Schisandra chinensis, Artemisia capillaris, and Aloe barbadensis, in mitigating chemically induced acute liver toxicities. Acetaminophen and carbon tetrachloride induced acute liver toxicity models in mice were utilized. Hepatic functional tests from serum collected at T24 and hepatic glutathione and superoxide dismutases from liver homogenates were evaluated. Histopathology analysis and merit of blending 3 standardized extracts were also confirmed. Statistically significant and dose-correlated inhibitions in serum ALT ranging from 52.5% (p = 0.004) to 34.6% (p = 0.05) in the APAP and 46.3% (p < 0.001) to 29.9% (p = 0.02) in the CCl4 models were observed for SAL administered at doses of 400–250 mg/kg. Moreover, SAL resulted in up to 60.6% and 80.2% reductions in serums AST and bile acid, respectively. The composition replenished depleted hepatic glutathione in association with an increase of hepatic superoxide dismutase. Unexpected synergistic protection from liver damage was also observed. Therefore, the composition SAL could be potentially utilized as an effective hepatic-detoxification agent for the protection from liver damage.Some botanicals have been reported to possess antioxidative activities acting as scavengers of free radicals rendering their usage in herbal medicine. Here we describe the potential use of “SAL,” a standardized blend comprised of three extracts from Liver diseases as a result of habitual repeated alcohol consumption, exposure to some xenobiotics, and/or drug interactions are some of the major causes of morbidity and mortality where variable symptoms manifested ranging from asymptomatic elevation of the liver enzyme to sudden hepatic failure. Liver is susceptible to alcohol-induced injury as both alcohol and its primary metabolite acetaldehyde produce reactive oxygen species (ROS) and hydroxyl radicals (OH), altering hepatic antioxidant defense system . While d4) are generally utilized to develop an animal model that mimics the human type of liver toxicity with similar mechanisms of actions. Acetaminophen  is a very safe and effective analgesic and antipyretic drug at therapeutic dosage. However, APAP overdose can cause severe liver toxicity characterized by depletion of GSH, protein adduct formation [4, a halogenated alkane with restricted usage as industrial chemical/solvent, is a well-known hepatotoxin that is widely used to induce acute toxic liver injury in a large range of laboratory animals. CCl4 toxicity is initiated by cytochrome P450s (CYP) primarily of 2E1 [3−), which can initiate lipid peroxidation and ultimately results in the overproduction of reactive oxygen species (ROS) and hepatocyte injuries [3OO−, a highly reactive species that could initiate the chain reaction of lipid peroxidation leading to cell death. Therefore, it could be inferred that, regardless of the chemical agents used to induce the hepatotoxicity, both the acetaminophen and carbon tetrachloride models share the critical step in oxidative stress induced by reactive oxygen species generated by excess intermediate metabolites leading to protein oxidation, lipid peroxidation, and DNA damage.Various xenobiotics are known to cause hepatotoxicity, among which acetaminophen (n-acetyl-p-aminophenol or APAP) and carbon tetrachloride  collected through legacy mining leading to the discovery of a composition designated as SAL which comprisedSchisandra chinensis, also known as Wuweizi and Wurenchum, is traditionally used for conditions of lung and kidney insufficiency. It is also indicated in cases of chronic cough and dyspnea, diarrhea, night sweats, wasting disorders, irritability, palpitations, and insomnia, as well as a general tonic for treating fatigue associated with illness [ Schisandra extracts preventing carbon tetrachloride induced hepatotoxicity and glutathione depletion and stimulating the activity of glutathione reductase [ Schisandra are lignans called schisandrins, which have energizing properties by increasing the activity of some enzymes which participate in the oxidative phosphorylation process and also increased superoxide dismutase and catalase activities in rat liver cytosol and were able to inhibit gossypol-induced superoxide anion generation in rat liver microsomes [ Schisandra fruit extracts has been reported in Chinese literature for the treatment of patients with hepatitis, in a clinically controlled trial resulting in 68% (72/107) and 44% (36/72) improvement in serum ALT levels within 4 weeks and 8 weeks [ illness . In modeeductase –14. The crosomes . The hep 8 weeks .Artemisia capillaris, with the common name “yinchen” or “yinchenhao” in Chinese depending on the different collection season, also known as “yinjin” in Korean, is one of the commonly used TCM included in various ancient Chinese dispensatories. The earliest record of Artemisia capillaris was recorded in Shen Nong Ben Cao Jing —a Chinese book of agriculture and medicinal plants—for treating jaundice, removing the dampness, and use as a diuretic. Both aqueous extracts and ethanol extracts have been reported with hepatoprotective efficacy in both in vitro assays and in vivo animal studies [ Artemisia capillaris [ studies , 18. CatAloe vera N-931 is a composition containing a unique combination of 1–4% aloesin and 96–99% 200 : 1 Aloe vera inner leaf gel powder with not less than 8% polysaccharides blended via a conventional method . Chromones isolated from various Aloe species have been reported to have diverse biological activity. A C-glycosyl chromone isolated from Aloe barbadensis demonstrates anti-inflammatory activity [activity  and antiactivity . Schisandra chinensis, Artemisia capillaris, and Aloe barbadensis was also evaluated.Here we implemented the most frequently used animal model with practical clinical implications such as acetaminophen and confirmed findings with the classic carbon tetrachloride induced hepatotoxicity model to assess the effect of the composition SAL in protecting liver from such damage. In addition, the merit of combining Artemisia capillaris extract, Schisandra chinensis extract, and Aloe vera composition, N-931. Artemisia capillaris extract was produced as 70% ethanol extracts of the aerial parts with no less than 3% chlorogenic acid. Schisandra chinensis extract was standardized as 70% ethanol extract of the fruits containing at least 2% total schisandrins. N-931 is a unique combination of 1–4% aloesin and 96–99% 200 : 1 Aloe vera inner leaf gel powder with not less than 8% polysaccharides provided by Aloecorp, Mexico. Schisandra extract, Artemisia extract, and Aloe N-931 were blended at a ratio of 4 : 8 : 3 to produce the standardized SAL composition containing no less than 0.2% schisandrins from Schisandra chinensis and 1.0% chlorogenic acid from Artemisia capillaris and N-931 with the above specifications.SAL is a novel synergistic composition containing ad libitum and were housed in a temperature controlled room (22.2°C) on a 12-hour light-dark cycle. All animal experiments were conducted according to institutional guidelines congruent with the guide for the care and use of laboratory animals and approved by IACUC with approval #SAL-441-14/15.Purpose bred female CD-1 mice, weighing 18–24 g, were purchased from USDA approved laboratory animal vendor  and acclimated upon arrival for a week. Individual cages were identified with a cage card indicating project number, test article, dose level, group, and animal number. The Harlan T7087 soft cob bedding was used and changed at least twice/week. Animals were provided with fresh water and rodent chow diet #T2018 4 induced hepatotoxicity model, CCl4  at a dose of 25 μL/kg dissolved in corn oil was administered intraperitoneally to overnight fasted CD-1 mice to induce toxicity [4 administration and +6 hr after induction. Materials were administered at (a) 400 mg/kg, 325 mg/kg, and 250 mg/kg of composition SAL for dose-response study; (b) 106.7 mg/kg, 213.3 mg/kg, 80 mg/kg, and 400 mg/kg of Schisandra chinensis, Artemisia capillaris, N-931, and composition SAL, respectively, for synergy determinations; (c) 300 mg/kg of each Schisandra chinensis, Artemisia capillaris, N-931, and composition SAL for functional tests and comparative study; and (d) 400 mg/kg and 50 mg/kg of composition SAL and UDCA, respectively, for activity confirmation tests. In total, the mice received 3 doses before the chemical induction and a dose after the chemical induction. 10% Tween-20 (Lot #0134C141 from Amresco) was used as a carrier vehicle for all the materials. Control mice with or without APAP or CCl4 received carrier vehicle only.A balanced therapeutic schedule was generated and optimized as follows to address prophylaxis and intervention: for APAP-induced hepatotoxicity model, APAP  at a dose of 400 mg/kg dissolved in warm saline  (heated to 60°C and cooled down to ambient temperature) was orally administered to overnight fasted CD-1 mice to induce toxicity [toxicity , 22, 23.Serum was isolated from blood drawn at T24 using serum separator tube after 30-minute room temperature clotting and spun at 3000 rpm for 10 minutes for ALT , AST (aspartate aminotransferase), total bilirubin, conjugated and unconjugated bilirubin, bile acid, total protein, albumin, globulin, and alkaline phosphatase monitoring in an automated colorimetric assay using Beckman Coulter AU2700 at Phoenix Laboratories .μM EDTA was added to ~0.2 g of ground tissue and homogenized for 1 min in ice bath using a homogenizer from Omni International. The mixture was then centrifuged for 15 min at 10,000 rpm at 4°C. A portion of the supernatant was used for SOD and protein analysis. The rest of supernatant was further processed as follows for GSH analysis. (B) GSH analysis: portion of the supernatant was mixed with the same volume of 100 mg/mL MPA solution to deproteinized in order to avoid interference from proteins. The mixture was allowed to stand at room temperature for 5 min after vortexing and then centrifuged for 15 min at 10,000 rpm at 4°C. The deproteinated supernatant was evaluated for GSH content. Glutathione (GSH) is a key intracellular tripeptide thiol that helps protecting cells from free radical damage by providing reducing equivalents for the reduction of lipid hydroperoxides. During this process, oxidized glutathione (GSSG) forms as a reaction product. GSH level has been used as indicative biomarkers of in vivo oxidant and oxidative stress level in cells and tissues. In this analysis, the sulfhydryl group of GSH reacts with DTNB ) to produce a a yellow colored 5-thio-2-nitrobenzoic acid (TNB) product. The amount of GSH in the deproteinated supernatant is determined via measurement of the absorbance of TNB at 410 nm. A Glutathione Assay Kit from CAYMAN Chemical Co., Inc. , was used for analysis. (C) SOD and protein analysis: superoxide dismutases (SODs) are metalloenzymes that catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide. SOD is considered one of the most important antioxidant enzymes in vivo. The SOD assay is a colorimetric assay, which utilizes a tetrazolium salt to measure the dismutation of superoxide radicals that were induced by xanthine oxidase and xanthine, and the activity of SOD in a given sample is quantified by the standard curve generated using the SOD standards. One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of superoxide radicals. A Superoxide Dismutase Assay Kit from CAYMAN Chemical Co., Inc., was used for analysis. The protein concentrations of the tissue homogenates were determined by assessing protein concentrations of the suppressants via a Pierce™ BCA Protein Assay Kit. (D) Materials and equipment: Homogenizer (cat number TH-01) from Omni International ; Hard Tissue Omni Tip™ Plastic Homogenizing Probes (7 mm × 110 mm) from Omni International ; Refrigerated Centrifuge (model number 5402) from Eppendorf ; and Microplate Reader (model number Synergy HT) from US Biotek  were used.Liver tissues were collected immediately after necropsy and were kept in dry ice until transferred to −80°C. Materials were then shipped to a contract laboratory  in dry ice for final specimen processing and biomarker analysis. (A) Sample preparation: Frozen tissue was ground to a course powder using a pulverizer. 1 mL of PBS containing 19.6 μm) slides were stained with haematoxylin/eosin and analyzed under the microscope system  with a magnification ×200.Liver samples were fixed in 10% buffered formaldehyde and embedded in paraffin wax for histological examination. All microsectioned  were considered as significant. When normality test failed, for nonparametric analysis, data were subjected to Mann-Whitney sum ranks for t-test and Kruskal-Wallis one-way ANOVA on ranks for ANOVA.Data were analyzed using SigmaPlot . The results are represented as mean ± standard deviation. Statistical significance among groups was calculated by means of single factor analysis of variance (ANOVA) and by 4 induced models. Mice were given orally the composition SAL at doses of 400 mg/kg, 325 mg/kg, and 250 mg/kg suspended in 10% Tween-20. As seen in p = 0.004), 48.5% (p = 0.007), and 34.6% (p = 0.05) inhibitions were observed for mice treated with doses of 400 mg/kg, 325 mg/kg, and 250 mg/kg SAL, respectively. Similarly, in the CCl4 group, dose-correlated inhibitions in serum ALT were observed for the composition. 46.3% (p < 0.001), 39.5% (p = 0.003), and 29.9% (p = 0.02) inhibitions were observed for mice treated with doses of 400 mg/kg, 325 mg/kg, and 250 mg/kg SAL, respectively. There was a 100% survival rate for all the groups in both models. Compared to the normal control animals that received 10% Tween-20, administration of APAP and CCl4 caused a 229- and 496-fold increase in serum ALT, respectively. The composition SAL has provided statistically significant protection from liver damage at a dosage level as low as 250 mg/kg as determined by serum ALT level when compared to vehicle treated diseased mice.The optimum dosage of the composition SAL that incurs significant liver protection was evaluated both in APAP and in CCl Schisandra, Artemisia, and N-931 at a dosage equivalent to each plant ratio in the composition of SAL as they appear in 4S : 8A : 3L at the highest dose tested (400 mg/kg). An average of 20% inhibition with 70–80% survival rates was observed for these plants at the given dose. Colby's equation [ Schisandra chinensis, Artemisia capillaris, and N-931 in both APAP and CCL4 model. As shown in  Schisandra, Artemisia, and N-931 was confirmed by their synergistic protection from liver damage caused by APAP and CCl4 induction.The efficacy of individual plants was tested includingequation  was util4 induced liver toxicity models were utilized to compare the liver protection activity of the SAL composition against its individual components at a dose of 300 mg/kg using reduced serum ALT level as a measure of efficacy. As seen in Figures  Schisandra, Artemisia, and N-931 compared to vehicle group, respectively. In this study, the lowest survival rate (50%) was observed for mice treated with Artemisia while survival rates of 90, 70, and 70 were observed for the composition, Schisandra, and N-931, respectively. Further substantiating the evidence observed in APAP model, the composition SAL showed greater liver protection than each individual component at a dose of 300 mg/kg in the CCl4 model using serum ALT as a measure of efficacy. Reductions of 37.0, 33.6, 37.4, and 34.2% were observed for SAL, Schisandra, Artemisia, and N-931, respectively. There was a 100% survival rate for all the groups in this model.Both APAP and CCl4 induced liver toxicity models were utilized to compare the liver protection activity of the composition of SAL (400 mg/kg) against pharmaceutical drug ursodeoxycholic acid (50 mg/kg) using liver panel data as a measure of efficacy. In this report only the CCl4 data have been depicted for the UDCA comparison. As depicted in Tables 4 model, 34.0, 44.5, 26.6 and 63.6% reductions in serums ALT, AST, bile acid, and direct bilirubin, respectively, were observed for mice treated with the composition , d-galactosamine, and dl-ethionine [4 induced hepatotoxicity [4 induced liver injury, the hepatoprotective effect of the major components isolated from Schisandrae is believed to be due to their inhibitory effect on lipid peroxidation and the binding of CC14-metabolites to lipids of liver microsomes [Hepatoprotective plant extracts have been traditionally used for treatments of liver diseases over centuries. Furthermore, in modern pharmacotherapy, mounting experimental and clinical evidences suggest the hepatoprotective nature of Schisandrae. For example, oral pretreatment of rats with a lignan-enriched extract from the fruit ofeductase . One of is study . In anot in rats . Similarthionine . Schisanthionine . The mectoxicity . Pretreatoxicity . In CCl4crosomes . Schisandra have been reported in Chinese literature with patients with hepatitis; in a clinically controlled trial involving 189 patients with chronic viral hepatitis B and elevated ALT levels, an ethanol extract of the fruits, containing 20 mg of lignans , resulted in 68% (72/107) and 44% (36/72) improvement in serum ALT levels within 4 weeks and 8 weeks, in patients receiving the extract and control group, respectively [More than 5000 case studies of hepatoprotective effects of fruit extracts fromectively . Schisandra extract, the physiological adaptogenic properties of extracts from Schisandra have also been reported. This mechanism will make the host prone to adapt to a state of nonspecific resistance which leads to biochemical changes at the time of exposure to harmful external or internal factors resulting in a more rapid and effective response to the stimuli [ Schisandra.Besides the description of hepatoprotective effect from stimuli . By doin Artemisia capillaries as the other active ingredient of the present composition. For example, when aqueous extract of A. capillaries was administered orally at a dose range of 50–100 mg/kg for 10 days to mice at which liver injuries were induced by oral administration of 30% alcohol  plus pyrazole , statistically significant reductions in ALT, AST, and malondialdehyde (MDA) levels in serum and liver tissues were observed for mice treated with the extract. In addition, it (a) moderated microvesicular steatosis and necrosis in hepatic histopathology; (b) replenished the antioxidant components including glutathione content and total antioxidant capacity and activities of glutathione peroxidase (GSH-Px) and catalase and SOD; (c) normalized levels of tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β) in hepatic tissues; and (d) attenuated the alterations of aldehyde dehydrogenase (ALDH) level in serum and hepatic gene expressions of ALDH and alcohol dehydrogenase (ADH) were the principal markers positively impacted suggesting both enhancement of antioxidant activities and modulation of proinflammatory cytokines as the possible mechanisms that could be involved during the hepatoprotective activity of A. capillaries [ A. capillaries for 7 days at a dose of 7.5 g/kg significantly attenuate serums ALT and AST and improved glutathione levels and enhanced the production of catalase and significantly attenuated the accumulation of thiobarbituric acid-reactive substances in both plasma and liver tissues compared with those of rats given AAPH alone [ A. capillaris (at 200 mg/kg) resulted in moderations in transaminase activities, MDA, and hydroxyproline concentrations [ A. capillaris on alcohol-induced hepatotoxicity in vitro. In this study, A. capillaris at a concentration range of 0.5–5 μg/mL inhibited the secretion of EtOH-induced interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α), IL-1α, and TNF-α-induced cytotoxicity and inhibited the EtOH-induced apoptosis of Hep G2 cells [ A. capillaris administered daily at a dose of 25 or 50 mg/kg for two weeks significantly reduced serum malondialdehyde and liver hydroxyproline levels and restored depleted glutathione content and glutathione peroxidase activity and attenuate cholestatic liver injury and collagen deposition and suppressed expression of fibrogenic factors suggesting the antifibrotic properties through both upregulation of antioxidant activities and downregulation of extracellular matrix protein production [In addition to treating various hepatic disorders in traditional oriental medicine, significant experimental reports have been documented to show the hepatoprotective activities ofillaries . In 2,2-PH alone . In an otrations . KnowingG2 cells . In a bi Aloe vera N-931, containing Aloe chromone aloesin and Aloe polysaccharide. Chromones isolated from various Aloe species have been reported to have diverse biological activity. Aloesin is a C-glycosylated 5-methylchromone with a potent antioxidation activity [ Aloe barbadensis was investigated using colorimetric assays, triple quadrupole and time-of-flight mass spectrometry, focusing on phenolic secondary metabolites in the different leaf portions, the outer green rind that contains aloesin was identified as the most active in radical scavenging activity, which is better than the inner parenchyma in stable radical DPPH test and ORAC assay. Further tests using isolated pure secondary metabolites confirmed that the 5-methylchromones aloesin were among the most active chromones [The third ingredient used in formulation of the SAL composition wasactivity , 31. In hromones . Aloe vera gel, have been utilized for varieties of human disease and suggested for liver protection, in part, because of their antioxidant activities. For instance, strong antioxidant activities have been reported for purified polysaccharides from Aloe barbadensis gel when tested in DPPH, hydroxyl, and alkyl radical scavenging assays [ Aloe plant age and function related study, polysaccharides from three-year-old Aloe extract were found showing the strongest radical scavenging activity (72.19%) which was significantly higher than that of synthetic antioxidants butylated hydroxytoluene (70.52%) and α-tocopherol (65.20%) at the same concentrations of 100 mg/L via DPPH assay [ A. vera have also been found to possess high antioxidant efficiency as demonstrated with a decrease in the oxidative stress marker MDA and an increase in the hepatic nonenzymatic antioxidant GSH and enzymatic antioxidant SOD in vivo in chronic alcohol-induced hepatotoxicity in mice [Moreover, polysaccharides, the major constituents ofg assays . SimilarPH assay . Polysac in mice .4. When the combinations of these three plant materials were tested, clearly interesting yet, an unexpected synergy was observed from the SAL composition which exceeded the predicted effect based on simply summing the effect observed for each individual ingredient at the given ratio. In fact, none of the individual ingredients showed liver protection activity at the magnitude equivalent to the one noted for the composition in both models separately. Furthermore, data from liver function test including AST, ALT, bile acid, total protein, total bilirubin, conjugated bilirubin, albumin, and total protein demonstrated that the composition has indeed liver protection activity when compared to the vehicle treated control animals with liver injury. This is supported by the comparison of the effect of SAL composition against a pharmaceutical drug Ursodeoxycholic acid (UDCA). Moreover, as reflected from data of the liver homogenate, the composition SAL also replenished the depleted hepatic glutathione in association with an increased activity in hepatic superoxide dismutase. Glutathione is a key intracellular tripeptide thiol that helps protecting cells from free radical damage by providing reducing equivalents for the reduction of lipid hydroperoxides. Similarly, SODs are metalloenzymes that catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide. As a result, SOD is considered one of the most important antioxidant enzymes in vivo. These phase II enzymes substantiate each other to provide the strong antioxidant activity of the composition. These findings were substantiated by the histopathological observations signifying a liver protection capability of SAL composition in both APAP and CCl4 models.Based on analyses of above results and data, it is reasonable to postulate that a composition comprised of these three plant materials possesses significant antioxidant activity and hence protects the liver from oxidative stress caused damage. To the best of our knowledge, these three plant extracts have never been reported to combine together before at specific ratios to formulate the SAL composition on the basis of literature search. In the present study, each component of the composition showed strong individual performances in modulating toxicities induced by the disclosed chemicals reinforcing the idea of combining these plant extracts for a better outcome in both models. This hypothesis needs to be confirmed in a way that the composition of SAL should demonstrate a boosted protection from liver damage elicited by both APAP and CCl Schisandra chinensis, Artemisia capillaris, and Aloe vera N-931 into the ratio of 4S : 8A : 3L provided a significantly enhanced liver protection activity to the composition. Therefore, the composition of SAL could potentially be considered as a mitigating agent for alcohol and/or chemical induced hepatotoxicity.Collectively, based on analyses of data from the hepatic function test, antioxidation biomarkers, and histopathological findings, we strongly believe that combining these traditionally well-known folk medicinal plants


Rapid and accurate risk stratification in patients with community-acquired pneumonia (CAP) is an unmet clinical need. Cortisol to dehydroepiandrosterone (DHEA) ratio was put forward as a prognostic marker in sepsis. We herein validated the prognostic value of the adrenal hormones DHEA, DHEA-Sulfate (DHEAS), cortisol/DHEA-, cortisol/DHEAS- and DHEA/DHEAS – ratios in patients with CAP.We assessed severity of illness using the pneumonia severity index (PSI) and measured adrenal hormone concentrations in 179 serum samples of prospectively recruited patients hospitalized with CAP. We calculated spearman rank correlation, logistic regression analysis and Kaplan Meier curves to study associations of adrenal hormones and outcomes.r = 0.24, p = 0.001), DHEAS , cortisol/DHEA , cortisol/DHEAS  and DHEA/DHEAS . In age and gender adjusted logistic regression analysis, cortisol  and DHEA , but not DHEAS and the different ratios were associated with all-cause mortality. The discriminatory accuracy of cortisol and DHEA in ROC analysis (area under the curve) was 0.74 and 0.61. In Kaplan Meier analysis, patients in the highest deciles of cortisol and DHEA (p = 0.005 and p = 0.015), and to a lesser extent of cortisol/DHEAS ratio (p = 0.081) had a higher risk of death.There was a significant correlation between PSI score and total cortisol (Cortisol, DHEAS and their ratios correlate with CAP severity, and cortisol and DHEA predict mortality. Adrenal function in severe pneumonia may be an important factor for CAP outcomes. Community-acquired pneumonia (CAP) is the most common sepsis-defining illness, and its treatment and prognosis has not changed since the 1950ies, while in-hospital mortality remained about 12% Therefore, several prognostic biomarkers have been evaluated in the last years Other surrogates of adrenal function like dehydroepiandrosterone (DHEA) also change upon inflammation Arlt et al. observed an increased cortisol to DHEA ratio in the most severely ill as compared to the least ill patients. Both cortisol and cortisol to DHEA ratio were of a similar prognostic accuracy In sepsis, cortisol to DHEA ratio was put forward as a prognostic marker In CAP, only one study evaluated cortisol and DHEA/-S levels for outcome prediction, but only with a relatively small sample size (n = 58), and without including assessment of CAP severity For the purpose of this study, we measured adrenal function parameters in remaining plasma samples of a prospective randomized trial in patients with CAP that was conducted from November 2003 through February 2005 at the University Hospital of Basel, a tertiary care hospital in Switzerland. The design of the original study has been described elsewhere Adult patients (>18 years) with CAP as their principal diagnosis on admission were eligible for the study. CAP was defined as a new infiltrate on chest x-ray accompanied by one or several acquired respiratory symptoms and signs  and the absence of a hospital stay within 14 days of admission. Exclusion criteria were cystic fibrosis, active tuberculosis, HIV infection with a CD4-cell count below 200, neutropenia of less than 500 G/l, chemotherapy with neutropenia between 500–100 G/l and an expected decrease below 500 G/l, and immunosuppression after organ transplantation. Baseline assessment included clinical data, vital signs, comorbidities, routine blood tests and the PSI. A follow-up telephone interview was performed after 6 weeks.Ethical approval was obtained from the local ethics committee (ethics committee Basel EKBB), and all included participants or their legal representatives gave written informed consent before inclusion into the study. This study adhered to the consolidate STROBE standards for the reporting of observational trials Serum samples were collected on admission and batch-measured. DHEA was analyzed at Bioanalytica AG, Luzern, Switzerland, by a routinely available ELISA-assay . For DHEAS and cortisol, we used the routinely available chemiluminescence immunoassay .Calculations were made with STATA 12.1  or Graph Pad Prism 5.0 .Correlation of hormones and PSI was calculated by Spearman rank correlation. For multi-group comparisons, Kruskal-Wallis analysis was performed. We calculated a logistic regression model adjusted for age and gender to assess associations of hormones and mortality after 6 weeks. Receiver operating characteristics (ROC) were performed and area under the curve (AUC) was calculated to assess the overall predictive accuracy of hormones. Finally, for graphical display, we also assessed time to death in Kaplan Meier curves; and used log rank tests to compare groups stratified by the highest decile of hormone levels.Detailed baseline characteristics of the study population are shown in The median cortisol level was 727 nmol/L (IQR 433–1238 nmol/L), median DHEA level was 7.6 nmol/L (IQR 5.5–11.0 nmol/L) and median DHEAS level was 2.1 umol/L (IQR 1.2–4.1umol/L). We found no significant difference of hormone levels according to gender.r = 0.24, p = 0.001), DHEAS  and the ratios cortisol/DHEA , cortisol/DHEAS  and DHEA/DHEAS  were significantly correlated with CAP severity as assessed by PSI.Total cortisol  and the ratios cortisol/DHEA (p = 0.010), cortisol/DHEAS (p<0.001) and DHEA/DHEAS (p = 0.023) were significantly increased with increasing CAP severity as assessed by PSI classes shown in .In logistic regression analysis adjusted for age and gender, cortisol  and DHEA  were associated with mortality. In addition, cortisol remained an independent predictor for mortality when adjusting for PSI score  and when adjusted for the inflammatory markers procalcitonin and white blood cell count . For DHEA the same association was also significant when adjusted for procalcitonin and white blood cell count , but not after adjustment for PSI . DHEAS and the other ratios were not associated with mortality after adjustment for PSI .ROC analysis showed AUCs of cortisol, DHEA and DHEAS of 0.74 (95% CI: 0.60–0.88), 0.61 (95% CI: 0.46–0.75) and 0.53 (95% CI: 0.39–0.67), respectively. AUC for cortisol/DHEA, cortisol/DHEAS and DHEA/DHEAS were 0.62 (95% CI: 0.49–0.75), 0.66 (95% CI: 0.53–0.79) and 0.60 (95% CI: 0.48–0.72), respectively. AUC for PSI was 0.72 (95% CI: 0.62–0.81).In Kaplan Meier analysis, a significant separation of groups stratified by the highest decile hormone level was observed for cortisol and DHEA , respectively, but not for cortisol/DHEAS (p = 0.08). DHEAS and the other ratios did not have significant results . The corresponding Kaplan Meier curves are shown in Our findings show that cortisol, DHEAS and their different ratios significantly correlate with CAP severity, while DHEA and cortisol are predictive for mortality. Thus, not only cortisol, but adrenal function in general and its possible intra-adrenal shift from DHEAS to cortisol production in severe pneumonia is an important factor for outcome and survival in CAP.The finding that high cortisol levels predict bad outcome and mortality confirms previous data In patients with a high severity of CAP, we found not only higher cortisol levels, but also lower levels of DHEAS compared to patients with a low CAP severity. Furthermore, in non-survivors, cortisol and DHEA levels were higher as compared to survivors.A similar dissociation between cortisol and DHEAS levels has been observed in sepsis and in stroke Low DHEAS levels were not only proposed as predictive marker for stroke and sepsis but were an even more sensitive marker than cortisol for the diagnosis of so-called relative adrenal insufficiency The pathophysiological mechanism why DHEAS is low in the presence of high cortisol in severely ill patients is currently unknown. A recent study found a biological activity for DHEAS, but not for DHEA, being able to enhance the activity of human neutrophils. Therefore, diminished levels of DHEAS could have adverse effects, especially in relation to susceptibility to bacterial infection Arlt et al. suggested the cortisol/DHEA ratio as a novel prognostic marker in septic shock, as they found a similar, significant dissociation between cortisol and DHEA in non-survivors and between the least ill and severest ill patients despite increased DHEA levels In addition, we observed not only a dissociation between cortisol and DHEAS but also between cortisol and DHEA levels in patients with progressive CAP severity. In our study we were able to confirm these data in CAP, as we found an elevated cortisol to DHEA ratio within increasing severity of CAP. Although this ratio missed significance for mortality prediction, there was a tendency for a higher cortisol to DHEA ratio. The mechanism for this imbalance of adrenal hormone synthesis is unknown.Kolditz et al. found elevated levels of cortisol, DHEA and an increased DHEA/DHEAS ratio in patients who were clinically instable 72 hours after enrolment, but only cortisol was predictive for mortality So far, only one study evaluated DHEA and DHEAS levels in CAP Kolditz et al., which for our data provided only little differences in Odds Ratios There are limitations to our study. First, it is a secondary analysis of remaining blood samples. However, we analyzed a predefined secondary endpoint in prospectively randomized trial patients. Second, DHEA/S levels were measured at the time of presentation, that is, at different times during the day, not taking into account the fact that DHEA/S- levels show a certain diurnal and individual variability In conclusion, this study found that adrenal function measured by DHEAS and cortisol is prognostic for severity in CAP, while DHEA and cortisol are predictors for mortality.


P = 0.007). No relationship was established between these predominant symptoms and the aeroallergens used in this study. Intermittent mild and moderate/severe AR were evident in 13.5% and 31.1%, while persistent mild and moderate/severe were seen in 20.3% and 35.1%, respectively. House dust mites allergen yielded the highest number of positive responses (22.6%) followed by tree pollen (16.8%). No relationship was observed between the allergens tested and AR severity. Majority of patients were oligosensitive (33.8%) and polysensitive (35.1%) and were not significantly associated with AR severity (P = 0.07). Most AR patients presenting for treatment in Abuja, Nigeria, had moderate-severe persistent AR and showed similar SPT sensitization pattern with countries having similar climatic conditions. Sensitization patterns were not related to ARIA classification or predominant AR symptoms.Allergic rhinitis (AR) is prevalent in Nigeria, though little information exists on the allergen. We assessed the clinical features of AR patients in our environment based on the allergic rhinitis impact on asthma (ARIA) classification. Only patients with positive skin prick test (SPT) were recruited. Seventy-four patients participated in the study. AR and asthma comorbidity were observed in 13.5%. The proportion of “sneezers-runners” was higher than “blockers” with significantly more “sneezers-runners” having persistent AR ( Allergic rhinitis (AR) is a recurrent or chronic allergen specific, IgE-mediated hypersensitivity disorder affecting the nasal lining and characterized by nasal congestion, rhinorrhea, sneezing, nasal itchiness, and/or postnasal drip . It is cSkin prick test (SPT) is a standardized test widely used in the diagnosis of suspected cases of IgE-mediated allergy. It is considered as the gold standard in the diagnosis of allergy . GeneralTo date, there has been no information regarding the common allergen from the Federal Capital Territory (FCT) of Nigeria, Abuja. The aim of this study is to identify the clinical profile of AR patients according to ARIA guidelines and investigate the common allergens in Abuja, Nigeria.This cross-sectional study was conducted for a period of 18 months (March 2014 to September 2015). Sample population was based on the AR patients referred to the allergy clinic, affiliated to Medicaid Radiodiagnostic Center, Wuse 2, Abuja. Patients were consecutively recruited (convenient sampling method) and fall within the age range of 5 to 65 years. They were patients with a positive history of nasal inflammation  for at least one-year duration. Patients' symptoms were categorized as “sneezers-runners” and “blockers” based on the predominant complaint [SPT was performed on patients who have stopped taking antihistamines at least 5 days before the test, while patients with severe dermatographism were excluded from the study. Informed consent was obtained from all patients. SPT was performed by the same investigator.Dermatophagoides pteronyssinus and Dermatophagoides farinae mix), molds , animal dander (cat hair and dog epithelium), and cockroach extracts. Of the pollen allergen used in this study, about 80% of the plants are present in Abuja environment. SPT was performed according to international guidelines [ørsholm., Denmark) placed at least 2 cm apart to avoid contamination. A positive reaction is a wheal ≥ 3 mm in diameter. Histamine hydrochloride (1%) and normal saline (0.9%) were used as positive and negative controls, respectively. Patients with negative skin prick test were excluded from the study.A total of 22 allergens were used in this study; these allergens make up the subtropical prick test batch of ALK-Abello, Denmark. These include tree pollen , weed pollen , grass pollen ), house dust mites , molds, animal dander, and cockroach extracts. SPSS 16 software (Chicago Illinois) was used in the analysis and A total of 96 new patients with suspected allergic rhinitis presented at the allergic clinic within the study period. Only 74 patients from these had a positive SPT result and were enrolled into the study. There were more females (56.8%) than males. The youngest was 5 years and the oldest was 65 years. Their mean age was 30.8 years (95% CI 26.7 to 34.9 years). Twenty-one respondents (28.4%) were categorized as children (5 to 17 years of age) with a male : female ratio of 1.3 : 1. The majority of the study population resided in Abuja Municipal Area Council (AMAC) territory which is basically the city center.P = 0.007). Based on ARIA guidelines, most patients (67.2%) had moderate-severe AR (intermittent and persistent) and this was significantly related to animal exposure (P = 0.035) and not to age, gender, or family history of atopy. There was no significant relationship between these predominant symptoms and any of the aeroallergens.According to predominant symptoms, the proportion of “sneezers-runners” was higher than “blockers” (56.8% versus 43.2%). “Sneezers-runners” tend to have persistent AR, while “blockers” symptoms were more intermittent (P = 0.005). Moderate-severe persistent AR was more common among “sneezers,” while moderate-severe intermittent AR was common with the “blockers.” There was no significant association between AR severity and the presence of asthma (P = 0.26) or family history of atopy (P = 0.19).There was a significant association between AR severity and the predominant complaints  by the pP = 0.03). Also, house dust mites was significantly related to a positive family history of atopy (P = 0.035). There was no significant difference between the positive skin tests and gender as well as history of asthma. Only 23 of the 74 patients (31.1%) had sensitivity to one allergen. There was no single allergen that has the tendency for monosensitization.House dust mites allergen yielded the highest number of positive responses (22.6%) followed by tree pollen (16.8%). Weed pollen allergen yielded the least (7.4%), while animal dander and fungi allergen both came to 13.1% each . The senFurthermore, no relationship was observed between the allergens tested and duration of AR (intermittent and persistent), as shown in Most of the patients with a positive SPT were in the persistent AR category (66.2%). The number of allergens which produced a positive skin response from each patient was closely distributed. The highest was reaction to 3 or more allergens (35.1%) followed by reaction to 2 allergens (33.8%) and then to one allergen (31.1%), as shown in The prevalence of AR is increasing worldwide, yet it remains underdiagnosed and undertreated especially in developing countries . A self-About a third of the children 33%) in this study reported persistence or recurrence of rhinitis symptoms after adenoidectomy. This was lower than was observed by Colavita et al.  but sign3% in thiP = 0.007). We could not establish any relationship between these predominant symptoms and the aeroallergens used in this study. Studies by Shah and Pawankar [We recorded a higher proportion of “sneezers-runners” to “blockers” similar to the findings by Lee et al.  and ShahPawankar  and Deb Pawankar  observedThis study observed that a majority of AR patients were categorized as moderate-severe persistent AR, according to ARIA classification, while the least were mild intermittent AR. This has been observed by most studies , 5, 12 wNo relationship was established between the type of AR and the allergen to which the patients were sensitized. This was similarly observed in a national, cross-sectional study of AR patients in Mexico . The mosOur study also revealed a tendency for multiple sensitizations for allergens among the patients. This was statistically significant in all except animal dander. Thus a patient with positive sensitization to house dust mites could also be sensitized against pollen, insect, or fungi allergens. This supports the argument that time of exposure  does not properly define AR patients  and alsoIn conclusion, most AR patients presenting for treatment in Abuja, Nigeria, have moderate-severe persistent AR and show similar SPT sensitization pattern with other countries having similar climatic conditions. Sensitization patterns are not related to ARIA classification or any predominant AR symptoms but rather may rely on the environmental condition of study area and genetic makeup of the study population.


Firmicutes, whereas the LH group had a higher relative abundance of Bacteroidetes. These changes at the phylum level reflected different dominant genus compositions. Compared with the LH group, changes of Firmicutes and Bacteroidetes were mainly due to a significant decrease of Prevotella in the HH group and were primarily attributable to significant decreases of Bacteroides and Prevotella as well as a significant increase of Catenibacterium in the T group. In conclusion, our results suggest that high altitude may contribute to shaping human gut microbiota. Genetic and dietary factors may also explain the different microbiota compositions between Tibetan and Chinese Han.Knowledge about the impact of altitude and ethnicity on human gut microbiota is currently limited. In this study, fecal microbiota from 12 Tibetans (T group), 11 Chinese Han living in Tibet (HH group) and 12 Chinese Han living in Shaanxi province (LH group) were profiled by 454 pyrosequencing. Analysis of UniFrac principal coordinates showed significant structural changes in fecal microbiota among the three groups. There were significant differences in the composition of fecal microbiota among the three groups at phylum and genus levels. At the phylum level, the fecal samples of HH and T groups had higher relative abundances of  The microbes in our bodies collectively make up to 100 trillion cells and they encode 100-fold more unique genes than our own genomeet al. (2010)Xylanibacter and Prevotella, which are related to cellulose and xylan hydrolysis, were exclusively present in gut microbiota of Burkina Faso childrenet al. (2013)et al. (2012)et al. (2011)47et al. (2008)et al. (2014)Different geographic origins of humans may result in diverse compositions of gut microbiota, due to distinctive life environments, genetic background or dietary habits. De Filippo t al. 201et al. et al. (2014)Tibet is a typical high altitude area in China and has special environment conditions, such as low barometric pressure, low temperature, low humidity and high radiation. The population in Tibet is mainly composed of Tibetans and Chinese Han. Indigenous Tibetans have been living at high altitude for many years. The hypoxia of altitude (hypobaric hypoxia) would thus have exerted substantial evolutionary selection pressure. Tibetans adapt well to the hypoxia environment because of their different genotypes formed by long time natural selection, called genetic adaptation141514151618Individuals from low-altitude populations (Han) suffer from a number of potential diseases specifically related to the low levels of oxygen, when they move to live at high altitude2111In total, 442,737 raw reads were obtained from all 35 fecal samples. After filtering, 294,698 high-quality sequences were produced, with an average of 6923 ± 1191 reads per sample. The total number of OTUs at the 97% similarity level was 22,913 . The ShaFirmicutes, Bacteroidetes, and Proteobacteria while the dominant classes of all groups were Clostridia, Bacteroidia and Erysipelotrichi. At the phylum level, the most predominant phylum was Firmicutes, contributing 75.31%, 90.45% and 92.77% of the fecal microbiota in LH, HH and T groups respectively, followed by Bacteroidetes, contributing 23.59%, 7.98% and 4.69% respectively. The microbial composition varied greatly among individuals. Relative abundances of the phyla were analyzed by using a metastats test which either incorporates a paired t-test or uses a paired Wilcoxon rank sum testFirmicutes was significantly more abundant in the fecal microbiota of HH and T groups than that in the LH group , while Bacteroidetes was significantly less abundant in the fecal microbiota of HH and T groups than that in the LH group . Actinobacteria differed between LH and HH groups (p = 0.044). Cyanobacteria and Fusobacteria exhibited significant differences between HH and T groups , while Verrucomicrobia differed between LH and T groups (p = 0.027).Overall microbiota compositions for each group at the phylum and class levels are shown in p < 0.05), including Acidaminococcus, Actinomyces, Blautia, Butyricimonas, Clostridium, Desulfovibrio, Helicobacter, Leuconostoc, Peptostreptococcaceae Incertae Sedis, Prevotellaceae uncultured, Prevotella, RC9 gut group and Rhodococcus. On the other hand, 3 genera were significantly lower in the HH group than in the LH group (p < 0.05), including Butyricimonas, Oscillospira and Sutterella. The relative abundances of 16 genera were significantly higher in HH group than in T group (p < 0.05), including Actinomyces, Alistipes, Bacteroides, Barnesiella, Blautia, Clostridium, Enterococcus, Faecalibacterium, Gordonibacter, Helicobacter, Holdemania, Mitsuokella, Peptostreptococcaceae Incertae Sedis, Rhodococcus, Ruminococcaceae Incertae Sedis and Staphylococcus. On the other hand, 5 genera were significantly lower in the HH group than in the T group (p < 0.05), including Allisonella, Christensenella, Oribacterium, Pseudobutyrivibrio and Solobacterium. The relative abundances of 6 genera were significantly higher in the LH group than in the T group (p < 0.05), including Alistipe, Bacteroides, Barnesiella, Prevotella, Staphylococcus and Sutterella. On the other hand, 8 genera were significantly lower in the LH group than in the T group (p < 0.05), including Butyrivibrio, Catenibacterium, Lactobacillus, Leuconostoc, Oscillibacter, Prevotellaceae uncultured, Slackia and Solobacterium.Relative abundances of major genera detected and statistical analyses among three groups are shown in A weighted UniFrac principal coordinates analysis (PCoA) was performed to compare the overall structure of gut microbiota of all samples, based on the relative abundance of OTUs (at a 97% similarity level). There was an obvious separation of HH and T groups, while individuals were scattered for the LH group. Pco1, Pco2 and Pco3 accounted for 22%, 12% and 10% of the total variations, respectively .Other analysis methods  for the beta-diversity produced similar results .Following the PCoA analysis, the statistical analyses showed that microbiota composition was significantly affected by ethnicity with various distance matrixes, but altitude significantly affected the microbiota composition only in weighted UniFrac, unweighted UniFrac and Abund_Jaccard analyses . These rp = 0.023). However, total bacterial numbers per 10 ng DNA did not differ between LH and T groups or between HH and T groups   (Firmicutes (p = 0.017)  . In cont= 0.021) . Further= 0.017) .Our results have provided additional evidence that the geographic origin contributes to shaping human gut microbiota, as previously reported34Firmicutes and Bacteroidetes are two prominent phyla in gut microbiota of humans and have received much attention. Our results showed that Tibetans had a high relative abundance of Firmicutes and a low relative abundance of Bacteroidetes compared with Chinese Han living at low altitude. However, there was no difference between Tibetans and Chinese Han living at the same high altitude area. These results from pyrosequencing were further verified by qPCR. Interestingly, these changes of the Firmicutes and Bacteroidetes at high altitude were similar to the shift observed in patients with obesityet al. (2014)The gut microbiota of Tibetans was compositionally distinct from Chinese Han living at high or low altitude in this study. Firmicutes and Bacteroidetes in the T group were mainly due to significant decreases in genera Bacteroides (4.2% vs 0.6%) and Prevotella (16.7% vs 3.8%) and a significant increase in genus Catenibacterium (0% vs 4.3%). Furet et al. (2010)Bacteroides and Prevotella were negatively correlated with energy intake. Tibetans have higher energy intakes than Chinese Han (13.7 MJ/d vs 11.1 MJ/d)Bacteroides and Prevotella observed in Tibetans. In addition, Tibetans had a high level of Pseudobutyrivibrio (26.8%). The metabolites of genus Pseudobutyrivibrio are mainly formate, butyrate and lactatePseudobutyrivibrio as a part of the human microbiome is not yet known. While both T and HH groups had a high ratio of F/B, the HH group was mainly due to significant increases in genera Faecalibacterium (21.3% vs 13.4%) and Bacteroides (6.1% vs 0.6%) and a significant decrease in genus Pseudobutyrivibrio (6.2% vs 26.8%) in comparison with the T group. Several genome-wide studies confirmed that several genes showed significantly different genotype frequencies between Tibetans and Chinese Han14159Compared with the LH group, the changes of the et al. 20BacteroidPrevotella (2.2% vs 16.7%). Prevotella is associated with consumption of a diet rich in carbohydratesIn order to further investigate the environmental factors at high altitude on compositions of gut microbiota, we also compared the gut microbiota between Chinese Han living at low altitude or at high altitude. The total bacteria of Chinese Han in the HH group were significantly decreased in comparison with the LH group. The decreased microbial load could weaken the intestinal ecosystem stabilityClostridium, Desulfovibrio, Bacteroides, Lactobacillus and Prevotella among the three groups. These genera of microbiota are all associated with production of short-chain fatty acids (SCFAs)3In our study, we found significant differences in genera Firmicutes and a low abundance of Bacteroidete. However, these differences were due to different genus compositions. Compared with the LH group, the changes of the Firmicutes and Bacteroidetes in the HH group were mainly due to a significant decrease in Prevotella. However, in the T group these changes were primarily attributable to significant decreases in Bacteroides and Prevotella and a significant increase in Catenibacterium. Whether these changes of genera could affect adaption to high altitude needs further large-scale studies, taking socioeconomic status, sanitation and exercise intensity into consideration.In summary, our study revealed that humans living at high altitude (Tibetans and Chinese Han) had a high abundance of We enrolled 12 healthy Tibetan adults (T group) and 12 healthy Han adults (HH group) living in Tibet  and 11 healthy Han adults (LH group) living in Shaanxi  for this study. The individuals were all males within a narrow range of age (18–25 years old). They did not have bowel diseases or metabolic diseases and had not taken any antibiotics or probiotics in the three months before the sampling dates. All Tibetan participants were living in Tibet for generations and never left Tibet. Chinese Han in the HH group had lived in Tibet for more than 4 years. Detailed information of each participant is shown in Genomic DNA was extracted from fecal samples by using the E.Z.N.A. stool DNA Kit  according to the manufacturer’s instructions. The amount of DNA was determined by Nanodrop ND-2000 . Integrity and size of DNA were checked by 1% (w/v) agarose gel electrophoresis. All DNA samples were stored at −20 °C until further processing.TM PicoGreen double-stranded DNA assay . For sequencing, amplicons from each reaction were mixed in equal amounts based on concentrations and subjected to an emulsion PCR as recommended by 454 Life Sciences by using Roche GS FLX Titanium emPCR Kits . Using the Sequencing Method Manual XLR70 kit , sequencing was performed on the Roche Genome Sequencer GS-FLX Titanium platform by the Majorbio Bio-Pharm Technology .The V1-V3 region of the 16S ribosomal RNA (rRNA) gene from each DNA sample was amplified using the bacterial universal forward primer 27F: 5′- AGAGTTTGATCCTGGCTCAG-3′ and the reverse primer 533R: 5′-TTACCGCGGCTGCTGGCAC-3′ containing the A and B sequencing adaptors. The cycling parameters were as follows: 2 min initial denaturation at 95 °C; 25 cycles of denaturation at 95 °C (30 s), annealing at 55 °C (30 s), elongation at 72 °C (30 s); and final extension at 72 °C for 5 min. Three separate PCR reactions of each sample were pooled for pyrosequencing. The PCR products were separated by 2% agarose gel electrophoresis and purified by using the AxyPrepDNA Gel extraction kit . Amplicon DNA concentrations were qualified by using a Quant-iThttp://sourceforge.net/projects/seqclean) and Mothur (http://www.mothur.org/wiki/Main_Page) were used for removing low quality sequences (i) that did not perfectly match the PCR primer at the beginning of a read , (ii) that did not match a barcode and PCR primer at the end of a read (pairwise identity less than 95%), (iii) that were shorter than 200 base pair in length, (iv) that contained ambiguous and homologous nucleotides, or (v) sequence quality score was less than 25 .Valid sequences were sorted into different samples according to barcodes. The programs Seqcln (http://www.arb-silva.de/) and similar sequences with a minimum pairwise identity of 97% were clustered into operational taxonomic units (OTUs) using the Mothur program (http://www.mothur.org/wiki/Cluster). OTUs that reached a 97% similarity level were used for diversity (Shannon), richness (Chao), Good’s coverage, and rarefaction curve analyses using the Mothur program. Taxonomical assignments of OTUs exhibiting 97% similarity were performed using the Mothur (http://www.mothur.org/wiki/Classify.seqs) which uses a RDP Naive Bayesian classifierhttp://metastats.cbcb.umd.edu/). The number of permutations used to calculate the P value  was set to 1000.The high-quality sequences were aligned in accordance with the SILVA alignment (Firmicutes and Bacteroidetes 16S rRNA gene from a clone library was diluted from 1 × 103 to 1 × 109 (copies/ml) to construct standard curves for the detection of total bacteria, Firmicutes and Bacteroidetes. PCR reactions were performed with CFX96 system  using SYBR Premix Ex TaqII . The data are represented as the mean ±SD of triplicate qPCR values.In order to further confirm the pyrosequencing data, real-time qPCR reactions were performed as previously describedAccession number: The sequence information in this paper has been deposited in the GenBank Sequence Read Archive with accession number SRP035344.How to cite this article: Li, L. and Zhao, X. Comparative analyses of fecal microbiota in Tibetan and Chinese Han living at low or high altitude by barcoded 454 pyrosequencing. Sci. Rep. 5, 14682; doi: 10.1038/srep14682 (2015).


Introduction. The association of prenatal growth with nutritional status, metabolic syndrome (MS), and insulin resistance (IR) was studied in school-age children. Methods. A retrospective cohort study was designed linking present data of children with perinatal records. 3325 subjects were enrolled. Anthropometry, blood pressure (BP), and pubertal status were assessed. Blood lipids, glucose, and insulin were measured. Linear associations were assessed using the Cochran-Armitage test. Odds ratios and nonlinear associations were computed. Results. 3290 children  were analyzed. Prevalence of obesity, stunting, MS, and IR was 16.0%, 3.6%, 7.3%, and 25.5%, respectively. The strongest positive association was between birth weight (BW) and obesity (OR 2.97 (95% CI 2.01–4.40) at BW ≥ 4,000 g compared to BW 2,500–2,999). The strongest inverse association was between birth length (BL) and stunting (OR 8.70 (95% CI 3.66–20.67) at BL < 48 cm compared to BL 52-53 cm). A U-shaped association between BL and BP ≥ 90th percentile was observed. Significant ORs were also found for MS and IR. Adjustments for present fat mass increased or maintained the most prenatal growth influences. Conclusions. Prenatal growth influences MS, IR, and nutritional status. Prenatal growth was more important than present body composition in determining these outcomes. Nutritional conditions during pregnancy, infancy, and childhood have been proposed to have a major impact on the development of obesity and related metabolic traits such as metabolic syndrome (MS) and insulin resistance (IR) –4.Birth weight (BW), placental weight, and measures of size at birth have all been related to risk factors for chronic disease in adult life , 5. BirtThe MS is a set of risk factors that may appear during childhood and have been closely linked to IR . ObesityWe recently studied 2,152 Chilean schoolchildren aged 10–15 years, assessing possible associations between MS and IR to prenatal growth; however, nutritional status indicators were not included in those analyses . The maiThis study aimed to investigate the associations of prenatal growth with MS and IR, including indicators of nutritional status, using a larger sample size than the previously reported.A retrospective cohort study of school-aged children, from 20 public schools managed by the Municipality of Puente Alto, Santiago, Chile, was designed. The public schools reach nearly 40% of all primary schools in Chile . All chiIn addition to sex, perinatal data used in this study were birth weight (BW), birth length (BL), and GA. They were determined at the maternity hospitals immediately after delivery by trained personnel , 18. InfThe criteria of Cook et al. were used to define MS in the children, when at least three out of five of its components were present, as defined by the following cut-off points : WC ≥ 902 in m) expressed in percentiles and z-scores [The evaluations at each school were performed by a trained nurse and a nutritionist. Height and weight were measured using a stadiometer and a beam-scale Seca, with an accuracy of 50 g, while being barefoot and lightly clothed. The final height and weight were the respective averages of three measurements. We calculated body mass index  for 12 hours prior to drawing of blood; noncompliers were asked to return another day for the blood sampling. A single laboratory at our university was used for all blood analyses. This laboratory has been credited by the standard ISO 15189 for medical laboratories .Venous blood samples were collected for determination of glucose  and insulin ; this method measures concentrations of insulin from 0.5 to 300 mUI/mL (sensitivity of 0.5 mUI/mL) with a coefficient of variation of 3.48% and 6.17% for concentrations of 23.51 mUI/mL and 62.49 mUI/mL, respectively. The formula developed by Matthews et al.  was usedSocioeconomic status was estimated using as a proxy the number of maternal years of formal education as collected at birth by the civil registry . This in t-test for averages were used. Prevalence of MS and its components was computed at each category of the perinatal variables. The presences of linear trends in this prevalence were assessed using the Cochran-Armitage test, while nonlinear associations were evaluated allowing quadratic terms in a logistic regression. Multivariate analysis was performed using logistic regression allowing specific adjustments for sex, Tanner stage, and %FM; those factors could be confounders which potentially influence the dependent variables [The prevalence of MS and its components were described as number of cases and percentage, while perinatal variables used mean and standard deviation (SD). To assess differences by sex, Pearson's chi-square test for proportions and independent samples Student'sariables , 23, 34. P values and values ≤ 0.05 were considered statistically significant. SAS software version 9.1 was used to compute statistical analysis.All data analyses were performed using 2-sidedParents or their representatives signed an informed consent form and boys/girls an informed acceptance form. The study was approved by the Ethics Committees of the School of Medicine (Pontificia Universidad Católica de Chile) and the National Fund for Science and Technology (FONDECYT).P < 0.001). Complete information on studied perinatal variables was obtained for 3290 children (98.9%), of whom 52% were females. There were no significant differences between those who participated or those who did not in terms of age, years of maternal education, BW, and BL.Initially 5,614 subjects were eligible for the study, 2,616 females and 2,998 males. A total of 3,325 children and adolescents who presented the signed informed consent forms were evaluated and 2289 refused to participate; from the latter 34.5% were females and 47.4% males (P = 0.039) (data not presented).Based on maternal education attainment, the socioeconomic status distribution in the total group of eligible subjects was medium 60.1%, basic 33.0%, and superior 6.9%. Proportions in the actually studied subjects were similar: 60.9%, 32.5%, and 6.6%, respectively. There were no major associations observed with the study outcomes except for short stature which was slightly more frequent for basic education than the other categories of maternal education (z-score) did not differ by sex. Among boys, mean BW and BL were higher than in females  being higher in males than in females. Most anthropometric characteristics assessed were slightly higher among females; only BMI  and GA with WC ≥ 90th percentile (P for trend of 0.0733). In addition, a U-shaped association between BL and BP ≥ 90th percentile was observed. Another association was found between BL and stunting (P for trend < 0.0001); this association was not included in Several associations were observed between perinatal variables and the outcome variables studied . PositivThe lowest prevalence of obesity and WC ≥ 90th percentile at school-age was observed within the 2500–2999 g BW category and was therefore considered as the reference level. This prevalence increased with BW categories, almost tripling the odds for obesity and almost doubling the odds for WC ≥ 90th percentile at the highest category of BW. Adjustments for %FM increased most ORs. Adjustment for %FM, sex, and Tanner stage for obesity maintained similar values. Adjustments for %FM and for %FM, sex, and Tanner stage showed a slight increase of OR values for WC ≥ 90th percentile in the <2500 g BW category.BW had an inverse association with the prevalence of stunting. Crude ORs in the 3500 g and over categories were protective; those values did not change after adjustments for %FM but disappeared after further adjustment for sex and Tanner stage.BW did not show an influence on BP ≥ 90th percentile when assuming the 2500–2999 g BW category as reference . HoweverBW had an inverse influence on IR prevalence. Crude ORs in the 3000 g and over categories were protective; those values were stable after adjustments.52-53 cm BL was the category with the lowest prevalence of BP ≥ 90th percentile at school-age and was considered as the reference level. Both, the lowest and highest categories of BL doubled the risk of BP ≥ 90th percentile, depicting a U-shaped behavior. Adjustments for %FM and for %FM, sex, and Tanner stage maintained these ORs only for BL < 48 cm.BL categories below 50 cm were protective for obesity. ORs declined and were more protective when adjusting for %FM. These associations disappeared when further adjusting for sex and Tanner stage.52-53 cm BL was the category with the lowest prevalence of stunting at school-age; BL ≥ 54 cm category did not present stunted children. OR values < 50 cm showed a risk of at least 5 times the observed in the reference category. Adjustments for %FM and for %FM, sex, and Tanner stage maintained these ORs.One BL category, that is, 48-49 cm, increased the risk for HOMA-IR just when adjusting for %FM, sex, and Tanner stage.GA ≥ 41 weeks was the category with the lowest prevalence of BP ≥ 90th percentile, MS, and TG ≥ 110 mg/dL at school-age and was considered the baseline level. Both, crude and adjusted ORs of prematurity, defined as GA ≤ 37 weeks, behaved in a similar way showing the highest figures for BP ≥ 90th percentile, MS, and TG ≥ 110 mg/dL. No associations were found for GA and IR.This is the first study focusing upon associations between perinatal factors and nutritional status, MS, and IR in Chilean children. We observed that prenatal growth influences obesity characteristics, MS, IR, and stunting. In general, prenatal growth seemed to be more important in predicting these associations than present body composition because adjustments for present fat mass increased or maintained the most prenatal growth influences. Small body size at birth was associated with elevated BP, IR, and MS, while a large body size at birth was associated with measurements of obesity. The undeniable role of prenatal growth on metabolic outcomes appears, even in school-aged children, whose metabolic damages are only developing.The finding of a U-shaped association between BL and BP ≥ 90th percentile is consistent with the well-documented associations between both low and high values of BW and BL with many perinatal health indicators in previous reports , 35.The associations of BW with a higher prevalence of obesity or increased adiposity have been observed in previous studies , 36. In BL was positively associated with obesity, probably reflecting the well-known previously reported finding that weight and height are positively correlated during adolescence but not during adulthood .GA was inversely associated with important metabolic risk factors, BP ≥ 90th percentile, TG ≥ 110 mg/dL, WC ≥ 90th percentile, and MS as a whole, in these children. As previously mentioned prematurity was defined in this study as ≤37 weeks following recent national recommendations . This faNational proportions of BL < 50 cm and ≥54 cm are now reaching 44% and 2.5%, respectively, meanwhile national proportions of BW < 3000 g and ≥4000 g are now reaching 21.8% and 9%, respectively , 19, 35.Most of these results confirmed observations of a previous study . HoweverThere are some weaknesses in our study that need to be addressed. The sample comes from all public schools in Puente Alto, the county with the highest number of inhabitants in Chile reaching more than 600,000 . Cases tBody composition at school-age would need to be associated with the same information at birth and also during pregnancy . MoreovePrenatal growth is related to MS, IR, and nutritional status in this large sample of 3290 Chilean children. Fetal growth seemed to be generally more important in these associations than in present body composition because adjustments for present fat mass increased or maintained the most prenatal growth influences.The most significant results may include association of anthropometry at birth with MS, obesity, BP ≥ 90th percentile, and stunting. Other results confirmed previous observations regarding high risk of obesity and high risk of WC ≥ 90th percentile in newborns who weighed ≥ 4000 g at delivery. BW had a strong negative association with BP ≥ 90th percentile as previously reported. Interestingly, BL < 50 cm was associated with at least ~30% reduction in the risk of obesity and at least five times increase in the OR of stunting. The lowest and highest categories of BL were associated with a 2-fold increase in the risk of high BP. On the other hand, children who were born ≤37 weeks exhibited higher risk for BP ≥ 90th percentile, hypertriglyceridemia, and MS.


Background. The aim of this study is to investigate the protective effects of hemin (the heme oxygenase-1 [OH-1] inducer) against nephrotoxic effects induced by cisplatin [cis-diamminedichloroplatinum II (CP)] in male rats. Methods. The evaluation was performed through monitoring renal redox parameters: lipid peroxidation (LPO), glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione reductase (GR), and reduced glutathione (GSH). The work also examined renal function tests (urea and creatinine), tissue proinflammatory mediator like nitric oxide (NO), and kidney cytopathology. Results. A single intraperitoneal dose of CP (10 mg/kg b.w.) caused significant elevation of blood urea, serum creatinine, and renal LPO and NO, along with significant decline of the activities of GPx and GR, but renal SOD activity and GSH level were statistically insignificant as compared to control group. Subcutaneous injection of hemin (40 µmol/kg b.w.) partially ameliorated CP-induced renal damage, based on suppression of blood urea, serum creatinine, the renal MDA and NO levels, and increased antioxidant capacity in CP-treated rats. The results of histopathological and ultrastructural investigations supported the renoprotective effect of hemin against CP-induced acute toxicity. Conclusion. The induction of HO-1 by hemin is a promising approach in the treatment of CP-induced nephrotoxicity. However, further preclinical studies are warranted to test effectiveness of CP/hemin on the outcome of tumor chemotherapy. Cisplatin (cis-diamminedichloroplatinum(II), CP) is a highly effective chemotherapeutic agent against a large spectrum of tumor types –3. HowevCP and hemin (powder) were purchased from Sigma Chemical Company, USA. Other chemical reagents were of high-quality analytical grade. Hemin was first dissolved in 0.1 M NaOH, titrated to pH 7.4 with 0.1 M HCl, and then diluted with normal saline (1 : 10 v/v), while CP was prepared in normal saline. ad libitum. They were allowed 1 week of acclimatization before the initiation of the experiment. Experimental protocol of this study complies with the NIH ethical guidelines for the manipulation and care of laboratory animals. Rats were randomly assigned into 4 groups (n = 6):saline group (control): rats received 3 mL/kg 0.09% NaCl, intraperitoneally (i.p.);CP group: rats received a single dose of CP, i.p. (10 mg/kg);μmol/kg hemin, subcutaneously (s.c.), 1 h following CP;CP+hemin group: rats received 40 μmol/kg hemin, s.c.hemin group: rats received 40 Healthy adult male rats (110–140 g) were obtained from animal house facility at King Saud University, Saudi Arabia. Rats were housed in polyethylene cages under controlled laboratory conditions and provided with standard rat chow and waterDoses, duration, and routes of exposure were chosen according to previously published reports , 22. AllSerum levels of urea and creatinine were measured spectrophotometrically using commercial diagnostic kits , according to the methods described by Tabacco et al.  and BartMalondialdehyde (MDA), an index of fatty acid oxidation, was estimated in quantifiable amounts using Thiobarbituric Acid Reactive Substances (TBARS) assay kit  according to the method of Ohkawa et al. . In thisμm-thick) were cut, stained with haematoxylin and eosin solutions, and examined under light microscope .Kidney taken from each animal was fixed in 10% formalin solution, dehydrated in ascending series of ethanol, and embedded in paraffin. Sections (4 n = 3 per group) were fixed in 3% glutaraldehyde in sodium phosphate buffer  for 3 h at 4°C. Postfixation was in cold 1% osmium tetroxide  for 1 h. After flushing in phosphate buffer, the tissue samples were dehydrated in graded ethanol solutions and embedded in Araldite . Ultrathin sectioning (80–100 nm) was carried out using Leica EM UC6  ultramicrotome. Sections were mounted on grids, double stained with 2% uranyl acetate and lead citrate, and viewed under Jeol JEM 1011 transmission electron microscope  at 80 kV.Small slices of kidney cortex . Data were presented as mean ± standard error (SE).All variables were compared using one-way analysis of variance (ANOVA) followed by LSD multiple range test. Differences at The results of biochemical analysis in all studied groups are shown in Representative light micrographs of renal cortex are illustrated in Renal cortical tissues of control animals revealed normal appearance of ultrastructural patterns of renal corpuscles and tubules Figures . ObviousKidneys are dynamic organs and represent one of the major homeostasis body systems; they are affected by diverse varieties of chemicals and drugs –36. Plat+2)/ferritin. BV formed in this reaction is rapidly recycled by the action of biliverdin reductase into bilirubin (BR) which has a higher free radical-scavenging activity and antiapoptotic properties [Development of therapies to prevent free radicals generation may influence renal oxidative damage and protect against irreversible cell damage and necrosis induced by CP. Excessive ROS production by CP causes antioxidant imbalance and leads to progressive LPO and antioxidant depletion. LPO adducts were previously shown to mediate glomerular lesions . In addioperties –49. In aoperties . Kim et operties  demonstrκB) which upregulates the transcription of the iNOS gene [Treatment with CP overdose induces nitrosative stress by NO and other nitrosylating agents and this was correlated with the expression of the inducible nitric oxide synthase (iNOS) protein . In the NOS gene , 62, 63.In conclusion, this study is the first to demonstrate the protective role of hemin (an HO-1 activator) against acute nephrotoxicity induced by CP. Treatment of hemin ameliorated renal ultrastructural changes and dysfunction, and this was associated with reduction of LPO, overstimulation of antioxidant capacity, and suppression of NO biosynthesis, the proinflammatory mediator. However, further preclinical studies are needed to verify whether coadministration of CP and hemin could affect the outcome of tumor chemotherapy.


Xenorhabdus indica KB-3, a well-known protease producer, was isolated from its entomopathogenic nematode symbiont Steinernema thermophilum. Since medium constituents are critical to the protease production, the chemical components of the selected medium (soya casein digest broth) were optimized by rotatable central composite design (RCCD) using response surface methodology (RSM). The effects of all five chemical components (considered as independent variables), namely tryptone, soya peptone, dextrose, NaCl, and dipotassium phosphate, on protease production (dependent variable) were studied, and it was found that tryptone and dextrose had maximum influence on protease production. The protease production was increased significantly by 66.31% under optimal medium conditions . To best of knowledge, there are no reports on optimization of medium component for protease production by X. indica KB-3 using RSM and their application in fibrinolysis. This study will be useful for industrial processes for production of protease enzyme from X. indica KB-3 for its application in the field of agriculture and medicine. Xenorhabdus is a unique genus of bacteria, which is symbiotically associated with entomopathogenic nematode of genus Steinernema. Xenorhabdus spp. are known to produce a plethora of secondary metabolite-like antibiotics , toxins (both endo and exo), and extracellular enzymes, namely, lipases, esterases, chitinase, phospholipases, and proteases. These metabolites not only have diverse chemical structures, but also have a wide range of bioactivities with medicinal and agricultural interests. Proteolytic enzymes are recognized by their catalytic type and are classified as aspartic, cysteine, metallo, serine, threonine, and several others that are yet to be classified. Most proteolytic enzymes are classified as metalloproteases. Several researchers have documented the secretion of metalloproteases by Xenorhabdus strains previously [ Xenorhabdus indica KB-3 isolated in the present study produces serralysin type of proteases [ Xenorhabdus [ X. indica KB-3 and its application in fibrinolysis.eviously –3. The Xroteases  like feworhabdus , 2, 5, 6orhabdus . Serratiorhabdus –10. Due orhabdus . For masorhabdus . On the orhabdus , 14. TheX. indica strain KB-3, a nematode symbiont, was isolated from Steinernema thermophilum obtained from the soil of Kerala, India. The strain had been identified to be X. indica according to its morphological and molecular characteristics [ X. indica KB-3 was maintained on nutrient agar (NA) slants supplemented with 0.04% (w/v) triphenyltetrazolium chloride and 0.025% (w/v) bromothymol blue and subcultured monthly [ X. indica KB-3 into 50 mL fresh soya casein digest medium [eristics . X. indi monthly . Seed cunutrient broth  supplemented with skim milk (1%) and gelatin (1%) separately,nutrient broth half strength  supplemented with skim milk (1%) and gelatin (1%) separately,soya casein digest medium  .For optimum production of protease enzyme, initially, five different media combinations were screened to find a suitable nutrient medium for protease enzyme production, which are as follows:The media were inoculated with 2% (v/v) seed culture into 50 mL of respective medium and incubated at 28°C, 150 ×g rpm for 24 h. Samples were withdrawn at intervals of 3 h up to 24 h and centrifuged at 10,000 rpm for 5 min. The cell pellet was discarded, and the supernatant was preserved at 4°C for enzyme analysis. X. indica KB-3. Before optimization, the important factors were screened by Plackett-Burman (PB) factorial design. After screening, optimization was done by rotatable central composite design (RCCD) using response surface methodology (RSM) by experimental design module of Design Expert-7 . Optimization of the chemical components and physical parameters was carried out separately assuming no interaction took place between these factors. Initially physical parameters were optimized using RSM separately (data not shown) followed by chemical optimization under optimal physical conditions .Tryptone, soya peptone, dextrose, NaCl, and dipotassium phosphate were considered as chemical components while pH, temperature, incubation time, and inoculum percentage were considered as physical parameters for protease production from X. indica KB-3. To determine the significant chemical factors for protease production, a total of five chemical components at three levels  were involved in the determination of chemical components: (1) tryptone, (2) soya peptone, (3) dextrose, (4) NaCl, and (5) dipotassium phosphate. Tables t-test.In preliminary studies, “one-factor-at-a-time” approach was used to evaluate various factors for their suitability to sustain good production of protease byA) tryptone, (B) soya peptone, (C) dextrose, (D) NaCl, and (E) dipotassium phosphate, were selected to find the optimum chemical components for protease enzyme from X. indica KB-3 using RCCD. The ranges and levels of the variables taken for RSM are listed in k + 2k + no, where k is the number of independent variables and no is the number of repetitions of the experiments at the centre point. A total of 50 sets of experiments including eight center points were conducted along with different combination of chemical components. Each numeric factor is varied over 5 levels , that is, plus and minus alpha , plus and minus one , and zero (center point). The response values (Y = protease activity) in each trial were the average of the triplicates. The full experimental plan along with coded variables is mentioned in All five chemical components,  for analysis of regression coefficient, prediction equations, and case statistics. The experimental results of RSM were fitted using the following second order polynomial equation:μL enzyme solution and 250 μL of 2% w/v azocasein (Megazyme) in water bath at 30°C for 30 min [The culture broth centrifuged at 10,000 rpm for 5 min was used as enzyme source. Protease activity was measured by incubating 150 r 30 min . After iExtracellular protease enzyme was extracted from cell suspension grown under optimized soya casein digest medium after 18 h of incubation by centrifugation at 10,000 rpm. Purification and characterization of protease enzyme were carried out as described previously .μL of the protease enzyme was inoculated onto the plate using agar well assay method. The plate was incubated for 5 h at 37°C and the presence of the clear hydrolytic zone was measured. A clear zone of fibrin hydrolysis confirms the presence and the potency of the fibrinolytic activity. Plasmin from human plasma (Sigma-Aldrich) was used as positive control.Fibrin degradation analysis was performed by modified method of Datta et al. (1995) and Mahajan et al. (2012) , 18. TheAmong different growth media, the highest proteolytic activity (955.68 U/mL) was observed in soya casein digest broth after 18 h, and 832 U/mL activity was observed with skim milk supplemented nutrient broth. Therefore, soya casein digest broth was selected for further optimization study using statistical approaches. Physical parameters were optimized first  followed by optimization of different chemical components of soya casein digest medium using COVT method, Plackett-Burman design, and response surface methodology (RSM). X. indica strain KB-3 was estimated to be 5 g/L, producing 1453 U/mL within 18 hrs . The optimum tryptone concentration for protease production was determined to be 5.0 g/L. Protease production increased consistently with the increase in tryptone concentration from 2.5 to 5.0 g/L and decreased with further increase in tryptone concentration .The optin 18 hrs . X. indica was studied in terms of NaCl concentration. The NaCl concentration was varied from 1 to 10 g/L and maximum protease production was observed at 6 g/L NaCl concentration , dextrose (C), and NaCl (D), having low P value  were identified to be the significant variables for protease production, but tryptone (A) and dipotassium phosphate (E) were identified as insignificant variables because of high P value. Tryptone is the main protein source of the medium, and during COVT experiments, it was found that the tryptone is one of the important factors and had major influence on protease activity, whereas potassium is a known inducer for alkaline proteases [t-test for ANOVA , soya peptone (B), dextrose (C), NaCl (D), and dipotassium phosphate (E), were examined through RSM following RCCD matrix. The experimental data along with the actual and predicted yield of protease obtained is given in RSM was used to evaluate the relationship between different independent variables and their interactive effects on protease production byY is the response (protease activity in U/mL) and A, B, C, D, and E are the coded values of the independent variables, namely, tryptone, soya peptone, dextrose, NaCl, and dipotassium phosphate, respectively.Following quadratic model of response equation in terms of coded variable and actual variables was obtained as follows:P < 0.05). The linear terms of A, C, and E were significant at P < 0.05 level. The result showed that linear effect of B and D as well as the interactive effects of AD, BD, and BE was not significant, and therefore these terms were removed from final model equation. After excluding insignificant model terms B and D, models become nonhierarchical. To avoid nonhierarchy, only interactive terms AD, BD, and BE were excluded and parameters were recalculated . The aptness of the model was checked by the coefficient of determination (R2), which was calculated to be 0.9747, indicating that 97.47% of the variability in the responses could be explained by the model. The computed F-value of 0.83 suggests insignificance of the lack of fit. The model was found to be highly significant and sufficient to exemplify the actual relationship between the response and the significant variables as indicated by the small model P value (P < 0.05), large lack-of-fit sum of square value (19313.66). The predicted coefficient of determination (predicted R2 = 0.9384) is in reasonable agreement with the adjusted coefficient of determination (adjusted R2 = 0.9612). Significance of fifteen model terms  and an adequate precision of 27.29 acknowledged low signal to noise ratio (a ratio greater than 4 is desirable)  .To analyze the effects of the independent variables and interactive effects of each independent variable for maximum protease production, 3D response surface curves and the 2D contour plots were drawn against two experimental variables while the other variables were maintained constant at its central level .α, the protease activity decreased. The 3D plot and its respective contour plot expedited the identification of the optimal levels of tryptone and dextrose. The optimal concentration of tryptone was around 5.71 g/L and dextrose was around 1.45 g/L.Figures  X. indica KB-3. The predicted protease activity was 1575.11 U/mL under the optimum conditions, that is, tryptone—5.71, soya peptone—4.9, dextrose—1.45, NaCl—6.08, and dipotassium phosphate—0.47 in g/L. The model was validated by comparing the observed and predicted value from model equation  of hydrolysis than that of plasmin , which cX. indica is a potent producer of proteolytic enzymes and medium composition can significantly affect their production. This is the first ever optimization study on alkaline metalloproteases produced from Xenorhabdus sp. This study was concentrated on the optimization of the medium composition for the protease production from X. indica KB-3 through central composite design falling under response surface methodology (RSM) by experimental design module of Design Expert-7 . The use of RSM empowers a better understanding of the possible interactions between all chemical component variables. The model developed in the present study supports the analysis of the influence of the chemical components on protease activity of X. indica KB-3. The CCD experiment was very helpful in deciding the optimal concentrations of individual chemical components and determining the interaction effects among the factors. Significant interaction between tryptone and soya peptone (AB), tryptone and dextrose (AC), tryptone and dipotassium phosphate (AE), soya peptone and dextrose (BC), dextrose and NaCl (CD), dextrose and dipotassium phosphate (CE), and NaCl and dipotassium phosphate (DE) revealed their importance in the production medium for maximum production of protease enzyme. The regression analysis showed that the interaction of different chemical variables opted in this study was significant. The model was validated by comparing the observed and predicted values at the optimal value and experiments were carried out under optimized condition. After optimization, an overall 66.31% increase in protease activity was achieved, which confirms the reliability of model for predicting the optimized condition. Xenorhabdus nematophila (NCBI accession number: gi/300724813) [Further purification and characterization of protease were carried out and approximately 8-fold purification was achieved. Characterization of protease, based on different pH and temperature, showed optimum protease activity at 34°C at pH 8.2. Different protease inhibitor study and MALDI-TOF/TOF analysis revealed its resemblance with serralysin type alkaline metalloproteases from X. indica. Area of zone of hydrolysis on fibrin plate was around 15 mm, which is almost equal to that of plasmin (16.5 mm). Therefore, it can be assumed that protease of the present study has stronger fibrinolytic activity like plasmin. The size of the clear hydrolytic zone did not change in the presence of plasminogen, suggesting that protease was a plasmin-like protease, which could directly degrade fibrin but not plasminogen activators such as urokinase (UK), streptokinase (SK), and tissue plasminogen activator (tPA). Therefore, secondary effects such as platelet activation related to plasmin formation could be avoided [ X. indica shares plasmin-like protease character, where it can be effectively used as new resource for thrombolytic agents and can be used to develop therapeutic agents for the treatment of thrombosis. The formulation may also find several biotechnological applications in ointments for topical application to control the skin inflammations.Fibrinolytic activity confirms the serralysin nature of purified alkaline metalloproteases of avoided . This is X. indica KB-3 using RSM and their use in fibrinolysis. The optimized medium resulted in 66.31% increase of protease production and the model is found efficient, simple, less time consuming, and importantly significant. This work will be useful for industrial processes for production of protease enzyme from X. indica KB-3 and it also expedites its use in the field of medicine.To the best of our knowledge, this is the first report on optimization of medium components for protease production by


We have observed different clinical responses to botulinum toxin A (BTX-A) in patients who had similar urodynamic parameters before the procedure. Furthermore, some bladders evaluated by cystography and cystoscopy during the procedure had different characteristics that could influence the outcome of the treatment. The aim of this study was to assess whether cystography and urodynamic parameters could help predict which patients with neurogenic detrusor overactivity (NDO) refractory to anticholinergics respond better to treatment with injection of BTX-A.In total, 34 patients with spinal cord injury were prospectively evaluated. All patients emptied their bladder by clean intermittent catheterization (CIC) and had incontinence and NDO, despite using 40 mg or more of intravesical oxybutynin and undergoing detrusor injection of BTX-A (300 IU). Pretreatment evaluation included urodynamic, and cystography. Follow-up consisted of urodynamic and ambulatory visits four months after treatment. The cystography parameters used were bladder shape, capacity and presence of diverticula. Urodynamic parameters used for assessment were maximum cystometric capacity (MCC), maximum detrusor pressure (MDP), compliance and reflex volume (RV).After injection of BTX-A, 70% of the patients had success, with 4 months or more of continence. Before the treatment, there were significant differences in most urodynamic parameters between those who responded successfully compared to those who did not. Patients who responded successfully had greater MCC (p = 0.019), higher RV (p = 0.041), and greater compliance (p = 0.043). There was no significant difference in the MDP (0.691). The cystography parameters were not significantly different between these groups bladder shape (p = 0.271), capacity (p > 0.720) and presence of diverticula (p > 0.999). Statistical analyses were performed using SPSS (version 20.0) and included Student’s t-test for two paired samples and Fisher’s exact test, with a significance threshold of 0.05.This study suggests that the cystography parameters evaluated cannot be used to help predict the response to injection of BTX-A in the treatment of refractory NDO. However, the urodynamic parameters were significantly different in patients who responded to the treatment, with the exception of the MDP. Botulinum toxin (BTX), which was described by Van Ermengem  in 1897,The treatment of neurogenic detrusor overactivity (NDO) with an injection of BTX-A into the detrusor muscle was introduced in 2000 . This thIn previous studies, we have observed different clinical responses to BTX-A in patients who had similar urodynamic parameters before the procedure . This stThirty-four patients with spinal cord injury who received injections of BTX-A between January 2012 and July 2013 participated of this prospective observational study. The sample size was determined by G*Power software, version 3.1.7  for two All procedures were performed in a hospital under general anesthesia. Antibiotics were administered orally, according to a urine culture, for seven days. The procedure performed on the fifth day of the antibiotic, all patients tested positive for bacteria. BTX- A  was diluted in sterile saline to a final concentration of 10 units/ml. Using a 19-Fr Storz cystoscope and 5 FR needle, a total of 300 IU (30 ml) was injected into 30 sites of the detrusor muscle, sparing the trigone region, as described in Schurch et al. . PatientOf the 34 patients, 23 were male and 11 were female. The mean age was 31.2 ± 10.3 years (mean ± standard deviation), with a range 19–55 years. There were 28 paraplegic and 6 tetraplegic patients and 25 traumatic and 9 non-traumatic cases. The mean time since SCI was 5.9 ± 4.4 years, with a maximum of 19 years and a minimum of 1 year. The patients tolerated all of the procedures and showed no acute complications related to the injections. Thirty patients (88.2%) were completely continent after the procedure. Six of these patients, however, showed little clinical response at four months and were considered unsuccessful. Four patients (11.8%) remained incontinent four months after treatment, although there were improvements in most urodynamic parameters and urinary losses. Twenty-four (70%) patients remained continent for more than 4 months and were considered successful. Twelve patients (35.2%) had an acontractile detrusor bladder after surgery. After four months, the following changes in the urodynamic parameters were observed: increased MCC (P <0.001), increased RV (p <0.001) and decreased MDP (p <0.001). The compliance did not change significantly (p = 0.366)  had no diverticula, eight patients had between 1 and 10 diverticula and 2 patients had more than 10 diverticula. By Fisher’s exact test, there was no association between the response and the presence of diverticula (p > 0.999), bladder shape (p <0.271) or bladder capacity (p = 0.720) Table .After 4 months of follow-up, twenty patients 58.8%) had reduced the dose of anticholinergics, five (14.7%) had discontinued their use and nine (26.5%) had not changed the dose . In this study, continence was observed over a period of more than 4 months in 70% of the patients undergoing treatment with BTX-A. In studies with similar populations of patients, the percentage of patients with continence after injection of the toxin ranged from 42 to 87% . Karsent;6. In thAlthough it has been reported that alterations can occur in the detrusor muscle of the NDO ,16, it wRegarding urodynamic parameters, we observed in this study that the bladders that had better compliance, greater capacity and increased reflex volume before treatment showed a better response to treatment. Furthermore, there was no significant change in compliance after surgery (p = 0.366), suggesting that compliance is related to alterations in the bladder wall, is unresponsive to drug therapy, and is, therefore, directly related to the treatment response. This is in contrast to a study conducted by Klaphajone J , in whicThis study suggests that the cystography parameters evaluated cannot be used to predict the response to BTX-A injection for the treatment of refractory NDO. It was observed in the urodynamic parameters, that patients whose bladders had higher cystometric capacity, greater reflex volume and greater compliance showed better results after treatment. The bladder compliance showed no significant improvement after the procedure, which is an important factor to be noted. A larger study with a multivariate analysis would be appropriate to clarify the results of this work.NDO: Neurogenic detrusor overactivity; BTX-A: Botulinum toxin A; BTX: Botulinum toxin; CIC: Clean intermittent catheterization; RV: Reflex volume; MDP: Maximum detrusor pressure; MCC: Maximum cystometric capacity.The authors declare that they have no competing interests.RAA conceived of the study and carried out the acquisition, analysis and interpretation of data and was involved in drafting the manuscript. IDA provided substantial contributions to the conception and design and revised the manuscript critically for important intellectual content. MDS participated in the study design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2490/14/66/prepub