients do increase mortality . PrescriThe low mortality associated with prescribed beta blockers was expected, considering the effect in reducing mortality in myocardial infarction with AF , and alsThe reduced mortality associated with prescription of statins is in line with evidence of positive effect of statins on diabetic patients . The morCrude mortality was higher in women than in men, in agreement with others’ findings . In an eA limitation of this study was the small sample size, which prevented us from detecting small differences, even though the analyses included almost 9,000 person-years at risk. Since this was an observational study of the association between prescription of drugs and mortality, the findings may have been subject to confounding by indication ,42. FurtA major strength of this study was that we were able to link clinical data from individual EPRs to data from national demographic and socioeconomic registers with less than 1% of information missing. In addition, an earlier study of the diagnoses recorded in the EPRs showed that less than 2% of all diagnoses per individual were missing . The useIn conclusion, we found significantly positive effects of both antiplatelets and anticoagulants on survival among individuals aged ≥80 years, which could imply that antiplatelets despite little effect in stroke prevention could be beneficial on survival in elderly diabetes patients with AF when anticoagulants cannot be prescribed. We also found gender differences, indicating a positive effect by digitalis on survival among women, and a negative effect by loop diuretics among men, both needing further attention. The decreased mortality risk associated with prescription of statins underlines the positive effect of statins in diabetes patients with AF, and treatment by statins could be as important as antithrombotics in these patients. More studies focusing on diabetes patients with AF are needed to confirm or reject our findings.The authors declare that they have no competing interest to disclose.JS and KS are the principal investigators of the database study, and contributed in the drafting of the manuscript. PW and ACC brought the idea of the specific study, PW performed the statistical analyses in discussion with ACC, PW drafted the manuscript in discussion with ACC. SEJ and MB contributed to the statistical analyses and to discussion of the statistical findings, MB especially on the Laplace regression method. All authors read and approved the final manuscript.


Bacillus sp., WBUNB009, isolated from a raw sewage canal in nitrate broth (Himedia M439) with a G+C content of 34.9% is reported. It removes 60.23% nitrate and 96% phosphate within 16 h at 37°C.The draft genome sequence  of a nitrate- and phosphate-removing  The contamination of water by nitrates due to anthropogenic activity is a potential hazard throughout the world. Nitrate-induced methemoglobinemia contributes to national infant death rate statistics . ApplicaPhosphorus is a scarce element with finite reserves on the earth and no synthetic method for its production. Phosphorus is extremely important for plant growth but only a fraction of it is utilized while the rest seeps into water bodies, causing eutrophication. High accumulation of residual phosphate in soil due to excessive use of fertilizers may enhance the downward movement of phosphate, which may eventually reach groundwater . MicrobiThis pure isolate with the ability to remove nitrate and phosphate was isolated from raw sewage canal water in nitrate broth with 2,000 ppm nitrate. This organism reaches a stationary phase at the 6th hour with a generation time of 21 min at 37°C. Its optimum growth is at 30°C and pH 9. The strain is available at the Microbial Culture Collection at the National Centre for Cell Sciences, Pune, India.This whole-genome sequence was obtained on a 316 chip using an Ion Torrent PGM instrument. The 3,322,254 reads were assembled using the MIRA assembler version 3.4.0. The coverage was 98× with 289 contigs generated. A total of 660 Mb data were sequenced, with 458 Mb data with quality values above 20. All contigs generated by the MIRA assembly were submitted to the RAST (Rapid Annotation using Subsystem Technology) annotation server (ppk), phosphoesterase, and exopolyphosphatase (ppx), are carried by ANFK01000043, and genes for phosphoglycerate transporter protein (pgtP) and pyrophosphatase (ppaX) are carried by ANFK01000004. The genes for phosphate transport system, consisting of phosphate transport system regulatory protein, phosphate transport ATP-binding protein (pstB), and phosphate transport system permease protein , are carried by ANFK01000021. The gene for urease domain protein is carried by ANFK01000002. The strain contains two Walker motifs, which are involved in phosphate transport.The genes responsible for nitrate metabolism, namely nitrite reductase [NAD(P)H] large subunit and small subunit, nitrate/nitrite transporter, and the respiratory nitrate reductase alpha, beta, gamma, and delta chains, are carried by ANFK01000040. The genes for nitrate/nitrite sensor proteins and the nitroreductase family proteins are carried by ANFK01000058. The genes responsible for phosphate metabolism, like polyphosphate kinase (ANFK00000000. The version described in this paper is the first version, ANFK01000000.This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number